After the publication of the first GWAS on RLS, a subsequent study by Xiong et al. (27), used human lymphoblastoid cell lines (LCL) as well as two different brain regions (thalamus and pons) from RLS patients for an expression study. A q-RT-PCR followed by western blot analysis showed that the patients who harbor the MEIS1 risk haplotype (GG/GG, rs12469063–rs2300478) express lower levels of MEIS1 mRNA and protein in LCL and thalamus (**Figure 1**). The authors argued that lower MEIS1 expression in a subset of individuals can contribute to the development of RLS symptoms (27).

Given the lower MEIS1 expression in a subgroup of patients, in vivo studies on mouse models with heterozygous Meis1 knockout were conducted (23, 28). Young male and female mice showed hyperactivity with no effect on anxiety-related behaviors, providing a potential animal model to study RLS (**Figure 1**). Furthermore, considering the age-related manifestation of RLS symptoms in human, effects of Meis1 haploinsufficiency was also studied in middle aged mice in a study by Salminen et al. (29). Meis1 haploinsufficiency was associated with a sex dependent increase in the activity more specific to the initial of the rest phase of animal, similar to the circadian rhythm of RLS symptoms observed in human patients. Effects on sensorimotor system in a sex dependent manner were also reported (29).

## THE LINKS BETWEEN MEIS1 AND IRON METABOLISM

Observations made using a Caenorhabditis elegans model showed that post developmental inactivation of Unc-62 (MEIS1 ortholog) makes it one of the 64 genes that increase the worm's life span. Unc-62 in the worms has 25% identity with MEIS1 and almost 80% identity with its homeodomain. Adding iron chelators in the culture media of the worms resulted in changes in the effect of Unc-62 inactivation on worms' lifespan, suggesting that Unc-62 is involved in iron metabolism. The link between Unc-62 and iron was also measured through its impact on the expression of ferritin. It was found that post developmental inactivation of Unc-62 using RNAi resulted in higher expression of ferritin, a protein that plays a key role in iron metabolism through its ability to store excess iron and to release it into a soluble and nontoxic form (30). Thalamus samples obtained from RLS patients also showed that individuals with the MEIS1 risk haplotype, who also have a reduced expression of MEIS1, show an increased expression of both ferritin (FTN) light and heavy chains. Furthermore, DMT1 gene expression was higher in these individuals. DMT1 is a proton-coupled metal transporter that carries iron from the extracellular domain to the cytoplasm (**Figure 1**). This finding also supports a link between MEIS1 and iron metabolism where DMT1 transports iron into the brain (31). More work remains to be done to clarify the exact role of MEIS1 in iron metabolism relevant to RLS (32). Recent

studies suggest that DMT1 is expressed in endosomes of brain capillary endothelial cells denoting the blood-brain barrier (BBB) (33). The best well-established neurobiological abnormality in RLS is reduced brain iron, despite a normal peripheral iron level. The fact that some, but not all RLS patients, respond to intravenous (IV) iron provides an opportunity to interrogate the underlying pathways differing between responders versus non-responders. This led some studies to focus their attention toward the iron uptake at the blood-brain barrier (34). A preliminary analysis of MEIS1 expression showed elevated MEIS1 levels in the microvasculature isolated from RLS brain tissue by comparison to the tissues of control individuals. Moreover, a cell culture model of the BBB showed that treatment with an iron chelator increased the MEIS1 expression, while iron loading conversely decreased MEIS1 expression (unpublished data presented in a review article on the links between iron and RLS by Connor et al.; the small sample size in this report indicates that more investigations remain to be done to further validate this observation) (35, 36). These data suggest a novel role for MEIS1 in the BBB that warrants further examination. There are also observations revealing peripheral hypoxia to be associated with RLS symptoms (37). Hypoxia pathway is activated in a number of cell types of RLS patients; this activation can result from or be related to cellular iron deficiency (38). Another study used LCLs and showed MEIS1 down regulation by RNAi techniques resulted in an increase in transferrin-2 receptor and ferroportin and a decrease in hepcidin mRNA expression (39, 40). The authors suggest that MEIS1 might control cellular iron transfer to mitochondria and cellular export of iron (40). Putting together all these findings, the data suggest that decreased acquisition of iron by the brain cells is an RLS related pathophysiology, for which a possible role for MEIS1 can be accounted.

### MEIS1 HAS AN ALLELE DEPENDENT CIS-REGULATORY FUNCTION IN TELENCEPHALON (A STUDY IN MICE AND ZEBRAFISH)

A cluster of highly conserved non-coding regions (HCNRs) in the MEIS1 locus suggests the presence of cis-regulatory elements (23). Considering that most variants found by GWAS are located in the regulatory regions, Spieler et al. conducted a study to identify the cis-regulatory role of the common intronic variants in MEIS1 HCNRs (23). They studied an RLS associated variant (rs12469063), which is in the HCNR 617 of MEIS1, in transgenic mice and zebrafish using a reporter assay. They found that in mice, rs12469063 lies within a region of high interspecies conservation with neural enhancer activity and has an allele-specific functional impact. This study found that the risk allele of rs12469063 decreases the enhancer activity of this region in LGE and MGE (lateral and medial ganglionic eminences). The effect of rs12469063 on MEIS1 enhancer function in the LGE/MGE region suggests that RLS may involve the basal ganglia because these regions give rise to the basal ganglia [also discussed in a review by Salminen et al. (41)]. The enhancer activity of HCNR harboring the RLS associated rs12469063 happens in this mouse model during development, which suggests its predisposition to RLS occurs during embryonic development (**Figure 1**). Affinity chromatography showed that CREB1 has higher binding affinity to the RLS risk allele compared to the protective allele of rs12469063. A reporter screen in the zebrafish confirms the enhancer activity of HCNR observed in the mice and found two more transcriptionally active enhancers to this region. HCNR 617 harboring rs12469063 is the only RLS-SNP dependent enhancer region (23, 42).

### MEIS1 REGULATES SKOR1

Expression studies of the RLS associated loci of BTBD9, MAP2K5, and SKOR1 (previously called LBXCOR1) did not find changes in their levels of expression in lymphoblasts or two brain regions (pons and thalamus) of RLS patients (43). However, RLS patients with the MEIS1 risk haplotype were observed to have a reduced expression of SKOR1, in addition to a reduced expression of MEIS1 (**Figure 1**) (43). Follow up studies using siRNA targeting MEIS1, electromobility shift assays and luciferase reporter assays suggested the expression of SKOR1 to be under the regulatory control of MEIS1. This transcription factor action of MEIS1 is due to its direct binding on two distinct promoter regions of SKOR1 (43). Hence the dysregulation of MEIS1 might predispose to RLS both directly and indirectly, possibly throughout its regulatory role on other genes like SKOR1. A new SNP reported in this study is 8.7 kb upstream SKOR1 ATG start site, which acts as a regulatory SNP (rSNP). The risk allele in this locus reduces the binding affinity of MEIS1 to the SKOR1 promotor and results in reduced expression of SKOR1 (43). SKOR1 acts as the transcriptional corepressor of LBX1, a homeodomain transcription factor. Skor1 expression in the Mice embryonic CNS is present in a certain subset of post-mitotic neurons generated posterior to the midbrain-hindbrain border. Skor1 is selectively expressed in the dorsal horn interneurons of developing spinal cord in Mice, where Lbx1 is required for proper specification. It is suggested that SKOR1 probably mediates the sensory inputs of RLS, among others. Despite the importance of SKOR1 in RLS genetic, only little is known regarding the actual function of this gene in RLS underlying pathways. The current literature only suggests that MEIS1 dysregulation may causes SKOR1 dysregulation possibly leading to the sensory phenotypes of RLS (44).

### MEIS1 AND OTHER SLEEP RELATED DISORDERS LIKE INSOMNIA, PLMS AND RBD

Insomnia is characterized by problems in falling asleep or maintaining asleep. With a heritability estimate of 38 and 59% in men and women, respectively, genetic factors must play a crucial role in insomnia. Recent genetic studies of insomnia using cases from the UK biobank showed that MEIS1 has the strongest association signal, suggesting MEIS1 may be a shared genetic risk factor for RLS and insomnia (45–47). Some reports argue that the phenotype overlap could only drive some, and not all of the MEIS1's association with insomnia, thus suggesting that MEIS1 has a pleiotropic effect on RLS and insomnia (45). However, other reports suggest that the association of MEIS1 with insomnia only comes from the inclusion of RLS cases (48). Such inconsistencies might be due to the heterogeneous phenotypic definition of insomnia itself which can lead to the inclusion of a substantial number of RLS cases. Furthermore, GWAS on periodic leg movement during sleep (PLMS), which are present in approximately 80% of RLS cases, also shows association with MEIS1 (49–53). This pleiotropic effect can arise from MEIS1's wide expression pattern during the development (54). So far, no genetic links between RBD (REM sleep behavior disorder) and MEIS1 have been reported.

### CONCLUSION

MEIS1 region is one of the several loci found to be associated with RLS genetic, which overall explain less than 10% of RLS heritability. The many roles for MEIS1 in development make the study of its role in RLS challenging. MEIS1 establishes motor neuron pool identity and their target-muscle connectivity (55), it also regulates the proximodistal limb axis development (21). This protein is highly expressed in dopaminergic neurons of the substantia nigra and red nucleus, though what it does in these cells remains unknown (22–24). Meanwhile, the biology of RLS is poorly understood, with the most consistent abnormality being altered iron homeostasis with brain iron deficiency (56–59). Data presented in this report suggest that the role of MEIS1 in RLS involves, among possibly other functions, altered iron homeostasis via altered transcriptional regulatory activity in RLS pathways. More in depth follow up studies on the function of MEIS1 with more focus on its regulatory role as a transcription factor might shed more light on the underlying pathways involved in RLS. To reach this goal, it will be essential to have access to RLS patients brain material carrying different genotypes of the RLS GWAS signals and to detailed clinical data to be used as covariates in the analyses. This combination would increase the likelihood of identifying elements that are critical to the onset and progression of RLS. This clinical data includes patient iron levels, their response to medication, age at onset, familial or sporadic RLS, presence or absence of PLMS, diagnosis by a physician and information about the patients' other health conditions. The next step could involve the use of model organisms to further validate and investigate RLS related mechanisms.

### AUTHOR CONTRIBUTIONS

FS contributed to the original concept of the article, did the literature search, wrote the original version of the manuscript and reviewed it as it progressed. PD and GR contributed to the original concept of the manuscript and reviewed the manuscript.

### 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 © 2019 Sarayloo, Dion and Rouleau. 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) and the copyright owner(s) 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.

# VPS35-Based Approach: A Potential Innovative Treatment in Parkinson's Disease

#### Simona Eleuteri 1 \* and Alberto Albanese1,2

1 Department of Neurology, Humanitas Research Hospital, Milan, Italy, 2 Department of Neurology, Catholic University, Milan, Italy

Several symptomatic treatments for Parkinson's disease (PD) are currently available. Still, the challenge today is to find a therapy that might reduce degeneration and slow down disease progression. The identification of pathogenic mutations in familial Parkinsonism (fPD) associated to the monogenic forms of PD provided pathophysiological insights and highlighted novel targets for therapeutic intervention. Mutations in the VPS35 gene have been associated with autosomal dominant fPD and a clinical phenotype indistinguishable from idiopathic PD. Although VPS35 mutations are relatively rare causes of PD, their study may help understanding specific cellular and molecular alterations that lead to accumulation α-synuclein in neurons of PD patients. Interacting with such mechanisms may provide innovative therapeutic approaches. We review here the evidence on the physiological role of VPS35 as a key intracellular trafficking protein controlling relevant neuronal functions. We further analyze VPS35 activity on α-synuclein degradation pathways that control the equilibrium between α-synuclein clearance and accumulation. Finally, we highlight the strategies for increasing VPS35 levels as a potential tool to treat PD.

Keywords: endosomal trafficking, retromer complex, therapeutic targets, Parkinson's disease, alpha-synucein, amyloid protein A (AA)

### INTRODUCTION

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting 1% of people over 65 and 4.3% of those older than 85 (1).

The clinical hallmark of PD is bradykinesia, in combination with rest tremor and rigidity (1). This phase is preceded by a preclinical stage, when neurodegenerative processes have commenced, but there are no evident symptoms or signs, and by a prodromal phase, when symptoms and signs are present, but are yet insufficient to define disease (2). The motor phenomenology is attributed to the selective loss of dopaminergic neurons (DNs) in the substantia nigra pars compacta (SNpc) resulting in degeneration of the nigrostriatal tract and dopamine deficiency (3).

The neuropathological hallmark of PD is the occurrence of intracellular inclusions, Lewy bodies (LBs) and Lewy neurites, mainly consisting of aggregated α-synuclein (4). A remarkably convincing set of data supports the view that accumulation and aggregation of α-synuclein in neurons may represent a key pathogenic event (5–9). Several approaches aim to reduce the accumulation and propagation of α-synuclein toxic aggregates in the brain (10–12) (**Figure 1**). Ideally, an intervention aimed to reduce α-synuclein should be performed in the preclinical disease phase or—at least—in the prodromal phase, although it may be argued that the early clinical phase may also provide a suitable time window.

#### Edited by:

Giuseppe De Michele, University of Naples Federico II, Italy

#### Reviewed by:

Darren John Moore, Van Andel Research Institute (VARI), United States Suzanne Lesage, Institut National de la Santé et de la Recherche Médicale (INSERM), France

#### \*Correspondence:

Simona Eleuteri<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Movement Disorders, a section of the journal Frontiers in Neurology

Received: 12 July 2019 Accepted: 18 November 2019 Published: 17 December 2019

#### Citation:

Eleuteri S and Albanese A (2019) VPS35-Based Approach: A Potential Innovative Treatment in Parkinson's Disease. Front. Neurol. 10:1272. doi: 10.3389/fneur.2019.01272

**50**

pathways. CMA, chaperone mediated autophagy; UPS, ubiquitin proteosome system; LAMP2A, lysosome-associated membrane protein 2A.

