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Sarcoidosis is a complex disease with autoimmune basis and still unknown etiology. We have screened for mutations in the cystic fibrosis conductance regulator (CFTR) gene and genotyped single-nucleotide polymorphisms in the tumor necrosis factor (TNF), interferon alpha-10 (IFNA10), IFNA17, and interferon gamma (IFNG) genes in 89 Greek patients with sarcoidosis and 212 control subjects to detect possible association between them and the risk for developing sarcoidosis. We have found a statistically significant increase (p = 6.1 x 10(-8)) of CFTR mutation carriers in the population of patients with sarcoidosis versus the control population. A difference was also noted within the group of patients with sarcoidosis where the ones with CFTR mutations suffered more frequently from dyspnea than those without (p = 5 x 10(-6)). Our study did not reproduce the associations previously noted with the TNF, IFNA10, IFNA17, and IFNG genes, which highlights the genetic complexity of the disorder and is in agreement with previous studies showing that CFTR might be an important factor in the clinical course of the disease.

Ο σκοπός της εργασίας ήταν η μελέτη της έκφρασης στο επίπεδο της μεταγραφής 8 μεταλλάξεων ματίσματος, που εδράζονται στις 5’ και 3’ περιοχές συρραφής εξονίων (θέσεις δότη και αποδέκτη), 2 παρερμηνεύσιμων μεταλλάξεων που βρίσκονται σε ρυθμιστικές περιοχές του εξονίου 12 και μίας μετάλλαξης τερματισμού του εξονίου 13 του CFTR mRNA. Όλες οι παραπάνω μεταλλάξεις επηρεάζουν τη σωστή παραγωγή ή και σταθερότητα του CFTR mRNA. Χρησιμοποιήθηκαν επιθηλιακά κύτταρα ρινικού βλεννογόνου 55 ασθενών ΚΙ και ετεροζυγωτών με τις υπό μελέτη μεταλλάξεις διότι αποτελεί ιστό εύκολα προσβάσιμο και όπου εκφράζεται το CFTR γονίδιο. Τα αποτελέσματα των μεταλλάξεων συγκρίθηκαν με ισάριθμα δείγματα φυσιολογικών μαρτύρων. Οι μεταλλάξεις c.621+3A>G, c.2751+2T>A, c.1898+1G>T, c.296+1G>C, 3120+1G>A που διαταράσσουν τις διατηρημένες αλληλουχίες ματίσματος στο 5’ των ιντρονίων, και η μετάλλαξη c.1525-1G>A η οποία διαταράσσει τη συντηρημένη αλληλουχία ματίσματος στο 3’ του ιντρονίου 9 καθώς και η ανερμηνεύσιμη μετάλλαξη p.Ε822Χ έχουν ως αποτέλεσμα δραστικά μειωμένη παραγωγή φυσιολογικών CFTR μεταγράφων (6-40%). Η μειωμένη ποσότητα ή μειωμένη σταθερότητα των CFTR μεταγράφων που παράγονται από τις παραπάνω μεταλλάξεις έχει ως αποτέλεσμα βαρύ κλινικό φαινότυπο στους ασθενείς. Αντίθετα οι μεταλλάξεις c.711+3A>G (5’ του ιντρονίου 5), και 2789+5G>A (5’ του ιντρονίου 14b) είναι ήπιες με αυξημένα ποσοστά φυσιολογικών μεταγράφων και ηπιότερο κλινικό φαινότυπο. Τέλος οι 2 παρερμηνεύσιμες μεταλλάξεις p.D565G και p.G576A, δρουν ως μεταλλάξεις ματίσματος προκαλώντας έλλειμμα του εξονίου 12, όπου εδράζονται. Επομένως παρερμηνεύσιμες μεταλλάξεις ή ακόμη και μεταλλάξεις συνώνυμες (χωρίς αλλαγή αμινοξέος) μπορεί να επηρεάσουν ρυθμιστικές αλληλουχίες ματίσματος (ESE και ESS) μέσα στα εξόνια καταλήγοντας σε παθολογικό μάτισμα και παραγωγή ποσοστού παθολογικών μεταγράφων μεγαλύτερου του 30-40%. Η δράση αυτή των παρερμηνεύσιμων ή συνώνυμων αλλαγών μπορεί να ερευνηθεί μόνο μελετώντας πρότυπο ματίσματος στο mRNA του CFTR γονιδίου σε ιστούς (όπως επιθηλιακά κύτταρα) όπου το γονίδιο εκφράζεται. Τα συμπεράσματα της μελέτης όσον αφορά τα προϊόντα που συνήθως παράγονται από τις μεταλλάξεις ματίσματος μας έδωσαν την δυνατότητα να σχεδιάσουμε ολιγονουκλεοτίδια ανιχνευτές για τον εύκολο και ταχύ χαρακτηρισμό αυτών των μεταλλάξεων στο CFTR γονίδιο χρησιμοποιώντας την πλατφόρμα του NanoChip® 400 Molecular Biology Workstation.

