Structure of the X-linked Kallmann syndrome gene and its homologous pseudogene on the Y chromosome.

The gene for the X-linked Kallmann syndrome (KAL), a developmental disorder characterized by hypogonadotropic hypogonadism and anosmia, maps to Xp22.3 and has a homologous locus, KALP, on Yq11. We show here that KAL consists of 14 exons spanning 120-200 kilobases that correlate with the distribution of domains in the predicted protein including four fibronectin type III repeats. The KALP locus reveals several large deletions and a number of small insertions, deletions and base substitutions which indicate it is a non-processed pseudogene. The sequence divergence between KAL and KALP in humans, and the chromosomal location of KAL homologous sequences in other primates, suggest that KALP and the steroid sulphatase pseudogene on Yq11 were involved in the same rearrangement event on the Y chromosome during primate evolution.

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein-stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

Five-year follow-up of patients with chronic C hepatitis and sustained virological response.

OBJECTIVE: to assess persistence of sustained viral response at 5 years of follow-up in patients with chronic viral hepatitis C treated with pegylated interferon and ribavirin. DESIGN: a descriptive study. PATIENTS: from August 2001 to May 2004, all patients treated at our center with pegylated interferon and ribavirin who achieved a sustained viral response were consecutively enrolled (93 patients). Demographic, histological, biochemical, and virological data were collected during treatment and 5 years after achievement of the sustained viral response. Eighty-six percent of patients enrolled (n = 80) attended the control visit at 5 years. RESULTS: mean age of enrolled patients was 41 years (standard deviation = 10 years), and 30.1% (n = 28) were women. Liver biopsy had been performed before treatment in 68.8% of patients (n = 64), showing no or mild fibrosis in 62.3% (F0 and F1) and significant fibrosis and cirrhosis in 37.7% (F ≥ 3). Genotype distribution was: 58.1% genotype 1 (n = 54); 8.6% genotype 2 (n = 8); 24.7% genotype 3 (n = 23); 7.5% genotype 4 (n = 7), and indeterminate in one patient. Only one patient experienced virological recurrence. All other patients had negative HCV RNA levels and, in the absence of other liver diseases, normal ALT levels. CONCLUSION: in patients treated with pegylated interferon and ribavirin with sustained viral response, long-term recurrence rate was very low.

A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression.

Eleven affected members of a large German-American family segregating recessively inherited, congenital, non-syndromic sensorineural hearing loss (SNHL) were found to be homozygous for the common 35delG mutation of GJB2, the gene encoding the gap junction protein Connexin 26. Surprisingly, four additional family members with bilateral profound SNHL carried only a single 35delG mutation. Previously, we demonstrated reduced expression of both GJB2 and GJB6 mRNA from the allele carried in trans with that bearing the 35delG mutation in these four persons. Using array comparative genome hybridization (array CGH), we have now identified on this allele a deletion of 131.4 kb whose proximal breakpoint lies more than 100 kb upstream of the transcriptional start sites of GJB2 and GJB6. This deletion, del(chr13:19,837,344-19,968,698), segregates as a completely penetrant DFNB1 allele in this family. It is not present in 528 persons with SNHL and monoallelic mutation of GJB2 or GJB6, and we have not identified any other candidate pathogenic copy number variation by arrayCGH in a subset of 10 such persons. Characterization of distant GJB2/GJB6 cis-regulatory regions evidenced by this allele may be required to find the 'missing' DFNB1 mutations that are believed to exist.

Stickler and branchio-oto-renal syndromes in a patient with mutations in EYA1 and COL2A1 genes.

