GJB2 Research Articles

Charcot-Marie-Tooth disease: genetic subtypes in the Sardinian population.

Charcot-Marie-Tooth disease (CMT) is characterised by great variability of genetic subtypes. This study aimed to assess the genetic subtypes of CMT disease in the Sardinian population. Genetic screening was performed for CMT cases (CMT1, CMT2, and hereditary neuropathy with susceptibility to pressure palsies [HNPP]). A total of 1,043 subjects (119 index cases) were evaluated. In CMT1 index cases (69/119; 58%), PMP22 duplication at 17p11.2 was the most frequent genetic diagnosis (60/69; 87%), followed by mutations in the GJB1 gene (5/69; 7.2%), in the SH3TC2 gene (3/69; 4.4%) and PMP22 Gly107Val point mutation (1/69; 1.4%). The CMT2 group (24/119; 20.1%) comprised 10/24 (41.6%) patients carrying MPZ gene Ser44Phe mutation, 6/24 (25%) with mutations in MFN2 and HSPB1, and 1/24 (4.2%) in GJB1 and LRSAM1. In the HNPP group (26/119; 21.9%), the majority of patients reported the PMP22 deletion (25/26; 96.2%). Further studies are needed to comprehend the overall picture of the disease in Mediterranean area.

Phenotypes and cellular effects of GJB1 mutations causing CMT1X in a cohort of 226 Chinese CMT families.

The aim of this study is to explore the phenotypic and genotypic features of X-linked Charcot-Marie-Tooth (CMT) disease in the mainland of China and to study the cellular effects of six novel Gap junction protein beta-1 variants. We identified 25 missense and 1 non-sense mutations of GJB1 in 31 unrelated families out of 226 CMT families. The frequency of GJB1 mutations was 13.7% of the total and 65% of intermediate CMT. Six novel GJB1 variants (c.5A>G, c.8G>A, c.242T>C, c.269T>C, c.317T>C and c.434T>G) were detected in six unrelated intermediate CMT families. Fluorescence revealed that HeLa cells transfected with EGFP-GJB1-V74M, EGFP-GJB1-L81P or EGFP-GJB1-L90P had diffuse endoplasmic reticulum staining, HeLa cells transfected with EGFP-GJB1-L106P had diffuse intracellular staining, and HeLa cells transfected with EGFP-GJB1-N2S had cytoplasmic and nuclear staining. The distribution of Cx32 in HeLa cells transfected with EGFP-GJB1-F145C was similar to that of those transfected with wild-type (WT). These six variants resulted in a higher percentage of apoptosis than did WT as detected by flow cytometry and Hoechst staining. In conclusion, mutation screening should be first performed in intermediate CMT patients, especially those with additional features. The novel GJB1 variants c.5A>G, c.8G>A, c.242T>C and c.269T>C are considered pathogenic, and c.317T>C and c.434T>G are classified as probably pathogenic.

Golgi-retained Cx32 mutants interfere with gene addition therapy for CMT1X

Numerous GJB1 gene mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X). GJB1 encodes connexin32 (Cx32), which forms trans-myelin gap junctions in Schwann cells. Most GJB1 mutations result in loss-of-function mechanisms, supporting the concept of gene replacement therapy. However, interactions between delivered wild type and endogenously expressed mutant Cx32 may potentially occur in the setting of gene replacement therapy. In order to screen for possible interactions of several representative CMT1X mutants with wild type Cx32 that may interfere with the functional gap junction formation, we established an in vitro screening method co-expressing in HeLa cells wild type Cx32 and one of eight different Cx32 mutants including A39P, A39V, T55I, R75W, M93V, L143P, N175D and R183S. Some of the Golgi-retained mutants hindered gap junction plaque assembly by Cx32 on the cell membrane, while co-immunoprecipitation analysis revealed a partial interaction of wild type protein with Golgi-retained mutants. Dye transfer studies confirmed that Golgi-retained R75W, M93V and N175D but not endoplasmic reticulum-retained T55I had a negative effect on wild type Cx32 function. Finally, in vivo intraneural delivery of the gene encoding the wild type Cx32 in mice bearing either the T55I or R75W mutation on Cx32 knockout background showed that virally delivered protein was correctly localized in mice expressing the endoplasmic reticulum-retained T55I whereas it did not traffic normally in mice expressing the Golgi-retained R75W. Thus, certain Golgi-retained Cx32 mutants may interfere with exogenously delivered Cx32. Screening for mutant-wild type Cx32 interactions should be considered prior to planning gene addition therapy for CMT1X.

