Truncating ribosomal protein S19 mutations and variable clinical expression in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare constitutional erythroblastopenia characterized by a specific defect in erythroid differentiation. Recently, mutations in the gene encoding ribosomal protein (RP) S19 were found in a subset of patients with the disease. To characterize further RPS19 mutations and to investigate genotype-phenotype relationships, we screened this gene for mutations in patients with DBA by direct sequencing and Southern-blot analysis. Four novel mutations were identified. A G120A nonsense mutation resulting in a stop at codon 33, a C302T nonsense mutation introducing a premature stop at codon 84, and a 327delG which results in a frame shift at codon 103. A fourth and more complex mutation (TT157-158AA, 160insCT) resulting in a Leu45Gln and a frame shift from codon 47 was found in three affected family members with variable phenotypes. The different clinical expression for identical mutations suggest the presence of other modulating factors for the disease. The mutations presented here further support the role of RPS19 in erythropoietic differentiation and proliferation.     

Genetic variation in α1-antichymotrypsin and its association with Alzheimer's disease

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by extracellular neuritic plaques and intracellular neurofibrillary tangles in brain parenchyma. Alpha-1-antichymotrypsin (ACT) is a component of plaque cores, can bind to Abeta, and has been proposed as a possible candidate gene for AD susceptibility. The genetic association between the ACT codon -17*A allele of the signal peptide polymorphism and AD has been shown in some, but not in all studies. One hypothesis is that the ACT codon -17*A allele is in linkage disequilibrium with unknown functional mutation(s) in the ACT gene. This study was undertaken to identify new mutation(s) in the ACT gene by PCR-SSCP-sequencing and, in conjunction with known mutations, to assess their role in affecting the risk of AD. A total of seven new point mutations were observed: 5'UTR(A-->G), Asp128Asn(G-->A), Ser250Ser(C-->T), Leu301Pro(T-->C), Thr324Thr(A-->G), G-->A in intron 4, and 3'UTR C-->A. Of these, mutations at codon 250, codon 324, intron 4 and 3'UTR showed a frequency of 1% or more. Of the known mutations, Thr-17Ala(A-->G), Lys76Lys(A-->G) and Leu241Leu(G-->A) occur at a polymorphic level. The ACT codon -17*A allele was associated with increased risk of AD (OR for AA vs TT: 1.71; 95% CI: 1.16-2.53; P=0.007), especially in the presence of the APOE*4 allele (OR for AA vs TT: 2.35; 95% CI: 1.13-4.85; P=0.02). The codon 241*A allele and the codon 250*T allele were associated with protective effects against AD (OR: 0.36; 95% CI: 0.13-0.86; P=0.02) (OR:0.39; 95% CI: 0.18-0.85; P=0.02). irrespective of the APOE*4 status. The codon 324*G allele was associated with a marginal protective effect (OR:0.57; 95% CI: 0.26-1.26; P=0.17). While the codon 241*A allele was in linkage disequilibrium with the codon -17*A allele, the codon 250*T and codon 324*G alleles were non-randomly associated with the codon -17*T allele. In contrast, the codon 76*G (OR:1.34; 95% CI: 0.92-1.95; P=0.13), codon 227*G (OR:3.96; 95% CI: 0.83-18.8; P=0.08) and intron 4*G (OR:1.47; 95% CI: 0.88-2.29; P=0.15) alleles were associated with a modest risk of AD, and all were in linkage disequilibrium with the codon -17*A allele. EH-based haplotype analysis showed that certain haplotypes are associated with either higher or lower risk of AD. Our data indicate that the ACT gene harbors several potentially important variable sites, which are associated with either an increased or decreased risk of AD. The non-random combination of risk and protective alleles may explain, in part, why the association studies regarding the ACT codon -17*A have been inconsistent, especially if the frequency of other ACT mutations varies between populations.     

Phenylalanine hydroxylase gene: novel missense mutation in exon 7 causing severe phenylketonuria.

By direct sequence analysis of 94 mutant phenylalanine hydroxylase alleles using polymerase chain reaction-based techniques, we identified a C to T transition in exon 7 of the human phenylalanine hydroxylase gene that is associated with RFLP haplotypes 1 and 4. A leucine for proline substitution at position 281 can be predicted from the nucleotide sequence of the mutant codon. Expression analysis in cultured mammalian cells after site-directed mutagenesis proved that the base substitution is a disease causing gene lesion. Dot-blot hybridization analysis using allele-specific oligonucleotides revealed that 25% of all mutant haplotype 1 alleles in the German population bear this mutation. In addition, this mutation could be detected on one mutant haplotype 4 allele. The fact that this mutation is associated with only 25% of all mutant haplotype 1 alleles suggests that multiple mutations may be associated with this haplotype. The occurrence of several different mutations would be in agreement with the clinical heterogeneity observed in the group of patients whose PKU alleles belong to haplotype 1.     

