Karina Lezirovitz

Prevalence of GJB2 (connexin-26) and GJB6 (connexin-30) mutations in a cohort of 300 Brazilian hearing-impaired individuals: implications for diagnosis and genetic counseling.

Objective: Hereditary nonsyndromic deafness is an autosomal recessive condition in about 80% of cases, and point mutations in the GJB2 gene (connexin 26) and two deletions in the GJB6 gene (connexin 30), del(GJB6-D13S1830) and del(GJB6-D13S1854), are reported to account for 50% of recessive deafness. Aiming at establishing the frequencies of GJB2 mutations and GJB6 deletions in the Brazilian population, we screened 300 unrelated individuals with hearing impairment, who were not affected by known deafness related syndromes. Methods: We firstly screened the most frequently reported mutations, c.35delG and c.167delT in the GJB2 gene, and del(GJB6-D13S1830) and del(GJB6-D13S1854) in the GJB6 gene, through specific techniques. The detected c.35delG and c.167delT mutations were validated by sequencing. Other mutations in the GJB2 gene were screened by single-strand conformation polymorphism and the coding region was sequenced when abnormal patterns were found. Results: Pathogenic mutations in GJB2 and GJB6 genes were detected in 41 individuals (13.7%), and 80.5% (33/41) presented these mutations in homozygosis or compound heterozygosis, thus explaining their hearing defect. The c.35delG in the GJB2 gene was the most frequent mutation (37/300; 12.4%), detected in 23% familial and 6.2% the sporadic cases. The second most frequent mutation (1%; 3/300) was the del(GJB6-D13S1830), always found associated with the c.35delG mutation. Nineteen different sequence variations were found in the GJB2 gene. In addition to the c.35delG mutation, nine known pathogenic alterations were detected c.167delT, p.Trp24X, p.Val37Ile, c.176_191del16, c.235delC, p.Leu90Pro, p.Arg127His, c.509insA, and p.Arg184Pro. Five substitutions had been previously considered benign polymorphisms: c.-15C>T, p.Val27Ile, p.Met34Thr, p.Ala40Ala, and p.Gly160Ser. Two previously reported mutations of unknown pathogenicity were found (p.Lys168Arg, and c.684C>A), and two novel substitutions, p.Leu81Val (c.G241C) and p.Met195Val (c.A583G), both in heterozygosis without an accompanying mutation in the other allele. None of these latter four variants of undefined status was present in a sample of 100 hearing controls. Conclusions: The present study demonstrates that mutations in the GJB2 gene and del(GJB6 D13S1830) are important causes of hearing impairment in Brazil, thus justifying their screening in a routine basis. The diversity of variants in our sample reflects the ethnic heterogeneity of the Brazilian population.

Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion

Background Split-hand/foot malformation (SHFM)—also known as ectrodactyly—is a congenital disorder characterised by severe malformations of the distal limbs affecting the central rays of hands and/or feet. A distinct entity termed SHFLD presents with SHFM and long bone deficiency. Mouse models suggest that a defect of the central apical ectodermal ridge leads to the phenotype. Although six different loci/mutations (SHFM1–6) have been associated with SHFM, the underlying cause in a large number of cases is still unresolved. Methods High resolution array comparative genomic hybridisation (CGH) was performed in patients with SHFLD to detect copy number changes. Candidate genes were further evaluated for expression and function during limb development by whole mount in situ hybridisation and morpholino knock-down experiments. Results Array CGH showed microduplications on chromosome 17p13.3, a locus previously associated with SHFLD. Detailed analysis of 17 families revealed that this copy number variation serves as a susceptibility factor for a highly variable phenotype with reduced penetrance, particularly in females. Compared to other known causes for SHFLD 17p duplications appear to be the most frequent cause of SHFLD. A ∼11.8 kb minimal critical region was identified encompassing a single gene, BHLHA9, a putative basic loop helix transcription factor. Whole mount in situ hybridisation showed expression restricted to the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock down of bhlha9 in zebrafish resulted in shortening of the pectoral fins. Conclusions Genomic duplications encompassing BHLHA9 are associated with SHFLD and non-Mendelian inheritance characterised by a high degree of non-penetrance with sex bias. Knock-down of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, indicating a role for this gene in limb development.