Monogenic PD subtypes represent a powerful tool for understanding the pathogenesis of PD and for developing treatment strategies. Mutations of the VPS35-retromer subunit have been recently shown to cause PD and to play a key role in neurodegenerative processes, as they control balance between degradation and recycling of fundamental proteins for neuronal survival (13). Moreover, this review outlines the genetic and biochemical evidence that pathogenic missense mutation and depletion of VPS35 affect dopamine (DA) neurons function at different levels. More recently, a direct link has been established between VPS35 and α-synuclein in a rodent PD model where VPS35 deficiency causes α-synuclein accumulation (14). This effect of VPS35 could unravel its key role in controlling macroautophagy, chaperone-mediated autophagy (CMA), and lysosomal function, which are the main degradation pathways of α-synuclein (15–17). A growing body of data converges to establish how VPS35 controls the transport and the localization of protein markers involved in α-synuclein degradation pathways, thus linking VPS35 to α-synuclein accumulation (18–21). An increase in VPS35 levels in PD mice rescues α-synuclein accumulation and induces neuroprotection (14), pointing to a possible role of VPS35 as a therapeutic

**Abbreviations:** AD, Alzheimer's disease; APP, amyloid protein precursor; ATG9A, autophagy-related protein 9A; CI-MPR, cation independent 6 mannose phosphate receptor; CMA, chaperone-mediated autophagy; CTSD, cathepsin D; DN, dopaminergic neurons; DAT, dopamine transporter; EE, Early endosome; LAMP2A, Lysosome-associated membrane protein 2A; PD, Parkinson's disease; SN, substantia nigra; TGN, trans-Golgi Network; WASH, Wiskott–Aldrich syndrome protein; VPS35, vacuolar sorting protein 35.

target for PD. Moreover, VPS35 could represent a diseasesmodifying target, and its neuroprotective role could be due to the control on neuronal signaling events (22), synaptic plasticity (23–25), trafficking of protein in dendritic spines (26), membrane dopamine transporter (DAT) recycling in DA neurons (27), and the regulation mitochondrial functions (28).

### PATHOGENIC ROLE OF VPS35 IN PD

Two independent studies with exome sequencing identified point mutations in the VPS35 gene causing an autosomal dominant form of PD (PARK17) (29, 30). The p.Asp620Asn (c.1858G>A) mutation has been proven to be pathogenic with a frequency ∼1.3% in familial cases and 0.3% in sporadic PD cases (31, 32). In addition, non-pathogenic VPS35 variants (33) and variant of uncertain significance (34) have been described. The VPS35 gene maps on chromosome 16q11.2 encompasses 29.6 kb and 17 exons (35) (**Figure 2**).

The clinical presentation of PARK17 PD patients bearing pathogenic VPS35 gene mutations is that of a typical clinically definite PD. The mean age at disease onset is 50.3 ± 7.3 years; cognitive and psychiatric features do not predominate, and levodopa response is sustained and not associated to severe dyskinesias (37).

To evaluate the pathogenic the role of VPS35 deletion and the VPS35–D620N mutation, different transgenic mouse models have been developed. Knockout mice with the deletion of the endogenous VPS35 at embryonic stage resulted in early lethality under embryonic day 10 (38). VPS35 heterozygote mice (expressing 50% of protein respect to the wild type) survived to adulthood (38) and did not presented any PD-relevant deficit up to 6 months of age. However, the neuropathological evaluation at late stage (12 months) in VPS35 heterozygote mice revealed DA neuronal loss in SNpc and increase in α-syn levels (16). VPS35DAT−CRE mice demonstrated as the depletion of VPS35 protein in SNpc-DA neurons at 2–3 months of age already showed a reduction in TH+ DA neurons and TH+ fibers in the striatum (28). To evaluate the effect of VPS35–D620N mutation, a viral-mediated gene transfer model in adult rat has been developed, showing that the expression of the mutant in the nigrostriatal pathway was sufficient to induce DA neuronal loss and axonal pathology (39). VPS35– D620N knockin (KI) mice (40) at 3 months of age have shown normal motor functions and no loss of striatal DA neurons (41, 42). However, it has been reported that KI mice showed increased capacity to evoke dopamine release in dorsolateral striatum, consistent with elevated extracellular dopamine (42). The evaluation of the effect of VPS35–D620 KI mice on PD pathology with chronic aging provided evidence that the increase in the endogenous D620N mutant levels has a gain of function or partial-negative dominant mechanism (43). Thus, the endogenous expression of VPS35–D620N is sufficient to recapitulate some features of PD pathology inducing (i) the progressive degeneration of the nigrostriatal pathway, (ii) modest motor deficit with age consistence, and (iii) widespread axonal damage (43). In VPS35–D620N KI mice, α-synuclein accumulation and LB pathology were not detected, but tau-positive pathology, characterized by the abnormal accumulation of early "pretangle," has been described (43). Moreover, VPS35–D620N KI mice or knockdown or knockout of VPS35 mice impacted LRRK2-mediated Rab protein phosphorylation (44) showing that VPS35 could play a major role in controlling LRRKK2 kinase activity and supporting the hypothesis that VPS35–D620N mutation resulted in a gain of function (44).

sorting protein 29 (VPS29; 20 kDa), vacuolar sorting protein 35 (VPS35; 92 kDa); the dimer of sinexin (SNXs). The N-terminal region (in blue) from the amino acid residues 1 to 172 [1–172] and C-terminal region (violet) from 307 to 796 (307–796) are important, respectively, for the interaction with VPS26 and VPS29 (36). The amino acid residues involved in the interaction with the SNXs are at N-terminal region (1–35, 37–54) and C-terminal region (307–796). A structural level VPS35 is a right-handed α-helix solenoid, and it is predicted to have 34 helices, 13 of which are in C-terminal. Different missense mutations have been identified in fPD. VPS35 variant of uncertain significance (VUS) are localized between exon 9 (P316S), exon 13 (R542W), and exon 14 (I560T, H599R, M607V), and the confirmed pathogenic mutation (D620N) is localized on the exon 15 (genes in light violet).

### VPS35 SUBUNIT OF THE RETROMER COMPLEX

The VPS35 gene encodes for a subunit of the retromer complex that is composed of 796 amino acids with a molecular weight of 92 kDa (45). The retromer is an evolutionary conserved complex of eukaryotic cells that structurally comprises two subcomplexes: (i) a cargo recognition complex VPS26–VPS29– VPS35 heterotrimer mainly involved in the transport of proteins and (ii) a membrane-targeting dimer of sorting nexin, important for the adhesion of the complex to the endosomal membrane (SNX1, SNX2, SNX5, SNX6, and SNX32) (45) (**Figure 2**). Mammals have two paralogues of VPS26 subunit (VPS26A and VPS26B) that compete for a single binding site on VPS35 (46). Two variants of VPS26A have been identified in a patient with familial PD (46).

The retromer complex represents the master "conductor" of the sorting in the endosomal network; it is localized at the early endosome (EE) and mainly controls the retrograde trafficking of cargo proteins from EE to the trans-Golgi network (TGN) or to the plasma membrane (**Figure 3**). Moreover, the retromer complex mediates vesicular transport from mitochondria to peroxisomes (47). This complex represents the main sorting hub that receives, dissociates, and sorts cargoes of different origin: (i) plasma membrane (recycling of membrane receptors), (ii) biosynthetic pathways (retrieval of trafficking from Golgi), and (iii) lysosomal pathway (cargoes direct to lysosomes) (48) (**Figure 3**). Overall, these activities control the homeostasis of transmembrane proteins at plasma membrane and endolysosomal levels and regulate receptor abundance, signaling receptors, adhesion molecules, and hydrolase receptors.

### THE RETROMER COMPLEX CONTROLS SYNAPTIC FUNCTION

It has been recently clarified that the retromer complex plays a key role in neurons, where it can, directly or indirectly, mediate essential neuronal functions through cargo selector accessory proteins (SNX27 and WASH complex). Thus, the retromer influences neuronal signaling events (such as downregulation of receptors activity) (22), synaptic plasticity (23–25), trafficking of protein in dendritic spines (26), and membrane DAT recycling in DA neurons (27). The VPS26-retromer subunit belonging to the β-arrestin family controls the downregulation and recycling

FIGURE 3 | Retromer complex functions. Retromer complex (in green) controls the sorting of cargoes (1) originate from plasma membrane (receptors in red), (2) direct to lysosome for degradation, and (3) trafficking between trans-Golgi Network (TGN) (3, 4). Figure was adapted from Servier Medical Art images (http://smart.servier. com/).

to cell surface of β2-adrenergic receptor A (22). This is a G-protein-coupled receptor characterized by a cyclical pattern of activation/inactivation through endosomal internalization: the retromer balances the recycling and the degradation of this receptor (49). The retromer could also influence neuronal activity indirectly, controlling function and localization of accessory proteins, such as SNX27 and WASH complex. The SNX27 protein controls recycling of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, involved in longterm potentiation, a process linked to learning and memory (23, 25). The D620N VPS35 pathogenic mutation impairs interaction with the WASH complex (15) and may lead to mistrafficking protein cargoes, such as the GluA1 glutamate receptors subunit, the Glut-1 glucose transporter (26, 49, 50) and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (51). DAT, which is strictly associated to the activity of DA neurons, undergoes constitutive internalization: its recycling is mediated by the retromer complex (27).

Therefore, retromer disruption via short hairpin RNAmediated VPS35 knockdown results in DAT depletion from plasma membrane and decrease in DAT recycling (27). In summary, the retromer influences trafficking of signaling receptors and controls rapid signaling events involved in synaptic plasticity and dendritic spine formation that are specifically relevant to DA neuron function (27).

### VPS35 INFLUENCES α-SYNUCLEIN ACCUMULATION

Recent studies highlighted the crucial role of the retromer complex in controlling the accumulation of α-synuclein in PD mouse models and in an in vitro model of α-synuclein spreading (14). Reduction in VPS35 levels or the expression of mutated VPS35 protein in PD mouse hippocampus displayed defective α-synuclein clearance resulting in widespread accumulation of aggregates (14). These data were confirmed in mouse models showing that VPS35 deficiency or a pathogenic VPS35 mutation leads to accumulation and aggregation of α-synuclein in the SN, accompanied by degeneration of DA neurons, reduction in DA levels, impairment of locomotor behavior, and alteration of lysosomal morphology (16). On the contrary, an excess of wild-type VPS35 expression rescues the accumulation of αsynuclein aggregates and leads to the reduction in neuronal loss and astrogliosis in a PD mouse model overexpressing αsynuclein (14). Using an in vitro model of cortical neurons, it has been demonstrated that an increase in wild-type VPS35 levels may counteract the propagation of α-synuclein aggregates from neuron to neuron (14). In keeping with this line of evidence, VPS35 ablation in Drosophila results in the accumulation of α-synuclein in lysosomes (17). Furthermore, silencing of endogenous VPS35 in an α-synuclein transgenic fly does not only promote the accumulation of human wild-type α-synuclein but also causes eye degeneration and motor disability (17). These data suggest that endosomal dysfunction caused by VPS35 deficiency impairs the ability of neurons to cope with the accumulation of α-synuclein, thus facilitating the spread of PD pathology.

### HOW DOES VPS35 CONTROL α-SYNUCLEIN ACCUMULATION?

The hypothesis that VPS35 could promote α-synuclein clearance is related to the observation of a central role of this retromer protein in controlling interaction and transport of key proteins in α-synuclein degradation pathways.

In normal conditions, the quality control network dealing with α-synuclein accumulation is efficient in promoting its refolding or degradation. In general, to induce α-synuclein degradation, a cell can activate the ubiquitin protein system or alternatively—in the presence of intracytoplasmatic aggregates and LBs—can enhance the endosome-lysosomal degradation system, macroautophagy, or CMA (52) (**Figure 4**). Recent studies highlighted that VPS35 can act at different levels of the αsynuclein degradation pathways (**Figure 5**). Particularly, VPS35 acts indirectly on lysosomal activity by sorting receptors of lysosomal hydrolases, including Sortilin and cation independent 6 mannose phosphate receptor (CI-MPR) (18). VPS35 regulates macroautophagy controlling the localization of WASH complex and the trafficking of ATG9a protein (15). Finally, VPS35 manages CMA by mediating the retrieval and transport of LAMP-2A receptor to lysosomes (16) (**Figure 5**).

### VPS35 Controls the Lysosomal Degradation Pathway

α-Synuclein enters through macroautophagy, CMA, and endosomal pathway into lysosome to be degraded by lysosomal hydrolases (53). VPS35 carries several cargoes involved in neurodegenerative disorders and more specifically in the clearance of α-synuclein (48). Two well-studied VPS35 cargoes are Sortilin (20, 21) and CI-MPR (18, 19), and VPS35–D620N mutant showed defects in sorting CI-MPR (54). These two cargoes operate as lysosomal hydrolase receptors, sorting acid hydrolases, such as cathepsins, from TGN to lysosomes. Aspartyl protease (cathepsin D) is the main lysosomal endopeptidase responsible for the degradation of α-synuclein (55). However, cysteine proteases (cathepsin B and L) have been shown to be effective in degraded α-synuclein (53). Direct interaction between α-synuclein and lysosomal proteases was shown in mice and human purified lysosomal extracts (53). CTSD-deficient mice showed accumulation of insoluble α-synuclein aggregates in the brain (55). Accordingly, CTSD-mutant brain from mice, sheep, and human showed selective synucleinopathy-like changes as well as α-synuclein accumulation and formation of ubiquitin-positive inclusions (55). Moreover, haploinsufficiency of CTSD resulted in the reduction in lysosomal functions and also acceleration of the propagation of LB pathology (56). Furthermore, recent studies in Drosophila have shown a direct link between VPS35 depletion and defects in CTSD trafficking and maturation (17) (**Figure 5**).

