Molecular and computational analyses of genes involved in mannose 6-phosphate independent trafficking.

The newly-synthesized lysosomal enzymes travel to the trans-Golgi network (TGN) and are then driven to the acidic organelle. While the best-known pathway for TGN-to-endosome transport is the delivery of soluble hydrolases by the M6P receptors (MPRs), additional pathways do exist, as showed by the identification of two alternative receptors: LIMP-2, implicated in the delivery of ß-glucocerebrosidase; and sortilin, involved in the transport of the sphingolipid activator proteins prosaposin and GM2AP, acid sphingomyelinase and cathepsins D and H. Disruption of the intracellular transport and delivery pathways to the lysosomes may result in lysosomal dysfunction, predictably leading to a range of clinical manifestations of lysosomal storage diseases. However, for a great percentage of patients presenting such manifestations, no condition is successfully diagnosed. To analyse if, in this group, phenotypes could be determined by impairments in the known M6P-independent receptors, we screened the genes that encode for LIMP-2 and sortilin. No pathogenic mutations were identified. Other approaches will be needed to clarify whether sortilin dysfunction may cause disease.

Mirandese language and genetic differentiation in Iberia: a study using X chromosome markers.

BACKGROUND: In the Iberian Peninsula, the Mirandese dialect, spoken in Miranda do Douro (Portugal) close to the north-eastern border with Spain, has attracted much attention. Aim, subjects and methods: This study focuses on providing further insight into the connections forged between Miranda do Douro and regions in the nearby Province of Zamora. This is in order to better assess the extent to which such relations could have been detained by the current patterns of genetic diversity of the populations, whilst contributing to refining the knowledge on patterns of micro-differentiation within the Peninsula. The genetic characterization of both populations was performed through the analysis of X-chromosomal markers: X-STRs and X-indels. RESULTS AND CONCLUSION: The results showed that Miranda do Douro tended to present slightly lower levels of diversity in comparison to the other studied regions, which can be a discreet sign of isolation of that population over the years that might have led the way to the preservation of a language not spoken anywhere else in the country. The analysis of X-STRs particularly brought to light the presence of a subtle population sub-structure at the micro-geographical area encompassing the north-eastern border, which seems to portray the importance of the political border as a mechanism withholding gene flow between the two countries.

Molecular characterization of maple syrup urine disease patients from Tunisia.

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1ß and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene.

Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patients.

The functional activity of lysosomal enzymes sialidase, ß-galactosidase and N-acetylaminogalacto-6-sulfate-sulfatase in the cell depends on their association in a multienzyme complex with cathepsin A. Mutations in any of the components of this complex result in functional deficiency thereby causing severe lysosomal storage disorders. Here, we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in ß-galactosidase; gene GLB1) in Portuguese patients. We performed molecular studies of the PPGB, NEU1 and GLB1 genes in biochemically diagnosed Portuguese patients. Gene expression was determined and the effect of each mutation predicted at protein levels. In the NEU1 gene, we found three novel missense mutations (p.P200L, p.D234N and p.Q282H) and one nonsense mutation (p.R341X). In the PPGB gene, we identified two missense mutations, one novel (p.G86V) and one already described (p.V104M), as well as two new deletions (c.230delC and c.991-992delT) that give rise to non-functional proteins. We also present the first molecular evidence of a causal missense mutation localized to the cathepsin A active site. Finally, in the GLB1 gene, we found six different mutations, all of them previously described (p.R59H, p.R201H, p.H281Y, p.W527X, c.1572-1577InsG and c.845-846delC). Seven novel mutations are reported here, contributing to our knowledge of the mutational spectrum of these diseases and to a better understanding of the genetics of the lysosomal multienzymatic complex. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling.

Origin and spread of a common deletion causing mucolipidosis type II: insights from patterns of haplotypic diversity.

