Genetic heterogeneity in propionic acidemia patients with alpha-subunit defects. Identification of five novel mutations, one of them causing instability of the protein

The inherited metabolic disease propionic acidemia (PA) can result from mutations in either of the genes PCCA or PCCB, which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl CoA-carboxylase. In this work we have analyzed the molecular basis of PCCA gene defects, studying mRNA levels and identifying putative disease causing mutations. A total of 10 different mutations, none predominant, are present in a sample of 24 mutant alleles studied. Five novel mutations are reported here for the first time. A neutral polymorphism and a variant allele present in the general population were also detected. To examine the effect of a point mutation (M348K) involving a highly conserved residue, we have carried out in vitro expression of normal and mutant PCCA cDNA and analyzed the mitochondrial import and stability of the resulting proteins. Both wild-type and mutant proteins were imported into mitochondria and processed into the mature form with similar efficiency, but the mature mutant M348K protein decayed more rapidly than did the wild-type, indicating a reduced stability, which is probably the disease-causing mechanism.     

Identification and expression of eight novel mutations among non-Jewish patients with Canavan disease.

Canavan disease is inherited as an autosomal recessive trait that is caused by the deficiency of aspartoacylase (ASPA). The majority of patients with Canavan disease are from an Ashkenazi Jewish background. Mutations in ASPA that lead to loss of enzymatic activity have been identified, and E285A and Y231X are the two predominant mutations that account for 97% of the mutant chromosomes in Ashkenazi Jewish patients. The current study was aimed at finding the molecular basis of Canavan disease in 25 independent patients of non-Jewish background. Eight novel and three previously characterized mutations accounted for 80% (40/50) of mutant chromosomes. The A305E missense mutation accounted for 48% (24/50) of mutant chromosomes in patients of western European descent, while the two predominant Jewish mutations each accounted for a single mutant chromosome. The eight novel mutations identified included 1- and 4-bp deletions (32 deltaT and 876 deltaAGAA, respectively) and I16T, G27R, D114E, G123E, C152Y, and R168C missense mutations. The homozygous 32 deltaT deletion was identified in the only known patient of African-American origin with Canavan disease. The heterozygosity for 876 deltaAGAA mutation was identified in three independent patients from England. Six single-base changes leading to missense mutations were identified in patients from Turkey (D114E, R168C), The Netherlands (I16T), Germany (G27R), Ireland (C152Y), and Canada (G123E). A PCR-based protocol is described that was used to introduce mutations in wild-type cDNA. In vitro expression of mutant cDNA clones demonstrated that all of these mutations led to a deficiency of ASPA and should therefore result in Canavan disease.     

A Swiss family with hemoglobin P Galveston beta117His leads to Arg, including two patients with hb P/beta thalassemia.

The mutant Hb P Galveston (beta117His leads to Arg) is observed in two heterozygotes for beta thalassemia and by itself does not cause clinical symptoms. Some of the physico-chemical properties of Hb P Galveston are identical to the onemical properties of Hb P Galveston are identical to the ones hemoglobin Zurich (beta 63 His leads to Arg) so that only a detailed analysis led to its proper identification.     

Identification and complementation of frameshift mutations associated with loss of cytadherence in Mycoplasma pneumoniae.

Mycoplasma pneumoniae cytadherence is mediated by a specialized, polar attachment organelle. Certain spontaneously arising cytadherence mutants (designated class I) lack HMW2, fail to localize the adhesin protein P1 to the attachment organelle, and exhibit accelerated turnover of proteins HMW1, HMW3, and P65. Insertional inactivation of hmw2 by Tn4001 results in a phenotype nearly identical to that of the class I mutants, suggesting that the latter may result from a defect in hmw2. In this study, the recombinant wild-type hmw2 allele successfully complemented a class I mutant when introduced by transposon delivery. Synthesis of recombinant HMW2 at wild-type levels resulted in reacquisition of hemadsorption and normal levels of HMW1, HMW3, and P65. Low-level production of HMW2 in some transformants resulted in only an intermediate capacity to hemadsorb. Furthermore, full restoration of HMW1 and P65, but not that of HMW3, was directly proportional to the amount of recombinant HMW2 produced, reflecting the importance of proper stoichiometry for certain cytadherence-associated proteins. The recombinant class I hmw2 allele did not restore cytadherence, consistent with a defect in hmw2 in this mutant. A frameshift was discovered in different oligoadenine tracts in hmw2 from two independent class I mutants. Finally, protein P28 is thought to be the product of internal translation initiation in hmw2. A transposon excision-deletion mutant produced a truncated HMW2 but no P28, consistent with this conclusion. However, this deletion mutant was hemadsorption positive, indicating that P28 may not be required for cytadherence.     

Identification of a mutation in the gene causing hyperkalemic periodic paralysis.

DNA from seven unrelated patients with hyperkalemic periodic paralysis (HYPP) was examined for mutations in the adult skeletal muscle sodium channel gene (SCN4A) known to be genetically linked to the disorder. Single-strand conformation polymorphism analysis revealed aberrant bands that were unique to three of these seven patients. All three had prominent fixed muscle weakness, while the remaining four did not. Sequencing the aberrant bands demonstrated the same C to T transition in all three unrelated patients, predicting substitution of a highly conserved threonine residue with a methionine in a membrane-spanning segment of this sodium channel protein. The observation of a distinct mutation that cosegregates with HYPP in two families and appears as a de novo mutation in a third establishes SCN4A as the HYPP gene. Furthermore, this mutation is associated with a form of HYPP in which fixed muscle weakness is seen.     

