Common Point Mutations Research Articles

THE COURSE OF HEPATIC STEATOSIS IN MCAD DEFICIENCY

Introduction: The occurrence of hepatic steatosis in MCAD deficiency has been well documented previously. Yet, the relationship of hepatic fat accumulation and regression to the metabolic disturbance is poorly understood. In this study, the course of hepatic steatosis and its relationship to plasma carnitine levels was studied in one individual. Background: A 7-month-old white male in previously good health presented with acute onset of lethargy and poor appetite. During the next day, he developed progressive vomiting and listlessness. His physical exam included hepatomegaly. Initial laboratory studies: hypoglycemia; metabolic acidosis; hypoketonuria; acylcarnitine profile - elevated levels of octanoyl and decenoyl carnitine with diminished acetylcarnitine. DNA testing for common single point mutation (A985G) in MCAD gene - homozygote. The child was resuscitated with intravenous fluids and his mental status returned to baseline status. He was discharged on carnitine supplementation (30 mg/kg/dose TID) and avoidance of prolonged fasting. Methods: On Day #1, Day #23, and Day #58 after presentation, the subject's hepatic fat fraction was assessed by MRI using a modification of the Dixon method. Scan parameters included TR = 9.3ms (in-phase) and 7.3ms (out-of-phase), TE = 4.2ms (in-phase) and 2.1ms (out-of-phase), α = 30°, slice thickness = 10mm, field of view = 32×16cm, matrix size = 256×128, and NEX = 8. Acquisition times were 5s and 4s for in-phase and out-of-phase scans respectively. Carnitine profiles were performed on Day #1 and Day #58. Results:Table Conclusion: Fat accumulation within the liver during the energy crisis in MCAD deficiency does not appear to be related to a carnitine deficient state. Carnitine supplementation did not have an immediate effect on the resolution of hepatic steatosis.

Clinical heterogeneity in respiratory chain complex III deficiency in childhood

Six children are presented with an isolated complex III deficiency in muscle tissue. More specifically, oxidation rates and ATP+CrP production rates from both pyruvate and succinate as substrates and/or the activity of decylubiquinol:cytochrome c oxidoreductase were all markedly reduced. Complex III deficiency was also present in liver of two patients tested, but could not be demonstrated in cultured fibroblasts of four patients tested. Mitochondrial DNA, extracted from muscle, was analyzed; no deletions or common point mutations were found. Four patients presented with a multi-organ disorder. Among these patients three presented at neonatal age with neurological signs and lactate elevation in blood and CSF, of whom two had severe neonatal Fanconi syndrome. One child, aged seven years, had encephalomyopathy, ophthalmoplegia, retinopathy and Wolff-Parkinson-White syndrome. The remaining two patients exhibited myopathy only, within the first year of life. Thus, like in other respiratory chain disorders, patients with complex III deficiency may present at any age and show variable symptoms and outcome, ranging from neonatal death to failure to thrive only. Apparently there are no clinical findings which are specific for complex III deficiency.

An Intracellular Trafficking Defect in Type I Cystinuria rBAT Mutants M467T and M467K

The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.

Temperature-programmed capillary electrophoresis for the analysis of high-melting point mutants in thalassemias.

The behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo- and heteroduplex, polymerase chain reaction (PCR)-amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the Tm's of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected Tm values. The deltaT applied is rather minute (1-1.5 degrees C) and the temperature gradient quite shallow (e.g., 0.05 degrees C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being high melters (in the temperature range of 60-62 degrees C) in presence of 6 M urea. Point mutants are fully resolved into a spectrum of four bands only when poly(N-acryloylaminopropanol) and hydroxyethylcellulose are used. However, the former offers the best separation capability at such high temperatures.

The Rate of CpG Mutation in AluRepetitive Elements within the p53 Tumor Suppressor Gene in the Primate Germline

Cytosine to thymine transition mutations at the CpG dinucleotide are the most common point mutations in cancer and genetic disease. We calculated the in vivorate of CpG mutation in the primate germline by deriving a primordial consensus sequence for an Alurepetitive element which inserted into intron 6 of the primate p53 gene 35 to 55 million years ago. Comparison of this primordial sequence to the Alusequence in intron 6 of present-day primates was used to determine the nature and rate of mutations which occurred during evolution. We estimate the half-life of a CpG nucleotide to be 24 to 60 million years, and the rate constant for mutation at this dinucleotide to be 1.2 × 10 −8to 2.9 × 10 −8years −1. These results were confirmed by the analysis of a second Alusequence in intron 10 of the p53 gene. The in vivomutation rate is at least 1250-fold slower than the in vitrochemical rate of 5-methylcytosine deamination in double-stranded DNA, showing that current estimates of CpG mutation repair have been significantly underestimated. Furthermore, the mutability of the CpG dinucleotide has led to the depletion of this dinucleotide from the vertebrate genome, and calculations in this study suggest that current levels of the CpG dinucleotide in the primate genome are very close to a steady state equilibrium in which the rate of CpG mutation is equal to the rate of CpG formation by random mutation.

Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from patients in New York City

From a collection of 367 isolates of Mycobacterium tuberculosis from patients in New York City in 1994, 45 isolates (12.3%) were resistant in vitro to 2 micrograms or more of streptomycin (SM) per ml. We further evaluated these isolates for levels of SM resistance and for mutations previously associated with resistance in the rpsL (S12 ribosomal protein) gene and the rrs (16S rRNA)-coding region. Twenty-four isolates, representing nine distinct patterns of susceptibility to antituberculosis drugs, were resistant to 500 micrograms of SM per ml and shared a common point mutation at nucleotide 128 in the rpsL gene. This mutation, which substitutes lysine for arginine in the S12 ribosomal binding protein, was not present in isolates with low-level SM resistance or in SM-susceptible control isolates. Among 20 isolates with low-level SM resistance, one possessed a substitution (C-->G865) in the 912 loop of the rrs gene. No mutations in the 530 loop of the rrs coding region were detected, suggesting the presence of an alternative SM resistance mechanism in 19 isolates. Single-strand conformation polymorphisms of mutants were readily detected by a nonradioactive gel screen.

Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system.

We describe a rapid and specific differential amplification system which can detect five of the most common cystic fibrosis mutations from a single cell. In the first round of the polymerase chain reaction (PCR), regions of exons 4, 10 and 11 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene containing the mutations delta F508, G551D, R553X, G542X and 621+1G > T were co-amplified in a single multiplex PCR. To identify potential contamination, we included external amplification primers for the polymorphic human tyrosine hydroxylase (HUMTH01) locus as a fingerprint for the sample. In the second round of PCR, detection of any of the five mutations was achieved using the amplification refractory mutation system (ARMS) in two separate reactions, each containing nested amplification primers for either wild type or mutant sequence. A separate second round PCR for the fingerprinting was performed with nested HUMTH01 primers. Using this procedure we have successfully and accurately detected five cystic fibrosis mutations in 30 single cells with a failed amplification rate of 7% and a contamination rate of 4.6% and that PCR failure or possible contamination will also be easily detected. This procedure allows detection of the five most common point mutations and small deletions responsible for cystic fibrosis from a single cell in < 8 h which could be applicable to preimplantation diagnosis in human embryos.

Screening for the Factor-V Arg 506 GIn Mutation in Patients with TlA and Stroke

A common point mutation in the factor-V gene at the exact site (Arg 506) where activated protein C (APC) normally cleaves and inactivates the Va pro-coagulant accounts for APC resistance and is the most important genetic risk factor for venous thrombosis. It has also been suggested as a possible risk factor for stroke. We determined the prevalence of the Arg 506 Gln mutation in 180 patients (mean age 65.3 years) with cerebrovascular disease (138 stroke and 42 TIA) and compared it with that in 70 age-matched controls (mean age 64.9 years). The mutation was no more common in patients than controls [15/180 (8.3%) vs. 5/70 (7.1 %), p = 0,6]. Within the patient group there was no relationship between the presence of the mutation and age of stroke or the occurrence of TIA or stroke. There was no association between the mutation and any particular stroke subtype (large-vessel, lacunar, cardioembolic or unknown). The factor-V Arg 506 Gln mutation is not an important risk factor for stroke and TIA in an unselected group of patients, and routine screening is not justified.

Did de novo MELAS common mitochondrial DNA point mutation (mtDNA 3243, A → G transition) occur in the mother of a proband of a Japanese MELAS pedigree?