Branchio-oto-renal (BOR) and Stickler (STL) syndromes are disorders that include hearing loss among their clinical features. STL syndrome type I (STL1) is a combination of ophthalmic, orofacial, articular, and auditory manifestations, caused by mutations in the COL2A1. BOR syndrome is an autosomal dominant trait encompassing branchial, otic and renal anomalies because of mutations in EYA1, SIX1 and SIX5. In this study, we have clinically and genetically diagnosed a proband that displayed STL1 and BOR syndromes. This patient and his younger brother exhibited hearing loss and cleft palate. Both siblings and their mother also showed myopia, congenital non-progressive vitreous anomaly and a flat face. Taken together, these clinical features are consistent with the diagnosis of a familial case of STL. Sequence analysis revealed in the three patients a novel COL2A1 mutation (c.1468_1475delinsT) that accounted for a STL1 phenotype. The proband also displayed pre-auricular pits, branchial fistulae and renal agenesis that define BOR syndrome. Interestingly, this patient carries an EYA1 mutation, p.R328X, which was not present in the two other patients or in his healthy father, supporting that the mutation arose de novo. In conclusion, this report highlights the importance of molecular testing and detailed clinical evaluation for the diagnosis of syndromes with overlapping phenotypic features.

Haplogroup analysis supports a pathogenic role for the 7510T>C mutation of mitochondrial tRNA(Ser(UCN)) in sensorineural hearing loss.

We ascertained a large North American family, LMG309, with matrilineal transmission of non-syndromic, progressive sensorineural hearing loss (SNHL). There was no history of aminoglycoside exposure, and penetrance was complete. We sequenced the entire mitochondrial genome and identified the previously reported 7510T>C transition in the tRNA(Ser(UCN)) gene. The 7510T>C was homoplasmic in all affected members. The LMG309 mitochondrial sequence belongs to an unnamed subgroup of mitochondrial haplogroup H. We demonstrate that the previously reported Spanish family S258 carries 7510T>C on a different mitochondrial sub-haplogroup, H1. We did not detect 7510T>C among 79 Caucasian haplogroup H control samples, including 11 from sub-haplogroup H1 and one from the same sub-haplogroup as LMG309. Our results provide strong genetic evidence that 7510T>C is a pathogenic mutation that causes non-syndromic SNHL.

Molecular and clinical characterisation of three Spanish families with maternally inherited non-syndromic hearing loss caused by the 1494C->T mutation in the mitochondrial 12S rRNA gene.

Mutations in the 12S rRNA gene of the mitochondrial genome are responsible for maternally inherited non-syndromic hearing loss (NSHL), and for increased susceptibility to the ototoxicity of aminoglycoside antibiotics. Among these mutations, 1555A-->G is the most prevalent in all populations tested so far. Recently, the 1494C-->T mutation was reported in two large Chinese pedigrees with maternally inherited NSHL. In this study, sequencing of the 12S rRNA gene in a Spanish family with maternally inherited NSHL showed the presence of the 1494C-->T mutation. An additional screening of 1339 unrelated Spanish patients with NSHL allowed the authors to find two other families with the mutation. Audiological data were obtained from 17 confirmed 1494C-->T carriers, which showed that the hearing loss was sensorineural, bilateral and symmetrical, with a remarkable variability in age of onset and severity. Three carriers were asymptomatic. Three affected carriers had a history of treatment with aminoglycoside antibiotics. The mitochondrial genome of one affected person from each of these three families was entirely sequenced, and it was established that they belong to different mitochondrial haplogroups (H, U5b, U6a). The study results further support the pathogenic role of 1494C-->T on hearing, and show that this mutation can be found in different Caucasian mitochondrial DNA backgrounds.

[Auditory neuropathy due to the Q829X mutation in the gene encoding otoferlin (OTOF) in an infant screened for newborn hearing impairment]. / Neuropatia auditiva secundaria a la mutación Q829X en el gen de la otoferlina (OTOF) en un lactante sometido a screening neonatal de hipoacusia.

We report an infant with auditory neuropathy secondary to the Q829X mutation in the gene encoding otoferlin (OTOF). Included in a universal newborn hearing screening program, the subject passed the otoacoustic emission (OAEs) test. Given that the infant had a familial history of deafness auditory brainstem response (ABR) testing was performed, revealing a profound hearing impairment. The genetic study confirmed that the subject was homozygous for the Q829X mutation in OTOF. The patient underwent a cochlear implant, obtaining satisfactory results. The moderately high prevalence of this mutation in the Spanish population could produce a significant false negative rate in newborn hearing screening programs using OAEs.