Mucolipidosis II (ML II alpha/beta), or I-cell disease, is a rare genetic disease in which activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent. GlcNAc-phosphotransferase is a multimeric enzyme encoded by two genes, GNPTAB and GNPTG. A spectrum of mutations in GNPTAB has been recently reported to cause ML II alpha/beta. Most of these mutations were found to be private or rare. However, the mutation c.3503_3504delTC has been detected among Israeli and Palestinian Arab-Muslim, Turkish, Canadian, Italian, Portuguese, Irish traveller and US patients. We analysed 44 patients who were either homozygous or compound heterozygous for this deletion (22 Italians, 8 Arab-Muslims, 1 Turk, 3 Argentineans, 3 Brazilians, 2 Irish travellers and 5 Portuguese) and 16 carriers (15 Canadians and 1 Italian) for three intragenic polymorphisms: c.-41_-39delGGC, c.18G>A and c.1932A>G as well as two microsatellite markers flanking the GNPTAB gene (D12S1607 and D12S1727). We identified a common haplotype in all chromosomes bearing the c.3503_3504delTC mutation. In summary, we showed that patients carrying the c.3503_3504delTC deletion presented with a common haplotype, which implies a common origin of this mutation. Additionally, the level of diversity observed at the most distant locus indicates that the mutation is relatively ancient (around 2063 years old), and the geographical distribution further suggests that it probably arose in a peri-Mediterranean region.

High prevalence of the GSTM3*A/B polymorphism in sub-Sarahan African populations

A 3-bp insertion/deletion polymorphism in intron 6 of GSTM3 (rs1799735, GSTM3*A/*B) affects the activity of the phase 2 xenobiotic metabolizing enzyme GSTM3 and has been associated with increased cancer risk. The GSTM3*B allele is rare or absent in Southeast Asians, occurs in 5-20 percent of Europeans but was detected in 80 percent of Bantu from South Africa. The wide genetic diversity among Africans led us to investigate whether the high frequency of GSTM3*B prevailed in other sub-Saharan African populations. In 168 healthy individuals from Angola, Mozambique and the São Tomé e Príncipe islands, the GSTM3*B allele was three times more frequent (0.74-0.78) than the GSTM3*A allele (0.22-0.26), with no significant differences in allele frequency across the three groups. We combined these data with previously published results to carry out a multidimensional scaling analysis, which provided a visualization of the worldwide population affinities based on the GSTM3 *A/*B polymorphism.

High prevalence of the GSTM3*A/B polymorphism in sub-Sarahan African populations.

A 3-bp insertion/deletion polymorphism in intron 6 of GSTM3 (rs1799735, GSTM3*A/*B) affects the activity of the phase 2 xenobiotic metabolizing enzyme GSTM3 and has been associated with increased cancer risk. The GSTM3*B allele is rare or absent in Southeast Asians, occurs in 5-20% of Europeans but was detected in 80% of Bantu from South Africa. The wide genetic diversity among Africans led us to investigate whether the high frequency of GSTM3*B prevailed in other sub-Saharan African populations. In 168 healthy individuals from Angola, Mozambique and the São Tomé e Príncipe islands, the GSTM3*B allele was three times more frequent (0.74-0.78) than the GSTM3*A allele (0.22-0.26), with no significant differences in allele frequency across the three groups. We combined these data with previously published results to carry out a multidimensional scaling analysis, which provided a visualization of the worldwide population affinities based on the GSTM3 *A/*B polymorphism.

Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III - identification of eight novel mutations.

Mucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. GlcNAc-phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (alpha, beta and gamma) encoded by two genes -GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610-1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT-PCR indicate the possible existence of feedback regulation mechanisms between alpha/beta and the gamma subunits.

In search of the pre- and post-neolithic genetic substrates in Iberia: evidence from Y-chromosome in Pyrenean populations.

The male-mediated genetic legacy of the Pyrenean population was assessed through the analysis of 12 Y-STR and 27 Y-SNP loci in a sample of 169 males from 5 main geographical areas in the Spanish Pyrenees: Cinco Villas (Western Pyrenees), Jacetania and Valle de Arán (Central Pyrenees) and Alto Urgel and Cerdaña (Eastern Pyrenees). In the Iberian context, the Pyrenean samples present some specificities, being characterizeded by a high proportion of chromosomes R1b1b2-M269 (including the usually uncommon R1b1b2d-SRY(2627) and R1b1b2c-M153 types) or I2a2-M26 and low proportions of other haplogroups. Our results indicate that an old pre-Neolithic substrate is preponderant in populations of the whole Pyrenean fringe. However, AMOVA revealed a high level of substructure within Pyrenean populations, partially explained by drift effects as well as by the signature of an ancient genetic differentiation between Western and Eastern Pyrenees.

