Amplification versus mutation as a mechanism for reversion of anHGPRT mutation

We have used a cloned cDNA for hypoxanthine-guanine phosphoribosyltransferase (HGPRT) to analyze the HGPRTgene and mRNA in an HGPRT-deficient mutant of Chinese hamster cells (RJK10) and its HGPRT-positive revertants. By Southern blot analysis, no DNA rearrangements were detected within the genes from any of the cell lines examined. However, four of five spontaneous revertants each contained 10-to 20-fold more copies of the HGPRTgene than did RJK10 or wild-type cells. In contrast, the gene was not amplified in four mutagen-induced revertants. The RJK10 mutation did not alter the size or concentration of HGPRT mRNA and representatives of the revertants contained the mRNA in amounts proportional to the number of genes they carried. Examples of clones with either stable or unstable gene amplification were identified and their HGPRT-positive phenotypes were shown to be dependent on the gene amplification. In a stably amplified revertant, the extra genes were found to be syntenic with the X chromosome marker glucose-6-phosphate dehydrogenase. In an unstable revertant only one of the 10 to 20 copies of the gene could be shown to be X linked. Thus, we found that RJK10 can revert by at least two distinct mechanisms: amplification of the HGPRTgene, which occurred spontaneously, or point mutation, which predominated after exposure to mutagens.     

KRAS mutation test

KRAS mutation is detected in around 40% of colorectal cancer (CRC). Recent clinical trials revealed the ineffectiveness of anti-EGFR antibodies in CRC patients with mutated KRAS. Consequently, KRAS test is now thought to be essential to the determination of indication for anti-EGFR antibody therapy in CRC. However, there are still the following problems to be solved. (1) Prospective studies are needed to validate these clinical trial results. We recently performed KRAS genotype-driven phase II clinical trial evaluating FOLFIR/FOLFILI plus cetuximab as second-line therapy in refractory advanced CRC. KRAS codon 12 and 13 mutations were detected in 45 out of 112 patients (40.2%) for whom cetuximab was not indicated. The relation of clinical response with KRAS mutation status is now under investigation. (2) The clinical significance of the KRAS mutations except for those of codons 12 and 13 remains unclear. Clinical evaluation is necessary for KRAS codon 15, 18, 61, 117 and 146 mutations. (3) The detection methods, which are divided into sequencing and non-sequencing ones, are not standardized. Non-sequencing methods are rapid and sensitive, but costly. Furthermore, to validate their ambiguous data, sequencing methods are needed. At the present time dideoxy sequencing method seems to be preferentially chosen when considering that it is widespread and general-purposive. (4) Heterogeneous distribution of KRAS mutation in CRC tissues. (5) Effects of other genes abnormalities such as BRAF and PIK3CA mutations, PTEN down-regulation and EGFR gene amplification on the efficacy of anti-EGFR antibodies in CRC patients with or without KRAS mutation.     

DHPLC mutation analysis of phenylketonuria

Denaturing high-performance liquid chromatography (DHPLC) is a sensitive and fast method for the detection of mutations which has been successfully used for mutation screening in several disease-related genes. Phenylketonuria (PKU, OMIM* 261600; McKusick 1986) is one of the most common autosomal recessive disorders in Europe. Mutations in the PAH gene mainly involve point mutations. In this study we report the successful use of DHPLC to analyse rapidly the complete coding sequence of the PAH gene in a total of 125 unrelated patients with PKU.     

TCOF1 mutation database: novel mutation in the alternatively spliced exon 6A and update in mutation nomenclature

Recently, a novel exon was described in TCOF1 that, although alternatively spliced, is included in the major protein isoform. In addition, most published mutations in this gene do not conform to current mutation nomenclature guidelines. Given these observations, we developed an online database of TCOF1 mutations in which all the reported mutations are renamed according to standard recommendations and in reference to the genomic and novel cDNA reference sequences (www.genoma.ib.usp.br/TCOF1_database). We also report in this work: 1) results of the first screening for large deletions in TCOF1 by Southern blot in patients without mutation detected by direct sequencing; 2) the identification of the first pathogenic mutation in the newly described exon 6A; and 3) statistical analysis of pathogenic mutations and polymorphism distribution throughout the gene.     

On modeling of mutation probabilities

A probability model is derived for DNA mutations and implemented in Java programming language to be accessible via Internet. Its time and space complexity is analyzed and optimized for a mutation probability matrix. Sample runs comparable to actual hereditary tests are presented. The implementation is useful for predicting mutations for laboratory use.     