Clinical and epidemiological characteristics of hereditary motor-sensory neuropathy 1X caused by the mutation c. 259C> G (p. P87A) in the GJB1 gene of patients from the Republic of Bashkortostan

Hereditary motor-sensory neuropathy 1X (НМСН 1X) is the second frequent form of hereditary motor-sensory neuropathies caused by mutations in the GJB1 gene (gap junction B1 type). The authors have established earlier that the с.259C>G (р.P87A) mutation is the most frequent cause of НМСН 1Х (92%) in patients from the Republic of Bashkortostan. To study in details the territorial ethnic distribution and clinical manifestations of the с.259C>G (р.P87A) in the GJB1 gene in patients with НМСН 1Х from the Republic of Bashkortostan. Clinical/neurological data were assessed in 52 patients (32 men and 20 women) from 13 families with this НМСН 1Х mutation in accordance to the diagnostic criteria of the European neuromuscular center. Twenty-three patients underwent standard electroneuromyographic study ('Nicolet Viking quest') using cutaneous electrodes. Data analysis was performed with Statistica ver.6.0 ('Stat Soft, Inc.', 2003) software. The с.259C>G (р.P87A) mutation was more frequent in Bashkir (61%) and Russian (31%) families from 6 areas of the Republic of Bashkortostan. The age-at-onset was 13.24±4.33 years in men. In women, the age-at-onset varied from 7 to 45 years, it was difficult to detect this parameter in several patients due to the absence of complaints and symptoms of disease. A comparative analysis revealed the higher degree of peripheral nerve lesions in men compared to women. There was the distinct difference in electrophysiological parameters (excitation spreading velocity and M-response amplitude) along motor fibers of the middle nerves between men and women that indicated the predominantly demyelinating character of the pathological process in men and the axonal character in women. Clear clinical/electrophysiological sex differences (intra- and inter family) were shown in patients with НМСН IX with the с.259C>G (р.P87A) mutation in the GJB1 gene. The disease was less severe and often with the absence of symptoms in women. Genetic testing for mutations in the GJB1 gene, including the с.259C>G (р.P87A) mutation, can be recommended to female patients with excitation spreading velocity >38m/s.

Clinico-Electrophysiological and Genetic Overlaps and Magnetic Resonance Imaging Findings in Charcot-Marie- Tooth Disease: A Pilot Study from Western India.

Background:Charcot–Marie–Tooth (CMT) disease is clinically and genetically heterogeneous. There are no published series describing clinical, electrophysiological, and genetic information on CMT from the Indian subcontinent. Magnetic resonance imaging (MRI) neurography technique provides useful information about the plexus and roots and can be employed in patients with CMT.Settings and Design:A prospective, observational study carried out at a tertiary care hospital in Western India.Subjects and Methods:CMT patients fulfilling the UK Genetic Testing Network criteria were included. They underwent clinical, electrophysiological, radiological, and multigene panel testing.Results:Totally 22 patients (19 males, 3 females; 18 sporadic and 4 familial cases) were studied. Pes cavus (19), hammer toes (16), and scoliosis was seen in 1 patient. Electrophysiology revealed motor predominant neuropathy with 15 demyelinating (10 uniform and 5 multifocal) and 7 axonal patterns. Thickened lumbosacral plexuses on MRI neurography were evident in 6/10 studied patients, all 6 having demyelinating neuropathy. Genetic analysis identified PMP22, GJB1, SH3TC2, HSPB1, SPTLC2, MPZ, AARS, and NEFH gene mutations.Conclusions:This small series documents the pattern of CMT neuropathies as seen in Western India. Clinico-electrophysiological and genetic diagnosis showed general concordance some overlaps and reiterated advantages of gene panel testing in this heterogeneous group of neuropathies. MRI neurography was useful as an additional investigation to detect nerve enlargement in patients with demyelinating neuropathies.

