Human genetics moves from clinic to bench--and back.
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Aging↗
Biomedical subjects
Xavier Estivill
Publications and source records attributed to Xavier Estivill.
Different mechanisms preclude mutant CLDN14 proteins from forming tight junctions in vitro.
Mutations in claudin 14 (CLDN14) cause nonsyndromic DFNB29 deafness in humans. The analysis of a murine model indicated that this phenotype is associated with degeneration of hair cells, possibly due to cation overload. However, the mechanism linking these alterations to CLDN14 mutations is unknown. To investigate this mechanism, we compared the ability of wild-type and missense mutant CLDN14 to form tight junctions. Ectopic expression in L mouse fibroblasts (LM cells) of wild-type CLDN14 protein induced the formation of tight junctions, while both the c.254T>A (p.V85D) mutant, previously identified in a Pakistani family, and the c.301 G>A (p.G101R) mutant, identified in this study through the screen of 183 Spanish and Greek patients affected with sporadic nonsyndromic deafness, failed to form such junctions. However, the two mutant proteins differed in their ability to localize at the plasma membrane. We further identified hitherto undescribed exons of CLDN14 that are utilized in alternative spliced transcripts. We demonstrated that different mutations of CLDN14 impaired by different mechanisms the ability of the protein to form tight junctions. Our results indicate that the ability of CLDN14 to be recruited to these junctions is crucial for the hearing process.
Alternative Splicing↗
Association of BDNF with restricting anorexia nervosa and minimum body mass index: a family-based association study of eight European populations.
Eating disorders (ED), such as anorexia nervosa (AN) and bulimia nervosa (BN), are complex psychiatric disorders where different genetic and environmental factors are involved. Several lines of evidence support that brain-derived neurotrophic factor (BDNF) plays an essential role in eating behaviour and that alterations on this neurotrophic system participates in the susceptibility to both AN and BN. Accordingly, intraventricular administration of BDNF in rats determines food starvation and body weight loss, while BDNF or its specific receptor NTRK2 knockout mice develop obesity and hyperphagia. Case-control studies also suggest a BDNF contribution in the aetiology of ED: we have previously reported a strong association between the Met66 variant within the BDNF gene, restricting AN (ANR) and minimum body mass index (minBMI) in a Spanish sample, and a positive association between the Val66Met and -270C/T BDNF SNPs and ED in six different European populations. To replicate these results, avoiding population stratification effects, we recruited 453 ED trios from eight European centres and performed a family-based association study. Both haplotype relative risk (HRR) and haplotype-based haplotype relative risk (HHRR) methods showed a positive association between the Met66 allele and ANR. Consistently, we also observed an effect of the Met66 variant on low minBMI and a preferential transmission of the -270C/Met66 haplotype to the affected ANR offspring. These results support the involvement of BDNF in eating behaviour and further suggest its participation in the genetic susceptibility to ED, mainly ANR and low minBMI.
Adult↗
Association of BDNF with anorexia, bulimia and age of onset of weight loss in six European populations.
Several genes with an essential role in the regulation of eating behavior and body weight are considered candidates involved in the etiology of eating disorders (ED), but no relevant susceptibility genes with a major effect on anorexia nervosa (AN) or bulimia nervosa (BN) have been identified. Brain-derived neurotrophic factor (BDNF) has been implicated in the regulation of food intake and body weight in rodents. We previously reported a strong association of the Met66 allele of the Val66Met BDNF variant with restricting AN (ANR) and low minimum body mass index in Spanish patients. Another single nucleotide polymorphism located in the promoter region of the BDNF gene (-270C>T) showed lack of association with any ED phenotype. In order to replicate these findings in a larger sample, we performed a case-control study in 1142 Caucasian patients with ED consecutively recruited in six different centers from five European countries (France, Germany, Italy, Spain and UK) participating in the 'Factors in Healthy Eating' project. We have found that the Met66 variant is strongly associated to all ED subtypes (AN, ANR, binge-eating/purging AN and BN), and that the -270C BDNF variant has an effect on BN and late age at onset of weight loss. These are the first two variants associated with the pathophysiology of ED in different populations and support a role for BDNF in the susceptibility to aberrant eating behaviors.
Age of Onset↗
Small marker chromosomes in two patients with segmental aneusomy for proximal 17p.
