Although there are no nationwide data on prevalence of PIDs in India, based on statistical projections it is estimated that the number of patients with PID is likely to be more than one million (4, 5). Hitherto, there was also paucity of published literature about PIDs from India as very few centers in the country had the clinical experience, laboratory facilities, and technical wherewithal for diagnosis and management of these conditions. As a result, an overwhelming majority of these patients continued to remain undiagnosed and untreated.

However, despite these limitations and challenges, India has advanced significantly in the field of PIDs in the last few years. The Indian Society for Primary Immune Deficiency (ISPID) was founded in 2011 in close liaison with the Foundation for Primary Immunodeficiency Diseases (FPID), USA. The Indian Council of Medical Research (ICMR) has also been at the forefront of activities related to PIDs in India. In this review, we highlight the progress in the field of PIDs in India over the last 25 years, the difficulties faced by clinicians across the country, the current state of PIDs in India and the future prospects.

#### STATUS OF PIDs IN INDIA PRIOR TO 2011

The first two cases of PIDs in India were reported in the year 1964—these were two case reports of boys with Wiskott–Aldrich syndrome (WAS) and agammaglobulinemia, respectively (6, 7). Subsequently, five families with ataxia telangiectasia were reported in the year 1973 (8). Since then several cases of PIDs have been reported from Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh and many other institutions across the country; however, the diagnosis was largely clinical and the immunological workup rather limited. Prior to 1992 (between 1975 and 1991), 59 cases of PIDs had been diagnosed in the country (5). Gupta et al. published their experience of the spectrum of PIDs seen at two large pediatric centers in the country [Advanced Pediatrics Centre, PGIMER, Chandigarh and the National Institute of Immunohaematology (NIIH) and B.J. Wadia Children's Hospital, Mumbai] and highlighted the difficulties in identification, evaluation (including molecular diagnosis) and management of these patients (5). The data were collected from 1992 onward. Between 1992 and 2010, a total of 153 and 122 patients had been diagnosed to have PIDs at Chandigarh and Mumbai respectively. There were important differences in spectrum of PIDs seen at both these centers. Antibody deficiency disorders [including X-linked agammaglobulinemia (XLA), common variable immunodeficiency (CVID), selective IgA deficiency with or without IgG2 subclass deficiency, isolated IgG2 subclass deficiency, and transient hypogammaglobulinemia of infancy] were the most common PIDs diagnosed in Chandigarh; however, the most common PID diagnosed at Mumbai was familial hemophagocytic lymphohistiocytosis (HLH). Cases of disorders of immune regulation (including HLH and autoimmune lymphoproliferative syndrome) were predominantly seen at Mumbai while only a few cases had been diagnosed at Chandigarh. Similarly, phagocyte defects [including neutropenia, leukocyte adhesion defect (LAD), and disorders of IFNγ-IL12 pathway] were predominantly being diagnosed at Mumbai with only a handful of cases at Chandigarh. On the other hand, WAS, hyper-IgE syndrome, ataxia telangiectasia, hereditary angioedema, and chronic mucocutaneous candidiasis were more common at Chandigarh. There could be several reasons for the difference in spectrum of PID seen at these two centers. These could be (1) difference in genetic constitution of the population in North and West India due to differences in racial ethnicities; (2) referral bias (the Mumbai centre is primarily a hemato-oncology unit and tends to attract more cases of HLH and phagocytic defects; the Chandigarh facility on the other hand is a pediatric center and sees the entire spectrum of PIDs); and (3) difference in rate of consanguineous marriages (much higher in Western and Southern India as compared to North India). The diagnosis of PIDs at both centers was being established based on clinical manifestations supplemented by few laboratory investigations including protein expression by flow cytometry and a few functional assays. Facilities for molecular analysis were limited at that time. However, both centers had been able to get some molecular analyses done on individual patients for diagnostic purposes through collaborative efforts with overseas laboratories specializing in PIDs. Major challenges in the diagnosis and management of cases of PID in India prior to 2011 were lack of awareness among general population and physicians, lack of appropriate diagnostic facilities and cost constraints in management of patients diagnosed to have PIDs especially with regard to access to immunoglobulin replacement and hematopoietic stem cell transplantation (HSCT).

### The FPID1

The FPID was co-founded by Dr. Sudhir Gupta and Dr. Abha Gupta in the USA. This foundation has the avowed aim of supporting education, early diagnosis, genetic counseling, therapy, and research in PID in both India and the USA. The FPID is spearheading efforts at establishing PID centers in India and is currently supporting activities at five institutions in the country, i.e., at PGIMER, Chandigarh; Sir Ganga Ram Hospital (SGRH), New Delhi; Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow; Nizam's Institute of Medical Sciences (NIMS), Hyderabad; and NIIH, Mumbai (Figure S1 in Supplementary Material). It is also supporting treatment of some patients with hypogammaglobulinemia in India by provision of intravenous immunoglobulin (IVIg) replacement and partial support toward HSCT.

#### Indian Society for Primary Immune Deficiency2

The ISPID was registered in March 2011. Very few developing countries have a dedicated society for PID—India is one of them. ISPID has been working toward increasing awareness regarding

1www.fpid.org.

2www.ispid.org.in.

PIDs and establishment of diagnostic support and research centers in the country. ISPID organizes a national and an international conference on PID every alternate year and a Continuing Medical Education program every year with the support of FPID, USA. Till date four such national and international conferences have been organized by the ISPID in India. The faculty at these conferences has consisted of world renowned scientists at the cutting edge of research in PID.

#### Indian Patients Society for Primary Immunodeficiency (IPSPI) 3

Indian Patients Society for Primary Immunodeficiency is a national non-profit organization for PIDs, which was established in 2004 and registered in 2005. It was the first patient society for PIDs in our country. It is based at Bhubaneshwar in Eastern India. IPSPI is affiliated with the International Patient Organization for Primary Immunodeficiencies. This organization works to improve the quality of life of individuals with PIDs in India.

#### Primary Immunodeficiency Patients Welfare Society (PIDPWS) 4

The second patient care society for PID in India was founded in April 2012 and registered in September 2012. Like the IPSPI, this society has also been established by parents of the children suffering from PIDs. PIDPWS is based at Bengaluru in South India and has been working for the improvement in quality of life of PID patients.

### STATUS OF PIDs IN INDIA AFTER 2011

Since the inception of ISPID in 2011, there has been a significant progress in the spread of awareness and setting up of diagnostic facilities for PIDs. The PGIMER, Chandigarh initiated a 3 years postdoctoral (DM) training course in Pediatric Clinical Immunology and Rheumatology in January 2014. This course is the first of its kind in India. The basic aim of this program is to train pediatric fellows and to provide them enough expertise in the field of immunology that can be disseminated across the country. Two fellows have already completed their training and are now working in Guwahati (East India) and Bengaluru (South India), respectively. In addition, six more trainees are currently pursuing this course at the institute. The institute has also started a 3 years postdoctoral (DM) training course for adult physicians in July 2014. Though several other institutes in the country have been running similar postdoctoral (DM) courses in Clinical Immunology (e.g., SGPGIMS, Lucknow and Jawaharlal Institute of Postgraduate Institute of Medical Education and Research, Puducherry) and in Rheumatology (e.g., Madras Medical College, Chennai; NIMS, Hyderabad; Christian Medical College, Vellore; and King George's Medical University, Lucknow). However, the major focus of these institutes has been clinical rheumatology rather than PIDs.

## CENTRE FOR ADVANCED RESEARCH (CAR) FOR PIDs IN INDIA

Clearly, PID care in India is still in its infancy, and we have a long way to go. There are very few centers in the country with clinical expertise and diagnostic facilities for PIDs. As a result, majority of patients with PIDs continue to remain undiagnosed. The ICMR helped set up the CAR in primary immunodeficiency disorders at PGIMER, Chandigarh in January 2015. The second such ICMR CAR in PID has recently been set up at the NIIH, Mumbai. The major objectives for these CARs are training of physicians/scientists, development and standardization of diagnostic facilities, establishment of normograms, and creation of state of the art facilities for diagnosis and management of PIDs.

### Asia Pacific Society for Immunodeficiencies (APSID)5

Asia Pacific Society for Immunodeficiencies was established in April 2015 by a group of Asian pediatricians and immunologists working in the field of PIDs. The aim of this society is to improve management of PIDs through increasing awareness and promoting collaboration among member countries. The APSID has been organizing Spring Schools for young clinicians who are in training for immunology and infectious diseases. The first conference of the APSID was held in 2016 in Hong Kong from 30th April to 1st May 2016. The APSID would provide a much needed fillip to PID-related activities across countries in the large Asia Pacific region which is home to ~60% of the world population.

### PUBLICATIONS ON PID FROM INDIA—A MAJOR THRUST IN LAST 6 YEARS

There has been a significant increase in the number and quality of publications on PID from India in the last 6 years. Publications prior to 2011 were mostly case reports and clinical reviews; however, the more recent ones contain a plethora of molecular and genetic details. This is a welcome change.

#### Search Strategy

The following search terms were used to find out the number of PubMed publications in PID in the last 50 years in India: India and Primary immune deficiency; Wiskott–Aldrich syndrome and India; Severe combined immunodeficiency and India; X-linked agammaglobulinemia and India; Chronic granulomatous disease and India; Neutropenia and India; Leucocyte adhesion defect and India; Hyper IgE syndrome and India; Hyper-IgM syndrome and India; Chédiak–Higashi syndrome and India; Hemophagocytic lymphohistiocytosis and India; DOCK-8 deficiency and India; Papillon–Lefevre syndrome and India; Ataxia telangiectasia and India; Mendelian susceptibility to mycobacterial diseases and India; Chronic mucocutaneous candidiasis and India; HSCT and Primary Immune deficiency and India; Intravenous immunoglobulin and India and Primary immunodeficiency.

5www.paed.hku.hk.

3www.ipspiindia.org.

4www.pidindia.net.

Based on this search strategy, we were able to access 311 publications on PID from India (**Figure 1**).

### IMMUNOGLOBULIN REPLACEMENT THERAPY IN INDIA

Immunoglobulin replacement therapy is the cornerstone of treatment for antibody deficiency diseases, arguably the most common clinically significant group of PIDs. Immunoglobulin can be administered by both intravenous and subcutaneous routes; however, subcutaneous immunoglobulin preparations are presently not being marketed in India. Cost of IVIg therapy is a major constraint in the management of children with PIDs in India, as also in other developing countries. However, with recent policy changes initiated by the Federal Government there has been a significant price reduction in IVIg formulations in India. A 5 g vial of IVIg presently costs around 120–140 USD. With the help of philanthropist organizations, FPID and several state governments, IVIg therapy is now being offered to many patients with hypogammaglobulinemia in India. The state government of Punjab (in North India) has taken a major step in this context and is freely providing IVIg to all patients with hypogammaglobulinemia. The state government of Karnataka (in South India) has also taken a major policy initiative and has agreed, in principle, to provide IVIg to patients with PIDs. Because of cost constraints, several of our patients with hypogammaglobulinemia are unable to get the recommended dose of IVIg. Suri et al. recently analyzed the trough levels of IgG in a cohort of 14 boys with XLA at a single center in Chandigarh and correlated the levels with the monthly replacement dose of IVIg and risk of infections (9). This study revealed that the mean dose of IVIg was 414 mg/kg/month and the mean trough IgG level of the entire cohort was 435 mg/dl. It was further observed that a median dose of IVIg of 397 mg/kg and a median trough IgG level of 354 mg/ dl was protective for majority of the children. These cutoff levels are much lower that what has hitherto been considered the norm in patients with hypogammaglobulinemia. However, these findings need to be corroborated by clinical results on long-term follow-up.

### HEMATOPOIETIC STEM CELL transplantation FOR PIDs

Management of children with PIDs requires a multidisciplinary approach and HSCT plays a major role in improving the outcome of patients with PID. HSCT provides a definitive cure for several primary immune deficiencies including severe combined immunodeficiency (SCID), WAS, chronic granulomatous disease (CGD), HLH, and many other immunodeficiency disorders (10). There are little published data regarding the outcome of HSCT for PID from India (11). The major hurdles so far have been the lack of awareness about diagnosis and availability of expertise for carrying out this procedure in the setting of PID. Even though many centers in India are now routinely performing HSCT for a variety of malignant diseases in both children and adults, there is only a minority

that have the requisite expertise for carrying out transplant for PIDs (Figure S1 in Supplementary Material). Kapoor and Raj have recently published the data on HSCT in PID from India (11). Approximately 100 transplants have so far been carried out in 10 centers across the country. The indications included SCID, HLH and WAS.

### VACCINES IN PID

Children with PIDs may not mount an immune response to a given vaccine (e.g., response to pneumococcal vaccine in children with XLA), and these vaccines may not be indicated (12). On the other hand, a live vaccine may be deleterious as once a live bacterium or virus from a vaccine strain is administered to these patients, it is very difficult to eradicate this organism from the body of an immunocompromised child (12). Oral live polio vaccine is still being used in India as a part of Polio Eradication and Endgame Strategic Plan and majority of children receive the first dose at birth. Subsequently, about three to four doses are given in the first year of life either as part of routine immunization or pulse polio immunization program (which is conducted on two designated days per year, and all children below 5 years are given a dose of oral polio on these days). Similarly, all babies born in India receive BCG vaccination at birth. This contrasts with the policy in some of the developed countries where BCG is either not administered at all or, if administered, it is deferred until results of T-cell receptor excision circle (TREC) assay are available. Similarly, OPV has been discontinued in most of the developed countries. Thus, babies born in India with a significant cell mediated immune defect are at risk of developing disseminated BCG infection as well as vaccine-associated paralytic poliomyelitis (VAPP) (4). Recently, a case was reported from India in whom VAPP caused by vaccine-derived poliovirus (VDPV) (strain type 2) was the first presenting manifestation of CVID (13). The recent update on VDPV from India (January 2015–May 2016) revealed three cases of VDPV (one each in XLA, CVID, and SCID) (14). The Jeffrey Modell Foundation in collaboration with Centers for Disease Control and Prevention, WHO—Global Polio Laboratory Network and The Task Force for Global Health recently conducted a surveillance to determine the prevalence of poliovirus excretion in immunodeficient children across 13 different countries (15). Twenty-three children with XLA from Chandigarh, India, were recruited in this multicenter study, and stool samples were cultured for detection of poliovirus excretion. While none of the patients from Chandigarh were excreting poliovirus, 13 patients from Iran, Tunisia, Turkey, and the Russian Federation were found to be doing so. These findings have implications for health planners and vaccinologists all over the world.

#### NEWBORN SCREENING (NBS) IN PID

Early diagnosis of PIDs is of paramount importance before these children acquire a significant infection (16). HSCT is a curative treatment for several PIDs and the best outcome can be achieved if the transplant is performed before these children acquire an infection (17). SCID is usually not apparent at birth and these children can only be detected by NBS before they get any major illness. NBS for SCID is usually done using TREC assay, which is a measure of thymic output of T cells and is a sensitive assay (18, 19). NBS for SCID is now being routinely carried in all states of USA and many other developed countries. However, the cost of TREC assay is a major constraint for its inclusion in the NBS program in India. In the current scenario, where routine NBS is not available even for more common disorders such as hypothyroidism, NBS for SCID in the near future appears far-fetched. Early screening for SCID will also prevent administration of BCG and other live vaccines to these patients. The FPID is actively considering pilot studies on NBS for SCID in India.

#### NON-INFECTIOUS COMPLICATIONS IN PID

Patients with PID are also predisposed to develop autoimmune, inflammatory, and allergic complications. While children with CGD have significant inflammatory disorders (20), autoimmune complications are characteristic of CVID and WAS. PIDs can also be associated with several malignancies. We recently reported a case of WAS with Hodgkin lymphoma from Chandigarh (21). A case of diffuse large B-cell lymphoma was reported in another child with WAS from Vellore in 2014 (22) and a case of multifocal extranodal non-Hodgkin lymphoma in a child with combined immunodeficiency and Epstein–Barr virus infection (23). We have also seen leiomyoma of liver in a boy with ataxia telangiectasia (unpublished data).

#### STATUS AND PROSPECTS OF PID INDIA

The spectrum of PIDs diagnosed at a single center in India (PGIMER, Chandigarh) is shown in **Figure 2**. It is apparent that this spectrum is perhaps no different from that in any other developed country. In addition, PGIMER and many other institutions in India have now developed the facility for genetic analysis for common immunodeficiency diseases. At Chandigarh, facilities for genetic testing for WAS, CGD, XLA, HLH (PRF-1), and X-linked hyper-IgM syndrome are in place. Similarly, genetic testing for HLH, LAD, and several other PIDs is available at NIIH, Mumbai. In addition, a prenatal screening program is being offered to patients with PIDs at PGIMER, Chandigarh as also at NIIH, Mumbai and SGRH, New Delhi (Figure S1 in Supplementary Material). This has been a major advance in the field of PIDs in India. An effort is being made to establish a PID registry for the country; however, it has not been established till date.

#### ETHNIC CONSTITUTION AND CONSANGUINITY IN INDIA

India has large variation in ethnicity across various geographic regions with six different major religions being followed in the country (24). There is a huge burden of consanguineous and endogamous marriages in India (25, 26). The figure varies from 1% in some states to as high as 38% in others. The rate of

consanguineous marriages is much higher in southern parts of India (e.g., 38% in Tamil Nadu, 29% in Andhra Pradesh, and 28% in Karnataka) as compared to northern states (27). Several autosomal recessive disorders (as for instance thalassemia) are more commonly reported from regions in India that have increased rate of consanguineous and endogamous marriages, data on PIDs are scare. Our own data from PGIMER, Chandigarh have shown that proportion of autosomal recessive CGD is higher than the X-linked CGD and this is likely due to the high incidence of endogamous and consanguineous marriages in this part of the world (28). However, the increased likelihood of finding an HLAmatched donor in immediate and extended families appears to be the "silver lining" amidst the baneful effects of consanguinity and endogamy (29, 30).

In conclusion, the outcome of patients with PIDs in India appears to be brighter with an increased awareness of these disorders among physicians and specialists. Availability of diagnostic facilities including candidate gene sequencing and prenatal diagnosis for common PIDs in few centers is also a big step forward from the last decade. HSCT is also being performed at more centers now. Although progress has been made during the last few years, we still have a long arduous journey ahead to provide PID patients in India the standard of care that they rightfully deserve.

## AUTHOR CONTRIBUTIONS

AJ and RP—initial draft of manuscript, editing of manuscript, revision of manuscript, and data collection. AR and SS—editing and critical revision of manuscript, data collection, and final approval of manuscript.

### FUNDING

Authors thankfully acknowledge Indian Council of Medical Research, New Delhi, India, and Department of Health Research, Ministry of Health and Family Welfare, Government of India, New Delhi, India, for funding vide Grant No. GIA/48/2014-DHR and the Foundation for Primary Immunodeficiency Diseases (FPID), USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2017.00714/ full#supplementary-material.

Figure S1 | Geographical map of India depicting PID centers across the country with different levels of care and facilities.

### REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Jindal, Pilania, Rawat and Singh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Primary immunodeficiency Diseases in Highly Consanguineous Populations from Middle east and north Africa: epidemiology, Diagnosis, and Care

*Hamoud Al-Mousa1,2,3\* and Bandar Al-Saud1,3\**

*1Department of Pediatrics, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 2Department of Genetics, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia, 3College of Medicine, Alfaisal University, Riyadh, Saudi Arabia*

#### *Edited by:*

*Mohamed-Ridha Barbouche, Institut Pasteur de Tunis, Tunisia*

#### *Reviewed by:*

*Kathleen Sullivan, Children's Hospital of Philadelphia, United States Craig Platt, Boston Children's Hospital, United States*

#### *\*Correspondence:*

*Hamoud Al-Mousa<EMAIL_ADDRESS>Bandar Al-Saud<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology*

*Received: 28 February 2017 Accepted: 24 May 2017 Published: 26 June 2017*

#### *Citation:*

*Al-Mousa H and Al-Saud B (2017) Primary Immunodeficiency Diseases in Highly Consanguineous Populations from Middle East and North Africa: Epidemiology, Diagnosis, and Care. Front. Immunol. 8:678. doi: 10.3389/fimmu.2017.00678*

Middle East and North Africa region (MENA)1 populations are of different ethnic origins. Consanguineous marriages are common practice with an overall incidence ranging between 20 and 50%. Primary immunodeficiency diseases (PIDs) are a group of heterogeneous genetic disorders caused by defects in the immune system that predisposes patients to recurrent infections, autoimmune diseases, and malignancies. PIDs are more common in areas with high rates of consanguineous marriage since most have an autosomal recessive mode of inheritance. Studies of PIDs in the region had contributed into the discovery and the understanding of several novel immunodeficiency disorders. Few MENA countries have established national registries that helped in estimating the prevalence and defining common PID phenotypes. Available reports from those registries suggest a predominance of combined immunodeficiency disorders in comparison to antibody deficiencies seen in other populations. Access to a comprehensive clinical immunology management services is limited in most MENA countries. Few countries had established advanced clinical immunology service, capable to provide extensive genetic testing and stem cell transplantation for various immunodeficiency disorders. Newborn screening for PIDs is an essential need in this population considering the high incidence of illness and can be implemented and incorporated into existing newborn screening programs in some MENA countries. Increased awareness, subspecialty training in clinical immunology, and establishing collaborating research centers are necessary to improve patient care. In this review, we highlight some of the available epidemiological data, challenges in establishing diagnosis, and available therapy for PID patients in the region.

Keywords: immunodeficiency, Middle East, North Africa, consanguinity, primary immunodeficiency, SCID, hematopoietic stem cell transplantation

1MENA countries include the following: Saudi Arabia, Kuwait, Bahrain, Qatar, United Arab Emirates, Oman, Yemen, Jordan, Iraq, Israel, Palestine, Syria, Lebanon, Iran, Turkey, Egypt, Sudan, Tunisia, Libya, Algeria, Morocco, and Mauritania.

## INTRODUCTION

Primary immunodeficiency diseases (PIDs) are a group of inherited heterogeneous disorders caused by monogenetic immune defects that predispose patients to infections (1). In addition, PID patients have non-infectious manifestation related to disturbed immune regulation that might cause lymphoproliferative and/or autoimmune manifestations (2). In 1952, Bruton described the first case of agammaglobulinemia (3). Since then, over 300 forms of PIDs have been described and characterized. The International Union of Immunological Societies PIDs Classification Committee (4) classified PID in to immunodeficiencies affecting cellular and humoral immunity, combined immunodeficiencies with associated or syndromic features, predominantly antibody deficiencies, disease of immune dysregulation, congenital defects of phagocyte number, function, or both, defects in intrinsic and innate immunity, autoinflammatory disorders, complement deficiencies, or phenocopies of PID. PIDs are considered to be rare disorders. Worldwide databases have shown geographical and racial variation in the epidemiology of PIDs. Published data from highly consanguineous population's like the Middle East/Northern Africa (MENA) region showed that PIDs are not uncommon. A consanguineous marriage is usually defined in clinical genetic as a marriage between two couples who are second cousins or closer (5). Consanguineous marriages are common practice in MENA region with an overall incidence ranging between 20 and 50% (6) [Bittles A. H. and Black M. L. (2015) Global Patterns & Tables of Consanguinity http://consang.net]. This has provided a background where autosomal recessive (AR) diseases are abundant. For example, there are 955 genetic diseases that have been identified in Arabs from the MENA region, of which 586 (60%) are reported to be recessive diseases (7). In addition to high rates of consanguinity, the large family size and the rapid population growth all are factors responsible for the high prevalence of rare genetic diseases in the MENA region (8). Here, we present a review of PIDs status in a highly consanguineous population from the MENA region with particular emphasis on epidemiology, diagnosis, and care.

#### MENA DEFINITION, POPULATION, AND ETHNICITY

The MENA region covers a surface area of nearly 15 million square kilometers from Morocco in the west to Iran in the east. The MENA region includes 22 countries and territories and accounts for 385 million people representing 6% of the world's population (9). The MENA region has an annual population growth rate of 1.8% compared to a 1.2% average global population growth rate (United Nations, Department of Economic and Social Affairs-Population Division, Population Estimates and Projections Section-World Population Prospects, 2015 Revision). The pediatric age group (0–14 years) represents 31.1% of the total population in the MENA region in comparison to 26.1% globally. The MENA population has a mix of Asian, Caucasian, Arab, and African racial ancestries. MENA region captures pan-ethnic geographically defined groups that include Arab, Persian, Turkish, Kurdish, Berber, Amazigh, Assyrian, Chaldean, Armenian and others.

## CONSANGUINITY

20% of world populations live in countries with a preference for consanguineous marriages (6). Among these, are the MENA region countries where consanguineous marriage is a normal practice for multiple sociocultural factors (10–15). The global consanguinity rate is 1–9% while it is 20–56% in the MENA region (4). PID population from the MENA region display a higher rate of consanguinity compared to their general population. In addition, AR inheritance is predominant. The T-B-NK+ SCID represents the most common SCID phenotype in 90, 87, and 50% of Saudi Arabia, Kuwait, and Egypt SCID patients, respectively. Family history suggestive of PID is common among patients in MENA region as captured in several registries at a rate of 30, 44, 48, 61, and 80% in Tunisia, Oman, Iran, Saudi Arabia, and Egypt, respectively (16–24). Moreover, a significant number of these AR PIDs were first described in patients living in the MENA region (25).

### EPIDEMIOLOGICAL DATA AND REGISTRIES

Ten countries from the MENA region have published epidemiological data in medical literature. These data result from a national registry or survey as those from Morocco (21), Tunisia (20), Israel (26), Kuwait (17), and Iran (22), or from a major referral centers in; Egypt (19), Turkey (23), Saudi Arabia (16), Qatar (24), and Oman (18). A total number of 4,918 patients were included. The male to female ratio ranged (1.1 to 2). The overall prevalence of PID was ranging from 0.81 to 30.5 in 100,000 populations (**Table 1**). In most registries more than 80% of patients were diagnosed at pediatric age group. The mean age at symptoms onset was within the first 2 years of life, except in Turkey, where it was at 4 years of age. That is probably explained by the fact that 70% of the patients of the Turkish registry are from a subgroup of predominantly antibody deficiencies while combined immunodeficiencies are commoner among other registries. Family history of PID was documented in seven registries and ranged from 17.2 to 61% (**Table 1**). Eight main PID categories based on IUIS classification were captured in various MENA registries as shown in **Figure 1A**. The disease distribution was variable among registries from the different MENA countries, where combined immunodeficiencies were the predominant category among patients registered in Saudi Arabia, Kuwait, Iran, Egypt, Israel, Tunisia, and Morocco. Predominantly antibodies deficiency was noticeably the commonest PID type among patients registered in Turkey. In addition, other PID categories were variably distributed in the registries, as, for example, congenital defect of phagocyte had a higher percentage of registered patients in both Oman and Tunisia while autoinflammatory disorder was higher in Turkey and Egypt in comparison to the other countries (**Figure 1B**).

#### DIAGNOSTIC FACILITIES

The extreme clinical, phenotypic, and genetic heterogeneity of many patients with PIDs represents significant diagnostic challenges to physicians in the MENA region. The capability to perform extensive immunologic investigations and mutation analysis is variable from one country to the other. In Saudi Arabia, an advanced clinical diagnostic immunology service at King Faisal Specialist Hospital and Research Centre (KFSHRC) had been established in the last 30 years that allowed access to diagnose most of the known PIDs. Extensive genetic testing that includes Sanger sequencing, Targeted next-generation sequencing PID gene panel, and whole exome sequencing is offered to affected patients (16, 27, 28). Sultan Qaboos University Hospital, the Royal Hospital, and Hamad General Hospital are the main PID centers in Oman and Qatar, respectively. AlSabah, Mubarak Al-Kabeer, and AlRahsid hospitals have PID centers in Kuwait. Genetic testing is performed through international diagnostic laboratories or through collaborative research facilities. The first immunology center in Turkey was established in 1972 at Hacettepe University in Ankara. There are now more than 10 immunology centers in different cities around Turkey with facilities to diagnose common PIDs (29). Departments of Pediatric Immunology at Uludag University Medical Faculty and Ege University Medical Faculty are the major centers reporting PID patients to European Society for Immunodeficiencies database from Turkey (23). Targeted next-generation sequencing PID genes panel and whole exome sequencing are offered to patients in Turkey. In Iran, there is at least one Medical University and immunology center at each of the 30 states of the country that could manage PIDs. National PID network is active in Iran, which allowed referring patients to the Research Center for Immunodeficiencies at Tehran University of Medical Sciences for further immunological and genetic testing (22). Queen Rania Children Hospital is the major pediatric immunology center in Jordan. In Lebanon, PID care is provided primarily by Pediatric infectious disease service at the American University of Beirut Medical center and few other medical centers. Several medical centers provide care and have diagnostic facilities to diagnose various types of PIDs in Israel with access to Sanger and whole exome sequencing (30). The Pasteur Institute of Tunisia is the main diagnostic facility in Tunisia with the ability to perform genetic testing for common PIDs. Ain Shams University center and Cairo University center are the main PID referral centers in Egypt; both are capable to diagnose major PID types while genetic testing is done at research diagnostic facilities in USA. In Algeria, there are five medical centers capable to diagnose and manage major types of PIDs. All are located in northern part of the country. Genetic testing for agammaglobulinemia, LAD, and MHC II deficiency is available and collaborates with research centers in France and USA to diagnose atypical PIDs. In other MENA countries, access is often limited because of either financial or personnel limitations.

#### PID CARE AND MANAGEMENT

Specialist clinical immunology services and hematopoietic stem cell transplantation (HSCT) facilities emerged in a few countries driven by the health demands of the population. In Saudi Arabia, an HSCT program for PID had been established in 1984 at KFSHRC in Riyadh. More than 500 PID cases had been transplanted with an average of 35–40 transplants per year (16, 31–33). 70% of HSCTs are from HLA matched related donors whereas the remaining donor sources were either unrelated umbilical cord blood or haploidentical bone marrow. Another two centers recently started performing HSCT for PID at National Guard Hospital, Riyadh and KFSHRC, Jeddah. There are five centers performing HSCT for PID in Turkey, in Ankara (Hacettepe and Ankara University.), in Izmir (Ege University), in Antalya and in Istanbul (Medical Park Hospital). In Iran, there are two HSCT centers in Tahran (Hematology, Oncology and Stem Cell Transplantation Research Centre and Children's Medical Center), and more than 50 HSCTs for PIDs had been performed over last 10 years. In Jordan, Queen Rania Children Hospital is the main transplanting center with an average of 8–10/year in addition to King Hussein Cancer Center (34). HSCTs are offered at several medical centers in Israel. Twenty eight HSCTs had been performed for PIDs in the last 8 years at bone marrow transplantation center of Tunis (27 genoidentical and 1 haploidentical). Sultan Qaboos University hospital in Oman performs two to four HSCTs for PID per year.

Figure 1 | (A) Distribution of primary immunodeficiency disorders in different primary immunodeficiency disease registries from the MENA region compared to the European Society for Immunodeficiencies registry. (B) Primary immunodeficiency disease prevalence in 100,000 inhabitants in the MENA region compared with counties from other world region.

A few HSCTs trials had been performed in Algeria, Egypt, and Morocco. Access to immunoglobulin replacement therapy and HSCT is very limited or unaffordable for many patients in some of the MENA countries.

#### NOVEL PID DISCOVERY

In the last three decades, more than 300 novel PIDs were discovered, and the list is continuously expanding. The inbred population of the MENA region provides a great opportunity to identify monogenic PIDs through novel next-generation sequencing technology. Studies of PIDs in the region had contributed into the discovery and the understanding of large numbers of these disorders. For example, more than 12 novel PID genes were discovered through studying patients from the MENA region in the last 2 years, which include DOCK2 (35), HOIP (36), IL-17RC (37), RORC (38), RLTPR (39), POLE2 (40), NEIL3 (41), TFRC (42), INO80 (43), LAT (44), MYSM1 (45), and CD70 (46).

### GENETIC COUNSELING AND DISEASE PREVENTION

In the MENA countries, the majority of patients has an AR mode of inheritance and come from families known to have the disease. Appropriate genetic counseling for affected families is an essential part of the management. In Saudi Arabia, Turkey, Iran, Israel, and Kuwait genetic counseling, prenatal and preimplantation genetic diagnosis and premarriage screening to identify carriers are offered to affected families. Such services are costly and require sophisticated diagnostic facilities not available in most of the MENA countries.

### EDUCATION AND TRAINING

Establishing a structural immunology educational and fellowship programs is essential to improve available immunology services in MENA region. In Saudi Arabia, allergy and immunology fellowship training program had been established since 1989. Structured clinical immunology training had been also established in Iran 30 years ago. A 3-year fellowship training is available in several universities in Turkey. Allergy and immunology fellowship program had been recently launched in Kuwait. Immunological centers at MENA region are actively involved in providing education sessions and training for general practitioners, pediatricians, and internists. Dedicated conferences and workshops on PID had been organized in various MENA countries aiming to increase the health providers' awareness.

### NEWBORN SCREENING FOR PID

Most patients with PIDs are asymptomatic at birth. Early diagnosis and initiation of therapy improve the outcome. SCID have been recognized as candidates for population-based newborn screening using the T-cell receptor recombination excision circle assay (TREC) and found to be cost-effective means to improve the quality and duration of life for children with SCID. The high disease incidence as seen in MENA region is a critical driving force that would affect the incremental cost-effectiveness ratio. Implementing such a program in the MENA region is possible in resource-rich countries. However, the health authorities should recognize the seriousness of the health problem and provide all required resources. Israel is the only MENA country performing universal SCID NBS. A NBS pilot study is ongoing in Saudi Arabia to identify the real incidence of SCID in Saudi population.

### DISCUSSION

The MENA region has a diverse ethnic background, but consanguineous marriages are common practice among this population. This practice is derived by cultural and socioeconomic interests aiming to strengthen family relations and structure. PIDs are more common in areas with high rates of consanguineous since most PIDs are inherited in an AR pattern and hence the observed increased incidence of combined immunodeficiencies among MENA populations in comparison to populations from the European countries (47) (**Figure 1A**). The higher incidence of combined immunodeficiency seen in Saudi Arabia in comparison to other MENA countries might be related to the availability of diagnostic facilities and to the fact that this was a single center registry. The lower rate of consanguineous marriage in Turkey had contributed to the reduced incidence of such disorders in their population. Moreover registries from the MENA region (16–21, 23, 24, 26) showed a high prevalence of PIDs when compared to the rest of the world (48–53) (**Figure 1B**). The variable low or high PID reported prevalence might be a reflection of the accessibility and the availability for diagnostic facilities in this population among MENA countries.

The majority of patients with PID in MENA region have a family history suggestive of PID. It would be cost effective to establish a family-based preventive program for PID in this region, which allows identifying all family carriers, offer genetic counseling, and premarriage screening. Affected couples can be offered preimplantation or prenatal genetic diagnosis. However, such program should respect social and religious beliefs.

A lot of excitements are observed among clinicians taking care of PID in MENA region, but they are confronted with lack of specialized centers dedicated to the care of PID patients, limited access to a diagnostic facilities and the costly burden for the therapeutic modalities. Molecular genetic testing is essential diagnostic tool for PIDs, it provides a definitive diagnosis, assists in genetic counseling, establishes the diagnosis in atypical cases, provides genotype–phenotype correlation and guide therapy for affected patients. Molecular genetic testing is complex, expensive, and unaffordable in most of MENA countries. It will be important to establish multicenter collaborations in the region to provide and share diagnostic facilities and therapy. This will require strategic support from heath authorities in the region. Governmental support and charity funding are essential to establish clinical services and ensure that patients can receive long term therapy such as immunoglobulins replacement therapy or stem cell transplantation.

Establishment of a MENA primary immunodeficiency network is essential to promote collaborations and experience sharing. Collaboration with various international research centers through implementing advanced molecular genetic testing had led into several novel genetic discoveries in the field of PID. Multicenter collaborations especially between clinicians and basic scientists can contribute in development of efficient PID research in the region. Clinicians' training is important to promote PID care in MENA region. This can be enforced through including clinical immunology in the undergraduate and postgraduate educational curriculum, implementing formal subspecialty training for pediatric and adult immunologist and through providing continuous medical education courses in clinical immunology.

This report highlights the need to improve awareness about PID in MENA region, enhances structural training in clinical immunology, and establishes national registries and centers of excellence in PID care and stem cell transplantation.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

HA-M and BA-S participated in data collection, analysis and interpretation, and drafting and final approval of manuscript.

#### ACKNOWLEDGMENTS

The authors are grateful for the invaluable information contributed by colleagues from all over the MENA region who inspired this review: Waleed Al-Herz, Salem Al-Tamemi, Mehdi Adeli, Adel Wahadnah, Nima Rizaie, Ghassan Dbaibo, Shereen Reda, Fethi Mellouli, Neslihan Karaca, Necil Kutukcular, and Rachida Boukari.

#### FUNDING

This work was supported by the National Science, Technology and Innovation Plan program in the Kingdom of Saudi Arabia (KACST: 14-MED 316-20).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

*Copyright © 2017 Al-Mousa and Al-Saud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Lessons from Genetic Studies of Primary immunodeficiencies in a Highly Consanguineous Population

*Mohamed-Ridha Barbouche1,2\*, Najla Mekki1,2, Meriem Ben-Ali1 and Imen Ben-Mustapha1,2*

*1 Laboratory of Transmission, Control and Immunobiology of Infection (LR11IPT02), Institut Pasteur de Tunis, Tunis, Tunisia, 2 Faculty of Medicine, Université de Tunis El Manar, Tunis, Tunisia*

During the last decades, the study of primary immunodeficiencies (PIDs) has contributed tremendously to unravel novel pathways involved in a variety of immune responses. Many of these PIDs have an autosomal recessive (AR) mode of inheritance. Thus, the investigation of the molecular basis of PIDs is particularly relevant in consanguineous populations from Middle East and North Africa (MENA). Although significant efforts have been made in recent years to develop genetic testing across the MENA region, few comprehensive studies reporting molecular basis of PIDs in these settings are available. Herein, we review genetic characteristics of PIDs identified in 168 patients from an inbred Tunisian population. A spectrum of 25 genes involved was analyzed. We show that AR forms compared to X-linked or autosomal dominant forms are clearly the most frequent. Furthermore, the study of informative consanguineous families did allow the identification of a novel hyper-IgE syndrome linked to phosphoglucomutase 3 mutations. We did also report a novel form of autoimmune lymphoproliferative syndrome caused by homozygous FAS mutations with normal or residual protein expression as well as a novel AR transcription factor 3 deficiency. Finally, we identified several founder effects for specific AR mutations. This did facilitate the implementation of preventive approaches through genetic counseling in affected consanguineous families. All together, these findings highlight the specific nature of highly consanguineous populations and confirm the importance of unraveling the molecular basis of genetic diseases in this context. Besides providing a better fundamental knowledge of novel pathways, their study is improving diagnosis strategies and appropriate care.

Keywords: primary immunodeficiencies, consanguinity, autosomal recessive, founder effect, genetic counseling

#### INTRODUCTION

The expression of recessive genes inherited from a common ancestor, in consanguineous populations, underlies higher frequency of otherwise rare genetic diseases (1–3). Indeed, the molecular basis of many monogenic disorders has been first characterized in highly inbred populations. Furthermore, in a significant proportion of cases, a strong founder effect for specific mutations has been reported in these settings; some mutations might be unique to a particular endogamous community (4, 5).

Primary immunodeficiencies (PIDs) are a heterogeneous group of genetic disorders of the immune system that predispose patients to infections, autoimmune diseases, lymphoproliferation,

#### *Edited by:*

*Waleed Al-Herz, Kuwait University, Kuwait*

#### *Reviewed by:*

*Hamoud Al-Mousa, King Faisal Specialist Hospital & Research Centre, Saudi Arabia Janet Chou, Boston Children's Hospital, United States*

*\*Correspondence:*

*Mohamed-Ridha Barbouche<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology*

*Received: 19 April 2017 Accepted: 12 June 2017 Published: 27 June 2017*

#### *Citation:*

*Barbouche M-R, Mekki N, Ben-Ali M and Ben-Mustapha I (2017) Lessons from Genetic Studies of Primary Immunodeficiencies in a Highly Consanguineous Population. Front. Immunol. 8:737. doi: 10.3389/fimmu.2017.00737*

and malignancy. The exact prevalence of PIDs worldwide is unknown but is expected to be more common in areas with high rates of consanguinity (6). The autosomal recessive (AR) forms of these disorders represent the most frequent mode of inheritance as compared to X-linked (XL) or autosomal dominant (AD) forms. Indeed, according to the 2015 IUIS classification of PIDs, 206 out of 289 known forms of PIDs are AR (7). The occurrence of the disease in the progeny of unaffected consanguineous parents is highly suggestive of an AR mode of inheritance. However, molecular studies are required to confirm the AR determinism of disease in these PIDs affected families.

Although significant efforts have been made in recent years to develop genetic testing across the Middle East and North Africa (MENA) region, very few comprehensive studies reporting molecular basis of PIDs in these settings are available. Herein, we review genetic characteristics of PIDs identified in a large series of inbred Tunisian (North-African) patients. We outline the molecular basis of disease in this population and its potential contribution to a better care through genetic counseling. Furthermore, we discuss the relevance of such studies in the discovery of new PIDs genes and novel modes of inheritance for known PIDs.

### PIDs DISTRIBUTION, PARENTAL CONSANGUINITY, AND FAMILIAL HISTORY

This review analyzes data collected from different genetic studies performed in a total of 168 PIDs patients belonging to 122 kindreds. This is at the best of our knowledge, one of the largest molecular studies of PIDs patients from highly consanguineous MENA populations along with the study from Saudi Arabia (8). The most frequently observed PIDs in this series include combined T and B cell immunodeficiencies and congenital defects of phagocyte that account for 52 (30.95%) and 37 (22%) patients, respectively, contrasting with data from European series showing the predominance of antibody deficiencies (9). Indeed, only 15.47% of the patients had predominantly antibody deficiencies. Similar observations have been reported in others series from MENA region (10, 11). This could be due to the less severe clinical phenotype and to the lack of adult physicians' awareness particularly with regard to common variable immunodeficiency. Moreover, the high frequency of particular AR forms of combined immunodeficiencies among North-African PIDs patients could also account for such findings. Indeed, major histocompatibility complex (MHC) class II deficiency is the most frequently reported PID entity in this series with 27 patients, and the majority of patients reported worldwide are of North-African origin (Algeria, Tunisia, and Morocco) (12). Other categories of PIDs included defects in intrinsic and innate immunity (23 cases), combined immunodeficiencies with associated or syndromic features (19 cases), and diseases of immune dysregulation (11 cases). Furthermore, few patients presenting rare PIDs with peculiar clinical and/or immunological phenotype did also undergo genetic characterization. Altogether, the pattern of PIDs distribution in patients with established molecular diagnosis in this series is representative of the actual distribution of PIDs reported by the few national registries available from MENA region (13).

Interestingly, Tunisian and other MENA region countries are unique with regard to high prevalence of consanguinity in the general population varying between 20 and 50% of all marriages (13). Consistently, the patients studied in this series show a high rate of parental consanguinity that reaches 61.9% accounting for the high frequency of family history (55.35%) including early deaths, similar clinical features, and/or previously identified PIDs in relatives. Consanguinity rate was particularly high in patients with Omenn syndrome (88.8%), phosphoglucomutase 3 (PGM3) deficiency (85.7%), leukocyte adhesion deficiency type 1 (LAD I) (76.4%), and MHC class II deficiency (70.3%) (**Table 1**). This is in accordance with the results obtained for one of the largest series of MHC class II North-African patients reporting a consanguinity rate of 81.8% (12), as well as for LAD I and Omenn syndrome in other patients series originating from MENA region (14–16).

### MOLECULAR STUDIES AND MODE OF INHERITANCE

In total, mutational studies of 25 candidate genes identified 58 different mutations as detailed in **Table 1**. Among them, 30 were novel (53.57%) with no records in three major databases including HGMD (The Human Gene Mutation Database), LOVD (Leiden Open Variation Database), and IDbases (databases for immunodeficiency-causing variations). Three mutations have been already reported for the first time in other patients from Tunisian origin (17–19) and nine mutations were recurrent. The identified mutations include 21 missense mutations, 10 nonsense mutations, 10 splice-site mutations, 12 deletions, 1 duplication, and 1 insertion. Interesting and rare mutational mechanisms included one complex mutation (2 pb insertion and 5 bp deletion) in *IGHM* gene (20), and one *de novo STAT1* mutation was identified in AD chronic mucocutaneous candidiasis disease (21). Mutations generating a premature stop codon were frequently observed in severe combined immunodeficiencies patients (71%), particularly those with *IL2RG* gene defect. All novel missense mutations were predicted to be possibly or probably damaging by Polyphen2 and/or SIFT algorithms. Furthermore, a deleterious effect was confirmed by appropriate functional testing for several gene mutations including *TCF3* (22), *AICDA* (23), *NCF2* (24), *FAS* (25), *STAT1* (21), *IL12B* (26), and *PGM3* (27).

These molecular studies confirm that the AR mode of inheritance is the most common in Tunisian patients accounting for 73% of all PIDs entities investigated. XL and AD modes were identified in only four and three different disorders, respectively. The deeply rooted tradition of parental consanguinity in the Tunisian general population, which remained relatively constant during the last four decades (28), has resulted in an elevated burden of AR PIDs since consanguinity favors the expression of recessive alleles (29). This is the case for two TABLE 1 | Mode of inheritance and molecular studies in the 170 Tunisian patients investigated.

Genetics of PIDs in a Consanguineous Population

#### TABLE 1 | Continued

*aThese mutations have been already reported for the first time in other patients from Tunisian origin.*

*XL, X-linked inheritance; AR, autosomal recessive inheritance; AD, autosomal dominant inheritance; SCID, severe combined immunodeficiencies; ALPS, autoimmune lymphoproliferative syndrome; AID, activation-induced cytidine deaminase; CGD, chronic granulomatous disease; MSMD, Mendelian susceptibility to mycobacterial disease; LAD, leukocyte adhesion deficiency; CMC, chronic mucocutaneous candidiasis; PID, primary immunodeficiency; MHC, major histocompatibility complex; LAD I, leukocyte adhesion deficiency type 1; HIGM, hyper-IgM syndrome; HIES, hyper-IgE syndrome.*

otherwise rare AR PIDs, namely, MHC class II deficiency and LAD I, diagnosed in fewer than 200 and 300 patients worldwide (12, 30) but accounting for 56 and 30 patients in Tunisia, respectively (31, 32). One practical implication related to this high frequency is to recommend, at least for the MHC class II deficiency, routine investigation of DR expression for North-African patients presenting symptoms suggestive of combined deficiency. Furthermore, for PIDs with more than one known mode of transmission, the AR trait was the most frequent or proportionately more represented in our settings than in other series and registries from non-consanguineous populations. Indeed, this mode of transmission accounted for most kindreds with chronic granulomatous disease (CGD) (93%), hyper-IgM syndrome (HIGM) (75%), and hyper-IgE syndrome (HIES) (71.42%). Consistently, among CGD patients, only one had a mutation in *CYBB* gene whereas the remaining patients (19/20) bear mutations in AR CGD genes. This is in accordance with previous reports showing a higher frequency of AR CGD in consanguineous populations (13, 33). In contrast, the XL form caused by *CYBB* gene mutation is the most common CGD form accounting for 70% of all patients according to the European registry data (34). Similar findings have also been observed in Tunisian patients with HIGM. Indeed, we demonstrate that 11 out of 15 patients are assigned to the AR form of the disease due to mutations in *AICDA* gene (20, 23). In contrast, the XL CD40L deficiency defines the most frequent type of HIGM in other series as it has been reported in 75% in North American and Asian patients (35), 42% in European patients (9), and 94% in Latin American patients (36). Thus, AR and XL forms for these diseases should be equally suspected in males originating from consanguineous regions. For HIES, PGM3 homozygous mutations account for 70% of Tunisian patients whereas the AD form due to mutations in signal transducer and activator of transcription 3 is the most common form reported worldwide (37).

#### IDENTIFICATION OF NOVEL GENE AND OF NOVEL MODE OF INHERITANCE FOR KNOWN GENES

Because a majority of PIDs are inherited as AR traits, the identification of patients with singular clinical and immunological phenotype within informative consanguineous families has tremendously contributed to the discovery of novel diseasecausing genes. Accordingly, we did recently identify *PGM3* gene defect due to homozygous hypomorphic mutations in patients from two Tunisian consanguineous families with hyper-IgE like syndrome (27). This gene has not been previously associated with human disease. The patients' clinical phenotype included classical features of HIES; however, they showed neurologic impairment with a developmental delay and psychomotor retardation (27). PGM3 enzyme plays an important role in the glycosylation pathway by catalyzing a key step in the synthesis of UDP-GlcNAc required for the biosynthesis of N-glycans (38). Thus, leukocytes from PGM3 deficient patients showed aberrant pattern of glycosylation due to altered PGM3 enzymatic activity (**Table 2**). This new congenital disorder of glycosylation accounts probably for the patients' clinical and immunological phenotype, although the underlying mechanisms remain to be fully understood. Concurrently, additional patients with heterogeneous clinical phenotypes carrying distinct *PGM3* mutations have also been described (39, 40). Given that some congenital disorders of glycosylation are treatable with supplements of enzyme substrates (41), such an approach in these patients with hypomorphic mutations and residual enzymatic activity could be proposed to improve their condition while waiting the generally difficult access to bone marrow transplantation in our settings. Many other gene discoveries have been previously made following the study of large consanguineous families from the MENA region. Indeed, as already reported by Barbouche and Eley (42), during the period 1994–2013, at least 21 novel underlying genes were first described in PIDs families living or originating from this region.

Families from areas with a high rate of consanguinity are important not only for the discovery of novel disease-causing genes but also for the identification of novel forms of known PIDs. Indeed, in autoimmune lymphoproliferative syndrome (ALPS), heterozygous germline mutations in the *FAS* gene inherited in an AD mode and associated with preserved protein expression are the most common cause of ALPS (25). These heterozygous mutations reported mainly in outbred human populations alter protein functioning by dominant-negative effect or by haploinsufficiency mechanisms (43–45). Very rare cases of total absence of Fas protein expression caused by homozygous *FAS*

TABLE 2 | Identification of novel gene and novel mode of inheritance for known genes in Tunisian consanguineous families. Identification strategy Mutation Protein expression Functional consequences Reference Novel gene *PGM3* Homozygosity mapping/linkage analysis and selector-based sequencing c.1018\_1020del (Hypomorphic) Reduced Reduced PGM3 enzymatic activity (27) c.248T>C (Hypomorphic) Novel mode of inheritance *FAS* Sanger sequencing c.1017A>G (Loss of function) Present Resistance to Fas-mediated cell death (25) c.581C>T (Loss of function) *TCF3* Whole exome sequencing c.808C>T (Loss of function) Absent Hypogammaglobulinemia (22)

mutations have been reported (46). Interestingly, we identified in two unrelated consanguineous Tunisian kindreds the first example of a human AR ALPS characterized by homozygous *FAS* gene mutations in either intracellular or extracellular domains associated with normal or residual Fas expression, respectively. Both mutations are associated with resistance to Fas-mediated cell-death (25) (**Table 2**).

In addition, we identified another mode of inheritance for the previously reported AD agammaglobulinemia due to heterozygous dominant-negative *de novo* mutation in transcription factor 3 (*TCF3*) gene (47). Indeed, we did recently report a patient with a homozygous nonsense mutation in *TCF3* gene, who presented with severe hypogammaglobulinemia, very low number of B cells and developed B-cell acute lymphoblastic leukemia (22) (**Table 2**).

Both novel modes of inheritance herein reported are supported by parents' consanguinity, familial history, and segregation of clinical and immunological features with the homozygous status. Such findings expand the spectrum of ALPS and TCF3 deficiency types and should prompt clinicians to search for such patients in highly endogamous populations for appropriate clinical and immunological follow-up.

### INVESTIGATIONS OF HOMOZYGOSITY MAPPING AND AGE ESTIMATION OF FOUNDER MUTATIONS

Another striking feature pinpointed in the study population is the high frequency of founder effects accounting for the recurrence of mutations, which were unique for several PIDs. The patterns of founder mutation distribution disclosed two types: those that are shared with other populations particularly from the North-African countries and those that are specific to Tunisia. One of the most illustrative PIDs examples for a regional founder effect is the AR MHC class II combined immunodeficiency that has been considered to be a "North-African disease." Indeed, we have identified a founder effect for the highly frequent c.338-25\_338del26 mutation (also known as 752delG-25) in the RFXANK gene resulting in a 26-bp deletion (31) that has been reported in other North-African studies (4, 5, 48). The founder event responsible for this mutation has been estimated to have arise approximately 2,250 years ago (12), a period concurrent to the Berber civilization. This is consistent with previous reports showing that the population of the North Africa particularly in Tunisia, Algeria, and Morocco has a common substantial genetic background and that founder mutations could be shared in some of these countries (49). Additional MHC class II mutations have been since reported in the Middle East and other world regions (50–52). Another recurrent mutation in *RAG1* gene (631delT) has been identified in nine Tunisian patients with Omenn syndrome; its description was limited to patients originating from North Africa as well (53–56) suggesting a possible founder effect for this variant. Furthermore, a recent study reported the recurrence of the same homozygous mutation (Q289X) in *CARD9* gene in eight Algerian and four Tunisian patients from seven unrelated families with deep dermatophytosis (57). Such finding was due to a founder effect with the common ancestor living approximately 975 years ago (57).

Interestingly, other identified founder effects seem to be limited to Tunisian regions (49). Indeed, a founder effect for c.298\_305del mutation in the *IL12B* gene has been reported in patients originating from the same Tunisian village. This mutation resulting in Mendelian susceptibility to mycobacterial disease is inherited as a common founder mutation arousing 1,100 years ago (26). In addition, we have recently reported a recurrent homozygous mutation in *NCF2* (c.257 + 2T>C) gene in 11 Tunisian patients CGD (24). The founder mutational event responsible of the recurrence of this mutation seems to be more recent since it was estimated to have occurred approximately 175 years ago (24).

Deleterious founder mutations have been reported in consanguineous populations to be the underlying cause of a large spectrum of monogenic AR diseases (28). In Tunisia, more than 300 genetic disorders were reported. Among them, 42% were associated to the presence of potential founder effect including PIDs (49, 58). Several clinical implications of the existence of the founder effect are the implementation of preventive approaches through genetic counseling and prenatal diagnosis in affected families. Furthermore, early genetic diagnosis in patients originating from the same geographical area will help propose HSCT, which is often the unique curative treatment, prior to the development of a severe clinical phenotype.

### CONCLUSION

This review shows evidence that AR forms of PIDs are frequent in our settings characterized by a high rate of consanguineous marriages. The presence of recurrent mutations and strong founder effects are shown to be common characteristics of PIDs patients from this population. Furthermore, these findings clearly demonstrate that the presence of an extended genetic homozygosity has the potential to reveal unusual patterns of inheritance. Thus, classical dominant disorders may assume a recessive pattern of inheritance in such population. Finally, it becomes obvious that PIDs in this region of the world is a relatively important health priority. The role of accurate molecular diagnosis and appropriate genetic counseling will help moderate the burden of PIDs and its associated costs for the community.

### AUTHOR CONTRIBUTIONS

M-RB, NM, MB-A, and IB-M did contribute to the collection, analysis, and interpretation of the data as well as editing of the manuscript and approval of the submitted version.

#### ACKNOWLEDGMENTS

The authors are grateful to all pediatricians and adult physicians for their collaboration. The list of collaborators is in alphabetic order: Aissaoui Lamia, Barsaoui Siheme, Bejaoui Mohamed, Ben-Ameur Houcine, Ben-Becher Saida, Ben-Jaballah Najla, Bouaziz Asma, Boughammoura Lamia, Boussetta Khadija, Denguezli Mohamed, Gannouni Souha, Garguah Taher, Guandoura Najoua, Gueddiche Neji, Hachicha Mongia, Hamzaoui Agnès, Harbi Abdelaziz, Hawwari Abbas, Helal Khaled, Jabnoun Sami, Mahfoudh Abdelmajid, Medhaffar Moez, Meherzi Ahmed, Mrabet Samir, Sammoud Azza, Sfar Tahar, Tebib Neji, and Zidi

#### REFERENCES

Fethi; the author would particularly like to thank the patients and their family members.

#### FUNDING

This work was supported by the Tunisian Ministry of Higher Education and Research.

Primary Immunodeficiency Registry. *J Clin Immunol* (2006) 26(6):519–32. doi:10.1007/s10875-006-9047-x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Barbouche, Mekki, Ben-Ali and Ben-Mustapha. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Cellular and Molecular Defects Underlying invasive Fungal infections—Revelations from endemic Mycoses

*Pamela P. Lee1 and Yu-Lung Lau1,2\**

*1 LKS Faculty of Medicine, Department of Paediatrics and Adolescent Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China, 2Shenzhen Primary Immunodeficiencies Diagnostic and Therapeutic Laboratory, The University of Hong Kong-Shenzhen Hospital (HKU-SZH), Shenzhen, China*

The global burden of fungal diseases has been increasing, as a result of the expanding number of susceptible individuals including people living with human immunodeficiency virus (HIV), hematopoietic stem cell or organ transplant recipients, patients with malignancies or immunological conditions receiving immunosuppressive treatment, premature neonates, and the elderly. Opportunistic fungal pathogens such as *Aspergillus, Candida, Cryptococcus, Rhizopus,* and *Pneumocystis jiroveci* are distributed worldwide and constitute the majority of invasive fungal infections (IFIs). Dimorphic fungi such as *Histoplasma capsulatum*, *Coccidioides* spp., *Paracoccidioides* spp., *Blastomyces dermatiditis*, *Sporothrix schenckii*, *Talaromyces (Penicillium) marneffei*, *and Emmonsia* spp. are geographically restricted to their respective habitats and cause endemic mycoses. Disseminated histoplasmosis, coccidioidomycosis, and *T. marneffei* infection are recognized as acquired immunodeficiency syndrome (AIDS)-defining conditions, while the rest also cause high rate of morbidities and mortalities in patients with HIV infection and other immunocompromised conditions. In the past decade, a growing number of monogenic immunodeficiency disorders causing increased susceptibility to fungal infections have been discovered. In particular, defects of the IL-12/IFN-γ pathway and T-helper 17-mediated response are associated with increased susceptibility to endemic mycoses. In this review, we put together the various forms of endemic mycoses on the map and take a journey around the world to examine how cellular and molecular defects of the immune system predispose to invasive endemic fungal infections, including primary immunodeficiencies, individuals with autoantibodies against interferon-γ, and those receiving biologic response modifiers. Though rare, these conditions provide importance insights to host defense mechanisms against endemic fungi, which can only be appreciated in unique climatic and geographical regions.

Keywords: endemic mycoses, primary immunodeficiencies, human immunodeficiency virus, histoplasmosis, coccidioidomycosis, paracoccidioidomycosis, blastomycosis, *Taloromyces marneffei*

#### INTRODUCTION

Endemic mycoses are infections caused by a diverse group of fungi that occupy specific ecologic niche in the environment (1). The major pathogenic fungi in this group, including *Blastomyces dermatiditis, Coccidioides immitis* and *C. posadasii*, *Paracoccidioides brasiliensis* and *P. lutzii, Histoplasma capsulatum, Sporothrix schenckii, Talaromyces marneffei* (formerly known as

#### *Edited by:*

*Antonio Condino-Neto, University of São Paulo, Brazil*

### *Reviewed by:*

*Pérsio Roxo-Junior, University of São Paulo, Brazil Satoshi Okada, Hiroshima University Graduate School of Biomedical & Health Sciences, Japan Shigeaki Nonoyama, National Defense Medical College, Japan*

> *\*Correspondence: Yu-Lung Lau<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology*

*Received: 15 March 2017 Accepted: 09 June 2017 Published: 28 June 2017*

#### *Citation:*

*Lee PP and Lau Y-L (2017) Cellular and Molecular Defects Underlying Invasive Fungal Infections— Revelations from Endemic Mycoses. Front. Immunol. 8:735. doi: 10.3389/fimmu.2017.00735*

**59**

*Penicillium marneffei*) and *Emmonsia* spp., belong to the phylum *Ascomycota* and are evolutionary related (2) (**Table 1**). They share the common characteristic of thermal dimorphism—they grow as saprophytic molds in the environment at temperatures ranging from 25 to 30°C, and undergo morphological switch to the yeast form, or spherules in *Coccidioides*, at body temperatures of mammalian hosts. The yeast form serves to accommodate intracellular growth within host phagocytes (3). Majority of these organisms are primary pathogens that are able to cause disease in healthy human individuals. However, they may cause severe, disseminated infections in immunocompromised hosts, such as patients with human immunodeficiency virus (HIV) infection, organ transplant, or hematopoietic stem cell transplant (HSCT) recipients, and those with autoimmune disorders receiving immunosuppressants (4–7). In particular, *T. marneffei* and *Emmonsia* spp. more typically cause disease in HIV-infected individuals (5–8). The HIV pandemic and the increasing use of immunosuppressive medications, such as calcineurin and tumor necrosis factor (TNF) inhibitors, have resulted in a rising trend of histoplasmosis and coccidioidomycosis in endemic regions (9, 10). Exposure to the specific environmental niche, either residential, occupational, or travel precedes the development of disease.

#### GEOGRAPHICAL DISTRIBUTION OF ENDEMIC MYCOSES

Endemic mycoses occur predominantly in specific climate zones. Coccidioidomycosis is present in semidesert areas, histoplasmosis and paracoccidioidomycosis (PCM) are prevalent in tropical regions while *T. marneffei* is endemic in subtropical regions, and blastomycosis belongs to temperate climates (6–8). Coccidioioidomycosis (11–13) and histoplasmosis (14–18) are widely distributed in the American continent and some tropical regions, while PCM is limited to Central and South America (19–21). *T. marneffei* is unique to Southeast Asia (22–24), and blastomycosis is found in North America, and Central and East Africa (25–27). *S. schenckii* is distributed around the world, mainly reported in those tropical and temperate zones with high humidity and mild temperatures (22–27°C) (28). Recently, an emerging thermally dimorphic fungus within the genus *Emmonsia* that is most closely related to *E. pasteuriana* has been recognized to be uniquely associated with in HIV infection in South Africa (29–31). The geographical distribution of endemic mycoses is shown in **Figure 1** (1, 12–14, 18–20, 27, 28, 32–39).

The biological niche is specific for each endemic fungus and knowledge of their natural habitat provides understanding about the risk factors for exposure to these pathogens (**Table 2**). *C. immitis* and *C. posadasii* are saprophytic fungi, which exist in their mycelial form in dry, alkaline soil in deserts with very low precipitation and extreme temperature variations (11–13). Coccidioidomycosis is most prevalent in Arizona and California in the United States (US) (40, 41). In contrast, *H. capsulatum* thrive in the tropical zones with high relative humidity, and its growth is favored by soil contaminated by bird and chicken excreta or bat guano, which creates an environment with high nitrogen content (14, 15). *P. brasiliensis* is also found in the tropical and very humid regions, especially in acidic soil where coffee and sugar canes are cultivated (20). PCM is prevalent in South America (Brazil, Columbia, Venezuela, Paraguay) and some regions of Central America and Mexico (19–21). *B. dermatitidis* exists in wet soils, and the most significant endemic epicenter is in Eastern US between the Ohio and Mississippi River valleys (25–27). *T. marneffei* is highly endemic in Thailand, Vietnam, Southern China, and other subtropical areas in Southeast Asia (22–24). Bamboo rats (*Rhizomys* spp. and *Cannomys* spp.) and soil from their burrows are important enzootic and environmental reservoirs of *T. marneffei*, respectively (39). *S. schenckii* is found in the soil containing decaying vegetation such as dead wood,

mosses, hay, and cornstalks. Sporotrichosis is also widely prevalent in warm-blooded animals including cats, dogs, armadillos, birds, and parrots, which constitute a source of zoonotic transmission (28). To date, disseminated emmonsiosis associated with HIV infection caused by the new *Emmonsia* spp. has only been described in South Africa (29–31).

## PATHOGENESIS AND CLINICAL MANIFESTATIONS

The acquisition of endemic fungi relates to human activities and climatic conditions that increase the risk of exposure to these organisms in susceptible individuals. Coccidioidomycosis, PCM, histoplasmosis, blastomycosis, and *T. marneffei* infection are acquired by the respiratory route. Occupational or recreational activities causing disturbance of the soil environment lead to aerosolization of the conidia, which could then be inhaled by exposed individuals to cause infection (1, 6–8, 14, 20, 22, 23, 27). In contrast, inhalation is not the major route by which *S. schenkii* is acquired. Instead, it typically occurs after traumatic inoculation or through microscopic breaks in the skin caused by pricks with plants, although the mode of transmission was not obvious in 60% of patients with sporotrichosis. Infection of the skin and subcutaneous tissues develops at the site of penetrating trauma and may spread to the muscles, fascia, cartilage, and bones (42, 43). The clinical features of these endemic mycoses are summarized in **Table 3**.

After entry to the body, inhaled conidia converts to the yeast form, which are taken up by tissue-resident macrophages. In most individuals, the pathogenic yeasts can be eliminated by the macrophages and the infection is usually asymptomatic or mild, and self-limiting in most cases. In patients whose immunity is compromised, the yeasts continue to proliferate in the macrophages, which if uncontained, systemic dissemination may occur *via* the reticuloendothelial system (12, 23, 44–46). HIV infection is the most important risk factor for disseminated endemic mycoses (5). Other risk factors include malignancy and immunosuppression, as listed in **Table 3** (4–7, 9, 10).

## ENDEMIC MYCOSES: INSIGHTS FROM THE HIV EPIDEMIC

Human immunodeficiency virus infection is the most common cause for disseminated or extrapulmonary forms of histoplasmosis, coccidioidomycosis, and *T. marneffei* infection, particularly in patients with profound T-cell lymphopenia (CD4+ lymphocytes <200/μL) (12, 47–50). They are considered as acquired immunodeficiency syndrome (AIDS)-defining conditions in the World Health Organization (WHO) clinical staging of HIV/AIDS for adults and adolescents (51). The association between PCM and

#### Table 2 | Endemic regions, natural habitats, and risk factors of exposure to endemic mycoses.

AIDS is relatively rare in contrast to the higher incidence of other systemic mycosis. HIV coinfection has been detected in about 5% of patients with PCM (52, 53). Although PCM and sporotrichosis are not included as AIDS-defining conditions, they are increasingly recognized as an emerging neglected opportunistic infections in HIV patients in Latin America (7, 53, 54). Systemic sporotrichosis with organ involvement or widespread cutaneous lesions may occur in these patients, causing a mortality rate of up to 30% (54, 55). Blastomycosis infrequently develops in HIV patients, but disease tends to be more severe with increased risk of central nervous system (CNS) involvement with high mortality (46, 56). The new species of *Emmonsia* spp. discovered in South

Table 3 | Clinical manifestations of endemic mycoses and risk factors for disseminated disease.

*HIV, human immunodeficiency virus; HSCT, hematopoietic stem cell transplant; IFN, interferon; TNF, tumor necrosis factor; PID, primary immunodeficiencies.*

Africa was found to cause disseminated infection almost exclusively in patients with AIDS (29–31).

The first case series of histoplasmosis in HIV-infected patients was described in 1982 in the US (57). Extrapulmonary or disseminated histoplasmosis became an AIDS-defining disease in 1987 (58). The increase in morbidity and mortality from histoplasmosis has been largely contributed by the HIV pandemic. In the US, the availability of highly active anti-retroviral therapy (HAART) and lipid formulations of amphotericin B, the increased awareness of the disease, and the development of rapid, non-invasive diagnostic methods led to decrease in the incidence and mortality associated with histoplasmosis in patients with AIDS (16). On the other hand, extrapulmonary or disseminated histoplasmosis is becoming an important health issue in the increasing number of patients receiving chemotherapy, solid organ or HSCT, immunosuppressive treatment especially TNF-α blockade (59), as well as a rare group of patients with primary immunodeficiencies (PIDs). In contrast, in endemic areas of Latin America histoplasmosis occurs in up to 25% of HIVinfected patients and represents the first manifestation of AIDS in up to 50–75% of patients (60, 61). Due to the lack of diagnostic facilities and algorithm, histoplasmosis is undiagnosed in many HIV-infected patients and is considered as an "invisible burden" of AIDS in less resourced countries (62, 63). These phenomena illustrate how demographics of histoplasmosis could be shaped by HIV burden and socioeconomic forces in different endemic regions.

Shortly after the description of increased susceptibility to histoplasmosis in AIDS, *Coccidioides* infection emerged as an important form of mycosis in patients with HIV infection (64–66). A prospective study at an Arizona HIV clinic in 1988 showed a cumulative incidence of active coccidioidomycosis of 25% during 41 months of follow-up, corresponding to an average annual incidence of 7.3% (67). In contrast, in a retrospective review at the same clinic during 2003–2008, only 11.3% of HIV-infected patients (*n* = 257) had coccidioidomycosis, and the annual incidence was only 0.9% when compared to the previous study (47). Both studies showed that CD4 count was the only predictor for developing active coccidioidomycosis; factors such as a history of coccidioidomycosis and duration of residence in an endemic area or age, sex, race, ethnicity, plasma HIV RNA level, or receipt of HAART were not associated with increased risk for coccidioidomycosis (11, 47, 67).

While histoplasmosis and coccidioidomycosis are well recognized as human pathogens long before the HIV epidemic, the importance of *T. marneffei* as a human disease was not recognized until the outbreak of HIV in Asia (22, 68, 69). In 1973, the first naturally occurring human case of *T. marneffei* infection was reported in an American minister with Hodgkin's disease who had been residing in Southeast Asia (70). No more than 50 cases were reported in the literature during the early 1980s (68–76). From 1988, *T. marneffei* infection was increasingly observed in patients with advanced HIV infection, initially in foreign visitors who have been to endemic regions, and later in local residents who were native to endemic parts of Thailand and Vietnam (22, 23, 77–79). In Northern Thailand, *T. marneffei* infection is the third most common opportunistic infection, accounting for 15–20% of all AIDS-related illness, after tuberculosis (TB) and cryptococcosis. *T. marneffei* infection is estimated to occur in 2.3% of new AIDS cases, compared with 0.39% for histoplasmosis (80). The trend of *T. marneffei* infection closely paralleled that of HIV, and in areas where reduction of HIV transmission and availability of HAART have improved, a decrease in the prevalence of *T. marneffei* infection has been observed (23, 81).

Together with *Emmonsia* spp., which causes disseminated infection almost exclusively in advanced HIV infection in South Africa (29–31), it is apparent that the epidemiology of coccidioidomycosis, histoplasmosis, and *P. marneffei* evolves with HIV epidemic. The close relationship between disease manifestation and severity with CD4+ cell count confirms the central importance of cell-mediated immunity against endemic fungi.

#### PIDs IN ENDEMIC MYCOSES: NEEDLES IN A HAYSTACK?

Primary immunodeficiencies are rare inborn errors of immunity. Defects of T-cell development and differentiation, phagocytic functions, and pathways involved in the innate recognition of pathogens and downstream signaling are associated with increased risk of fungal infections, the most common being candidiasis and aspergillosis. Endemic mycoses are rarely described in patients with PID, and little is known about the spectrum of PID associated with increased susceptibility to endemic fungi. On the other hand, as individuals who are apparently healthy can also develop disease caused by endemic fungi, recognition of those who may have an underlying PID could be a challenge.

*Talaromyces marneffei* infection is mostly seen in advanced HIV infection with CD4+ cell count <100/μL, and in fact, up to 80% or more of the cases have CD4+ count <50/μL (22, 81, 82). Only small proportion of disseminated *T. marneffei* infection occurs in patients with secondary immunodeficiencies (22, 23, 83, 84). It is otherwise rare in healthy persons, especially in children. The close epidemiological relationship between HIV and *T. marneffei*, and the fact that *T. marneffei* is an AIDS-defining illness (51) suggests that individuals who are HIV negative and without secondary immunodeficiencies may have underlying immune defects that are unrecognized. A systematic review by Lee et al (85) on more than 500 articles published in English and Chinese from 1970 to 2011 on penicilliosis revealed 32 patients aged 3 months to 16 years with *T. marneffei* infection but without known HIV infection. Twenty-four patients (75%) had disseminated disease, and 55% died of *T. marneffei* infection. Eight patients had PID or blood disorders, while four others had abnormal immune functions. Immune evaluations of the remaining patients were unstated. This observation highlights the knowledge gap in the immunological susceptibility to *T. marneffei.*

Two systematic reviews on PID in histoplasmosis and coccidioidomycoses were recently published. Lovell et al. (86) summarized all published cases of histoplasmosis in patients with PID up to August 2015 and revealed 47 patients with underlying PID, defined either molecularly or clinically. Together with the four patients described in their report, more than 50 PID patients have been documented to have *Histoplasma* infection. Disseminated histoplasmosis occurred in 68% of cases, and two deaths occurred because of progressive disease. Another systematic literature search on disseminated coccidioidomycosis yielded 370 case reports, and 8 cases of PID were identified (87). The frequency of PID underlying endemic mycoses is unknown. Given the rarity of PID, the proportion of PID accounting for disseminated endemic mycoses is likely to be small. However, the cellular and molecular defects of these PID can provide important mechanistic insights into host defense mechanisms against endemic fungi. More importantly, disseminated or extrapulmonary forms of endemic mycoses can be utilized as unique indicators for PIDs, which is of particular relevance to clinicians working in respective endemic areas (88).

## PIDs UNDERLYING FUNGAL INFECTIONS

Host immune response toward fungal pathogens is initiated by the recognition of invading fungi *via* pattern recognition receptors (PRRs) expressed on neutrophils, monocytes, macrophages, and dendritic cells (DCs). Receptor-mediated signaling induces downstream events such as cytokine and chemokine release, phagocytosis, and respiratory burst ultimately leading to fungal killing (89–93). In addition, the cytokine responses shape the induction of Th-1 and Th-17 adaptive immune response. IL-12 drives IFN-γ production by T-helper 1 (Th1) cells, which is crucial for phagocyte activation. On the other hand, IL-1β, IL-6, and IL-23 promotes Th17 differentiation (89–91). The various PRRs that recognize fungal pathogen-associated molecular patterns and the downstream signaling pathways leading to induction of Th1 and Th17 response are shown in **Figure 2A**.

Defects of the dectin-1/CARD9-MALT1-BCL10 signaling pathway are associated with chronic mucocutaneous candidiasis (CMC) (94). Patients with CARD9 deficiency have defects in Th17 differentiation and impaired neutrophil killing (95), and they are susceptible to CMC (96), deep dermatophytoses (97), and invasive fungal infection, particularly *Candida* meningitis. Other monogenic disorders causing CMC include autosomal recessive (AR) IL-17 receptor A (IL17RA), AR IL-17RC, AR ACT1, and autosomal-dominant (AD) IL-17F deficiencies (98). These patients display deficiency of IL-17F and IL-17A/F (*IL-17F* mutations) or dysfunctional responses to IL-17A, IL-17A/F, and IL-17F (*IL17RA*, *IL-17RC*, and *ACT1* mutations). In patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, high plasma titers of neutralizing autoantibodies against IL-17A, IL-17F, and IL-22 can be detected, as a result of the lack of AIRE expression in the thymus causing impaired T-cell tolerance (99). However, endemic mycoses have not been reported in patients with these genetic defects.

Opportunistic fungal infections are common in patients with severe combined immunodeficiencies (SCIDs) and phagocytic disorders. Infants with classical SCID often have recurrent or persistent mucosal and/or cutaneous candidiasis involving the orodigestive tract, genital area, nails, and skin. *Pneumocystis jiroveci* pneumonia (PCP) and invasive fungal infections (IFIs) including systemic candidiasis and aspergillosis are often lifethreatening (100, 101). Interestingly, endemic mycoses have not been described in SCID babies, at least in the English literature. It may be due to the fact that it is uncommon for infants to be exposed to the natural habitats containing those endemic fungi, but further epidemiological data would be required to address this. Numerical and functional defects of phagocytes such as severe congenital neutropenia, chronic granulomatous disease (CGD), and leukocyte adhesion deficiency (LAD) are major groups of PIDs predisposing to systemic candidiasis and invasive aspergillosis (102–104). Filamentous fungi other than *Aspergillus* causing pulmonary infections in CGD include *Geosmithia argillacea* and *Trichosporon inkin*. Osteomyelitis can also be caused by rare non-*Aspergillus* filamentous fungi, including *Cladophialophora arxii*, *Inonotus tropicalis*, *Scedospoium apiospermum*, *Penicillium piceum*, and *P. variotii*. Cerebral abscesses caused by dematiaceous molds such as *Exophiala* spp., *Phaeoacremonium* spp., and *Alternaria* spp. have been reported (105–107). Interestingly, endemic mycoses have not been reported in CGD and LAD.

In the following sections, we highlight the spectrum of PIDs, which are associated with increased susceptibility to disseminated endemic mycoses. The genetic defects are summarized in **Figure 2B**.

### Combined Immunodeficiencies: CD40 Ligand, Nuclear Factor Kappa B (NF-**κ**B) Essential Modulator (NEMO), and DOCK8 Deficiencies

Mucosal candidiasis is common in combined immunodeficiencies e.g. CD40 ligand (CD40L) deficiency, NEMO (IKBG) deficiency, IKBA gain-of-function (GOF) mutation and DOCK8 deficiency, in addition to a broad range of viral, bacterial, and IFI (102, 103). CD40L is expressed on activated T-cells and signals through NEMO/NF-κB to induce IL-12 production. CD40L deficiency, also known as hyper-IgM syndrome, is inherited in an X-linked recessive manner. Patients are susceptible to opportunistic infections including PCP, cryptosporidiosis, and mycobacterial infections due to impaired interaction between T-cells and antigen-presenting cells (APCs). In addition, the failure of B-cell immunoglobulin (Ig) isotype switching results in markedly low serum IgG and IgA, while IgM is elevated (108, 109). Disseminated and cutaneous forms of histoplasmosis have been reported in eight patients in patients with X-linked hyper-IgM disorder (**Table 4**). Five cases had disseminated histoplasmosis, while two had lymphadenitis and one had cutaneous involvement only (86, 110–115). All of them responded well to antifungal therapy, and only two patients had recurrent histoplasmosis (86, 111). One case of PCM was reported in Brazil (116). It was shown that mature DCs from patients with CD40L deficiency exhibited markedly reduced IL-12 and increased IL-10 production in response to *P. brasiliensis* and *C. albicans* compared with normal controls, and T cells had significantly reduced IFN-γ production when cocultured with their DCs, whereas IL-4 and IL-5 production was increased. In contrast, T-cell proliferation and generation of TGF-β and IL-17 were comparable with normal controls. These findings suggested that the absence of CD40L during monocyte/ DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with CD40L deficiency (117). Four cases of *T. marneffei* infection were reported in CD40L deficiency (118–120). One patient who had disseminated *T. marneffei* infection had rapid deterioration due to late diagnosis and died, and CD40L deficiency was diagnosed after he passed away (120).

One patient with NEMO deficiency was reported to have persistent nodal histoplamosis at the age of 52 years. Symptoms including worsening dyspnea and intermittent night sweats that lasted for 1 year, and imaging studies revealed perihilar mass and mediastinal lymphadenopathy. Positive culture of *H. capsulatum* was obtained from paratracheal lymph node biopsy. He responded well to posaconazole (86). A patient with DOCK8 deficiency had miliary pneumonia caused by *H. capsulatum*. She also had numerous infectious caused by viruses (molluscum contagiosum, recurrent herpes zoster, cutaneous human papilloma

#### Figure 2 | Continued

Signaling pathways in innate recognition of fungal pathogens and differentiation of CD4+ T helper cells. (A) Pathogen-associated molecular patterns (PAMPs) expressed by fungi are recognized by host pattern recognition receptors (PRRs), including toll-like receptors (TLRs), C-type lectin receptors (CLRs) [e.g., dendritic cell (DC)-specific ICAM3-grabbing non-integrin (DC-SIGN), Dectin-1, Dectin-2, MINCLE, and mannose receptor] and complement receptor 3 (CR3). TLRs and CLRs activate multiple intracellular signaling pathways upon binding to specific fungal PAMPs, including β-glucans, chitin, *O*-linked mannan and *N*-linked mannan, and nucleic acids. These signals activate canonical or non-canonical nuclear factor-κB and the NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome. The integration of simultaneously activated PRRs occurs at the level of intracellular adaptors and transcription factors shared between overlapping pathways. The resulting cytokine responses shape the activation of adaptive immune response. Induction of IL-12 drives IFN-γ production by T-helper 1 (Th1) cells, which is crucial for phagocyte activation. Induction of IL-1β, IL-6, and IL-23 promotes Th17 differentiation. Regulatory T-cells (Treg) act as host-driven homeostatic response to keep inflammation under control. (B) Th1 and Th17 differentiation. Polarization of naive T cells into Th1 leads to IFNγ production, and its signaling is mediated through the Janus kinase (JAK)–signal transducer and activator of transcription 1 (STAT1) pathway, leading to transcription of IFNγ-inducible genes. IL-6 and IL-21 upregulate the expression of the retinoic acid-related orphan receptor RORγt and RORα, leading to expression of the inducible component of the IL-23 receptor (IL-23R) and further Th17 development. IL-17A and IL-17F produced by Th17 cells augments neutrophil production in the bone marrow and their recruitment to the site of infection. IL-17A, IL-17F, and IL-22 promote production of antimicrobial peptides in epithelial cells. Molecules in which genetic defects have been identified to be associated with increased susceptibility to endemic mycoses are marked in bold red.

virus infection), and dermatitis with *Staphylococcus aureus* superinfection (86, 121).

### GATA2 Deficiency

A syndrome of monocytopenia with susceptibility to nontuberculous mycobacterial (NTM) infections, often termed "MonoMAC," is caused by haploinsufficiency of the hematopoietic transcription factor GATA2 (122). Majority of the patients have monocytopenia, natural killer (NK), and B lymphocytopenia, while CD4 lymphocytopenia and neutropenia are also common but less marked. Affected individuals are susceptible to a broad range of viral (human herpes virus and human papillomavirus), disseminated NTM, bacterial, and fungal infections (123, 124). In a cohort of 57 patients with *GATA2* mutations evaluated at the National Institutes of Health in the US (124), severe fungal infections were observed in 16%, including invasive aspergillosis (9%), disseminated histoplasmosis (5%), and recalcitrant mucosal candidiasis (5%). Another patient with GATA2 deficiency and disseminated histoplasmosis was reported by Lovell et al. (86). All the patients diagnosed to have GATA deficiency with disseminated histoplamosis were adults (86, 124). Apart from infections, other clinical features of GATA2 deficiency include congenital lymphedema, pulmonary alveolar proteinosis, and predisposition to myelodysplastic syndrome or acute myeloid leukemia, but considerable clinical heterogeneity exists.

### Inborn Errors of IFN-**γ**-Dependent Immunity

Host defense against intracellular bacterial and fungal pathogens depends on effective cell-mediated immunity, which is coordinated by APC and T-lymphocytes (89, 90, 125). Following phagocytosis, macrophages, monocytes, and DCs secrete IL-12p70, a heterodimer of IL-12p40 (IL12B) and IL-12p35 (IL-12A) that stimulates T and NK cells through its receptor IL-12R, a heterodimer of IL-12Rβ1 and IL-12Rβ2. IL-12Rβ1 is bound to tyrosine kinase 2 (TYK2), and IL-12Rβ2 is bound to Janus kinase-2 (JAK2). IL-12 receptor signaling induces phosphorylation, dimerization, and nuclear translocation of signal transducer and activator of transcription-4 (STAT4) to induce IFN-γ production in T-cells and NK cells, and drives Th1 polarization of CD4+ T-cells. Binding of IFN-γ to its heterodimeric receptor consisting of IFN-γR1 and IFN-γR2 leads to signal transducer and activator of transcription 1 (STAT1) phosphorylation by Janus kinase 1 (JAK1) and JAK2. The phospho-STAT1 (p-STAT1) homodimer translocates to the nucleus and modifies gene expression regulated by the γ-regulated sequencing, resulting in phagocyte activation including production of bactericidal ROS by NADPH oxidase, further IL-12 production, and killing of intracellular pathogens (126, 127). IL-12 production is augmented by a T-cell dependent pathway through interaction of CD40 on the surface of APC with CD40L expressed on activated T-cells (128). The signaling pathway is shown in **Figure 2B**.

IL-23 shares the p40 component with IL-12p70, and IL-12Rβ1 combines with IL-23 receptor (IL-23R) to form the IL-23R complex (129). IL-23R signaling leads to STAT3/STAT4 heterodimer phosphorylation by TYK2 and JAK2, supporting the proliferation of Th17 cells, which are critical mediators of immunity at the mucosal surface (130). Activated Th17 cells produce IL-17 and IL-22, which induce antimicrobial peptide production in epithelial cells, and the recruitment and activation of inflammatory cells, especially neutrophils (131). These effector functions are critical in the control of mycobacteria, fungi, and bacterial pathogens such as *salmonella* (131–133).

Genetic defects of the IFN-γ-dependent immunity are collectively known as the Mendelian susceptibility to mycobacterial disease (MSMD) (126, 127). These disorders encompass defects of IFN-γ production or response to IFN-γ, caused by mutations in *IL12B*, *IL12RB1*, *ISG15, NEMO*, *IFNGR1*, *IFNGR2*, *STAT1*, *NEMO*, *IRF8*, and *CYBB.* Altogether, they constitute 18 genetic etiologies of MSMD based on the mode of inheritance, complete or partial defect, expression of the mutant allele, and the functional aberrations. Mycobacterial infection is the sole infectious phenotype in some of these disorders (AD IRF8 deficiency, AR ISG15 deficiency), while others have increased susceptibility to a broader range of pathogens (126, 127). Endemic fungal infections have been reported in AR IL12Rβ1, AR IFN-γR1, and AD GOF STAT1 defects.

#### AR IL12R**β**1 Deficiency

Autosomal recessive IL12Rβ1 deficiency is the most common form of MSMD, accounting for approximately half of the cases in which a genetic cause has been identified (126, 127, 134). Patients with IL12Rβ1 deficiency are recognized by their susceptibility to

Table 4 | Endemic mycoses in CD40 ligand deficiency.

*Location of residence is indicated wherever information is available.*

*PCP, Pneumocystis jiroveci pneumonia.*

mycobacterial infections (*M. bovis BCG*, NTM, and *M. tuberculosis*) and non-typhoidal salmonellosis of unusual severity or frequency, but some patients are also susceptible to *Candida*, *Klebsiella*, *Nocardia*, *Leishmania*, *Histoplasma*, *Coccicioides*, *and Paracoccidioides* (134). Peripheral blood mononuclear cells of these patients do not respond to IL-12 and IL-23, resulting in impaired IFN-γ production by T and NK cells. The development of IL-17-producing T-cells is also impaired, due to defect of the IL-23R complex, which is composed of IL-12Rβ1 (135–137). This accounts for the susceptibility to develop CMC observed in 23% of patients (134–137).

The seven reported cases of coccidioidomycosis, PCM, and histoplasmosis in patients with IL12Rβ1 deficiency are summarized in **Table 5** (134, 138–141). The age at which disseminated mycoses developed varied from childhood to adulthood, some with past history of mycobacterial infection and salmonellosis. Two cases had recurrence disease, but could be controlled by antifungal treatment.

#### AR IFN**γ** Receptor Deficiency

IFN-γR1 and IFN-γ2 are the ligand-binding and transducing receptor chains of the INF-γ receptor, respectively. Biallelic null mutations in the *IFNGR1* gene result in AR complete IFNγR1 deficiency, which is characterized by high plasma concentration of IFN-γ and a lack of response to IFNγ *in vitro* (142, 143). These patients have early onset, life-threatening disseminated mycobacterial infections and the overall prognosis is poor with high rate of fatality. Other infections caused by viruses (cytomegalovirus, respiratory syncytial virus, varicellar zoster virus, human herpes virus 8) and bacteria (*Listeria monocytogenes*) have been described. In AR partial IFN-γR1 deficiency, the clinical phenotype is less severe (144). Apart from mycobacterial infections, bacterial, viral, and parasitic organisms have been reported (126, 127, 142, 144).

Autosomal dominant partial IFN-γR1 deficiency is caused by mono-allelic mutations affecting exon 6 and exon 7. A hotspot mutation, 818del4, accounts for over 80% of patients with AD IFN-γR1 deficiency (126, 127). In contrast with AR IFN-γR1 deficiency, these is an increase in IFN-γR1 protein expression on the cell surface, due to the accumulation of truncated IFN-γR1 receptors lacking the recycling domain. Despite the presence of receptors encoded by the wild-type *IFNGR1* allele, the nonfunctioning IFN-γR1 protein impedes the normal function of IFN-γR1 dimers by negative dominance and impairs the response to IFN-γ *in vitro* (145). The clinical features are less severe than those seen in patients in AR complete IFN-γR1 deficiency. Most

Table 5 | Endemic mycoses in IL12RB1 deficiency and INFGR1 deficiency.

(*Continued*)

#### TABLE 5 | Continued

*Location of residence is indicated wherever information is available.*

*IFN, interferon; MAC, Mycobacterium avium complex.*

patients have BCG or NTM infections, and salmonella infection was reported in only 5% of cases (126, 127, 142). Disseminated histoplasmosis (146) and coccidioidomycosis (147) were reported in two patients with IFN-γR1 deficiency and both had a refractory or relapsing course (**Table 5**). Our group diagnosed AR IFN-γ receptor 1 deficiency in a Burmese infant suffering from disseminated *T. marneffei* infection, and she eventually died of salmonellosis (manuscript in preparation).

IFN-γR2 deficiency is less common than IFN-γR1 deficiency. Similarly, AR complete or partial IFN-γR2 deficiency cause increased susceptibility to mycobacterial infections (148–150); other infections are rare and include salmonellosis in one patient and cytomegalovirus disease in three patients (126), but mycosis has not been reported. AD form of partial IFN-γR2 deficiency was diagnosed in a patient with mild BCG disease, and clinical penetrance is very low (151).

#### AD STAT1 Defect

Signal transducer and activator of transcription 1 (STAT1) is a transcription factor involved in cellular responses mediated by type I (IFNα/β), type II (IFN-γ), and type III (IFN-λ) IFNs (152). AR complete STAT1 deficiency is characterized by the absence of STAT1 expression and abolished cellular response to IFN-γ as well as IFN-α/β and IFN-λ, resulting in severely impaired antimycobacterial and antiviral immunity (153, 154). Patients with complete STAT1 deficiency caused by null mutations have increased susceptibility to mycobacterial, viral, and bacterial infections, whereas biallelic hypomorphic mutations in AR partial STAT1 deficiency are associated with milder clinical severity (153–155). AD partial STAT1 deficiency with mono-allelic loss-of-function (LOF) *STAT1* mutation predispose to mycobacterial infection (156); in contrast, AD GOF *STAT1* mutation is recognized as the most common cause of CMC disease, accounting for half of the cases (157–159). Majority of the mutations affect the coiled-coil domain or DNA-binding domain of STAT1 (159). They increase STAT1 phosphorylation by impairing nuclear dephosphorylation. They are GOF for the STAT1-dependent cytokines including IFN-α, IFN-β, IFN-γ, and IL-27, which repress Th17 development, accounting for the low numbers of IL-17-producing T-cells in these patients (160).

In addition to CMC, invasive mycoses caused by a variety of yeasts (e.g., *Cryptococcus*), molds (*Aspergillus*, *Fusarium*), and endemic fungi (*Histoplasma, Coccidioides, T. marneffei*) have been reported in patients with GOF *STAT1* mutations, as summarized in **Table 6** (161, 162). The two patients who developed disseminated coccidioidomycosis during childhood had progressive disease, which persisted into teenage despite intensive treatment; one had spinal cord compression and one died of overwhelming coccidioidomycosis at 17 years. Of note, the latter patient did not have CMC or other unusual infections, implying that coccidioidomycosis could be the sole infection in patients with GOF STAT1 defect. Three patients had disseminated histoplasmosis and two of them had recurrent disease. All of them responded well to antifungal treatment (161).

Table 6 | Endemic mycoses in AD signal transducer and activator of transcription 1 defect.

(*Continued*)

#### TABLE 6 | Continued

*Location of residence is indicated wherever information is available.*

*BAL, bronchoalveolar lavage; CMC, chronic mucocutaneous candidiasis; CNS, central nervous system; DM, diabetes mellitus; EBV, Epstein–Barr virus; IFN, interferon; PML, progressive multifocal leukoencephalopathy; SVC, superior vena cava.*

In Hong Kong, five pediatric patients were diagnosed to have *T. marneffei* infection from 1983 to 2009 in a single center (74, 85, 162, 163). One patient was lost to follow-up after complete recovery from *T. marneffei* infection (163), while the remaining four patients underwent thorough immunological investigations and genetic studies, and all were found to have GOF STAT1 defect (162). They all had CMC, and two had recurrent sinopulmonary infections, herpes virus infections (cytomegalovirus, Epstein– Barr virus and varicella zoster), TB, and disseminated aspergillosis (85, 162). The patient with chronic relapsing *T. marneffei* infection first reported by Yuen et al. in 1986 was subsequently investigated when his son was referred for CMC, and both were confirmed to have AD GOF STAT1 defect in 2015.

An increased incidence of herpes virus infections as well as TB and NTM infections (e.g., *M. bovis BCG*) has been observed in patients with GOF STAT1 defect, and it was thought that the enhancement of signaling downstream to IFN-α/IFN-β and IFN-γ caused by GOF *STAT1* mutations could lead to exhaustion of virus-specific T-cells and refractory response to IFN-γ. Other clinical manifestations of GOF *STAT1* mutations include autoimmunity (e.g., type 1 diabetes mellitus, thyroid disease, autoimmune cytopenia, and hepatitis), vascular aneurysms, and malignancies, particularly squamous cell carcinoma of the esophagus (159, 160, 164).

#### Inborn Errors of Immunity Associated with Impaired Th-17-Mediated Immunity

Autosomal-dominant hyper-IgE syndrome caused by LOF *STAT3* mutation, also known as Job's syndrome, is characterized by recurrent staphylococcal cold abscesses, pneumonia, and eczema. In addition, patients often display joint hyperextensibility, skeletal abnormalities and pathological fractures, delayed dental deciduation, coronary artery aneurysms, brain lesions, and Chiari's malformation (165, 166). Pneumonia is typically caused by *S. aureus, Haemophilus influenzae*, or *Streptococcus pneumoniae*, and is often complicated by pneumoatocele formation. Approximately 20% of patients with AD hyper-IgE syndrome develop invasive infection caused by Aspergillus, which has angioinvasive properties with tendency to cause hematogenous dissemination (167–170). STAT3 promotes the expression of the gene encoding the retinoic acid receptor-related orphan receptor-γt (RORγt), and important transcription factor that drives differentiation of naïve CD4+ T-cells to Th17 cells. Dominant negative *STAT3* mutation leads to impaired RORγt induction in response to IL-1β, IL-6, and IL-23, causing severe deficiency of IL-17-producing effector cells (171, 172).

Three cases of coccidioidomycosis, 10 cases of histoplasmosis and two cases of *T. marneffei* infection were reported in patients with hyper-IgE syndrome, as summarized in **Table 7** (85, 87, 173–183). The three cases of hyper-IgE syndrome with coccidioidomycosis all had CNS involvement (87, 173, 174), while those with IL-12Rβ1 deficiency (139), IFN-γR1 deficiency (147), and GOF STAT1 defect (161) mainly had lymphadenopathy and osteomyelitis, so it appears that there is a predilection for *Coccidioides* to disseminate to the CNS in hyper-IgE syndrome. In contrast, disseminated histoplasmosis occurred in IL-12Rβ1 deficiency (141), IFN-γR1 deficiency (146), and GOF STAT1 defect (161), but 7 out of 10 cases of histoplasmosis in hyper-IgE syndrome involved the aerodigestive tract only (175–182). Two cases of *T. marneffei* infection were reported in hyper-IgE syndrome (85, 183).

To summarize, the susceptibility to endemic mycoses in CD40L deficiency, IL12Rβ1 deficiency, and IFN-γR1 deficiency highlights the critical role of the IL-12/IFN-γ crosstalk in macrophage activation and killing of these endemic fungi, while the deficiency of Th17 cells in patients with GOF STAT1 defect and AD hyper-IgE syndrome puts them at risk for both CMC and IFIs, and they frequently have CMC due to impaired mucosal immunity against *C. albicans*. Defective oxidative burst alone, as in CGD, is not sufficient to cause increased risk to endemic mycoses, suggestive that other mechanisms of phagosomal killing may compensate for the lack of NADPH oxidase activity to control these endemic fungi, distinguishing them from many other invasive fungi to which CGD patients are susceptible.

### PROTECTIVE IMMUNITY AGAINST ENDEMIC MYCOSES CONFERRED BY CYTOKINES: INSIGHTS FROM BIOLOGICS AND ANTI-IFN-**γ** AUTOANTIBODIES

The development of biologic response modifiers (BRMs), such as monoclonal antibodies and receptor antagonists that target

*Location of residence and mutation of STAT3 gene are provided wherever information is available.*

pro-inflammatory cytokines and their receptors has led to major advances in the treatment of autoimmune and malignant disorders. However, they have the potential to suppress host immune response and increase the risk of infections. The use of biologics is associated with a small but important risk of IFI. Histoplasmosis is the most common IFI associated with TNF-α inhibitors (10, 59, 184, 185). In a survey of infectious disease specialists, histoplasmosis was second only to *S. aureus* as the cause of serious infection complicating anti-TNF and other BRM (184). In most cases, patients reside in areas where the fungus is endemic and have received other immunosuppressants concurrently. Up to 2% of patients receiving BRMs will develop coccidioidomycosis if they reside in an endemic region (186). The American Academy of Pediatrics (AAP) Committee on Infectious Diseases recommends that patients on BRM should be enquired about epidemiologic risk factors and possible exposures to histoplasmosis and coccidioidomycosis, which have symptoms and signs that significantly overlap with TB. If there is suspicion of signs or symptoms compatible with acute histoplasmosis or coccidioidomycosis, BRM should be discontinued immediately and patients will require evaluation with a combination of chest radiography and serologic, antigen detection, and culture tests, which are best conducted in consultation with an infectious diseases expert (186, 187).

Other targeted therapies have also been implicated as risk factors for endemic mycoses. In Hong Kong, four cases of disseminated *T. marneffei* infection were diagnosed in adult hematology patients receiving anti-CD20 monoclonal antibodies (rituximab and obinutuzumab) and kinase inhibitors (84, 188, 189). The observation is revealing, as the importance of B-lymphocytes and humoral immune response against fungus is not well defined, and *T. marneffei* infection has so far not been reported in patients with congenital agammaglobulinemia. Depletion of B-lymphocytes may lead to profound deficiency in the production of neutralizing antibodies against key virulence factors of *T. marneffei*. Kinase inhibitors such as ruxolitinib and sorafenib are increasingly used in treating hematological malignancies, solid tumors, psoriasis, and alopecia areata. Ruxolitinib is a selective JAK1 and JAK2 inhibitor that interferes with the IFN-γ and its downstream JAK-STAT signaling. Sorafenib is a multi-kinase inhibitor that exhibits immunomodulatory effect by impairing T-lymphocyte proliferation, production of IFN-γ and other pro-inflammatory cytokines, NK cell, and DC functions. The suppression of IFN-γ signaling pathway poses risk to develop *T. marneffei* infection (84). It would be important for clinicians to have a high index of suspicion on *T. marneffei* infection in patients receiving these targeted therapies to avoid delay in diagnosis and treatment.

Autoantibody against IFN-γ has been reported to be associated with adult-onset immunodeficiency in patients from Asian countries (190–195). Disseminated NTM is the most common clinical presentation. In a cross-sectional, case-control study conducted in Chiang Mai, Thailand showed that patients with opportunistic infections including disseminated NTM, disseminated *T. marneffei* infection, melioidosis and non-typhoidal Salmonellosis had anti-IFN-γ autoantibody level above 99th percentile of cut-off for healthy individuals, and the level of autoantibody in patients who had active opportunistic infection was relatively higher than those without active infection (193). Similar observations were also reported in Hong Kong and Taiwan (190, 192, 194, 195). HLA class II molecules HLA-DRB1\*15:02–HLA-DQB1\*05:01 and HLA-DRB1\*16:02–HLA-DQB1\*05:02 are specifically associated with anti-IFN-γ autoantibodies and NTM (196, 197). The high frequency of such alleles in Southeast Asia might account for the relatively high prevalence this condition in the Asian population. The study by Lin et al. showed that anti-IFN-γ autoantibody from patients recognizes an epitope at the C terminus of IFN-γ, and binding of the autoantibody neutralizes IFNγ-induced signaling. This epitope displays a high degree of sequence homology to the Aspergillus Noc2 protein. It was postulated that in the warm and humid environment of Southeast Asia where exposure to Aspergillus species is common in everyday life, some individuals might develop anti-IFN-γ autoantibodies due to molecular mimicry (198). The co-evolution of anti-IFN-γ autoantibody production as the susceptibility trait amongst Southeast Asians, and the high prevalence of *T. marneffei*, NTM, and mellioidosis in this region is a unique combination not observed in the rest of the world, and it is so interesting that exposure to a common environmental fungal agent could indirectly induce susceptibility to other pathogens.

#### FUTURE PERSPECTIVES

The understanding about inborn errors of immunity predisposing to endemic mycoses is limited. First, exposure to these environmental fungal pathogens is often a chance event that depends on the natural habitat and climate, as well as the circumstances and activities in which the individual is engaged. Thus, the number of cases of endemic mycoses associating with PID is likely to be low. The proportion of patients with PID amongst those with disseminated endemic mycoses is unknown, due to the lack of information about the population incidence (i.e., the "denominator"). Second, some forms of endemic mycoses, particularly *T. marneffei* are prevalent in less resourced countries where well-developed clinical service for PID is lacking, and diagnosis is often delayed or missed. Third, the global health impact of these geographically restricted endemic fungi is probably less than those opportunistic fungal pathogens of worldwide distribution causing high disease burden in immunocompromised patients (e.g., *Candida*, *Aspergillus*, *P. jiroveci*, *Cryptococcus*, and *Rhizopus*), and they probably generate less attractions and interests in the global public and scientific community (7, 199, 200). Regional and global effort in establishing registries on disseminated endemic mycoses is crucial, in order to collect patient demographic data and determine their true population incidence.

While endemic mycoses are geographically restricted to certain regions, clinicians looking after patients with PID or acquired immunodeficiencies should gain knowledge about these rare fungal infections so that appropriate advice can be given to their patients when planning for travels, and to have heightened awareness of such diagnostic possibility when they return from endemic areas. Climate, environment, and exposure (behavior) are the "triad" that determine the risk of endemic fungal infection in susceptible hosts. High-risk activities that increase the chance of exposure to fungal conidia should be avoided, otherwise, precautionary measures should be taken.

The discovery of PID predisposing to endemic mycoses is a fascinating journey, as it illuminates the key molecules and signaling pathways that are crucial in host defense against this group of dimorphic fungi, which are closely related in phylogeny. As disseminated coccidioidomycosis, histoplasmosis, PCM, and *T. marneffei* infection are recognized as AIDS-defining illness, they should also be regarded as indicator diseases for PID in individuals who are HIV-negative, and without known risk factors and secondary immunosuppression, particularly in children. There is a need to design an algorithm to evaluate such patients, with stepwise immunological investigations. A detailed history on previous infections, CMC, autoimmune manifestations, and family history should be taken. We recommend a basic panel of immunological evaluations including Ig pattern (IgG, IgA, IgM, and IgE), lymphocyte subset, and nitroblue tetrazolium, or dihydrorhodamine tests to assess oxidative burst activity. These patients should be assessed by immunologists. Abnormal results obtained during acute illness should be repeated upon full recovery. A systematic approach will facilitate clinicians to identify patients who warrant candidate gene studies or functional

#### REFERENCES

delineation of the pathways involved in immune recognition, T-cell activation and differentiation, cytokine signaling, and phagocytic killing. The presence of anti-IFN-γ autoantibody should be excluded. Functional evaluation of the IL-12/IFN-γ axis, STAT1 phosphorylation studies, and Th17 enumeration will be particularly relevant in this context. The utilization of next-generation sequencing techniques may lead to discoveries of novel monogenic disorders causing unique susceptibility to endemic mycoses.

### AUTHOR CONTRIBUTIONS

PL wrote the article. Y-LL provided the conceptual framework and reviewed the manuscript.

#### FUNDING

The authors would like to thank the Hong Kong Society for the Relief of Disabled Children for funding the molecular testing of primary immunodeficiency disorders for patients in Hong Kong and others referred to the Asian Primary Immunodeficiency Network.

neglect to priority. *PLoS Negl Trop Dis* (2014) 8:e3100. doi:10.1371/journal. pntd.0003100

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer, PR-J, and handling editor declared their shared affiliation and the handling editor states that the process nevertheless met the standards of a fair and objective review.

*Copyright © 2017 Lee and Lau. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Uses of Next-Generation Sequencing Technologies for the Diagnosis of Primary immunodeficiencies

#### *Michael Seleman† , Rodrigo Hoyos-Bachiloglu† , Raif S. Geha† and Janet Chou\*†*

*Division of Immunology, Boston Children's Hospital, Boston, MA, United States*

Primary immunodeficiencies (PIDs) are genetic disorders impairing host immunity, leading to life-threatening infections, autoimmunity, and/or malignancies. Genomic technologies have been critical for expediting the discovery of novel genetic defects underlying PIDs, expanding our knowledge of the complex clinical phenotypes associated with PIDs, and in shifting paradigms of PID pathogenesis. Once considered Mendelian, monogenic, and completely penetrant disorders, genomic studies have redefined PIDs as a heterogeneous group of diseases found in the global population that may arise through multigenic defects, non-germline transmission, and with variable penetrance. This review examines the uses of next-generation DNA sequencing (NGS) in the diagnosis of PIDs. While whole genome sequencing identifies variants throughout the genome, whole exome sequencing sequences only the protein-coding regions within a genome, and targeted gene panels sequence only a specific cohort of genes. The advantages and limitations of each sequencing approach are compared. The complexities of variant interpretation and variant validation remain the major challenge in wide-spread implementation of these technologies. Lastly, the roles of NGS in newborn screening and precision therapeutics for individuals with PID are also addressed.

#### *Edited by:*

*Mohamed-Ridha Barbouche, Institut Pasteur de Tunis, Tunisia*

#### *Reviewed by:*

*Steven M. Holland, National Institutes of Health, United States Yu Lung Lau, University of Hong Kong, Hong Kong*

#### *\*Correspondence:*

*Janet Chou<EMAIL_ADDRESS>
*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology*

*Received: 01 March 2017 Accepted: 05 July 2017 Published: 24 July 2017*

#### *Citation:*

*Seleman M, Hoyos-Bachiloglu R, Geha RS and Chou J (2017) Uses of Next-Generation Sequencing Technologies for the Diagnosis of Primary Immunodeficiencies. Front. Immunol. 8:847. doi: 10.3389/fimmu.2017.00847*

Keywords: primary immunodeficiency, next-generation sequencing, whole exome sequencing, gene panels, genomics

## INTRODUCTION

Primary immunodeficiencies (PIDs) are genetic diseases that affect the development and/or function of the immune system, thereby increasing the susceptibility to infectious pathogens, autoimmunity (1), and malignancies (2). Classically, PIDs have been considered monogenic disorders that follow the principles of Mendelian inheritance. However, advances in DNA sequencing technologies have facilitated the identification of multigenic and somatic causes of PIDs and have revealed the wide phenotypic variability of these diseases (3, 4).