1. Molecular genetics of RhD blood group 2. Mutational and pathological mechanisms underlying human mutations 3. Genetic basis of chronic pancreatitis and cystic fibrosis
Abstract: The wide clinical spectrum of the ABCB4 gene (ATP-binding cassette subfamily B member 4) deficiency syndromes in humans includes low phospholipid-associated cholelithiasis (LPAC), intrahepatic cholestasis of pregnancy (ICP), oral contraceptives-induced cholestasis (CIC), and progressive familial intrahepatic cholestasis type 3 (PFIC3). No ABCB4 mutations are found in a significant proportion of patients with these syndromes. In the present study, 102 unrelated adult patients with LPAC (43 patients) or CIC/ICP (59 patients) were screened for ABCB4 mutations using DNA sequencing. Heterozygous ABCB4 point or short insertion/deletion mutations were found in 37% (16/43) of the LPAC patients and in 27% (16/59) of the ICP/CIC patients. High-resolution gene dosage methodologies were used in the 70 negative patients. Here, we describe for the first time ABCB4 partial or complete heterozygous deletions in 7% (3/43) of the LPAC patients, and in 2% (1/59) of the ICP/CIC patients. Our observations urge to systematically test patients with LPAC, ICP/CIC, and also children with PFIC3 for the presence of ABCB4 deletions using molecular tools allowing detection of gross rearrangements. In clinical practice, a comprehensive ABCB4 alteration-screening algorithm will permit the use of ABCB4 genotyping to confirm the diagnosis of LPAC or ICP/CIC, and allow familial testing. An early diagnosis of these biliary diseases may be beneficial because of the preventive effect of ursodeoxycholic acid on biliary complications. Further comparative studies of patients with well-characterized genotypes (including deletions) and phenotypes will help determine whether ABCB4 mutation types influence clinical outcomes.
Abstract: Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disorder, is caused by mutations in PKD1 or PKD2. The molecular diagnosis of ADPKD is complicated by extensive allelic heterogeneity and particularly by the presence of 6 highly homologous sequences of PKD1 exons 1-33. Here, we screened PKD1 and PKD2 for both conventional mutations and gross genomic rearrangements in up to 700 unrelated ADPKD patients-the largest patient cohort to date-by means of direct sequencing followed by quantitative fluorescent multiplex PCR or array-comparative genomic hybridization. This resulted in the identification of the largest number of new pathogenic mutations (n = 351) in a single publication, expanded the spectrum of known ADPKD pathogenic mutations by 41.8% for PKD1 and by 23.8% for PKD2, and provided new insights into several issues, such as the population-dependent distribution of recurrent mutations compared to founder mutations and the relative paucity of pathogenic missense mutations in the PKD2 gene. Our study, together with others, highlights the importance of developing novel approaches for both mutation detection and functional validation of non-definite pathogenic mutations to increase the diagnostic value of molecular testing for ADPKD.
Abstract: BACKGROUND: The RhD blood group system exemplifies a genotype-phenotype correlation by virtue of its highly polymorphic and immunogenic nature. Weak D phenotypes are generally thought to result from missense mutations leading to quantitative change of the D antigen in the red blood cell membrane or intracellularly. STUDY DESIGN AND METHODS: Different sets of polymerase chain reaction primers were designed to map and clone a deletion involving RHD Exon 10, which was found in approximately 3% of approximately 2000 RHD hemizygous subjects with D phenotype ambiguity. D antigen density was measured by flow cytometry. Transcript analysis was carried out by 3'-rapid amplification of complementary DNA ends. Haplotype analysis was performed by microsatellite genotyping. RESULTS: A 5405-bp deletion that removed nearly two-thirds of Intron 9 and almost all of Exon 10 of the RHD gene was characterized. It is predicted to result in the replacement of the last eight amino acids of the wild-type RhD protein by another four amino acids. The mean RhD antigen density from two deletion carriers was determined to be only 30. A consensus haplotype could be deduced from the deletion carriers based on the microsatellite genotyping data. CONCLUSION: The currently reported deletion was derived from a common founder. This deletion appears to represent not only the first large deletion associated with weak D but also the weakest of weak D alleles so far reported. This highly unusual genotype-phenotype relationship may be attributable to the additive effect of three distinct mechanisms that affect mRNA formation, mRNA stability, and RhD/ankyrin-R interaction, respectively.
Abstract: Clustered mutations may be broadly defined as the presence of two or more mutations within a spatially localized genomic region on a single chromosome. Known instances vary in terms of both the number and type of the component mutations, ranging from two closely spaced point mutations to tens or even hundreds of genomic rearrangements. Although clustered mutations can represent the observable net result of independent lesions sequentially acquired over multiple cell cycles, they can also be generated in a simultaneous or quasi-simultaneous manner within a single cell cycle. This review focuses on those mechanisms known to underlie the latter type. Both gene conversion and transient hypermutability are capable of generating closely spaced multiple mutations. However, a recently described phenomenon in human cancer cells, known as 'chromothripsis', has provided convincing evidence that tens to hundreds of genomic rearrangements can sometimes be generated simultaneously via a single catastrophic event. The distinctive genomic features observed in the derivative chromosomes, together with the highly characteristic junction sequences, point to non-homologous end joining (NHEJ) as being the likely underlying mutational mechanism. By contrast, replication-based mechanisms such as microhomology-mediated break-induced replication (MMBIR) which involves serial replication slippage or serial template switching probably account for those complex genomic rearrangements that comprise multiple duplications and/or triplications.
Abstract: A considerable number of RHD alleles responsible for weak and partial D phenotypes have been identified over the past decade. Two particular concerns, namely, 1) that red blood cells of these phenotypes may cause anti-D immunization when transfused to D- recipients and 2) that serologic determination of these phenotypes is often doubtful, make genetic analysis of the RHD gene highly desirable.
Abstract: The SPINK1 gene, encoding the human pancreatic secretory trypsin inhibitor, is one of the major genes involved in predisposition to chronic pancreatitis (CP). In this study we have assessed the potential functional impact of 11 SPINK1 promoter variants by means of both luciferase reporter gene assay and electrophoretic mobility shift assay (EMSA), using human pancreatic COLO-357 cells as an expression system. The 11 promoter variants were found to be separable into three distinct categories on the basis of the reporter gene assay results viz loss-of-function, gain-of-function and functionally neutral. These findings, which were validated by EMSA, concurred with data from previous deletion studies and DNase I footprinting assays. Further, binding sites for two transcription factors, HNF1 and PTF1, were newly identified within the SPINK1 promoter by virtue of their being affected by specific variants. Combining the functional data with epidemiological data (derived by resequencing the SPINK1 promoter region in French, German and Indian CP patients and controls), then allowed us to make meaningful inferences as to each variant's likely contribution to CP. We conclude that only the three promoter variants associated with a loss-of-function (ie, -53C>T, -142T>C and -147A>G) are likely to be disease-predisposing alterations.
Abstract: OBJECTIVES:: The aim of this study was to evaluate whether variations in the glycoprotein 2 gene (GP2) may potentially affect the risk of chronic pancreatitis. METHODS:: Six hundred sixty-one French white patients (idiopathic chronic pancreatitis, n = 590; familial chronic pancreatitis, n = 42; hereditary pancreatitis, n = 29), 445 Dravidian patients from India (tropical calcific pancreatitis, n = 306; idiopathic chronic pancreatitis, n = 139), and 962 unrelated healthy subjects (French white, n = 500; Dravidian, n = 462) participated in this case-control association study. The entire coding sequence of the GP2 gene was searched for conventional genetic variations by direct sequencing, whereas all 12 exons of the GP2 gene were screened for copy number variations by quantitative fluorescent multiplex-polymerase chain reaction. RESULTS:: Only 3 rare missense mutations (p.A137T, p.E250D, and p.V432M; only p.E250D was not detected in any control subjects) and 3 common synonymous polymorphisms (c.348C>T, c.714G>C, and c.1275A>G) were identified. The c.348C>T and c.1275A>G variations were found to be contradictorily associated with the disease (ranging from protective effects to disease-predisposing effects) in the French white and Indian populations. CONCLUSION:: The paucity of patient-specific missense mutations and contradictory findings with respect to 2 common polymorphisms in the 2 contrasting populations suggest that the GP2 gene is unlikely to play a major role in the etiology of chronic pancreatitis.
Abstract: We have recently found that a common synonymous single nucleotide polymorphism (SNP), c.1275A>G, in exon 9 of the glycoprotein 2 (GP2) gene was significantly underrepresented in French idiopathic chronic pancreatitis patients 20years old or younger at disease onset than in the control population. To further investigate to this preliminary genetic finding, we characterized the functionality of c.1275A>G in the context of a minigene system. Bioinformatics analysis predicted that c.1275A>G could lead to disruption/generation of exonic splicing enhancer hexamers within exon 9 of the GP2 gene. Minigene analysis revealed that both the wild-type and mutant sequences expressed a full-length transcript and a short transcript lacking exon 9. Quantitation of the relative amount of the two transcripts indicated that the fraction of the full-length transcript derived from c.1275A>G is much lower than that derived from the wild-type (51.9% vs 77.4%). Extinction of two splicing factors (SF2/ASF and SC35) by RNA interference also affected c.1275A>G more seriously than the wild-type in terms of exon 9 skipping. Exon 9 skipping was presumed to cause a loss of GP2 function. This study represents the first detailed analysis of any variation in the GP2 gene and gives some support to the putative association of c.1275A>G with disease protection.
