Breed differences in allele frequency of the dopamine receptor D4 gene in dogs.

We previously reported that the dog dopamine receptor D4 (DRD4) gene is polymorphic as observed in humans, and four alleles were identified based on the number and/or order of the 12 and 39 bp sequences located in the homologous region of human DRD4. To assess the diversity of the DRD4 gene in dogs we examined the allelic variations in four breeds (beagle, golden retriever, Shetland sheepdog, and shiba) employing the polymerase chain reaction (PCR). As a result, we found three novel alleles and determined the DNA sequences of these alleles. The beagle shared four alleles, including 396, 435, 447a, and 447b, with the 435 (52.6%) and 447a (39.5%) alleles being common. The golden retriever had the 435 and 447a alleles, and the 435 allele was frequent (73.3%). In the Shetland sheepdog, the 435, 447a, and 498 alleles were observed, of which the 447a allele was most frequent (82.5%). The shiba had five alleles-447a, 447b, 486, 498, and 549-and the 447b allele was most common (55.4%). These findings suggest that the allele frequency varied among the four dog breeds, and analysis of the DRD4 polymorphism may therefore be useful for elucidating the relationships among dog breeds.     

Protease inhibitor system in horses: classification and detection of a new allele

A method of horizontal thin layer polyacrylamide gel electrophoresis at acid pH has been developed for the separation of the prealbumins in equine plasma. Using this method, it has been possible to split the S allele into two, S₁ and S₂, bringing the total number of prealbumin alleles in Thoroughbred horses to eight. The gene frequencies of these eight alleles in Australian Thoroughbreds are presented. All eight prealbumin types exhibit antiprotease activity and therefore, it is suggested that the name prealbumin (Pr) should be abandoned in favour of protease inhibitor (Pi) although at this stage it is not known whether this incorporates the Pi1 and Pi2 described by Juneja et al. (1979).     

Mitochondrial DNA mutations in human neoplasia

Many models of tumour formation have been put forth so far. In general they involve mutations in at least three elements within the cell: oncogenes, tumour suppressors and regulators of telomere replication. Recently numerous mutations in mitochondria have been found in many tumours, whereas they were absent in normal tissues from the same individual. The presence of mutations, of course, does not prove that they play a causative role in development of neoplastic lesions and progression; however, the key role played by mitochondria in both apoptosis and generation of DNA-damaging reactive oxygen species might indicate that the observed mutations contribute to tumour development. Recent experiments with nude mice have proven that mtDNA mutations are indeed responsible for tumour growth and exacerbated ROS production. This review describes mtDNA mutations in main types of human neoplasia.     

Persistently elevated frequency of spontaneous mutations in progeny of CHO clones surviving X-irradiation: association with delayed reproductive death phenotype

Certain clonal progeny of Chinese hamster ovary (CHO) cells surviving X-irradiation demonstrate pleomorphic changes including a persistently decreased cloning efficiency, a dominant phenotype we have termed delayed reproductive death (Chang and Little, 1991, 1992b). We now report that cells from these progeny clones show a persistently elevated frequency of spontaneous mutations at the hprt locus for up to 95–100 population doubling post-irradiation. Mutant fractions as high as 10⁻³ were scored, more than two orders of magnitude higher than those observed in clonal progeny of non-irradiated cells studied in parallel. These results are discussed in terms of the hypothesis that radiation induces a type of genetic instability among some surviving cells that results in a heritable mutator phenotype, and that this instability may also be involved in the phenomenon of delayed reproductive death.     

ALFRED: An allele frequency database for anthropology

The deluge of data from the human genome project (HGP) presents new opportunities for molecular anthropologists to study human variation through the promise of vast numbers of new polymorphisms (e.g., single nucleotide polymorphisms or SNPs). Collecting the resulting data into a single, easily accessible resource will be important to facilitate this research. We created a prototype Web-accessible database named ALFRED (ALelle FREquency Database, http://alfred.med.yale.edu/alfred/) to store and make publicly available allele frequency data on diverse polymorphic sites for many populations. In constructing this database, we considered many different concerns relating to the types of information needed for anthropology, population genetics, molecular genetics, and statistics, as well as issues of data integrity and ease of access to data. We also developed links to other Web-based databases as well as procedures for others to make links to the data in ALFRED. Here we present an overview of the issues considered and provisional solutions, as well as an example of data already available. It is our hope that this database will be useful for research and teaching in a wide range of fields, and that colleagues from various fields will contribute to making ALFRED an important resource for many studies as yet unforeseen.     

Seven novel KIT mutations in horses with white coat colour phenotypes

White coat colour in horses is inherited as a monogenic autosomal dominant trait showing a variable expression of coat depigmentation. Mutations in the KIT gene have previously been shown to cause white coat colour phenotypes in pigs, mice and humans. We recently also demonstrated that four independent mutations in the equine KIT gene are responsible for the dominant white coat colour phenotype in various horse breeds. We have now analysed additional horse families segregating for white coat colour phenotypes and report seven new KIT mutations in independent Thoroughbred, Icelandic Horse, German Holstein, Quarter Horse and South German Draft Horse families. In four of the seven families, only one single white horse, presumably representing the founder for each of the four respective mutations, was available for genotyping. The newly reported mutations comprise two frameshift mutations (c.1126_1129delGAAC; c.2193delG), two missense mutations (c.856G>A; c.1789G>A) and three splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). White phenotypes in horses show a remarkable allelic heterogeneity. In fact, a higher number of alleles are molecularly characterized at the equine KIT gene than for any other known gene in livestock species.     

