Allele separation facilitates interpretation of potential splicing alterations and genomic rearrangements.
Mutations that alter normal splice patterns and genomic rearrangements are common causes of hereditary diseases including hereditary nonpolyposis colorectal cancer. However, abnormal transcripts can be difficult to detect and interpret because splicing patterns are often heterogeneous even in normal cells. Standard techniques including sequencing and Southern hybridization fail to detect some genomic rearrangements. We show here that separation of alleles in somatic cell hybrids, through "conversion" technology, considerably facilitates the interpretation of abnormal splicing patterns and the detection of genomic rearrangements. We detected novel mutations in MLH1 in each of four hereditary nonpolyposis colorectal cancer patients. The genomic mutations were CAG>CAA predicting Q346Q; GAG>AAG predicting E102K; a>g at nucleotide 1559-2 at intron 13, and a tandem duplication involving exons 7-12. By separating the two alleles, we showed that one allele produced only abnormal transcript or no transcript whereas the other allele produced only normal transcript. These results allowed pathogenicity to be unambiguously assigned to the mutations and increased the sensitivity of genomic testing.
Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.
Hidewaki
H
Nakagawa
Hai
H
Yan
Janet
J
Lockman
Heather
H
Hampel
Kenneth W
KW
Kinzler
Bert
B
Vogelstein
Albert
A
De La Chapelle
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics*; D004268:DNA-Binding Proteins; D004171:Diploidy; D015321:Gene Rearrangement; D015894:Genome, Human; D006238:Haploidy; D006801:Humans; D051379:Mice; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497172:MSH2 protein, human; C497173:Msh2 protein, mouse; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein