Generation and characterization of mice with null mutation of the chloride intracellular channel 1 gene

CLIC1 belongs to a family of highly conserved and widely expressed intracellular chloride ion channel proteins existing in both soluble and membrane integrated forms. To study the physiological and biological role of CLIC1 in vivo, we undertook conditional gene targeting to engineer Clic1 gene knock‐out mice. This represents creation of the first gene knock‐out of a vertebrate CLIC protein family member. We first generated a Clic1 Knock‐in (Clic1^FN) allele, followed by Clic1 knock‐out (Clic1^−/−) mice by crossing Clic1^FN allele with TNAP‐cre mice, resulting in germline gene deletion through Cre‐mediated recombination. Mice heterozygous or homozygous for these alleles are viable and fertile and appear normal. However, Clic1^−/− mice show a mild platelet dysfunction characterized by prolonged bleeding times and decreased platelet activation in response to adenosine diphosphate stimulation linked to P2Y₁₂ receptor signaling. genesis 48:127–136, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular mechanisms of Bartter syndrome caused by mutations in the BSND gene

Barttin, a gene product of BSND, was identified as a fourth gene responsible for Bartter syndrome. The co-expression of barttin with CLC-K chloride channels has been demonstrated to dramatically induce the expression of CLC-K current. However, it remains unknown how barttin interacts with CLC-K channels in mammalian cells and how the mutations of barttin lead to Bartter syndrome. In an attempt to clarify the effect of barttin expression on CLC-K2 cellular localization, we examined the expression of the CLC-K2 chloride channel and barttin, solely and in combination, in transient and stable expression systems in mammalian cells. In addition, we generated several stable cell lines expressing mutant barttins to clarify the consequence of the previously reported barttin mutations in Bartter syndrome. In immunocytochemistry, CLC-K2 was retained in the Golgi in the absence of barttin expression, but delivered to the plasma membrane when barttin was present. Barttin was coprecipitated with CLC-K2, suggesting a protein-protein interaction. Disease-causing mutant barttins, especially R8L, were retained intracellularly, but their binding ability to CLC-K2 was preserved. This led to a retention of CLC-K2 in intracellular organelles with barttin, and a loss of plasma membrane localization. The stability of the CLC-K2 protein was also markedly increased by coexpression with barttin. These results clarified that barttin determined cellular localization of CLC-K2 by protein-protein interaction. Thus, the mislocalization of CLC-K2 was identified as the molecular pathogenesis of Bartter syndrome by mutant barttins.     

Ala397Asp mutation of myosin VIIA gene segregating in a Spanish family with type-Ib Usher syndrome

In the current study, 12 Spanish families affected by type-I Usher syndrome, that was previously linked to chromosome 11q, were screened for the presence of mutations in the N-terminal coding portion of the motor domain of the myosin VIIA gene by single-strand conformation polymorphism analysis of the first 14 exons. A mutation (Ala397Asp) segregating with the disease was identified, and several polymorphisms were also detected. It is presumed that the other USH1B mutations in these families could be located in the unscreened regions of the gene. Received: 10 November 1997 / Accepted: 15 March 1998     

A "de novo" mutation of the LDL-receptor gene as the cause of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a common genetic disorder caused by mutations of the LDL-receptor gene and transmitted as a co-dominant trait. However, there are some forms of hypercholesterolemia which have a recessive type of transmission. We have identified a subject with the clinical phenotype of heterozygous FH whose parents had normal plasma lipid values, suggesting a recessive type of transmission. The analysis of the LDL-receptor gene revealed that the patient was heterozygous for a G>C transversion in exon 4, which results in a serine for cysteine substitution at position 88 (C88S) of the receptor protein. Since this novel mutation was not found in the proband's parents and non-paternity was excluded, we concluded that the patient was a carrier of a "de novo" mutation. Haplotype analysis of LDL-receptor locus indicated that this "de novo" mutation occurred in the paternal germ line. The C88S mutation is the likely cause of LDL-receptor defect as it was present in the proband's hypercholesterolemic son and was not found in 200 chromosomes of control subjects.     

