Allelic dropout in long QT syndrome genetic testing: a possible mechanism underlying false-negative results.

BACKGROUND: Genetic testing for congenital long QT syndrome (LQTS) has been performed in research laboratories for the past decade. Approximately 75% of patients with high clinical probability for LQTS have a mutation in one of five LQTS-causing cardiac channel genes. Possible explanations for the remaining genotype-negative cases include LQTS mimickers, novel LQTS-causing genes, unexplored regions of the known genes, and genetic testing detection failures. OBJECTIVES: The purpose of this study was to explore the possibility of allelic dropout as a possible mechanism underlying false-negative test results. METHODS: The published primers currently used by many research laboratories to conduct a comprehensive analysis of the 60 translated exons in the KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) genes were analyzed for the presence of common intronic single nucleotide polymorphisms (SNPs). Repeat mutational analysis, following primer/amplicon redesign using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, was performed on a cohort of 541 consecutive, unrelated patients referred for LQTS genetic testing. RESULTS: Common (>1% minor allele frequency) intronic SNPs were discovered within the primer sequences of five of 60 translated exons. Following primer redesign to eliminate the possibility of allelic dropout, four previously genotype-negative index cases were found to possess LQTS-causing mutations: R591H-KCNQ1 and R594Q-KCNQ1 for exon 15 and E229X-KCNH2 in two unrelated cases. Repeat examination of these two amplicons in 400 reference alleles did not identify these or any additional amino acid variants. CONCLUSION: Allelic dropout secondary to intronic SNP-primer mismatch prevented the discovery of LQTS-causing mutations in four cases. Considering that many LQTS genetic testing research laboratories have used these primers, patients who reportedly are genotype negative may benefit from re-examination of those regions susceptible to allelic dropout due to primer-disrupting SNPs, particularly exon 15 in KCNQ1 and exon 4 in KCNH2.

Spectrum and prevalence of mutations from the first 2,500 consecutive unrelated patients referred for the FAMILION long QT syndrome genetic test.

BACKGROUND: Long QT syndrome (LQTS) is a potentially lethal, highly treatable cardiac channelopathy for which genetic testing has matured from discovery to translation and now clinical implementation. OBJECTIVES: Here we examine the spectrum and prevalence of mutations found in the first 2,500 unrelated cases referred for the FAMILION LQTS clinical genetic test. METHODS: Retrospective analysis of the first 2,500 cases (1,515 female patients, average age at testing 23 +/- 17 years, range 0 to 90 years) scanned for mutations in 5 of the LQTS-susceptibility genes: KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6). RESULTS: Overall, 903 referral cases (36%) hosted a possible LQTS-causing mutation that was absent in >2,600 reference alleles; 821 (91%) of the mutation-positive cases had single genotypes, whereas the remaining 82 patients (9%) had >1 mutation in > or =1 gene, including 52 cases that were compound heterozygous with mutations in >1 gene. Of the 562 distinct mutations, 394 (70%) were missense, 428 (76%) were seen once, and 336 (60%) are novel, including 92 of 199 in KCNQ1, 159 of 226 in KCNH2, and 70 of 110 in SCN5A. CONCLUSION: This cohort increases the publicly available compendium of putative LQTS-associated mutations by >50%, and approximately one-third of the most recently detected mutations continue to be novel. Although control population data suggest that the great majority of these mutations are pathogenic, expert interpretation of genetic test results will remain critical for effective clinical use of LQTS genetic test results.

Genomic analysis of Hox clusters in the sea lamprey Petromyzon marinus.

The sea lamprey Petromyzon marinus is among the most primitive of extant vertebrates. We are interested in the organization of its Hox gene clusters, because, as a close relative of the gnathostomes, this information would help to infer Hox cluster organization at the base of the gnathostome radiation. We have partially mapped the P. marinus Hox clusters using phage, cosmid, and P1 artificial chromosome libraries. Complete homeobox sequences were obtained for the 22 Hox genes recovered in the genomic library screens and analyzed for cognate group identity. We estimate that the clusters are somewhat larger than those of mammals (roughly 140 kbp vs. 105 kbp) but much smaller than the single Hox cluster of the cephalochordate amphioxus (at more than 260 kb). We never obtained more than three genes from any single cognate group from the genomic library screens, although it is unlikely that our screen was exhaustive, and therefore conclude that P. marinus has a total of either three or four Hox clusters. We also identify four highly conserved non-coding sequence motifs shared with higher vertebrates in a genomic comparison of Hox 10 genes.