Abstract: Osteoporosis is characterized by low bone mineral density and structural deterioration of bone tissue, leading to an increased risk of fractures. It is the most common metabolic bone disorder worldwide, affecting one in three women and one in eight men over the age of 50. In the past 15 years, a large number of genes have been reported as being associated with osteoporosis. However, only in the past 4 years we have witnessed an accelerated pace in identifying and validating osteoporosis susceptibility loci. This increase in pace is mostly due to large-scale association studies, meta-analyses, and genome-wide association studies of both single nucleotide polymorphisms and copy number variations. A comprehensive review of these developments revealed that, to date, at least 15 genes (VDR, ESR1, ESR2, LRP5, LRP4, SOST, GRP177, OPG, RANK, RANKL, COLIA1, SPP1, ITGA1, SP7, and SOX6) can be reasonably assigned as confirmed osteoporosis susceptibility genes, whereas, another >30 genes are promising candidate genes. Notably, confirmed and promising genes are clustered in three biological pathways, the estrogen endocrine pathway, the Wnt/beta-catenin signaling pathway, and the RANKL/RANK/OPG pathway. New biological pathways will certainly emerge when more osteoporosis genes are identified and validated. These genetic findings may provide new routes toward improved therapeutic and preventive interventions of this complex disease.
Abstract: Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1-2 (n = 1) or exons 5-7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5' and/or 3' breakpoints located within Alu repeat elements. In addition, we identified the 5' breakpoint of a previously reported deletion of exons 1-2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.
Abstract: Over the last 20 years since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more than 1,600 different putatively pathological CFTR mutations have been identified. Until now, however, copy number mutations (CNMs) involving the CFTR gene have not been methodically analyzed, resulting almost certainly in the underascertainment of CFTR gene duplications compared with deletions. Here, high-resolution array comparative genomic hybridization (averaging one interrogating probe every 95 bp) was used to analyze the entire length of the CFTR gene (189 kb) in 233 cystic fibrosis chromosomes lacking conventional mutations. We succeeded in identifying five duplication CNMs that would otherwise have been refractory to analysis. Based upon findings from this and other studies, we propose that deletion and duplication CNMs in the human autosomal genome are likely to be generated in the proportion of approximately 2-3:1. We further postulate that intragenic gene duplication CNMs in other disease loci may have been routinely underascertained. Finally, our analysis of +/-20 bp flanking each of the 40 CFTR breakpoints characterized at the DNA sequence level provide support for the emerging concept that non-B DNA conformations in combination with specific sequence motifs predispose to both recurring and nonrecurring genomic rearrangements. Hum Mutat 31:1-8, 2010. (c) 2010 Wiley-Liss, Inc.
Abstract: Gene conversion is a specific type of homologous recombination that involves the unidirectional transfer of genetic material from a ‘donor’ sequence to a highly homologous ‘acceptor’. We have recently reviewed the molecular mechanisms underlying gene conversion, explored the key part that this process has played in fashioning extant human genes, and performed a meta-analysis of gene-conversion events known to have caused human genetic disease. Here we shall briefly summarize some of the latest developments in the study of pathogenic gene conversion events, including (i) the emerging idea of minimal efficient sequence homology (MESH) for homologous recombination, (ii) the local DNA sequence features that appear to predispose to gene conversion, (iii) a mechanistic comparison of gene conversion and transient hypermutability, and (iv) recently reported examples of pathogenic gene conversion events.
Abstract: The number of reported germline mutations in human nuclear genes, either underlying or associated with inherited disease, has now exceeded 100,000 in more than 3,700 different genes. The availability of these data has both revolutionized the study of the morbid anatomy of the human genome and facilitated "personalized genomics." With approximately 300 new "inherited disease genes" (and approximately 10,000 new mutations) being identified annually, it is pertinent to ask how many "inherited disease genes" there are in the human genome, how many mutations reside within them, and where such lesions are likely to be located? To address these questions, it is necessary not only to reconsider how we define human genes but also to explore notions of gene "essentiality" and "dispensability."Answers to these questions are now emerging from recent novel insights into genome structure and function and through complete genome sequence information derived from multiple individual human genomes. However, a change in focus toward screening functional genomic elements as opposed to genes sensu stricto will be required if we are to capitalize fully on recent technical and conceptual advances and identify new types of disease-associated mutation within noncoding regions remote from the genes whose function they disrupt.
Abstract: Osteoporosis is the most common metabolic bone disorder worldwide. At least 15 genes (e.g., ESR1, LRP5, SOST, OPG, RANK and RANKL) have been confirmed as osteoporosis susceptibility genes, and another 30 have been highlighted as promising susceptibility genes. Notably, these genes are clustered in three biological pathways: the estrogen endocrine pathway, the Wnt/β-catenin signaling pathway and the RANK/RANKL/osteoprotegerin (OPG) pathway. In this article, using data pertaining to these three biological pathways as examples, we illustrate possible principles of personalized therapy for osteoporosis. In particular, we propose to use inhibitors (e.g., denosumab) of the RANK/RANKL/OPG signaling pathway to circumvent resistance to estrogen-replacement therapy: a novel idea resulting from the consideration of a mechanistic link between the estrogen endocrine pathway and the RANK/RANKL/OPG signaling pathway. In addition, we call for more attention to be focused on rare variants of major effects in future studies.
Abstract: Genomic rearrangements in inherited disease and cancer involve gross alterations of chromosomes or large chromosomal regions and can take the form of deletions, duplications, insertions, inversions or translocations. The characterization of a considerable number of rearrangement breakpoints has now been accomplished at the nucleotide sequence level, thereby providing an invaluable resource for the detailed study of the mutational mechanisms which underlie genomic recombination events. A better understanding of these mutational mechanisms is vital for improving the design of mutation detection strategies. At least five categories of mutational mechanism are known to give rise to genomic rearrangements: (i) homologous recombination including non-allelic homologous recombination (NAHR), gene conversion, single strand annealing (SSA) and break-induced replication (BIR), (ii) non-homologous end joining (NHEJ), (iii) microhomology-mediated replication-dependent recombination (MMRDR), (iv) long interspersed element-1 (LINE-1 or L1)-mediated retrotransposition and (v) telomere healing. Focussing on the first three of these general mechanisms, we compare and contrast their hallmark characteristics, and discuss the role of various local DNA sequence features (e.g. recombination-promoting motifs, repetitive sequences and sequences capable of non-B DNA formation) in mediating the recombination events that underlie gross genomic rearrangements. Finally, we explore how studies both at the level of the gene (using the neurofibromatosis type-1 gene as an example) and the whole genome (using data derived from cancer genome sequencing studies) are shaping our understanding of the impact of genomic rearrangements as a cause of human genetic disease.
Abstract: A variety of DNA sequence motifs including inverted repeats, minisatellites, and the chi recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the chi-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome.
Abstract: Chronic pancreatitis (CP) is a persistent inflammation of the pancreas. Over the past 12 years, genetic studies of hereditary, familial, and idiopathic forms of CP have made great progress in defining the disease pathogenesis. Identification of gain-of-function missense and copy number mutations in the cationic trypsinogen gene (PRSS1) and loss-of-function variants in both the pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) genes has firmly established the pivotal role of prematurely activated trypsin within the pancreas in the etiology of CP. Loss-of-function variants in the cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-sensing receptor (CASR) genes also increase the risk of CP. Here, we review recent developments in this rapidly evolving field, highlight the importance of gene-gene and gene-environment interactions in causing the disease, and discuss the opportunities and challenges in identifying novel genetic factors that affect susceptibility/resistance to CP.
Abstract: One of the causes of chronic pancreatitis is the duplication and triplication of a approximately 605 kb segment containing the trypsinogen locus. Employing array-comparative genomic hybridization, we fully characterized the triplication copy number mutation (CNM) and found it to be part of a complex rearrangement that also contains a triplicated approximately 137 kb segment and 21 bp sequence tract. This triplication allele therefore constitutes a gain of two tandemly arranged composite duplication blocks, each comprising a copy of the approximately 605 kb segment, a copy of the inverted approximately 137 kb segment and a copy of the inverted 21 bp sequence tract. As such, it represents the first characterization of a human complex triplication CNM at the DNA sequence level. All triplications and duplications identified were found to arise from a common founder chromosome. A two-step process is proposed for the generation of this highly unusual triplication CNM. Thus, the first composite duplication block is envisaged to have been generated by break-induced serial replication slippage during mitosis. This duplication would have provided the sequence homology required to promote non-allelic homologous recombination (NAHR) during meiosis which would then, in a second step, have generated the complex triplication allele. Our data provide support for the view that many human germline copy number variants arise through replication-based mechanisms during the premeiotic mitotic divisions of germ cells. The low copy repeats thereby generated could then serve to promote NAHR during meiosis, giving rise to amplified DNA sequences which would themselves predispose to further recombinational events during both mitosis and meiosis.
Abstract: Data from diverse organisms suggests that transient hypermutability is a general mutational mechanism with the potential to generate multiple synchronous mutations, a phenomenon probably best exemplified by closely spaced multiple mutations (CSMMs). Here we have attempted to extend the concept of transient hypermutability from somatic cells to the germline, using human inherited disease-causing multiple mutations as a model system. Employing stringent criteria for data inclusion, we have retrospectively identified numerous potential examples of pathogenic CSMMs that exhibit marked similarities to the CSMMs reported in other systems. These examples include (1) eight multiple mutations, each comprising three or more components within a sequence tract of <100 bp; (2) three possible instances of "mutation showers"; and (3) numerous highly informative "homocoordinate" mutations. Using the proportion of CpG substitution as a crude indicator of the relative likelihood of transient hypermutability, we present evidence to suggest that CSMMs comprising at least one pair of mutations separated by </=100 bp may constitute signatures of transient hypermutability in human genes. Although this analysis extends the generality of the concept of transient hypermutability and provides new insights into what may be considered a novel mechanism of mutagenesis underlying human inherited disease, it has raised serious concerns regarding current practices in mutation screening.Hum Mutat 30:1-14, 2009. (c) 2009 Wiley-Liss, Inc.
