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Biomedical subjects

R J Desnick

Publications and source records attributed to R J Desnick.

At least 73 records · Page 4Linked to original sources
PubMed1999

Cerebral glucose metabolism in type I alpha-N-acetylgalactosaminidase deficiency: an infantile neuroaxonal dystrophy.

Cerebral glucose metabolism was investigated in a 4.8-year-old boy with alpha-N-acetylgalactosaminidase deficiency using 2-[18F]fluoro-2-deoxy-D-glucose and positron emission tomography (PET). In comparison to normal values for age, the overall cerebral glucose metabolism was reduced and the regional cerebral glucose metabolism was decreased in proportion to the degree of atrophy. In the supratentorial cortical regions, the hypometabolism was asymmetric. However, the level of regional cerebral glucose metabolism in all cortical regions excluded a persistent vegetative state. In the lentiform nucleus and the head of the caudate, comparatively increased regional cerebral glucose metabolism was documented, similar to findings in neurodegenerative disorders with active epilepsy. In contrast, the infratentorial structures (cerebellar hemispheres, brain stem, mesencephalon, and hypothalamus), which are predominantly affected by the atrophic process, showed distinct and symmetric hypometabolism. Thus, the 2-[18F]-fluoro-2-deoxy-D-glucose PET scans provided additional insight into and correlation of the functional and structural disturbances in type I alpha-N-acetylgalactosaminidase deficiency, in addition to documenting the hypometabolism due to brain atrophy.

Blood Glucose↗
PubMed1999

Determination of bone markers in pycnodysostosis: effects of cathepsin K deficiency on bone matrix degradation.

Pycnodysostosis (Pycno) is an autosomal recessive osteosclerotic skeletal dysplasia that is caused by the markedly deficient activity of cathepsin K. This lysosomal cysteine protease has substantial collagenase activity, is present at high levels in osteoclasts, and is secreted into the subosteoclastic space where bone matrix is degraded. In vitro studies revealed that mutant cathepsin K proteins causing Pycno did not degrade type I collagen, the protein that constitutes 95% of organic bone matrix. To determine the in vivo effects of cathepsin K mutations on bone metabolism in general and osteoclast-mediated bone resorption specifically, several bone metabolism markers were assayed in serum and urine from seven Pycno patients. Two markers of bone synthesis, type I collagen carboxy-terminal propeptide and osteocalcin, were normal in all Pycno patients. Tartrate-resistent acid phosphatase, an osteoclast marker, was also normal in these patients. Two markers that detect type I collagen telopeptide cross-links from the N and C termini, NTX and CTX, respectively, were low in Pycno. A third marker which detects a more proximal portion of the C terminus of type I collagen in serum, ICTP, was elevated in Pycno, a seemingly paradoxical result. The finding of decreased osteoclast-mediated type I collagen degradation as well as the use of alternative collagen cleavage sites by other proteases, and the accumulation of larger C-terminal fragments containing the ICTP epitope, established a unique biochemical phenotype for Pycno.

Acid Phosphatase↗
PubMed1999

Identification and expression of mutations in the hydroxymethylbilane synthase gene causing acute intermittent porphyria (AIP).

BACKGROUND: Acute intermittent porphyria (AIP), an autosomal dominant inborn error, results from the half-normal activity of the heme biosynthetic enzyme hydroxymethylbilane synthase (EC 4.3.1.8; HMB-synthase). This disease is characterized by acute, life-threatening neurologic attacks that are precipitated by various drugs, hormones, and other factors. The enzymatic and/or biochemical diagnosis of AIP heterozygotes is problematic; therefore, efforts have focused on the identification of HMB-synthase mutations so that heterozygotes can be identified and educated to avoid the precipitating factors. In Spain, the occurrence of AIP has been reported, but the nature of the HMB-synthase mutations causing AIP in Spanish families has not been investigated. Molecular analysis was therefore undertaken in nine unrelated Spanish AIP patients. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and family members of nine unrelated Spanish families with AIP. The HMB-synthase gene was amplified by long-range PCR and the nucleotide sequence of each exon was determined by cycle sequencing. RESULTS: Three new mutations, a missense, M212V; a single base insertion, g4715insT; and a deletion/insertion, g7902ACT-->G, as well as five previously reported mutations (G111R, R116W, R149X R167W, and R173W) were detected in the Spanish probands. Expression of the novel missense mutation M212V in E. coli revealed that the mutation was causative, having <2% residual activity. CONCLUSIONS: These studies identified the first mutations in the HMB-synthase gene causing AIP in Spanish patients. Three of the mutations were novel, while five previously reported lesions were found in six Spanish families. These findings enable accurate identification and counseling of presymptomatic carriers in these nine unrelated Spanish AIP families and further demonstrate the genetic heterogeneity of mutations causing AIP.

