Genetic analysis of osteogenesis imperfecta in a large Brazilian cohort.

INTRODUCTION: Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder caused by disruption of type I collagen synthesis. Previous Brazilian molecular OI studies have been restricted to case reports or small cohorts. The Brazilian OI Network (BOIN) is a multicenter study collecting clinical OI treatment data from five reference centers in three regions of Brazil. OBJECTIVE: To describe the molecular analysis of a large cohort of OI registered at BOIN. METHODS: Targeted next-generation sequencing (NGS) was performed at a centralized laboratory with the Ion Torrent platform, covering 99.6 % of the coding regions of 18 OI-associated genes. Clinical information was obtained from a clinical database. RESULTS: We included 156 subjects in the molecular analyses. Variants were detected in 121 subjects: 65 (53.7 %) in COL1A1, 42 (34.7 %) in COL1A2, 2 (1.7 %) in IFITM5, one (0.8 %) in CRTAP, three (2.5 %) in P3H1, two (1.7 %) in PPIB, four (3.3 %) FKBP10, one (0.8 %) in SERPINH1, and one (0.8 %) in TMEM38B. Ninety-one distinct variants were identified, of which 26 were novel. Of the 107 variants identified in COL1A1 and COL1A2, 24.5 % cause mild OI, while the remaining 75.5 % cause moderate, severe, or lethal OI, of which 49.3 % are glycine to serine substitutions. A single variant in FKBP10 (c.179A>C; p.Gln60Pro) was found in three unrelated and non-consanguineous participants living in the same geographic area in Northeast Brazil, suggesting a possible founder effect. CONCLUSION: Consistent with the literature, 88.4 % of the subjects had a variant in the COL1A1 and COL1A2 genes, with 10 % inherited in an autosomal recessive manner. Notably, one variant in FKBP10 with a potential founder effect requires further investigation. Data from this large cohort improves our understanding of genotype-phenotype correlations for OI in Brazil.

A Multicentric Brazilian Investigative Study of Copy Number Variations in Patients with Congenital Anomalies and Intellectual Disability.

Genomic imbalances are the most common cause of congenital anomalies (CA) and intellectual disability (ID). The aims of this study were to identify copy number variations (CNVs) in 416 patients with CA and ID from 5 different genetics centers within 4 different states by using the Multiplex Ligation-dependent Probe Amplification (MLPA) technique and to apply the chromosomal microarray (CMA) methodology in selected cases. The samples were analyzed by MLPA kits P064, P036, P070 and P250. Positive results were found in 97/416 (23.3%) patients. CMA was applied in 14 selected cases. In 6/14 (42.85%) patients, CMA detected other copy number variations not detected by the MLPA studies. Although CMA is indispensable for genotype refinement, the technique is still unfeasible in some countries as a routine analysis due to economic and technical limitations. In these cases, clinical evaluation followed by karyotyping and MLPA analysis is a helpful and affordable solution for diagnostic purposes.

Mucopolysaccharidosis type II: identification of 30 novel mutations among Latin American patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Mucolipidosis II and III alpha/beta in Brazil: analysis of the GNPTAB gene.

UNLABELLED: Mucolipidosis II and III (MLII and MLIII) alpha/beta are rare autosomal recessive lysosomal storage diseases (LSDs) caused by pathogenic variations in the GNPTAB gene. GNPTAB gene codes for the α and ß subunits of phosphotransferase, the enzyme responsible for synthesis of the mannose-6-phosphate (M6P) marker that directs lysosomal enzymes to the lysosome. OBJECTIVES: The objective of this study is to identify sequence variations of the GNPTAB gene in Brazilian patients with MLII and MLIII alpha/beta. METHOD: Sequencing of the GNPTAB gene was performed in samples of gDNA extracted from the peripheral blood of patients with MLII/III diagnosed at a national reference center for LSDs. RESULTS: Twelve unrelated patients, from several regions of Brazil, were included in this study. Only one was born of consanguineous parents. All patients were found to carry at least one nonpathogenic variation. Nine causal sequence variations were found: c.242G>T (p.W81L); c.1123C>T (p.R375X); c.1196C>T (p.S399F); c.1208T>C (p.I403T); c.1514G>A (p.C505Y); c.1759C>T (p.R587X); c.2808A>G (p.Y937_M972del, novel mutation); c. 2269_2273delGAAAC (p.E757KfsX2, novel mutation); and c.3503_3504delTC (p.L1168QfsX5). Both pathogenic variations were identified in 8 of 12 patients; in four patients, only one pathogenic variation was identified. Mutation c.3503_3504delTC, located in exon 19, was the most frequent pathogenic variation found (n=11/24 alleles). The deleterious effect of the c.2808A>C mutation on splicing was confirmed by cDNA analysis. DISCUSSION/CONCLUSIONS: Our findings confirm that the GNPTAB gene presents broad allelic heterogeneity and suggests that, in Brazilian ML II and III patients, screening for mutations should begin at exon 19 of the GNPTAB gene. Further analyses will be conducted on patients in whom both pathogenic mutations have not been found in this study.

