The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function.

BACKGROUND: Dystroglycan (Dg) is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC) which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys) WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. RESULTS: We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. CONCLUSION: Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

Differential expression of CYP3A12 and CYP3A26 mRNAs in canine liver and intestine.

The cytochrome P450 (CYP) 3A family is often considered the most important CYP subfamily with regard to drug metabolism in people. Certainly, CYP3A4 contributes to poor oral bioavailability of a number of drugs, and a tremendous amount of effort has been made in attempting to find an appropriate model system to predict the oral bioavailability of candidate drugs. The dog is a species widely used as a preclinical model for the evaluation of drug safety and pharmacokinetics. Compared with other species, little information is available on the tissue distribution of CYP enzymes. The purpose of this study was to determine the level of messenger RNA (mRNA) expression of the canine CYP3A subfamily (CYP3A12 and CYP3A26) in the liver and duodenum. Overall, expression of CYP3A mRNA was greater in the liver than in the duodenum. Hepatic expression of CYP3A26 was greater than CYP3A12 in all dogs, with CYP3A26 comprising 75.2% of the hepatic mRNA CYP3A pool. Conversely, duodenal expression of CYP3A12 was greater than CYP3A26 in all dogs, with CYP3A12 comprising 99.8% of the duodenal mRNA CYP3A pool. In summary, these results represent an important step toward the systematic comparison of human and canine CYP3A enzymes, particularly in relation to oral bioavailability of substrate drugs.

Haplotypes vs single marker linkage disequilibrium tests: what do we gain?

The genetic dissection of complex diseases represents a formidable challenge for modern human genetics. Recently, it has been suggested that linkage disequilibrium (LD) based methods will be a powerful approach for delineating complex disease genes. Most proposed LD test statistics search for association between a single marker and a putative trait locus. However, the power of a single marker association test may suffer because LD information contained in flanking markers is ignored. Intuitively, haplotypes (which can be regarded as a collection of ordered markers) may be more powerful than individual, unorganised markers. In this study, we derive the analytical tools based on standard chi-square statistics to directly investigate and compare the power between multilocus haplotypes and single marker LD tests. More specifically, novel formulas are obtained in order to calculate expected haplotype frequencies of unlimited size. This study demonstrates that the use of haplotypes can significantly improve the power and robustness of mapping disease genes. Additionally, we detail how the power of haplotype based association tests are affected by important population genetic parameters such as the genetic distance between markers and disease locus, mode of disease inheritance, age of trait causing mutation, frequency of associated marker allele, and level of initial LD. Finally, published data from the Hereditary Hemochromatosis disease region is used to illustrate the utility of haplotypes.

Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping.

High-throughput methods for assaying DNA variation require two important steps: (i) discriminating the variation and (ii) detecting the signal. In this report, we describe a novel SNP genotyping method that we refer to as melting curve analysis of SNPs (McSNP). McSNP combines a classic approach for discriminating alleles, restriction enzyme digestion, with a more recent method for detecting DNA fragments, melting curve analysis. Melting curve analysis is performed by slowly heating DNA fragments in the presence of the dsDNA-specific fluorescent dye SYBR Green I. As the sample is heated, fluorescence rapidly decreases when the melting temperature of a particular fragment is reached. We show that it is possible to determine the composition of simple mixtures of DNA fragments, such as those that result from restriction enzyme digestions of short PCR products. McSNP is well suited for high-throughput genotyping because 96 samples can be analyzed and automatically scored in 20 min. Our results clearly demonstrate that McSNP is a simple, inexpensive, and accurate means of genotyping SNP variation.

The haplotype linkage disequilibrium test for genome-wide screens: its power and study design.