Heterogeneity of Hereditary Hearing Loss in Iran: a Comprehensive Review.

A significant contribution to the causes of hereditary hearing impairment comes from genetic factors. More than 120 genes and 160 loci have been identified to be involved in hearing impairment. Given that consanguine populations are more vulnerable to most inherited diseases, such as hereditary hearing loss (HHL), the genetic picture of HHL among the Iranian population, which consists of at least eight ethnic subgroups with a high rate of intermarriage, is expected to be highly heterogeneous. Using an electronic literature review through various databases such as PubMed, MEDLINE, and Scopus, we review the current picture of HHL in Iran. In this review, we present more than 39 deafness genes reported to cause non-syndromic HHL in Iran, of which the most prevalent causative genes include GJB2, SLC26A4, MYO15A, and MYO7A. In addition, we highlight some of the more common genetic causes of syndromic HHL in Iran. These results are of importance for further investigation and elucidation of the molecular basis of HHL in Iran and also for developing a national diagnostic tool tailored to the Iranian context enabling early and efficient diagnosis of hereditary hearing impairment.

Efficiency of hearing screening combined with gene mutation screening in neonates with high-risk of hearing impairment

Objective To investigate the significance of hearing screening combined with gene screening for neonates with high-risk of hearing impairment. Methods Neonates admitted to the Neonatal Department of Guangdong Women and Children Hospital between July 2013 and June 2014 were enrolled in this study. They were divided into high-risk group (with high-risk for hearing impairment) (n=3 129), and control group (n=5 106). Neonate hearing screening was carried out using otoacoustic emission and automated auditory brainstem response. Blood samples were collected using a standard protocol for detecting the mutations of four common deafness genes, including GJB2, GJB3, SLC26A4 and mitochondrial 12s rRNA. Chi-square test was used to compare the differences of the pass rate of hearing screening and positive rate of gene mutations between the two groups. Results The rates of failure on otoacoustic emission, automated auditory brainstem response or both in the high-risk group were 11.92% (373/3 129), 10.32% (323/3 129) and 4.83% (151/3 129), respectively, higher than those in the control group [5.03%(257/5 106), 6.56%(335/5 106) and 2.02% (103/5 106)] (χ2=130.265, 37.354 and 51.196, all P=0.000). In the high-risk group, the overall positive rate of gene mutations was 5.63% (176/3 129), and the GJB2 and SLC26A4 gene mutation rates were 3.04% (95/3 129) and 2.40% (75/3 129)], all higher than the control group [3.15% (161/5 106), 2.04% (104/5 106) and 1.06% (54/5106)] (χ2=30.301, 8.216 and 22.517, all P 0.05]. The rates of failure on otoacoustic emission and automated auditory brainstem response of the neonates with deafness gene mutations were 9.50% (32/337) and 10.39% (35/337), respectively, higher than the neonates without [1.14% (90/7 898) and 1.29% (102/7 898)] (χ2=154.621 and 163.399, both P=0.000). Conclusion Combined hearing screening is of clinical significance for neonates with high-risk of hearing impairment. Key words: Hearing disorders; Deafness; Connexins; Membrane transport proteins; Mutation; Genetic predisposition to disease; Neonatal screening

Mutation Analysis of Gap Junction Protein Beta 1 and Genotype-Phenotype Correlation in X-linked Charcot-Marie-Tooth Disease in Chinese Patients.

