A HS6ST2 gene variant associated with X‐linked intellectual disability and severe myopia in two male twins

X‐linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X‐linked heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3′‐phosphate, 5′‐phosphosulfate to the sixth position of the N‐sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered “deleterious” by in‐silico tools. An in‐vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.


Funding information
Italian Fiscal Contribution "5x1000" 2015 devolved to Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms.
WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the Xlinked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3 0 -phosphate, 5 0 -phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.

K E Y W O R D S
HS6ST2, intellectual disability (ID), syndromic myopia, whole exome sequencing (WES)

| INTRODUCTION
Intellectual disability (ID) is defined as a neurodevelopmental disorder with onset before 18 years of age, characterized by IQ ≤ 70 and deficit in at least two adaptive behaviors (eg, communication, self-care). 1 ID occurs in approximately 1% to 3% of the population 2 and has a variety of environmental and genetic causes, which are frequently present in combination with one another. 3 Mild ID is typically associated with environmental features, while genetic factors have a major influence in moderate, severe, and profound ID 4 ; however, the identification of factors contributing to ID etiology is complicated by the extreme heterogeneity of this condition. Numeric and structural chromosomal aberrations, as well as variants in at least 800 proteincoding genes, 3 explain approximately half of ID diagnoses, while the etiological factors underlying the remaining cases are unknown. Mutations in genes influencing ID can give rise to both syndromic and non-syndromic forms of the condition. Syndromic ID includes a highly heterogeneous group of phenotypes where ID occurs together with a constellation of other features. 5 ID gene density is particularly high on the X chromosome, with more than 10% of all ID-related proteincoding genes mapping to the X, establishing the condition referred to as X-linked ID (XLID) that predominantly occurs in males 6 ; overall, 30% more males are affected than females. 7 Here, we report two monozygotic male twins of a triplet pregnancy with clinical diagnosis of syndromic ID combined with severe myopia and mild dysmorphic features.
High myopia is a prominent refractive error. Gupta et al. reviewed the candidate genes related with inherited myopia and reported only two forms of non-syndromic condition, one involving ZNF644 and the other related to SCO2 gene. 8 High myopia and severe ID, presenting as isolated manifestations without other relevant dysmorphic or malformative features, have never been previously associated with a known clinical condition. Conversely, they have been identified in many syndromes, such as Donnai-Barrow or Cohen syndromes, as part of a broad range of clinical signs affecting many parts of the body.
Genetic investigation of the triplets and their immediate family revealed a mutation in the heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene on the X chromosome, predicted to detrimentally influence protein function. in vitro assays confirmed the deleterious effect of the mutation on HS6ST2 enzyme activity. Hence, mutations in HS6ST2 represent a potential new cause of syndromic XLID.

| Study aim, design, and setting
The aim of this study was to screen for the genetic basis of the phenotype of two identical twins presenting at our hospital with ID and severe myopia. Following clinical examination, whole exome sequencing (WES) and functional analysis of the potential causal mutation were performed. The study setting was the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy. Appropriate informed consent for WES analysis for research purposes was obtained from all family members.
Parental approval was also obtained for the inclusion of the genetic data of all family members in a scientific publication.

| Whole exome sequencing
Genomic DNA was extracted from peripheral blood samples of all family members using a QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). A SureSelect Clinical Research Exome V5 kit (Agilent, Santa Clara, CA) was used to perform next-generation sequencing (NGS) analyses of DNA samples (2 μg). Libraries were analyzed by paired-end NGS (NextSeq 550; Illumina, San Diego, CA).
Autosomal and X-linked recessive transmissions were first investigated as potential inheritance patterns. De novo variants were also considered. Accordingly, we set the variant calling parameters as follows: PassFilter = ON; Quality>30; Read Deph>8; Alt Variant Freq>25. We first focused our analysis on consensus splice-site changes, non-synonymous variants, and insertions/deletions in exonic regions. We also assumed that the alternative allele should have a frequency lower than 0.01 (based on Exome Aggregation Consortium [ExAC] all frequency) or that it should be absent from the ExAC, 1000 Genomes, dbSNP132, COSMIC and ClinVar databases. Output files from VariantStudio are provided as Supporting Information Appendix S1, S2, and S3 (autosomal recessive, X-linked recessive and de novo variants, respectively).

| Bioinformatic analyses
In silico prediction of the functional effect of the c.916G>C mutation on the HS6ST2 gene was performed using the "Mutation Taster" tool.
At the protein level, the consequences of the Gly306Arg substitution on HS6ST2 tertiary structure were evaluated using Phyre2 software (Protein Homology-fold Recognition Server; www.sbg.bio.ic.ac.uk/ phyre2/). Wild-type and mutated HS6ST2 3D structures were modelled. 9 These analyses were implemented using Phyre investigator, a workbench analysis tool with additional features for investigation of models created using Phyre2. All *.pdb files generated using Phyre2 were loaded and visualized with ChemDraw (version 8; Cambridge Software; PerkinElmer, Inc., Waltham, Massachusetts).

