A cohort study of neurodevelopmental disorders and/or congenital anomalies using high resolution chromosomal microarrays in southern Brazil highlighting the significance of ASD.

Chromosomal microarray (CMA) is the reference in evaluation of copy number variations (CNVs) in individuals with neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and/or autism spectrum disorder (ASD), which affect around 3-4% of the world's population. Modern platforms for CMA, also include probes for single nucleotide polymorphisms (SNPs) that detect homozygous regions in the genome, such as long contiguous stretches of homozygosity (LCSH). These regions result from complete or segmental chromosomal homozygosis and may be indicative of uniparental disomy (UPD), inbreeding, population characteristics, as well as replicative DNA repair events. In this retrospective study, we analyzed CMA reading files requested by geneticists and neurologists for diagnostic purposes along with available clinical data. Our objectives were interpreting CNVs and assess the frequencies and implications of LCSH detected by Affymetrix CytoScan HD (41%) or 750K (59%) platforms in 1012 patients from the south of Brazil. The patients were mainly children with NDDs and/or congenital anomalies (CAs). A total of 206 CNVs, comprising 132 deletions and 74 duplications, interpreted as pathogenic, were found in 17% of the patients in the cohort and across all chromosomes. Additionally, 12% presented rare variants of uncertain clinical significance, including LPCNVs, as the only clinically relevant CNV. Within the realm of NDDs, ASD carries a particular importance, owing to its escalating prevalence and its growing repercussions for individuals, families, and communities. ASD was one clinical phenotype, if not the main reason for referral to testing, for about one-third of the cohort, and these patients were further analyzed as a sub-cohort. Considering only the patients with ASD, the diagnostic rate was 10%, within the range reported in the literature (8-21%). It was higher (16%) when associated with dysmorphic features and lower (7%) for "isolated" ASD (without ID and without dysmorphic features). In 953 CMAs of the whole cohort, LCSH (≥ 3 Mbp) were analyzed not only for their potential pathogenic significance but were also explored to identify common LCSH in the South Brazilians population. CMA revealed at least one LCSH in 91% of the patients. For about 11.5% of patients, the LCSH suggested consanguinity from the first to the fifth degree, with a greater probability of clinical impact, and in 2.8%, they revealed a putative UPD. LCSH found at a frequency of 5% or more were considered common LCSH in the general population, allowing us to delineate 10 regions as potentially representing ancestral haplotypes of neglectable clinical significance. The main referrals for CMA were developmental delay (56%), ID (33%), ASD (33%) and syndromic features (56%). Some phenotypes in this population may be predictive of a higher probability of indicating a carrier of a pathogenic CNV. Here, we present the largest report of CMA data in a cohort with NDDs and/or CAs from the South of Brazil. We characterize the rare CNVs found along with the main phenotypes presented by each patient and show the importance and usefulness of LCSH interpretation in CMA results that incorporate SNPs, as well as we illustrate the value of CMA to investigate CNV in ASD.

Association between mutations in the FMR1 gene and ovarian dysfunction in Brazilian patients.

OBJECTIVE: Our study aimed to identify mutations in the FMR1 gene in a group of Brazilian women diagnosed with primary ovarian insufficiency (POI). METHODS: This cross-sectional study included patients aged under 40 years with confirmed POI from a convenience sample of patients seen from June 2017 to December 2018 at a University Hospital in Curitiba, Brazil. Genomic DNA was extracted and analyzed using FragilEase(tm) PCR kits (PerkinElmer), a commercially available test that enables the quantification of CGG trinucleotide repeat expansions in the FMR1 gene. RESULTS: A total of 52 patients with an average age of 35.8±3.97 years were included. Fifty (96.1%) had normal alleles with 18 to 43 CGG repeats. The most frequent CGG-repeat sizes were 28 and 30. Two patients (3.8%) presented mutations in the FMR1 gene. The first had alleles with 19/97 CGG repeats, was categorized as a premutation carrier for FXS, and had a son with cognitive impairment. The second had alleles with 21/45 CGG repeats and was described as belonging to the gray zone. CONCLUSIONS: In our study, 3.8% of the females with POI had mutations in the FMR1 gene. The most frequent allele sizes were 28 and 30 CGG repeats.

Etiology of intellectual disability in individuals from special education schools in the south of Brazil.

