Deletions within COL11A1 in Type 2 stickler syndrome detected by multiplex ligation-dependent probe amplification (MLPA).

BACKGROUND: COL11A1 is a large complex gene around 250 kb in length and consisting of 68 exons. Pathogenic mutations in the gene can result in Stickler syndrome, Marshall syndrome or Fibrochondrogenesis. Many of the mutations resulting in either Stickler or Marshall syndrome alter splice sites and result in exon skipping, which because of the exon structure of collagen genes usually leaves the message in-frame. The mutant protein then exerts a dominant negative effect as it co-assembles with other collagen gene products. To date only one large deletion of 40 kb in the COL11A1, which was detected by RT-PCR, has been characterized. However, commonly used screening protocols, utilizing genomic amplification and exon sequencing, are unlikely to detect such large deletions. Consequently the frequency of this type of mutation is unknown. CASE PRESENTATIONS: We have used Multiplex Ligation-Dependent Probe Amplification (MLPA) in conjunction with exon amplification and sequencing, to analyze patients with clinical features of Stickler syndrome, and have detected six novel deletions that were not found by exon sequencing alone. CONCLUSION: Exon deletions appear to represent a significant proportion of type 2 Stickler syndrome. This observation was previously unknown and so diagnostic screening of COL11A1 should include assays capable of detecting both large and small deletions, in addition to exon sequencing.

A new case of GABA transaminase deficiency facilitated by proton MR spectroscopy.

BACKGROUND: Deficiency of 4-aminobutyrate aminotransferase (GABA-T) is a rare disorder of GABA catabolism, with only a single sibship reported. We report on a third case, a Japanese female infant with severe psychomotor retardation and recurrent episodic lethargy with intractable seizures, with the diagnosis facilitated by proton magnetic resonance (MR) spectroscopy ((1)H-MRS). METHODS: Neuroimaging was performed at the first episode of lethargy. For (1)H-MRS, locations were placed in the semioval center and the basal ganglia. Quantification of metabolite concentrations were derived using the LCModel. We confirmed the diagnosis subsequently by enzyme and molecular studies, which involved direct DNA sequence analysis and the development of a novel multiplex ligation-dependent probe amplification test. RESULTS: (1)H-MRS analysis revealed an elevated GABA concentration in the basal ganglia (2.9 mmol/l). Based on the results of quantitative (1)H-MRS and clinical findings, GABA-T deficiency was suspected and confirmed in cultured lymphoblasts. Molecular studies of the GABA-T gene revealed compound heterozygosity for a deletion of one exon and a missense mutation, 275G>A, which was not detected in 210 control chromosomes. CONCLUSIONS: Our results suggest that excessive prenatal GABA exposure in the central nervous system (CNS) was responsible for the clinical manifestations of GABA transaminase deficiency. Our findings suggest the dual nature of GABA as an excitatory molecule early in life, followed by a functional switch to an inhibitory species later in development. Furthermore, quantitative (1)H-MRS appears to be a useful, noninvasive tool for detecting inborn errors of GABA metabolism in the CNS.

An overview of L-2-hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype-phenotype study.

L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.

Clinical and histomolecular endometrial tumor characterization of patients at-risk for Lynch syndrome in South of Brazil.

Lynch syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in one of the mismatch repair (MMR) genes: MLH1, MSH2, MSH6 and PMS2. Clinically, Lynch syndrome is characterized by early onset (45 years) of colorectal cancer (CRC), as well as extra-colonic cancer. Male and female carriers of Lynch syndrome-associated mutations have different lifetime risks for CRC and in women endometrial cancer (EC) may be the most common tumor. Whenever Amsterdam criteria are not fulfilled, the currently recommended laboratory screening strategies involve microsatellite instability testing and immunohistochemistry staining of the tumor for the major MMR proteins. The aim of this study was to estimate the frequency of MMR deficiencies in women diagnosed with EC who are at-risk for Lynch syndrome. Thirty women diagnosed with EC under the age of 50 years and/or women with EC and a first degree relative diagnosed with a Lynch syndrome-associated tumor were included. To assess MMR deficiencies four methods were used: multiplex PCR, Single Strand Conformation Polymorphism, Immunohistochemistry and Methylation Specific-Multiplex Ligation-dependent Probe Amplification. Twelve (40%) patients with EC fulfilling one of the inclusion criteria had results indicative of MMR deficiency. The identification of 5 women with clear evidence of MMR deficiency and absence of either Amsterdam or Bethesda criteria among 10 diagnosed with EC under the age of 50 years reinforces previous suggestions by some authors that these women should be considered at risk and always screened for Lynch syndrome after informed consent.

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria.

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.

MLPA diagnostics of complex microbial communities: relative quantification of bacterial species in oral biofilms.

