Niemann-Pick disease: a frequent missense mutation in the acid sphingomyelinase gene of Ashkenazi Jewish type A and B patients.

Although the A and B subtypes of Niemann-Pick disease (NPD) both result from the deficient activity of acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) and the lysosomal accumulation of sphingomyelin, they have remarkably distinct phenotypes. Type A disease is a fatal neurodegenerative disorder of infancy, whereas type B disease has no neurologic manifestations and is characterized primarily by reticuloendothelial involvement and survival into adulthood. Both disorders are more frequent among individual of Ashkenazi Jewish ancestry than in the general population. The recent isolation and characterization of cDNA and genomic sequences encoding ASM has facilitated investigation of the molecular lesions causing the NPD subtypes. Total RNA was reverse-transcribed, and the ASM cDNA from an Ashkenazi Jewish type A patient was specifically amplified by the polymerase chain reaction (PCR). Molecular analysis of the PCR products revealed a G----T transversion of nucleotide 1487, which occurred at a CpG dinucleotide and predicted an Arg----Leu substitution in residue 496. Hybridization of PCR-amplified genomic DNA with allele-specific oligonucleotides indicated that the proband was homoallelic for the Arg----Leu substitution and that both parents and several other relatives were heterozygous. This mutation was detected in 32% (10 of 31) of the Ashkenazi Jewish NPD type A alleles studied and occurred in only 5.6% (2 of 36) of ASM alleles from non-Jewish type A patients. Of interest, the Arg----Leu substitution occurred in one of the ASM alleles from the two Ashkenazi Jewish NPD type B patients studied and in none of the ASM alleles of 15 non-Jewish type B patients. In contrast, the mutation was not present in 180 ASM alleles from normal individuals of Ashkenazi Jewish descent. These findings identify a frequent missense mutation among NPD patients of Ashkenazi Jewish ancestry that results in neuronopathic type A disease when homoallelic and can result in the nonneuronopathic type B phenotype when heteroallelic. The identification of this ASM mutation in Ashkenazi Jewish patients should facilitate the prevention of NPD in this population by carrier detection with molecular diagnostic techniques.     

A founder mutation in the MPL gene causes congenital amegakaryocytic thrombocytopenia (CAMT) in the Ashkenazi Jewish population

Congenital amegakaryocytic thrombocytopenia (MIM #604498) (CAMT) is a rare inherited disease presenting as severe thrombocytopenia in infancy. Untreated, many CAMT patients develop aplastic anemia within the first decade of life; the only effective treatment of CAMT is bone marrow transplantation. CAMT is the result of the presence of homozygous or compound heterozygous mutations in the thrombopoietin receptor-encoding gene, MPL. We report here the identification and characterization of a founder mutation in MPL in the Ashkenazi Jewish (AJ) population. This mutation, termed c.79+2T>A, is a T to A transversion in the invariant second base of the intron 1 donor splice site. Analysis of a random sample of 2018 individuals of AJ descent revealed a carrier frequency of approximately 1 in 75. Genotyping of six loci adjacent to the MPL gene in the proband and in the 27 individuals identified as carriers of the c.79+2T>A mutation revealed that the presence of this mutation in the AJ population is due to a single founder. The observed carrier frequency predicts an incidence of CAMT in the AJ population of approximately 1 in 22,500 pregnancies. The identification of this mutation will enable population carrier testing and will facilitate the identification and treatment of individuals homozygous for this mutation.     

A novel ancestral splicing mutation in the multidrug resistance protein 2 gene causes Dubin-Johnson syndrome in Ashkenazi Jewish patients.

Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by chronic conjugated hyperbilirubinemia due to the absence or dysfunction of the multidrug resistance protein 2 (MRP2). We previously identified two distinct ancestral mutations causing DJS in 22 unrelated Iranian and five unrelated Moroccan Jewish patients, respectively. In this study we identified and characterized the mutation causing DJS in Ashkenazi Jewish patients and assessed a possible founder effect. Sequencing of all 32 exons of the MRP2 gene identified a novel IVS8+4A-->G mutation in three unrelated homozygotes. Haplotype analysis using four intragenic dimorphisms disclosed a founder effect for the mutation. RT-PCR and real time PCR analysis of mRNA from one patient revealed three splice variants all leading to frameshifts and predicting premature termination codons. The main splice variant was a consequence of the use of a cryptic donor splice site inside exon 8. Liver biopsy in one patient revealed complete absence of MRP2 from the canalicular membrane of hepatocytes. In conclusion, our results provide strong evidence that an ancestral IVS8+4A-->G mutation causes DJS in Ashkenazi Jewish patients by abolishing normal splicing of intron 8 leading to aberrantly spliced products that predict truncation of MRP2.     

