rSNP_Guide: An integrated database‐tools system for studying SNPs and site‐directed mutations in transcription factor binding sitesPonomarenko, Julia V.; Orlova, Galina V.; Merkulova, Tatyana I.; Gorshkova, Elena V.; Fokin, Oleg N.; Vasiliev, Gennady V.; Frolov, Anatoly S.; Ponomarenko, Mikhail P.
doi: 10.1002/humu.10116pmid: 12325018
Since the human genome was sequenced in draft, single nucleotide polymorphism (SNP) analysis has become one of the keynote fields of bioinformatics. We have developed an integrated database‐tools system, rSNP_Guide (http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/), devoted to prediction of transcription factor (TF) binding sites, alterations of which could be associated with disease phenotype. By inputting data on alterations in DNA sequence and in DNA binding pattern of an unknown TF, rSNP_Guide searches for a known TF with alterations in the recognition score calculated on the basis of TF site's sequence and consistent with the input alterations in DNA binding to the unknown TF. Our system has been tested on many relationships between known TF sites and diseases, as well as on site‐directed mutagenesis data. Experimental verification of rSNP_Guide system was made on functionally important SNPs in human TDO2and mouse K‐ras genes. Additional examples of analysis are reported involving variants in the human γA‐globin (HBG1), hsp70(HSPA1A), and Factor IX (F9) gene promoters. Hum Mutat 20:239–248, 2002. © 2002 Wiley‐Liss, Inc.
Identification of a family with nonspecific mental retardation (MRX79) with the A140V mutation in the MECP2 gene: Is there a need for routine screening?Winnepenninckx, Birgitta; Errijgers, Vanessa; Hayez‐Delatte, France; Reyniers, Edwin; Frank Kooy, R.
doi: 10.1002/humu.10130pmid: 12325019
Mutations in the methyl‐CpG‐binding protein 2 (MECP2) cause Rett syndrome, a severe neurodevelopmental disorder occurring predominantly in females. Male patients with Rett syndrome are extremely rare, as the Rett‐causing mutations in the MECP2 gene are usually lethal in hemizygous males. However, different mutations in the same gene were reported to cause mental retardation, both in sporadic non‐syndromic males as well as in syndromic families with disease manifestation in carrier females. The majority of the reported MECP2 mutations in mentally retarded patients cause amino acid substitutions and, especially in isolated cases, discrimination between a disease‐causing mutation and a rare polymorphism is not obvious and the significance of each individual variation should be verified. We mapped a new non‐syndromic X‐linked family (MRX79) to the chromosomal region Xq27.3‐Xq28 and identified an A140V mutation in the MEPC2 gene in all patients with the disease haplotype. In addition to data published by others, this suggests that A140V is a recurrent mutation (and not a polymorphism) found in patients with X‐linked mental retardation. Hum Mutat 20:249–252, 2002. © 2002 Wiley‐Liss, Inc.
Novel mutations and SNPs identified in CCR2 using a new comprehensive denaturing gradient gel electrophoresis assayPetersen, Desiree C.; Laten, Annette; Zeier, Michele D.; Grimwood, Ashraf; Rensburg, Estrelita Janse van; Hayes, Vanessa M.
doi: 10.1002/humu.10111pmid: 12325020
A single nucleotide polymorphism (SNP) at codon 64 in the CC chemokine receptor 2 gene (CCR2 V64I) has been associated with a dominant effect of delaying disease progression from human immunodeficiency virus‐1 (HIV‐1) infection to acquired immunodeficiency syndrome (AIDS). The objective of our study was to design a comprehensive mutation detection assay for the entire coding region of the CCR2A and CCR2B gene transcripts, including all relevant splice site junctions and to identify novel mutations and SNPs within our predominantly African‐based population, which could influence an individual's susceptibility to HIV‐1 infection and/or progression to AIDS. The mutation detection assay, based on denaturing gradient gel electrophoresis (DGGE), allowed for the complete analysis of five individuals per denaturing gel. Our study cohort consisted of 102 HIV seropositive patients and 144 HIV seronegative controls from the diverse South African population. Application of the CCR2‐DGGE assay resulted in the detection of two previously reported CCR2 polymorphisms, namely CCR2 V64I and CCR2 N260N, and 11 novel mutations, including seven SNPs occurring at high allelic frequencies within specific population groups of South Africa. The large number of novel mutations/SNPs identified, using the CCR2‐DGGE assay, indicates the importance for comprehensive analysis of all candidate genes in host susceptibility to HIV‐1 infection, specifically in the under‐studied African‐based populations. Hum Mutat 20:253–259, 2002. © 2002 Wiley‐Liss, Inc.
Abnormal mRNA splicing resulting from consensus sequence splicing mutations of ATP7BLoudianos, G.; Lovicu, M.; Dessi, V.; Tzetis, M.; Kanavakis, E.; Zancan, L.; Zelante, L.; Galvèz‐Galvèz, C.; Cao, A.
doi: 10.1002/humu.10121pmid: 12325021
More than 200 Wilson disease (WD) disease‐causing mutations have been defined to date. Missense mutations are largely prevalent while splice‐site mutations are limited in number. Most reside in the splice donor or acceptor sites and only a minority are detected in splicing consensus sequences. Furthermore, only a few splicing mutations have been studied at the RNA level to date. In this study, using the RT‐PCR method we performed the molecular characterization of four consensus splice‐site mutations identified by DNA analysis in patients with WD. One of them, previously described 1707+3insT, occurred at position 3 in the donor splice site of intron 4, while the other three, 2122‐8T>G, 2866‐6T>G, and 3061‐12T>A, are novel and occurred in the acceptor splice sites of introns 7, 12, and 13, respectively. Analysis revealed a prevalently abnormal splicing in the samples carrying the mutations compared to the normal controls. Comparison of RNA splicing with normal controls in liver and lymphocytes further suggests that abnormal splicing of the WD gene is also present and differentially regulated in normal tissues. The results produced in this study strongly suggest that DNA mutations residing in the consensus sequence of WD gene splice sites result in the WD phenotype by interfering with the production of the normal WD protein. Further studies are necessary to better quantify the amount of different transcripts produced by these mutations, and establish their correlation with the disease phenotype. Hum Mutat 20:260–266, 2002. © 2002 Wiley‐Liss, Inc.