BACKGROUND: Intellectual Disability (ID) is characterized by significant limitations that affect intellectual functioning, adaptive behavior, and practical skills which directly interfere with interpersonal relationships and the environment. In Western countries, individuals with ID are overrepresented in the health system, often due to associated comorbidities, and its life-time cost places ID as one of the most expensive conditions of all diagnoses in the International Classification of Diseases. Most of the people affected (75%) live in low-income countries, suffer from malnutrition, lack health care, and do not have access to adequate treatment. The aim of this study was to obtain an estimate of the diagnostic status as well as the prevalence of familial ID among individuals with serious (moderate or severe) ID in a region of the State of Santa Catarina, investigating attendees of special education schools of the Florianópolis Macroregion. METHODS: This was a cross-sectional study conducted between August 2011 and August 2014, through a semi-structured screening questionnaire for the collection of relevant developmental, clinical, familial and educational data, applied in an interview to guardians of students of special education schools of the macroregion of Florianópolis. RESULTS: The participant special schools enrolled close to 1700 students during the study period and the questionnaire was applied to 849 (50.5%). The male to female ratio of the participants was 1.39:1. Clear etiologic explanations were relatively scarce (24%); most diagnoses referring only to the type and the degree of impairment and for the majority (61.4%) the cause was unknown. About half were sporadic cases within their families (considering three generations). For 44.2% at least one other case of an ID-related condition in the extended family was mentioned, with 293 (34.5%) representing potential familial cases. CONCLUSION: Here we describe the epidemiological profile, the available diagnostics, etiology, family history and possible parental consanguinity of participants with ID of special education schools in the South of Brazil. The main results show the need for etiological diagnosis and uncover the relevance of potential hereditary cases in a population where consanguineous unions have a relatively low frequency (0,6%) and highlight the need for public health actions.

Molecular analysis of FMR1 gene in a population in Southern Brazil: Comparison of four methods.

OBJECTIVES: Fragile X syndrome (FXS) is caused by expansion of the number of cytosine-guanine-guanine (CGG) repeats in the regulatory region of the gene fragile X mental retardation 1 (FMR1). The molecular diagnoses of FXS can be performed using two tests based on two different techniques, namely polymerase chain reaction (PCR) and Southern blotting (SB). However, both of these techniques have limitations. The purpose of this study was to evaluate the performance of the commercial FragilEase™ PCR kit for FXS diagnosis comparing to other laboratory methods. DESIGN: and methods: This study had a retrospective design. We analyzed the performance of the FragilEase™ PCR kit using 90 DNA samples from patients with clinical suspicion of FXS or a family history of the syndrome using capillary electrophoresis and compared with the results obtained for the same samples using PCR, SB, and AmplideX FMR1 PCR. RESULTS: FragilEase™ PCR kit displayed high concordance with the results obtained using PCR, SB, and AmplideX FMR1 PCR regarding the detection of normal, intermediate/gray zone, premutation, and full mutation alleles, as well as female homozygosity and mosaicism. The replicate sizes found using the FragilEase™ PCR assay varied on average by two CGG repeats. CONCLUSION: FragilEase™ PCR, as well as other commercially available kits, efficiently detect FMR1 mutations and simplify the workflow in laboratories that performing FXS diagnoses.

Copy Number Variations in a Cohort of 420 Individuals with Neurodevelopmental Disorders From the South of Brazil.

Chromosomal microarray (CMA) is now recommended as first tier for the evaluation in individuals with unexplained neurodevelopmental disorders (ND). However, in developing countries such as Brazil, classical cytogenetic tests are still the most used in clinical practice, as reflected by the scarcity of publications of microarray investigation in larger cohorts. This is a retrospective study which analyses the reading files of CMA and available clinical data from 420 patients from the south of Brazil, mostly children, with neurodevelopmental disorders requested by medical geneticists and neurologists for diagnostic purpose. Previous karyotyping was reported for 138 and includes 17 with abnormal results. The platforms used for CMA were CYTOSCAN 750K (75%) and CYTOSCAN HD (25%). The sex ratio of the patients was 1.625 males :1 female and the mean age was 9.5 years. A total of 96 pathogenic copy number variations (CNVs), 58 deletions and 38 duplications, were found in 18% of the patients and in all chromosomes, except chromosome 11. For 12% of the patients only variants of uncertain clinical significance were found. No clinically relevant CNV was found in 70%. The main referrals for chromosomal microarrays (CMA) were developmental delay (DD), intellectual disability (ID), facial dysmorphism and autism spectrum disorder (ASD). DD/ID were present in 80%, facial dysmorphism in 52% and ASD in 32%. Some phenotypes in this population could be predictive of a higher probability to carry a pathogenic CNV, as follows: dysmorphic facial features (p-value = < 0.0001, OR = 0.32), obesity (p-value = 0.006, OR = 0.20), short stature (p-value = 0.032, OR = 0.44), genitourinary anomalies (p-value = 0.032, OR = 0.63) and ASD (p-value = 0.039, OR = 1.94). The diagnostic rate for CMA in this study was 18%. We present the largest report of CMA data in a cohort with ND in Brazil. We characterize the rare CNVs found together with the main phenotypes presented by each patient, list phenotypes which could predict a higher diagnostic probability by CMA in patients with a neurodevelopmental disorder and show how CMA and classical karyotyping results are complementary.

