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PubMed1999

Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase.

During biosynthesis of bacteriochlorophyll or chlorophyll, three protein subunits of 140, 70, and 42 kDa interact to insert Mg2+ into protoporphyrin IX. The semidominant Chlorina-125, -157, and -161 mutants in barley are deficient in this step and accumulate protoporphyrin IX after feeding on 5-aminolevulinate. Chlorina-125, -157, and -161 are allelic to the recessive xantha-h mutants and contain G559A, G806A, and C271T mutations, respectively. These mutations cause single amino acid substitutions in residues that are conserved in all known primary structures of the 42-kDa subunit. In vitro complementation and reconstitution of Mg-chelatase activity show that the 42-kDa subunits are defective in the semidominant Chlorina mutants. A mutated protein is maintained in the Chlorina plastids, unlike in the xantha-h plastids. Heterozygous Chlorina seedlings have 25-50% of the Mg-chelatase activity of wild-type seedlings. Codominant expression of active and inactive 42-kDa subunits in heterozygous Chlorina seedlings is likely to produce two types of heterodimers between the strongly interacting 42-kDa and 70-kDa subunits. Reduced Mg-chelatase activity is explained by the capacity of heterodimers consisting of mutated 42-kDa and wild-type 70-kDa protein to bind to the 140-kDa subunit. The 42-kDa subunit is similar to chaperones that refold denatured polypeptides with respect to its ATP-to-ADP exchange activity and its ability to generate ATPase activity with the 70-kDa subunit. We hypothesize that the association of the 42-kDa subunit with the 70-kDa subunit allows them to form a specific complex with the 140-kDa subunit and that this complex inserts Mg2+ into protoporphyrin IX.

Amino Acid Sequence↗
PubMed1996

Compound heterozygosity for nonsense and missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa.

Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DNA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5.

Aged↗
PubMed2004

Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies.

OBJECTIVE: There has been considerable interest in understanding determinants of the diverse cardiac phenotypes associated with heterozygous NKX2.5 mutations. We hypothesized that analysis of functional properties of NKX2.5 mutant proteins would result in the ability to classify mutations according to function in a scheme that would help to clarify genotype-phenotype correlations. We analyzed missense mutations in the conserved homeodomain. METHODS: We studied in vitro biochemical characteristics, including nuclear localization, DNA binding, transcriptional activation and protein-protein interaction with transcriptional partners (GATA4, TBX5 and NKX2.5 itself), of eight homeodomain missense mutations. Associated phenotypes include atrioventricular (AV) block (98% penetrance), atrial septal defect (83% penetrance), and additional varied heart malformations. RESULTS: Mutations were present at varied homeodomain locations in the putative nuclear localizing signal (1), helix 2 (1), a turn between helix 2 and 3 (1) and helix 3 (5); a spectrum of biochemical phenotypes was observed. All mutants localized to the nuclei but some exhibited anomalous nuclear distribution. While all mutants exhibited markedly decreased DNA binding and reduced transcriptional activation, interaction with transcriptional partners was varied. CONCLUSION: Each mutant protein had a unique spectrum of observed properties, but our data show that while dominant negative properties could be demonstrated in vitro, the best correlation with clinical phenotypes resulted from the markedly reduced DNA binding shared by all eight homeodomain mutations. This suggests that the principle determinant of the two most common phenotypes associated with homeodomain missense mutations is the total dose of NKX2.5 capable of binding to DNA.

Cell Nucleus↗
PubMed2004

Prophylactic bilateral nephrectomies in two paediatric patients with missense mutations in the WT1 gene.

BACKGROUND: Denys-Drash syndrome (DDS) is associated with mutations of the Wilms' tumour 1 (WT1) gene, and is characterized by pseudohermaphroditism, a progressive glomerulopathy, and the development of Wilms' tumour. More than 90% of patients with DDS who carry constitutional intragenic WT1 mutations are at high risk (90%) for the development of Wilms' tumour. WT1 is a signalling protein with 90% of WT1 mutations occurring in the WT1 zinc finger region as single nucleotide polymorphisms, the majority of which are missense mutations. METHODS: Constitutional DNA was extracted from peripheral blood. Direct sequencing and restriction enzymes were employed to analyse mutations. RESULTS: Two children, 46XY males who had evidence of pseudohermaphroditism, hypogonadism and renal failure with a glomerulopathy atypical for DDS, but no Wilms' tumour or nephroblastomatosis, on investigation, prior to transplant, were identified with missense mutations in the WT1 gene, in exons 8 and 9, respectively. The decision to do prophylactic nephrectomies was based on the genetic identification of WT1 mutations supporting a diagnosis of incomplete DDS, with the potential for increased risk of malignancy with the development of Wilms' tumour. The nephrectomy specimens demonstrated nephrogenic rests (nephroblastomatosis), which have a potential for malignant transformation. CONCLUSIONS: WT1 missense mutations in exons 8 and 9 can be regarded as having the potential for malignant change supporting prophylactic nephrectomy in apparent incomplete DDS patients with end-stage renal disease.