Background:Among patients with Charcot–Marie–Tooth disease (CMT), the X-linked variant (CMTX) caused by gap junction protein beta 1 (GJB1) gene mutation is the second most frequent type, accounting for approximately 90% of all CMTX. More than 400 mutations have been identified in the GJB1 gene that encodes connexin 32 (CX32). CX32 is thought to form gap junctions that promote the diffusion pathway between cells. GJB1 mutations interfere with the formation of the functional channel and impair the maintenance of peripheral myelin, and novel mutations are continually discovered.Methods:We included 79 unrelated patients clinically diagnosed with CMT at the Department of Neurology of the Chinese People's Liberation Army General Hospital from December 20, 2012, to December 31, 2015. Clinical examination, nerve conduction studies, and molecular and bioinformatics analyses were performed to identify patients with CMTX1.Results:Nine GJB1 mutations (c.283G>A, c.77C>T, c.643C>T, c.515C>T, c.191G>A, c.610C>T, c.490C>T, c.491G>A, and c.44G>A) were discovered in nine patients. Median motor nerve conduction velocities of all nine patients were < 38 m/s, resembling CMT Type 1. Three novel mutations, c.643C>T, c.191G>A, and c.610C>T, were revealed and bioinformatics analyses indicated high pathogenicity.Conclusions:The three novel missense mutations within the GJB1 gene broaden the mutational diversity of CMT1X. Molecular analysis of family members and bioinformatics analyses of the afflicted patients confirmed the pathogenicity of these mutations.

Construction of HaCaT cell lines stably expressing the human GJB6 gene by using a Tet-On lentiviral vector and their identification

Objective To construct HaCaT cell lines stably expressing the wild type human GJB6 gene or its mutant by using a Tet-On lentiviral vector, and to lay an experimental foundation for studies on pathogenesis of hidrotic ectodermal dysplasia. Methods The wild-type human GJB6 gene and its mutant (A88V) were amplified by PCR, and then inserted into the Tet - on lentivirus plasmid to construct recombinant lentivirus vectors. The recombinants were identified by gene sequencing and enzymatic digestion. Cultured HaCaT cells were classified into three groups to be transfected with a negative control lentiviral vector (NC group), the lentivirus vector expressing the wild-type human GJB6 gene (WT group), or the lentivirus vector expressing the mutant human GJB6 gene (MU group). Puromycin was used to select HaCaT cell clones stably expressing the GJB6 gene which encodes the connexin 30 (Cx30) protein. The selected HaCaT cell clones were cultured with or without tetracycline for 48 hours, thereafter, real-time PCR (RT-PCR) was performed to detect GJB6 gene mRNA expression, Western-blot analysis to measure expressions of Cx30 and FLAG-tag proteins, and cell counting kit 8 (CCK8) assay to evaluate cellular proliferative activity. Results Enzymatic digestion and gene sequencing showed that recombinant lentivirus plasmids were successfully constructed. RT-PCR showed evidently increased mRNA expression of the GJB6 gene in stably transfected HaCaT cells. Moreover, the expression abundance of the GJB6 gene was 112.369 times higher in the WT group induced by tetracycline than in that without tetracycline treatment (P 0.05). Conclusion HaCaT cell lines which stably express the wild - type GJB6 gene or its mutant (A88V) are successfully constructed. Key words: Ectodermal dysplasia; Connexins; GJB6 gene; HaCaT cells; Tet-on

Частота носительства в российской популяции мутаций в генах GJB2 и GALT, ассоциированных с развитием нейросенсорной тугоухости и галактоземии

Статья продолжает цикл работ, посвященных определению частоты носительства в российской популяции мутаций, ассоциированных с развитием распространенных моногенных заболеваний. Целью исследования было установление частоты распространенных в российской популяции мутаций в генах GJB2 и GALT у доноров первичной кроводачи. При генотипировании 1000 доноров первичной кроводачи, идентифицирующих себя как русских и постоянно проживающих на территории Российской Федерации, обнаружены 37 носителей мутаций в гене GJB2, ассоциированных с развитием нейросенсорной тугоухости (частота в выборке составила 3,7 %, или 1 : 27), и 6 носителей мутаций в гене GALT, ассоциированных с развитием галактоземии (частота в выборке — 0,6 %, или 1 : 167). Выявлен 1 случай сочетанного носительства мутаций, и, таким образом, всего обнаружены 42 носителя мутаций в генах GJB2 и GALT (частота в выборке — 4,2 %, или 1 : 24).

