Identification of novel variants in Iranian consanguineous pedigrees with nonsyndromic hearing loss by next‐generation sequencing

Abstract Background The extremely high genetic heterogeneity of hearing loss due to diverse group of genes encoding proteins required for development, function, and maintenance of the complex auditory system makes the genetic diagnosis of this disease challenging. Up to now, 121 different genes have been identified for nonsyndromic hearing loss (NSHL), of which 76 genes are responsible for the most common forms of NSHL, autosomal recessive nonsyndromic hearing loss (ARNSHL). Methods After excluding mutations in the most common ARNSHL gene, GJB2, by Sanger sequencing, genetic screening for a panel of genes responsible for hereditary hearing impairment performed in 9 individuals with ARNSHL from unrelated Iranian consanguineous pedigrees. Results One compound heterozygote and eight homozygote variants, of which five are novel, were identified: CDH23:p.(Glu1970Lys), and p.(Ala1072Asp), GIPC3:p.(Asn82Ser), and (p.Thr41Lys), MYO7A:p.[Phe456Phe]; p.[Met708Val], and p.(Gly163Arg), TECTA:p.(Leu17Leufs*19), OTOF:c.1392+1G>A, and TRIOBP:p.(Arg1068*). Sanger sequencing confirmed the segregation of the variants with the disease in each family. Conclusion Finding more variants and expanding the spectrum of hearing impairment mutations can increase the diagnostic value of molecular testing in the screening of patients and can improve counseling to minimize the risk of having affected children for at risk couples.


| INTRODUC TI ON
Hearing loss is the most common sensory disorder in humans. It is estimated that 360 million people worldwide are suffering from hearing loss. 1 The frequency of congenital deafness ranges from 1 to 2 per 1000 in Western countries, while in Iran it reaches to 1 in 166; in other words, the prevalence of deafness in Iran is estimated to be 2-3 times higher than the other parts of the world. 2,3 Iran as one of the consanguinity belt countries, with 38.6% rate of consanguineous marriage which is culturally and socially favored, among the world's most heterogeneous populations, has received a great deal of attention as a potential risk factor for many autosomal recessive disorders including autosomal recessive nonsyndromic hearing loss (ARNSHL). 4,5 Genetic forms of deafness responsible for more than half of hearing loss cases have been shown to have diverse etiologies, and it is estimated that approximately 1% of all human genes are involved in the biology of hearing. 6 Congenital hearing loss is the second most common disorder following intellectual impairment in Iran. 3 Malfunctions of the cochlea and inner ear due to dysfunction of proteins involved in mechanisms related to the adhesion of hair cells, intracellular transport, neurotransmitter release, ionic homeostasis, and cytoskeleton of hair cells can cause hearing impairment. A defect in any part of these mechanisms can cause the disease. 7 The extremely genetic heterogeneity of deafness can be due to the complexity of the auditory system, which requires coordination of multiple processes controlled by the interaction of various proteins coded by several hundred genes. 6,8 Up to now, 121 genes have been implicated in the pathogenesis of nonsyndromic deafness in which about 76 genes have been reported to cause ARNSHL (https://hered itary heari ngloss.org/). Causative genes can be classified by their molecular function, homeostasis, hair cell structure, transcription factors, cytokinesis, extracellular matrix, mitochondrial, and other/unknown. 6,9 Previous studies have shown mutations in GJB2, SLC26A4, and TECTA genes as the most common cause of NSHL in the Iranian population followed by MYO15A, ILDR1, TMC1, PJVK, LRTOMT, MYO7A, OTOF, and MARVELD2. 10 In spite of tremendous heterogeneity, recently in a cohort of 302 GJB2-negative Iranian probands with ARNSHL, over half of all genetic diagnoses (52%) have been shown to be due to the causative variants in only five genes (SLC26A4, MYO15A, MYO7A, CDH23, and PCDH15). 1 In the remaining pedigrees, mutations in 35 other genes including GIPC3, TECTA, OTOF, and TRIOBP were identified. 1 In the present study, 9 unrelated Iranian families with at least one affected individual who were negative for mutations in GJB2 were screened by next-generation sequencing (NGS) for 127 known deafness genes. In this report, variants in 6 different genes including three variants in MYO7A, two variants in CDH23 and GIPC3, and one variant in TECTA, OTOF, and TRIOBP were identified.

