Mutations in HYAL2, Encoding Hyaluronidase 2, Cause a Syndrome of Orofacial Clefting and Cor Triatriatum Sinister in Humans and Mice

Orofacial clefting is amongst the most common of birth defects, with both genetic and environmental components. Although numerous studies have been undertaken to investigate the complexities of the genetic etiology of this heterogeneous condition, this factor remains incompletely understood. Here, we describe mutations in the HYAL2 gene as a cause of syndromic orofacial clefting. HYAL2, encoding hyaluronidase 2, degrades extracellular hyaluronan, a critical component of the developing heart and palatal shelf matrix. Transfection assays demonstrated that the gene mutations destabilize the molecule, dramatically reducing HYAL2 protein levels. Consistent with the clinical presentation in affected individuals, investigations of Hyal2-/- mice revealed craniofacial abnormalities, including submucosal cleft palate. In addition, cor triatriatum sinister and hearing loss, identified in a proportion of Hyal2-/- mice, were also found as incompletely penetrant features in affected humans. Taken together our findings identify a new genetic cause of orofacial clefting in humans and mice, and define the first molecular cause of human cor triatriatum sinister, illustrating the fundamental importance of HYAL2 and hyaluronan turnover for normal human and mouse development.


Introduction
Orofacial clefts, which include cleft lip (CL) with or without cleft palate (CLP) and cleft palate alone (CP), collectively referred to as CL/P, are amongst the most common birth defects in all populations worldwide with many syndromic and non-syndromic forms described. The majority of cases are of unknown molecular cause [1]. The average incidence of CL/P is one in 700 newborns, with wide variability across racial and ethnic groups and socioeconomic status [2,3]. The frequency of CL/P also differs by gender and laterality, there being a 2:1 male to female ratio for CLP, and a 1:2 male to female ratio for CP [2,4]. Orofacial clefts arise from a failure of the intricate molecular and cellular processes that regulate bilateral fusion of the future lip and palate during craniofacial development [1]. They are clinically categorized dependent upon the absence (non-syndromic CL/P; 70% of cases) or presence (syndromic CL/P; 30% of cases) of additional congenital anomalies [5,6]. For non-syndromic CL/P, 17 genetic risk loci have been defined and replicated with genome wide association studies worldwide [7][8][9][10][11]. As well as environmental influences, more recent evidence implicates non-coding or regulatory genomic regions in non-syndromic CL/P [12][13][14], perhaps explaining why resequencing of candidate genes or exome sequencing strategies rarely identify mutations in these cases [15]. For syndromic forms of CL/P, >300 different syndromes have been described in which CL/P is a prominent feature [16]. Congenital heart disease (CHD) is commonly associated with syndromic CL/P with a reported incidence of 13%-27% [15,[17][18][19][20][21][22]. Cor triatriatum is a rare congenital cardiac anomaly reported in 0.1-0.4% of individuals with congenital heart disease [23,24]. It is characterized by division of either the left (cor triatriatum sinister) or less commonly the right atrium (cor triatriatum dexter) [25,26]. To date, no molecular cause has been identified for cor triatriatum in humans. In the current study, we identify mutations in the gene HYAL2 as a new genetic cause of orofacial clefting in humans and mice and describe the first molecular basis for cor triatriatum sinister in humans. Our findings

