Presence Of Rare Alleles Research Articles

DNA barcodes of Saudi Arabian birds: Implications for species identification and diversity analysis

ObjectivesDNA barcoding using cytochrome c oxidase I (COI) gene is an effective tool for species identification with additional power of measuring molecular diversity and phylogenetic inference. In this study, we evaluated the performance of COI gene sequences for species identification and molecular diversity analysis of some Saudi Arabian birds. MethodsWe sequenced the 694 base pair segment of COI gene from 5 samples of white cheeked bulbul (Pycnonotus leucogenys), 4 samples of black scrub robin (Cercotrichas podobe), and 2 samples of crested lark (Galerida cristata), all belonged to the same Order. We also included all the COI sequences available in the GenBank for these birds with the aim of studying molecular diversity across geographies. For species identification, we enriched our dataset by including COI barcodes of other Saudi Arabian bird species, including Arabian partridge (Alectoris melanocephala), houbara bustard (Chlamydotis undulata macqueenii), green bee-eater (Merops orientalis), laughing dove (Streptopelia senegalensis), namaqua dove (Oena capensis) and collared dove (Streptopelia decaocto), representing different genera. ResultsWhite-cheeked bulbul from Saudi Arabia showed more diversity than the same birds from Iraq. The mean nucleotide difference and nucleotide diversity were 5.20 and 0.009 respectively with as Tajima’s D score of 1.6549 indicating the scarcity of rare alleles. The specimens of black scrub robin from Saudi Arabia and Djibouti showed mixed phylogeny with only two segregating sties and a near zero Tajima’s D score indicating no major selection. Out of 18 samples of crested lark, 13 birds from 4 different geographical regions showed identical sequences, while 3 birds from Russia and 1 from Cyprus grouped in a separate cluster and 1 crusted lark from Djibouti differed from all. The mean nucleotide difference and nucleotide diversity were 0.6993 and 0.0014, while a negative Tajima’s D of −1.1956 indicated the presence of rare alleles. Phylogenetic analysis of 9 different species of Saudi Arabian birds showed the discriminatory power of COI barcodes as all the different species grouped separately in specific clusters. ConclusionsCOI barcodes are not only indispensable tools for species identification but can also be used for analyzing molecular diversity and phylogenetic inference.

Effect of a common missense variant in LIPA gene on fatty liver disease and lipid phenotype: New perspectives from a single-center observational study.

Lysosomal acid lipase deficiency (LAL‐D) is an autosomal recessive disease characterized by hypoalphalipoproteinemia, mixed hyperlipemia, and fatty liver (FL) due to mutations in LIPAse A, lysosomal acid type (LIPA) gene. The rs1051338 single‐nucleotide polymorphism (SNP) in LIPA gene, in vitro, could adversely affect the LAL activity (LAL‐A). Nonalcoholic fatty liver disease (NAFLD) is often associated with metabolic syndrome, and the diagnosis requires the exclusion of excess of alcohol intake and other causes of hepatic disease. The aim of the study was to evaluate the impact of rs1051338 rare allele on lipid phenotype, severity of FL, and LAL‐A in patients suffering from dyslipidemia associated with NAFLD. We selected 74 subjects with hypoalphalipoproteinemia or mixed hyperlipemia and evaluated transaminases, liver assessment with controlled attenuation parameter (CAP), LAL‐A, rs1051338 SNP genotype. The presence of rare allele caused higher levels of triglycerides and hepatic transaminase and lower levels of high‐density lipoprotein cholesterol (HDL‐C). Multivariate analysis highlighted independent association between rare allele and FL severity in subjects with NAFLD. The rs1051338 SNP may modulate FL severity and atherogenic dyslipidemia in patients suffering from NAFLD.

True Colors: Commercially-acquired morphological genotypes reveal hidden allele variation among dog breeds, informing both trait ancestry and breed potential.