A novel founder mutation in the RNASEL gene, 471delAAAG,is associated with prostate cancer in Ashkenazi Jews

HPC1/RNASEL was recently identified as a candidate gene for hereditary prostate cancer. We identified a novel founder frameshift mutation in RNASEL, 471delAAAG, in Ashkenazi Jews. The mutation frequency in the Ashkenazi population, estimated on the basis of the frequency in 150 healthy young women, was 4% (95% confidence interval [CI] 1.9%-8.4%). Among Ashkenazi Jews, the mutation frequency was higher in patients with prostate cancer (PRCA) than in elderly male control individuals (6.9% vs. 2.4%; odds ratio = 3.0; 95% CI 0.6-15.3; P=.17). 471delAAAG was not detected in the 134 non-Ashkenazi patients with PRCA and control individuals tested. The median age at PRCA diagnosis did not differ significantly between the Ashkenazi carriers and noncarriers included in our study. However, carriers received diagnoses at a significantly earlier age, compared with patients with PRCA who were registered in the Israeli National Cancer Registry (65 vs. 74.4 years, respectively; P<.001). When we examined two brothers with PRCA, we found a heterozygous 471delAAAG mutation in one and a homozygous mutation in the other. Loss of heterozygosity was demonstrated in the tumor of the heterozygous sib. Taken together, these data suggest that the 471delAAAG null mutation is associated with PRCA in Ashkenazi men. However, additional studies are required to determine whether this mutation confers increased risk for PRCA in this population.     

Identification of a founder mutation in the protoporphyrinogen oxidase gene in variegate porphyria patients from chile

Variegate porphyria (VP; OMIM 176200) is characterized by a partial defect in the activity of protoporphyrinogen oxidase (PPO), the seventh enzyme of the porphyrin-heme biosynthetic pathway. The disease is usually inherited as an autosomal dominant trait displaying incomplete penetrance. In an effort to characterize the spectrum of molecular defects in VP, we identified 3 distinct mutations in 6 VP families from Chile by PCR, heteroduplex analysis, automated sequencing, restriction enzyme digestion and haplotyping analysis. The mutations consisted of 2 deletions and 1 missense mutation, designated 1239delTACAC, 1330delT and R168H. The occurrence of the missense mutation R168H had been reported previously in American, German and Dutch VP families, suggesting that this may represent a frequent recurrent mutation. Interestingly, the mutation 1239delTACAC was found in patients from 4 unrelated families living in different parts of Chile, suggesting that it might represent a common mutation in Chile. Haplotype analysis using 15 microsatellite markers which closely flank the PPO gene on chromosome 1q22, spanning approximately 21 cM, revealed the presence of R168H on different haplotypes in 6 VP patients from 3 unrelated families. In contrast, we found the occurrence of 1239delTACAC on the same chromosome 1 haplotype in 11 mutation carriers from 4 unrelated families with VP. These findings are consistent with R168H representing a hotspot mutation and 1239delTACAC existing as a founder mutation in the PPO gene. Our data comprise the first genetic studies of the porphyrias in South America and will streamline the elucidation of the genetic defects in VP patients from Chile by allowing an initial screening for the founder mutation 1239delTACAC.     

Homozygous nonsense mutation in the FOXE3 gene as a cause of congenital primary aphakia in humans

Congenital primary aphakia (CPA) is a rare developmental disorder characterized by the absence of lens, the development of which is normally induced during the 4th-5th wk of human embryogenesis. This original failure leads, in turn, to complete aplasia of the anterior segment of the eye, which is the diagnostic histological criterion for CPA. So far, the genetic basis for this human condition has remained unclear. Here, we present the analysis of a consanguineous family with three siblings who had bilateral aphakia, microphthalmia, and complete agenesis of the ocular anterior segment. We show that a null mutation in the FOXE3 gene segregates and, in the homozygous state, produces the mutant phenotype in this family. Therefore, this study identifies--to our knowledge, for the first time--a causative gene for CPA in humans. Furthermore, it indicates a possible critical role for FOXE3 very early in the lens developmental program, perhaps earlier than any role recognized elsewhere for this gene.     

