Gap junction beta 1 (<Emphasis Type="Italic">GJB1</Emphasis>) gene mutations in Italian patients with X-linked Charcot-Marie-Tooth disease

X-linked Charcot-Marie-Tooth disease (CMT1X) is a peripheral neuropathy transmitted in a dominant manner and caused by mutations in the Connexin 32 (Cx32) gene (GJB1, gap junction beta 1). Here we report the mutation analysis of the GJB1 gene in 76 subjects with possible CMT1 and absence of 17p11.2 duplication, and in 38 CMT2 patients without mutations in CMT2-associated-genes, selected from a cohort of 684 patients with peripheral sensory-motor neuropathy. The analysis was performed by direct sequencing of the coding sequence and exon/intron boundaries of the GJB1 gene. The mutation screening identified 22 mutations in GJB1, eight of which have not been previously published: six point mutations (c.50C > G, c.107T > A, c.545C > T, c.545C > G, c.548G > C, c.791G > T) and two deletions (c.84delC, c.573_581delCGTCTTCAT). The GJB1 mutation frequency (19.3%) and the clinical heterogeneity of our patients suggest searching for GJB1 mutations in all CMT cases without the 17p11.2 duplication, regardless of the gender of the proband, as well as in CMT2 patients with possible X-linked inheritance.     

Low prevalence of Connexin 26 (GJB2) variants in Pakistani families with autosomal recessive non-syndromic hearing impairment

The Pakistani population has become an important resource for research on autosomal recessive non-syndromic hearing impairment (ARNSHI) due to the availability of large extended and highly consanguineous pedigrees. Here is presented the first report on the prevalence of gap junction beta-2 (GJB2) variants in Pakistan. One hundred and ninety-six unrelated Pakistani families with ARNSHI were recruited for a study on the genetics of NSHI. DNA sequencing of the GJB2 coding region was done on two affected individuals per family. Evolutionary conservation and predicted effect on the protein product were studied in order to hypothesize whether or not a variant was potentially deleterious. Homozygous putatively functional GJB2 variants were identified in 6.1% of families. None of the putatively functional GJB2 variants were observed in the compound heterozygous state. The six putatively causative variants noted were 231G > A(W77X), 71G > A(W24X), 167delT, 95G > A(R32H), 358-360delGAG(delE120), and 269T > C(L90P), with 231G > A(W77X) and 71G > A(W24X) being the most common. In addition, five benign polymorphisms, 380G > A(R127H), 457G > A(V153I), 493C > T(R165W), 79G > A(V27I), and 341 A > G(E114G), were identified within this population. In a few individuals, benign polymorphisms were observed to occur on the same haplotype, namely [457G > A(V153I); 493C > T(R165W)] and [79G > A(V27I); 341 A > G(E114G)]. The spectrum of GJB2 sequence variants in Pakistan may reflect shared origins of hearing impairment alleles within the Indian subcontinent. The high degree of consanguinity within Pakistan may have maintained the GJB2 prevalence at a much lower rate than within India and other populations.     

Two novel mutations in the <Emphasis Type="Italic">EPM2A </Emphasis>gene in a Korean patient with Lafora's progressive myoclonus epilepsy

 The progressive myoclonus epilepsy of the Lafora type (LD; MIM 254780) is a rare autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration, and the presence of periodic acid-Schiff-positive polyglucosan inclusions (Lafora bodies). Mutations in the EPM2A gene have recently been found to cause LD and about 30 or more mutations have been reported thus far. LD is relatively common in countries of the Mediterranean Basin, the Middle East, India, and Pakistan. Although a few sporadic cases with the typical LD phenotype have also been reported in the Far East including Korea and Japan, a recent effort to find mutations in Japanese LD families was not successful. In the present study, we report two novel mutations in a Korean girl with LD; a 1-bp insertion mutation (c.223insC; G75fsX107) in exon 1 and a missense mutation (c.559A>G; T187A) in exon 3 of the EPM2A gene. To our knowledge, this is the first report of a genetically confirmed case of LD in Koreans and also in the Far East. Received: September 2, 2002 / Accepted: November 8, 2002 Acknowledgments This work was supported by the fund from the Center for Functional Analysis of Human Genome (FG-2–1-02), Korea. The third and sixth authors contributed equally to this work. Correspondence to:J.-W. Kim     