Child↗
PubMed2005

Early onset, non fluctuating spinocerebellar ataxia and a novel missense mutation in CACNA1A gene.

Mutations in the brain-specific P/Q type Ca2+ channel alpha1 subunit gene, CACNA1A, have been identified in three clinically distinct disorders, spinocerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2), and familial hemiplegic migraine type 1 (FHM1). SCA6 is associated with small expansions of a CAG repeat at the 3' end of the gene, while point mutations are mostly responsible for its two allelic disorders, FHMI and EA2. From the electrophysiological point of view, while FHMI mutations lead to a gain of function [Tottene A, Fellin T, Pagnutti S, Luvisetto S, Striessnig J, Fletcher C, et al. Familial hemiplegic migraine mutations increase Ca2+ influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc Natl Acad Sci 99 (20) (2002) 13284-13289.], EA2 mutations usually generate a loss of channel function [Guida S, Trettel F, Pagnutti S, Mantuano E, Tottene A, Veneziano L, et al. Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2. Am J Hum Genet 68 (3) (2001) 759-764, Wappl E, Koschak A, Poteser M, Sinnegger MJ, Walter D, Eberhart A, et al. Functional consequences of P/Q-type Ca2+ channel Cav2.1 missense mutations associated with episodic ataxia type 2 and progressive ataxia. J Biol Chem 277 (9) (2002) 6960-6966.]. In the present study, we describe a child affected by permanent non-fluctuating limb and trunk ataxia with a quite early age of onset. Interestingly, the size of the CACNA1A triplet repeat region in the patient is within the normal range while he carries a novel de novo missense mutation in this gene, p.R1664Q. Although functional data are not available, based on the literature data indicating that severe reductions in P/Q-type channel activity favour episodic and/or progressive ataxic symptoms [Wappl E, Koschak A, Poteser M, Sinnegger MJ, Walter D, Eberhart A, et al. Functional consequences of P/Q-type Ca2+ channel Cav2.1 missense mutations associated with episodic ataxia type 2 and progressive ataxia. J Biol Chem 2002;277(9):6960-6966.], we hypothesize that the functional consequence of the mutation here identified is a partial loss of the Ca channel function. In conclusion, the clinical and molecular findings reported here suggest the opportunity to screen for point mutation in this gene, even patients with a clinical phenotype for some aspects slightly different from the typical picture more commonly associated to SCA6, EA2 or FHM1 diseases.

Age of Onset↗
PubMed1996

Compound heterozygosity for nonsense ans missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa.

Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of a nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5.

Aged↗
PubMed2003

Somatic and germline mosaicism for a R248C missense mutation in FGFR3, resulting in a skeletal dysplasia distinct from thanatophoric dysplasia.

In this communication, we report the identification of a mosaic R248C missense mutation in the IgII-III linker region of the gene encoding the fibroblast growth factor receptor-3 (FGFR3), in an individual who manifests a skeletal dysplasia and epidermal hyperplasia. By means of Denaturing High Performance Liquid Chromatography (DHPLC), we determined that 25% of her lymphocytes are heterozygous for this particular missense mutation in FGFR3, and that 12.5% of her lymphocyte-derived genomic DNA encodes a cysteine residue at this position. The proposita has disproportionate short stature, radial head dislocation, coxa vara, and bowing of some of the long bones, associated with an S-shaped deformity of the humerus, accompanied by widespread acanthosis nigricans in the integument. These features do not match any previously described skeletal dysplasia. Further, the proposita's only pregnancy ended in the delivery of a fetus manifesting a lethal short-limbed dwarfism with pulmonary hypoplasia, strongly suggestive of an undiagnosed thanatophoric dysplasia. These findings confirm the proposita to be a somatic and germline mosaic for this particular missense mutation in FGFR3. Thus far, all reported FGFR3 R248C mutations have resulted in thanatophoric dysplasia type I (TDI).

