Ultra-rare Mutations Research Articles

An ultra-rare mutation (C.181C>T) in GALNS gene associated with Morquio syndrome: A case report

MotivationOne of the rare inherited diseases is the mucopolysaccharidoses disorders (MPS) associated with accumulation of glycosaminoglycan in several organs, leading to abnormalities in musculoskeletal, respiratory, cardiac, neurological, ophthalmological, otolaryngological, and gastrointestinal. Nowadays, an increasing number of patients with MPS are reaching adulthood and are involved in family planning due to improvements in diagnosis, multi-disciplinary care, and therapies such as enzyme replacement therapy and hematopoietic stem cell transplantation. Knowledge on skeletal deformity in MPS remains relatively uncommon and includes a few case reports. To assess more, we present a case report on musculoskeletal deformity in one subject with MPS. CaseThis case revealed that subject with MPS has generalized osteoporosis, Degenerative Joint Disease (DJD) of both hips, general skeletal deformity alongside normal mental status. On physical examination, disproportionate short stature, skeletal dysplasia, osteoporosis, prognathism, negative ulnar variance, angle blunting and ulnar styloid were detected. In the present research work, C.181C>T (p.Arg61Trp) variant of GALNS gene was found through whole exome sequencing and the mutation was confirmed using Sanger sequencing method, which is associated with MPS4A phenotype. ConclusionsPathogenic variant in the GALNS gene associated with hereditary Mucopolysaccharidosis IVA, (MPS4A, Morquio syndrome A) which encodes N-acetylgalactosamin-sulfate sulfatase, may be useful as a biomarker to early diagnosis and treatment.

Broadening targeted therapy in cystic fibrosis

Medicine There are more than 1700 mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene that cause cystic fibrosis. Some mutations occur frequently, whereas others affect very few or individual patients. Modulators have been developed to target specific CFTR mutations classified according to their functional impact on the encoded protein. In a Perspective, Manfredi et al. discuss the emerging view that many CFTR mutations have pleiotropic effects and so more patients could benefit from modulator therapy but do not receive it. Moreover, individuals with ultrarare CFTR mutations are often not indicated to receive these targeted drugs. The authors outline the approaches needed to broaden the personalization of these modulators to treat more patients. Science , this issue p. [220][1] [1]: /lookup/doi/10.1126/science.aaw0553

Abstract 4156: Multiple rare germline variants in a thyroid cancer family

Abstract Thyroid cancer is the 12th most common cancer in the USA. Four main types exist: the majority (85-90%) is papillary thyroid cancer (PTC). Familial form of PTC represents 5 to 15 % of all thyroid cancers. We have access to 155 PTC families including clinical data and biological samples. Our aim was to search for highly penetrant gene variants in 17 PTC families. Data from one of the families in which PTC is segregating in three generations are described here. Whole genome sequencing was performed in three family members affected by PTC. Sanger sequencing at germline DNA and RNA levels followed by TOPO cloning was used to study the effects of mutations. We found 4 different either novel or ultra-rare mutations in the ITGAD, AARS and PDPR genes on chromosome 16 (Table). All mutations segregated perfectly with PTC in family members (n=4) and were absent in unaffected members (n=3). We Sanger sequenced cDNA from two of the PTC patients and cloned the products into the pCR 2.1-® TOPO vector. Based on the cDNA sequencing and cloning results, all of the mutations will create a premature stop codon leading to a truncated protein. We will verify the effects of the mutations by protein analysis as well. In addition, structural variation analysis will be done to exclude large genomic rearrangements. The germline events leading to PTC are neither well known nor extensively researched. When occurring in families PTC regularly shows autosomal dominant inheritance with incomplete penetrance. Only a few of probable mutations have been reported. Nevertheless, our finding of the truncating mutations in 3 genes on chromosome 16 that are cosegregating with PTC in a 3-generation family is surprising. Even more so: is the localization of the three genes in one chromosome coincidental? Preliminary findings in some of the other 16 PTC families suggest that the occurrence of more than one truncating mutation is not a unique event. We look forward to answer the question whether combination of two or more mutant genes can be responsible for one disease segregating in families. Citation Format: Taina T. Nieminen, Daniel F. Comiskey, Yanqiang Wang, Sandya Liyanarachchi, Pamela Brock, Paivi Peltomaki, Huiling He, Albert de la Chapelle. Multiple rare germline variants in a thyroid cancer family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4156.

