Laís Ribeiro Mota

Cystic fibrosis: Identification and frequency of mutations in a mixed population from a low-income region in Northeastern Brazil

Current evidence points to the importance of a more personalized approach that takes into account the ethnicity of a population in the diagnosis of Cystic Fibrosis (CF). Recent studies have indicated that use of gene sequencing is a reliable approach to diagnose CF. Although this approach is extremely relevant, it is costly. The rational use of health resources is highly recommended, especially in low-incomeareas.Brazil is a country of continental dimensions and with great ethnic-racial heterogeneity. The state of Bahia, located in the Northeast of Brazil, presents a population with a high degree of miscegenation due to the characteristics of its ancestry: African (47.3%), European (36.4%), and Amerindian (16.3%). The population of this state is approximately 15.2 million, most of it with low income, and only two medical centers provide clinical care for CF patients; these centers are part of the public health system, which has universal coverage in this country. Our center is a multidisciplinary clinic, located at the teaching Hospital of the Federal University of Bahia. Since January 2008, we have enrolled and prospectively followed patients with CF from 0 to 20 years of age. Between 2012 and 2017, 50 patients with confirmed CF diagnosis by two positive sweat tests were submitted to molecular analysis for screening of seven mutations by conventional methods using peripheral blood samples (polymerase chain reaction and enzymatic digestion), or by next-generation sequencing of oral mucosa samples using Illumina HiSeq platform whenever it was not possible to determine the patients’ genotype through conventional analysis. Of the patients studied, 26 (52%) were male, 48 (96%) were nonwhite, median age of 6 years. Through conventional molecular techniques, five mutations were identified, allowing us to ascertain the genotype of 25 patients. The sequencing study identified 14 new mutations (including a new variant, not yet described in the literature), expanding to 43 (86%) the number of patients with known genotype. These findings allowed us to predict that by using a 20 mutation panel, we would be able to complete diagnostic genotyping from approximately 90% of our patients. Three patients had no mutant allele identified and whereas additional four patients presented with only one mutation. Genotypes and mutation frequencies are described in Table 1. Advances in precision medicine require knowledge of the genotypic profiles of CF patients. However, the routine performance of genetic testing is not a reality for the vastmajority of CF care centers in Brazil. Data from the Brazilian Registry of Cystic Fibrosis (REBRAFC) from 2015 indicated that only 46.2% from 3857 patients were submitted to any genetic study. Thus, it expansion in use of genetic testing is warranted to better diagnose CF. To optimize diagnosis without significantly increasing cost, we predict that development of a panel of mutations more specific to each region is highly recommended, since the use of a single panel may not be equally sensitive, particularly in regions with major heterogeneity in ethnicity such as Brazil. The results of our study revealed that despite the high degree of miscegenation of the study population, with 96% non-white patients, the initial investigation of only F508del mutation determined the genotype of approximately 30% of patients. This finding corroborates with the proposal of an initial investigation searching only for F508del mutation, as suggested for patients from the Brazilian southeast. In our population, subsequent investigation for G542X and 3120 + 1G>A mutations expanded the genotype knowledge to 50% of patients. Thus, the establishment of a step wise protocol, with the research of only three mutations, can optimize the use of public health resources and increase the access to molecular diagnosis techniques. The other mutations, found only through sequencing, can be incorporated into a specific panel for this region of Brazil, reserved only for patients whose genotype could not be determined by the initial investigation. Identifying the genotypes of our patients allowed us to discover that 42% of these were eligible for precision medicine treatment: 14 (28%) patients F508del/F508del—Lumacaftor/Ivacaftor or Tezacaftor/Ivacaftor combination; 3 (6%) patients were suitable to use Ivacaftor and 3 (6%) patients for the use of the association Tezacaftor/ Ivacaftor or Ivacaftor. Furthermore, due to low availability of other diagnostic resources such as CFTR function evaluation or periodic dosing of fecal elastase to investigate pancreatic insufficiency, genotype determination has firmly contributed to improve treatment strategies for our patients.

Description of rare mutations and a novel variant in Brazilian patients with Cystic Fibrosis: a case series from a referral center in the Bahia State

Knowledge of the genetic profile of Cystic Fibrosis (CF) contributes to a better understanding of the genotype/phenotype relationship, particularly in mixed populations such as in Brazil. To describe clinical data of CF patients with rare or not yet observed CFTR gene mutations in Brazil. It was a case series of CF patients followed-up at a referral center. Clinical and laboratory data were obtained through medical records. Molecular analysis of the mutations was performed by conventional methods and/or by next-generation sequencing. Ten patients were studied, seven had five pathogenic mutations without previous description in Brazil (Q1100P, Y109C, A107P, E1409K and K162E), one of which has not yet been reported in patients with CF (A107P). Among the seven patients, three (two siblings) had the second mutant allele of rare occurrence among Brazilians patients (G1069R and 2307insA). Three other patients also had at least one rare variant (V201M, S466X and G1069R). The age of the CF diagnosis ranged from 1 to 190 months in the ten cases and the main clinical manifestations were respiratory symptoms and difficulty in gaining weight. All but one patient presented clinical and/or laboratory data compatible with pancreatic insufficiency. The identification of rare or not yet described CFTR mutations in patients with CF in Brazil highlights the high genetic heterogeneity in this population. Knowledge of the genotypic profile of Brazilian CF patients can contribute to the development of specific mutation panels for the genetic investigation targeting each region of the country, as well as helping to understand the complex genotype/phenotype relationship, especially in mixed populations.