Linkage analysis of neurofibromatosis type 1

Summary In order to investigate the molecular basis of phenylketonuria (PKU) in the Polish population, we screened 44 mutant chromosomes from PKU probands for six known mutations, frequently occurring in western European countries, by polymerase chain reaction amplification of their genomic DNA and hybridization with allele-specific oligonucleotides. Our results show that the majority (66%) of all PKU alleles are characterized by three different mutations: in codon 408 (56.8%), codon 158 (6.8%) and codon 261 (2.27%). Of the mutant haplotype 2 alleles, 96% were linked to the mutation in codon 408. Out of five mutant haplotype 4 alleles, three showed the codon 158 mutation, and out of four mutant haplotype 1 alleles, one had the codon 261 mutation. In two families, MspI digests revealed an additional 13.5-kb band similar in length to that previously reported. However, analysis of exon 9 excluded the presence of the T to C transition originally described, indicating a new MspI variant in the Polish population.     

Characterization of a novel mutation causing hepatic lipase deficiency among French Canadians

Individuals with hepatic lipase (HL) deficiency are often characterized by elevated levels of triglycerides (TGs) and cholesterol. The aim of the present study was to characterize the molecular defect leading to severe HL deficiency in a Québec-based kindred. In the proband and two of her brothers, the very low to undetectable HL activity resulted from compound heterozygosity for two rare HL gene mutations, a previously unknown missense mutation in exon 5 designated A174T and the previously reported T383M mutation in exon 8 of the HL gene. The mutation at codon 174 resulted in the substitution of alanine for threonine, a polar amino acid, in a highly conserved nonpolar region of the protein involved in the catalytic activity of the enzyme. The severe HL deficiency among the three related compound heterozygotes was associated with a marked TG enrichment of LDL and HDL particles. The two men with severe HL deficiency also presented with abdominal obesity, which appeared to amplify the impact of HL deficiency on plasma TG-rich lipoprotein levels. Our results demonstrated that HL deficiency in this Québec kindred is associated with an abnormal lipoprotein-lipid profile, which may vary considerably in the presence of secondary factors such as abdominal obesity.     

Homozygous CYP17A1 mutation (H373L) identified in a 46,XX female with combined 17α-hydroxylase/17,20-lyase deficiency

Background: Defects in cytochrome P450c17 are uncommon forms of congenital adrenal hyperplasia caused by CYP17A1 mutations. An H373L mutation in the CYP17A1 gene has been identified in Japanese and Chinese patients. This mutation impairs 17α-hydroxylase and 17,20-lyase activity. Case: A 23-year-old Korean female (46,XX) presented with absent spontaneous puberty and hypertension. Hormonal findings were consistent with combined 17α-hydroxylase/17,20-lyase deficiency. Very high levels of progesterone and 11-deoxycorticosterone were detected, coincident with normal 17-hydroxysteroid levels. Plasma levels of dehydroepiandrosterone, androstenedione and testosterone were extremely low. Mutation analysis of the CYP17A1 gene identified a homozygous missense mutation changing His (CAC) to Leu (CTC) at codon 373. This mutation is known to completely abolish both 17α-hydroxylase and 17,20-lyase activity. The patient's nonconsanguineous parents were heterozygous for this mutation. Of note, her serum steroid levels indicated decreased, but still present, 17α-hydroxylase activity in vivo. Conclusion: We detected a homozygous H373L mutation in a patient with combined 17α-hydroxylase/17,20-lyase deficiency. Our findings demonstrate minimally preserved 17α-hydroxylase activity in vivo and contribute to our knowledge of the regional prevalence of this mutation in Northeast Asia.     

Human triosephosphate isomerase deficiency resulting from mutation of Phe-240.

Triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketolisomerase [E.C.5.3.1.1]) deficiency is an autosomal recessive disorder that typically results in chronic, nonspherocytic hemolytic anemia and in neuromuscular impairment. The molecular basis of this disease was analyzed for one Hungarian family and for two Australian families by localizing the defects in TPI cDNA and by determining how each defect affects TPI gene expression. The Hungarian family is noteworthy in having the first reported case of an individual, A. Jó., who harbors two defective TPI alleles but who does not manifest neuromuscular disabilities. This family was characterized by two mutations that have never been described. One is a missense mutation within codon 240 (TTC [Phe]-->CTC [Leu]), which creates a thermolabile protein, as indicated by the results of enzyme activity assays using cell extracts. This substitution, which changes a phylogenetically conserved amino acid, may affect enzyme activity by disrupting intersubunit contacts or substrate binding, as deduced from enzyme structural studies. The other mutation has yet to be localized but reduces the abundance of TPI mRNA 10-20-fold. Each of the Australian families was characterized by a previously described mutation within codon 104 (GAG [Glu]-->GAC [Asp]), which also results in thermolabile protein.     

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PI*Q0 alleles and one deficient PI*M allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.     

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.     

Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy.

