Efficient mapping of mendelian traits in dogs through genome-wide association

With several hundred genetic diseases and an advantageous genome structure, dogs are ideal for mapping genes that cause disease. Here we report the development of a genotyping array with approximately 27,000 SNPs and show that genome-wide association mapping of mendelian traits in dog breeds can be achieved with only approximately 20 dogs. Specifically, we map two traits with mendelian inheritance: the major white spotting (S) locus and the hair ridge in Rhodesian ridgebacks. For both traits, we map the loci to discrete regions of <1 Mb. Fine-mapping of the S locus in two breeds refines the localization to a region of approximately 100 kb contained within the pigmentation-related gene MITF. Complete sequencing of the white and solid haplotypes identifies candidate regulatory mutations in the melanocyte-specific promoter of MITF. Our results show that genome-wide association mapping within dog breeds, followed by fine-mapping across multiple breeds, will be highly efficient and generally applicable to trait mapping, providing insights into canine and human health.     

Anion sensitivity and spectral tuning of middle- and long-wavelength-sensitive (MWS/LWS) visual pigments

The long-wavelength-sensitive (LWS) opsins form one of four classes of vertebrate cone visual pigment and exhibit peak spectral sensitivities (λ(max)) that generally range from 525 to 560 nm for rhodopsin/vitamin-A(1) photopigments. Unique amongst the opsin classes, many LWS pigments show anion sensitivity through the interaction of chloride ions with a histidine residue at site 197 (H197) to give a long-wavelength spectral shift in peak sensitivity. Although it has been shown that amino acid substitutions at five sites (180, 197, 277, 285 and 308) are useful in predicting the λ(max) values of the LWS pigment class, some species, such as the elephant shark and most marine mammals, express LWS opsins that possess λ(max) values that are not consistent with this 'five-site' rule, indicating that other interactions may be involved. This study has taken advantage of the natural mutation at the chloride-binding site in the mouse LWS pigment. Through the use of a number of mutant pigments generated by site-directed mutagenesis, a new model has been formulated that takes into account the role of charge and steric properties of the side chains of residues at sites 197 and 308 in the function of the chloride-binding site in determining the peak sensitivity of LWS photopigments.     

Identification of common variants influencing risk of the tauopathy progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a movement disorder with prominent tau neuropathology. Brain diseases with abnormal tau deposits are called tauopathies, the most common of which is Alzheimer's disease. Environmental causes of tauopathies include repetitive head trauma associated with some sports. To identify common genetic variation contributing to risk for tauopathies, we carried out a genome-wide association study of 1,114 individuals with PSP (cases) and 3,247 controls (stage 1) followed by a second stage in which we genotyped 1,051 cases and 3,560 controls for the stage 1 SNPs that yielded P ≤ 10(-3). We found significant previously unidentified signals (P < 5 × 10(-8)) associated with PSP risk at STX6, EIF2AK3 and MOBP. We confirmed two independent variants in MAPT affecting risk for PSP, one of which influences MAPT brain expression. The genes implicated encode proteins for vesicle-membrane fusion at the Golgi-endosomal interface, for the endoplasmic reticulum unfolded protein response and for a myelin structural component.     

MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis

The increased burden of chronic kidney and end-stage kidney diseases (ESKD) in populations of African ancestry has been largely unexplained. To identify genetic variants predisposing to idiopathic and HIV-1-associated focal segmental glomerulosclerosis (FSGS), we carried out an admixture-mapping linkage-disequilibrium genome scan on 190 African American individuals with FSGS and 222 controls. We identified a chromosome 22 region with a genome-wide logarithm of the odds (lod) score of 9.2 and a peak lod of 12.4 centered on MYH9, a functional candidate gene expressed in kidney podocytes. Multiple MYH9 SNPs and haplotypes were recessively associated with FSGS, most strongly a haplotype spanning exons 14 through 23 (OR = 5.0, 95% CI = 3.5-7.1; P = 4 x 10(-23), n = 852). This association extended to hypertensive ESKD (OR = 2.2, 95% CI = 1.5-3.4; n = 433), but not type 2 diabetic ESKD (n = 476). Genetic variation at the MYH9 locus substantially explains the increased burden of FSGS and hypertensive ESKD among African Americans.     

Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA)

Iron deficiency is usually attributed to chronic blood loss or inadequate dietary intake. Here, we show that iron deficiency anemia refractory to oral iron therapy can be caused by germline mutations in TMPRSS6, which encodes a type II transmembrane serine protease produced by the liver that regulates the expression of the systemic iron regulatory hormone hepcidin. These findings demonstrate that TMPRSS6 is essential for normal systemic iron homeostasis in humans.     

An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis

Using advanced gene targeting methods, generating mouse models of cancer that accurately reproduce the genetic alterations present in human tumors is now relatively straightforward. The challenge is to determine to what extent such models faithfully mimic human disease with respect to the underlying molecular mechanisms that accompany tumor progression. Here we describe a method for comparing mouse models of cancer with human tumors using gene-expression profiling. We applied this method to the analysis of a model of Kras2-mediated lung cancer and found a good relationship to human lung adenocarcinoma, thereby validating the model. Furthermore, we found that whereas a gene-expression signature of KRAS2 activation was not identifiable when analyzing human tumors with known KRAS2 mutation status alone, integrating mouse and human data uncovered a gene-expression signature of KRAS2 mutation in human lung cancer. We confirmed the importance of this signature by gene-expression analysis of short hairpin RNA-mediated inhibition of oncogenic Kras2. These experiments identified both a pattern of gene expression indicative of KRAS2 mutation and potential effectors of oncogenic KRAS2 activity in human cancer. This approach provides a strategy for using genomic analysis of animal models to probe human disease.     

Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome.

Rabbit beta-globin complementary DNA (cDNA) has been inserted into SV40 DNA in place of the gene coding for the virus' major capsid protein, VP1. The recombinant genome, SVGT5-RabetaG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the beta-globin coding sequence. Cells propagating SVGT5-RabetaG produce substantial quantities of rabbit beta-globin polypeptide.