and a patient with a novel homozygous variant (panel p2) [31]. (**D**) Transmission electron microscopy-based cupromeronic blue staining. GAG chains are curved and maintain close contact with attached collagen fibrils in the skin specimens from a healthy control volunteer (panels c-a, c-b); conversely, they are linear and stretch from the outer surface of collagen fibrils to adjacent fibrils in skin specimens from a patient with heterozygous variants Pro281Leu/ Tyr293Cys (panel p1), a patient with a novel homozygous variant (panels p2a and p2b) and another patient with heterozygous variants Pro281Leu/ Tyr293Cys (panel p3) [31]. (**E**) Schematic representations of collagen fibrils and GAG chains. Decorin core protein binds to D bands of collagen fibrils both in normal skin and in affected skin. GAG chains composed of DS adhere to collagen fibrils along D bands, beginning from the core protein (panels a and c), whereas GAG chains composed of CS extend linearly and perpendicularly to collagen fibrils from the core protein (panels b and d) [31]. Putative spatial disorganization of collagen fibril networks in the skin of patients (panel e) (A, reproduced from Miyake et al. *Hum. Mutat.* **2010**, *31*, 1233–1239, with permission from Wiley-Liss, Inc.; B–E, reproduced from Hirose et al. *Biochim. Biophys. Acta Gen. Subj.* **2019**, *1863*, 623–631, with permission from Elsevier, Inc.).

#### **5. Animal Model-Based Findings**

Knockout (*Chst14*−/−) mice were generated through homologous recombination that targeted the only coding exon 1 (i.e., exon 1) of *Chst14* [35,36]. F2 mice showed reduced weight and/or length and reduced bone volume/thickness/density of their lumbar vertebrae [35]. *Chst14*−/− mice showed reduced neurogenesis and diminished proliferation of neural stem cells, accompanied by increased expression of glutamate aspartate transporter and epidermal growth factor, compared with findings in both wild-type (*Chst14*+/+) mice and other knockout (*Chst11*−/−) mice, a model for chondroitin 4-*O*-sulfotransferase 1 (C4ST1) deficiency [36]. *Chst14*−/− mice also had a smaller body mass (Figure 5A, B), reduced fertility, a kinked tail, and increased skin fragility compared with their wild-type (*Chst14*+/+) littermates; however, brain weight and gross anatomy were not affected [36,37]. Schwann cells from *Chst14*−/− mice formed longer processes in vitro and exhibited greater proliferation than those from *Chst14*+/+ mice. Functional recovery and axonal regrowth in *Chst14*−/− mice were initially accelerated after femoral nerve transection and suture; after 3 months, these characteristics were similar to those in *Chst14*+/+ littermates. These findings suggested that DS, synthesized by Chst14/D4st1, might be of limited importance for neural development; moreover, it might contribute to the regeneration-restricting environment in the adult mammalian nervous system [38]. Only a few adult *Chst14*−/− mice were generated because of perinatal lethality in most of the homozygous mice; these adult *Chst14*−/− mice showed significantly shorter crown-rump length, compared with wild-type or heterozygous mice (Figure 5A–C) [37,38]. The placentas of *Chst14*−/− fetuses showed a reduced weight, alterations in the vascular structure and ischemic and/or necrotic-like changes (Figure 5D–G). Transmission electron microscopy of homozygous placentas demonstrated an abnormal capillary basement membrane structure in the placental villus, compared with wild-type or heterozygous placentas (Figure 5H–J). These findings showed that DS was essential for placental vascular development and perinatal fetal survival. In addition, DS was suggested to be related to the structure and/or function of capillary basement membranes, which constitutes an extracellular matrix [37].

C4st1, encoded by *Chst11*, transfers a sulfate group from 3 -phosphoadenosine 5 -phosphosulfates to the C4 position of GalNAc residues in chondroitin (Figure 3A). *Chst11*-mutant mice died within 6 hours of birth due to respiratory failure, severe dwarfism and chondrodysplasia (i.e., abnormalities in the cartilage growth plate and chondrocyte columns) [39]. Furthermore, marked reductions in the content and 4-*O*-sulfation of CS, as well as the downregulation of bone morphogenetic protein signaling and upregulation of transforming growth factor-β, have also been observed in *Chst11*-mutant mice. These findings suggest that Chst11/C4st1 and the 4-*O*-sulfation of CS chains are essential for early development and for bone morphogenetic protein and transforming growth factor-β in signaling pathways in cartilage. When considered in the context of *Chst14*−/− mouse phenotypes, the above findings suggest that 4-*O*-sulfation is required for the maturation of both CS and DS; moreover, CS and DS exert distinct functions in the development of cartilage and skin, respectively. However, the DS content in *Chst11*-mutant mice remains to be elucidated [39].

**Figure 5.** Mouse model for mcEDS-*CHST14*. (**A**) Numbers (percentages) of each type at embryonic (E18.5) and postnatal (adult) ages. Markedly greater numbers of homozygous fetuses are observed, compared with homozygous adults. (**B**) General appearances of wild-type (*Chst14*+/+), heterozygous (*Chst14*+/−) and homozygous (*Chst14*−/−) fetuses [37]. (**C**) Crown-rump length of each type of fetus (bar: 5 mm). Homozygous mice demonstrate significantly shorter crown-rump length, compared with wild-type or heterozygous mice [37]. (**D**) Appearance of each type of placenta (bar: 5 mm) [37]. (**E**) Homozygous placentas exhibit appearances indicative of hypoxia (left) and necrosis (right) [37]. (**F**) Microphotographs of the chorionic plate side of the placentas (bar: 1 mm). Homozygous placentas demonstrate smaller vascular diameters, compared with wild-type or heterozygous placentas [37]. (**G**) Weight of each type of placenta (mean + standard error of the mean). (**H**) Transmission electron microscopy of capillary basement membrane in the labyrinth zone of each type of placenta (bar: 200 nm). Arrows indicate capillary basement membrane [37]. (**I**) Homozygous placentas show a significantly

thinner capillary basement membrane, compared with wild-type or heterozygous placentas [37]. (**J**) Homozygous placentas show structural abnormalities (arrows) in capillary basement membrane (bar: 0.2 μm) [37]. \* *p* < 0.05, \*\* *p* < 0.01, compared with wild-type; # *p* < 0.05, ## *p* < 0.01, compared with heterozygous; one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post hoc test. (A–J, reproduced from Yoshizawa et al. *Glycobiology* **2018**, *28*, 80–89, with permission from Oxford University Press, Inc.).

Biosynthesis of DS requires D4st1, dermatan sulfate epimerase 1 (DS-epi1) and dermatan sulfate epimerase 2 (DS-epi2), which are encoded by *Chst14*, *Dse* and *Dsel*, respectively [40–43]. Furthermore, D4ST1 interacts directly with DS-epi1, but not with DS-epi2, to form a hetero-complex that is required for the formation of IdoUA blocks in DS chains [44]. *DS-epi1*−/− mice showed greater skin fragility compared with wild-type littermates, due to altered collagen fibril morphology [45]; this phenotype was similar to that of *Chst14*−/− mice [38]. The numbers of IdoUA blocks were dramatically reduced in DS side chains of decorin-PG and biglycan-PG from the skin of *DS-epi1*−/− mice [45], which suggests that DS-epi1 mainly synthesizes IdoUA blocks in vivo. *DS-epi1*−/− embryos and newborn mice showed kinked tails, which is a common feature in *Chst14*−/− mice [38]; the *DS-epi1*−/− embryos and newborn mice also showed significantly thicker epidermal layers through histological staining, compared with heterozygous or wild-type littermates [45,46]. Immunohistochemical staining of epidermal layers in *DS-epi1*−/− newborns showed increased expression of keratin 5 in the basal layer and keratin 1 in the spinous layer [46]. Furthermore, a small portion of *DS-epi1*−/− embryos showed an abdominal wall defect with herniated intestines, exencephaly and spina bifida. Defective collagen structure in the dermis and imbalanced keratocyte maturation were presumed to cause developmental defects in *DS-epi1*−/− mice [46]. These observations indicate that *DS-epi1*−/− mice may constitute a useful model of mcEDS-*DSE*. *DS-epi2*−/− mice displayed no significant defects and DS-epi1 compensated for the absence of DS-epi2 in most tissue, which indicated that DS-epi1 is the major contributor of epimerase activity [47]. DS-epi2 exhibits higher expression than DS-epi1 in developing the mouse brain [48]. Although CS/DS chains in the brains of *DS-epi2*−/− newborn mice demonstrated a 38% reduction in IdoUA content, compared with wild-type littermates, the brains of adult knockout mice showed normal extracellular matrix features [47]. *DS-epi1*−/−/*DS-epi2*−/− mice experienced perinatal death with variable phenotypes at late embryological stages and birth; these phenotypes included umbilical hernia, exencephaly, and a kinked tail, as well as complete loss of IdoUA residues in CS/DS chains [49]. However, a minority of embryos exhibited normal lung, bone, and cartilage features.

These findings indicate that DS, DS-PGs, DS-epi1 and/or DS-epi2 are important in early embryonic development and perinatal survival [49].

#### **6. Ongoing Projects and Future Perspectives**

Our group established a multicenter collaboration network to promote comprehensive basic research on mcEDS. In this network, genetic testing is provided as part of routine medical care covered by national health insurance, using a custom next generation sequencing-based panel that includes all EDS-related genes and genes for hereditary connective tissue disorders [50]. Whole exome sequencing is performed to identify other potential causative genes for mcEDS in patients with similar features who do not exhibit pathogenic variants in *CHST14* or *DSE*. Biochemical abnormalities of the extracellular matrix are investigated (e.g., various types of collagen and DS-PGs, including decorin and biglycan), using patient skin fibroblasts. Furthermore, mass spectrometry is performed to identify appropriate serum/plasma biomarker(s) that may be useful in the diagnosis or surveillance of disease progression. A subsequent pathological approach includes an elucidation of the structural alterations of collagen fibril networks and GAG chains of decorin in skin specimens from patients with mcEDS-*DSE*. Determination of the crystal structure of D4ST1 would be useful for understanding the effects of common missense variants (e.g., p.(Pro281Leu)) in *CHST14*. A technical breakthrough in the efficient generation of knockout mice for *Chst14* (e.g., CRISPR/cas9) is needed to continue mouse-based phenotypic and pathophysiological investigations. Considering the differences in the phenotypes between patients with mcEDS and *Chst14*−/− or *DS-epi1*−/− mice, experiments using other models that could reflect phenotypic and pathophysiological abnormalities in patients with mcEDS, such as induced pluripotent stem cells, would be particularly useful. All of these approaches will be valuable for further elucidating the critical roles of DS and DS-PGs, including decorin and biglycan, in the multisystem development and maintenance of connective tissues; in addition, they provide fundamental evidence for future etiology-based therapies, such as adeno-associated virus-based gene therapy.

