MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing.

Germline pathogenic variants in the DNA mismatch repair genes (MMR): MLH1, MSH2, MSH6, and PMS2, are causative of Lynch syndrome (LS). However, many of the variants mapping outside the invariant splice site positions (IVS ± 1, IVS ± 2) are classified as variants of unknown significance (VUS). Three such variants (MLH1 c.588+5G>C, c.588+5G>T and c.677+5G>A) were identified in 8 unrelated LS families from Argentina, Brazil and Chile. Herein, we collected clinical information on these families and performed segregation analysis and RNA splicing studies to assess the implication of these VUS in LS etiology. Pedigrees showed a clear pattern of variant co-segregation with colorectal cancer and/or other LS-associated malignancies. Tumors presented deficient expression of MLH1-PMS2 proteins in 7/7 of the LS families, and MSI-high status in 3/3 cases. Moreover, RNA analyses revealed that c.588+5G>C and c.588+5G>T induce skipping of exon 7 whereas c.677+5G>A causes skipping of exon 8. In sum, we report that the combined clinical findings in the families and the molecular studies provided the evidences needed to demonstrate that the three MLH1 variants are causative of LS and to classify c.588+5G>C and c.677+5G>A as class 5 (pathogenic), and c.588+5G>T as class 4 (likely-pathogenic). Our findings underline the importance of performing clinical and family analyses, as well as RNA splicing assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives.

Identificação de doenças genéticas na Atenção Primária à Saúde: experiência de um município de porte médio no Brasil / Identification of genetic diseases in Primary Health Care: experience of a medium county in Brazil / Identificación de enfermedades genéticas en la Atención Primaria de Salud: experiencia de un municipio mediano en Brasil

Problema: Embora individualmente raras, somadas, as doenças genéticas têm prevalência global estimada de 31,5 a 73,0 por 1.000 indivíduos. Além disto, doenças genéticas e defeitos congênitos representam a segunda causa de mortalidade infantil no Brasil. Diante deste cenário, foi instituída a Política Nacional de Atenção Integral às Pessoas com Doenças Raras no Sistema Único de Saúde. Esta política prevê funções específicas para Atenção Primária à Saúde (APS) que incluem diagnóstico precoce e mapeamento de pessoas com ou sob-risco de desenvolver doenças genéticas raras e/ou defeitos congênitos para encaminhamento regulado. Essa experiência objetivou colaborar com o desenvolvimento de métodos para o reconhecimento de indivíduos com ou sob-risco de desenvolver doenças genéticas na APS. Métodos: Através de visitas domiciliares e por meio do preenchimento de uma ficha específica, realizou-se busca ativa de casos de doença genética e/ou defeito congênito em uma amostra probabilística aleatória, representativa de uma Unidade de Saúde da Família de um município brasileiro de porte médio. Resultados: Foram investigados 295 domicílios, totalizando 1.160 indivíduos e 238 casais. A média de filhos por casal foi de 2,7, a frequência de consanguinidade foi 3,8% e de abortamento espontâneo foi 8,7%. Foram identificadas 29 pessoas (2,5%) com doenças congênitas, 11 (0,9%) com deficiências auditivas, 10 (0,9%) com deficiência mental e 6 (0,5%) com déficits visuais importantes. Atraso no desenvolvimento neuropsicomotor foi relatado em 8,8% das crianças e adolescentes. Doze indivíduos (1%) possuíam câncer e 9,6% relataram história familiar positiva para câncer. Conclusão: Os profissionais da APS estão em posição privilegiada para identificar e organizar uma rede de cuidados para indivíduos com doenças genéticas e/ou defeitos congênitos. A utilização sistemática de instrumentos que facilitem o reconhecimento de fatores de risco e de situações suspeitas pode ser uma estratégia útil a ser incorporada pela APS.