A perspective on the history of the Iberian gypsies provided by phylogeographic analysis of Y-chromosome lineages.

The European Gypsies, commonly referred to as Roma, are represented by a vast number of groups spread across many countries. Although sharing a common origin, the Gypsy groups are highly heterogeneous as a consequence of genetic drift and different levels of admixture with surrounding populations. With this study we aimed at contributing to the knowledge of the Roma history by studying 17 Y-STR and 34 Y-SNP loci in a sample of 126 Portuguese Gypsies. Distinct genetic hallmarks of their past and migration route were detected, namely: an ancestral component, shared by all Roma groups, that reflects their origin in India (H1a-M82; approximately 17%); an influence from their long permanence in the Balkans/Middle-East region (J2a1b-M67, J2a1b1-M92, I-M170, Q-M242; approximately 31%); traces of contacts with European populations preceding the entrance in the Iberian Peninsula (R1b1c-M269, J2b1a-M241; approximately 10%); and a high proportion of admixture with the non-Gypsy population from Iberia (R1b1c-M269, R1-M173/del.M269, J2a-M410, I1b1b-M26, E3b1b-M81; approximately 37%). Among the Portuguese Gypsies the proportion of introgression from host populations is higher than observed in other groups, a fact which is somewhat unexpected since the arrival of the Roma to Portugal is documented to be more recent than in Central or East Europe.

Molecular analysis of mucopolysaccharidosis type IIIB in Portugal: evidence of a single origin for a common mutation (R234C) in the Iberian Peninsula.

Mucopolysaccharidosis type IIIB (Sanfilippo B disease) is a rare autosomal recessive disorder caused by defective alpha-N-acetylglucosaminidase (NAGLU). We examined the NAGLU gene in 11 MPS IIIB Portuguese patients, having identified five novel (M1K, W147X, G304V, S522P, and R533X) and four previously reported mutations (W168X, R234C, R565W and R643C). R234C attained the high prevalence of 32% of the mutated alleles. Because R234C had already been reported to be common in Spanish patients, a haplotypic analysis was conducted to address the question of its origin in the Iberian Peninsula. Three neutral markers were studied that allowed for the identification of the probable founder haplotype (174-234-G) on which R234C arose. The sharing of the ancestral haplotype by Portuguese and Spanish patients clearly implied a common origin of the mutation in Iberia, through an event that was inferred to have been rather recent. Therefore, the reconstructed history of R234C explains the high incidence of the mutation in Iberian patients with Sanfilippo B disease.

Do the distribution patterns of polymorphisms at the thiopurine S-methyltransferase locus in sub-Saharan populations need revision? Hints from Cabinda and Mozambique.

Genetic data on the thiopurine S-methyltransferase (TPMT) polymorphism were obtained in population samples from Cabinda and Mozambique (located in the western and eastern coasts of sub-Saharan Africa, respectively). The overall frequency of TPMT-deficient alleles was 5.6% in Mozambique and 6.3% in Cabinda. Accordingly, one out of the 103 individuals from Cabinda tested had a genotype associated with TPMT deficiency, yielding a frequency that is threefold higher than heretofore reported in any population. In addition, in both Cabinda or Mozambique, TPMT*8 accounted for a significant proportion of non-functional alleles (nearly 40% in Cabinda). Since the substitution defining TPMT*8 seems to be highly specific of sub-Saharan Africa populations and given the fact it has not been integrated into the set of single nucleotide polymorphisms routinely tested for TPMT, a re-design of molecular screenings should be considered in the future in order to avoid serious underestimates of TPMT deficiency when the enzymatic profiles in populations are unknown.

Dissecting the genetic history of São Tomé e Príncipe: a new window from Y-chromosome biallelic markers.

Twenty biallelic Y chromosome markers were analyzed in Angolares, Forros and Tongas, three population groups from the African archipelago of São Tomé e Príncipe. While most male lineages belonged to sub-Saharan haplogroups, the component of European origin added up 23.9% in the archipelago. This contrasts with the reported absence of European mtDNA lineages, and the combined findings testify to a strong sex-biased admixture process during the long-lasting colonial period in São Tomé e Príncipe. Furthermore, the male mediated European component was clearly found to be out of proportion to the small demographic impact of the Portuguese on the islands, reflecting high variance in the reproductive success of the individuals that contributed to its peopling. The male portion of European ancestry was 33.3% in Forros, 27.3% in Tongas and approximately two-fold less, 14.5%, in Angolares. The Angolares also showed the lowest haplogroup diversity and the most reduced number of different haplogroups. The current results reinforce our previous evidence pointing to remarkable restrictions in gene flow between Angolares and other São Tomean inhabitants, in agreement with their considerable isolation and confinement to the south-eastern tip of São Tomé until recently.

