Molecular genetic analysis of factor XI deficiency: identification of five novel gene alterations and the origin of type II mutation in Portuguese families.

Coagulation factor XI (FXI) deficiency is an inherited autosomal recessive mild bleeding disorder. In this study, we report the molecular genetic analysis of FXI deficiency in six unrelated families of Portuguese origin. The Jewish type II mutation was found in two families, of seemingly Portuguese origin. Haplotype analysis in these families demonstrated that this mutation is of Jewish origin. In the remaining families, five novel FXI mutations have been identified. Two of these mutations (FXI IVS K -10T-->A and FXI 1026G-->T, cd 324) affect the FXI pre-mRNA splicing. A further two (FXI 307 ins AAGCAAT, cd 85 and FXI 1072 del A, cd 340) introduce frameshifts leading to premature termination codons. The FXI splicing mutation, 1026G-->T cd 324, was found in compound heterozygosity with missense mutation FXI K518N. Analysis of the FXI mRNA from the latter genotype demonstrated new donor splice site usage. All reported mutations most likely result in functional null-alleles. In addition, three novel polymorphisms have been identified: at nt -138 in intron A, at codon D125 in exon 5 and at codon T249 in exon 8.

Ethnic heterogeneity of the factor XIII Val34Leu polymorphism.

A polymorphism in the coagulation factor XIII gene (FXIII Val34Leu) has been recently described to confer protection for arterial and venous thrombosis and to predispose to intracerebral hemorrhage. At present it is known that FXIII Val34Leu is prevalent in Caucasians, but information upon its distribution in different ethnic groups is scarce. We investigated the prevalence of FXIIIVal34Leu in 450 unrelated subjects of four ethnic groups: 97 Caucasians (Brazilians of European descent and Portuguese), 149 Blacks (Brazilians, and Africans from Cameroon, Zaire and Angola), 40 Asians (Japanese descendents) and 164 Amerindians from South America. PCR amplification of exon 2 of FXIII gene followed by MseI restriction-digestion was employed to define the genotypes. FXIIIVal34Leu was detected in 44.3% of the Caucasians, in 28.9% of the Blacks, in 2.5% of the Asians and in 51.2% of the Amerindians. These data confirm that FXIII Val34Leu is highly prevalent in Caucasians and indicate that it is rarer in populations of African origin. The very high frequency among Amerindians indicates that FXIII Val34Leu is not absent among Asians, and since it has a very low prevalence in Japanese, a heterogeneity in its distribution in Asia may be inferred. Taken together, our data showed that FXIII Val34Leu exhibits a significant ethnic heterogeneity, a finding that is relevant for studies relating this polymorphism with thrombotic and bleeding disorders.

Analysis of the essential sequences of the factor VIII gene in twelve haemophilia A patients by single-stranded conformation polymorphism.

We report the analysis by single-stranded conformation polymorphism of the essential sequences of the factor VIII(FVIII) gene (total length about 14 kb) including the entire coding sequence, flanking intronic sequences and the putative regulatory sequences 5' to the gene, in twelve unselected haemophilia A patients of Portuguese origin. Direct sequencing of the fragments with an altered migration pattern led to the identification of the disease-producing mutations in five patients. Three of these mutations, namely a 1 bp insertion in a motif of eight consecutive A residues at codon 1439 (FVIIIPorto3); a C to T transition at codon 1966 (Arg-->Stop), found in an inhibitor-positive patient (FVIIIMontijo); and a G to A transition at codon 479 (Gly-->Arg; FVIIIPorto1), have been reported in other ethnic groups. The two novel mutations are the substitution of AG by GG at the 3' end of intron 4 (FVIIILisboa1) destroying the invariant splice acceptor sequence, and a G to A transition at codon 1948 resulting in an aspartic acid substitution for glycine (FVIIIPorto2).

[Molecular biology in cardiology: what is and where is the mutated gene?]. / Biologia molecular em cardiologia: qual é e onde está o gene mutado?

In this paper the role of molecular biology research and development in establishing etiology and pathogenesis of cardiovascular disease is discussed. Several examples of our own practice in the field (namely, Di George sequence, hereditary protein S deficiency, and hyperhomocysteinaemia) are provided as an illustration of the currently applied strategies.

