An unusual splice defect in the mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Grey cattle

Tyrolean Grey cattle represent a local breed with a population size of approximately 5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

LUPA: a European initiative taking advantage of the canine genome architecture for unravelling complex disorders in both human and dogs

The domestic dog offers a unique opportunity to explore the genetic basis of disease, morphology and behaviour. Humans share many diseases with our canine companions, making dogs an ideal model organism for comparative disease genetics. Using newly developed resources, genome-wide association studies in dog breeds are proving to be exceptionally powerful. Towards this aim, veterinarians and geneticists from 12 European countries are collaborating to collect and analyse the DNA from large cohorts of dogs suffering from a range of carefully defined diseases of relevance to human health. This project, named LUPA, has already delivered considerable results. The consortium has collaborated to develop a new high density single nucleotide polymorphism (SNP) array. Mutations for four monogenic diseases have been identified and the information has been utilised to find mutations in human patients. Several complex diseases have been mapped and fine mapping is underway. These findings should ultimately lead to a better understanding of the molecular mechanisms underlying complex diseases in both humans and their best friend.

Microphthalmia in Texel sheep is associated with a missense mutation in the paired-like homeodomain 3 (PITX3) gene

Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina's OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G>C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia.

A deletion in the N-myc downstream regulated gene 1 (NDRG1) gene in Greyhounds with polyneuropathy

The polyneuropathy of juvenile Greyhound show dogs shows clinical similarities to the genetically heterogeneous Charcot-Marie-Tooth (CMT) disease in humans. The pedigrees containing affected dogs suggest monogenic autosomal recessive inheritance and all affected dogs trace back to a single male. Here, we studied the neuropathology of this disease and identified a candidate causative mutation. Peripheral nerve biopsies from affected dogs were examined using semi-thin histology, nerve fibre teasing and electron microscopy. A severe chronic progressive mixed polyneuropathy was observed. Seven affected and 17 related control dogs were genotyped on the 50k canine SNP chip. This allowed us to localize the causative mutation to a 19.5 Mb interval on chromosome 13 by homozygosity mapping. The NDRG1 gene is located within this interval and NDRG1 mutations have been shown to cause hereditary motor and sensory neuropathy-Lom in humans (CMT4D). Therefore, we considered NDRG1 a positional and functional candidate gene and performed mutation analysis in affected and control Greyhounds. A 10 bp deletion in canine NDRG1 exon 15 (c.1080_1089delTCGCCTGGAC) was perfectly associated with the polyneuropathy phenotype of Greyhound show dogs. The deletion causes a frame shift (p.Arg361SerfsX60) which alters several amino acids before a stop codon is encountered. A reduced level of NDRG1 transcript could be detected by RT-PCR. Western blot analysis demonstrated an absence of NDRG1 protein in peripheral nerve biopsy of an affected Greyhound. We thus have identified a candidate causative mutation for polyneuropathy in Greyhounds and identified the first genetically characterized canine CMT model which offers an opportunity to gain further insights into the pathobiology and therapy of human NDRG1 associated CMT disease. Selection against this mutation can now be used to eliminate polyneuropathy from Greyhound show dogs.

Effective population size of an indigenous Swiss cattle breed estimated from linkage disequilibrium

Effective population size is an important parameter for the assessment of genetic diversity within a livestock population and its development over time. If pedigree information is not available, linkage disequilibrium (LD) analysis might offer an alternative perspective for the estimation of effective population size. In this study, 128 individuals of the Swiss Eringer breed were genotyped using the Illumina BovineSNP50 beadchip. We set bin size at 50 kb for LD analysis, assuming that LD for proximal single nucleotide polymorphism (SNP)-pairs reflects distant breeding history while LD from distal SNP-pairs would reflect near history. Recombination rates varied among different regions of the genome. The use of physical distances as an approximation of genetic distances (e.g. setting 1 Mb = 0.01 Morgan) led to an upward bias in LD-based estimates of effective population size for generations beyond 50, while estimates for recent history were unaffected. Correction for restricted sample size did not substantially affect these results. LD-based actual effective population size was estimated in the range of 87-149, whereas pedigree-based effective population size resulted in 321 individuals. For conservation purposes, requiring knowledge of recent history (<50 generations), approximation assuming constant recombination rate seemed adequate.