Abstract: Hereditary pancreatitis, an autosomal dominant disease with approximately 80% penetrance, can be caused by both 'gain-of-function' missense and copy number mutations in the cationic trypsinogen gene (PRSS1). Here we demonstrate a heterozygous hybrid PRSS2 (encoding anionic trypsinogen)/PRSS1 gene in a French white family with hereditary pancreatitis, by means of quantitative fluorescent multiplex PCR and RT-PCR analyses. The hybrid gene, in which exons 1 and 2 are derived from PRSS2 and exons 3-5 from PRSS1, apparently resulted from a non-allelic homologous recombination (NAHR) event between the chromosome 7 homologs or sister chromatids during meiosis. Interestingly, this hybrid gene causes the disease through a combination of its inherent 'double gain-of-function' effect, acting simultaneously as a 'quantitative' copy number mutation and a 'qualitative' missense mutation (i.e. the known disease-causing p.N29I mutation). Our finding reveals a previously unknown mechanism causing human inherited disease, enriches the lexicon of human genetic variation and goes beyond the known interaction between copy number variations (CNVs) and single nucleotide substitutions in health and disease. Our finding should also stimulate more interest in analyzing both types of genetic variation whenever one tries to determine the contribution of a specific locus to a given disease phenotype.
Abstract: BACKGROUND & AIMS: We have recently reported that the triplication of a approximately 605 kilobase segment containing the PRSS1 (encoding cationic trypsinogen) and PRSS2 (encoding anionic trypsinogen) genes causes hereditary pancreatitis. Here we went further to investigate whether this copy number mutation could account for some unidentified French white patients with idiopathic chronic pancreatitis (ICP) or familial chronic pancreatitis (FCP) as well as Indian patients with tropical calcific pancreatitis (TCP). METHODS: Patients and controls were screened by means of previously described quantitative fluorescent multiplex polymerase chain reaction and/or genotyping of the microsatellite marker rs3222967. RESULTS: The approximately 605 kilobase triplication and a novel duplication (confirmed by fluorescence in situ hybridization) of the trypsinogen locus were detected in 10 and 2 of 202 ICP patients, respectively (age of disease onset, <or=20 years) but were absent in 282 French controls. In addition, the duplication mutation was found in 2 of 1044 ICP patients whose age of disease onset was >20 years. However, the 2 trypsinogen copy number mutations were observed in neither 103 FCP patients nor 268 Indian TCP patients. CONCLUSIONS: Our findings revealed the molecular basis of 6% of the young ICP patients and further demonstrated that chronic pancreatitis is a genomic disorder. Our findings also add to the mounting evidence showing that trypsinogen gene mutations do not appear to play an important role in the pathogenesis of TCP in the Indian population. Finally, a dividend of this study is that we have provided convincing evidence to show that all 5 previously described copy number variations involving PRSS1 or/and PRSS2 are artifacts.
Abstract: Extensive genetic studies of chronic pancreatitis over the past decade have highlighted the importance of a tightly regulated balance between activation and inactivation of trypsin within the pancreas to disease susceptibility and resistance. The recent identification of chymotrypsin C (CTRC) as enzyme Y, which was proposed to protect the pancreas by degrading prematurely activated trypsinogen within the pancreas 20 years ago, made CTRC an excellent candidate gene for disease-association studies. Here, we analyzed all eight exons of the CTRC gene for conventional genetic variants and copy number variations (CNVs) by direct sequencing and quantitative fluorescent multiplex PCR, respectively, in a total of 287 French white patients (idiopathic x 216; familial x 42; hereditary x 29). While no CNVs were found in any of the 287 subjects, 20 conventional variations including a nonsense mutation (p.W55X), a microdeletion mutation (p.K247_R254del) and nine missense mutations were found in the 216 patients with idiopathic chronic pancreatitis (ICP). Except for two common polymorphisms, all the remaining 18 mutational events represent rare variations, with a minor allele frequency of 0-0.3% in the control population. All these rare variants were always found more frequently in the ICP patients than in the controls, and their combined frequency in the ICP patients (26/216; 12.0%) is significantly different from that in the controls (4/350; 1.1%) (OR = 11.8 [3.9-40.6]), chi (2) = 31.58, P < 10(-6)). This genetic finding, when considered in the perceived role of CTRC in eliminating prematurely activated trypsin, indicated that CTRC is a new pancreatitis susceptibility gene.
Abstract: BACKGROUND: LINE-1 (long interspersed element-1) or L1-mediated retrotransposition is a potent force in human genome evolution and an occasional cause of human genetic disease. Since the first report of two de novo L1 insertions in the F8 gene causing hemophilia A, more than 50 L1-mediated retrotranspositional events have been identified as causing human genetic disease. However, a significant bias has generally militated against the detection of these pathological events at autosomal loci. Based upon this and other observations, we surmised that some previously unresolved cystic fibrosis chromosomes might carry hitherto undetected L1-mediated retrotranspositional insertions at the CFTR locus. This study represents an attempt to identify such mutational events. METHODS: 100 previously unresolved cystic fibrosis chromosomes were carefully reanalyzed using quantitative high-performance liquid chromatography (QHPLC). RESULTS: Two simple Alu insertions were identified in the CFTR gene, within exons 16 and 17b respectively. CONCLUSIONS: Our findings have not only revealed a previously unknown mutational mechanism responsible for cystic fibrosis but also represent an important addition to the already diverse spectrum of known CFTR gene mutations. Experience with the CFTR gene suggests that pathological L1-mediated retrotranspositional events may also have been overlooked at other gene loci and should always be considered in cases that appear to be refractory to analysis.
Abstract: In 1996, shortly after a locus for hereditary pancreatitis had been mapped to chromosome 7q35, an apparent gain-of-function missense mutation, p.R122H, in the cationic trypsinogen gene (PRSS1) was identified. Thereafter, the search for chronic pancreatitis-associated genetic factors has been largely focused on one form of genetic variation, namely, single nucleotide substitutions (SNSs). Only very recently has another type of genetic variation - copy number variations (CNVs) - been found to cause the disease. First, we identified duplication and triplication of an approximately 605 kb segment on chromosome 7q35 in French white patients with hereditary or idiopathic chronic pancreatitis. These alterations increased the copy number of PRSS1 as well as PRSS2, which encodes anionic trypsinogen. Second, we characterized a hybrid trypsinogen gene, in which exons 1 and 2 were derived from PRSS2 and exons 3 to 5 from PRSS1. Interestingly, this hybrid gene had two independent gain-of-function effects: increased trypsinogen gene copy number and it contained the p.N29I pancreatitis-causing missense mutation. Lastly, we identified two loss-of-function copy number mutations (deletions) in the SPINK1 gene, which encodes pancreatic secretory trypsin inhibitor (PSTI). Particularly, in one family with chronic pancreatitis, deletion of the complete SPINK1 gene was co-inherited with a CFTR missense mutation (p.L997F), revealing another layer of complexity between CNV and SNS interactions in the determination of a given disease phenotype. These findings represent a further demonstration of how studies of CNVs have altered the landscape of genetic research in the past few years and offer a fresh glimpse into the exciting realm of human CNVs.
Abstract: Hemochromatosis is predominantly associated with the HFE p.C282Y homozygous genotype, which is carried by approximately 1 person in 200 in Northern European populations. However, p.C282Y homozygosity is often characterized by incomplete penetrance. Here, we describe the case of a woman who had a major structural alteration in the HFE gene. Molecular characterization revealed an Alu-mediated recombination leading to the loss of the entire HFE gene sequence. Although homozygous for the HFE deleted allele, the woman had a phenotype similar to that seen in most women homozygous for the common p.C282Y mutation. Contrasting with previously reported results in Hfe knockout and Hfe knockin mice, our report gives further evidence that progression of the disease depends on modifying factors.
Abstract: Gene conversion, one of the two mechanisms of homologous recombination, involves the unidirectional transfer of genetic material from a 'donor' sequence to a highly homologous 'acceptor'. Considerable progress has been made in understanding the molecular mechanisms that underlie gene conversion, its formative role in human genome evolution and its implications for human inherited disease. Here we assess current thinking about how gene conversion occurs, explore the key part it has played in fashioning extant human genes, and carry out a meta-analysis of gene-conversion events that are known to have caused human genetic disease.
Abstract: The goal of this experiment was to identify the presence of genetic variants in the adenosine receptor genes and assess their relationship to infarct size in a population of patients with ischemic cardiomyopathy. Adenosine receptors play an important role in protecting the heart during ischemia and in mediating the effects of ischemic preconditioning. We sequenced DNA samples from 273 individuals with ischemic cardiomyopathy and from 203 normal controls to identify the presence of genetic variants in the adenosine receptor genes. Subsequently, we analyzed the relationship between the identified genetic variants and infarct size, left ventricular size, and left ventricular function. Three variants in the 3'-untranslated region of the A(1)-adenosine gene (nt 1689 C/A, nt 2206 Tdel, nt 2683del36) and an informative polymorphism in the coding region of the A3-adenosine gene (nt 1509 A/C I248L) were associated with changes in infarct size. These results suggest that genetic variants in the adenosine receptor genes may predict the heart's response to ischemia or injury and might also influence an individual's response to adenosine therapy.Clinical Pharmacology & Therapeutics (2007) 82, 435-440; doi:10.1038/sj.clpt.6100331; published online 29 August 2007.