Problem: Although individually rare, when added together, genetic diseases have an estimated overall prevalence of 31.5 to 73.0 per 1,000 individuals. In addition, genetic diseases and birth defects represent the second cause of infant mortality in Brazil. In this context, the National Policy on Comprehensive Care of People with Rare Diseases was established in the Brazilian National Health System. This policy provides specific Primary Health Care (PHC) assignments that includes early diagnosis and mapping people with or at risk of developing rare genetic disease and/or birth defects for regulated referral. This experience aimed to collaborate with developing methods for recognizing individuals with or at risk of developing genetic diseases in PHC. Methods: Through home visits and filling out a specific form, an active search for cases of genetic disease and/or birth defect was carried out in a random probabilistic sample, representative of a Family Health Unit in a Brazilian medium-sized county. Results: A total of 295 households were surveyed, totalling 1,160 individuals and 238 couples. The mean number of children per couple was 2.7, the inbreeding rate was 3.8% and the frequency of miscarriage was estimated in 8.7%. Twenty-nine individuals (2.5%) with congenital disorders, 11 (0.9%) with hearing impairment, 10 (0.9%) with mental disability, and 6 (0.5%) with significant visual deficits were identified. Neuropsychomotor developmental delay was presented in 8.8% of the children and teenagers. Twelve individuals (1%) had cancer and 9.6% reported a positive family history of cancer. Conclusion: PHC professionals are in a privileged position to identify and organize a care network for individuals with genetic diseases and/or birth defects. The systematic use of instruments that facilitate the recognition of risk factors and suspicious situations can be a useful strategy to be incorporated by PHC.

Genética na atenção primária à saúdeRev Bras Med Fam Comunidade. Rio de Janeiro, 2020 Jan-Dez; 15(42):23472INTRODUÇÃOEstima-se que existam cerca de 6 a 7 mil doenças genéticas diferentes e, embora a maior parte destas sejam individualmente raras, somadas, as doenças genéticas têm prevalência global estimada de 31,5 a 73,0 por 1.000 indivíduos.1 Doenças genéticas podem ser congênitas ou podem se manifestar ao longo da vida, impactando nas diferentes faixas etárias: alterações cromossômicas estão presentes em cerca de 50% dos abortamentos espontâneos de primeiro trimestre; entre recém-nascidos, 3 a 5% apresentam algum defeito congênito, determinado total ou parcialmente por fatores genéticos; deficiências físicas, intelectuais e sensoriais em crianças e jovens frequentemente fazem parte do quadro clínico de síndromes genéticas; nos adultos, aproximadamente 5 a 10% dos cânceres possuem forte componente genético hereditário.1,2 Doenças genéticas podem ser hereditárias, sendo transmitidas ao longo das gerações de uma família, ou não hereditárias, ocorrendo por "mutação de novo".2 São sabidamente fatores de risco para doenças genéticas idades materna e/ou paterna avançadas, consanguinidade e história familiar positiva para doenças genéticas previamente reconhecidas.2-5 Além destes, a exposição de gestantes a teratógenos é fator de risco para defeitos congênitos.6Problema: Aunque individualmente raras, las enfermedades genéticas combinadas tienen prevalencia general estimada de 31.5 a 73.0 por 1,000 individuos. Además, enfermedades genéticas y anomalías congénitas representan la segunda causa de mortalidad infantil en Brasil. Ante este escenario, se instituyó la Política Nacional de Atención Integral a Personas con Enfermedades Raras en el Sistema Único de Salud. Esta Política proporciona funciones específicas para Atención Primaria de Salud (APS) que incluyen diagnóstico precoz y mapeo de personas con o en riesgo de desarrollar enfermedades genéticas raras y/o anomalías congénitas para derivación regulada. Esta experiencia tuvo como objetivo colaborar con el desarrollo de métodos para el reconocimiento de individuos con o en riesgo de desarrollar enfermedades genéticas en la APS. Método: Mediante visitas domiciliarias y completando un formulario específico, se realizó búsqueda activa de casos de enfermedades genéticas y/o defectos congénitos en una muestra probabilística aleatoria, representativa de una Unidad de Salud Familiar en un municipio brasileño de tamaño mediano. Resultados: Se investigaron un total de 295 hogares, 1,160 personas y 238 parejas. El número promedio de hijos por pareja fue de 2.7, la frecuencia de consanguinidad 3.8% y el aborto espontáneo 8.7%. Se identificaron 29 personas (2.5%) con enfermedades congénitas, 11 (0.9%) con discapacidad auditiva, 10 (0.9%) con discapacidad mental y 6 (0.5%) con déficits visuales significativos. Se informó retraso psicomotor en 8.8% de los niños y adolescentes. Doce personas (1%) tenían cáncer y 9.6% reportaron antecedentes familiares de cáncer. Conclusión: Los profesionales de la APS están en posición privilegiada para identificar y organizar una red de atención para personas con enfermedades genéticas y/o defectos congénitos. El uso sistemático de instrumentos que facilitan el reconocimiento de factores de riesgo y situaciones sospechosas puede ser una estrategia útil para la APS.

