Screening for exonic copy number mutations at MSH2 and MLH1 by MAPH.
BACKGROUND
Exonic deletions in MSH2 and MLH1 are significant contributors to the mutation spectrum in HNPCC, and heterozygous changes in exon copy number are not detected by conventional mutation screening methods.


OBJECTIVE
We aimed to develop methods for screening copy number changes in all the exons of the MLH1 and MSH2 genes using a single multiplex amplifiable probe hybridisation (MAPH) assay.


METHODS
We developed a probe set consisting of probes from the 19 exons of MLH1 and 16 exons of MSH2, and 3 control probes, and applied it to screening for deletions and duplications using fluorescent detection of amplified fragments.


RESULTS
We tested 73 DNA samples from controls and 50 from HNPCC patients in whom no point mutations had been found, and detected 10 copy number changes among the patient samples. A deletion of about 1.4 kb including exon 3 of MSH2 was confirmed by amplification of a junction fragment, and was shown to be the result of an unequal recombination between intronic Alu elements.


CONCLUSIONS
MAPH can detect exonic copy number changes in MLH1 and MSH2 in DNA from HNPCC patients. Since finding an exonic deletion or duplication makes full sequence analysis unnecessary, it may be most cost-effective to pre-screen samples by MAPH or MLPA before screening for point mutations.
Akrami
Seyed Mohammad
SM
Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK. john.armour@nottingham.ac.uk
Dunlop
Malcolm G
MG
Farrington
Susan M
SM
Frayling
Ian M
IM
MacDonald
Fiona
F
Harvey
John F
JF
Armour
John A L
JA
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D016022:Case-Control Studies; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005091:Exons; D016368:Frameshift Mutation; D018628:Gene Dosage; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein