Disease-associated variants of Gap Junction Beta 2 protein (GJB2) in the deaf population of Southern Punjab of Pakistan

Hearing impairment (HI) is a highly heterogeneous genetic disorder and is classified into nonsyndromic (without any other clinical manifestations) and syndromic (if combined with other clinical presentations) forms. Variations in GJB2 gene are the leading cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) in several populations worldwide. This study was carried out to investigate the prevalence of GJB2 variations in severe-to-profound hearing impaired families of Southern Punjab of Pakistan. Ten families segregating ARNSHL were recruited from different areas of the region. Sanger sequencing of GJB2 coding region was carried out. In two out of ten families, NM_004004:c.*71G>A (p.(Trp24*)) and NM_004004:c.358_360del (p.(Glu120del)) homozygous variants were identified as the cause of hearing loss. Our study showed that GJB2-related hearing loss accounts for at least 20% of all cases with severe-to-profound hearing loss in the Southern Punjab population of Pakistan.


Introduction
Deafness is a prevalent sensory disorder affecting 1 in 650 infants worldwide, with a prevalence of 3.5/1000 in teenagers and 2.7/1000 in children making it the most common hereditary sensory impairment [1].
It is a multifactorial disorder associated with environmental and genetic causes. About 50-60% of deafness is due to genetic factors. Clinically, deafness can be classified into nonsyndromic and syndromic deafness. Nonsyndromic deafness is isolated deafness with no other clinical manifestations, while syndromic deafness is associated with other metabolic or physiological conditions. Nonsyndromic deafness has a prevalence of 70% and is highly heterogeneous [2]. In case of nonsyndromic deafness, the most common inheritance pattern is autosomal recessive. Until now, 87 autosomal recessive nonsyndromic loci and 77 genes have a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 been identified. Out of 87 DFNB loci and 77 genes, 45 loci and 31 genes were localized in Pakistani families (https://hereditaryhearingloss.org/recessive-loci). Consanguineous marriages are very common in Pakistan, making this population a valuable source for studying genetic disorders [3]. Moreover, it is observed that the rate of hearing loss in Pakistan is about 1.6/1000 live births, greater than the global prevalence i.e., 1 in 1000 live births [4]. It is also estimated that up to 50% of hearing loss is due to GJB2 variants in prelingual deafness [5]. In Pakistan, more than 50% ARNSHL is associated with GJB2 [6]. It is found that genetic alterations in GJB2 are also the most primary cause of nonsyndromic hearing loss in South Asia [7]. Up to 38% of the GJB2 frequency has been reported in north of Iran [8]. Several variations in the GJB2 gene have been detected and reported; some are recurrent variations while others are not so prevalent. The variant spectrum diverges significantly among populations and demonstrates ethnic biases, e.g., c.35delG is common among caucasoids with 1 in 51 carrier rate [9] while carrier rate of c.235delC is 1-2% in the Japanese [10]. Carrier rate of 7.5% of c.167delT is reported in the Ashkenazi Jews [11] and carrier rate of 11.6% of p.Val37Ile in Taiwan [12]. The overall prevalence of GJB2 variants in different populations worldwide is significantly high, thereby highlighting the clinical significance of this gene for genetic testing.
Southern Punjab is a loosely defined territory in Punjab Province of Pakistan that encompasses the civil divisions of Bahawalpur, Multan, and Dera Ghazi Khan. It accounts for around 52% of the province's total land and 32% of its population. It has a population of more than 34,743,590 inhabitants. To the best of our knowledge, the prevalence of GJB2 in hearing impaired population of Southern Punjab of Pakistan has not been studied. Therefore, the present study was conducted to determine the prevalence of GJB2 gene in Southern Punjab of Pakistan. Sequencing of coding exon of GJB2 revealed two disease-associated variants; NM_004004:c. � 71G>A (p.(Trp24 � )) in NKDF01 and NM_004004:c.358_360del (p.(Glu120del)) in NKDF08. This research will help in genetic counselling of these families to avoid carrier to carrier or carrier to affected marriages, which will result in a decrease in the deaf population in Southern Punjab. Moreover, GJB2 variants profiling for the hearing impaired population of Southern Punjab of Pakistan will be a valuable resource for the development of molecular genetic screening tests in the future.

Cohort ascertainment
This research work was approved by the Institutional Research Ethics Committee (IREC) of Govt. College University Faisalabad, Pakistan. For this study, 10 punjabi severe to profound deaf families with consanguineous marriages were recruited from Dera Ghazi Khan and Rajanpur districts of South Punjab of Pakistan. Written consent forms were signed by all participants after receiving information about the study. A total of 37 deaf individuals and 72 normal siblings/parents were enrolled for GJB2 sequencing.

Phenotype characterization
Multiple family members, including elders, were interviewed to obtain medical history and rule out the environmental and syndromic deafness. A physical examination was also carried out to confirm signs and symptoms of night blindness, goiter and skin pigmentation for some of the affected participants. Two affected and one normal individual from each family were subjected to otoscopic examination and pure tone audiometry was performed at various threshold levels ranging from 250 Hz to 4 kHz (Table 1). Tandem gait and Romberg tests were performed to assess the vestibular function in two affected and one normal individuals of each family.