Direct-to-consumer canine genetic testing is becoming increasingly popular among dog owners. The data collected therein provides intriguing insight into the current status of morphological variation present within purebred populations. Mars WISDOM PANELTM data from 11,790 anonymized dogs, representing 212 breeds and 4 wild canine species, were evaluated at genes associated with 7 coat color traits and 5 physical characteristics. Frequencies for all tested alleles at these 12 genes were determined by breed and by phylogenetic grouping. A sub-set of the data, consisting of 30 breeds, was divided into separate same-breed populations based on country of collection, body size, coat variation, or lineages selected for working or conformation traits. Significantly different (p ≤ 0.00167) allele frequencies were observed between populations for at least one of the tested genes in 26 of the 30 breeds. Next, standard breed descriptions from major American and international registries were used to determine colors and tail lengths (e.g. genetic bobtail) accepted within each breed. Alleles capable of producing traits incongruous with breed descriptions were observed in 143 breeds, such that random mating within breeds has probabilities of between 4.9e-7 and 0.25 of creating undesirable phenotypes. Finally, the presence of rare alleles within breeds, such as those for the recessive black coloration and natural bobtail, was combined with previously published identity-by-decent haplotype sharing levels to propose pathways by which the alleles may have spread throughout dog breeds. Taken together, this work demonstrates that: 1) the occurrence of low frequency alleles within breeds can reveal the influence of regional or functional selection practices; 2) it is possible to visualize the potential historic connections between breeds that share rare alleles; and 3) the necessity of addressing conflicting ideals in breed descriptions relative to actual genetic potential is crucial.

Functional status and severity of osteoarthritis in elderly is associated to the polymorphism of TNFA gene

BackgroundOsteoarthritis (OA) is a major musculoskeletal disease with high prevalence in the elderly. The study of genetic polymorphisms of inflammatory mediators involved in OA may contribute to the elucidation of the complex pathophysiology of this disease and identification of susceptibility individuals.AimThis study aimed to evaluate the association between polymorphism at tumor necrosis factor alpha gene (SNP - 308 G/A TNFA) with presence, severity and functional status of osteoarthritis in elderly.MethodsThis study was characterized as case-control and encompassed 257 physically independent elderly (Mean Age: 68.55 ± 5.2; Minimum age: 60 and Maximum age: 82) were recruited. After this selection, the groups were divided in: 92 elderly individuals with osteoarthritis (case group) and 165 without the disease (control group).MethodsThe individuals were genotyped by the TaqMan real-time PCR system. The subjects were classified based on the degree of radiological impairment according to the criteria of Kellgren-Laurence and regarding functional impairment using the WOMAC and LEQUESNE questionnaires.ResultsTNFA gene polymorphic individuals (subjects harboring allele A) are more affected by OA (χ2 = 8.7, p = 0.003), once they have major radiological lesion both in hip (Fisher-Freeman-Halton Test = 3.9, p = 0.04) and knee (Fisher-Freeman-Halton Test = 4.0, p = 0.04) as well as worse functional status assessed by the Lequesne questionnaire (Mann-Whitney, p = 0.04). At the multivariate analysis, after adjustment for age, gender, body mass index, the presence of rare allele for TNFA (allele A) increases the susceptibility to OA development [OR: 1.87 (95% CI: 1.1–3.2)].ConclusionWe conclude that the SNP − 308 G/A of TNFA gene may affect osteoarthritis susceptibility, severity and functional status of individuals with osteoarthritis.

Correlation of follicle-stimulating hormone receptor gene Asn 680 Ser (rs6166) polymorphism with female infertility.

Background and Aims:Female infertility is a complex multifactorial, and polygenic disease associated with genetic factors plays an essential role in its formation and follicle development, oocyte maturation, and steroidogenesis regulation in the ovary. The aim here is too study the genetic association between follicle-stimulating hormone receptor (FSHR) Asn680Ser; (rs6166) gene polymorphism with female Infertility in our population.Methods:In this prospective case-control study, we enrolled 106 infertile and 164 unrelated healthy control individuals. Genomic DNA was extracted from the 5 ml of venous blood using the modified salting-out method. A polymerase chain reaction-amplified exon 10 of FSHR and purified PCR products were sequenced on an ABI 3730XL DNA sequencer. The data were analyzed statistically.Results:We found that the presence of rare allele ”G” and heterozygous and common homozygous genotypes significantly increased the risk of female infertility. No significant change in the FSHR 191756 G >A genotype frequency was observed, regardless of chromosomal integrity. The genotype frequency distribution of locus 680 was consistent with the Hardy–Weinberg Equilibrium (HWE) in both groups (P > 0.05).Conclusion:No significant differences were found in allelic variants frequency and genotype distribution between each category of subjects when analyzing the FSHR SNPs in the exonic region (P value >0.05). FSHR Asn680Ser polymorphisms and female infertility (P > 0.05). Variations in FSHR gene have an essential influence on ovarian function and can account for several defects of female fertility. FSHR Asn680Ser (rs6166) gene polymorphism is associated with female infertility and can be used as a relevant molecular biomarker to identify the risk of infertility in our population. This finding can be important for disease pathogenesis.