Genetic studies
In order to map the chromosomal location of the disease gene we undertook a genome-wide SNP study in affected members of Family 1 assuming that a recessive founder mutation was responsible. This identified a single notable shared autozygous region of 10.18Mb on chromosome 3p21.31 (rs6441961-rs2201057; chr3:46310893-56499374 [hg38]) as likely to correspond to the disease locus. In support of this, multipoint linkage analysis with Simwalk2 [30] assuming autosomal recessive inheritance, full penetrance and a disease allele frequency of 0.0001, achieved a LOD MAX = 10.37 corresponding to the autozygous interval. To identify the causative mutation, whole exome sequence analysis of a single affected individual (XII:3) was undertaken (Otogenetics Corporation). After filtering for call quality, potential pathogenicity, population frequency and localization within the candidate interval, we identified only a single likely deleterious variant located in the HYAL2 gene (HYAL2 chr3:g.50320047T>C [hg38]; Western blots were performed on lysates prepared from MEFs deficient in HYAL2 that were transfected with NM_003773.4:c.443A>G; p.K148R; PolyPhen = probably damaging (1); SIFT = deleterious (0.03) MutationTaster = disease causing (probability 0.999997) Fig 1B). The variant was validated by Sanger dideoxy sequencing, which was also used to confirm co-segregation of the variant within the extended Amish pedigree (Fig 1A). Seven heterozygous carriers of the c.443A>G variant were identified in 532 Amish control chromosomes examined, indicating an allele frequency of approximately 0.013 in the population. The variant was not listed in the Exome Variant Server (NHLBI GO Exome Sequencing Project (ESP), Seattle, WA; http://evs. gs.washington.edu/EVS/), 1000 Genomes browser (http://browser.1000genomes.org/index. html) or the Exome Aggregation Consortium (ExAC) database (http://exacbroadinstitute.org/ ). Interestingly, HYAL2 had recently been identified as a candidate gene as a cause of short stature and facial dysmorphism in a study of multiplex consanguineous families [27]. This study undertook genetic studies in 33 heterogeneous families with dysmorphic and other systemic clinical manifestations, and identified a sequence alteration in HYAL2 as a possible cause of the clinical features in a single family comprising two affected individuals ( Fig 1A, Family 2 Fig 1A and 1B), absent in 817 ethnically matched individuals, was identified as the candidate genetic cause. This variant (rs781999115) was not listed in the Exome Variant Server or the 1000 Genomes browser but two carriers were reported on the ExAC database. The genomic autozygous region (chr3:46310893-54379802 [hg38]) common to both family cohorts is 8.0Mb and predicted to contain 270 RefSeq alignment-supported transcripts (S1 Fig).The p.K148R and p.P250L substitutions decrease protein stability.
HYAL2 encodes hyaluronidase 2, a membrane localized protein [33] with weak activity toward HA [34], which is an extracellular matrix glycosaminoglycan that is abundant during development. To assess the impact of p.K148R and p.P250L on the HYAL2 protein, the mutations were each introduced into a human HYAL2 expression construct and transiently transfected into MEFs deficient in HYAL2. Western blot analysis revealed a profound effect of the p.K148R and p.P250L mutations on protein levels, resulting in 11 and 20 fold reductions, respectively, compared to wild type ( Fig 1C).

Evaluation of craniofacial features and palate in Hyal2 -/mice
Consistent with the facial dysmorphism in the human affected individuals, Hyal2 -/mice exhibited a short broad nose, increased interorbitary space, and Wormian bones in the interfrontal suture [35]. To determine if the CL/P seen in the human affected individuals was also present in Hyal2 -/mice, we examined the time of pre-weaning lethality described to affect 2/3 of knockouts [35,36]. Among 201 progeny of Hyal2 +/intercrosses followed from birth to weaning, 16 died at postnatal day (P)1, 16 died between P2-P9, and 16 Hyal2 -/mice survived (S1 Table). Assuming all pups that died were Hyal2 -/-, 48 of 201 progeny (24%) would be Hyal2 -/-, approximating the Mendelian frequency for an autosomal recessive condition. We empty vector, pCMV6-HYAL2, pCMV6-HYAL2K148R, or pCMV6-HYAL2P250L. An arrow indicates HYAL2. HYAL2 levels shown in the graph were quantified by imaging the chemiluminescent signal using a BioRad ChemiDoc. The columns represent the average level (x 10 6 light units) of HYAL2 ± SEM (n = 4). Significance was determined using the student's T test. *** indicates p<0.0001. also studied ninety E18.5 embryos from Hyal2 +/intercrosses and found two Hyal2 -/and one Hyal2 +/embryo(s) that had died at approximately E15.5. Taking these intrauterine deaths into account, the proportion of Hyal2 +/+ mice still exceeded that of Hyal2 -/mice (30% vs 24%, S1 Table). Although this difference did not reach significance, it suggested reduced survival of Hyal2 -/and Hyal2 +/embryos during early gestation. CL/P was a likely cause of death in Hyal2 -/mice at P1 and possibly between P2-P9, whereas heart defects were the more probable cause at E15.5. Although most deceased pups were cannibalized before their genotype could be verified, four found dead at P1, without milk in their stomachs, were identified as Hyal2 -/-. Micro-CT studies of these pups showed an underdeveloped and underossified viscerocranium compared to littermate controls (Fig 2Ai-ii). Several central palate bones were underdeveloped and the vomer did not fuse centrally or form a head that articulated with the maxilla (Fig 2Aiii-vi). The ethmoid bone was nearly absent (Fig 2Aiii   Height, weight and OFC Z-scores were calculated using a Microsoft Excel add-in to access growth references based on the LMS method [31] using a reference European population [32] doi:10.1371/journal.pgen.1006470.t001 Evaluation of the heart in Hyal2 -/mice Valvular thickening and atrial dilatation are found in all Hyal2 -/mice, although the severity of the phenotype varies [36]. Cor triatriatum sinister has been detected in 50% of Hyal2 -/mice ( [37] and Fig 2B). It is conceivable that deaths in the progeny of Hyal2 +/intercrosses during embryogenesis and between P2-P9 observed are due to more severe (congenital) cardiac anomalies in Hyal2 -/mice. The cardiac findings in the five individuals with the HYAL2 K148R variant display significant similarities including one individual with cor triatriatum sinister and two further individuals with dilated coronary sinuses indicative of a PLSVC (see Table 1 and Fig 2C). This provides convincing evidence of the importance of HYAL2 for normal heart development in humans and mice [36,37] and identified mutation of HYAL2 as the first described molecular mechanism for cor triatriatum sinister in humans.