Abstract: Genomic rearrangements are a well-recognized cause of genetic disease and can be formed by a variety of mechanisms. We report a complex rearrangement causing severe hemophilia A, identified and further characterized using a range of PCR-based methods, and confirmed using array-comparative genomic hybridization (array-CGH). This rearrangement consists of a 15.5-kb deletion/16-bp insertion located 0.6 kb from a 28.1-kb deletion/263-kb insertion at Xq28 and is one of the most complex rearrangements described at a DNA sequence level. We propose that the rearrangement was generated by distinct but linked cellular responses to double strand breakage, namely break-induced replication (BIR) and a novel model of break-induced serial replication slippage (SRS). The copy number of several genes is affected by this rearrangement, with deletion of part of the Factor VIII gene (F8, causing hemophilia A) and the FUNDC2 gene, and duplication of the TMEM185A, HSFX1, MAGEA9, and MAGEA11 genes. As the patient exhibits no clinically detectable phenotype other than hemophilia A, it appears that the biological effects of the other genes involved are not dosage-dependent. This investigation has provided novel insights into processes of DNA repair including BIR and the first description of SRS during repair in a pathological context. Hum Mutat 28(12),1198-1206, 2007. (c) 2007 Wiley-Liss, Inc.
Abstract: The 3' untranslated regions (3' UTRs) of human protein-coding genes play a pivotal role in the regulation of mRNA 3' end formation, stability/degradation, nuclear export, subcellular localisation and translation, and hence are particularly rich in cis-acting regulatory elements. One recent addition to the already large repertoire of known cis-acting regulatory elements are the microRNA (miRNA) target sites that are present in the 3' UTRs of many human genes. miRNAs post-transcriptionally down-regulate gene expression by binding to complementary sequences on their cognate target mRNAs, thereby inducing either mRNA degradation or translational repression. To date, only one disease-associated 3' UTR variant (in the SLITRK1 gene) has been reported to occur within a bona fide miRNA binding site. By means of sequence complementarity, we have performed the first systematic search for potential miRNA-target site mutations within a set of 79 known disease-associated 3' UTR variants. Since no variants were found that either disrupted or created binding sites for known human miRNAs, we surmise that miRNA-target site mutations are not likely to represent a frequent cause of human genetic disease.
Abstract: Variants of the SPINK1 gene encoding pancreatic secretory trypsin inhibitor have been described in association with chronic pancreatitis (CP). These alterations are believed to cause a loss of function by either impairing the trypsin inhibitory activity or reducing expression. Here we report two novel SPINK1 variants in exon 1 that affect the secretory signal peptide. The disease-associated c.41T>G (p.L14R) alteration was found in two European families with autosomal dominant hereditary pancreatitis, whereas the c.36G>C (p.L12F) variant was identified as a frequent alteration in subjects of African descent. The functional effects of both alterations and the previously reported c.41T>C (p.L14P) variant were characterized by activity assays and Western blots of wild-type and mutant SPINK1 expressed in human embryonic kidney 293T and Chinese hamster ovary cells. Alterations p.L14R and p.L14P destined the inhibitor for rapid intracellular degradation and thereby abolished SPINK1 secretion, whereas the p.L12F variant showed no detrimental effect. The results provide the first clear experimental demonstration that alterations that markedly reduce SPINK1 expression are associated with classic hereditary pancreatitis. Therefore, these variants should be classified as severe and regarded as disease-causing rather than disease-modifiers.
Abstract: LINE-1 or L1 has driven the generation of at least 10% of the human genome by mobilising Alu sequences. Although there is no doubt that Alu insertion is initiated by L1-dependent target site-primed reverse transcription, the mechanism by which the newly synthesised 3' end of a given Alu cDNA attaches to the target genomic DNA is less well understood. Intrigued by observations made on 28 pathological simple Alu insertions, we have sought to ascertain whether microhomologies could have played a role in the integration of shorter Alu sequences into the human genome. A meta-analysis of the 1624 Alu insertion polymorphisms deposited in the Database of Retrotransposon Insertion Polymorphisms in Humans (dbRIP), when considered together with a re-evaluation of the mechanism underlying how the three previously annotated large deletion-associated short pathological Alu inserts were generated, enabled us to present a unifying model for Alu insertion into the human genome. Since Alu elements are comparatively short, L1 RT is usually able to complete nascent Alu cDNA strand synthesis leading to the generation of full-length Alu inserts. However, the synthesis of the nascent Alu cDNA strand may be terminated prematurely if its 3' end anneals to the 3' terminal of the top strand's 5' overhang by means of microhomology-mediated mispairing, an event which would often lead to the formation of significantly truncated Alu inserts. Furthermore, the nascent Alu cDNA strand may be 'hijacked' to patch existing double strand breaks located in the top-strand's upstream regions, leading to the generation of large genomic deletions.
Abstract: The long interspersed element-1 (LINE-1 or L1) retrotransposition has altered the human genome in many ways. In particular, recent in vitro studies have demonstrated that the retrotranspositional insertion of L1 elements has resulted in significant genomic deletions. Here we provide evidence for its operation in the human genome by identifying a approximately 46-kb pathological genomic deletion in the PDHX gene directly linked to the insertion of a full-length L1 element, in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. Both the deduced bottom and top strand cleavage sites in the PDHX gene coincide with the consensus L1 endonuclease (EN) target sequence 5'-TTTT/A-3', while the full-length L1 element is followed by a 67-bp poly(A) tail. Interestingly, two hairpin structures, potentially formed by the inverted repeats present immediately 5' to the top strand nick site and 3' to the bottom strand nick site, may have facilitated the accessibility of L1 EN to the target sequences and also brought the two otherwise distantly located sequences into close proximity. Since the L1 element inserted in the PDHX gene is full-length, we favor the model of the template jumping as opposed to that of the microhomology-mediated end-joining for linking the 5' end of the nascent L1 copy to its genomic target. Our finding not only serves as an important complement to the in vitro approaches to studying L1 retrotransposition, but also reveals a novel mechanism causing human genetic disease.
Abstract: Variations in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor (PSTI)) are associated with chronic pancreatitis. We have recently determined the functional consequences of three missense mutations that occurred within the signal peptide sequence of PSTI by Western blotting analysis of wild-type and mutant PSTI expressed in Chinese hamster ovary cells. Here, this approach was extended to analyze seven missense mutations (p.N34S, p.G48E, p.D50E, p.Y54H, p.P55S, p.R65Q and p.R67C) occurring within the mature peptide of PSTI. This analysis enabled us to classify these missense mutations into three categories. The first category comprises the p.N34S and p.P55S polymorphisms, both of which occur in evolutionarily non-conserved residues, involve amino-acid substitutions with similar physicochemical properties, and do not cause any significant reduction in terms of PSTI mature peptide expression. The second category contains only the p.R65Q missense mutation, which occurs in a well-conserved residue, involves the substitution of a positively charged amino acid by a non-charged one, and causes a approximately 60% reduction of protein expression. The third category comprises p.G48E, p.D50E, p.Y54H, and p.R67C, all of which occur in strictly conserved residues, involve charged amino acids, and cause complete or nearly complete loss of PSTI expression. Having excluded the possibility that the reduced protein expression may have resulted from reduced transcription or unstable mRNA, we surmise that these missense mutations probably cause intracellular retention of their respective mutant proteins. This is suggestive of a potential unifying pathological mechanism underlying both the signal peptide and mature peptide mutations.European Journal of Human Genetics (2007) 15, 936-942; doi:10.1038/sj.ejhg.5201873; published online 13 June 2007.
Abstract: BACKGROUND: Unlike the standard RHD+ or RHD- alleles, serologic determination of weak or partial D alleles is often not clear-cut. Most importantly, rare weak D alleles, not typed by serology, are prone to alloimmunization when transfused with D+ blood. Although more than 100 RHD variants have currently been reported, many more rare alleles probably remain to be identified. STUDY DESIGN AND METHODS: To identify novel unusual RHD alleles, genomic DNA samples were collected from 333 blood donors or recipients in western France. All displayed ambiguity for D phenotype as determined by routinely used serologic reagents and analyzed by means of denaturing high-performance liquid chromatography (DHPLC) analysis in parallel with direct sequencing. RESULTS: For the first time it has been established that a reliable DHPLC-based approach potentiates the rapid screening of the entire RHD gene-coding sequence. In so doing, a total of 12 novel RHD alleles were identified. Except for the null allele that is in trans with a Weak D type 4 allele, the predicted effects of the other new alleles on gene expression correlated well with the discrepant routine D phenotype results. In particular, the carrier of the p.Leu214Phe missense mutation developed alloanti-D antibodies after transfusion of D+ blood. CONCLUSION: The identification of 12 novel RHD alleles represents a significant addition to the known repertoire of unusual RHD variants and, at the same time, serves to deepen our understanding of the molecular basis of weak and partial D. The accurate molecular typing of RHD alleles would allow to reduce the alloimmunization risk.
Abstract: Quantitative fluorescent multiplex PCR (QFM-PCR) was established in order to make possible the rapid and efficient mutational analysis of the pancreatic secretory trypsin inhibitor (SPINK1) gene. Using QFM-PCR, a novel heterozygous deletion encompassing the entire SPINK1 gene was identified in one of nine newly recruited French Caucasian families with chronic pancreatitis. The breakpoints were fully characterized and the approximately 30kb deletion was termed c.1-15969_c.240+7702del30588bp. Whilst sequences with the potential to form non-B DNA structures were found to span both the 5' and 3' deletion breakpoints, the generation of this gross deletion is potentially explicable in terms of non-homologous end-joining facilitated by the presence of a 1-bp microhomology at the two ends. The SPINK1 gene deletion identified in the index patient was also detected in her affected father and paternal uncle but not in 50 healthy French Caucasians. Remarkably, in all three affected individuals, the SPINK1 deletion was found to be co-inherited with a heterozygous p.L997F missense mutation in the unlinked CFTR gene, a lesion previously reported to be associated with a variety of cystic fibrosis-related diseases including idiopathic pancreatitis. Given that the SPINK1 deletion constitutes a clear-cut disease-causing factor, it may be that the CFTR missense mutation acts as a disease modifier in the context of this particular family.