A frequent variant in the Japanese population determines quasi-Mendelian inheritance of rare retinal ciliopathy

Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e., a 3.5-fold enrichment; p-value = 9.2 × 10−5). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation in trans with the Alu insertion, but might also associate with disease in combination with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.

Ultra-rare C3 mutation leading to a dominant familial C3 glomerulopathy phenotype

Ultra-rare C3 mutation leading to a dominant familial C3 glomerulopathy phenotype

Ultra-rare mutations in SRCAP segregate in Caribbean Hispanic families with Alzheimer disease.

Objective:To identify rare coding variants segregating with late-onset Alzheimer disease (LOAD) in Caribbean Hispanic families.Methods:Whole-exome sequencing (WES) was completed in 110 individuals from 31 Caribbean Hispanic families without APOE ε4 homozygous carriers. Rare coding mutations segregating in families were subsequently genotyped in additional families and in an independent cohort of Caribbean Hispanic patients and controls. SRCAP messenger RNA (mRNA) expression was assessed in whole blood from mutation carriers with LOAD, noncarriers with LOAD, and healthy elderly controls, and also from autopsied brains in 2 clinical neuropathologic cohort studies of aging and dementia.Results:Ten ultra-rare missense mutations in the Snf2-related CREBBP, activator protein (SRCAP), were found in 12 unrelated families. Compared with the frequency in Caribbean Hispanic controls and the Latino population in the Exome Aggregation Consortium, the frequency of SRCAP mutations among Caribbean Hispanic patients with LOAD was significantly enriched (p = 1.19e-16). mRNA expression of SRCAP in whole blood was significantly lower in mutation carriers with LOAD, while the expression in whole blood and in the brain was significantly higher in nonmutation carriers with LOAD. Brain expression also correlated with clinical and neuropathologic endophenotypes.Conclusions:WES in Caribbean Hispanic families with LOAD revealed ultra-rare missense mutations in SRCAP, a gene expressed in the brain and mutated in Floating-Harbor syndrome. SRCAP is a potent coactivator of the CREB-binding protein and a regulator of DNA damage response involving ATP-dependent chromatin remodeling. We hypothesize that increased expression in LOAD suggests a compensatory mechanism altered in mutation carriers.

Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations

Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.

Complexity of mechanisms among human proprotein convertase subtilisin-kexin type 9 variants.

There are many reports of human variants in proprotein convertase subtilisin-kexin type 9 (PCSK9) that are either gain-of-function (GOF) or loss-of-function (LOF), with downstream effects on LDL cholesterol and cardiovascular disease (CVD) risk. However, data on particular mechanisms have only been minimally curated. GOF variants are individually ultrarare, affect all domains of the protein, act to reduce LDL receptor expression through several mechanisms, are a minor cause of familial hypercholesterolemia, have been reported mainly within families, have variable LDL cholesterol-raising effects, and are associated with increased CVD risk mainly through observational studies in families and small cohorts. In contrast, LOF variants can be either ultrarare mutations or relatively more common polymorphisms seen in populations, affect all domains of the protein, act to increase LDL receptor expression through several mechanisms, have variable LDL cholesterol-lowering effects, and have been associated with decreased CVD risk mainly through Mendelian randomization studies in epidemiologic populations. There is considerable complexity underlying the clinical concept of both LOF and GOF variants of PCSK9. But despite the underlying mechanistic heterogeneity, altered PCSK9 secretion or function is ultimately correlated with plasma LDL cholesterol level, which is also the driver of CVD outcomes.

Abstract 2395: Direct assessment of sequence heterogeneity in human cancers by Duplex Sequencing

