Augusto Abade

A tale of two islands: population history and mitochondrial DNA sequence variation of Bioko and São Tomé, Gulf of Guinea

The hypervariable segment I of the control region of the mtDNA was sequenced in 45 unrelated individuals from Bioko and 50 from São Tomé, two islands in the Gulf of Guinea that have had very different settlement patterns: Bioko was colonized around 10000 BP, while São Tomé was first settled by the Portuguese, who brought African slaves to the island. Two different patterns of sequence variation are evident and are also clearly a consequence of their very different demographic histories. The Bubi present a low genetic diversity and it is likely that the island was colonized by a small number of individuals with small later migration. São Tomeans might be considered a subset of a mainland African population relocated to the island. They present high genetic diversity with a high number of sequences being shared with many continental populations. This study, with knowledge of the population history in island populations, strengthens the genetic approach to unravel past demographic events.

Genetic Structure and Origin of Peopling in The Azores Islands (Portugal): The View from mtDNA

The Azores islands (Portugal), uninhabited when discovered by Portuguese navigators in the fifteenth century, are located in the Atlantic Ocean 1500 km from the European mainland. The archipelago is formed by nine islands of volcanic origin that define three geographical groups: Eastern (S. Miguel and Sta. Maria), Central (Terceira, Faial, Pico, Graciosa and S. Jorge) and Western (Flores and Corvo). To improve the genetic characterisation of the Azorean population, and to clarify some aspects related to the history of settlement, a study of mtDNA was conducted in the population of the archipelago. The HVRI region was sequenced and specific RFLPs were screened in 146 samples obtained from unrelated individuals with Azorean ancestry (50 from the Eastern group, 60 from the Central group, and 37 from the Western group). Samples were classified into haplogroups based on the information obtained from both sequencing and RFLP analysis.

A new PKLR gene mutation in the R-type promoter region affects the gene transcription causing pyruvate kinase deficiency.

Mutations in the PKLR gene responsible for pyruvate kinase (PK)‐deficient anaemia are mainly located in the coding regions: 11 are in the splicing sites and, recently, three mutations have been described in the promoter region. We now report a novel point mutation A→G on nucleotide 72, upstream from the initiation codon of the PKLR gene, in four Portuguese PK‐deficient patients. This new regulatory mutation occurs within the most proximal of the four GATA motifs (GATA‐A element) in the R‐type promoter region. In two patients who were homozygous for this mutation, a semiquantitative reverse transcription polymerase chain reaction (PCR) procedure was used to evaluate the amount of R‐PK mRNA transcript in the reticulocytes. The mRNA level was about five times lower than in normal controls, demonstrating that the PKLR gene transcription is severely affected, most probably because the −72A→G point mutation disables the binding of the erythroid transcription factor GATA‐1 to the GATA‐A element. Supporting these data, the two patients homozygous for the −72A→G mutation had severe haemolytic anaemia and were transfusion dependent until splenectomy. Two other patients who were compound heterozygous for this mutation and the previously described missense mutation 1456C→T had a mild condition.

PK-LR gene mutations in pyruvate kinase deficient Portuguese patients.

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK‐LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G → C on exon 12 and two 5′ splice donor site (GT) mutations on intron 8 [IVS8(+2)T → G] and intron 10 [IVS10(+1)G → C]. Two previously described missense mutations, 1456C → T and 993C → A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G → C or IVS8(+2)T → G.

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. [End Page 431]

Evidence for population sub‐structuring in São Tomé e Príncipe as inferred from Y‐chromosome STR analysis

Seven Y‐chromosome STR loci, DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 have been analysed in population samples of Angolares, Forros and Tongas, three ethnic groups from the African archipelago of São Tomé e Príncipe (Gulf of Guinea). Complete typings were obtained for 103 chromosomes, which belonged to 79 different haplotypes. The mean heterozygosity per locus in the overall São Tomean sample was 0.566, with the highest value found among Forros and the lowest among Angolares. Angolares also showed the lowest level of haplotype diversity. On average, the mean pairwise difference between two random haplotypes from Angolares, Forros and Tongas was 4.69, 6.74 and 6.23 repeats, respectively. The genetic distances were found to be statistically significant between Angolares and Forros or Tongas. In accordance, AMOVA revealed that the percentage of variation attributable to differences among groups was only significant when we distinguished between Angolares and non‐Angolares. Globally, these results indicate that, with respect to the pool of male lineages of São Tomé e Príncipe, some genetic sub‐structuring does exist, basically determined by the Angolares ethnic group.

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 β-thalassemia carriers and in normal individuals of Portuguese origin. Sixty five β-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 β-thal carriers linear regression models revealed a strong statistical significant association for HBG2 (XmnI) rs7482144 (β=0.455; P=5.858×10(-7)), and nominal significance for BCL11A rs766432 (β=0.215; P=0.029) and HMIP rs9399137 (β=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 β-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.

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.

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 Betica376G/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 SantaMaria376G/542T allele was found on haplotype (+/–/–/+/–/+/201) and 10 G6PD variants on RFLP haplotypes (–/–/+/+/–/–), (–/–/+/+/–/+) and (–/–/+/+/+/+).

G6PD Deficient Alleles and Haplotype Analysis of Human G6PD Locus in São Tomé e Príncipe (West Africa)

ABSTRACT 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.