Mutations in the gene encoding smooth muscle cell alpha actin (ACTA2) have recently been shown to cause familial thoracic aortic aneurysms leading to type A dissections (TAAD) and predispose to premature stroke and coronary artery disease. In order to further explore the role of ACTA2 variations in the pathogenesis of TAAD, we sequenced the coding regions of this gene in 40 unrelated German patients with TAAD (with (n=21) or without (n=19) clinical features suggestive of Marfan syndrome). All patients had previously tested negative for mutations in the FBN1 and TGFBR2 genes. We identified three novel ACTA2 mutations and mapped them on a three-dimensional model of actin. Two mutations affect residues within (M49V) or adjacent to (R39C), the DNAse-I-binding loop within subdomain 2 of alpha actin. They were observed in families with recurrent aortic aneurysm (R39C) or aortic dissection (M49V). The third mutation causes an exchange in the vicinity of the ATP-binding site (G304R) in a patient thought to have isolated TAAD. None of the affected individuals had clinical features typical for Marfan syndrome, and no case of premature stroke or coronary artery disease was reported from the affected families. In conclusion, we underscore the role of ACTA2 mutations in nonsyndromic TAAD and suggest that ACTA2 should be included in the genes routinely investigated for syndromic and nonsyndromic TAAD. Detailed clinical investigations of additional families are warranted to further explore the full range of phenotypic signs associated with the three novel mutations described here.
ACTA2; marfan syndrome; TAAD; aneurysm; dissection; actin
The hypothesis that females of socially monogamous species obtain indirect benefits (good or compatible genes) from extra-pair mating behaviour has received enormous attention but much less generally accepted support. Here we ask whether selection for adult survival and fecundity or sexual selection contribute to indirect selection of the extra-pair mating behaviour in socially monogamous coal tits (Periparus ater). We tracked locally recruited individuals with known paternity status through their lives predicting that the extra-pair offspring (EPO) would outperform the within-pair offspring (WPO). No differences between the WPO and EPO recruits were detected in lifespan or age of first reproduction. However, the male WPO had a higher lifetime number of broods and higher lifetime number of social offspring compared with male EPO recruits, while no such differences were evident for female recruits. Male EPO recruits did not compensate for their lower social reproductive success by higher fertilization success within their social pair bonds. Thus, our results do not support the idea that enhanced adult survival, fecundity or within-pair fertilization success are manifestations of the genetic benefits of extra-pair matings. But we emphasize that a crucial fitness component, the extra-pair fertilization success of male recruits, has yet to be taken into account to fully appreciate the fitness consequences of extra-pair matings.
adult survival selection; extra-pair paternity; lifetime reproductive success; local recruitment; longevity; Parus ater
In this retrospective study, we examined changes in decision-making for and against the predictive genetic test for Huntington's disease including 478 persons at risk who had undergone genetic counselling in one centre in Germany between 1993 and 2004. At the outset of the counselling procedure the majority of subjects (71%) wanted to make use of the test, yet the actual demand of the predictive test result declined from 67 to 38% over the years. In addition, the time interval between counselling session and blood withdrawal was reduced, as determined by the counselees: in 2000–2004 the majority of persons at risk made the appointment for blood withdrawal after the shortest possible time span. Demographic factors of the cohort remained comparatively stable in the investigated time period. An association was evident between the ratio of test usage and the counselling person. These and other possible factors influencing the time flow of predictive DNA testing are discussed. Further studies are necessary to investigate whether changes of test demand rates are a general phenomenon.
genetic testing; genetic counselling; Huntington's disease; predictive testing
Mutations in the SPG4 gene (spastin) and in the SPG3A gene (atlastin) account for the majority of 'pure' autosomal dominant form of hereditary spastic paraplegia (HSP). Recently, mutations in the REEP1 gene were identified to cause autosomal dominant HSP type SPG31. The purpose of this study was to determine the prevalence of REEP1 mutations in a cohort of 162 unrelated Caucasian index patients with 'pure' HSP and a positive family history (at least two persons per family presented symptoms).
162 patients were screened for mutations by, both, DHPLC and direct sequencing.
Ten mutations were identified in the REEP1 gene, these included eight novel mutations comprising small insertions/deletions causing frame shifts and subsequently premature stop codons, one nonsense mutation and one splice site mutation as well as two missense mutations. Both missense mutations and the splice site mutation were not identified in 170 control subjects.
In our HSP cohort we found pathogenic mutations in 4.3% of cases with autosomal dominant inheritance. Our results confirm the previously observed mutation range of 3% to 6.5%, respectively, and they widen the spectrum of REEP1 mutations.
