Whole exome sequencing reveals a biallelic frameshift mutation in GRXCR2 in hearing impairment in Cameroon

Abstract Background Hearing impairment (HI) genes are poorly studied in African populations. Methods We used whole exome sequencing (WES) to investigate pathogenic and likely pathogenic (PLP) variants in 10 individuals with HI, from four multiplex families from Cameroon, two of which were previously unresolved with a targeted gene enrichment (TGE) panel of 116 genes. In silico protein modelling, western blotting and live imaging of transfected HEK293 cells were performed to study protein structure and functions. Results All PLP variants previously identified with TGE were replicated. In one previously unresolved family, we found a homozygous frameshift PLP variant in GRXCR2 (OMIM: 615762), NM_001080516.1(GRXCR2):c.251delC p.(Ile85SerfsTer33), in two affected siblings; and additionally, in 1/80 unrelated individuals affected with non‐syndromic hearing impairment (NSHI). The GRXCR2‐c.251delC variant introduced a premature stop codon, leading to truncation and loss of a zinc‐finger domain. Fluorescence confocal microscopy tracked the wild‐type GRXCR2 protein to the cellular membrane, unlike the mutated GRXCR2 protein. Conclusion This study confirms GRXCR2 as a HI‐associated gene. GRXCR2 should be included to the currently available TGE panels for HI diagnosis.


| INTRODUCTION
Hearing impairment (HI) is the most common sensory disorder, affecting nearly 500 million people worldwide (WHO Media Centre, 2018). The highest incidence rate is found in sub-Saharan Africa (SSA): up to 6 per 1000 compared to about 1 per 1000 in Europeans or Nord Americans (Olusanya et al., 2014). About 50% of congenital HI cases in high-income countries are due to genetic causes (Schrijver, 2004). Variants in more than 150 genes have been associated with congenital HI (The Molecular Otolaryngology and Renal Research Laboratories, The University of Iowa, 2016), with common mutations in GJB2 and GJB6 associated with up to 50% non-syndromic HI (NSHI) in Europeans and Asians (Chan & Chang, 2014). But mutations in GJB2 and GJB6 and other connexins genes have rarely been found in Africans with NSHI ( Yan et al., 2016). Moreover, the prevalence of autosomal recessive non-syndromic hearing impairment (ARNSHI) due to pathogenic and likely pathogenic (PLP) variants, selected from the ClinVar and Deafness Variation Databases, and gnomAD database, was estimated at 5.2 per 100,000 individuals for Africans/African Americans, compared to the highest prevalence of 96.9 per 100,000 individuals for Ashkenazi Jews (Chakchouk et al., 2019). This knowledge deficit in genetic cause of HI in populations of African ancestry is likely to hinder our current understanding of hearing pathophysiology, refinement of gene-disease pairs and clinical validity curation (DiStefano et al., 2019).
We previously used a TGE panel of 116 genes (OtoSCOPE®) to successfully resolve the genetic causes in 7/9 selected multiplex families, segregating ARNSHI from Cameroon (Lebeko et al., 2016). In the present study, we use whole exome sequencing (WES) to investigate the causative variants in the two unresolved families, while exploring its sensitivity in detecting variants found in two other families through TGE. We further used in silico analyses to predict disruptions in protein folding and partner interactions, and in vivo cellular assays to explore and visualize protein localization.

| Patient participants
Four multiplex Cameroonian families of 10 members affected with ARNSHI were selected for WES, all previously investigated with TGE performed at the Molecular Otolaryngology and Renal Research Laboratories, Carver College of Medicine, University of Iowa, Iowa City, USA, as previously reported (Lebeko et al., 2016). In order to explore the sensitivity of WES, these families included two in which no causative variants were found using a TGE panel of 116 genes (OtoSCOPE ® ), and two other families in which the causative variants were known.
Unrelated participants with sporadic and familial NSHI (n = 80) were investigated for allele frequency (AF) of potential PLP variants found with WES. These participants were recruited in Cameroon (n = 57) and South Africa (n = 23). DNA samples were extracted from peripheral blood samples, and all participants did not have PLP variants in GJB2 and GJB6, following direct Sanger sequencing performed at the Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, South Africa, as previously reported Wonkam et al., 2013).

