Restriction sites containing CpG show a higher frequency of polymorphism in human DNA

doi:10.1016/0092-8674(84)90081-3

Cell

Volume 36, Issue 1, January 1984, Pages 131-138

https://doi.org/10.1016/0092-8674(84)90081-3 Get rights and content

Abstract

Unique loci in the human genome were examined with restriction enzymes in order to detect restriction fragment length polymorphisms (RFLPs). Of 31 arbitrary loci, nine were detectably polymorphic, reflecting ten polymorphic restriction sites. Nine of the ten polymorphic sites were revealed with two restriction enzymes, Msp I and Taq I, whose recognition sequences have in common the dimer sequence CpG. The cytosines in the CpG sequence are known to be frequently methylated in mammals, and the occurrence of significant variation in Msp I and Taq I sites supports the view that methylated cytosine residues are hotspots for mutation in mammalian DNA.

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Identification of mutations in Colombian patients affected with Fabry disease
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Citation Excerpt :
To date, more than 600 mutations have been described including missense, nonsense and splice-site mutations, as well as gene rearrangements (Ashley et al., 2001; Schirinzi et al., 2008; Shabbeer et al., 2006). Most of the described mutations are private (with some few exceptions found in several unrelated subjects) and are usually associated with modifications in CpG dinucleotides, known hotspots for the disease (Barker et al., 1984; Cooper and Youssoufian, 1988). In this study, we describe the first clinical and genetic analysis of nine Colombian FD patients.
Fabry Disease (FD) is an X-linked inborn error of glycosphingolipid catabolism, caused by a deficiency of the lisosomal α-galactosidase A (AGAL). The disorder leads to a vascular disease secondary to the involvement of kidney, heart and the central nervous system. The mutation analysis is a valuable tool for diagnosis and genetic counseling. Although more than 600 mutations have been identified, most mutations are private. Our objective was to describe the analysis of nine Colombian patients with Fabry disease by automated sequencing of the seven exons of the GLA gene. Two novel mutations were identified in two patients affected with the classical subtype of FD, in addition to other 6 mutations previously reported. The present study confirms the heterogeneity of mutations in Fabry disease and the importance of molecular analysis for genetic counseling, female heterozygotes detection as well as therapeutic decisions.
Suicidal function of DNA methylation in age-related genome disintegration
2009, Ageing Research Reviews
Citation Excerpt :
The mutagenic activity of cytosine methylation was studied in detail, and it is commonly accepted that CG methylation sites are hotspots for most C > T and G > A (G:C > A:T) transition mutations in human genes (Cooper and Krawczak, 1989). CG-containing restriction sites are often polymorphic and have a high frequency of 5mC > T mutations in both Esherichia coli and human DNA (Barker et al., 1984; Coulondre et al., 1985). Most single-base substitutions (BS) that are responsible for genetic diseases and cancer are localized at CG sites in human genes (Cooper and Youssoufian, 1988; Mazin, 1994a; Walsh and Xu, 2006).
This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC > T transitions that induce 40–70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.
Clinical outcomes in Menkes disease patients with a copper-responsive ATP7A mutation, G727R
2008, Molecular Genetics and Metabolism
Menkes disease is a fatal neurodegenerative disorder of infancy caused by defects in an X-linked copper transport gene, ATP7A. Evidence from a recent clinical trial indicates that favorable response to early treatment of this disorder with copper injections involves mutations that retain some copper transport capacity. In three unrelated infants, we identified the same mutation, G727R, in the second transmembrane segment of ATP7A that complemented a Saccharomyces cerevisiae copper transport mutant, consistent with partial copper transport activity. Quantitative reverse transcription-polymerase chain reaction studies showed approximately normal levels of ATP7A_G727R transcript in two patients’ fibroblasts compared to wild-type controls, but Western blot analyses showed markedly reduced quantities of ATP7A, suggesting post-translational degradation. We confirmed the latter by comparing degradation rates of mutant and wild-type ATP7A via cyclohexamide treatment of cultured fibroblasts; half-life of the G727R mutant was 2.9 h and for the wild-type, 11.4 h. We also documented a X-box binding protein 1 splice variant in G727R cells—known to be associated with the cellular misfolded protein response. Patient A, diagnosed 6 months of age, began treatment at 228 days (7.6 months) of age. At his current age (2.5 years), his overall neurodevelopment remains at a 2- to 4-month level. In contrast, patient B and patient C were diagnosed in the neonatal period, began treatment within 25 days of age, and show near normal neurodevelopment at their current ages, 3 years (patient B), and 7 months (patient C). The poor clinical outcome in patient A with the same missense mutation as patient A and patient B with near normal oucomes, confirms the importance of early medical intervention in Menkes disease and highlights the critical potential benefit of newborn screening for this disorder.
Na<inf>v</inf> 1.5/R1193Q polymorphism is associated with both long QT and Brugada syndromes
2006, Canadian Journal of Cardiology
