Mutations In Human Genome Research Articles

αIIbβ3 Variants Defined By Next Generation Sequencing: Implications for Predicting Variants Likely to Cause Glanzmann Thrombasthenia and Alloimmune Disorders

# Authors contributed equally to this work.~ Currently at Genomics England Ltd, London, United KingdomNext generation sequencing is transforming our understanding of human genetic variation and is becoming a routine part of human genetic analysis. The identification of millions of new variants, which are mainly rare and assessing their implications for human health presents new challenges to researchers and clinicians. We have analyzed missense variants in the ITGB2A and ITGB3 genes obtained from whole exome and whole genome sequencing (WES & WGS) data from 5 databases: The Human Genome Mutation Database, the 1000 Genomes project, the UK10K Whole Exome Sequencing project, the UK10K Whole Genome Sequencing project, and The National Heart, Lung and Blood Institute Exome Sequencing Project. Together, these encompass variants of the platelet αIIbβ3 integrin receptor from ~32,000 alleles derived from 16,108 individuals. We identified 111 missense variants that have previously been associated with Glanzmann thrombasthenia (GT), 20 variants associated with alloimmune thrombocytopenia, and 5 variants associated with aniso/macrothrombocytopenia. None of the GT variants were found in the last four databases, indicating that they have minor allele frequencies (MAF) less than ~0.01%, attesting to both their rarity and the likelihood that they entered the population within the last ~2,500 years.We also identified 114 novel missense variants in ITGB2A affecting ~11% of the amino acids and 68 novel missense variants in ITGB3 affecting ~9% of the amino acids. 96% of the novel variants had MAF <0.1%, indicating their rarity. Based on sequence conservation, MAF, and/or location of the substituted residue on a complete model of αIIbβ3 that suggested a possible effect on protein folding, we selected three novel variants (αIIb P943A and P176H, and β3 C547G) that affect amino acids previously associated with GT for expression in HEK 293 cells. Both αIIb P176H and β3 C547G severely affected αIIbβ3 expression, whereas αIIb P943A had only a partial effect on expression and no effect on DTT-induced fibrinogen binding. We were not surprised that the latter variant did not have a severe effect on expression or function because it has an MAF (0.46%) that is much higher than the MAFs of the other GT-causing variants.To estimate the percentage of the 114 novel identified variants that are likely to be deleterious we used 3 different algorithms, CADD, Polyphen 2-HDVI, and SIFT. The algorithms showed moderate concordance in their rankings of the likelihood that a variant is deleterious. To compare their predictive powers, we performed receiver operating characteristic (ROC) analysis based on their ability to discriminate confirmed GT missense variants (positive controls) from alloantigens (negative controls); the area under the curve (AUC) values were 0.91, 0.88, and 0.90, respectively. At cutoff values that achieved greater than 95% sensitivity for each algorithm: 1) the specificity values were 75%, 65%, and 60%, and 2) the percentages of novel αIIb+β3 missense variants predicted to be deleterious were 43%, 56%, and 58%. Polyphen 2-HDVI and SIFT identified αIIb P176H and β3 C547G as highly likely to be deleterious and αIIb P943A as much less likely to be deleterious, whereas CADD did not differentiate them in the same way. We conclude that ~1.1% of individuals in the populations studied carry at least one missense variant in αIIb or β3 and that 0.6% carry a variant that might be deleterious and therefore may result in a hemorrhagic GT-like phenotype. The rarity of almost all of the novel missense variants identified indicates that they entered the population recently. Despite having detailed knowledge of the structure and function of αIIbβ3, it is difficult to predict with certainty the impact of any single missense variant. This will pose serious challenges as more individuals undergo WES and WGS; we anticipate that linkage to health record data, as will happen for the UK 100,000 Genomes project, will aid clinical interpretation. Finally, “hypomorphic” gene variants that produce only a partial decrease in expression, such as αIIb P943A, may contribute to the wide variation in αIIbβ3 surface expression observed in the healthy population. DisclosuresNo relevant conflicts of interest to declare.