| Control participants
Ethnically matched control participants (n = 100) were recruited in Cameroon from an apparently healthy group of blood donors, without a personal or family history of HI, at Central Hospital of Yaounde, Cameroon.

| Whole exome sequencing
A total of 10 individuals with ARNSHI from four unrelated multiplex families underwent WES. DNA was extracted from peripheral blood samples as previously reported | 3 of 10 WONKAM et Al. . Using the SureSelect Human All Exon 50 Mb kit (Agilent Technologies, Inc.), which covers the exonic sequences of ≈24 000 genes corresponding to 50 Mb of genomic DNA, library preparation and sequencing on the Illumina HiSeq 2000 were performed at the Institute of Human Genetics, Helmholtz Zentrum München, Germany. The Burrows-Wheeler Alignment tool (version 0.7.5) was used to align the reads to the human genome assembly hg19 (GRCh37

| Genotyping of targeted variants
To investigate any other potentially causative and secondary variants in GRXCR2, which were found through WES to have a PLP variant in one family, the entire coding region of GRXCR2 was screened using direct Sanger sequencing in 80 patients with NSHI, and 100 Cameroonian controls, at the Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, South Africa. Primers were designed to amplify all three exons of GRXCR2. PCR amplification was confirmed by gel electrophoresis and products were cleaned up using FastAp and Endonuclease I. BigDye ® Terminator v3.1 Cycle Sequencing mix was used for the sequencing reaction according to the manufacturer's guidelines (Thermo Fisher Scientific). Sequencing products were resolved on 3130xl Genetic Analyser ABI Prism using capillary electrophoresis and resultant electropherograms were analysed using DNAStar software (Applied Biosystems).

| In silico pathogenicity prediction of genomic variants
The GRXCR2 cDNA sequence (ENST00000377976.2) was extracted from the Ensemble genome browser and manually edited by deleting the cytosine at position 251 of the cDNA sequence. The altered sequence was then interrogated using two online programs, namely ExPASy (https://web.expasy. org/trans late/) and EMBOSS Transeq (http://www.ebi. ac.uk/Tools/ st/emboss_trans eq/). This was done to predict the effect of the identified novel frameshift deletion on the translated protein sequence. To infer the importance of this variant across species, a multiple sequence alignment of higher primates was extracted from Ensemble (http://ensem bl.org).

Cell line and transfection
The HEK293 Human Embryonic Kidney cell line was maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 200 units/ml penicillin and 100 μg/ml streptomycin. Transient transfections were performed using X-tremeGENE™ HP DNA Transfection Reagent according to the manufacturer's instructions (Roche). Cells were plated at 3.5 × 10 4 cells/well in 35 mm diameter dishes 16 h before transfection. Cells were transfected with 1 µg of pCMV6-GRXCR2-WT or pCMV6-GRXCR2-MT and cultured for 30 h before total protein harvest. For microscopy visualization, cells were plated as before, and transfected with 2 µg pCMV-GRXCR2WT or pCMV-GRXCR2MT or pEGFP-C1 and incubated for 48 h before being prepared for viewing.

| Visualization using confocal microscopy
Live viewing was performed 48 hours following transfection, using a Zeiss LSM8800 with Airyscan confocal microscope (Zeiss). The detector of the confocal was a photo multiplier tube (PMT) and allowed detection of the green fluorescence signal through the Argon laser at 488 nm. Images were visualized and processed using the ZEN Black Software (latest version) provided by Zeiss (Zeiss).

| Description of families selected for WES
The pedigrees of multiplex families selected for WES are shown in Figure S1. All affected members presented with autosomal recessive bilateral sensorineural non-syndromic HI, with variable severity.  (Table 1). In one family, novel variants were identified through WES in PAX3 and MYO7A, but were excluded on account of their possible syndromic manifestation as well as their suspected dominant pattern of inheritance, both of which did not fit the ARNSHI phenotype observed in the family. No causative variants were identified for one family through TGE and WES.  (Figure 1c). There was an evolutionary conservation of this base across eight primate species (Figure 1d).