A case of keratitis-ichthyosis-deafness syndrome complicated by cutaneous squamous cell carcinoma: mutation analysis of the GJB2 gene and literature review

Objective To analyze mutations in the GJB2 gene in a Chinese patient with keratitis-ichthyosis-deafness (KID) syndrome complicated by cutaneous squamous cell carcinoma. Methods Clinical data were collected from a patient with KID syndrome complicated by cutaneous squamous cell carcinoma. Peripheral blood samples were obtained from the patient and her parents, and DNA was extracted from these blood samples. PCR was performed to amplify the exon 2 of the GJB2 gene followed by direct DNA sequencing. Results A mutation (c.148G > A) was identified at position 148 in exon 2 of the GJB2 gene, which caused a codon change from GAC to AAC and resulted in the substitution of aspartate by asparagine at position 50 in the connexin26 (Cx26) protein (p.Asp50Asn). In addition, another mutation (c.79G > A) , which led to the substitution of valine by isoleucine at codon 27 in Cx26 (p.Val27Ile) , was found at position 79 in exon 2 of the GJB2 gene. Neither of the two mutations was detected in the patient's parents. Literature review revealed that 13 cases of KID syndrome complicated by cutaneous squamous cell carcinoma had been reported in abroad, and the mutation c.148G > A was detected in the GJB2 gene in all the 7 cases finally diagnosed by gene sequencing. Conclusion GJB2 gene mutations may be responsible for the clinical phenotype of KID syndrome in this Chinese patient, and the mutation c.148G > A may be related to the development of cutaneous squamous cell carcinoma. Key words: Mutation; Carcinoma, squamous cell; GJB2 gene; Keratitis-ichthyosis-deafness syndrome

Clinical and genetic spectra in a series of Chinese patients with Charcot–Marie–Tooth disease

The aim of this study was to determine the clinical features and frequencies of genetic subtypes in a series of patients with Charcot-Marie-Tooth (CMT) disease from Eastern China. Patients were divided into three subtypes, CMT1, CMT2 and hereditary neuropathy with liability to pressure palsy (HNPP), according to their electrophysiological manifestations. Multiplex ligation-dependent probe analysis (MLPA) was performed to detect duplications/deletions in the PMP22 gene. The coding regions and splice sites of the GJB1, MPZ, MFN2 and GDAP-1 genes were determined by direct sequencing. Among the 148 patients in the study, 37.2% of the cases had mutations in genes assessed. The mutation detection rate was higher in patients with family histories than in spontaneous cases. PMP22 duplication (13.5%) was predominant in this group of patients, followed by PMP22 deletion (11.5%), and point mutations in GJB1 (8.8%), MPZ (2.0%) and MFN2 (0.7%). Three novel mutations (c.151T>C and c.310 A>G in GJB1 and c.1516 C>G in MFN2) were detected. A small deletion in PMP22 exon 4 was detected in a patient with severe CMT1. Genetic tests have great value in CMT patients with family histories. The frequency of PMP22 duplications was lower in Asian patients than in others. We suggest that genetic testing strategies in CMT patients should be primarily based on electromyography data.

