Near Fixation of 374l Allele Frequencies of the Skin Pigmentation Gene SLC45A2 in Africa

The L374F polymorphism of the SLC45A2 gene, encoding the membrane-associated transporter protein that plays an important role in melanin synthesis, has been suggested to be associated with skin color in human populations. In this study, the detailed distribution of the 374f and 374l alleles has been investigated in 2,581 unrelated subjects from 36 North, East, West, and Central African populations. We found once more the highly significant (p < 0.001) correlation coefficient (r = 0.957) cline of 374f frequencies with degrees of latitude in European and North African populations. Almost all the African populations located below 16° of latitude are fixed for the 374l allele. Peul, Toucouleur, and Soninké populations have 374l allele frequencies of 0.06, 0.03, and 0.03, respectively.     

A Decreasing Gradient of 374F Allele Frequencies in the Skin Pigmentation Gene SLC45A2, from the North of West Europe to North Africa

The 374F mutation in the SLC45A2 gene, encoding the membrane-associated transporter protein (MATP) that plays an important role in melanin synthesis, has been suggested to be associated with skin color in Caucasians. In this study, the detailed distribution of the 374F allele has been investigated in 2063 unrelated subjects from 18 European and three North African populations. The highest allele frequency is observed in Denmark (0.980), and the lowest frequencies are observed in Tunisia (0.610) and in Morocco (0.691). A significant latitudinal cline in 374F allele frequencies was observed, ranging from the north of West Europe to North Africa (r = 0.869). The results confirm that the distribution of the 374F allele may reflect the ultraviolet radiation level and can be associated with skin color variation in these regions.     

Association of polymorphisms in the Chr18q11.2 locus with tuberculosis in Chinese population

A GWAS study has reported that two single nucleotide polymorphisms (SNPs) were associated with predisposition to tuberculosis (TB) in African populations. These two loci represented the long-waited GWAS hits for TB susceptibility. To determine whether these two SNPs are associated with TB in Chinese population, we attempted an replication in a cohort of over one thousand Chinese TB patients and 1,280 healthy controls using melting temperature shift allele-specific genotyping analysis. We found that only SNP rs4331426 was significantly associated with TB in Chinese population (p = 0.011). However, the effect was opposite. The G allele of the SNP in Chinese population is a protective allele (OR = 0.62, 95 % CI 0.44–0.87), while it was the risk allele for African population (OR = 1.19, 95 % CI 1.12–1.26). No significance was found for SNP rs2335704. The results provided an independent support for a role in susceptibility to TB for SNP rs4331426. However, it also indicated that direct predisposition element to TB and the association effects may vary across ethnic groups.     

Isolation of a major genetic interaction associated with an extreme phenotype using assorted F2 populations in rice

Detection of quantitative trait loci (QTLs) is dependent on the materials used in the analysis, as different combinations of parental materials may lead to different outcomes in QTLs for the same trait. On the other hand, an extreme phenotype associated with a given trait implies the potential involvement of a particular allele in various allelic interactions. A genetic factor associated with such an extreme phenotype may frequently be identified from various genetic populations consisting of different parental combinations. In this study, we attempted to uncover the genetic factor associated with extremely early heading date in rice, using various F2 populations. Heading date in rice has been characterized by at least 19 QTLs, from which 12 genes have been identified. A58, a rice strain with an extremely early heading date, is adapted to Hokkaido, the northernmost limit of rice cultivation. Six F2 populations derived from crosses of A58 with six other strains displayed a range of heading dates. Genotyping using 19 QTL markers indicated that the A58 allele of the Ghd7 locus was present in most F2 individuals exhibiting extremely early heading dates. This analysis also demonstrated that when the wild-type Ehd1 allele was present, the Ghd7 allele from A58 accelerated floral induction. The results of this study demonstrate that assorted F2 populations are valuable materials for comprehensive genotyping to explore major genetic factors for extreme phenotypes, and that this methodology is broadly applicable to other unknown traits.     

Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing

Individual genotyping of single nucleotide polymorphisms (SNPs) remains expensive, especially for linkage disequilibrium mapping strategies involving high-throughput SNP genotyping. On one hand, current methods may suit scientific and laboratory needs in regard to accuracy, reproducibility/robustness, and large-scale application. On the other hand, a cheaper and less time-consuming alternative to individual genotyping is the use of SNP allelefrequencies determined in DNA pools. We have developed an accurate and reproducible protocol for allele frequency determination using Pyrosequencing technology in large genomic DNA pools (374 individuals). The measured correlation (R2) in large DNA pools was 0.980. In the context of disease-associated SNPs studies, we compared the allele frequencies between the disease (e.g., type 2 diabetes and obesity) and control groups detected by either individual genotyping or Pyrosequencing of DNA pools. In large pools, the variation between the two methods was 1.5 +/- 0.9%. It may be concluded that the allele frequency determination protocol could reliably detect over 4% differences between populations. The method is economical in regard to amounts of DNA, PCR, and primer extension reagents required. Furthermore, it allows the rapid determination of allelefrequency differences in case/control groups for association studies and susceptibility gene discovery in complex diseases.     

