The principles of molecular diagnosis of recessive forms of prelingual non-syndromic hearing loss

The GJB2 gene defects are the most frequent cause of autosomal recessive non-syndromic hearing loss (DFNB1). Epidemiological data suggest that 35delG is the most prevalent mutation found in 88% of mutated alleles. Another mutations - 313del14 was found in 7% of mutated alleles. The other mutations were identified only in single families. Following the analysis of distribution of GJB2 mutations in the Polish population we propose an algorithm for molecular diagnosis of DFNB1. We propose to screen all patients affected with prelingual non-syndromic deafness for 35delG mutation using ASO or multiplex AS-PCR methods. The presence of 35delG on two alleles confirms DFNB1. The identification of heterozygous 35delG mutation requires additional GJB2 analysis including 313del14 mutation detection and en exon 2 direct sequencing. To determinate the frequency of digenic (GJB2/GJB6) background of DFNB we screened 17 patients with heterozygous 35delG mutation for deletion of 342 kb in GJB6 gene. No such mutation was detected in the analyzed group.     

GJB2 and GJB6 mutations in children with congenital cytomegalovirus infection

Congenital cytomegalovirus (CMV) infection is a leading cause of sensorineural hearing loss (SNHL) in children. Whether connexin mutations are factors in the development of CMV-related hearing loss has not been explored. We examined gap junction protein beta-2 (GJB2) and gap junction protein beta-6 (GJB6) mutations in 149 children with congenital CMV infection and 380 uninfected neonates. Mutations in GJB2 and GJB6 were assessed by nucleotide sequencing and polymerase chain reaction (PCR) methods, respectively. The study population was predominantly African American, and 4.3% of the subjects were carriers of a connexin 26 mutation. The overall frequency of GJB2 mutations was significantly higher in the group of children with CMV infection and hearing loss (21%) compared with those with CMV infection and normal hearing (3%, p = 0.017) and the group of uninfected newborns (3.9%, p = 0.016). Eight previously reported mutations (M34T, V27I, R127H, F83L, R143W, V37I, V84L, G160S), and four novel mutations (V167M, G4D, A40T, and R160Q) were detected. None of the study children had the 342-kb deletion (delGJB6-D13S1830) in GJB6, which suggests that this mutation does not play a role in hereditary deafness in the African American population. Although GJB2 mutations were detected in children with and without CMV-related hearing loss, those with hearing loss had a higher frequency of GJB2 mutations.     

GJB2基因及其诊断筛查技术研究进展

耳聋基因GJB2定位于13q11-q12,编码connexin26(CX26)蛋白.在常染色体隐形遗传的非综合征性耳聋中,有50%的患者存在着GJB2基因的突变,然而在不同种族中,GJB2基因的突变位点也是不同的.35delG是欧美人群主要的突变形式;突变位点167delT在犹太耳聋人群中多见;而在亚裔人群中,235delC突变占有极大的比例.由于GJB2基因在遗传性耳聋中的特殊地位,因此对于GJB2基因的诊断及筛查技术就显得尤为重要.在新生儿听力筛查基础上,融入耳聋易患基因分子水平筛查,在早期发现和干预先天性听力损失方面发挥着重要作用.     

The R245X mutation of PCDH15 in Ashkenazi Jewish children diagnosed with nonsyndromic hearing loss foreshadows retinitis pigmentosa