Abstract: The 3' regulatory regions (3' RRs) of human genes play an important role in regulating mRNA 3' end formation, stability/degradation, nuclear export, subcellular localization and translation and are consequently rich in regulatory elements. Although 3' RRs contain only approximately 0.2% of known disease-associated mutations, this is likely to represent a rather conservative estimate of their actual prevalence. In an attempt to catalogue 3' RR-mediated disease and also to gain a greater understanding of the functional role of regulatory elements within 3' RRs, we have performed a systematic analysis of disease-associated 3' RR variants; 121 3' RR variants in 94 human genes were collated. These included 17 mutations in the upstream core polyadenylation signal sequence (UCPAS), 81 in the upstream sequence (USS) between the translational termination codon and the UCPAS, 6 in the left arm of the 'spacer' sequence (LAS) between the UCPAS and the pre-mRNA cleavage site (CS), 3 in the right arm of the 'spacer' sequence (RAS) or downstream core polyadenylation signal sequence (DCPAS) and 7 in the downstream sequence (DSS) of the 3'-flanking region, with 7 further mutations being treated as isolated examples. All the UCPAS mutations and the rather unusual cases of the DMPK, SCA8, FCMD and GLA mutations exert a significant effect on the mRNA phenotype and are usually associated with monogenic disease. By contrast, most of the remaining variants are polymorphisms that exert a comparatively minor influence on mRNA expression, but which may nevertheless predispose to or otherwise modify complex clinical phenotypes. Considerable efforts have been made to validate/elucidate the mechanisms through which the 3' untranslated region (3' UTR) variants affect gene expression. It is hoped that the integrative approach employed here in the study of naturally occurring variants of actual or potential pathological significance will serve to complement ongoing efforts to identify all functional regulatory elements in the human genome.
Abstract: Gross genomic rearrangements involving deletions in the CFTR gene have recently been found to account for approximately 20% of unidentified cystic fibrosis (CF) chromosomes in both French and Italian patients. Using QMPSF and walking quantitative DHPLC, six novel mutations (three simple deletions, two complex deletions with short insertions of 3-6 bp, and a complex deletion with a 182 bp inverted downstream sequence) were characterized by screening 274 unidentified CF chromosomes from 10 different countries. These lesions increase the total number of fully characterized large CFTR genomic rearrangements involving deletions to 21. Systematic analysis of the 42 associated breakpoints indicated that all 21 events were caused by nonhomologous recombination. Whole gene complexity analysis revealed a significant correlation between regions of low sequence complexity and the locations of the deletion breakpoints. Known recombination-promoting motifs were noted in the vicinity of the breakpoints. A total of 11 simple deletions were potentially explicable in terms of the classical model of replication slippage. However, the complex deletions appear to have arisen via multiple mechanisms; three of the five complex deletions with short insertions and both examples of large inverted insertions (299 and 182 bp, respectively) can be explained by either a model of serial replication slippage in cis (SRScis) or SRS in trans (SRStrans). Finally, the nature and distribution of large genomic rearrangements in the CFTR gene were compared and contrasted with those of two other genes, DMD and MSH2, with a view to gaining a broader understanding of DNA sequence context in mediating the diverse underlying mutational mechanisms.
Abstract: Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.
Abstract: Mutations and polymorphisms in the SPINK1 gene, which encodes trypsin's physiological inhibitor, pancreatic secretory trypsin inhibitor, have been found to be associated with chronic pancreatitis. However, to date, all currently reported SPINK1 variants are either single-nucleotide substitutions or microinsertions/deletions. It is possible that large genomic rearrangements at this locus may underlie certain cases of chronic pancreatitis. However, such events, if indeed they exist, may have been overlooked by conventional PCR-based techniques. Here we attempted to screen all four exons as well as the promoter region of the SPINK1 gene for large genomic deletions by means of quantitative high-performance liquid chromatography analysis. Of the 47 pancreatitis families (not carrying any known PRSS1, SPINK1 and CFTR variants/mutations after screening the coding regions by our previously established denaturing high-performance liquid chromatography methods), one family was suggested to carry a large genomic deletion in the SPINK1 gene. The aberrant chromosomal junction was encapsulated by long-range PCR and the breakpoints were determined by direct sequencing of the rearranged fragment. A 2-bp short direct repeat was present at the deletion breakpoints; this simple deletion (c.1-320_c.55+961del1336 bp) can thus in principle be explained by replication slippage. Identification of this lesion has not only expanded the SPINK1 mutational spectrum but also served to identify a novel mutational mechanism causing chronic pancreatitis.
Abstract: LINE-1 (L1) elements are the most abundant autonomous non-LTR retrotransposons in the human genome. Having recently performed a meta-analysis of L1 endonuclease-mediated retrotranspositional events causing human genetic disease, we have extended this study by focusing on two key issues, namely, mutation detection bias and the multiplicity of mechanisms of target gene disruption. Our analysis suggests that whereas an ascertainment bias may have generally militated against the detection of autosomal L1-mediated insertions, autosomal L1 direct insertions could have been disproportionately overlooked owing to their unusually large size. Our analysis has also indicated that the mechanisms underlying the functional disruption of target genes by L1-mediated retrotranspositional events are likely to be dependent on several different factors such as the type of insertion (L1 direct, L1 trans-driven Alu, or SVA), the precise locations of the inserted sequences within the target gene regions, the length of the inserted sequences, and possibly also their orientation.
Abstract: In an attempt both to catalogue 3' regulatory region (3' RR)-mediated disease and to improve our understanding of the structure and function of the 3' RR, we have performed a systematic analysis of disease-associated variants in the 3' RRs of human protein-coding genes. We have previously analysed the variants that have occurred in two specific domains/motifs of the 3' untranslated region (3' UTR) as well as in the 3' flanking region. Here we have focused upon 83 known variants within the upstream sequence (USS; between the translational termination codon and the upstream core polyadenylation signal sequence) of the 3' UTR. To place these variants in their proper context, we first performed a comprehensive survey of known cis-regulatory elements within the USS and the mechanisms by which they effect post-transcriptional gene regulation. Although this survey supports the view that RNA regulatory elements function within the context of specific secondary structures, there are no general rules governing how secondary structure might exert its influence. We have therefore addressed this question by systematically evaluating both functional and non-functional (based upon in vitro reporter gene and/or electrophoretic mobility shift assay data) USS variant-containing sequences against known cis-regulatory motifs within the context of predicted RNA secondary structures. This has allowed us not only to establish a reliable and objective means to perform secondary structure prediction but also to identify consistent patterns of secondary structural change that could potentiate the discrimination of functional USS variants from their non-functional counterparts. The resulting rules were then used to infer potential functionality in the case of some of the remaining functionally uncharacterized USS variants, from their predicted secondary structures. This not only led us to identify further patterns of secondary structural change but also several potential novel cis-regulatory motifs within the 3' UTRs studied.
Abstract: Hereditary pancreatitis has been reported to be caused by 'gain-of-function' missense mutations in the cationic trypsinogen gene (PRSS1). Here we report the triplication of a approximately 605-kb segment containing the PRSS1 gene on chromosome 7 in five families with hereditary pancreatitis. This triplication, which seems to result in a gain of trypsin through a gene dosage effect, represents a previously unknown molecular mechanism causing hereditary pancreatitis.
Abstract: Although gross insertions (>20 bp) comprise <1% of disease-causing mutations, they nevertheless represent an important category of pathological lesion. In an attempt to study these insertions in a systematic way, 158 gross insertions ranging in size between 21 bp and approximately 10 kb were identified using the Human Gene Mutation Database (www.hgmd.org). A careful meta-analytical study revealed extensive diversity in terms of the nature of the inserted DNA sequence and has provided new insights into the underlying mutational mechanisms. Some 70% of gross insertions were found to represent sequence duplications of different types (tandem, partial tandem, or complex). Although most of the tandem duplications were explicable by simple replication slippage, the three complex duplications appear to result from multiple slippage events. Some 11% of gross insertions were attributable to nonpolyglutamine repeat expansions (including octapeptide repeat expansions in the prion protein gene [PRNP] and polyalanine tract expansions) and evidence is presented to support the contention that these mutations are also caused by replication slippage rather than by unequal crossing over. Some 17% of gross insertions, all >or=276 bp in length, were found to be due to LINE-1 (L1) retrotransposition involving different types of element (L1 trans-driven Alu, L1 direct, and L1 trans-driven SVA). A second example of pathological mitochondrial-nuclear sequence transfer was identified in the USH1C gene but appears to arise via a novel mechanism, trans-replication slippage. Finally, evidence for another novel mechanism of human genetic disease, involving the possible capture of DNA oligonucleotides, is presented in the context of a 26-bp insertion into the ERCC6 gene.
Abstract: The now-classical model of replication slippage can in principle account for both simple deletions and tandem duplications associated with short direct repeats. Invariably, a single replication slippage event is invoked, irrespective of whether simple deletions or tandem duplications are involved. However, we recently identified three complex duplicational insertions that could also be accounted for by a model of serial replication slippage. We postulate that a sizeable proportion of hitherto inexplicable complex gene rearrangements may be explained by such a model. To test this idea, and to assess the generality of our initial findings, a number of complex gene rearrangements were selected from the Human Gene Mutation Database (HGMD). Some 95% (20/21) of these mutations were found to be explicable by twin or multiple rounds of replication slippage, the sole exception being a double deletion in the F9 gene that is associated with DNA sequences that appear capable of adopting non-B conformations. Of the 20 complex gene rearrangements, 19 (seven simple double deletions, one triple deletion, two double mutational events comprising a simple deletion and a simple insertion, six simple indels that may constitute a novel and non-canonical class of gene conversion, and three complex indels) were compatible with the model of serial replication slippage in cis; the remaining indel in the MECP2 gene, however, appears to have arisen via interchromosomal replication slippage in trans. Our postulate that serial replication slippage may account for a variety of complex gene rearrangements has therefore received broad support from the study of the above diverse series of mutations.
