SNPs in human miRNA genes affect biogenesis and function.

MicroRNAs (miRNAs) are 21-25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.

Somatic microindels in human cancer: the insertions are highly error-prone and derive from nearby but not adjacent sense and antisense templates.

Somatic microindels (microdeletions with microinsertions) have been studied in normal mouse tissues using the Big Blue lacI transgenic mutation detection system. Here we analyze microindels in human cancers using an endogenous and transcribed gene, the TP53 gene. Microindel frequency, the enhancement of 1-2 microindels and other features are generally similar to that observed in the non-transcribed lacI gene in normal mouse tissues. The current larger sample of somatic microindels reveals recurroids: mutations in which deletions are identical and the co-localized insertion is similar. The data reveal that the inserted sequences derive from nearby but not adjacent sequences in contrast to the slippage that characterizes the great majority of pure microinsertions. The microindel inserted sequences derive from a template on the sense or antisense strand with similar frequency. The estimated error rate of the insertion process of 13% per bp is by far the largest reported in vivo, with the possible exception of somatic hypermutation in the immunoglobulin gene. The data constrain possible mechanisms of microindels and raise the question of whether microindels are 'scars' from the bypass of large DNA adducts by a translesional polymerase, e.g. the 'Tarzan model' presented herein.

Mutagenic and recombinagenic responses to defective DNA polymerase delta are facilitated by the Rev1 protein in pol3-t mutants of Saccharomyces cerevisiae.

Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase delta that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol eta, Pol zeta, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol delta. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases.

Familial deletion within NLGN4 associated with autism and Tourette syndrome.

Neuroligin 4 (NLGN4) is a member of a cell adhesion protein family that appears to play a role in the maturation and function of neuronal synapses. Mutations in the X-linked NLGN4 gene are a potential cause of autistic spectrum disorders, and mutations have been reported in several patients with autism, Asperger syndrome, and mental retardation. We describe here a family with a wide variation in neuropsychiatric illness associated with a deletion of exons 4, 5, and 6 of NLGN4. The proband is an autistic boy with a motor tic. His brother has Tourette syndrome and attention deficit hyperactivity disorder. Their mother, a carrier, has a learning disorder, anxiety, and depression. This family demonstrates that NLGN4 mutations can be associated with a wide spectrum of neuropsychiatric conditions and that carriers may be affected with milder symptoms.

Identification of high risk DISC1 structural variants with a 2% attributable risk for schizophrenia.

The causes of schizophrenia remain elusive. In a large Scottish pedigree, a balanced translocation t(1;11) (q42.1;q14.3) disrupting the DISC1 and DISC2 genes segregates with major mental illness, including schizophrenia and unipolar depression. A frame-shift carboxyl-terminal deletion was reported in DISC1 in an American family, but subsequently found in two controls. A few common structural variants have been associated with less than a 2-fold increased risk for schizophrenia, but replication has not been uniform. No large scale case-control mutation study has been performed. We have analyzed the regions of likely functional significance in the DISC1 gene in 288 patients with schizophrenia and 288 controls (5 megabases of genomic sequence analyzed). Six patients with schizophrenia were heterozygous for ultra-rare missense variants not found in the 288 controls (p=0.015) and shown to be ultra-rare by their absence in a pool of 10,000 control alleles. We conclude that ultra-rare structural variants in DISC1 are associated with an attributable risk of about 2% for schizophrenia. In addition, we confirm that two common structural variants (Q264R and S704C) elevate the risk for schizophrenia slightly (odds ratio 1.3, 95% CI: 1.0-1.7). DISC1 illustrates how common/moderate risk alleles suggested by the HapMap project might be followed up by resequencing to identify genes with high risk, low frequency alleles of clinical relevance.

Analysis of highly conserved regions of the 3'UTR of MECP2 gene in patients with clinical diagnosis of Rett syndrome and other disorders associated with mental retardation.

