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PubMed1996

A genetic screen for mutations affecting embryogenesis in zebrafish.

Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for developmental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pigmentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.

Alleles↗
PubMed2005

The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe.

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 80 patients from 72 families by means of a PCR-based mutation screening strategy, consisting of heteroduplex analysis, restriction enzyme digest, DNA single strand electrophoresis, and direct sequencing. For a subset of patients mutation screening with DHPLC was established which turned out to be as fast and as sensitive as the more conventional methods. Fifteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in smaller previous studies, p.A150P (65%), p.A175D (11%) and p.N335K (8%) were the most common mutated alleles, followed by c.360_363delCAAA, p.R60X, p.Y204X, and c.865delC. Eight novel mutations were identified in eight families with HFI: a small indel mutation (c.1044_1049delTTCTGGinsACACT), two small deletions (c.345_372del28; c.841_842delAC), two splice site mutations (c.113-1G>A, c.799+2T>A), one nonsense mutation (c.612T>G (p.Y204X)), and two missense mutations (c.532T>C (p.C178R), c.851T>C (p.L284P)). By mutation screening for the three most common ALDOB mutations by DHPLC in 2,000 randomly selected newborns we detected 21 heterozygotes. Based on these data and after correction for less common and private ALDOB mutations, HFI prevalence in central Europe is estimated to be 1:26,100 (95% confidence interval 1: 12,600-79,000).

Chromatography, High Pressure Liquid↗
PubMed2000

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes).

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.

Animals↗
PubMed1992

Screening for mutations in the open reading frame and promoter of the beta-amyloid precursor protein gene in familial Alzheimer's disease: identification of a further family with APP717 Val-->Ile.

Following the identification of mutations in the beta-amyloid precursor protein (APP) gene in familial, early onset Alzheimer's disease (AD), we have developed a screening protocol using single strand conformation analysis (SSCA) to screen exon 17 for the known mutations within APP. In addition, we used this protocol to screen the other seventeen exons of APP and a three hundred and thirty base pair regulatory region of the promoter for new mutations in 9 families with early onset AD. Exons 16 and 17, which encode the deposited beta-amyloid peptide, were screened in a further 10 families. Our screening procedure identifies all the reported mutations within APP. While we have identified a further family with APP717 Val-->Ile, we did not find any previously undescribed mutations. Screening of other exons of APP in 2 families in which we have previously reported mutations at APP717, failed to reveal other sequence abnormalities supporting the hypothesis that the mutations at APP717 cause the disease in these families. These data suggest that mutations in APP are a rare cause of familial early onset AD (3/21 families tested) and that within APP most, possibly all, mutations which cause AD are in exon 17.

Adult↗
PubMed1996

Screening for mutations in the LDL receptor gene and apolipoprotein B-100 gene in 218 patients with severe hypercholesterolemia.

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol > 260 mg/dl) living in the Cologne area were screened for mutations in the LDL receptor gene and apolipoprotein B-100 gene. In the LDL receptor gene Southern blotting was used for detection of major DNA rearrangements and the single-strand conformation polymorphism (SSCP) method was used to screen for micro-deletions and insertions and single base alterations. The Arg3500-->Glu mutation, which is the only relevant mutation in the apolipoprotein B-100 gene causing hypercholesterolemia, was detected by a modified PCR and restriction enzyme digestion. Three different major rearrangements, all of which were deletion, were found in the LDL receptor gene. The SSCP screening was started with exon 4. In 20 cases an abnormal fragment pattern was observed. The apolipoprotein B-100 mutation was detected in 15 patients. In summary, by the combined analysis of major rearrangements, micro-deletions, insertions and single base alterations in the LDL receptor gene and the Arg3500-->Glu mutation in the apolipoprotein B-100 gene, mutations causing or probably causing hypercholesterolemia could be detected in 38 of the 218 studied patients. The expansion of SSCP screening to other exons of the LDL receptor gene will greatly increase the identification of mutations causing hypercholesterolemia.

Apolipoprotein B-100↗
PubMed1999

Fast and reliable screening of mutations in human tumors: use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA).

Human tumor samples were screened for point mutations by adapting a mobility-shift assay to automated DNA sizing. This screen identifies the type of point mutation and relative amount of mutated DNA sequences present in a sample. Test samples having known hypoxanthine-guanine phosphoribosyl transferase (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplification, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modified strands, using an automated DNA sequencer. Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA strands in a sequence-dependent manner, resulting in composition-dependent electrophoretic mobility. Thus, point mutations are identified as shifts in mobility between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified, even at fourfold dilution with control DNA.

Animals↗
PubMed2006

Evaluation of the role of RET polymorphisms/haplotypes as modifier loci for MEN 2, and analysis of the correlation with the type of RET mutation in a series of Spanish patients.