A multitude of molecular methods are currently used for identification and characterization of oral biofilms or for community profiling. However, multiplex PCR techniques that are able to routinely identify several species in a single assay are not available. Multiplex Ligation-dependent Probe Amplification (MLPA) identifies up to 45 unique fragments in a single tube PCR. Here we report a novel use of MLPA in the relative quantification of targeted microorganisms in a community of oral microbiota. We designed 9 species specific probes for: Actinomyces gerencseriae, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans, Lactobacillus acidophilus, Rothia dentocariosa, Streptococcus mutans, Streptococcus sanguinis and Veillonella parvula; and genus specific probes for selected oral Streptococci and Lactobacilli based on their 16S rDNA sequences. MLPA analysis of DNA pooled from the strains showed the expected specific MLPA products. Relative quantification of a serial dilution of equimolar DNA showed that as little as 10 pg templates can be detected with clearly discernible signals. Moreover, a 2 to 7% divergence in relative signal ratio of amplified probes observed from normalized peak area values suggests MLPA can be a cheaper alternative to using qPCR for quantification. We observed 2 to 6 fold fluctuations in signal intensities of MLPA products in DNAs isolated from multispecies biofilms grown in various media for various culture times. Furthermore, MLPA analyses of DNA isolated from saliva obtained from different donors gave a varying number and intensity of signals. This clearly shows the usefulness of MLPA in a quantitative description of microbial shifts.

Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia.

By assessment of the methylation status of 25 candidate tumor suppressor genes (TSGs) in 119 acute myeloid leukemia (AML) patients and 5 controls, we aimed to determine whether simultaneous methylation of multiple TSGs exerts prognostic impact. Methylation-specific multiplex ligation probe amplification (MS-MLPA) revealed methylation of at least one TSG in 59/119 patients, while no methylation was found in controls. Methylation of different TSGs within patients was substantially correlated (intra-class correlation; 0.38). ESR1 methylation (34/119) strongly predicted concurrent methylation of other genes, OR 7.33 (95%CI 4.13-12.99). A Cox regression model that included the three most frequently methylated TSGs ESR1, CDKN2B/p15 and IGSF4, showed ESR1 to have opposite effects on overall survival (OS) compared with the other two, HR 0.22 (95% CI 0.09-0.53) and HR 1.66 (95% CI 0.73-3.79), HR 1.61 (95%CI 0.66-3.93). By assessment of CDKN2B/p15 and IGSF4 methylation, patients with methylation at multiple loci can be identified. Accumulation of methylation aberrancies is much more pronounced in ESR1 methylated patients. When combined, the methylation status of ESR1, CDKN2B/p15 and IGSF4 enable identification of patient subgroups with large differences in OS (p <0.0001). This study shows that methylation profiling allows risk stratification in AML. In addition, ESR1 methylation may reflect a biological pathway that leads to hypermethylation of multiple genes, which is reflected by methylation of IGSF4 and/or CDKN2B/p15.

Genetic subtyping of Fanconi anemia by comprehensive mutation screening.

Fanconi anemia (FA) is a recessively inherited syndrome with predisposition to bone marrow failure and malignancies. Hypersensitivity to cross-linking agents is a cellular feature used to confirm the diagnosis. The mode of inheritance is autosomal recessive (12 subtypes) as well as X-linked (one subtype). Most genetic subtypes have initially been defined as "complementation groups" by cell fusion studies. Here we report a comprehensive genetic subtyping approach for FA that is primarily based on mutation screening, supplemented by protein expression analysis and by functional assays to test for pathogenicity of unclassified variants. Of 80 FA cases analyzed, 73 (91%) were successfully subtyped. In total, 92 distinct mutations were detected, of which 56 were novel (40 in FANCA, eight in FANCC, two in FANCD1, three in FANCE, one in FANCF, and three in FANCG). All known complementation groups were represented, except D2, J, L, and M. Three patients could not be classified because proliferating cell cultures from the probands were lacking. In cell lines from the remaining four patients, immunoblotting was used to determine their capacity to monoubiquitinate FANCD2. In one case FANCD2 monoubiquitination was normal, indicating a defect downstream. In the remaining three cases monoubiquitination was not detectable, indicating a defect upstream. In the latter four patients, pathogenic mutations in a known FA gene may have been missed, or these patients might represent novel genetic subtypes. We conclude that direct mutation screening allows a molecular diagnosis of FA in the vast majority of patients, even in cases where growing cells from affected individuals are unavailable. Proliferating cell lines are required in a minority (<15%) of the patients, to allow testing for FANCD2 ubiquitination status and sequencing of FANCD2 using cDNA, to avoid interference from pseudogenes.

MS-MLPA: an attractive alternative laboratory assay for robust, reliable, and semiquantitative detection of MGMT promoter hypermethylation in gliomas.

Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2.

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.

Cytogenetic instability in ovarian epithelial cells from women at risk of ovarian cancer.

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

Epigenetic events of disease progression in head and neck squamous cell carcinoma.

