Relevance of Connexin 26 (GJB2) Gene Mutations With Congenital Nonsyndromic Sensorineural Hearing Loss in Lraqi Deafness Patients.

Objective: This study aimed to detect the frequency of the three most common mutations of GJB2 in nonsyndromic sensorineural deafness for Iraqi population. Method: The current case-control study was conducted from January 2018 to November 2019 at ENT Departments from middle Euphrates region of Iraq. The study was included 95 deaf patients group (55 males and 40 females) their age range between 11-40 years old and 21.5 ± 6.3 year (mean ± SD). and 110 healthy control group, their ages range between 10-40 years old and 20.1 ± 5.9 year (mean ± SD), these two groups were matched in age and gender. In order to detect c.35delG, 235delC and 167delT mutations in GJB2 gene, we were employed the polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) technique. Results: From 95 deaf patients with non syndromic hearing loss (NSHL), were participated in this study. The c.35delG was the main frequent mutation encountered with GJB2 gene, of the 95 patients, 38(40%) were heterozygous and the others 57(60%) were homozygous genotypes. The second degree mutation in GJB2 gene was c.235delC mutation. Which from the 95 deaf patients, 35 (36.8%) were carried out homozygous, 5 (5.3%) were carried out heterozygous and 55(57.9) of the study individuals were appeared wild genotypes. None of the 95 deaf patients were showed the c.167delT mutation, while Connexin 26 studied mutations were not detected in healthy control group. Conclusion: Our data conclude that the GJB2 c.35delG and c.235delC gene mutations were the main cause of congenital hearing loss in Iraqi deaf population.


Introduction
Deafness is the complete or partial loss of hearing ability, which affects 6 -8% in the populations of developing countries in any ages and with different levels of severity. It is consequent to societies and individuals, which are highly affected by the age of manifestation and its severity. When it appears early in childhood and comprises severe defect, it causes signi cant profound limitations of speech aquizition and following by psychosocial defects. While in elderly, it affects the life features by the social isolation of an individual (1) .
Congenital Hearing loss is the most common form of sensory defect in human beings, with about 1/1000 infants born with a severe hearing de cit (2) . Inherited hearing loss was divided into two types: the 1st type is syndromic and the 2nd type is non syndromic. The syndromic type, which accounts 30% of inherited congenital deafness , is associated with other clinical characteristics . The non-syndromic type, which represents the other 70%, is only clinical appearance of deafness (3) . Regarding to the non-syndromic hearing loss (NSHL), autosomal recessive is the major common form that accounting 75% to 85% of deaf cases (4,5) .
More than 100 genes may be implicated in NSHL, but the higher risk of mutations were found in the protein of gap junction, connexin 26 (GJB2). Which is one of a large family of proteins that forms gap junction in close to every cell type. This junction is consisted of multimeric connexons, that allows the molecules to pass from cell to another cell. The connexons are contained of connexins, which are differ in cell speci city and their properties of gating (6) .
The gene that encodes the gap junction protein GJB2 was located on chromosome 13q11-12 and was reported to have up to 50% mutations in all patients from various populations. In spite of numerous mutations were recognized (35delG, 235delC, 167delT, V37I, 109G-A, etc), but 235delC was found to be the most common GJB2 mutation in East Asians populations, c.35delG in Caucasians and 167delT predominant in Ashkenazi Jews (7)(8)(9)(10) .
There is no molecular study of GJB2 gene was recorded in Iraqi population with non syndromic sensorineural hearing loss. for that reason, this study was aimed to investigate the common c.35delG, c.235delC and c.167delT mutations in the gene of connexin 26 (GJB2) in Iraqi deaf patients.