### VPS35 Controls Macroautophagy

Macroautophagy has a profound importance in the clearance of intracytoplasmatic aggregate-prone proteins as well as α-synuclein in PD and huntingtin in Huntington's disease (57). The treatment with rapamycin, which stimulates

autophagy, enhanced the clearance of aggregate-prone proteins (57). Dysregulation of the autophagy pathway has been observed in the brain of PD patients and in PD mouse models (58), and the overexpression of α-synuclein caused mislocalization of autophagic proteins and impaired macroautophagy (59).

VPS35 controls the endosomal recruitment of WASH complex, a scaffolding protein localized at the EE (15). WASH complex regulates (i) the autophagic process of the autophagosome development (15), (ii) the formation of actin patch on endosomes to promote protein sorting (60), and (iii) endosome-to-cell surface recycling (61). Recently, it has been demonstrated that retromer interacts directly with FAM21– WASH complex controlling its correct localization (15). VPS35– D620N mutant destabilized this interaction, impairing WASH complex recruitment to endosomes (15, 62). The mislocalization of WASH complex induces defects in autophagosome formation, perturbing protein sorting, and specifically altering ATG9A trafficking (15) (**Figure 5**). ATG9A is a multipass transmembrane protein that acts early in the autophagic pathway and controls LC3-positive compartment interaction with autophagosome (15). Moreover, parkin PD mutations could impair the ubiquitination of VPS35, inducing the regulation of retromerdependent sorting (63). Thus, parkin deficiency leads to the reduction in retromer-associated WASH complex cargo in mouse brains (63).

### VPS35 Controls CMA

The clearance of misfolded proteins and aggregates by CMA is essential for the normal cellular functions and its efficiency

decline with age. Defects in CMA have been detected in PD patient (64). Therefore, the protein levels of CMA markers (LAMP2A and hsc70) were significantly reduced in SNpc and amygdala of PD patients (64). CMA is controlling the degradation of cytosolic proteins (as well as α-synuclein and LRRK2) carrying the recognition motif (KFERQ) (41). CMA presents multiple steps: (i) the recognition of KFERQ motif by HSPA8/HSC70 which target the substrate to lysosomes, (ii) the binding of the substrate to LAMP2A receptor, (iii) formation of substrate–translocation complex with membranebound LAMP2A, (iv) substrate degradation by lysosomal enzymes, and (v) disassembly of translocation complex and degradation of multimeric LAMP2A to be recycled (41). Recent studies have been shown that LAMP2A receptor trafficking from endosome to Golgi is altered in mice with reduced VPS35 levels or VPS35–D620N mutations (**Figure 5**). These mice showed alterations in lysosomal morphology with an increase in LAMP1A, reduction in LAMP2A vesicles, and an a relative increase in α-synuclein accumulation (16) (**Figure 5**).

### DISCUSSION AND OUTLOOK

Pathogenic mutations in different PD-related genes with autosomal dominant or autosomal recessive Mendelian inheritance (LRRK2, VPS35, DNAJC13, SYNJ1, TMEM230,

RAB39B) are involved in endosomal trafficking, indicating that DA neurons are specifically susceptible to endosomal dysfunctions (36). Neuropathological and neuroanatomical studies have demonstrated that the impairment of cellular trafficking may preferentially affect SNc DA neurons (36, 65). VPS35 mutations identified in PD lead to retromer dysfunctions (29, 30). Recent studies highlighted the interplay between VPS35 and LRRK2, two endosomal proteins associate to late-onset PD, showing a common pathway for the sorting of proteins through TGN and endolysosomal system (66–68). The loss of VPS35 or LRRK2 in Drosophila has been reported to affect synaptic recycling (68). Furthermore, the overexpression of VPS35 or VPS26 retromer subunits ameliorated the pathogenic mutant LRRK2 eye phenotype, protecting from locomotor deficit (67). VPS35–D620N KI mice or knockdown of VPS35 mice impacted LRRK2-mediated Rab protein phosphorylation (44) supporting the hypothesis that VPS35–D620N mutation resulted in a gain of function (44).

Different studies have shown that VPS35 has a key role in controlling α-synuclein accumulation in different PD models (14, 16, 17) even if the development of VPS35–D620N KI mice lack the presence of α-synuclein accumulation (43). Here, we review evidence that the increase in VPS35 levels in neurons represents a potential therapeutic target for the treatment of PD. Boosting VPS35 functions could help DA neurons to degrade α-synuclein toxic aggregates and prevent neuronal loss (**Figure 5**).

Despite the understanding on VPS35 association to PD pathology, very little is known about the molecular mechanisms involved in the control of α-synuclein accumulation and in a possible neuroprotective role of VPS35 from α-synucleininduced toxicity. The retromer complex is mainly involved in neurodegenerative disorders through the control of synaptic functions, trafficking, and localization of cargo proteins that are essential for neuronal homeostasis and α-synuclein accumulation vs. clearance. Therefore, VPS35 mutations could cause PD by acting on the α-synuclein degradation pathways (15–17, 69). A more detailed knowledge on the molecular mechanisms by which VPS35 promotes α-synuclein clearance could serve to clarify the role of VPS35-retromer protein in PD and to identify alternative and efficient new potential therapeutic targets downstream to VPS35 action. Besides, validation of new VPS35 based targets could be tested in PD mouse models before being developed in the clinical setting. VPS35 has a wide spectrum of actions within neurons, many of which are potentially relevant for the pathophysiology of PD, particularly synaptic functions (22–24, 26, 48) and control of DAT recycling (27) (**Figure 6**). Moreover, VPS35 controls mitochondrial function: VPS35 deficiency disrupts the mitophagic process, promoting mitochondrial fragmentation and DA neuron loss (28).

However, the role of VPS35 could also be played in a more general level on the control of accumulation/degradation of misfolded proteins, hence representing a potential target for different neurodegenerative disorders, such as PD and AD. Indeed, dysregulation of retromer transport is also strongly implicated in AD (70–73). Alterations of retromer activity in AD mouse models leads to pronounced neurodegeneration (deficit in post-synaptic glutamatergic neurotransmission) and deficiency in development of hippocampal-dependent memory (impairment of long-term potentiation) (36, 65) compatible with the role of retromer complex in controlling synaptic functions (22–24, 26). Moreover, different studies addressing VPS35 loss of function reported an elevation of endogenous Aβ peptide levels, in keeping with a VPS35 role in controlling amyloid protein precursor (APP) sorting and processing (28, 70–73).

New research may seek to develop a VPS35-based therapy. A possible therapeutic strategy to boost VPS35 functions could be a drug promoting the stabilization of retromer complex. Earlier studies identified chemical molecules capable of stabilizing the retromer complex by binding the complex in a bind site between VPS35–VPS29 (74). The stabilization of the retromer complex resulted in the reduction in complex degradation and in the increase in VPS35 and retromer subunit levels in cell lines (74). Starting from this observation, it could be possible to identify a lead compound able to combine the effect on the

### REFERENCES

target with the possibility to cross the blood–brain barrier, to be tested in a clinical trial on PD patients. On the other hand, a second disease-modifying approach to boost VPS35 functions could be the delivery of VPS35 gene in the brain by adenoassociated-2-viral vectors (AAV2). The gene therapy approach will allow the introduction of VPS35 gene in the cells that result in the upregulation of VPS35 expression. Recently, it has been demonstrated that AAV2 treatment represents promising human therapeutic applications, with increasing evidence for safety and efficacy (75). First, AAV2 is the most effective in delivery genes in vivo for the presence of the primary adenovirus receptor in human cells that allow easy infection with a high yield of transgene expression. Moreover, currently, there are many clinical trials using AAV2 applications, and doses and routes of administration are already established. Finally, the elucidation of the molecular mechanism used by VPS35 to control neuronal functions, neuroprotection, and αsynuclein clearance could be addressed and may provide a new way to identify new therapeutic strategies in PD and other neurodegenerative disorders where neurotoxic aggregates build up. The other challenges will be to overcome in the translational therapeutic pipeline, including biomarker development, clinical trial strategies, and understanding the potential utility of VPS35 as therapeutic target in different PD backgrounds (sporadic and genetic PD cases).

A clinically related question is when a potentially diseasemodifying therapy should be prescribed. Patients who are in the early symptomatic phase are thought to have a significant proportion of viable DA neurons. The diverse functions of VPS35 and its ability to counteract α-synuclein accumulation would justify testing VPS35-based approach from the prodromal until the early symptomatic phase of PD.

### AUTHOR CONTRIBUTIONS

SE and AA conceived the study, wrote, and revised the manuscript. SE generated the figures.

### FUNDING

This work was supported by Fondazione Umberto Veronesi (grant to SE) and by Fondazione Grigioni (grant to AA).

mechanism: tracking studies using a novel fluorophore-coupling approach. J Neurosci. (2017) 37:9438–52. doi: 10.1523/JNEUROSCI.3885-16.2017

**Conflict of Interest:** 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 © 2019 Eleuteri and Albanese. 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) and the copyright owner(s) 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.

# Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia

Michaela Pogoda1†, Franz-Joachim Hilke1,2†, Ebba Lohmann3,4,5, Marc Sturm1 , Florian Lenz 1 , Jakob Matthes 1 , Francesc Muyas 1,6,7, Stephan Ossowski 1,6,7 , Alexander Hoischen8,9, Ulrike Faust 1 , Ilnaz Sepahi 1 , Nicolas Casadei 1,10, Sven Poths 1 , Olaf Riess 1,10, Christopher Schroeder 1 \* and Kathrin Grundmann1

1 Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany, 2 Department of Dermatology, Venereology and Allergology, Charité - Universitätsmedizin Berlin, Berlin, Germany, 3 Behavioral Neurology and Movement Disorders Unit, Department of Neurology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey, 4 Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany, 5 German Center for Neurodegenerative Diseases, Tübingen, Germany, 6 Bioinformatics and Genomics Program, Centre for Genomic Regulation, The Barcelona Institute of Science and Technology, Barcelona, Spain, 7 Universitat Pompeu Fabra, Barcelona, Spain, 8 Department of Human Genetics, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 9 Radboud Institute of Molecular Life Sciences, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 10 DFG NGS Competence Center Tübingen, Tübingen, Germany

#### Edited by:

Matthew James Farrer, University of Florida, United States

#### Reviewed by:

Pedro Chana, Universidad de Santiago de Chile, Chile Jong-Min Kim, Seoul National University Bundang Hospital, South Korea

#### \*Correspondence:

Christopher Schroeder christopher.schroeder@ med.uni-tuebingen.de

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Movement Disorders, a section of the journal Frontiers in Neurology

Received: 05 June 2019 Accepted: 02 December 2019 Published: 18 December 2019

#### Citation:

Pogoda M, Hilke F-J, Lohmann E, Sturm M, Lenz F, Matthes J, Muyas F, Ossowski S, Hoischen A, Faust U, Sepahi I, Casadei N, Poths S, Riess O, Schroeder C and Grundmann K (2019) Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia. Front. Neurol. 10:1332. doi: 10.3389/fneur.2019.01332 Background: This study's aim was to investigate a large cohort of dystonia patients for pathogenic and rare variants in the ATM gene, making use of a new, cost-efficient enrichment technology for NGS-based screening.

Methods: Single molecule Molecular Inversion Probes (smMIPs) were used for targeted enrichment and sequencing of all protein coding exons and exon-intron boundaries of the ATM gene in 373 dystonia patients and six positive controls with known ATM variants. Additionally, a rare-variant association study was performed.

Results: One patient (0.3%) was compound heterozygous and 21 others were carriers of variants of unknown significance (VUS) in the ATM gene. Although mutations in sporadic dystonia patients are not common, exclusion of pathogenic variants is crucial to recognize a potential tumor predisposition syndrome. SmMIPs produced similar results as routinely used NGS-based approaches.

Conclusion: Our results underline the importance of implementing ATM in the routine genetic testing of dystonia patients and confirm the reliability of smMIPs and their usability for germline screenings in rare neurodegenerative conditions.

Keywords: dystonia, ATM, NGS, ataxia-telangiectasia, MIPs

## INTRODUCTION

Ataxia-telangiectasia (A-T) is a rare autosomal recessively inherited disease usually characterized by ataxia, neuro-motor impairment, ocular or cutaneous telangiectasia, high risk of malignancies and immunodeficiency (1–3). A-T patients are often extraordinarily sensitive to ionizing radiation, contraindicating radiation therapy for them as a standard therapy in case of cancer (4, 5). The disease is caused by mutations of the Ataxia telangiectasia mutated (ATM) gene located on chromosome 11q22-23 (6). ATM encodes the 350 kDa ATM protein, a nuclear

**61**

serine/threonine-protein kinase which is crucial in the cellular response to DNA damage such as double-strand breaks (7, 8). Classic A-T is caused by biallelic truncating ATM mutations which lead to a total loss of ATM protein, resulting in an impaired cell cycle (9). Since variants in ATM are known to confer cancer risk in heterozygous carriers (10), and at the same time cause increased sensitivity of the patients to toxic effects of ionizing radiation (4, 5), identifying ATM mutation carriers can be highly relevant for adequate treatment and regular cancer control examinations.

Furthermore, recent reports describe non-classic forms of A-T or "variant A-T" (11, 12). In these cases, some residual ATM kinase function is maintained and the phenotype is highly variable, including incomplete or atypical phenotypes, e.g., ataxia plus extrapyramidal symptoms or choreoathetosis lacking the classical hallmarks, often masking the correct diagnosis (11, 13). Some of these ATM mutations manifest as pure generalized or focal dystonia (14).

