Cis-acting mutation affecting GJA5 transcription is underlying the Melanotic within-feather pigmentation pattern in chickens.

Melanotic (Ml) is a mutation in chickens that extends black (eumelanin) pigmentation in normally brown or red (pheomelanin) areas, thus affecting multiple within-feather patterns [J. W. Moore, J. R. Smyth Jr, J. Hered. 62, 215-219 (1971)]. In the present study, linkage mapping using a back-cross between Dark Cornish (Ml/Ml) and Partridge Plymouth Rock (ml+/ml+ ) chickens assigned Ml to an 820-kb region on chromosome 1. Identity-by-descent mapping, via whole-genome sequencing and diagnostic tests using a diverse set of chickens, refined the localization to the genomic region harboring GJA5 encoding gap-junction protein 5 (alias connexin 40) previously associated with pigmentation patterns in zebrafish. An insertion/deletion polymorphism located in the vicinity of the GJA5 promoter region was identified as the candidate causal mutation. Four different GJA5 transcripts were found to be expressed in feather follicles and at least two showed differential expression between genotypes. The results showed that Melanotic constitutes a cis-acting regulatory mutation affecting GJA5 expression. A recent study established the melanocortin-1 receptor (MC1R) locus and the interaction between the MC1R receptor and its antagonist agouti-signaling protein as the primary mechanism underlying variation in within-feather pigmentation patterns in chickens. The present study advances understanding the mechanisms underlying variation in plumage color in birds because it demonstrates that the activity of connexin 40/GJA5 can modulate the periodic pigmentation patterns within individual feathers.

Mutations Upstream of the TBX5 and PITX1 Transcription Factor Genes Are Associated with Feathered Legs in the Domestic Chicken.

Feathered leg is a trait in domestic chickens that has undergone intense selection by fancy breeders. Previous studies have shown that two major loci controlling feathered leg are located on chromosomes 13 and 15. Here, we present genetic evidence for the identification of candidate causal mutations at these loci. This was accomplished by combining classical linkage mapping using an experimental cross segregating for feathered leg and high-resolution identical-by-descent mapping using whole-genome sequence data from 167 samples of chicken with or without feathered legs. The first predicted causal mutation is a single-base change located 25 kb upstream of the gene for the forelimb-specific transcription factor TBX5 on chromosome 15. The second is a 17.7-kb deletion located ∼200 kb upstream of the gene for the hindlimb-specific transcription factor PITX1 on chromosome 13. These mutations are predicted to activate TBX5 and repress PITX1 expression, respectively. The study reveals a remarkable convergence in the evolution of the feathered-leg phenotype in domestic chickens and domestic pigeons, as this phenotype is caused by noncoding mutations upstream of the same two genes. Furthermore, the PITX1 causal variants are large overlapping deletions, 17.7 kb in chicken and 44 kb in pigeons. The results of the present study are consistent with the previously proposed model for pigeon that feathered leg is caused by reduced PITX1 expression and ectopic expression of TBX5 in hindlimb buds resulting in a shift of limb identity from hindlimb to more forelimb-like identity.

Population-based identity-by-descent mapping combined with exome sequencing to detect rare risk variants for schizophrenia.

Genome-wide association studies (GWASs) are highly effective at identifying common risk variants for schizophrenia. Rare risk variants are also important contributors to schizophrenia etiology but, with the exception of large copy number variants, are difficult to detect with GWAS. Exome and genome sequencing, which have accelerated the study of rare variants, are expensive so alternative methods are needed to aid detection of rare variants. Here we re-analyze an Irish schizophrenia GWAS dataset (n = 3,473) by performing identity-by-descent (IBD) mapping followed by exome sequencing of individuals identified as sharing risk haplotypes to search for rare risk variants in coding regions. We identified 45 rare haplotypes (>1 cM) that were significantly more common in cases than controls. By exome sequencing 105 haplotype carriers, we investigated these haplotypes for functional coding variants that could be tested for association in independent GWAS samples. We identified one rare missense variant in PCNT but did not find statistical support for an association with schizophrenia in a replication analysis. However, IBD mapping can prioritize both individual samples and genomic regions for follow-up analysis but genome rather than exome sequencing may be more effective at detecting risk variants on rare haplotypes.