Assessment of genetic structure of wild populations of mrigal carp,Cirrhinus cirrhosusby microsatellite DNA markers

The genetic structure of mrigal carp, Cirrhinus cirrhosus was analysed using six microsatellite DNA markers (MFW1, MFW2, MFW17, Bgon22, Barb54, and Bgon17) to detect genetic variations among fish samples from the three rivers, the Halda, the Padma, and the Jamuna in Bangladesh. All the populations showed polymorphism at all loci except Bgon17. Genetic richness of the Halda and Padma populations was indicated by the presence of rare alleles, two in Halda at MFW2 (160 bp) and MFW17 (201 bp), and one in Padma at Bgon22 (96 bp). The highest average number of allele (2.8) was observed in the Halda followed by the Padma (2.6) and the Jamuna (2.4). The observed heterozygosity (Hₒ) was highest (0.400) in the Padma followed by the Halda (0.383) and the Jamuna (0.377). The highest (0.0257) population differentiation (FST) value was observed between the Halda and Padma populations and the lowest (0.0143) between the Padma and Jamuna populations. The highest gene flow (Nₘ) was recorded between the Padma and Jamuna populations (17.1756) and the lowest between the Halda and Padma populations (9.4832). The highest (0.037) genetic distance (D) was observed between the Halda and Padma populations and the lowest (0.0195) between the Jamuna and Padma populations. The results indicated a low level of genetic variation regarding allelic richness and heterozygosity among wild stocks of C. cirrhosus and indicated it might be advisable to take necessary measures for the conservation and enhancement of C. cirrhosus genetic resources.

Conservation genetics of an ex situ population of Primula reinii var. rhodotricha, an endangered primrose endemic to Japan on a limestone mountain

Primula reinii var. rhodotricha is a perennial herb endemic to the limestone slope of Mt. Buko, located approximately 50 km northwest of central Tokyo, Japan. In recent years, its natural population size has decreased markedly due to limestone mining, and this species has been assigned to the ‘Critically Endangered (CR)’ category on the latest Japanese Red List. Although a remnant population of this species has been protected by a mining company outside their historical distribution range on Mt. Buko, the ex situ conservation of this endangered plant has been difficult because of insufficient low seed production. The genetic status of ex situ P. reinii var. rhodotricha and related species were investigated to develop an effective conservation plan for this species. Microsatellite analysis indicated that the ex situ population harbors lower genetic diversity than sister taxa, providing molecular evidence for the recent critical status designation of the ex situ population, whereas the presence of rare alleles may imply further potential for seed reproduction by outcrossing. Therefore, an appropriate propagation strategy that considers genetic diversity is needed for restoration and recovery of this critically endangered ex situ primrose population.

Addressing Beacon re-identification attacks: quantification and mitigation of privacy risks.

The Global Alliance for Genomics and Health (GA4GH) created the Beacon Project as a means of testing the willingness of data holders to share genetic data in the simplest technical context—a query for the presence of a specified nucleotide at a given position within a chromosome. Each participating site (or “beacon”) is responsible for assuring that genomic data are exposed through the Beacon service only with the permission of the individual to whom the data pertains and in accordance with the GA4GH policy and standards.While recognizing the inference risks associated with large-scale data aggregation, and the fact that some beacons contain sensitive phenotypic associations that increase privacy risk, the GA4GH adjudged the risk of re-identification based on the binary yes/no allele-presence query responses as acceptable. However, recent work demonstrated that, given a beacon with specific characteristics (including relatively small sample size and an adversary who possesses an individual’s whole genome sequence), the individual’s membership in a beacon can be inferred through repeated queries for variants present in the individual’s genome.In this paper, we propose three practical strategies for reducing re-identification risks in beacons. The first two strategies manipulate the beacon such that the presence of rare alleles is obscured; the third strategy budgets the number of accesses per user for each individual genome. Using a beacon containing data from the 1000 Genomes Project, we demonstrate that the proposed strategies can effectively reduce re-identification risk in beacon-like datasets.