However, the frequency of ATM mutations in different cohorts of dystonia patients is not well-described. Therefore, we tested a cohort of 373 dystonia patients for ATM alterations.

We applied single molecule Molecular Inversion Probes (smMIPs) for targeted enrichment and sequencing of all protein coding exons and exon-intron boundaries of ATM. SmMIPs represent a cost-efficient and fast high-throughput technique to identify sequence variation in genes containing many exons (15). SmMIPs are oligonucleotide probes possessing two sequences complementary to defined genomic target regions (16–21). These complementary sequences are located at the 3′ -end and at the 5 ′ -end of the probe and hybridize to the single-stranded sample DNA upstream and downstream of the chosen target region of variable length. The gap between the complementary sequences is filled with the copy of the DNA target region by a polymerase, circularizing the probe in an additional ligation reaction.

The circular smMIP-target molecule can then be amplified in a PCR reaction and is, after a single library purification step, ready for sequencing. One main advantage of smMIPs is that probes can be designed in a modular way, tiling all relevant regions as closely as necessary, covering both DNA strands if desired (22). This is especially relevant for the avoidance of artifactual DNA sample damage, since artifacts usually only occur randomly in one strand (17, 23). Another asset of smMIPs is the usage of a unique single molecule (sm) molecular identifier (UMI = unique molecular identifier), a sequence of (in our case) 8 random bases in the probe that is individual for 48 ≈ 65,500 molecules. Since every smMIP molecule with an individual UMI-tag can only hybridize to one genomic DNA fragment, the UMI sequence can be used to retrace all originally different DNA molecules and to correct for PCR duplicates in the bioinformatical analysis. Thus, an accurate representation of the diversity of DNA molecules in the sample allows the sensitive detection of variants, even at low frequencies (17, 23, 24). Therefore, it is also most suitable for especially cost-efficient, reliable germline mutation screenings in large cohorts. An asset of the technology is that smMIPs can be designed in a customized way (22) and can be used for massively parallel resequencing of many thousands of target regions (25). In addition, most of the chemistry is independent of a specific supplier.

### METHODS

Informed consent was obtained from all patients. All samples were taken in accordance with the local Ethical Committee (# 847/2017BO). Genomic DNA was isolated from blood of 373 dystonia patients of Caucasian origin, who had been examined by specialists in movement disorders according to the current clinical criteria (26). Inclusion criteria were as follows: various degrees of dystonia as defined by published clinical criteria and a clinical course compatible with primary dystonia without features indicating secondary dystonia.

### Probe Design and Pooling

The whole smMIPs protocol was only slightly modified according to established protocols (beside the protocol, a detailed scheme of the methodology and workflow can be found in 15, 17, 21).

We designed smMIPs to screen the patients' DNA using the open source tool MIPGen (22). The design resulted in 190 smMIPs spanning all 62 protein coding exons of the ATM gene [transcript ENST00000278616, Ensemble (27)], covering both the sense and anti-sense strand of the DNA. Probes were synthesized by IDT (Integrated DNA Technologies; Coralville, USA). Upon arrival, all smMIPs were pooled in an equimolar manner. Six random samples were processed and sequenced to assess probe performance. Using the read depth of all individual probes, the pool was rebalanced twice, in order to improve uniformity of coverage of the target regions (17, 28) (see Supplementary Material for further information).

### Targeted Enrichment and Amplification

One hundred nanogram isolated DNA were used as input for the targeted capture of genomic regions of interest, and incubated for 21 h with smMIPs, using a ratio of 1:800 (DNA molecules:smMIPs), adding a polymerase and ligase for gapfilling and circularization. Subsequently, a 1-h exonuclease digestion was performed to remove any linear DNA. Thus, only circular smMIPs were amplified in a PCR using a highfidelity polymerase. A small amount of each PCR product was categorized semi-quantitatively on an agarose gel, and the remaining PCR products were pooled accordingly to obtain equimolar representation of all samples. The pools were cleaned up for Illumina sequencing in one step using XP Ampure beads and the DNA concentration and fragment size was measured using a Qubit 2.0 device and an Agilent TapeStation 2200 to calculate the molarity of each pool. Samples were sequenced on a HiSeq2500 platform with 2 × 125 cycles (paired-end) and a target of 500 clusters per smMIP per sample.

### Sequencing Analyses and Statistics

Sequencing data were analyzed using an adapted in-house pipeline (available on https://github.com/imgag/megSAP, version 0.1-663-ged5a95d). Briefly, all sequences were identified by the UMI to allow later grouping and correction for PCR duplicates. Reads were aligned and mapped using paired-end

CADD-scores (Combined Annotation-Dependent Depletion) (35, 36) are not available for intronic variants and deletions. \*Evidence in literature for pathogenicity (14), but missing functional analyses.

reads (29), PCR duplicates were used to correct random PCR or sequencing errors labeling their base-quality to 0 to avoid calling falls-positive variants. BAM-files were generated, and variants called using the tool FreeBayes, version 1.1.0 (30). Variants were annotated and saved in GSvar-format.

For rare-variant association, a case-control study was performed (31). As a control cohort, vcf-files of the 1000 genomes project (32) were downloaded for 404 patients with European descent (populations Italian, Spanish, British, and Utah with European ancestry; Finnish not included). In order to obtain a representative and unbiased control cohort, we chose a public database for control data. Only target regions that were covered in both groups, case and control, were analyzed. All variants were subsequently filtered by the following criteria.

For filtering the variants, allele frequencies in the 1000 genomes database and in the ExAC database were necessitated to be below 1% and all variants needed to be reported <50 times in our in-house database. Synonymous and intronic variants (cut-off ± 8 bp) were excluded as well as variants that had been classified as benign or likely benign in our diagnostic inhouse database. All variants were classified according to slightly modified guidelines (33, 34).

Statistical analyses were performed using JMP software version 13.0.0 (SAS Institute, Cary, NC). For rare-variant association, a Fisher's exact test was performed (α = 0.05).

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher. The data (vcf-files) of the control cohort were obtained from the 1000 genomes project (32) and are publicly available.

### RESULTS

The median target region read depth for all samples (n = 373) was 218×, ranging from 34× for one sample to 840× in the sample with the highest coverage. The 20×-coverage of all target regions was 98.0%. Note that these figures only comprise condensed reads (no PCR duplicates) which were shown to be sufficient for allele frequencies typical in germline analyses (17).

Among 373 dystonia patients, we found 21 different variants in 22 of the dystonia patients (**Table 1**). Of these, 20 were of uncertain clinical significance (VUS) (33). Ten of them had high in silico prediction of pathogenicity [Combined Annotation-Dependent Depletion, CADD-Score > 20, (35, 36)]. One variant was classified as pathogenic (p. (Val2716Ala), class 5). There was no significant difference from the control group from the 1000 genomes project (p > 0.05 for both VUS or predicted pathogenic variants).

Among the dystonia patients, one patient was found with two variants in the ATM gene: one was pathogenic and one a VUS with evidence for pathogenicity (p. (Val2617Ala), p. (Ser2860del)). Clinical examination had produced highly elevated AFP levels, familial aggregation of malignancies and a deterioration of the dystonic symptoms that were largely insensitive to treatment. This patient was recently published (14). In contrast, we found no biallelic pathogenic or likely pathogenic variants in the 1000 genomes control cohort.

All pathogenic variants reported for the **six** positive controls were also detected using smMIPs. The comparison of all variants showed no discrepancy between smMIPs and the diagnostic results.

### DISCUSSION

### Dystonia

Although we found no statistical association of ATM variants between dystonia patients and a control group, we found one patient with a pathogenic variant (p. (Val2617Ala)) and a VUS with evidence for pathogenicity (p. (Ser2860del)) among 373 dystonia patients. In accordance with other published data on the patient (14), our study confirms the efficiency of smMIPs as a diagnostic screening tool (15) and moreover the importance of identifying ATM as rare underlying cause in dystonia (37, 38). The genetic causes of dystonia, a rare disease, are very heterogeneous (39), and many genes have been identified in recent times. Among these genetic causes, ATM sequence variations are a crucial factor for patients regarding cancer susceptibility (10), disease progression and radiation toxicity (3, 4, 40, 41). Thus, we stress the importance of including ATM to the general screening for causes of dystonia.

### smMIPs

Due to the usage of UMIs, smMIPs increase the confidence in variants without the necessity of a high coverage, because PCR duplicates can be used to correct for artifacts (17, 23). We successfully established smMIPs in a screening for ATM germline variants in dystonia patients. All pathogenic variants that had been detected with different diagnostic NGS approaches in six control patients (and one dystonia patient) were confirmed, validating smMIPs as a sensitive, competitive methodology. They provide a straight-forward wet lab protocol, with customizable chemistry, flexible probe design, sensitive variant detection, and cost-efficient sample processing, especially for high-throughput genetic testing (15, 21, 28, 42). Thus, they constitute a convenient tool for panel-based genetic testing.

### CONCLUSION

Since ATM mutations confer a higher risk of developing cancer and radiation toxicity, it is crucial to detect ATM-variants as the underlying cause in any dystonic patient. In our study, we found smMIPs to be as sensitive as other NGS-based approaches while being highly cost-efficient and flexible.

### DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. The data (vcf-files) of the control cohort were obtained from the 1000 genomes project and are publicly accessible here: http://www.internationalgenome.org/ data#download.

### ETHICS STATEMENT

All experimental protocols in this study were reviewed and approved by the Ethics Committee of the Eberhard Karls University of Tübingen and of the Medical Faculty of the University Hospital Tübingen. All patients gave written informed consent in accordance with the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.

### AUTHOR CONTRIBUTIONS

The research project was conceptualized by OR, CS, F-JH, EL, and KG. Sample acquisition was accomplished by EL, KG, and UF. Methodology was implemented and supported by AH, F-JH, MP, NC, and SP. The computational analyses were developed and/or conducted by CS, MS, FL, SO, FM, and JM. Variant classification and (statistical) analyses were conducted by MP, IS, and SO. All authors revised and approved the final manuscript.

### FUNDING

The study was funded by the Exzellenzinitiative Zukunftskonzept Universität Tübingen, project ID D.27.13047.

### ACKNOWLEDGMENTS

Illumina sequencing was performed by the DFG NGS Competence Center Tübingen (NCCT, Tübingen, Germany). We would also like to acknowledge all patients and their families who contributed to this study, as well as the clinicians and the technical staff. We acknowledge support by the Deutsche Forschungsgemeinschaft and the Open Access Publishing Fund of the University of Tübingen. This manuscript has been part of an unpublished Master Thesis (43).

### REFERENCES

Brit J Radiol. (1983) 56:343–5. doi: 10.1259/0007-1285-56- 665-343

Bioinformatics. (2014) 30:2670–2. doi: 10.1093/bioinformatics/b tu353

**Conflict of Interest:** 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 © 2019 Pogoda, Hilke, Lohmann, Sturm, Lenz, Matthes, Muyas, Ossowski, Hoischen, Faust, Sepahi, Casadei, Poths, Riess, Schroeder and Grundmann. 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) and the copyright owner(s) 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.

# Can the Cognitive Phenotype in Neurofibromatosis Type 1 (NF1) Be Explained by Neuroimaging? A Review

Eloïse Baudou1,2 \*, Federico Nemmi 2 , Maëlle Biotteau2 , Stéphanie Maziero2,3 , Patrice Peran2 and Yves Chaix 1,2

1 Children's Hospital, Purpan University Hospital, Toulouse, France, 2 ToNIC, Toulouse NeuroImaging Center, University of Toulouse, Inserm, UPS, Toulouse, France, 3 Octogone-Lordat, University of Toulouse, Toulouse, France

Neurofibromatosis type 1 (NF1) is one of the most frequent monogenetic disorders. It can be associated with cognitive dysfunctions in several domains such as executive functioning, language, visual perception, motor skills, social skills, memory and/or attention. Neuroimaging is becoming more and more important for a clearer understanding of the neural basis of these deficits. In recent years, several studies have used different imaging techniques to examine structural, morphological and functional alterations in NF1 disease. They have shown that NF1 patients have specific brain characteristics such as Unidentified Bright Objects (UBOs), macrocephaly, a higher volume of subcortical structures, microstructure integrity alterations, or connectivity alterations. In this review, which focuses on the studies published after the last 2 reviews of this topic (in 2010 and 2011), we report on recent structural, morphological and functional neuroimaging studies in NF1 subjects, with special focus on those that examine the neural basis of the NF1 cognitive phenotype. Although UBOs are one of the most obvious and visible elements in brain imaging, correlation studies have failed to establish a robust and reproducible link between major cognitive deficits in NF1 and their presence, number or localization. In the same vein, the results among structural studies are not consistent. Functional magnetic resonance imaging (fMRI) studies appear to be more sensitive, especially for understanding the executive function deficit that seems to be associated with a dysfunction in the right inferior frontal areas and the middle frontal areas. Similarly, fMRI studies have found that visuospatial deficits could be associated with a dysfunction in the visual cortex and especially in the magnocellular pathway involved in the processing of low spatial frequency and high temporal frequency. Connectivity studies have shown a reduction in anterior-posterior "long-range" connectivity and a deficit in deactivation in default mode network (DMN) during cognitive tasks. In conclusion, despite the contribution of new imaging techniques and despite relative advancement, the cognitive phenotype of NF1 patients is not totally understood.

