Polymorphic variations influencing fetal hemoglobin levels: association study in beta-thalassemia carriers and in normal individuals of Portuguese origin.

Three major loci have been associated with HbF levels, including -158C/T (XmnI) at HBG2 promoter region, and several polymorphisms at BCL11A intron-2 and HBS1L-MYB (HMIP) intergenic region. Mutations in the KLF1 gene were recently associated with increased HbF levels. This study aims to evaluate whether genetic variability at these loci influences HbF levels in ß-thalassemia carriers and in normal individuals of Portuguese origin. Sixty five ß-thalassemia carriers, HbF levels ranging from 0.2% to 9.5%, and 60 individuals with normal hematological parameters, HbF levels ranging from 0.2% to 7.4%, were selected for this study. In ß-thal carriers linear regression models revealed a strong statistical significant association for HBG2 (XmnI) rs7482144 (ß=0.455; P=5.858×10(-7)), and nominal significance for BCL11A rs766432 (ß=0.215; P=0.029) and HMIP rs9399137 (ß=0.209; P=0.011). In normal individuals, a case (HbF>2%; n=15) vs. control (HbF<1.7%; n=45) model, showed nominal significant associations for BCL11A SNPs rs11886868 (OR=4; P=0.001), rs766432 (OR=3.7; P=0.002) and rs7606173 (OR=0.36; P=0.032). KLF1 rs3817621 was not found associated with HbF levels. Our results suggest that in Portuguese ß-thal carriers the HBG2 XmnI polymorphism is strongly associated with HbF levels. In normal individuals, BCL11A polymorphisms, but not HMIP or HBG2 (XmnI) loci, are nominally associated with HbF expression.

Distribution of the - 13910C>T polymorphism in the general population of Portugal and in subjects with gastrointestinal complaints associated with milk consumption.

BACKGROUND: The - 13910C>T polymorphism has been associated with lactase persistence (LP) in European populations. AIM: To assess - 13910C>T genotypes across Portugal and in adult individuals with unspecific gastrointestinal complaints associated with milk consumption. SUBJECTS AND METHODS: This study genotyped - 13910C>T in the general population from Northern (n = 64), Central (n = 70) and Southern (n = 65) Portugal and in 40 subjects with gastrointestinal symptoms. Additionally, the concordance was evaluated between breath-hydrogen test and - 13910C>T genotypes in 65 samples. RESULTS: An overall frequency of 0.349 for the LP - 13910*T allele was estimated in the general population, with a noticeable decrease in the South (0.269) compared with North (0.383) and Centre (0.393). Among the symptomatic group, the frequency of the - 13910*T allele (0.363) was not significantly different from the general population. A 94% concordance was found between the breath-hydrogen and the molecular tests. CONCLUSIONS: This study suggests that (i) the distribution of the LP polymorphism is not uniform across the country, (ii) genotyping - 13910C>T is a good diagnostic tool for lactase status in the Portuguese population and (iii) self-reported gastrointestinal complaints are not good predictors of the LP status, implying that a significant part of those complaints may not be related to hypolactasia.

Testing hierarchical levels of population sub-structuring: the Azores islands (Portugal) as a case study.

The Azores archipelago (Portugal) is formed by nine islands whose relative positions define them as three geographical groups: Eastern (S. Miguel and Sta. Maria), Central (Terceira, Faial, Pico, Graciosa and S. Jorge) and Western (Flores and Corvo). Using the father's surname of 187,398 individuals living on the nine Azorean Islands, a population analysis based on inter-island relationship and hierarchical organization was conducted. The relation between islands was investigated using summary statistics, analysis of molecular variance (AMOVA) as well as graphical methods. When the values of heteronymy were contrasted with values of gene diversity based on haplogroup frequencies of the Y chromosome, it was possible to verify that Graciosa and Sta. Maria appeared to have the least diverse populations, and that Flores, despite its smaller population size and geographical isolation, has considerably higher levels of diversity. As for inter-island relationships, the difficulty of directly interpreting summary statistics values was evidenced. The AMOVA revealed that only 0.77% of the variation in surnames can be attributed to among-island variation, a value that, although small, can be considered significant. Application of Malécot's model revealed that geographic distance has an important impact in the genetic structure of the archipelago. Monmonier's maximum-difference algorithm demonstrated that the most isolated island of the archipelago appears to be Graciosa, followed by the islands of the Western group and by Sta. Maria. After integrating values of summary statistics with results from AMOVA and graphical methods, a more accurate genetic profile of the Azores, highly supported by genetic data, has emerged.