Sarcoidosis is a complex disease with autoimmune basis and still unknown etiology. We have screened for mutations in the cystic fibrosis conductance regulator (CFTR) gene and genotyped single-nucleotide polymorphisms in the tumor necrosis factor (TNF), interferon alpha-10 (IFNA10), IFNA17, and interferon gamma (IFNG) genes in 89 Greek patients with sarcoidosis and 212 control subjects to detect possible association between them and the risk for developing sarcoidosis. We have found a statistically significant increase (p=6.1×10-8) of CFTR mutation carriers in the population of patients with sarcoidosis versus the control population. A difference was also noted within the group of patients with sarcoidosis where the ones with CFTR mutations suffered more frequently from dyspnea than those without (p=5×10-6). Our study did not reproduce the associations previously noted with the TNF, IFNA10, IFNA17, and IFNG genes, which highlights the genetic complexity of the disorder and is in agreement with previous studies showing that CFTR might be an important factor in the clinical course of the disease. © 2010, Mary Ann Liebert, Inc.

Presented here is a genome sequence of an individual human. It was produced from ∼32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2–206 bp), 292,102 heterozygous insertion/deletion events (indels)(1–571 bp), 559,473 homozygous indels (1–82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information., Author Summary We have generated an independently assembled diploid human genomic DNA sequence from both chromosomes of a single individual (J. Craig Venter). Our approach, based on whole-genome shotgun sequencing and using enhanced genome assembly strategies and software, generated an assembled genome over half of which is represented in large diploid segments (>200 kilobases), enabling study of the diploid genome. Comparison with previous reference human genome sequences, which were composites comprising multiple humans, revealed that the majority of genomic alterations are the well-studied class of variants based on single nucleotides (SNPs). However, the results also reveal that lesser-studied genomic variants, insertions and deletions, while comprising a minority (22%) of genomic variation events, actually account for almost 74% of variant nucleotides. Inclusion of insertion and deletion genetic variation into our estimates of interchromosomal difference reveals that only 99.5% similarity exists between the two chromosomal copies of an individual and that genetic variation between two individuals is as much as five times higher than previously estimated. The existence of a well-characterized diploid human genome sequence provides a starting point for future individual genome comparisons and enables the emerging era of individualized genomic information., Comparison of the DNA sequence of an individual human from the reference sequence reveals a surprising amount of difference.

It is now clear that tyrosine kinases represent attractive targets for therapeutic intervention in cancer. Recent advances in DNA sequencing technology now provide the opportunity to survey mutational changes in cancer in a high-throughput and comprehensive manner. Here we report on the sequence analysis of members of the receptor tyrosine kinase (RTK) gene family in the genomes of glioblastoma brain tumors. Previous studies have identified a number of molecular alterations in glioblastoma, including amplification of the RTK epidermal growth factor receptor. We have identified mutations in two other RTKs: ( i ) fibroblast growth receptor 1, including the first mutations in the kinase domain in this gene observed in any cancer, and ( ii ) a frameshift mutation in the platelet-derived growth factor receptor-α gene. Fibroblast growth receptor 1, platelet-derived growth factor receptor-α, and epidermal growth factor receptor are all potential entry points to the phosphatidylinositol 3-kinase and mitogen-activated protein kinase intracellular signaling pathways already known to be important for neoplasia. Our results demonstrate the utility of applying DNA sequencing technology to systematically assess the coding sequence of genes within cancer genomes.