A case of Usher syndrome type IIA caused by a rare USH2A homozygous frameshift variant with maternal uniparental disomy (UPD) in a Chinese family

Abstract Usher syndrome encompasses a group of genetically and clinically heterogeneous autosomal recessive disorders with hearing deficiencies and retinitis pigmentosa. The mechanisms underlying the Usher syndrome are highly variable. In the present study, a Chinese family with Usher syndrome was recruited. Whole exome sequencing (WES), Sanger sequencing, homozygosity mapping, short tandem repeat (STR) analysis and segregation analysis were performed. Functional domains of the pathogenic variant for USH2A were analysed. We identified a homozygous frameshift variant c.99_100insT (p.Arg34Serfs*41) in the USH2A gene in the proband that showed discordant segregation in the father. Further homozygosity mapping and STR analysis identified an unusual homozygous variant of proband that originated from maternal uniparental disomy (UPD). The p.Arg34Serfs*41 variant produced a predicted truncated protein that removes all functional domains of USH2A. The variant was not included in the 1000 Human Genomes Project database, ExAC database, HGMD or gnomAD database, but was included in the ClinVar databases as pathogenic. Although USH2A is an autosomal recessive disease, the effects of UPD should be informed in genetic counselling since the recurrence risk of an affected child is greatly reduced when the disease is due to the UPD mechanism. To test potential patients, WES, combined with STR analysis and homozygosity mapping, provides an accurate and useful strategy for genetic diagnosis. In summary, our discoveries can help further the understanding of the molecular pathogenesis of Usher syndrome type IIA to advance the prevention, diagnosis and therapy for this disorder.


| INTRODUC TI ON
Usher syndrome consists of a group of genetically and clinically heterogeneous autosomal recessive disorders with sensorineural hearing deficiencies and progressive retinitis pigmentosa (RP).
Diseases under the umbrella term Usher syndrome include Usher syndrome type I, II and III. 1,2 Usher syndrome type II includes USH2A, USH2C and USH2D. Usher syndrome type IIA (USH2A locus, OMIM 276901) is caused by mutations of the USH2A gene (OMIM 608400). 3 This gene maps to the chromosome 1q41 and encodes a protein containing 5202 amino acids that contain a pentaxin motif, laminin EGF motifs and numerous fibronectin type III domains. 4 The protein is localized in the basement membrane and has a vital role for development and homeostasis in the inner ear and retina.
Homozygosity has long been known to be related to rare often devastating Mendelian disorders and imprinting diseases. 5 Uniparental disomy (UPD) is the inheritance of both copies of one chromosome from only one parent, without the inheritance of a representative copy from the other parent. 6,7 Euploidy can result from aneuploid gametes if monosomic rescue, trisomic rescue or gametic complementational restore of normal ploidy occur during early human development. Detecting UPD is a practical diagnostic approach for rare Mendelian disorders and imprinting disorders caused by homozygosity. [7][8][9] The relationship between the variants in the Usher syndrome-associated genes and the resultant Usher syndrome phenotypes in the patients is highly variable. The causality and genetic mechanism of Usher syndrome type IIA have not been well documented. In the present study, we identified a rare homozygous frameshift mutation in the gene USH2A that originated from maternal UPD by whole exome sequencing (WES) and homozygosity mapping in a Chinese pedigree with Usher syndrome.

| Sanger validation and segregation analysis
PCR amplification was performed for mutation validation. 13,23 Primer pairs M567-USH-99 were designed using the Primer3 program with genomic DNA sequences containing the NM_206933.2 variant: c.99_100insT in the USH2A gene (Table 1). PCR amplification was performed using the aforementioned primer pair USH2A-99, and the amplified PCR products were then sequenced using the Sanger method on an ABI-3500DX sequencer 24,25 using primer USH2A-99L (Table 1). Ethnically matched unrelated control samples were also sequenced as described above. Testing of the variant in the mother and father ensued.