Feasibility of Low-Throughput Next Generation Sequencing for Germline DNA Screening

Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS). Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1-12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases. The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤ 0.15, strand biases >-35, or genotype quality scores ≤ 80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤ 5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity). Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.

A modified panel of sentinel congenital anomalies for potential use in mutation epidemiology based on birth defects registry data.

Since 1983, several authors have used panels of "sentinel" congenital anomalies that might serve as indicators of the human genome mutation rate. The current study suggests a considerably updated panel, and applies it to public health birth defects registry data to determine the potential number of de novo cases. Data were taken from deliveries in 1999-2009 from the Texas Birth Defects Registry, an active surveillance program. Cases with one of the conditions or syndromes in the panel were identified using codes and text searches. Frequencies and birth prevalence were calculated for the overall panel and subcategories within it. Of the 60 conditions appearing in previous papers on sentinel phenotypes, 21 (35%) were used in the current study along with 27 new phenotypes. We found 1,694 cases. Of those, 1,100 exhibited phenotypes thought to arise de novo in at least 90% of the cases ("all/almost all" subpanel), and 594 considered de novo in roughly 50-90% of cases ("most" subpanel). Chromosomal deletion disorders were present in 523 cases and imprinting disorders in 243. After adjusting for maternal age, occurrence of cases in the total panel, "most" subpanel, and imprinting disorders subpanel were significantly associated with paternal age. Our panel of sentinel phenotypes differs from previous panels due to evolved knowledge of genetic disorders, different approaches with respect to interviewing, and different operational definitions. It is hoped that using an overall panel as well as subpanels may maximize statistical power as well as suggest potential mechanisms.

In vitro screening of embryos by whole-genome sequencing: now, in the future or never?

What are the analytical and clinical validity and the clinical utility of in vitro screening of embryos by whole-genome sequencing? At present there are still many limitations in terms of analytical and clinical validity and utility and many ethical questions remain. Whole-genome sequencing of IVF/ICSI embryos is technically possible. Many loss-of-function mutations exist in the general population without serious effects on the phenotype of the individual. Moreover, annotations of genes and the reference genome are still not 100% correct. We used publicly available samples from the 1000 Genomes project and Complete Genomics, together with 42 samples from in-house research samples of parents from trios to investigate the presence of loss-of-function mutations in healthy individuals. In the samples, we looked for mutations in genes that are associated with a selection of severe Mendelian disorders with a known molecular basis. We looked for mutations predicted to be damaging by PolyPhen and SIFT and for mutations annotated as disease causing in Human Genome Mutation Database (HGMD). More than 40% of individuals who can be considered healthy have mutations that are predicted to be damaging in genes associated with severe Mendelian disorders or are annotated as disease causing. The analysis relies on current knowledge and databases are continuously updated to reflect our increasing knowledge about the genome. In the process of our analysis several updates were already made. At this moment it is not advisable to use whole-genome sequencing as a tool to set up health profiles to select embryos for transfer. We also raise some ethical questions that have to be addressed before this technology can be used for embryo selection. N/A.

Human genome mutation and rearrangement studies – the way to investigate monogenic and complex disease pathogenesis

The summarized results of 25-year studies of department of human genomics of IMBG NASU are presented. The investigations were focused on identification of molecular genetic nature of human genome coding and non-coding region mutations (genetic polymorphisms) and rearrangements, their spectrum, and origin in Ukrainian population. The role of genome heterogeneity in some severe monogenic and complex disorder pathogenesis has been shown. The data concerning correlation between certain determinator gene mutations and phenotypical manifestation of most common in Ukraine monogenic diseases have been demonstrated. Moreover, the role of modifying genes in specific clinical phenotype variations has been shown. Origin of particular mutant alleles and main mechanisms of their frequency maintenance in Ukrainian population have been investigated. The data about association of some polymorphic variants with infertility, cardiovascular diseases (ischemic stroke) as well as mass infectious diseases (hepatitis C, AIDS) outcome and standard therapy efficiency have been presented. The first results and prospects for candidate genes of neurodegenerative disorders and intellectual disability search using whole genome CNVs screening are shown.