A genotype-phenotype correlation for GJB2 (connexin 26) deafness.

INTRODUCTION: Mutations in GJB2 are the most common cause of non-syndromic autosomal recessive hearing impairment, ranging from mild to profound. Mutation analysis of this gene is widely available as a genetic diagnostic test. OBJECTIVE: To assess a possible genotype-phenotype correlation for GJB2. DESIGN: Retrospective analysis of audiometric data from people with hearing impairment, segregating two GJB2 mutations. SUBJECTS: Two hundred and seventy seven unrelated patients with hearing impairment who were seen at the ENT departments of local and university hospitals from Italy, Belgium, Spain, and the United States, and who harboured bi-allelic GJB2 mutations. RESULTS: We found that 35delG homozygotes have significantly more hearing impairment, compared with 35delG/non-35delG compound heterozygotes. People with two non-35delG mutations have even less hearing impairment. We observed a similar gradient of hearing impairment when we categorised mutations as inactivating (that is, stop mutations or frame shifts) or non-inactivating (that is, missense mutations). We demonstrated that certain mutation combinations (including the combination of 35delG with the missense mutations L90P, V37I, or the splice-site mutation IVS1+1G>A, and the V37I/V37I genotype) are associated with significantly less hearing impairment compared with 35delG homozygous genotypes. CONCLUSIONS: This study is the first large systematic analysis indicating that the GJB2 genotype has a major impact on the degree of hearing impairment, and identifying mild genotypes. Furthermore, this study shows that it will be possible to refine this correlation and extend it to additional genotypes. These data will be useful in evaluating habilitation options for people with GJB2 related deafness.

[Prevalence of the 35delG mutation in the GJB2 gene, del (GJB6-D13S1830) in the GJB6 gene, Q829X in the OTOF gene and A1555G in the mitochondrial 12S rRNA gene in subjects with non-syndromic sensorineural hearing impairment of congenital/childhood onset]. / Prevalencia de las mutaciones 35delG en el gen GJB2, del (GJB6-D13S1830) en el gen GJB6, Q829X en el gen OTOF y A1555G en el gen del ARNr 12S mitocondrial en sujetos con hipoacusia neurosensorial no sindrómica de inicio congénito o en la infancia.

INTRODUCTION: The most frequent mutations responsible for non-syndromic hearing impairment in the Spanish population are the 35delG mutation in the connexin 26 gene (GJB2), the del(GJB6-D13S1830) deletion in the connexin 30 gene (GJB6), the Q829X mutation in the otoferlin gene (OTOF), and the A1555G mutation in the 12S rRNA gene of the mitochondrial genome. PATIENTS AND METHODS: Screening for these mutations was performed on 38 patients from Cantabria with non-syndromic sensorineural hearing impairment of congenital/childhood onset. RESULTS: The A1555G mutation was detected in homoplasmy in 9 patients (23.7%). Three individuals were heterozygous for the 35delG mutation (7.9%). The heterozygous del(GJB6-D13S1830) deletion was present in one case (2.6%). One subject was homozygous for the Q829X mutation (2.6%). CONCLUSIONS: These four mutations are present in 36.8% of all cases of non-syndromic hearing impairment in our population.

Clinical (but not cognitive) recovery in schizophrenia through the experience of fictional cinema.