The focus of human genetics continues to shift toward the dissection of complex phenotypes. Integral to these endeavors is the development of powerful analytical tools. To this end, we propose a novel method designated the haplotype linkage disequibrium (LD) test for identifying diseases genes. The basic structure of the haplotype test statistic is a chi-square in which haplotypes, as opposed to individual marker data, are compared between cases and controls. Specifically, we performed power calculations and demonstrate that the use of haplotypes improves the power of mapping disease genes. We show that this approach can be used for initial genome-wide screens in mapping disease genes. Furthermore, we investigated the factors influencing statistical power of the method and discussed basic principals underlying study design. Published data from the Hereditary Hemochromatosis region was used to illustrate the utility of the haplotype test. Also discussed is its relationship with linkage disequibrium.

The effect that genotyping errors have on the robustness of common linkage-disequilibrium measures.

The rapid development of a dense single-nucleotide-polymorphism marker map has stimulated numerous studies attempting to characterize the magnitude and distribution of background linkage disequilibrium (LD) within and between human populations. Although genotyping errors are an inherent problem in all LD studies, there have been few systematic investigations documenting their consequences on estimates of background LD. Therefore, we derived simple deterministic formulas to investigate the effect that genotyping errors have on four commonly used LD measures-D', r, Q, and d-in studies of background LD. We have found that genotyping error rates as small as 3% can have serious affects on these LD measures, depending on the allele frequencies and the assumed error model. Furthermore, we compared the robustness of D', r, Q, and d, in the presence of genotyping errors. In general, Q and d are more robust than D' and r, although exceptions do exist. Finally, through stochastic simulations, we illustrate how genotyping errors can lead to erroneous inferences when measures of LD between two samples are compared.

Interaction between the melanocortin-1 receptor and P genes contributes to inter-individual variation in skin pigmentation phenotypes in a Tibetan population.

The melanocortin-1 receptor (MC1R) and P gene product are two important components of the human pigmentary system that have been shown to be associated with red hair/fair skin and cause type II oculocutaneous albinism, respectively. However, their contribution to inter-individual variation at the population level is not well defined. To this end, we genotyped 3 single nucleotide polymorphisms (SNPs) in the MC1R gene (Arg67Gln, Gln163Arg, Val92Met) and 2 SNPs in the P gene (IVS 13-15 and Gly780Gly) in 184 randomly ascertained Tibetan subjects, whose skin color was measured as a quantitative trait by reflective spectroscopy. Single locus analyses failed to demonstrate an association between any of the 5 SNPs and skin pigmentation. However, when an epistatic model was applied to the data, a significant gene-gene interaction was identified between Val92Met in MCIR and IVS13-15 in the P gene (F=2.43; P=0.0105). We also discuss the possible mechanisms of how gene interactions arise in signal transduction pathways.

Ethnic-affiliation estimation by use of population-specific DNA markers.

During the past 10 years, DNA analysis has revolutionized the determination of identity in a forensic context. Statements about the biological identity of two human DNA samples now can be made with complete confidence. Although DNA markers are very powerful for distinguishing among individuals, most offer little power to distinguish ethnicity or to support any statement about the physical characteristics of an individual. Through a search of the literature and of unpublished data on allele frequencies we have identified a panel of population-specific genetic markers that enable robust ethnic-affiliation estimation for major U.S. resident populations. In this report, we identify these loci and present their levels of allele-frequency differential between ethnically defined samples, and we demonstrate, using log-likelihood analysis, that this panel of markers provides significant statistical power for ethnic-affiliation estimation. In addition to their use in forensic ethnic-affiliation estimation, population-specific genetic markers are very useful in both population- and individual-level admixture estimation and in mapping genes by use of the linkage disequilibrium created when populations hybridize.

Estimating African American admixture proportions by use of population-specific alleles.