**Acknowledgments:** We thank all the collaborators engaged in this project. We are also grateful to the patients and their families for participating in the studies described here. We thank Ryan Chastain-Gross, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The drafting and revision of this review was supported by the Practical Research Project for Rare/Intractable Diseases #105 from the Japan Agency for Medical Research Development (AMED) (T.K., S.M., T.Y., N.M., and S.Y.); Grant-in-Aid for Young Scientists (B) (16K19396) (T.Y.); Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#16K08251, #19K07054) (S.M.) (#19K08745) (T.Y.); Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#19H03616) (T.K., S.M., T.W., T.Y., N.M., and S.Y.); Grant-in Aid for Research Center for Pathogenesis of Intractable Diseases from the Research Institute of Meijo University (S.M. and S.Y.).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **Defining the Clinical, Molecular and Ultrastructural Characteristics in Occipital Horn Syndrome: Two New Cases and Review of the Literature**

**Aude Beyens 1,2, Kyaran Van Meensel 1, Lore Pottie 1, Riet De Rycke 3,4,5, Michiel De Bruyne 3,4,5, Femke Baeke 3,4,5, Piet Hoebeke 6, Frank Plasschaert 7, Bart Loeys 8, Sofie De Schepper 2, Sofie Symoens 1 and Bert Callewaert 1,\***

Received: 28 June 2019; Accepted: 11 July 2019; Published: 12 July 2019

**Abstract:** Occipital horn syndrome (OHS) is a rare connective tissue disorder caused by pathogenic variants in ATP7A, encoding a copper transporter. The main clinical features, including cutis laxa, bony exostoses, and bladder diverticula are attributed to a decreased activity of lysyl oxidase (LOX), a cupro-enzyme involved in collagen crosslinking. The absence of large case series and natural history studies precludes efficient diagnosis and management of OHS patients. This study describes the clinical and molecular characteristics of two new patients and 32 patients previously reported in the literature. We report on the need for long-term specialized care and follow-up, in which MR angiography, echocardiography and spirometry should be incorporated into standard follow-up guidelines for OHS patients, next to neurodevelopmental, orthopedic and urological follow-up. Furthermore, we report on ultrastructural abnormalities including increased collagen diameter, mild elastic fiber abnormalities and multiple autophagolysosomes reflecting the role of lysyl oxidase and defective ATP7A trafficking as pathomechanisms of OHS.

**Keywords:** occipital horn syndrome; Ehlers–Danlos syndrome type IX; Menkes syndrome; ATP7A; review; copper transport; elastic fiber; collagen

#### **1. Introduction**

Occipital horn syndrome (OHS, OMIM#304150), previously known as Ehlers–Danlos syndrome type IX or X-linked cutis laxa, is a rare disorder characterized by prominent connective tissue abnormalities including cutis laxa, hernias, joint laxity and bladder diverticula, and pathognomonic exostoses with "occipital horns" or downward pointing exostoses situated in the tendinous insertions of the sternocleidomastoid and trapezius muscles. In addition, patients may show mild to moderate intellectual disability [1–6]. Being allelic with Menkes disease (MD, OMIM#30011), OHS is considered the milder end of the phenotypic spectrum due to pathogenic variants in *ATP7A* [4] that encodes a copper transporter. Several clinical features in both disorders are related to the malfunctioning of cupro-enzymes, including lysyl oxidase, dopamine ß-hydroxylase, tyrosinase and cytochrome C oxidase [7–10].

ATP7A mediates (1) copper transport from the gastrointestinal tract into the bloodstream, (2) intracellular delivery of copper to cupro-enzymes in the secretory pathway and (3) efflux of excess copper from the cell [11,12]. Intracellular ATP7A trafficking is essential for copper homeostasis. Under basal low copper conditions, ATP7A is found in the trans-Golgi network, where it is essential for cupro-enzyme biogenesis. At higher copper concentrations, it reversibly localizes to the plasma membrane and post-Golgi compartments, where it is responsible for the extrusion of excess copper from the cell [13–15]. The *ATP7A* gene maps to Xq13.3 and spans a genomic region of ~140 kb. Its predominant transcription product contains 23 exons [16–20]. ATP7A belongs to the large P-type ATPase family, which are ATP-driven membrane pumps essential in the maintenance of electrochemical gradients (such as Na+/K+-, H+/K+- and Ca2+-pumps), as well as lipid and cationic homeostasis [21], including ATP7A and ATP7B, both important for copper homeostasis [13,22]. P-type ATPases share a structural core containing a transmembrane (TM) domain responsible for transport and an N-, P- and A- soluble catalytic domain, respectively involved in nucleotide binding, phosphorylation and dephosphorylation [23]. The transmembrane domain of ATP7A contains eight transmembrane helices (TM1–TM8). Six amino terminal domains contain metal-binding CXXC motifs (MBD1–MBD6) [24,25].

MD is mostly caused by truncating variants, including nonsense variants, frameshift variants and splice-site variants that lead to out of frame transcripts and large deletions, which result in very low to absent ATP7A levels [12,26]. On the contrary, the milder symptoms in OHS are the consequence of pathogenic variants in *ATP7A*, mostly "leaky" splice-site variants resulting in exon-skipping, which permit the production of a small amount of normal ATP7A mRNA [6,13,27–30]. This low amount of normal mRNA can result in residual protein activity and allow effective cation transfer to some copper-dependent enzymes. ATP7A levels as low as 2–5% have been shown to be sufficient to result in the milder OHS phenotype [31]. Connective tissue abnormalities in OHS have been attributed to a decreased activity of lysyl oxidase (LOX), a cupro-enzyme that normally deaminates lysine and hydrolysine residues in the first step of collagen crosslinking [7,32]. Patients may also encounter mild neurological signs and dysautonomia due to partial defects in dopamine-ß-hydroxylase enzyme activity, that converts dopamine to norepinephrine, a crucial neurotransmitter in norepinephrinergic neurons. As such, patients with OHS typically have low to normal levels of serum copper and ceruloplasmin together with abnormal plasma and cerebrospinal fluid (CSF) catecholamine levels [3,33,34].

Due to the rarity of OHS and the limited number of cases scattered in literature, the natural history and clinical phenotype remain incompletely studied. This study reports the clinical, molecular and ultrastructural data for two new cases of OHS. In addition, we performed a clinical review of 32 previously described patients [6,7,11,27–31,35–51].

#### **2. Materials and Methods**

#### *2.1. Patients*

Informed consent was obtained from subject 1 and his parents, and from subject 2. Additional consent was given to publish the clinical pictures in Figure 1. Clinical information was obtained by evaluating both patients at our center with the use of a detailed clinical checklist. We performed a literature review of 32 OHS patients with confirmed *ATP7A* pathogenic variants in the Pubmed and Embase databases, only including studies with ample clinical data. Selection of the reports was undertaken independently by two different authors (A.B and K.V.M). Clinical data from the literature were evaluated using the same methodology (Supplementary Table S1). This study was conducted in accordance with the Declaration of Helsinki and approved by the Ghent University Hospital ethics committee (registration number B670201319316).

#### 2.1.1. Clinical Reports

Case 1

Subject 1 (F1:II-1) was born at term after an uncomplicated pregnancy. At birth, his weight was 2400 g, length 45.5 cm and head circumference 32.4 cm. Developmental milestones were achieved normally. At three years of age, he presented with micturition problems evolving to urinary retention, which led to the discovery of giant bladder diverticula. Over subsequent years, several diverticulectomies were performed, followed by continent diversion and intermittent catheterization.

Clinical examination at 11 years of age showed mild facial dysmorphism (Figure 1) with a flat face, deep-set eyes, a long narrow nose, large ears and mild webbing of the neck. His weight was 24.9 kg (P1, −2.37 SD), height 139.2 cm (P26, −0.649 SD) and head circumference 56.2 cm (P98, +2.1 SD). Skeletal abnormalities included joint hyperlaxity of the wrists, metacarpophalangeal joints and fingers, a flat back, marked bony thickening of the proximal radius and tibia, genua valga and pedes plani. There was skin webbing between the second and third toe. His skin was soft and mildly hyperextensible with fine wrinkles over the abdomen and dorsum of hands and feet. He had no hair abnormalities. Neurological examination was normal. He had recurrent inguinal hernias. He reported bruising easily, but no impaired wound healing. He suffered from chronic joint pain. The patient attended normal education.

Additional examinations included a total skeletal radiography and MRI/MRA (Figure 2). X-rays show severe skeletal dysplasia, occipital horns, undertubulation of bony structures and broadening of the ventral end of the first left rib, the distal end of both claviculae and the scapular neck. The long bones showed bowing with mid-diaphyseal broadening. There was bilateral luxation of the radial heads, bilateral overgrowth of the ulnar coronoid processes and prominent trochanter minor of both femurs. We observed a short fibula and bilateral coxa valga with rounded iliac wings. The metatarsals were broad. The vertebrae showed platyspondyly. Brain MRI and angiography showed tortuous intracranial arteries. Echocardiography, lung radiography and spirometry were normal. Serum copper and ceruloplasmin levels were within normal ranges (89.6μg/dL (70–140) and 0.287 g/L (0.2–0.6), respectively).

**Figure 1.** Clinical characteristics in subjects F1:II-1 and F2:II-1. Subject F1:II-1 displays mild facial dysmorphism with a flat face, deep-set eyes, a long and narrow nose, large ears and mild webbing of the neck. Craniofacial features in patient F2:II-1 are more pronounced and include dolichocephaly, a high forehead, biparietal narrowing, downslanting eyes, a convex nasal ridge, midfacial hypoplasia, micrognathia and low-set ears. No hair abnormalities were observed. Both have lax and hyperextensible skin, marked joint hypermobility and deformities of the proximal radius.

**Figure 2.** Total skeletal radiography and MRI/MRA studies in subject F1:II-1 showing severe skeletal dysplasia with occipital horns, undertubulation of bony structures and broadening of the ventral end of the first left rib, both claviculae and scapular neck, bowing of the long bones with mid-diaphyseal broadening, bilateral luxation of the radial heads, bilateral overgrowth of the ulnar coronoid processes, prominent trochanter minor of both femurs, short fibula, bilateral coxa valga with rounded iliac wings and broad metatarsals. Brain MRI and angiography showed tortuous intracranial arteries and normal brain structure.

#### Case 2

Subject 2 (F2:II-1) was evaluated at the age of 30. He was born at term after an uncomplicated pregnancy. He developed an inguinal hernia at six weeks of age. Since then, he had multiple inguinal and femoral hernias requiring repeated mesh repairs. Starting from two years of age, he had severe bladder problems, including recurrent urinary tract infections and urinary retention, due to multiple bladder diverticula. After more than 20 surgical bladder repairs, he started self-catheterization at 8 years of age. Aged 20 years, he underwent left nephrectomy for a non-functioning kidney. He reported a mild motor delay and walked at two years of age. Joint hypermobility resulted in repeated shoulder and elbow subluxations. He was of normal intelligence and pursued higher education. He suffered from migraine, worsening musculoskeletal pain, fatigue, and depression for which he was treated with gabapentin and amitriptyline. He had a history of mitral valve prolapse, orthostatic hypotension, palpitations and a prolonged QT interval on electrocardiogram. Finally, he had a longstanding history of upper and lower gastrointestinal symptoms including nausea, post-prandial vomiting and chronic diarrhea, possibly in the context of dysautonomia. He was diagnosed with asthma.

His height was 169 cm, his weight was 50.6 kg and his head circumference was 58.5 cm. Craniofacial features consisted of dolichocephaly, a high forehead, biparietal narrowing, downslanting eyes, a convex nasal ridge, midfacial hypoplasia, micrognathia and low-set ears. He had lax and hyperextensible skin (Figure 1). He had no hair abnormalities. Skeletal abnormalities included occipital horns, a loss of sagittal spine curvature, scoliosis, pectus excavatum, and restricted elbow extension. He had bowing of the ulna and radius, bilateral exostoses of the radii and tibiae, bilateral rounding of the iliac wings, cranial displacement of the right humeral head and genua vara. Neurological examination and brain

MRI were normal. Serum copper and ceruloplasmin levels were 8.3 μmol/L (11–20) and 0.13 g/L (0.20–0.50), respectively.

