Connexin-30 deletion analysis in connexin-26 heterozygotes

Mutations in the Connexin-26 gene (Cx 26, GJB2) are the most common cause of hereditary nonsyndromic sensorineural hearing loss (SNHL). DNA analysis of the Cx 26 gene in deaf or hard-of-hearing individuals frequently demonstrates heterozygosity despite the fact that most mutations are known to be recessive. A 342-kb deletion in a gene adjacent to Cx 26, the Connexin-30 gene (Cx 30, GJB6), has been reported to cause deafness in the homozygous state or in combination with heterozygous mutations in Cx 26 (digenic inheritance). We have analyzed deaf or hard-of-hearing Cx 26 heterozygotes and individuals with no mutations in Cx 26 for this Cx 30 deletion. We found that 4/20 (20%) of the Cx 26 heterozygotes are heterozygous for this deletion and that no individuals were homozygous for the Cx 30 deletion. Cx 30 deletion analysis is recommended for all individuals with nonsyndromic SNHL following Cx 26 sequencing that does not demonstrate two recessive mutations.     

Multiplex detection of common mutations in the Connexin-26 gene

Hearing impairment that results from inherited genetic defects occurs in approximately 1/2,000 live births. Mutations in the Connexin-26 gene have been shown to be a major contributor to prelingual, nonsyndromic, autosomal recessive deafness in many populations. The most common mutations in this gene are 35delG, 167delT, 235delC, M34T, and W77X. We describe a nonisotopic, single-tube, polymerase chain reaction (PCR) multiplex system for the detection of these common mutations. The method presented is reliable, simple, and low in cost.     

A PCR-RFLP test for simultaneous detection of two single-nucleotide insertions in the Connexin-26 gene promoter

Comparisons of Connexin-26 (GJB2) gene sequences available in the GenBank data base indicate the presence of a polymorphism in the promoter, but no easy method is available for the detection of this polymorphism. We have developed a PCR-RFLP test for simultaneous detection of two single nucleotide insertions (G and A) in the GJB2 promoter. The test is based on amplification of a 146-bp DNA fragment, which was digested with Mae I to detect the G insertion in the promoter. A similar digestion with Hinf I detects the A insertion. The test was validated using direct DNA sequencing of amplified DNA from 33 samples. After validation, we have used it to investigate DNA samples from 160 control subjects and 51 unrelated patients with nonsyndromic autosomal recessive deafness. All of the samples analyzed using the PCR test and DNA sequencing were found to contain both the G and A insertions in the GJB2 gene promoter. This PCR test will be useful in studying the prevalence of these two insertions in other populations.     

Absence of mutations in GJB2 (Connexin-26) gene in an ethnic group of southwest Iran

BACKGROUND:

The common GJB2 gene mutation (35delG) has been previously reported from Iranian patients that were affected with nonsyndromic autosomal recessive deafness. We, therefore, for the first time, investigated the prevalence and frequency of the GJB2 gene mutation in the Iranian deaf population with Arabian origins.

MATERIALS AND METHODs:

We amplified and sequenced the entire coding sequence of the GJB2 gene from 61 deaf patients and 26 control subjects.

RESULT:

None of the analyzed samples revealed deafness-associated mutation.

CONCLUSION:

This finding differs from several reports from Iran as we have focused on the GJB2 gene that possesses various mutations as the cause of congenital recessive deafness.     

Genetic laboratory practices related to testing of the GJB2 (Connexin-26) gene in the United States in 1999 and 2000

Given the rapidly growing area of molecular genetic laboratory testing, we sought to assess changing issues over a 2-year period of time pertaining to the availability of testing for GJB2 mutations associated with non-syndromic hearing loss. Laboratory assessments carried out by telephone interviews with directors or other key personnel revealed variations among laboratories in informed consent practices, evaluation of test requests for appropriateness, and the reporting of results. From 1999 to 2000, referral patterns shifted as did sources for reimbursement, policies regarding evaluation of incoming test requests, and reporting procedures. We propose that these results reflect changes occurring as a result of a new test moving from the research to the clinical phase.     

Cloning and expression analysis of p26 gene in Artemia sinica

The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the α-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expression of the p26 gene at different developmental stages ofA. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development.     

Impact of Genetic Counseling and Connexin-26 and Connexin-30 Testing on Deaf Identity and Comprehension of Genetic Test Results in a Sample of Deaf Adults: A Prospective,Longitudinal Study

Using a prospective, longitudinal study design, this paper addresses the impact of genetic counseling and testing for deafness on deaf adults and the Deaf community. This study specifically evaluated the effect of genetic counseling and Connexin-26 and Connexin-30 genetic test results on participants'' deaf identity and understanding of their genetic test results. Connexin-26 and Connexin-30 genetic testing was offered to participants in the context of linguistically and culturally appropriate genetic counseling. Questionnaire data collected from 209 deaf adults at four time points (baseline, immediately following pre-test genetic counseling, 1-month following genetic test result disclosure, and 6-months after result disclosure) were analyzed. Four deaf identity orientations (hearing, marginal, immersion, bicultural) were evaluated using subscales of the Deaf Identity Development Scale-Revised. We found evidence that participants understood their specific genetic test results following genetic counseling, but found no evidence of change in deaf identity based on genetic counseling or their genetic test results. This study demonstrated that culturally and linguistically appropriate genetic counseling can improve deaf clients'' understanding of genetic test results, and the formation of deaf identity was not directly related to genetic counseling or Connexin-26 and Connexin-30 genetic test results.     