A defect in cholesterol esterification in Niemann-Pick disease (type C) patients.

The demonstration of a defect of cholesterol esterification in a mutant strain of BALB/c mice with an attendant reduction of sphingomyelinase activity [Pentchev, P. G., Boothe, A. D., Kruth, H.S., Weintroub, H., Stivers, J. & Brady, R. O. (1984) J. Biol. Chem. 259, 5784-5791] prompted us to examine the capacity of cultured human Niemann-Pick fibroblasts to esterify exogenously derived cholesterol. Cholesterol was supplied to cell cultures in the form of native or chemically modified, positively charged low density lipoprotein or as non-lipoprotein cholesterol. Cholesterol esterification was not impaired in cell cultures derived from patients with type A or B Niemann-Pick disease. However, esterification of exogenously administered cholesterol was deficient in 20 type C Niemann-Pick cell lines that were available for testing. Fluorescence histochemical staining of unesterified cholesterol in type C cells suggested that these cells were able to internalize and lysosomally process lipoprotein cholesterol. Acyl-CoA:cholesterol acyltransferase activity did not appear deficient in type C cell extracts. The error in cholesterol esterification may provide an opportunity for probing the molecular lesion in this disorder and may afford a useful and reliable means for establishing diagnosis.     

Identification of a common mutation (Gly194Cys) in both Arab Moslem and Ashkenazi Jewish patients with dihydrolipoamide dehydrogenase (E3) deficiency: Possible beneficial effect of vitamin therapy

Dihydrolipoamide dehydrogenase (E3) deficiency with a clinical phenotype and genotype (Gly194Cys homozygous) previously identified only in Ashkenazi Jewish patients, was diagnosed in two Palestinian Arab siblings and two unrelated Ashkenazi Jewish patients. While three of the four patients died in childhood without specific treatment, the surviving patient at age 18 years may have benefited from long-term daily supplementation with a cocktail of riboflavin, biotin, coenzyme Q and carnitine.     

Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.     

A K19E missense mutation in the plasminogen gene is a common cause of familial hypoplasminogenaemia.

The prevalence of familial plasminogen deficiency in Scotland has recently been calculated at 2.9/1000. However, little is known of the molecular genetic background and the frequency of plasminogen gene mutations in most cases of inherited plasminogen deficiency. Having previously identified 28 unrelated subjects with familial plasminogen deficiency from a cohort of 9611 blood donors, we have now reviewed 19 of these 28 subjects and screened the plasminogen gene in 15 subjects with hypoplasminogenaemia (plus five relatives) and four subjects with dysplasminogenaemia for mutations and polymorphisms. A missense mutation K19E in the plasminogen gene was found in 13 of the 15 propositi with hypoplasminogenaemia, in one of these in a homozygous manner. In two subjects with hypoplasminogenaemia, two new mutations (P353A and R471X) were identified. These three different mutations, if inherited in a homozygous or compound-heterozygous manner, may be associated with the development of ligneous conjunctivitis. In four subjects with dysplasminogenaemia, three heterozygous mutations (C548G, n = 1; A601T, n = 1; G693R, n = 2) were found. None of the propositi with plasminogen deficiency developed venous thrombosis at any time. In conclusion, the K19E mutation in the plasminogen gene is a common cause of hypoplasminogenaemia in Scotland, with an estimated prevalence of around 0.14%.     

Identification of a new missense mutation in Japanese phenylketonuric patients

Summary A new missense mutation in the phenylalanine hydroxylase (PAH) gene was identified in 20/30 members of the families of 10 unrelated Japanese phenylketonuria (PKU) patients from Kyushu island. The point mutation was present in 20 of 40 mutant alleles. This was proved by DNA sequence analysis after polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide (ASO) hybridization. This point mutation, an A to G transition at the first base of codon 276 in exon 7, resulted in an amino acid substitution. Methionine was replaced by valine and the mutation was found to be associated with restriction fragment length polymorphism (RFLP) haplotype 4 in the investigated patients. The mutation was not found in 24 unrelated Caucasian patients from different countries. These findings may indicate a founder effect in the transmission of the mutation.     