#### *2.2. Molecular Analysis*

For individual 1, genomic DNA was extracted from blood leukocytes using standard procedures. Array comparative genomic hybridization (180 k arrayCGH, Agilent Technologies, Santa Clara, CA, USA) showed a 300 kb deletion of chromosome band 1q31.1 (1q31.1.1q31.1(188407205-188707983)x1)(hg19/GRCh37), which does not contain any known genes. *FBLN5* (OMIM#219100, GenBank RefSeq accession number NM\_006329.3), *LTBP4* (OMIM#613177, NM\_003573.2) and *ATP7A* (OMIM#304150, NM\_000052.7) gene sequencing was performed without the identification of a pathogenic mutation. Total RNA was extracted from cultured fibroblasts using the RNeasy Kit (Qiagen, Hilden, Germany) and cDNA was synthesized with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). *ATP7A* cDNA analysis was performed followed by confirmation with long-range RT-PCR and next-generation sequencing (Miseq, Illumina, San Diego, CA, USA). For individual 2, array comparative genome hybridization was normal after which *ATP7A* gene sequencing (GenBank RefSeq accession number NM\_000052.7) was performed.

#### *2.3. Transmission Electron Microscopy (TEM)*

For transmission electron microscopy, 3 mm skin fragments from both individuals and an age- and sex-matched control were immersed in a fixative solution of 2.5% glutaraldehyde and 4% formaldehyde in Na-Cacodylate buffer 0.1 M, placed in a vacuum oven for 30 min and left rotating for 3 hours at room temperature. This solution was later replaced with fresh fixative and samples were left rotating over night at 4 ◦C. After washing, samples were post-fixed in 1% OsO4 with K3Fe(CN)6 in 0.1 M NaCacodylate buffer, pH 7.2. After washing in double-distilled H2O, samples were subsequently dehydrated through a graded ethanol series, including bulk staining with 2% uranyl acetate at the 50% ethanol step, followed by embedding in Spurr's resin. To select the area of interest on the block and in order to have an overview of the phenotype, semi-thin sections were first cut at 0.5 μm and stained with toluidine blue. Ultrathin sections of a gold interference color were cut using an ultramicrotome (Leica EM UC6, Wetzlar, Germany), followed by post-staining in a Leica EM AC20 for 40 min in uranyl acetate at 20 ◦C and for 10 min in lead stain at 20 ◦C. Sections were collected on formvar-coated copper slot grids. Grids were viewed with a JEM 1400plus transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV.

#### *2.4. Collagen Biochemical Analysis*

Fibroblast cultures were started from skin biopsies from individuals 1 and 2, and a matched control. Biochemical (pro)collagen sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE analysis) was performed on the medium and cellular fractions of the cultured skin fibroblasts. When confluent, cells were labeled with 14C-proline [52]. After SDS-PAGE separation, further processing of the gels was performed as described previously by our group [53].

#### **3. Results**

#### *3.1. Clinical Characteristics*

We describe the clinical characteristics of two new individuals with OHS and 32 patients described in the literature (Table 1, Supplementary Table S1). All patients were male, except for patient F17:II-2. The mean reported age of patients was 19.6 years (mean 17, range 1–50). Disease-related mortality was observed in 5 out of 34 patients. The mean age at death was 21.2 years and causes included respiratory failure (post-surgery apnea), gastric perforation and diffuse intravascular coagulation due to a large cephalhematoma at birth.

**Table 1.** Overview of the phenotypic presentation in occipital horn syndrome.

Initial presentations leading to the diagnosis are presented in Table 2. One-third of patients presented with neurological features including hypotonia, developmental delay, and seizures. Nine individuals presented with connective tissue problems such as cephalhematoma and inguinal hernia. Other presenting symptoms included bladder problems such as bladder diverticula, urinary tract infections and skeletal problems.

**Table 2.** Initial presentations leading to the diagnosis in occipital horn syndrome.

"CTD": connective tissue disease.

Patients displayed variable craniofacial features, including a long face (6/13, 46%), large ears (5/13, 38%), sagging cheeks (5/11, 45%) and hair abnormalities (14/19, 74%). Trichoscopy confirmed pili torti in 7 out of 10 examined individuals. Connective tissue abnormalities were reported in all subjects. Cutis laxa was present in the majority of patients (25/26, 94%), as well as inguinal hernias (15/25, 63%). An umbilical hernia and hiatal hernia were observed in only one patient each (F25:II-1 and F15:II-1, respectively). Wound healing was reported to be normal after trauma or surgery. Occipital horns were reported in nearly all cases (25/26, 96%). Other recurrent skeletal manifestations included hammer-shaped claviculae (9/19, 47%), scoliosis (8/21, 38%), pectus deformities (13/23, 57%), radial/tibial exostoses (6/16, 38%), coxa valga (6/16, 38%) and genua valga (6/17, 35%). Dislocations, mainly of the radial heads and joint hypermobility were present in about two-thirds of individuals, 12/20 (60%) and 16/26 (62%), respectively. Less frequently reported skeletal manifestations include bowing of the long bones, mid-diaphyseal broadening, metaphyseal spurring and rounding of the iliac wings. Osteopenia or osteoporosis was reported in four patients and fractures were observed in two subjects.

Mild, mainly motor, developmental delay, was observed in 17 subjects (17/30, 57%). Intellectual disability was present in an equal number of patients (17/33, 52%) of whom seven had mild, four had moderate and three had severe intellectual disability. In three patients, the severity of intellectual disability was not specified. Several subjects displayed muscle hypotonia, especially at a young age (before the age of two). Seizures were documented in five patients (5/25, 20%). One individual was diagnosed with a spontaneous intracerebral hemorrhage at the age of 15, resulting in aphasia and spastic quadriparesis. MRI brain studies were performed in 11 patients and documented to be abnormal in 7 subjects, showing mild atrophy and/or delayed myelination. Intracranial tortuosity was present in two-thirds of patients (7/11, 64%). Tortuosity of extracranial arteries was found in three cases and was located in the cervical, splenic and hepatic arteries. Aneurysm formation was found in five patients, mainly affecting the same arteries. One individual was diagnosed with a type A aortic dissection at the age of 39, which was successfully surgically manage. Two patients showed dilatation of the large veins. Orthostatic hypotension, temperature instability, chronic diarrhea, and other symptoms of dysautonomia were seen in a high number of patients (13/15, 87%). Severe urological complications are an important concern in OHS, with bladder diverticula in 83% (25/30) of reported patients, of which

three were eventually diagnosed with a bladder rupture. Recurrent urinary tract infections based on incomplete emptying of the bladder were seen in 74% (17/23) of patients. One-third of patients had vesicourethral reflux (VUR), sometimes complicated by secondary renal problems. Asthma was reported in four cases. Low levels of serum copper and ceruloplasmin were found in two-thirds of cases, 20/28 (71%) and 20/27 (74%), respectively.

#### *3.2. Molecular Characteristics*

After normal *ATP7A* gene sequencing, cDNA analysis showed a deletion of exon 10 in subject F1:II-1. Subsequent confirmation with long-range RT-PCR revealed a deep intronic c.2407-516G>T variant, underlying the exon 10 skipping, likely due to the activation of a cryptic splice acceptor. In patient F2:II-1, *ATP7A* gene sequencing identified a hemizygous c.2917-4A>G splice-site variant in *ATP7A*.

A total of 16 different *ATP7A* pathogenic variants were reported (Figure 3); these included seven splice-site, four missense and two frameshift variants, two deep intronic variants and one deletion. In eight patients, the specific variant was not reported, including a 98-bp deletion in the regulatory region (F9:II-1), a deep intronic intron six splice donor site variant (F12:II-1) and a duplication in exon 11 and 12 (F23:II-1). Nonsense ATP7A variants were not observed. Pathogenicity information and CADD scores can be found in Supplementary Table S2. We did not observe any clear genotype-phenotype correlation and there was no correlation with serum copper and ceruloplasmin levels (Supplementary Table S3).

**Figure 3.** ATP7A pathogenic variants reported in this study. ATP7A consists of six amino terminal metal binding domains (MBD1-MBD6), a transmembrane domain containing eight transmembrane helices (TM1-TM8) and an N-, P- and A- soluble catalytic domain. The pathogenic variants identified in subjects F1:II-1 and F2:II-1 are indicated in bold and outlined.

#### *3.3. Transmission Electron Microscopy*

Transmission electron microscopy (TEM) findings in skin samples of subjects F1:II-1, F2:II-1 and a matched control are shown in Figure 4. Collagen fibrils in the dermis of the control sample are regularly organized in curvilinear bundles with uniform diameters. In the dermis of both OHS patients, the collagen fiber diameter appears to be larger and more variable. The fibrils are more loosely packed (especially in F2:II-1) but retain a normal shape. TEM of a control elastic fiber demonstrates a solid and dense core of elastin surrounded by a sparse mantle of microfibrils. In both individuals, the elastin core is surrounded by a debris extending outside the elastic fiber with loss of the microfibrillar organization. In addition, the elastin core in subject F2:II-1 is almost completely disrupted and difficult to discriminate. Fibroblast abnormalities are observed in both patients, with multiple autophagolysomes containing sickle-shaped electron dense deposits (Supplementary Figure S1). The mitochondria, Golgi complex, and endoplasmatic reticulum appear normal.

**Figure 4.** Transmission electron microscopy findings in occipital horn syndrome. Transmission electron microscopy (TEM) findings in skin samples of patients F1:II-1 and F2:II-1 and a matched control showing collagen (column 1), elastic fibers (column 2) and a fibroblast (column 3). col = collagen; ef = elastic fiber; fb = fibroblast. Magnification for column 1: ×15,000, column 2: ×8000 and column 3: ×4000.

#### *3.4. Collagen Biochemistry*

Considering the collagen abnormalities observed in both individuals with transmission electron microscopy, SDS-PAGE analysis of the intracellular and secreted (pro)collagen proteins produced by skin fibroblasts was performed for subjects 1 and 2 and a matched control. In both cases, we observed normal SDS-PAGE migration patterns (Supplementary Figure S2).

#### **4. Discussion and Conclusions**

The absence of large case series and natural history studies precludes efficient diagnosis and management of OHS patients. This study describes the clinical and molecular characteristics of two new patients and 32 patients previously reported in the literature. OHS is considered a mild but rare allelic variant of Menkes disease. OHS patients show prominent connective tissue abnormalities and have a better prognosis and survival rate than patients with MD [3], with low childhood mortality (1/34) in OHS. However, there is still significant mortality in (young) adults, often related to gastrointestinal, respiratory, or bleeding complications, such as an intestinal perforation due to a gastric ulcer and (post-surgery) apnea.

The clinical characteristics of OHS are listed in Table 2. Craniofacial features include mild facial dysmorphism with a long face, large ears and sagging cheeks in about half of reported patients. Hair abnormalities, including coarse hair and pili torti, are frequently present. Ubiquitous connective tissue manifestations in OHS include increased skin laxity and inguinal hernia.

Concerning the skeletal features, the pathognomonic occipital horns are present in all patients but one, which is probably an age-dependent discrepancy [41,54,55]. Other less specific skeletal abnormalities include hammer-shaped claviculae and radial/tibial exostoses. Scoliosis, pectus deformities, coxa and genua valga, joint luxations, mainly of the radial heads and general joint hypermobility are frequent but non-specific. These skeletal problems may limit daily activities and often require multiple surgical interventions.

Urological complications, mostly giant bladder diverticula and vesicourethal reflux, are found in over 80% of subjects. Secondary problems such urinary tract infections, urinary retention, bladder rupture, or VUR-mediated renal failure, which are all related to the bladder diverticula, are of great concern. Surgical interventions removing the diverticula often fail, with frequent relapse of the diverticula. Self-catheterization, vesicostomy, or continent diversion are often required for proper emptying of the bladder.