We have produced mutations in a cloned Escherichia coli 23S rRNA gene at positions G2252 and G2253. These sites are protected in chemical footprinting studies by the 3' terminal CCA of P site-bound tRNA. Three possible base changes were introduced at each position and the mutations produced a range of effects on growth rate and translational accuracy. Growth of cells bearing mutations at 2252 was severely compromised while the only mutation at 2253 causing a marked reduction in growth rate was a G to C transversion. Most of the mutations affected translational accuracy, causing increased readthrough of UGA, UAG and UAA nonsense mutations as well as +1 and -1 frameshifting in a lacZ reporter gene in vivo. C2253 was shown to act as a suppressor of a UGA nonsense mutation at codon 243 of the trpA gene. The C2253 mutation was also found not to interact with alleles of rpsL coding for restrictive forms of ribosomal protein S12. These results provide further evidence that nucleotides localized to the P site in the 50S ribosomal subunit influence the accuracy of decoding in the ribosomal A site.     

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.     

Haplotype distribution and mutations at the PAH locus in Croatia

Summary Restriction fragment length polymorphism (RFLP) haplotypes and mutations at the phenylalanine hydroxylase (PAH) locus have been studied in 25 unrelated families from Croatia. The results of RFLP analysis demonstrated that 80% of the mutant alleles were associated with three haplotypes (1, 2 and 4). Eight mutations were detected on the background of six mutant haplotypes, comprising 68% of phenylketonuria (PKU) alleles in Croatia. The mutation in codon 408 was most frequent, as was the haplotype 2 allele with which it was associated. These data are in accordance with formerly published population genetic analyses at the PAH locus, and with studies revealing the molecular basis of the phenotypic heterogeneity of PKU. The codon 281 mutation was more frequent in Croatia than previously observed in other populations.     

Rare variants of blood proteins in human populations

Rare variants of blood proteins occur, due to mutations (mutant alleles) in monomorphic loci encoding various proteins. A number of authors studied the distribution of these variants in human populations using the method of electrophoresis. The population of USA, South America, Japan, Europe was analysed. 1334 rare variants (1.0.10(-3)) were discovered out of 1,329,558 alleles (test locus in 664,779 individuals). 7 mutant alleles (3.6.10(-6)) were found among 1,957,305 alleles. The low frequency of occurrence of mutations in the loci encoding rare blood protein variants, when testing the speed of mutagenicity and its alteration, necessitates electrophoresis of blood proteins to be done in large scales. A method was proposed, based on accounting rare variants in children with congenital disorders, which are supposed to have a heavy load of mutations. The data collected demonstrated that the majority of rare variants in a given generation were obtained from parents. Accumulation of rare protein variants at low concentrations, as neutral alleles, in conditions of low mutation frequency in monomorphic loci takes place in the population. Comparison of frequencies of rare variants among healthy newborns and the children with congenital disorders revealed their identity (1.0.10(-3)), as compared to 1.05.10(-3)). Simplification of the method for scoring mutations judging by rare blood protein variants, which is necessary for monitoring for gene mutations in human populations, stimulates development of novel approaches.     

Structure of the SLC7A7 gene and mutational analysis of patients affected by lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of cationic amino acid transport caused by mutations in the SLC7A7 gene. We report the genomic structure of the gene and the results of the mutational analysis in Italian, Tunisian, and Japanese patients. The SLC7A7 gene consists of 10 exons; sequences of all of the exon-intron boundaries are reported here. All of the mutant alleles were characterized and eight novel mutations were detected, including two missense mutations, 242A-->C (M1L) and 1399C-->A (S386R); a nonsense mutation 967G-->A (W242X); two splice mutations IVS3 +1G-->A and IVS6 +1G-->T; a single-base insertion, 786insT; and two 4-bp deletions, 455delCTCT and 1425delTTCT. In addition, a previously reported mutation, 1625insATCA, was found in one patient. It is noteworthy that 242A-->C causes the change of Met1 to Leu, a rare mutational event previously found in a few inherited conditions. We failed to establish a genotype/phenotype correlation. In fact, both intrafamilial and interfamilial phenotypic variability were observed in homozygotes for the same mutation. The DNA-based tests are now easily accessible for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.     

Relationships between mutations affecting protein kinase and accumulation of energy reserves in Saccharomyces cerevisiae

Abstract Independently discovered mutations which alter cyclic-AMP dependent protein kinase activity in Saccharomyces cerevisiae are analysed in relation to trehalose and glycogen storage. The defective trehalose and glycogen accumulation in strains which bear the glc1 mutation results from abnormal activation of trehalase by a protein kinase which has partially lost its cAMP dependence. Cells bearing the bcy1 mutation produce an altered protein kinase due to extremely low levels of the cAMP-binding protein. This altered kinase activates trehalase, resulting in low trehalose contents in these cells. In cell-free extracts of control strains (S288C and 7Q-2D), which produce normal levels of glycogen and trehalose, the enzyme trehalase is mainly found in an inactive, cryptic form. Each of the haploid strains containing one of the mutant genes (glc1, glc4-1 and bcy1) is defective in both trehalose and glycogen accumulation and exhibits low activation ratios of trehalase by protein kinase. Genetic complementation experiments clearly establish that the bcy1 mutation involves a different gene to that altered by the glc1 mutation, since the resulting diploid behaved normally. Strain AM9-10D, previously classified as wild-type (normal for bcy1 ), is defective in the accumulation of trehalose and glycogen and exhibits almost all trehalose in the active form.     

Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl-terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus-Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.