Mutations associated with variant phenotypes in ataxia-telangiectasia.

We have identified 14 families with ataxia-telangiectasia (A-T) in which mutation of the ATM gene is associated with a less severe clinical and cellular phenotype (approximately 10%-15% of A-T families identified in the United Kingdom). In 10 of these families, all the homozygotes have a 137-bp insertion in their cDNA caused by a point mutation in a sequence resembling a splice-donor site. The second A-T allele has a different mutation in each patient. We show that the less severe phenotype in these patients is caused by some degree of normal splicing, which occurs as an alternative product from the insertion-containing allele. The level of the 137-bp PCR product containing the insertion was lowest in two patients who showed a later onset of cerebellar ataxia. A further four families who do not have this insertion have been identified. Mutations detected in two of four of these are missense mutations, normally rare in A-T patients. The demonstration of mutations giving rise to a slightly milder phenotype in A-T raises the interesting question of what range of phenotypes might occur in individuals in whom both mutations are milder. One possibility might be that individuals who are compound heterozygotes for ATM mutations are more common than we realize.     

Mutations and sequence variants in the testis-determining region of the Y chromosome in individuals with a 46,XY female phenotype

The testis-determining gene SRY (sex determining region, Y) is located on the short arm of the Y chromosome and consists of a single exon, the central third of which is predicted to encode a conserved motif with DNA binding/bending properties. We describe the screening of 26 patients who presented with 46,XY partial or complete gonadal dysgenesis for mutations in both the SRY open reading frame (ORF) and in 3.8 kb of Y-specific flanking sequences. DNA samples were screened by using the fluorescence-assisted mismatch analysis (FAMA) method. In two patients, de novo mutations causing complete gonadal dysgenesis were detected in the SRY ORF. One was a nonsense mutation 5′ to the HMG box, whereas the other was a missense substitution located at the C terminus of the conserved motif and identical to one previously detected in an unrelated patient. In addition, two Y-specific polymorphisms were found 5′ to the SRY gene, and a sequence variant was identified 3′ to the SRY polyadenylation site. No duplications of the DSS region in 20 of these patients were detected. Received: 18 November 1996 / Revised: 13 December 1996     

Gaucher disease: heterologous expression of two alleles associated with neuronopathic phenotypes.

To investigate the molecular basis for the distinct neuronopathic phenotypes of Gaucher disease, acid beta-glucosidases expressed from mutant DNAs in Gaucher disease type 2 (acute) and type 3 (subacute) patients were characterized in fibroblasts and with the baculovirus expression system in insect cells. Expression of the mutant DNA encoding a proline-for-leucine substitution at amino acid 444 (L444P) resulted in a catalytically defective, unstable acid beta-glucosidase in either fibroblasts from L444P/L444P homozygotes or in insect cells. This mutation was found to be homoallelic in subacute neuronopathic (type 3) Gaucher disease. In comparison, expression of the mutant cDNA encoding an arginine-for-proline substitution at amino acid 415 (P415R) resulted in an inactive and unstable protein in insect cells. This allele was found only in a type 2 patient with the L444P/P415R genotype. The substantial variation in the type 3 phenotype (L444P homozygotes) suggests the complex nature of the molecular basis of phenotypic variation in Gaucher disease. Yet, the association of neuronopathic phenotypes with alleles producing severely compromised (L444P) or functionally null (P415R) enzymes indicates that the effective level of residual activity at the lysosome is likely to be a major determinant of the severity of Gaucher disease.     

Wolbachia variant that induces two distinct reproductive phenotypes in different hosts

Wolbachia is an intracellular endosymbiont that induces a variety of reproductive alterations in diverse arthropods. The almond moth, Cadra cautella, is double infected with two Wolbachia variants, wCauA and wCauB, and expresses complete cytoplasmic incompatibility (CI). The individual contribution of wCauA and wCauB to the expression of CI are unclear, however, because the two variants have not been separated in this host. The effect of wCauA is of particular interest because it induces male killing when transferred into the Mediterranean flour moth, Ephestia kuehniella. In the present study, we generated C. cautella infected with only wCauA by treating double-infected insects with tetracycline. Single-infected C. cautella exhibited strong CI, demonstrating that wCauA induces two distinct reproductive phenotypes in different hosts: CI in C. cautella and male killing in E. kuehniella. CI was also observed in the cross of double-infected males and single-infected females. Comparison of the single- and double-infected insects by real-time quantitative polymerase chain reaction suggested that the wCauA density is not affected much by the presence or absence of wCauB.     