We report a nine-year-old girl (patient 1934) and a five-year-old boy (patient 2170) with small, de novo supernumerary marker chromosomes (SMCs) derived from proximal 17p. The clinical features of patient 1934 include developmental delay, triangular face, prominent forehead, low set ears, dental abnormalities, a high arched palate, long, flexible fingers, and joint laxity. Patient 2170 is affected with developmental delay, oral-motor dyspraxia/verbal apraxia, thick upper and lower lips, bilateral fifth finger clinodactyly, joint laxity and mild hypotonia. G-banded chromosome analysis of patient 1934 revealed mosaicism for a SMC in 72% of peripheral lymphocytes analyzed, whereas analysis of patient 2170 identified a smaller SMC present in 100% of cells analyzed. Fluorescence in situ hybridization (FISH) studies demonstrated that both of the SMCs derived from 17p10-p11.2. Using FISH and array-CGH analysis, the proximal breakpoints mapped within the centromere and the distal breakpoints were both located within the Smith-Magenis syndrome (SMS) common deletion region. We compare the clinical characteristics of our patients with those previously reported to have either SMC including 17p or duplications of proximal 17p in an effort to further delineate the phenotype of trisomy 17p10-p11.2 and to elucidate genotype-phenotype correlations.
Abnormalities, Multiple↗
Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4).
Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.
Amino Acid Sequence↗
Characterization of the segmental duplication LCR7-20 in the human genome.
Our previous study described the amplification of a genomic sequence containing exon 9 of CFTR in the human genome. Here we report that this CFTR sequence is part of a large duplicated sequence unit, provisionally named LCR7-20. Through successive screening of two human chromosome 7-specific cosmid libraries to construct a cosmid contig, we assembled two sequenced BAC clones into a single contig containing a prototypic LCR7-20 unit. Subsequent searches of existing human genome sequences identified additional six copies of LCR7-20-like sequences with more than 90% sequence homology. Additional genomic clones containing LCR7-20-like sequences were then isolated from total genomic BAC and PAC libraries. Restriction fragment analysis and limited sequencing data indicated that there could be around 30 copies of LCR7-20-like sequences in the human genome and that the average region of homology could extend over 120 kb. As indicated by fluorescence in situ hybridization analysis, LCR7-20-like sequences are dispersed on different chromosomes, mainly in the centromeric and pericentromeric regions, and some may exist in tandem copies. Our study also indicates that many genomic regions containing LCR7-20's either have been misassembled or are missing in current versions of the human genome sequence.
Blotting, Southern↗
Human mitochondrial transcription factor B1 as a modifier gene for hearing loss associated with the mitochondrial A1555G mutation.
Phenotypic expression of the deafness-associated homoplasmic A1555G mutation in the mitochondrial 12S rRNA gene varies from profound congenital hearing loss to normal hearing. It has been shown that this variability in clinical expression in most patients is due to the complex inheritance of multiple nuclear-encoded modifier genes. Human mitochondrial transcription factor B1 (TFB1M) has been proposed as a candidate for being such a modifier, since it methylates adenine residues in the adjacent loop of the A1555G mutation in the 12S rRNA gene. Polymorphic markers within and adjacent to the TFB1M gene were genotyped in 214 individuals from 41 multiplex families with the A1555G mutation of Spanish, Italian, and Arab-Israeli origin. Multipoint non-parametric linkage analysis of all families combined revealed an NPL score of 1.7 (P = 0.05), and a Lod score of 1.4 (P = 0.04). Linkage disequilibrium by the Transmission Disequilibrium Test at D6S1577, a microsatellite adjacent to TFB1M, showed preferential non-transmission of an allele to affected individuals with chi2 = 8.76; P = 0.003. Sequence analysis of the coding region of the gene and testing of all intragenic SNPs did not reveal a putative causative mutation. These data provide suggestive evidence that TFB1M is a nuclear-encoded modifier gene for phenotypic expression of the A1555G mutation, and that the effect may occur through a regulatory or splicing mutation.
Chromosomes, Human, Pair 6↗
Phenotype of non-syndromic deafness associated with the mitochondrial A1555G mutation is modulated by mitochondrial RNA modifying enzymes MTO1 and GTPBP3.