Identification of a novel mitochondrial mutation in Dupuytren's disease using multiplex DHPLC

Dupuytren's disease is a familial fibroproliferative disorder of late onset affecting the hands. It is extremely common in individuals of Northern European extraction. Genetic studies have yet to identify the genes involved in the formation of the disease. Mitochondria play a critical role in cell metabolism and apoptosis. It is known that defective mitochondria generate abnormally high levels of reactive oxygen species by means of electron leak and that antioxidant enzyme activities decrease with age in skin fibroblasts. Respiratory function of mitochondria is also impaired in aging human tissues. Oxidative stress and production of free radicals may be important factors in the pathogenesis of Dupuytren's disease. Mitochondrial genes are also included in the regulation of apoptosis. Diseased tissue contains large numbers of myo- fibroblasts, which disappear by apoptosis during normal wound healing. High numbers of mitochondria have been observed in fibroblasts derived from diseased tissue. In the light of this evidence, the mitochondrial genome represents a potential location for candidate susceptibility genes for this late-onset disorder. In this study, the authors investigated the presence of mutations within the mitochondrial genome in 40 subjects; 20 Caucasian Dupuytren's disease patients with a maternally transmitted inheritance pattern and 20 control subjects were matched for age, sex, and race using a multiplex denaturing high-performance liquid chromatography approach. A hitherto unknown heteroplasmic mutation located within the mitochondrial 16s rRNA region was evident in 90 percent of patients and absent from all control subjects (p < 0.001; chi2 = 16.1). This mutation may be important in the pathogenesis of Dupuytren's disease.     

A benign sickle-cell disease in a Saudi subject with beta zero-thalassemia and glucose-6-phosphate dehydrogenase deficiency

Sickle-cell disease with raised fetal hemoglobin is found relatively frequently in the eastern part of the Arabian Peninsula. In contrast to the severe and sometimes life-threatening complications of sickle-cell disease in the black population, Saudi Arabs homozygotes for HbS gene exhibit a mild course for this disease. Here we present a Saudi sickle-cell patient with an unusually low fetal hemoglobin level. Moreover, this individual has beta 0-thalassemia and a deficiency in the enzyme glucose-6-phosphate dehydrogenase. Clinical and hematological examinations revealed a remarkably benign condition. This observation is potentially important since most of the mild clinical symptoms of sickle-cell disease have been attributed to high fetal hemoglobin. Clearly in this case, other factors are operating and may be also operating in those patients with high fetal hemoglobin.     

Identification of a novel IVD mutation in a consanguineous family with isovaleric acidemia

Isovaleric acidemia (IVA) is a rare autosomal recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase encoded by IVD gene. In this case study we report the first Saudi IVA patients from a consanguineous family with a novel transversion (p.G362V) and briefly discuss likely phenotype–genotype correlation of the disease in the Saudi population. We explored the functional consequences of the mutation by using various bioinformatics prediction algorithms and discussed the likely mechanism of the disease caused by the mutation.     

Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90-95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients.     

Four novel GALC gene mutations in two Chinese patients with Krabbe disease

Krabbe disease (OMIM #245200) is a rare autosomal recessive leukodystrophy caused by deficiency of galactocerebrosidase (GALC) activity. We identified four novel mutations of the GALC gene in two unrelated Chinese families with Krabbe disease: one insertion mutation, c.1836_1837insT, and one nonsense mutation, c.599C>A (p.S200X), in an infantile patient, and one deletion mutation, c.1911+1_1911+5delGTAAG, and one missense mutation, c.2041G>A, in an adult late-onset patient. This is the first identification of GALC mutations in the Chinese population.     

Identification of 99 novel mutations in a worldwide cohort of 1,056 patients with a nephronophthisis-related ciliopathy

Nephronophthisis-related ciliopathies (NPHP-RC) are autosomal-recessive cystic kidney diseases. More than 13 genes are implicated in its pathogenesis to date, accounting for only 40 % of all cases. High-throughput mutation screenings of large patient cohorts represent a powerful tool for diagnostics and identification of novel NPHP genes. We here performed a new high-throughput mutation analysis method to study 13 established NPHP genes (NPHP1–NPHP13) in a worldwide cohort of 1,056 patients diagnosed with NPHP-RC. We first applied multiplexed PCR-based amplification using Fluidigm Access-Array? technology followed by barcoding and next-generation resequencing on an Illumina platform. As a result, we established the molecular diagnosis in 127/1,056 independent individuals (12.0 %) and identified a single heterozygous truncating mutation in an additional 31 individuals (2.9 %). Altogether, we detected 159 different mutations in 11 out of 13 different NPHP genes, 99 of which were novel. Phenotypically most remarkable were two patients with truncating mutations in INVS/NPHP2 who did not present as infants and did not exhibit extrarenal manifestations. In addition, we present the first case of Caroli disease due to mutations in WDR19/NPHP13 and the second case ever with a recessive mutation in GLIS2/NPHP7. This study represents the most comprehensive mutation analysis in NPHP-RC patients, identifying the largest number of novel mutations in a single study worldwide.     

Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport.

P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.