The sole presence of the testis-determining region of the Y chromosome (SRY) in 46,XX patients is associated with phenotypic variability.

Four cases of XX patients with testis development are reported. The aim of this study was to describe their clinical features and to see if there was any relationship between phenotypes and the presence of Y material. Several human Y-derived sequences including the SRY probe were used to analyze the DNA of the patients. Yp material including the pseudo-autosomal region and SRY was detected. The cases reported in this study confirm that XX true hermaphrodites cannot be distinguished from XX males on the basis of their genotypes. There is no relationship between clinical and anatomical phenotypes and the presence of Y material. SRY does not warrant a complete and normal testis differentiation. Although similar in some features with Klinefelter's syndrome patients, XX males exhibit specific clinical manifestations due to the lack of Y-specific genes.     

Familial acromegaly - case study of two sisters with acromegaly

In the majority of cases, acromegaly is sporadic. However, it can also occur in a familial setting as a component of MEN-1, MEN-4, Carney complex (CNC) or as the extremely rare syndrome of isolated familial somatotropinoma (IFS), the latter belonging to familial isolated pituitary adenomas (FIPA). The diagnosis of IFS is based on the recognition of acromegaly/gigantism in at least two family members, given that the family is not affected by MEN-1, MEN-4 or CNC. The authors present a case study of two sisters: a 56 year-old patient (case no. 1) and a 61 year-old patient (case no. 2). In both sisters, acromegaly was recognised in the course of pituitary macroadenoma. Neither of the sisters showed features of MEN-1, MEN-4 or Carney complex. The authors suppose that the presented cases are manifestations of IFS. However, this diagnosis has not been confirmed yet because of the poor availability of genetic tests.     

New phenotypes associated with mucAB: alteration of a MucA sequence homologous to the LexA cleavage site.

Most mutagenesis by UV and many chemicals in Escherichia coli requires the products of the umuDC operon or an analogous plasmid-derived operon mucAB. Activated RecA protein is also required for, or enhances, this process. MucA and UmuD proteins share homology with the LexA protein, suggesting that they might interact with the RecA protein as LexA does. We used oligonucleotide-directed mutagenesis to alter a site in MucA homologous to the Ala-Gly cleavage site of LexA. The mutation, termed mucA101(Glu26), results in a change of Gly26 of MucA to Glu26. A lexA(Def) recA441 umuC122::Tn5 strain carrying a mucA101(Glu26)B+ plasmid did not exhibit the greatly increased frequency of spontaneous mutagenesis in response to RecA activation that a strain carrying a mucA+B+ plasmid did but retained a basal recA-dependent ability to confer increased spontaneous mutagenesis that was independent of the state of RecA activation. These results are consistent with a model in which RecA plays two distinct roles in mutagenesis apart from its role in the cleavage of LexA. A pBR322-derived plasmid carrying mucA+B+, but not one carrying mucA101(Glu26)B+, inhibited the UV induction of SOS genes, suggesting that MucA+ and MucA(Glu26) proteins may have different abilities to compete with LexA for activated RecA protein. The spectrum of UV-induced mutagenesis was also altered in strains carrying the mucA101(Glu26) mutation. These results are consistent with the hypothesis that activated RecA protein interacts with wild-type MucA protein, possibly promoting proteolytic cleavage, and that this interaction is responsible for facilitating certain mutagenic processes.     

Nucleotide sequence of the traD region in the Escherichia coli F sex factor

The complete nucleotide sequence has been determined of a 3635-bp region, extending from the HpaI site in traT, at F coordinate 90.3 kb, to beyond the end of traD, of the F sex factor plasmid of Escherichia coli K-12. This region contains the C-terminal coding part of traT and the entire traD gene. An open reading frame (ORF) of 2148 bp within the sequence confirms that traD encodes an 81.4-kDa cytoplasmic membrane protein. The TraD protein has several regions with an unusually high pI (greater than 10), suggesting that they may correspond to the DNA-binding domains. Several other ORFs were detected within the region including the gene (ORF1) for a 26.3-kDa protein and ORF2, probably corresponding to traI, which continues to the end of the sequence. An ORF for an 8.5-kDa protein preceded by an excellent promoter and ribosome-binding site is present in the region following traD but on the opposite strand. This promoter is thought to correspond to the major RNA polymerase binding site in this region, implying that traI does not have its own promoter. The lack of a typical terminator following traD and ORF1 and the translational coupling provided by overlapping stop and start codons is consistent with this conclusion.     

RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.     

Single nucleotide polymorphisms in the mitochondrial control region are associated with metabolic phenotypes and oxidative stress

Objective

To identify the mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) in the control region and elucidate their role in metabolic phenotypes and oxidative stress.

Methods

A total of 861 nondiabetic subjects were enrolled, including 250 impaired fasting glucose (IFG) and 370 obese subjects (body mass index [BMI] > 25 kg/m²). Antioxidant status presented as total free thiol level was determined from serum samples. DNA was extracted from peripheral blood leucocytes, and the sequences were analyzed using the DNASTAR software. SNPs were identified by comparison with the Cambridge Reference Sequence.

Results

After adjusting odds ratios for age, sex, and BMI, the selected independently significant SNPs indicated 4 susceptible SNPs: SNP-16126C and SNP-16261T, which were related to abdominal obesity (P = 0.009; 0.06); SNP-16390A, related to hypertension (HTN) (P = 0.007); and SNP-16092C, related to decreased antioxidant capacity (P = 0.015). In the obese subgroup, 3 susceptible SNPs included SNP-16189C and SNP-16260T, which showed significantly higher IFG prevalence (P = 0.016 and 0.024, respectively), and SNP-16519C, which was significantly higher in the HTN group (P = 0.036). As to protective SNPs, 5 protective SNPs were identified in all subjects but only one SNP-16093C is consistent in obese group, which showed a significantly lower prevalence in patients with abdominal obesity and was associated with a higher antioxidant status (P < 0.001).

Conclusion

SNPs in the mtDNA control region are associated with metabolic phenotypes and oxidative stress markers. Some SNPs are relating to the interaction between obesity and genetic factors. The beneficial effects of these protective SNPs were insignificant and some susceptible SNPs became dominant within the obese subgroup. Subjects harboring these SNPs should avoid excessive weight gain.     

The IL-17F sequence variant is associated with susceptibility to tuberculosis

The interleukin (IL)-17 gene plays a key role in host defence against infections from microbes, including Mycobacterium tuberculosis. Genetic factors contribute to host defence. However, whether genetic variation in IL-17 is associated with altered susceptibility to tuberculosis is unknown. A total of 596 pulmonary tuberculosis (PTB) patients, 176 extra-pulmonary tuberculosis (EPTB) patients, and 622 control patients from a Chinese Han population were recruited. Two single-nucleotide polymorphisms (SNPs) in IL-17F (rs1889570 and rs763780) and one SNP in IL-17A (rs2275913) were genotyped using the SNaPshot technique. Of the three SNPs in the IL-17 gene tested, there was an increased frequency of the rs1889570 G allele and the rs763780 C allele in the PTB patients and an increased frequency of the rs763780 C allele in the EPTB patients compared with the control patients. There were also significant differences in the distribution of the rs763780 genotype between the PTB and EPTB patients and the controls. The patients who had the CT/TT genotype of the rs763780 SNP were more susceptible to tuberculosis, compared to the CC genotype. There was no significant difference observed between the IL-17 SNPs when the PTB and EPTB patients were compared. Genetic variation in IL-17F is associated with altered susceptibility to tuberculosis and may provide valuable information in the development of tuberculosis.     

Analysis of the sequence and gene products of the transfer region of the F sex factor.