Delivering genetic education and genetic counseling for rare diseases in rural Brazil.

Brazil is the largest country in Latin America, with an ethnically diverse, Portuguese-speaking and predominantly Roman Catholic population of some 194 million. Universal health care is provided under the Federal Unified Health System (Sistema Único de Saúde) but, as in many other middle and low income countries, access to medical genetics services is limited in rural and remote regions of the country. Since there is no formally recognized Genetic Counseling profession, genetic counseling is provided by physicians, trained either in medical genetics or a related clinical discipline. A comprehensive medical genetics program has been established in Monte Santo, an inland rural community located in the state of Bahia in Northeast Brazil, with high prevalences of a number of autosomal recessive genetic disorders, including non-syndromic deafness, phenyketonuria, congenital hypothyroidism and mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). Genetic education, counseling and treatment are locally provided, with a neonatal screening program for MPSVI currently under trial.

Types of marriages, population structure and genetic disease.

A high occurrence rate of consanguineous marriages may favour the onset and increased frequency of autosomal recessive diseases in a population. The population of Monte Santo, Bahia, Brazil, has a high frequency of rare genetic diseases such as mucopolysaccharidosis type VI, whose observed frequency in this population is 1:5000, while the incidence of this disease recorded in other regions of the world varies from 1:43,261 in Turkey to 1:1,505,160 in Switzerland. To verify the influence of consanguineous marriage on the increased frequency of observed genetic diseases in this population, the population structure and frequency of different types of marriage during different time periods were evaluated. A total of 9765 marriages were found in an analysis of parish marriage records from the city. Over three periods, 1860-1895, 1950-1961 and 1975-2010, the inbreeding rates were 37.1%, 13.2% and 4.2% respectively. Although there was a high rate of inbreeding, endogamic marriages were the dominant marriage type in all three periods. In the most recent period, there was an increase in the number of exogamous marriages and those among immigrants, but most of these occurred among individuals from cities that neighbour Monte Santo. The low rate of migration and high frequency of endogamic and consanguineous marriages show that growth of this population is predominantly internal and could explain the occurrence, and increase in frequency, of recessive genetic diseases in the city.

Distribution of SDF1-3'A polymorphisms in three different ethnic groups from Brazil.

A mutation described as a G-to-A transition has been reported in SDF-1 gene (SDF1-3'A), being prevalent in all ethnic groups, except in Africans. This mutation is associated with the onset of AIDS progression. Our aim was to identify the frequency of this allele in different groups from Brazil: Tiriyó and Waiampi Amerindian tribes (Asian ancestry); selected blood donors from Joinville (German descendents); and from Salvador (predominance of African and Portuguese mixture). SDF1-3'A was screened by PCR/RFLP with MspI enzyme. Our results showed a high allelic frequency in Tiriyó tribe (0.24) and Joinville population (0.21), and a frequency of 0.17 and 0.05 in the Salvador population and in the Waiampi tribe, respectively. There was no statistical difference among the allelic frequencies in the studied ethnic groups, except in the Waiampi. Due to the great genetic diversity among Brazilian population and the lack of studies on SDF1-3'A allele, our study of this allelic frequency in these different Brazilian ethnic groups could be important to identification of biomarker for therapeutic support in progression to AIDS and a molecular marker for analysis of evolutionary relationships among human populations.

Clinical and biochemical studies in mucopolysaccharidosis type II carriers.

