Single Heterozygous Mutation Research Articles

DJ-1 (PARK7) mutations are less frequent than Parkin (PARK2) mutations in early-onset Parkinson disease.

Mutations in the Parkin gene (PARK2) are the most commonly identified cause of recessively inherited early-onset Parkinson disease (EOPD) but account for only a portion of cases. DJ-1 (PARK7) was recently reported as a second gene associated with recessively inherited PD with a homozygous exon deletion and a homozygous point mutation in two families. To investigate the frequency of DJ-1 mutations, the authors performed mutational analysis of all six coding exons of DJ-1 in 100 EOPD patients. For the detection of exon rearrangements, the authors developed a quantitative duplex PCR assay. Denaturing high performance liquid chromatography analysis was used to screen for point mutations and small deletions. Further, Parkin analysis was performed as previously described. The authors identified two carriers of single heterozygous loss-of-function DJ-1 mutations, including a heterozygous deletion of exons 5 to 7 and an 11-base pair deletion, removing the invariant donor splice site in intron 5. Interestingly, both DJ-1 mutations identified in this study were found in the heterozygous state only. The authors also detected a polymorphism (R98Q) in 1.5% of the chromosomes in both the patient and control group. In the same patient sample, 17 cases were detected with mutations in the Parkin gene. Mutations in DJ-1 are less frequent than mutations in Parkin in EOPD patients but should be considered as a possible cause of EOPD. The effect of single heterozygous mutations in DJ-1 on the nigrostriatal system, as described for heterozygous changes in Parkin and PARK6, remains to be elucidated.

Novel Mutant Vasopressin-neurophysin II Gene Associated with Familial Neurohypophyseal Diabetes Insipidus

We describe a novel missense mutant of arginine vasopressin (AVP)-dependent neurohypophyseal diabetes insipidus in an autosomal dominant family. A 54-year-old woman was admitted to our hospital because of thyroidectomy for thyroid cancer. After thyroidectomy she was found to have hypernatremia and polyuria and polydipsia both of which had been present from childhood. She had no obstructive hydronephrosis. Her father, father's younger sister and her third son also had polyuria and polydipsia. Basal plasma AVP concentration at normal plasma osmolality was normal but did not respond to increased plasma osmolality despite hyperosmolality during infusion of hypertonic saline infusion, indicating that plasma AVP secretion was impaired. Sodium concentration in urine and urine osmolality were low and increased after nasal administration of DDAVP. There was a diminished but bright signal of pituitary posterior gland on magnetic resonance T1 weighted image. Molecular genetic analysis demonstrated that the patient and her son had a single heterozygous missense mutation (G-->A) at nucleotide 1829 in 1 AVP allele, yielding an abnormal AVP precursor with lacking Glu-47 in its neurophysin II moiety. The abnormal AVP precursor may be related to the impaired AVP secretion.

Mutation in the CHAC gene in a family of autosomal dominant chorea-acanthocytosis.

Although mutations in the CHAC gene have been identified in autosomal recessive chorea-acanthocytosis (AR-ChAc), the molecular basis of autosomal dominant ChAc (AD-ChAc) remains to be determined. The authors investigated abnormalities in the CHAC gene in an AD-ChAc family with mRNA and sequencing analyses of mRNA and genomic DNA. A novel single heterozygous mutation in the last nucleotide of exon 57 of the CHAC gene, which could cause skipping of the exon, was detected in affected siblings.

How much phenotypic variation can be attributed to parkin genotype?

To establish phenotype-genotype correlations in early-onset parkinsonism, we have compared the phenotype of a large series of 146 patients with and 250 patients without parkin mutations. Although no single sign distinguished the groups, patients with mutations had significantly earlier and more symmetrical onset, dystonia more often at onset and hyperreflexia, slower progression of the disease, and a tendency toward a greater response to levodopa despite lower doses. After forward stepwise multiple logistic regression analysis, dystonia at onset and brisk reflexes were not longer significantly different but were correlated with age at onset rather than the presence of the parkin mutation. Age at onset in carriers of parkin mutations varied as did the rate of progression of the disease: the younger the age at onset the slower the evolution. The genotype influenced the phenotype: carriers of at least one missense mutation had a higher United Parkinson's Disease Rating Scale motor score than those carrying two truncating mutations. The localization of the mutations was also important because missense mutations in functional domains of parkin resulted in earlier onset. Patients with a single heterozygous mutation had significantly later and more asymmetrical onset and more frequent levodopa-induced fluctuations and dystonia than patients with two mutations.

Double mutations in klotho and osteoprotegerin gene loci rescued osteopetrotic phenotype.

