Nadine Cambisano

Highly effective SNP-based association mapping and management of recessive defects in livestock

The widespread use of elite sires by means of artificial insemination in livestock breeding leads to the frequent emergence of recessive genetic defects, which cause significant economic and animal welfare concerns. Here we show that the availability of genome-wide, high-density SNP panels, combined with the typical structure of livestock populations, markedly accelerates the positional identification of genes and mutations that cause inherited defects. We report the fine-scale mapping of five recessive disorders in cattle and the molecular basis for three of these: congenital muscular dystony (CMD) types 1 and 2 in Belgian Blue cattle and ichthyosis fetalis in Italian Chianina cattle. Identification of these causative mutations has an immediate translation into breeding practice, allowing marker assisted selection against the defects through avoidance of at-risk matings.

Variants modulating the expression of a chromosome domain encompassing PLAG1 influence bovine stature

We report mapping of a quantitative trait locus (QTL) with a major effect on bovine stature to a ∼780-kb interval using a Hidden Markov Model–based approach that simultaneously exploits linkage and linkage disequilibrium. We re-sequenced the interval in six sires with known QTL genotype and identified 13 clustered candidate quantitative trait nucleotides (QTNs) out of >9,572 discovered variants. We eliminated five candidate QTNs by studying the phenotypic effect of a recombinant haplotype identified in a breed diversity panel. We show that the QTL influences fetal expression of seven of the nine genes mapping to the ∼780-kb interval. We further show that two of the eight candidate QTNs, mapping to the PLAG1-CHCHD7 intergenic region, influence bidirectional promoter strength and affect binding of nuclear factors. By performing expression QTL analyses, we identified a splice site variant in CHCHD7 and exploited this naturally occurring null allele to exclude CHCHD7 as single causative gene.

A QTL with major effect on milk yield and composition maps to bovine Chromosome 14

Abstract. A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand2-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect.

Serial translocation by means of circular intermediates underlies colour sidedness in cattle

Colour sidedness is a dominantly inherited phenotype of cattle characterized by the polarization of pigmented sectors on the flanks, snout and ear tips. It is also referred to as ‘lineback’ or ‘witrik’ (which means white back), as colour-sided animals typically display a white band along their spine. Colour sidedness is documented at least since the Middle Ages and is presently segregating in several cattle breeds around the globe, including in Belgian blue and brown Swiss. Here we report that colour sidedness is determined by a first allele on chromosome 29 (Cs29), which results from the translocation of a 492-kilobase chromosome 6 segment encompassing KIT to chromosome 29, and a second allele on chromosome 6 (Cs6), derived from the first by repatriation of fused 575-kilobase chromosome 6 and 29 sequences to the KIT locus. We provide evidence that both translocation events involved circular intermediates. This is the first example, to our knowledge, of a phenotype determined by homologous yet non-syntenic alleles that result from a novel copy-number-variant-generating mechanism.

A Deletion in the Bovine FANCI Gene Compromises Fertility by Causing Fetal Death and Brachyspina

Fertility is one of the most important traits in dairy cattle, and has been steadily declining over the last decades. We herein use state-of-the-art genomic tools, including high-throughput SNP genotyping and next-generation sequencing, to identify a 3.3 Kb deletion in the FANCI gene causing the brachyspina syndrome (BS), a rare recessive genetic defect in Holstein dairy cattle. We determine that despite the very low incidence of BS (<1/100,000), carrier frequency is as high as 7.4% in the Holstein breed. We demonstrate that this apparent discrepancy is likely due to the fact that a large proportion of homozygous mutant calves die during pregnancy. We postulate that several other embryonic lethals may segregate in livestock and significantly compromise fertility, and propose a genotype-driven screening strategy to detect the corresponding deleterious mutations.

A missense mutation accelerating the gating of the lysosomal Cl−/H+-exchanger ClC-7/Ostm1 causes osteopetrosis with gingival hamartomas in cattle

Chloride-proton exchange by the lysosomal anion transporter ClC-7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.

