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1.
Oncotarget ; 6(36): 38777-88, 2015 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-26472283

RESUMEN

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes and tumor suppressors. Loss-of-function mutations give rise to hereditary paragangliomas/pheochromocytomas and hereditary leiomyomatosis and renal cell carcinoma. Inactivation of SDH and FH results in an abnormal accumulation of their substrates succinate and fumarate, leading to inhibition of numerous α-ketoglutarate dependent dioxygenases, including histone demethylases and the ten-eleven-translocation (TET) family of 5-methylcytosine (5 mC) hydroxylases. To evaluate the distribution of DNA and histone methylation, we used immunohistochemistry to analyze the expression of 5 mC, 5-hydroxymethylcytosine (5 hmC), TET1, H3K4me3, H3K9me3, and H3K27me3 on tissue microarrays containing paragangliomas/pheochromocytomas (n = 134) and hereditary and sporadic smooth muscle tumors (n = 56) in comparison to their normal counterparts. Our results demonstrate distinct loss of 5 hmC in tumor cells in SDH- and FH-deficient tumors. Loss of 5 hmC in SDH-deficient tumors was associated with nuclear exclusion of TET1, a known regulator of 5 hmC levels. Moreover, increased methylation of H3K9me3 occurred predominantly in the chief cell component of SDH mutant tumors, while no changes were seen in H3K4me3 and H3K27me3, data supported by in vitro knockdown of SDH genes. We also show for the first time that FH-deficient smooth muscle tumors exhibit increased H3K9me3 methylation compared to wildtype tumors. Our findings reveal broadly similar patterns of epigenetic deregulation in both FH- and SDH-deficient tumors, suggesting that defects in genes of the TCA cycle result in common mechanisms of inhibition of histone and DNA demethylases.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Citosina/análogos & derivados , Fumarato Hidratasa/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Paraganglioma/genética , Feocromocitoma/genética , Tumor de Músculo Liso/genética , Succinato Deshidrogenasa/genética , 5-Metilcitosina/análogos & derivados , Neoplasias de las Glándulas Suprarrenales/enzimología , Núcleo Celular/metabolismo , Citosina/metabolismo , Fumarato Hidratasa/deficiencia , Fumarato Hidratasa/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Inmunohistoquímica , Oxigenasas de Función Mixta/metabolismo , Paraganglioma/enzimología , Paraganglioma/patología , Feocromocitoma/enzimología , Feocromocitoma/patología , Proteínas Proto-Oncogénicas/metabolismo , Tumor de Músculo Liso/enzimología , Succinato Deshidrogenasa/deficiencia , Succinato Deshidrogenasa/metabolismo
2.
J Mol Diagn ; 17(5): 590-6, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26162331

RESUMEN

The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. We have developed a sensitive, mutation-specific, and fast alternative for next-generation sequencing-mediated noninvasive prenatal diagnosis using a PCR-based method. For this proof-of-principle study, noninvasive fetal paternally inherited mutation detection was performed using cell-free DNA from maternal plasma. Preferential amplification of the paternally inherited allele was accomplished through a personalized approach using a blocking probe against maternal sequences in a high-resolution melting curve analysis-based assay. Enhanced detection of the fetal paternally inherited mutation was obtained for both an autosomal dominant and a recessive monogenic disorder by blocking the amplification of maternal sequences in maternal plasma.


Asunto(s)
Análisis Mutacional de ADN/métodos , Padre , Enfermedades Genéticas Congénitas/diagnóstico , Embarazo/sangre , Diagnóstico Prenatal/métodos , Análisis Químico de la Sangre/métodos , ADN/análisis , ADN/sangre , Femenino , Enfermedades Genéticas Congénitas/genética , Mutación de Línea Germinal , Humanos , Patrón de Herencia , Masculino , Madres , Mutación , Linaje , Polimorfismo de Nucleótido Simple
3.
Fam Cancer ; 14(2): 247-57, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25604157

RESUMEN

Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6% for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6%) of the patients and pathogenic mosaic variants in 2 (1%). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7% of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.