Klotho gene mutant mice (klotho mice, also called kl/kl) exhibit osteopetrosis in the metaphysis of femora and tibiae and die within 3 months. We previously showed by semiquantitative RT-PCR that osteoprotegerin (opg) expression levels in klotho mice were about 2-fold higher than those in wild-type mice in the bone marrow, spleen, and lung. To examine whether the high osteoprotegerin expression levels account for the osteopetrotic phenotype in the klotho homozygous mutant mice in vivo, we made double mutant mice by crossing klotho mutant and osteoprotegerin-deficient mice. Micro computed tomography analysis in the two-dimensional sagittal planes of the metaphyses and cross-sections of femoral midshaft revealed that the abnormally high fractional trabecular bone volume in klotho homozygous mice (kl/kl; 29.71%), which was about 4-fold higher compared with that of wild-type [klotho (+/+) opg (+/+)] mice (7.81%), was rescued by the coexistence of heterozygous mutation in opg gene locus (+/-; 8.36%). Single heterozygous mutation in the opg gene locus alone (without klotho mutation) did not show phenotype (trabecular bone volume, 5.84%; not significantly different from wild type). High levels of osteoprotegerin mRNA expression in the bone marrow in klotho mutant mice were reduced by the heterozygous mutation in the opg gene locus. Furthermore, high osteoprotegerin protein levels in klotho mutant mice were also reduced by the heterozygous mutations in opg gene locus. Thus, elevated levels of osteoprotegerin in mutant mice contribute at least in part to reveal the osteopetrotic phenotype in klotho mice.

Fine structure mapping of CIAS1: identification of an ancestral haplotype and a common FCAS mutation, L353P.

Familial cold autoinflammatory syndrome (FCAS) is an autosomal dominant inflammatory disease with a high degree of penetrance that is characterized by episodes of rash, arthralgia, fever, conjunctivitis, and leukocytosis after generalized exposure to cold. FCAS was previously mapped to a 10-cM region on chromosome 1q44, and subsequently the gene ( CIAS1) responsible for FCAS was identified. In this paper, we describe the physical and genetic mapping of the FCAS locus, and we report a large ancestral haplotype and a new disease-causing mutation. A BAC contig of approximately 3 Mb was developed and subsequently used for high throughput sequencing. We identified a critical region of 4 cM using rare crossover events in four large North American FCAS families. An unusually large shared haplotype (40 cM) was identified in three of the four families. We found a single heterozygous missense mutation (T1058C=L353P) in exon 3 of CIAS1 in all four families that is responsible for the large majority of FCAS cases described in the literature. We also report a comprehensive list of intragenic single nucleotide polymorphisms. The data provided here will assist others researching the 1q44 region and will aid clinicians in the diagnosis of FCAS.

Homozygosity for Multiple Contiguous Single-Nucleotide Polymorphisms as an Indicator of Large Heterozygous Deletions: Identification of a Novel Heterozygous 8-kb Intragenic Deletion (IVS7–19 to IVS15–17) in a Patient with Glycogen Storage Disease Type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.

CNGA3 Mutations in Hereditary Cone Photoreceptor Disorders

We recently showed that mutations in the CNGA3 gene encoding the alpha-subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258 additional independent families with hereditary cone photoreceptor disorders. CNGA3 mutations were detected not only in patients with the complete form of achromatopsia but also in incomplete achromats with residual cone photoreceptor function and (rarely) in patients with evidence for severe progressive cone dystrophy. In total, mutations were identified in 53 independent families comprising 38 new CNGA3 mutations, in addition to the 8 mutations reported elsewhere. Apparently, both mutant alleles were identified in 47 families, including 16 families with presumed homozygous mutations and 31 families with two heterozygous mutations. Single heterozygous mutations were identified in six additional families. The majority of all known CNGA3 mutations (39/46) are amino acid substitutions compared with only four stop-codon mutations, two 1-bp insertions and one 3-bp in-frame deletion. The missense mutations mostly affect amino acids conserved among the members of the cyclic nucleotide gated (CNG) channel family and cluster at the cytoplasmic face of transmembrane domains (TM) S1 and S2, in TM S4, and in the cGMP-binding domain. Several mutations were identified recurrently (e.g., R277C, R283W, R436W, and F547L). These four mutations account for 41.8% of all detected mutant CNGA3 alleles. Haplotype analysis suggests that the R436W and F547L mutant alleles have multiple origins, whereas we found evidence that the R283W alleles, which are particularly frequent among patients from Scandinavia and northern Italy, have a common origin.

