Identification of mutations in the tumor suppressor gene TP53 has implications for the molecular epidemiology and for the molecular pathology of human cancer. We have developed and evaluated an arrayed primer extension assay for covering both strands of a region of the coding sequence containing more than 95% of the mutations described so far in TP53. On average, 97.5% of the arrayed TP53 gene sequence can be analyzed from either sense or antisense strands, and 81% from both strands. A patient DNA sample is amplified and annealed to arrayed primers, which then promote DNA polymerase extension reactions with four fluorescently labeled dideoxynucleotides. The TP53 gene chip spans exons 2-9 plus two introns from both strands. The performance of the assay was evaluated by using freshly extracted genomic DNA, as well as DNA extracted from archival (paraffin-embedded) DNA samples. The arrayed primer extension-based TP53 gene test provides an accurate and efficient tool for DNA sequence analysis of this frequently mutated gene for both research and clinical applications.APEX ͉ oligonucleotide array ͉ chip T he evidence is growing that specific mutations in the TP53 gene can represent important factors for the prognosis of cancer and for the response to various types of cytotoxic therapy. Furthermore, patterns of TP53 mutations have differed considerably from one type of cancer to the other (1-4). However, screening for TP53 mutations gene has yet to become a routine in clinical or epidemiological practice, mainly because current detection technologies are labor-intensive and have prohibitive costs for large-scale prospective studies. Another strong limitation to routine analysis of TP53 mutations resides in the fact that many tumors contain an excess of wild-type TP53 as compared with mutant, resulting from the presence of intact alleles in tumor as well as in noncancer cells (stroma, inflammatory cells, blood vessels).In this report we describe the development of an arrayed primer extension (APEX) assay for the rapid and sensitive detection and identification of mutations in the TP53 gene. APEX is a genotyping and resequencing technology that combines the advantages of Sanger dideoxy sequencing with the parallelization and high-throughput potential of the microarray format. A DNA sample is amplified, fragmented enzymatically, and annealed to arrayed primers, promoting sites for templatedependent DNA polymerase extension reactions by using four fluorescently labeled dideoxynucleotides. Each base is probed with two primers, one for the sense and another for the antisense strand (5). GENORAMA imaging system and genotyping software (Asper Ltd., Tartu, Estonia, www.asperbio.com) were used for imaging and semiautomatic sequence analysis (Fig. 1).The principle of sequencing by primer extension on oligonucleotide array has been successfully applied for the systematic identification of all common TP53 mutations in human cancers. The TP53 microarray presented here spans exons 2-9 [containing more than 98% of all mutations descr...

Background In addition to whole exomes, large gene panels of clinically associated genes are used as high‐throughput sequencing tests in many clinical centers, but their clinical utility has been much less investigated. Materials and Methods Here we report the results of the 501 first unselected cases for whom TruSight One panel (Illumina Inc., San Diego, California) was sequenced as a clinical diagnostic test for a variety of indications in our department. The analysis was restricted to virtual subpanels based on referral forms, where doctors were asked to list candidate genes or select one from predefined larger panels. Results A probable or definite pathogenic finding was reported in 26.3% of cases. In 238 samples for whom 1 to 9 genes were requested for analysis, the diagnostic yield was significantly higher compared to other 263 cases for whom larger subpanels were requested (31.5% vs 21.7%, respectively, P = .016). Detected mutations included single nucleotide variants, small insertions and deletions, and larger copy number variants. Out of 157 reported mutations, 67 were previously undescribed. Conclusion The clinical utility of large gene panel sequencing in the context of other genetic diagnostic tests is discussed in detail.

Parkin mutations account for the majority of familial and sporadic early onset Parkinson's disease (EOPD) cases with a known genetic association. More than 100 mutations have been described in the Parkin gene that includes homozygous, compound heterozygous, and single heterozygous mutations. We have designed a Parkin mutation genotyping array (gene chip) that includes published Parkin sequence variants and allows their simultaneous detection. The chip was validated by screening 85 PD cases and 47 controls previously tested for Parkin mutations. Similar genotyping microarrays have been developed for other genetically heterogeneous diseases including age-related macular degeneration. Here, we show the utility of a genotyping array for Parkinson's disease by analysis of 60 subjects from the Genetic Epidemiology of Parkinson Disease (GEPD) study that includes 15 early-onset PD case probands and 45 relatives.

Background: Ribose 2'-O-methylation, the most common nucleotide modification in mammalian rRNA, is directed by the C/D box small nucleolar RNAs (snoRNAs). Thus far, more than fifty putative human rRNA methylation guide snoRNAs have been identified. For nine of these snoRNAs, the respective ribose methylations in human 28S rRNA have been only presumptive.