Spastic Paraplegia, Optic Atrophy, and Neuropathy Is Linked to Chromosome 11q13

We report an autosomal recessive neurodegenerative disorder in 25 white members from a large inbred Brazilian family, 22 of whom were evaluated clinically. This condition is characterized by (1) subnormal vision secondary to apparently nonprogressive congenital optic atrophy; (2) onset of progressive spastic paraplegia in infancy; (3) onset of progressive motor and sensory axonal neuropathy in late childhood/early adolescence; (4) dysarthria starting in the third decade of life; (5) exacerbated acoustic startle response; and (6) progressive joint contractures and spine deformities. Motor handicap was severe, and all patients were wheelchair bound after 15 years old. We performed a genome‐wide screen including 25 affected individuals and 49 of their unaffected relatives. Linkage was detected at 11q13 region with a maximum logarithm of odds score of +14.43, obtained with marker D11S1883. The candidate region, which lies between D11S1908 and D11S1889, encompasses ∼4.8Mb and has more than 100 genes and expressed sequences. We propose the acronym SPOAN (spastic paraplegia, optic atrophy, and neuropathy) for this complex syndrome. Ann Neurol 2005;57:730–737

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.

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.

Spastic Paraplegia, Optic Atrophy, and Neuropathy: New Observations, Locus Refinement, and Exclusion of Candidate Genes

SPOAN is an autosomal recessive neurodegenerative disorder which was recently characterized by our group in a large inbred Brazilian family with 25 affected individuals. This condition is clinically defined by: 1. congenital optic atrophy; 2. progressive spastic paraplegia with onset in infancy; and 3. progressive motor and sensory axonal neuropathy. Overall, we are now aware of 68 SPOAN patients (45 females and 23 males, with age ranging from 5 to 72 years), 44 of which are presented here for the first time. They were all born in the same geographic micro region. Those 68 patients belong to 43 sibships, 40 of which exhibit parental consanguinity. Sixty‐one patients were fully clinically evaluated and 64 were included in the genetic investigation. All molecularly studied patients are homozygotes for D11S1889 at 11q13. This enabled us to reduce the critical region for the SPOAN gene from 4.8 to 2.3 Mb, with a maximum two point lod score of 33.2 (with marker D11S987) and of 27.0 (with marker D11S1889). Three genes located in this newly defined critical region were sequenced, but no pathogenic mutation was detected. The gene responsible for SPOAN remains elusive.

A novel locus for split-hand/foot malformation associated with tibial hemimelia (SHFLD syndrome) maps to chromosome region 17p13.1-17p13.3

Split-hand/foot malformation (SHFM) associated with aplasia of long bones, SHFLD syndrome or Tibial hemimelia-ectrodactyly syndrome is a rare condition with autosomal dominant inheritance, reduced penetrance and an incidence estimated to be about 1 in 1,000,000 liveborns. To date, three chromosomal regions have been reported as strong candidates for harboring SHFLD syndrome genes: 1q42.2–q43, 6q14.1 and 2q14.2. We characterized the phenotype of nine affected individuals from a large family with the aim of mapping the causative gene. Among the nine affected patients, four had only SHFM of the hands and no tibial defects, three had both defects and two had only unilateral tibial hemimelia. In keeping with previous publications of this and other families, there was clear evidence of both variable expression and incomplete penetrance, the latter bearing hallmarks of anticipation. Segregation analysis and multipoint Lod scores calculations (maximum Lod score of 5.03 using the LINKMAP software) using all potentially informative family members, both affected and unaffected, identified the chromosomal region 17p13.1–17p13.3 as the best and only candidate for harboring a novel mutated gene responsible for the syndrome in this family. The candidate gene CRK located within this region was sequenced but no pathogenic mutation was detected.

Reevaluation of a large family defines a new locus for X-linked recessive pure spastic paraplegia (SPG34) on chromosome Xq25

Herein, we report a new locus, named SPG34, for a pure form of X-linked hereditary spastic paraplegia (HSP) in a large Brazilian family followed by our group since 1976 [1]. In 2002, a study of seven patients suggested linkage to Xq22.2 [2]. We now were able to perform a more comprehensive clinical and molecular evaluation, including five patients not previously ascertained, and reassigned the putative locus to Xq25. After Institutional Review Board approval, we genotyped 12 affected individuals (aged 24 to 79 years), one unaffected 60-year-old man, and 11 women (aged 40 to 81), seven of which were obligate carriers (Fig. 1). Neurological examination was performed in 11 of the 12 affected men and in all obligate women carriers. Age of onset varied from 12 to 25 years but was sometimes difficult to be determined. The clinical phenotype was stereotyped, and shuffling gait was the first recognized clinical sign. The disease was invariably progressive, and after two decades of onset, patients usually need support to walk; after three to four decades, they are usually wheelchair-bound. In upper limbs, strength was never affected, even late in life, but tendon reflexes were brisk. Lower limbs spasticity was progressive and debilitating. Babinski sign, ankle clonus, and brisk reflexes were frequently present. Lower limb vibratory sensibility was commonly reduced after the sixth decade of life; spontaneous lower limb pain was also a common complaint. No urinary or bowel sphincter dysfunction were ever reported. The SPG34 locus encompasses a 14 cM region at Xq24– q25, in which 69 genes and bona fide transcripts have been assigned. Using a candidate gene approach to try to identify the causative sequence abnormality of SPG34, we fully sequenced AIFM1, which encodes a mitochondrial flavoprotein essential for apoptotic nuclear disassembly [3], but no mutation was detected. Therefore, the molecular basis for SPG34 remains unknown and we will sequence additional candidate genes that are highly expressed in the central nervous system. The calculated logarithm of the odds score of 4.13 at marker DXS8057 was much higher than in the previous study [2], and additional markers definitely excluded Xq22.2. No other X-linked HSP have been so far assigned to this region, which defines the fourth locus for X-linked HSP. Differently from the other two well-characterized X-linked HSP, SPG1 and SPG2 [4, 5], SPG34 is not associated to mental retardation. Evidences for the SPG16 [6] locus, located in a large region at Xq11.2–q23 overlapping SPG2 locus, are weaker and based only in two small families. In short, we identified in a large multigenerational family with pure spastic paraplegia a new locus (named SPG34) at Xq25 for an X-linked pure form of HSP, with onset in the Neurogenetics (2008) 9:225–226 DOI 10.1007/s10048-008-0130-8

Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea.

BackgroundCulturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.MethodsOrgans of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα). Immunofluorescence assays were conducted for phenotype characterization.ResultsThe TGFα group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells.ConclusionsDissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.

Is autosomal recessive deafness associated with oculocutaneous albinism a “coincidence syndrome”?

AbstractHearing impairment is frequently found associated with pigmentary disorders in many syndromes. However, total oculocutaneous albinism (OCA) associated with deafness has been described only once, by Ziprkowski and Adam (Arch Dermatol 89:151-155, 1964) in an inbred family. A syndrome associating deafness and OCA was suggested by the authors, but two separate recessive genes segregating in this inbred group were also proposed later by Fraser (OMIM # 220900). Combined deafness and total OCA were also observed by us in a family originally reported to be nonconsanguineous but in which haplotyping showed evidence of a common ancestry: the proband was affected by both diseases, one of his sisters had only OCA and another sister had only deafness. Both the proband and his deaf sister were found to be homozygotes for the 35delG mutation (GJB2 gene), the most frequent cause of hereditary deafness. Linkage analysis with markers close to the four known OCA loci excluded linkage to OCA1, OCA2, and OCA3, and homozygosity in markers near OCA4 locus was observed. Sequencing of the corresponding gene (MATP) revealed a c.1121delT mutation, which leads to a stop codon at position 397 (L374fsX397). Clearly, the combined occurrence of deafness and albinism in this pedigree was due to mutations in two different genes, showing autosomal recessive inheritance. We speculate that the putative syndrome reported by Ziprkowski and Adam might have resulted from the co-occurrence of autosomal recessive deafness and albinism in the same pedigree, as suggested by Fraser.