[Y chromosome and male infertility]. / Cromossoma Y e infertilidade masculina.

Spermatogenesis is a complex physiological process characterized by an orderly proliferation and differentiation of germ cell types, from diploid spermatogonial stem cells to haploid spermatids, and after spermiogenesis to spermatozoa. It is known that male reproductive capacity is deficient in about one half of infertile couples. Effective treatment is available only for a small fraction of these infertile men. In most cases, the cause of male infertility is unknown. Aetiologically, male infertility may result from genetic and non-genetic causes. Among the genetic factors known to disturb spermatogenesis, chromosomal aberrations, involving autosomes and/or sex chromosomes, are well known. However, molecular genetic causes of idiopathic male infertility have been accumulating in the last years, predominantly for the Y chromosome. Localization of genes that control spermatogenesis on Yq11 was first proposed, on cytogenetic evidence, by Tiepolo and Zuffardi in 1976. Since then, many subsequent reports using molecular genetics methods have supported the initial proposal by detecting submicroscopic interstitial deletions on Yq11, present in patients with idiophatic azoospermia and a cytogenetically normal Y chromosome. Recently, four spermatogenesis candidate Y-linked genes (or gene families), RBM, DAZ, SPGY and TSPY have been cloned and characterized. In the euchromatic Y chromosome long arm three intervals--AZFa, AZFb and AZFc--were also defined. All of them may contain genes necessary for normal spermatogenesis. All the four genes have a testis-specific expression and three of them (RBM, DAZ and SPGY) code for ribonucleoproteins with a single RNA recognition motif. Candidate genes with in AZFb are some members of the RBM gene family and within AZFc are the DAZ and SPGY gene family. While AZFc deletions are associated with azoospermia or with severe oligoteratoasthenozoospermia, microdeletions involving AZFa or AZFb cause azoospermia only.

[Diagnosis of Lepore hemoglobinopathy with DNA analysis]. / Diagnóstico da hemoglobinopatia de Lepore por análise do DNA.

We illustrate the application of recombinant DNA methods (namely Southern blotting) for the genotype diagnosis of haemoglobin Lepore Boston: the use of the restriction endonucleases PstI and XbaI along with a beta globin gene specific probe make it possible to detect a deletion of approximately 7kb which typically characterizes the delta beta Boston gene. The impact of using such methods in the prenatal diagnosis of major haemoglobinopathies is discussed.

[The molecular basis of dominantly inherited beta-thalassemia]. / Base molecular de uma beta-talassémia de transmissão dominante.

In this study, we sought to clarity the molecular basis of a dominant inherited beta-thalassemia, found in heterozygosity in a northern Portuguese family with thalassemia intermedia. We characterized: i) the alpha-globin gene cluster structure; ii) the beta-globin gene cluster haplotype; and iii) the beta-thalassemia mutation. The alpha-globin gene cluster was structurally normal. The G-->T transversion at codon 121 of the beta-globin gene was found in the affected individuals in association with Orkin's haplotype V. This is an uncommon, though ubiquitous, mutation. Which has also been found, in association with different haplotypes, in several distant populations. It has only been observed in this three-generation family, in the Portuguese population. We suggest a mechanism to explain the genotype/phenotype correlation.

[The prevention of hereditary erythrocytic diseases]. / Profilaxia das doenças hereditárias do eritrócito.

The authors report the importance of not only all over the world but also in Portugal and, particularly, in Dona Estefânia Hospital. Some considerations are made about the usefulness of molecular biology methods in prenatal diagnosis. With this tool can also be do the origins and migrations of populations, which contributes to the knowledge of aspects of our history. Finally, they present consensual attitudes which should adopt regarding these chronic diseases, with special emphasis to the prophylactic aspects.

[Mucoviscidosis with respiratory symptomatology in the neonatal period]. / Mucoviscidose com sintomatologia respiratória no período neonatal.

A case of cystic fibrosis presented in the neonatal period with respiratory symptomatology associated with early pancreatic insufficiency is reported. The CFTR gene molecular analysis was found to be a compound heterozygotes for delta F508 and G542X. The rarity of this mode of presentation and the inclusion of this entity in the differential diagnosis for neonatal respiratory distress syndrome is emphasised. The pathogenesis and some therapeutic aspects carried out in our patient, which might have improved the life expectancy of patients with this disease, are discussed.

