Fabiana Alexandrino

Molecular genetics study of deafness in Brazil: 8-year experience.

Hereditary hearing loss is a complex disorder that involves a large number of genes. In developed countries, 1 in 1,000 children is born with deafness severe enough to require special education services, and about 60% of the cases of isolated deafness have a genetic origin. Although more than 100 genes for hearing loss are known currently, only a few are routinely tested in the clinical practice. In this study, we present our findings from the molecular diagnostic screening of the GJB2 and GJB3 genes, del(GJB6‐D13S1830) and del(GJB6‐D13S1854) deletions in the GJB6 gene, Q829X mutation in the otoferlin gene (OTOF) and, the A1555G and A7445G mutations in the mitochondrial genome over an 8‐year period. Mutations analysis in the previously mentioned genes and mutations was performed on 645 unrelated Brazilian patients with hearing loss who fell into two different testing groups. Different mutations in the GJB2 gene were responsible for most of cases studied, but deletions in the GJB6 gene as well as mitochondrial mutations were also found. While most cases of hearing loss in this country are due to environmental factors, the genetic etiology of deafness will increasingly be determined as more genetic tests become available.

Genética molecular da deficiência auditiva não-sindrômica

One in every 1,000 newborn suffers from congenital hearing impairment. More than 60% of the congenital cases are caused by genetic factors. In most cases, hearing loss is a multifactorial disorder caused by both genetic and environmental factors. Molecular genetics of deafness has experienced remarkable progress in the last decade. Genes responsible for hereditary hearing impairment are being mapped and cloned progressively. This review focuses on non-syndromic hearing loss, since the gene involved in this type of hearing loss have only recently begun to be identified.

G59S mutation in the GJB2 (connexin 26) gene in a patient with Bart–Pumphrey syndrome

The connexins are a family of proteins whose major function is as part of the gap junctions of cell-to-cell channels. They are expressed in several tissues including brain, skin, and cochlea. Mutations in connexins play a major role in nonsyndromic deafness, but have been also described in individuals with variable dermatological features [Kelsell et al., 2001]. Initially, the connexin 26 (GJB2) gene was implicated in autosomal recessive forms of hearing loss, but was later found to be involved in both recessive and dominant forms of genetic deafness [Chaib et al., 1994; Petit et al., 2001]. The most common mutation associated with recessive deafness is 35delG within the GJB2 gene [Denoyelle et al., 1997], which accounts for up 70% of the connexin 26 mutations observed in European and American Caucasian populations [Green et al., 1999; Gasparini et al., 2000]. In addition, there are other mutations such as nonsense mutations, deletions, and insertions within the GJB2 gene that result in hearing loss. Heterozygous missense mutations in GJB2 were found in several conditions associating deafness and hyperkeratosis: D66H in mutilating keratoderma with sensorineural deafness (Vohwinkel’s syndrome, OMIM 124500) [Maestrini et al., 2002], G59A and R75Q in palmoplantar hyperkeratosis with deafness (PPKD, OMIM 148350) [Heathcote et al., 2000; Uyguner et al., 2002], as well as D50Y and D50N in keratitisichthyosis-deafness syndrome (KID, OMIM 148210), and in hystrix like-ichthyosis-deafness syndrome (HID, OMIM 602540) [Richard et al., 2002; van Geel et al., 2002; Yotsumoto et al., 2003]. Richard et al. [2002] also described two sporadic cases of KID syndrome with G12R and S17F missense mutations, each patient showing a different mutation. GJB2 is contiguous to another connexin gene, GJB6, which encodes connexin 30. Together they form the DFBN1 locus. Large deletions in GJB6 extending to the GJB2 gene may result in a monogenic or digenic pattern of prelingual deafness. Missense mutations in the GJB6 gene cause hidrotic ectodermal dysplasia (Clouston syndrome), an autosomal dominant disorder characterized by hair and nail defects, palmoplantar hyperkeratosis, and other variable findings. Mutations in the GJB3 (connexin 31) gene also cause both autosomal recessive and autosomal dominant forms of nonsyndromic deafness, as well as erythrokeratodermia variabilis, an autosomal dominant genodermatosis with variable clinical presentation including hyperkeratosis. Bart–Pumphrey syndrome (BPS, OMIM 149200) is a rare autosomaldominantdisorder characterizedbycongenitaldeafness, palmoplantar hyperkeratosis, knuckle pads, and leukonychia. To date, only a few families with this disorder have been reported [Schwann, 1963; Bart and Pumphrey, 1967; Crosbyand Vidurrizaga,1976; Ramer etal., 1994; Oliveiraetal., 2003]. Richard et al. [2004] recently reported BPS mutation. We present here a 26-year-old male patient with BPS who was submitted to molecular analysis of the connexin 26, 30, and 31 genes. He presented with congenital sensorineural deafness. Verrucous-like nodes on the interphalangeal, metacarpophalangeal, and metatarsophalangeal joints of the dorsa of the hands and feet (knuckle pads, Fig. 1) as well as palmoplantar hyperkeratosis started at age 3 years. Nail shape and size were normal, and no leukonychia was observed. Anthropometrical data were within normal ranges and no signs of cognitive impairment were detected. An audiogram and a brainstem evoked response audiometry (BERA) were performed, both showing severe sensorineural hearing loss. His father showed a similar phenotype. Clinical presentation and hystologic findings of this family were previously reported by Oliveira et al. [2003]. Since the father was not willing to participate in molecular studies, blood specimens were collected only from the propositus with approval by the appropriate institutional review board and informed consent of the subject. DNA samples were extracted from whole blood by standard techniques. The GJB2 gene was PCR amplified using the two pairs of primers [Denoyelle et al., 1997; Kelsell et al., 1997], and GJB3 gene was amplified using five pairs of primers described elsewhere [Xia et al., 1998]. For mutation analysis, the single coding regions of the GJB2 and GJB3 genes were sequenced from PCR products using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI PRISM/PE Biosystems, Foster City, CA) and the products resolved on ABI PRISM 377 (Perkin Elmer, Boston, MA). In order to examine the deletion of the GJB6 gene, we amplified the breakpoint junction of the (GJB6-D13S1830)del by PCR [del Castillo et al., 2002]. Analysis and sequencing of GJB3 did not show any abnormality and the (GJB6-D13S1830)del in the GJB6 gene was not found. However, sequencing of GJB2 revealed a G to A substitution at nucleotide 175. This nucleotide substitution corresponds to a heterozygous glycine to serine change at codon 59 (G59S) (Fig. 2). This amino acid substitution was not found in more than 200 individuals with isolated deafness and in over 100 individuals without hearing loss tested in our service. This substitution was also not listed in the Human Gene Mutation Database [2004]. A mutation in the same codon (59) was reported by Heathcote et al. [2000] who proposed that G59A was responsible for the syndrome of hearing loss and hyperkeratosis observed in their study. This codon is located in the first extracellular loop (E1) of connexin 26 (Fig. 3), a region that is highly conserved among all connexins sequenced to date. Most syndromic forms of deafness-hyperkeratosis are associated with mutations in the first extracellular domain of Grant sponsor: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Molecular genetics of non-syndromic deafness