Characterization of a novel Nav1.5 channel mutation, A551T, associated with Brugada syndrome

Brugada syndrome is a life-threatening, inherited arrhythmia disorder associated with autosomal dominant mutations in SCN5A, the gene encoding the human cardiac Na⁺ channel α subunit (Nav1.5). Here, we characterized the biophysical properties of a novel Brugada syndrome-associated Nav1.5 mutation, A551T, identified in a proband who was successfully resuscitated from an episode of ventricular fibrillation with sudden collapse. Whole-cell currents through wild-type (WT) Nav1.5 and mutant (A551T) channels were recorded and compared in the human embryonic kidney cell line HEK293T transfected with SCN5A cDNA and SCN1B cDNA, using the patch-clamp technique. Current density was decreased in the A551T mutant compared to the WT. In addition, the A551T mutation reduced Nav1.5 activity by promoting entry of the channel into fast inactivation from the closed state, thereby shifting the steady-state inactivation curve by -5 mV. Furthermore, when evaluated at -90 mV, the resting membrane potential, but not at the conventionally used -120 mV, both the percentage, and rate, of channel recovery from inactivation were reduced in the mutant. These results suggest that the DI-DII linker may be involved in the stability of inactivation gating process. This study supports the notion that a reduction in Nav1.5 channel function is involved in the pathogenesis of Brugada syndrome. The structural-functional study of the Nav1.5 channel advances our understanding of its pathophysiolgocial function.     

Homozygosity mapping identifies a GALK1 mutation as the cause of autosomal recessive congenital cataracts in 4 adult siblings

Objective

Monogenic congenital cataract is one of the most genetically heterogeneous ocular conditions with almost 30 different genes involved in its etiology. In adult patients, genotype–phenotype correlations are troubled by eye surgery during infancy and/or long-term ocular complications. Here, we describe the molecular diagnosis of GALK1 deficiency as the cause of autosomal recessive congenital cataract in a family from Costa Rica.

Methods

Four affected siblings were included in the study. All of them underwent eye surgery during the first decade but medical records were not available. Congenital cataract was diagnosed by report. Molecular analysis included genome wide homozygosity mapping using a 250 K SNP Affymetrix microarray followed by PCR amplification and direct nucleotide sequencing of candidate gene.

Results

Genome wide homozygosity mapping revealed a 6 Mb region of homozygosity shared by two affected siblings at 17q25. The GALK1 gene was included in this interval and direct sequencing of this gene revealed a homozygous c.1144C>T mutation (p.Q382*) in all four affected subjects.

Conclusions

This work demonstrates the utility of homozygosity mapping in the retrospective diagnosis of a family with congenital cataracts in which ocular surgery at early age, the lack of medical records, and the presence of long term eye complications, impeded a clear clinical diagnosis during the initial phases of evaluation.     

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit

Calcium-activated chloride channels (CaCCs) are major regulators of sensory transduction, epithelial secretion, and smooth muscle contraction. Other crucial roles of CaCCs include action potential generation in Characean algae and prevention of polyspermia in frog egg membrane. None of the known molecular candidates share properties characteristic of most CaCCs in native cells. Using Axolotl oocytes as an expression system, we have identified TMEM16A as the Xenopus oocyte CaCC. The TMEM16 family of "transmembrane proteins with unknown function" is conserved among eukaryotes, with family members linked to tracheomalacia (mouse TMEM16A), gnathodiaphyseal dysplasia (human TMEM16E), aberrant X segregation (a Drosophila TMEM16 family member), and increased sodium tolerance (yeast TMEM16). Moreover, mouse TMEM16A and TMEM16B yield CaCCs in Axolotl oocytes and mammalian HEK293 cells and recapitulate the broad CaCC expression. The identification of this new family of ion channels may help the development of CaCC modulators for treating diseases including hypertension and cystic fibrosis.     

Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome

Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.     