Mitochondrial DNA (mtDNA) polymorphism is a possible factor contributing to the maternal parent-of-origin effect in multiple sclerosis (MS) susceptibility.
Methods and Findings
In order to investigate the role of mtDNA variations in MS, we investigated six European MS case-control cohorts comprising >5,000 individuals. Three well matched cohorts were genotyped with seven common, potentially functional mtDNA single nucleotide polymorphisms (SNPs). A SNP, nt13708 G/A, was significantly associated with MS susceptibility in all three cohorts. The nt13708A allele was associated with an increased risk of MS (OR = 1.71, 95% CI 1.28–2.26, P = 0.0002). Subsequent sequencing of the mtDNA of 50 individuals revealed that the nt13708 itself, rather than SNPs linked to it, was responsible for the association. However, the association of nt13708 G/A with MS was not significant in MS cohorts which were not well case-control matched, indicating that the significance of association was affected by the population structure of controls.
Taken together, our finding identified the nt13708A variant as a susceptibility allele to MS, which could contribute to defining the role of the mitochondrial genome in MS pathogenesis.
Atopic dermatitis (AD) is a common inflammatory skin disorder, affecting up to 15% of children in industrialized countries. Toll-interacting protein (TOLLIP) is an inhibitory adaptor protein within the toll-like receptor (TLR) pathway, a part of the innate immune system that recognizes structurally conserved molecular patterns of microbial pathogens, leading to an inflammatory immune response.
In order to detect a possible role of TOLLIP variation in the pathogenesis of AD, we screened the entire coding sequence of the TOLLIP gene by SSCP in 50 AD patients. We identified an amino acid exchange in exon 6 (Ala222Ser) and a synonymous variation in exon 4 (Pro139Pro). Subsequently, these two variations and four additional non-coding polymorphisms (-526 C/G, two polymorphisms in intron 1 and one in the 3'UTR) were genotyped in 317 AD patients and 224 healthy controls.
The -526G allele showed borderline association with AD in our cohort (p = 0.012; significance level after correction for multiple testing 0.0102). Haplotype analysis did not yield additional information. Evaluation of mRNA expression by quantitative real-time polymerase chain reaction in six probands with the CC and six with the GG genotype at the -526 C/G locus did not reveal significant differences between genotypes.
Variation in the TOLLIP gene may play a role in the pathogenesis of AD. Yet, replication studies in other cohorts and populations are warranted to confirm these association results.
Wegener granulomatosis (WG) belongs to the heterogeneous group of systemic vasculitides. The multifactorial pathophysiology of WG is supposedly caused by yet unknown environmental influence(s) on the basis of genetic predisposition. The presence of anti-neutrophil cytoplasmic antibodies (ANCA) in the plasma of patients and genetic involvement of the human leukocyte antigen system reflect an autoimmune background of the disease. Strong associations were revealed with WG by markers located in the major histocompatibility complex class II (MHC II) region in the vicinity of human leukocyte antigen (HLA)-DPB1 and the retinoid X receptor B (RXRB) loci. In order to define the involvement of the 6p21.3 region in WG in more detail this previous population-based association study was expanded here to the respective 3.6 megabase encompassing this region on chromosome 6. The RXRB gene was analysed as well as a splice-site variation of the butyrophilin-like (BTNL2) gene which is also located within the respective region. The latter polymorphism has been evaluated here as it appears as a HLA independent susceptibility factor in another granulomatous disorder, sarcoidosis.
150–180 German WG patients and a corresponding cohort of healthy controls (n = 100–261) were used in a two-step study. A panel of 94 microsatellites was designed for the initial step using a DNA pooling approach. Markers with significantly differing allele frequencies between patient and control pools were individually genotyped. The RXRB gene was analysed for single strand conformation polymorphisms (SSCP) and restriction fragment length polymorphisms (RFLP). The splice-site polymorphism in the BTNL2 gene was also investigated by RFLP analysis.
A previously investigated microsatellite (#18.104.22.168, Santa Cruz genome browser (UCSC) May 2004 Freeze localisation: chr6:31257596-34999883), which was used as a positive control, remained associated throughout the whole two-step approach. Yet, no additional evidence for association of other microsatellite markers was found in the entire investigated region. Analysis of the RXRB gene located in the WG associated region revealed associations of two variations (rs10548957 pallelic = 0.02 and rs6531 pallelic = 5.20 × 10-5, OR = 1.88). Several alleles of markers located between HLA-DPB1, SNP rs6531 and microsatellite 22.214.171.124 showed linkage disequilibrium with r2 values exceeding 0.10. Significant differences were not demonstrable for the sarcoidosis associated splice-site variation (rs2076530 pallelic = 0.80) in our WG cohort.
