[Show abstract][Hide abstract] ABSTRACT: Mitochondrial DNA (mtDNA) mutations are an important cause of human disease. Most mtDNA mutations are found in heteroplasmy, in which the proportion of mutant vs. wild-type species is believed to explain some of the observed high phenotypic heterogeneity. However, homoplasmic mutations also observe phenotypic heterogeneity, which may be in part due to undetected low levels of heteroplasmy. In the present report, we have developed two assays, using DHPLC and Pyrosequencing (Biotage AB, Uppsala, Sweden), for reliably and accurately detecting low-level mtDNA heteroplasmy. Using these assays we have identified a three-generation family segregating two mtDNA mutations in heteroplasmy: the deafness-related m.1555A>G mutation in the 12S rRNA gene (MTRNR1) and a new variant (m.15287T>C) in the cytochrome b gene (MTCYB). Both heteroplasmic mtDNA mutations are transmitted through generations in a random manner, thus showing differences in mutation load between siblings within the family. In addition, the developed assays were also used to screen a group of deaf subjects of unknown etiology for the presence of heteroplasmy for both mtDNA variants. Two additional heteroplasmic m.1555A>G samples, previously considered as homoplasmic, and two deaf subjects carrying m.15287T>C variant were identified, thus confirming the high specificity and reliability of the approach. The development of assays for reliably detecting low-level heteroplasmy, together with the study of heteroplasmic mtDNA transmission, are essential steps for a better knowledge and clinical management of mtDNA diseases.
Human Mutation 02/2008; 29(2):248-57. DOI:10.1002/humu.20639 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A screen for TBX1 gene mutations identified two mutations in patients with some features compatible with the 22q11.2-deletion syndrome but with no deletions. One is a de novo missense mutation and the other is a 5' untranslated region (5'UTR) C>T change that affects a nucleotide with a remarkable trans-species conservation. Computer modelling shows that the 5'UTR change is likely to affect the mRNA structure and in vitro translation experiments demonstrate that it produces a twofold increase in translation efficiency. Recently, duplications in the 22q11.2 region were reported in patients referred for fragile-X determination because of cognitive and behavioural problems. Because the 5'UTR nucleotide change may be a functional equivalent of a duplication of the TBX1 gene, we decided to screen 200 patients who had been referred for fragile-X determination and 400 healthy control individuals. As a result, we found the 5'UTR mutation to be present in three patients with mental retardation or behavioural problems and absent in control individuals of the same ethnic background. This observation suggests that it may be reasonable to screen for such mutation among patients with unspecific cognitive deficits and we provide an easy and quick way to do it with an amplification refractory mutation system (ARMS) approach. To our knowledge, this is the first human mutation showing that TBX1 is a candidate causing mental retardation associated with the 22q11.2 duplication syndrome.
European Journal of HumanGenetics 07/2007; 15(6):658-63. DOI:10.1038/sj.ejhg.5201819 · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fragile X syndrome is the most common form of inherited mental retardation. It is caused by the increase in length of a stretch of CGG triplet repeats within the FMR1 gene. A full mutation (> 200 repeats) leads to methylation of the CpG island and silencing of the FMR1 gene. We present here two sisters that are compound heterozygotes for a full mutation and a 53 repeat intermediate allele, one of them showing mental retardation and clinical features of an affected male (speech delay, hyperactivity, large ears, prominent jaw, gaze aversion), while the other is borderline normal (mild delay). Southern blot and FMRP expression analysis showed that the sister with mental retardation had the normal FMR1 gene totally methylated and no detectable protein, while her sister had 70% of her cells with the normal FMR1 gene unmethylated and normal FMRP levels. We found that the observed phenotypic differences between both sisters who are cytogenetically normal, are caused by extreme skewed X-chromosome inactivation. Analysis of the extended family showed that most of the other female family members that carry a pre-mutation or a full mutation showed some degree of skewing in their X-chromosome inactivation. The presence of several family members with skewed X inactivation and the direction and degree of skewing is inconsistent with a mere selection during development, and suggests a genetic origin for this phenomenon.