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant cancer syndrome, which is divided into three subtypes: MEN 2A, MEN 2B and familial medullary thyroid cancer (FMTC). Approximately 92% of MEN 2 cases are caused by mutations in exons 10, 11, 13-16 of the RET proto-oncogene. There exists inter- and intra-familial phenotypic variability among the MEN 2 families, even when the disease is caused by the same RET mutation, suggesting a role for genetic modifiers, such as polymorphisms/haplotypes. We have sought to determine the frequency and position of RET germline mutations in a cohort of 114 Spanish probands with any sign of MEN 2, and to search for putative modifier loci. Mutational screening of RET revealed 9 different mutations, present in 26 of the 114 probands (22.8%). In addition, distributions of 8 RET polymorphisms and the haplotypes comprising them, were studied in the context of the families positive for RET mutational screening, in order to evaluate them as genetic modifiers. The relationship between RET mutation type and presence of a polymorphism/haplotype was analyzed. The relationship between the presence of pheochromocytoma (PC) and/or hiperparathyroidism (HPT) in carriers of the same RET mutation, and the genotype for the specific variants was also studied. The results derived from those analyses revealed no associations of any variant/haplotype to a specific mutation or to the clinical presentation. Nevertheless, these observations do not permit us to exclude the possible role of other variants in RET or other related genes, in the final presentation of the disease.

Alleles↗
PubMed2003

Characterization of a Saccharomyces cerevisiae mutant with pseudohyphae and cloning of a gene complementing the mutation.

Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis. Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells. Few reports exist of haploid cells growing as pseudohyphae in liquid culture. Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall. Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype. Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change.

Cell Wall↗
PubMed2003

Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism.

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highly G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem and GenePhor, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem and 20% gels at 15 degree C, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

DNA Mutational Analysis↗
PubMed2000

MEN1 gene mutation analysis in Italian patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial syndrome characterized by parathyroid, enteropancreatic and pituitary tumors. The gene responsible for this syndrome is localized at chromosomal 11q13 region and DNA markers from this region cosegregate with the disease. The recent identification of the MEN1 gene, encoding for a protein termed menin of 610 amino acids, allowed mutational screening to be performed both in affected families and sporadic cases. To date many different heterozygous mutations, spreading across all the encoding sequence, have been identified in MEN 1 patients with no apparent mutational hot spots or genotype-phenotype correlation. To analyze the genetic alterations of the MEN1 gene occurring in Italian patients we performed mutational screening by Denaturant Gradient Gel Electrophoresis followed by sequencing of exons 2-10 of the MEN1 gene in 27 Italian MEN 1 families and in five sporadic cases. We identified 17 different heterozygous mutations in 60% of analyzed cases. Twelve of these mutations are novel. Two mutations each occurred twice in unrelated families but no evidence of genotype-phenotype correlation can be established for these families. The extension of genetic diagnosis to asymptomatic family members allowed the identification of 10 MEN1 mutant gene carriers, one newly described and nine previously detected by linkage analysis with DNA markers from the 11q13 region. Our findings add new information to the diversity of mutations occurring in the MEN1 gene and confirm that the mutational screening of MEN 1 is a useful approach to detect individuals at higher risk of developing MEN 1-associated tumors.

DNA Mutational Analysis↗
PubMed1995

Screening for mutations in exon 4 of the LDL receptor gene in a German population with severe hypercholesterolemia.

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol > 260 mg/dl) living in the Cologne area were screened for mutations in the 3 half of exon 4 of the low density lipoprotein (LDL) receptor gene by the single-strand conformation polymorphism (SSCP) method. The analysed fragment was 242 bp in length and comprised approximately 6% of the coding region. In 11 patients an abnormal SSCP pattern was observed. Two of the abnormal fragment patterns were identical. The results of the SSCP screening could be confirmed by direct DNA sequencing. Three of the ten different mutations were previously described (3 bp deletion: codon 197; Asp200-->Gly; Glu207-->stop). Of the newly identified mutations there were two deletions, two insertions, one combined insertion and deletion mutation and two single base pair substitutions [1 bp deletion: G in codon 197; 37 bp deletion: T in codon 196-208 or AT in 196-207 and GA in codon 208; 18 bp insertion: codon 201-206; 8 bp insertion: codon 155-156 and GA in codon 157; 6 bp insertion (codon 196-197) and 5 bp deletion (codon 199, C in codon 198 and G in codon 198 or 200); Asp200-->Tyr; Asp203-->Val]. The 8-bp insertion was detected in a second unrelated individual. The analysis of the functional consequences of the mutations indicates that all mutations were causative of the LDL cholesterol elevation.

Base Sequence↗
PubMed1998

A new technique of screening gene mutation--fluorescent fingerprinting.