Patients And Methods
The current case-control study was conducted from January 2018 to November 2019 at ENT Departments from middle Euphrates region of Iraq. The study was included the consent forms approved by medical ethics committee in Faculty of Medicine, Kufa University, Iraq. The sample size was calculated by using the online software OSSE (online sample size estimator) (11) . Ninety ve patients were diagnosed with bilateral nonsyndromic sensorineural hearing loss (NSHL), they were (55 males and 40 females) their ages between 11-40 years old and 21.5 ± 6.3 year (mean ± SD). Among them, 75 were sporadic cases of hearing loss, compatible with recessive inheritance while the other 20 probands were from families with more than one sib of non-syndromic hearing loss, age and gender were matched with control group which were consisted of 110 voluntary individuals unrelated to patients, they were selected from general population and apparently healthy, their ages ranged between 10-40 years old and 20.1 ± 5.9 year (mean ± SD). Only individuals free from signs and symptoms of any chronic diseases such as DM, cardiac diseases, hypertension, renal diseases or others were selected to participate in this study.
Consent information was obtained from all participants relatives and patients younger than 18 years from their parents. Assessment was included a complete case history, using Pure Tone Audiometry and physical examination, the inclusion criteria was the ultimately nding of examinations with NSHL.While the exclusion criteria was acquired hearing loss related to environmental causes and patients who diagnosed with syndromic hearing loss were excluded from current study.

Audiology
All study participants underwent pure-tone audiometry by using diagnostic audiometer instrument (Siemens Danplex DA 74 clinical diagnostic audiometer, USA) in a soundproof room. Which averages pure-tone of more than 25 dBHL (dBHL at 500, 1000, and 2000 Hz) were de nite as hearing loss according to the hearing loss classi cation (12) . Profound 91 to equipment limits (12) The hearing impairment degrees were ranged from moderate to profound in current study.

Mutation Analysis
One ml of venous blood was collected from all individuals participated in this study. Genomic DNA was isolated and puri ed from peripheral blood lymphocytes by using ReliaPrep™ Blood gDNA Miniprexp System (Promega) according to the standard protocol of the manufacturer.
The DNA was extracted then used in the screening of selected mutations. Polymerase Chain Reaction-Restriction fragment length polymorphism (PCR-RFLP) analyses were applied to detect the three GJB2 common mutations that described previously and as appeared in Table 1 Wild type PCR products were cut by the enzymes in c.35delG and c.235delC genes and showed two bands in gel electrophoresis, whereas 167delT mutated gene was uncut and produced single band when applied by electrophoresis in 2% agarose gel stained with ethidium bromide, and the results were recognized by gel documentation instrument.

Results
The study was involved three mutations in connexin 26 (GJB2) gene related with non syndromic autosomal recessive deafmute in 95 patients. Among them, 55 (57.9%) were males and 40 (42.1%) were females. The results of questionnaire were appeared that endogamy marriage between parents of deaf patients is more common which accounts (80%), and there is a family history in the mother's family (39%) or the father's family (42%). As well as, 89/95 (94%) of the patients' parents have normal hearing. The presented data for ve of remaining sex patients was appeared that their paternal and maternal grandparents have normal hearing. hence, from the interview outcomes were considered all patients as having autosomal recessive non syndromic hearing loss.
A total of 95 deaf patients with NSHL who recruited in this study, were included 75 simplex proband which refers to sporadic patients whose families have no one suffering from hearing loss and 20 multiplex proband which means that there is at least one rst or second degree deaf relative in their families. Clinical characteristics of deaf patients were summarized in Table 2. Hearing tests con rmed that the level of hearing loss was severe to profound in 91 patients. The remaining 4 patients showed moderate hearing loss as appeared in Table 2.
The c.35delG was recorded as the mainly frequent encountered mutation in GJB2 gene, all ninety ve patients were appeared clearly mutation in this gene either heterozygous genotype (one allele mutant copy) or homozygous genotype (two allele mutant copies). Of the ninety ve deafness sensorineural patients, thirty eight (40%) were found to have heterozygous genotype while the other fty seven (60%) were appeared homozygous genotype of the tested mutation as in gure (1).
On the other hand, c235delC mutation was represented as the second degree mutation in GJB2 gene. Where from the ninety ve deaf patients, thirty ve (36.8%) were carried out homozygous genotype (two allele mutant copies) and ve (5.3%) were carried out heterozygous genotype (one allele mutant copy). However, 55(57.9) of the study individuals were showed wild genotype for the tested mutation as in gure (2).
None of the 95 deaf patients showed the c.167delT mutation. As well as there were no mutations appeared in control group. The frequency of the detected mutations is summarized in Table 3.