Identity by descent mapping of HCV spontaneous clearance in populations of diverse ancestry.

Background: Acute infection with hepatitis C virus (HCV) affects millions of individuals worldwide. Host genetics plays a role in spontaneous clearance of the acute infection which occurs in approximately 30% of the individuals. Common variants in GPR158, genes in the interferon lambda (IFNL) cluster, and the MHC region have been associated with HCV clearance in populations of diverse ancestry. Fine mapping of those regions has identified some key variants and amino acids as potential causal variants but the role of rare variants in those regions and in the genome, in general, has not been explored. We aimed to detect haplotypes containing rare variants related to HCV clearance using identity-by-descent (IBD) haplotype sharing between unrelated cases/case pairs and case/controls pairs in 3,608 individuals with European and African ancestry. Results: We detected 1,711,832 and 5,678,043 and individual pairs of IBD segments in the European and African ancestry individuals, respectively. As expected, individuals of African descent had more, and shorter segments compared to Europeans. We did not detect any significant IBD signals in the known associated gene regions. Conclusions: IBD is based on sharing of haplotypes and is most powerful in populations with a shared founder or recent common ancestor. For the complex trait of HCV clearance, we used two outbred, global populations that limited our power to detect IBD associations. Overall, in this population-based sample we failed to detect rare variations associated with HCV clearance in individuals of European and African ancestry.

The crest phenotype in domestic chicken is caused by a 197 bp duplication in the intron of HOXC10.

The Crest mutation in chicken shows incomplete dominance and causes a spectacular phenotype in which the small feathers normally present on the head are replaced by much larger feathers normally present only in dorsal skin. Using whole-genome sequencing, we show that the crest phenotype is caused by a 197 bp duplication of an evolutionarily conserved sequence located in the intron of HOXC10 on chromosome 33. A diagnostic test showed that the duplication was present in all 54 crested chickens representing eight breeds and absent from all 433 non-crested chickens representing 214 populations. The mutation causes ectopic expression of at least five closely linked HOXC genes, including HOXC10, in cranial skin of crested chickens. The result is consistent with the interpretation that the crest feathers are caused by an altered body region identity. The upregulated HOXC gene expression is expanded to skull tissue of Polish chickens showing a large crest often associated with cerebral hernia, but not in Silkie chickens characterized by a small crest, both homozygous for the duplication. Thus, the 197 bp duplication is required for the development of a large crest and susceptibility to cerebral hernia because only crested chicken show this malformation. However, this mutation is not sufficient to cause herniation because this malformation is not present in breeds with a small crest, like Silkie chickens.

Association of NOD2 Mutations with Aggressive Periodontitis.

Aggressive periodontitis (AgP) is characterized by rapid alveolar bone destruction and tooth loss early in life, and its etiology remains unclear. To explore the genetic risk factors of AgP, we performed genome-wide single-nucleotide polymorphism genotyping for identity-by-descent mapping and identified 32 distinct candidate loci, followed by whole exome sequencing with 2 pedigrees of AgP consisting of 3 cases and 1 control in 1 family and 2 sibling cases in the other. After variant filtering procedures and validation by targeted Sanger sequencing, we identified 2 missense mutations at 16q12 in NOD2 (p.Ala110Thr and p.Arg311Trp), which encodes nucleotide-binding oligomerization domain protein 2. We further examined 94 genetically unrelated AgP patients by targeted sequencing of NOD2 and found that 2 patients among them also carried the p.Arg311Trp variant. Furthermore, we found 3 additional missense mutations in this gene (p.His370Tyr, p.Arg459Cys, and p.Ala868Thr). These mutations either had not been previously observed or are extremely rare (frequency <0.001) in Asian populations. NOD2 plays a crucial role in innate immunity as an intracellular receptor initiating nuclear factor κB-dependent and mitogen-activated protein kinase-dependent gene transcription. These results demonstrated NOD2 as a novel gene involved in AgP.