A new technique called fluorescent fingerprinting (FF) was set up and used to screen and detect gene mutation from a large number of samples by using fluorescent labelled ddNTP and ABI-373A sequencer. By comparing the colour ladders of several lanes from the same sequencing region of different samples, any band with a different colour from its neighbours or a pattern displacement could be easily found out in the Macintosh screen which indicates a gene mutation. Then, the sequencing peak-chromatograms were made out only for the selected samples.

DNA Fingerprinting↗
PubMed1996

Use of the single strand conformation polymorphism method for rapid screening for mutations in the low density lipoprotein receptor gene in patients with familial hypercholesterolemia: effect on plasma lipid levels of different classes of mutations.

The single strand conformational polymorphism (SSCP) method was used to screen patients with familial hypercholesterolemia (FH) for mutations in the 3' part of exon 4 of the low density lipoprotein receptor (LDLR) gene. In 311 patients, six previously described mutations were identified in 29 apparently unrelated individuals (9.3%); three of the mutations are null alleles producing no protein, while the other three lead to production of a defective protein. In the patients where no mutation was detected mean total plasma cholesterol levels were 9.4 mmol/l, compared to 11.3 mmol/l in those individuals with a mutation creating a null allele, and 11.2 mmol/l in those with a mutation that resulted in the production of a defective protein (p < 0.001). These data reinforce observations of others that specific mutations in the LDL receptor gene are associated with different effects on plasma lipids, and indicate that the phenotype is influenced by the genotype.

Adult↗
PubMed2006

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Animal Diseases↗
PubMed2001

Genomic organization of the dysferlin gene and novel mutations in Miyoshi myopathy.

OBJECTIVE: Mutations in the skeletal muscle gene dysferlin cause two autosomal recessive forms of muscular dystrophy: Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B). The purpose of this study was to define the genomic organization of the dysferlin gene and conduct mutational screening and a survey of clinical features in 21 patients with defined molecular defects in the dysferlin gene. METHODS: Genomic organization of the gene was determined by comparing the dysferlin cDNA and genomic sequence in P1-derived artificial chromosomes (PACs) containing the gene. Mutational screening entailed conformational analysis and sequencing of genomic DNA and cDNA. Clinical records of patients with defined dysferlin gene defects were reviewed retrospectively. RESULTS: The dysferlin gene encompasses 55 exons spanning over 150 kb of genomic DNA. Mutational screening revealed nine novel mutations associated with MM. The range of onset in this patient group was narrow with a mean of 19.0 +/- 3.9 years. CONCLUSION: This study confirms that the dysferlin gene is mutated in MM and LGMD2B and extends understanding of the timing of onset of the disease. Knowledge of the genomic organization of the gene will facilitate mutation detection and investigations of the molecular biologic properties of the dysferlin gene.

Adolescent↗
PubMed1999

Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis. Identification and characterization of six novel mutations associated with familial PCT.

The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able to detect the underlying UROD mutation in 10 previously characterized DNA samples as well as a new mutation in each of six previously unexamined PCT patients. The six novel UROD mutations comprise three missense mutations (M01T, F229L, and M324T), two splice mutations (IVS3-2A-->T and IVS5-2A-->G) leading to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual catalytic activity, whereas the two missense mutants retained some residual activity. Furthermore, the missense mutants exhibited a considerable increase in thermolability. The six new mutations bring to a total of 29 the number of disease-related mutations in the UROD gene. The DGGE assay presented greatly improves the genetic diagnosis of fPCT and HEP, thereby facilitating the detection of familial UROD deficient patients as well as the discrimination between familial and sporadic PCT cases.

Alleles↗
PubMed2003

Mutations in the CACNA1F and NYX genes in British CSNBX families.

X-linked congenital stationary night blindness (CSNBX) is a genetically and phenotypically heterogeneous non-progressive disorder, characterised by impaired night vision but grossly normal retinal appearance. Other more variable features include reduction in visual acuity, myopia, nystagmus and strabismus. Genetic mapping studies by other groups, and our own studies of British patients, identified key recombination events indicating the presence of at least 2 disease genes on Xp11. Two causative genes (CACNA1F and NYX) for CSNBX have now been identified through positional cloning strategies. In this report, we present the results of comprehensive mutation screening in 14 CSNBX families, three with mutations in the CACNA1F gene and 10 with mutations in the NYX gene. In one family we failed to identify the mutation after testing RP2, RPGR, NYX and CACNA1F. NYX gene mutations are a more frequent cause of CSNBX, although there is evidence for founder mutations. Our report of patient population mutation screening for both CSNBX genes, and our exclusion of RP2 and RGPR, indicates that mutations in CACNA1F and NYX are likely to account for all CSNBX.

Calcium Channels↗