G.P.326 - Deletion of the P2 promoter of the GJB1 gene confirms X-linked Charcot–Marie–Tooth disease in a large family

G.P.326 - Deletion of the P2 promoter of the GJB1 gene confirms X-linked Charcot–Marie–Tooth disease in a large family

Intraneural GJB1 gene delivery improves nerve pathology in a model of X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common inherited neuropathy caused by mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32). Clinical studies and disease models indicate that neuropathy mainly results from Schwann cell autonomous, loss-of-function mechanisms; therefore, CMT1X may be treatable by gene replacement. A lentiviral vector LV.Mpz-GJB1 carrying the GJB1 gene under the Schwann cell-specific myelin protein zero (Mpz) promoter was generated and delivered into the mouse sciatic nerve by a single injection immediately distal to the sciatic notch. Enhanced green fluorescent protein (EGFP) reporter gene expression was quantified and Cx32 expression was examined on a Cx32 knockout (KO) background. A gene therapy trial was performed in a Cx32 KO model of CMT1X. EGFP was expressed throughout the length of the sciatic nerve in up to 50% of Schwann cells starting 2 weeks after injection and remaining stable for up to 16 weeks. Following LV.Mpz-GJB1 injection into Cx32 KO nerves, we detected Cx32 expression and correct localization in non-compact myelin areas where gap junctions are normally formed. Gene therapy trial by intraneural injection in groups of 2-month-old Cx32 KO mice, before demyelination onset, significantly reduced the ratio of abnormally myelinated fibers (p = 0.00148) and secondary inflammation (p = 0.0178) at 6 months of age compared to mock-treated animals. Gene delivery using a lentiviral vector leads to efficient gene expression specifically in Schwann cells. Restoration of Cx32 expression ameliorates nerve pathology in a disease model and provides a promising approach for future treatments of CMT1X and other inherited neuropathies.

Regulation of migration and invasion by Toll-like receptor-9 signaling network in prostate cancer.

Toll-like receptors (TLRs) play an important role in tumorigenesis and progress of prostate cancer. However, the function and mechanism of Toll-like receptor-9 (TLR9) in prostate cancer is not totally understood. Here, we found that high expression of TLR9 was associated with a higher probability of lymph node metastasis and poor prognosis. Further in vitro functional study verified that silence of TLR9 inhibited migration and invasion of PC-3 cells, indicating expression of TLR9 involving in the migration and invasion of cancer cells. The data of microarray exhibited silence of TLR9 induced 205 genes with larger than 2-fold changes in expression levels, including 164 genes down-regulated and 41 genes up-regulated. Functional Gene Ontology (GO) processes annotation demonstrated that the top three scores of molecular and cellular functions were regulation of programmed cell death, regulation of locomotion and response to calcium ion. TLR9 signaling network analysis of the migration and invasion related genes identified several genes, like matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9), chemokine receptor 4 (CXCR4) and interleukin 8 (IL8), formed the core interaction network based on their known biological relationships. A few genes, such as odontogenic ameloblast-associated protein (ODAM), claudin 2 (CLDN2), gap junction protein beta 1 (GJB1) and Rho-associated coiled-coil containing protein kinase 1 pseudogene 1 (ROCK1P1), so far have not been found to interact with the other genes. This study provided the foundation to discover the new molecular mechanism in signaling networks of invasion and metastasis in prostate cancer.