Selection in action: dissecting the molecular underpinnings of the increasing muscle mass of Belgian Blue Cattle

BackgroundBelgian Blue cattle are famous for their exceptional muscular development or “double-muscling”. This defining feature emerged following the fixation of a loss-of-function variant in the myostatin gene in the eighties. Since then, sustained selection has further increased muscle mass of Belgian Blue animals to a comparable extent. In the present paper, we study the genetic determinants of this second wave of muscle growth.ResultsA scan for selective sweeps did not reveal the recent fixation of another allele with major effect on muscularity. However, a genome-wide association study identified two genome-wide significant and three suggestive quantitative trait loci (QTL) affecting specific muscle groups and jointly explaining 8-21% of the heritability. The top two QTL are caused by presumably recent mutations on unique haplotypes that have rapidly risen in frequency in the population. While one appears on its way to fixation, the ascent of the other is compromised as the likely underlying MRC2 mutation causes crooked tail syndrome in homozygotes. Genomic prediction models indicate that the residual additive variance is largely polygenic.ConclusionsContrary to complex traits in humans which have a near-exclusive polygenic architecture, muscle mass in beef cattle (as other production traits under directional selection), appears to be controlled by (i) a handful of recent mutations with large effect that rapidly sweep through the population, and (ii) a large number of presumably older variants with very small effects that rise slowly in the population (polygenic adaptation).

Frequency of mosaicism points towards mutation-prone early cleavage cell divisions.

It has recently become possible to directly estimate the germ-line de novo mutation (DNM) rate by sequencing the whole genome of father-mother-offspring trios, and this has been conducted in human1–5, chimpanzee6, birds7 and fish8. In these studies DNMs are defined as variants that are heterozygous in the offspring while being absent in both parents. They are assumed to have occurred in the germ-line of a parent and to have been transmitted to the offspring via the sperm or oocyte. This definition assumes that detectable mosaïcism in the individual in which the mutation occurred is negligible. However, instances of mosaïcism are well-documented in humans and other organisms, including ruminants9,10. We herein take advantage of the unique pedigree structure of cattle to show that mosaïcism associated with DNMs is a common occurrence, and that this should be taken into account to accurately estimate the mutation rate in this and possibly other species. It suggests that early cleavage cell divisions are particularly mutation-prone, and that the recurrence risk of DNM-dependent disorders in sibs may be higher than generally assumed.

On the use of the transmission disequilibrium test to detect pseudo-autosomal variants affecting traits with sex-limited expression

We herein describe the realization of a genome-wide association study for scrotal hernia and cryptorchidism in Norwegian and Belgian commercial pig populations. We have used the transmission disequilibrium test to avoid spurious associations due to population stratification. By doing so, we obtained genome-wide significant signals for both diseases with SNPs located in the pseudo-autosomal region in the vicinity of the pseudo-autosomal boundary. By further analyzing these signals, we demonstrate that the observed transmission disequilibria are artifactual. We determine that transmission bias at pseudo-autosomal markers will occur (i) when analyzing traits with sex-limited expression and (ii) when the allelic frequencies at the marker locus differ between X and Y chromosomes. We show that the bias is due to the fact that (i) sires will preferentially transmit the allele enriched on the Y (respectively X) chromosome to affected sons (respectively daughters) and (ii) dams will appear to preferentially transmit the allele enriched on the Y (respectively X) to affected sons (respectively daughters), as offspring inheriting the other allele are more likely to be non-informative. We define the conditions to mitigate these issues, namely by (i) extracting information from maternal meiosis only and (ii) ignoring trios for which sire and dam have the same heterozygous genotype. We show that by applying these rules to scrotal hernia and cryptorchidism, the pseudo-autosomal signals disappear, confirming their spurious nature.

389 A Candidate Gene Study of Rare Monogenic Disorders With IBD-Like Phenotype Identified Rare Variants in XIAP Gene in a Cohort of Early-Onset IBD Patients

frameshift variant(rs5743293) had the strongest effect in Group6 (OR=5.52, p=1.07E-16) yet no effect in Group2(OR=1.12, p=0.78), with a p-value of 1.43E-7 for test of heterogeneity; the ATG16L1 T300A variant showed a strong effect in Group4(OR=0.589, p=1.68E-9), with weak to no effect observed in other groups(p for heterogeneity: 5.33E-5). An unbiased Immunochip-wide analysis with these subgroups compared to non-IBD controls identified a putative novel CD association with PRKCQ. Three PRKCQ SNPs contributed individually to different subgroups (rs113912197 to Group2, OR=24.47, p=8.18E-7; rs112123005 to Group3, OR=5.98, p=8.63E-6 and rs661985 to Group1, OR=1.34, p=4.70E-4). LD between these SNPs is weak(R2<0.1), suggesting they are independent signals. PRKCQ codes for nPKC-θ, a serine-protein kinase that mediates non-redundant functions in T-cell receptor signaling and plays an important role in the development of T-helper 2 cells following immune and inflammatory responses. PRKCQ is also a known locus for Rheumatoid Arthritis and Type I diabetes, making it functionally highly relevant. Conclusion: Using SOM, we identified 6 serologically-defined subgroups within CD. These subgroups have distinct clinical characteristics and different genetic load. Known CD loci also show dramatically different effects in these subgroups, indicating they are genetically more homogenous.