| Screening of GRXCR2 for additional variants in HI patients through Sanger sequencing
Identification of the identified novel variant in a second unrelated case (Figure 1b) prompted the sequencing of the other two exons of GRXCR2 in 80 patients with NSHI from Cameroon and South Africa. The sociodemographic and phenotypic description of these patient are summarized in Tables S1 and S2. Sixty-five percent (65%) of the patients were males (n = 52) with 70% of patients reported as having a prelingual onset of hearing loss. Up to 47.5% (n = 38) were reported as having a family history of hearing loss, with most of this group being from Cameroon. All the Cameroonian patients had sensorineural NSHI of various degrees, symmetrical in most cases (n = 40). No other PLP variants were found in GRXCR2, but two polymorphisms were identified in patients and controls (Table S3).

| GRXCR2 protein analysis
Protein sequence prediction by ExPASy and EMBOSS Trans revealed that the GRXCR2-c.251delC p.(Ile85SerfsTer33) altered the amino acid sequences after p.Arg84 as well as the introduction of a premature stop codon at position 117 of the amino acid sequence (Figure 1e). In silico analysis of the secondary structure of the GRXCR2 protein depicted the predicted domains and their positions along the protein sequence, showing the mutant GRXCR2 protein to be a relatively small protein with mostly random coiling with few helices and beta sheets (Figure 1f). The mutant sequence could not be interrogated in PSIPRED due to its small size. The predicted stability was explored (Figure 1g) according to identified IDRs, which are regions that can alter their state from structured to unstructured as a protein prepares to or carries out its functions, particularly in binding with other proteins. GRXCR2 was found to have three distinct protein binding regions corresponding with high confidence of IDRs. Truncation of the protein leads to the loss of the C-terminus IDR which includes the cysteine-rich region, which is predicted to fold into a zinc-finger structure known to act in PPIs (Figure 1g).

| Confirmation of premature stop codon using western blotting
To confirm the predicted truncation of the mutant protein, western blotting was used. The DDK-tag located on the C-terminal end of the protein could be detected in the wildtype (WT) protein, showing a predicted size of 24 kDa, but the same was not present in the mutant (MT) protein, indicating premature truncation (Figure 1h).

| Live imaging shows disruption of protein transport and localization of WT compared to MT GRXCR2 proteins
The WT and MT proteins could be visualized via N-terminal GFP tags, using confocal microscopy in HEK293 cells. As expected, cells transfected with the empty vector showed strong and uniform pattern of expression throughout the HEK293 cells (Figure 1i). On the other hand, the WT-GTP-tagged GRXCR2 protein was preferentially localized outside of the nucleus and predominantly in the cytoplasm, with some punctate staining close to the periphery of the cell membranes (Figure 1j). This suggests that the WT protein is expressed in the cytoplasm, where it is confined, and potentially shuttled to the periphery close to the membrane. Since these cells were visualized live to preserve the GFP, a DNA-specific stain could not be used  to locate the nuclei. Interestingly, and in contrast to the GFP-GRXCR2-WT protein, the GRXCR2-MT protein lacked any particular localization within the cells and showed a distribution similar to that observed for the empty vector, although at a seemingly lower intensity (Figure 1k). , often with lower pick-up rate compared to European and Asian populations. The present study is therefore a rare attempt to use WES to investigate HI in multiplex families from SSA (Cameroon), showing the efficiency of this approach in being specific enough to identity variants in known genes, but equally sensitive enough to identify variants in novel HI genes. The present report from an understudied African population is important for improving the disease-gene pair curation, globally. Indeed, PLP variants in GRXCR2 were reported only twice: the GRXCR2-c.714dupT mutation was identified in a Pakistani family segregating ARNSHI (Imtiaz et al., 2014), and GRXCR2-c.65A>G was recently reported in Chinese proband with HI (Wu et al., 2020). Therefore, this study provides additional evidence to confirm GRXCR2 as an ARNSHI gene in humans. Interestingly, null mutations in GRXCR2 in mice result in early onset progressive hearing loss without vestibular dysfunction (Avenarius et al., 2018). The GRXCR2-c.251delC mutation reported in the present study is absent from gnomAD v3.0, which includes >20,000 African exomes or genomes. Generally, the occurrence of frameshift mutations in this gene is very rare (cumulative minor allele frequency (CMAF) in gnomAD overall = 4.89 × 10 −5 ; CMAF in African = 7.14 × 10 −5 ), and there are no homozygote carriers for any frameshift mutation (though we cannot exclude the possibility of compound heterozygote carriers). Based on gnomAD, the frequency of biallelic carriers for GRXCR2 frameshift mutations is exceptionally rare at 2.19 × 10 −9 . The NHIBI Exome Sequencing Project reports a single heterozygous carrier of the GRXCR2-c.251delC variant reported in this paper, in an African American individual (http:// evs.gs.washi ngton.edu/EVS/), suggesting that investigating sub-Saharan Africans will have implications for the African diaspora. Transcript of the inner hear indicates that 100 s of HI genes are still to be discovered (Hertzano & Elkon, 2012).