In this work we explored the role of the 3'UTR of the MECP2 gene in patients with clinical diagnosis of RTT and mental retardation; focusing on regions of the 3'UTR with almost 100% conservation at the nucleotide level among mouse and human. By mutation scanning (DOVAM-S technique) the MECP2 3'UTR of a total of 66 affected females were studied. Five 3'UTR variants in the MECP2 were found (c.1461+9G>A, c.1461+98insA, c.2595G>A, c.9961C>G and c.9964delC) in our group of patients. None of the variants found is located in putative protein-binding sites nor predicted to have a pathogenic role. Our data suggest that mutations in this region do not account for a large proportion of the RTT cases without a genetic explanation.

A novel mutation in JARID1C/SMCX in a patient with autism spectrum disorder (ASD).

We describe a nondysmorphic patient with developmental delay and autism spectrum disorder who has a missense mutation in the Jumonji AT-rich interactive domain 1C (JARID1C) gene. This child first presented at 30 months of age with stereotyped and repetitive behaviors, impairment in social reciprocity and in the use of multiple nonverbal behaviors, and developmental delay primarily in the language domain. A diagnosis of autism was made and subsequently confirmed at the current age of 47 months. Cytogenetic and fragile X studies were normal. Mutational analysis revealed a novel missense mutation in exon 16 of the JARID1C gene that results in an arginine to tryptophan substitution at amino acid 766 (R766W). Sequence alignment analysis with multiple available eukaryotic sequences including the homologous proteins of mouse and zebrafish demonstrated that the affected amino acid is conserved. JARID1C has not previously been implicated in autism susceptibility. Recent novel molecular evidence suggests that it is a histone demethylase specific for di- and trimethylated histone 3 lysine 4 (H3K4) and functions as a transcriptional repressor by fostering REST-mediated neuronal gene regulation. The JARID1C-regulated genes SCN2A, CACNA1H, BDNF, and SLC18A1 have previously been associated with autism and cognitive dysfunction. This patient brings the total number of reported JARID1C mutations to 14. This presentation both extends the range of neurocognitive phenotypes attributable to mutations in this gene and illustrates the importance of molecular studies and DNA sequence analysis for accurate diagnosis of monogenic causes of autism.

Neurexin 1alpha structural variants associated with autism.

Neurexins are presynaptic membrane cell-adhesion molecules which bind to neuroligins, a family of proteins that are associated with autism. To explore the possibility that structural variants in the neurexin alpha genes predispose to autism, the coding regions and associated splice junctions of the neurexin 1alpha gene were sequenced in 116 Caucasian patients with autism and 192 Caucasian controls. Five ultra-rare structural variants including a predicted splicing mutation were found in patients with autism and absent in 10,000 control alleles. Only one ultra-rare structural variant was found in controls (5/116 vs. 1/192; P=0.03, Fisher's exact test, one-sided). In the context of all available data, the ultra-rare structural variants of the neurexin 1alpha gene are consistent with mutations predisposing to autism.

Detection of ultrarare somatic mutation in the human TP53 gene by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification.

The detection of ultra-rare mutation in the presence of excess amounts of normal genomic DNA is highly advantageous in a number of circumstances, including: 1) identification of minimal residual disease for improved cancer chemotherapy; 2) measurement of mutation load to assess environmental mutagen exposure or endogenous DNA repair; and 3) prenatal diagnosis of paternally-derived mutations within fetal cells in the maternal circulation. Bidirectional pyrophosphorolysis activated polymerization allele-specific amplification (Bi-PAP-A) utilizes two opposing 3'-terminal blocked oligonucleotides (P(*)s) with 1 nucleotide overlap at their 3' termini. The selectivity of Bi-PAP-A derives from the serial coupling of pyrophosphorolysis and DNA polymerization. A total of 13 Bi-PAP-A assays were developed and validated for the human p53 gene (TP53). The sensitivity and specificity of each assay were determined with mutated and wild-type DNA templates, respectively. Bi-PAP-A has a sensitivity of one molecule for most assays and a selectivity (sensitivity:specificity) greater than 1:10(7)-1:10(9) for four of all six mutation types. Four assays with high selectivity were used to detect rare somatic mutations in blood white cells. The silent g.13147C>G (p.R156) mutation was present at an estimated frequency of 1.1 x 10(-7). The g.14523A>T (p.E285V), g.14487G>C (p.R273P), and g.14060G>C (p.G245R) mutations were undetectable with frequencies less than 2.0 x 10(-8). We conclude that Bi-PAP-A is a general and rapid method for detecting ultra-rare mutations.