Hypofibrinogenemia due to Novel 316 Asp → Tyr Substitution in the Fibrinogen Bβ Chain

We investigated the molecular basis of hypofibrinogenemia in a woman with a plasma fibrinogen of 1.0 mg/mL. After sequencing the coding region and intronic boundaries of all three fibrinogen genes a single heterozygous GAC-->TAC mutation was identified at codon 316 of the Bbeta gene. This Asp-->Tyr substitution segregated with the hypofibrinogenemia in the only other affected family member. Examination by SDS-PAGE, isoelectric focussing, reverse phase chromatography and electrospray ionisation (ESI) mass spectrometery, failed to detect expression of the new Bbeta chain in purified plasma fibrinogen. The absence of the variant chain was confirmed by ESI tryptic mapping; while the [M + 1 H] and [M + 2 H] ions of the affected peptide (MGPTELLIEMEDWK) were clearly visible at 1,692 and 847 m/z, there were no new signals (1,741 or 871 m/z) that would at indicate expression of the variant in plasma. Asp 316 and its gamma chain homologue (Asp 252) are conserved in all known species and this is the first report of a mutation at either of these. The residue appears to be critical in maintaining the structure of the five stranded sheet that forms the dominant structural feature of the D domains.

Fibrinogen Brescia: Hepatic Endoplasmic Reticulum Storage and Hypofibrinogenemia Because of a γ284 Gly→Arg Mutation

The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass inclusions. The hepatic inclusion bodies were weakly periodic acid-Schiff diastase-positive, and on immunoperoxidase staining reacted specifically with anti-fibrinogen antisera. Coagulation investigations revealed low functional and antigenic fibrinogen together with a prolonged thrombin time of 37 seconds (normal, 17 to 22 seconds) suggestive of a hypodysfibrinogenemia. DNA sequencing of all three fibrinogen genes showed a single heterozygous mutation of GGG (Gly)-->CGG (Arg) at codon 284 of the gamma-chain gene. However, examination of purified fibrinogen chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, and isoelectric focusing, failed to show any evidence of the mutant gamma(Br) chain in plasma fibrinogen. This finding was substantiated by electrospray ionization mass spectrometry, which showed only a normal gamma (and Bbeta) chain mass, but a large increase in the portion of their disialo isoforms. We speculate that misfolding of the variant protein causes hepatic retention and the subsequent hypofibrinogenemia, and that the functional defect (dysfibrinogenemia) results from hypersialylation of otherwise normal Bbeta and gamma chains consequent to the liver cirrhosis. These conclusions were supported by studies on six other family members with hypofibrinogenemia, and essentially normal clotting times, who were heterozygous for the gamma284 Gly-->Arg mutation.

Enlarged vestibular aqueduct: a radiological marker of pendred syndrome, and mutation of the PDS gene.

Although the textbook view of Pendred syndrome is that of an autosomal recessive condition characterized by deafness and goitre, it is increasingly clear that not all such patients present this classical clinical picture. Malformations of the inner ear, specifically enlargement of the vestibular aqueduct, are common in Pendred syndrome and mutations in the PDS (Pendred Syndrome) gene have been recorded in patients presenting with deafness and vestibular aqueduct dilatation only, without other features of Pendred syndrome. Since this is the most common radiological malformation of the cochlea in deaf patients, we investigated what proportion of such cases were due to mutation of the PDS gene. We assessed 57 patients referred with radiological evidence of vestibular aqueduct enlargement, by history, clinical examination, perchlorate discharge test and molecular analysis of the PDS locus. Forty-one patients (72%) had unequivocal evidence of Pendred syndrome. The finding of a single heterozygous mutation at the PDS gene in a further eight was strongly suggestive of a critical role for pendrin, the protein product of the PDS gene, in the generation of enlarged vestibular aqueducts in at least 86% (49/57 cases) of patients with this radiological malformation. Securing the diagnosis of Pendred syndrome may be difficult, especially in the single case. Goitre is an inconstant finding, and the perchlorate discharge test, although helpful, is of diagnostic value only if abnormal. Enlargement of the vestibular aqueduct should be considered as the most likely presentation of Pendred syndrome and should prompt specific investigation of that diagnostic possibility. Pendred syndrome might henceforth be recharacterized as deafness with enlargement of the vestibular aqueduct, which is sometimes associated with goitre.

A mutation detected in DNA polymerase delta cDNA from Novikoff hepatoma cells correlates with abnormal catalytic properties of the enzyme.

Tumor development is characterized by accumulation of mutations. Such mutations, if induced by carcinogens in DNA polymerase genes, would confer mutator properties on the DNA replication machinery, even at later stages of development. To investigate whether DNA polymerase delta can be mutated, we compared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibition characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalytic subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prepared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the polymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygous mutation (CGG --> CAG) has been detected in nucleotide position 1948 (codon 648) of the polymerase delta gene from Novikoff cells, resulting in an Arg to Gln change. Position 648 lies just proximal to the conserved region VI, which is part of the "fingers" subdomain of alpha-like polymerases. This subdomain is involved in dNTP binding. Upon comparison of biochemical characteristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K(50) values for nucleotide analogs (e.g. butylphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fidelity was decreased when primer templates containing O(6)-methylguanine were used, and (iv) the activity of DNA polymerase delta from Novikoff tumor cells was less stimulated by lactate dehydrogenase than the enzyme from normal cells. The altered biochemical characteristics of DNA polymerase delta from Novikoff cells suggest mutator properties. We conclude that the point mutation detected in the cDNA might be causally related to the observed changes in inhibition characteristics and copying fidelity.

Missense mutation of amylin gene (S20G) in Japanese NIDDM patients.

Many studies suggest that amylin, which is cosecreted with insulin from islet beta-cells, is a biologically active peptide and modulates plasma glucose levels. We therefore scanned the amylin gene for mutations in 294 Japanese NIDDM patients by single-strand conformational polymorphism, and we found a single heterozygous missense mutation (Ser-->Gly at position 20: S20G mutation) in 12 NIDDM patients (frequency 4.1%). None of the 187 nondiabetic subjects or 59 IDDM patients had the mutation. Of 12 patients carrying the mutation, 8 were diagnosed as having NIDDM at a relatively early age (< or = 35 years), and they had severe diabetes and strong family histories of late-onset NIDDM. On the other hand, the remaining four patients were diagnosed as having NIDDM after age 51, and they had mild diabetes without family histories of diabetes. In high-performance liquid chromatography analysis, a small amount (16%) of amylin immunoreactivity appeared in the position corresponding to normal amylin and a much larger amount (84%) appeared in the position corresponding to mutant amylin. These findings suggest that the S20G mutation of the amylin gene may play a partial role in the pathogenesis of early-onset NIDDM in the Japanese population and may also provide an important model to investigate the true physiological action of amylin.

The Gly40Ser Mutation in the Human Glucagon Receptor Gene Associated With NIDDM Results in a Receptor With Reduced Sensitivity to Glucagon

The pancreatic islet hormone, glucagon, stimulates hepatic glucose production and has also been shown to potentiate glucose-induced insulin secretion. Because glucagon is a key regulator of glucose homeostasis, its receptor, which mediates the actions of glucagon, was considered a candidate gene involved in the pathogenesis of NIDDM. We have previously reported that a single heterozygous missense mutation in exon 2 of the glucagon receptor gene, which changes a glycine to a serine (Gly40Ser), is associated with NIDDM in a French population. In the present study, the signaling properties of this mutant receptor were examined in baby hamster kidney cells and rat insulinoma cells (RIN-5AH) stably transfected with either the wild type or Gly40Ser mutant human glucagon receptor cDNAs. Competition assays using (125)I-labeled glucagon were performed, and in both cell types, the Gly40Ser mutant receptor was found to bind glucagon with an approximately threefold lower affinity compared with the wild type receptor. In both cell types, the production of cAMP in response to glucagon was decreased in cells expressing the mutant receptor compared with those expressing the wild type. Finally, glucagon-stimulated insulin secretion by RIN cells expressing the mutant receptor was decreased such that the dose-response curve was shifted to the right in comparison to that obtained with cells expressing the wild type receptor. These results indicate that this single-point mutation located in the extracellular region of the glucagon receptor decreases the sensitivity of target tissues to glucagon.

Functional properties of a heterozygous mutation (Arg 1174 → Gln) in the tyrosine kinase domain of the insulin receptor from a Type A insulin resistant patient

We analysed the biochemical properties of insulin receptors of a Type A insulin resistant patient with a single heterozygous point mutation substituting Gln for Arg 1174. Insulin binding capacity and affinty to Epstein-Barr virus transformed lymphocytes was normal. Quantitative analysis of autophosphorylation and substrate phosphorylation of soluble insulin receptors isolated from patient cells revealed no differences in the basal state whereas in the presence of insulin autophosphorylation activity was only 30% of control receptors. The stimulation of substrate phosphorylation and down-regulation of receptors on patient cells after chronic exposure to insulin was diminished when compared to controls. We conclude that the heterozygous Arg 1174 mutation does not perturb basal kinase activity but specifically interferes with the kinase activation by insulin and that the mutation has a dominant negative effect on the wild type kinase.