A simple and rapid quantitative method of detection of the common achondroplasia mutation: analysis in mismatch repair deficient cells

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 97% of cases, it is caused by a recurrent point mutation, a G to A substitution at nucleotide position 1138 (G1138A) of the fibroblast growth factor receptor 3 gene. Although this is an autosomal dominant condition, more than 90% of all mutations occur sporadically making this one of the most mutagenic sites in the human genome. The reasons for the high spontaneous G1138A mutation rate are not known. This investigation was performed by developing a simple and rapid semi-quantitative allele specific PCR based assay capable of reliably detecting more than 25 mutant G1138A copies in a pool of 300 000 wild type molecules. Using this assay, the G1138A mutation frequency was measured in cell lines deficient in mismatch repair (LoVo, SW48) and comparing it with controls. No differences were found in the frequency of this point mutation between the mismatch repair deficient and wild type cell lines.     

A Simple and Rapid Quantitative Method of Detection of the Common Achondroplasia Mutation: Analysis in Mismatch Repair Deficient Cells

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 97% of cases, it is caused by a recurrent point mutation, a G to A substitution at nucleotide position 1138 (G1138A) of the fibroblast growth factor receptor 3 gene. Although this is an autosomal dominant condition, more than 90% of all mutations occur sporadically making this one of the most mutagenic sites in the human genome. The reasons for the high spontaneous G1138A mutation rate are not known. This investigation was performed by developing a simple and rapid semi-quantitative allele specific PCR based assay capable of reliably detecting more than 25 mutant G1138A copies in a pool of 300 000 wild type molecules. Using this assay, the G1138A mutation frequency was measured in cell lines deficient in mismatch repair (LoVo, SW48) and comparing it with controls. No differences were found in the frequency of this point mutation between the mismatch repair deficient and wild type cell lines.__________From Genetika, Vol. 41, No. 8, 2005, pp. 1137–1141.Original English Text Copyright © 2005 by Grewal.This article was submitted by the author in English.     

Achondroplasia is defined by recurrent G380R mutations of FGFR3.

Genomic DNA from 154 unrelated individuals with achondroplasia was evaluated for mutations in the fibroblast growth factor receptor 3 (FGFR3) transmembrane domain. All but one, an atypical case, were found to have a glycine-to-arginine substitution at codon 380. Of these, 150 had a G-to-A transition at nt 1138, and 3 had a G-to-C transversion at this same position. On the basis of estimates of the prevalence of achondroplasia, the mutation rate at the FGFR3 1138 guanosine nucleotide is two to three orders of magnitude higher than that previously reported for tranversions and transitions in CpG dinucleotides. To date, this represents the most mutable single nucleotide reported in the human genome. The homogeneity of mutations in achondroplasia is unprecedented for an autosomal dominant disorder and may explain the relative lack of heterogeneity in the achondroplasia phenotype.     

Two novel arrangements of the human fetal globin genes: G gamma-G gamma and A gamma-A gamma.

We describe two novel arrangements of the human fetal globin gene region: one chromosome with two linked A gamma genes (A gamma-A gamma) and two chromosomes with two linked G gamma genes (G gamma-G gamma). The gamma genes of these three chromosomes were cloned and the unusual 5' A gamma gene and one of the unusual 3' G gamma genes were partially sequenced. Both of these unusual genes differ from the genes normally found at their respective locations by a nucleotide substitution at the site of the single coding region difference between normal G gamma and A gamma genes. In both cases, the substitution is identical to the nucleotide found at that position in the normal neighboring gene. The unusual 3' G gamma gene also differs from normal A gamma genes at two other nucleotide positions, but both differences appear to be "private" or exclusive to this particular gene. These unusual fetal globin gene arrangements could have arisen from point mutations or from gene conversions of limited extent, the boundaries of which have been determined for all three chromosomes.     

Rapid detection of common southeast Asian beta-thalassemia mutations by nonisotopic multiplex PCR-SSCP analysis

This report describes the detection of seven beta-thalassemia mutations common in Southeast Asia by amplifying three short PCR fragments in two separate tubes, followed by single-strand conformation polymorphism (SSCP) analysis in single lanes. These mutations are -28 A --> G, codon 17 A --> T, IVS1 + 5 G --> C, codon 41/42 -CTTT, codon 43 G --> T, codon 71/72 + A, and IVS2 + 654 C --> T, and account for 70% to over 95% of the cases in this region. This rapid nonisotopic method was also found capable of detecting other mutations within the amplified fragments. It is simple, rapid, and cheap, and thus suitable for carrier screening and prenatal diagnosis in Southeast Asia.     