INTRODUCTION: One of the criticisms of rehabilitation techniques is their limited application to the patient's daily life. In the past, cinema has been used as a psychiatric rehabilitation tool, with the primary objective of facilitating training in social abilities and communication. In this study, we consider the use of film not only as a clinical recovery tool but also as a novel cognitive recovery tool for additional rehabilitation not only for communication and social abilities but also for all of the basic cognitive and social cognition processes. METHODS: In this randomized clinical trial, 48 patients with schizophrenia were assigned to an experimental or control group. Both of the groups received treatment sessions that included viewing episodes of the television series The Sopranos. Next, the experimental group participated in a structured cognitive training session that featured questions and exercises based on the episodes. The control group participated in an idea-sharing session (of the same duration and frequency) about what the group members saw in the episode. RESULTS: At the end of the treatment, both the positive and negative clinical symptoms of the experimental group improved significantly compared with the control group. However, this improvement was not observed in basic or social cognitive functions. DISCUSSION: A brief intervention based on transforming the activities of daily life can be an effective tool for psychiatric rehabilitation. However, the study's current characteristics and sample did not produce benefits in cognitive parameters.

Isolation of microsatellites from the spinal muscular atrophy (SMA) candidate region on chromosome 5q and linkage analysis in Spanish SMA families.

A locus responsible for autosomal recessive spinal muscular atrophy (SMA) on chromosome 5q11.2-q13.3 has been mapped to a critical interval delimited by markers D5S435 and D5S557. By a modification of the Vectorette-(GT)n method, we have isolated three polymorphic CA repeats from two YACs of the SMA region. Two of them (D5S1417 and D5S1416) map within the SMA critical region, and the other (D5S1415) is centromeric to D5S435. Linkage analysis in Spanish SMA families with eleven markers showed that in our families the disease is linked to this region and confirmed that the novel markers are tightly linked to the SMA locus. The most likely order of markers was 5cen-(D5S63/D5S1356)-(D5S125/D5S465)- (D5S435/D5S1417/D5S1416/D5S557)-D5S610- D5S112-D5S127-5qter, with odds against alternative orders > 1,000:1. Genetic distances are in agreement with those previously published. However, the recombination fraction between D5S610 and D5S112 is remarkably greater than expected from the physical distance, suggesting a hot spot for recombination in this region. Our results from haplotype and multipoint analyses show that the SMA locus must lie between D5S465 and D5S112, and lend further support to the current location of the SMA locus.

Characterization of the promoter of the human KAL gene, responsible for the X-chromosome-linked Kallmann syndrome.

We report on the first characterization of the human KAL promoter (pKAL), based on the analysis of a 2-kb fragment of the 5' flanking region. As determined by primer extension, transcription of the human KAL gene is initiated at two different sites in the quail embryonic neuroretina QNR/D cell line. The promoter region is G+C rich and contains a CCAAT box, two binding sites for the SP1 transcription factor and two AP2-binding sites, but no TATA box. It also shares a motif with several neural-specific genes. The ability of four deletion mutants to drive transcription of the heterologous chloramphenicol acetyltransferase (CAT)-encoding gene was determined in transfection experiments. The mutant containing the KAL sequence from nt +2 to -435 demonstrated a tissue-specific, although weak, transcriptional activity only in the quail embryonic neuroretina K2 and QNR/D cell lines. Longer constructs did not confer any activity. Therefore, we suggest that this 437-bp segment of pKAL constitutes a neural-specific promoter which could be negatively controlled by upstream sequences.

Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity.

The Escherichia coli K-12 sheA gene encodes a pore-forming hemolysin that is secreted to the medium by a hitherto unidentified mechanism. To study SheA secretion, we constructed fusions between SheA and the mature form of the periplasmic enzyme beta-lactamase, and performed site-directed mutagenesis on these constructs. The SheA-Bla and Bla-SheA hybrid proteins displayed hemolytic activity and were efficiently exported to the extracellular medium. Our results with mutant hybrid proteins show that secretion of SheA is independent of its cytolytic activity, that secretion is paralleled by a transient leakage of periplasmic contents to the extracellular medium, and that deletion of the 11 C-terminal residues of SheA has no effect on its secretion and cytolytic activity.

Association analysis of genotypic frequencies of matrilin-1 gene in patients with osteoarthritis.