One in every 1,000 newborn suffers from congenital hearing impairment. More than 60% of the congenital cases are caused by genetic factors. In most cases, hearing loss is a multifactorial disorder caused by both genetic and environmental factors. Molecular genetics of deafness has experienced remarkable progress in the last decade. Genes responsible for hereditary hearing impairment are being mapped and cloned progressively. This review focuses on non-syndromic hearing loss, since the gene involved in this type of hearing loss have only recently begun to be identified.

Use of Escherichia coli BOX-PCR fingerprints to identify sources of fecal contamination of water bodies in the State of São Paulo, Brazil.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) is one of the commonest methods used to identify sources of fecal contamination of water systems. In this work, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) was used to discriminate Escherichia coli strains originating from different animals and water sources, and the suitability of the technique for bacterial source tracking (BST) was evaluated. A total of 214 strains from humans, 150 strains from animals, 55 strains from sewage and 77 strains from water bodies were analyzed by the BOX-PCR technique. When maximum similarity between the fingerprints was used, a correct classification rate of 84% was achieved for strains from human and animal sources. Furthermore, 95% of the strains found in sewage were classified as being from human sources by at least one of the four classification tools used. Classification of the strains found in water bodies in the State of São Paulo was based on the fingerprints obtained for human and animal sources. Most of the sampling sites appeared to be affected by mixed sources of fecal contamination. The use of BOX-PCR for BST could be especially valuable in developing countries, where simplicity and cost are important considerations.