Keywords: NF1, cognitive phenotype, cerebral substrate, brain imagery, fMRI

#### Edited by:

Matthew James Farrer, University of Florida, United States

#### Reviewed by:

Andrea Domenico Praticò, University of Catania, Italy Maria Augusta Montenegro, Campinas State University, Brazil

> \*Correspondence: Eloïse Baudou<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Pediatric Neurology, a section of the journal Frontiers in Neurology

Received: 10 June 2019 Accepted: 11 December 2019 Published: 14 January 2020

#### Citation:

Baudou E, Nemmi F, Biotteau M, Maziero S, Peran P and Chaix Y (2020) Can the Cognitive Phenotype in Neurofibromatosis Type 1 (NF1) Be Explained by Neuroimaging? A Review. Front. Neurol. 10:1373. doi: 10.3389/fneur.2019.01373

## INTRODUCTION

In recent years, the cognitive and behavioral phenotypes of Neurofibromatosis 1 (NF1) have been well described in affected children. Despite some heterogeneity, which is not yet clearly understood in the absence of established genotype-phenotype correlation, the main cognitive characteristics that have been highlighted are:

Therefore, different cognitive domains are affected, which suggests that different brain networks are involved in the physiopathology of NF1.

However, NF1 patients present specific neuroimaging features. Among them, Unidentified Bright Objects (UBOs) are the best known and are suggested as diagnostic criteria by some authors (8). However, the presence of UBOs does not explain the cognitive and behavioral phenotype in NF1 disease (9, 10). A recent study using multimodal neuroimaging including structural, diffusion and resting state functional magnetic resonance imaging (fMRI) (11) showed that NF1 patients and healthy controls can be differentiated using neuroimaging that combines the measurement of gray matter volume, fractional anisotropy and mean diffusivity. This suggests a complex physiopathology involving gray and white matter abnormalities.

The links between behavioral and cognitive phenotypes and the cerebral substratum are poorly understood. However, the number of brain imaging studies has considerably increased since the reviews of literature by Payne et al. (9) and Hachon et al. (10).

Based on the evidences established in the two previous reviews, we pursued an examination of the literature since 2010 concerning brain imaging in NF1 disease. The aim of this literature review is to find out whether in 2019, nearly 10 years after the last reviews, there is a clearer understanding of the link between cognitive and behavioral patterns and NF1 cerebral physiopathology in children with NF1.

## REVIEW

## Structural Level

### Summary of the Findings Before 2010

On average, NF1 patients have a larger brain volume than the general population, with macrocephalia in 50% of the cases. This difference in brain volume is predominant in white matter (WM), and less significant in gray matter (GM) (12). Up until now, results from brain imaging studies have been unable to clearly indicate any correlation between total brain volume, WM volume or GM volume and the neuropsychological profile. For example, the majority of studies report an NF1-related increase in corpus callosum (CC) volume (13, 14), sometimes with a positive correlation with learning disability (13) and sometimes a negative correlation with ADHD (14) (**Table 1**).

Billingsley et al. highlighted focal architectural abnormalities in NF1 (15, 16). They showed a link between language disability/low academic achievement (reading and/or mathematics) and reduced asymmetry of the left/right planum temporale (**Table 1**). They also found a positive interaction between an atypically structured right inferior frontal gyrus and language level (**Table 1**). These studies suggest an atypical lateralization of linguistic functions in NF1 subjects.

### Post-2010 Studies: From the Macrostructural to the Microstructural Level

In an NF1 group, Huijbregts et al. (17) found a larger WM volume associated with a larger volume of all subcortical structures (hippocampus, thalami, striatum, amygdala, accumbens nucleus) and a lower GM density in frontal and parietal regions (**Table 1**). Positive correlations were found between cognitive abilities and social skills, and the volume of subcortical structures:

Violante et al. (20) also found that subcortical structures (thalami, right caudate, middle CC) had larger volumes in a group of 14 NF1 children aged 8 to 16 years compared to 14 controls. In this study, the authors confirmed a larger whole brain volume (+10%) with a greater difference between WM (+20%) and GM (+8%) compared to controls. They analyzed the volume variation in more detail according to the function of the region and found a greater difference in bilateral frontal and temporal regions and in the left parietal region for lobar WM volumes. They observed less distinctive gyrification in NF1 subjects, without any difference in

**Abbreviations:** ACC, anterior cingulate cortex; AD, axial diffusion; ADC, apparent diffusion coefficient; ADHD, Attention Deficit and Hyperactivity Disorder; ASD, Autism Spectrum Disorder; ATR, anterior thalamic radiation; BRIEF, Behavioral Rating Inventory of Executive Function; CBCL, Child Behavior Check List; CC, corpus callosum; DLPFC, dorsolateral prefrontal cortex; DEX, Dysexecutive Questionnaire; DMN, default mode network; DTI, diffusion tensor imaging; FA, fractional anisotropy; FDG, fluorodeoxyglucose; FEF, frontal eye fields; FLAIR, Fluid-Attenuated Inversion Recovery; fMRI, functional magnetic resonance imaging; GABA, gamma-aminobutyric acid; GM, gray matter; IQ, intelligence quotient; JLO, Judgment of Line Orientation; MD, mean diffusivity; MRI, magnetic resonance imaging; MRS, magnetic resonance spectroscopy; NAA, N-acetyl-aspartate; NF1, Neurofibromatosis type 1; PET, positron emission tomography; RD, radial diffusion; ROI, region of interest; SRS, Social Responsiveness Scale; UBOs, unidentified bright objects; WM, white matter.

 In human phylogenic evolution, an increase in brain volume is associated with an increase in cortical gyrification (21). However, in NF1 patients, gyrification is not proportional to brain volume.

Aydin et al. (18) found a higher CC volume in NF1 children (**Table 1**). This study focused on micro- and macrostructural measurements of the CC (midsagittal CC area measurements, fractional anisotropy (FA), and absolute diffusion coefficient (ADC) values of the genu and splenium of the CC). Negative correlations were shown between the ADC values of the genu of the CC and the arithmetic and digit span scores and between the FA values in the genu and coding scores in children with NF-1.

Karlsgodt et al. (22) compared 14 young adults with NF1 and 12 healthy controls using Diffusion Tensor Imaging (DTI) analyses. These authors confirmed an increase in WM volume in NF1 patients and showed an alteration in WM integrity in the anterior thalamic radiation (ATR). This study specifically showed a decrease in FA and an increase in ADC and radial diffusivity (RD) and, to a lesser extent, in axial diffusivity (AD) in the ATR. This pattern suggests an increase in diffusivity due to reduced myelination and reduced axonal organization. More recently, Koini et al. (19) showed a correlation between a decrease in executive functions (inhibitory control evaluated by a sustained attention test) and modifications in microstructure parameters (a decrease in FA and an increase in mean diffusivity, RD and AD) in the ATR (**Table 1**). It is noteworthy that there was no link between these alterations and the presence of thalamic UBOs.

#### **Summary**

In comparison to the general population, NF1 subjects present an increase in brain volume that is more pronounced in WM than in GM. However, cortical gyrification is proportionally less compared to healthy subjects. Similarly, the volume of the CC, the thalamus and the striatum is larger in the NF1 population. At the microstructural level, a decrease in FA and an increase in mean diffusivity seem to be systematic, possibly due to an alteration in myelination. Finally, a link can be established between abnormalities in the ATR and the executive dysfunction observed in NF1 subjects. Characteristics of the main neuroimaging studies that show a link between structural features and cognitive functions are indicated in **Table 1**.

 However, they are not considered to be a criterion in the diagnosis of NF1. UBOs are hyperintensities on T2-weighted or Fluid-Attenuated Inversion Recovery (FLAIR) MRI sequences, without mass effect, and without contrast enhancement. We will interchangeably use the term UBOs and T2-hyperintensities in the following paragraphs. UBOs are found in approximately 70% of NF1 subjects (23), but only between 0.8 and 2.2% in the general population, depending on the study. UBOs can be discrete or diffuse. They are principally located in basal ganglia, thalami, cerebellum and brainstem. In less than 20% of the cases UBOs are supratentorial and hemispheric. They tend to regress with age, at least for those located in the basal ganglia and the brainstem (24). There are different hypotheses concerning their nature: e.g., low grade tumor, hamartoma, heterotopias or a modification in the water content of myelin with dysplastic glial cells. Some authors used MRI to try to clarify the microstructural nature of UBOs. Using DTI, they showed an increase in ADC (25, 26) and a decrease in FA (27). The only anatomo-pathological study of UBOs was conducted by Dipaolo et al. (28). Their histological analysis showed that UBOs result from a vacuolar and spongiotic alteration in WM caused by intramyelinic edema. However, a limitation of their study is the heterogeneity of the NF1 population investigated, since one subject was born prematurely and another received chemotherapy for fibrosarcoma.

The most important question concerning UBOs is their possible involvement in cognitive impairment and their impact on learning, especially in NF1 children. The literature does not provide a definitive answer, even though some studies have highlighted the importance of the location of UBOs in cognition rather than their numbers, with a possible link between thalamic location and cognitive impairment (IQ, attention span) (29). Feldman et al. (30) showed a link between a decrease in T2-hyperintensities in basal ganglia (thalami) and IQ point gain (**Table 2**).

### Post 2010 Studies

### **Morphologic neuroimaging and the relationship with cognitive phenotype**

In a large cohort, Sabol et al. (8) confirmed the presence of UBOs in 73.5% of 162 NF1 children aged 2 to 18 years, vs. 4.3% of 163 healthy controls. This provides excellent specificity for the diagnosis of NF1 when UBOs are present (specificity: 98%, sensitivity: 81% before 7 years of age). This study confirmed that the basal ganglia were the most frequent location of T2 hyperintensities and that they decrease with age. Payne et al. (31) highlighted this decrease in T2-hyperintensities through a longitudinal study in which the authors presented cognitive (IQ) and structural neuroimaging data (UBOs) (**Table 2**). They showed a decrease of 35% in T2-hyperintensities over an 18-year period, with differences in progression depending on the type of lesion (discrete lesions decreased and diffuse lesions remained unaltered) and the location (deep lesions in the basal ganglia, cerebellum, and brainstem decreased while hemispheric lesions remained unaltered). A decrease in UBOs was associated with an increase in IQ only on the third assessment, while IQ remained stable in subjects without T2-hyperintensities.

Piscitelli et al. (32) showed a relationship between cerebellum T2-hyperintensities and the neurocognitive profile. Subjects with cerebellar UBOs (31 out of 49 NF1 children in the study) presented worse scores on verbal IQ, full-scale IQ and visuospatial tests (reasoning and memory) than subjects without cerebellar hyperintensities (18 out of 49 NF1 children in the study). However, Roy et al. (33) showed no relationship between executive functions, evaluated with a test or a questionnaire, and the presence, number or location of T2-hyperintensities (**Table 2**).

### **Diffusion imaging for a better understanding of UBOs**

MRI, especially DTI sequences, has been used in several studies to further the understanding of the microstructure of UBOs. Ferraz-Filho et al. (34) showed a decrease in FA values in the bilateral cerebellum and thalami in NF1 patients, regardless

TABLE 2 | Correlation between the MRI analysis of UBOs and neuropsychological findings: main characteristics of the neuroimaging studies included.

IQ, intellectual quotient; MRI, magnetic resonance imaging; NF1, neurofibromatosis type 1; r, range; SD, standard deviation; T2H, T2 hyperintensity; UBOs, unidentified bright objects; WISC-III, Wechsler Intelligence Scale for Children III.

of the occurrence of UBOs in the thalami. This suggests that microstructural abnormalities can be present even if there are no hyperintensities in the brain. In a second study (35) with 27 NF1 subjects on whom 2 MRI examinations were performed between 1 and 5 years, the authors confirmed a decrease in T2 hyperintensities with a non-linear pattern of progression after the first decade of life. During the first decade of life, hyperintensities can remain stable or increase in number. The author found a reduction in the mean FA in UBOs regions and in regions where UBOs have disappeared (i.e., thalami, cerebellum and basal ganglia).

To better understand the microstructural modifications in UBOs sites, Billiet et al. (36) combined DTI analysis with other MRI-based techniques such as multi-exponential T2 relaxation, diffusion kurtosis imaging or neurite orientation dispersion and density imaging. They compared these parameters in 17 NF1 subjects, within UBOs sites and in contralateral normalappearing WM. The authors found a lower FA, greater mean diffusivity (MD), RD and AD, and a longer T2 time for intracellular and extracellular water in UBOs in comparison to contralateral normal appearing WM. The authors considered that these results might have been related to intramyelinic edema. Ertan et al. (37) analyzed DTI parameters (FA, MD, AD and RD) in regions of interest in 14 NF1 subjects and 14 healthy controls, comparing UBOs sites and normal appearing sites. The decrease in FA was found in GM and WM UBOs, but mostly in WM. Previous studies suggested that a combined decrease in FA and increase in AD and RD could be explained by a combination of myelin damage and axonal disturbance. Tractography showed WM fiber integrity in 15 UBOs out of 18.