Inherited Risk Factors for Hemorrhagic Complications in the 1st Trimester of Pregnancy

Purpose: The purpose of the study was to assess the role of genetic risk factors in the development of retrochorial hematoma. Materials and methods: Genotyping of four polymorphisms of the folate cycle (MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G) and eight polymorphisms of hemostasis system (F2 G20210A, F5 G1691A, F7 G10976A, F13 G103T, FGB G-455A, ITGA2 C807T, ITGB3 T1565S, SERPINE1-675 5G/4G) was performed to identify genetic risk factors of retrochorial hematoma accompanied by bleeding in the I trimester of pregnancy. The study was conducted among 238 pregnant women with retrochorial hematoma and 67 pregnant women without retrochorial hematoma. Results: The risk of retrochorial hematoma increases in the presence of rare alleles of polymorphic loci of proconvertin F7 gene (sensitivity 62,61 (56,12-68,77), specificity 16,42 (8,49-27,48), PPV 72,68 (66,04-78,66)) and fibrin stabilizing factor F13 gene (sensitivity 73,11 (67-78,63), specificity 5,97 (1,65-14,59), PPV 73,42 (67,31-78,93)), while the presence of polymorphic alleles of these genes in homozygous state is the most unfavorable combination. The chance of developing a retrochorial hematoma increases 5.5 times with the combination of F7 G10976A (genotype G/A and A/A) and F 13 G103T (genotype G/T and T/T). Conclusion: Since the genotype G/A or A/A of G10976A F7 gene polymorphism and genotype G/T or T/T of G103T F13 polymorphism are associated with a predisposition to hypocoagulation.

Diversity and spatial genetic structure of a natural population of Theobroma speciosum (Malvaceae) in the Brazilian Amazon

The quantification of genetic diversity and intrapopulation spatial genetic structure (SGS) of tree species are important aspects for in and ex situ conservation practices. In this study we seek to understand the importance of conservation areas by quantifying the genetic diversity and the spatial genetic structure of a natural population of Theobroma speciosum. Within this population, 49 adults and 51 subadults were genotyped for five microsatellite loci. The results showed that adults and subadults have similar levels of genetic diversity and inbreeding (adults: A= 10.4, Ae = 10.3, F= 0.68, subadults: A= 10.6, Ae= 10.6, F= 0.57). Genetic diversity was spatially structured within the population, and the results suggest that near-neighbor trees up to a distance of 70 m are likely related. SGS is likely the result of short-distance seed dispersal, the short-distance range of pollinators, and infrequent breaches of the self-incompatible mating system. Considering the high demographic density of the species and size of the study area, as well as the high average number of alleles per locus and the presence of rare alleles, we believe that the study population is an excellent resource for in situ genetic conservation of T. speciosum. The study area is also a useful resource for collecting germplasm for ex situ conservation and seed collection, either for breeding programs used in the restoration of degraded areas or forest improvement.

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima’D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

Heterozygosity increases microsatellite mutation rate.

Whole genome sequencing of families of Arabidopsis has recently lent strong support to the heterozygote instability (HI) hypothesis that heterozygosity locally increases mutation rate. However, there is an important theoretical difference between the impact on base substitutions, where mutation rate increases in regions surrounding a heterozygous site, and the impact of HI on sequences such as microsatellites, where mutations are likely to occur at the heterozygous site itself. At microsatellite loci, HI should create a positive feedback loop, with heterozygosity and mutation rate mutually increasing each other. Direct support for HI acting on microsatellites is limited and contradictory. I therefore analysed AC microsatellites in 1163 genome sequences from the 1000 genomes project. I used the presence of rare alleles, which are likely to be very recent in origin, as a surrogate measure of mutation rate. I show that rare alleles are more likely to occur at locus-population combinations with higher heterozygosity even when all populations carry exactly the same number of alleles.

Genetic variability of Scots pine (Pinus sylvestris L.) in maternal regions of provenance

Abstract The analysis of chosen selected Scots Pine (Pinus sylvestris L.), populations representing different seed regions: 107 (Międzyzdroje), 305 (Woziwoda), 206 (Strzałowo), 208 (Białowieża), 504 (Bolesławiec), 606 (Józefów) were performed using 10 isoensyme markers: Gdh (E.C.1.4.1.2), Sdh-A, Sdh-B (E.C.1.1.1.25), Pgd-B (E.C.1.1.1.44), Mdh-A, Mdh-C (E.C.1.1.1.37), Got-A, Got-B, Got-C (E.C.2.6.1.1), Dia-C (E.C.1.8.1.4). There were calculated following genetic parameters: allelic frequencies, observed and expected heterozygosities, and Wright’s fixation indexes. In populations, the results of analysis indicated presence of rare alleles. In all study populations, the average effective number of alleles per locus was 1.46 and was lower than expected number of alleles per locus of 1.93. The results for the effective number of alleles of the population per locus were as follows: Strzałowo and Białowieża 1.54, Bolesławiec 1.48, Jozefów 1.44, Świnoujście 1.42, Woziwoda 1.35. Average observed heterozygosity in the studied populations was calculated at the level of 0.26 and it was lower than the expected heterozygosity at 0.28. For populations, the level of heterozygosities were as follow: Strzałowo 0.35, Bolesławiec and Białowieża 0.25, Józefów 0.27, Świnoujście 0.23, Woziwoda 0.26. In particular loci level of heterozygosity was different, as the most heterozygous Mdh-C locus was estimated, while minimum Got-C. Significant differences in allele frequency of Hardy-Weinberg deviation equilibrium were found in 10 cases: Strzałowo (Got-C), Bolesławiec (Got-B), Białowieża (Got-B, Gdh), Józefów (Mdh-C), Międzyzdroje (Got-B, Got-C, Mdh-C), Woziwoda (Got-C, Gdh). All studied populations had a lower effective number of alleles per locus (Ne) comparing to the observed number of alleles (Na). Wright’s fixation indices were negative for populations: Strzałowo (-0.09), Józefów (-0.03), Międzyzdroje (-0.01) and positive for: Białowieża (0.12), Woziwoda and Bolesławiec (0.07). The extremely high Wright’s fixation index (0.12) was observed for Białowieża population.

Glacial refugia and modern genetic diversity of 22 western North American tree species.

North American tree species, subspecies and genetic varieties have primarily evolved in a landscape of extensive continental ice and restricted temperate climate environments. Here, we reconstruct the refugial history of western North American trees since the last glacial maximum using species distribution models, validated against 3571 palaeoecological records. We investigate how modern subspecies structure and genetic diversity corresponds to modelled glacial refugia, based on a meta-analysis of allelic richness and expected heterozygosity for 473 populations of 22 tree species. We find that species with strong genetic differentiation into subspecies had widespread and large glacial refugia, whereas species with restricted refugia show no differentiation among populations and little genetic diversity, despite being common over a wide range of environments today. In addition, a strong relationship between allelic richness and the size of modelled glacial refugia (r(2) = 0.55) suggest that population bottlenecks during glacial periods had a pronounced effect on the presence of rare alleles.

Association of interleukin 1 receptor antagonist (IL1RN) gene polymorphism with recurrent pregnancy loss risk in the North Indian Population and a meta-analysis.

An appropriate ratio of interleukin 1 beta to interleukin 1 receptor antagonist (IL1Ra) is required for successful pregnancy. Our objective was to study the genetic association between IL1RN variable numbers of tandem repeat (VNTR) polymorphism and recurrent pregnancy loss (RPL). To analyze the association between IL1RN VNTR allele and RPL, we investigated the IL1RN VNTR polymorphism in 136 RPL patients and in 200 healthy control women. Meta-analysis on this polymorphism was conducted to support our findings. PCR based approach was used to analyze IL1RN VNTR polymorphism and it was further confirmed by sequencing. Systematic review and meta-analysis was done using electronic database (Pub-Med, Google Scholar and Ovid) up to February 27, 2013. This meta-analysis was assessed by comprehensive meta-analysis software version 2. For meta-analysis 549 cases and 1,450 controls were included. The frequency of IL1RN genotype 2/2 was significantly higher in RPL compared to control group (AORs 3.10, 95 % CI 1.58-6.11, p = 0.001). The presence of rare allele also increased the risk of RPL significantly (ORs 1.63, 95 % CI 1.16-2.29, p = 0.004). The meta-analysis stratified by ethnicity showed that individuals with allele 2 had increased risk of RPL (OR 1.29, 95 % CI 1.04-1.61, p = 0.01), in Asians population by using fixed model. However the data of the present study clearly suggests that IL1RN VNTR polymorphism is a genetic risk factor for pregnancy loss in the study population.