Usher syndrome is a frequent cause of the combination of deafness and blindness due to retinitis pigmentosa (RP). Five genes are known to underlie different forms of Usher syndrome type I (USH1). In the Ashkenazi Jewish population, the R245X mutation of the PCDH15 gene may be the most common cause of USH1 (Ben-Yosef T, Ness SL, Madeo AC, Bar-Lev A, Wolfman JH, Ahmed ZM, Desnick RK, Willner JP, Avraham KB, Ostrer H, Oddoux C, Griffith AJ, Friedman TB N Engl J Med 348: 1664-1670, 2003). To estimate what percentage of Ashkenazi Jewish children born with profound hearing loss will develop RP due to R245X, we examined the prevalence of the R245X PCDH15 mutation and its carrier rate among Ashkenazi Jews in Israel. Among probands diagnosed with nonsyndromic hearing loss not due to mutations of connexin 26 (GJB2) and/or connexin 30 (GJB6), and below the age of 10, 2 of 20 (10%) were homozygous for the R245X mutation. Among older nonsyndromic deaf individuals, no homozygotes were detected, although one individual was heterozygous for R245X. The carrier rate of the R245X mutation among the normal hearing Ashkenazi population in Israel was estimated at 1%. Ashkenazi Jewish children with profound prelingual hearing loss should be evaluated for the R245X PCDH15 mutation and undergo ophthalmologic evaluation to determine whether they will develop RP. Rehabilitation can then begin before loss of vision. Early use of cochlear implants in such cases may rescue these individuals from a dual neurosensory deficit.     

Identification of an ancestral haplotype of the 35delG mutation in the GJB2 (connexin 26) gene responsible for autosomal recessive non-syndromic hearing loss in families from the Eastern Black Sea Region in Turkey

Mutations in the GJB2 gene have been shown to be the major cause of autosomal recessively inherited, prelingual, non-syndromic hearing loss. 35delG was found to be the most frequent mutation among Caucasians. In this study, we performed haplotype analysis of two large families with autosomal recessive non-syndromic hearing loss (totally 33 affected, 37 unaffected) from Trabzon (a city from the Eastern Black Sea region) by using polymorphic markers close to the 35delG mutation region, and identified a common haplotype, "2-6-4". The frequency of the mutant chromosomes having the 2-6-4 haplotype was compared between the Eastern Black Sea region and the other regions of Turkey and the difference was found to be significant (chi squared = 5.13/df = 1/p = 0.023). Also, when the frequency of mutant and wild type chromosomes having the 2-6-4 haplotype was compared in the Eastern Black Sea region, a statistically significant difference was observed in the mutant chromosomes (chi squared = 7.46/df = 1/p < or = 0.01). The results of this study demonstrate that the ancestral haplotype of the chromosomes bearing 35delG mutation in the Eastern Black Sea region is "2-6-4".     

西北地区751例新生儿耳聋基因突变筛查

目的初步了解西北地区新生儿常见耳聋基因突变类型和携带率,探讨耳聋基因突变筛查对于辅助诊断和防治新生儿遗传性耳聋的临床价值。方法采集西北地区751例新生儿的足跟血,采用15项遗传性耳聋基因检测试剂盒(微阵列芯片法)对中国人群常见4种耳聋基因15个突变位点进行筛查。结果在751例新生儿中,检测到39例新生儿携带耳聋基因突变,总突变携带率为5.18%。其中GJB2基因突变19例,突变携带率为2.53%;SLC26A4基因突变16例,突变携带率为2.12%;线粒体12SrRNA 1555A>G均质突变4例,突变携带率为0.53%。新疆出生的新生儿279例中,检测到耳聋基因突变11例,突变携带率3.95%;甘肃省出生的新生儿277例中,检测到耳聋基因突变12例,突变携带率4.32%;陕西省出生的新生儿178例中,检测到耳聋基因突变12例,突变携带率6.74%;青海省出生的新生儿17例中,检测到耳聋基因突变4例,突变携带率23.52%。结论西北地区新生儿耳聋基因突变携带率偏高,但是GJB2基因235 del C位点的突变携带率偏低,可能与西北地区的地域特征和人口遗传学特点有关。新生儿耳聋基因筛查对于听力筛查具有很好的互补作用,可以从基因水平发现可能出现迟发性耳聋和药物敏感性耳聋的高危新生儿。     

Connexin 26 <Emphasis Type="Italic">(GJB2)</Emphasis> Mutations Associated with Non-Syndromic Hearing Loss (NSHL)

Objective

To determine the prevalence and spectrum of Connexin 26 (GJB2) mutations in pre-lingual non-syndromic hearing loss (NSHL) patients in authors’ centre and to review the data of Indian patients from the literature.