### **Multimodal approach**

Barbier et al. (38) compared spectroscopic imaging in a multivoxel approach in basal ganglia and thalami in 25 NF1 children aged 8 to 15 years divided into two groups, one without UBOs (UBOs − group: 10 subjects) and one with UBOs (UBOs + group: 15 subjects). These authors found lower N-acetyl-aspartate (NAA)/creatinine, NAA/Choline and NAA/myoInositol ratios and a higher MyoInositol/Choline ratio in the right lateral thalamus in the UBOs + group, compared with the UBOs − group. These results could suggest a thalamic dysfunction that affects the thalamo-cortico-frontal loops related to neural and/or astroglial abnormalities. In a multimodal approach that combines spectroscopy MRI and DTI, Nicita et al. (39) analyzed spectroscopy imaging and 2 DTI parameters (ADC and FA) for 4 regions of interest (the caudate nucleus, the globus pallidus, the putamen and the thalamus) in 14 NF1 subjects aged 8 to 31 years and 8 healthy controls. The authors found (1) lower NAA, NAA/choline and NAA/creatinine ratios regardless of the subject's age (under or above 18 years of age) and the presence or absence of UBOs when the NF1 subjects were compared with the controls; (2) and a higher ADC without FA changes in UBOs subjects and subjects under 18 years of age. The presence of metabolic and microstructural abnormalities was an indication of axonal damages associated with an increase in myelin turnover in areas of intramyelinic edema, especially in young subjects. Interestingly, the subjects in this study manifested no developmental delay or cognitive deficits. These results somewhat contradict those of Rodrigues et al. (40), who found a preservation of NAA values but an increase in MyoInositol/Creatinine and Choline/Creatinine ratios in the basal ganglia with the use of a larger sample (42 NF1 subjects aged 4 to 24 years and 25 healthy controls) regardless of the UBOs status (presence or absence). Lastly, Violante et al. (41) used magnetic resonance spectroscopy (MRS) and [11C]-flumazenil PET, to compare 14 NF1 adults and 13 matched controls. These authors found a lower gamma-aminobutyric acid (GABA) concentration in the visual cortex and the frontal eye fields (FEF) (11.5 and 22% respectively), and a reduction in the binding of GABAA receptors in the left parieto-occipital cortex, midbrain and thalami, which were not explained by a lower GM volume. Only a correlation between GABA concentration and GABAA receptor density was found.

### Summary

UBOs are present in almost ¾ of the NF1 children, with the basal ganglia being the most frequent anatomical location. UBOs decrease in number after the first decade and this decrease is associated by cognitive improvement. This decrease affects discrete lesions (vs. diffuse) and deep lesions (vs. hemispheric). The microstructural studies found lower FA and higher MD, RD, AD, and mean T2 time, which supports the notion of a myelin edema. These microstructural abnormalities persist after UBOs regression, which indicates that structural abnormalities exist with or without macroscopic lesions (UBOs). Studies using MRS confirm these results but are contradictory with regards to the axonal damage associated with myelin damage. Characteristics of main neuroimaging studies showing a link between UBOs and cognitive functions are indicated in the **Table 2**.

### Functional Level

### Summary of the Findings Before 2010

Few studies used functional brain imaging to investigate NF1 physiopathology before 2010. Studies with Positron Emission Tomography (PET) scans that utilized [18F] fluorodeoxyglucose (FDG) usually included a small number of subjects (<30). They suggested thalamic hypometabolism in 9 NF1 children [Kaplan et al. (42)] and 29 NF1 adults [Buchert et al. (43)] compared to matching healthy controls.

Four studies published before 2010 used functional MRI (fMRI). In 2003, Billingsley's team (16) compared brain activation during a phonologic and an orthographic task in NF1 children and healthy controls (**Table 3**). They showed greater activation in the right hemisphere during the phonologic task in the NF1 children. They also reported greater involvement of posterior regions (middle temporal and occipital regions) than frontal regions during the orthographic tasks in the same group. The authors interpreted these results as compatible with a "disconnection" between anterior and posterior brain regions in the NF1 population, related to the WM damage that exists in this disease. In 2004, Billingsley et al. (44), the same 2 groups were compared during a letter and number identification task presented under 3 different conditions: baseline condition, mirror condition (targets were inverted) and rotation condition (targets were rotated at varying degrees). The authors found higher brain activity in posterior regions (middle temporal, parietal and occipital cortices) than in anterior regions (frontal cortices) in the NF1 subjects compared to the controls during visual-spatial analysis. They suggested that the functional abnormalities observed in this study could be related to structural abnormalities that were previously reported by the same team in these regions. Lastly, Clements-Stephens et al. (45) (**Table 3**) showed an inefficient right hemisphere network and more significant involvement of the left hemisphere in an NF1 group during a JLO task. The authors also found decreased activation in the primary visual cortex of the NF1 sample in comparison to the healthy controls. Shilyansky et al. (46) (**Table 3**) found lower activation in NF1 subjects within several cortical and subcortical regions of the right hemisphere dorsolateral prefrontal cortex, FEF and striatum during a visuospatial working memory task.

### Post-2010 Studies

### **PET**

To our knowledge, since 2010 only one PET study by Apostolova et al. (54) using PET FDG in a large population (compared with previousstudies) of 50 adult NF1 patients and 50 controls showed a single 11.2 ml cluster of reduced FDG uptake in the thalamus of NF1 patients compared with the control.

### **fMRI**

Several research lines related to fMRI studies have been developed.

The relationship between cerebral dysfunction and cognitive deficit. Some studies have tried to link cerebral dysfunction and deficient cognitive processes in NF1. For example, North's team tried to correlate executive deficit, one of the characteristic deficits in NF1, and brain dysfunction. Pride et al. (47) (**Table 3**), using a Go/No-Go task to explore response inhibition, showed reduced activation compared to controls in the pre-motor and presupplementary motor area, the right anterior cingulate cortex, the right inferior frontal gyrus, the inferior occipital gyrus and the left fusiform gyrus. The literature identifies a relationship between impulsivity and sustained attention deficit, and hypoactivation of the right inferior frontal gyrus, known to be involved in the inhibition response (55). In a second study, Pride et al. (48) (**Table 3**) used an region of interest (ROI) approach focused on the attentional networks to identify hypoactivation in the exogenous attention system or bottom-up or ventral attention system during an auditory attention task. This network included the bilateral temporoparietal junctions and the anterior cingulate cortex. Moreover, the authors showed a correlation between brain activation level in the right inferior frontal gyrus and attention scores during the task.

Functional connectivity. Due to the known abnormalities in the macro and micro structures of white matter, several groups have investigated the possible abnormalities in functional connectivity of the brain of NF1 subjects. In adults, Ibrahim et al. (49) (**Table 3**) showed reduced recruitment of the left dorsolateral prefrontal cortex (DLPFC) and parietal cortex during a visuospatial working memory task, which confirms previous results by Shilyansky et al. (46). The authors also found differences in the task-related functional connectivity between NF1 subjects and control subjects: during a visuospatial working memory task they observed greater connectivity between bilateral parietal regions and the visual cortex and lower connectivity between the posterior cingulate cortex and the left temporal region in NF1 subjects compared to controls. These connectivity differences suggest an inactivation deficit of the default mode network (DMN) in NF1 subjects, an inactivation that usually occurs during cognitive tasks. This confirms the results of a previous study in children conducted by Loitfelder et al. (50) (**Table 3**). In this study, functional imaging data were collected during resting state fMRI (rs-fMRI) and cognitive and social skills were evaluated with parent questionnaires (BRIEF, DEX, SRS, Social Skills Rating system: SSRS, CBCL). Among the more significant results, the authors showed an increase in connectivity between the left ventral anterior cingulate cortex and the frontal cortex, between the left amygdala and the posterior cingulate cortex/precuneus, and between the left orbito-frontal cortex and the homolateral pallidum in NF1 children compared to controls. Using rs-fMRI, Tomson et al. (56) found a reduction in posteroanterior "long distance" connectivity in NF1 subjects compared to controls along with a less organized DMN and visual network.

ADHD is frequent in the NF1 population and Jonas et al. (53) consequently explored the hypothesis of a deficit in reward processing since this has been shown in ADHD patients without NF1 (**Table 3**). Neuroimaging revealed reduced neuronal activity in the regions involved in the reward circuitry (anterior cingulate, paracingulate, supramarginal, and angular gyri) and a different blood oxygenation level dependent (BOLD) signal development across ages between NF1 subjects and controls, especially in frontal regions, with a decrease in neural activity related to an increase in age in the controls and an increase in neural activity related to an increase in age in the patients with NF1.

Multimodal approach. Violante et al. (51) focused on the analysis of visuoperceptual deficit in NF1 (**Table 3**). To this end, the authors used fMRI with a block design, including a rest period and a different visual stimulus presentation that stimulated either magnocellular or parvocellular pathways: M stimuli (25 cycles per degree, 18 Hz, low contrast: 18%) and P stimuli (2 cycles per degree, 2 Hz, high contrast: 100%). They used an ROI approach that focused on the occipital lobe: areas V1, V2, and V3. The main results were: a functional deficit for low-level stimuli, magno- or parvocellular, in children as well as adults; greater hypoactivation in the extrastriate cortex (V2 and V3) of the dorsal pathway, and abnormal activation during low-level M stimuli (suggesting an interference by deficient deactivation) in the DMN (medial prefrontal cortex, anterior cingulate cortex (ACC), the posterior parietal cortex and parietal cortex) related to ADHD frequency in this population. To pursue the physiopathological analysis of the visual cortex in NF1 patients, the same team conducted a study that combined fMRI, spectroscopy (GABA/Creatinine and Glutamate/Creatinine ratios in the occipital cortex) and genetic analysis (57). Eighteen NF1 children and 26 controls aged 7 to 19 years performed a simple fMRI visual task in which they had to press a button whenever a target disappeared.

TABLE 3 | Correlation between fMRI analysis and neuropsychological findings, main characteristics of the neuroimaging studies included.

ACC, anterior cingulate cortex; Cr, creatinine; DLPFC, dorsolateral prefrontal cortex; DMN, default mode network; EEG, electroencephalogram; FEF, frontal eye field; fMRI, functional magnetic resonance imaging; GABA, gamma-aminobutyric acid; IFG, inferior frontal gyrus; IQ, intellectual quotient; IOG, inferior occipital gyrus; IPS, intraparietal sulcus; JLO, judgement line orientation; MRS, magnetic resonance spectroscopy; NF1, neurofibromatosis type 1; PCC, posterior cingulate cortex; pre-SMA, pre-supplementary motor area; r, range; SD, standard deviation; STG, superior temporal gyrus, OFC, orbitofrontal cortex, vACC, ventral anterior cingulate cortex.

Through the results, the authors were able to show a reduction in the GABA/Creatinine ratio in NF1 subjects compared to the controls but no difference in the Glutamate/Creatinine ratio, which suggests an alteration in the balance between excitatory and inhibitory mechanisms with altered inhibition in the NF1 occipital cortex. The authors also found a correlation between mutation type and GABA level suggesting a role of neurofibromin in GABAergic neurotransmission. Lastly, they showed a negative correlation between GABA/Creatinine and BOLD level with no difference between the NF1 subjects and the controls. The same team [**Table 3**; Ribeiro et al. (52)], using combined high-density electroencephalography, MRS and fMRI with a Go/No Go task, examined the neural mechanisms of impulsive behavior in NF1. During the Go/No Go task in visual modality, NF1 subjects made more errors of omission and had a faster reaction time, which confirms the impulsive phenotype. This behavior was correlated with a decrease in GABA/Creatinine ratio found in the medial frontal cortex (including the pre-motor area, the supplementary motor area and the ACC). However, the decrease in this same ratio in the occipital regions was not correlated with the behavioral data. Regarding evoked potentials, an early component corresponding to early visual processing and a later component in the frontal regions matched the inhibitory response that was altered in NF1 subjects. However, in this study, there was no link between genetic findings and altered GABAergic neurotransmission in frontal regions.

Evaluation of the therapeutic care of NF1 patients. In conclusion with regards to functional neuroimaging, two research teams used fMRI as a means to evaluate NF1 patient therapy. The first research, conducted by Charbernaud et al. (58), did not include a control group, which limits the interpretation of the results. In this phase 1, open label trial including 7 children aged 10 to 15 years treated with lovastatin for 12 weeks, an MRI was performed 1 day prior to the start of treatment and on the last day of treatment. The authors compared functional activity in rs-fMRI on the first neuroimaging and functional activity in the resting block between periods of visual stimuli on the second MRI, which they considered as a "pseudo rs-fMRI." The main result was an increase in anterior-posterior long-range connectivity and a decrease in short-range connectivity as observed in normal development. A second preliminary study, conducted by Yoncheva et al. (59) in 16 NF1 children aged 8 to 15 years with a deficit in working memory, evaluated the impact of cognitive training (Cogmed Training) in 25 sessions for 6 and 10 weeks. An rs-fMRI was performed before and after training. Four rs-fMRI indices previously used in typically developing children were analyzed: the amplitude of low frequency fluctuations (ALFF) and the fractional amplitude of low frequency fluctuations (fALFF) that characterize intrinsic neural activity, regional homogeneity (ReHo) that characterizes local synchronization and voxel mirrored homotopic connectivity (VMHC) that reflects interhemispheric synchronization. After training a reduction in fALFF in the cerebellum (left cerebellum I to IV and right cerebellum V) and in the thalamus (right and left), the authors observed a reduction in ReHo in the right middle frontal gyrus and an increase in ReHO in the left

fractional anisotropy; FEF, frontal eye field; FG, fusiform gyrus; GABA, gamma-aminobutyric acid; HG, Heschl's gyrus; IFG, inferior frontal gyrus; IOG, inferior occipital gyrus; JLO, judgement line orientation; NF1, neurofibromatosis type 1; OFC, orbitofrontal cortex; PC, posterior cerebellum; PCC, posterior cingulate cortex; pre-SMA, pre-supplementary motor area; SLF, superior longitudinal fasciculus; STG, superior temporal gyrus; T2H, T2 hyperintensity, WM, white matter.

fusiform gyrus. This was a preliminary study that showed the possibility to record neural activity changes after training, but this did not enable us to distinguish a developmental effect vs. a specific effect of training because of the absence of a control group.

### Summary

Functional neuroimaging studies have primarily explored two main characteristics of the NF1 cognitive phenotype: executive function deficit and visuospatial deficit.

The deficit in executive functions is characterized by deficient inhibitory control and a deficit in exogenous attention depending on the external stimuli. It appears to be associated with a dysfunction in the right inferior frontal areas and the middle frontal areas (pre-motor area, supplementary motor area and ACC). A GABAergic deficit has also been shown for these regions.

Concerning the visuospatial deficit, studies have suggested a dysfunction in the visual cortex (V2-V3) and especially in the magnocellular pathway involved in the processing of low spatial frequency and high temporal frequency. This dysfunction could be associated with a disruption in excitatory-inhibitory balance, which involves neurofibromin, with a decrease in inhibitory GABA in the occipital cortex in NF1.