MELAS is a major maternally inherited mitochondrial (mt) encephalomyopathy of which 80% of cases are associated with mtDNA point mutation (mtDNA 3243, A → G transition) which exists under heteroplasmic conditions with wild-type mtDNA. The origin of this mutation remains obscure in the reported pedigrees. I analyzed this mutation in a Japanese MELAS pedigree by PCR. The proband had typical MELAS features. The proband's mother was oligosymptomatic (fatigability, nerve deafness and diabetes mellitus). The proband's maternal grandmother was diagnosed as having senile dementia of the Alzheimer type clinically. The brother of the proband's mother was healthy. The ratios of this mutation in muscle and leukocytes of the proband and his mother were 89%, 36%, 79% and 10%, respectively. There were no mutations in muscle and leukocytes of the proband's maternal grandmother and his mother's brother. These results showed the possibility that this mutation occurred in the proband's mother.

A common mutation site in the β-galactosidase gene originates in Puerto Rico

Several mutation sites have been found in the β-galactosidase gene of patients with GMI gangliosidosis. In a previous report we found a common point mutation site in American patients with GMl gangliosidosis resulting in a 208Arg → Cys amino acid substitution. From the patients' family history, we suggested that this mutation may have come to South and North America via Puerto Rico. Four new patients with infantile GM1 gangliosidosis have been analyzed with allele-specific hybridization. Two siblings from Puerto Rico of Spanish ancestry are homozygous for this mutation. Another patient also from Puerto Rico is heterozygous for this allele, and another black patient does not have this mutation. These results support our initial hypothesis that this mutation has probably arisen in Puerto Rico.

A mutational hot spot in the p53 gene is associated with hepatoblastomas.

Hepatoblastomas generally appear in children aged 2 or 3 years old and arise from apparently normal, non-cirrhotic liver. To elucidate any possible role of p53 mutations in their genesis, we amplified and sequenced exons 5 to 8 of the p53 gene in 10 cases of hepatoblastoma. Somatic mutations were detected in 9 cases, in eight of which a common point mutation at the first-base position of codon 157 was found, resulting in an amino-acid substitution of phenylalanine for valine. Two missense mutations in codon 244, and one each in codons 273 and 279, were also found, with 3 hepatoblastomas having double mis-sense mutations. Out of the total of 12 mutations, 11 were G-to-T transversions. One was a G-to-A transition and guanines were always present on the transcribed strand. Furthermore, p53 over-expression was immunohistochemically observed in 7 out of 9 cases with p53 gene mutations, although the staining pattern was focal and heterogeneous. The findings suggest that particular environmental mutagens may be involved in mutagenesis of the p53 gene in some cases of hepatoblastomas and that p53 mutations at a specific site may play an important role in the genesis of this disease.

Robust nonradioactive oligonucleotide ligation assay to detect a common point mutation in the CYP2D6 gene causing abnormal drug metabolism

A new nonradioactive oligonucleotide ligation assay for the detection of a common point mutation in the CYP2D6 gene is presented. The assay takes advantage of simultaneous time-resolved fluorescence measurements of lanthanide-labeled probes and the specificity of T4-DNA ligase in combination with the polymerase chain reaction. This strategy makes it possible to perform the assay using both the wild-type-specific and mutant-specific probes simultaneously, securing an internal control in each reaction. We show that the allele-specific ligation part of the assay can be performed with great accuracy over a wide range of temperatures, salt concentrations, and T4-DNA ligase concentrations. This eliminates the risk of false-positive or false-negative reactions due to variations in these factors. Because the assay is simple to perform, is very reliable, and can be partly automated, we conclude that it is well-suited for analysis in a routine laboratory.

Amyotrophic lateral sclerosis: human challenge for neuroscience.

Amyotrophic lateral sclerosis: human challenge for neuroscience.

A single missense mutation in codon 918 of the RET proto-oncogene in sporadic medullary thyroid carcinomas.

The RET proto-oncogene is expressed in human medullary thyroid carcinoma and pheochromocytoma. Recently germline mutations of the RET proto-oncogene were reported in four syndromes (MEN 2A, MEN 2B, familial medullary thyroid carcinoma and Hirschprung's disease) and somatic mutation was also found in sporadic medullary thyroid carcinoma. To determine the incidence of RET mutations in medullary thyroid carcinoma in Japan, we investigated 14 medullary thyroid carcinomas (comprising 1 case of MEN 2A, 1 case of MEN 2B, 2 cases of familial medullary thyroid carcinoma and 10 cases of sporadic). Tumors from all cases were screened by PCR-SSCP on exons 10 and 11. DNA sequencing on these exons was performed for the hereditary medullary thyroid carcinoma cases. The PCR products of exon 16 from tumor DNA were analyzed by means of Fok1 restriction enzyme digestion analysis and mutations confirmed by DNA sequencing. We found no structural abnormalities in either exon 10 or exon 11 in any of the cases examined, but in four of 10 sporadic cases we detected a common point mutation at codon 918 (ATG to ACG) in exon 16, where methionine was replaced with threonine. Our results support the theory that a point mutation of exon 16 of the RET proto-oncogene may be related to the oncogenesis of sporadic medullary thyroid carcinomas. However, further studies on the entire RET proto-oncogene are needed to clarify the relationship between its expression and thyroid tumorigenesis.