Molecular characterization of five porcine candidate genes for drip loss in pork

Drip loss is the loss of fluid from a piece of meat without mechanical force and represents an important meat quality trait. Previous work revealed a quantitative trait locus (QTL) for drip loss in pork in an experimental Duroc x Pietrain (DUPI) F2 family on SSC 5. Based on functional data indicating their possible involvement in water holding capacity and their expression in skeletal muscle, we selected five positional candidates (ACO2, ADSL, CBY1, KCNJ4, PLA2AG6) out of 130 predicted genes in the QTL interval for further analysis. We performed a mutation analysis of all coding exons and discovered 204 polymorphisms. We genotyped 39 single nucleotide polymorphisms (SNPs) in 192 Pietrain pigs with extreme drip loss phenotypes and detected a possible association with drip loss for one non-coding SNP in the ADSL gene (ss107793818, p(raw) = 0.021). Correspondingly, ADSL diplotypes were associated with drip loss and pH1 of M. longissimus dorsi. However, after correction for multiple testing, none of the tested SNPs were significantly associated with drip loss. One possible explanation for these results is that one of the QTL-alleles from the experimental DUPI family may be fixed or nearly fixed in the tested Pietrain population.

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy

The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP).

First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

Background Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. Methods and Findings We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. Conclusions We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.

Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

Age-modulated association between prefrontal NAA and the BDNF gene

Brain-derived neurotrophic factor (BDNF) has been implicated in the pathophysiology of psychiatric and neurological disorders and in the mechanisms of antidepressant pharmacotherapy. Psychiatric and neurological conditions have also been associated with reduced brain levels of N-acetyl-aspartate (NAA), which has been used as a putative marker of neural integrity. However, few studies have explored the relationship between BDNF polymorphisms and NAA levels directly. Here, we present data from a single-voxel proton magnetic resonance spectroscopy study of 64 individuals and explore the relationship between BDNF polymorphisms and prefrontal NAA level. Our results indicate an association between a single nucleotide polymorphism (SNP) within BDNF, known as rs1519480, and reduced NAA level (p = 0.023). NAA levels were further predicted by age and Asian ancestry. There was a significant rs1519480 × age interaction on NAA level (p = 0.031). Specifically, the effect of rs1519480 on NAA level became significant at age ⩾34.17 yr. NAA level decreased with advancing age for genotype TT (p = 0.001) but not for genotype CT (p = 0.82) or CC (p = 0.34). Additional in silico analysis of 142 post-mortem brain samples revealed an association between the same SNP and reduced BDNF mRNA expression in the prefrontal cortex. The rs1519480 SNP influences BDNF mRNA expression and has an impact on prefrontal NAA level over time. This genetic mechanism may contribute to inter-individual variation in cognitive performance seen during normal ageing, as well as contributing to the risk for developing psychiatric and neurological conditions.

Social Cognition, Face Processing, and Oxytocin Receptor Single Nucleotide Polymorphisms in Typically Developing Children

Recent research has provided evidence of a link between behavioral measures of social cognition (SC) and neural and genetic correlates. Differences in face processing and variations in the oxytocin receptor (OXTR) gene have been associated with SC deficits and autism spectrum disorder (ASD) traits. Much work has examined the qualitative differences between those with ASD and typically developing (TD) individuals, but very little has been done to quantify the natural variation in ASD-like traits in the typical population. The present study examines this variation in TD children using a multidimensional perspective involving behavior assessment, neural electroencephalogram (EEG) testing, and OXTR genotyping. Children completed a series of neurocognitive assessments, provided saliva samples for sequencing, and completed a face processing task while connected to an EEG. No clear pattern emerged for EEG covariates or genotypes for individual OXTR single nucleotide polymorphisms (SNPs). However, SNPs rs2254298 and rs53576 consistently interacted such that the AG/GG allele combination of these SNPs was associated with poorer performance on neurocognitive measures. These results suggest that neither SNP in isolation is risk-conferring, but rather that the combination of rs2254298(A/G) and rs53576(G/G) confers a deleterious effect on SC across several neurocognitive measures. Copyright 2014. Published by Elsevier Ltd.