| HS6ST2 site-directed mutagenesis
The mammalian pReceiver-M12 Ampr vector containing the full length

| Mutation analysis by NGS
Using HS6ST2 have been identified and exhibit tissue-specific localization: full length HS6ST2 is expressed in the eyes and brain during embryonic development, and a short form of the gene, hHS6ST-2S, encoding 40 fewer amino acids, has been detected in the ovary, placenta, and fetal kidney. 12 Both isoforms catalyze the same sulfation reaction. 13 The novel G>C transversion in HS6ST2 identified here changes a GGC codon, encoding a non-polar glycine residue, to a CGC, which encodes a positively charged arginine. The variant was present in the affected triplets (FII-1 and FII-2), carried by the healthy mother, and absent in both the father and the unaffected brother (FII-3) (Figure 1).
Sanger sequencing confirmed the findings of NGS and the segregation of the transversion in the family. The "Mutation Taster" in silico prediction tool classified the c.916G>C substitution as deleterious, with a score of 1.000 (values close to 1 indicate a high "security" of prediction). This substitution has never been reported in the main databases of nucleotide variants, including dbSNP, 1000 Genomes, and Exac (Appendix S1).

| In silico 3D modeling of the HS6ST2-G306R protein
The

| In vitro assay of HS6ST2-G306R activity
To test whether G306R variant affects HS6ST2 enzyme expression and/or activity, HEK293 cells were transiently transfected with the empty vector (mock control) and with plasmids expressing wild-type (HS6ST2-WT) or G306R mutant (HS6ST2-G306R) FLAG-HS6ST2. We first analyzed the expression of recombinant wild-type and mutant HS6ST2 proteins in HEK293 cells. Protein expression was undetectable in mock-transfected cells but was evident in the HS6ST2-WT and HS6ST2-G306R transfected cells. As shown in Figure 3A, the expression levels of the wild-type and mutant proteins were very similar.

| DISCUSSION
In this study, WES was performed to characterize genetically two affected monozygotic twins with an inconclusive clinical diagnosis. We Severe myopia is arbitrarily defined as short sightedness greater than six diopters. 14 It is usually an isolated disorder; however, more rarely, it can be associated with syndromic conditions (eg, Stickler syndrome). 15   Altered HS6ST2 expression has been identified in numerous human cancers, 17 and is reported to participate in the pathogenesis of osteoarthritis and Kashin-Beck disease. 18 23 HS have been specifically noted in eye development, cranial axon guidance, and motor neuron migration. 24,25 In a mouse model, abnormally low sulfation of HS allows normal retinal neurogenesis and optic fissure closure; however, it leads to defective optic disc and stalk development. 24 Adult mutant animals develop optic nerve aplasia/hypoplasia and exhibit retinal degeneration. 24 Although myopia is not directly related to optic nerve aplasia/ hypoplasia, but rather occurs if the eyeball is too long or the cornea is too curved, a link may exist between the mutation detected in the HS6ST2 gene and the severe myopia observed in the affected children. FIGURE 3 Expression and activity of HS6ST2 in mock, wild type and G306R mutant cells. HEK293 cells were transiently transfected with empty vector (mock), or with plasmids expressing wild-type FLAG-HS6ST2 (WT), or mutant FLAG-G306R (G306R), and then subjected to immunoblot analysis (A), and the HS6ST2 assay (B and C). A, representative immunoblot image of HS6ST2 expression in mock, wild type and G306R mutant cells. β-actin was used as loading control. B and C, HS6ST2 activity in the cell extracts of mock, WT and G306R cells. Enzyme activity was assayed as described in Materials and Methods in the absence (B) or presence (C) of 10 mM dithiothreitol (+ DTT). In all cases, the reaction rate was linear with enzyme protein and incubation time. Data are the mean AE SD of three individual assays. Statistical significance was assessed using two-tailed Student's t test; **P < 0.01 In conclusion, we identified a novel c.916G>C variant in the HS6ST2 gene. The mutation caused a significantly reduction in HS6ST2 6-O-sulfotransferase activity and was associated with a previously undescribed form of syndromic XLID with severe congenital myopia. Although the phenotype of the probands in this study is weakly similar to BWB, the underlying genetic cause differs. A hypothesis could suggest possible loci and pathway heterogeneity in BWB syndrome. Alternatively, HS6ST2 may cause a novel clinical condition and be pivotal for the development of myopia and cognitive impairment. Although the current report describes only a single pedigree, it could be used by clinicians to facilitate recognition of similar families and to provide a more accurate genetic counseling.