Methods

Sanger sequencing of entire coding region contained in single exon (Exon 2) of GJB2 gene in 15 patients of NSHL.

Results

GJB2 mutations were found in 40% (6/15) of NSHL patients, out of which mono-allelic were 33.3% (2/6). Bi-allelic GJB2 mutations were identified in 4 of 6 patients. Most common GJB2 mutation identified was c.71G?>?A(p.W24X), comprising 30% of the total GJB2 mutant alleles. Six studies involving 1119 patients with NSHL were reviewed and 4 of them have reported c.71G?>?A(p.W24X) as the commonest mutation while 2 studies found c.35delG as the commonest. GJB2 mutations accounted for 10.9%–36% cases of NSHL. Sixteen other mutations in GJB2 gene were reported in Indian patients out of which 6 mutations other than c.71G?>?A(p.W24X) viz., c.35delG, c.1A?>?G(p.M1V), c.127G?>?A(p.V43 M), c.204C?>?G(p.Y86X), c.231G?>?A(p.W77X) and c.439G?>?A(p.E147K) were identified in the present study.

Conclusions

Connexin 26 (GJB2) mutations are responsible for 19.4% of NSHL in Indian population. The c.71G?>?A(W24X) and c.35delG were the most prevalent GJB2 mutations accounting for 72.2% (234 of 324 total mutated alleles from 7 studies) and 15.4% (50 of 324 total mutated alleles from 7 studies) respectively. Thus, screening of these two common mutations in GJB2 gene by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) would greatly help in providing easy genetic diagnosis and help in genetic counseling of the families with NSHL.

     

111 例未通过听力筛查新生儿常见耳聋基因突变位点分析

目的了解未通过听力筛查新生儿耳聋基因突变情况。方法随机选取听力筛查未通过、经听觉脑干诱发电位(ABR)测试为感音神经性耳聋患儿111例,收集足跟血血片,提取基因组DNA后,检测GJB2、SLC26A4和线粒体12 Sr RNA基因中的11个热点的突变,分析听力损失程度与突变的关联。结果 111例新生儿中,共检出携带耳聋基因突变24例(21.6%)。其中,GJB 2基因突变14例(12.6%),包括235 del C单杂合突变5例,235 del C和299_300 del AT复合杂合突变5例,以及235del C纯合突变、299_300del AT单杂合突变、176_191del16和235del C复合杂合突变、299_300del AT和508_511 dup AACG复合杂合突变各1例;SLC 26 A 4基因突变10例(9.0%),包括IVS 7-2 AG单杂合突变2例,1226 GA单杂合突变3例,2168 AG单杂合突变2例,IVS7-2AG和2168 AG复合杂合突变3例。本组耳聋患儿中未检出线粒体基因突变。结论未通过听力筛查新生儿中,超过1/5检测到聋基因突变,并以GJB2基因突变最常见,实施热点致聋基因检测可以提高耳聋的病因诊断率。     