Abstract: Serial replication slippage in cis (SRScis) provides a plausible explanation for many complex genomic rearrangements that underlie human genetic disease. This concept, taken together with the intra- and intermolecular strand switch models that account for mutations that arise via quasipalindrome correction, suggest that intrachromosomal SRS in trans (SRStrans) mediated by short inverted repeats may also give rise to a diverse series of complex genomic rearrangements. If this were to be so, such rearrangements would invariably generate inversions. To test this idea, we collated all informative mutations involving inversions of >or=5 bp but <1 kb by screening the Human Gene Mutation Database (HGMD; www.hgmd.org) and conducting an extensive literature search. Of the 21 resulting mutations, only two (both of which coincidentally contain untemplated additions) were found to be incompatible with the SRStrans model. Eighteen (one simple inversion, six inversions involving sequence replacement by upstream or downstream sequence, five inversions involving the partial reinsertion of removed sequence, and six inversions that occurred in a more complicated context) of the remaining 19 mutations were found to be consistent with either two steps of intrachromosomal SRStrans or a combination of replication slippage in cis plus intrachromosomal SRStrans. The remaining lesion, a 31-kb segmental duplication associated with a small inversion in the SLC3A1 gene, is explicable in terms of a modified SRS model that integrates the concept of "break-induced replication." This study therefore lends broad support to our postulate that intrachromosomal SRStrans can account for a variety of complex gene rearrangements that involve inversions.
Abstract: Diverse long interspersed element-1 (LINE-1 or L1)-dependent mutational mechanisms have been extensively studied with respect to L1 and Alu elements engineered for retrotransposition in cultured cells and/or in genome-wide analyses. To what extent the in vitro studies can be held to accurately reflect in vivo events in the human genome, however, remains to be clarified. We have attempted to address this question by means of a systematic analysis of recent L1-mediated retrotranspositional events that have caused human genetic disease, with a view to providing a more complete picture of how L1-mediated retrotransposition impacts upon the architecture of the human genome. A total of 48 such mutations were identified, including those described as L1-mediated retrotransposons, as well as insertions reported to contain a poly(A) tail: 26 were L1 trans-driven Alu insertions, 15 were direct L1 insertions, four were L1 trans-driven SVA insertions, and three were associated with simple poly(A) insertions. The systematic study of these lesions, when combined with previous in vitro and genome-wide analyses, has strengthened several important conclusions regarding L1-mediated retrotransposition in humans: (a) approximately 25% of L1 insertions are associated with the 3' transduction of adjacent genomic sequences, (b) approximately 25% of the new L1 inserts are full-length, (c) poly(A) tail length correlates inversely with the age of the element, and (d) the length of target site duplication in vivo is rarely longer than 20 bp. Our analysis also suggests that some 10% of L1-mediated retrotranspositional events are associated with significant genomic deletions in humans. Finally, the identification of independent retrotranspositional events that have integrated at the same genomic locations provides new insight into the L1-mediated insertional process in humans.
Abstract: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR/ABCC7). Despite the extensive and enduring efforts of many CF researchers over the past 14 years, up to 30% of disease alleles still remain to be identified in some populations. It has long been suggested that gross genomic rearrangements could account for these unidentified alleles. To date, however, only a few large deletions have been found in the CFTR gene and only three have been fully characterized. Here, we report the first systematic screening of the 27 exons of the CFTR gene for large genomic rearrangements, by means of the quantitative multiplex PCR of short fluorescent fragments (QMPSF). A well-characterized cohort of 39 classical CF patients carrying at least one unidentified allele (after extensive and complete screening of the CFTR gene by both denaturing gradient gel electrophoresis and denaturing high-performance liquid chromatography) participated in this study. Using QMPSF, some 16% of the previously unidentified CF mutant alleles were identified and characterized, including five novel mutations (one large deletion and four indels). The breakpoints of these five mutations were precisely determined, enabling us to explore the underlying mechanisms of mutagenesis. Although non-homologous recombination may be invoked to explain all five complex lesions, each mutation appears to have arisen through a different mechanism. One of the indels was highly unusual in that it involved the insertion of a short 41 bp sequence with partial homology to a retrotranspositionally-competent LINE-1 element. The insertion of this ultra-short LINE-1 element (dubbed a "hyphen element") may constitute a novel type of mutation associated with human genetic disease.
Abstract: The human pancreas secretes two major trypsinogen isoforms, cationic and anionic trypsinogen. To date, 19 genetic variants have been identified in the cationic trypsinogen gene (PRSS1) of patients with hereditary, familial, or sporadic chronic pancreatitis. A common feature of cationic trypsinogen mutants studied so far is an increased propensity for autocatalytic activation (autoactivation). This is thought to lead to premature intrapancreatic digestive protease activation. In contrast, no pancreatitis-associated mutations have been found in the anionic trypsinogen gene (PRSS2), suggesting that this isoform might play a relatively unimportant role in pancreatitis. To challenge this notion, here we describe the unique properties of the E79K cationic trypsinogen mutation (c.235G>A), which was identified in three European families affected by sporadic or familial pancreatitis cases. In vitro analysis of recombinant wild-type and mutant enzymes revealed that catalytic activity of E79K trypsin was normal, and its inhibition by pancreatic secretory trypsin inhibitor was unaffected. Although the E79K mutation introduces a potential new tryptic cleavage site, autocatalytic degradation (autolysis) of E79K-trypsin was also unchanged. Furthermore, in contrast to previously characterized disease-causing mutations, E79K markedly inhibited autoactivation of cationic trypsinogen. Remarkably, however, E79K trypsin activated anionic trypsinogen two-fold better than wild-type cationic trypsin did, while the common pancreatitis-associated mutants R122H or N29I had no such effect. The observations not only suggest a novel mechanism of action for pancreatitis-associated trypsinogen mutations, but also highlight the importance of interactions between the two major trypsinogen isoforms in the development of genetically determined chronic pancreatitis.
Abstract: Mutations in the serine protease inhibitor Kazal type 1 gene (SPINK1) encoding pancreatic secretory trypsin inhibitor (PSTI) have recently been found to be associated with chronic pancreatitis. Nevertheless, knowledge of severe mutations is particularly scarce, both in terms of number and in the extent of clinical information. The aim of this study was to expand the known spectrum of such mutations. 46 unrelated families, each including at least two pancreatitis patients and carrying neither cationic trypsinogen (PRSS1) mutations nor the frequent SPINK1 N34S mutation, participated in this study. The four exons and their flanking sequences of the SPINK1 gene were screened by denaturing high performance liquid chromatography analysis (DHPLC); and mutations were identified by direct sequencing. A heterozygous microdeletion mutation (c.27delC), which occurs within a symmetric element, was identified in two families. In one family, c.27delC showed segregation with the disease across two generations, with a penetrance of up to 75%. But in the other family, however, the same mutation manifested as a low-penetrance susceptibility factor. In addition, a novel heterozygous splicing mutation, c.87+1G>A (G>A substitution at nucleotide +1 of intron 2) was found in one family with familial pancreatitis. Our results also helped to resolve the sharply differing views about PSTI's role in pancreatitis.
Abstract: Several genetic factors have been well known to predispose one to chronic pancreatitis (CP). However, little is known about the genetic factors that may provide a protective effect against the disease. Having found a nonsense mutation (c.111C>A; Y37X) and a splicing mutation (IVS2+1G>A) in the cationic trypsinogen gene (protease, serine, 1; PRSS1) in alcoholics without the development of CP, but not in alcoholics with CP and patients with hereditary or idiopathic CP, we propose that while "gain of function" mutations in the PRSS1 gene predispose one to pancreatitis, "loss of function" mutations in the gene may protect one against the disease.
Abstract: An allegedly novel human trypsinogen cDNA clone termed trypsinogen hL was recently reported in the Biological & Pharmaceutical Bulletin (2003; 26: 361-364). This "new" trypsinogen sequence was isolated by PCR using human lung cDNAs as template and was presumed to be a new member of the human trypsinogen family. However, trypsinogen hL does not match any sequence in the human genome; whereas it is 100% identical to mouse trypsinogen 7. Thus, trypsinogen hL is not of human origin, and its cloning from a human cDNA library was clearly a result of contamination with mouse genetic material. Publication of this cloning artifact could have been avoided by a simple BLAST search of GenBank or other sequence databanks.
Abstract: The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.
Abstract: In the last 5 years, mutations in three genes, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the pancreatic secretory trypsin inhibitor (PSTI) gene, have been found to be associated with chronic pancreatitis (CP). In this study, using established mutation screening methods, we systematically analysed the entire coding sequences and all exon/intron junctions of the three genes in 39 patients with idiopathic CP (ICP), with a view to evaluating the relative contribution of each gene to the aetiology of the disease. Our results demonstrate that, firstly, 'gain-of-function' mutations in the PRSS1 gene may occasionally be found in an obvious ICP subject. Secondly, presumably 'loss-of-function' mutations in the PSTI gene appear to be frequent, with a detection rate of at least 10% in ICP and, finally, abnormal CFTR alleles are common: at least 20% of patients carried one of the most common CFTR mutations, and about 10% of patients were compound heterozygotes, having at least one 'mild' allele. Thus, in total, about 30% of ICP patients carried at least one abnormal allele in one of the three genes, and this is the most conservative estimate. Moreover, a trans-heterozygous state with sequence variations in the PSTI/CFTR genes was found in three patients. However, an association between the 5T allele in intron 8 of the CFTR gene and ICP remains unproven.