| PCR amplification and genotyping for short tandem repeat (STR)
Short tandem repeat genotype analysis was carried out in accordance with the relevant provisions of the Technical Specification for Paternity Appraisal by China (SF/Z JD0105001-2018) with the GoldenEye™ kit. PCR reactions were carried out according to the manufacturer's instructions using the extracted genomic DNA samples. The PCR amplification cycle was performed in an "Applied Biosystems Veriti ® 96-Well Thermal Cycler" machine. Then, the amplified product was mixed with the sample mixture for capillary electrophoresis using the 3500DX Gene Analyzer (Applied Biosystems Inc). 24 Genotype analysis for STR profiles was performed by using the software of GeneMapper ® ID-X 1.5.

| Homozygosity mapping
The homozygosity mapping analysis was performed using the Illumina ASA (Asian Screening Array) (Genmed, Inc). To do this, we used a PLINK software, Illumina chip matching reagents and an Illumina instrument (Multi-Sample BeadChip Alignment Fixture).
By applying the PLINK software, we screened loci with a missing rate < 1%, minor allele frequency (MAF) > 5%, and Hardy-Weinberg equilibrium P > 1e-3, and set one window for every 50 SNPs and slid 5 SNPs for each section with r 2 < .5 in two SNPs. If a region spanning 2.5 Mb contained continuous homozygous SNPs, then the region was considered to be a possible homozygous region (run of homozygosity, ROH). Thus, the SNPs selected using these methods were relatively independent and more common in the whole genome. 26

| Proband and clinical characteristics
The patient ( Figure 1A, II: 1) was a 5-year-old Chinese boy. He had been clinically diagnosed with hearing loss since age 5 years. A pure-tone audiometry testing was performed which indicated binaural moderate to severe deafness with sloping audiograms that included increased thresholds across all frequencies ( Figure 1B  3 domains ( Figure 2D). The variant c.99_100insT (p.Arg34Serfs*41) causes a loss of all functional domains ( Figure 2D)

| Discordant segregation of the c.99_100insT variant in the proband's father
Sanger sequencing was performed for the co-segregation analysis. As expected, the heterozygous mutation was identified in the proband's mother ( Figure 2B), M566, I:2). But surprisingly, the proband's father (M565, I:1) had a wild-type genotype ( Figure 2C) instead of the expected heterozygous genotype. Thus, this variant c.99_100insT (p.Arg34Serfs*41) was not inherited from the father.

| Homozygous variant c.99_100insT is due to upd from proband's mother
Given that the mutant allele should come from the proband's father or arise de novo, we perform STR analysis to confirm the paternity by using 20 STR markers, including 19 autoso-  Note: μ = 0.0005; p and q are the frequencies of the alleles.

| D ISCUSS I ON
In this study, we described a homozygous, pathogenic frameshift There are five possibilities for the presence of discordant segregation in this pedigree: no paternity or sampling errors, de novo mutation, heterozygous micro-deletion and UPD. UPD is described in roughly 3300 cases so far and has been linked to clinical phenotypes due to imprinting disorders or recessive diseases, including schizophrenia, cardiovascular disease and cognitive impairment. 5,7,34 Detecting UPD is a useful diagnostic approach for uncovering rare A pure-tone audiometry testing in the proband indicated the binaural moderate to severe deafness with sloping audiograms that included increased thresholds across all frequencies ( Figure 1B). An air-bone gap value was great than 10 dB in the both ears, meaning a problem in the outer or middle ears of the proband ( Figure 1B).

ACK N OWLED G EM ENTS
The authors thank the patient and family members for supporting

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
JF was in charge of the idea, project design of the study. JF and H. L.
conducted sampling. Ji. F., S S. and J. C. performed DNA extraction, PCR amplification and sequencing. S S. involved in reading the puretone audiogram. Ji. F. and J. F. performed STR and data analysis. J. F. wrote and revised the manuscript.

E TH I C A L A PPROVA L
The study has been approved by the Ethics Committee of Southwest Medical University. The informed consent form was obtained from the members of the family or guardian.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used for the analyses in this report are available from the corresponding author on reasonable request.