Journeys into the genome of cancer cells

I come from a family in which there have been no scientists or doctors. I was interested, however, in biology at school and started my scientific career by training in medicine at Oxford University and Guys Hospital, London. Practising as a doctor reinforced my curiosity about the biological processes underlying human disease. As a consequence, I pursued a clinical vocation in histopathology, a discipline that couples exposure to the sights and smells of the autopsy room with a daily journey into the often beautiful, sometimes ugly world of healthy and diseased human tissues under the microscope. After an introduction to general histopathology in Nick Wright's department at the Hammersmith Hospital, London, I completed my postgraduate medical training in neuropathology with Peter Lantos at the Maudsley Hospital, London.Peering at the nuclei of cancer cells under the microscope, for me it was a matter of fascination that hidden within them were the key events converting normal cells into cancer cells, and frustration because they were out of reach. Many of the tissue samples examined by pathologists are from cancers. The clonal theory of cancer development and the general role of DNA mutations in generating cancer cell clones had been established by 1986 when I was working as a junior pathologist. Indeed, the first mutated cancer gene, HRAS , had recently been identified through application of the, then new, technologies of recombinant DNA technology. Peering at the nuclei of cancer cells under the microscope, for me it was a matter of fascination that hidden within them were the key events converting normal cells into cancer cells, and frustration because they were out of reach. So, I took 3 years break from medicine to study for a PhD, learning the methods and thinking of molecular oncology in Colin Cooper's laboratory at the Institute …

A new era in the discovery of de novomutations underlying human genetic disease

Editorial text Germline mutations arise anew during meiosis in every generation. Such spontaneously occurring genetic alterations are termed de novo mutations and serve to describe those heritable mutations that neither parent possessed or transmitted. Thus, de novo mutations denote mutations that arose in the gametes of the parents as distinct from post-zygotic somatic mutations that arise during embryonic development. Studies of de novo mutations in the human genome have been very challenging owing to past technological limitations. However, the advent of highthrough put next-generation sequencing (NGS) technologies has ushered in a new era in the study of de novo mutations. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can now be performed on parentoffspring trios to identify de novo point mutations in the entire genome or within protein-coding regions, respectively [1-3]. Although genome-wide de novo copy number variants (CNVs) were previously studied using microarrays [4-7], the study of de novo point mutations or single nucleotide variants (SNVs) required the advent of largescale sequencing, which was not feasible using Sanger sequencing [1-3]. As with inherited mutations, de novo mutations range in size from point mutations and small indels of multiple bases to much larger CNVs and structural rearrangements [8]. In this article, we focus on de novo point mutations/SNVs and their implications for our understanding of the etiology of common complex diseases as well as rare Mendelian diseases. The availability of NGS has allowed first estimations of the human genome mutation rate as well as a glimpse of

Loaded Dice for Human Genome Mutation

Not all bases in the human genome are equally prone to chance mutations. Michaelson etal. show that individuals can acquire clusters of de novo DNA changes and propose that the likelihood of single base pair change differs across three orders of magnitude.

Genetic Diagnosis of Inherited Bleeding Disorders in 1250 Probands From India: A Single Centre Experience

