Pharmacogenetic profile of a South Portuguese population: results from the pilot study of the European Health Examination Survey in Portugal.

BACKGROUND: The genetic inter-individual variability of drug response can lead to therapeutic failure or adverse drug reactions (ADRs). The aims of this study were to assess the pharmacogenetic profile of a South Portuguese population according to established dosing guidelines for commonly prescribed drugs and to compare it with that of previously genotyped populations. METHODS: A cross-sectional study was developed in the context of the Portuguese Component of the European Health Examination Survey (EHES). A total of 47 pharmacogenetically relevant variants in 23 different genes were genotyped in 208 participants. Allelic and genotypic frequencies were calculated, and the pharmacogenetic profile of the participants was defined. A comparative analysis was conducted through electronic database search. Pairwise Fst calculations were performed to assess the genetic distance between populations. RESULTS: We found a significant small differentiation between the Portuguese regional populations regarding CYP2C9 rs1057910, CYP2D6 rs3892097, MTHFR rs1801133 and F5 rs6025. When consid-ering 4 HapMap populations, ADH1B rs2066702, ADH1B rs1229984, NAT2 rs1799931 and VKORC1 rs9923231 displayed a significant population differentiation. We found that 18.9% of the participants are intermediate or poor metabolizers for at least 3 drugs simultaneously and that 84.6% of the participants have at least one therapeutic failure or ADR risk allele for the considered drugs. CONCLUSIONS: There is a high prevalence of risk alleles associated with an altered drug metabolism regarding drugs largely used by the South Portuguese population. This knowledge contributes to the prediction of their clinical efficacy and/or toxicity, optimizing therapeutic response while improving cost-effectiveness.

Genetic variation in CD36, HBA, NOS3 and VCAM1 is associated with chronic haemolysis level in sickle cell anaemia: a longitudinal study.

Chronic haemolysis stands out as one of the hallmarks of sickle cell anaemia, a clinically heterogeneous autosomal recessive monogenic anaemia. However, the genetic architecture of this sub-phenotype is still poorly understood. Here, we report the results of an association study between haemolysis biomarkers (serum LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants of ten candidate genes in a series of 99 paediatric SS patients (median current age of 9.9 yr) followed up in two general hospitals in Greater Lisboa area (median follow-up per patient of 5.0 yr). Although in a large number of tests a seemingly significant (i.e. P < 0.05) association was observed, the following ones were confirmed upon correction for multiple comparisons: (i) an increased serum LDH level was associated with haplotype 7 within VCAM1 gene; (ii) a lower total bilirubin was associated with the 3.7-kb deletion at HBA gene, rs2070744_T allele at NOS3 gene, and haplotype 9 within VCAM1 promoter; and (iii) a diminished reticulocyte count was associated with the 3.7-kb deletion at HBA, whereas an increased count was associated with rs1984112_G allele at CD36 gene. On the whole, our findings suggest a complex genetic architecture for the sickle cell anaemia haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.

Novel large deletions in the human alpha-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environment.

Globin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human alpha-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the alpha-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the alpha-globin distal regulatory elements and/or the complete alpha-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the alpha-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of alpha-globin genes but it is not associated with a complete absence of alpha-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment.

Non-classical hereditary hemochromatosis in Portugal: novel mutations identified in iron metabolism-related genes.

The most frequent genotype associated with Hereditary hemochromatosis is the homozygosity for C282Y, a common HFE mutation. However, other mutations in HFE, transferrin receptor 2 (TFR2), hemojuvelin (HJV) and hepcidin (HAMP) genes, have also been reported in association with this pathology. A mutational analysis of these genes was carried out in 215 Portuguese iron-overloaded individuals previously characterized as non-C282Y or non-H63D homozygous and non-compound heterozygous. The aim was to determine the influence of these genes in the development of iron overload phenotypes in our population. Regarding HFE, some known mutations were found, as S65C and E277K. In addition, three novel missense mutations (L46W, D129N and Y230F) and one nonsense mutation (Y138X) were identified. In TFR2, besides the I238M polymorphism and the rare IVS5 -9T-->A mutation, a novel missense mutation was detected (F280L). Concerning HAMP, the deleterious mutation 5'UTR -25G-->A was found once, associated with Juvenile Hemochromatosis. In HJV, the A310G polymorphism, the novel E275E silent alteration and the novel putative splicing mutation (IVS2 +395C-->G) were identified. In conclusion, only a few number of mutations which can be linked to iron overload was found, revealing their modest contribution for the development of this phenotype in our population, and suggesting that their screening in routine diagnosis is not cost-effective.

Mutational spectrum of delta-globin gene in the Portuguese population.