Long contiguous stretches of homozygosity detected by chromosomal microarrays (CMA) in patients with neurodevelopmental disorders in the South of Brazil.

BACKGROUND: Currently, chromosomal microarrays (CMA) are recommended as first-tier test in the investigation of developmental disorders to examine copy number variations. The modern platforms also include probes for single nucleotide polymorphisms (SNPs) that detect homozygous regions in the genome, such as long contiguous stretches of homozygosity (LCSH) also named runs of homozygosity (ROH). LCHS are chromosomal segments resulting from complete or segmental chromosomal homozygosity, which may be indicative of uniparental disomy (UPD), consanguinity, as well as replicative DNA repair events, however also are common findings in normal populations. Knowing common LCSH of a population, which probably represent ancestral haplotypes of low-recombination regions in the genome, facilitates the interpretation of LCSH found in patients, allowing to prioritize those with possible clinical significance. However, population records of ancestral haplotype derived LCSH by SNP arrays are still scarce, particularly for countries such as Brazil where even for the clinic, microarrays that include SNPs are difficult to request due to their high cost. METHODS: In this study, we evaluate the frequencies and implications of LCSH detected by Affymetrix CytoScan® HD or 750 K platforms in 430 patients with neurodevelopmental disorders in southern Brazil. LCSH were analyzed in the context of pathogenic significance and also explored to identify ancestral haplotype derived LCSH. The criteria for considering a region as LCSH was homozygosis ≥3 Mbp on an autosome. RESULTS: In 95% of the patients, at least one LCSH was detected, a total of 1478 LCSH in 407 patients. In 2.6%, the findings were suggestive of UPD. For about 8.5% LCSH suggest offspring from first to fifth grade, more likely to have a clinical impact. Considering recurrent LCSH found at a frequency of 5% or more, we outline 11 regions as potentially representing ancestral haplotypes in our population. The region most involved with homozygosity was 16p11.2p11.1 (49%), followed by 1q21.2q21.3 (21%), 11p11.2p11.12 (19%), 3p21.31p21.2 (16%), 15q15 1q33p32.3 (12%), 2q11.1q12.1 (9%), 1p33p32.3 (6%), 20q11.21q11.23 (6%), 10q22.1q23.31 (5%), 6p22.2p22 (5%), and 7q11.22q11.23 (5%). CONCLUSIONS: In this work, we show the importance and usefulness of interpreting LCSH in the results of CMA wich incorporate SNPs.

15 STR loci frequencies in the population from Santa Catarina, Southern Brazil.

Allele frequencies for 15 short tandem repeats (STR) loci were determined with a sample of 3000 unrelated individuals from the population of Santa Catarina, Southern Brazil. The loci are most commonly used in forensic and paternity testing, being analyzed by the Powerplex 16 (Promega) commercial kit. The data shows that most polymorphic loci were Penta E and FGA. The distributions of the genotypes in the evaluated loci are in Hardy-Weinberg equilibrium. Comparative analyses between our population data and other Brazilian populations are presented. The calculated forensic parameters showed that the loci are useful for the solution of forensic problems in Brazilian Southern region.

Infecção pelo Papilomavírus humano (HPV) em mulheres de Florianópolis, Santa Catarina / Human Papillomavirus (HPV) infection in women of Florianópolis, Santa Catarina, Brazil

Introdução: a infecção pelo papilomavírus humano (HPV) é a doença sexualmente transmissível mais frequente no mundo e apresenta um amploespectro de manifestações, desde a infecção assintomática até o carcinoma invasivo. Estima-se que pelo menos 50% das pessoas sexualmente ativas venham a adquirir esta infecção em algum momento de suas vidas. Objetivo: determinar a prevalência da infecção pelo HPV e identificar a frequência dos grupos virais de baixo e alto risco oncogênico em mulheres atendidas para consulta ginecológica de rotina em Florianópolis, bem como verificar a associação entre a presença do vírus e alguns fatores de risco. Métodos: trata-se de estudo transversal, de caráter descritivo. Foram avaliadas 100 mulheres atendidas em ambulatório para consulta ginecológica de rotina, selecionadas aleatoriamente. Todas tiveram amostra cervical submetida ao teste de DNA-HPV pelo método de Captura Híbrida II® e responderam a um questionário sobre fatores de risco. Resultados: das 100 mulheres analisadas, 21 (21%) apresentaram positividade para o DNA-HPV. O HPV de alto risco oncogênico esteve presente em 71% e o de baixo risco em 52% das amostras positivas, incluindo as infecções mistas. A infecção pelo vírus de alto risco foi associada às pacientes com início de atividade sexual precoce. Outros fatores avaliados como idade, escolaridade, número de parceiros sexuais, paridade, uso de anticoncepcionais orais, doença sexualmente transmissível e tabagismo não mostraram relação com a infecção pelo HPV. Conclusão: a prevalência de HPV na população estudada foi de 21%, com 71% para vírus de alto risco e 52% para vírus de baixo risco oncogênico, incluindo infecções mistas. A detecção do vírus de alto risco foi associada ao início precoce de atividade sexual.