Association of the OCA2 Polymorphism His615Arg with Melanin Content in East Asian Populations: Further Evidence of Convergent Evolution of Skin Pigmentation

The last decade has witnessed important advances in our understanding of the genetics of pigmentation in European populations, but very little is known about the genes involved in skin pigmentation variation in East Asian populations. Here, we present the results of a study evaluating the association of 10 Single Nucleotide Polymorphisms (SNPs) located within 5 pigmentation candidate genes (OCA2, DCT, ADAM17, ADAMTS20, and TYRP1) with skin pigmentation measured quantitatively in a sample of individuals of East Asian ancestry living in Canada. We show that the non-synonymous polymorphism rs1800414 (His615Arg) located within the OCA2 gene is significantly associated with skin pigmentation in this sample. We replicated this result in an independent sample of Chinese individuals of Han ancestry. This polymorphism is characterized by a derived allele that is present at a high frequency in East Asian populations, but is absent in other population groups. In both samples, individuals with the derived G allele, which codes for the amino acid arginine, show lower melanin levels than those with the ancestral A allele, which codes for the amino acid histidine. An analysis of this non-synonymous polymorphism using several programs to predict potential functional effects provides additional support for the role of this SNP in skin pigmentation variation in East Asian populations. Our results are consistent with previous research indicating that evolution to lightly-pigmented skin occurred, at least in part, independently in Europe and East Asia.     

Evidence for recent positive selection at the human AIM1 locus in a European population

Two missense polymorphisms (E272K and L374F) of the AIM1 locus,encoding a melanocyte differentiation antigen, were shown tohave a clear association with human ethnicities. These two nonpathogenicsingle nucleotide polymorphisms (SNPs) may be associated withhuman pigmentation variation. In this study, we investigatedsequence variation in the coding region and exon-flanking sequenceand found low genetic variation only in subjects of Europeandescent. All four statistical tests applied to the 7.55-kb regionsurrounding the L374F polymorphism detected statistically significantdeviations from selective neutrality in Europeans. In addition,haplotype analysis revealed that one haplotype carrying 374Fwas overrepresented in this population, and the low rate ofvariation, with some features of selective sweeps, was shownto be statistically significant. These results suggest thatpositive selection recently has been acting or has acted onat least this region of the melanogenic gene and that an advantageoushaplotype spread rapidly in Europe.     

DNA Pooling: a tool for large-scale association studies

DNA pooling is a practical way to reduce the cost of large-scale association studies to identify susceptibility loci for common diseases. Pooling allows allele frequencies in groups of individuals to be measured using far fewer PCR reactions and genotyping assays than are used when genotyping individuals. Here, we discuss recent developments in quantitative genotyping assays and in the design and analysis of pooling studies. Sophisticated pooling designs are being developed that can take account of hidden population stratification, confounders and inter-loci interactions, and that allow the analysis of haplotypes.     

Replication of Type 2 Diabetes Candidate Genes Variations in Three Geographically Unrelated Indian Population Groups

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p<5.5E−04) with T2D susceptibility in combined population. Interestingly, SNP rs7903146 of the TCF7L2 gene passed the genome wide significance threshold (combined P value = 2.05E−08) in the studied populations. We also observed the association of rs7903146 with blood glucose (fasting and postprandial) levels, supporting the role of TCF7L2 gene in blood glucose homeostasis. Further, we noted that the moderate risk provided by the independently associated loci in combined population with Odds Ratio (OR)<1.38 increased to OR = 2.44, (95%CI = 1.67–3.59) when the risk providing genotypes of TCF7L2, HHEX, ENPP1 and FTO genes were combined, suggesting the importance of gene-gene interactions evaluation in complex disorders like T2D.     