AbstractAbstract 1127Congenital defects of platelets or plasma proteins involved in haemostasis generally lead to bleeding disorders. Von Willebrand disease (VWD) and hemophilia A and B are the most common while defects in other plasma coagulation proteins and platelet factors are relatively rare with an incidence of <1: 1–2 million. Despite significant advancements in the world, state of the art care is inaccessible to a vast majority of patients in developing countries. Genetic diagnosis aimed at offering carrier detection and prenatal diagnoses thus remains an important part of the management of these patients. From the year 2000, we have systematically established cost-effective genetic diagnoses services for not only hemophilia A and B but for other rare bleeding disorders [Factor (F) 1, F2, F5F8, F7, F10, F11 and F13] and platelet disorders [Glanzmann Thrombasthenia, Bernard Soulier Syndrome, Wiskott Aldrich Syndrome] as well. Our approach for molecular diagnosis includes screening the target genes for common mutations first, followed by point mutation screening by PCR and conformation sensitive gel electrophoresis and DNA sequencing, as needed, after that. For large genes such as F8 (186kb), ITG2B and ITG3A (65kb), we employ multiplex amplification of the coding regions to simplify the mutation detection protocols. Alternately, linkage analysis has also been established for some diseases such as hemophilia A, hemophilia B and Glanzmann Thrombasthenia to assist in those families where no mutation is found. Using these validated protocols, we have compiled genetic mutations in 1246 probands from 803 families with various bleeding disorders (table 1). These does not include patients with VWD for which we have only now initiated genetic evaluation. Mutations were identified in 762 families (95%). Of these, 358 were unique mutations, of which 103 (29%) are novel. We were not able to characterize disease causing mutation in a proportion (5 %, n=41/803) of families which is similar to the published literature. The frequency and distribution of the various mutations (Table 1) for all the disorders is similar to the spectrum reported in the human genome mutation database. Based on the availability of this molecular data, we have successfully offered carrier diagnosis to 334 probands from 271 unrelated families while prenatal diagnosis was carried out on 124 probands from 113 unrelated families. The development of efficient diagnostic algorithms for each of the bleeding disorder has helped to limit the turnaround time for genetic diagnoses to an average of 3–7 days at a cost of about $100-150 per sample for reagents and disposables. In conclusion, this report is the largest single-centre molecular data from a cohort of patients with bleeding disorders. The availability of such vast amount of genetic information and robust techniques for molecular diagnosis for different disorders of hemostasis contributes significantly to the effective management of these patients with bleeding disorders in India.Table 1Bleeding disorderTotal samples analyzedUnique families analyzedTotal reported mutationsNovel mutationsIntron22 inversionIntron1 inversionMissenseNonsenseFrame shiftSplice siteMajor deletionsNo mutations identifiedFactor VIII deficiency856479995347% (n=227)3% (n=13%)16% (n=76)10% (n=46)12% (n=57)3% (n=12)2% (n=10)6% (n=27)Factor IX deficiency153120473166% (n=79)7% (n=8)8% (n=9)3% (n=3)10% (n=12)2% (n=2)Glanzmann Thrombasthenia605832057 (n=34)14% (n=8)17% (n=10)5% (n=3)06.9% (n=4)Wiskott Aldrich Syndrome482912110% (n=3)24% (n=7)24% (n=7)14% (n=4)3 % (n=1)31% (n=9)Bernard-Soulier syndrome282721156% (n=15)4% (n=1)048% (13)00Fibrinogen deficiency282621315% (n=4)031% (n=8)58% (n=15)00Factor VII deficiency231411250% (n=7)29% (n=4)14% (n=2)14% (n=2)00Factor XI deficiency121251100% (n=12)0017% (n=2)00Factor XIII deficiency9911036% (n=4)9% (n=1)18% (n=2)36% (n=4)00Combined Factor V+VIII deficiency994011% (n=1)056% (n=5)33% (n=3)00Factor II deficiency887288% (n=7)0025% (n=2)00Factor × deficiency7780100% (n=7)0014 (n=1)00Factor V deficiency554040% (n=2)0060% (n=3)00Total124680325510325175100572341 Disclosures:No relevant conflicts of interest to declare.

Next generation sequencing (NGS) in the UK steroid resistant nephrotic syndrome (SRNS) study reveals complex genetic heterogeneity

This national cohort was developed within the rare renal disease registry (RaDaR) with detailed clinical phenotyping. Sequencing was undertaken for (470) genes in the first 24 patients with SRNS (either acquired or congenital) as a pilot project. Nimblegen Roche multiplex capture array (12×135KB) was used to capture exons from genomic DNA and sequencing was undertaken on an Illumina GAII in-house. Data was processed using CLC Genomics workbench software for mapping against human genome (hg19/GRch37) and detecting SNPs (single nucleotide polymorphisms) and DIPs (deletion insertion polymorphisms). Any variations were checked against dbSNP (132) and the Human Genome Mutation Database (professional version). All amino acids changes were further analysed in a prediction tool MutPred (www.mutpred.mutdb.org) for probability of pathogenicity. 97% of reads mapped to the reference sequence and 76% of reads were on target genes. On average, 3400 SNPs were found per patient, 185 being non-synonymous with approximately 10 novel per patient. An average total of 2 DIPs were found per patient. SNP and DIP analysis revealed several known disease causing variations in Nephrin, Podocin, PLCe1 but also novel mutations in these and other genes. In one patient an isolated COQ2 nephropathy (single previous case report) was revealed without other organ involvement. In families, addition of single heterozygote mutations on top of homozygous disease causing mutations altered phenotype causing early onset of disease. All variations considered clinically significant were verified with Sanger sequencing with no false positives identified. All novel variations were evaluated within MutPred and several were identified as being probably pathogenic, warranting further investigation. Although knowledge of genes involved in this condition has evolved rapidly in the last ten years, prevalence in the UK cohort has never been tested before. This study suggests a detection-of-mutation rate in sporadic disease of approximately 25% and in familial of 40% and also reveals that tri-allelic hits are not uncommon and may alter phenotype. Genotype - phenotype analysis of the extended cohort will allow for development of prognostic variants which could ultimately be used in the early clinical setting.

Prothrombin Mumbai causes severe prothrombin deficiency due to a novel Cys90Ser mutation

Dear Editor, Prothrombin (coagulation factor II) is a zymogen which is proteolytically cleaved to form its active derivative, thrombin, a serine protease, in the penultimate step of the coagulation cascade. The prothrombin gene is located on chromosome 11 (11p11-q12) [1]. Homozygous prothrombin deficiency is a rare autosomal recessive bleeding disorder occurring approximately in one of two million individuals. The Human Genome Mutation Database shows reports of only 32 mutations causing prothrombin deficiency [2]. The molecular basis is diverse, and mutations or polymorphisms in f2 gene are known to lead to both thrombosis and bleeding [3, 4]. A 5-month-old female child from Nepal, born of nonconsanguinous marriage, was referred with acute intracranial hemorrhage (Fig. 1a). Since birth, the baby had few episodes of bleeding ranging from hematemesis, umbilical stump bleed, and right-sided subdural hematoma. During these episodes, the baby was given an injection of vitamin K, fresh frozen plasma, and whole blood. Coagulation investigations done during these episodes showed consistently deranged prothrombin time (PT) and activated partial thromboplastin time (APTT); however, no reports of factor assays were available. Both the parents were clinically normal with neither any history of bleeding nor thrombosis during their life time. Blood samples of the patient and parents were collected in 3.2 % tri-sodium citrate after an informed consent. All the coagulation tests were performed in a semi-automated coagulometer using commercial reagents and calibrators (Diagnostica Stago, Asnieres, France). All the 14 exons and their flanking intronic regions were amplified using 12 sets of primers as described earlier [5]. The amplicons were sequenced using BigDye Terminator cycle sequencing kit in a ABI 3130XL genetic analyzer (Applied Biosystems, Foster City, CA, USA). The primary amino acid sequences of prothrombin from Homo sapiens (NC_000011.9), Pan troglodytes (NC_006478.2), Macaca mulatta (NC_007871.1), and Mus musculus (NC_000068.6) were obtained from NCBI gene [6]. Multiple sequence alignment was performed with ClustalW [7] to study the conservation of amino acids mutated by missense variations. Ensembl Genome Browser [8] was used to refer to the reported variations in the prothrombin gene. PolyPhen, Sorting Intolerant from Tolerant (SIFT), and Panther were used to predict the severity of the amino acid substitution on the protein function. The coagulation parameters of the patient were as follows: PT 27 s/control 14.5 s, APTT 40 s/control 29 s, thrombin time (TT) 18 s/control 15 s, fibrinogen 222 g/dL (normal range, 150–400 mg/dL). Mixing studies were normal for PT, APTT, and TT. Factor II (prothrombin) was found to be <1% on assay, with other factors being within normal limits. PCR amplification of all 14 exons followed by DNA sequencing in the patient showed a homozygous c.G269C variation in exon 4 of the patient. Both the parents were B. Kulkarni :K. Ghosh : S. Shetty (*) National Institute of Immunohaematology (I.C.M.R.), 13th floor, New building, K.E.M. Hospital Campus, Parel, Mumbai 400012, India e-mail: shrimatishetty@yahoo.com