Identification of the second common Jewish Gaucher disease mutation makes possible population-based screening for the heterozygous state.

Gaucher disease is an autosomal recessive glycolipid storage disease characterized by a deficiency of glucocerebrosidase. The disease is most common in persons of Ashkenazi Jewish ancestry and the most common mutation, accounting for about 75% of the mutant alleles in this population, is known to be an A----G substitution at cDNA nucleotide (nt) 1226. Screening for this disease has not been possible because nearly 25% of the mutant alleles had not been identified, but linkage analysis led to the suggestion that most of these could be accounted for by a single mutation. We now report the discovery of this mutation. The insertion of a single nucleotide, a second guanine at cDNA nt 84 (the 84GG mutation), has been detected in the 5' coding region of the glucocerebrosidase gene. The amount of mRNA produced is shown to be normal but since the frameshift produced early termination, no translation product is seen. This finding is consistent with the virtual absence of antigen found in patients carrying this mutation. The 84GG mutation accounts for most of the previously unidentified Gaucher disease mutations in Jewish patients. The common Jewish mutation at nt 1226, the 84GG mutation, and the less-common mutation at nt 1448 accounted for 95% of all of the Gaucher disease-producing alleles in 71 Jewish patients. This now makes it possible to screen for heterozygotes on a DNA level with a relatively low risk of missing couples at risk for producing infants with Gaucher disease.     

Identification of a common mutation (R245H) in Sanfilippo A patients from the Netherlands

We have identified a common mutation (R245H) in the sulphamidase gene of Sanfilippo syndrome type A (mucopolysaccharidosis type IIIA, MPS IIIA) patients from The Netherlands. Allele-specific oligonucleotide hybridization was used to determine the incidence of this mutation in 45 unrelated MPS IIIA patients from different regions of The Netherlands. R245H was present in 51 alleles, representing 56.7% of the total allelic population. Of 39 patients, for whom we have uniform clinical details, 13 MPS IIIA patients who were homozygous for this common mutation had a more uniform but severe clinical phenotype than the remaining 21 or 5 patients, containing respectively one or no R245H alleles. The R245H allele had a higher prevalence in western rather than eastern regions of The Netherlands.     

Identification of an inframe deletion and a missense mutation in the factor XIIIA gene in two Turkish patients

We report two novel mutations in factor XIIIA (FXIIIA) gene that caused congenital factor XIII deficiency in two unrelated patients. The first alteration, a missense mutation Leu235Arg in exon 6 of FXIIIA gene, is located in the putative calcium-binding part of the core domain of the enzyme. Replacement of non-polar hydrophobic leucine residue with positively charged arginine residue is likely to effect protein folding thus destabilizing the molecule. The second mutation is a 3-bp deletion in exon 14 of FXIIIA gene. This deletion is located in beta barrel 2 domain of the protein and results in translation of an aberrant FXIIIA molecule that lacks lysine residue either at positions 677 or 678. As this inframe deletion is located in a direct repetetive sequence of AAGAAG, that codes for two lysine residues, the exact location of deletion could not be detected.     

Liddle's syndrome caused by a novel missense mutation (P617L) of the epithelial sodium channel beta subunit

OBJECTIVE: The aim of the study was to search for mutations of SCNN1B and SCNN1G in an Italian family with apparently dominant autosomal transmission of a clinical phenotype consistent with Liddle's syndrome. METHODS: Genetic analysis was performed in the proband, his relatives, and 100 control subjects. To determine the functional role of the mutation identified in the proband, we expressed the mutant or wild-type epithelial sodium channel in Xenopus laevis oocytes. RESULTS: A novel point mutation, causing an expected substitution of a leucine residue for the second proline residue of the conserved PY motif (PPP x Y) of the beta subunit was identified in the proband. The functional expression of the mutant epithelial sodium channel in X. laevis oocytes showed a three-fold increase in the amiloride-sensitive current as compared with that of the wild-type channel. CONCLUSION: This newly identified mutation adds to other missense mutations of the PY motif of the beta subunit of the epithelial sodium channel, thus confirming its crucial role in the regulation of the epithelial sodium channel. To our knowledge, this is the first report of Liddle's syndrome in the Italian population, confirmed by genetic and functional analysis, with the identification of a gain-of-function mutation not previously reported.     