Study of newborn hearing and genetic screening in Jinan

In this study, we employed newborn hearing screening and gene screening concurrently to explore the hearing loss associated with mutations in the city of Jinan. A total of 3 288 newborns born between March 2013 and December 2013 in Jinan Maternity and Child Care Hospital received hearing concurrent genetic screening. Transiently evoked otoacoustic emissions (TEOAE) was used in rooming-in newborns, while TEOAE and auto auditory brainstem response (AABR) was used in infants in neonatal intensive care unit (NICU). Two drops of heel blood were harvested with filter paper. Nine mutations [GJB2 (235delC, 35delG, 299delAT, 176del16), SLC26A4 (IVS7-2A>G,2168 A>G), GJB3 (538 C>T), 12SrRNA (1555 A>G, 1494C>T)] of 4 frequent genes associated with Chinese hearing loss were determined by gene chip in these dried blood samples. Among 3 288 newborns, 363 cases failed to pass the hearing screening, and 36 cases of these 363 newborns carried mutations, with a carrier rate of 9.91%. 2 925 cases passed the hearing screening, of which 113 carried mutations, with a carrier rate of 3.86%. There was a significantly statistic difference (χ2=8.67, P=0.000) in carrier rate between two groups. 149 (4.53%) infants were detected to carry at least one mutation allele,among which 113 cases passed the hearing screening and 36 cases failed. Seven cases were diagnosed to have hearing loss. Homozygous GJB2 mutation was detected in 2 cases, compound heterozygous GJB2 mutation was detected in 1 case, and heterozygous GJB2 mutation in 88 cases. There were 91 cases carried GJB2 mutations totally, with a total rate of 2.76%. There were 40 cases were detected to carry heterozygous SLC26A4 mutation, with a carrier rate of 1.22%. Nine cases had heterozygous GJB3 mutation, with a carrier rate of 0.27%. Six cases had homogeneous mitochondria 12SrRNA mutation, and 1 had heterogeneous mutations. There were 7 cases totally, with a total rate of 0.21%. 142 infants with gene mutation should be follow-up. A follow-up system in infants, passed hearing screening,with single heterozygous mutation and mutations associated with drug-induced hearing loss, can help to detect infants with hearing defects early and effectively prevent late-onset hearing impairment.

A start codon CMT1X mutation associated with transient encephalomyelitis causes complete loss of Cx32.

X-linked Charcot-Marie-Tooth disease (CMTX1) results from numerous mutations in the GJB1 gene encoding the gap junction protein connexin32 (Cx32) and is one of the commonest forms of inherited neuropathy. Owing to the expression of Cx32 not only in Schwann cells but also in oligodendrocytes, a subset of CMT1X patients develops central nervous system (CNS) clinical manifestations in addition to peripheral neuropathy. While most GJB1 mutations appear to cause peripheral neuropathy through loss of Cx32 function, the cellular mechanisms underlying the CNS manifestations remain controversial. A novel start codon GJB1 mutation (p.Met1Ile) has been found in a CMT1X patient presenting with recurrent episodes of transient encephalomyelitis without apparent signs of peripheral neuropathy. In order to clarify the functional consequences of this mutation, we examined the cellular expression of two different constructs cloned from genomic DNA including the mutated start codon. None of the cloned constructs resulted in detectable expression of Cx32 by immunocytochemistry or immunoblot, although mRNA was produced at normal levels. Furthermore, co-expression with the other major oligodendrocyte connexin, Cx47, had no negative effect on GJ formation by Cx47. Finally, lysosomal and proteasomal inhibition in cells expressing the start codon mutant constructs failed to recover any detection of Cx32 as a result of impaired protein degradation. Our results indicate that the Cx32 start codon mutation is equivalent to a complete loss of the protein with failure of translation, although transcription is not impaired. Thus, complete loss of Cx32 function is sufficient to produce CNS dysfunction with clinical manifestations.

MALDI-TOF-MS based diagnosis and analysis of newborn hereditary deafness screening in Wuxi, Jiangsu Province

Objective To investigate the hot spots of genetic mutations that are responsible for hearing loss of newborns and the carrier frequencies of varies mutations, and also, to set up a clinical pattern for newborn hearing concurrent genetic screening in Wuxi. Methods A total of 2 796 newborns participated in this study were all born in Wuxi Maternity and Child Health Care Hospital during 2013-2014. Twenty hot mutation spots of four genes (i.e. GJB2, GLB3, SLC26A4, 12s rRNA) were chosen according to the latest epidemiologic study and were analyzed with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry ( MALDI-TOF-MS). OAE and AABR methods were both used to help with the diagnosis of newborn hearing loss. Results 158 of 2 796 newborns tested (5.65%) were positive with at least one hot mutation spot. The number of positively tested single site mutation were 152 (5.44%), while the number of positively tested compound heterozygous mutations were 6 (0.21%). The positive mutation sites in GJB2 and SLC26A4 ranked 88.61% among all the positive mutation sites tested. Mutation site 235delC within GJB2 gene were hottest positive site with a number of 73 newborns, followed by IVS7-2A>G within SLC26A4 (18.99%, 30/158). Three 1555A>G homozygous mutations were also found in 12s rRNA. Four deafness newborns were found with newborn hearing concurrent genetic screening. Conclusions In comparison to the conventional detection methods, there could be more detection sites, greater coverage , and high throughput in MALDI-TOF-MS, and it would provid valuable guidance for newborns in the future. Key words: Hearing loss; Connexins; Membrane transport proteins; Mutations; Spectrometry, mass, matrix-Assisted laser desorption-ionization; Neonatal screening