Trait linked microsatellite marker based diversity analysis for yield attributing traits in some bengal landraces of rice (Oryza sativaL.)

Forty one diverse rice genotypes collected from West Bengal have been studied to assess genetic diversity using five SSR markers linked with QTL of yield attributing traits. Genotypes were grown at the Gayeshpur Regional Research Station (New Alluvial Zone), Bidhan Chandra Krishi Viswavidyalaya, West Bengal, India. Results shown that Polymorphic pattern were generated by all five SSR primers which altogether produced 14 reproducible alleles. Most of the markers generated two reproducible alleles whereas RM-3 and RM-16 generated three and four alleles respectively. The percentage of highest frequency alleles among the markers ranging from 70.73 % (RM-16) to 95.12 % (RM-5) where as PIC values varied from 0.1488 (RM-3) to 0.314 (RM-130). RM-16 generated maximum number of alleles although PIC value was maximum in case of RM-130. These two markers could be used for diversity analysis related to yield attributes in rice. The dendogram revealed 7 clusters at a cut-off similarity coefficient level of 0.64. At the similarity coefficient level of 0.35 and 0.62 Kumargore and Oradhan respectively were distinct where as rest of the genotypes grouped into 5 major clusters. On the basis of yield performance as well as presence of rare alleles of the used markers, Tulaipanji, Kumargore, Butadhan, Oradhan can be considered further as parental material for development of high yielding varieties of rice.

Association of maternal and fetal MTHFR A1298C polymorphism with the risk of pregnancy loss: a study of an Indian population and a meta-analysis

To study the genetic association between methylenetetrahydrofolate reductase (MTHFR) A1298 polymorphism and recurrent pregnancy loss (RPL). Prospective case-control study, systematic review, and meta-analysis using an electronic database up to July 27,2012. Meta-analysis of four studies on RPL and three studies on spontaneously aborted embryos, including the present study. A total of 129 RPL patients and 202 healthy control women with successful pregnancy were analyzed including 40 spontaneously aborted embryos and 40 aborted embryos as control samples. For meta-analysis, 1,080 case and 709 control subjects were included of RPL and 375 case and 384 control samples of spontaneously aborted embryos. Blood was collected by peripheral venous punctures, and spontaneously aborted embryos were collected by curettage or manual vacuum aspiration. Meta-analysis was done on the basis of heterogeneity of the studies. Genotyping was done by polymerase chain reaction (PCR)-restriction-fragment-length polymorphism (RFLP). DNA sequencing was used to ascertain PCR-RFLP results. Age-adjusted odds ratios were calculated by logistic regression analysis. Meta-analysis on this polymorphism was conducted to support our findings. We found that presence of rare allele "C" and heterozygous and rare homozygous genotypes significantly increased the risk of RPL. No significant change in the fetal MTHFR A1298C genotype frequency was observed, regardless of chromosomal integrity. Meta-analysis of A1298C polymorphism on both RPL and in spontaneously aborted embryos showed significantly increased risk in the carriers of AC and CC genotypes. The data of the present study clearly suggests that MTHFR A1298C polymorphism is a genetic risk factor for pregnancy loss.

UGT1A1, UGT1A6 and UGT1A7 genetic analysis: repercussion for irinotecan pharmacogenetics in the São Miguel Island Population (Azores, Portugal).