Crouzon syndrome: mutations in two spliceoforms of FGFR2 and a common point mutation shared with Jackson-Weiss syndrome.

Dominant mutations in the fibroblast growth factor receptor 2 (FGFR2) gene have been recently identified as causes of four phenotypically distinct craniosynostosis syndromes, including Crouzon, Jackson-Weiss, Pfeiffer, and Apert syndromes. These data suggest that the genetics of the craniosynostosis syndromes is more complex than would be expected from their simple autosomal-dominant inheritance pattern. Identical mutations in the FGFR2 gene have been reported to cause both Pfeiffer and Crouzon syndrome phenotypes. We now report the finding of a mutation in exon IIIc of the FGFR2 gene in a kindred affected with Crouzon syndrome (C1043 to G; Ala344Gly) that is identical to the mutation previously associated with Jackson-Weiss syndrome. We also report finding in a Crouzon kindred a mutation in the 3' end of exon IIIu (formerly referred to as exon 5, exon 7, or exon U) (A878 to C; Gln289Pro) which encodes the amino terminal portion of the Ig-like III domain of the FGFR2 protein. This exon is common to both the FGFR2 and the KGFR spliceoforms of the FGFR2 gene, unlike all previously reported Crouzon mutations, which have been found only in the FGFR2 spliceoform. These findings reveal further unexpected complexity in the molecular genetics of these craniosynostosis syndromes. The data implies that second-site mutations in FGFR2 itself (outside of exon IIIc) or in other genes may determine specific aspects of the phenotypes of craniosynostosis syndromes.

Identification of a mutational hostspot in the type II collagen gene (COL2A1): A common point mutation in two unrelated individuals with kniest dysplasia

Identification of a mutational hostspot in the type II collagen gene (COL2A1): A common point mutation in two unrelated individuals with kniest dysplasia

Frequent Constitutional C to T Mutations in CGA-Arginine Codons in the RB1 Gene Produce Premature Stop Codons in Patients with Bilateral (Hereditary) Retinoblastoma

We have shown previously that the most common point mutation in the RB1 gene in retinoblastoma tumours is a C-->T transition and that the majority of these occur in CGAarg codons. As a result of this mutation, a TGAstop codon is generated directly. We have analysed the 14 CGAarg codons in the RB1 gene for mutations in 113 patients with bilateral retinoblastoma. At 6 of these sites, C-->T mutations in CGA codons alter a restriction enzyme site which makes their identification relatively straightforward. It was necessary, however, to analyse the other 8 CGA codons using single-strand conformation polymorphism (SSCP) analysis. A total of 18 C-->T mutations were found, which represents 16% of all patients. Of these 13 (73%) were at two particular CGA codons in exon 8 (codon 251) and exon 17 (codon 552). During the course of the SSCP analysis, mutations were identified in 7 other individuals. Thus, 20-25% of all mutations can be identified by a relatively quick survey of the CGA codons in the RB1 gene, which has important implications for genetic screening programmes. All of the mutations in the RB1 gene in these bilaterally affected patients would be predicted to result in the absence of a functional protein.

Screening of human bladder tumors and urine sediments for the presence of H-ras mutations.

A series of 111 human bladder tumors were screened using oligonucleotide mutant specific probes, restriction enzyme analysis and single-stranded confirmation polymorphism (SSCP) for the presence of H-ras activation events. Thirty-three tumors were found to harbor H-ras mutations where a glycine to valine (G-->T) change in codon 12 was the most common point mutation recorded (26 tumors). Additional mutations involved glycine to cysteine at codon 13 (2 tumors) and glutamine to arginine/lysine/leucine at codon 61 (3/1/1 tumors, respectively). Ambiguous signals recorded with oligonucleotide probes were further analyzed using SSCP analysis revealing the presence of H-ras mutations in restricted regions of some tumors. The apparent sensitivity of SSCP enabled us to extend this study to DNA isolated from urine sediments where 4 of the 9 patients studied showed representation of mutant H-ras. Our study demonstrates a sensitive, non-invasive assay for the screening of urine-borne cells, with no requirement for prior knowledge of the mutational change at the H-ras locus.