Spontaneous mutation in Big Blue mice from fetus to old age: tissue-specific time courses of mutation frequency but similar mutation types

Transgenic mouse mutation detection systems permit rapid determination of the frequency and type of mutations allowing direct examination of mutational markers for aging, neurodegeneration, and cancer. The Big Blue transgenic mouse mutation detection system was used to determine the frequency and nature of spontaneous mutations versus age in multiple tissue types. Nuclear DNA was extracted from whole fetus at 13.5 days postcoitus (dpc) and from six tissues postbirth (cerebellum, forebrain, thymus, liver, adipose tissue, and male germline) of Big Blue transgenic mice at four ages: 10 days and at 3, 10, and 25 months postbirth. Forty million total plaque-forming units (pfu) were screened. The time course of mutation frequency with age had a significantly different shape in different tissues (P < 10(-6)). By 13.5 dpc, the whole fetus mutation frequency had already started increasing from the theoretical zero at conception to a value that was about one-half the mid-adulthood (3-10 months) average. From 10 days to 3 months, mutation frequency increased significantly in liver (P = 0.007) and showed an increasing trend in cerebellum, forebrain, and thymus. From 3 to 10 months, there was no significant change in mutation frequency in any tissue examined. From 10 to 25 months, the mutation frequency increased significantly in liver (P < 10(-6)) and adipose tissue (P = 0.002), but not in the other tissues examined (cerebellum, forebrain, and male germline). It is of interest that the mutation frequency in the male germline is consistently the lowest, remaining essentially unchanged in old age. The spectrum of mutation types was unaltered with age, tissue type and gender, although, as previously reported, tandem GG-->TT mutations are tissue specific and show significant increases with age and certain hotspots (Buettner VL et al. [1999]: Environ Mol Mutagen 33:320-324; Hill KA et al. [2003]: Mutat Res 534:173-186). The spectrum of mutation types was generally the same for all tissue types, despite the tissue-specific increases in mutation frequency with age. These data provide a useful reference for future studies of endogenous and exogenous mutagenesis.     

A novel hepcidin mutation

BackgroundThe bioactive peptide hormone hepcidin-25 regulates iron levels by inhibiting iron transport to plasma via ferroportin. Hepcidin-25 is synthesized in the liver where the 84 amino acids pro-hepcidin is cleaved into the bioactive hepcidin-25. A patient admitted to the hospital presented with infertility and fatigue.MethodsGenomic DNA was purified from whole blood using the Maxwell 16 system (Promega). MLPA analysis was performed to detect large genomic rearrangements using the SALSA MLPA kit # P347, Hemochromatosis (MRC Holland, Holland). Plasma hepcidin measurements were performed using liquid chromatography/tandem mass spectrometry (LC-MS/MS).ResultsA novel HAMP mutation (homozygous one base deletion in c.215delG, p.Cys72Serfs*?) was detected. The deletion in nucleotide 215 causes a frameshift altering the predicted protein sequence from cysteine13 in mature peptide. Whether this leads to nonsense mediated decay of the mRNA or synthesis of an aberrant peptide in unknown, but bioactive hepcidin-25 was undetectable in plasma.The patient had massive iron overload with ferritin up to 8360 µg/L. He was anaemic with a Hb at 7.0 mmol/L (11.3 g/dL) and suffered from hypogonadotropic hypogonadism with a total testosterone of 1.2 nmol/l. Continued treatment with venesection and gonadotropins led to reduced fatigue, reduction in iron overload, a normalized Hb and improvement of semen quality.ConclusionA novel hepcidin mutation was detected in a patient with massive iron overload, fatigue and hypogonadotropic hypogonadism.     

Central mutation databases--a review

The Internet has been a key component in the coordination of the diverse group of scientists involved in the Human Genome Project. Nowhere has this contribution been more critical than in the maintenance and exchange of information about genetic variation and mutation. Whereas the majority of DNA sequence is generated and stored by a relatively few sites, a far greater number of researchers investigate the variations in that sequence from sites scattered worldwide. It falls to central databases to utilize the Internet to assemble data from these sites and make them available to the greater human genomic community.     

Pig-a gene mutation database

Mutations in the X-linked phosphatidylinositol glycan, class A gene (Pig-a) lead to loss of glycosylphosphatidylinositol (GPI) anchors and GPI-anchored proteins from the surface of erythrocytes and other mammalian cells. The Pig-a gene mutation assay quantifies in vivo gene mutation by immunofluorescent labeling and flow cytometry to detect the loss of GPI-anchored proteins on peripheral blood erythrocytes. As part of the regulatory acceptance of the assay, a public database has been created that provides detailed information on Pig-a gene mutation assays conducted in rats and mice. A searchable version of the database is available through a website designed and hosted by the University of Maryland School of Pharmacy. Currently, the database contains only mouse and rat data, but it is anticipated that it will expand to include data from other species, including humans. A major purpose in developing the database was to aid in the preparation of a Retrospective Performance Analysis and Detailed Review Paper required for Organisation for Economic Co-operation and Development Test Guideline acceptance. We anticipate, however, that it also will be useful for accessing and comparing Pig-a data to data from other assays and for conducting quantitative assessments of Pig-a gene mutation responses. Environ. Mol. Mutagen., 60:759–762, 2019. © 2019 Wiley Periodicals, Inc.     