Mutations and haplotype diversity in 70 Portuguese G6PD-deficient individuals: an overview on the origin and evolution of mutated alleles.

G6PD deficiency mutational profile and haplotype diversity using 6 RFLPs (FokI/PvuII/BspHI/PstI/BclI/NlaIII) and a (CTT)(n) microsatellite, were investigated in 70 G6PD-deficient Portuguese individuals. All but one G6PD A-(376G/202A) variants (44/45) have a single haplotype (+/+/-/+/-/+/195). G6PD Betica(376G/968C) alleles (n=10) have a single RFLP haplotype (+/-/-/+/-/+) and 4 different (CTT)(n) repeats. Age estimates based on microsatellite variation suggest that Betica mutation arose 900 generations ago. G6PD SantaMaria(376G/542T) allele was found on haplotype (+/-/-/+/-/+/201) and 10 G6PD variants on RFLP haplotypes (-/-/+/+/-/-), (-/-/+/+/-/+) and (-/-/+/+/+/+).

Y-chromosome diversity in central Portugal reveals signatures of ancient maritime expansions.

The genetic diversity of human populations in Portugal results from several different demographic events that occurred in distinct prehistorical and historical periods. The main objective of this study was to examine if patterns of Y-chromosome diversity explained by ancient maritime Mediterranean expansions can be observed in Coimbra district (central-west region of Portugal). A total of 125 male DNA samples were typed for 16 Y-SNPs and eight Y-STRs using standard molecular methodologies. Thirteen different haplogroups were identified, being the typical Western European haplogroup R1b1b2-M269 the most common (57.6%), followed by J-M304 (16%) and E1b1b1-M35 (12%). Haplogroup J-M304, whose origin maps to the Middle East, showed significant differences when comparisons were made between Coimbra district and the innermost region of Beira Interior (p = 0.022), in the same geographic area of the country, but not with the Portuguese regions of Alentejo (p = 0.165) and Algarve (p = 0.254), with known evidences of Mediterranean influence. Y-STR analysis revealed in Coimbra district several haplotypes previously associated with ancient maritime Mediterranean expansions. These findings suggest that maritime routes in the first millennium B.C. may have been important for introduction of new male lineages in Central Portugal.

G6PD deficient alleles and haplotype analysis of human G6PD locus in São Tomé e Príncipe (West Africa).

A population sample from São Tomé e Príncipe (West Africa) was screened for the G6PD-deficient variants A- (376G/202A), Betica (376G/968C), and Santa Maria (376G/542T). G6PD locus haplotype diversity was also investigated using six intragenic RFLPs (FokI, PvuII, BspHI, PstI, BclI, NlaIII) and a (CTT)n microsatellite 18.61 kb within the G6PD locus. The estimated frequencies of the G6PD*B normal allele, the G6PD*A variant (376G), and the G6PD*A- allele were 0.698, 0.194, and 0.108, respectively. G6PD variants Betica and Santa Maria were not found. Similar levels of microsatellite diversity were found on variants G6PD*B and G6PD*A (H = 0.61 and 0.68, respectively), indicating a similar age for both alleles. All G6PD*A- alleles share the RFLP-microsatellite haplotype ++(-)+(-)+/195, the same haplotype described in nearly all the *A-alleles from sub-Saharan, Mexican Mestizo, and Portuguese populations, consistent with a single and recent origin of the G202A mutation on this *A haplotype.

Genetic structure of Flores island (Azores, Portugal) in the 19th century and in the present day: evidence from surname analysis.

