mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.

Even with advent of next generation sequencing complete sequencing of large disease-associated genes and intronic regions is economically not feasible. This is the case of cystic fibrosis transmembrane conductance regulator (CFTR), the gene responsible for cystic fibrosis (CF). Yet, to confirm a CF diagnosis, proof of CFTR dysfunction needs to be obtained, namely by the identification of two disease-causing mutations. Moreover, with the advent of mutation-based therapies, genotyping is an essential tool for CF disease management. There is, however, still an unmet need to genotype CF patients by fast, comprehensive and cost-effective approaches, especially in populations with high genetic heterogeneity (and low p.F508del incidence), where CF is now emerging with new diagnosis dilemmas (Brazil, Asia, etc). Herein, we report an innovative mRNA-based approach to identify CFTR mutations in the complete coding and intronic regions. We applied this protocol to genotype individuals with a suspicion of CF and only one or no CFTR mutations identified by routine methods. It successfully detected multiple intronic mutations unlikely to be detected by CFTR exon sequencing. We conclude that this is a rapid, robust and inexpensive method to detect any CFTR coding/intronic mutation (including rare ones) that can be easily used either as primary approach or after routine DNA analysis.

Bacterial transfer of large functional genomic DNA into human cells.

Efficient transfer of chromosome-based vectors into mammalian cells is difficult, mostly due to their large size. Using a genetically engineered invasive Escherichia coli vector, alpha satellite DNA cloned in P1-based artificial chromosome was stably delivered into the HT1080 cell line and efficiently generated human artificial chromosomes de novo. Similarly, a large genomic cystic fibrosis transmembrane conductance regulator (CFTR) construct of 160 kb containing a portion of the CFTR gene was stably propagated in the bacterial vector and transferred into HT1080 cells where it was transcribed, and correctly spliced, indicating transfer of an intact and functional locus of at least 80 kb. These results demonstrate that bacteria allow the cloning, propagation and transfer of large intact and functional genomic DNA fragments and their subsequent direct delivery into cells for functional analysis. Such an approach opens new perspectives for gene therapy.

Maternal effect: an additional mechanism maintaining balanced polymorphisms of haemoglobinopathies?

The malarial hypothesis presupposes that carriers of sickle cell trait and beta-thalassaemia are born in accordance with Mendelian inheritance and later favoured by an environmental factor. However, an eventual distortion favouring the transmission of the mutant allele is also a plausible hypothesis. The Mendelian proportion was tested in the progeny of 201 sickle cell trait (AS) and 138 beta-thalassaemia (AT) probands married to individuals with normal haemoglobin (AA). An excess of births of heterozygotes in the offspring of the 107 AS mothers (144 AS:89 AA, chi2=12.98; p<0.001) and 95 AT mothers (117 AT:66 AA, chi2=14.21; p<0.001) married to homozygous normal fathers was observed, but not for births to the reverse parental combinations (103 AS: 101 AA, chi2=0.019; p=0.89 and 57 AT:42 AA, chi2=2.27; p=0.13). Such data reveal a statistically significant maternal effect favouring the transmission of haemoglobin S and beta-thalassaemia alleles. Obviously, prezygotic and postzygotic mechanisms of distortion cannot be ignored and need further investigation. However, the selective embryonic survival of AS and AT conceptuses of heterozygotic mothers, with an excess of unrecognized very early embryonic deaths among the AA conceptuses is a very attractive hypothesis that should also be taken into consideration.

[Community genetics and hemophilia in a Brazilian population]. / Genética comunitária e hemofilia em uma população brasileira.

Hemophilia is an important hemorrhagic disease in Brazil, affecting about 1 out of every 10,000 males. Patient's self-perception of hemophilia and interaction with the community are relevant to the clinical management of this disease. We investigated several social, psychological, and community aspects of hemophilia in a Brazilian population (Campinas, São Paulo State), interviewing 30 hemophiliac males, a control sample comprised of 73 non-hemophiliac brothers, and 641 individuals from the community. According to our results, more severe social disability in the hemophiliac patient was related to economic factors, mainly unemployment; however, no difference was found in relation to marital status, reproduction, or education. Self-perception of changes in health and lifestyle by individuals with hemophilia showed frequent self-stigmatization, along with depression, anxiety, and insecurity. The community showed a widespread lack of familiarity with hemophilia (49%), viewing people with hemophilia with the kinds of prejudices often observed in relation to people with infectious diseases, like AIDS. The paper concludes by recommending that a community-based program be implemented to improve the social adjustment status of individuals with hemophilia.

[Genetico-epidemiological and molecular investigation of G-6-PD deficiency in a Brazilian community]. / Investigação genético-epidemiológica e molecular da deficiência de G-6-PD em uma comunidade brasileira.

