Denaturing HPLC-based assay for detection of ATRX gene mutations.

human leukocytes cause activating mutations in the K-ras protooncogene. Nakajima T. K-ras gene point mutation in neo-genetic lesions of subpleural fibrotic lesions: either an early genetic event in lung cancer development or a non-specific genetic change during the inflammatory reparative process. Topographic analysis of K-ras mutations in histologically normal lung tissues and tumours of lung cancer patients.ing for p53 and K-ras mutations in whole-gut lavage in chronic inflammatory bowel disease.ras mutations in sera of patients with colorectal neoplasias and long-standing inflammatory bowel disease.ras mutation in Helicobacter pylori-associated chronic gastritis in patients with and without gastric cancer. Int To the Editor: In 2003, we described (1) a broad-range denaturing gradient gel elec-trophoresis method for mutation scanning of the entire open reading frame and canonical splice sites of the ATRX gene (OMIM 300032), a zinc finger transcriptional regulator undergoing X inactivation and probably involved in chromatin remodel-ing, DNA methylation, and gene expression in mammalian development (2, 3). Mutations affecting the ATRX gene lead to the ␣-thalassemia/men-tal retardation syndrome (ATR-X syndrome; OMIM 301040). We now propose a simpler, rapid mutational approach based on dena-turing HPLC (DHPLC) (4), with which we were able to confirm all of the nucleotide variations described in our first report (1) and to detect 5 other mutations in 7 of 15 unrelated Italian patients with a clinical suspicion of ATR-X syndrome. Segregation of the syndrome was sporadic in all but 2 individuals. X-inactivation status at the human androgen receptor locus was tested in all patients' mothers as described previously (5). PCR primers (Table 1 of the Data Supplement that accompanies the online version of this letter at http:// www.clinchem.org/content/vol51/ issue7/) were designed to amplify all 35 exons and the consensus splicing sites of the ATRX gene (Entrez Gene ID 546). A total of 44 reactions were performed at a single annealing temperature (57 °C) with Optimase DNA polymerase in 1ϫ buffer (both from Transgenomic); exon 9 was amplified as 10 overlapping fragments. Hetero-duplex formation was obtained by mixing together, denaturing, and gradually reannealing equimolar quantities of PCR products for patients and controls. DHPLC analysis was performed with the WAVE TM 3500HT System (Transgenomic). Each crude PCR product (50 ␮L) was eluted by a 2.5-min run at 3 different analysis temperatures, and the buffer gradients were chosen as suggested by the Navigator software (Trans-genomic; Table 1 of the online Data Supplement). Wild-type controls were included in each run. For the …