Glucuronidation reactions, catalyzed by uridine-diphosphate glucuronosyltransferase (UGT) enzymes, constitute a detoxification process that adds glucuronic acid to endogenous and exogenous compounds, aiding their excretion. UGT1A proteins have been implicated as risk factors for both the development of cancer and adverse drug effects. Here, we assess the genome of 469 individuals from São Miguel Island (Azores, Portugal) in order to determine the frequencies of polymorphisms and haplotypes in UGT1A1, UGT1A6, and UGT1A7, the co-occurrence of reduced enzyme activity UGT1A variants related to irinotecan toxicity, and to calculate the extent of linkage disequilibrium (LD) in the genomic region encompassing these genes. Allelic analysis disclosed the presence of rare alleles - UGT1A1*36 and UGT1A1*37--only found in individuals of African descent, and UGT1A7*4. These alleles confirm our previous results on the São Miguel Island genetic background. We identified five different genotypes in UGT1A1 and UGT1A6 and nine in UGT1A7. Haplotype analysis showed that three haplotypes constituted approximately 80% of the allelic variants. Interestingly, haplotype 3 (UGT1A1*28-UGT1A6*2-UGT1A7*3), with a frequency of 0.235, gathers the three alleles encoding the low-function UGT isoforms. Additionally, LD indicates a strong interaction between functional polymorphisms related to the alteration of the UGT enzyme activity. In summary, the results demonstrate a high variability of alleles and haplotypes, which have important roles in modifying expression and activity of UGTs. The data presented here could improve the understanding of the predisposition to cancers and susceptibility to the adverse effects of irinotecan in the São Miguel Island population.

Genetic Polymorphisms of the Wnt Receptor LRP5 are Differentially Associated with Trochanteric and Cervical Hip Fractures

Epidemiological studies suggest that cervical and trochanteric hip fractures have different pathogenesis. We tested the hypothesis that genetic factors have different influences on both types of fractures. Ten polymorphisms of genes known to play an important role in skeletal homeostasis [estrogen receptor alpha (ESR1), aromatase (CYP19A1), type I collagen (COL1A1), and lipoprotein receptor-related protein 5 (LRP5)] were analyzed in 471 Spanish patients with fragility hip fractures. Two polymorphisms of the LRP5 gene (rs7116604 and rs3781600) were associated with the type of fracture (P=0.0085 and 0.0047, respectively). The presence of rare alleles at each locus was associated with trochanteric fractures over cervical fractures (OR=1.7 in individuals with at least one rare allele at rs7116604 or rs3781600 loci in comparison with the common homozygotes). Considering individuals bearing the four common alleles as reference, the OR for trochanteric fractures was 1.6 in those with one or two rare alleles and 7.5 in those with three or four rare alleles (P for trend=0.0074), which is consistent with an allele-dosage effect. There were no significant differences in the frequency distributions of the ESR1, CYP19A1, and COL1A1 genotypes between trochanteric and cervical fractures in either the original group or an extended group of 818 patients. These results suggest that LRP5 alleles influence the type of hip fractures. They support the view that different genetic factors are involved in cervical and trochanteric fractures, which should be taken into consideration in future genetic association studies.

Comparison of genetic association strategies in the presence of rare alleles

In the quest for the missing heritability of most complex diseases, rare variants have received increased attention. Advances in large-scale sequencing have led to a shift from the common disease/common variant hypothesis to the common disease/rare variant hypothesis or have at least reopened the debate about the relevance and importance of rare variants for gene discoveries. The investigation of modeling and testing approaches to identify significant disease/rare variant associations is in full motion. New methods to better deal with parameter estimation instabilities, convergence problems, or multiple testing corrections in the presence of rare variants or effect modifiers of rare variants are in their infancy. Using a recently developed semiparametric strategy to detect causal variants, we investigate the performance of the model-based multifactor dimensionality reduction (MB-MDR) technique in terms of power and family-wise error rate (FWER) control in the presence of rare variants, using population-based and family-based data (FAM-MDR). We compare family-based results obtained from MB-MDR analyses to screening findings from a quantitative trait Pedigree-based association test (PBAT). Population-based data were further examined using penalized regression models. We restrict attention to all available single-nucleotide polymorphisms on chromosome 4 and consider Q1 as the outcome of interest. The considered family-based methods identified marker C4S4935 in the VEGFC gene with estimated power not exceeding 0.35 (FAM-MDR), when FWER was kept under control. The considered population-based methods gave rise to highly inflated FWERs (up to 90% for PBAT screening).