Detecting gradients of asymmetry in site-specific substitutions in mitochondrial genomes

During mitochondrial replication, spontaneous mutations occur and accumulate asymmetrically during the time spent single stranded by the heavy strand (DssH). The predominant mutations appear to be deaminations from adenine to hypoxanthine (A --> H, which leads to an A --> G substitution) and cytosine to thymine (C --> T). Previous findings indicated that C --> T substitutions accumulate rapidly and then saturate at high DssH, suggesting protection or repair, whereas A --> G accumulates linearly with DssH. We describe here the implementation of a simple hidden Markov model (HMM) of among-site rate correlations to provide an almost continuous profile of the asymmetry in substitution response for any particular substitution type. We implement this model using a phylogeny-based Bayesian Markov chain Monte Carlo (MCMC) approach. We compare and contrast the relative asymmetries in all 12 possible substitution types, and find that the observed transition substitution responses determined using our new method agree quite well with previous predictions of a saturating curve for C --> T transition substitutions and a linear accumulation of A --> G transitions. The patterns seen in transversion substitutions show much lower among-site variation, and are nonlinear and more complex than those seen in transitions. We also find that, after accounting for the principal linear effect, some of the residual variation in A --> G/G --> A response ratios is explained by the average predicted nucleic acid secondary structure propensity at a site, possibly due to protection from mutation when secondary structure forms.     

Type II hereditary angio-oedema associated with two mutations in one allele of the C1-inhibitor gene around the reactive-site coding region.

The polymerase chain reaction and nucleotide sequence analysis have been used to characterise two point mutations in the eighth exon of one allele of the C1-inhibitor gene in a kindred with type II hereditary angio-oedema (HAE). The mutations comprise a G to A substitution at C1-inhibitor gene nucleotide 16789 and an upstream C to T substitution at nucleotide position 16765. This represents the first report of these two mutations in the same C1-inhibitor allele in type II HAE. The molecular genetic pathogenesis of HAE is discussed in the light of these findings.     

Spontaneous spectinomycin resistance mutations of the chloroplast rrn16 gene in Daucus carota callus lines

Bioballistic transformation of carrot Daucus carota L. callus cultures with a plasmid containing the aadA (aminoglycoside 3'-adenyltransferase) gene and subsequent selection oftransformants on a selective medium containing spectinomycin (100-500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3' end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.     

Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides

Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, we introduce the correction of point mutations in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, we generated three cell lines with one hprt point mutation each. These cell lines were treated with specific single-stranded 45 base phosphothioate modified oligonucleotides and selected by HAT medium. The surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C --> T; position 151, C --> T; and position 400, G --> A). The correction efficiency was very low but reproducible. We suggest that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.     

Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains

Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools. Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors. We show here that in E. coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system. Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient. A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.     

Molecular analysis of hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiencies: novel mutations and the spectrum of Japanese mutations

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.     

Molecular genetics of the glucose-6-phosphate dehydrogenase (G6PD) Mediterranean variant and description of a new G6PD mutant, G6PD Andalus1361A

Glucose-6-phosphate dehydrogenase (G6PD; E.C.1.1.1.49) deficiency is the most common human enzymopathy; more than 300 different biochemical variants of the enzyme have been described. In many parts of the world the Mediterranean type of G6PD deficiency is prevalent. However, G6PD Mediterranean has come to be regarded as a generic term applied to similar G6PD mutations thought, however, to represent a somewhat heterogeneous group. A C----T mutation at nucleotide 563 of G6PD Mediterranean has been identified by Vulliamy et al., and the same mutation has been found by De Vita et al. in G6PD Mediterranean, G6PD Sassari, and G6PD Cagliari. The latter subjects had an additional mutation, at nucleotide 1311, that did not produce a coding change. We have examined genomic DNA of five patients--four of Spanish origin and one of Jewish origin--having enzymatically documented G6PD Mediterranean. All had both the mutation at nucleotide 563 and that at nucleotide 1311. A sixth sample, resembling G6PD Mediterranean kinetically but with a slightly rapid electrophoretic mobility, was designated G6PD Andalus and was found to have a different mutation, a G----A transition at nucleotide 1361, producing an arginine-to-histidine substitution. These studies suggest that G6PD Mediterranean is, after all, relatively homogeneous.     

Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta

Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.     

The molecular anatomy of spontaneous germline mutations in human testes

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10³ to >10⁴ times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10⁻⁶) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change.     