BRCA1 and BRCA2 rearrangements in Brazilian individuals with Hereditary Breast and Ovarian Cancer Syndrome.

Approximately 5-10% of breast cancers are caused by germline mutations in high penetrance predisposition genes. Among these, BRCA1 and BRCA2, which are associated with the Hereditary Breast and Ovarian Cancer (HBOC) syndrome, are the most frequently affected genes. Recent studies confirm that gene rearrangements, especially in BRCA1, are responsible for a significant proportion of mutations in certain populations. In this study we determined the prevalence of BRCA rearrangements in 145 unrelated Brazilian individuals at risk for HBOC syndrome who had not been previously tested for BRCA mutations. Using Multiplex Ligation-dependent Probe Amplification (MLPA) and a specific PCR-based protocol to identify a Portuguese founder BRCA2 mutation, we identified two (1,4%) individuals with germline BRCA1 rearrangements (c.547+240_5193+178del and c.4675+467_5075-990del) and three probands with the c.156_157insAlu founder BRCA2 rearrangement. Furthermore, two families with false positive MLPA results were shown to carry a deleterious point mutation at the probe binding site. This study comprises the largest Brazilian series of HBOC families tested for BRCA1 and BRCA2 rearrangements to date and includes patients from three regions of the country. The overall observed rearrangement frequency of 3.44% indicates that rearrangements are relatively uncommon in the admixed population of Brazil.

Genetic ancestry analysis in non-alcoholic fatty liver disease patients from Brazil and Portugal.

AIM: To study the association between genetic ancestry, non-alcoholic fatty liver disease (NAFLD) metabolic characteristics in two cohorts of patients, from Brazil and Portugal. METHODS: We included 131 subjects from Brazil [(n = 45 with simple steatosis (S. Steatosis) and n = 86 with nonalcoholic steatohepatitis (NASH)] and 90 patients from Portugal (n = 66, S. Steatosis; n = 24, NASH). All patients had biopsy-proven NAFLD. In histologic evaluation NAFLD activity score was used to assess histology and more than 5 points defined NASH in this study. Patients were divided into two groups according to histology diagnosis: simple steatosis or non-alcoholic statohepatitis. Genetic ancestry was assessed using real-time polymerase chain reaction. Seven ancestry informative markers (AT3-I/D, LPL, Sb19.3, APO, FY-Null, PV92, and CKMM) with the greatest ethnic-geographical differential frequencies (≥ 48%) were used to define genetic ancestry. Data were analyzed using R PROJECTS software. Ancestry allele frequencies between groups were analyzed by GENEPOP online and the estimation of genetic ancestry contribution was evaluated by ADMIX-95 software. The 5% alpha-error was considered as significant (P < 0.05). RESULTS: In the Brazilian sample, NASH was significantly more frequent among the elderly patients with diabetes (NASH 56 ± 1.1 years old vs S. Steatosis 51 ± 1.5 years old, P = 3.7 x 10(-9)), dyslipidemia (NASH 63% vs S. Steatosis 37%, P = 0.009), higher fasting glucose levels (NASH 124 ± 5.2 vs S. Steatosis 106 ± 5.3, P = 0.001) and Homeostatic Model of Assessment index > 2.5 [NASH 5.3 (70.8%) vs S. Steatosis 4.6 (29.2%) P = 0.04]. In the Portuguese study population, dyslipidemia was present in all patients with NASH (P = 0.03) and hypertension was present in a larger percentage of subjects in the S. Steatosis group (P = 0.003, respectively). The genetic ancestry contribution among Brazilian and Portuguese individuals with NASH was similar to those with S. Steatosis from each cohort (Brazilian cohort: P = 0.75; Portuguese cohort: P = 0.97). Nonetheless, the genetic ancestry contribution of the Brazilian and Portuguese population were different, and a greater European and Amerindian ancestry contribution was detected in the Portuguese population while a higher African genetic ancestry contribution was observed in Brazilian population of both NASH and S. Steatosis groups. CONCLUSION: There was no difference between the genetic ancestry contribution among Brazilian and Portuguese individuals with NASH and S. Steatosis from each cohort.