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ABSTRACT: Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease caused by mutations in the NF1 gene. The mutation rate of NF1 is one of the highest known for human genes and the mutational analysis has revealed a wide variety of changes, a significant proportion of which affect normal pre-mRNA splicing. Here, we describe two truncating mutations in exon 37 of NF1, the recurrent c.6792C>A and the novel c.6799C>T change, that occur in cis and segregate with NF1 in a large family. The double mutation induces defective splicing of exon 37 and thus, we performed quantitative comparisons of transcripts harboring single (c.6792C>G or c.6792C>A) and double (c.6792C>A and c.6799C>T) mutations to assess their effects on exon 37 splicing. Skipping of exon 37 was greater and there were fewer mutant full-length transcripts in samples with the double mutation than in those carrying single mutations. Thus, the combination of the c.6792C>A and c.6799C>T mutations augmented exon 37 skipping. These findings suggest that, in addition to the previously described exonic splicing enhancer in the c.6791_6795 region, c.6799 lies within an additional regulatory element that influences the splicing of exon 37.
Clinical Genetics 08/2012; · 3.13 Impact Factor
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ABSTRACT: Germline mutations in the SMARCB1 gene cause familial schwannomatosis, a condition characterized by the presence of multiple schwannomas, although mutations in SMARCB1 have also been associated with rhadboid tumor predisposition syndrome 1 (RTPS1). Both schwannomatosis and RTPS1 are autosomal dominant conditions that predispose individuals to develop distinct types of tumors. We clinically and genetically characterized two families with schwannomatosis associated with SMARCB1 mutations. Eight affected members of these families developed different numbers of schwannomas and/or meningiomas at distinct ages, evidence that meningiomas are variably expressed in this condition. We identified two germline mutations in SMARCB1 associated with the familial disease, c.233-1G>A and the novel c.207_208dupTA mutation, which both proved to affect the main SMARCB1 isoforms at the RNA level distinctly. Interestingly, the c.207_208dupTA mutation had no effect on the coding sequence, pre-mRNA splicing or the level of expression of the SMARCB1 isoform 2. Furthermore, SMARCB1 isoforms harboring a premature termination codon were largely eliminated via the nonsense-mediated mRNA decay pathway. Our results highlight the importance of RNA-based studies to characterize SMARCB1 germline mutations in order to determine their impact on protein expression and gain further insight into the genetic basis of conditions associated with SMARCB1 mutations.
Neurogenetics 07/2012; 13(3):267-74. · 3.35 Impact Factor
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Expert Review of Molecular Diagnostics 09/2011; 11(7):671-3. · 4.86 Impact Factor
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ABSTRACT: Spinal muscular atrophy (SMA) is caused by loss or mutations of the survival motor neuron 1 gene (SMN1). Its highly homologous copy, SMN2, is present in all SMA cases and is a phenotypic modifier. There are cases where asymptomatic siblings of typical SMA patients possess a homozygous deletion of SMN1 just like their symptomatic brothers or sisters. Plastin 3 (PLS3) when over expressed in lymphoblasts from females has been suggested to act as a genetic modifier of SMA. We studied PLS3 expression in four Spanish SMA families with discordant siblings haploidentical for the SMA locus. We excluded PLS3 as a possible modifier in two of our families with female discordant siblings. In the remaining two, we observed small differences in PLS3 expression between male and female discordant siblings. Indeed, we found that values of PLS3 expression in lymphoblasts and peripheral blood ranged from 12 to 200-fold less than those in fibroblasts. These findings warrant further investigation in motor neurons derived from induced pluripotential stem cells of these patients.
Neuromuscular Disorders 06/2011; 21(6):413-9. · 2.80 Impact Factor
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ABSTRACT: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by absence of or mutations in the survival motor neuron1 gene (SMN1). All SMA patients have a highly homologous copy of SMN1, the SMN2 gene. Severe (type I) SMA patients present one or two SMN2 copies, whereas milder chronic forms (type II-III) usually have three or four SMN2 copies. SMN2 dosage is important to stratify patients for motor function tests and clinical trials. Our aim was to compare three methods, marker analysis, real-time quantitative polymerase chain reaction using the LightCycler instrument, and multiple ligation-dependent probe amplification (MLPA), to characterize their accuracy in quantifying SMN2 genes. We studied a group of 62 genetically confirmed SMA patients, 54 with homozygous absence of exons 7 and 8 of SMN1 and 8 with SMN2-SMN1 hybrid genes. A complete correlation using the three methods was observed in 32 patients (51.6%). In the remaining 30 patients, discordances between the three methods were found, including under or overestimation of SMN2 copies by marker analysis with respect to the quantitative methods (LightCycler and MLPA) because of lack of informativeness of markers, 3' deletions of SMN genes, and breakpoints in SMN2-SMN1 hybrid genes. The technical limitations and advantages and disadvantages of these methods are discussed. We conclude that the three methods complement each other in estimating the SMN2 copy number in most cases. However, MLPA offers additional information to characterize SMA cases with particular rearrangements such as partial deletions and hybrid genes.