Manifestações clínicas da mutação F508del: uma série de casos de pacientes com fibrose cística

Introducao: a Fibrose Cistica (FC) e a doenca autossomica recessiva mais comum e letal na populacao de origem caucasoide. Causada por mutacoes no gene que codifica a proteina CFTR, no qual ja existem mais de 2.000 mutacoes identificadas, sendo a mutacao F508del a mais frequente. Esta doenca apresenta-se de forma multissistemica com quadro clinico altamente variado e com consideravel diversidade na gravidade e na progressao da doenca. Alguns estudos correlacionam os sintomas ao genotipo dos pacientes. Objetivo: descrever o genotipo e apresentacao clinica dos pacientes homozigotos ou heterozigotos para esta mutacao F508del. Metodologia: foi realizada a descricao de uma serie de casos de pacientes diagnosticados com FC que apresentam a mutacao F508del, acompanhados pelo Ambulatorio Multidisciplinar de FC do Complexo Hospitalar Universitario Prof. Edgard Santos do Estado da Bahia. Resultados: Dez (45,4%) criancas eram homozigotos para a mutacao e 12/22 (54,5%) heterozigotos. As principais manifestacoes clinicas que levaram ao diagnostico foram: insuficiencia pancreatica (95,4%), sintomas respiratorios (85,2%), dificuldade em ganhar peso (88,5%), esteatorreia (73,3%), e ritmo intestinal alterado (53,8%). A idade de inicio dos sintomas (mediana 0,16 anos) e do diagnostico (mediana 0,58 anos) foram precoces, refletindo a gravidade da doenca. Conclusoes: Conclui-se que as caracteristicas clinicas e laboratoriais dos pacientes descritos foram semelhantes aos relatados na literatura e destacam a associacao entre insuficiencia pancreatica e o genotipo dos pacientes, enfatizando a importância do estudo genetico na determinacao do prognostico dos pacientes, em especial em populacoes altamente miscigenadas, como no Brasil.

A modified MS-PCR approach to diagnose patients with Prader-Willi and Angelman syndrome

Prader-Willi (PWS) and Angelman (AS) syndromes are clinically distinct neurodevelopmental genetic diseases with multiple phenotypic manifestations. They are one of the most common genetic syndromes caused by non-Mendelian inheritance in the form of genomic imprinting, and can be attributable to the loss of gene expression due to imprinting within the chromosomal region 15q11-q13. Clinical diagnosis of PWS and AS is challenging, and the use of molecular and cytomolecular studies is recommended to help in determining the diagnosis of these conditions. The methylation analysis is a sensible approach; however there are several techniques for this purpose, such as the methylation-sensitive polymerase chain reaction (MS-PCR). This study aims to optimize the MS-PCR assay for the diagnosis of potential PWS and AS patients using DNA modified by sodium bisulfite. We used the MS-PCR technique of PCR described by Kosaki et al. (1997) adapted with betaine. All different concentrations of betaine used to amplify the methylated and unmethylated chromosomal region 15q11-q13 on the gene SNRPN showed amplification results, which increased proportionally to the concentration of betaine. The methylation analysis is a technically robust and reproducible screening method for PWS and AS. The MS-PCR assures a faster, cheaper and more efficient method for the primary diagnosis of the SNRPN gene in cases with PWS and AS, and may detect all of the three associated genetic abnormalities: deletion, uniparental disomy or imprinting errors.

7 Frequency of five CFTR gene mutations in miscegenated cystic fibrosis population of the Brazilian Northeast

Introduction There are more than 1,900 known mutations in the CFTR gene. In the state of Bahia, in the northeastern area of Brazil, there are high rates of miscegenation, principally white/black miscegenation. The knowledge about genetics of cystic fibrosis (CF) in this population is scarce. Objective To determine the prevalence of five CFTR gene mutations in cystic fibrosis patients being assisted at Professor Edgard Santos Teaching Hospital in Salvador, Brazil. Methods It was a cross-sectional study involving 47 patients of 0–20 years of age with cystic fibrosis. The molecular analysis for DF508, G542X, G551D, R553X and 3120+1G→A mutations was performed by standard methods. Results The median age of the patients was 5.6 years at the time of diagnosis. The allele frequency of DF508 and G542X mutations was 23.9% (21/88) and 2.7 (2/74), respectively. So far the mutations G551D, R553X and 3120+1G→A have been studied in 21 patients. Three patients had the splicing mutation 3120+1G→A and the other mutations were not observed. Conclusion The diagnosis of the disease was late. The frequency of the DF508 mutation was lower than that one found in Caucasian populations, but the frequency of G542X mutation was similar to the one observed in this population. The 3120+1G→A mutation was found in three patients.