The phenotype of increased Hb A2 typical of beta-thalassaemia (beta-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a delta-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of delta-globin mutations in the Portuguese population we performed a mutational analysis of the delta-globin gene in a group of 51 Portuguese beta-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have delta-globin structural abnormalities. The heterozygosity for the beta(+)IVS-I-6T-->C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical beta-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common beta-thal mutation and the delta(+)Cd27G-->T mutation (Hb A2-Yialousa; HBD:c.82G>T). One of them also presented a novel delta-globin gene promoter mutation,-80G-->A (HBD:c.-130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have delta-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified - Hb A2' (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [delta64(E8)(Gly-->Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different delta-globin mutations were found, being two of them new molecular defects. All delta-alleles identified were found linked to the expected beta-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a beta-thal allele.

Hb Evora [alpha2-35 (B16), Ser-->Pro], a novel hemoglobin variant associated with an alpha-thalassemia phenotype.

We report a novel mutation in the alpha2-globin gene, codon 35 (T-->C), detected in two unrelated Portuguese families. This mutation gives rise to a previously undescribed hemoglobin (Hb) variant, which we named Hb Evora. This variant seems to be responsible for the alpha-thalassemia phenotype present in its carriers. It cannot be detected by conventional laboratory techniques, probably because of its highly unstable nature.

Hemoglobin Loves Park [beta68 (E12) Leu-->Phe]: report of five cases including one originating from a de novo mutation.

Hemoglobin (Hb) Loves Park [beta68 (E12) Leu-->Phe] was identified in a 2-year-old Portuguese boy with anemia, microcytosis, and hypochromia. This Hb variant was detected by isoelectric focusing and quantified by reverse-phase high-performance liquid chromatography (HPLC) (48.4%), and the DNA mutation was identified by HBB (beta-globin gene) sequencing. Hematological and biochemical analyses performed on his parents revealed normal hematological parameters and normal hemoglobin and globin chain profiles. DNA sequence analysis of the HBB gene of both parents showed the absence of the Hb Loves Park mutation. Study of the haplotypes in the beta-globin gene cluster confirmed parenthood. Moreover, paternity was confirmed by the study of nine short tandem repeats (STRs) and four variable-number tandem repeat (VNTRs) loci. The most likely explanation for these results is that the Hb Loves Park mutation has occurred de novo in this family. The original American cases of Hb Loves Park, from a family of Italian origin, which were never published, as well as two additional cases, are also included in this report. Functional studies revealed that Hb Loves Park is stable and has a decreased oxygen affinity.

Human alpha2-globin nonsense-mediated mRNA decay induced by a novel alpha-thalassaemia frameshift mutation at codon 22.

We describe a novel alpha-thalassaemia determinant in a 3-year-old girl presenting a mild microcytic and hypochromic anaemia, and normal haemoglobin A2 level. Molecular studies revealed heterozygosity for a novel microdeletion (-C) at codon 22 of the alpha2-globin gene. As the frameshift mutation generates a premature translation termination codon at position 48/49, we investigated the effect of the nonsense codon on the alpha2-globin gene expression. Although it does not affect RNA splicing, the premature nonsense codon induces accelerated mRNA degradation. To our knowledge, this is the first time the nonsense-mediated mRNA decay has been reported to occur in human alpha-globin mRNA.

Clinical and laboratory effects of hydroxyurea in children and adolescents with sickle cell anemia: a Portuguese hospital study.

Our aim was to assess the efficacy and safety of hydroxyurea (HU) in children with severe forms of sickle cell anemia followed in a Portuguese hospital. We carried out an open-label uncontrolled prospective study, which included children with severe forms of sickle cell anemia. Hydroxyurea was started at 15 mg/kg/day and increased to a maximum dose of 25 mg/kg/day. Patients were monitored to assess compliance, clinical and hematological response and toxicity. Nine children and adolescents, five girls and four boys, with a median age of 13 years (range 8 to 16) were enrolled in the study during a period of 24 months. All patients completed at least 15 months of therapy. Hb F was significantly increased, from a mean of 7.0 +/- 3.9% to 13.7 +/- 5.3% (p = 0.028). Clinically, all patients responded significantly with a reduction of 80% in the number of vaso-occlusive crises (VOC), 69% in hospital admissions, 76% in hospitalization days and 67% in transfusion requirements, without significant toxicity. We concluded that, in our population, HU proved to be effective in increasing Hb F levels, and in decreasing hospitalizations for VOC and transfusion requirements with no major side effects. Long-term clinical follow-up is important to certify benefit maintenance.

The role of HFE mutations on iron metabolism in beta-thalassemia carriers.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism characterized by increased iron absorption and progressive storage resulting in organ damage. HFE gene mutations C282Y and H63D are responsible for the majority of HH cases. A third HFE mutation, S65C, has been associated with the development of a mild form of hemochromatosis. The beta-thalassemia trait is characterized by mild, ineffective erythropoiesis that can induce excess iron absorption and ultimately lead to iron overload. The aim of this study was to evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. Serum iron (P=0.029) and transferrin saturation (P=0.009) were increased in beta-thalassemia carriers heterozygous for H63D mutation. The number of subjects carrying C282Y or S65C mutations was too low to conclude their effect on the iron status. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.