Bacterial conjugation results in the transfer of DNA of either plasmid or chromosomal origin between microorganisms. Transfer begins at a defined point in the DNA sequence, usually called the origin of transfer (oriT). The capacity of conjugative DNA transfer is a property of self-transmissible plasmids and conjugative transposons, which will mobilize other plasmids and DNA sequences that include a compatible oriT locus. This review will concentrate on the genes required for bacterial conjugation that are encoded within the transfer region (or regions) of conjugative plasmids. One of the best-defined conjugation systems is that of the F plasmid, which has been the paradigm for conjugation systems since it was discovered nearly 50 years ago. The F transfer region (over 33 kb) contains about 40 genes, arranged contiguously. These are involved in the synthesis of pili, extracellular filaments which establish contact between donor and recipient cells; mating-pair stabilization; prevention of mating between similar donor cells in a process termed surface exclusions; DNA nicking and transfer during conjugation; and the regulation of expression of these functions. This review is a compendium of the products and other features found in the F transfer region as well as a discussion of their role in conjugation. While the genetics of F transfer have been described extensively, the mechanism of conjugation has proved elusive, in large part because of the low levels of expression of the pilus and the numerous envelope components essential for F plasmid transfer. The advent of molecular genetic techniques has, however, resulted in considerable recent progress. This summary of the known properties of the F transfer region is provided in the hope that it will form a useful basis for future comparison with other conjugation systems.     

Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the late region of the genome.

Two mutants of simian virus 40, dl-1261 and dl-1262, have deletions that map between coordinated 0.90 and 0.95 (Cole et al., J. Virol 24:277--294, 1977). Both affect the structure of the two minor proteins VP2 and VP3. The precise location and size of the deletions have now been determined by nucleotide sequence analysis. Mutant dl-1261 is deleted of 54 base pairs, is temperature sensitive for the protein defined by the D complementation group, and promotes the synthesis of shorter VP2 and VP3 polypeptides. Mutant dl-1262 is viable irrespective of temperature and has a deletion of 36 base pairs, 23 of which overlap the deletion in dl-1261. Since these mutants produce normal VP1, the deleted regions probably have no function in the splicing of precursor RNA to the VP1 mRNA.     

Identical germ-line mutations in the triosephosphate isomerase alleles of two brothers are associated with distinct clinical phenotypes

We describe here a new stop mutation at triosephosphate isomerase (TPI) position 145 in a Hungarian family for which the first mutation (240 Phe-->Leu) was published earlier. The entire genomic TPI locus (exons, introns and promoter) was sequenced and found to be identical in the two compound-heterozygote brothers. Both brothers have the same well-compensated level of non-spherocytic hemolytic anemia and very high levels of the TPI substrate dihydroxyacetonephosphate (DHAP), but only one brother manifests neurologic disorders. Differences in nonsense-mediated mRNA decay may be at the basis of the differences in phenotype expression although it cannot be excluded the interaction with a modifier gene. Based on our earlier results, the development of neurodegeneration may be decisively modulated by the cellular environment of the mutant proteins initiating the process of focal apoptosis of neurons in glycolytic, peroxisomal and prion-induced neurological diseases.     

Multiple phenotypes associated with Myc-induced transformation of chick embryo fibroblasts can be dissociated by a basic region mutation.

Chimaeric alleles were constructed to assay the biological functions of an N-terminal deletion and C-terminal mutations which were found in a naturally occurring mutant of feline vMyc, T17. The mutant alleles were assayed for their ability to transform chick embryo fibroblasts in vitro by a number of criteria, namely the ability to induce morphological transformation, an accelerated growth rate and growth in soft agar. Feline cMyc could transform the avian cells, whilst T17 vMyc could not, and the N-terminal deletion was responsible for conferring the primary transformation defect on the mutant protein. The C-terminal mutations which consist of a point mutation adjacent to the nuclear localisation signal and a point mutation/amino acid insertion within the basic region (BR) could, however, dissociate the Myc-induced parameters of transformation. This effect was a specific function of the BR mutation alone, and the mutation could be transferred into avian cMyc with comparable biological consequences. The BR mutation did not disrupt the sequence specific DNA binding activity of the protein in vivo, despite exerting a biological effect. These data suggest a novel phenotype where the mutation may affect a subset of Myc-regulated genes through altered DNA binding specificity or protein-protein interactions.     

Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, two unique complex rearrangements consisting of contiguous small insertions and deletions were found in exons 1 and 2 causing L45R/H46S and L120X, respectively. CONCLUSIONS: These studies further define the heterogeneity of mutations causing Fabry disease, permit precise carrier identification and prenatal diagnosis in these families, and facilitate the identification of candidates for enzyme replacement therapy.