Prevalent connexin 26 gene (GJB2) mutations in Japanese

The gene responsible for DNFB1 and DFNA3, connexin 26 (GJB2), was recently identified and more than 20 disease causing mutations have been reported so far. This paper presents mutation analysis for GJB2 in Japanese non-syndromic hearing loss patients compatible with recessive inheritance. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population, with three mutations, 235delC, Y136X, and R143W, especially frequent. Of these three mutations, 235delC was most prevalent at 73%. Surprisingly, the 35delG mutation, which is the most common GJB2 mutation in white subjects, was not found in the present study. Our data indicated that specific combinations of GJB2 mutation exist in different populations.     

Connexin26 gene ( GJB2): prevalence of mutations in the Chinese population

The connexin26 gene ( GJB2) has been shown to be responsible for DFNB1 and DFNA3 (Autosomal Recessive Hereditary Nonsyndromic Deafness Locus 1 and Autosomal Dominant Hereditary Nonsyndromic Deafness Locus 3). Two hundred ten independently ascertained Chinese probands with nonsyndromic hearing loss (NSHL) were evaluated for mutations in GJB2, including 43 probands from families with more than one sib with NSHL, likely indicating dominant inheritance, and sporadic cases of NSHL, compatible with recessive inheritance. Of the 210 probands, 43 (20%) were homozygous or heterozygous for mutations in GJB2. Four different mutations were identified: 35delG, 109G-A, 235delC, and 299-300delAT. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population. Of these four mutations, 235delC was the most prevalent at 93%; yet the 35delG mutation, which is the most common GJB2 mutation in Caucasian subjects (Europeans and Americans), was found in low frequency in the present study. It appears from our limited data and reports from other East Asians that 235delC is the most prevalent GJB2 mutation in these populations. GJB2 mutations are consistent with ethnic predilections.     

Catalog of 86 single-nucleotide polymorphisms (SNPs) in three uridine diphosphate glycosyltransferase genes: <Emphasis Type="Italic">UGT2A1</Emphasis>, <Emphasis Type="Italic">UGT2B15</Emphasis>, and <Emphasis Type="Italic">UGT8</Emphasis>

We report here three high-density maps of variations found among 48 Japanese individuals in three uridine diphosphate glycosyltransferase (UGT) genes, UGT2A1, UGT2B15, and UGT8. A total of 86 single-nucleotide polymorphisms (SNPs) were identified through systematic screening of genomic regions containing these genes: 8 in 5′ flanking regions, 7 in coding regions, 67 in introns, 3 in 3′ untranslated regions, and 1 in a 3′ flanking region. We also discovered 14 variations of other types. Of the 86 SNPs, 63 (73%) were considered to be novel on the basis of comparison of our data with the Database of SNPs (dbSNP) of the National Center for Biotechnology Information. Among the seven SNPs identified in exonic sequences, five were non-synonymous changes that would result in amino-acid substitutions. The collection of SNPs derived from this study will serve as an additional resource for studies of complex genetic diseases and responsiveness to drug therapy. Received: June 12, 2002 / Accepted: June 13, 2002     

A novel dominant missense mutation--D179N--in the GJB2 gene (Connexin 26) associated with non-syndromic hearing loss

Mutations of the GJB2 gene, encoding Connexin 26, are the most common cause of hereditary congenital hearing loss in many countries, and account for up to 50% of cases of autosomal-recessive non-syndromic deafness. By contrast, only a few GJB2 mutations have been reported to cause an autosomal-dominant form of non-syndromic deafness. We report on a family from southern Italy in whom dominant, non-syndromic, post-lingual hearing loss is associated with a novel missense mutation in the GJB2 gene. Direct sequencing of the gene showed a heterozygous G-->A transition at nucleotide 535, resulting in an aspartic acid to asparagine amino acid substitution at codon 179 (D179N). This mutation occurred in the second extracellular domain (EC2), which would seem to be very important for connexon-connexon interaction.     