Previous reports describe a mild neurological phenotype limited to slight generalized muscle weakness and symptoms of dysautonomia [3,27], however, more detailed case studies reveal some conspicuous observations including developmental delay and intellectual disability in two-thirds of subjects and documented seizures in five individuals. Nevertheless, neurological affliction remains rather mild and normal intelligence is possible. Symptoms of dysautonomia, including chronic diarrhea, temperature instability, and orthostatic hypotension are present in almost 90% of patients and can be disabling.

Brain imaging studies are rarely performed in OHS patients and are often reported as normal. However, intracranial arterial tortuosity are noted in approximately two thirds of patients. Extracranial arterial tortuosity is less frequently observed, but has been reported in the cervical, splenic and hepatic vasculature. Aneurysm formation may complicate pre-existing tortuosity and may affect the arterial and venous circulation.

OHS is often diagnosed upon the identification of connective tissue anomalies, including cutis laxa, joint hypermobility with hypotonia, mild intellectual impairment and bladder diverticula. Later on, the pathognomonic occipital horns become more evident. The initial differential diagnosis includes other forms of cutis laxa, including *FBLN5*- and *LTBP4*-related cutis laxa (OMIM#219100 and #613177) [56] and the dermatosparaxis type of Ehlers–Danlos syndrome (EDS type VIIC, OMIM#225410) [57], which may present with cutis laxa, hypotonia and bladder diverticula, but with normal mental development. However, in OHS there is no early onset emphysema as in *FBLN5*- and *LTBP4*- related cutis laxa, nor easy bruising or severe skin fragility as in the dermatosparaxis type of EDS. *ATP6V0A2*-related cutis laxa (OMIM#219200) may also present with mild intellectual impairment but does not commonly show bladder diverticula or exostoses. Later on the exostoses can be mistaken for hereditary multiple exostoses due to defects in *EXT1* or *EXT2* (OMIM#133700 and #133701) [58].

Concerning follow-up, routine evaluation of neurodevelopment and early intervention with physiotherapy is recommended, as well as regular urological evaluation, including video urodynamic studies of bladder function. Any pain, or vascular or neurological symptoms that may relate to exostoses should be promptly investigated. To date, there are no data on a possible risk for chondrosarcomas in exostoses although these have been described in hereditary multiple exostoses [59]. It could be recommended to perform MR angiography and echocardiography from puberty onwards. Indeed, aortic root dilatation and dissection, and an increased risk for intracranial bleeds and ischemic complications have been described in other related connective tissue disorders such as Ehlers–Danlos, arterial tortuosity, Marfan and some cutis laxa syndromes. Furthermore, spirometry might detect asthma at an early stage as it was reported in four individuals. Prolonged vigilance for apnea is necessary following surgery.

The molecular data confirmed that pathogenic variants in *ATP7A* causing OHS mainly included splice-site variants, while missense and frameshift variants were less frequently observed. This observation may fit the hypothesis that a low amount of normal *ATP7A* mRNA and residual ATP7A functioning is sufficient to prevent the more severe MD [13,27,29,31,60]. No mutational hot spots were observed (Figure 3). Importantly, nearly all pathogenic variants are located in the endofacial domains and only two affect a transmembrane domain or cytosolic loop, respectively. This highlights the critical role of the transmembrane domains forming the channel for copper translocation [13]. Variants in OHS mainly affect residues implicated in ATPase-coupled copper transport. Some of these variants are predicted to inhibit the catalytic dephosphorylation leading to permanent distribution of the protein to the plasma membrane and cytosolic vesicles [14,15,21,61,62].

Our transmission electron microscopy data in skin samples of OHS individuals show larger diameters of collagen fibrils that were more loosely packed compared to those of the control sample. Elastic fibers were affected to a lesser extent but are fragmented and/or surrounded by extracellular matrix debris. OHS is associated with decreased activity of the copper dependent lysyl oxidase (LOX), that oxidates lysyl and hydroxylysyl residues in collagen and lysyl residues in elastin to aldehydes [7]. These aldehyde residues are than condensed to form cross-links, essential for collagen fibril stabilization and the integrity and elasticity of mature elastin [32,63]. Overall, this would not affect the amount or migration of soluble collagens from the medium or cellular fraction upon electrophoresis but will affect the assembly in the extracellular matrix. Also, lysyl oxidase may form cross-links with the N-propeptide domain of collagen type V, essential in collagen diameter regulation [64,65]. Fibroblasts synthesize only low amounts of type V collagen which is difficult to evaluate by electrophoresis [66,67]. Indeed, irregular fibril diameters are reminiscent of classic Ehlers–Danlos syndrome, despite the absence of cauliflower fibrils [68].

The observation of major fibroblast abnormalities, with multiple autophagolysosomes containing sickle-shaped electron dense deposits, is novel and might point to defective trafficking and trapping of proteins needing cuproenzyme-dependent processing in the post-Golgi network [14,15,21].

In conclusion, this study sheds light on the natural history of OHS by describing two new patients and 32 patients reported in the literature. Mortality in adulthood warrants long-term specialized care and follow-up, incorporating MR angiography, echocardiograpy and spirometry into standard follow-up guidelines for OHS patients, next to orthopedic and urological follow-up. Ultrastructural abnormalities including increased collagen diameter, mild elastic fiber abnormalities, and multiple autophagolysosomes reflect the role of LOX and defective ATP7A trafficking in the pathomechanism of OHS.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/10/7/528/s1; Supplemental Figure S1: Fibroblast abnormalities in OHS. Magnification: x4000, x8000 and x15000; Supplemental Figure S2: SDS-PAGE collagen analysis in patients F1:II-1 and F2:II-1 and a matched control. Panel A: medium procollagen fraction on fibroblasts in culture for three days. Panel B: cellular collagen fraction; Supplemental Table S1. Clinical characteristics of all patients. '"+": present, "-": absent, "M": male, "F": female, "y": year(s), "mo": month(s), "d":day(s), "CVA": cerebrovascular accident, "DIC": diffuse intravascular coagulation, "FTT": failure to thrive, "GER": gastro-esophageal reflux, "SC": subcutaneous, "UTI": urinary tract infection; Supplemental Table S2. Pathogenicity information for the ATP7A variants in this study. The pathogenicity classification used in this table is based on the Alamut®mutation analysis software, which uses the following prediction tools: Align GVGD (Huntsman Cancer Institute, University of Utah) combines the biophysical characteristics of amino acids and protein multiple sequence alignments to where missense substitutions in genes of interest fall from enriched deleterious to enriched neutral. SIFT predicts whether amino acid substitution affects protein function. The MutationTaster prediction program integrates information from different biomedical databases, evolutionary conservation, splice-site changes and loss of protein features. Protein conservation is based on MARRVEL®multiple protein alignment. The splice prediction predicts the chance of variants having

an impact on alternative splicing. Gnomad allele frequencies and Clinvar data can also be found. In the last column, CADD scores are depicted (Kircher et al., 2014). "NS": not specified, "NA": not applicable; Supplemental Table S3. Copper and ceruloplasmin levels in all patients.

**Author Contributions:** Conceptualization, A.B. and B.C.; Data curation, A.B., K.V.M., P.H., F.P., B.L., S.S. and B.C.; Formal analysis, K.V.M.; Funding acquisition, B.C.; Investigation, A.B., K.V.M., L.P., R.D.R., M.D.B., F.B., S.S. and B.C.; Methodology, A.B., L.P., R.D.R., S.S. and B.C.; Project administration, B.C.; Resources, K.V.M., R.D.R., P.H., F.P., B.L., S.S. and B.C.; Supervision, S.D.S., S.S. and B.C.; Validation, A.B., K.V.M. and B.C.; Visualization, A.B., L.P. and R.D.R.; Writing—original draft, A.B.; Writing—review and editing, A.B., K.V.M., L.P., R.D.R., M.D.B., F.B., P.H., F.P., B.L., S.D.S., S.S. and B.C.

**Funding:** This work is supported by the Special Research Fund, Flanders of Ghent University (grant 01N04516C to B.C.) and the European Academy of Dermatovenereology (EADV project PPRC-2018-50 to B.C.). B.C. and B.L. are senior clinical investigators of the Research Foundation Flanders.

**Acknowledgments:** We are thankful to all the families and patients for their interest and participation. We want to thank Zoë Malfait and Lisa Caboor for their technical support. The Center for Medical Genetics and Department of Dermatology of the Ghent University Hospital are members of the European Reference Network (ERN)-Skin. The Centers for Medical Genetics of both Ghent and Antwerp are members of VASCERN.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Characterization of Two Novel Intronic Variants A**ff**ecting** *Splicing* **in** *FBN1***-Related Disorders**

### **Carmela Fusco 1,\*, Silvia Morlino 2, Lucia Micale 1, Alessandro Ferraris 2, Paola Grammatico 2 and Marco Castori 1**

Received: 19 April 2019; Accepted: 7 June 2019; Published: 10 June 2019

**Abstract:** *FBN1* encodes fibrillin 1, a key structural component of the extracellular matrix, and its variants are associated with a wide range of hereditary connective tissues disorders, such as Marfan syndrome (MFS) and mitral valve–aorta–skeleton–skin (MASS) syndrome. Interpretations of the genomic data and possible genotype–phenotype correlations in *FBN1* are complicated by the high rate of intronic variants of unknown significance. Here, we report two unrelated individuals with the *FBN1* deep intronic variants c.6872-24T>A and c.7571-12T>A, clinically associated with MFS and MASS syndrome, respectively. The individual carrying the c.6872-24T>A variant is positive for aortic disease. Both individuals lacked ectopia lentis. In silico analysis and subsequent mRNA study by RT-PCR demonstrated the effect of the identified variant on the splicing process in both cases. The c.6872-24T>A and c.7571-12T>A variants generate the retention of intronic nucleotides and lead to the introduction of a premature stop codon. This study enlarges the mutation spectrum of *FBN1* and points out the importance of intronic sequence analysis and the need for integrative functional studies in *FBN1* diagnostics.

**Keywords:** fibrillin 1; Marfan syndrome; MASS syndrome; mRNA; splicing

#### **1. Introduction**

The *FBN1* gene is located in 15q21.1 and includes 65 exons which encode for fibrillin 1, a large glycoprotein constituted by 47 cysteine-rich epidermal growth factor (EGF)-like domains and seven motifs homologous to the binding protein for transforming growth factor beta (TGFβ) [1,2]. Fibrillin 1 is a major structural component of the extracellular matrix microfibrils and thus, gives stability and elasticity to many tissues [2]. Variants in *FBN1* cause a wide range of autosomal dominant heritable connective tissue disorders, including Marfan syndrome (MFS; OMIM 154700), mitral valve–aorta–skeleton–skin syndrome (MASS syndrome; OMIM 604308), Marfan lipodystrophy syndrome (OMIM 616914), isolated autosomal dominant ectopia lentis (OMIM 129600), Weill–Marchesani syndrome type 2 (OMIM 608328), acromicric dysplasia (OMIM 102370), geleophysic dysplasia type 2 (OMIM 614185), stiff skin syndrome (OMIM 184900), and autosomal dominant thoracic aortic aneurysms and dissections [3].

To date, over 1800 different *FBN1* germline variants have been identified so far (UMD-*FBN1*, http://www.umd.be/FBN1/) [4,5] and only a few genotype–phenotype correlations are available [6,7]. For example, nonsense, frameshift, and some splicing variants appear correlated with a more severe skin and skeletal phenotype, as compared to in-frame variants [8]. Other studies suggest that variants affecting or creating cysteine residues are more commonly associated with ectopia lentis, while *null*

alleles typically combine with an increased rate of aortic events in young age and thoracic deformities [7]. In the suspicion of a *FBN1*-related disorder, the identification of the causative variant is not only relevant for diagnosis confirmation and genetic counseling but is also increasingly useful for personalized medicine approaches, comprising preventive drug treatment and aortic surgery planning [9].

