Linkage analysis with restriction fragment length polymorphisms for the gene for type II procollagen (8). Alleles of the variable-number tandem. repeat at the 3' end of COL2AI (11) were identified as PCR products (Fig. 1). Logarithm-of-odds (lod) scores were calculated with the LIPED program (12).Isolation of Cosmid Clones and DNA Sequencing. The procedure for isolating cosmid clones has been described (13). Genomic DNA was isolated from 2 x 108 cultured human skin fibroblasts and the DNA was partially digested with restriction endonuclease Sph I (Boehringer Mannheim). The DNA was fractionated by gel electrophoresis. Fragments ranging from 25 to 35 kilobases (kb) in length were electroeluted from the gel and were cloned into the cosmid vector pJB8 (Amersham), previously engineered to receive Sph I-Sph I fragments of about 25-35 kb. Clones were screened by filter hybridization using a partial genomic clone of COL2AJ as a probe. DNA from positive clones was isolated by cesium chloride centrifugation. The clones were analyzed by digestion with several restriction endonucleases and Southern blotting with probes for the gene. For subcloning, the cosmid DNA was digested with Sph I, and two fragments of 12 and 14 kb were isolated by agarose gel electrophoresis and electroelution. The two Sph I fragments were digested with EcoRI and the resulting fragments of 2-7 kb were isolated by agarose gel electrophoresis and then subcloned into the plasmid pUC19 (Boehringer Mannheim). The plasmid subclones were used for sequencing of double-stranded DNA by the dideoxynucleotide method with the use of specifically designed oligonucleotide primers based on the cDNA and genomic DNA sequences (14-16).Allele-Specific Oligonucleotide Hybridization. Genomic DNA was used as template for the PCR (17). The 5' primer was an 18-mer with a sequence corresponding to a sequence in intron 39 of COL2AJ, and the 3' primer was an 18-mer with a sequence complementary to a sequence in intron 40 (16) (Fig. 2). DNA obtained from the PCR was used for hybridization with allele-specific oligonucleotides (17). DNA blots were prehybridized for 2 hr at 650C in 6x standard saline citrate (SSC)/5x Denhardt's solution/0.5% SDS containing denatured salmon sperm DNA at 0.25 mg/ml. Hybridization was performed at 37°C for 15 hr. The filters were washed three times in 2x SSC for 15 min at room temperature and then for 2 min at 56°C (ASO-normal probe) or at 57°C (ASO-mutant probe). RESULTSLinkage analysis in the family (Fig. 1) §To whom reprint requests should be addressed.

Expression of guanylyl cyclase C mRNA in lymph nodes is associated with recurrence of colorectal cancer in patients with stage II disease. Analysis of guanylyl cyclase mRNA expression by RT-PCR may be useful for colorectal cancer staging.

A significantly increased risk of synchronous colorectal, small-bowel, gastric, and esophageal cancers and metachronous lung, prostate, and urinary tract neoplasms is clearly demonstrated. After the diagnosis of colorectal carcinoid tumors, patients should undergo appropriate screening and surveillance for cancer at these sites.

No abstract