Studies that specifically address the issue of connectivity show an altered neural connectivity in NF1 subjects compared to controls with a reduction in anterior-posterior "long-range" connectivity and a deficit in deactivation in DMN during cognitive tasks.

Abnormalities observed in brain activity can serve as a basis to evaluate the efficacy of new therapeutics that could be used in NF1 subjects over the next few years. Preliminary studies suggest the possibility to use this technique as a biomarker in future treatment trials.

Characteristics of the main neuroimaging studies showing a link between functional features and cognitive functions are indicated in the **Table 3**.

### CONCLUSION

Although the link between the specific cognitive and behavioral features of NF1 and cerebral characteristics is not totally clear at present, comprehension of the neural basis has improved thanks to emerging neuroimaging methods. Executive dysfunction in NF1 children seems to be associated with a dysfunction in the right inferior frontal areas and the middle frontal areas and an alteration in microstructural integrity (DTI) in ATR.

### REFERENCES

Visuospatial deficit appears to be correlated with a dysfunction in the visual cortex (V2-V3) and especially in the magnocellular pathway involved in the processing of low spatial frequency and high temporal frequency. Moreover, connectivity studies show an altered neural connectivity with a reduction in anteriorposterior "long-range" connectivity and a deficit in deactivation in DMN during cognitive tasks. Therefore, functional MRI has become a widely used technique over the past years and might be helpful in the understanding of the cerebral basis of NF1 cognitive phenotype. However, at present there are inconsistencies in the findings of several studies with regard to morphological and macrostructural neuroimaging brain features (UBOs, megalencephaly, higher volume of sub-cortical structures) in NF1. Therefore, it is difficult to find a conclusive link between these features and neurocognitive phenotype (**Figure 1**).

Recently, a study by our group using a multimodal approach involving measures of gray matter volume, fractional anisotropy, and mean diffusivity highlighted a NF1 brain signature (11). Considering that studies using a monomodal approach have failed to explain the cognitive phenotype in NF1, in the future, the development of multimodal approaches could help to clarify the relationships with NF1 phenotype and evaluate the efficacy of specific therapeutics. Moreover, the cognitive phenotype of NF1 subjects is extremely variable from one individual to another. This heterogeneity is probably multifactorial resulting from genetic and environmental factors in such a way that an approach exclusively based on neuroimaging cannot entirely explain the cognitive phenotype. The study of the impact of various factors that influence the cognitive phenotype (genetic, environmental, etc.) remains an indispensable complement to the neuroimaging approach in NF1.

### AUTHOR CONTRIBUTIONS

YC wrote the first draft of the manuscript. EB translated and completed a part of the manuscript, and drew up the tables. FN made critical amendments and gave essential feedback for the manuscript. MB developed and formatted the figure and helped to improve tables. SM gave essential feedback for the manuscript. PP made critical amendments especially for the figure. All authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

We would like to thank SARL TTP-AMPLUS for his careful checking of the English language and the reviewers for their constructive comments.

of the neuropsychological profile of children with NF1. Child Neuropsychol. (2018) 24:558–74. doi: 10.1080/09297049.2017.13 13970

3. Payne JM, Hyman SL, Shores EA, North KN. Assessment of executive function and attention on children with neurofibromatosis type 1: relationships between cognitive measures and real-world behavior. Child Neuropsychol. (2011) 17:313–29. doi: 10.1080/09297049.2010.542746

thalamus in neurofibromatosis type 1: correlation with T2 hyperintensities. Neuroradiology. (2011) 53:141–8. doi: 10.1007/s00234-010-0776-4

**Conflict of Interest:** 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 © 2020 Baudou, Nemmi, Biotteau, Maziero, Peran and Chaix. 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) and the copyright owner(s) 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.

# A Novel Homozygous Variant in the Fork-Head-Associated Domain of Polynucleotide Kinase Phosphatase in a Patient Affected by Late-Onset Ataxia With Oculomotor Apraxia Type 4

Rosa Campopiano1 , Rosangela Ferese1 , Fabio Buttari 1 , Cinzia Femiano1 , Diego Centonze1,2, Francesco Fornai 1,3, Francesca Biagioni 1 , Maria Antonietta Chiaravalloti 1 , Mauro Magnani 4 , Emiliano Giardina5,6, Anna Ruzzo4 and Stefano Gambardella1,4 \*

1 IRCCS Neuromed, Pozzilli, Italy, 2 Dipartimento di Medicina dei Sistemi, Università di Roma Tor Vergata, Rome, Italy, 3 Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy, 4 Department of Biomolecular Sciences, University of Urbino "Carlo Bo", Urbino, Italy, 5 Department of Biomedicine and Prevention, University of Rome "Tor Vergata", Rome, Italy, 6 Molecular Genetics Laboratory UILDM, Santa Lucia Foundation, Rome, Italy

#### Edited by:

Matthew James Farrer, University of Florida, United States

#### Reviewed by:

Mario Reynaldo Cornejo-Olivas, National Institute of Neurological Sciences, Peru Luca Marsili, University of Cincinnati, United States

\*Correspondence: Stefano Gambardella<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurogenetics, a section of the journal Frontiers in Neurology

Received: 07 June 2019 Accepted: 02 December 2019 Published: 15 January 2020

#### Citation:

Campopiano R, Ferese R, Buttari F, Femiano C, Centonze D, Fornai F, Biagioni F, Chiaravalloti MA, Magnani M, Giardina E, Ruzzo A and Gambardella S (2020) A Novel Homozygous Variant in the Fork-Head-Associated Domain of Polynucleotide Kinase Phosphatase in a Patient Affected by Late-Onset Ataxia With Oculomotor Apraxia Type 4. Front. Neurol. 10:1331. doi: 10.3389/fneur.2019.01331 Ataxia with oculomotor apraxia (AOA) is a clinical syndrome featuring a group of genetic diseases including at least four separate autosomal-recessive cerebellar ataxias. All these disorders are due to altered genes involved in DNA repair. AOA type 4 (AOA4) is caused by mutations in DNA repair factor polynucleotide kinase phosphatase (PNKP), which encodes for a DNA processing enzyme also involved in other syndromes featured by microcephaly or neurodegeneration. To date, only a few AOA4 patients have been reported worldwide. All these patients are homozygous or compound heterozygous carriers for mutations in the kinase domain of PNKP. In this report, we describe a 56 years old patient affected by AOA4 characterized by ataxia, polyneuropathy, oculomotor apraxia, and cognitive impairment with the absence of dystonia. The disease is characterized by a very late onset (50 years) when compared with other AOA4 patients described so far (median age of onset at 4 years). In this proband, Clinical Exome Analysis through Next Generation Sequencing (NGS) consisting of 4,800 genes, identified the PNKP homozygous mutation p.Gln50Glu. This variant, classified as a likely pathogenic variant according to American College of Medical Genetics (ACMG) guidelines, does not involve the kinase domain but falls in the fork-head-associated (FHA) domain. So far, mutations in such a domain were reported to associate only with a pure seizure syndrome without the classic AOA4 features. Therefore, this is the first report of patients carrying a mutation of the FHA domain within the PNKP gene which expresses the clinical phenotype known as the AOA4 syndrome and the lack of any seizure activity. Further studies are required to investigate specifically the significance of various mutations within the FHA domain, and it would be worth to correlate these variants with the age of onset of the AOA4 syndrome.

Keywords: ataxia with oculomotor apraxia, PNKP gene, clinical exome, late onset, neurogenetics

## INTRODUCTION

Mutations in the DNA repair factor polynucleotide kinase phosphatase (PNKP) have been linked to multiple distinct human inherited syndromes. These include (i) microcephaly with seizures (MCSZ, MIM 613402, also known as early infantile epileptic encephalopathy-10), which is characterized by microcephaly, early-onset, intractable seizures, and developmental delay (1), (ii) progressive cerebellar atrophy and polyneuropathy (2), and (iii) ataxia with oculomotor apraxia type 4 (AOA4) (3, 4).

Ataxia with oculomotor apraxia (AOA), is a group of at least four autosomal-recessive cerebellar ataxia syndromes. They are related to genes which provide instructions for making proteins that are involved in repairing damaged DNA: (i) APTX (aprataxin), responsible for AOA1 (MIM 208920), a progressive syndrome associated with hypoalbuminemia and elevated levels of cholesterol (5–7); (ii) SETX (senataxin), responsible for AOA2 or SCAR1 (MIM 606002), a progressive ataxia occurring later than AOA1, characterized by increased alpha-fetoprotein levels (8, 9), (iii) PIK3R5 (phosphoinositide-3-kinase, regulatory subunit 5), responsible for AOA3 (MIM 615217), with clinical features similar to AOA2; (iv) PNKP (polynucleotide kinase 3′ phosphatase) responsible for AOA4 (MIM 616267) characterized by ataxia, oculomotor apraxia, peripheral neuropathy, and dystonia (3).

These syndromes impact cerebellar function and result in a profound loss of motor control characterized by cerebellar degeneration, apraxia, and oculomotor apraxia (abnormal saccadic eye movement). Very often these symptoms occur in the presence of dystonia and peripheral neuropathy (5, 10).

AOA4 is characterized by prominent dystonia which spontaneously attenuates during the course of the disease. Muscle wasting in the hands and feet and neuropathy are also common and lead to tetraplegia and short atrophic hands and feet. Usually, cognitive function is not affected, although some people may present intellectual disability. AOA diseases are characterized by an early onset which occurs in the first decade; strikingly, AOA4 is characterized by the earliest disease onset among AOAs which occurs at about 4 years of age (11).

PNKP is a DNA processing enzyme in which the C-terminal catalytic domain contains a fused bimodal phosphatase and kinase domain, with a fork-head-associated (FHA) domain at its N-terminus (12).

The FHA domain of PNKP is important for interaction with either the XRCC1 or XRCC4 scaffold proteins, which are required for assembling single-strand break repair (SSBR) or dominant pathway for DNA double-strand break repair (DSBR) (NHEJ, non-homologous end-joining) components respectively (13–15). In fact, PNKP is important for both the SSBR/Base excision repair (BER) and NHEJ pathways (12).

To date, only a few patients with mutations in PNKP have been reported worldwide. Substantial variation exists among affected individuals with PNKP mutations, as individuals diagnosed with MCSZ show no neurodegeneration, while individuals with AOA4 show pronounced neurodegeneration.

Among AO4 patients, the different phenotypes associated with mutations in PNKP do not seem to relate to either the type or the location of the mutation (3). So far, all PNKP mutations related to AOA4 have been described in the kinase domain.

In this report, we describe a patient affected by AOA4 related to a homozygous mutation in PNKP, with very late onset at 50 years. Despite the occurrence of AOA4 phenotype, this variant falls within the FHA domain, which was never reported before in AOA4 patients.

### MATERIALS AND METHODS

### Genetic Testing

The pedigree of the proband's family is shown in **Figure 1A**. After genetic counseling, written informed consent was obtained, and clinical exome sequencing (4,800 human genes) was performed including 100 genes related to Ataxia and or Spastic Paraplegia (Clinical Exome Solution, SOPHiA genetics) on MiSeq platform (Illumina) (16).

SOPHiA DDM (SOPHiA genetics) was used for annotation and characterization of variants. Sequence analysis identified the homozygous mutation NM\_007254.3:c.[148C>G]; NP\_009185.2: p. (Gln50Glu) (rs756746191:C>G).

In the FHA domain PNKP (OMIM #605610), resulting in the replacement of glutamine with glutamic acid in the protein at position 50. The variant was confirmed by Sanger sequencing (ABI 3130xl Genetic Analyzer, Applied Biosystem) (**Figure 1B**). No mutations in other ataxia-related genes were identified. The variant has been submitted to Clinical Variant (ClinVar Accession Number SUB6349616).

The frequency of the variant is not known either in the ExAC database (http://evs.gs.washington.edu/EVS) or in the 1,000 Genomes database (http://browser.1000genomes.org). The prediction analysis in silico reveals a damaging effect in three out of four tools used [Mutation taster (http://www.mutationtaster. org), SIFT (http://sift.jcvi.org), and Polyphen-2 (http://genetics. bwh.harvard.edu/pph2)]. The PROVEAN (http://provean.jcvi. org/index.php) tool alone predicts it as neutral. The analysis using the PhyloP (http://compgen.bscb.cornell.edu/phast/) and GERP (http://mendel.stanford.edu/sidowlab/downloads/gerp/ index.html) tools gave respectively a positive score (1) and an neutral rate (NR) equal to 5.4 indicating that both the wild type nucleotide and the amino acid are conserved in mammals.

The presence of p.Gln50Glu was evaluated in II:2 (54 years old brother) and II:3 (49 years old sister), who are asymptomatic heterozygous carriers (**Figure 1A**).

According to ACMG guidelines, which consider data on familial segregation, bioinformatic analysis, and allele frequencies, p. (Gln50Glu) has been classified as a likely pathogenic variant.

### RESULTS

A 56-year-old Italian male (II:1) was referred to the Neurology Unit of the Scientific Institute for Research and Healthcare Neuromed in Pozzilli (IS) Italy, with a 6 years history of distal weakness and tactile hypoesthesia of the legs and arms, cramps, urinary urgency, dysarthria, and mild dysphagia. He was born at term by unrelated parents that died late in life. His comorbidity

Sequence analysis is shown for proband (II:1) who is homozygous for variant and brother (II:2) and his sister (II:3) who is heterozygous carrier of the same variant.

consists of allergic bronchial asthma which was routinely treated with beta2-agonists and corticosteroids (**Figure 1A**).