The introduction of next generation sequencing (NGS) technologies in molecular diagnostics improved turnaround time and costs but did not significantly affect the clinical interpretation of variants of uncertain significance, such as intronic nucleotide changes which potentially affect splicing. The burden associated with uncertain results related to these variants is still high in MFS and related disorders, as point variants possibly impacting FBN1 pre-mRNA splicing seem to account for ~10% of reported molecular findings in MFS [10]. In these cases, optimal clinical interpretation should integrate molecular findings with customized studies exploring variant effects at the transcriptional or translational levels.

Here, we report two unrelated individuals with MFS and MASS syndrome, respectively, which were associated with different intronic variants in *FBN1*. Characterization of the effects of these variants on mRNA allowed us to support their pathogenicity and, therefore, to use them for patient and family management.

#### **2. Materials and Methods**

All investigated subjects signed informed consent for molecular testing and research use of biological and related clinical data. Molecular testing on cDNA and mRNA in individual 1 and 2 were carried out within the routine clinical diagnostic activities of the Division of Medical Genetics of Foundation IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo (Italy). The results of this work are part of a larger research project approved by our local ethics committee (approval protocol no. GTB12001).

#### *2.1. Clinical Description: Individual 1*

This was a 27-year-old man, the second child of unrelated parents. His father was 50-year-old, 191 cm tall and affected by dilatation of the aortic root and ascending aorta, which required surgery at 45 years. The mother and sister were healthy, and 172 and 161 cm tall, respectively. The proband was born at term after an uneventful pregnancy. At birth, he presented respiratory distress, which needed intensive care support for a few days. The neonatal period was otherwise normal, as well as psychomotor development, scholarship, and mentation. At 11 years, progressive scoliosis required an orthopedic corset for some years. The patient suffered from recurrent spontaneous pneumothorax with four episodes between 12 and 14 years of age. For this, he successfully underwent pleurodesis at 14 years. At 18 years, the previous diagnosis of aortic disease in the father prompted full cardiologic assessment in the proband who showed dilatation of the aortic root and mitral valve prolapse. Since then, he was under preventive pharmacologic therapy. Current treatment includes losartan 50 mg/day (single dose) and nebivolol 50 mg/day (single dose), without significant side effects. The last heart ultrasound, at 27 years, showed a stable dilatation of the aortic root (48 mm; Z score 171.13 = 4.66 SD [11]) with normal ascending aorta (34 mm), minimal insufficiency of the aortic valve, prolapse of the anterior mitral leaflet with minimal insufficiency, and mild insufficiency of the tricuspid valve. Dilatation of the aortic root (48 × 43 mm) was also confirmed by angio-computer tomography. A total spine MRI at 23 years showed bilateral dural ectasias of the lumbar spine. A recent ophthalmologic exam excluded lens dislocation and revealed mild myopia (−2 diopters on the right and −1.5 diopters on the left).

Physical examination included height 196 cm, weight 72 kg, arm span 198 cm, arm span/height ratio 1.01 (normal), bilateral positive wrist sign, bilateral negative thumb sign, apparent enophthalmos, down slanting palpebral fissures, malar hypoplasia, high-arched palate, severe scoliosis, pectus carinatum, bilateral valgus deformity of the elbow, bilateral metatarsus varus, some mildly atrophic

post-surgical scars of the thorax, and striae rubrae of the back (Figure 1A–D). The combination of aortic root dilatation (Z-score > 2 SD) and 10 points of systemic features lead to the clinical suspect of MFS.

**Figure 1.** Clinical features. Individual 1 showing high-arched palate (**A**), pectus carinatum (**B**), scoliosis (**C**) and arachnodactyly (**D**). Lumbar spine MRI of Individual 2 demonstrating bilateral dural ectasias at axial (**E**), coronal (**F**), and sagittal (**G**) views. Asterisks indicate the dilated dural sac.

#### *2.2. Clinical Description: Individual 2*

Individual 2 was a 48-year-old, nullipara woman, the seventh child of a sibship of eleven. All brothers and sisters were healthy, but two brothers were described with tall stature (~194 cm). The mother was 165 cm tall and died at 65 years due to rupture of a cerebral aneurysm. The father was 175 tall and died at 78 years due to a stroke. Family history was negative for aortic disease. The proband was sent to clinical genetics assessment by the neurosurgeon, who first evaluated the woman for chronic back pain, diffuse spondylosis, and marked dural ectasias of the lumbar spine (Figure 1E–G). Relevant additional clinical features included moderately severe scoliosis since her teens, severe myopia (−6 diopters on the right and −8 dipters on the left), and chronic fatigue. A heart ultrasound repeatedly showed normal aortic diameters, prolapse, and mild insufficiency of the mitral valve. At 47 years, aortic root diameter was 36 mm (Z score 1.26 SD [11]). Routine ophthalmologic assessments always excluded lens dislocations. Physical examination showed height 181 cm, weight 80 kg, arm span 188 cm, arm span/height ratio 1.038 (normal), bilaterally positive wrist and thumb signs, hypermobility of fingers (but the Beighton score was 0/9), elbow limitation on both sides, enophthalmos, down slanting palpebral fissures, retrognathia, high-arched palate, striae distensae

of the shoulders. The presence of 10 points of systemic features and an aortic root diameter <2 SD suggested a diagnosis of the MASS syndrome. Positive family history for tall stature and absence of aortic disease in relatives were in accordance with the diagnosis. Molecular testing in Individual 2 was carried out with a multigene panel approach including extended intronic regions around exon–intron junctions (see below).

#### *2.3. Genomic DNA Extraction*

Genomic DNA was extracted from individuals' peripheral bloods by using Bio Robot EZ1 (Qiagen, Hilden, Germany), according to standard procedures. The DNA was quantified with Nanodrop 2000 C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.4. Genetic Testing (Sanger Sequencing): Individual 1*

Molecular analysis for individual 1 was performed by Sanger sequencing. Primers were designed using the Primer 3 Output program (http://frodo.wi.mit.edu/primer3/) to amplify extended intronic sequences and exon–intron flanking sequences of *FBN1* (RefSeq NM\_000138). Primers were checked both by BLAST and BLAT against the human genome to ensure specificity. Amplified products were subsequently purified and sequenced with a ready reaction kit (BigDye Terminator v1.1 Cycle, Applied Biosystems, Foster City, CA, USA). Fragments were then purified using DyeEx plates (Qiagen, Hilden, Germany) and resolved on an automated sequencer (3130xl Genetyc analyzer DNA Analyzer, ABI Prism, Foster City, CA, USA). Sequences were analyzed using the Sequencer software (Gene Codes, Ann Arbor, MI, USA). To investigate familial segregation, both parents were sequenced for the identified variant.

#### *2.5. Genetic Testing (Next-Generation Sequencing): Individual 2*

Individual 2's DNA underwent sequencing with a HaloPlex gene panel (Agilent Technologies, Santa Clara, CA, USA) designed to selectively capture known genes associated with syndromic and non-syndromic thoracic aneurysms and/or Mafanoid habitus, including: *ACTA2* (NM\_001141945), *BGN* (NM\_001711.5), *CBS* (NM\_000071.2), *COL3A1* (NM\_000090), *COL4A1* (NM\_001303110), *COL5A1* (NM\_000093), *COL5A2* (NM\_000393), *DLG4* (NM\_001365.4), *EFEMP2* (NM\_016938), *ELN* (NM\_000501), *EMILIN* (NM\_007046.3), *FBN1* (NM\_000138), *FBN2* (NM\_001999), *FLNA* (NM\_001110556), *FOXE3* (NM\_012186), *GATA5* (NM\_080473), *LOX* (NM\_002317), *MAT2A* (NM\_005911), *LTBP3* (NM\_001130144.2), *MAT2A* (NM\_005911.5), *MED12* (NM\_005120.2), *MFAP5* (NM\_003480), *MYH11* (NM\_001040113), *MYLK* (NM\_053025), *NOTCH1* (NM\_017617), *PLOD1* (NM\_001316320), *PRKG1* (NM\_006258), *SKI* (NM\_003036), *SLC2A10* (NM\_030777), *SMAD2* (NM\_005901.5), *SMAD3* (NM\_005902), SMAD4 (NM\_005359), *TAB2* (NM\_015093), *TGFB2* (NM\_001135599), *TGFB3* (NM\_003239), *TGFBR1* (NM\_001306210), *TGFBR2* (NM\_001024847), *UPF3B* (NM\_080632.2), and *ZDHHC9* (NM\_016032.3). Targeted fragments were then sequenced on a MiSeq platform (Illumina, San Diego, CA, USA) using a MiSeq Reagent kit V3 300 cycles flow cell. Data analysis was performed considering the frequency, impact on the encoded protein, conservation, and expression of variants using distinct tools, as appropriate ANNOVAR (http://annovar.openbioinformatics.org/en/latest/), dbSNP (https://www.ncbi.nlm.nih.gov/snp), 1000 Genomes (http://www.internationalgenome.org), ExAC (http://exac.broadinstitute.org). Selected variants are interpreted according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMGG/AMP) [12]. The identified variant was confirmed by Sanger sequencing. We checked the selected variant in the UMD-*FBN1* database, a computerized database that currently contains information about the published mutations of the *FBN1* gene.

#### *2.6. Variant Designation*

Nucleotide variant nomenclature follows the format indicated in the Human Genome Variation Society (HGVS, http://www.hgvs.org) recommendations. DNA variant numbering system refers to cDNA. Nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1.

#### *2.7. In Silico Prediction*

In silico analysis of identified intronic variants was conducted by running three independent algorithms for splice signal detection: NetGene2 (http://www.cbs.dtu.dk/services/NetGene2/), Berkeley Drosophila Genome Project (BDGP, http://www.fruitfly.org/seq\_tools/splice.html), and Human Splicing Finder (HSF, http://www.umd.be/HSF3/).

#### *2.8. RNA Extraction and Reverse Transcription–Polymerase Chain Reaction*

Total RNA was extracted from lymphocytes fraction isolated from peripheral blood. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany), treated with DNase-RNase free (Qiagen, Hilden, Germany), quantified by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), and reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Primers used for DNA amplification and reverse transcription–polymerase chain reaction are reported in Table 1.

**Table 1.** Primers used for DNA amplification reverse transcription of *FBN1* fragments carrying the investigated variants (RefSeq NM\_000138) in individual 1 and individual 2.

#### **3. Results**

#### *3.1. Individual 1*

Molecular testing was carried out on the Individual 1 DNA by Sanger sequencing of the *FBN1* gene, and this gave normal results. Multiple ligation-dependent probe amplification analysis for *FBN1* intragenic deletions/insertions resulted normal. Given the convincing clinical picture and positive family history, re-analyzing raw data from the Sanger sequence of extended intronic sequences was carried out and revealed a novel *FBN1* heterozygous splicing variant c.6872-24T>A, p.(?) (Figure 2A). Co-segregation with the disease was demonstrated on the father's DNA.

Computational predictions conducted using NetGene2, BDGP, and HSF revealed that the intronic c.6872-24T>A variant might influence the splicing process by differentially affecting canonical versus cryptic splice site utilization. The newly detected variant was predicted to insert 22 nucleotides (nt) of intron 56 into the mature mRNA (Figure 2B). This should give rise to frameshift and a PTC (p.(Asp2291Glyfs\*9)), leading to the loss of the TGFβ binding protein homologous motifs, the last seven cysteine-rich EGF-like domains, and the FibuCTDIII-like motif.