Characterization of a mutation commonly associated with persistent stuttering: evidence for a founder mutation

Stuttering is a disorder that affects the fluency of speech. It has been shown to have high heritability and has recently been linked to mutations in the GNPTAB gene. One such mutation, Glu1200Lys, has been repeatedly observed in unrelated families and individual cases. Eight unrelated individuals carrying this mutation were analyzed in an effort to distinguish whether these arise from repeated mutation at the same site, or whether they represent a founder mutation with a single origin. Results show that all 12 chromosomes carrying this mutation share a common haplotype in this region, indicating that it is a founder mutation. Further analysis estimated the age of this allele to be ～ 572 generations. Construction of a cladogram tracing the mutation through our study sample also supports the founder mutation hypothesis.     

A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism

Leydig cell hypoplasia (LCH) is a rare form of male pseudohermaphroditism caused by inactivating mutations in the luteinizing hormone receptor gene (LHCGR). The majority of LHCGR mutations are located in the coding sequence, resulting in impairment of either LH/CG binding or signal transduction. We report a Chinese family with two siblings (46, XY and 46, XX) carrying a missense mutation (c. 455 T>C, p. Ile152Thr) and a splice site mutation (c. 537‐3 C>A). Computational analysis of the missense mutation in the three‐dimensional structural model predicted it might influence the distribution of hydrogen bonds and intermolecular contacts between the hormone and receptor. Consistent with these findings, in vitro mutant analysis revealed a marked impairment of human chorionic gonadotropin binding and signal transduction. The splice‐acceptor mutation (c. 537‐3 C>A) resulted in abnormal splicing of LHCGR mRNA, skipping exon 7. This report expands the genotypic spectrum of LHCGR mutations, with relevant implications for the molecular analysis of this gene. © 2009 Wiley‐Liss, Inc.     

Influence of missense mutation and silent mutation of LHbeta-subunit gene in Japanese patients with ovulatory disorders

The frequency of variant LHbeta containing two point mutations (T(986)-C and T(1008)-C) and its relationship to reproductive disorders differ widely between ethnic groups. In a Japanese population, variant luteinizing hormone (LH) correlates with ovulatory disorders. Here we examined the relationship between two missense mutations and five silent mutations (C(894)-T, G(1018)-C, C(1036)-A, C(1098)-T and C(1423)-T) in the LHbeta gene, and ovulatory disorders. We studied 43 patients with ovulatory disorders, 79 patients with normal ovulatory cycles, and 23 healthy men who agreed to join our DNA analysis. PCR-amplified LHbeta-subunit gene sequences were compared with a base sequence of wild-type LH reported after direct sequencing. The highest frequency (0.945) of novel allele was observed at the position of the C(1036)-A transition. No homozygotes for wild-type LHbeta (C(1036)) were identified. The frequency of novel allele in patients with polycystic ovary syndrome, endometriosis, premature ovarian failure and luteal insufficiency was significantly different from that of healthy women. The frequencies of novel alleles (C(894)-T, C(1098)-T and C(1423)-T) in patients with ovulatory disorders were significantly higher than those with normal ovulatory cycles. The mean incidence of point mutation in patients with ovulatory disorders was higher than in those with normal ovulatory cycles. Among patients with variant LH, five silent mutations were identified in 87.5% of patients with ovulatory disorders, whereas only a few silent mutations were identified in patients with normal ovulatory cycles. In a Japanese population, five silent mutations of variant LH could have influenced two missense mutations and/or other unknown missense mutations, causing ovulatory disorders.     

A novel missense mutation of doublecortin: mutation analysis of Korean patients with subcortical band heterotopia

The neuronal migration disorders, X-linked lissencephaly syndrome (XLIS) and subcortical band heterotopia (SBH), also called "double cortex", have been linked to missense, nonsense, aberrant splicing, deletion, and insertion mutations in doublecortin (DCX) in families and sporadic cases. Most DCX mutations identified to date are located in two evolutionarily conserved domains. We performed mutation analysis of DCX in two Korean patients with SBH. The SBH patients had mild to moderate developmental delays, drug-resistant generalized seizures, and diffuse thick SBH upon brain MRI. Sequence analysis of the DCX coding region in Patient 1 revealed a c.386 C>T change in exon 3. The sequence variation results in a serine to leucine amino acid change at position 129 (S129L), which has not been found in other family members of Patient 1 or in a large panel of 120 control X-chromosomes. We report here a novel c.386 C>T mutation of DCX that is responsible for SBH.