On neurological examination, he had cerebellar dysarthria, gait ataxia (the patient was able to walk without support but with moderate gait instability), mild bilateral dysmetria, oculomotor apraxia, distal weakness of the arms and legs, distal hypoesthesia, absent tendon reflexes. The International Cooperative Ataxia Rating Scale (ICARS) total score was 40/100. Electroneuromyography (ENMG) indicated the presence of a sensory-motor axonal polyneuropathy. Motor evoked potentials (MEPs) were characterized by increased central motor conduction time. Somatosensory evoked potentials (SEP) were not measurable due to a lack of reproducibility of peripheral responses. Brain Magnetic Resonance Imaging (MRI) at the age of 55 showed marked subtentorial atrophy without spinal cord alteration. In detail, Axial T1-weighted showed

cerebellar and brainstem atrophy, Sagittal MRI scan showed global cerebellar atrophy, and Axial FLAIR MRI sequences showed no dentate nucleus hyperintensity (**Figure 2**). The neuropsychological assessment showed impairment of shortterm memory, attention and constructive apraxia. Nothing relevant was found on body Computer Tomography (CT) scan. There were no alterations in the liquor analyzed after lumbar puncture. A second neurological examination performed after 6 months showed no significant deterioration, suggesting a slow progression of the disease.

Blood tests showed mild hyper-CKemia (624 UI/l), hypercholesterolemia (263 mg/dl), hight alfa-fetoprotein (10.85 ng/ml), and low albumin level (3.4 g/dl). BMI index was 24.22. LDL increased level (173 mg/dl, nv < 130) confirmed data by Scheiss et al. (4), who reported increased LDL and IgE levels in AOA4 patients. The immunoglobulin dosage was not performed. High-density lipoprotein (HDL), triglycerides, thyroid hormones, vitamins, autoimmune screen, anti-ganglioside, and anti-neuronal antibodies were normal.

### DISCUSSION

A variety of human neurologic diseases are caused by inherited defects in DNA repair, underscoring the critical requirement for genome stability in this tissue (17). An example is provided by the defective enzymatic activity of the genes responsible for ataxia with AOA, a subgroup involving cerebellar ataxia, axonal neuropathy, oculomotor apraxia, and extrapyramidal features. Genes responsible for this condition provide instructions for making proteins that are involved in repairing damaged DNA. DNA damage that is not repaired makes the cell unstable and can lead to cell death, producing a severe effect in the cerebellum, as witnessed by the occurrence of ataxia with oculomotor apraxia (18).

Recently described in a Portuguese family (19), AOA4 is a complex, progressive movement disorder characterized by phenotypic heterogeneity like other AOAs. In most patients, the first symptom is dystonia, which spontaneously attenuates during the course of the disease. Other features include cerebellar ataxia, oculomotor apraxia, and polyneuropathy. Cognitive impairment is rare, while a few patients at later stages of the disease present abnormal values of alfa-fetoprotein, albumin, and cholesterol.

In this report, we describe the later onset of AOA4 in a patient that did not experience dystonia but a classic cerebellar syndrome with gait ataxia, mild dysmetria, oculomotor apraxia, and cerebellar dysarthria and dysphagia. Sensorialmotor axonal polyneuropathy was also present. Cerebellar ataxia was compatible with cerebellar atrophy detected at brain MRI (20) (**Figure 2**). Dysphagia could be explained by the involvement of the nucleus ambiguous in the brainstem atrophy detected at MRI. However, considering the concomitance of cerebellar dysarthria, the presence of dysphagia is likely to be due to the cerebellar involvement. Similarly, brainstem atrophy could be further investigated concerning the potential involvement of pre-cerebellar nuclei. Mild hyper-CKemia, although non-specific, is likely to be due to the motor component of polyneuropathy.

The AO4 patient here described is unique considering his age of onset at 50. In fact, age of onset of AO4 patients ranges from 1 to 9 years (with a mean of 4.3). So far, only a German woman was described with a delayed onset although she was much younger (23-year-old), compared with the present case report. Additionally, this 23 year old diagnosed patient possessed an AOA phenotype which was combined with a cerebellar pilocytic astrocytoma (21).

The present paper reports a patient who is a carrier of the homozygous mutation NM\_007254.3:c.[148C>G]; NP\_009185.2: p. (Gln50Glu) (rs756746191:C>G) in the FHA domain PNKP. To date, multiple distinct human syndromes have been linked to mutations in PNKP, leading to microcephaly or neurodegeneration (17). PNKP is a multifunctional DNA repair enzyme important for both the SSBR/BER and NHEJ pathways (12), and this dual functionality potentially explains the presence of both microcephaly and neurodegeneration in patients with certain inherited PNKP mutations (22).

In support of this, it's well-known that different domains of the protein are linked to different diseases. The kinase domain of PNKP is associated with neurodegeneration associated with AOA4, as witnessed by the variants p.G375W, p.T408del, p.R439fs, p.T442fs, p.Thr424fs∗ 49, and p.Tyr515∗ (21). Mutations responsible for MCSZ are mainly located in the phosphatase domain (p.L176F and p.E326) but spread along the whole gene. In detail, an MCSZ patient carries the compound heterozygote genotype p.G292R/p.A55S, falling respectively in the phosphatase and FHA domains; while another MCSZ patient is homozygous for p.T424GfsX48 in the kinase domain (a variant which was also reported in a compound heterozygous AOA4 patient) (2, 23).

Remarkably, so far variants in the FHA domain have never been associated with AOA4 and even with neurodegeneration or microcephaly, as they have been solely reported in individuals who manifest only seizures (p.P20S, p.A55S) (11).

The genetic analysis performed in this paper has limitations. In fact, although the sequencing analysis has been carried out on 4,800 human genes including 100 genes related to Ataxia and Spastic paraplegia, these can't rule out the involvement of other genes related to clinical phenotype. Probably, a more extensive approach like whole exome or whole genome sequencing would guarantee more informative data. Moreover, segregation analysis is not fully informative. In fact, the age of the brother and the sister of the proband, both asymptomatic heterozygous carriers, can't rule out the appearance of clinical symptoms in the next years.

The present paper describes the first variant in the FHA domain leading to neurodegeneration and appearing with an AOA4 phenotype. Further studies are required to establish the role of mutations in different PNKP domains with a special emphasis of site-specificity within the FHA domain, and to identify and understand the role of modifier genes (24). This is required to explain the molecular mechanisms leading to the onset of AOA4 phenotype and to understand whether a late disease onset is a typical feature of AOA4 when this is produced by FHA domain specific mutations.

### AUTHOR CONTRIBUTIONS

SG and RF interpretation of data and drafting of manuscript. FF and DC acquisition of data, analysis and interpretation of data, and critical revision. RC, RF, FBi, and MC acquisition of data, analysis and interpretation of data. FBu and CF neurological history and interpretation of clinical data. MM, EG, and AR analysis and interpretation of data and critical revision. SG study conception and design, analysis and interpretation of data, and drafting of manuscript.

### REFERENCES

### FUNDING

This work was supported by the Italian Ministry of Health (Current Research 2019-2023: Identification of New Variants and/or New Genes Responsible for Ataxia and Spastic Paraplegia).

### ACKNOWLEDGMENTS

We thank the patients and their family for participating in the study.

24. Leuzzi V, D'Agnano D, Menotta M, Caputi C, Chessa L, Magnani M. Ataxia-telangiectasia: a new remitting form with a peculiar transcriptome signature. Neurol. Genet. (2018) 4:e228. doi: 10.1212/NXG.00000000000 00228

**Conflict of Interest:** 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 © 2020 Campopiano, Ferese, Buttari, Femiano, Centonze, Fornai, Biagioni, Chiaravalloti, Magnani, Giardina, Ruzzo and Gambardella. 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) and the copyright owner(s) 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.

# Outcomes of Bone Marrow Mononuclear Cell Transplantation for Neurological Sequelae Due to Intracranial Hemorrhage Incidence in the Neonatal Period: Report of Four Cases

Nguyen Thanh Liem1 \*, Truong Linh Huyen1 , Le Thu Huong2 , Ngo Van Doan2 , Bui Viet Anh1 , Nguyen Thi Phuong Anh2 and Dang Thanh Tung2

1 Cellular Manufacturing Department, Vinmec Research Institute of Stem Cell and Gene Technology, Hanoi, Vietnam, 2 Vinmec Times City General Hospital, Hanoi, Vietnam

#### Edited by:

Matthew James Farrer, University of Florida, United States

#### Reviewed by:

Wang-Tso Lee, National Taiwan University Hospital, Taiwan Juan Dario Ortigoza-Escobar, Pediatric Research Hospital Sant Joan de Déu, Spain

> \*Correspondence: Nguyen Thanh Liem<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Pediatric Neurology, a section of the journal Frontiers in Pediatrics

Received: 13 June 2019 Accepted: 12 December 2019 Published: 24 January 2020

#### Citation:

Liem NT, Huyen TL, Huong LT, Doan NV, Anh BV, Anh NTP and Tung DT (2020) Outcomes of Bone Marrow Mononuclear Cell Transplantation for Neurological Sequelae Due to Intracranial Hemorrhage Incidence in the Neonatal Period: Report of Four Cases. Front. Pediatr. 7:543. doi: 10.3389/fped.2019.00543 Aim: The aim of this study was to present primary outcomes of autologous bone marrow mononuclear cell (BMMNC) transplantation to improve neurological sequelae in four children with intracranial hemorrhage (ICH) incidence during the neonatal period.

Methods: GMFM88 and modified Ashworth score were used to assess motor function and muscle spasticity before BMMNC transplantation and after transplantation. Brain MRI was performed to evaluate brain morphology before and after BMMNC transplantation. Bone marrow were harvested from anterior iliac crest puncture and BMMNCs were isolated using Ficoll gradient centrifugation. The microbiological testing, cell counting, and hematopoietic stem cell (hHSC CD34+ cell) analysis were performed, following which BMMNCs were infused intrathecally.

Results: Improvement in motor function was observed in all patients after transplantation. In addition, muscle spasticity was reduced in all four patients.

Conclusion: Autologous BMMNC transplantation may improve motor function and reduce muscle spasticity in children with ICH incidence during the neonatal period.

Keywords: intracranial hemorrhage, neurological sequelae, bone marrow mononuclear cells, autologous, neonate

### INTRODUCTION

Intracranial hemorrhage (ICH), including extra-ventricular hemorrhage and intra-ventricular hemorrhage, is a severe condition in neonates, especially in preterm babies with high morbidity and mortality. It severely affects the central nervous system because the neonatal period is a critical window for brain development (1, 2).

Intra-ventricular hemorrhage is the common type of ICH in premature newborns. The incidence of intra-ventricular hemorrhage among infants born between 24 and 31 complete weeks in China in 2013–2014 was 15.4% (3), whereas the rate of severe intra-ventricular hemorrhage among infants born between 22 and 31 weeks of gestation at hospitals in California, United States, between 2005 and 2015 was 7.7% (4). Different mechanisms are cited to explain the etiology of intra-ventricular hemorrhage in premature babies, including fragility of the germinal matrix vasculature, disturbance in the cerebral blood flow, and coagulation disorders. Many factors such as a low gestational age and birth weight, intrauterine infection, vaginal delivery, low Apgar score, severe respiratory distress syndrome, pneumothorax, hypoxia, hypercapnia, seizures, patent ductus arteriosus, and thrombocytopenia are thought to be risk factors associated with intra-ventricular hemorrhage (5–7).

ICH occurred not only in preterm infants but also in full-term babies related to brain trauma, vascular malformation, vitamin K deficiency, and many other conditions (1, 8–13).

ICH in neonates results in severe neurodevelopmental adverse effects. Bolisetty et al. showed that infants born at 23 to 28 weeks' gestation with grade III–IV intra-ventricular hemorrhage had high rates of developmental delay (17.5%), cerebral palsy (30%), deafness (8.6%), and blindness (2.2%) (2). According to Pierrat et al., the overall rate of cerebral palsy in infants born at 24– 26, 27–31, and 32–34 weeks of gestation was 6.9, 4.3, and 1%, respectively (14).

Traditional treatments of neurological sequelae after ICH in newborns have limited effect. Recently, stem cell transplantation is being applied in the management of neurological impairment in children, resulting in promising outcomes (15–21). In 2018, Ahn et al. published the first report using stem cell transplantation for severe intra-ventricular hemorrhage to reduce neurological complications (22). However, to our knowledge there have been no publications using stem cell transplantation in the management of neurological sequelae after ICH incidence in the neonatal period.

The aim of this report is to present outcomes of bone marrow mononuclear cell (BMMNC) transplantation in four children with neurological sequelae due to ICH incidence during the neonatal period.

### PATIENTS AND METHODS

### Patient's Enrollment

The study was approved by the Hospital ethical committee and performed at Vinmec International Hospital in Hanoi, Vietnam. The parents were carefully educated about anticipated benefits and potential side effects of the procedures and provided written informed consent according to the Good Clinical Practice and the Helsinki Declaration. The general data collected before the transplantation consisted of age, gender, past medical history, clinical findings on admission, and brain MRI manifestations.

Prior to the treatment, the patients were thoroughly examined by an experienced physiotherapist using Gross Motor Function Classification System (GMFCS), Gross Motor Function Measure-88 (GMFM-88) scores, and modified Ashworth scale, and by an experienced psychologist using Denver II scale (23–25).

Four children with neurological sequelae due to ICH incidence in the neonatal period were enrolled in this study. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

## BMMNC Procedure

### Bone Marrow Aspiration

Bone marrow was harvested through anterior iliac crest puncture under general anesthesia in the operating theater. The volume collected depended on the patients' body weight as follows: 8 ml/kg for patients under 10 kg [80 ml + (body weight in kg – 10) × 7 ml] for patients above 10 kg (18).

### BMMNC Isolation, Characterization, and Preparation for Transplantation