| Patients and ethics statement
In this study, nine Iranian families with at least one hearing impaired member who was referred to the Department of Medical Genetics, DeNA Laboratory, Tehran, Iran, were investigated. All clinical data of hearing impaired patients in these families were obtained at DeNA Laboratory using a uniform questionnaire according to ACMG guidelines for the etiologic diagnosis of congenital hearing loss, included consanguinity and hearing status of the parents and siblings, age of onset, one or both ears deafness, syndromic or nonsyndromic deafness, presence of accompanying symptoms such as visual anomalies, endocrine abnormalities, thyroid disorders, skin problems, exposure to environmental factors like taking drugs or drinking alcohol during pregnancy, and intrauterine infections. 11 The hearing impaired individuals in these pedigrees had no obvious vestibular dysfunction, retinal degeneration, or report of other anomalies, suggesting that the families are suffering from nonsyndromic deafness. Evaluation of the deaf patients showed prelingual bilateral nonsyndromic sensorineural hearing loss in all cases. Medical investigations included otoscopy and physical examination by an otolaryngologist and a geneticist. According to audiological evaluations, the severity of deafness varied among patients, ranging from mild to profound (Table 1).
In all cases, deaf patients had consanguineous normal parents, suggesting autosomal recessive deafness. Written informed consent for genetic testing was obtained from the adult patients or from their parents in case the patients were under 18 years of age. Some cases were sporadic, while other families had history of multiple affected members with hearing loss. Pedigrees are shown in Figure 1.

| DNA extraction
Blood samples were collected from families including 12 patients and 32 normal individuals (

| Targeted next-generation sequencing and in silico analysis
All families were negative for mutations in GJB2. A custom-designed NimbleGen chip capturing 127 genes involved in HL based on the deafness variation database (DVD) (http://deafn ess-varia tiond ataba se.org/letter) followed by next-generation sequencing was employed to do genetic screening in proband in each family. List of the genes included in this panel is provided as Table S1. In general, the test examined >95% of the target genes with sensitivity >99%.

| Segregation analysis
The

| RE SULTS
This study assessed a total of 9 ARNSHL Iranian families, 9 index cases and their 35 relatives, to confirm the diagnosis of the ARNSHL disease (  Possible causative variants in each family are summarized in Table 2. A total of 10 variants in 6 distinct genes (CDH23, GIPC3, MYO7A, TRIOBP, TECTA, and OTOF) in 9 recessive pedigrees ( Table 2) were identified. Among them, five variants were previously reported and the other 5 variants were novel. The 10 identified variants included 6 missense, 1 nonsense, 1 intronic, 1 frameshift, and 1 synonymous variant which predicted to affect on splicing by Human Splicing Finder (Table 3).
Sanger sequencing on available family members revealed that these variants segregate with the disease in each family (Table 2 and Figure 1). The in silico pathogenicity predictions for each variant using SIFT, Polyphen2, and MutationTaster software are shown in Table 3. We showed that this variant is segregating with the disease in this family by investigating her normal parents (I-1 and I-2) and her sister      improve speech and language development. 16,17 Nonsyndromic hearing loss is the second most common disorder after intellectual disability in Iran, affecting one in 16 individuals.

