A Complex Genomic Rearrangement Involving the Endothelin 3 Locus Causes Dermal Hyperpigmentation in the Chicken

Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals

A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus

Objectives: Ncf1 polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases in animal models. The human NCF1 gene is very complex with both functional and non-functional gene copies and genotyping requires assays specific for functional NCF1 genes. We aimed at investigating association and function of the missense single nucleotide polymorphism (SNP), rs201802880 (here denoted NCF1-339) in NCF1 with systemic lupus erythematosus (SLE). Methods: We genotyped the NCF1-339 SNP in 973 Swedish patients with SLE and 1301 controls, using nested PCR and pyrosequencing. ROS production and gene expression of type 1 interferon-regulated genes were measured in isolated cells from subjects with different NCF1-339 genotypes. Results: We found an increased frequency of the NCF1-339 T allele in patients with SLE, 11% compared with 4% in controls, OR 3.0, 95% CI 2.4 to 3.9, p=7.0×10-20. The NCF1-339 T allele reduced extracellular ROS production in neutrophils (p=0.004) and led to an increase expression of type 1 interferon-regulated genes. In addition, the NCF1-339 T allele was associated with a younger age at diagnosis of SLE; mean age 30.3 compared with 35.9, p=2.0×1-6. Conclusions: These results clearly demonstrate that a genetically controlled reduced production of ROS increases the risk of developing SLE and confirm the hypothesis that ROS regulate chronic autoimmune inflammatory diseases

NCF1-339 polymorphism is associated with altered formation of neutrophil extracellular traps, high serum interferon activity and antiphospholipid syndrome in systemic lupus erythematosus

Objectives: A single nucleotide polymorphism in the NCF1 gene (NCF1-339, rs201802880), encoding NADPH oxidase type II subunit NCF1/p47phox, reducing production of reactive oxygen species (ROS) is strongly associated with the development of systemic lupus erythematosus (SLE). This study aimed at characterising NCF1-339 effects on neutrophil extracellular trap (NET) formation, type I interferon activity and antibody profile in patients with SLE. Methods: Neutrophil NET-release pathways (n=31), serum interferon (n=141) and finally antibody profiles (n=305) were investigated in SLE subjects from Lund, genotyped for NCF1-339. Then, 1087 SLE subjects from the rheumatology departments of four Swedish SLE centres, genotyped for NCF1-339, were clinically characterised to validate these findings. Results: Compared with patients with normal-ROS NCF1-339 genotypes, neutrophils from patients with SLE with low-ROS NCF1-339 genotypes displayed impaired NET formation (p<0.01) and increased dependence on mitochondrial ROS (p<0.05). Low-ROS patients also had increased frequency of high serum interferon activity (80% vs 21.4%, p<0.05) and positivity for anti-β2 glycoprotein I (p<0.01) and anticardiolipin antibodies (p<0.05) but were not associated with other antibodies. We confirmed an over-representation of having any antiphospholipid antibody, OR 1.40 (95% CI 1.01 to 1.95), anti-β2 glycoprotein I, OR 1.82 (95% CI 1.02 to 3.24) and the antiphospholipid syndrome (APS), OR 1.74 (95% CI 1.19 to 2.55) in all four cohorts (n=1087). Conclusions: The NCF1-339 SNP mediated decreased NADPH oxidase function, is associated with high interferon activity and impaired formation of NETs in SLE, allowing dependence on mitochondrial ROS. Unexpectedly, we revealed a striking connection between the ROS deficient NCF1-339 genotypes and the presence of phospholipid antibodies and APS