Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. * 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. * 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression

Concept, Design and Implementation of a Cardiovascular Gene-Centric 50 K SNP Array for Large-Scale Genomic Association Studies

A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a “cosmopolitan” tagging approach to capture the genetic diversity across ∼2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions

Meta-Analysis of the INSIG2 Association with Obesity Including 74,345 Individuals: Does Heterogeneity of Estimates Relate to Study Design?

The INSIG2 rs7566605 polymorphism was identified for obesity (BMI≥30 kg/m2) in one of the first genome-wide association studies, but replications were inconsistent. We collected statistics from 34 studies (n = 74,345), including general population (GP) studies, population-based studies with subjects selected for conditions related to a better health status (‘healthy population’, HP), and obesity studies (OB). We tested five hypotheses to explore potential sources of heterogeneity. The meta-analysis of 27 studies on Caucasian adults (n = 66,213) combining the different study designs did not support overall association of the CC-genotype with obesity, yielding an odds ratio (OR) of 1.05 (p-value = 0.27). The I2 measure of 41% (p-value = 0.015) indicated between-study heterogeneity. Restricting to GP studies resulted in a declined I2 measure of 11% (p-value = 0.33) and an OR of 1.10 (p-value = 0.015). Regarding the five hypotheses, our data showed (a) some difference between GP and HP studies (p-value = 0.012) and (b) an association in extreme comparisons (BMI≥32.5, 35.0, 37.5, 40.0 kg/m2 versus BMI<25 kg/m2) yielding ORs of 1.16, 1.18, 1.22, or 1.27 (p-values 0.001 to 0.003), which was also underscored by significantly increased CC-genotype frequencies across BMI categories (10.4% to 12.5%, p-value for trend = 0.0002). We did not find evidence for differential ORs (c) among studies with higher than average obesity prevalence compared to lower, (d) among studies with BMI assessment after the year 2000 compared to those before, or (e) among studies from older populations compared to younger. Analysis of non-Caucasian adults (n = 4889) or children (n = 3243) yielded ORs of 1.01 (p-value = 0.94) or 1.15 (p-value = 0.22), respectively. There was no evidence for overall association of the rs7566605 polymorphism with obesity. Our data suggested an association with extreme degrees of obesity, and consequently heterogeneous effects from different study designs may mask an underlying association when unaccounted for. The importance of study design might be under-recognized in gene discovery and association replication so far

Is there selection in favour of heterozygotes in families with merosin-deficient congenital muscular dystrophy?

Merosin-deficient congenital muscular dystrophy is an autosomal recessive neuromuscular disorder caused by partial or total absence of laminin-2 (merosin) in the skeletal muscle. Affected children have severe weakness, hypotonia at birth, high creatine kinase (CK) levels (more than 10 times normal) and are not able to walk or stand unsupported. Linkage and mutation analysis demonstrated that the gene encoding for the laminin-alpha2 chain, mapped on chromosome 6q22-23, is invariably responsible for this form of congenital muscular dystrophy. We investigated the pattern of inheritance of the haplotypes associated with the mutated allele in 29 informative merosin-deficient families, using tightly linked informative polymorphic microsatellite markers. This allowed us to identify heterozygous individuals from normal homozygotes, who are clinically, pathologically and biochemically indistinguishable. By linkage analysis, we found a statistically significant increase in the number of heterozygous individuals carrying either the paternal or the maternal haplotypes associated with the mutated allele. This could suggest a selection in favour of the alleles carrying mutations at the laminin alpha2-chain locus

Mutations in the laminin alpha2-chain gene in two children with early-onset muscular dystrophy.

We investigated two children who presented with delayed motor milestones. The first was a girl who was referred at 20 months because of developmental delay. She walked at 28 months and currently, aged 5 years, is independently mobile but has difficulty rising from the floor or going upstairs. The second was also a girl who presented at 6 weeks of age with hypotonia. Her motor milestones were delayed and she walked at the age of 2 years and 8 months and is currently independently mobile at the age of 3 years. Serum creatine kinase was elevated and a muscle biopsy showed dystrophic changes in both children. Immunohistochemistry of the laminin alpha2 chain of merosin was very similar in both cases: using a C-terminal antibody that recognizes an 80 kDa fragment, there was a mild reduction in expression on most fibres, while the staining with another antibody that recognizes a 300 kDa fragment showed a very marked reduction. Mutational analysis of the laminin alpha2 chain gene in the first patient showed that one of the two alleles had a de novo single nucleotide deletion at position 5702, causing a frameshift. In the other allele, we identified two point mutations present in cis; one was a G-->C transition at position +5 while the second was a T-->C transition at position +6 of the conserved donor splicing consensus sequence of introns 37 and 63, respectively. Transcription analysis of the corresponding cDNA region did not show any alternative splicing occurring as a result of these splice site mutations. Therefore, these mutations probably affect the splicing efficiency. Interestingly, the second child carried in both alleles the same two splicing consensus sequence mutations found in cis in the first patient. Our data provide further evidence that mutations in the laminin alpha2 chain gene are responsible not only for the severe form of congenital muscular dystrophy with onset at birth, but also for milder phenotypes, with later onset, in which the synthesis of a partially functional protein, or of a normal protein but in reduced quantity, is possible. The finding that these two unrelated patients had the same unusual mutation in common might suggest that this is a relatively commonly allele responsible for partial merosin deficiency in the UK

Prenatal diagnosis in merosin-deficient congenital muscular dystrophy

Prenatal diagnosis was carried out in five merosin-deficient congenital muscular dystrophy (CMD) families. We studied both laminin-alpha 2 chain expression in trophoblast using immunocytochemistry and linkage analysis to the LAMA2 locus. In four families there was good agreement between the immunocytochemistry and linkage analysis results: in one case the trophoblast was negative for LAMA2 expression and haplotype analysis suggested the foetus was affected; in the other three cases the laminin-alpha 2 chain expression was normal and foetuses were found to be carriers. In the remaining family, a case of partial laminin-alpha 2 chain expression, the immunostaining of the trophoblast was weaker compared to the control. Linkage analysis, however, could not be performed because of maternal DNA contamination. After termination of pregnancy, the foetal muscle was studied and suggested weak laminin-alpha 2 chain expression. The haplotype analysis however showed that the foetus was probably a carrier, unless a double recombinant event had occurred. We conclude that a combination of immunocytochemistry and linkage analysis can be used for the prenatal diagnosis of merosin deficient CMD. The results are easy to interpret in families with total absence of the protein, while caution is required when dealing with families where partial expression occurs

The protein defect in congenital muscular dystrophy

--