| D ISCUSS I ON
This relatively high incidence of hearing loss may be explained by high consanguinity rate in Iran. 18 Consanguineous marriage is frequent among Asian, African, and Latin American communities due to various factors such as their tradition, culture, and religion. Large pedigrees are also frequent in these communities. 19 Consanguineous marriage in Middle Eastern countries is ranging from 20% to 70%.
Iran with consanguinity rates of 38% of all marriages, ranging from 15.9% in the northern provinces to 47.0% in the eastern provinces, accounts as one of the countries with high levels of consanguinity. 5 Single gene autosomal recessive inheritance is responsible for the majority of hereditary hearing loss cases. 19 Consanguineous matings have long been known as a key etiologic factor in the prevalence of genetic disorders through making disease-causing recessive genes, inherited from a common ancestor, homozygous. In other words, the probability of inheritaning a similar deleterious recessive allele from both parents increases. 16 It is confirmed in various reports that the deafness is more common among children of consanguineous marriages. In two epidemiological Saudi Arabian surveys, the prevalence of SNHL has been shown to be 66% and 36.6%, respectively, out of which about 45% and 47% of the children had consanguineous parents. 19 In an Indian case-control study, the rates of affected children with consanguineous and nonrelated parents have been shown to be 48% and 28%, respectively. 17 Parental consanguinity was shown to be more common in Qatari families with hearing impaired patients compared to ones with normal hearing children, 60.5% versus 25.3%. 20 In a largescale study in Oman, it was found that 70% of the hearing impaired children had blood relative parents. 21 The parental consanguinity rate of hearing impaired patients was measured to be over 60% in several reports from Iran. [22][23][24][25] Nowadays with the advent of NGS, identification of molecular defects involved in HL has been accelerated.
We have studied nine Iranian families, comprising at least one affected individual with nonsyndromic bilateral autosomal recessive prelingual hearing loss. We assessed a total of 9 index cases and their 35 relatives, to confirm the diagnosis of the ARNSHL disease.
All deaf probands were born to consanguineous parents. Targeted   38 Previous studies have shown that a single amino acid change, even in nonconserved residues, in 1 C2 domain severely affects protein stability and localization. This could explain the profound deafness phenotype due to the severe effect of the splice site variant on C2C and downstream domains of the protein. 39 The OTOF gene (DFNB9) is mainly expressed in cochlear inner hair cells and is necessary for synaptic exocytosis at the auditory ribbon synapse.
Because of the expected good outcomes of cochlear implantation for patients with OTOF mutations, it is important to perform mutation screening for OTOF to select the appropriate intervention. 40 To date, more than 100 pathogenic variants including missense, nonsense, frameshift, splice site, deletion, and duplication have been found in various populations, nearly a third of which are from the Middle East, especially Pakistan and Turkey. Studies in Iran suggested that ARNSHL due to OTOF gene mutations ranges from 0.7% to 2.6%.
There are few reports of splice site mutations. 39 Hearing loss due to OTOF mutations is characterized by abnormal inner hair cell function and dyssynchrony of neural transmission of the auditory signal from the inner ear to the auditory nerve and brainstem. 41 The OTOF gene, located on chromosome 2p23.1, encodes a membrane-anchored cytosolic protein, otoferlin, with several isoforms. 42,43 It is believed that variants affecting the long isoform of this gene cause ARNSHL. 41 The long isoform, which is thought to be required for normal hearing, involves 48 coding exons which contains six C2 domains (C2A-C2F) and a transmembrane domain (TM). 43,44 Otoferlin plays a role in the calcium-dependent fusion of vesicles to the plasma membrane. 43

| CON CLUS ION
In this study, a total of 10 variants in the patients were identified.
Among them, five mutations were previously reported and the other five variants were novel. Accurate identification of causative mutations plays a key role in affected families to offer them preimplantation genetic diagnosis (PGD), prenatal diagnosis (PND), or further therapy strategies. Besides finding more mutations and new genes provides the possibility to do further studies on the pathophysiology of this disease and identify the involved pathways and mechanisms.

ACK N OWLED G M ENTS
We thank the families for their contribution to this study. This research received no specific grant from any funding agency, commercial, or not for profit sectors. Masoud Garshasbi https://orcid.org/0000-0002-5508-7903