Introduction: the Human Papillomavirus (HPV) infection is the most frequent sexually transmitted disease in the world and has many clinical manifestations,from asymptomatic infection to invasive carcinoma. At least 50% of sexually active people will have this infection in their life. Objective:to determine the prevalence of HPV and identify the frequency of high risk and low risk virus groups in women attending gynecologic routine as wellas evaluate the association between the presence of these viruses and some risk factors. Methods: this is a cross-sectional study. A hundred randomized selected women who attended the gynecologic service for routine test were evaluated. All of them had cervical specimens tested for DNA-HPV by the Hybrid Capture II assay and answered a questionnaire to acess risk factors. Results: twenty-one (21%) out of 100 analyzed women showed positivity for DNA-HPV. High risk HPV was detected in 71% and low risk HPV in 52% of positive specimens, including mixed infections. Infection with high risk types was associated with younger age at first sexual intercourse. Other evaluated risk factors such as age, level of education, number of sexual partners, parity, use of oral contraceptives, sexually transmitted disease and smoking did not show relation to HPV infection. Conclusion: the prevalence of HPV in the studied population was 21%, with 71% of high risk virus and 52% of low risk types, including mixed infections. High risk HPV detection was associated with younger age at first sexual intercourse.

High proportion of hepatitis C virus genotypes 1 and 3 in a large cohort of patients from Southern Brazil.

Hepatitis C virus (HCV) isolates have been divided into six genotypes (1 to 6). The duration of hepatitis C standard treatment is 48 weeks for patients infected with HCV genotype 1 vs 24 weeks for those infected with genotypes 2 and 3. A total of 1544 HCV isolates from chronic patients living in the southern Brazilian states of Rio Grande do Sul (RS, n=627) and Santa Catarina (SC, n=917) were genotyped by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. In RS, 338 (53.9%; 95% CI 50.0-57.8%), 34 (5.4%; 95% CI 3.8-7.4%) and, 255 (40.7%; 95% CI 36.9-44.6%) samples were from genotypes 1, 2, and 3, respectively. In SC, 468 (51%; 95% CI 47.8-54.2%), 26 (2.9%; 95% CI 1.9-4.1%) and, 423 (46.1%; 95% CI 42.9-49.3%) samples were from genotypes 1, 2, and 3, respectively. Genotyping results were confirmed by direct nucleotide sequencing of PCR products derived from 68 samples, without any discrepancy between PCR-RFLP and nucleotide sequencing methods. In conclusion, almost half of the hepatitis C patients from South of Brazil are infected by genotypes 2 and 3 and, these results have important consequential therapeutic implications as they can be treated for only 24 weeks, not 48.

High proportion of hepatitis C virus genotypes 1 and 3 in a large cohort of patients from Southern Brazil

Hepatitis C virus (HCV) isolates have been divided into six genotypes (1 to 6). The duration of hepatitis C standard treatment is 48 weeks for patients infected with HCV genotype 1 vs 24 weeks for those infected with genotypes 2 and 3. A total of 1544 HCV isolates from chronic patients living in the southern Brazilian states of Rio Grande do Sul (RS, n = 627) and Santa Catarina (SC, n = 917) were genotyped by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. In RS, 338 (53.9 percent; 95 percent CI 50.0 - 57.8 percent), 34 (5.4 percent; 95 percent CI 3.8 - 7.4 percent) and, 255 (40.7 percent; 95 percent CI 36.9 - 44.6 percent) samples were from genotypes 1, 2, and 3, respectively. In SC, 468 (51 percent; 95 percent CI 47.8 - 54.2 percent), 26 (2.9 percent; 95 percent CI 1.9 - 4.1 percent) and, 423 (46.1 percent; 95 percent CI 42.9 - 49.3 percent) samples were from genotypes 1, 2, and 3, respectively. Genotyping results were confirmed by direct nucleotide sequencing of PCR products derived from 68 samples, without any discrepancy between PCR-RFLP and nucleotide sequencing methods. In conclusion, almost half of the hepatitis C patients from South of Brazil are infected by genotypes 2 and 3 and, these results have important consequential therapeutic implications as they can be treated for only 24 weeks, not 48.