新生儿重症监护病房新生儿耳聋基因致病变异携带者筛查的横断面调查

背景：我国耳聋发病率高与耳聋基因致病变异的携带率高有关,目前缺乏对NICU新生儿耳聋基因致病变异携带者的筛查数据。 目的：调查NICU新生儿中耳聋基因GJB2 和SLC26A4致病变异的携带率。 设计：横断面研究。 方法：纳入2016年1月至2021年12月在复旦大学附属儿科医院NICU住院、入院日龄≤28 d,且出院前完成高通量测序的新生儿,排除生后耳聋相关基因诊断阳性者。从病历系统中截取患儿的性别、胎龄、出生体重;从测序数据库中提取GJB2 基因和SLC26A4基因的检测结果、患儿人类表型标准用语信息。携带率（%）=杂合致病或可能致病（P/LP）变异例数/总研究对象人数。检索PubMed、Embase和万方数据库,纳入既往报道中国NICU人群、新生儿人群和孕妇人群中GJB2 基因和/或SLC26A4基因P/LP变异携带情况的文献,并行复习。 主要结局指标：GJB2 基因和SLC26A4基因的P/LP变异携带率。 结果：纳入14 924例新生儿,男8 587例（57.5%）,女6 337例,胎龄（35.6±3.7）周,出生体重（2 711.7±887.1）g。携带GJB2 基因P/LP变异的患儿2 009例（13.462%）,共检出18种杂合P/LP变异,其中c.109G>A最常见（10.902%）,其次为c.235del（1.749%）、c.299_300del（0.409%）、c.176_191del（0.154%）、c.508_511dup（0.074%）和c.257C>G（0.034%）。携带SLC26A4基因P/LP变异的患儿305例（2.044%）,共检出31种杂合P/LP变异,携带率最高的6种依次为c.919-2A>G（1.139%）、c.2168A>G（0.181%）、c.1226G>A（0.100%）、c.1229C>T（0.094%）、c.1174A>T（0.080%）和c.1003T>C（0.047%）。 结论：建议将GJB2 基因上的c.109G>A、c.508_511dup和c.257C>G以及SLC26A4基因的c.1003T>C位点纳入NICU新生儿耳聋基因致病变异携带者筛查。     

Nonsyndromic sensorineural deafness--analysis of etiology in relatives

We present the results of complex clinical examination of children affected with sensorineural hearing loss. The siblings (minimum two) were born from unaffected parents and came from twelve families. Molecular studies confirmed genetic background of hearing loss in 6 families and enabled identification of GJB2 mutations in investigated probants.     

Aspects génétiques de la surdité

Hearing loss is the most common sensory disability with an incidence of one over 1000 newborns. Hearing loss may be caused by environmental and genetic factors; inherited causes are assumed in two thirds of cases. There is a great clinical and genetic heterogenicity. All inheritance modes have been described. Mutations in the GJB2 gene, which encodes connexin 26, are mainly responsible for sensorineural deafness resulting in prelingual non syndromic autosomal recessive phenotypes DFNB1. The 35delG mutation of this gene is very frequent (70% of the cases). Thus, 35delG is, with the delta F508 mutation of the CFTR gene, the most frequent human pathogenic mutation known. Hearing loss might also be associated with other clinical features. Some of these syndromes, including hearing loss, have to be looked for systematically because of their frequency, of their possible clinical presentation as an isolated hearing loss and of the possibility of a medical treatment.     

Hypogonadotrophic hypogonadism associated with prelingual deafness due to a connexin 26 gene mutation

In Mediterranean countries, almost half the incidence of non-syndromic congenital hearing loss is caused by mutations in the gap junction (GJ) connexin 26 gene (GJB2/DFNB1 locus). In this form of deafness the cochlear defect is usually isolated. We describe here the first case of hypogonadotrophic hypogonadism in association with this particular cochlear defect. The male patient had moderate deafness inherited from his deaf parents. All family members had a homozygous 35delG mutation in the connexin 26 gene. This mutation accounts for 70% of all connexin 26 gene mutations. The patient was referred to a paediatric endocrinology unit at 11 years of age for moderate growth retardation. Growth rate was normal until 11 years. The patient then presented delayed puberty (testicular volume 4 ml, penis length 4 cm) and did not undergo the usual pubertal growth spurt. LH and FSH secretory responses to GnRH at the age of 14.5 years (bone age 13.5 years), were: LH baseline level 1.1 IU/l, peak 34 IU/l; FSH baseline level 1.8 IU/l, peak 5.7 IU/l. Testosterone concentration was <0.11 ng/ml. From 11 to 14 years old, testosterone concentration ranged from 0.11 to 0.2 ng/ml. Anti-Mullerian hormone (AMH) level was 38.6 ng/ml (normal for Tanner stage I), cortisol 109 ng/ml, and ACTH 37 pg/ml., Karyotype was 46 XY. On MRI analysis, the anterior pituitary and olfactory bulbs were normal. These data were consistent with partial hypogonadotrophic hypogonadism of hypothalamic origin, and the patient was treated with testosterone. This report supports the possible involvement of connexins in puberty initiation. Connexins may play a part in the co-ordination and synchronisation of GnRH release.     