Great expectations: using massively parallel sequencing to solve inherited disorders

The mutation discovery process for monogenic disorders is undergoing an exciting revolution. Previously dependent on linkage mapping and carefully selected gene-candidate approaches, identifying disease-causing mutations was both time consuming and frustrating. The advent of the new and emerging massively parallel sequencing technologies is driving unbiased discovery of novel disease-causing mutations at an unprecedented rate. How long will it be before the process is fully automated and the job of molecular geneticist is changed forever? We comment on the mutation-discovery strategies compatible with this technology and demonstrate that while the modern gene jockey might need to trade in the horse for something a bit faster, the finish line for interpreting human genetic variation is still a long way ahead. A recent comprehensive and highly recommended review covers the technical capabilities of massively parallel (also called next-generation) sequencing for the field of genetics [1]. Mutation-discovery applications using massively parallel sequencing survey whole-genome, wholeexome, regionor candidate-gene groupspecific DNA templates. Each strategy has strengths and weaknesses that must be considered and matched with the genetics and biology of the clinical phenotype to be studied. Whole-exome sequencing is already the method of choice for most mutation discovery efforts. Commercially available chip-based and in-solution array enrichment platforms make this protocol amenable to high-throughput automation and standardization. There are companies that offer this protocol as a fee for service, including downstream sequence analysis [101]. Currently, 86% of the 100,000 mutations cataloged in the human genome mutation database affect coding regions [2], which are captured by the whole-exome arrays. Even considering the biases, which have driven the discovery of these mutations so far, it is predicted that exome sequencing will be successful most of the time. Exome sequencing is already having an impact on patient care; in one study, mutations in SLC26A3 that are normally associated with congenital chloride-losing diarrhea (Online Mendelian Inheritance in Man [OMIM] 214700) were identified in a patient with a diagnosis of an atypical Bartter syndrome (OMIM 607364) [3]. These findings led to the re-examination of the patient to confirm the diagnosis of chloride-losing diarrhea and the subsequent identification of five additional mutations in SLC26A3 in a cohort of 39 individuals diagnosed with Bartter syndrome. In a similar study, 11 novel mutations in DHODH associated with the recessive disorder Miller syndrome (OMIM 263750) Mark Corbett

Single nucleotide polymorphisms in FAS and FASL and sensitivity to gefitinib in patients with advanced non-small cell lung cancer.