ORAI1 genetic polymorphisms associated with the susceptibility of atopic dermatitis in Japanese and Taiwanese populations

Atopic dermatitis is a chronic inflammatory skin disease. Multiple genetic and environmental factors are thought to be responsible for susceptibility to AD. In this study, we collected 2,478 DNA samples including 209 AD patients and 729 control subjects from Taiwanese population and 513 AD patients and 1027 control subject from Japanese population for sequencing and genotyping ORAI1. A total of 14 genetic variants including 3 novel single-nucleotide polymorphisms (SNPs) in the ORAI1 gene were identified. Our results indicated that a non-synonymous SNP (rs3741596, Ser218Gly) associated with the susceptibility of AD in the Japanese population but not in the Taiwanese population. However, there is another SNP of ORAI1 (rs3741595) associated with the risk of AD in the Taiwanese population but not in the Japanese population. Taken together, our results indicated that genetic polymorphisms of ORAI1 are very likely to be involved in the susceptibility of AD.     

Undetected genotyping errors cause apparent overtransmission of common alleles in the transmission/disequilibrium test

The transmission/disequilibrium test (TDT), a family-based test of linkage and association, is a popular and intuitive statistical test for studies of complex inheritance, as it is nonparametric and robust to population stratification. We carried out a literature search and located 79 significant TDT-derived associations between a microsatellite marker allele and a disease. Among these, there were 31 (39%) in which the most common allele was found to exhibit distorted transmission to affected offspring, implying that the allele may be associated with either susceptibility to or protection from a disease. In 27 of these 31 studies (87%), the most common allele appeared to be overtransmitted to affected offspring (a risk factor), and, in the remaining 4 studies, the most common allele appeared to be undertransmitted (a protective factor). In a second literature search, we identified 92 case-control studies in which a microsatellite marker allele was found to have significantly different frequencies in case and control groups. Of these, there were 37 instances (40%) in which the most common allele was involved. In 12 of these 37 studies (32%), the most common allele was enriched in cases relative to controls (a risk factor), and, in the remaining 25 studies, the most common allele was enriched in controls (a protective factor). Thus, the most common allele appears to be a risk factor when identified through the TDT, and it appears to be protective when identified through case-control analysis. To understand this phenomenon, we incorporated an error model into the calculation of the TDT statistic. We show that undetected genotyping error can cause apparent transmission distortion at markers with alleles of unequal frequency. We demonstrate that this distortion is in the direction of overtransmission for common alleles. Therefore, we conclude that undetected genotyping errors may be contributing to an inflated false-positive rate among reported TDT-derived associations and that genotyping fidelity must be increased.     

The association of apolipoprotein E gene polymorphisms with cerebral palsy in Chinese infants

Apolipoprotein E (APOE, protein; ApoE, gene) is a lipid transport protein abundantly present in brain cells. Previous studies have suggested that there is an association between genetic variants of ApoE and susceptibility to cerebral palsy (CP). The purpose of this study was to explore whether the ApoE gene is involved in the etiology of CP in the Chinese population. In this study, 350 CP patients and 242 healthy control children were recruited. Genomic DNA was prepared from venous blood and all five single nucleotide polymorphisms (SNPs) in ApoE (rs769446, rs405509, rs121918399, rs429358, and rs190853081) were detected by the MassARRAY platform-based genotyping approach. The SHEsis program was used to analyze the genotyping data, and we systemically analyzed the association of the ApoE SNPs with different subtypes of CP. No significant association was detected between the e4 identified by the C allele of rs429358 and CP, but there were significant differences in allelic frequencies between the CP patients and controls at rs769446 (P = 0.005, P = 0.025 after Bonferroni correction), as well as between the CP patients with preterm birth (<34 gestational weeks) and controls at rs769446 (P = 0.001, P = 0.005 after Bonferroni correction). A haplotype consisting of the five SNPs rs769446(C), rs405509(C), rs121918399(C), rs429358(T), and rs190853081(G) was associated with a decreased risk of CP (P = 0.002 after Bonferroni correction). However, we found no significant association between any of the other three SNPs and CP based on different subgroup analyses. This study provides the first evidence that ApoE gene polymorphisms are a potential risk factor for CP in the Chinese population.     

Genetic variation and structure of the endangered Lady Fern Athyrium viridescentipes based on ubiquitous genotyping

To clarify the genetic status and provide effective information for the conservation of Athyrium viridescentipes, a critically endangered fern species with only 103 individuals remaining in the wild, we conducted ubiquitous genotyping to determine the genotypes of all remnant individuals of the target species. We analyzed the genetic variation of the 103 known individuals in four populations by using 13 microsatellite loci. The genotypes of single spores from a sporophytic individual were also determined in order to reveal the breeding system of this species. The level of allelic variation in A. viridescentipes was significantly lower than that of closely related Athyrium species. The genetic composition of the four populations was rather similar. Sixty-nine individuals (67%) possessed an identical pattern in the allele combinations at 13 microsatellite loci. The mean pairwise F (ST) among four populations was 0.018. The segregated pattern of alleles, determined by single-spore genotyping, revealed that allelic recombination occurs through meiosis. The results indicate that this species contains a low level of genetic variation, has low population differentiation, and maintains populations by sexual reproduction. These findings could lead to more effective conservation programs, the selection of the most appropriate individuals for ex situ conservation efforts, and separate management of extant populations.     