**Figure 2.** Molecular analyses of individual 1. (**A**) Sanger sequencing in an unaffected (unrelated) control (CTRL), the proband and his father of the *FBN1* region spanning the 56 to 57 exon–intron junction. The *FBN1* genomic variant is indicated by an arrow. (**B**) *S*chematic representation of *FBN1* region including 55 to 57 exons and introns. Rectangles represent exons; thin horizontal lines represent introns. The position of the identified variant is indicated with an asterisk. Wild type and abnormal transcripts generated by the c.6872-24T>A splicing variant are shown. (**C**) Total RNA of a normal (unrelated) control, the proband, and his father were used for RT-PCR of the *FBN1* transcript. The same amounts of patient's and control cDNA were PCR amplified. PCR products were analyzed by electrophoresis on 3.5% agarose gel. M, DNA marker; C, control; P, proband; F, father. Affected individuals (lane P and F) present both the wild-type mRNA (lower band) and aberrantly spliced transcript (upper band). (**D**) Sanger sequencing of the *FBN1* transcript, including exons 56 to 57 in an unaffected (unrelated) control (C), the proband (P), and his father (F). Variant position on mRNA level was annotated (r.6871\_6872ins6872-22\_6872-1). The intronic region was reported at the bottom of the Sanger chromatograms of all affected individuals.

The effect of the *FBN1* splice site variant c.6872-24T>A at the mRNA level was ascertained by in vitro RT-PCR amplification of *FBN1* RNA of Individual 1 and his father. The sequencing of the RT-PCR products spanning the 55 to 57 exons of *FBN1* showed the insertion of 22 nts of intron 56–57 in the *FBN1* mRNA of Individual 1 and his father (r.6871\_6872ins6872-22\_6872-1) (Figure 2C,D). Electropherogram showed a significant reduction of the peaks corresponding to the mutated allele. The aberrantly spliced transcript is not present in the expressed sequence tag database (EST, https://www.ncbi.nlm.nih.gov/dbEST/index.html) of *FBN1*. Following available standards for the interpretation of sequence variants [12], the *FBN1* c.6872-24T>A variant was classified as likely pathogenic and submitted to the Leiden Open Variation Database (LOVD, https://www.lovd.nl; accession number: # 00229824).

#### *3.2. Individual 2*

Targeted NGS analysis revealed the *FBN1* heterozygous c.7571-12T>A variant in intron 61 (Figure 3A). No other candidate variant was found in the remaining genes. The c.7571-12T>A variant was not reported in the UMD-*FBN1* and was absent in the proband's healthy sister. Both parents died, and the other brothers and sisters were not available for genetic testing.

**Figure 3.** Molecular analyses of individual 2. (**A**) Sanger sequencing in an unaffected (unrelated) control and the proband of the *FBN1* region spanning 61 to 62 exon–intron junction. The *FBN1* genomic variant is indicated by an arrow. (**B**) *S*chematic representation of *FBN1* region including 61 to 62 exons and introns. Rectangles represent exons; thin horizontal lines represent introns. The position of the identified variant is indicated with an asterisk. Abnormal transcripts generated by the c.7571-12T>A splicing variant. (**C**) Total RNA of a normal (unrelated) control and the proband were used for RT-PCR of the *FBN1* transcript. The same amounts of patient and control cDNA were PCR amplified. PCR products were analyzed by electrophoresis on 3.5% agarose gel. M, DNA marker; C, control; P, the proband. M, DNA marker; C, control; P, proband; F, father. Affected individual (lane P) presents both wild-type (lower band) and aberrant transcript (upper band). (**D**) Sanger sequencing of the *FBN1* transcript, including exons 61 to 62 in an unaffected (unrelated) control (C) and the proband (P). Variant position on mRNA level was annotated (r.7570\_7571ins7571-10\_7571-1). The intronic region was reported at the bottom of the Sanger chromatogram of the affected individual.

The splicing predictor tools showed that the variant might alter the splicing process. In particular, all predicted with high probability that the c.7571-12T>A intronic variant activates a cryptic acceptor splice site within intron 61 that, in turn, leads to an inefficient recognition of the canonical splice site (Figure 3B) and insertion of intronic nucleotides in the *FBN1* mRNA. Sequence analysis on cDNA confirmed the c.7571-12T>A variant causes a shift from canonical toward cryptic splicing that leads to the inclusion of 10 nts of the 61 to 62 intronic sequence (r.7570\_7571ins7571-10\_7571-1) (Figure 3C,D) and a frameshift which generate a PTC in exon 61 (p.(Asp2524Argfs\*5)). The truncated protein loses the last seven cysteine-rich EGF-like domains and the FibuCTDIII-like motif. Electropherogram showed a reduction of the peaks corresponding to the mutated allele. The sequence of the alternative mRNA was absent in the EST database. The *FBN1* c.7571-12T>A variant was therefore interpreted as likely pathogenic and submitted to LOVD (accession number: #00229832).

#### **4. Discussion**

In this work, we added two novel intronic variants to the *FBN1* mutational repertoire and demonstrated their effect at the mRNA level. Our findings highlight the impact of variants affecting non-coding DNA in MFS and related disorders, and the opportunity to re-consider the standard diagnostic approaches, at least, in specific cases. In particular, molecular studies revealed that the novel c.6872-24T>A and c.7571-12T>A *FBN1* variants induce aberrant pre-mRNA splicing by the generation of a new cryptic acceptor site that outcompetes the canonical splice site and generate exonization of intronic sequences. This, in turn, results in a frameshift with the introduction of a PTC. In both cases, PTC is predicted to produce a truncated fibrillin 1 with a loss of critical C-terminal domains of fibrillin 1 [13].

The mechanism(s) by which *FBN1* splicing variants might exert their pathogenic effect is (are) complex, and both dominant negative and loss-of-function effects have been proposed based on the predicted consequences on mRNA, and protein structure and function(s) [14–16]. In splicing variants generating out-of-frame deletions/insertions, the introduction of a PTC and the synthesis of a truncated protein are assumed. However, PTC might elicit nonsense-mediated mRNA decay (NMD), with a variable proportion of the mutated allele acting as a *null* allele. If NMD affects ~100% of the transcripts, haploinsufficiency is the leading molecular pathogenesis. Conversely, the production of a truncated fibrillin 1 due to complete NMD escape, can impact its function due to truncated protein production. In proteins with pleiotropic effects and distinct domains with different functions, such as fibrillin 1 [3], the length of the synthesized truncated protein and the differential expression of the gene in the different tissues are the leading modulator factors of its residual functions. A mixed pathogenesis should also be considered for PTC that allow only in part NMD. In splicing variants leading to in-frame deletions/insertions, the predicted consequence might mirror the dominant negative effect attributed to point variants [17].

 For example, Wang et al., by reporting a small case series of familial aortic aneurysms and dissection, studied the effect of the *FBN1* c.5917+6T>C variant in HEK293 cells and concluded that skipping of exon 47 seems to associate with a higher rate of cardiovascular involvement [18]. Wypasek and coworkers reported an adult with MFS, severe cardiac involvement but normally placed lens, and the *FBN1* c.1589-9T>A affecting splicing by in silico and in vitro investigations [19]. Recently, a further MFS patient with full cardiac, ocular, and skeletal phenotype has been reported with the *FBN1* c.2678-15C>A variant affecting splicing by minigene approaches [20].

In our study, both variants are predicted to result in a truncated protein. Although we did not further investigate their effect, both transcripts are likely destined, at least in part, to NMD, as the PTC falls in more than 50 nts upstream of the last exon–exon junction within the mRNA [21]. This might be hypothesized by the differences in peak heights in the electropherograms of *FBN1* transcripts (Figures 2 and 3). This result suggests that a portion of aberrantly *FBN1*-spliced transcripts could be degraded by NMD process in both cases. *FBN1* haploinsufficiency due to NMD-mediated degradation of aberrant mRNA might be the leading candidate molecular mechanism causing disease for the novel c.6872-24T>A and c.7571-12T>A variants. However, we cannot exclude that a proportion of the mutated transcripts escapes NMD at least in blood cells. Therefore, both NMD and the synthesis of a truncated protein may result from the identified causative variants. In Individual 1 the predicted truncated protein (p.(Asp2291Glyfs\*9)) losses the seven TGFβ binding protein homologous motifs, the last seven cysteine-rich EGF-like domains, and the FibuCTDIII-like motif; while in Individual 2, protein truncation should occur later (p.(Asp2524Argfs\*5)) preserving the loss of TGFβ binding protein homologous motifs. Individual 1 (and his affected father) showed severe aortic disease, a feature that was not observed in Individual 2, and both individuals lack ectopia lentis.

The reasons underlying the different clinical diagnoses in these two patients are hard to define. We can speculate that in Individual 1 aortic involvement might be, at least in part, due to the loss of TGFβ binding domain, which, in turn, is preserved in the truncated protein predicted in Individual 2 (MASS syndrome), who showed normal aorta. In fact, perturbed binding with TGFβ results in the alteration of elasticity and microfibrils stability, two factors primary influencing aortic wall integrity [22]. A trend toward an increased risk for ectopia lentis was reported in cysteine-destroying or cysteine-creating variants [2,15], a mechanism not easily evoked in our two Individuals. Therefore, our observation testifies once more the difficulties in tracing consistent genotype–phenotype correlations in MFS and the need for international collaborations working towards this aim.

In this work, we highlighted the importance of including extended intronic regions in the analysis of *FBN1* for clinical purposes. This remains valid in the era of NGS and should be considered at the stage of (virtual) multigene panel design, as well as during variant prioritization and post-analytical phase. Such an approach also needs the availability of additional tests validated for diagnostics and able to explore variant effects at the mRNA or post-transcriptional levels for supporting the clinical interpretation of the genomic data. Therefore, this work points out the need for introducing functional investigations in the diagnostic workflow of at least selected disorders or gene panels, for which a high rate of intronic variants with a potential pathogenic effect is expected. The description of further individuals with splicing variants with documented effects at the various post-genomic levels seems critical for improving our knowledge on this issue.

**Author Contributions:** Formal analysis, C.F.; Funding acquisition, L.M.; Investigation, C.F. and L.M.; Resources, S.M., A.F. and P.G.; Supervision, M.C.; Writing—original draft, C.F., P.G. and M.C.; Writing—review and editing, C.F., S.M., L.M., A.F., P.G. and M.C.

**Funding:** This work was supported by the Ricerca Corrente 2018–2021 Program from the Italian Ministry of Health.

**Acknowledgments:** The authors thank the families for their kind availability in sharing the findings within the scientific community The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

**Conflicts of Interest:** All authors declare no conflict of interest concerning this work.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Genotypic Categorization of Loeys-Dietz Syndrome Based on 24 Novel Families and Literature Data**

**Letizia Camerota 1,**†**, Marco Ritelli 2,**†**, Anita Wischmeijer 3, Silvia Majore 4,5, Valeria Cinquina 2, Paola Fortugno 6, Rosanna Monetta 1,6, Laura Gigante 7,8,**

**Marfan Syndrome Study Group Tor Vergata University Hospital** ‡**, Federica Carla Sangiuolo 7,8, Giuseppe Novelli 7,8,9, Marina Colombi 2,\* and Francesco Brancati 1,6,\***

Received: 27 August 2019; Accepted: 21 September 2019; Published: 28 September 2019

**Abstract:** Loeys-Dietz syndrome (LDS) is a connective tissue disorder first described in 2005 featuring aortic/arterial aneurysms, dissections, and tortuosity associated with craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations. Heterozygous mutations in 6 genes (*TGFBR1*/*2, TGFB2*/*3, SMAD2*/*3*), encoding components of the TGF-β pathway, cause LDS. Such genetic heterogeneity mirrors broad phenotypic variability with significant differences, especially in terms of the age of onset, penetrance, and severity of life-threatening vascular manifestations and multiorgan involvement, indicating the need to obtain genotype-to-phenotype correlations for personalized management and counseling. Herein, we report on a cohort of 34 LDS patients from 24 families all receiving a molecular diagnosis. Fifteen variants were novel, affecting the *TGFBR1* (6), *TGFBR2* (6), *SMAD3* (2), and *TGFB2* (1) genes. Clinical features were scored for each distinct gene and matched with literature data to strengthen genotype-phenotype correlations such as more severe vascular manifestations in *TGFBR1*/*2*-related LDS. Additional features included spontaneous pneumothorax in *SMAD3*-related LDS and cervical spine instability in *TGFB2*-related LDS. Our study broadens the clinical and molecular spectrum of LDS and indicates that a phenotypic continuum emerges

as more patients are described, although genotype-phenotype correlations may still contribute to clinical management.

