Claudin-19 mutations and clinical phenotype in Spanish patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsorption in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease.

Disentangling reasons for low Y chromosome variation in the greater white-toothed shrew (Crocidura russula).

Y chromosome variation is determined by several confounding factors including mutation rate, effective population size, demography, and selection. Disentangling these factors is essential to better understand the evolutionary properties of the Y chromosome. We analyzed genetic variation on the Y chromosome, X chromosome, and mtDNA of the greater white-toothed shrew, a species with low variance in male reproductive success and limited sex-biased dispersal, which enables us to control to some extent for life-history effects. We also compared ancestral (Moroccan) to derived (European) populations to investigate the role of demographic history in determining Y variation. Recent colonization of Europe by a small number of founders (combined with low mutation rates) is largely responsible for low diversity observed on the European Y and X chromosomes compared to mtDNA. After accounting for mutation rate, copy number, and demography, the Y chromosome still displays a deficit in variation relative to the X in both populations. This is possibly influenced by directional selection, but the slightly higher variance in male reproductive success is also likely to play a role, even though the difference is small compared to that in highly polygynous species. This study illustrates that demography and life-history effects should be scrutinized before inferring strong selective pressure as a reason for low diversity on the Y chromosome.

Origin and genetic differentiation of Three Mexican Native Groups (Purépechas, Triquis and Mayas): contribution of CODIS-STRs to the history of human populations of Mesoamerica

BACKGROUND: CODIS-STRs in Native Mexican groups have rarely been analysed for human identification and anthropological purposes. AIM:To analyse the genetic relationships and population structure among three Native Mexican groups from Mesoamerica.SUBJECTS AND METHODS: 531 unrelated Native individuals from Mexico were PCR-typed for 15 and 9 autosomal STRs (Identifiler™ and Profiler™ kits, respectively), including five population samples: Purépechas (Mountain, Valley and Lake), Triquis and Yucatec Mayas. Previously published STR data were included in the analyses. RESULTS:Allele frequencies and statistical parameters of forensic importance were estimated by population. The majority of Native groups were not differentiated pairwise, excepting Triquis and Purépechas, which was attributable to their relative geographic and cultural isolation. Although Mayas, Triquis and Purépechas-Mountain presented the highest number of private alleles, suggesting recurrent gene flow, the elevated differentiation of Triquis indicates a different origin of this gene flow. Interestingly, Huastecos and Mayas were not differentiated, which is in agreement with the archaeological hypothesis that Huastecos represent an ancestral Maya group. Interpopulation variability was greater in Natives than in Mestizos, both significant.CONCLUSION: Although results suggest that European admixture has increased the similarity between Native Mexican groups, the differentiation and inconsistent clustering by language or geography stresses the importance of serial founder effect and/or genetic drift in showing their present genetic relationships.

Frequency and impact of autosomal dominant polycystic kidney disease in the Seychelles (Indian Ocean).

BACKGROUND: As little such data is available in African populations, we investigated the prevalence of ADPKD and the impact of the disease in the Seychelles islands, where approximately 65% of the population is of African descent and 30% of Caucasian or mixed descent. METHODS: Prevalent cases were identified over a 3-year period by requesting all the doctors in the country (most of them are employed within a national health system) to refer all presumed or confirmed cases and by systematically examining the family members of all confirmed cases. The diagnosis was based on standard criteria including ultrasonographic findings and family history. RESULTS: Forty-two cases were identified in this population of 74,331 inhabitants, a total prevalence (per 100,000 total population) of 57 (95% CI, 41-76). All but one of the cases were of Caucasian descent so that the prevalence rates of the disease in the populations of Black and Caucasian descents were respectively 2 (0-11) and 184 (132-249). The prevalence rates of the gene(s) carriers were estimated to be 75 (45-117) in the total population respectively 6 (0-33) and 236 (140-372) in the Black and Caucasian populations. Haplotype analysis in 58 cases from three families showed a common DNA fragment in all affected individuals. Cases had significantly higher blood pressure compared to the general population and 21% had serum creatinine higher than 120 mumol/l. Among the established pedigrees, mean age of death between 1960 and 1995 (haemodialysis was introduced in 1992) was younger in subjects with than those without ADPKD (50.5 vs 67.7 years; P < 0.001). CONCLUSIONS: In the Seychelles, ADPKD clusters in the Caucasian population (possibly a founder effect), is rare in individuals of black descent, and is associated with substantial clinical and survival impact.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

Mutation hot spots in 5q31-linked corneal dystrophies.