We analyzed the European genetic contribution to 10 populations of African descent in the United States (Maywood, Illinois; Detroit; New York; Philadelphia; Pittsburgh; Baltimore; Charleston, South Carolina; New Orleans; and Houston) and in Jamaica, using nine autosomal DNA markers. These markers either are population-specific or show frequency differences >45% between the parental populations and are thus especially informative for admixture. European genetic ancestry ranged from 6.8% (Jamaica) to 22.5% (New Orleans). The unique utility of these markers is reflected in the low variance associated with these admixture estimates (SEM 1.3%-2.7%). We also estimated the male and female European contribution to African Americans, on the basis of informative mtDNA (haplogroups H and L) and Y Alu polymorphic markers. Results indicate a sex-biased gene flow from Europeans, the male contribution being substantially greater than the female contribution. mtDNA haplogroups analysis shows no evidence of a significant maternal Amerindian contribution to any of the 10 populations. We detected significant nonrandom association between two markers located 22 cM apart (FY-null and AT3), most likely due to admixture linkage disequilibrium created in the interbreeding of the two parental populations. The strength of this association and the substantial genetic distance between FY and AT3 emphasize the importance of admixed populations as a useful resource for mapping traits with different prevalence in two parental populations.

A polymorphism in the promoter region of catalase is associated with blood pressure levels.

Catalase is an important antioxidant enzyme that detoxifies H2O2 into oxygen and water and thus limits the deleterious effects of reactive oxygen species (ROS). Because chronic exposure to excess ROS may contribute to vascular damage, we investigated whether genetic variation in catalase was associated with susceptibility to essential hypertension (EHYT) in 324 individuals (at least 50 years old) who were randomly sampled from an isolated population living in Xiangchang, China. They were screened for genetic variation in the promoter of catalase by direct sequencing. In total, four single nucleotide polymorphisms (SNPs) were identified. The association between the SNPs and EHYT was investigated by a linear regression model under phenotypic selection; in our analyses, we used both SBP>150 mmHg and SBP>160 mmHg as thresholds. A SNP 844 bp upstream of the start codon (SNP-844) demonstrated strong evidence of association with EHYT (SBP>150 mmHg: F=5.09, P=0.008; SBP>160 mmHg: F=7.13, P=0.002). This is the first study to implicate genetic variation in catalase in susceptibility to EHYT and suggests that polymorphisms in promoter regions may be particularly relevant to the study of complex diseases.

Y-Chromosome evidence for a northward migration of modern humans into Eastern Asia during the last Ice Age.

The timing and nature of the arrival and the subsequent expansion of modern humans into eastern Asia remains controversial. Using Y-chromosome biallelic markers, we investigated the ancient human-migration patterns in eastern Asia. Our data indicate that southern populations in eastern Asia are much more polymorphic than northern populations, which have only a subset of the southern haplotypes. This pattern indicates that the first settlement of modern humans in eastern Asia occurred in mainland Southeast Asia during the last Ice Age, coinciding with the absence of human fossils in eastern Asia, 50,000-100,000 years ago. After the initial peopling, a great northward migration extended into northern China and Siberia.

Estimating African American admixture proportions by use of population-specific alleles

We analyzed the European genetic contribution to 10 populations of Africans descent in the United States (Maywood, Illinois; Detroit; New York; Philadelphia; Pittsburgh; Baltimore; Charleston, South Carolina; New Orleans; and Houston) and in Jamaica, using nine autosomal DNA markers. These markers either are population-specific or show frequency differences >45 percent between the parental populations and are thus especially informative for admixture. European genetic ancestry ranged from 6.8 percent (Jamaica) to 22.5 percent (New Orleans). The unique utility of these markers is reflected in the low variance associated with these admixture estimates (SEM 1.3 percent -2.7 percent). We also estimated the male and female European contribution to African Americans. on the basis of informative mtDNA (haplogroups H and L) and Y Alu polymorphic markers. Results indicate a sex-biased gene flow from Europeans, the male contribution being substantially greater that the female contribution. mtDNA haplogroups analysis shows no evidence of a significant maternal Amerindian contribution to any of the 10 populations. We detected significant nonrandom association between two markers located 22 cM apart (FY-null and AT3), most likely due to admixture linkage disequilibrium created in the interbreeding of the two parental populations. The strength of this association and the substantial genetic distance between FY and AT3 emphasize the importance of admixed populations as a useful resources for mapping traits with different prevalence in two parental populations (AU)