Pharmacogenetically relevant polymorphisms in Portugal.

OBJECTIVE: Most drugs are developed based on data from European-derived 'reference' populations; however, clinically relevant DNA polymorphisms often demonstrate population-specific patterns of allele frequencies. Given that the knowledge of the frequency distribution of functional polymorphisms in a population may guide national planning for selection of therapeutic options, in the present study we examined the allele frequencies of enzymes responsible for drug disposition in Portugal. PATIENTS & METHODS: Using PCR- and Pyrosequencing-based methods, the current study assessed the frequencies of 15 key polymorphisms from genes encoding enzymes involved in Phases I, II and III of drug metabolism, DNA repair and intracellular metabolism in 135 healthy individuals from Portugal. RESULTS: Allele frequencies were derived for cytochrome P450 (CYP)2C9*2 (13.2%), CYP2C9*3 (8%), CYP2C19*2 (14%), CYP3A4*1B (7%), CYP3A5*3C (87.5%), glutathione S-transferase (GST)M1*0 (77.9%), GSTP1 313A>G (33%), inosine triphosphatase 94C>A (7%), UDP-glucuronosyltransferase (UGT)1A1*28 (28%), UGT1A1 -3156G>A (23%), ATP-binding cassette (ABC)B1 1236C>T (46%), ABCB1 2677G>A/T (2 and 42%), ABCG2 421C>A (8%), excision repair cross-complementing rodent repair deficiency 2 2251A>C (3%) and thymidylate synthetase 1494del (31%). CONCLUSION: Although, on the whole, the frequency distributions among the Portuguese fitted the patterns commonly found in other Europeans well, evidence for some degree of African influence was observed. This is the most comprehensive study on pharmacogenetically relevant variations in Portugal to date, and the baseline of pharmacogenetic data might be important for determining policy guidelines for cancer prevention and drug treatments in the Portuguese population.

Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a defect in the iduronate-2-sulfatase gene (IDS). Alternative splicing of the IDS gene can occur and the underlying regulatory mechanism may be rather complex. Nevertheless, little information is available on the role of variations at the IDS locus in the splicing process. Here we report that splice mutations at the IDS locus are an important source of MPS II pathogenicity, accounting for almost 56% of Portuguese cases. Among 16 unrelated Portuguese MPS II patients, 15 different mutations were identified: six intronic splice mutations (c.104-2AG, c.241-2A>G, c.241-1G>A, c.418+1G>A, c.880-8AG and c.1181-1G>C); two exonic splice mutations (c.1006G>lC and c.1122C>T); five missense mutations (D269V, D69V, D148N, R88C and P86L); one nonsense mutation (Q465Ter); one total IDS gene deletion; and one rearrangement involving a IDS gene inversion. Furthermore, nine of the 15 detected mutations affected the usual splicing pattern at the locus. Some of them are responsible for dramatic changes in the splicing mechanism. For example, the substitution mutation, c.418+1G>A, revealed the presence of an exonic sequence inside intron 3. Our study provides evidence that the IDS locus is prone to splicing mutations and that such susceptibility is particularly high in exon 3 and neighbouring regions. Consequently, mutation screening of the IDS gene cannot be restricted to gDNA examination. Unless cDNA analysis is also conducted, misclassifications as silent or missense mutations can be produced and even uncharacteristic splice-site mutations can be misinterpreted as classic splicing defects that may generate severe, unconventional splicing alterations.