[Neonatal screening of hemoglobinopathies in a population residing in Portugal]. / Rastreio neonatal de hemoglobinopatias numa população residente em Portugal.

The primary objective of newborn screening of hemoglobinopathies is the early identification of infants with sickle cell disease, as they are at increased clinical risk. Other goals include the identification of other types of clinically significant hemoglobinopathies and the detection of heterozygous carriers followed by the screening and counselling of family members. We performed a pilot study for the neonatal screening of hemoglobinopathies in 400 samples of cord blood taken from a maternity in Lisbon. We did not find any newborn with sickle cell disease. Six samples were from sickle cell heterozygotes, the respective families were studied and informed. We looked for the presence of alpha-thalassemia at birth in 100 consecutive samples of cord blood, by the presence of Hb Bart's, abnormal red blood cell indices and alpha-globin genotype. The results show an incidence of 10% of alpha-thalassemia (-alpha) carriers and 4% of triple alpha-globin gene carriers. The authors discuss the feasibility of neonatal screening of hemoglobinopathies in a Portuguese-speaking population consisting of a low prevalence of Hb S trait autoclonous group and a high prevalence immigrant minority.

Determining oxidative and non-oxidative genotoxic effects driven by estuarine sediment contaminants on a human hepatoma cell line.

Estuarine sediments may be reservoirs of hydrophilic and hydrophobic pollutants, many of which are acknowledged genotoxicants, pro-mutagens and even potential carcinogens for humans. Still, studies aiming at narrowing the gap between ecological and human health risk of sediment-bound contaminant mixtures are scarce. Taking an impacted estuary as a case study (the Sado, SW Portugal), HepG2 (human hepatoma) cells were exposed in vitro for 48 h to extracts of sediments collected from two areas (urban/industrial and Triverine/agricultural), both contaminated by distinct mixtures of organic and inorganic toxicants, among which are found priority mutagens such as benzo[a]pyrene. Comparatively to a control test, extracts of sediments from both impacted areas produced deleterious effects in a dose-response manner. However, sediment extracts from the industrial area caused lower replication index plus higher cytotoxicity and genotoxicity (concerning total DNA strand breakage and clastogenesis), with emphasis on micronucleus induction. On the other hand, extracts from the rural area induced the highest oxidative damage to DNA, as revealed by the FPG (formamidopyrimidine-DNA glycosylase) enzyme in the Comet assay. Although the estuary, on its whole, has been classified as moderately contaminated, the results suggest that the sediments from the industrial area are significantly genotoxic and, furthermore, elicit permanent chromosome damage, thus potentially being more mutagenic than those from the rural area. The results are consistent with contamination by pro-mutagens like polycyclic aromatic hydrocarbons (PAHs), potentiated by metals. The sediments from the agriculture-influenced area likely owe their genotoxic effects to metals and other toxicants, probably pesticides and fertilizers, and able to induce reactive oxygen species without the formation of DNA strand breakage. The findings suggest that the mixtures of contaminants present in the assayed sediments are genotoxic to HepG2 cells, ultimately providing a useful approach to hazard identification and an effective line-of-evidence in the environmental monitoring of anthropogenically-impacted coastal ecosystems.

Human hepatoma cells exposed to estuarine sediment contaminant extracts permitted the differentiation between cytotoxic and pro-mutagenic fractions.

Complex toxicant mixtures present in estuarine sediments often render contaminant screening unfeasible and compromise determining causation. HepG2 cells were subjected to bioassays with sediment extracts obtained with a series of progressively polar solvents plus a crude extract. The sediments were collected from an impacted area of an estuary otherwise regarded as pristine, whose stressors result mostly from aquaculture effluents and hydrodynamic shifts that enhance particle deposition. Compared to a reference scenario, the most polar extracts yielded highest cytotoxicity while higher genotoxicity (including oxidative damage) was elicited by non-polar solvents. While the former caused effects similar to those expected from biocides, the latter triggered effects compatible with known pro-mutagens like PAHs, even though the overall levels of toxicants were considered of low risk. The results indicate that the approach may constitute an effective line-of-evidence to infer on the predominant set of hazardous contaminants present in complex environmental mixtures.