Hb Yaoundé [beta134(H12)Val-->Ala] in association with Hb C [beta6(A3)Glu-->Lys] in a Caucasian Portuguese family.

Hb Yaoundé [beta134(H12)Val-->Ala] is a rare, silent and asymptomatic hemoglobin (Hb) variant. It was previously reported in a Black man from Cameroon, in association with Hb Kenitra [beta69(E13)Gly-->Arg], and was subsequently found and described as Hb Mataro in a sub-Saharan child. To date, Hb Yaoundé has not been described in Caucasian people and molecular studies have never been performed. Here we describe a three-generation Caucasian Portuguese family in whom Hb Yaoundé was found in association with Hb C [beta6(A3)Glu-->Lys] (in the proband) and in a heterozygous state (in the proband's mother). The Hb studies of the proband's hemolysate, performed by isoelectric focusing (IEF) and low-pressure cation exchange chromatography, only revealed an Hb variant identified as Hb C by comparison with a control. However, analysis performed by reversed-phase high-performance liquid chromatography (HPLC) showed two different beta chain variants and a complete absence of the normal beta chain. This uncommon Hb variant was identified as Hb Yaoundé by DNA sequencing analysis of the beta-globin gene (codon 134, GTG-->GCG). The beta-globin gene haplotypes were determined in all family members using polymerase chain reaction (PCR)-based methodology; Hb Yaoundé was found to be associated with the Mediterranean haplotype II. This study is the first description of Hb Yaoundé in Caucasian individuals, and its association with a Mediterranean haplotype supports the hypothesis of an independent genetic origin other than African.

Genetic studies suggest a novel Portuguese origin for hemoglobin Porto Alegre.

Molecular studies performed in Portuguese and Brazilian cases of hemoglobin Porto Alegre [beta9 Ser->Cys] revealed that the mutation is in association with the Mediterranean haplotype I and framework 1 and that it is also in cis with an undescribed intragenic polymorphism (codon 27, GCC->GCT). Based upon these findings, and reinforced by historical data, we suggest that hemoglobin Porto Alegre originated from a single mutational event in the Portuguese population and was then spread to South America, namely to Brazil.

The beta-thalassemia mutation/haplotype distribution in the moroccan population.

The present study compiles the results of our own research and of a prior study on beta-thalassemia (thal) in Morocco, comprising a total of 187 beta-thalassemic chromosomes. Six major mutations: (beta0) codon 39 (C --> T), (beta+) IVS-I-6 (T --> C), (beta0) frameshift codon (FSC) 6 (-A), (beta0) FSC 8 (-AA), (beta0) IVS-I-1 (G --> A) and (beta+) -29 (A --> G) account for 75.7% of the independent chromosomes studied. A regional predominance was observed (Gharb and West regions) for the (beta+) IVS-I-6 (T --> C) mutation. Despite an observed heterogeneity of molecular anomalies, a direct method of diagnosis of the prevalent mutations is feasible in this population. The distributions of mutations and haplotypes are in conformity with the geographical location of Morocco and the historical links with both the Mediterranean communities that have successively interspersed with the Berbers, the Phoenicians, the Carthaginians, the Romans, the Arabs, the population of the Iberian Peninsula and, to a lesser degree, the Vandals and the Byzantines and permanently, with the Sub-Saharan Africans. In the adult population, the levels of fetal hemoglobin (Hb) in heterozygotes vary from trace quantities to 2.38 g/dL of total Hb. With the exception of the (beta0) codon 39 (C --> T) nonsense mutation, no statistically significant correlation was found, neither between mutation and Hb F levels, nor gender and Hb F levels in heterozygotes. The genetic markers for Hb F increase, located within cis active sites such as the XmnI site at -158 bp of the Ggamma-globin gene and the AT(X)T(Y) repeat region at -540 bp of the beta-globin gene, were assessed. The polymorphism XmnI shows linkage disequilibrium with haplotypes III, IV and IX, as previously observed in the Algerian, Sicilian and Portuguese beta-thal populations. Contrary to what has previously been reported for a population of beta-thal carriers of European descent, this sample does not show a statistically significant correlation between Hb F levels and the presence of the genetic markers XmnI restriction site at -158 bp of the Ggamma-globin gene and AT(X)T(Y) alleles at 5' of the beta-globin gene.

Spectrum of beta thalassemia mutations and HbF levels in the heterozygous Moroccan population.