Somatic microindels: analysis in mouse soma and comparison with the human germline.

Microindels, defined as mutations that result in a colocalized microinsertion and microdeletion with a net gain or loss of between 1 and 50 nucleotides, may be an important contributor to cancer. We report the first comprehensive analysis of somatic microindels. Our large database of mutations in the lacI transgene of Big Blue((R)) mice contains 0.5% microindels, 2.8% pure microinsertions, and 11.5% pure microdeletions. There appears to be no age, gender, or tissue-type specificity in the frequency of microindels. Of the independent somatic mutations that result in a net in-frame insertion or deletion, microindels are responsible for 13% of protein expansions and 6% of protein contractions. These in-frame microindels may play a crucial role in oncogenesis and evolution via "protein tinkering" (i.e., modest expansion or contraction of proteins). Four characteristics suggest that microindels are caused by unique mechanisms, not just simple combinations of the same mechanisms that cause pure microinsertions and pure microdeletions. First, microinsertions and microdeletions commonly occur at hotspots, but none of the 30 microindels are recurrent. Second, the sizes of the deletions and insertions in microindels are larger and more varied than in pure microdeletions and pure microinsertions. Third, microinsertions overwhelmingly repeat the adjacent base (97%) while the insertions in microindels do so only infrequently (17%). Fourth, analysis of the sequence contexts of microindels is consistent with unique mechanisms including recruitment of translesion DNA synthesis polymerases. The mouse somatic microindels have characteristics similar to those of human germline microindels, consistent with similar causative mechanisms in mouse and human, and in soma and germline.

Database of somatic mutations in EGFR with analyses revealing indel hotspots but no smoking-associated signature.

We created an Epidermal Growth Factor Receptor (EGFR) Mutation Database (http://www.cityofhope.org/cmdl/egfr_db) that curates a convenient compilation of somatic EGFR mutations in non-small-cell lung cancer (NSCLC) and associated epidemiological and methodological data, including response to the tyrosine kinase inhibitors Gefitinib and Erlotinib. Herein, we analyze 809 mutations collected from 26 publications. Four super hotspots account for 70% of reported mutations while two-thirds of 131 unique mutations have been reported only once and account for only 11% of reported mutations. Consistent with strong biological selection for gain of function, the reported mutations are virtually all missense substitutions or in-frame microdeletions, microinsertions, or microindels (colocalized insertion and deletion with a net gain or loss of 1-50 nucleotides). Microdeletions and microindels are common in a region of exon 19. Microindels, which account for 8% of mutations, have smaller inserted sequences (95% are 1 to 5 bp) and are elevated 16-fold relative to mouse somatic microindels and to human germline microindels. Microdeletions/microindels are significantly more frequent in responders to Gefitinib or Erlotinib (P = 0.003). In addition, EGFR mutations in smokers do not carry signatures of mutagens in cigarette smoke. Otherwise, the mutation pattern does not differ significantly with respect to gender, age, or tumor histology. The EGFR Mutation Database is a central resource of EGFR sequence variant data for clinicians, geneticists, and other researchers. Authors are encouraged to submit new publications with EGFR sequence variants to be included in the database or to provide direct submissions via The WayStation submission and publication process (http://www.centralmutations.org).

Candidate gene analyses by scanning or brute force fluorescent sequencing: a comparison of DOVAM-S with gel-based and capillary-based sequencing.

For epidemiological and diagnostic applications, detection of virtually all mutations is desired. Herein, blinded analyses of DOVAM-S (Detection Of Virtually All Mutations-SSCP), a robotically enhanced multiplex SSCP method, demonstrate that all of 525 mutations (391 unique) are detected by the method. In addition, the costs of DOVAM-S, gel-based fluorescent sequencing and capillary-based fluorescent sequencing are compared. The relative cost effectiveness of gel-based and capillary-based sequence analysis depends on throughput and whether depreciation and service are considered. DOVAM-S reduces the cost of candidate gene analyses relative to brute force sequencing by about threefold.