Prospects for genetic hearing loss screening: 35delG mutation tracking in a newborn population

OBJECTIVES: To investigate the prevalence of the 35delG mutation in a newborn population, with specific molecular testing, and to evaluate the prospects for genetic neonatal screening for hearing impairment. POPULATION AND METHOD: 233 newborn were evaluated at the Hospital de Base de Sao Jose do Rio Preto, SP, for molecular analysis of the 35delG mutation in the connexin 26 gene, with the reaction technique in allele-specific polymerase chain reaction, after genomic DNA extraction from umbilical cord blood. RESULTS: Five heterozygotes were identified, obtaining a prevalence of 2.24% of 35delG mutation carriers in the study population. CONCLUSION: Using the molecular test allowed for the identification of the 35delG mutation in the study population with the possibility of being used as a complement to neonatal audiometric screening as being simple, fast, and easily to perform with low costs.

Newborn hearing screening and genetic testing in 8974 Brazilian neonates

OBJECTIVE An early diagnosis has been a priority in the audiological practice. Identifying hearing loss until 3 months old through Universal Newborn Hearing Screening and intervention before 6 months old, minimize the impact of auditory loss in the health and communication development of these children. However, in the clinical practice, despite the help of the risk indicators in the audiological and etiological diagnosis, the integrated services have come up against the challenge of determining the causes of auditory loss, bearing in mind that approximately 50% of the subjects who have congenital loss do not show risk factors in their clinical history. The current research aims introduce together etiologic and audiological diagnosis of newborns. METHODS We eluted dried blood spots from paper and performed genetic testing for 35delG mutation in 8974 newborns that were also screened for transient otoacoustic emissions (TOAE). In addition, the A1555G and A827G mutations in the MTRNR1 mitochondrial gene were screened in all newborns. RESULTS We have found 17 individuals who failed in TOAE. Among them, we detected 4 homozygous newborns for 35delG mutation and 3 individuals with A827G mutation in the MTRNR1 mitochondrial gene. The frequency of 35delG carriers was 0.94% [84/8974]. In all 17 individuals who failed in OAE no other mutation besides those mentioned above was found. CONCLUSIONS The results greatly contribute to the public health area indicating the etiologic diagnosis, allowing family counseling as well as the early rehabilitation treatment or surgical intervention. Over time that will help to reduce the costs of rehabilitation considerably.

Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment.

Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.

Connexin mutations in Brazilian patients with skin disorders with or without hearing loss.

The connexins are a family of proteins whose major function is as part of the gap junctions of cell‐to‐cell channels. They are expressed in several tissues including brain, skin, and cochlea. Mutations in connexin genes play a major role in non‐syndromic sensorineural deafness, but have been also described in individuals with variable dermatological features. In recent years, many genes responsible for hereditary skin diseases have been discovered. These genes may encode different proteins that participate in the terminal differentiation of the epidermis. Therefore alteration or absence of these proteins causes a keratinization disorder. It has been demonstrated that distinct germline mutations within six connexin (Cx) genes GJB2 (Cx26), GJB6 (Cx30), GJB3 (Cx31), GJA1 (Cx43), GJB4 (Cx30.3), and GJB5 (Cx31.1), may cause sensorineural hearing loss and various skin disease phenotypes. The crucial functional importance of each of these connexins in the mentioned ectodermic tissues is reflected by the finding that genetic defects in their genes produce a wide spectrum of genetic disorders comprising sensorineural hearing loss, disorders of cornification of the skin, hair, and nails, and keratitis. Here, we report on different mutations in the connexin genes in individuals with or without hearing loss and different skin disorders illustrating the clinical and genetic heterogeneity of the condition.

Molecular study in Brazilian cochlear implant recipients

The most common form of non‐syndromic autosomal recessive deafness (NSRD) is caused by mutations in the GJB2 gene. Recently, a deletion truncating the GJB6 gene, called del(GJB6‐D13S1830) has also been described normally accompanying mutations in another allele of the GJB2 gene. Among all the mutations described to date, 35delG in the GJB2 gene is the most common. Preliminary data suggest that pathologic changes due to GJB2 mutations do not affect the spiral ganglion cells, which are the site of stimulation of the cochlear implant. Besides, the survival of the spiral ganglion cells is believed to be an important determinant of the outcome after surgery. Therefore, we have studied 49 non‐syndromic deaf patients with unknown etiologies in order to determine the prevalence of GJB2 and GJB6 gene mutations in patients undergoing cochlear implantation surgery. Also, the molecular studies were performed using polymerase chain reaction amplification and direct sequencing. As a result, we found 19 individuals with GJB2 mutation including one new mutation (K168R), one patient homozygous for the del(GJB6‐D13S1830). These results establish that genetic screening can provide an etiologic diagnosis, and may help with prognosis after cochlear implantation, as has been hypothesized in previous studies.