One step closer to fixing association studies: evidence for age- and gender-specific allele frequency variations and deviations from Hardy-Weinberg expectations in controls

Association studies are the most powerful method available for identifying modest gene effects in complex disorders, but they often produce inconsistent results. With the rapidly growing SNP databases, haplotype maps and high throughput genotyping, the use of association studies is expected to increase; therefore, it is critical and timely that the problems with study design are identified and fixed. We questioned if unrecognized allele and genotype frequency variations in controls could be responsible for some of the inconsistent association findings. We performed a population genetic study of apolipoprotein E (APOE) and cytochrome P450 2D6 (CYP2D6) in 1,748 individuals ranging in age from newborns to centenarians. Although APOE and CYP2D6 are two of the most commonly used candidate genes, this is the first study to examine age- and gender-specific frequency distributions over the entire age spectrum, using a large, ethnically and geographically uniform population. We found significant, previously unrecognized variations in APOE allele frequencies, and deviations from Hardy-Weinberg expectations in CYP2D6 genotype frequencies starting at birth. The allele frequency variations within controls were larger than some reported case-control differences. We demonstrate that unrecognized frequency fluctuations in controls are a serious and potentially common confounder whose impact on association studies has not been appreciated, and one that can be addressed with proper study design. We recommend that population genetic studies be performed on commonly used candidate markers and that rigorous standards be applied for case-control matching.     

Revised transthyretin Ile 122 allele frequency in African-Americans

The transthyretin (TTR) Ile 122 variant is associated with cardiac amyloidosis in individuals of African descent. To determine the prevalence of the allele encoding TTR Ile 122 in African-Americans, we have used PCR and restriction analysis to test DNA from African-Americans from various geographic areas, and found an allele frequency of 66/3376 (0.020), which is higher than the value we previously reported in a much smaller pilot study. Our data indicate that this TTR variant is present at equal frequency in African-Americans throughout the U.S., and suggest that this mutation may be a common, often unrecognized cause of cardiac disease in African-Americans. Received: 23 January 1996     

Characterization of mutations in patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.     

Importance of allele frequency estimates in epidemiological studies

This study addresses the issue of appropriate allelic frequency estimates in epidemiological studies. Reasons for imprecise estimate of allele frequency may be population stratification, and lack of power of many published studies to define true allele frequencies in the general population. As an example of the lack of power of epidemiological studies, we plot the frequency of GSTM1 deletion versus sample size for the 79 studies from the GSEC pooled analysis. The estimate of allele frequency derived from small groups of controls deviates more from the true frequency than the estimate derived from larger studies. We discuss the possible consequences of not properly defining allele frequencies in the population. This may reflect on the conduct of association studies, on assessment of the effects of multigenic mechanisms, and on the determination of genetic diversity.     

A new allele of glucose phosphate isomerase in dogs

Summary. A new variant of red cell glucose phosphate isomerase (GPI), designated GPI-C, was observed in the chow-chow breed of dog. GPI-C could be separated from the previously reported dog GPI variants (A and B), both by starch gel electrophoresis and by isoelectric focusing (pH 3–10). Family data supported the hypothesis that GPI-C is controlled by a third codominant allele ( GPI C ). GPI C occurred as a rare allele in the Dutch chow-chow population.     

Power for genetic association studies with random allele frequencies and genotype distributions

One of the first and most important steps in planning a genetic association study is the accurate estimation of the statistical power under a proposed study design and sample size. In association studies for candidate genes or in fine-mapping applications, allele and genotype frequencies are often assumed to be known when, in fact, they are unknown (i.e., random variables from some distribution). For example, if we consider a diallelic marker with allele frequencies of 0.5 and 0.5 and Hardy-Weinberg proportions, the three genotype frequencies are often assumed to be 0.25, 0.50, and 0.25, and the statistical power is calculated. Unfortunately, ignoring this source of variation can inflate the estimated power of the study. In the present article, we propose averaging the estimates of power over the distribution of the genotype frequencies to calculate the true estimate of power for a fixed allele frequency. For the usual situation, in which allele frequencies in a population are not known, we propose placing a prior distribution on the allele frequency, taking advantage of any available genotype information. This Bayesian approach provides a more accurate estimate of power. We present examples for quantitative and qualitative traits in cohort studies of unrelated individuals and results from an extensive series of examples that show that ignoring the uncertainty in allele frequencies can inflate the estimated power of the study. We also present the results from case-control studies and show that standard methods may also overestimate power. As discussed in this article, the approach of fixing allele frequencies even if they are not known is the common approach to power calculations. We show that ignoring the sources of variation in allele frequencies tends to result in overestimates of power and, consequently, in studies that are underpowered. Software in C is available at http://www.ambrosius.net/Power/.     

A second KRT71 allele in curly coated dogs

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant ¹⁵¹Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein‐changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C‐terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs.     

A new allele of glucose phosphate isomerase in dogs

A new variant of red cell glucose phosphate isomerase (GPI), designated GPI-C, was observed in the chow-chow breed of dog. GPI-C could be separated from the previously reported dog GPI variants (A and B), both by starch gel electrophoresis and by isoelectric focusing (pH 3-10). Family data supported the hypothesis that GPI-C is controlled by a third codominant allele (GPI C). GPI C occurred as a rare allele in the Dutch chow-chow population.