e13529 Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) play a key role in the management of advanced non-small cell lung cancer (NSCLC). Numerous investigations have demonstrated an association between the presence of somatic mutations in EGFR and the sensitivity to gefitinib or erlotinib in NSCLC. Since genetic polymorphisms are more prevalent than mutations in human genome, some of them may be more practical as genetic biomarkers to predict the sensitivity to EGFR-TKIs. The FAS-FASL system plays crucial role in apoptotic signaling in many tumor cells. Here, we assessed the possibility that single nucleotide polymorphisms (SNPs) in FAS and FASL could be used to predict the sensitivity to gefitinib in patients with advanced NSCLC. Methods: Candidate polymorphic loci approach was used to determine the functional polymorphisms. SNPs in the promoter region of the FAS (−1377G/A and −670A/G) and FASL (−844T/C) were assessed in 88 advanced NSCLC patients treated with gefitinib. The Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The association between genotypes and sensitivity to gefitinib was validated by statistical tests. Results: For FAS -1377G/A, the clinical benefit rates (CBR) of gefitinib in advanced NSCLC patients were 31.3%, 61.1% or 74.3% (p = 0.014) in AA, GA or GG genotypes, respectively. For FAS −670A/G, the CBR were 32.0%, 78.0% or 57.9% (p = 0.001) in GG, AG or AA genotypes. For FASL - 844T/C, the CBR were 65.2%, 57.1% or 50% (p = 0.276) in CC, CT or TT genotypes. FAS -670 GG genotype carriers also had a significantly shorter survival time after the treatment of gefitinib than variation allele carriers (AG or AA genotypes), the overall survival time were 26 and 47 months (p = 0.012), respectively. Conclusions: FAS -1377G/A and -670A/G polymorphisms may be predictive for sensitivity to gefitinib in patients with advanced NSCLC. No significant financial relationships to disclose.

A Novel COL7A1 Gene Mutation in an Iranian Individual Suffering Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is a heritable skin disorder with dominant and recessive genetic patterns. Numerous studies underline that both forms are caused by mutations of the COL7A1 gene, which encodes collagen type VII. It has been reported that most mutations detected in the recessive disease form are nonsense mutations or small insertions or deletions leading to frameshift and premature translational termination, which tend to produce severe phenotypes. In contrast, missense mutations causing amino acid substitutions, which result in variable phenotypes, predominate in the dominant form of dystrophic epidermolysis bullosa. Genomic DNA from the patient and parents was subjected to PCR amplification of the coding region of the COL7A1 gene. Direct sequencing of the PCR products revealed a homozygous single-base deletion in the patient (c.6269-6270delC). The parents were heterozygous for the same mutation. This deletion is a novel mutation in the human COL7A1 gene based on comparisons with the Human Genome Mutation Database. To our knowledge, this is the first report of dystrophic epidermolysis bullosa in an Iranian patient confirmed by molecular diagnosis.

Mutation analysis of EXT1 and EXT2 Genes in a Korean family with multiple exostoses

A 35-year-old female patient diagnosed clinically as multiple exostosis visited the hospital for infertility evaluation and treatment. She had an operation in pelvis, humerus, tibia and femur in 1993. An extended pedigree analysis showed three of her siblings and several cousins have suffered from the same disease with a typical autosomal dominant pattern of inheritance. So she wanted a genetic test for her disease before having a child. For mutation analysis, DNAs were extracted from the patient and her brother. All exons and exon-intron boundaries of EXT1 and EXT2 genes were amplified by polymerase chain reactions. The PCR products were directly sequenced and analyzed by ABI genetic analyzer. A single base pair deletion c.2241delC in the exon 6 of EXT1 gene was detected in both patient and her brother. Generation of a premature stop codon resulting from frameshift of codons might be a causative of the disease. According to the human genome mutation data base (HGMD), the mutation detected is not previously documented.

Genome-Wide Analysis of Human Disease Alleles Reveals That Their Locations Are Correlated in Paralogous Proteins

The millions of mutations and polymorphisms that occur in human populations are potential predictors of disease, of our reactions to drugs, of predisposition to microbial infections, and of age-related conditions such as impaired brain and cardiovascular functions. However, predicting the phenotypic consequences and eventual clinical significance of a sequence variant is not an easy task. Computational approaches have found perturbation of conserved amino acids to be a useful criterion for identifying variants likely to have phenotypic consequences. To our knowledge, however, no study to date has explored the potential of variants that occur at homologous positions within paralogous human proteins as a means of identifying polymorphisms with likely phenotypic consequences. In order to investigate the potential of this approach, we have assembled a unique collection of known disease-causing variants from OMIM and the Human Genome Mutation Database (HGMD) and used them to identify and characterize pairs of sequence variants that occur at homologous positions within paralogous human proteins. Our analyses demonstrate that the locations of variants are correlated in paralogous proteins. Moreover, if one member of a variant-pair is disease-causing, its partner is likely to be disease-causing as well. Thus, information about variant-pairs can be used to identify potentially disease-causing variants, extend existing procedures for polymorphism prioritization, and provide a suite of candidates for further diagnostic and therapeutic purposes.