**Keywords:** hereditary connective tissue disorders; Loeys-Dietz syndrome; Ehlers-Danlos syndrome; arterial aneurysms; *TGFBR1*; *TGFBR2*; *SMAD2*; *SMAD3*; *TGFB2*; *TGFB3*

#### **1. Introduction**

Loeys–Dietz syndrome (LDS) is a rare hereditary connective tissue disorder (HCTD) with an autosomal dominant inheritance characterized by a widespread systemic involvement. The disorder was first described in 2005 and in its most typical presentation it features aortic/arterial aneurysms and/or dissections, as well as arterial tortuosity in association with variable craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations [1,2]. LDS is caused by pathogenic variants in transforming growth factor β (TGF-β) signaling pathway-related genes, i.e., *TGFBR1*, *TGFBR2*, *SMAD2*, *SMAD3*, *TGFB2,* and *TGFB3*, which alter the physiological development and function of the extracellular matrix, leading to cardiovascular and multisystem abnormalities [3]. Mutations in the genes encoding the TGF-β receptor subunits (*TGFBR1* and *TGFBR2*) were firstly identified and an initial clinical classification of LDS type I and type II was proposed based on the presence of typical craniofacial features in LDS type I [2]. Over the years, the discovery of novel disease-causative genes, eased by the advent of next-generation sequencing techniques, paved the way for novel genotype-phenotype correlations. For example, the identification of a subset of LDS patients with pathogenic variants in *SMAD3* displaying high frequency of osteoarthritis, prompted some authors to define the resulting phenotype as "aneurysms-osteoarthritis syndrome" or LDS type III [4]. McCarrick and coworkers argued that this phenotype fits within the LDS spectrum based on the analysis of large cohorts of mutated patients [5]. In particular, the authors concluded that, in the absence of formal diagnostic criteria for LDS, a pathogenic variant in any of the six LDS disease-causative genes in combination with the presence of arterial aneurysm or dissection or a positive family history should be considered sufficient for the diagnosis of LDS [5]. This stimulated the study of genotype-phenotype correlations, which are of paramount value for correct management of patients and to plan appropriate prevention programs in clinical practice. Even if wide interand intrafamilial variability in the distribution and severity of clinical features is observed, a more accurate gene-based categorization of LDS may have a great clinical impact in early diagnosis and management and guide treatment strategies for patients and family members with this life-threatening condition. Of note, the diagnosis of patients with LDS prompting molecular testing is not always straightforward, since the spectrum of clinical manifestations is broad and often overlaps other HCTDs, including Marfan syndrome (MFS), Shprintzen-Goldberg syndrome, some types of cutis laxa, Ehlers-Danlos syndromes (EDS) (particularly the vascular type), arterial tortuosity syndrome, congenital contractual arachnodactyly, and biglycan (*BGN*)-associated aortic aneurysm syndrome. Additionally, in some cases further genetic testing may be indicated for the differential diagnosis with other hereditary thoracic aortic disorders caused by pathogenic variants in distinct genes including *NOTCH1*, *ACTA2*, *MYH11*, *MYLK*, *PRKG1*, *MAT2A*, *FOXE3*, *MFAP5*, and *LOX* [6]. Herein, we describe 34 patients from 24 families with LDS in which we identified the genetic defect in one of the known genes; 15 variants were novel, expanding the mutational repertoire. We further strengthened genotype-phenotype correlations by fitting our data with available studies in literature and updated the gene-to-phenotype categorization of LDS.

#### **2. Patients and Methods**

#### *2.1. Patients*

Twenty-four index patients and 10 relatives with LDS, mostly of Italian origin but two from Philippines and Sri Lanka, were evaluated from 2010 to 2018 in specialized outpatient clinics for the diagnosis of HCTDs, namely: (*i*) the Ehlers-Danlos Syndrome and Inherited Connective Tissue Disorders Clinic at the University Hospital Spedali Civili of Brescia, (*ii*) the Medical Genetics Unit of the Sant'Orsola-Malpighi Hospital of Bologna, (*iii*) the Clinical Genetics Unit of the Regional Hospital of Bolzano, (*iv*) the Centre of Expertise for Marfan syndrome and Marfan-related disorders at Policlinico Tor Vergata University Hospital of Rome and (*v*) the Medical Genetics Unit, Department of Life, Health, and Environmental Sciences of the University of L'Aquila.

#### *2.2. Molecular Investigations*

Molecular analyses were performed in the laboratory of genetic testing at the Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia. Mutational screening was achieved on genomic DNA purified from peripheral blood leukocytes of affected and unaffected family members by standard procedures. All exons and their intron-flanking regions of *TGFBR1* (NM\_004612.4, NP\_004603.1), *TGFBR2* (NM\_003242.5, NP\_0033233.4), *SMAD*3 (NM\_003242.5, NP\_005893.1), and *TGFB2* (NM\_001135599.3, NP\_001129071.1) were PCR amplified by using optimized genomic primers (Supplementary Table S1, primers were designed by using the Primer Express software v. 3.0.1 (Thermo Fisher Scientific, South San Francisco, CA, USA) and purchased by Metabion International AG, Planneg, Germany), which were analyzed for the absence of known variants using the GnomAD database [7]. After enzymatic cleanup of the PCR products, all fragments were sequenced in both orientations using the Big Dye Terminator Cycle Sequencing kit protocol (Thermo Fisher Scientific, South San Francisco, CA, USA) followed by capillary electrophoresis on the ABI3130XL Genetic analyzer. The sequences were analyzed with the Sequencher 5.0 software (Gene Codes Corporation, Ann Arbor, MI, USA) and variants were annotated according to the Human Genome Variation Society (HGVS) nomenclature with the Alamut Visual software version 2.11 (Interactive Biosoftware, Rouen, France), which was also used for splice site prediction, since it includes four different prediction algorithms: SpliceSiteFinder-like, MaxEntScan, NNSplice, and GeneSplicer. When available, fresh blood samples were collected in patients carrying variants affecting canonical splice sites. In order to verify the effect on splicing, RT-PCR was carried out on total RNA extracted from patients' whole blood stabilized in Paxgene tubes following the manufacturer's protocol by using the Paxgene Blood RNA Extraction Kit (PreAnalytiX, Qiagen, Hilden, Germany). In particular, amplification of cDNA covering exons 2–4 of *TGFBR2* (TGFBRex2-forw: 5 -GTGGCTGTATGGTAAGAGA-3 and TGFBR2ex4-rev: 5 -CCAGGTTGAACTCAGCTTCTG-3 ) and exons 6-8 of *SMAD3* (SMAD3ex6-forw: 5 -CCTAGGGCTGCTCTCCAATG-3 and SMAD3ex8-rev: 5 -GTGCACATTCGGGTCAACTG-3 ) was performed, respectively, and followed by Sanger sequencing of the RT-PCR products. All identified variants were submitted to the Leiden Open Variation Database (LOVD) [8].

#### *2.3. Genotype-Phenotype Analysis and Literature Review*

The medical records of 34 patients with a molecularly proven diagnosis of LDS were accessed and their phenotypes were defined using the Human Phenotype Ontology (HPO) terms by a careful review of clinical notes. Phenotypic categories were created based on the number of observations in the patient's cohort and their relationship to LDS. In reporting the frequency of clinical features of our case series, the used denominator refers to the number of subjects for which information on a specific feature was available (Table 1). We reviewed the medical literature on the clinical manifestations of patients with a confirmed genetic diagnosis of LDS, i.e., patients from large cohorts harboring mutations in one of the six known disease-causative genes. The primary search was completed in the PubMed database and limited to articles in the English language literature without restrictions on the date of publication. Keywords used were "Loeys-Dietz syndrome" AND "genotype", "Loeys-Dietz syndrome" AND "phenotype" as well as "Loeys-Dietz syndrome" AND "review." A secondary search was performed to identify pertinent articles cited in those selected in the primary search. We tried to identify all papers for which genotype-phenotype correlation data were available, focusing on reviews and large cohort studies.

#### *2.4. Ethical Compliance*

This study was approved by the relevant Ethical Authorities of the: Policlinico Tor Vergata University Hospital (Progetto di Ricerca PGR00229, R.S. 204/16); University of L'Aquila, Medical Genetics Section, Department of Life, Health, and Environmental Sciences (PGR00229, prot. 20251); and local Ethical Committees. This study was performed in agreement with the principles of the 1964 Helsinki Declaration. All subjects (or their legally authorized representative) enrolled into the genetic study provided written informed consent.

**Table 1.** Comparison of the clinical features observed in previously reported and LDS patients described in this cohort for each distinct

gene.

#### **3. Results**

#### *3.1. Demographic Data and Genotype-Phenotype Analysis of LDS Patient's Cohort*

We report the clinical and genetic features of 34 individuals from 24 families. The female-to-male ratio of affected probands was 1/1. The mean age at diagnosis of the 24 unrelated probands was 26 years (range 1–51 years). Thirteen of them had a positive family history, while 11 were sporadic. Ten individuals were diagnosed upon familial segregation. The detailed clinical and molecular features of each affected individual are summarized in Supplementary Table S2. The overall frequency of selected clinical features observed in our cohort, categorized for each distinct gene as compared to in the literature, is shown in Table 1. An overview of characteristic features observed in the patients of our cohort is presented in Figure 1.

**Figure 1.** Clinical and instrumental findings observed in our Loeys-Dietz syndrome (LDS) patients.

(**a**–**c**) Proband of family 1 (p.Asp400Gly,*TGFBR1*), aged 7 years manifesting dolichocephaly (**a**), milia (**b**) and enamel defect of permanent dentition. (**d**,**e**) Family 4 (p.Gly271Asp, *TGFBR1*), proband (**d**) and his brother (**e**) outlining intrafamilial variability of palpebral fissures, horizontal in the proband (**d**) and downslanted in his brother (**e**) in the presence of severe milia in both. (**f**) CT-angiography of the abdomen in the proband of family 7 (p.Gly353Arg, *TGFBR1*), aged 23 years, showing aneurysm of the abdominal aorta (asterisk) with intimal blister flap (arrow head). (**g**) Proband of family 15 (p.Asp522Asn, *TGFBR2*), aged 36 years: CT-angiography of head and neck showing fusiform aneurysm of the left subclavian artery (asterisk), middle cerebral artery stenosis (arrow heads), diffuse tortuosity of vertebral arteries (area within the circle). (**h**–**k**) Proband of family 16 (p.Asp446Asn, *TGFBR2*), aged 7 years, displays asymmetry of iliac crest, thin and translucent skin with highly visible subcutaneous venous reticulum (**h**); Limited function of the feet after corrective surgery for bilateral clubfoot (**i**); hind foot deformity with bilateral pronated valgus pes planus (**j**); X-ray frontal view performed at 6 years shows the displaced heart in the left thorax due to severe pectus excavatum and scoliosis with lumbar left rotation and asymmetric iliac wings (**k**). (**l**–**n**), Proband of family 24 (p.Phe160Leufs\*14, *TGFB2*), aged 48 years featuring facial asymmetry and dysmorphism (highly arched eyebrows, hypertelorism, bilateral exophthalmos, bifid nasal tip, long philtrum with thin upper lip, micrognathia) in addition to premature aging appearance (**l**); Joint laxity of the thumb (**m**) and the knee (**n**); Note the thin and translucent skin with visible subcutaneous veins.