Preferential occurrence of 1-2 microindels.

Microindels are unique, infrequent mutations that result in inserted and deleted sequences of different sizes (between one and 50 nucleotides) at the same nucleotide position. Little is known about the mutational mechanisms that are responsible for these mutations. From our database of 6,016 independent somatic mutational events in the lacI gene in Big Blue mice, we assembled the 30 microindels (0.5%) for analysis. Microindels with one nucleotide inserted and two nucleotides deleted (1-2 microindels) accounted for seven (23%) of the microindels observed, with the remaining microindels distributed among 21 other combinations of insertion and deletion sizes. A preferential occurrence of 1-2 microindels (20%) was also observed in human germline transmitted mutations in the Human Gene Mutation Database (HGMD). An examination of the sequence flanking the mouse 1-2 microindels did not reveal obvious site specificity or associated secondary structure. A detailed examination of 1-2 microindels did not reveal the features typical of pure microinsertion and microdeletion events, but rather suggested a unique mutational mechanism. The 1 bp insertion in 1-2 microinsertions, and pure 1 bp insertions show distinct features. The mechanism for 1-2 microindels is not obviously a simple combination of pure microinsertion and microdeletion events. The dramatic enhancement of 1-2 microindels requires explanation. We speculate that certain error-prone polymerases may be responsible for the preferential occurrence of 1-2 microindels in both somatic tissues and germ cells. It is estimated that a human adult carries roughly 400 billion somatic 1-2 microindels with the potential to predispose to cancer.

Multiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions.

Large heterozygous chromosomal deletions and gene duplications are important classes of mutations that are generally missed by standard PCR amplification and sequencing. Multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP), a derivative of PAP, was utilized to detect these types of mutations. PAP is a method for nucleic acid amplification in which 3' blocked oligonucleotides (P*) are activated by pyrophosphorolysis when annealed to the target template and subsequently extended. A key advantage to this technology is that PAP reactions produce little or no primer-dimer or false priming. As a result of this enhanced specificity, MD-PAP is easy to optimize. Herein, we utilize MD-PAP to determine gene dosage of each exon of the human factor IX gene by comparison with one endogenous internal control from the ATM gene. Estimated dosage is proportional to the actual template copy number over a minimum dynamic range from 1 to 16 copies. A blinded analysis detected 100% of 43 heterozygous deletions of exons in the human factor IX gene.

High frequency of neurexin 1beta signal peptide structural variants in patients with autism.

Neuroligins are postsynaptic membrane cell-adhesion molecules which bind to beta-neurexins, a family of proteins that act as neuronal cell surface receptors. To explore the possibility that structural variants in the beta-neurexin genes predispose to autism, the coding regions and associated splice junctions of three beta-neurexin genes were scanned with detection of virtually all mutations-SSCP (DOVAM-S) in 72 Caucasian patients with autism. In addition, segments of the neurexin 1beta gene were sequenced in 131 additional Caucasian and 61 Afro-American patients with autism from South Carolina and the Midwest. Two putative missense structural variants were identified in the neurexin 1beta gene in four Caucasian patients with autism and not in 535 healthy Caucasian controls (4/203 vs. 0/535, P=0.0056). Initial family data suggest that incomplete penetrance may occur. In addition, no structural variant was found in the neurexin 2beta gene and the neurexin 3beta gene. In the context of all available data, we conclude that mutations of the neurexin 1beta gene may contribute to autism susceptibility.

Pyrophosphorolysis-activatable oligonucleotides may facilitate detection of rare alleles, mutation scanning and analysis of chromatin structures.

Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of pyrophosphorolysis-mediated activation of a pyrophosphorolysis-activatable oligonucleotide (P*) followed by extension of the activated oligonucleotide. Herein, we demonstrate that genetically engineered DNA polymerases greatly improve the efficiency of PAP, making it a practical technique for detection of rare mutations. We also show that P* oligonucleotides have the novel and unexpected property of high sensitivity to mismatches throughout at least the 16 3'-terminal nucleotides. Thus, PAP constitutes a technology platform of potential utility whenever high specificity is required along the length of an oligonucleotide.

Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases.

DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.