American Journal of Medical Genetics Part A 10/2003; 122A(2):108-14. DOI:10.1002/ajmg.a.20160 · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pendred syndrome is an autosomal-recessive disorder characterized by congenital sensorineural hearing loss combined with goiter. This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS/SLC26A4) has been mapped to chromosome 7q22-q31 and encodes a chloride-iodide transport protein. Mutations in this gene are also a cause of non-syndromic autosomal recessive hearing impairment (DFNB4). We have analyzed the PDS/SLC26A4 gene in Spanish and Italian families and we have detected five novel mutations (X781W, T132I, IVS2-2A>G, Y556H and 406del5).
Human Mutation 07/2002; 20(1):77-8. DOI:10.1002/humu.9043 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pendred syndrome is an autosomal-recessive disorder characterized by congenital sensorineural hearing loss combined with goiter. This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS/SLC26A4) has been mapped to chromosome 7q22-q31 and encodes a chloride-iodide transport protein. Mutations in this gene are also a cause of non-syndromic autosomal recessive hearing impairment (DFNB4). We have analyzed the PDS/SLC26A4 gene in Spanish and Italian families and we have detected five new mutations (X871M, T132I, IVS1-2A>G, Y556H and 406del5).
Human Mutation 12/2001; 18(6):548. DOI:10.1002/humu.1238 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pendred syndrome is a recessive inherited disorder that consists of developmental abnormalities of the cochlea, sensorineural hearing loss, and diffuse thyroid enlargement (goiter). This disorder may account for up to 10% of cases of hereditary deafness. The disease gene (PDS) has been mapped to chromosome 7q22-q31, and encodes a chloride-iodide transport protein. We performed mutation analysis of individual exons of the PDS gene in one Spanish family that shows intrafamilial variability of the deafness phenotype (two patients with profound and one with moderate-severe deafness). We identified a new splice-site mutation affecting intron 4 of the PDS gene, at nucleotide position 639+7. RNA analysis from lymphocytes of the affected patients showed that mutation 639+7A-->G generates a new donor splice site, leading to an mRNA with an insertion of six nucleotides from intron 4 of PDS. Since the newly created donor splice site is likely to compete with the normal one, variations of the levels of normal and aberrant transcripts of the PDS gene in the cochlea may explain the variability in the deafness presentation.
Human Mutation 12/1999; 14(6):520-6. DOI:10.1002/(SICI)1098-1004(199912)14:6<520::AID-HUMU11>3.0.CO;2-K · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to >/=30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments.
The American Journal of Human Genetics 12/1999; 65(5):1349-58. DOI:10.1086/302642 · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hearing impairment affects one infant in 1000 and 4% of people aged younger than 45 years. Congenital deafness is inherited or apparently sporadic. We have shown previously that DFNB1 on chromosome 13 is a major locus for recessive deafness in about 80% of Mediterranean families and that the connexin-26 gene gap junction protein beta2 (GJB2) is mutated in DFNB1 families. We investigated mutations in the GJB2 gene in familial and sporadic cases of deafness.
We obtained DNA samples from 82 families from Italy and Spain with recessive non-syndromic deafness and from 54 unrelated participants with apparently sporadic congenital deafness. We analysed the coding region of the GJB2 gene for mutations. We also tested 280 unrelated people from the general populations of Italy and Spain for the frameshift mutation 35delG.
49% of participants with recessive deafness and 37% of sporadic cases had mutations in the GJB2 gene. The 35delG mutation accounted for 85% of GJB2 mutations, six other mutations accounted for 6% of alleles, and no changes in the coding region of GJB2 were detected in 9% of DFNB1 alleles. The carrier frequency of mutation 35delG among people from the general population was one in 31 (95% CI one in 19 to one in 87).
Mutations in the GJB2 gene are a major cause of inherited and apparently sporadic congenital deafness. Mutation 35delG is the most common mutation for sensorineural deafness. Identification of 35delG and other mutations in the GJB2 gene should facilitate diagnosis and counselling for the most common genetic form of deafness.