Haplotype analysis of the human collectin placenta 1 (<Emphasis Type="Italic">hCL-P1</Emphasis>) gene

 Collectins are a family of C-type lectins found in vertebrates. These proteins have four regions, a relatively short N-terminal region, a collagen-like region, an alpha-helical coiled coil, and a carbohydrate recognition domain. Collectins are involved in host defense through their ability to bind carbohydrate antigens on microorganisms. Type A scavenger receptors are classical-type scavenger receptors that also have collagen-like domains. We previously described a new scavenger receptor, collectin from placenta [collectin placenta 1 (CL-P1)]. CL-P1 is a type II membrane protein with all four regions. We found that CL-P1 can bind and phagocytize both bacteria and yeast. In addition to that, it reacts with oxidized low-density lipoprotein (LDL) but not with acetylated LDL. These results suggest that CL-P1 might play important roles in host defenses and/or atherosclerosis formation. One rational strategy to study the role of CL-P1 in these pathological conditions would be to perform a haplotype association study using human samples. As a first step for this strategy, we analyzed the haplotype structure of the CL-P1 gene. By sequencing the CL-P1 gene in ten Japanese volunteers, we identified five single-nucleotide polymorphisms (SNPs) with a minor allele frequency of at least 29%. To obtain SNPs in the 5′-upstream region of the gene, we screened a total of 20 SNPs described in the database and finally picked up one SNP for the present study. Thus, a total of six SNPs, one in the 5′-upstream region, two in intron 2, one in exon 5, and two in exon 6, were used to analyze the haplotype structure of the gene, with DNAs derived from 54 individuals (108 alleles). The analysis revealed that only two of six SNPs showed significant linkage disequilibrium (r ² > 0.5) with each other. This haplotype information may be useful in disease-association studies in which a contribution of the CL-P1 gene has been suspected, especially in immunological disturbance or atherosclerosis. Two SNPs in exon 6, both leading to amino acid substitutions, could be candidates for influencing disease susceptibility. Received: September 12, 2002 / Accepted: November 12, 2002 Present address Department of Public Health, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Tel. +81-43-226-2067; Fax +81-43-226-2070 e-mail: ahata@med.m.chiba-u.ac.jp Acknowledgments We are grateful to Katsura Nakanishi and Ayako Sado for their technical assistance. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (C) “Medical Genome Science” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Correspondence to:A. Hata     

Mutations in the connexin26/GJB2 gene are the most common event in non-syndromic hearing loss among the German population

Congenital sensorineural hearing loss affects approximately 1/1,000 live births. Mutations in the gene encoding connexin26 (GJB2) have been described as a major cause of genetic nonsyndromic hearing impairment. Additionally, another gap junction gene, connexin30 (GJB6), was found to be responsible for hereditary hearing loss. We have studied 134 patients with severe to profound hearing loss or deafness and 13 patients with mild to moderate nonsyndromic sensorineural hearing loss in order to evaluate the prevalence of connexin26 and connexin30 mutations in Germany. Mutations in the connexin26 gene were found in 30 patients (22%) with profound to severe hearing impairment whereas only one novel single nucleotide polymorphism (396G-->A) in the connexin30 gene was detected. Among the 13 patients with mild to moderate hearing loss neither mutations in the connexin26 nor in the connexin30 gene could be detected. These results demonstrate that mutations in the connexin26 gene are also a frequent cause of hereditary non-syndromic hearing loss in Germany. Therefore a screening of mutations in the connexin26 gene should be performed in every case of non-syndromic hearing loss of unknown origin.     

Comparison of <Emphasis Type="Italic">SYBR Green I</Emphasis> and <Emphasis Type="Italic">TaqMan</Emphasis> real-time PCR formats for the analysis of <Emphasis Type="Italic">her2</Emphasis> gene dose in human breast tumors

We compared two technologies of real-time PCR (with the use of fluorescent SYBR Green I dye and specific TaqMan probe) for quantification of the dose of her2 gene in breast tumors. The maximum increase in the gene dose in TaqMan and SYBR Green I analyses was 10-and 5-fold, respectively. In was found that TaqMan and SYBR Green I technologies allow detection of the matrix in amounts corresponding to 1–100 and 2.5–40.0 ng genomic DNA, respectively. Tenfold increase in the gene dose leads to incorrect evaluation of multiplication ratio in the SYBR Green I analysis. These results suggest that TaqMan technology is more preferable for correct evaluation of her2 gene dose. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 2, pp. 201–205, February, 2008     

Deafness resulting from mutations in the GJB2 (connexin 26) gene in Brazilian patients