External quality survey results of newborn deafness gene mutations in China

To evaluate the external quality survey results of newborn hereditary deafness gene mutations to improve and enhance the testing quality of hereditary deafness gene mutations. Each of 30 voluntarily participating laboratories testing newborn hereditary deafness gene mutations received 15 lots of quality control blood spots in this survey at May 2013, including mitochondria DNA 12SrRNA 1555A>G (201311-201315), SLC26A4 IVS7-2A>G (201321-201325) and GJB2 235delC (201331-201335). The testing results, methods, equipments and reagent information were submitted for analyses of Clinet EQA, Microsoft Excel 2007 and SPSS 13.0. The rates of correction (number of correct results/total number of submitted results) were used for evaluating the performance of laboratories. Among them, 24 laboratories submitted the testing results of mitochondria DNA 12SrRNA 1555A>G and the rate of submission was 80.0% (24/30). The rates of correction for each lot were 95.8% (23/24), 95.8% (23/24), 100% (24/24), 95.8% (23/24) and 95.8% (23/24) respectively and the overall rate was 96.7% (116/120). And 23 laboratories submitted the other two kinds of genetic test results, the rate of submission was 76.7% (23/30); the rates of correction for each lot of SLC26A4 IVS7-2A>G genetic test were 95.7% (22/23), 95.7% (22/23), 100% (23/23), 95.7% (22/23) and 95.7% (22/23) respectively and the overall rate was 96.5% (111/115); the genetic test results of GJB2 235delC were all correct (23/23). In this survey, 2/3 laboratories employed fluorescent polymerase chain reaction (PCR) and the remainder microarray chips. The survey of newborn deafness gene mutation testing results is generally satisfactory. Only mitochondria DNA 12SrRNA and SLC26A4 have some errors. The laboratories of gene testing should improve their quality control system, correct mistakes during the test period without any delay and boost the rate of correction for newborn hereditary deafness gene testing.

A novel mutation in GJB1 (c.212T>G) in a Chinese family with X-linked Charcot–Marie–Tooth disease

Gap junction protein beta 1 (GJB1) gene mutations lead to X-linked Charcot–Marie–Tooth (CMTX) disease. We investigated a Chinese family with CMTX and identified a novel GJB1 point mutation. Clinical and electrophysiological features of the pedigree were examined, and sequence alterations of the coding region of GJB1 that encode connexin32 were determined by direct sequencing. Sequence alignment of the mutation site was performed using Clustal W. Mutation effects were analysed using PolyPhen-2, SIFT and Mutation Taster software. The three-dimensional structures of the mutant and wild-type proteins were predicted by modeling with SWISS MODEL online software. The affected family members displayed typical Charcot–Marie–Tooth phenotypes, but phenotypic heterogeneity was observed. Nerve conduction velocities of all affected patients were slow. Sequencing of GJB1 revealed a heterozygous T>G missense mutation at nucleotide 212 in the proband, the proband’s mother and the proband’s daughter. The affected male sibling of the proband displayed a hemizygous missense mutation with T>G transition at the identical position on the GJB1 gene. This mutation resulted in an amino acid change from isoleucine to serine that was predicted to lead to tertiary structural alterations that would disrupt the function of the GJB1 protein. A novel point mutation in GJB1 was detected, expanding the spectrum of GJB1 mutations known to be associated with CMTX.