Characterization of a splicing mutation in the factor VIII gene at the RNA level.

Haemophilia A is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. The identification and characterization of naturally occurring disease-producing mutations allows the recognition of new mechanisms of pathogenesis in haemophilia A. Analysis of the illegitimately transcribed FVIII mRNA in a severely affected patient has revealed that the A-->G transition at position -2 of the acceptor splice site of intron 4 results in the skipping of exon 5 in 90% of the processed pre-mRNA. Another minor mRNA species arising from the skipping of exons 4 and 5 has also been observed. The skipping of exon 5 predicts the removal of the corresponding 13 amino acids from the A1 domain of FVIII. A novel missense mutation, C329S, in exon 8 of FVIII gene has been identified in another patient.

DNA polymorphisms associated with the factor VIII:C gene in the Portuguese population.

We have determined the allele frequencies of seven restriction fragment length polymorphisms within or close to factor VIII:C gene in the Portuguese population. The allele frequency of all studied intra- and extragenic biallelic polymorphisms does not differ significantly from those found in other European or Asian populations. On the contrary, the distribution of the different alleles of the TaqI RFLP at the DXS52 locus revealed similarity only with Algerians. We observed a correspondence between the TaqI and BclI alleles at this locus.

Beta-thalassaemia in Cubans: novel allele increases the genetic diversity at the HBB locus in the Caribbean.

In order to establish the molecular basis of beta-thalassaemia in Cubans, a total of 75 unrelated individuals, with beta-thalassaemia major (7), Hb S-beta-thalassaemia (28), Hb C-beta-thalassaemia (1), and beta-thalassaemia trait (39) yielding 82 beta-thalassaemia alleles, were analyzed. Seventeen different point mutations were identified accounting for 93% of the beta-thalassaemia alleles studied, revealing a high genetic heterogeneity at the HBB locus in this population. The more prevalent mutations, namely, CD 39 (C --> T) (30.5%), -29 (A --> G) (13.4%), IVS-I-110 (G --> A) (8.5%), and IVS-II-1 (G --> A) (8.5%), reflect the Mediterranean and African predominant ancestry of the extant Cuban population. We also report the identification of a novel allele, IVS-I-108 (T --> C), that possibly activates a cryptic branch site, in a beta-thalassaemia carrier with no other molecular defect within the beta-globin gene and its proximal promoter. This study shows that prenatal diagnosis of beta-thalassaemia should be feasible in about 60% of at-risk pregnancies by direct detection of selected point mutations. However, due to the wide spectrum of mutations, and in order to offer fully informative prenatal diagnosis to more than 87% of at-risk couples, the screening for beta-thalassaemia mutations in Cubans should be performed by using a general point mutation detection method, such as DGGE (denaturing gradient gel electrophoresis).

A novel mosaic Bantu/Benin/Bantu beta s haplotype found in several African populations.

In a survey of the chromosomal backgrounds associated with the sickle cell gene in Portuguese-speaking populations from Europe and Africa, a discordance between the classical haplotype and the predicted allele at the RsaI polymorphism 5' to the beta globin gene was observed in four patients. Extensive typing of the corresponding beta s chromosomes at simple polymorphic repeat motifs revealed a novel "extended" haplotype that appeared to be a mosaic of (1) a Bantu-type DNase I hypersensitive site 2 within the beta globin gene cluster locus control region, (2) a Benin 5' subhaplotype, and (3) a Bantu 3' subhaplotype. We propose two alternative schedules for the generation of yet another chromosomal background of the sickle cell gene.

A fertile male with cystic fibrosis: molecular genetic analysis.

A family study is presented in which the father of a girl with severe cystic fibrosis (CF) was also found to have CF but was mildly affected. He was diagnosed with three positive sweat tests including one after suppression with fludrocortisone. Genetic analysis showed that he is a compound heterozygote with the delta F508 CF mutation associated with haplotype B and a second CF mutation associated with haplotype C. In this unusual, fertile CF male, the late age of diagnosis (30 years) and the mild clinical picture suggest that the compound genotype (delta F508/other CF mutation) determines a much less severe form of the disease which might have gone unnoticed in the absence of a severely affected child. The implications of these findings for genetic counselling of families with CF are discussed.