Patterns of somatic mutation in human cancer genomes

AACR Centennial Conference: Translational Cancer Medicine-- Nov 4-8, 2007; Singapore PL02-05 All cancers are due to abnormalities in DNA. The availability of the human genome sequence has led to the proposal that resequencing of cancer genomes will reveal the full complement of somatic mutations and hence all the cancer genes. To explore the nature of the information that will be derived from cancer genome sequencing we have sequenced the coding exons of the family of 518 protein kinases, ~1.3Mb DNA per cancer sample, in 210 cancers of diverse histological types. Despite the screen being directed toward the coding regions of a gene family that has previously been strongly implicated in oncogenesis, the results indicate that the majority of somatic mutations detected are “passengers”. There is considerable variation in the number and pattern of these mutations between individual cancers, indicating substantial diversity of processes of molecular evolution between cancers. The imprints of exogenous mutagenic exposures, mutagenic treatment regimes and DNA repair defects can all be seen in the distinctive mutational signatures of individual cancers. This systematic mutation screen and others have previously yielded a number of cancer genes that are frequently mutated in one or more cancer types and which are now anticancer drug targets (for example BRAF , PIK3CA , and EGFR ). However, detailed analyses of the data from our screen additionally suggest that there exist a large number of additional “driver” mutations which are distributed across a substantial number of genes. It therefore appears that cells may be able to utilise mutations in a large repertoire of potential cancer genes to acquire the neoplastic phenotype. However, many of these genes are employed only infrequently. These findings may have implications for future anticancer drug development.

Association of the EGFR gene polymorphisms with clinical outcomes in patients with advanced non-small cell lung cancer (NSCLC) treated with gefitinib

14516 Background: Mutations in the EGFR gene appear to confer sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Genetic polymorphisms are more prevalent than mutations in human genome. We evaluated the association of polymorphisms in the EGFR gene with the clinical outcomes in the patients with advanced NSCLC treated with gefitinib, an EGFR-TKI. Methods: Candidate polymorphic loci approach was used to determine the functional polymorphisms within the EGFR gene range. Four most representative tagging single nucleotide polymorphisms (SNPs), one well-known CA simple sequence repeat (CA-SSR) in intron 1, and several common SNPs in the region encoding tyrosine kinase domain of EGFR were included in our study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and directly DNA sequencing approach were used to determine the genotypes of 84 advanced NSCLC patients treated with gefitinib. Results: Rs2293347 in exon 25 and CA-SSR in intron 1 were addressed by the candidate loci approach. For rs2293347, the clinical benefit rates were 71.2% and 37.5% (P = 0.0043) in wild-type (G/G genotype) and variation allele carriers (A/G or A/A genotypes). The progression-free survival were 11 months and 3 months (P = 0.0018), respectively, while the results for overall survival were similar. For CA-SSR, the clinical benefit rate was 88.5% in patients carrying at least one shorter CA repeat allele [≤16 (CA)n], and 48.3% in patients carrying two longer CA repeat alleles [>16 (CA)n] (P = 0.0005). However, CA- SSR polymorphism was not found to be associated with the progression-free survival and overall survival. Conclusions: Rs2293347 and CA-SSR polymorphisms may be predictive for clinical outcomes in patients with advanced NSCLC treated with gefitinib. No significant financial relationships to disclose.

Patterns of somatic mutation in human cancer genomes.

Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.

Designing a Novel Molecular Beacon for Surface-Immobilized DNA Hybridization Studies

Designing a Novel Molecular Beacon for Surface-Immobilized DNA Hybridization Studies