title: Somatic mutations, allele loss, and DNA methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) gene reveals association with early age of diagnosis in colorectal cancer patients.
abstract: BACKGROUND
  The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.
  
  
  METHODS
  We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.
  
  
  RESULTS
  Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15:1, a ratio significantly higher than the expected 2:1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.
  
  
  CONCLUSIONS
  Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.
authors.affiliation: Georgia Health Sciences University Cancer Center, Georgia Health Sciences University, Augusta, Georgia, United States of America.
authors.forename: Austin Y
authors.initials: AY
authors.lastname: Shull
authors.forename: Megan L
authors.initials: ML
authors.lastname: Clendenning
authors.forename: Sampa
authors.initials: S
authors.lastname: Ghoshal-Gupta
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Farrell
authors.forename: Hima V
authors.initials: HV
authors.lastname: Vangapandu
authors.forename: Larry
authors.initials: L
authors.lastname: Dudas
authors.forename: Brent J
authors.initials: BJ
authors.lastname: Wilkerson
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Buckhaults
mesh_terms: D000328:Adult; D000367:Age Factors; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D022981:Allelic Imbalance; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D006801:Humans; D019656:Loss of Heterozygosity; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D053842:Microsatellite Instability; D008875:Middle Aged; D009154:Mutation; D059645:Mutation Rate; D009367:Neoplasm Staging; D025521:Tumor Suppressor Proteins; D055815:Young Adult
chemical_list: C477880:CSMD1 protein, human; D008565:Membrane Proteins; D025521:Tumor Suppressor Proteins