Molecular analysis of British facioscapulohumeral dystrophy families for 4q DNA rearrangements

Facioscapulohumeral muscular dystrophy is an important autosomaldominant neuromuscular disorder that has been localised to 4q35.We have analysed our extensive panel of 45 families with a newDNA marker p13E-11. The findings, based on multiply informativeindividual meioses and multipoint mapping, suggest that probep13E-11is the closest marker for the disorder and it is likelyto be located proximal to the disease locus as are all the otherpresent markers. In nine of the ten new mutations studied, anew smaller EcoRl fragment which was not present in either ofthe parents was detected, indicating that a de novo DNA rearrangmentis indeed associated with the development of the disease state.However, in view of the difficulty in defining the size of over30kb alleles and the recombinant events observed with p13E-11,we suggest that it should be used in combination with anotherVNTR marker until a close distal flanking marker for this conditionis identified or the gene itself is isolated.     

Association of the TGF-�� receptor genes with abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a multifactorial condition. The transforming growth factor β (TGF-β) pathway regulates vascular remodeling and mutations in its receptor genes, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysm (TAA). The TGF-β pathway may be involved in aneurysm development in general. We performed an association study by analyzing all the common genetic variants in TGFBR1 and TGFBR2 using tag single nucleotide polymorphisms (SNPs) in a Dutch AAA case–control population in a two-stage genotyping approach. In stage 1, analyzing 376 cases and 648 controls, three of the four TGFBR1 SNPs and nine of the 28 TGFBR2 SNPs had a P<0.07. Genotyping of these SNPs in an independent cohort of 360 cases and 376 controls in stage 2 confirmed association (P<0.05) for the same allele of one SNP in TGFBR1 and two SNPs in TGFBR2. Joint analysis of the 736 cases and 1024 controls showed statistically significant associations of these SNPs, which sustained after proper correction for multiple testing (TGFBR1 rs1626340 OR 1.32 95% CI 1.11–1.56 P=0.001 and TGFBR2 rs1036095 OR 1.32 95% CI 1.12–1.54 P=0.001 and rs4522809 OR 1.28 95% CI 1.12–1.46 P=0.0004). We conclude that genetic variations in TGFBR1 and TGFBR2 associate with AAA in the Dutch population. This suggests that AAA may develop partly by similar defects as TAA, which in the future may provide novel therapeutic options.     

Genetic variation in myosin IXB is associated with ulcerative colitis

BACKGROUND & AIMS: Common germline genetic variation in the 3' region of myosin IXB (MYO9B) has been associated recently with susceptibility to celiac disease, with a hypothesis that MYO9B variants might influence intestinal permeability. These findings suggested the current study investigating a possible further role for MYO9B variation in inflammatory bowel disease. METHODS: Eight single-nucleotide polymorphisms (SNPs) were selected to tag common haplotypes from the 35-kb 3' region of MYO9B. These included the strongest celiac disease-associated variants reported in a Dutch cohort. These SNPs were studied in 3 independently collected and genotyped case-control cohorts of European descent (UK, Dutch, and Canadian/Italian), comprising in total 2717 inflammatory bowel disease patients (1197 with Crohn's disease, 1520 with ulcerative colitis) and 4440 controls. RESULTS: Common variation in MYO9B was associated with susceptibility to inflammatory bowel disease in all 3 cohorts examined (most associated SNP, rs1545620; meta-analysis P = 1.9 x 10(-6); odds ratio, 1.2), with the same alleles showing association as reported for celiac disease. CONCLUSIONS: MYO9B genetic variants predispose to inflammatory bowel disease. Interestingly, rs1545620 is a nonsynonymous variant leading to an amino acid change (Ala1011Ser) in the third calmodulin binding IQ domain of MYO9B. Unlike previous variants (in other genes) reported to predispose to inflammatory bowel disease, the association at MYO9B was considerably stronger with ulcerative colitis, although weaker association with Crohn's disease also was observed. These data imply shared causal mechanisms underlying intestinal inflammatory diseases.     

From genome-wide association studies to disease mechanisms: celiac disease as a model for autoimmune diseases

Celiac disease is characterized by a chronic inflammatory reaction in the intestine and is triggered by gluten, a constituent derived from grains which is present in the common daily diet in the Western world. Despite decades of research, the mechanisms behind celiac disease etiology are still not fully understood, although it is clear that both genetic and environmental factors are involved. To improve the understanding of the disease, the genetic component has been extensively studied by genome-wide association studies. These have uncovered a wealth of information that still needs further investigation to clarify its importance. In this review, we summarize and discuss the results of the genetic studies in celiac disease, focusing on the "non-HLA" genes. We also present novel approaches to identifying the causal variants in complex susceptibility loci and disease mechanisms.     