Assessment of D216H DYT1 polymorphism in a Chinese primary dystonia patient cohort

The D216H single-nucleotide polymorphism (SNP) (rs1801968) in DYT1 exon 4 has been suggested to be a genetic modifier in primary dystonia.

Identification of SNPs in the cystic fibrosis interactome influencing pulmonary progression in cystic fibrosis

There is growing evidence that the great phenotypic variability in patients with cystic fibrosis (CF) not only depends on the genotype, but apart from a combination of environmental and stochastic factors predominantly also on modifier gene effects. It has been proposed that genes interacting with CF transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) are potential modifiers. Therefore, we assessed the impact of single-nucleotide polymorphisms (SNPs) of several of these interacters on CF disease outcome. SNPs that potentially alter gene function were genotyped in 95 well-characterized p.Phe508del homozygous CF patients. Linear mixed-effect model analysis was used to assess the relationship between sequence variants and the repeated measurements of lung function parameters. In total, we genotyped 72 SNPs in 10 genes. Twenty-five SNPs were used for statistical analysis, where we found strong associations for one SNP in PPP2R4 with the lung clearance index (P ≤ 0.01), the specific effective airway resistance (P ≤ 0.005) and the forced expiratory volume in 1 s (P ≤ 0.005). In addition, we identified one SNP in SNAP23 to be significantly associated with three lung function parameters as well as one SNP in PPP2R1A and three in KRT19 to show a significant influence on one lung function parameter each. Our findings indicate that direct interacters with CFTR, such as SNAP23, PPP2R4 and PPP2R1A, may modify the residual function of p.Phe508del-CFTR while variants in KRT19 may modulate the amount of p.Phe508del-CFTR at the apical membrane and consequently modify CF disease.

Molecular characterization of the equine collagen, type IX, alpha 2 (COL9A2) gene on horse chromosome 2p16-->p15

The mammalian collagen, type IX, alpha 2 gene (COL9A2) encodes the alpha-2 chain of type IX collagen and is located on horse chromosome 2p16-->p14 harbouring a quantitative trait locus for osteochondrosis. We isolated a bacterial artificial chromosome (BAC) clone containing the equine COL9A2 gene and determined the complete genomic sequence of this gene. Cloning and characterization of equine COL9A2 revealed that the equine gene consists of 32 exons spanning approximately 15 kb. The COL9A2 transcript encodes a single protein of 688 amino acids. Thirty two single nucleotide polymorphisms (SNPs) equally distributed in the gene were detected in a mutation scan of eight unrelated Hanoverian warmblood stallions, including one SNP that affects the amino acid sequence of COL9A2. Comparative analyses between horse, human, mouse and rat indicate that the chromosomal location of equine COL9A2 is in agreement with known chromosomal synteny relationships. The comparison of the gene structure and transcript revealed a high degree of conservation towards the other mammalian COL9A2 genes. We chose three informative SNPs for association and linkage disequilibrium tests in three to five paternal half-sib families of Hanoverian warmblood horses consisting of 44 to 75 genotyped animals. The test statistics did not reach the significance threshold of 5% and so we could not show an association of COL9A2 with equine osteochondrosis.

ASSOCIATON TESTS THAT ACCOMMODATE GENOTYPING ERRORS

High-throughput SNP arrays provide estimates of genotypes for up to one million loci, often used in genome-wide association studies. While these estimates are typically very accurate, genotyping errors do occur, which can influence in particular the most extreme test statistics and p-values. Estimates for the genotype uncertainties are also available, although typically ignored. In this manuscript, we develop a framework to incorporate these genotype uncertainties in case-control studies for any genetic model. We verify that using the assumption of a “local alternative” in the score test is very reasonable for effect sizes typically seen in SNP association studies, and show that the power of the score test is simply a function of the correlation of the genotype probabilities with the true genotypes. We demonstrate that the power to detect a true association can be substantially increased for difficult to call genotypes, resulting in improved inference in association studies.