Evaluation of hearing in the Hyal2 -/mice
Although conductive hearing loss was seen in most of the affected human subjects in this study, one affected individual was found to have profound sensorineural hearing loss. To assess hearing in Hyal2 -/mice (8-12 weeks of age), the auditory brainstem response (ABR) was evaluated (S4 Fig). Hyal2 -/mice exhibited a significantly higher threshold at all frequencies tested, (mean +/-95% confidence interval for Hyal2 -/and Hyal2 +/+ mice were 17.6 +/-7.16 and 5.00 +/-3.88dB respectively; Mann-Whitney U-test U = 1433, p<0.001), indicating hearing loss in 100% of Hyal2 -/mice. The ABR test parameters did not differentiate conductive and sensorineural hearing loss, but because these were performed on adult mice, severe craniofacial defects were excluded, although minor defects may be present. The presence of hearing loss in mice without severe palate defects suggests more studies are needed to determine whether HYAL2 has a direct role in the development and/or maintenance of normal hearing.

HA accumulation in Hyal2 -/deficiency
Previous studies of Hyal2 -/mice with a complete absence of HYAL2 activity have demonstrated levels of circulating HA that are 19 fold higher than that of control mice at 12 weeks of age and accumulation of HA in the heart and lung [36] as well as other tissues [37]. In the current study, the HA levels were clearly elevated throughout the nasopharynx and in the palate (S3J, S3K, S3N and S3O Fig). It remains unclear to which degree this reflects species differences, but this finding is consistent with our transfection studies which show residual HYAL2 is present. This level of HYAL2 may be sufficient for constitutive turnover, but inadequate during development when rapid turnover of high levels of HA in the provisional matrix is required.