The frequency of a disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase in sudden infant death syndrome.

A number of rare inherited metabolic disorders are known to lead to death in infancy. Deficiency of medium-chain acyl CoA dehydrogenase has, on clinical grounds, been related particularly to sudden infant death syndrome. The contribution of this disorder to the etiology of sudden infant death syndrome is still a matter of controversy. The present study investigated 120 well-defined cases of sudden infant death syndrome in order to detect the frequency of the most common disease-causing point mutation in the gene coding for medium-chain acyl-CoA dehydrogenase (G985) compared with the frequency in the general population. A highly specific polymerase chain reaction assay was applied on dried blood spots. No over-representation of homo- or heterozygosity for G985 appears to exist in such a strictly defined population, for which reason it may be more relevant to look at a broader spectrum of clinical presentations of inherited metabolic disorders and examine a wider range of sudden death in infancy.

PRENATAL DIAGNOSIS OF CONGENITAL ADRENAL HYPERPLASIA DUE TO 21-HYDROXYLASE DEFICIENCY

Prenatal diagnosis of the classic forms of the congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency has progressed at the same time as the advances made in the field of both endocrinology and molecular genetics. In 1975, prenatal diagnosis of CAH was first made in the second trimester on the basis of raised levels of 17-hydroxyprogesterone (17-OHP) in the amniotic fluid (AF). Later an indirect prenatal diagnosis based on the genetic tracking of CAH was greatly facilitated by its linkage to HLA. By 1979, to obviate the need for corrective genital surgery the virilized genitalia in CAH female fetuses, a prenatal treatment was proposed by giving dexametfiasone to the mother in early pregnancy, and later was shown to be successful. Rapidly, there was a demand for an early prenatal diagnosis of the affection. This was rendered possible by the advances in molecular biology associated with the practice of chorionic villus biopsy. Prenatal diagnosis was then done in the first trimester of the pregnancy, by either HLA typing using HLA-DNA probes or direct studies of the genetic abnormalities of the C4-CYP21 locus. In practice, four methods have been, and can still be used : measurement of AF levels of 17-OHP, HLA typing, molecular studies of the C4-CYP21 genes, all in association with fetal sex determination (measurement of AF testosterone levels, karyotype and more recently detection of Y-chromosome specific sequences). Steroid analysis remains a simple and safe method in the absence of prenatal treatment and the only informative method when there is no previous family study. Nowadays, an early and accurate prenatal diagnosis can be achieved by molecular genetic studies on DNA extracted from chorion villus samples, but it requires the previous study of the nuclear family. The choice between HLA typing and direct C4-CYP21 loci studies depend on the possibilities and expertise of a given laboratory. HLA typing is still more often used with the improvement of using molecular studies instead of serological methods because almost all families are then informative. Nevertheless, HLA typing is unreliable in some circumstances: in case of a recombination event (1 % in our studies of over 250 families) or de novo mutation(s) and when the index case is deceased or not available. Studies of the C4-CYP21 gene locus by Southern blotting and the CYP21B gene mutations by PCR method have not these disadvantages. In our hands, Southern blotting study of the duplicated C4-CYP21 region has shown some complex genetic rearrangements and is fully informative method for prenatal diagnosis in about 60% of CAH families. The 9 most common point mutations of the CYP21B gene causing CAH (and also present in the CYP21A pseudogene) can be detected after specific PCR amplification of the functional CYP21B gene. In these conditions, almost all families are informative in our experience. Moreover, using this direct genetic analysis associated with the possibility to detect the heterozygotes in a non related-CAH population, a prenatal diagnosis could be done in a family without a previous CAH affected child. A prenatal diagnosis in the first trimester is required in order to stop unnecessary prenatal treatments (if unaffected female fetuses and all males fetuses), or when the parents wish to interrupt the pregnancy in case of a CAH affected fetus. In our experience using molecular genetics of the CYP21-C4 loci and sex determination by PCR method (SRY, Y-chromosome repeated sequences) or karyotype, prenatal diagnosis has been successfully done in 23 pregnancies at risk 1:4. In one case, there was no previous family study because the index c;>se was dead; in an other family, the only informations were the state of heterozygoty of the parents: one being a relative of a characterized CAH family, the other being detected by a systematic ACTH test and confirmed by mutation studies.