Spectral and spectral-temporal heart rate analysis in newborns

Spectral (in 3-min segments) and spectral-temporal (in 36-s windows with a 9-s step) analyses were carried out on the basis of cardiointerval recordings of newborns (full-term and preterm) during 24 h. It was found that each of the 3-min intervals of the 24-h cardiointerval recordings in the newborns had a unique spectrum; the spectra could be combined into several types. During 24 h, a spectrum with a marked LF component prevailed in the newborns. A spectrum with a peak in the HF region recorded in the newborns in the range from 0.4 to ～1 Hz was an indicator of the rhythmic pattern of the variation of cardiointervals related to respiratory waves. Owing to the diversity of patterns of the spectral behavior of cardiointervals in a newborn during 24 h, no single short segment of the cardiointerval recording could serve as an unambiguous characteristic of the newborn’s physiological state. The actual heart rate variability pattern could be seen by means of the spectraltemporal analysis, visualizing the dynamics of conversion of one spectral type to another, i.e., the change of the dominant system of regulation.     

Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions

A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome. The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method. The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5′ and 3′ ends of the maize 26 S rDNA have been located by comparison with the E. coli sequence. The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA. In all cases, there are numerous regions of 70% or higher homology.     

PA26 is a candidate gene for heterotaxia in humans: identification of a novel PA26-related gene family in human and mouse

Heterotaxia is an aetiologically heterogeneous condition caused by an abnormal left-right axis formation, resulting in reversed left-right polarity of one or more organ systems. In a patient with heterotaxia and a de novo reciprocal translocation t(6;18)(q21;q21), we found that the PA26 gene was disrupted by the 6q21 breakpoint. Northern blot analysis showed decreased expression of the PA26 gene in an Epstein-Barr virus-transformed cell line of this patient. During early embryogenesis of Xenopus, the orthologue of PA26, XPA26 is exclusively expressed in the notochord, a midline structure. This further supports a possible role of PA26 in human situs determination. Mutation analysis of human PA26 gene in 40 unrelated individuals with unexplained heterotaxia failed to identify mutations, indicating that PA26 mutations are not a frequent cause of heterotaxia in humans. Analysis of the PA26 gene structure resulted in the identification of a novel PA26-related gene family, which we have named the sestrin family, and which comprises three closely related genes in human and in mouse.     

Variability of polymorphic gene markers in Buryat and Russian newborns from Ulan-Ude]

Variability of ten polymorphic loci (ABO, RhD, PGD, ACP, PGM1, GLO, ESD, ADA, GC, TF) was studied in 326 Buryat and 310 Russian newborns from Ulan-Ude city. Marked differences between two groups were observed in the distributions of allelic frequencies of ABO, RhD, PGD, ACP, PGM1, GLO, ESD, GC loci. Genetic similarities between Buryat and other mongoloids were estimated. Close similarity was observed between Buryat, Mongols, Yakut and Kyzyl.     

The genome sequence of Pseudoplusia includens single nucleopolyhedrovirus and an analysis of p26 gene evolution in the baculoviruses

Background

Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated.

Results

The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX – Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins.

Conclusions

PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1323-9) contains supplementary material, which is available to authorized users.     

Connexin 26 expression and mutation analysis in epidermal disease

Gap junctional communication has a key role in the co-ordination of keratinocyte differentiation. Multiple connexins are expressed in the epidermis and mutations in four of these connexins are associated with disorders of keratinisation. Specific autosomal dominant Cx26 mutations have been associated with syndromes of skin disease and hearing loss. Here we describe the characterization of a new Cx26 polyclonal antibody raised against the cytoplasmic region of the protein. It has been used to investigate Cx26 protein localization in epidermal disease and in the study of mutant Cx26 proteins.     

Connexin- and Pannexin-Based Channels in Normal Skeletal Muscles and Their Possible Role in Muscle Atrophy

Precursor cells of skeletal muscles express connexins 39, 43 and 45 and pannexin1. In these cells, most connexins form two types of membrane channels, gap junction channels and hemichannels, whereas pannexin1 forms only hemichannels. All these channels are low-resistance pathways permeable to ions and small molecules that coordinate developmental events. During late stages of skeletal muscle differentiation, myofibers become innervated and stop expressing connexins but still express pannexin1 hemichannels that are potential pathways for the ATP release required for potentiation of the contraction response. Adult injured muscles undergo regeneration, and connexins are reexpressed and form membrane channels. In vivo, connexin reexpression occurs in undifferentiated cells that form new myofibers, favoring the healing process of injured muscle. However, differentiated myofibers maintained in culture for 48 h or treated with proinflammatory cytokines for less than 3 h also reexpress connexins and only form functional hemichannels at the cell surface. We propose that opening of these hemichannels contributes to drastic changes in electrochemical gradients, including reduction of membrane potential, increases in intracellular free Ca²⁺ concentration and release of diverse metabolites (e.g., NAD⁺ and ATP) to the extracellular milieu, contributing to multiple metabolic and physiologic alterations that characterize muscles undergoing atrophy in several acquired and genetic human diseases. Consequently, inhibition of connexin hemichannels expressed by injured or denervated skeletal muscles might reduce or prevent deleterious changes triggered by conditions that promote muscle atrophy.