Mutations in the BIGH3 gene on chromosome 5q31 cause four distinct autosomal dominant diseases of the human cornea: granular (Groenouw type I), Reis-Bücklers, lattice type I, and Avellino corneal dystrophies. All four diseases are characterized by both progressive accumulation of corneal deposits and eventual loss of vision. We have identified a specific recurrent missense mutation for each type of dystrophy, in 10 independently ascertained families. Genotype analysis with microsatellite markers surrounding the BIGH3 locus was performed in these 10 families and in 5 families reported previously. The affected haplotype could be determined in 10 of the 15 families and was different in each family. These data indicate that R555W, R124C, and R124H mutations occurred independently in several ethnic groups and that these mutations do not reflect a putative founder effect. Furthermore, this study confirms the specific importance of the R124 and R555 amino acids in the pathogenesis of autosomal dominant corneal dystrophies linked to 5q.

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf (Canis lupus) population

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central-southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise F(ST) values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first-generation migrants indicates that migration has been unidirectional and male-biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long-range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25-2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8-16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.

Distribution of the transposable elements bilbo and gypsy in original and colonizing populations of Drosophila subobscura

Background: Transposable elements (TEs) constitute a substantial amount of all eukaryotic genomes. They induce an important proportion of deleterious mutations by insertion into genes or gene regulatory regions. However, their mutational capabilities are not always adverse but can contribute to the genetic diversity and evolution of organisms. Knowledge of their distribution and activity in the genomes of populations under different environmental and demographic regimes, is important to understand their role in species evolution. In this work we study the chromosomaldistribution of two TEs, gypsy and bilbo, in original and colonizing populations of Drosophilasubobscura to reveal the putative effect of colonization on their insertion profile.Results: Chromosomal frequency distribution of two TEs in one original and three colonizingpopulations of D. subobscura, is different. Whereas the original population shows a low insertionfrequency in most TE sites, colonizing populations have a mixture of high (frequency ¿ 10%) andlow insertion sites for both TEs. Most highly occupied sites are coincident among colonizingpopulations and some of them are correlated to chromosomal arrangements. Comparisons of TEcopy number between the X chromosome and autosomes show that gypsy occupancy seems to becontrolled by negative selection, but bilbo one does not. Conclusion: These results are in accordance that TEs in Drosophila subobscura colonizing populations are submitted to a founder effect followed by genetic drift as a consequence of colonization. This would explain the high insertion frequencies of bilbo and gypsy in coincident sites of colonizing populations. High occupancy sites would represent insertion events prior to colonization. Sites of low frequency would be insertions that occurred after colonization and/orcopies from the original population whose frequency is decreasing in colonizing populations. Thiswork is a pioneer attempt to explain the chromosomal distribution of TEs in a colonizing specieswith high inversion polymorphism to reveal the putative effect of arrangements in TE insertionprofiles. In general no associations between arrangements and TE have been found, except in a fewcases where the association is very strong. Alternatively, founder drift effects, seem to play aleading role in TE genome distribution in colonizing populations.

A Diagnostic view of the Genetics of CASADIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL, OMIM #125310) is an inherited vascular disease. The main symptoms include migraineous headache, recurrent strokes and progressive cognitive impairment. CADASIL is caused by mutations in the NOTCH3 gene which result in degeneration of vascular smooth muscle cells, arteriolar stenosis and impaired cerebral blood flow. The aims of this study were assessment of the genetic background of Finnish and Swedish CADASIL patients, analysis of genetic and environmental factors that may influence the phenotype, and identification of the optimal diagnostic strategy. The majority of Finnish CADASIL patients carry the p.Arg133Cys mutation. Haplotype analysis of 18 families revealed a region of linkage disequilibrium around the NOTCH3 locus, which is evidence for a founder effect and a common ancestral mutation. Despite the same mutational background, the clinical course of CADASIL is highly variable between and even within families. The association of several genetic factors with the phenotypic variation was investigated in 120 CADASIL patients. Apolipoprotein E allele 4 was associated with earlier occurrence of strokes, especially in younger patients. Study of a pair of monozygotic twins with CADASIL revealed environmental factors which may influence the phenotype, i.e. smoking, statin medication and physical activity. Knowledge of these factors is useful, since life-style choices may influence the disease progression. The clinical CADASIL diagnosis can be confirmed by detection of either the NOTCH3 mutation or granular osmiophilic material by electron microscopy in skin biopsy, although the sensitivity estimates have been contradictory. Comparison of these two methods in a group of 131 diagnostic cases from Finland, Sweden and France demonstrated that both methods are highly sensitive and reliable.

Combining genetic and demographic data for the conservation of a Mediterranean habitat-forming species

The integration of ecological and evolutionary data is highly valuable for conservation planning. However, it has been rarely used in the marine realm, where the adequate design of marine protected areas (MPAs) is urgently needed. Here, we examined the interacting processes underlying the patterns of genetic structure and demographic strucuture of a highly vulnerable Mediterranean habitat-forming species (i.e. Paramuricea clavata (Risso, 1826)), with particular emphasis on the processes of contemporary dispersal, genetic drift, and colonization of a new population. Isolation by distance and genetic discontinuities were found, and three genetic clusters were detected; each submitted to variations in the relative impact of drift and gene flow. No founder effect was found in the new population. The interplay of ecology and evolution revealed that drift is strongly impacting the smallest, most isolated populations, where partial mortality of individuals was highest. Moreover, the eco-evolutionary analyses entailed important conservation implications for P. clavata. Our study supports the inclusion of habitat-forming organisms in the design of MPAs and highlights the need to account for genetic drift in the development of MPAs. Moreover, it reinforces the importance of integrating genetic and demographic data in marine conservation.

21-Hydroxylase deficiency in Brazil

We determined the frequency of large rearrangements and point mutations in 130 Brazilian patients with 21-hydroxylase deficiency and correlated genotype with phenotype. The frequency of CYP21 deletions was lower (4.4%) than in most of the previous series described, whereas the frequency of large gene conversions was similar to the frequency reported in the literature (6.6%). The most frequent point mutations were I2 splice (41.8% in salt wasting - SW), I172N (32.6% in simple virilizing - SV) and V281L (40.2% in the late onset form - LO). The frequency of the nine most common point mutations was similar to that reported for other countries. The 93 fully genotyped patients were classified into 3 mutation groups based on the degree of enzymatic activity (A<2%, B @ 2%, C>20%). In group A, 62% of cases presented the SW form; in group B, 96% the SV form, and in group C, 88% the LO form. We diagnosed 80% of the affected alleles after screening for large rearrangements and 15 point mutations. To diagnose these remaining alleles we sequenced the CYP21 gene of one patient with the SV form and identified a heterozygous G->A transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region and was also found in a compound heterozygous state in 4 other patients. The mutation G424S presented a linkage disequilibrium with CYP21P and C4A gene deletions and HLA DR17, suggesting a probable founder effect. Search for the G424S mutation in other populations will reveal if it is restricted to the Brazilian patients or if it has a wider ethnic distribution.

Genomic analysis of Brazilian patients with Fabry disease

Fabry disease is an X-linked lysosomal disorder due to a-galactosidase A deficiency that causes storage of globotriaosylceramide. The gene coding for this lysosomal enzyme is located on the long arm of the X chromosome, in region Xq21.33-Xq22. Disease progression leads to vascular disease secondary to involvement of kidney, heart and the central nervous system. Detection of female carriers based solely on enzyme assays is often inconclusive. Therefore, mutation analysis is a valuable tool for diagnosis and genetic counseling. Many mutations of the a-galactosidase A gene have been reported with high genetic heterogeneity, being most mutations private found in only one family. The disease is panethnic, and estimates of incidence range from about 1 in 40,000 to 60,000 males. Our objective was to describe the analysis of 6 male and 7 female individuals belonging to 4 different Fabry disease families by automated sequencing of the seven exons of the a-galactosidase gene. Sequencing was performed using PCR fragments for each exon amplified from DNA extracted from peripheral blood. Three known mutations and one previously described in another Brazilian family were detected. Of 7 female relatives studied, 4 were carriers. Although the present study confirms the heterogeneity of mutations in Fabry disease, the finding of the same mutation previously detected in another Fabry family from our region raises the possibility of some founder effect, or genetic drift. Finally, the present study highlights the importance of molecular analysis for carrier detection and genetic counseling.

Haplotype analysis suggest common founders in carriers of the recurrent BRCA2 mutation, 3398delAAAAG, in French Canadian hereditary breast and/ovarian cancer families

Affiliation: Faculté de médicine, Université de Montréal

Caractérisation clinique et génétique d’une famille canadienne-française atteinte de la neuropathie héréditaire sensitive avec rétinite pigmentaire et ataxie