Asunto(s)
Poliposis Adenomatosa del Colon/genética , Genes APC , Mosaicismo , Mutación , Adolescente , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple
4.
Hum Mutat ; 34(11): 1519-28, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23929686

RESUMEN

De novo germline variants in several components of the SWI/SNF-like BAF complex can cause Coffin-Siris syndrome (CSS), Nicolaides-Baraitser syndrome (NCBRS), and nonsyndromic intellectual disability. We screened 63 patients with a clinical diagnosis of CSS for these genes (ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCB1, and SMARCE1) and identified pathogenic variants in 45 (71%) patients. We found a high proportion of variants in ARID1B (68%). All four pathogenic variants in ARID1A appeared to be mosaic. By using all variants from the Exome Variant Server as test data, we were able to classify variants in ARID1A, ARID1B, and SMARCB1 reliably as being pathogenic or nonpathogenic. For SMARCA2, SMARCA4, and SMARCE1 several variants in the EVS remained unclassified, underlining the importance of parental testing. We have entered all variant and clinical information in LOVD-powered databases to facilitate further genotype-phenotype correlations, as these will become increasingly important because of the uptake of targeted and untargeted next generation sequencing in diagnostics. The emerging phenotype-genotype correlation is that SMARCB1 patients have the most marked physical phenotype and severe cognitive and growth delay. The variability in phenotype seems most marked in ARID1A and ARID1B patients. Distal limbs anomalies are most marked in ARID1A patients and least in SMARCB1 patients. Numbers are small however, and larger series are needed to confirm this correlation.


Asunto(s)
Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Cara/anomalías , Estudios de Asociación Genética , Deformidades Congénitas de la Mano/diagnóstico , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Micrognatismo/diagnóstico , Micrognatismo/genética , Complejos Multiproteicos/genética , Cuello/anomalías , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Exones , Facies , Orden Génico , Humanos , Proteínas Nucleares/genética , Fenotipo , Proteína SMARCB1 , Factores de Transcripción/genética
5.
Breast Cancer Res Treat ; 134(1): 219-27, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22297469

RESUMEN

The MUTYH gene is involved in base excision repair. MUTYH mutations predispose to recessively inherited colorectal polyposis and cancer. Here, we evaluate an association with breast cancer (BC), following up our previous finding of an elevated BC frequency among Dutch bi-allelic MUTYH mutation carriers. A case­control study was performed comparing 1,469 incident BC patients (ORIGO cohort), 471 individuals displaying features suggesting a genetic predisposition for BC, but without a detectable BRCA1 or BRCA2 mutation (BRCAx cohort), and 1,666 controls. First, for 303 consecutive patients diagnosed before age 55 years and/or with multiple primary breast tumors, the MUTYH coding region and flanking introns were sequenced. The remaining subjects were genotyped for five coding variants, p.Tyr179Cys, p.Arg309Cys, p.Gly396Asp, p.Pro405Leu, and p.Ser515Phe, and four tagging SNPs, c.37-2487G>T, p.Val22Met, c.504+35G>A, and p.Gln338His. No bi-allelic pathogenic MUTYH mutations were identified. The pathogenic variant p.Gly396Asp and the variant of uncertain significance p.Arg309Cys occurred twice as frequently in BRCAx subjects as compared to incident BC patients and controls (p=0.13 and p=0.15, respectively). The likely benign variant p.Val22Met occurred less frequently in patients from the incident BC (p=0.03) and BRCAx groups (p=0.11), respectively, as compared to the controls. Minor allele genotypes of several MUTYH variants showed trends towards association with lobular BC histology. This extensive case­control study could not confirm previously reported associations of MUTYH variants with BC, although it was too small to exclude subtle effects on BC susceptibility.


Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Lobular/genética , ADN Glicosilasas/genética , Estudios de Casos y Controles , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Genes BRCA1 , Genes BRCA2 , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Persona de Mediana Edad , Mutación , Países Bajos
6.
Hum Mutat ; 31(11): 1205-15, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20725929

RESUMEN

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A4G/p.Tyr179Cys and c.1187G4A/p.Gly396Asp (previously c.494A4G/p.Tyr165Cys and c.1145G4A/p.Gly382Asp).However, for a substantial fraction of the detected variants, the clinical significance remains uncertain,compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database (www.lovd.nl/MUTYH) with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants,respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.


Asunto(s)
ADN Glicosilasas/genética , Bases de Datos Genéticas , Variación Genética , Poliposis Adenomatosa del Colon/genética , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , ADN Glicosilasas/química , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Datos de Secuencia Molecular , Mutación , Países Bajos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína
7.
PLoS One ; 4(11): e7987, 2009 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-19956719

RESUMEN

BACKGROUND: Mitochondrial succinate dehydrogenase (SDH) is a component of both the tricarboxylic acid cycle and the electron transport chain. Mutations of SDHD, the first protein of intermediary metabolism shown to be involved in tumorigenesis, lead to the human tumors paraganglioma (PGL) and pheochromocytoma (PC). SDHD is remarkable in showing an 'imprinted' tumor suppressor phenotype. Mutations of SDHD show a very high penetrance in man and we postulated that knockout of Sdhd would lead to the development of PGL/PC, probably in aged mice. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conventional knockout of Sdhd in the mouse, removing the entire third exon. We also crossed this mouse with a knockout of H19, a postulated imprinted modifier gene of Sdhd tumorigenesis, to evaluate if loss of these genes together would lead to the initiation or enhancement of tumor development. Homozygous knockout of Sdhd results in embryonic lethality. No paraganglioma or other tumor development was seen in Sdhd KO mice followed for their entire lifespan, in sharp contrast to the highly penetrant phenotype in humans. Heterozygous Sdhd KO mice did not show hyperplasia of paraganglioma-related tissues such as the carotid body or of the adrenal medulla, or any genotype-related pathology, with similar body and organ weights to wildtype mice. A cohort of Sdhd/H19 KO mice developed several cases of profound cardiac hypertrophy, but showed no evidence of PGL/PC. CONCLUSIONS: Knockout of Sdhd in the mouse does not result in a disease phenotype. H19 may not be an initiator of PGL/PC tumorigenesis.


Asunto(s)
Mutación , Paraganglioma/genética , Feocromocitoma/genética , ARN no Traducido/genética , Succinato Deshidrogenasa/genética , Animales , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Genotipo , Heterocigoto , Masculino , Ratones , Ratones Noqueados , Fenotipo , ARN Largo no Codificante
8.
Hum Mutat ; 30(12): 1703-12, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19842214

RESUMEN

We evaluated massive parallel sequencing and long-range PCR (LRP) for rare variant detection and allele frequency estimation in pooled DNA samples. Exons 2 to 16 of the MUTYH gene were analyzed in breast cancer patients with Illumina's (Solexa) technology. From a pool of 287 genomic DNA samples we generated a single LRP product, while the same LRP was performed on 88 individual samples and the resulting products then pooled. Concentrations of constituent samples were measured with fluorimetry for genomic DNA and high-resolution melting curve analysis (HR-MCA) for LRP products. Illumina sequencing results were compared to Sanger sequencing data of individual samples. Correlation between allele frequencies detected by both methods was poor in the first pool, presumably because the genomic samples amplified unequally in the LRP, due to DNA quality variability. In contrast, allele frequencies correlated well in the second pool, in which all expected alleles at a frequency of 1% and higher were reliably detected, plus the majority of singletons (0.6% allele frequency). We describe custom bioinformatics and statistics to optimize detection of rare variants and to estimate required sequencing depth. Our results provide directions for designing high-throughput analyses of candidate genes.


Asunto(s)
ADN/genética , Variación Genética/genética , Análisis de Secuencia de ADN/métodos , Alelos , Frecuencia de los Genes/genética , Genoma Humano/genética , Humanos , Desnaturalización de Ácido Nucleico/genética , Reacción en Cadena de la Polimerasa
9.
Int J Cancer ; 121(6): 1386-9, 2007 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-17520677

RESUMEN

Germline mutations in nuclear genes encoding mitochondrial enzymes fumarate hydratase (FH) and succinate dehydrogenase (subunits SDHB/C/D) have been implicated in the development of tumor syndromes referred to as hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary paragangliomatosis (HPGL), respectively. FH and SDH are operating in the tricarboxylic acid cycle (the TCA cycle, the Krebs cycle). In the FH and SDH deficient tumors, accumulation of the substrates, fumarate and succinate, has been shown to cause stabilization of hypoxia inducible factor 1 alpha (HIF1 alpha). According to recent studies, HIF1 alpha could contribute to the hypoxia induced genomic instability seen in many cancers, through repression of mismatch repair (MMR) protein MSH2. In this study, in agreement with previous works, we found HIF1 alpha to be moderately or highly stabilized in 67% (16/24) and 77% (48/62) of HLRCC tumors and SDHB/C/D paragangliomas (PGL) and pheochromocytomas (PHEO), respectively. In addition, a set of 54 other familial and nonfamilial PGLs/PHEOs were studied. Moderately or highly stabilized HIF1 alpha was present in 68% (26/38) of the PGLs but in PHEOs (n = 16) no such pattern was observed. We then analyzed the suggested link between HIF1 alpha stabilization and MSH2 repression, in HLRCC and HPGL tumor material. No microsatellite instability (MSI) or lack of MSH2 expression was, however, observed. Thus we failed to provide in vivo evidence for the proposed link between HIF1 alpha stabilization and functional MMR deficiency, in TCAC deficient tumors.


Asunto(s)
Fumarato Hidratasa/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inestabilidad de Microsatélites , Síndromes Neoplásicos Hereditarios/genética , Succinato Deshidrogenasa/deficiencia , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Ciclo del Ácido Cítrico/fisiología , Fumarato Hidratasa/genética , Mutación de Línea Germinal , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Leiomiomatosis/genética , Leiomiomatosis/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Síndromes Neoplásicos Hereditarios/metabolismo , Paraganglioma/genética , Paraganglioma/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Succinato Deshidrogenasa/genética , Análisis de Matrices Tisulares
10.
BMC Med Genet ; 7: 1, 2006 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-16405730

RESUMEN

BACKGROUND: Germline mutations of the SDHD, SDHB and SDHC genes, encoding three of the four subunits of succinate dehydrogenase, are a major cause of hereditary paraganglioma and pheochromocytoma, and demonstrate that these genes are classic tumor suppressors. Succinate dehydrogenase is a heterotetrameric protein complex and a component of both the Krebs cycle and the mitochondrial respiratory chain (succinate:ubiquinone oxidoreductase or complex II). METHODS: Using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing to analyse genomic DNA from peripheral blood lymphocytes, here we describe the mutation analysis of the SDHB and SDHC genes in 37 patients with sporadic (i.e. no known family history) head and neck paraganglioma and five pheochromocytoma and/or paraganglioma families. RESULTS: Two sporadic patients were found to have a SDHB splice site mutation in intron 4, c.423+1G>A, which produces a mis-spliced transcript with a 54 nucleotide deletion, resulting in an 18 amino acid in-frame deletion. A third patient was found to carry the c.214C>T (p.Arg72Cys) missense mutation in exon 4 of SDHC, which is situated in a highly conserved protein motif that constitutes the quinone-binding site of the succinate: ubiquinone oxidoreductase (SQR) complex in E. coli. Together with our previous results, we found 27 germline mutations of SDH genes in 95 cases (28%) of sporadic head and neck paraganglioma. In addition all index patients of five families showing hereditary pheochromocytoma-paraganglioma were found to carry germline mutations of SDHB: four of which were novel, c.343C>T (p.Arg115X), c.141G>A (p.Trp47X), c.281G>A (p.Arg94Lys), and c.653G>C (p.Trp218Ser), and one reported previously, c.136C>T, p.Arg46X. CONCLUSION: In conclusion, these data indicate that germline mutations of SDHB and SDHC play a minor role in sporadic head and neck paraganglioma and further underline the importance of germline SDHB mutations in cases of familial pheochromocytoma-paraganglioma.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Mutación de Línea Germinal , Neoplasias de Cabeza y Cuello/genética , Proteínas Hierro-Azufre/genética , Proteínas de la Membrana/genética , Paraganglioma/genética , Feocromocitoma/genética , Subunidades de Proteína/genética , Succinato Deshidrogenasa/genética , Adolescente , Adulto , Anciano , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sitios de Empalme de ARN
11.
Oncogene ; 23(23): 4076-83, 2004 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-15064708

RESUMEN

Germline mutations in succinate dehydrogenase subunits B, C and D (SDHB, SDHC and SDHD), genes encoding subunits of mitochondrial complex II, cause hereditary paragangliomas and phaeochromocytomas. In SDHB (1p36)- and SDHC (1q21)-linked families, disease inheritance is autosomal dominant. In SDHD (11q23)-linked families, the disease phenotype is expressed only upon paternal transmission of the mutation, consistent with maternal imprinting. However, SDHD shows biallelic expression in brain, kidney and lymphoid tissues (Baysal et al., 2000). Moreover, consistent loss of the wild-type (wt) maternal allele in SDHD-linked tumours suggests expression of the maternal SDHD allele in normal paraganglia. Here we demonstrate exclusive loss of the entire maternal chromosome 11 in SDHD-linked paragangliomas and phaeochromocytomas, suggesting that combined loss of the wt SDHD allele and maternal 11p region is essential for tumorigenesis. We hypothesize that this is driven by selective loss of one or more imprinted genes in the 11p15 region. In paternally, but not in maternally derived SDHD mutation carriers, this can be achieved by a single event, that is, non-disjunctional loss of the maternal chromosome 11. Thus, the exclusive paternal transmission of the disease can be explained by a somatic genetic mechanism targeting both the SDHD gene on 11q23 and a paternally imprinted gene on 11p15.5, rather than imprinting of SDHD.


Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Proteínas de la Membrana/genética , Paraganglioma/genética , Impresión Genómica , Humanos , Hibridación Fluorescente in Situ , Pérdida de Heterocigocidad , Modelos Genéticos , Succinato Deshidrogenasa
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