Hearing loss in children with osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue. Progressive hearing loss is one of the principal symptoms of OI, affecting about 50% of adult patients. Hearing loss may also occur in childhood and results in additional disability in education and psychosocial adaptation and aggravates the physical handicap. This can be avoided by appropriate otological and audiological treatment. In a nationwide search, 254 Finnish patients with OI were identified indicating a prevalence of 4.9/100 000. Of the 60 children, 45 aged between 4 and 16 years accepting to participate the study on hearing, were evaluated by a questionnaire and clinical audiometry. Hearing loss was defined as pure tone average (PTA_{0.5–2 kHz}) more than 20 dB hearing level (HL). A clinical geneticist determined the type of OI among the 45 patients. Two sporadic OI cases with conductive hearing loss were ascertained (4.4%): An 11-year-old girl with type IV OI with a PTA_{0.5–2 kHz} of 35/40 dB HL and a 15-year-old boy with type IV OI with a PTA_{0.5–2 kHz} of 27/18 dB HL. In addition, a 6-year-old girl with familial OI type I had either a congenital sensorineural deafness or early progressive deafness with PTA_{0.5–2 kHz} of 97/103 dB HL, probably of unrelated aetiology. Conclusion Hearing loss in children with osteogenesis imperfecta is less frequent than generally suspected. Nevertheless, it is recommended that audiometry is performed in children with osteogenesis imperfecta even without symptoms of hearing loss at the age of 10 years, and repeated every 3 years thereafter. Received: 13 October 1999 and in revised forms: 20 December 1999, 13 January 2000 and 24 January 2000 / Accepted: 25 January 2000     

Genetic deafness in a preterm infant with a critical postnatal course.

OBJECTIVE: We present a case of deafness in a preterm infant with several predisposing factors of an acquired hearing impairment that, however, turned out to have a genetic cause. We describe the severe postnatal course and review the relevant literature. DESIGN: Case report. SETTING: University-based tertiary neonatal intensive care unit. PATIENT: Preterm infant (gestational age, 26/37; wks). MEASUREMENTS AND MAIN RESULTS: A preterm infant exhibited hearing impairment after a complicated clinical course with pneumothoraces, a hemodynamically relevant patent ductus arteriosus, treatment with potentially ototoxic drugs, intraventricular hemorrhage, and periventricular leukomalacia. Despite the absence of a family history for deafness, genetic testing was performed. Surprisingly, genetic analysis revealed the presence of two compound heterozygous mutations in the patient's GJB2 gene as the cause for his early-onset nonsyndromic deafness. CONCLUSION: To elucidate the nature of a hearing disorder, it is worthwhile to consider a genetic cause, despite the fact that it may seem unlikely in a severely sick preterm infant with numerous risk factors for a postnatally acquired hearing impairment and without a positive family history.     

Unauffälliges UNHS-Screening bei einem homozygoten Träger der 35delG-Mutation im Connexin 26 kodierenden gjb2-Gen

Background

Some cases of recessive nonsyndromic deafness are caused by a mutation in the connexin 26 (Cx26) coding GJB2 gene, mostly 35delG mutations in Western countries. Typically Cx26 mutation-associated hearing impairments are permanent, bilateral, and moderate to severe. However, some variations regarding these factors have been reported.

Case report

A male newborn child received a pass in a TEOAE-based universal newborn hearing screening (UNHS). Though the mother was told about the pass, she was suspicious that her child might be hearing impaired. In response further testing was carried out. A bilateral severe hearing impairment was confirmed by the age of 12 weeks with absent DPAOE and no ABR.