This paper reports on a study of the G-6-PD deficiency in Bragança Paulista, São Paulo State, Brazil. A total of 4,621 male blood donors were investigated over a 36-month period. Of these, 80 had the G-6-PD deficiency. Molecular analysis was performed on 70 unrelated G-6-PD deficients through DNA amplification followed by digestion with restriction enzymes and single strand conformation polymorphism analysis (SSCP). In 98.6%, the G-6-PD A- (202 G<--A) mutation was observed through digestion of exon 4 with Nla III. The presence of an uncommon mutation in exon 9 was also observed through SSCP. No case of the Mediterranean variant was observed. These results indicate that the A- (202G<--A) variant, almost exclusive, was introduced into the community not only by individuals of African origin, but also by European immigrants, mainly Italian, Spanish, and Portuguese. The Italian contribution in terms of the G-6-PD Mediterranean variant was smaller than its contribution to beta thalassemia, probably due to the Northern Italian origin of these immigrants.

Community genetics: a new discipline and its application in Brazil.

Community genetics is a new discipline which aims to provide genetic services to the community as a whole. As a science, community genetics encompasses all research needed to develop and evaluate its application. There is no question that the development of community genetics is necessary in Brazil. The implementation of such programs in our country, especially for hemoglobinopathies, has been recommended by the World Health Organization and other international organizations. Apart from the need for and appeal of community genetics programs, some aspects require serious review. This article discusses various cultural, social, psychological, and economic factors that can make genetic screening an invasion of individual privacy.

[Hemoglobin screening: response of a Brazilian community to optional programs]. / Triagem de hemoglobinopatias: resposta de uma comunidade brasileira a programas opcionais.

The efficiency and the viability of three hemoglobin screening programs were investigated. They were offered on a voluntary basis to a Brazilian population and started with the analysis of blood donors, pregnant women and students. The hemoglobin screening was done through optional exams which included electrophoresis of hemoglobin and complementary hematological tests. A total of 13,670 people were tested over a period of 39 months and a total of 644 individuals with hereditary hemoglobin disorders were detected - 4. 7% of the samples examined. The programs showed satisfactory indicators of viability and efficiency, expressed by the significative proportion of exams performed among the probands and their relatives.

[Community-based programs for hereditary hemoglobinopathies in Brazilian high school students]. / Programa comunitário de hemoglobinopatias hereditárias em população estudantil brasileira.

This study tested the viability and efficiency of a hereditary hemoglobinopathy program in which a Brazilian community the town of Bragança Paulista, SP (Brazil)-was involved, were tested focusing school students. The screening of the hemoglobinopathies was done through optional exams for patients by using electrophoresis of hemoglobins and complementary hematological tests. A total of 1,171 individuals, including, 1,118 pupils and 53 of their relatives were tested over a period of 24 months. A total of 47 individuals with hereditary hemoglobin changes were detected-4.0% of the samples examined. The community was fairly receptive to the program which showed a general level of acceptance of 55.4%. The study aroused the interest of the community and motivated the implantation of a special service for diagnosis, genetic counseling and treatment of the hemoglobinopathy carriers in the community where it was undertaken.

[Sickle cell disease as a public health problem in Brazil]. / A anemia falciforme como problema de Saúde Pública no Brasil.

Sickle cell anemia is the most prevalent hereditary disease in Brazil. However, the Brazilian literature registers no investigations into the public health aspects of the disease. This present study investigates the way of life of 80 adult patients (49 women and 31 men) with a diagnosis of sickle cell anemia, at a blood center in Brazil. The late diagnosis of the disease was one of the most significant aspects observed in this group of patients. It was also observed that the dominant problem faced by adult patients with sickle cell anemia is of an economic nature, mainly due to lack of professional opportunities. However, patients can well undertake economic activities under adequate medical supervision, according to their own limitations and potentialities. The psychotherapeutic orientation was well accepted by patients regardless of sex. It is concluded that there exists need for the establishment of community programs for early diagnosis and medical, social and psychological orientation for sickle cell anemia patients in Brazil.

[Thalassemia and Hansen's disease]. / Talassemia e hanseníase.

The beta-thalassemia trait was investigated among 165 Brazilians who were unmixed Italian descendants (80 Virchowian patients and 85 normal controls, composed of universitary students). The frequency of the beta-thalassemia trait was 6.25% among the Virchowian patients and 5.88% in the control group. In spite of the similar geographical distribution of both hanseniasis and the gene for beta-thalassemia in Asia, the present data does not support the hypothesis that hanseniasis might have contributed to maintain high prevalence of this allele by selection favouring beta-thalassemia trait.