OBJECTIVE: It has been suggested that genotypic variation in the gene which encodes the matrilin-1 (MATN-1) protein may be involved in the development of hip osteoarthritis (OA). We compared genotype frequencies of the MATN-1 gene (1p35) in patients with OA and controls to determine if there is any association between the MATN-1 genotype and OA. METHODS: 73 OA patients and 53 controls from a rheumatology ambulatory center and a university hospital were studied. They were unrelated subjects. Controls were free of clinical OA. OA was defined according to the American College of Rheumatology criteria. The MATN-1 microsatellite in the 3'untranslated region was amplified by PCR. The size of the amplification products was determined by capilar electrophoresis in a DNA Genetic Analizer Genotypic distribution was compared by the chi2 test. RESULTS: We identified 4 alleles according to their basepair (bp) length: A1 = 110 bp; A2 = 108 bp; A3 = 106 bp and A6 = 104 pb. Six genotypes were found, with an observed heterozygosity of 0.48. The most frequent genotype in OA and controls was A1/A1 (43.8% and 43.4%, respectively). No significant difference in genotype distribution was found between OA - even when discriminating by the affected joint - and controls. CONCLUSION: We did not find any difference in the MATN-1 genotype distribution in OA patients and controls. To our knowledge, this would be the first time a MATN-1 allele of 104 bp (A6) has been identified These results do not support a role of the MATN-1 genotypes in the occurrence of clinical OA.

Isolation and characterization of the gene responsible for the X chromosome-linked Kallmann syndrome.

Kallmann de Morsier Syndrome is defined by the association of an hypogonadism with an anosmia. The hypogonadism is due to a deficiency of GnRH (gonadotropin-releasing hormone). Olfactory bulbs and tracts are underdeveloped in the patients. Embryological studies have indicated that the migration of GnRH neurons and the axonal extension of olfactory neurons, which both originate in the olfactory epithelium during embryogenesis, were impaired in a fetus affected by X-linked Kallmann Syndrome. By a positional cloning strategy, we have isolated the KAL gene, responsible for the X-linked form of the disease. The gene consists of 14 exons. A highly homologous pseudogene on the Y chromosome has been characterized. The KAL gene encodes a putative secreted protein of 680 amino acids, which contains four fibronectin type III repeats and a four disulphide core motif. The former motif is usually associated with adhesion function. The latter has been described in protein with antiprotease activity. We have isolated the chicken KAL homologue and studied its expression by in situ hybridization during late embryonic development. The gene is expressed in various neuronal populations of the central nervous system, including mitral cells of the olfactory bulbs. We suggested that the KAL protein might be involved in late neuronal differentiation.

Sensorineural hearing loss and Mondini dysplasia caused by a deletion at locus DFN3.

OBJECTIVE: To study a family with inner ear malformations and sensorineural hearing loss. DESIGN: Clinical, radiological, and genetic study of the members of a family with different degrees of sensorineural hearing loss. RESULTS: The males in the family manifested profound congenital hearing loss with severe inner ear malformations, while the only affected female had progressive hearing loss that had begun during puberty. Computed tomography showed inner ear malformations in both males, with enlarged internal auditory meatus and Mondini dysplasia. Genetic analysis disclosed a microdeletion at the locus DFN3 on chromosome X. CONCLUSION: A familial Mondini dysplasia is associated to a microdeletion at the deafness locus DFN3.

[Non-syndromic familial hearing impairment transmitted by mitochondrial inheritance]. / Hipoacusia familiar no sindrómica transmitida por herencia mitocondrial.