OBJECTIVE: To examine the promoter methylation status of the 22 cancer genes and their contribution to disease progression in 6 head and neck squamous cell carcinoma (HNSCC) cell lines. DESIGN: A panel of 41 gene probes, designed to interrogate 35 unique genes with known associations to cancer including HNSCC, was interrogated for alterations in gene copy number and aberrant methylation status (22 genes) using the methylation-specific multiplex ligation-dependent probe amplification assay. SUBJECTS: Primary (A) and recurrent or metastatic (B) HNSCC cell lines UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B are described. RESULTS: Nine genes, TIMP3, APC, KLK10, TP73, CDH13, IGSF4, FHIT, ESR1, and DAPK1, were aberrantly methylated. The most frequently hypermethylated genes were APC and IGSF4, observed in 3 of 6 cell lines, and TP73 and DAPK1, observed in 2 of 6. For KLK10 and IGSF4, TIMP3 and FHIT, and TP73, in UMSCC-11B, UMSCC-17B, and UMSCC-81B, respectively, promoter hypermethylation was a disease progression event, indicating complete abrogation of tumor suppressor function for KLK10, IGSF4, and TIMP3 and gene silencing of 1 of 2 copies of TP73. Hypermethylation of IGSF4, TP73, CDH13, ESR1, DAPK1, and APC were primary events in UMSCC-17A. CONCLUSIONS: Gene silencing through promoter hypermethylation was observed in 5 of 6 cell lines and contributed to primary and progressive events in HNSCC. In addition to genetic alterations of gains and losses, epigenetic events appear to further undermine a destabilized genomic repertoire in HNSCC.

Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.

Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA-probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA-probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader-Willy syndrome, Angelman syndrome or acute myeloid leukemia.

Screening for large mutations of the NF2 gene.

Neurofibromatosis 2 (NF2) is a genetic disorder caused by mutational inactivation of the NF2 gene and is characterized by bilateral vestibular schwannomas, spinal tumors, and other benign tumors of the nervous system. Previously, we found intragenic NF2 mutations in 99 of 188 unrelated NF2 patients by exon-scanning-based methods. Tumor analysis of 22 de novo NF2 patients led to the identification of 12 additional constitutive NF2 mutations. The remaining 77 patients were further examined for large alterations using the newly developed gene dosage assay multiplex ligation-dependent probe amplification (MLPA). One deletion of a single exon, seven deletions of multiple exons, seven deletions involving the 3' or 5' end of the NF2 gene, four deletions involving the whole NF2 gene, and one duplication of three exons were detected. For 47 of the 77 patients, mRNA of adequate quality could be obtained, enabling transcript analysis, which confirmed eight alterations detected by MLPA. In addition, in one family, the mRNA analysis detected an insertion of two exons of another gene. Thus, deletions, duplications, and insertions affecting the NF2 gene were found in 21 cases, which is 11% of the 188 unrelated NF2 patients studied, 16% of the 132 mutations identified, and 27% of the 77 cases in which no intragenic small mutations were detected by exon scanning. The combination of multiple screening techniques facilitated a mutation-detection rate of 100% for the 21 inherited cases in this study.

Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques.

Large deletions in the APC (adenomatous polyposis coli) gene, causing familial adenomatous polyposis (FAP), cannot easily be detected by conventional mutation-detection techniques. Therefore, we have developed two independent quantitative methods for the detection of large deletions, encompassing one or more exons, of APC. Multiplex ligation-dependent probe amplification (MLPA) is performed in one reaction for the initial quantification of all APC exon copy numbers. Subsequently, quantitative real-time PCR (QRT-PCR) is used to verify the results obtained in the MLPA reaction. The identification of a deletion of the whole APC gene in a patient with classical FAP is described. The mutation was detected with the two quantitative methods and further verified on chromosomal level by the use of FISH (fluorescence in situ hybridization) on metaphase spreads. Furthermore, a large deletion covering exons 11-13 of the APC gene was detected in two apparently unrelated families. This deletion was further verified and characterized with long-range PCR. The MLPA test ensures a sensitive high-throughput screening for large deletions of the APC gene and can easily be implemented in the diagnostic testing for FAP.

A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2.

We recently described a CHO DSBR mutant belonging to the XRCC7 complementation group (XR-C2) that has the interesting phenotype of being radiosensitive, but having only a modest defect in VDJ recombination. This cell line expresses only slightly reduced levels of DNA-PKcs but has undetectable DNA-PK activity. Limited sequence analyses of DNA-PKcs transcripts from XR-C2 revealed a point mutation that results in an amino acid substitution of glutamic acid for glycine six residues from the C-terminus. To determine whether this single substitution was responsible for the phenotype in XR-C2 cells, we introduced the mutation into a DNA-PKcs expression vector. Whereas transfection of this expression vector significantly restores the VDJ recombination deficits in DNA-PKcs-deficient cells, radioresistance is not restored. Thus, expression of this mutant form of DNA-PKcs in DNA-PKcs- deficient cells substantially recapitulates the phenotype observed in XR-C2, and we conclude that this single amino acid substitution is responsible for the non-homologous end joining deficits observed in XR-C2.

Brca2 (XRCC11) deficiency results in radioresistant DNA synthesis and a higher frequency of spontaneous deletions.

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.