Maria Teresa Balester de Mello Auricchio

Novel OTOF mutations in Brazilian patients with auditory neuropathy.

The OTOF gene encoding otoferlin is associated with auditory neuropathy (AN), a type of non-syndromic deafness. We investigated the contribution of OTOF mutations to AN and to non-syndromic recessive deafness in Brazil. A test for the Q829X mutation was carried out on a sample of 342 unrelated individuals with non-syndromic hearing loss, but none presented this mutation. We selected 48 cases suggestive of autosomal recessive inheritance, plus four familial and seven isolated cases of AN, for genotyping of five microsatellite markers linked to the OTOF gene. The haplotype analysis showed compatibility with linkage in 11 families (including the four families with AN). Samples of the 11 probands from these families and from seven isolated cases of AN were selected for an exon-by-exon screening for mutations in the OTOF gene. Ten different pathogenic variants were detected, among which six are novel. Among the 52 pedigrees with autosomal recessive inheritance (including four familial cases of AN), mutations were identified in 4 (7.7%). Among the 11 probands with AN, seven had at least one pathogenic mutation in the OTOF gene. Mutations in the OTOF gene are frequent causes of AN in Brazil and our results confirm that they are spread worldwide.

Multilocus Analyses of Seven Candidate Genes Suggest Interacting Pathways for Obesity‐Related Traits in Brazilian Populations

We investigated whether variants in major candidate genes for food intake and body weight regulation contribute to obesity‐related traits under a multilocus perspective. We studied 375 Brazilian subjects from partially isolated African‐derived populations (quilombos). Seven variants displaying conflicting results in previous reports and supposedly implicated in the susceptibility of obesity‐related phenotypes were investigated: β2‐adrenergic receptor (ADRB2) (Arg16Gly), insulin induced gene 2 (INSIG2) (rs7566605), leptin (LEP) (A19G), LEP receptor (LEPR) (Gln223Arg), perilipin (PLIN) (6209T > C), peroxisome proliferator‐activated receptor‐γ (PPARG) (Pro12Ala), and resistin (RETN) (−420C > G). Regression models as well as generalized multifactor dimensionality reduction (GMDR) were employed to test the contribution of individual effects and higher‐order interactions to BMI and waist‐hip ratio (WHR) variation and risk of overweight/obesity. The best multilocus association signal identified in the quilombos was further examined in an independent sample of 334 Brazilian subjects of European ancestry. In quilombos, only the PPARG polymorphism displayed significant individual effects (WHR variation, P = 0.028). No association was observed either with the risk of overweight/obesity (BMI ≥ 25 kg/m2), risk of obesity alone (BMI ≥ 30 kg/m2) or BMI variation. However, GMDR analyses revealed an interaction between the LEPR and ADRB2 polymorphisms (P = 0.009) as well as a third‐order effect involving the latter two variants plus INSIG2 (P = 0.034) with overweight/obesity. Assessment of the LEPR‐ADRB2 interaction in the second sample indicated a marginally significant association (P = 0.0724), which was further verified to be limited to men (P = 0.0118). Together, our findings suggest evidence for a two‐locus interaction between the LEPR Gln223Arg and ADRB2 Arg16Gly variants in the risk of overweight/obesity, and highlight further the importance of multilocus effects in the genetic component of obesity.

Unexpected genetic heterogeneity in a large consanguineous Brazilian pedigree presenting deafness

Nonsyndromic autosomal recessive deafness accounts for 80% of hereditary deafness. To date, 52 loci responsible for autosomal recessive deafness have been mapped and 24 genes identified. Here, we report a large inbred Brazilian pedigree with 26 subjects affected by prelingual deafness. Given the extensive consanguinity found in this pedigree, the most probable pattern of inheritance is autosomal recessive. However, our linkage and mutational analysis revealed, instead of an expected homozygous mutation in a single gene, two different mutant alleles and a possible third undetected mutant allele in the MYO15A gene (DFNB3 locus), as well as evidence for other causes for deafness in the same pedigree. Among the 26 affected subjects, 15 were homozygous for the novel c.10573delA mutation in the MYO15A gene, 5 were compound heterozygous for the mutation c.10573delA and the novel deletion c.9957_9960delTGAC and one inherited only a single c.10573delA mutant allele, while the other one could not be identified. Given the extensive consanguinity of the pedigree, there might be at least one more deafness locus segregating to explain the condition in some of the subjects whose deafness is not clearly associated with MYO15A mutations, although overlooked environmental causes could not be ruled out. Our findings illustrate a high level of etiological heterogeneity for deafness in the family and highlight some of the pitfalls of genetic analysis of large genes in extended pedigrees, when homozygosity for a single mutant allele is expected.