Discussion
The molecular basis of human CLP is incompletely understood, despite its frequent occurrence and associated morbidity. Our findings demonstrate that a deficiency of the HA-degrading enzyme HYAL2 is a novel cause of syndromic CLP in humans and SMCP in mice, and define the first molecular explanation for cor triatriatum sinister in humans. All but one affected individual exhibited orofacial clefting, and craniofacial dysmorphism was a consistent finding. Myopia and other ocular abnormalities, pectus excavatum, single palmar creases, cor triatriatum sinister, a persistent left superior vena cava and other cardiac features were variably present. Our studies show that Hyal2 -/mice universally exhibit facial dysmorphism, an underdevelopment of the viscerocranium and cardiac valve thickening as well as variably penetrant hearing loss, partial or SMCP (83%), cor triatriatum sinister (50%) and atrial dilatation (50%). The significant parity between the human and mouse phenotypes provides strong evidence that HYAL2-deficiency is the cause of these developmental abnormalities.
Our data indicate that the differences between the human and mouse phenotypes may be largely explained by the complete absence of HYAL2 in Hyal2 -/mice, while the destabilizing and deactivating p.K148R and p.P250L HYAL2 substitutions identified in human affected individuals lead primarily to profound reductions in HYAL2 levels, which may still permit limited residual functionality. In surviving Hyal2 -/mice progressive accumulation of HA in the circulation, pulmonary fibrosis, and cardiac dysfunction lead to premature heart failure [36], while the absence of elevated circulating HA in the affected Amish individuals may partially or completely protect them from these cardiopulmonary complications. The residual activity associated with the amino acid substitutions in human subjects likely supports ongoing constitutive turnover of HA, but is insufficient for the rapid and regulated turnover required during development. Further support for this is provided by our study of Hyal2 +/mice where a small number showed developmental abnormalities in the absence of elevated circulating HA [36]. However, this may be an overly simplistic interpretation as circulating HA levels are approximately 10-20 fold higher in the mouse, despite similar rates of turnover in mice and humans [38]. The identification of additional HYAL2-deficient individuals would undoubtedly provide a more complete understanding of the phenotypic spectrum associated with HYAL2-deficiency.
We identified SMCP as a likely cause of early pre-weaning lethality in Hyal2 -/mice. The underdeveloped viscerocranium likely weakens the palate, and in combination with the facial dysmorphism, presents significant feeding difficulties for Hyal2 -/suckling pups. Neurological abnormalities could also contribute to death, as the cribriform plate of the ethmoid is important in supporting the olfactory bulb and in preventing the leakage of cerebrospinal fluid into the nasal cavity. The bones of the viscerocranium have not yet been examined in affected individuals with the HYAL2 variant, but defects of the vomer are common in affected individuals with CLP [39]. The identification of developmental abnormalities in a small number of heterozygous (Hyal2 +/-) mice suggests that haploinsufficiency of HYAL2 may also pose the risk of a similar outcome in humans. Consistent with the notion that altered HYAL2 levels may affect palatal development, decreased HA has previously been associated with increased risk for CP in the Tbx1 -/mouse [40], whereas increased HA has been associated with CP in Sim2 -/mice [41]. Thus, HYAL2 variants may be considered as candidates, even in the heterozygous form, to confer risk for these developmental abnormalities. However screening a cohort of non-syndromic CL/P individuals, provided little evidence for a major role for HYAL2 in this cohort (personal communication-P. Stanier). The cohort consisted of 380 individuals (176 with unilateral or bilateral cleft lip and palate, 38 with cleft lip only, 121 with cleft palate only and 46 with submucous cleft palate) of a mixture of ethnicities the majority of which (72%) were of white European origin recruited from the North East Thames region of the UK. Approximately 22% of cases were familial with affected first or second-degree relatives. While this study cohort is too small to draw definitive genotype-phenotype conclusions, this finding remains consistent with HYAL2 mutation being likely only associated with a rare, autosomal recessive syndrome.
Eloquent studies in mice deficient in HA synthesis (Has2 -/-) demonstrated that high molecular mass HA was essential for epithelial to mesenchymal transition (EMT) in the developing heart, whereas HA oligosaccharides inhibited EMT and promoted angiogenesis [42][43][44]. HYAL2 could modulate the size of HA to inhibit EMT and promote differentiation, in which case its deficiency would result in increased levels of high molecular mass HA, an overproduction of mesenchymal cells, and a decrease in differentiation. Indeed, the thickened cardiac valves and excess mesenchymal cells in some parts of the head of Hyal2 -/mice, suggest that EMT is upregulated in Hyal2 -/mice, and that HYAL2 normally plays a role in inhibiting this process. In histological sections, encompassing the nasopharynx and secondary palate, there is an abundance of cartilage but a deficiency in bone. The deficient ossification in the skull of Hyal2 -/mice, and underdevelopment of the viscerocranium, suggests a failure in the development of osteoblasts, as has been seen in Tbx22 -/mice [45].
In addition to the direct effects of HA on signaling pathways, it is possible that an overabundance of HA in Hyal2 -/tissues leads to alterations in the gradients of morphogens or signal transduction factors, impacting development. Clearly further studies are needed to investigate this possibility. Treatment strategies for this developmental disorder deserve further research, as HA synthesis can be inhibited using 4-methylumelliferone, an FDA-approved drug in some parts of the world [46]. Further, commercial preparations of human hyaluronidase that function at a neutral pH could be used to remove extracellular HA; these enzymes are available for human use [47], and formulations that are stable in the circulation are under development [48]. With the Hyal2 -/mouse model already available, testing of such strategies may enable the development of a pre-natal treatment for this malformation syndrome. Taken together, our findings highlight hyaluronidase enzymes as playing a vital role in both human and mouse development, and in particular have revealed a previously unrecognized pathway involved in the pathogenesis of orofacial clefting, myopia and cor triatriatum sinister in humans, which may ultimately be amenable to treatment therapies.