The Lancet 03/1998; 351(9100):394-8. DOI:10.1016/S0140-6736(97)11124-2 · 45.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hearing loss involves both genetic and environmental factors. A mutation (A1555G) in the mtDNA has been associated with aminoglycoside-induced and nonsyndromic sensorineural deafness. The pathological significance of this mutation in Caucasoid families has not been established, and its relationship with antibiotic treatment is not well understood. We studied 70 Spanish families with sensorineural deafness (36 congenital and 34 late onset) for the mtDNA A1555G mutation. The A1555G mutation was found in 19 families with maternally transmitted deafness but not in the other 51 families or in 200 control subjects. In 12 families all the patients with the A1555G mutation who received aminoglycosides became deaf, representing 30.3% of the deaf patients in these families. None of the deaf patients from seven other families received aminoglycosides. Overall, only 17.7% of the patients with deafness and the A1555G mutation had been treated with aminoglycosides. The age at onset of deafness was lower (median age 5 years, range 1-52 years) in those treated with aminoglycosides than in those who did not receive antibiotics (median age 20 years, range 1-65 years) (P < .001). The mtDNA of these families belongs to haplotypes common in Europeans. These data indicate that the A1555G mutation accounts for a large proportion of the Spanish families with late-onset sensorineural deafness, that the A1555G mutation has an age-dependent penetrance for deafness (enhanced by treatment with aminoglycosides), and that mtDNA backgrounds probably do not play a major role in disease expression.
The American Journal of Human Genetics 01/1998; 62(1):27-35. DOI:10.1086/301676 · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-syndromic neurosensory autosomal recessive deafness (NSRD) is the most common form of genetic hearing loss. Previous studies defined at least 15 human NSRD loci. Recently we demonstrated that DFNB1, located on the long arm of chromosome 13, accounts for approximately 80% of cases in the Mediterranean area. Further analysis with additional markers now identifies several recombinants which narrow the candidate region to approximately 5 cM, encompassed by markers D13S141 and D13S232 and including several ESTs and candidate genes, including the connexin26 (GJB2) gene. Analysis of PCR products from our affected patients' DNA shows two frameshift mutations in the connexin26 gene. Deletion of a G within a stretch of six Gs at position 35 of the GJB2 cDNA (mutation 35delG) leads to premature chain termination and is present in 63% of NSRD chromosomes, demonstrating linkage to chromosome 13. Deletion of a T at position 167 of GJB2 (mutation 167delT), also resulting in premature chain termination, was detected in another patient. Four neutral sequence polymorphisms were also identified. These findings are in agreement with a recent study showing that mutations in the connexin26 gene are associated with genetic forms of deafness in three Pakistani families and that GJB2 is DFNB1. Connexin26 is a member of a large family of proteins involved in formation of gap junctions, which are involved in electrical synapses and the direct transfer of small molecules and ionic currents between neighboring cells. The identification of GJB2 as the DFNB1 gene should provide a better understanding of the biology of normal and abnormal hearing, help form the basis for diagnosis and may facilitate development of strategies for treatment of this common genetic disorder.
Human Molecular Genetics 10/1997; 6(9):1605-9. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies show a susceptibility locus (DFNB1) responsible for non-syndromic neurosensory autosomal-recessive deafness (NSRD) mapping to the pericentromeric region of chromosome 13q. In order to better understand the frequency with which DFNB1 is the gene for deafness in our patient population and the role of DFNB1 in Caucasians, we performed a genetic linkage study with four microsatellite markers linked to DFNB1 in a total of 48 independent Mediterranean families, of which 30 and 18 were of Italian and Spanish descent, respectively. A maximum two-point lod score of 7.28 was found with marker D13S115 at a recombination frequency of theta 0.1. Significant lod scores were also obtained for D13S143, D13S292 and D13S175. Genetic heterogeneity was confirmed using the HOMOG program which indicated absence of linkage to DFNB1 in approximately 21% of the sample. This study clearly demonstrates that DFNB1 plays an important role in 79% of Mediterranean families with NSRD. Furthermore, results from multipoint analysis predict that the DFNB1 gene maps between markers D13S175 and D13S115 which are separated by approximately 14.2 cM.
European Journal of HumanGenetics 03/1997; 5(2):83-8. · 4.23 Impact Factor