Mapping QTL affecting a systemic sclerosis-like disorder in a cross between UCD-200 and red jungle fowl chickens

Abstract

Systemic sclerosis (SSc) or scleroderma is a rare, autoimmune, multi-factorial disease characterized by early microvascular alterations, inflammation, and fibrosis. Chickens from the UCD-200 line develop a hereditary SSc-like disease, showing all the hallmarks of the human disorder, which makes this line a promising model to study genetic factors underlying the disease. A backcross was generated between UCD-200 chickens and its wild ancestor – the red jungle fowl and a genome-scan was performed to identify loci affecting early (21 days of age) and late (175 days of age) ischemic lesions of the comb. A significant difference in frequency of disease was observed between sexes in the BC population, where the homogametic males were more affected than females, and there was evidence for a protective W chromosome effect. Three suggestive disease predisposing loci were mapped to chromosomes 2, 12 and 14. Three orthologues of genes implicated in human SSc are located in the QTL region on chromosome 2, TGFRB1, EXOC2-IRF4 and COL1A2, as well as CCR8, which is more generally related to immune function. IGFBP3 is also located within the QTL on chromosome 2 and earlier studies have showed increased IGFBP3 serum levels in SSc patients. To our knowledge, this study is the first to reveal a potential genetic association between IGFBP3 and SSc. Another gene with an immunological function, SOCS1, is located in the QTL region on chromosome 14. These results illustrate the usefulness of the UCD-200 chicken as a model of human SSc and motivate further in-depth functional studies of the implicated candidate genes.

Introduction

Systemic sclerosis (SSc) or scleroderma is a rare autoimmune disease of the skin and internal organs such as the gastrointestinal tract, lungs, kidneys and heart. It is characterized by microvascular damage, immune dysfunction, and excessive deposition of extracellular matrix (ECM) (Chizzolini, 2007). The aetiology of SSc is poorly understood but it is generally accepted that both environmental and genetic factors contribute to the disease. Some recent genetic studies have identified association between several genes and SSc in humans, including common allelic variants in Exocyst complex component 2-Interferon regulatory factor 4 (EXOC2-IRF4), Cluster of differentiation 247 (CD247), Transforming growth factor beta receptor I (TGFBR1), Collagen alpha 2(I) chain (COL1A2) and Secreted protein acidic and rich in cystein (SPARC) (Pannu et al., 2006, Radstake et al., 2010, Zhou et al., 2003). However, not all of them could be confirmed in independent studies (Agarwal, 2010, Lagan et al., 2005). Some possible reasons for the different results might be clinical heterogeneity within patients diagnosed with SSc, small effects of single genes on SSc predisposition, and low disease incidence, meaning that effects can be missed unless very large cohorts are examined.

Genetic studies in animal models can provide a valuable complement to human studies because experimental crosses can be generated. Animal models also enable in-depth experimental validation and exploration of the molecular basis of identified genes (Andersson and Georges, 2004). One of the best animal models for SSc is the University of California at Davis-line 200 chicken (UCD-200). UCD-200 chickens develop a hereditary disease displaying all the disease characteristics of human SSc, including the typical microvascular alterations, perivascular mononuclear cell infiltration, fibrosis of skin and viscera, and autoantibody production, e.g. anti-nuclear antibodies (ANA), and anti-endothelial cell antibodies (AECA) (Beyer et al., 2010, Gershwin et al., 1981). There are clear genetic, immunological and environmental influences on disease outcome in similarity with the human condition including a difference in disease frequency between the sexes (Beyer et al., 2010, Gershwin et al., 1981, Wick et al., 2006). Chickens are ideal for genetic studies since large numbers of offspring can rapidly be produced from each pair of parents, and phenotypic characteristics are easily assessed. Another advantage of the UCD-200 chicken model of SSc compared to other animal models for SSc, is the opportunity to study the disease in the embryo, i.e. long before clinical and immunological symptoms emerge. In common with many autoimmune diseases, women tend to be more affected with SSc than men, but in crosses including the UCD-200 line, males have a higher disease incidence (Selmi et al., 2006, Wick et al., 2006). However, in both chickens and humans it is the homogametic sex that is more affected; male birds are homogametic ZZ and female birds are ZW.

One to two weeks after hatching, UCD-200 birds manifest typical comb lesions with a current incidence of 97.5% (Wick et al., 2006). Disease progression starts with erythema and oedema of the comb leading to necrosis and, in most cases, loss of the comb. These lesions are considered equivalent to fingertip ulcers in human SSc. Some of the animals also show ischemic lesions of toes or develop dermal lesions at the neck. In the inflammatory phase of the disease, T-cell receptor (TCR)γ/δ+/CD3+/MHC class II− T-cells prevail in the stratum papillare, while TCRα/β+/CD3+/CD4+/MHC class II+ T-cells predominate in the deeper dermis. The early inflammatory stage subsequently progresses to a chronic stage characterized by fibrosis with excessive accumulation of collagen types I, III, and VI. Visceral involvement occurs in the oesophagus, small intestine, lungs, and kidneys with early vascular alterations and inflammation, followed by fibrosis (Nguyen et al., 2000, Wick et al., 2006).

Earlier crosses of UCD-200 chickens with healthy controls resulted in only healthy birds in the F₁ generation, suggesting a recessive autosomal mode of gene action. F₂ progeny (F₁ × F₁) showed a low incidence (4%) of affected individuals. This suggests that multiple loci and interactions between loci may be important, as for a typical single recessive gene with full penetrance, 25% of F₂ individuals are expected to be diseased. However, the number of genes involved should be limited because a high incidence of scleroderma was attained after relatively few generations of selective breeding (Wick et al., 2006). An F₂ design is generally more powerful compared to a backcross (BC) design, because both F₁ parents should be heterozygous at the causative loci. However, if the disorder is dependent on interactions between several loci and the incidence of disease is low in the F₂, as in this case, a BC can be more powerful if it generates more affected individuals. For example, if a trait is caused by recessive alleles at four unlinked loci, in an F₂ design (1/4)⁴ = 1/256 of the progeny would be diseased, whereas (1/2)⁴ = 1/16 of the progeny would be affected in a BC design. Backcrossing F₁ chickens to UCD-200 chickens, resulted in approximately 50% of the chickens being affected (Abplanalp et al., 1990). Therefore, in the present study, a backcross population was constructed for analyzing SSc.

Here, we report results aimed at dissecting the genetic architecture underlying avian SSc in the UCD-200 chicken population. A genome-wide scan for individual and interacting Quantitative Trait Loci (QTL) was performed to identify novel loci involved in the disorder.