The search of a genetic basis for noise-induced hearing loss (NIHL)

Background and aim: Knowledge about the genetic factors responsible for noise-induced hearing loss (NIHL) is still limited. This study investigated whether genetic factors are associated or not to susceptibility to NIHL. Subjects and methods: The family history and genotypes were studied for candidate genes in 107 individuals with NIHL, 44 with other causes of hearing impairment and 104 controls. Mutations frequently found among deaf individuals were investigated (35delG, 167delT in GJB2, Δ_rm;(GJB6- D13S1830), Δ_rm;(GJB6- D13S1854) in GJB6 and A1555G in MT-RNR1 genes); allelic and genotypic frequencies were also determined at the SNP rs877098 in DFNB1, of deletions of GSTM1 and GSTT1 and sequence variants in both MTRNR1 and MTTS1 genes, as well as mitochondrial haplogroups. Results: When those with NIHL were compared with the control group, a significant increase was detected in the number of relatives affected by hearing impairment, of the genotype corresponding to the presence of both GSTM1 and GSTT1 enzymes and of cases with mitochondrial haplogroup L1. Conclusion: The findings suggest effects of familial history of hearing loss, of GSTT1 and GSTM1 enzymes and of mitochondrial haplogroup L1 on the risk of NIHL. This study also described novel sequence variants of MTRNR1 and MTTS1 genes.

Frequency and Origins of Hemoglobin S Mutation in African-Derived Brazilian Populations

ABSTRACT Africans arrived in Brazil as slaves in great numbers, mainly after 1550. Before the abolition of slavery in Brazil in 1888, many communities, called quilombos, were formed by runaway or abandoned African slaves. These communities are presently referred to as remnants of quilombos, and many are still partially genetically isolated. These remnants can be regarded as relicts of the original African genetic contribution to the Brazilian population. In this study we assessed frequencies and probable geographic origins of hemoglobin S (HBB*S) mutations in remnants of quilombo populations in the Ribeira River valley, São Paulo, Brazil, to reconstruct the history of African- derived populations in the region. We screened for HBB*S mutations in 11 quilombo populations (1,058 samples) and found HBB*S carrier frequencies that ranged from 0% to 14%. We analyzed β-globin gene cluster haplotypes linked to the HBB*S mutation in 86 chromosomes and found the four known African haplotypes: 70 (81.4%) Bantu (Central Africa Republic), 7 (8.1%) Benin, 7 (8.1%) Senegal, and 2 (2.3%) Cameroon haplotypes. One sickle cell homozygote was Bantu/Bantu and two homozygotes had Bantu/Benin combinations. The high frequency of the sickle cell trait and the diversity of HBB*S linked haplotypes indicate that Brazilian remnants of quilombos are interesting repositories of genetic diversity present in the ancestral African populations.

Genomic ancestry of rural African-derived populations from Southeastern Brazil

xMany Africans were brought to Brazil as slaves. The runaway or abandoned slaves founded isolated communities named quilombos. There are many quilombo remnants in Vale do Ribeira region in the southern part of São Paulo State. The aim of our study was to contribute to understanding the origins of these populations, through admixture studies.

Multilocus Family-Based Association Analysis of Seven Candidate Polymorphisms with Essential Hypertension in an African-Derived Semi-Isolated Brazilian Population

Background. It has been widely suggested that analyses considering multilocus effects would be crucial to characterize the relationship between gene variability and essential hypertension (EH). Objective. To test for the presence of multilocus effects between/among seven polymorphisms (six genes) on blood pressure-related traits in African-derived semi-isolated Brazilian populations (quilombos). Methods. Analyses were carried out using a family-based design in a sample of 652 participants (97 families). Seven variants were investigated: ACE (rs1799752), AGT (rs669), ADD2 (rs3755351), NOS3 (rs1799983), GNB3 (rs5441 and rs5443), and GRK4 (rs1801058). Sensitivity analyses were further performed under a case-control design with unrelated participants only. Results. None of the investigated variants were associated individually with both systolic and diastolic BP levels (SBP and DBP, respectively) or EH (as a binary outcome). Multifactor dimensionality reduction-based techniques revealed a marginal association of the combined effect of both GNB3 variants on DBP levels in a family-based design (P = 0.040), whereas a putative NOS3-GRK4 interaction also in relation to DBP levels was observed in the case-control design only (P = 0.004). Conclusion. Our results provide limited support for the hypothesis of multilocus effects between/among the studied variants on blood pressure in quilombos. Further larger studies are needed to validate our findings.

AGG interspersion patterns in the CGG repeat of the FMR1 gene and linked DXS548/FRAXAC1 haplotypes in Brazilian populations