OBJECTIVE: To determine the clinical features and mode of inheritance of hearing impairment observed in several members of a Spanish family with putative genetic susceptibility to ototoxicity induced by aminoglycoside antibiotics. MATERIALS AND METHODS: In 14 patients belonging to the same family, an interview, otological examination and audiometry were carried out. Three of these patients also underwent a study of brainstem auditory-evoked potentials. A genetic study was made of 11 patients and 9 unaffected relatives to determine the mode of inheritance of the hearing impairment and to detect associated mutations. RESULTS: Most of the patients developed hearing loss before the age of 8 years, particularly for high frequencies. In almost all of them, there was no further evolution. Six patients had previous exposure to ototoxic drugs. Three patients had sudden hearing loss. In 2 of the 3 patients examined for brainstem auditory-evoked potentials, the hearing impairment was of cochlear origin. The genetic study detected A1555G mutation in the mitochondrial DNA of all the maternal relatives studied. CONCLUSIONS: Mitochondrially-inherited sensorineural hearing loss should be suspected in families with a maternal transmission of the disorder and more than one relative with aminoglycoside-induced hearing loss.

[Audiometric features of familial hearing impairment transmitted by mitochondrial inheritance (A1555G)]. / Características audiométricas de la hipoacusia familiar transmitida por herencia mitocondrial (A1555G).

OBJECTIVE: To examine the audiometric patterns of familial hearing impairment due to the A1555G mutation in the mitochondrial DNA. PATIENTS AND METHODS: We include 55 subjects with the A1555G mutation from 6 unrelated families, affected by nonsyndromic sensorineural hearing loss and residing in Cantabria. The A1555G mutation was found in homoplasmy in all the families, except in one family, in which it was in heteroplasmy. Aside from standard history taking and general otolaryngological examination, pure tone audiometry was carried out in all patients. RESULTS: Hearing loss was developed by most of the patients. The auditory defect was a slowly progressive bilateral symmetrical sensorineural hearing loss, affecting mainly the high frequencies. In patients in which aminoglycoside ototoxicity could be excluded, hearing loss usually ranged from mild to moderate, with a late onset. In 17 cases there were previous history of treatment with a ototoxic drugs, and most of them developed severe hearing loss. One of them was deaf-mute. No audometric differences between families with the homoplasmic and the heteroplasmic A1555G mutation were observed. CONCLUSIONS: Patients with the A1555G mutation generally present bilateral symmetrical sensorineural hearing loss, ranging from mild to moderate, slowly progressive, which is obvious approximately in the second decade of life and affects specially the high frequencies. Hearing loss severity is increased by treatment with aminoglycosides.

[Incidence of A1555G mutations in the mitochondrial DNA and 35delG in the GJB2 gene (connexin-26) in families with late onset non-syndromic sensorineural hearing loss from Cantabria]. / Incidencia de las mutaciones A1555G en el ADN mitocondrial y 35delG en el gen GJB2 (conexina 26) en familias con hipoacusia neurosensorial postlocutiva no sindrómica en Cantabria.

INTRODUCTION: Sensorineural deafness is a very common disorder in humans, which affects approximately 10% of the population. Genetic causes are suggested to be responsible for more than half of the cases. The A1555G mutation in the mitochondrial 12S rRNA gene and the 35delG mutation in the GJB2 gene are the most common mutations for sensorineural deafness in the Spanish population. METHODS: A genetic study was carried out in order to determine the frequency of the mutations A1555G in the mitochondrial DNA and 35delG in the connexin-26 gene in 21 patients from 21 non-consanguineous unrelated families affected by late-onset bilateral non-syndromic sensorineural hearing loss from Cantabria. RESULTS: The A1555G mutation was found in 6 patients. Five of these 6 patients had been treated with aminoglycosides. In all of them the auditory impairment affected mainly the high frequencies. The 35delG mutation was not found in any of the patients. CONCLUSIONS: The A1555G mutation in the mitochondrial DNA has been found to be the most common amongst the Cantabrian population. The A1555G mutation should be suspected in those members of families affected by sensorineural hearing impairment with a maternal inheritance pattern and ototoxicity from treatment with aminoglycoside antibiotics. The 35delG mutation in the GJB2 gene does not seem to be a major cause of deafness in families with late-onset non-syndromic sensorineural hearing loss in our area.