Abstract The extent of heterogeneity in DNA sequence within human cancers has been difficult to fully assess, due to the absence of methods with sufficient sensitivity to detect minority variants. For example, next-generation sequencing could in principle detect sub-clonal mutations at any genomic position; however due to amplification errors and inaccuracies in the sequencing platform itself, such approaches are limited to detection of mutations present in >1% of cells. We have overcome this limitation by developing an approach, Duplex Sequencing, which improves the accuracy of next-generation sequencing by >100,000 fold. Duplex Sequencing is based upon separately tagging and sequencing the two strands of single molecules of duplex DNA. True mutations are present at the same position in both DNA strands and are complementary, whereas artifacts arising from amplification or sequencing errors are seen in only one strand. The calculated error rate of our approach is less than one artifactual mutation per billion nucleotides sequenced (ref. 1). We have also developed an enrichment approach based on sequential rounds of hybridization to biotinylated probes which enables efficient sequencing of targeted regions of the genome (ref. 2). Duplex Sequencing thereby enables exceptionally sensitive detection of sequence heterogeneity within any set of genes. We have now applied Duplex Sequencing to study of sequence heterogeneity in both normal and malignant samples. In acute myeloid leukemia (AML), we find multiple sub-clonal mutations which fall below the detection limit of conventional approaches, some of which encode mutations that can drive chemotherapy resistance and cancer progression. Our results indicate that prior studies have under-estimated the burden of sub-clonal mutations in AML by more than 1,000-fold. We have additionally studied chronic myeloid leukemia (CML); treatment of CML with inhibitors directed against the Abl kinase represents the prototypical targeted cancer therapy. With Duplex Sequencing, we find that Abl mutations are extremely uncommon at the time of CML diagnosis. In contrast, refractory patients who fail therapy frequently possess multiple sub-clonal mutations within the Abl kinase, most of which are below the resolution of conventional approaches but are readily detected with our method. In ongoing work, we have found multiple sub-clonal mutations within human prostate cancer and colon cancer, suggestive of widespread intratumor heterogeneity that may limit the efficacy of single-agent therapy in these diseases.

Abstract A29: Detection of ultra-rare mutations during breast stem cell tumor progression by Duplex Sequencing

Abstract Most studies have mainly investigated clonal mutations due to the high background error frequencies of conventional next generation sequencing (NGS) methods. We have established a new method termed Duplex Sequencing (DS) that detects mutations with unprecedented accuracy and enables us to detect sub-clonal and rare mutations as well as clonal mutations. Using DS, we present our first genome-wide analysis of mitochondrial (mt) DNA mutations during the transformation of human normal breast stem cells to tumorigenic cells. We used a model system to dissect sequential steps in the progression of normal stem cells to tumorigenic cells. Significant differences have been found among normal stem cells, immortal cells, and tumorigenic cells. These cells differ in putative breast cancer stem cell (CSC) populations, mtDNA copy numbers, and the frequencies and types of mutations. We found that putative CSCs and mtDNA copy numbers increase as normal stem cells become tumorigenic cells. The vast majority of mutations identified in our study are stochastic (rare mutations) and the frequency of rare mutations is lower in transformed cells than in normal stem cells. This lower rare mutation load in transformed cells induces lower scores of predicted pathogenicity in transformed cells than in normal stem cells. C>T/G>A and A>G/T>C transitions are the major mutation types in normal stem cells, while only C>T/G>A are the major mutation types in transformed cells. Using DS, we detected a total of 1220 rare mutations and 678 of these variants have not been reported previously. The mtDNA mutation spectra of breast tumorigenic cells correlate with published nuclear DNA mutation signatures, which are found in the majority of tumors. We have identified mutations that can serve as potential biomarkers that indicate the different stages of breast carcinogenesis. These significant differences in mutation profiles could aid in characterizing different phenotypes between normal stem cells and transformed cells. Citation Format: Eun Hyun Ahn, Kensen Hirohata, Seung Hyuk Lee, Joon Yup Kim, Brendan F. Kohrn, Edward J. Fox, Chia-Cheng Chang, Lawrence A. Loeb. Detection of ultra-rare mutations during breast stem cell tumor progression by Duplex Sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A29.

G.P.87: Prospects for single antisense oligonucleotide-induced multiple exon skipping for rare non-hotspot mutations in Duchenne muscular dystrophy

Antisense oligonucleotides (AONs) for Duchenne muscular dystrophy (DMD) are designed to induce skipping of a single exon during pre-mRNA splicing, thus restoring the transcript’s open reading frame and consequently synthesis of the deficient dystrophin protein. Although this approach is mutation-dependent, the majority of mutations cluster in a hotspot region from exon 45 to 53, allowing application of one AON to a subpopulation of patients with grouped mutations. Some mutations however are ultra-rare with only few patients worldwide, and require the skipping of an exon outside the hotspot region. Combining these ultra-rare mutations by applying multiple exon skipping is therefore attractive. The feasibility of multiple exon skipping using a cocktail of AONs has been demonstrated, but a drawback is the increased complexity of the development pathway and manufacturing costs. We therefore studied the possibility of designing a single AON capable of inducing simultaneous skipping of multiple exons. The repetitive nature of the central rod domain in dystrophin implies the occurrence of homologous stretches within the transcript. Indeed, in the exon 10 to 40 region several exon pairs were identified sharing such a homologous stretch, which we explored as a potential dual target for a single AON. In-frame exon combinations that would mimic a Becker-like mutation associated with a relatively mild phenotype and that would apply to a considerable patient subpopulation (up to ∼15%) were selected. In vitro proof-of-concept was obtained for healthy donor as well as for DMD patient-derived muscle cells and resulted in novel dystrophin expression. Results demonstrating the applicability in the mdx mouse model in vitro and in vivo will be presented. The data support further (pre) clinical development of such multiple exon skipping AONs, not only for more rare mutations that are not targeted by current single skip programs but also for mutations in the hotspot region.