Genetic Testing and Molecular Biomarkers 05/2011; 15(9):587-94. · 1.11 Impact Factor
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ABSTRACT: Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, the presence of pseudogenes, and the wide variety of possible lesions. We developed a method for detecting germline mutations by combining an original RNA-based cDNA-PCR mutation detection method and denaturing high-performance liquid chromatography (DHPLC) with multiplex ligation-dependent probe amplification (MLPA). The protocol was validated in a cohort of 56 blood samples from NF1 patients who fulfilled NIH diagnostic criteria, identifying the germline mutation in 53 cases (95% sensitivity). The efficiency and reliability of this approach facilitated detection of different types of mutations, including single-base substitutions, deletions or insertions of one to several nucleotides, microdeletions, and changes in intragenic copy number. Because mutational screening for minor lesions was performed using cDNA and the characterization of mutated alleles was performed at both the RNA and genomic DNA level, the analysis provided insight into the nature of the different mutations and their effect on NF1 mRNA splicing. After validation, we implemented the protocol as a routine test. Here we present the overall unbiased spectrum of NF1 mutations identified in 93 patients in a cohort of 105. The results indicate that this protocol is a powerful new tool for the molecular diagnosis of NF1.
The Journal of molecular diagnostics: JMD 03/2011; 13(2):113-22. · 3.48 Impact Factor
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ABSTRACT: Neurofibromatosis type 1 is a common autosomal dominant disorder with full penetrance and variable expression. The condition predisposes individuals to the development of malignant nervous system tumours, most frequently Malignant Peripheral Nerve Sheath Tumours (MPNSTs). Previous studies indicate that genetic factors other than mutations in NF1 may be responsible for the condition's variable expression.
Here we present data from a pair of monozygotic twins affected by Neurofibromatosis type 1 resulting from a de novo mutation. Both twins developed a left sciatic plexiform neurofibroma that evolved into MPNST at a similar age and they also developed pulmonary metastasis at the same age. Other concordant traits between the twins were: macrocephaly, psychomotor delay, café-au-lait spots, cutaneous neurofibromas, retroperitoneal, pleural and paraspinal neurofibromas. The main discordant features observed were tibial pseudoarthrosis, pectus carinatum, osteoporosis and thymus hyperplasia.
This is the first report of monozygotic twins with Neurofibromatosis type 1 that develop MPNSTs, the localization and chronological evolution of which, and its metastasis, is concordant in both twins. These cases suggest that the events involved in the transformation of benign plexiform neurofibromas to MPNSTs in Neurofibromatosis type 1, follow a spatiotemporally programme that is influenced by heritable factors other than NF1 mutations.
BMC Cancer 01/2010; 10:407. · 3.01 Impact Factor
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Laura Alías,
Sara Bernal,
Pablo Fuentes-Prior,
María Jesus Barceló,
Eva Also,
Rebeca Martínez-Hernández,
Francisco J Rodríguez-Alvarez,
Yolanda Martín,
Elena Aller,
Elena Grau,
Ana Peciña,
Guillermo Antiñolo,
Enrique Galán,
Alberto L Rosa,
Miguel Fernández-Burriel,
Salud Borrego,
José M Millán, Concepción Hernández-Chico,
Montserrat Baiget,
Eduardo F Tizzano
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ABSTRACT: Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II-III) predominated in males (p<0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1-SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN-SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.
Human Genetics 01/2009; 125(1):29-39. · 5.07 Impact Factor
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ABSTRACT: Pendred syndrome is an autosomal recessive disorder characterized by sensorineural hearing loss, with malformations of the inner ear, ranging from enlarged vestibular aqueduct (EVA) to Mondini malformation, and deficient iodide organification in the thyroid gland. Nonsyndromic EVA (ns-EVA) is a separate type of sensorineural hearing loss showing normal thyroid function. Both Pendred syndrome and ns-EVA seem to be linked to the malfunction of pendrin (SLC26A4), a membrane transporter able to exchange anions between the cytosol and extracellular fluid. In the past, the pathogenicity of SLC26A4 missense mutations were assumed if the mutations fulfilled two criteria: low incidence of the mutation in the control population and substitution of evolutionary conserved amino acids. Here we show that these criteria are insufficient to make meaningful predictions about the effect of these SLC26A4 variants on the pendrin-induced ion transport. Furthermore, we functionally characterized 10 missense mutations within the SLC26A4 ORF, and consistently found that on the protein level, an addition or omission of a proline or a charged amino acid in the SLC26A4 sequence is detrimental to its function. These types of changes may be adequate for predicting SLC26A4 functionality in the absence of direct functional tests.