Abstract: BACKGROUND: R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365 through 366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. MATERIALS AND METHODS: DNA samples containing either the known c.365G>A or c.365 through 366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC) assay. RESULTS: DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C). CONCLUSIONS: A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.
Abstract: Over the past 5 years, several gain-of-function missense mutations in the human cationic trypsinogen gene (PRSS1, OMIM 276000) have been associated with hereditary and/or sporadic pancreatitis. This study reports a new pancreatitis-associated mutation--R116C (CGT > TGT: c.346C > T)--in the gene.
Abstract: Several missense mutations, including R122H, N29I, K23R, A16V and D22G, in the cationic trypsinogen gene (PRSS1), have been associated with certain forms of hereditary pancreatitis (HP). Their occurrence in the idiopathic chronic pancreatitis (ICP) and whether novel mutations could be identified in PRSS1 remain to be further evaluated. These were addressed by the mutational screening of the entire coding sequence and the intronic/exonic boundaries of the PRSS1 gene in 221 ICP subjects, using a previously established denaturing gradient gel electrophoresis technique. Among the known PRSS1 mutations, only the R122H was detected in a single subject and the A16V in two subjects in the cohort, strengthening that HP-associated PRSS1 mutations are rare in ICP. Additional missense mutations, including P36R, E79K, G83E, K92N and V123M, were identified once separately. By analogy with the known PRSS1 mutations, predisposition to pancreatitis by some of them, particularly the V123M autolysis cleavage site mutation, is suspected. Functional analysis is expected to clarify their possible medical consequences.
Abstract: Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires.
Abstract: Since the identification in 1996 of a "gain of function" missense mutation, R122H, in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis, continued screening of this gene in both hereditary and sporadic pancreatitis has found more disease-associated missense mutations than expected. In addition, functional analysis has yielded interesting findings regarding their underlying mechanisms resulting in a gain of trypsin. A critical review of these data, in the context of the complicated biogenesis and complex autoactivation and autolysis of trypsin(ogen), highlights that PRSS1 mutations cause the disease by various mechanisms depending on which biochemical process they affect. The discovery of these mutations also modifies the classical perception of the disease and, more importantly, reveals fascinating new aspects of the molecular evolution and normal physiology of trypsinogen. First, activation peptide of trypsinogen is under strong selection pressure to minimize autoactivation in higher vertebrates. Second, the R122 primary autolysis site has further evolved in mammalian trypsinogens. Third, evolutionary divergence from threonine to asparagine at residue 29 in human cationic trypsinogen provides additional advantage. Accordingly, we tentatively assign, in human cationic trypsinogen, the strongly selected activation peptide as the first-line and the R122 autolysis site as the second-line of the built-in defensive mechanisms against premature trypsin activation within the pancreas, respectively, and the positively selected asparagine at residue 29 as an "amplifier" to the R122 "fail-safe" mechanism.
Abstract: Hereditary pancreatitis (HP) is an autosomal dominant disease. Two heterozygous missense mutations, R122H (R117H) and N29I (N21I), in the cationic trypsinogen gene have been clearly associated with HP. The 'self-destruct' model proposed for the R122H mutation is discussed in connection with the existing theory of pancreatitis, and the basic biochemistry and physiology of trypsinogen, with particular reference to R122 as the primary autolysis site of the cationic trypsinogen. Two different genetic mechanisms are identified which cause the R122H mutation, and gene conversion is the likely cause of the N29I mutation. A unifying model, which highlights an indirect impairment on the R122 autolysis site is hypothesised for the N29I mutation. Possible predisposition to pancreatitis by additional DNA variants in the gene, such as the A16V signal peptide cleavage site mutation and the K23R activation peptide cleavage site mutation is suspected, but not proven. Evidence of genetic heterogeneity of HP is reviewed and cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations detected in HP families are re-evaluated. Finally, large scale association studies are expected to clarify the additional variants' role in pancreatitis and to identify new HP genes.
Abstract: The N21I missense mutation in the cationic trypsinogen gene is the second most frequent mutation in hereditary pancreatitis (HP). In this article, we suggest that the N21I mutation most likely arose as a gene conversion event in which the functional anionic trypsinogen gene acted as the donor sequence. This hypothesis is supported by the unique presence of Ile at residue 21 of the anionic gene amongst the several highly homologous trypsinogen genes; a single unbroken tract of nucleotides of up to 113 bp flanking the I21 residue in the anionic trypsinogen gene; and the presence of a chi-like sequence in the 5' proximity and a palindromic sequence in the 3' vicinity of the N21I mutation. Furthermore, a multiple alignment of the partial amino acid sequence of vertebrate trypsins around residue 21 indicated that N21 and I21 may represent advantageously selected mutations of the two functional human trypsinogen genes in evolutionary history. These observations, which are complementary to the previous findings, provide further insights into the genetic mechanism and pathogenic role of the N21I mutation in HP.
Abstract: Historically, trypsinogens/trypsins have been one of the most extensively studied enzyme models of protein structure and function. They have received renewed attention after the identification of mutations in the cationic trypsinogen gene as being associated with hereditary pancreatitis. A survey of the literature revealed five cloned cDNAs, but only three protein products of human trypsinogens, and their nomenclature has been confusing. The availability of the complete genomic sequencing of the human trypsinogen gene family made it possible to provide a systematic review of the genes, cDNAs, and protein products of human trypsinogens and to clarify some controversial issues. Further, the confusing coexistence of two systems for naming the cationic trypsinogen mutations is addressed.
Abstract: Over the past decade, gene conversion has been shown increasingly to be a cause of human disease. Through this process, a functional gene is converted into a mutant by a homologous, nonfunctional one. In this article, we demonstrate that gene conversion is a likely cause of the mutations of the human cationic trypsinogen (PRSS1) gene that are associated with hereditary or sporadic pancreatitis, including the R122H (CGC>CAT: c.365-366 GC>AT), N29I (AAC>ATC: c.86A>T), and A16V (GCC>GTC: c.47C>T) missense mutations. This hypothesis is strongly supported by four lines of observation. First, human group I trypsinogen genes are tandemly repeated and share a high sequence homology between them. Secondly, a possible donor sequence for each variant is present in the PRSS1 gene's paralog(s). Thirdly, there exist uninterrupted sequence tracts ranging from 30 to 114 bp in the putatively converted regions. Finally, Chi-like and palindromic sequences are found in the vicinity of these missense mutations. This theory, if correct, will make the pancreatitis-associated PRSS1 mutations a unique example, as it shows that a functional gene may be converted by several paralogs, and that such an event may even occur between two functional genes (i.e., the N29I mutation), resulting in disease. This adds further to the diversity of genetic mechanisms underlying human disease. In addition, this genetic finding provides, for the first time, concrete evidence of the contribution made by gene conversion to the molecular evolution of the human trypsinogen family.
Abstract: The R domain of the cystic fibrosis transmembrane conductance regulator (CFTR) was originally defined as 241 amino acids, encoded by exon 13. Such exon/intron boundaries provide a convenient way to define the R domain, but do not necessarily reflect the corresponding functional domain within CFTR. A two-domain model was later proposed based on a comparison of the R-domain sequences from 10 species. While RD1, the N-terminal third of the R domain is highly conserved, RD2, the large central region of the R domain has less rigid structural requirements. Although this two-domain model was given strong support by recent functional analysis data, the simple observation that two of the four main phosphorylation sites are excluded from RD2 clearly indicates that RD2 still does not satisfy the requirements of a "functional R domain." Nevertheless, knowledge of the CFTR structure and function accumulated over the past decade and reevaluated in the context of a comprehensive sequence comparison of 15 CFTR homologues made it possible to define such a "functional R domain," i.e., amino acids C647 to D836. This definition is validated primarily because it contains all of the important potential consensus phosphorylation sequences. In addition, it includes the highly charged motif from E822 to D836. Finally, it includes all of the deletions/insertions in this region. This definition also aids in understanding the effects of missense mutations occurring within this domain.
Abstract: O6-methylguanine-DNA-methyltransferase (MGMT) plays a very important role in the cellular resistance to nitrosoureas drugs. Inhibition of MGMT might be a useful approach in tumor chemotherapy. In this study, the depletion of MGMT activity by retroviral-mediated antisense RNA transfection were reported. Three retroviral vectors expressing MGMT antisense RNA were constructed and transfected into HeLa S3 cells. The difference of MGMT mRNA, MGMT activity as well as cellular resistance to ACNU before and after transfection were observed. It was found that antisense RNA targeting 5' region and whole length of MGMT mRNA could partially deplete MGMT activity and enhance killing effects of ACNU. However, 3' region antisense RNA had no effect on MGMT modulation.
Abstract: Previous studies have demonstrated that O6-methylguanine-DNA methyltransferase (MGMT) is a major contributor to tumor cellular resistance toward chloroethylnitrosoureas. To further clarify the effect of MGMT gene expression on cellular chemosensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)-3-(2-chloroethyl)-3-nitrosourea (ACNU) in human cells, a repair-deficient human tumor cell line (HeLa MR) was transfected with a human MGMT expression vector (pSV2MGMT-neo). Multiple unique transfectants were isolated which exhibited variable levels of MGMT mRNA by Northern hybridization analysis. Vector-transfected controls were generated simultaneously. Transfectants expressing high levels of MGMT activity showed an increased resistance to ACNU-induced cytotoxicity. Furthermore, the levels of the protective effect against ACNU correlated generally with the levels of introduced MGMT expression. This study further provided direct evidence of MGMT contribution to ACNU resistance in human tumor cells. Based on the results presented here, we also discussed the perspective of the clinical utility of MGMT cDNA transfer and expression.
