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.

SummaryThe 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.All the analyses performed support the idea that, in the whole group of islands, the majority of mtDNA lineages originated from the Iberian Peninsula, mainly from Portugal (mainland). However contributions from other European populations, especially from Northern Europe, cannot be disregarded. The values obtained for the various diversity parameters in the Azores archipelago indicate that the Azorean population, as a whole, does not exhibit the typical characteristics of an isolated population. The analysis of genetic data by groups of islands showed that the Western group exhibited particular features. The distribution of haplogroups in the Western group is very atypical, being significantly different from what is observed in the Eastern and Central groups. Furthermore, the diversity values are, in general, lower than those observed in other populations used for comparison. African haplogroups were found in all the groups of islands. Therefore the presence of Moorish and African slaves on the islands, as reported in historical sources, is supported by the mtDNA genetic data, especially in the Eastern group. The presence of Jews in the Central group is also supported by the mtDNA data. Neither historical nor genetic data (phylogeography of mtDNA) supports the idea of a differential settlement history for the Western group; however, it is represented in the phylogenies as an isolated branch. The effect of genetic drift, induced by the reduced population size since peopling occurred, has led to a very atypical distribution of haplogroups/haplotypes in this group of islands.We cannot ignore the influence of biodemographic and genetic processes, namely founder effect, genetic drift, migration, and even recent mutational events in the mtDNA lineages of the Azorean populations. Nevertheless, a great part of the variation in the Azorean mtDNA can be explained by the settlement history.

Summary. 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 A3G 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 272A3G 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 272A3G 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 1456C3T had a mild condition.

Summary.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 0 splice donor site (GT) mutations on intron 8 [IVS8(þ2)T → G] and intron 10 [IVS10(þ1)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(þ1)G → C or IVS8(þ2)T → G.Keywords: pyruvate kinase deficiency, human PK-LR gene mutations, haemolytic anaemia, splice site, Portugal.Pyruvate kinase (PK EC 2.7.1.40) deficiency is the most common enzyme abnormality in the erythrocyte glycolytic pathway causing hereditary nonspherocytic haemolytic anaemia (HNSHA). The PK-deficient anaemia is transmitted as an autosomal recessive disorder with a heterogenous clinical phenotype, ranging from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring exchange transfusion. Splenectomy is effective in decreasing haemolysis in most patients. Heterozygotes are usually asymptomatic, with a 30-50% decrease in the PK activity levels (Miwa et al, 1979). PK deficiency was first described by Valentine et al (1961) and since then more than 400 cases have been reported, with the biochemical characteristics of the residual enzyme differing markedly between patients. The International Committee for Standardization in Haematology (ICSH) established the standard methods for enzymatic studies; however, the low activity, different stability and the hybrid tetramers formed in the compound heterozygous causes difficulty in characterization of the PK variants (Miwa et al, 1979). A molecular approach, identifying the gene mutations, enables a better characterization of the PK-deficient patients with a more precise genotype/phenotype correlation.It is also useful for diagnosis among transfusion-dependent patients with chronic haemolytic anaemia and prenatal diagnosis.Four PK isoenzymes have been described in mammals, M1, M2, L and R, which are tissue specific and differ in their enzymatic properties (Imamura & Tanaka, 1972). In mammals there are only two different PK genes: the PK-M gene that codes for the isoenzymes M1 (muscle and brain) and M2 (fetal and most adult tissues) using alternative RNA splicing (Noguchi et al, 1986) and the PK-LR gene, that codes for the L-type (liver) and R-type (erythrocyte) isoenzymes using two different tissue-specific promoters (Noguchi et al, 1987). More than 70 mutations have been identified in the coding and flank...

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.