Congenital deafness occurs in approximately 1 in 1000 live births. In developed countries about 60% of hearing loss is genetic. However, in Brazil most cases of hearing loss are due to environmental factors, such as congenital infections (mainly rubella), perinatal anoxia, kernicterus and meningitis. Recently, it has been demonstrated that the GJB2 gene is a major gene underlying congenital sensorial deafness. Mutations in this gene cause 10-20% of all genetic sensory hearing loss. One specific mutation, 35delG, accounts for the majority of mutant alleles. The extent of the hearing impairment varies from mild/moderate to profound, even within the patients homozygous for the common 35delG mutation. There may also be progression with age. Mutation analysis in the GJB2 gene was performed on 36 families (group A) presenting with at least one individual with non-syndromic deafness (NSD). An unselected series of 26 deaf individuals referred by other services where the environmental factors were not completely excluded was also part of the study (group B). Mutations in the GJB2 gene were found in 22% (eight patients) of the families tested in group A, and 11.5% (three patients) of individuals within group B. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.     

Different functional consequences of two missense mutations in the GJB2 gene associated with non‐syndromic hearing loss

Mutations in the GJB2 gene, which encodes the gap junction (GJ) protein connexin26 (Cx26), are the most common cause of inherited non‐syndromic hearing loss (NSHL). We identified two missense mutations, p.D46E (c.138T>G) and p.T86R (c.257C>G), of GJB2 in Korean HL families. The novel p.D46E mutation exhibited autosomal dominant inheritance, while the p.T86R mutation, which is exclusively found in Asians, segregated with an autosomal recessive pattern. Thus, we sought to elucidate the pathogenic nature of such different inherited patterns of HL. We studied protein localization and gap junction functions in cells transfected with wild‐type or mutant Cx26 tagged with fluorescent proteins, which allowed visual confirmation of homozygous or heterozygous mutant GJs. The Cx26‐D46E mutant was targeted to the plasma membrane, but this mutant protein failed to transfer Ca²⁺ or propidium iodide intercellularly, suggesting disruption of both ionic and biochemical coupling. Heterozygous GJs also showed dysfunctional intercellular couplings and hemichannel opening, confirming the dominant‐negative nature of the p.D46E mutation. The Cx26‐T86R mutant protein did not form GJs, since the mutated protein was confined in the cytoplasm and not transported to the cell membrane. When Cx26‐T86R was co‐expressed with Cx26‐WT, ionic and biochemical coupling was normal, consistent with the recessive nature of the mutation. These studies revealed distinct pathogenic mechanisms of two GJB2 mutations identified in Korean families. © 2009 Wiley‐Liss, Inc.     

Prevalence of Deafness‐Associated Connexin‐26 (GJB2) and Connexin‐30 (GJB6) Pathogenic Alleles in a Large Patient Cohort from Eastern Sicily

Mutations in the gene encoding the gap junction protein connexin 26 (GJB2) and connexin 30 (GJB6) have been shown to be a major contributor to prelingual, sensorineural, nonsyndromic deafness. The aim of this study was to characterize and establish the prevalence of GJB2 and GJB6 gene alterations in 196 patients affected by sensorineural, nonsyndromic hearing loss, from Eastern Sicily. We performed sequence analysis of GJB2 and identified sequence variants in 68 out of 196 patients (34.7%); (28 homozygous for c.35delG, 22 compound heterozygous and 11 with only one variant allele). We found 12 different allelic variants, the most prevalent being c.35delG, which was found on 89 chromosomes (65.5%), followed by other alleles with different frequencies (p.E47X, c.‐23+1G>A, p.L90P, p.R184W, p.M34T, c.167delT, p.R127H, p.M163V, p.V153I, p.W24X, and p.T8M). Importantly, for the first time we present the frequency and spectrum of GJB2 mutations in NSHL patients from Eastern Sicily. No alterations were found in the GJB6 gene, confirming that alterations in this gene are uncommon in our geographic area. Note that 65.3% and 23.5% of our patients, respectively were found to be negative or carriers by GJB2 molecular screening. This emphasizes the need to broaden the genetic analysis to other genes involved in hearing loss.     

Chromosome pairing in allotetraploid hybrids of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> revealed by genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.     