Congenital lamellar ichthyosis in Tunisia is caused by a founder nonsense mutation in the TGM1 gene

Lamellar ichthyosis (LI, MIM# 242300) is a severe autosomal recessive genodermatosis present at birth in the form of collodion membrane covering the neonate. Mutations in the TGM1 gene encoding transglutaminase-1 are a major cause of LI. In this study molecular analysis of two LI Tunisian patients revealed a common nonsense c.788G>A mutation in TGM1 gene. The identification of a cluster of LI pedigrees carrying the c.788G>A mutation in a specific area raises the question of the origin of this mutation from a common ancestor. We carried out a haplotype-based analysis by way of genotyping 4 microsatellite markers and 8 SNPs flanking and within the TGM1 gene spanning a region of 6 Mb. Haplotype reconstruction from genotypes of all members of the affected pedigrees indicated that all carriers for the mutation c.788G>A harbored the same haplotype, indicating common ancestor. The finding of a founder effect in a rare disease is essential for the genetic diagnosis and the genetic counselling of affected LI pedigrees in Tunisia.     

Disruption of clh-1, a chloride channel gene, results in a wider body of Caenorhabditis elegans

We cloned the clh-1 gene coding for a putative ClC chloride channel in Caenorhabditis elegans. The gene product exhibited a high degree of homology with human ClC-1 and ClC-2. The clh-1 gene was predominantly expressed in the hypodermis, including seam cells. Null mutations of clh-1 caused a significantly wider body and an abnormal alae structure. High osmolarity in the culture medium restored the normal body width of the clh-1 mutants. These results suggest that the clh-1 gene contributes to maintenance of the body width through regulation of osmolarity.     

Characterization of a murine gene homologous to the bovine CaCC chloride channel

The bovine CaCC protein is a putative Ca2+-dependent Cl- channel of airway epithelial cells. Therefore, CaCC proteins could contribute to transepithelial Cl- transport and accordingly modify the phenotype of cystic fibrosis (CF) patients. We have identified a murine EST containing a full-length cDNA coding for a 902-amino-acid protein highly homologous to bovine CaCC. The murine gene (mCaCC) maps to chromosome 3 at the H2-H3 band and is expressed, as indicated by Northern blot analysis, in mouse skin and kidney but not in brain, heart, lung or testis. RT-PCR indicates a low expression in tracheal epithelial cells. Heterologous expression of mCaCC in Xenopus oocytes elicits membrane currents that are anion-selective and inhibited by DIDS and by niflumic acid, a blocker of the endogenous chloride current in oocytes. The identification of genes belonging to the CaCC family will help to evaluate their role as ion channels or channel regulators and their actual contribution to epithelial chloride transport.     

Pyridoxine-dependent epilepsy in Tunisia is caused by a founder missense mutation of the ALDH7A1 gene

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder characterized by seizures and therapeutic response to pharmacological dose of pyridoxine. Mutations in the ALDH7A1 gene, encoding α-aminoadipic semialdehyde (α-AASA) dehydrogenase (antiquitin), have been reported to cause PDE in most patients. In this study molecular analysis of seven PDE Tunisian patients revealed a common missense c.1364T > C mutation in the ALDH7A1 gene. The identification of a cluster of PDE pedigrees carrying the c.1364T > C mutation in a specific area raises the question of the origin of this mutation from a common ancestor. We carried out a genotype-based analysis by way of genotyping a new generated microsatellite marker within the ALDH7A1 gene. Genotype reconstruction of all affected pedigree members indicate that all c.1364T > C mutation carriers harbored the same allele, indicating a common ancestor. The finding of a founder effect in a rare disease is essential for the genetic diagnosis and the genetic counseling of affected PDE pedigrees in Tunisia.     

A PCR-RFLP assay for the A716T mutation in the WFS1 gene,a common cause of low-frequency sensorineural hearing loss

Nonsyndromic low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of hearing loss that affects frequencies at 2,000 Hz and below. Recently, we reported five different heterozygous missense mutations in the Wolfram syndrome gene, WFS1, found to be responsible for LFSNHL in six families. One of the five mutations, A716T, may be a common cause of LFSNHL, as it has been reported in three families to date (Bespalova et al., 2001; Young et al., 2001). We have developed a PCR-based restriction fragment-length polymorphism (RFLP) assay to detect the A716T mutation in a simple, specific test. This method was evaluated with DNA samples from a family in which the A716T mutation was segregating with LFSNHL. This simple assay successfully detected the presence of the A716T mutation in all of the individuals predicted to be affected, based on audiologic results. Therefore, this assay can be routinely used for initial screening of the A716T mutation in patients with LFSNHL, before screening the entire coding region of the WFS1 gene.