Frequency of the thiopurine S-methyltransferase alleles in the ancient genetic population isolate of Sardinia.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an enzyme involved in the normal metabolic inactivation of thiopurine drugs. Patients with intermediate or no TPMT activity are at risk of toxicity after receiving standard doses of thiopurine drugs and it was shown that inter-individual differences in response to these drugs is largely determined by genetic variation at the TPMT locus. OBJECTIVE: This study was designed to investigate in the Sardinian population the frequency distribution of four of the most common variants accounting for TPMT deficiency and to conduct comparative analyses with other populations in order to obtain insights into the main factors that have shaped diversity at the TPMT locus in Sardinia. METHODS: DNA was extracted in 259 Sardinians and the frequencies of allelic variants of TPMT were determined using polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: Among the 259 Sardinians genotyped, 6.95% were found to be heterozygous for one of four TPMT variants screened; for each variant the frequency estimate was 1.74%, 0.58%, 0.39% and 0.77% for TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C respectively. CONCLUSIONS: Although Sardinia does not show reduced diversity at the TPMT locus, the spectrum of TPMT allele frequencies affords evidence of remarkable influence of genetic drift and founder effects throughout its population history. In the broad context of the European TPMT diversity, the Sardinians come out as outliers, an observation consistent with previous genetic inferences that Sardinia has features of a genetic isolate.

Analysis of Y-chromosome variability and its comparison with mtDNA variability reveals different demographic histories between islands in the Azores Archipelago (Portugal).

We determined the Y-chromosomal composition of the population of the Azores Islands (Portugal), by analyzing 20 binary polymorphisms located in the non-recombining portion of the Y-chromosome (NRY), in 185 unrelated individuals from the three groups of islands forming the Archipelago (Eastern, Central and Western). Similar to that described for other Portuguese samples, the most frequent haplogroups were R1(xR1b3f) (55.1%), E(xE3a) (13%) and J (8.6%). Principal components analysis revealed a Western European profile for the Azorean population. No significant differences between Azores and mainland Portugal were observed. However, the haplogroup distribution across the three groups of islands was not similar (P<0.003). The Western group presented differences in the frequencies of haplogroups R1, E(xE3a) and I1b2 (27.3%, 22.7% and 13.6%, respectively) when compared to the other two groups. An assessment of the NRY variability, and its comparison with mitochondrial DNA (mtDNA) variability, was further evidence of the differential composition of males during the settlement of the three groups of islands, contrary to what has been previously deduced for the female settlers using mtDNA data.

Tracing the origin of the most common thiopurine methyltransferase (TPMT) variants: preliminary data from the patterns of haplotypic association with two CA repeats.

Thiopurine methyltransferase (TPMT) is an essential enzyme for normal metabolism of thiopurine drugs. In humans TPMT activity is largely dependent upon genetic variation at the TPMT locus, with TPMT*3A and TPMT*3C being the most frequent mutant alleles associated with reduced activity. TPMT*3C is a widespread allele reaching the highest frequencies in Africans, whereas TPMT*3A is virtually restricted to Caucasian descendent populations. To estimate the time of origin of these two alleles, we analyzed the levels of diversity at two CA repeats flanking the TPMT gene. In accordance to its pattern of geographical distribution, the study of the decay in linkage disequilibrium over time indicated that TPMT*3A was the younger allele. The estimated age was 5700 years, which coincides with the Neolithic, a period characterized by major population expansion that could have been responsible for the spread of TPMT*3A from its place of origin, maybe a western Eurasian population. TPMT*3C was found to have arisen earlier, roughly 14000 years ago, which could explain the worldwide dispersal of TPMT*3C.

Pattern of mtDNA variation in three populations from São Tomé e Príncipe.

We have analysed the matrilineal genetic composition of three self-reported ethnic groups from São Tomé e Príncipe (Gulf of Guinea), an African archipelago whose settlement begun in the late fifteenth century. Sequence data from the hypervariable segments I (HVS-I) and II (HVS-II) were obtained for 30 Angolares, 35 Forros and 38 Tongas. The repertory of mtDNA lineages in São Tomé e Príncipe denoted a fully African maternal pool, primarily arisen from a Central/Southwestern substratum. The absence of any lineages of putative European descent means that the European impact at the mitochondrial pool was virtually nil. Angolares showed a clear reduction of mtDNA diversity and a slight genetic differentiation relative to Tongas or Forros, whereas the latter two groups did not present any signs of genetic boundaries between each other. The data obtained here reinforce the depiction of genetic substructuring in São Tomé e Príncipe previously derived from Y-chromosome STRs. In addition, the crossing of mtDNA and Y-STR information led to the inference that the female mediated gene flow within the archipelago was less restricted than the male, a pattern that could be framed in the cultural traditions and socio-historical interactions among the groups.