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.     

Association of a missense mutation of the laminin alpha2 gene with tuberculoid type of leprosy in Indonesian patients

Leprosy, an infection caused by Mycobacterium leprae, has a specific tropism for the myelinating Schwann cells of peripheral nerves. Recently, the G domain of laminin alpha2 has been shown to be a mediator for M. leprae to bind to alpha-dystroglycan in Schwann cells. In order to analyse the association of leprosy with the mediator, three genetic polymorphisms encoding the G domain of the laminin alpha2 chain were analysed by direct sequencing in 53 leprosy patients and 58 healthy contact individuals from Indonesia. There was no significant difference in the incidence of the polymorphisms between patients and non-patients. Remarkably, it was found that a missense mutation (T7809C) substituting valine with alanine (V2587A) was found to be more frequent in the tuberculoid type than in the lepromatous type leprosy. It is supposed that this missense mutation is one of the determinant factors in the early onset of peripheral nerve damage in Indonesian tuberculoid leprosy patients.     

A missense mutation of the nitric oxide synthase (eNOS) gene (Glu298Asp) in five patients with coronary artery disease--case reports.

The authors report five patients with a missense mutation of the endothelial nitric oxide synthase (eNOS) gene (Glu298Asp) who have angiographically proven coronary artery disease (CAD). They compare their clinical findings and coronary arteriographic characteristics. They conclude that these case reports show that this mutation is not solely responsible for development of CAD. Diabetes mellitus, smoking, and hyperlipidemia are other risk factors.     

Identification of novel mutations in the NPC1 gene in German patients with Niemann-Pick C disease

Niemann–Pick disease type C (NPC) is an inherited neuro degenerative disorder associated with intracellular cholesterol trafficking defects. Mutations in two distinct genes, NPC1 and HE1, have recently been shown to cause this disease. We have analysed the NPC1 gene in five German patients with NPC from four unrelated families. We identified a total of five novel mutations in the coding region of the NPC1 gene (G231V, D874V, I642M, I1094T and R116stop). All affected individuals displayed compound heterozygosity. The mutated alleles were transmitted by the nonaffected parents with the exception of one patient, in whom a de novo mutation (G231V) had occurred. Interestingly, the G231V/P237S NPC1 genotype in this individual is associated with an early-onset form of NPC. In contrast, we found that the D874V/D948N genotype, observed in another NPC patient, is characterized by a late onset of clinical symptoms that presents with a pronounced white-matter disease. Our results will contribute to defining the association between the clinical phenotypes and the genetic abnormalities in Niemann–Pick C disease.     

A common 2 bp deletion mutation in the glucose-6-phosphatase gene in Indian patients with glycogen storage disease type Ia

This study reports a novel mutation which may be prevalent in Indian patients with glycogen storage disease type Ia.     

A missense mutation in the previously undescribed gene Tmhs underlies deafness in hurry-scurry (hscy) mice

Mouse deafness mutations provide valuable models of human hearing disorders and entry points into molecular pathways important to the hearing process. A newly discovered mouse mutation named hurry-scurry (hscy) causes deafness and vestibular dysfunction. Scanning electron microscopy of cochleae from 8-day-old mutants revealed disorganized hair bundles, and by 50 days of age, many hair cells are missing. To positionally clone hscy, 1,160 F(2) mice were produced from an intercross of (C57BL/6-hscy x CAST/EiJ) F(1) hybrids, and the mutation was localized to a 182-kb region of chromosome 17. A missense mutation causing a critical cysteine to phenylalanine codon change was discovered in a previously undescribed gene within this candidate interval. The gene is predicted to encode an integral membrane protein with four transmembrane helices. A synthetic peptide designed from the predicted protein was used to produce specific polyclonal antibodies, and strong immunoreactivity was observed on hair bundles of both inner and outer hair cells in cochleae of newborn +/+ controls and +/hscy heterozygotes but was absent in hscy/hscy mutants. Accordingly, the gene was given the name "tetraspan membrane protein of hair cell stereocilia," symbol Tmhs. Two related proteins (>60% amino acid identity) are encoded by genes on mouse chromosomes 5 and 6 and, together with the Tmhs-encoded protein (TMHS), comprise a distinct tetraspan subfamily. Our localization of TMHS to the apical membrane of inner ear hair cells during the period of stereocilia formation suggests a function in hair bundle morphogenesis.