High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype–phenotype correlationCitron, Michael; Godmilow, Lynn; Ganguly, Tapan; Ganguly, Arupa
doi: 10.1002/humu.10119pmid: 12325022
Hemophilia A (HEMA) is an X‐linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular genetic testing for the factor VIII gene is challenging due to its large size. Here we present results of high throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon‐intron boundaries of the factor VIII gene. The results of mutation analysis on 89 hemophiliac males showed presence of a disease‐causing mutation in 80 individuals (90%, 95% CI of 82%–95%). Seven out of nine mutation‐negative individuals were severe cases of hemophilia A with < 1% factor VIII protein in the blood. The correlation of phenotype with genotype as observed in this study was not absolute. This finding is supported by similar observations in the international database for hemophilia A mutations (HAMSTeRS). This issue raises the importance of genotypes at other loci that can act as modifiers for the phenotype. Thirty‐four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations. The mutations cover the full spectrum including rearrangements, deletions, frameshift, and point mutations. The novel missense mutations require careful evaluation. Prediction of a mutation as the disease‐causing allele was made from the nature of the substitution and the degree of conservation of the mutated amino acid among species that have diverged in evolution. In some cases segregation analysis of the mutation with disease condition was performed when other family members were available. Hum Mutat 20:267–274, 2002. © 2002 Wiley‐Liss, Inc.
Characterization of a novel CYP2A7/CYP2A6 hybrid allele (CYP2A6*12) that causes reduced CYP2A6 activityOscarson, Mikael; McLellan, Roman A.; Asp, Vendela; Ledesma, MariCarmen; Ruiz, Maria Luisa Bernal; Sinues, Blanca; Rautio, Arja; Ingelman‐Sundberg, Magnus
doi: 10.1002/humu.10126pmid: 12325023
The human CYP2A6 enzyme metabolizes certain drugs and pre‐carcinogens and appears to be the most important enzyme for nicotine metabolism. At present, more than 10 different allelic variants are known that cause abolished or decreased enzyme activity. Genetic polymorphism in this gene might be of particular importance for an individual's need for nicotine and for susceptibility to lung and/or liver cancer. We have identified a new CYP2A6 allele (CYP2A6*12) which carries an unequal crossover between the CYP2A6 and CYP2A7 genes in intron 2. This results in a hybrid allele where the 5′ regulatory region and exons 1–2 are of CYP2A7 origin and exons 3–9 are of CYP2A6 origin, resulting in 10 amino acid substitutions compared to the CYP2A6*1 allele. Phenotyping with the CYP2A6 substrate coumarin indicates that it causes reduced CYP2A6 activity in'vivo. Furthermore, when expressed in mammalian COS‐1 cells, the enzyme variant catalyzed 7‐hydroxylation of coumarin at a rate approximately 60% of that of the wild‐type enzyme. The CYP2A6*12 allele was present at an allele frequency of 2.2% among Spaniards, but was absent in Chinese. Hum Mutat 20:275–283, 2002.© 2002 Wiley‐Liss, Inc.
PTPN11 Mutations in Noonan syndrome type I: detection of recurrent mutations in exons 3 and 13Maheshwari, M.; Belmont, J.; Fernbach, S.; Ho, T.; Molinari, L.; Yakub, I.; Yu, F.; Combes, A.; Towbin, J.; Craigen, W. J.; Gibbs, R.
doi: 10.1002/humu.10129pmid: 12325025
We surveyed 16 subjects with the clinical diagnosis of Noonan Syndrome (NS1) from 12 families and their relevant family members for mutations in PTPN11/SHP2 using direct DNA sequencing. We found three different mutations among five families. Two unrelated subjects shared the same de novo missense substitution in exon 13 (S502T); an additional two unrelated families had a mutation in exon 3 (Y63C); and one subject had the amino acid substitution Y62D, also in exon 3. None of the three mutations were present in ethnically matched controls. In the mature protein model, the exon 3 mutants and the exon 13 mutant amino acids cluster at the interface between the N′ SH2 domain and the phosphatase catalytic domain. Six of eight subjects with PTPN11/SHP2 mutations had pulmonary valve stenosis while no mutations were identified in those subjects (N = 4) with hypertrophic cardiomyopathy. An additional four subjects with possible Noonan syndrome were evaluated, but no mutations in PTPN11/SHP2 were identified. These results confirm that mutations in PTPN11/SHP2 underlie a common form of Noonan syndrome, and that the disease exhibits both allelic and locus heterogeneity. The observation of recurrent mutations supports the hypothesis that a special class of gain‐of‐function mutations in SHP2 give rise to Noonan syndrome. Hum Mutat 20:298–304, 2002. © 2002 Wiley‐Liss, Inc.
An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLCMatin, Maryam M.; Baumer, Alessandra; Hornby, David P.
doi: 10.1002/humu.10118pmid: 12325026
We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion‐pair reversed‐phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader‐Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite‐deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods. Hum Mutat 20:305–311, 2002. © 2002 Wiley‐Liss, Inc.