Correction: Ultra-Rare Mutation in Long-Range Enhancer Predisposes to Thyroid Carcinoma with High Penetrance

An additional grant from the National Cancer Institute was incorrectly omitted from the Funding Statement. The number of the grant is: R01 CA151979.

Ultra-Rare Mutation in Long-Range Enhancer Predisposes to Thyroid Carcinoma with High Penetrance

Thyroid cancer shows high heritability but causative genes remain largely unknown. According to a common hypothesis the genetic predisposition to thyroid cancer is highly heterogeneous; being in part due to many different rare alleles. Here we used linkage analysis and targeted deep sequencing to detect a novel single-nucleotide mutation in chromosome 4q32 (4q32A>C) in a large pedigree displaying non-medullary thyroid carcinoma (NMTC). This mutation is generally ultra-rare; it was not found in 38 NMTC families, in 2676 sporadic NMTC cases or 2470 controls. The mutation is located in a long-range enhancer element whose ability to bind the transcription factors POU2F and YY1 is significantly impaired, with decreased activity in the presence of the C- allele compared with the wild type A-allele. An enhancer RNA (eRNA) is transcribed in thyroid tissue from this region and is greatly downregulated in NMTC tumors. We suggest that this is an example of an ultra-rare mutation predisposing to thyroid cancer with high penetrance.

Abstract 1789A: Duplex DNA sequencing detects subclonal and ultra-rare mutations in human tumors.

Abstract Next-Generation DNA Sequencing now allows us to sequence billions of nucleotides in a single experiment and to detect germ-line mutations and driver mutations in human cancer. However, a major limitation is the error rate of approximately 1% to 0.1%. These scattered errors limit its utility in sequencing genetically heterogeneous mixtures such as human cancers or viruses. We have developed a method termed "Duplex Sequencing” that allows for the detection of rare mutations in DNA with unprecedented accuracy. This approach greatly lowers errors by independently sequencing each of the two strands of single DNA molecules. As the two strands are complementary, true mutations are scored only if they are present at the same position and are complementary. Moreover, small deletions and insertions can be easily identified. In contrast, sequencing errors, which occur from PCR - amplification of DNA containing damaged nucleotides, are present in only one strand and thus are not scored as mutations. To demonstrate the power of duplex sequencing we measured the background error frequency in human mitochondrial DNA obtained from cortical regions of the brain of aged normal humans; an overall mutation frequency was 3 X 10-5. These mutations are not random, but rather, show an inordinately large frequency of mutations in the D-loop of the mitochondrial genome. The spectrum of mutations is strikingly different from that reported in the literature using next-generation sequencing. Prior reports have indicated a high frequency of G->T; however, we find that these are likely technical artifacts arising from oxidative DNA damage. In contrast, by Duplex Sequencing, we find a predominance of G->A and T->C single base substitutions. The G->A and T->C mutations could result from cytidine deamination or errors during replication by the mitochondrial DNA polymerase gamma. In order to establish the background mutation frequency in nuclear DNA by Duplex Sequencing we first captured a small portion of the genome, the exons of the replicative DNA polymerases. Sequencing captured DNA from normal human tissues yielded a background mutation frequency of 10-6 to 1O-7 indicating that the error rate of duplex sequencing is less than one to ten mutations in ten million nucleotides. Current effort is to sequence nuclear DNA from different human cancers and the corresponding normal tissues. This result should indicate the distribution of subclonal mutations in human tumors. Subclonal mutations could delineate tumor cell lineage, account for the geographic heterogeneity of mutations in human tumors and serve as a biomarker for the presence of drug resistant cells prior to or during chemotherapy. Citation Format: Lawrence A. Loeb, Edward J. Fox, Scott R. Kennedy, Jessica Kuong, Michael W. Schmitt, Jesse J. Salk. Duplex DNA sequencing detects subclonal and ultra-rare mutations in human tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1789A. doi:10.1158/1538-7445.AM2013-1789A

Detection of ultra-rare mutations by next-generation sequencing

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

Analysis of cancer mutation signatures in blood by a novel ultra-sensitive assay: monitoring of therapy or recurrence in non-metastatic breast cancer.