Phenotypic expression of the deafness-associated mitochondrial A1555G mutation in the 12S rRNA gene is influenced by aminoglycosides and complex inheritance of nuclear-encoded modifier genes. The position of a major nuclear modifier gene has been localized to chromosome 8p23.1, but the identification of this gene has remained elusive. Recently, we identified a second modifier gene, mitochondrial transcription factor B1 (TFB1M), involved in mitochondrial rRNA modification. In the present study, we tested three genes involved in mitochondrial tRNA or rRNA modification, and two genes associated with non-syndromic deafness, for linkage and linkage disequilibrium (LD) in 214 DNA samples from Spanish, Italian, and Arab-Israeli families with maternally inherited non-syndromic hearing loss. The multipoint non-parametric linkage analysis and transmission disequilibrium test testing were done using all families combined as well as divided based on linkage to the chromosome 8 locus and ethnicity. Two genes, MTO1 and GTPBP3, showed strongly suggestive linkage and significant LD results. Since both genes, as well as TFB1M, are involved in the process of mitochondrial RNA modification, it appears that the modification of mitochondrial RNA is an important regulatory pathway in the phenotypic expression of the deafness-associated mitochondrial A1555G mutation. This conclusion was supported by comparing linkage results of simulated genotypes with actual results for the four genes involved in mitochondrial RNA modification.
Carrier Proteins↗
Different CFTR mutational spectrum in alcoholic and idiopathic chronic pancreatitis?
OBJECTIVE: Cystic fibrosis transmembrane conductance regulator (CFTR) mutations are responsible for cystic fibrosis (CF) and have been postulated as a predisposing risk factor to chronic pancreatitis (CP), but controversial results demand additional support. We have therefore investigated the role of the CFTR gene in a cohort of 68 CP patients. METHODS: We have performed the CFTR gene analysis using 2 screening techniques. Fragments showing abnormal migration patterns were characterized by sequencing. Patients were classified in alcoholic (ACP) (n = 37) and idiopathic (ICP) (n = 31) chronic pancreatitis. Clinical features of CP and CF were evaluated. RESULTS: Sixteen mutations/variants were identified in 27 patients (40%), most of them (35%) presenting a single CFTR mutant gene. The 1716G/A variant showed the highest frequency accounting for 22% in ICP and 5% in ACP, in contrast with other more common mutations such as F508del found in 8% of ACP and the 5T variant identified in 7% of patients. Acute pancreatitis, abdominal pain, tobacco, pancreatic calcifications, and pancreatic pseudocysts showed significant higher values in ACP than ICP patients. No significant differences were found between patients with and without CFTR mutations. CONCLUSIONS: Apart from reinforcing previous findings our data highlight the increased susceptibility of CFTR heterozygous to developing CP. Heterozygosity, combined with other factors, places these individuals at greater risk.
Adult↗
Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.
Base Sequence↗
Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: a multicenter study.
Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.
Connexin 26↗
Identification and characterization of a novel splice-site mutation in a patient with Wiskott-Aldrich syndrome.
Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by immunodeficiency, eczema, and thrombocytopenia with small platelets. A wide spectrum of mutations in the Wiskott-Aldrich syndrome protein ( WASP) gene have been identified as causative of the disease. In the present paper, we report on a family with a boy affected by WAS, with a splice-site mutation caused by a T to G substitution in the +2 position of intron 6 (IVS6+2T>G). Expression studies performed in COS-7 and U-937 cells showed that the mutation affected the normal splicing process. As a consequence, an abnormally long transcript of 38 nucleotides is generated. Such missplicing is probably due to the activation of a cryptic splice donor site located 38 nt downstream of exon 6. The translation of such aberrant mRNA will produce a truncated protein with a premature stop at codon 190. Thus, a novel splice-site mutation is reported in a patient with a mild WAS phenotype.
Animals↗
Phosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life.
Calcipressin 1 is an endogenous inhibitor of calcineurin, which is a serine/threonine phosphatase under the control of Ca(2+) and calmodulin. Calcipressin 1 is encoded by DSCR1, a gene on human chromosome 21 with seven exons, exons 1-4 are alternative first exons (isoforms 1-4). We show that calcipressin 1 isoform 1 has an N-terminal coding region longer than that previously described, and this generates a new polypeptide of 252 amino acids. This polypeptide is able to interact with calcineurin A and to inhibit NF-AT-mediated transcriptional activation. We demonstrate for the first time that endogenous calcipressin 1 exists as a complex together with the calcineurin A and B heterodimer. Calcipressin 1 is a phosphoprotein that increases its capacity to inhibit calcineurin when phosphorylated at the FLISPP motif, and this phosphorylation also controls the half-life of calcipressin 1 by accelerating its degradation. Additionally, we have also detected further phosphorylation sites outside the FLISPP motif and these contribute to the complex phosphorylation pattern of calcipressin 1. Taking all these results into consideration we suggest that phosphorylation of calcipressin 1 is involved in the regulation of the phosphatase activity of calcineurin and can therefore act as a modulator of calcineurin-dependent cellular pathways.