We have recently described in this journal the distribution of CGG repeats of the FMR1 gene, the allelic frequencies at DXS548 and FRAXAC1 microsatellite loci, and DXS548/FRAXAC1 haplotype frequencies in African-derived, European-derived, and Amerindian populations from South America [Mingroni-Netto et al., 2002]. The CGG repeat sequences and AGG interspersion patterns were not available at that time. A number of haplotype studies in affected males with the fragile X syndrome pointed to two main pathways for the origin of the fragile X mutation in European-derived populations. The two most frequent DXS548/FRAXAC1 haplotypes linked to the fragile X mutation have been found to be associated with CGG repeat structures in the general population, which would favor expansions: the haplotype 2-1 (204/158 bp) is associated to long repeats with uninterrupted CGG arrays, leading to predisposed chromosomes, which would increase in size gradually; the haplotype 6-4 (196/152 bp) appears associated to smaller repeats particularly prone to expansions because of the loss of AGG interruptions at the 30 end of the repeat, also giving rise to long uninterspersed CGG arrays [Eichler et al., 1996]. In the city of São Paulo, Brazil, we observed that these two haplotypes were the most frequent among fragile X patients [MingroniNetto et al., 1999]. More recently, Crawford et al. [2000a] found that the two most prevalent haplotypes in African-American fragile X patients were 4-4 (200/152 bp) and 3-4 (202/152 bp). In normal African-American populations, the haplotype 4-4 was linked to CGG repeats devoid of AGG interruptions near the 50 end of the repeat [Crawford et al., 2000b]. These findings suggested that the pathways leading to fragile X mutations in Africans might be distinct from those leading to mutations in Europeans. To investigate the pathways leading to fragile X mutations in Africans, we expanded our previous sample of partially isolated communities of African ancestry (quilombos) investigating the populations of Abobral, Pedro Cubas, Galvão, and São Pedro in the Ribeira River Valley, state of São Paulo, South-Easthern Brazil (Fig. 1). Runaway slaves who reached the region between 1750 and 1850 founded these communities. A predominantly European-derived sample of 55 unrelated non-carrier brothers of fragile X males, collected in the city of São Paulo, was included in the study for comparison. All individuals were genotyped for DXS548 and FRAXAC1 alleles, as described by Mingroni-Netto et al. [2002]. Allelic frequencies at the two loci are shown in Table I. The frequencies of DXS548/FRAXAC1 haplotypes are in Table II. For AGG interruption analysis, a subset of 32 AfricanBrazilian males was selected, including individuals from Pontal, Cajual, and Rio das Rãs, previously studied by Mingroni-Netto et al. [2002]. Repeats on haplotypes known to be in linkage disequilibrium with the fragile X mutation either in Europeans (2-1 and 6-4) or in African-Americans (4-4 and 34) were preferentially sequenced, as well as those associated to uncommon haplotypes in European-derived populations. The 55 alleles from São Paulo city population were all sequenced, regardless of the linked haplotypes. In brief, the CGG repeat and surrounding DNA sequences were amplified from genomic DNA by Pfx polymerase (Invitrogen, Carlsbad, CA) with primers c and f [Fu et al., 1991]. In most samples, the forward and reverse strands were sequenced. Whenever sequencing of only one strand was obtained, the pattern was validated by re-sequencing. In the instances of rare or not previously reported patterns, sequencing was performed at least twice with each primer. After purification, approximately 60 ng of PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Forster City, CA), in an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Forster City, CA). CGG repeat interspersion patterns are presented in Figure 2. Haplotype 2-1 was not found among the four AfricanBrazilian populations from the state of São Paulo. In one sample, from Rio das Rãs, [Mingroni-Netto et al., 2002], this haplotype was linked to a 23 CGG repeat with one AGG interruption at the 50 end (Fig. 2a). In the predominantly Europeanderived sample from São Paulo city, the 2-1 haplotype was associated to four alleles with high repeat numbers ranging from 39–48 repeats. The 39 repeat had three interruptions: 9þ 9þ 9þ 9 (9 representing the number of CGG’s, and the signalþ one AGG interruption). The other alleles had two AGG interruptions: 9þ 9þ 22, 9þ 9þ 23, and 9þ 9þ 28 (Fig. 2b). Haplotype 6-4 was relatively frequent in the African-derived communities (8.3%–19.6%). It was less frequent in São Paulo city (1.8%, present study and 8.3%, Mingroni-Netto et al., 2002]. This is not a rare haplotype in populations hereto studied [Chiurazzi et al., 1996; Bonaventure et al., 1998; Jara Claudia B. Angeli and Leonardo P. Capelli equally contributed to this study.

Role of the mitochondrial mutations, m.827A>G and the novel m.7462C>T, in the origin of hearing loss.

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m.1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m.827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m.7472insC mutation and three probands presented a novel variant, m.7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

A novel missense mutation p.L76P in the GJB2 gene causing nonsyndromic recessive deafness in a Brazilian family

Mutations in the GJB2 gene, encoding connexin 26 (Cx26), are a major cause of nonsyndromic recessive hearing loss in many countries. We report here on a novel point mutation in GJB2, p.L76P (c.227C>T), in compound heterozygosity with a c.35delG mutation, in two Brazilian sibs, one presenting mild and the other profound nonsyndromic neurosensorial hearing impairment. Their father, who carried a wild-type allele and a p.L76P mutation, had normal hearing. The mutation leads to the substitution of leucine (L) by proline (P) at residue 76, an evolutionarily conserved position in Cx26 as well as in other connexins. This mutation is predicted to affect the first extracellular domain (EC1) or the second transmembrane domain (TM2). EC1 is important for connexon-connexon interaction and for the control of channel voltage gating. The segregation of the c.227C>T (p.L76P) mutation together with c.35delG in this family indicates a recessive mode of inheritance. The association between the p.L76P mutation and hearing impairment is further supported by its absence in a normal hearing control group of 100 individuals, 50 European-Brazilians and 50 African-Brazilians.