Discussion

Assuming a valid UNHS test result, this would be the first case of a rapid progression in a Cx26-associated hearing impairment. Considering the fact that some members of that family were known to be hearing impaired, a two-step screening (OAE/ABR) should have been favored in the first place. Anyway this case stresses the importance of a thorough and timely audiologic examination in cases of parental supposition that the child may not respond to acoustic stimuli properly.     

Hearing evaluation in children aged 3-6 years in day-care centers

OBJECTIVE: To estimate the prevalence of deafness in children 3-6 years old in day-care centers of S?o José do Rio Preto, SP, Brazil. METHODS: We used free-field audiometry to screen 103 children from 8-selected day-care centers. Children with abnormal examination results were referred to the speech therapy outpatient service to undergo pure-tone conventional audiometry. We adopted the WHO classification for degree of deafness. RESULTS: Audiometric dysfunctions were present in 10 children (9.70%; SD=0.96) with one boy showing left ear mild conductive hearing loss (mean LE=35 dB), while 9 children (8.73%; SD=2.78) showed alterations in air-conduction threshold in 4000, 6000 or 8000 sharp frequencies. Out of 102 children (99.03%; SD=0.96), 55 boys (53.39%; SD=4.9) and 47 girls (45.64%; SD=4.9) presented no hearing loss according to the WHO criteria. CONCLUSIONS: The prevalence of 9.7% audiometric dysfunctions found in this study indicates that deafness prevention programs should be organized.     

Genetic causes of nonsyndromic hearing loss

Explosive progress is being made in genetic studies of hearing and deafness from the clinical and basic research perspectives. Greater than half of hearing loss is estimated to have a genetic basis. Recent studies of hearing and deafness have identified a dozen genes that cause nonsyndromic hearing disorders. Deafness can be inherited in an autosomal recessive, autosomal dominant, X-linked, or mitochondrial manner. Mutations in one gene, connexin 26 (encoding the gap junction protein beta 2), may be responsible for half of all autosomal recessive nonsyndromic deafness. With new mandates for hearing screening programs for newborns in many states, for the first time, the new information on the genetics of hearing loss can be used to diagnose the cause of hearing loss in some children and to understand better the molecular biology of hearing.     

Auditory profile in children recovering from bacterial meningitis

In the present study BERA profile of 30 post-meningitic children was compared with 15 normal children of the same age and it was observed that 36.6% children in the age range of 6 months to 36 months were found to have varying degree of sensorineural deafness. Severe bilateral sensorineural hearing loss (>80 dB) was observed in 6.6% children and moderate (40–80 dB) hearing loss in 30% of children. Abnormalities were bilateral in both the samples of children with severe hearing loss (>80 dB) whereas among 9 children who had moderate hearing loss abnormalities were bilateral in one patient and unilateral in the remaining 8 children. A relationship between higher incidence of sensorineural deafness and younger age of children, and occurence of seizures during meningitis were noted. But no relationship was observed with either sex, hydrocephalus, subdural effusion or with low CSF sugar and high CSF proteins.     

Deafness: ever heard of it? Delayed recognition of permanent hearing loss

Records of approximately 1,000 children seen for evaluation of developmental delay during the period July 1979 to December 1985 were reviewed; 46 children with permanent hearing loss were identified. Age at diagnosis of deafness and factors contributing to delay in diagnosis were sought. Mean age at diagnosis of profound congenital deafness was 24 months. Lesser degrees of congenital hearing loss were not diagnosed until 48 months of age. High-risk medical history or physical anomalies associated with embryologic abnormalities of the auditory system that should have triggered a prompt search for deafness went unheeded in most instances. In 40% of subjects, the author was the first to diagnose hearing loss. For two thirds of this subgroup, audiologic referral was prompted by medical, physical, or developmental findings rather than clinically evident hearing loss during physical examination. Adherence to specific historical, physical, or developmental risk criteria, regardless of the examiner's subjective impression of how well the child seems to hear, would have permitted the timely diagnosis of hearing impairment in all children in this series.