Proceedings of the National Academy of Sciences 12/2008; 105(47):18608-13. · 9.68 Impact Factor
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ABSTRACT: Pendred syndrome (PS) and DFNB4, a non-syndromic sensorineural hearing loss with enlargement of the vestibular aqueduct (EVA), are caused by mutations in the SLC26A4 gene. Both disorders are recessive, and yet only one mutated SLC26A4 allele, or no mutations, are identified in many cases. Here we present the genetic characterization of 105 Spanish patients from 47 families with PS or non-syndromic EVA and 20 families with recessive non-syndromic hearing loss, which segregated with the DFNB4 locus. In this cohort, two causative SLC26A4 mutations could be characterized in 18 families (27%), whereas a single mutated allele was found in a patient with unilateral hearing loss and EVA in the same ear. In all, 24 different causative mutations were identified, including eight novel mutations. The novel p.Q514K variant was the most prevalent mutation in SLC26A4, accounting for 17% (6/36) of the mutated alleles identified in this study, deriving from a founder effect. We also characterized a novel multiexon 14 kb deletion spanning from intron 3 to intron 6 (g.8091T_22145Cdel). This study also revealed the first case of a de novo recessive mutation p.Q413P causing PS that arose in the proband's paternal allele, the maternal one carrying the p.L445W. The relevance of our results for genetic diagnosis of PS and non-syndromic EVA hearing loss is discussed.
European Journal of HumanGenetics 03/2008; 16(8):888-96. · 4.40 Impact Factor
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ABSTRACT: Autosomal recessive spinal muscular atrophy (SMA) is a disease resulting from mutations in the telomeric survival motor neuron gene ( SMN1). In our sample of 150 Spanish SMA families, 87% of patients had homozygous deletions of SMN1. To identify patients who retained a single SMN1 copy, SMN dosage analysis was performed by a fluorescent quantitative PCR assay. In five out of 19 patients tested we detected one SMN1 copy. An extensive SMN gene analysis in these patients led to identification of four intragenic mutations, including two novel ones: a frameshift mutation in exon 6 (773insC) and a splice site mutation in intron 6 (c.867+2T-->G). Two previously described mutations were also found: a deletion in exon 3 (430del4), identified in several Spanish patients, and a frequently occurring mutation in exon 6 (813ins/dup11), reported in several populations. Although the spectrum of intragenic mutations is small, only 27 reported up to now, identification of three mutations found exclusively in the Spanish population indicates that the occurrence of different intragenic mutations depends on the ethnic origin of SMA patients. In the remaining patient, who had a single SMN1 copy and three SMN2 copies, we found that the SMN1 allele was non-functional; the patient did not show any SMN1 transcript. Sequencing of the SMN promoter regions revealed various differences between promoters of the patient's four SMN genes, in particular a change in the length of a polyA run removing a putative YY1 binding site, which may affect the expression of SMN genes.
Human Genetics 03/2002; 110(3):257-63. · 5.07 Impact Factor
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ABSTRACT: Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6 - centromeric exon 7 - telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6 - centromeric exon 7 - telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.
Human Genetics 01/2001; 108(3):222-229. · 5.07 Impact Factor
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Laura Alías,
Sara Bernal,
Pablo Fuentes-Prior,
María Jesús Barceló,
Eva Also,
Rebeca Martínez-Hernández,
Francisco J. Rodríguez-Álvarez,
Yolanda Martín,
Elena Aller,
Elena Grau,
Ana Peciña,
Guillermo Antiñolo,
Enrique Galán,
Alberto L. Rosa,
Miguel Fernández-Burriel,
Salud Borrego,
José M Millán, Concepción Hernández-Chico,
Montserrat Baiget,
Eduardo F Tizzano
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ABSTRACT: 11 pages, 2 figures, 3 tables.-- PMID: 19050931 [PubMed].-- Available online Dec 3, 2008. Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II–III) predominated in males (p < 0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1–SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN–SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein. This work was supported by CIBERER (to L.A. and E. Aller), GENAME Project (to S. Bernal, R.M.H., F.J.M.A., E.G. and A.P.), and FIS05-2416 (to E. Also); Grants: FIS 05-2416 (E.F.T.) and GENAME Project (E.F.T., C·H.C., J.M.M., S. Borrego, J.C., M.R.) We wish to thank the consenting parents and patients who made this study possible. Peer reviewed