BackgroundTumor DNA has been shown to be present both in circulating tumor cells in blood and as fragments in the plasma of metastatic cancer patients. The identification of ultra-rare tumor-specific mutations in blood would be the ultimate marker to measure efficacy of cancer therapy and/ or early recurrence. Herein we present a method for detecting microinsertions/deletions/indels (MIDIs) at ultra-high analytical selectivity. MIDIs comprise about 15% of mutations.Methods and FindingsWe describe MIDI-Activated Pyrophosphorolysis (MAP), a method of ultra-high analytical selectivity for detecting MIDIs. The high analytical selectivity of MAP is putatively due to serial coupling of two rare events: heteroduplex slippage and mis-pyrophosphorolysis. MAP generally has an analytical selectivity of one mutant molecule per >1 billion wild type molecules and an analytical sensitivity of one mutant molecule per reaction. The analytical selectivity of MAP is about 100,000-fold better than that of our previously described method of Pyrophosphorolysis Activated Polymerization-Allele specific amplification (PAP-A) for detecting MIDIs. The utility of this method is illustrated in two ways. 1) We demonstrate that two EGFR deletions commonly found in lung cancers are not present in tissue from four normal human lungs (107 copies of gDNA each) or in blood samples from 10 healthy individuals (107 copies of gDNA each). This is inconsistent, at least at an analytical sensitivity of 10−7, with the hypotheses of (a) hypermutation or (b) strong selection of these growth factor-mutated cells during normal lung development leads to accumulation of pre-neoplastic cells with these EGFR mutations, which sometimes can lead to lung cancer in late adulthood. Moreover, MAP was used for large scale, high throughput “gene pool” analysis. No germline or early embryonic somatic mosaic mutation was detected (at a frequency of >0.3%) for the 15/18 bp EGFR deletion mutations in 6,400 individuals, suggesting that early embryonic EGFR somatic mutation is very rare, inconsistent with hypermutation or strong selection of these deletions in the embryo. 2) The second illustration of MAP utility is in personalized monitoring of therapy and early recurrence in cancer. Tumor-specific p53 mutations identified at diagnosis in the plasma of six patients with stage II and III breast cancer were undetectable after therapy in four women, consistent with clinical remission, and continued to be detected after treatment in two others, reflecting tumor progression.ConclusionsMAP has an analytical selectivity of one part per billion for detection of MIDIs and an analytical sensitivity of one molecule. MAP provides a general tool for monitoring ultra-rare mutations in tissues and blood. As an example, we show that the personalized cancer signature in six out of six patients with non-metastatic breast cancer can be detected and that levels over time are correlated with the clinical course of disease.

Evidence for X-Chromosomal Schizophrenia Associated with microRNA Alterations

BackgroundSchizophrenia is a severe disabling brain disease affecting about 1% of the population. Individual microRNAs (miRNAs) affect moderate downregulation of gene expression. In addition, components required for miRNA processing and/or function have also been implicated in X-linked mental retardation, neurological and neoplastic diseases, pointing to the wide ranging involvement of miRNAs in disease.Methods and FindingsTo explore the role of miRNAs in schizophrenia, 59 microRNA genes on the X-chromosome were amplified and sequenced in males with (193) and without (191) schizophrenia spectrum disorders to test the hypothesis that ultra-rare mutations in microRNA collectively contribute to the risk of schizophrenia. Here we provide the first association of microRNA gene dysfunction with schizophrenia. Eight ultra-rare variants in the precursor or mature miRNA were identified in eight distinct miRNA genes in 4% of analyzed males with schizophrenia. One ultra-rare variant was identified in a control sample (with a history of depression) (8/193 versus 1/191, p = 0.02 by one-sided Fisher's exact test, odds ratio = 8.2). These variants were not found in an additional 7,197 control X-chromosomes.ConclusionsFunctional analyses of ectopically expressed copies of the variant miRNA precursors demonstrate loss of function, gain of function or altered expression levels. While confirmation is required, this study suggests that microRNA mutations can contribute to schizophrenia.

Detection of ultrarare somatic mutation in the humanTP53gene 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.