Straightforward inference of ancestry and admixture proportions through ancestry-informative insertion deletion multiplexing

Ancestry-informative markers (AIMs) show high allele frequency divergence between different ancestral or geographically distant populations. These genetic markers are especially useful in inferring the likely ancestral origin of an individual or estimating the apportionment of ancestry components in admixed individuals or populations. The study of AIMs is of great interest in clinical genetics research, particularly to detect and correct for population substructure effects in case-control association studies, but also in population and forensic genetics studies. This work presents a set of 46 ancestry-informative insertion deletion polymorphisms selected to efficiently measure population admixture proportions of four different origins (African, European, East Asian and Native American). All markers are analyzed in short fragments (under 230 basepairs) through a single PCR followed by capillary electrophoresis (CE) allowing a very simple one tube PCR-to-CE approach. HGDP-CEPH diversity panel samples from the four groups, together with Oceanians, were genotyped to evaluate the efficiency of the assay in clustering populations from different continental origins and to establish reference databases. In addition, other populations from diverse geographic origins were tested using the HGDP-CEPH samples as reference data. The results revealed that the AIM-INDEL set developed is highly efficient at inferring the ancestry of individuals and provides good estimates of ancestry proportions at the population level. In conclusion, we have optimized the multiplexed genotyping of 46 AIM-INDELs in a simple and informative assay, enabling a more straightforward alternative to the commonly available AIM-SNP typing methods dependent on complex, multi-step protocols or implementation of large-scale genotyping technologies.     

Analysis of Association Between the MUC4 g.8227C>G Polymorphism and Production Traits in Italian Heavy Pigs Using a Selective Genotyping Approach

In pigs, susceptibility to enterotoxigenic Escherichia coli (ETEC) K88 strains (locus F4bcR) is determined by a dominant allele, with the recessive allele determining resistance. The susceptible allele also appeared to be associated with higher growth rate even with discordant results. A single nucleotide polymorphism (SNP) in exon 7 of the mucin 4 (MUC4) gene (DQ848681:g.8227C>G), shown to be in close linkage disequilibrium with the F4bcR locus, has been used as marker to identify susceptible pigs, substituting invasive villous adhesion tests. We herein analyzed this SNP in Italian local breeds and applied a selective genotyping approach in Italian Large White, Italian Landrace, and Italian Duroc comparing allele frequency distribution in groups of pigs with extreme estimated breeding values (EBV) for average daily gain (ADG) and backfat thickness (BFT) to evaluate if this marker is associated with these traits. Allele G (associated with susceptibility to ETEC) was associated with higher ADG and BFT in Italian Large White (P?=?6.66E-04 and P?=?0.012, respectively) and higher ADG in Italian Landrace (P?=?7.23E-12). This polymorphism was poorly informative in Italian Duroc. Antagonistic associations of the MUC4?g.8227C>G alleles on susceptibility to ETEC and growth performances evidence the complexity of applying marker assisted selection in pig breeding.     

Estimating effects of rare haplotypes on failure time using a penalized Cox proportional hazards regression model

Background

POU5F1 expression is required to maintain stem cell pluripotency and for primordial germ cells to retain proliferative capability in embryonic development. Recent evidence suggests that POU5F1 may also be a testicular germ cell carcinoma (TGCC) oncogene, and POU5F1 variation may influence TGCC risk. As an important first step to a genetic association study, we sought to identify all common sequence variants in an 11.3 kb region containing POU5F1, and to describe the linkage disequilibrium patterns, using DNA from individuals of African-descent (AD) and European-descent (ED).

Results

A higher number of polymorphisms was observed in the AD (n = 102) versus ED (n = 82) population. Among the 41 observed haplotypes, 21 (51%) and 12 (29%) were unique to the AD and ED populations, respectively, while 8 (20%) were observed in both. The number of tagging polymorphisms necessary to explain at least 80% of common variation (minor allele frequency ≥ 0.10) due to the remaining untyped polymorphisms was 17 for an AD and 10 for an ED population, providing a 4.0- and 7.0-fold gain in genotyping efficiency for characterizing nucleotide variation, respectively.

Conclusion

POU5F1 is highly polymorphic, however a smaller subset of polymorphisms can tag the observed genetic variation with little loss of information.     

Tagging Single Nucleotide Polymorphisms in the IRF1 and IRF8 Genes and Tuberculosis Susceptibility

Genes encoding IRF1 and IRF8 protein have been proposed as candidate tuberculosis susceptibility genes. In order to elucidate whether the IRF1 and IRF8 variants were associated with tuberculosis susceptibility, we conducted a case-control study consisting of 495 controls and 452 ethnically matched cases with tuberculosis in a Chinese population. Seven haplotype tagging single-nucleotide polymorphisms (tagSNPs) (rs2057656; rs2706381; rs2070724; rs2070721; rs2549008; rs2549007; rs2706386) from HapMap database were analyzed, which provided an almost complete coverage of the genetic variations in the IRF1 gene. Fifteen tagSNPs (rs12924316; rs182511; rs305080; rs2292980; rs925994; rs424971; rs16939967; rs11117415; rs4843860; rs9926411; rs8064189; rs12929551; rs10514611; rs1044873; rs6638) were observed in the IRF8 gene. All these tagSNPs were genotyped by SNPstream genotyping and SNaPshot typing. None of the seven tagSNPs was individually associated with tuberculosis in the IRF1 gene. In the IRF8 gene, interestingly, we found that three tagSNPs (rs925994 and rs11117415 located in the intron region; rs10514611 located in the 3′UTR) were associated with risk of tuberculosis after Bonferroni correction. Per allele OR was 1.75 (95% CI 1.35∼2.27, P = 0.002), 4.75 (95% CI 2.16∼10.43, P = 0.002) and 3.39 (95% CI 1.60∼7.20, P = 0.015) respectively. Luciferase reporter gene assay showed that the construct that contained the non-risk allele C of rs10514611 showed significantly higher luciferase activity than did the risk T allele (P<0.01), which implied rs10514611 was a potential functional SNP site. Our results indicated that the IRF8 gene might participate in genetic susceptibility to tuberculosis in a Chinese population.     

Identification of QTL associated with resistance to bacterial spot race T4 in tomato

Bacterial spot of tomato (Solanum lycopersicum L.), caused by several Xanthomonas sp., is a serious but difficult disease to control by chemical means. Development of resistance has been hindered by emergence of races virulent to tomato, by the quantitative inheritance of resistance, and by a low correlation between seedling assays and resistance in the field. Resistance to multiple races, including race T4, has been described in the S. lycopersicum var. cerasiformae accession PI 114490. We used molecular markers to identify associations with quantitative trait loci (QTL) in an elite inbred backcross (IBC) population derived from OH 9242, PI 114490 and Fla. 7600, a breeding line with tomato accession Hawaii 7998 (H7998) in its pedigree. Race T4 resistance has also been described in the advanced breeding lines Fla. 8233, Fla. 8517, and Fla. 8326, and a selective genotyping approach was used to identify introgressions associated with resistance in segregating progeny derived from crosses with these lines. In the IBC population, loci on chromosomes 11 and 3, respectively, explained as much as 29.4 and 4.8% of resistance variation. Both these loci were also confirmed by selective genotyping: PI 114490 and H7998 alleles on chromosome 11 each provided resistance. The PI 114490 allele on chromosome 3 was confirmed in the Fla. 8517 population, and an allele of undetermined descent was confirmed at this locus in the Fla. 8326 population. A chromosome 12 allele was associated with susceptibility in the Fla. 8517 population. Additional loci contributing minor effects were also implicated in the IBC population or by selective genotyping. Selection for the major QTL in a marker-directed phenotyping approach should significantly improve the efficiency of breeding for resistance to bacterial spot race T4, although as yet undetected QTL would be necessary to carry out strict marker assisted selection.     

When phenotypes do not match genotypes--unexpected phenotypic diversity and potential environmental constraints in Icelandic stickleback

Divergent lateral plate phenotypes in stickleback represent one of only a few cases known, where a single gene underlies the phenotype under divergent selection between different habitats. However, the selection pressures leading to the repeated loss of lateral plates in freshwater are still not well understood. By genotyping 838 individuals from 9 independently colonized lakes and 1 marine population in Iceland, we found 1) that only in some lakes are phenotypes associated with the expected genotype and 2) that the independent repeated occurrence of a rarely described plate phenotype is expressed in the absence of an allele that is usually associated with this phenotype. This suggests that either other genes such as modifiers might be under divergent selection between lakes or that lateral plate expression in these populations is restricted due to environmental constraints.