Acanthosis Nigricans↗
PubMed2003

Heterogeneity in residual function of MeCP2 carrying missense mutations in the methyl CpG binding domain.

Rett syndrome is a neurodevelopmental disorder with severe mental retardation caused by mutations in the MECP2 gene. Mutations in the MECP2 gene are also associated with other genetic disorders, including X linked mental retardation in males. Missense mutations identified so far are present primarily in the methyl CpG binding domain (MBD) of MECP2. Here, the functional significance of 28 MBD missense mutations identified in patients were analysed by transient expression of the mutant proteins in cultured cells. The effects of mutations were evaluated by analysis of the affinity of MeCP2 to pericentromeric heterochromatin in mouse L929 cells and on transcriptional repressive activity of MeCP2 in Drosophila SL2 cells. These analyses showed that approximately one-third (9/28) of MBD missense mutations showed strong impairment of MeCP2 function. The mutation of the R111 residue, which directly interacts with the methyl group of methyl cytosine, completely abolished MeCP2 function and mutations affecting beta-sheets and a hairpin loop have substantial functional consequences. In contrast, mutations that showed marginal or mild impairment of the function fell in unstructured regions with no DNA interaction. Since each of these mutations is known to be pathogenic, the mutations may indicate residues that are important for specific functions of MeCP2 in neurones.

Animals↗
PubMed1997

A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction.

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Acid alpha-glucosidase deficiency: identification and expression of a missense mutation (S529V) in a Japanese adult phenotype.

We report a missense mutation in an adult Japanese patient with acid alpha-glucosidase (GAA) deficiency. A TC to GT transition at nucleotides 1585-1586, was identified. This transition resulted in an amino acid substitution of Ser-529 to Val (S529V) in exon 11. We also have demonstrated that the S529V mutation abolishes the catalytic activity of the enzyme. Our data suggest that this mutation is the cause of the clinical manifestation known as adult-onset GAA deficiency. The missense mutation described here is a new mutation, and the first identified in Japanese patients with GAA deficiency.

Adult↗
PubMed2005

Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass-associated missense mutations in LRP5 affect canonical Wnt signaling.

The low-density-lipoprotein receptor-related protein 5 (LRP5), a coreceptor in the canonical Wnt signaling pathway, has been implicated in human disorders of low and high bone mass. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome, and heterozygous missense mutations in families segregating autosomal dominant high bone mass (HBM) phenotypes have been identified. We expressed seven different HBM-LRP5 missense mutations to delineate the mechanism by which they alter Wnt signaling. None of the mutations caused activation of the receptor in the absence of ligand. Each mutant receptor was able to reach the cell surface, albeit at differing amounts, and transduce exogenously supplied Wnt1 and Wnt3a signal. All HBM mutant proteins had reduced physical interaction with and reduced inhibition by DKK1. These data suggest that HBM mutant proteins can transit to the cell surface in sufficient quantity to transduce Wnt signal and that the likely mechanism for the HBM mutations' physiologic effects is via reduced affinity to and inhibition by DKK1.

Animals↗
PubMed1998

Functional characterization of missense mutations in ATP7B: Wilson disease mutation or normal variant?

Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.

Adenosine Triphosphatases↗
PubMedDate not supplied

Acute intermittent porphyria: novel missense mutations in the human hydroxymethylbilane synthase gene.

PURPOSE: To identify mutations in families with acute intermittent porphyria, an autosomal dominant inborn error of metabolism that results from the half-normal activity of the third enzyme in the heme biosynthetic pathway, hydroxymethylbilane synthase. METHODS: Mutations were identified by direct solid phase sequencing. RESULTS: Two novel missense mutations E80G and T78P and three previously reported mutations, R173W, G111R, and the splice site lesion, IVS1+1, were detected, each in an unrelated proband. The causality of the novel missense mutations was demonstrated by expression studies. CONCLUSION: These findings provide for the precise diagnosis of carriers in these families and further expand the molecular heterogeneity of AIP.