A comprehensive hematological and molecular analysis of 57 beta thalassemic heterozygotes, 28 homozygotes, 18 double heterozygotes, 3 compound heterozygotes beta thal/beta S and one compound heterozygote beta thal/Hb Newcastle, in 46 Moroccan families with at least one beta thalassemia patient is reported. Six major mutations: beta(0)39 (C-->T), beta(0)FsCD8(-AA), beta(+)IVS1,nt6 (T-->C) and beta(0)IVS1,nt1 (G-->A), beta(0)FsCD6 (-A) and beta(+)-29 (A-->G) cap site account for 75% of the 86 independent beta thal chromosomes studied. For the first time, an extensive mutation/haplotype study has been performed on the Moroccan population, and data are consistent with the geographical location of the country and historical links with both the Mediterranean and the Sub-Saharan Africa communities. Despite the heterogeneous spectrum of mutations, good genetic counseling can be offered to the carrier population. This study focuses on the analysis of fetal hemoglobin levels in beta thalassemic heterozygotes and its correlation with beta globin cluster polymorphic markers in this population. Fetal hemoglobin levels in heterozygotes vary from trace quantities to 17.9% (2.38 g/dl) of total hemoglobin in the adult. No statistically significant correlation was found, either between genders and HbF levels, or between the mutation and the HbF level, with the exception of mutation beta(0)FSCD6(-A). We have examined the alpha globin genotype and the beta globin genotype of heterozygotes, namely, the extended haplotype, which includes the XmnI site at -158 bp of the Ggamma gene and the microsatellite (AT)(x)T(y) at -540 bp of the beta globin gene. In this sample, we confirm the existence of linkage disequilibrium between the C-->T variation at -158 bp of Ggamma globin gene (XmnI(+)) and Orkin's haplotypes III, IV, or IX (the 5' subhaplotype class A). At 5' beta globin gene, we observe exclusively the allele (AT)(7)T(7). In the beta thalassemic heterozygotes studied, no correlation of those genetic markers with HbF levels is observed.

Asymptomatic homozygous deletional beta(0)-thalassemia in an African individual.

Homozygosity or compound heterozygosity for beta(0)-thalassemia mutations most commonly results in a transfusion-dependent thalassemia major phenotype. In this report, we describe a 55-year-old male, from Guinea-Bissau, that had been asymptomatic and never transfused until being admitted to hospital with anemia, fever, splenomegaly, and asthenia. Following hospital admission, HIV-2 and Mycobacterium tuberculosis infections were diagnosed, and biochemical and molecular studies revealed homozygosity for beta(0)-thalassemia. At the molecular level, this is the first description of homozygosity for the beta(0)-Black 1,393-bp deletion. In this case, the complete absence of beta-globin gene expression seems to be compensated by an unusually high fetal globin gene expression (Hb F 96%). Beta-globin haplotyping results were compatible with the propositus being homozygous for the Black 2 haplotype and for the absence of the XmnI polymorphism at -158 of (G)gamma-globin gene (-/-). Co-inheritance of genetic factors usually associated with high Hb F levels was not detected. Otherwise, the propositus is a heterozygote for the alpha-globin gene 3.7-kb deletion that is a beneficial modulating factor but not sufficient to explain this extremely mild phenotype. This unusual genotype/phenotype association is discussed in terms of the mechanisms underlying hemoglobin switching during development.

Fetal hemoglobin elevation in Hb Lepore heterozygotes and its correlation with beta globin cluster linked determinants.

We have analysed, at the hematological and molecular level, 51 Hb lepore heterozygotes and three compound heterozygotes for Hb Lepore and HbS (HbLep/HbS) in 26 unselected Portuguese families. The Lepore Boston variant was present in one family, in association with classical haplotype V. All of the other Lepore alleles present haplotype III in association with XmnI (+)5' of (G)gamma gene, in tight linkage disequilibrium to the major mutation found in the Portuguese population, the Lepore Baltimore variant ( delta(68Leu)-beta(84Thr)). The three compound heterozygotes are the first HbLep/HbS individuals reported in the literature, with the Lepore Baltimore mutation linked to haplotype III. In agreement with other studies, these Lepore Baltimore heterozygotes have higher HbF (1.4-14.1% of total hemoglobin) than published cases of Lepore Boston (0.8-5.4%), which is associated with XmnI(-). Among the Lepore Baltimore heterozygotes, the (AT)xTy repeat region at -540 bp of the beta globin gene in trans to the Lepore chromosome, can account for much of the variability in HbF level. The allele (AT)7T7 is associated with lower HbF, and (AT)9T5 is associated with higher HbF. As we previously reported for beta thalassemic carriers, we observe in Lepore Baltimore carriers an effector in trans, linked to the (AT)xTy sequence, acting as an HPFH (Hereditary Persistence of Fetal Hemoglobin) determinant.