The island of Flores is the most westerly of the Azores archipelago (Portugal). Despite its marked geographic isolation and reduced population size, biodemographic and genetic studies conducted so far do not support the idea that its population constitutes a genetic isolate. In this study we conducted a surname analysis of the Flores population for two time periods: the second half of the 19th century and the present day. Our main purposes were (1) to biodemographically and genetically characterize the island, taking into account the strong reduction in population observed from the middle of the 19th century to the present day; and (2) to analyze the influence that the effective population size and geographic distance have on the genetic structure of populations. For both periods analyzed, all indicators of diversity revealed a high level of surname diversity. Our results are in accordance with the diversity estimates obtained from both monoparental genetic markers located in the Y chromosome and frequencies of mtDNA haplogroups. Contrary to what could be expected, considering the strong reduction of population in the last 150 years, we observed that diversity was maintained and that microdifferentiation decreased. Both observations support a higher openness of parishes as a consequence of the increase in communication routes. From the first to the second period analyzed, a change in surname composition is evident, although the more frequent surnames in Flores are almost the same for both periods and some of them are reported to be surnames present in the first settlers of Flores. This result testifies to the impact of founders on the present-day gene pool of Flores island and allows us to infer that the genetic characterization of the present-day population of Flores could provide reliable information about the history of the peopling of the Azores.

Understanding differences between phylogenetic and pedigree-derived mtDNA mutation rate: a model using families from the Azores Islands (Portugal).

We analyzed the control region of the mitochondrial DNA (mtDNA) from maternally related individuals originating from the Azores Islands (Portugal) in order to estimate the mutation rate of mtDNA and to gain insights into the process by which a new mutation arises and segregates into heteroplasmy. Length and/or point heteroplasmies were found at least in one individual of 72% of the studied families. Eleven new point substitutions were found, all of them in heteroplasmy, from which five appear to be somatic mutations and six can be considered germinal, evidencing the high frequency of somatic mutations in mtDNA in healthy young individuals. Different values of the mutation rate according to different assumptions were estimated. When considering all the germinal mutations, the value of the mutation rate obtained is one of the highest reported so far in family studies. However, when corrected for gender (assuming that the mutations present in men have the same evolutionary weight of somatic mutations because they will inevitably be lost) and for the probability of intraindividual fixation, the value for the mutation rate obtained for HVRI and HVRII (0.2415 mutations/site/Myr) was in the upper end of the values provided by phylogenetic estimations. These results indicate that the discrepancy, that has been reported previously, between the human mtDNA mutation rates observed along evolutionary timescales and the estimations obtained using family pedigrees can be minimized when corrections for gender proportions in newborn individuals and for the probability of intraindividual fixation are introduced. The analyses performed support the hypothesis that (1) in a constant, tight bottleneck genetic drift alone can explain different patterns of heteroplasmy segregation and (2) in neutral conditions, the destiny of a new mutation is strictly related to the initial proportion of the new variant. Another important point arising from the data obtained is that, even in the absence of a paternal contribution of mtDNA, recombination may occur between mtDNA molecules present in an individual, which is only observable if it occurs between mtDNA types that differ at two or more positions.

Determination of human caucasian mitochondrial DNA haplogroups by means of a hierarchical approach.

In this paper we propose a hierarchical approach that allows the screening of mitochondrial DNA (mtDNA) haplogroups in populations that have essentially West Eurasian mtDNA backgrounds but that could have some non-West Eurasian contributions. To develop and validate this scheme, we used data on 18 coding region polymorphisms (17 analyzed by RFLP analysis and 1 by sequencing) and sequences of hypervariable segment I (HVSI) of the mtDNA control region from the Azores Islands (Portugal) population. The proposed scheme allows the characterization of almost all West Eurasian and African major clusters by means of RFLPs. Furthermore, the scheme includes information on situations in which sequencing is pertinent to defining a particular haplogroup. The validity of the scheme is ensured by (1) using relatively stable polymorphic positions, (2) screening more than one position to define a specific haplogroup, and (3) typing confirmatory positions. Dubious samples can be resolved by sequencing. The robustness of this approach was assessed by sequencing all samples for HVSI, taking advantage of the previously established relationships between RFLPs and control region sequence polymorphisms. The use of this hierarchical approach avoids the screening of unnecessary control region polymorphisms and therefore results in a more rapid and cost-efficient screening than one in which all polymorphic positions are analyzed. Even if this approach leads to a lower level of phylogeographic resolution than the sequencing of all samples, it allows us to define population movements on a continental level and can be applied, unlike sequencing all samples, with a low cost in any laboratory.