Discussion
The GJB2 gene which encodes connexins 26, these proteins are dependent in both terms of expression and localization. Within the cochlea, connexins 26 form heterometric gap junctions which obviously contribute to the up keeping homeostasis of cochlea. The mutation of this gene as heterozygous or homozygous form in uenced on the actions of the gap junctions and therefore prevent the conversion of the mechanical signal to the electrical signal. Hence deafness was installed as symmetrical and bilateral, most often severe or profound (13) .
This study was investigated three GJB2 mutations in NSHL from middle Euphrates region of Iraq. The distribution of connexin 26 (GJB2) mutations widely differs among ethnicities. The results were revealed that the c. 35delG was evident in 65 (68.4%) which was agreed with (70%) prevalence of the c.35delG mutation recorded in Cuba and (74%) in Spain for studying patients by Yenitse et al from Cuba and Spain (14) . As well as, The GJB2 c.35delG mutation was recorded in (60%) of North European and Turkish who suffer from hereditary deafness (15,16) . It was arranged among the highest mutation in GJB2 when compared with its frequency in other Arabic populations rates. The GJB2 mutations account in Algeria 40%, in Lebanon 33.3%, in Palestine 23%, in Tunisia 17% and in Jordanian 16.9%. However, this mutation very rare in Asian patients and it is frequently encountered GJB2 mutation in Caucasians (17) .
Another less frequent frameshift mutation, c.235delC was reported in this study. Which detected in 40(42.1%) from all NSHL patients including 35 (36.8%) homozygous and 5 (5.3%) Heterozygous genotypes of mutant alleles. The c.235delC mutation was found as the most common mutation cause premature protein termination in patients suffering from hearing loss in East and Southeast Asia, while lower frequencies was recorded in Oceania and Europe (18)(19)(20) . It was also presented in Japanese, Korean, and Mongolian populations (8,(21)(22)(23) . for that reason, mutation in GJB2 was a signi cantly contributed to the recessive inheritance NSHL in the Chinese population, as also appeared in another ethnic group (24) .
The third mutation c.167delT in GJB2 gene, which was not appeared in our study subjects, was mainly presented in Ashkenazi Jews (7) . Also, this mutation was detected in a Palestinians group from Bethlehem (2) , which representing that the variant of GJB2 mutant allele frequency also may be marked in the groups of the similar population.
Number of studies demonstrate a shared sources of the mutation in GJB2, such as the R143W mutation in a numerous families in a Ghanaian village and the 167delT mutation in Ashkenazi Jews (7,25) .
The variation between various studies in type of GJB2 mutations and terms of frequency associated with NSHL may be due to several causes that include: sample size (large sample size increase the probability for detecting rare mutations), selection criteria of the patients investigated, accuracy of genotyping method that employed and consanguineous marriage rate.

Conclusion
Connexin 26 (GJB2) , c.35delG and c.235delC gene mutations was a causative agent for congenital hearing loss in Iraqi population. These data can support the screening programs of audiometric recognizing neonates with congenital inherited hearing loss in Iraqi endogamy parents.      Restriction analysis by ApaI enzyme for PCR product of c.235delC mutation in GJB2 gene in 2% agarose gel electrophoresis. Lane M: DNA Ladder. Lane 1, 2, 3, 5, 6, 7, 9 and 10 are wild genotype and Lane 4, and 8 are homozygous genotype of mutant alleles.