Amino Acid Substitution↗
PubMed2001

Analyses of the effects that disease-causing missense mutations have on the structure and function of the winged-helix protein FOXC1.

Five missense mutations of the winged-helix FOXC1 transcription factor, found in patients with Axenfeld-Rieger (AR) malformations, were investigated for their effects on FOXC1 structure and function. Molecular modeling of the FOXC1 forkhead domain predicted that the missense mutations did not alter FOXC1 structure. Biochemical analyses indicated that, whereas all mutant proteins correctly localize to the cell nucleus, the I87M mutation reduced FOXC1-protein levels. DNA-binding experiments revealed that, although the S82T and S131L mutations decreased DNA binding, the F112S and I126M mutations did not. However, the F112S and I126M mutations decrease the transactivation ability of FOXC1. All the FOXC1 mutations had the net effect of reducing FOXC1 transactivation ability. These results indicate that the FOXC1 forkhead domain contains separable DNA-binding and transactivation functions. In addition, these findings demonstrate that reduced stability, DNA binding, or transactivation, all causing a decrease in the ability of FOXC1 to transactivate genes, can underlie AR malformations.

Amino Acid Sequence↗
PubMed2000

Novel missense mutations in the glutamate dehydrogenase gene in the congenital hyperinsulinism-hyperammonemia syndrome.

OBJECTIVES: The objectives of this study were to clarify the involvement of the glutamate dehydrogenase gene in congenital hyperinsulinemia-hyperammonemia syndrome (CHHS) and the relationships between the mutation of the gene and clinical severity. STUDY DESIGN: Five unrelated Japanese patients (3 girls and 2 boys) with CHHS were investigated. All patients had convulsions or loss of consciousness resulting from hypoglycemia at less than 1 year of age. We examined mutations of the glutamate dehydrogenase gene using genomic or reverse-transcriptase polymerase chain reactions, followed by direct sequencing. RESULTS: We identified heterozygous missense mutations in all patients. Three patients had a previously identified mutation (C-->T at nt 1506) at codon 445 in the allosteric domain. Two novel missense mutations were identified in the other patients. These mutations included a change of A-->C at nt 1059 and a change of G-->A at nt 966, within the catalytic domain of the glutamate dehydrogenase gene. The locus of the mutations was not associated with the severity of hypoglycemia. CONCLUSIONS: Our results suggest that structural aberrations of not only the allosteric domain but also the catalytic domain of the glutamate dehydrogenase protein, caused by missense mutations, can result in the development of CHHS.

Adenine↗
PubMed2000

A missense mutation in the OCTN2 gene associated with residual carnitine transport activity.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease can present early in life with hypoketotic hypoglycemia and acute metabolic decompensation, or later in life with skeletal or cardiac myopathy. Mutations abolishing the function of OCTN2, an organic cation/carnitine transporter with twelve putative transmembrane spanning domains, were recently demonstrated in patients with early- and late-onset (up to seven years of age) presentation of this syndrome. Most of the reported mutations are null alleles. Here we evaluate the OCTN2 gene in a male patient who presented at seven years of age with severe dilated cardiomyopathy. Plasma carnitine levels were undetectable and carnitine transport by his fibroblasts was reduced to about 3% of normal controls. This patient was homozygous for a single base pair change in exon 8 of the OCTN2 gene (1354G>A) converting the codon for Glu 452 to Lys (E452K) in the predicted intracellular loop between transmembrane domains 10 and 11. Stable expression of the mutant E452K-OCTN2 cDNA in Chinese hamster ovary (CHO) cells caused a partial increase in carnitine transport to 2-4% of the levels measured in the wild type transporter. This reduced transport activity was associated with normal Km toward carnitine (3.1 +/- 1.1 microM), but markedly reduced Vmax. These results indicate that primary carnitine deficiency can be caused by mutations encoding for carnitine transporters with residual activity, and that the E452K affects a domain not involved in carnitine recognition.

Adult↗
PubMed1999

McArdle's disease associated with homozygosity for the missense mutation Gly204Ser of the myophosphorylase gene in a Spanish patient.

We studied a pateint whose clinical, morphological and biochemical findings were consistent with McArdle's disease. Molecular genetic studies revealed that the patient did not harbor the common Arg49Stop mutation and was homozygous for the Gly204Ser mutation. Until now, no patient having the missense mutation in the two alleles has been documented.

Adult↗