Abstract: This trial is based on the strategy of reversing O6-methylguanine-DNA methyltransferase (MGMT)-mediated nimustine resistance by depleting MGMT activity with streptozotocin (STZ) pretreatment. Eight patients with recurrent malignant gliomas refractory to previous nimustine chemotherapy were entered in this study. Patients received STZ (2g/m2) followed one hour with nimustine (2-3 mg/Kg) via the ipsilateral carotid artery. After 1-2 cycles of therapy, 3 patients responded, 4 stabilized, and 1 failed. Toxic effects were generally tolerated. The preliminary results indicated that nimustine-resistant tumor cells in vivo could also be sensitized by modulation of MGMT activity.
Abstract: O6-methylguanine-DNA methyltransferase (MGMT) gene expression in 6 Mer+ (HeLa S3, SMMC-7721, SGC-7901, B-239, AGZY83-a, and Cc801) and 2 Mer- (SHG-44, and HeLa MR) human tumor cell lines was examined. Southern blot analysis revealed no deletion, amplification, or rearrangement of the MGMT gene in these cell lines. However, approximately 1.0 kb transcripts were detected in the 6 Mer+ cell lines but not in the 2 Mer- cell lines by Northern blot analysis. Furthermore, a rough correlation between MGMT activity and mRNA level in these cell lines was observed. These results suggest that transcriptional regulation of the MGMT gene is the molecular basis of the absence of MGMT activity in Mer- cell lines.
Abstract: The overcoming effect of O6-benzylguanine on O6-methylguanine-DNA methyltransferase (MGMT)-mediated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro was evaluated. Depletion of MGMT activity in Mer+ HeLa S3 cells by O6-benzylguanine was dose-dependent and a complete loss of MGMT activity was achieved at a concentration of 0.5 microM. The cytotoxic potential of BCNU on MGMT proficient HeLa S3 (1.10 pmol/mg of protein), SMMC-7721 (0.72 pmol/mg of protein) and Cc801 (0.39 pmol/mg of protein) was greatly enhanced when cells were exposed to 10 microM O6-benzylguanine for 1 h, but there was a lack of potentiation of BCNU sensitivity in Mer- HeLa MR cells due to its nearly undetectable level of MGMT. There existed a correlation between the extent of enhancement and the amount of MGMT activity. The intensity of enhancement expressed as dose modifying factor = IC50 (BCNU alone)/IC50 (10 microM O6-benzylguanine + BCNU) was 4.56, 3.89, 3.67 and 0.97 in HeLa S3, SMMC-7721, Cc801 and HeLa MR cells respectively. The results further demonstrated that O6-benzylguanine may have potential utility as an adjuvant in combination chemotherapy with chloroethylnitrosourea agents.
Abstract: O6-Methylguanine DNA methyltransferase (O6-MT) activity and cellular sensitivity to nitrosourea drugs of 10 kinds of tumor cell strains derived from Chinese patients were measured by 3H radioactivity and colony-forming ability, respectively. The results in vitro showed that nimustine (Nim) 25 micrograms.ml-1 and carmustine (Car) 20 micrograms.ml-1 exhibited specific killing effects on Mer-phenotype tumor cells characterized by low O6-MT activity. In vivo both Nim and Car (25 mg.kg-1.wk-1 x 4 wk, ip) had specific curative ability to Mer- tumor cells implanted in nude mice. These findings suggested that assay of O6- MT activity in tumor biopsy could be used as a predictable guide to human tumor chemotherapy with nitrosourea compounds.
Abstract: Our previous studies have indicated that O6-methyl-guanine-DNA methyltransferase (MGMT) is a key factor determining tumor cellular resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). This study describes the modulation of MGMT-mediated ACNU resistance by O6-benzylguanine pretreatment. The ACNU sensitivity of MGMT proficient human tumor HeLa S3, SMMC-7721, and Cc801 cells in tissue culture was markly enhanced by 10 mm O6-benzylguanine, and a correlation between the extent of enhancement and the level of MGMT activities was observed. A single i.p. injection of 100 mg/kg of O6-benzylguanine caused a complete inhibition of MGMT activities in HeLa S3 tumor xenografts and combination of O6-benzylguanine with ACNU (7.5 mg/kg) significantly inhibited HeLa S3 tumor growth. The results demonstrated that O6-benzylguanine could be used as a potential adjuvant in combination chemotherapy with ACNU to treat MGMT proficient tumors.
Abstract: The distribution of O6-methylguanine-DNA methyltransferase (MGMT) activity in extracts of tumors from 74 patients was measured. The results demonstrated that there was considerable variation of MGMT activity in different human tumor tissues as well as in different individuals. The mean values (X +/- SD, pmol/mg of protein) in breast cancer, stomach cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, esophageal carcinoma, brain tumors, colon carcinoma and malignant melanoma were 1.071 +/- 0.374 (9), 0.515 +/- 0.107 (5), 0.509 +/- 0.251 (5), 0.461 +/- 0.227 (24), 0.329 +/- 0.246 (5), 0.273 +/- 0.376 (5), 0.244 +/- 0.175 (14), 0.242 +/- 0.308 (5) and 0.201 +/- 0.161 (2) respectively. It was notable that six samples (1/24 non-small cell lung cancer, 3/5 esophageal carcinoma, 1/14 brain tumors and 1/5 colon carcinoma) did not have any detectable level of MGMT activity. Activity of glutamine pyruvic transaminase (GPT) was also measured in the same extracts used for the assay of MGMT activity. The activity of GPT in these samples with undetectable level of MGMT activity was similar to those with significant MGMT activity. These results further strengthen the assumption that a certain fraction of human tumors are Mer-.
Abstract: The relationship between O6-methylguanine-DNA methyltransferase (O6-MT) activity and the sensitivity of 4 kinds of human tumor cell lines to bis-chloroethylnitrosourea (BCNU) was evaluated. The results demonstrated that cellular resistance to BCNU was linearly correlated with O6-MT activity, suggesting that O6-MT plays an important role in repairing DNA damage induced by BCNU. Furthermore, the depletion of O6-MT activity by streptozotocin (STZ) pretreatment and its effect on the cell's sensitivity to BCNU were investigated. O6-MT activity could be efficiently reduced, and sensitivity to BCNU was subsequently increased significantly. There was also a linear correlation between depletion of O6-MT activity and enhancement of BCNU's cytotoxic effects. These results indicate that O6-MT might constitute the molecular basis of cellular resistance to BCNU, and a combination of STZ and BCNU may result in better therapeutic effects.
Abstract: Osteoporosis is characterised by comprised bone strength predisposing to an increased risk of fracture. It is a complex trait that is influenced by many genetic variants and their interactions with environmental factors. In the past four to five years, we have witnessed an accelerated pace in identifying and validating osteoporosis susceptibility loci, which was largely attributable to the use of genome-wide association studies (GWAS). At least 15 genes (i.e. VDR, ESR1, ESR2, LRP5, LRP4, SOST, GRP177, OPG, RANK, RANKL, COLIA1, SPP1, ITGA1, SP7 and SOX6) may be reasonably assigned as confirmed osteoporosis genes; and new osteoporosis-associated genes are being increasingly reported. Notably, confirmed and promising osteoporosis genes are clustered in three biological pathways, the estrogen endocrine pathway, the Wnt/β-catenin signalling pathway and the RANKL/RANK/OPG pathway. The ultimate promise of osteoporosis genetics is not only to better understand the disease process, but more importantly, to lead better therapeutic and preventive interventions.
Abstract: Genomic rearrangements involve gross alterations of chromosomes or large chromosomal regions and can take the form of deletions, duplications, insertions, inversions or translocations. The characterisation of a considerable number of rearrangement breakpoints has now been accomplished at the nucleotide sequence level, thereby providing an invaluable resource for the detailed study of the mutational mechanisms which underlie genomic recombination events. At least five categories of mutational mechanism are known to give rise to genomic rearrangements: (i) homologous recombination including nonallelic homologous recombination (NAHR), gene conversion, single strand annealing (SSA) and break-induced replication (BIR); (ii) nonhomologous end joining (NHEJ); (iii) microhomology-mediated replication-dependent recombination (MMRDR); (iv) long interspersed element 1 (LINE-1 or L1)-mediated retrotransposition and (v) telomere healing. We compare and contrast the hallmark characteristics of the first three mutational mechanisms and discuss the recent developments with respect to the ratio of deletions to duplications in vivo.
Abstract: Historically, trypsinogen has been among the most extensively studied models of protein structure and function. It has received renewed attention since the identification of a gain-of-function missense mutation in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis in 1996; a finding gave strong support to the then century-old hypothesis that pancreatitis is an autodigestive disease in which prematurely activated trypsin within the pancreas was thought to play a pivotal role. Whereas gain-of-function PRSS1 missense mutations cause chronic pancreatitis through a negative effect on trypsin lysis and a positive effect on trypsinogen autoactivation, duplication and triplication copy number mutations of the trypsinogen locus cause the disease through a gene-dosage effect. By contrast, loss-of-function variations in the PRSS1 and PRSS2 (encoding anionic trypsinogen) genes protect against chronic pancreatitis. The study of pathogenic PRSS1 mutations also shed lights on the evolution of trypsinogen genes.
Abstract: Information obtained by comparing cystic fibrosis transmembrane conductance regulator sequences has been used to refine the domain structure of the protein, to better understand the effects of the many mutations identified in typical and atypical cystic fibrosis patients, and to develop better animal models for cystic fibrosis studies.