#### *3.2. Molecular Findings*

In this study, 24 different variants in four distinct LDS-related genes were identified in the proband of each family by direct sequencing (Table 2). All variants were deposited in LOVD. The majority of variants affected the *TGFBR1* (9/24) and *TGFBR2* (10/24) genes, four families showed variants in *SMAD3*, and 1 patient harbored a *TGFB2* variant. No variants were identified in *SMAD2* and *TGFB3*. Fifteen variants affecting *TGFBR1*, *TGFBR2*, *SMAD3*, and *TGFB2* were novel, while nine in *TGFBR1*, *TGFBR2*, *SMAD3* were previously reported either in the literature or in the ClinVar database (Supplementary Table S2 and Table 2).

Among novel variants, 10 missense substitutions and one in-frame deletion of two highly conserved amino acid residues p.(Lys232\_Ile233del) were evaluated for their putative pathogenicity through different *in silico* prediction algorithms, all predicted them as being high impacting variants. Given that they are: (i) located in critical and well-established functional domains without benign variation; (ii) absent in publicly available population databases, (iii) predicted as deleterious my multiple lines of computational evidence and based on the matching of the clinical phenotypes, all these variants were classified as pathogenic (class 5) according to the American College of Medical Genetics and Genomics (ACMG) guidelines [9].

Likewise, the novel c.480del variant in *TGFB2* was classified as pathogenic (class 5), since it leads to frameshift and formation of a premature termination codon p.(Phe160Leufs\*14) that likely activates nonsense-mediated mRNA decay (NMD).

For two out of three variants affecting splice sites, we obtained fresh blood samples and demonstrated an effect on the splicing process, corroborating their pathogenicity. In particular, the c.263+6C>T variant in *TGFBR2* (rs758501054, Minor allele frequency in ExAC, MAF = 0.000008) was classified as likely pathogenic (class 4) according to the ACMG, since it creates a new splice donor site 6 bases downstream of the wild-type donor with retention of 4 nucleotides of intron 3, formation of a stop codon (p.Arg114\*) and activation of NMD. The c.1009+1G>A variant in *SMAD3* was classified as pathogenic (class 5), since it abolishes the canonical splice donor site of exon 7 causing in-frame exon skipping (p.Arg292\_Gly337del). Lastly, although RNA was not available to investigate its effect, the c.862\_871+8del pathogenic variant (class 5) variant in *SMAD3,* thought formally a frameshift variant, likely leads to abnormal splicing as well (i.e., in-frame exon 6 skipping), as the canonical splice donor site is lost.

#### **4. Discussion**

Since the relatively recent description of LDS in 2005, six disease-causative genes have been identified, namely *TGFBR1* (MIM #190181) [1], *TGFBR2* (MIM#190182) [1], *SMAD2* (MIM #601366) [10], *SMAD3* (MIM #603109) [4], *TGFB2* (MIM #190220) [11], and *TGFB3* (MIM#190230) [12]. This advised the subdivision of LDS into multiple classes based on the causative gene (LDS1-5) providing a general indication of the spectrum of disease severity, from most to least severe form: LDS1=LDS2>LDS3>LDS4>LDS5 [6]. Still, there are not enough data on LDS caused by heterozygous mutations in *SMAD2* to place this form (LDS6) in this spectrum [13]. Recently, it has been suggested that mutations in genes different from *TGFBR1*/2 may give rise to a phenotypic continuum hard to categorize in clear-cut genotype-phenotype correlations [3,6]. In this study, we performed a retrospective multi-center study on clinical and mutational analyses in 24 novel LDS families with 34 patients. Furthermore, a systematic overview of LDS patient's features for each mutated gene was conducted to verify if observed genotype-phenotype correlations fitted current knowledge.

Overall, we noticed significant overlaps for *TGFBR1*/*2*, *SMAD3,* and *TGFB2* patients (Table 1), while emerging genotype-phenotype correlations are described below, subdivided for each gene.

#### *4.1. TGFBR1*/*2 Genes*

According to previous studies, most of the identified mutations affected *TGFBR1* (9/24) and *TGFBR2* (10/24) genes. While nearly all mutated patients displayed ascending/aortic root aneurysm, severe arterial involvement was recorded in half. Early aortic dissection was more rarely reported together with arterial tortuosity. Interestingly, we noticed a high number of different arteries being affected. These findings highlight the need for extended arterial imaging, as was already recommended [5]. Valve abnormalities, in particular mitral valve prolapse and insufficiency, affected nearly half of the patients; isolated mitral valve prolapse was also present in seven out of 10 *TGFBR2* patients. As previously described, craniofacial features may be considered to be characteristics of *TGFBR1*/*2*-LDS including dolichocephaly, hypertelorism, malar hypoplasia and highly arched palate, while abnormal palate/bifid uvula was not as frequently found as described in the literature. Recurrent skeletal anomalies included scoliosis, pes planus, long slender fingers, marfanoid habitus, and pectus deformity. Hernias were of different types (diaphragmatic, inguinal, or umbilical) and together with joint laxity were registered in half of patients. Cutaneous findings appeared extremely frequent including translucent skin, easy bruising and striae; together with facial milia, described in half mutated patients, skin features appeared as extremely useful handle for diagnosis [14]. Overall, our data confirm broad overlap between LDS phenotypes caused by *TGFBR1*/*2* pathogenic variants, as previously assessed [6]. Also, in four families the presence of significant craniofacial malformations correlated with a higher risk of vascular manifestations [5,6]. Finally, two children had tooth abnormalities, in particular enamel defects, a possibly underestimated features in LDS in line with the role of TGF-β signaling in dental and enamel formation [15].

#### *4.2. SMAD3 Gene*

Among our three *SMAD3*-mutated families, early osteoarthritis, considered a hallmark of this subset of patients, was described only in one, although it segregated in the proband and his father. In line with our data, despite early studies reported osteoarthritis in all *SMAD3* patients [4], others noticed a lower incidence [16]. Interestingly, this family presented very mild cardiovascular anomalies, with only mitral valve prolapse registered in the proband. Generally, *SMAD3*-LDS patients displayed a less severe degree of cardiovascular complications. In fact, despite all the presence of thoracic aortic (and renal arteries) aneurysms, dissection was reported only once, as well as arterial tortuosity and varices. Additional clinical findings overlapped with those recorded in other LDS patients, including recurrent craniofacial dysmorphism such as hypertelorism, malar hypoplasia, micrognathia and highly arched palate, cleft palate and/or uvula, cutaneous features with translucent skin, striae and facial milia,

hernias and skeletal manifestation like pes planus, pectus deformity, marfanoid habitus, and scoliosis. It is worth noting that one subject had spontaneous pneumothorax, recorded in a single *SMAD3* patient in the literature [3]. The causes of spontaneous pneumothorax in LDS have not been clearly elucidated but its association with LDS independently from the genetic subtype as well as the occurrence in other HCTD such as Marfan syndrome argues in favor of a common role for TGF-beta signaling. Several lines of evidence based on mouse models, knockout for genes encoding members of the TGF-beta pathway, support this observation (reviewed in reference [17]). In particular, perturbed TGF-beta signaling was implicated in abnormal pulmonary alveolarization and alveolar destruction, leading with age to emphysema and spontaneous lung rupture (i.e., primary spontaneous pneumothorax).

#### *4.3. TGFB2 Gene*

We identified only one patient with a *TGFB2* pathogenic variant, whose cardiovascular features consisted in mitral valve prolapse and insufficiency, mild arterial tortuosity and varicose veins, in the absence of aneurysms. Additional features included bifid uvula, striae, translucent and hyperextensible skin, facial milia, hernias, joint hypermobility, pectus deformity, scoliosis, and pes planus observed in other LDS genetic subtypes. In line with previous observations, this patient presented the mildest end of LDS phenotypic spectrum [18]. However, it should be noted that phenotypic variability ranging from mild to severe expression was also recorded in *TGFB2*-LDS [19]. Further broadening the clinical picture of this genetic subtype, she also showed cervical spine instability, which was not previously registered in association with *TGFB2*, although it has been commonly encountered in LDS [3]. Interestingly, in Tgfb2 heterozygous knockout mice severe skeletal defects are also observed including skull base and vertebral malformations, in line with the human phenotype [20]. These data corroborate the importance to investigate and diagnose malformation of cervical spine and/or instability, which should be carefully monitored to prevent severe complications in these patients.

#### *4.4. Other Genes*

No mutations were identified in the latest described LDS genes, namely *SMAD2* and *TGFB3*. Still, limited numbers are available in literature about genotype-phenotype correlation. To date, 9 (likely) pathogenic variants in *SMAD2* have now been described in 15 subjects displaying a broad range of features, including aneurysms, tortuosity of the entire arterial tree, and coronary artery dissections, even in the absence of prominent connective tissue characteristics [13]. Concerning *TGFB3*, 15 different variants were reported in 56 individuals presenting with phenotypic overlap between LDS and MFS [12].

#### **5. Conclusions**

In this work, we broadened the clinical and molecular spectrum of LDS, corroborated, and expanded previously delineated genotype-phenotype correlations, paving the way for a gene-based classification of different disease subtypes. Larger cohort screenings are needed to unravel the thorough clinical and molecular repertoires in LDS to accurately establish diagnostic criteria, define genotype-phenotype correlations, and collect natural history data for clinical prognostication.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/10/10/764/s1, Table S1: Primers, Table S2: Clinical and molecular features of LDS patients described in this cohort.

**Author Contributions:** All authors fulfill the criteria for authorship. F.B. and M.C. conceived the study and wrote the manuscript drafted by L.C. and M.R. who organized data contents, reviewed the literature and performed the tables; F.B. and L.C. prepared the figure; F.B., L.C. and L.G. performed clinical evaluation and follow-up of families 1, 2, 3, 4, 5, 10, 20; F.C.S. and G.N. performed genetic counseling and reviewed molecular data for families 1, 2, 3, 4, 5, 10, 20; A.W. performed clinical evaluation, follow-up and genetic counseling for families 6, 8, 16, 17, 18, 19, 21, 22, 23; S.M. performed clinical evaluation and follow-up of families 7 and 15; M.C. and M.R. performed genetic counseling and follow-up for families 9,11, 12, 13, 14, 22, 24; M.C., M.R. and V.C. carried out molecular investigations and RNA studies; R.M. and P.F. reviewed, analyzed and interpreted molecular data; L.C. submitted the study. All authors discussed, read, and approved the final manuscript.

**Funding:** F.B. receives funding from the University of L'Aquila (Fondi RIA 2019 and Fondi Ricerca Premiale 2017), the Italian Ministry of Health (Ricerca Finalizzata GR-2013-02356227) and the Undiagnosed Disease Network Italy established at Istituto Superiore di Sanità, Italy (Farmindustria, contributo incondizionato FAC. X7E).

**Acknowledgments:** The authors wish to thank all patients for their cooperation during the diagnostic process. M.R, V.C., and M.C. thank the Fazzo Cusan family for its generous support. We thank Domenica Taruscio and Marco Salvatore from the Istituto Superiore di Sanità (Italy) for their constant support.

**Conflicts of Interest:** All authors declare that there is no conflict of interest concerning this work.

#### **References**

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