Frequent intragenic rearrangements of DPYD in colorectal tumours.

Dihydropyrimidine dehydrogenase is a crucial enzyme for the degradation of 5-fluorouracil (5FU). DPYD, which encodes dihydropyrimidine dehydrogenase, is prone to acquire genomic rearrangements because of the presence of an intragenic fragile site FRA1E. We evaluated DPYD copy number variations (CNVs) in a prospective series of 242 stage I-III colorectal tumours (including 87 patients receiving 5FU-based treatment). CNVs in one or more exons of DPYD were detected in 27% of tumours (deletions or amplifications of one or more DPYD exons observed in 17% and 10% of cases, respectively). A significant relationship was observed between the DPYD intragenic rearrangement status and dihydropyrimidine dehydrogenase (DPD) mRNA levels (both at the tumour level). The presence of somatic DPYD aberrations was not associated with known prognostic or predictive biomarkers, except for LOH of chromosome 8p. No association was observed between DPYD aberrations and patient survival, suggesting that assessment of somatic DPYD intragenic rearrangement status is not a powerful biomarker to predict the outcome of 5FU-based chemotherapy in patients with colorectal cancer.

Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers.

BACKGROUND: Genomic rearrangements at the fragile site FRA1E may disrupt the dihydropyrimidine dehydrogenase gene (DPYD) which is involved in 5-fluorouracil (5-FU) catabolism. In triple-negative breast cancer (TNBC), a subtype of breast cancer frequently deficient in DNA repair, we have investigated the susceptibility to acquire copy number variations (CNVs) in DPYD and evaluated their impact on standard adjuvant treatment. METHODS: DPYD CNVs were analysed in 106 TNBC tumour specimens using multiplex ligation-dependent probe amplification (MLPA) analysis. Dihydropyrimidine dehydrogenase (DPD) expression was determined by immunohistochemistry in 146 tumour tissues. RESULTS: In TNBC, we detected 43 (41%) tumour specimens with genomic deletions and/or duplications within DPYD which were associated with higher histological grade (P=0.006) and with rearrangements in the DNA repair gene BRCA1 (P=0.007). Immunohistochemical analysis revealed low, moderate and high DPD expression in 64%, 29% and 7% of all TNBCs, and in 40%, 53% and 7% of TNBCs with DPYD CNVs, respectively. Irrespective of DPD protein levels, the presence of CNVs was significantly related to longer time to progression in patients who had received 5-FU- and/or anthracycline-based polychemotherapy (hazard ratio=0.26 (95% CI: 0.07-0.91), log-rank P=0.023; adjusted for tumour stage: P=0.037). CONCLUSION: Genomic rearrangements in DPYD, rather than aberrant DPD protein levels, reflect a distinct tumour profile associated with prolonged time to progression upon first-line chemotherapy in TNBC.

Multi-ethnic cytochrome-P450 copy number profiling: novel pharmacogenetic alleles and mechanism of copy number variation formation.

To determine the role of CYP450 copy number variation (CNV) beyond CYP2D6, 11 CYP450 genes were interrogated by multiplex ligation-dependent probe amplification and quantitative PCR in 542 African-American, Asian, Caucasian, Hispanic and Ashkenazi Jewish individuals. The CYP2A6, CYP2B6 and CYP2E1 combined deletion/duplication allele frequencies ranged from 2 to 10% in these populations. High-resolution microarray-based comparative genomic hybridization (aCGH) localized CYP2A6, CYP2B6 and CYP2E1 breakpoints to directly oriented low-copy repeats. Sequencing localized the CYP2B6 breakpoint to a 529-bp intron 4 region with high homology to CYP2B7P1, resulting in the CYP2B6*29 partial deletion allele and the reciprocal, and novel, CYP2B6/2B7P1 duplicated fusion allele (CYP2B6*30). Together, these data identified novel CYP450 CNV alleles (CYP2B6*30 and CYP2E1*1Cx2) and indicate that common CYP450 CNV formation is likely mediated by non-allelic homologous recombination resulting in both full gene and gene-fusion copy number imbalances. Detection of these CNVs should be considered when interrogating these genes for pharmacogenetic drug selection and dosing.

Dihydropyrimidine dehydrogenase deficiency caused by a novel genomic deletion c.505_513del of DPYD.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disorder of the pyrimidine degradation pathway. In a patient presenting with convulsions, psychomotor retardation and Reye like syndrome, strongly elevated levels of uracil and thymine were detected in urine. No DPD activity could be detected in peripheral blood mononuclear cells. Analysis of the gene encoding DPD (DPYD) showed that the patient was homozygous for a novel c.505_513del (p.169_171del) mutation in exon 6 of DPYD.

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis.

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.

Detailed analysis of 22q11.2 with a high density MLPA probe set.

The presence of chromosome-specific low-copy repeats (LCRs) predisposes chromosome 22 to deletions and duplications. The current diagnostic procedure for detecting aberrations at 22q11.2 is chromosomal analysis coupled with fluorescence in situ hybridization (FISH) or PCR-based multiplex ligation dependent probe amplification (MLPA). However, there are copy number variations (CNVs) in 22q11.2 that are only detected by high-resolution platforms such as array comparative genomic hybridization (aCGH). We report on development of a high-definition MLPA (MLPA-HD) 22q11 kit that detects copy number changes at 37 loci on the long arm of chromosome 22. These include the 3-Mb region commonly deleted in DiGeorge/velocardiofacial syndrome (DGS/VCFS), the cat eye syndrome (CES) region, and more distal regions in 22q11 that have recently been shown to be deleted. We have used this MLPA-HD probe set to analyze 363 previously well-characterized samples with a variety of different rearrangements at 22q11 and demonstrate that it can detect copy number alterations with high sensitivity and specificity. In addition to detection of the common recurrent deletions associated with DGS/VCFS, variant and novel chromosome 22 aberrations have been detected. These include duplications within as well as deletions distal to this region. Further, the MLPA-HD detects deletion endpoint differences between patients with the common 3-Mb deletion. The MLPA-HD kit is proposed as a cost effective alternative to the currently available detection methods for individuals with features of the 22q11 aberrations. In patients with the relevant phenotypic characteristics, this MLPA-HD probe set could replace FISH for the clinical diagnosis of 22q11.2 deletions and duplications.

Large germline deletions and duplication in isolated cerebral cavernous malformation patients.

Cerebral cavernous malformations (CCM) are vascular lesions that predispose to headaches, seizures, and hemorrhagic stroke. Hereditary CCMs are usually associated with the occurrence of multiple CCMs and occur with a frequency of 1:2,000 to 1:10,000. In this study, eight isolated cases with multiple CCMs but no CCM1-3 point mutation were analyzed using the multiplex ligation-dependent probe amplification assay. Four genomic rearrangements were identified including a previously unreported large duplication within the CCM1 gene and a novel deletion involving the entire coding region of the CCM2 gene. Consequently, systematic screening for CCM deletions/duplications is recommended.