Transmission of human mtDNA heteroplasmy in the Genome of the Netherlands families: support for a variable-size bottleneck

Although previous studies have documented a bottleneck in the transmission of mtDNA genomes from mothers to offspring, several aspects remain unclear, including the size and nature of the bottleneck. Here, we analyze the dynamics of mtDNA heteroplasmy transmission in the Genomes of the Netherlands (GoNL) data, which consists of complete mtDNA genome sequences from 228 trios, eight dizygotic (DZ) twin quartets, and 10 monozygotic (MZ) twin quartets. Using a minor allele frequency (MAF) threshold of 2%, we identified 189 heteroplasmies in the trio mothers, of which 59% were transmitted to offspring, and 159 heteroplasmies in the trio offspring, of which 70% were inherited from the mothers. MZ twin pairs exhibited greater similarity in MAF at heteroplasmic sites than DZ twin pairs, suggesting that the heteroplasmy MAF in the oocyte is the major determinant of the heteroplasmy MAF in the offspring. We used a likelihood method to estimate the effective number of mtDNA genomes transmitted to offspring under different bottleneck models; a variable bottleneck size model provided the best fit to the data, with an estimated mean of nine individual mtDNA genomes transmitted. We also found evidence for negative selection during transmission against novel heteroplasmies (in which the minor allele has never been observed in polymorphism data). These novel heteroplasmies are enhanced for tRNA and rRNA genes, and mutations associated with mtDNA diseases frequently occur in these genes. Our results thus suggest that the female germ line is able to recognize and select against deleterious heteroplasmies.Heteroplasmy (intra-individual variation) in mitochondrial DNA (mtDNA) plays an important role in mtDNA-related diseases and has also been implicated in aging and cancer (Greaves et al. 2012; Wallace 2012; Chinnery and Hudson 2013; Lombès et al. 2014). Most mtDNA mutations that cause diseases due to defects in mitochondrial function exist as heteroplasmies and only cause disease symptoms when the frequency of the mutant allele exceeds a particular threshold (Wallace and Chalkia 2013). Below this threshold, individuals are asymptomatic, presumably because there are sufficient functional mitochondria for normal metabolism. Changes in the frequency of pathogenic mutations during the transmission of heteroplasmies from mothers to offspring can thus play an important role in the disease risk of the offspring. However, most of our knowledge concerning the dynamics of heteroplasmy transmission comes from studies of pathogenic mutations (Monnot et al. 2011; Shen et al. 2012; de Laat et al. 2013; Wallace and Chalkia 2013), which in blood have been shown to decrease over time and hence may not accurately reflect the overall level of such pathogenic mutations within an individual (Poulton and Morten 1993; ‘t Hart et al. 1996; Rahman et al. 2001; Rajasimha et al. 2008). Mouse models have also been utilized (Cree et al. 2008; Fan et al. 2008; Freyer et al. 2012; Ross et al. 2013), but to date, there have been only a few studies of normal patterns of heteroplasmy transmission in humans (Sekiguchi et al. 2003; Goto et al. 2011; Sondheimer et al. 2011; Guo et al. 2013; Rebolledo-Jaramillo et al. 2014), including studies of oocytes and placenta (Marchington et al. 1997, 2002; Jacobs et al. 2007), and several questions remain.For example, although it is clear that a bottleneck occurs during the transmission of mtDNA genomes from mothers to offspring, the size of the bottleneck remains a contentious issue. Previous estimates of the effective number of transmitted mtDNA genomes range widely, from eight to 200 (Brown et al. 2001; Guo et al. 2013; Rebolledo-Jaramillo et al. 2014). However, all previous studies have assumed a constant size for the bottleneck across individuals; the effect of allowing the bottleneck size to vary among individuals has not been investigated. Moreover, it has been suggested that mtDNA genomes may not behave as independent entities but instead are organized into discrete units called “nucleoids,” each of which contains 5–10 mtDNA genomes (Jacobs et al. 2000; Cao et al. 2007; Khrapko 2008), although recently it has been suggested that the number may be much smaller, on the order of one mtDNA genome per nucleoid (Kukat et al. 2011). Each nucleoid is thought to be homoplasmic for mtDNA genome sequences; thus, mtDNA heteroplasmy at the cellular level would reflect nucleoids that are homoplasmic for different sequence variants. Nucleoid structures within cells have been studied microscopically and biochemically (Bogenhagen 2012), and nucleoid-based models have been found to provide a better fit to the segregation of heteroplasmic mtDNA genomes in cell lines than do simple bottleneck models in some studies (Cao et al. 2007; Khrapko 2008), but not in others (Cree et al. 2008). However, to date, nucleoid-based models have not been investigated in the transmission of mtDNA heteroplasmy from mothers to offspring.Another issue is the degree to which negative (or purifying) selection may act on deleterious variants during the transmission of mtDNA heteroplasmy. There are conflicting results and views as to whether changes in the frequency of a heteroplasmic mutation from mother to offspring are governed solely by genetic drift, or whether there is an additional role for negative (purifying) selection (Jenuth et al. 1997; Durham et al. 2006; Stewart et al. 2008a,b; Wonnapinij et al. 2008; Wallace and Chalkia 2013; Rebolledo-Jaramillo et al. 2014). Negative selection during heteroplasmy transmission, as evidenced by a decrease in the frequency of presumably deleterious heteroplasmic variants in offspring compared to mothers, must operate on the female germ line and/or early in development after fertilization, and hence differs from negative selection operating on homoplasmic variants that reduce viability or fertility (Holt et al. 2014). The opportunities for, and extent of, such negative selection during heteroplasmy transmission in humans remain largely unknown.Here, we utilize the Genomes of the Netherlands (GoNL) project (Boomsma et al. 2014; Genome of the Netherlands Consortium 2014), consisting of whole-genome sequence data from blood samples from 250 families, to carry out the largest study to date (to our knowledge) of the dynamics of heteroplasmy transmission across the entire mtDNA genome. We utilize the data on changes in minor allele frequency (MAF) from mothers to offspring at heteroplasmic sites to compare different models for the inheritance of mtDNA genomes, and we analyze the data for evidence of negative selection during heteroplasmy transmission.