Identification of two novel <Emphasis Type="Italic">RECQL4 </Emphasis>exonic SNPs and genomic characterization of the IVS12 minisatellite

 Rothmund-Thomson syndrome is a rare autosomal recessive disorder characterized by a widely heterogeneous clinical presentation. Only a subset of clinically diagnosed patients carry RECQL4 gene mutations, probably because of their genetic heterogeneity and/or the complexity of molecular testing. We here describe the polymorphic sites of the RECQL4 gene that detail its genomic structure and may be of interest as modulators of the splicing process and gene expression. We characterized two novel and one already described single-nucleotide polymorphism in the coding region of the RECQL4 gene, which were shown by the exonic splicing enhancer (ESE) score matrix to fall into high-score motifs recognized by serine/arginine-rich proteins. We also describe the genomic structure of a G-C rich minisatellite flanking the 3′ splice site of IVS12 in the helicase domain of the RECQL4 gene, which may enhance mutations such as those described at the IVS12 acceptor site. RECQL4 polymorphic sites may be useful for identifying alleles associated with missplicing and, more generally, in cancer-susceptibility association studies. Received: October 24, 2002 / Accepted: November 21, 2002 Acknowledgments This study was supported by the AIRC (Associazione Italiana per la Ricerca sul Cancro): 2001 grant to L.L. Correspondence to:L. Larizza     

Mutation analysis of the GJB2 (connexin 26) gene in Egypt

Fifty to eighty percent of autosomal recessive deafness is due to mutations in the GJB2 gene encoding connexin 26. Among Caucasians, the c.35delG mutation in this gene accounts for up to 30 to 70% of all cases with early childhood deafness. In this study, we present the analysis of the GJB2 gene in 159 Egyptians from 111 families with non-syndromic mild to profound hearing impairment. An additional family with Vohwinkel syndrome, a combination of hearing impairment and palmoplantar keratoderma with constriction of the digits, was also included. We used direct sequencing analysis to detect all possible coding GJB2 variants in this population. The presence of the g.1777179_2085947del mutation (hereafter called del(GJB6-D13S1830)) was also investigated as it was shown to be the second most common mutation causing non-syndromic prelingual hearing impairment in Spain. Sequencing analysis of one randomly chosen individual per family revealed that the c.35delG mutation was present in 24 out of 222 chromosomes (10.8%), making it the most frequent mutation in the GJB2 gene in Egypt. Five other mutations were already described previously [p.Thr8Met, p.Val37Ile, p.Val153Ile, c.333_334delAA, c.1-3172G>A (commonly designated as IVS1+1G>A)]. This study also revealed three other novel gene variants resulting in amino acid substitutions (p.Phe142del, p.Asp117His, p.Ala148Pro). In contrast with most populations, the del(GJB6-D13S1830) mutation upstream of the GJB2 gene was not present in this Egyptian population. A dominant mutation at a highly conserved residue, p.Gly130Val, was found in the family with Vohwinkel syndrome.     

C-<Emphasis Type="Italic">erb</Emphasis>B-2 oncogene and <Emphasis Type="Italic">P21</Emphasis><Superscript><Emphasis Type="Italic">WAF/CIP1</Emphasis></Superscript> tumor suppressor gene expression as prognostic factors in canine mammary adenocarcinomas

To evaluate the use of c-erbB-2 oncogene and p21 ^WAF1/CIP1 suppressor gene products as the prognosis markers for canine mammary tumors, expression of these gene products were examined immunohistochemically using tumor tissues and clinical data from 96 dogs with malignant mammary tumors. Semiquantitative data was compared with histopathological grades, proliferating cell nuclear antigen (PCNA)-positive index, and clinicopathological matters. The expression c-erbB-2 protein was found in the cellular membrane and cytoplasm of neoplastic epithelial cells, and the positive index had no significant relation to the histopathological features and PCNA-positive index, except for the individual age of affected dogs (P < 0.05). The product of p21 ^WAF1/CIP1 was mostly found in cytoplasm and occasionally in the nucleus of neoplastic cells. The quantitative data had significant association to the malignancy grade and size of tumors (P < 0.05). However, that had no significant relationship to the PCNA-positive index. The present study concluded that both gene products could not apply as the direct markers to evaluate the prognosis of canine mammary tumors. The detection of c-erbB-2 product may be partly beneficial to the differential diagnosis of epithelial type of mammary cancer. The use of p21 ^WAF1/CIP1 product in prognosis of canine mammary cancer needs further investigation.