Brugada syndrome (BS) and long QT syndrome (LQTS) are electrical disorders with a genetic background. They are revealed on surface electrocardiograms as either right bundle branch block and ST segment elevation in the right pericardial leads (V1, V2 and V3) in BS or as a long QTc interval on all 12 leads of the electrocardiogram in LQTS. Both BS and LQTS can lead to syncope and even sudden death. The R1193Q SCN5A variant was recently associated with LQTS, BS and cardiac conductance disease.
The aim of the present study was to screen the SCN5A gene from two patients – one with BS and the other showing signs of a BS/LQTS phenotype – for mutations, and to characterize the effect of the mutations on channel function using the patch clamp technique.
A heterozygous mutation (R1193Q) was identified on the SCN5A gene in both patients. The R1193Q polymorphism was absent in 100 unrelated control alleles, suggesting that it has a low frequency in the French Canadian population. Mutant R1193Q expressed in tsA201 cells, which was studied using the patch clamp technique, had a persistent sodium current that could account for the QTc prolongation. A shift of steady-state inactivation toward more hyperpolarized voltages was observed that could explain the BS phenotype.
The R1193Q polymorphism is definitively associated with cardiac electrical abnormalities.
Le syndrome de Brugada (SB) et le syndrome du QT long (SQTL) sont des troubles électriques d’origine génétique. Ils se manifestent à l’électrocardiogramme de surface par un bloc de branche droit et un sus-décalage du segment ST dans les dérivations péricardiques droites (V1, V2 et V3) dans le cas du SB et par un allongement de l’intervalle QTc dans les 12 dérivations de l’électrocardiogramme dans le cas du SQTL. Les deux syndromes peuvent provoquer une syncope et parfois même entraîner la mort subite. La variante R1193Q du gène SCN5A a été associée dernièrement au SQTL, au SB et à des troubles de la conduction électrique dans le cœur.
La présente étude avait pour buts d’examiner le gène SCN5A chez deux patients, l’un atteint du SB et l’autre montrant des signes d’un phénotype du SB ou du SQTL, à la recherche de mutations et de caractériser l’effet de ces mutations sur le fonctionnement des canaux ioniques à l’aide de la technique du « patch clamp ».
Une mutation hétérozygote (R1193Q) a été découverte sur le gène SCN5A chez les deux patients. Toutefois, l’absence du polymorphisme R1193Q dans 100 allèles témoins, non liés porte à croire que la fréquence de la mutation est peu élevée dans la population canadienne française. La mutation R1193Q qui s’exprimait dans les cellules tsA201, étudiée à l’aide de la technique du « patch clamp », produisait un courant sodique continuel, susceptible d’être mis en cause dans l’allongement de l’intervalle QTc. Par ailleurs, un déplacement de l’inactivation à l’état d’équilibre vers une plus grande hyperpolarisation des différences de potentiel pourrait expliquer le phénotype du SB.
Le polymorphisme R1193Q est, sans contredit, associé à des anomalies de la conduction électrique dans le cœur.
Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2
2006, Blood
Citation Excerpt :
Among 6 families with the 149 + 5G>A mutation, 4 different haplotypes were identified, indicating at least 4 independent origins for this mutation (Table 3). These data are consistent with distinct geographic origins for many of these families and suggest a recurring mutation, perhaps due to a mutation hotspot at a CpG dinucleotide.17 The haplotypes of the 2 families (B9 and 9II-5, Zhang et al6) with the I136T missense mutation in MCFD2 diverge at both the BZ1 and D2S2227 markers (Table 3), suggesting that this mutation also arose independently.
Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.
Analbuminemia in a Swiss family is caused by a C → T transition at nucleotide 4446 of the albumin gene
2005, Clinical Biochemistry
To define the molecular defect that causes analbuminemia in an apparently healthy boy, son of non-consanguineous Swiss parents.
Total DNA, extracted from peripheral blood samples from the proband and from both parents, was PCR-amplified using oligonucleotide primers designed to amplify the 14 exons of the human albumin gene and the flanking intron regions. The products were screened for mutations by single-strand conformation polymorphism (SSCP) and heteroduplex analyses (HA) either directly or after digestion with restriction enzymes. The combination of these methods identified the abnormal fragment, which was then sequenced.
DNA sequence analysis identified in the homozygous proband a C → T transition at nucleotide 4446. The mutation changes the codon CGA for Arg 114 to a stop codon TGA, resulting in premature termination and is therefore responsible for the analbuminemic trait. The same mutation has been previously reported to cause analbuminemia in an American female. The putative protein product would have a length of 113 residues. The parents were found to be heterozygous for the mutation.
Gel-based mutation detection and DNA sequencing confirmed the diagnosis of congenital analbuminemia in the proband. Our results show that the combination of SSCP and HA represents a powerful tool to study the molecular defects causing analbuminemia in humans.

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^∗: Present address: Collaborative Research, Lexington, MA.

^†: Present address: Institut fur Genetik, Universitat Dusseldorf.

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Cell

ArticleRestriction sites containing CpG show a higher frequency of polymorphism in human DNA

Abstract

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Method for detection of specific RNAs in agarose gels by transfer to diazobenxymethyl-paper and hybridization with DNA probes

Analysis of the regions flanking the human insulin gene and sequence of an Alu family member

Nucl. Acids. Res.

Polymorphic DNA region adjacent to the 5′ end of the human insulin gene

Article
Restriction sites containing CpG show a higher frequency of polymorphism in human DNA