The aim of the study was to characterize clinically and biochemically mucopolysaccharidosis type II (MPS II) heterozygotes. Fifty-two women at risk to be a carrier, with a mean age of 34.1 years (range 16-57 years), were evaluated through pedigree analysis, medical history, physical examination, measurement of iduronate sulfatase (IDS) activities in plasma and in leukocytes, quantification of glycosaminoglycans (GAGs) in urine, and analysis of the IDS gene. Eligibility criteria for the study also included being 16 years of age or older and being enrolled in a genetic counselling programme. The pedigree and DNA analyses allowed the identification of 40/52 carriers and 12/52 non-carriers. All women evaluated were clinically healthy, and their levels of urinary GAGs were within normal limits. Median plasma and leukocyte IDS activities found among carriers were significantly lower than the values found for non-carriers; there was, however, an overlap between carriers' and non-carriers' values. Our data suggests that MPS II carriers show lower plasma and leukocyte IDS activities but that this reduction is generally associated neither with changes in levels of urinary GAGs nor with the occurrence of clinical manifestations.

A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics.

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU). We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis. Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein. Detailed kinetic analyses of PAH expressed in E. coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein. Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000. This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency. Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin.

Ten novel mutations in the phenylalanine hydroxylase gene (PAH) observed in Brazilian patients with phenylketonuria.

In the present study we report on the identification of ten novel mutations in the phenylalanine hydroxylase (PAH) gene of Brazilian patients with phenylketonuria (PKU): IVS5-54A>G, IVS6+17G>T, E205A, F240S, K274E, I318T, L321L, C357G, IVS11+17G>A and S411X. These mutations were detected during the characterization of the PAH genotypes of 115 patients with PKU from the southeast region of Brazil. The results obtained confirm the high heterogeneity of the PAH gene and provide information about the distribution of PKU mutations in the Brazilian population.

Raine dysplasia: a Brazilian case with a mild radiological involvement.

We report a preterm male infant, the first child of a young consanguineous couple, whose physical examination revealed craniofacial disproportion with microcephaly, wide fontanelles, exophthalmos, low nasal root and hypoplastic nose, long philtrum, small mouth, high arched and narrow palate, micrognathia, dysplastic, low-set and rounded ears, short neck and, arthrogryposis. Postmortem findings included hypoplastic lungs. Radiological examinations showed mild and localized increased of bone density in the cranial vault and skull base and facial bones and undermodelled in the long bones. The above findings are characteristics of Raine dysplasia but the case reported here presents a mild bone involvement with only a localized bone sclerosis and absence of prenatal fractures. We discuss the possibility that this case represents an allelic mutation of the Raine gene. The consanguinity of the parents reinforces the hypothesis of autosomal recessive inheritance for this entity.

Syndrome of psychomotor retardation, bulbous nose, and epilepsy (Hernandez syndrome): a Brazilian case.

A new case of Hernandez syndrome is described in a 16-year-old Brazilian girl. The syndrome consists mainly of psychomotor retardation, epilepsy, a bulbous nose and obesity.

A viable fetus presenting 68, XX[73]/69, XXX[27] triploid mosaicism

A triploidia é uma anomalia cromossômica comum encontrada em 1 a 2 por cento das gestaçöes clinicamente reconhecidas e em cerca de 15 a 20 por cento dos abortos espontâneos de causa cromossômica. Em aproximadamente 5 por cento dos casos, uma aneuploidia pode estar também associada (Boué et al., 1985). Descrevemos um recém-nascido do sexo feminino, prematuro (30 semanas de idade gestacional), com microcefalia, dismorfias faciais e alteraçöes de membros, que foi a óbito com 1 dia de vida por insuficiência respiratória. O exame anátomo-patológico da placenta revelou alteraçöes compatíveis com degeneraçäo molar. A necrópsia da criança näo evidenciou malformaçöes internas. A análise citogenética de 100 metástases, obtidas a partir de cultura de tecido renal, evidenciou cariótipo 68,XX[73]/69,XXX[27]. Apenas 9 casos de triploidia 68,XX foram descritos anteriormente, sendo 7 em abortos, 1 em feto de 21 semanas e 1 em recém-nascido a termo. Consideramos que este estudo seja o primeiro da literatura relatando a ocorrência de mosaicismo 69,XXX/68,XX em um recém-nascido vivo. Os autores discutem os achados clínicos e os possíveis mecanismos envolvidos nesta aberraçäo cromossômica.