Increased risk of idiopathic chronic pancreatitis in cystic fibrosis carriers.

Cystic fibrosis (CF) is a recessive disease caused by mutations of the CF transmembrane conductance regulator (CFTR) gene. The risk of idiopathic chronic pancreatitis (ICP) is increased in individuals who have CFTR genotypes containing a CF-causing mutation plus a second pathogenic allele. It is unknown whether the risk of ICP is increased in CF carriers who have one CF-causing mutation plus one normal allele. In this study, 52 sporadic cases of ICP were ascertained through the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer. Individuals with pathogenic cationic trypsinogen mutations were excluded. DNA was comprehensively tested for CFTR mutations using a robotically enhanced, multiplexed, and highly redundant form of single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing. Fifteen subjects had a total of 18 pathogenic CFTR alleles. Eight subjects had common CF-causing mutations. This group included seven CF carriers in whom the second CFTR allele was normal (4.3 times the expected frequency, P=0.0002). Three subjects had compound heterozygotes genotypes containing two pathogenic alleles (31 times the expected frequency, P<0.0001). A variant allele of uncertain significance (p.R75Q) was detected in eight of the 52 ICP subjects and at a similar frequency (13/96) in random donors. ICP differs from other established CFTR-related conditions in that ICP risk is increased in CF carriers who have one documented normal CFTR allele. Having two CFTR mutations imparts a higher relative risk, while having only one mutation imparts a higher attributable risk.

Mutation rates in the dystrophin gene: a hotspot of mutation at a CpG dinucleotide.

An analysis of mutations was performed in 141 Duchenne muscular dystrophy (DMD) patients previously found to be negative for large deletions by standard multiplex PCR assays. Comprehensive mutation scanning of all coding exons, adjacent intronic splice regions, and promoter sequences was performed by DOVAM-S, a robotically enhanced, high throughput method that detects essentially all point mutations. Samples negative for point mutations were further analyzed for duplications by multiplex amplifiable probe hybridization (MAPH). Presumptive causative mutations were detected in 90% of the patients (70% protein truncating point mutations, 13% duplications, and 7% deletions not detected by the standard multiplex screening method). A total of 40 of the mutations are putatively novel. Most duplications involve multiple exons with an average and median size of about 160 and 153 kb, respectively. This is the first analysis of the absolute and relative rates of point mutations in the dystrophin gene. Relative to microdeletions (0.68 x 10(-9) per bp per generation), transitions at CpG dinucleotides are enhanced 150-fold while complex indels, the least common mutation type, are less frequent than microdeletions by a factor of five. The frequency of microdeletions and microinsertions at mononucleotide repeats increases exponentially with length. When compared to the well-studied human factor IX gene (F9), the results are similar, with two exceptions: a hotspot of mutation in the dystrophin gene (c.8713C>T/p.R2905X) at a CpG dinucleotide and an altered size distribution of microdeletions. The hotspot reflects a difference in the underlying pattern of mutation, while the altered size distribution of microdeletions reflects certain abundant sequence motifs within the dystrophin coding sequence (relative to factor IX).

Detection of heterozygous deletions and duplications in the MECP2 gene in Rett syndrome by Robust Dosage PCR (RD-PCR).

Fifty to eighty percent of Rett syndrome (RTT) cases have point mutations in the gene encoding methyl-CpG-binding protein-2 (MECP2). A fraction of MECP2 negative classical RTT patients has large heterozygous deletions. Robust Dosage PCR (RD-PCR) assays were developed as a rapid, convenient and accurate method to detect large heterozygous deletions and duplications. A blinded analysis was performed for 65 RTT cases from Portugal by RD-PCR in the coding exons 2-4 of the MECP2 gene. Neither the patients with point mutations nor the non-classical RTT patients without point mutation had a deletion or duplication. One of remaining eight female patients with classical RTT without point mutation had a heterozygous deletion. This is the first report of a deletion spanning the entire MECP2 gene. The deletion was confirmed by Southern blotting analysis and the deletion junction was localized 37 kb upstream from exon 1 and 18 kb downstream from exon 4. No duplications were detected. Our results suggest that RD-PCR is an accurate and convenient molecular diagnostic method.