Amino Acid Motifs↗
Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements.
The sequence of the mouse genome allows one to compare the conservation of synteny between the human and mouse genome and exploration of regions that might have been involved in major rearrangements during the evolution of these two species (evolutionary genome rearrangements). Recent segmental duplications (or duplicons) are paralogous DNA sequences with high sequence identity that account for about 3.5-5% of the human genome and have emerged during the past approximately 35 million years of evolution. These regions are susceptible to illegitimate recombination leading to rearrangements that result in genomic disorders or genomic mutations. A catalogue of several hundred segmental duplications potentially leading to genomic rearrangements has been reported. The authors and others have observed that some chromosome regions involved in genomic disorders are shuffled in orientation and order in the mouse genome and that regions flanked by segmental duplications are often polymorphic. We have compared the human and mouse genome sequences and demonstrate here that recent segmental duplications correlate with breaks of synteny between these two species. We also observed that nine primary regions involved in human genomic disorders show changes in the order or the orientation of mouse/human synteny segments, were often flanked by segmental duplications in the human sequence. We found that 53% of all evolutionary rearrangement breakpoints associate with segmental duplications, as compared with 18% expected in a random location of breaks along the chromosome (P<0.0001). Our data suggest that segmental duplications have participated in the recent evolution of the human genome, as driving forces for evolutionary rearrangements, chromosome structure polymorphisms and genomic disorders.
Animals↗
DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly.
The protein kinase DYRK1A is distributed throughout the nucleoplasm, accumulating in speckle-like regions. We have found that this punctuated nuclear distribution is determined by the contribution of several elements. Although the nuclear import is mediated by two distinct nuclear localization signals, one at the N-terminus and the other located in the linker region, between subdomains X and XI of the catalytic domain, the accumulation in speckles that are SC35 positive depends on a sequence motif that is located C-terminal to the kinase domain and comprises a histidine tail. A similar sequence is also responsible for the targeting of cyclin T1. Therefore the histidine-rich region represents a novel splicing speckle targeting signal. Moreover, overexpression of DYRK1A induces speckle disassembly. Such disassembly is DYRK1A activity specific, since the overexpression of a DYRK1A kinase inactive mutant, the paralogous DYRK1B or a chimeric protein DYRK1B that has been directed to the speckles via the DYRK1A targeting signal, leaves the SC35 speckle pattern untouched. Thus DYRK1A protein kinase may play a role in regulating the biogenesis of the splicing speckle compartment.
Animals↗
Genomic inversions of human chromosome 15q11-q13 in mothers of Angelman syndrome patients with class II (BP2/3) deletions.
Parental submicroscopic genomic inversions have recently been demonstrated to be present in several genomic disorders. These inversions are genomic polymorphisms that facilitate misalignment and abnormal recombination between flanking segmental duplications. Angelman syndrome (AS; MIM 105830) is associated with specific abnormalities of chromosome 15q11-q13, with about 70% of cases being mother-of-origin 4 Mb deletions. We present here evidence that some mothers of AS patients with deletions of the 15q11-q13 region have a heterozygous inversion involving the region that is deleted in the affected offspring. The inversion was detected in the mothers of four of six AS cases with the breakpoint 2-3 (BP2/3) 15q11-q13 deletion, but not in seven mothers of AS due to paternal uniparental disomy (UPD) 15. We have identified variable inversion breakpoints within BP segmental duplications in the inverted AS mothers, as well as in AS deleted patients. Interestingly, the BP2-BP3 region is inverted in the mouse draft genome sequence with respect to the human draft sequence. The BP2-BP3 chromosome 15q11-q13 inversion was detected in four of 44 subjects (9%) of the general population (P<0.004). The BP2/3 inversion should be an intermediate estate that facilitates the occurrence of 15q11-q13 BP2/3 deletions in the offspring.
Angelman Syndrome↗
Human chromosome 7: DNA sequence and biology.
DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.
Animals↗