PCR-induced sequence alterations hamper the typing of prehistoric bone samples for diagnostic achondroplasia mutations

Achondroplasia (ACH) is a skeletal disorder (MIM100800) with an autosomal dominant Mendelian inheritance and complete penetrance. Here we report the screening of ancient bone samples for diagnostic ACH mutations. The diagnostic G-->A transition in the FGFR3 gene at cDNA position 1138 was detected in cloned polymerase chain reaction (PCR) products obtained from the dry mummy of the Semerchet tomb, Egypt (first dynasty, approximately 4,890-5,050 BP [before present]), and from an individual from Kirchheim, Germany (Merovingian period, approximately 1,300-1,500 BP), both of which had short stature. However, these mutations were also reproducibly observed in four ancient control samples from phenotypically healthy individuals (false-positives), rendering the reliable molecular typing of ancient bones for ACH impossible. The treatment of a false-positive DNA extract with uracil N-glycosylase (UNG) to minimize type 2 transitions (G-->A/C-->T) did not reduce the frequency of the false-positive diagnostic ACH mutations. Recently, it was suggested that ancient DNA extracts may induce mutations under PCR. Contemporary human template DNA from a phenotypically healthy individual was therefore spiked with an ancient DNA extract from a cave bear. Again, sequences with the diagnostic G-->A transition in the FGFR3 gene were observed, and it is likely that the false-positive G-->A transitions result from errors introduced during the PCR reaction. Amplifications in the presence of MnCl(2) indicate that position 1138 of the FGFR3 gene is particularly sensitive for mutations. Our data are in line with previously published results on the occurrence of nonrandom mutations in PCR products of contemporary human mitochondrial HVRI template DNA spiked with ancient DNA extracts.     

Single nucleotide polymorphisms in the open reading frame of the stearoyl-CoA desaturase gene and resulting genetic variants in Canadian Holstein and Jersey cows.

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) from their saturated counterparts in the mammary gland and adipose tissue of ruminant animals. We hypothesize that single nucleotide polymorphisms (SNPs) in the SCD gene account for some of the differences in SCD activity, and consequently for some of the variations in CLA and MUFA content of milk fat between Holsteins and Jersey cows and within these two breeds. We analyzed the open reading frame of the SCD gene of 44 Holsteins and 48 Jerseys for SNPs by sequencing. Three SNPs: 702A --> G, 762T --> C and 878C --> T were identified in both breeds and a further SNP, 435G --> A, was unique to Holsteins. The SNPs characterized four different genetic variants in Holsteins: A (G(435)A(702)T(762)C(878)), A1 (A(435)A(702)T(762)C(878)), B (G(435)G(702)C(762)T(878)) and B1 (A(435)G(702)C(762)T(878)), with only variants A and B in Jerseys. SNP 878C --> T resulted in a non-synonymous codon change while the rest resulted in synonymous codon changes giving rise to two protein variants, A having alanine and B having valine. Allele A was the most prevalent in the two breeds. These differences may, therefore, contribute to existing variations in CLA and fat content between and within Canadian Holstein and Jersey cows.     

A:T --> G:C base pair substitutions occur at a higher rate than other substitution events in Pms2 deficient mouse cells

The mismatch repair pathway involves multiple proteins that are required to correct DNA polymerase generated mismatches before they become mutations. It has been shown recently, that the predominant base-pair substitution events leading to loss of endogenous Aprt activity in Pms2 null mouse cells are A:T --> G:C mutations (Oncogene 21 (2002) 1768, Oncogene 21 (2002) 2840). To determine if this observation could be explained by an increased rate of A:T --> G:C mutations relative to other base-pair substitutions, we developed a reversion assay to examine G:C --> A:T, C:G --> A:T, and A:T --> G:C mutations within mouse Aprt in a Pms2 null mouse kidney cell line. The results demonstrated a 6-50-fold increase in the rate of the A:T --> G:C mutations relative to the other base-pair substitutions. Additional work demonstrated that growth of the Pms2 null cells in antioxidant containing medium reduced the rate of the A:T --> G:C mutations. The results are discussed with regards to the role of mismatch repair proteins in preventing base-pair substitutions, including those induced by oxidative stress.     

Distinct pathogenic mechanisms of various RARS1 mutations in Pelizaeus-Merzbacher-like disease

Mutations of the genes encoding aminoacyl-t RNA synthetases are highly associated with various central nervous system disorders. Recurrent mutations, including c.5 AG, p.D2 G; c.1367 CT, p.S456 L; c.1535 GA, p.R512 Q and c.1846_1847 del, p.Y616 Lfs*6 of RARS1 gene, which encodes two forms of human cytoplasmic arginyl-t RNA synthetase(h Arg RS), are linked to Pelizaeus-Merzbacher-like disease(PMLD) with unclear pathogenesis. Among these mutations, c.5 AG is the most extensively reported mutation, leading to a p.D2 G mutation in the N-terminal extension of the long-form h Arg RS. Here, we showed the detrimental effects of R512 Q substitution and ΔC mutations on the structure and function of h Arg RS, while the most frequent mutation c.5 AG, p.D2 G acted in a different manner without impairing h Arg RS activity. The nucleotide substitution c.5 AG reduced translation of h Arg RS m RNA, and an upstream open reading frame contributed to the suppressed translation of the downstream main ORF. Taken together, our results elucidated distinct pathogenic mechanisms of various RARS1 mutations in PMLD.