Genetic analysis
Genomic DNA extraction. 10 ml of blood was collected from each person that was properly labelled and stored in 50 ml of Sterilin 1 polypropylene tubes containing 400 ul of 0.5M EDTA. The genomic DNA was isolated from all blood samples using the non-organic method [13]. Isolated genomic DNA's quantity and quality was determined by gel electrophoresis and spectrophotometer (Bio-Rad, Hercules, CA).
Sanger sequencing. DNA of one affected from each pedigree was subjected to Sanger sequencing. Primers (5'-TGTGCATTCGTCTTTTCCAG-3' and 5-'GGGAAATGCTAGCGACT GAG-3') were designed, synthesized, and were subsequently used for amplification of the coding exon of GJB2. ExoSAp treatment was applied to the amplified products and sequencing was done using the Big Dye (Big Dye Terminator v3.1 Cycle Sequencing Biosystems 1 Kit). Genetic Analyzer 3730 (Applied Biosystems Inc) was used to run the sequencing products and data was analyzed with Chromas software version 2.6.6 (Technelysium Pty Ltd).
Physical examination did not exhibit the signs and symptoms of skin pigmentation, goiter and night blindness in any individual of this family. Furthermore, deafness in this family was not segregating with any other abnormality. No vestibular dysfunction was observed by tandem gait and rhomberg tests. Pure tone audiometry at 250, 500, 1000, 2000 and 4000 Hz for individuals IV:9 and IV:10 revealed severe to profound hearing loss (Fig 2a).
Sanger sequencing of GJB2 gene identified a recurrent disease associated variant NM_004004:c. � 71G>A (p.(Trp24 � )) in deaf individuals of this family (Fig 1a). This nonsense variant was segregating in all the affected individuals of the family.
Hearing impairment was segregating without any other anomaly in the affected persons of this family. The signs and symptoms of goiter, skin pigmentation and night blindness in any individual of this family were not recognized. Moreover, tandem gait and romberg tests did not reveal the vestibular dysfunction. Pure tone audiometry demonstrated a severe to profound hearing loss in individuals IV:10 and IV:11 at 250, 500, 1000, 2000 and 4000 Hz (Fig  2b). Sanger sequencing revealed an in-frame deletion of three nucleotides i.e., c.358_360del (p. (Glu120del)) segregating with disease phenotype in this family (Fig 1b).

Discussion
Variations in GJB2 are the leading cause of hearing loss in Pakistani families [6]. Different studies had reported that c. � 231G>A (p.Trp77 � ) and c. � 71G>A (p.Trp24 � ) are the most prevalent variants of GJB2 gene in Pakistani population [14][15][16]. Our results are also consistent with the previously published data which suggests that GJB2 sequence variations are the primary contributor of deafness in Pakistani individuals. We did not perform the CNV analysis on the deaf families, however sanger sequencing revealed a c. � 71G>A (p.(Trp24 � )) change in NKDF01 family out of ten severe to profound hearing loss families. Hence, suggests a prevalence of 10% in this population. p.Trp24 � frequency is also high in the Indian population reaching up to 95% [17][18][19]. Previously, it has been reported that both moderate to severe and profound hearing loss are associated with p.Trp24 � in the Pakistani deaf population [14,20]. Hearing thresholds for affected individuals IV:9 and IV:10 of NKDF01 are also consistent with previous findings (Fig 2a). Another GJB2 variant c.358_360del (p.(Glu120del)) was observed in NKDF08 family, which exhibited a prevalence of 10% in Southern Punjab. Previously, this change was reported in only one Pakistani family [14]. However, it is the third and second most common change in Iran and Turkey respectively [21,22]. Moreover, phenotypic variability of mild and profound hearing loss was noticed in p.(Glu120del) individuals [23]. The common change in the Southern Punjab of Pakistan may be p.(Glu120del), but further sequencing from this region is required to confirm it. Because only two families were diagnosed with GJB2 variants, the remaining families will be investigated further and subjected to exome sequencing to identify the causative genes.

Conclusions
Our results suggest that the prevalence of GJB2 related hearing loss in severe to profound deaf families is high in Southern Punjab, i.e., 20%. Sequencing of coding exon of GJB2 revealed two disease-associated variants; NM_004004:c. � 71G>A (p.(Trp24 � )) in NKDF01 and NM_004004:c.358_360del (p.(Glu120del)) in NKDF08 out of ten families. As published earlier, p.Trp24 � is the common change in the Pakistani families, while p.Glu120del has been identified in only one Pakistani family. Determination of reported variants along with other frequent variants will help in genetic counselling and family planning for these families, which will result in a decrease in the deaf population in Southern Punjab. Early detection of common GJB2 variants in infants will enable us to adopt the multiple interventional strategies in time. Moreover, GJB2 variants profiling for the hearing impaired population of Southern Punjab of Pakistan will be a valuable resource for the development of molecular genetic screening tests in the future.
Supporting information S1 Table. Clinical manifestation in families subjected to GJB2 sequence analysis.