Ethics statement
Signed consent was obtained for all individuals and the study was approved by the institutional review board at the University of Arizona (reference 10-0050-01) and the Great Ormond Street Hospital and the Institute of Child Health Research Ethics Committee (REC No. 08H0713/46). Animals were euthanized by isoflurane overdose and embryos were collected for gross morphological or histological studies. All procedures were in compliance with the Canadian Council on Animal Care and followed a protocol approved by the University of Manitoba Animal Care Committee.

Genetic studies
Blood samples for DNA extraction were obtained with informed consent from all participating family members (institutional review board-approved research protocols UoA:10-0050-10 and KFSHRC RAC#2080006). A genome-wide SNP screen was undertaken in the affected siblings (Family 1: XII:3; XIII:5; XII:7; XII:9; XII:12, and Family 2: V:3; VI:2) using Illumina Human-CytoSNP-12 v2.1 or Axiom SNP Chip arrays for autozygosity mapping of regions of >1Mb using AutoSNPa [49]. Whole exome sequencing of genomic DNA from Family 1 was performed at Otogenetics Corporation (Norcross, GA, USA) using the Agilent SureSelect Human All ExonV4 (51Mb) enrichment kit with a paired-end (2 × 100) protocol at a mean coverage of 30X. For Family 2, we used a TruSeq Exome Enrichment kit (Illumina, San Diego, CA) with samples prepared as an Illumina sequencing library enriched for the desired targets using the Illumina Exome Enrichment, with captured libraries sequenced using an Illumina HiSeq 2000 Sequencer. Sequence reads were aligned to the human genome reference sequence [hg19] and read alignment, variant calling, and annotation were performed by DNAnexus (DNAnexus Inc, Mountain View, CA). Intronic variants not predicted to affect splicing or regulation were excluded and SIFT [50], PolyPhen-2 [51], and MutationTaster [52] was used to predict the impact of any identified amino acid substitution on the protein structure and function and to predict and prioritize potential disease causing sequence alterations. The presence of the variants were confirmed in the transcript by bidirectional dideoxy Sanger sequencing performed on an ABI3730 XL capillary sequencer (Applied Biosystems), which was also used to confirm its co-segregation within the respective pedigrees.

Experimental animals
Hyal2 -/-(null) mice and littermate controls (Hyal2 +/or Hyal2 +/+ ) were obtained from heterozygous intercrosses of mice maintained on an outbred (C129; CD1; C57BL/6) background as viable Hyal2 -/mice have not been obtained on an inbred background [35]. Embryos were considered E0.5 the morning a vaginal plug was discovered. Genotyping was performed as described [36]. Animals were euthanized by isoflurane overdose and embryos were collected for gross morphological or histological studies. All procedures were in compliance with the Canadian Council on Animal Care and followed a protocol approved by the University of Manitoba Animal Care Committee.

Animal imaging, hearing analysis and histological studies
Ultrasound imaging was performed on mice anesthetized with 2% isoflurane, using a Visual Sonics Vevo 2100 ultrasound equipped with a 40 MHz transducer. Micro-CT imaging was performed at 9μm resolution with a Skyscan 1176 micro-CT and images were reconstructed and analyzed using Bruker's NRecon, Data Viewer, CT Analyzer, or CT Vox software. ABR testing was performed as described previously [53]. Skeletal staining of neonatal mice was as described in [54]. For histological studies, tissues were fixed in 1% cetylpyridinium chloride in 10% buffered formalin, decalcified if appropriate, imbedded in paraffin, and 5μm sections were prepared. Sections were stained with H & E for analysis of morphology. HA was detected using the HA binding protein (HABP) following established protocols [36].

HA and hyaluronidase assays
HA levels were determined using a DuoSet enzyme linked immunosorbent assay (ELISA) development kit (R & D Systems).

Transient transfection and western blot analysis
Transfections of HYAL2-expressing vectors were into mouse embryonic fibroblasts (MEFs) derived from a Hyal2 -/embryo. Cells and culture medium were collected 48 hours post-transfection and the β-galactosidase activity was determined using O-nitrophenol-β-D-galactopyranoside as a substrate [55]. Protein concentrations in the lysates were determined using a BioRad protein assay kit based on the Bradford method, and β-galactosidase levels were used to normalize for transfection efficiency in all assays. Western blot analysis was as described previously, but using polyclonal anti-HYAL2 (Proteintech) [56].