Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification

Abstract

Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n ¼ 162) and FAP (n ¼ 74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n ¼ 122; FAP, n ¼ 24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes.

Analysis of copy number changes on chromosome 16q in male breast cancer by multiplex ligation-dependent probe amplification

“…Values between 0.7 and 1.3 were regarded normal. 13,14 Whole arm loss was defined as copy number loss of 475% of all the probes, as defined before using array-comparative genomic hybridization. 15 Partial loss on the long arm of the chromosome was defined as any probe showing copy number loss.…”

“…To define smallest regions of overlap (SRO) between areas of copy number loss we analyzed the cases according to the previous definition, with an additional threshold defining retention 0.8-1.2, 14,16 values between 0.7-0.8 and 1.2-1.3 were regarded as gray areas.…”

Molecular profiling of invasive breast cancer by multiplex ligation-dependent probe amplification-based copy number analysis of tumor suppressor and oncogenes

“…If this mean value was below 0.7 the respective gene was defined as lost, a value between 0.7 and 1.3 was defined as normal, a value between 1.3 and 2.0 as low-level amplification and values 42.0 as high-level amplified as previously established. 31,32 Statistics Statistics were performed using SPSS statistical software. Data were dichotomized as follows:…”

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

“…For example, this pathogenic mechanism has been described in alpha-thalassaemia (MIM] 141800) [Mathew et al, 1983;Borriello et al, 1994] and Duchenne and Becker muscular dystrophies (DMD, MIM] 310200; BMD, MIM] 300376) where it is responsible for approximately 70% of cases [Kunkel, 1986]. More recently, it was reported to play a relevant role in familial cancer predisposition syndromes, such as hereditary nonpolyposis colorectal cancer (HNPCC; MIM] 114500) [Charbonnier et al, 2000]; familial adenomatous polyposis coli (FAP; MIM] 175100); and familial breast and ovarian cancer (MIM] 113705; MIM] 600185) [Bunyan et al, 2004]. Gross genomic rearrangements can range from extra copies of entire chromosomes to deletions or duplications of single exons.…”