Since a microsatellite flanking the RXRB gene and two intragenic polymorphisms are associated significantly with WG on chromosome 6p21.3, further investigations should be focussed on extensive fine-mapping in this region by densely mapping with additional markers such as SNPs. This strategy may reveal even deeper insights into the genetic contributions of the respective region for the pathogenesis of WG.
Parkinson's disease (PD) is the second most common neurodegenerative disorder. Recently, mutations in the PINK1 (PARK6) gene were shown to rarely cause autosomal-recessively transmitted, early-onset parkinsonism. In order to evaluate whether PINK1 contributes to the risk of common late-onset PD we analysed PINK1 sequence variations. A German (85 patients) and a Norwegian cohort (90 patients) suffering from late-onset PD were screened for mutations and single nucleotide polymorphisms (SNPs) in the PINK1 gene. Both cohorts consist of well-characterized patients presenting a positive family history of PD in ~17%. Investigations were performed by single strand conformation polymorphism (SSCP), denaturating high performance liquid chromatography (DHPLC) and sequencing analyses. SNP frequencies were compared by the χ2 test
Several common SNPs were identified in our cohorts, including a recently identified coding variant (Q115L) in exon 1. Genotyping of the Q115L variation did not reveal significant frequency differences between patients and controls. Pathogenic mutations in the PINK1 gene were not identified, neither in the German nor in the Norwegian cohort.
Sequence variation in the PINK1 gene appears to play a marginal quantitative role in the pathogenesis of the late-onset form of PD, in German and Norwegian cohorts, if at all.
Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, e.g. with α(1)-antitrypsin, proteinase 3 and with the HLA-DPB1 locus. A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseCγ2 (PLCγ-2) gene, corresponding to human PLCγ-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2 (HRL1 and 2, respectively) and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLCγ-2 gene.
Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort.
This report does not reveal any hints for an involvement of the PLCγ-2 gene in the pathogenesis of WG in our case-control study.
Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR). HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6.
Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out.
We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6) resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702]), three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298]) and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]). All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H).
HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.
The δ and γ subunits of the cGMP-phosphodiesterase (PDE6D, PDE6G) genes were screened in order to identify mutations causing generalised progressive retinal atrophy (gPRA) in dogs. In the PDE6D gene, single nucleotide polymorphisms (SNP) were observed in exon 4, in introns 2 and 3 and in the 3' untranslated region (UTR) of different dog breeds. In the coding region of the PDE6G gene, exclusively healthy Labrador Retrievers showed an A → G transition in exon 4 without amino acid exchange. SNP were also observed in introns 1 and 2 in different dog breeds. The different SNP were used as intragenic markers to investigate the involvement of both genes in gPRA. The informative substitutions allowed us to exclude mutations in the PDE6D and PDE6G genes as causing retinal degeneration in 15 of the 22 dog breeds with presumed autosomal recessively transmitted (ar) gPRA.
cGMP-phosphodiesterase; canine; generalised progressive retinal atrophy; SNP; retinitis pigmentosa; SSCP
Since contradictory results have been reported, we reanalysed the 77C→G transition in exon 4 of the protein-tyrosine phosphatase receptor-type C (PTPRC also known as CD45) in a large cohort of German MS patients and controls. Different isoforms of the protein are expressed, depending on alternative splicing of exons 4 (CD45RA), 5 (CD45RB) and 6 (CD45RC) (CD45RO, exons 4–6 spliced out). The 77C→G transition does not change the amino acid sequence, but it is probably part of a motif necessary for splicing leading to the isoform CD45RA. The expression of CD45RA is increased in 77C/G heterozygous individuals. The aim of the study was to clarify the importance of the PTPRC 77C→G transition in our German cohort of MS patients.
PCR products of exon 4 were digested using endonuclease MspI. The resulting restriction fragments of the wildtype C allele are 198 and 62 bp in length. In the G allele an additional restriction site is present yielding fragments of 114 and 84 bp.
The G allele was identified in 10 of the 347 controls (1.4%) and in 7 of 454 MS patients (0.8%; Table 1). No homozygous individuals were found either in the control or in the patient group. Genetic association between the PTPRC 77C→G transition and MS susceptibility was excluded in the MS cohort. In addition, subgrouping patients according to differences in the clinical course of MS or according to HLA-DRB1*15 status did not yield significant differences.
The 77C→G transition in exon 4 of the PTPRC gene may contribute to MS susceptibility only in very few families, if at all, but it is not relevant for the majority of MS cases, including virtually all German patients.