| DISCUSSION
In the present study, both in silico and in vitro investigations (Figure 1e-k) support the contribution of the GRXCR2c.251delC variant in HI in the patients investigated. The GRXCR2 gene is expressed in the inner ear during development (Schraders et al., 2010). The GRXCR2 protein belongs to the glutaredoxin domain-containing family. It is a paralog of GRXCR1 and both have been shown to be required for stereocilia bundle development, organization and maintenance (Avenarius et al., 2018). Pirouette mouse models showed variants in GRXCR1 to result in hearing loss with vestibular dysfunction (Odeh et al., 2010). A similar syndromic phenotype was observed in humans who carried pathogenic variants in the GRXCR1 gene (Schraders et al., 2010). The differences observed in the phenotype of variants of GRXCR1 and GRXCR2 suggest that the proteins they encode might have slightly distinct roles in the stereocilia maturation pathway (Avenarius et al., 2018;Liu et al., 2018). It is most likely that PPIs mediated by the domain missing in the protein encoded by the GRXCR2-c.251delC variant are essential for the protein's functionality, with GRXCR1, particularly in the formation of homodimers or other protein complexes (Avenarius et al., 2018). Functional interaction between the GRXCR2-and GRXCR1-encoded proteins could be similar to that seen in the Connexin family between GJB2-and GJB6encoded proteins, where their heterodimerization is needed for functionality (Xu & Nicholson, 2013), and deserves additional exploration.
We could not identify PLP variants in one multiplex family. This could be due to a complex structural change which was not detected by WES, or variants in other parts of the genome. The closest approach to whole-genome sequencing (WGS) was whole-genome SNP mapping, which was able to resolve 100% of 30 families with HI from Pakistan (Shafique et al., 2014). However, this might not be as effective in a population of African ancestry, which was poorly represented in the data that informed the development of panels used (Lek et al., 2016).
This study has a few limitations: firstly, the GRXCR2c.251delC variant was not investigated in parents or other family members due to lack of available DNA. Secondly, in our functional studies, the use of GRXCR2-specific antibodies could have increased accuracy of both localization and protein expression levels, and future study should demonstrate that the mutation caused a truncated protein via inability to detect the tag on the C-terminus of the protein. Moreover, the identification of the same homozygous mutation in patients from non-consanguineous unrelated families suggests the presence of a founder effect or a mutational hotspot that could be investigated in future. Despite these limitations, the study has provided sufficient data to support the implication of GRXCR2 in HI in Cameroonian patients investigated.

| CONCLUSIONS
This study has provided additional evidence to confirm GRXCR2 as a HI-associated gene. GRXCR2 should be included to the currently available TGE panels for HI diagnosis. Additionally, the study showed that WES is as sensitive as TGE in detecting variants in known HI genes, and emphasizes the urgent need to use WES to enhance HI genes and discovery of variants in populations of African ancestry.