La complexité de l’étude des neuropathies héréditaires provient de leur hétérogénéité clinique et génétique et de la diversité des fibres composant les nerfs périphériques. Cette complexité se reflète dans les nombreuses classifications différentes. Les neuropathies héréditaires se classifient entre autres selon leur mode de transmission et leur atteinte sensitive, autonomique et motrice. Les neuropathies héréditaires sensitives et autonomiques (NHSA) se présentent avec une perte de la sensation distale aux membres, accompagnée d’autres manifestations selon le type de NHSA. L’étude des NHSA est facilitée lorsqu’il existe des grappes de familles originaires de régions du Québec où des effets fondateurs pour des maladies récessives ont déjà été identifiés. Nous avons recruté une grande famille canadienne-française originaire de Paspébiac dans la Baie-des-Chaleurs dans laquelle nous avons identifié quatre cas atteints d’une neuropathie héréditaire sensitive avec rétinite pigmentaire et ataxie (NHSRPA). Nous avons émis l’hypothèse que nous étions en présence d’une nouvelle forme de neuropathie héréditaire sensitive récessive à effet fondateur. Afin d’identifier la position chromosomique du gène muté responsable de la NHSRPA, nous avons tout d’abord complété un criblage du génome en génotypant des marqueurs microsatellites «single tandem repeat» (STR) sur des individus clés et nous avons ensuite procédé à une analyse de liaison génétique paramétrique. Ces études nous ont permis de lier cette famille au chromosome 1 et de définir un premier intervalle candidat de 6,7 Mb. Grâce à un génotypage de marqueurs «single nucleotide polymorphism» (SNP), nous avons réduit l’intervalle candidat à 5,3 Mb au locus 1q32,2-q32,3. Cette région contient 44 gènes candidats. Une revue plus fine de la littérature a fait ressortir qu’une famille espagnole et une américaine de souche hollandaise souffrant de la même maladie avaient déjà été liées au même locus. L’origine possiblement basque de notre famille gaspésienne nous a poussé à comparer l’haplotype porteur avec celui de la famille espagnole qui, quoi que gitane, provient du pays basque espagnol. Ces travaux ont démontré le partage d’une région de 203 kb. Afin de rétrécir davantage notre intervalle candidat, nous avons comparé les haplotypes des cas entre les deux familles et nous avons identifié un dernier intervalle candidat de 60 SNP au locus 1q32,3. Cette région ne contient que quatre gènes candidats dont le plus intéressant est le gène «activating transcription factor» (ATF3). À ce jour, aucune mutation n’a été trouvée dans le gène ATF3 et les gènes FAM71A, BATF3 et NSL1. Des expériences supplémentaires sont nécessaires afin d’identifier le gène muté responsable de la NHSRPA.

Pathogenèse moléculaire de la neuropathie sensitive et motrice héréditaire avec agénésie du corps calleux

La neuropathie sensitive et motrice héréditaire avec agénésie du corps calleux (NSMH/ACC) se traduit par une atteinte neurodégénérative sévère associée à des anomalies développementales dans le système nerveux central et du retard mental. Bien que rare dans le monde, ce désordre autosomique récessif est particulièrement fréquent dans la population Québécoise du Canada Français du fait d’un effet fondateur. L’unique étude réalisée sur la mutation québécoise du gène qui code pour le co-transporteur de potassiumchlore 3 (KCC3) a montré qu’il y a une perte de fonction de la protéine. Cependant, la maladie est également retrouvée hors du Québec et il reste encore à élucider les pathomécanismes mis en jeu. Nous avons donc séquencé les 26 exons du gène KCC3 chez des individus recrutés dans le monde entier et suspectés d’être atteints de la maladie. Nous avons ainsi identifié trois nouvelles mutations. L’étude fonctionnelle de ces mutations nous a confirmé la perte de fonction systématique des co-transporteurs mutés. Puisque l’inactivation de KCC3 se produit majoritairement via l’élimination de segments peptidiques en C-terminus, nous avons concentré notre attention sur l’identification des interactions qui s’y produisent. À l’aide d’approches double hybride, pull-down et immunomarquage, nous avons déterminé que KCC3 interagit avec la créatine kinase CK-B et que cette interaction est perturbée par les mutations tronquantes. De plus, l’utilisation d’un inhibiteur de créatine kinase inactive KCC3, ce qui démontre qu’il existe bien un lien fonctionnel et pathologique entre KCC3 et ses partenaires C-terminaux. Nous avons aussi identifié des anomalies majeures de localisation membranaire des KCC3 mutés. Que KCC3 soit tronqué ou pleine longueur, sa distribution subcellulaire est affectée dans des cellules en culture, dans les ovocytes de Xenopes et dans des échantillons de cerveau de patients. La perte d’interaction entre KCC3 et CK-B et/ou les défauts de transit intracellulaire de KCC3 sont donc les mécanismes pathologiques majeurs de la NSMH/ACC.