Amino Acid Sequence↗
PubMed1999

Twenty novel mutations in the alpha-galactosidase A gene causing Fabry disease.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A). The nature of the molecular lesions in the alpha-Gal A gene in 30 unrelated families was determined to provide precise heterozygote detection, prenatal diagnosis, and define genotype-phenotype correlations. MATERIALS AND METHODS: Genomic DNA was isolated from affected males and/or carrier females from 30 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and automated sequencing. RESULTS: Twenty new mutations were identified, each in a single family: C142R, G183D, S235C, W236L, D244H, P259L, M267I, I289F, Q321E, C378Y, C52X, W277X, IVS4(+4), IVS6(+2), IVS6(-1), 35del13, 256del1, 892ins1, 1176del4, and 1188del1. In the remaining 10 unrelated Fabry families, 9 previously reported mutations were detected: M42V, R112C, S148R, D165V, N215S (in 2 families), Q99X, C142X, R227X, and 1072del3. Haplotype analysis using markers closely flanking the alpha-Gal A gene indicated that the two patients with the N215S lesion were unrelated. The IVS4(+4) mutation was a rare intronic splice site mutation that causes Fabry disease. CONCLUSIONS: These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis, and help delineate phenotype-genotype correlations in this disease. </AB

Alternative Splicing↗
PubMed1998

Congenital erythropoietic porphyria successfully treated by allogeneic bone marrow transplantation.

The long-term biochemical and clinical effectiveness of allogenic bone marrow transplantation (BMT) was shown in a severely affected, transfusion-dependent 18-month-old female with congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis resulting from mutations in the uroporphyrinogen III synthase (URO-synthase) gene. Three years post-BMT, the recipient had normal hemoglobin, markedly reduced urinary porphyrin excretion, and no cutaneous lesions with unlimited exposure to sunlight. The patient was homoallelic for a novel URO-synthase missense mutation, G188R, that expressed less than 5% of mean normal activity in Escherichia coli, consistent with her transfusion dependency. Because the clinical severity of CEP is highly variable, ranging from nonimmune hydrops fetalis to milder, later onset forms with only cutaneous lesions, the importance of genotyping newly diagnosed infants to select severely affected patients for BMT is emphasized. In addition, the long-term effectiveness of BMT in this patient provides the rationale for future hematopoietic stem cell gene therapy in severely affected patients with CEP.

Bone Marrow Transplantation↗
PubMed1998

Genomic structure, chromosomal localization, and expression of human cathepsin W.

A 12 kb genomic fragment containing the entire open reading frame of the human cathepsin W was isolated and the genomic organization of this papain-like protease gene was determined. The 3.8 kb gene was mapped by fluorescence in situ hybridization to chromosome 11q13.1. The gene contained ten exons with introns ranging from 81 to 1119 bp. Four of the nine introns and a 5' untranslated exon were conserved when compared to related genes such as cathepsins L, K and S, whereas there was no similarity to the genomic organization of cathepsins B or C. In contrast to conserved splice site locations in other cysteine protease family members, the cathepsin W gene contained five unique locations. Furthermore, human cathepsin W cDNA was expressed, and was found to be localized within the rough endoplasmic reticulum in transiently transfected Cos-7 and Hela cells.

Cathepsin W↗
PubMed1998

Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility.

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.

Amino Acid Sequence↗
PubMed1998

Prenatal diagnosis and outcome of mosaicism for a de novo unbalanced translocation identified in amniocytes.

Mosaicism for an unbalanced reciprocal translocation was identified in cultured amniocytes of a 16-week-old fetus; mos46,XX,der(4)t(4;5)(q34;q12)/46,XX. Parental karyotypes were normal, indicating a de novo origin of the unbalanced translocation in the fetus. The additional chromosomal material on the der(4) was derived from chromosome 5 as demonstrated by both GTG banding and fluorescence in situ hybridization with a chromosome 5 paint. Two subsequent amniocenteses, at 18 and 20 weeks, confirmed the presence of the abnormal cell line. A percutaneous umbilical blood sample (PUBS) contained only normal cells, 46,XX, and a high resolution ultrasound revealed no fetal abnormalities or growth retardation. The pregnancy was continued and a normal female was born at term. No evidence of the unbalanced translocation cell line was found in cord blood or placental samples at birth. The finding of mosaicism for an unbalanced translocation at amniocentesis is rare, and is associated with a high risk of fetal abnormality. This case illustrates the importance of follow-up studies by PUBS and high-resolution ultrasound for further assessing the risk of phenotypic abnormality.

Adult↗
PubMed1998

Identification of a novel point mutation (S65T) in alpha-galactosidase A gene in Chinese patients with Fabry disease. Mutations in brief no. 169. Online.

Fabry disease is an X-linked inborn error of sphingolipid catabolism resulting from deficient enzyme activity of alpha-galactosidase A. The molecular defects of human alpha-galactosidase A gene causing Fabry disease have been characterized, including gene rearrangement and point mutations, which show the genetic heterogeneity in Fabry disease. To characterize the molecular defects of these patients, each exon of alpha-galactosidase A gene including intron-exon junctions were PCR amplified using biotin-labelled primer and sequenced using magnetic beads solid-phase sequencing. A G to C transversion was identified in the last nucleotide of exon 1 in two unrelated Chinese patients. This mutation obliterates an EcoN1 restriction site. Family studies show close linkage with the affected family members. Screening of 100 alleles (22 males, 39 females) of unrelated normal Chinese can not find this mutation. This mutation not only changes the amino acid from serine to threonine, but also likely cause splicing defects. To our knowledge, this is the first report of mutation in Chinese patients with Fabry disease, and a novel mutation causing Fabry disease not reported in literature previously.

Adult↗
PubMed1998

Effect of the delta-aminolevulinate dehydratase polymorphism on the accumulation of lead in bone and blood in lead smelter workers.

Lead inhibition of the zinc metalloenzyme delta-aminolevulinate dehydratase (ALAD) is one of the most sensitive indicators of blood lead levels. ALAD is polymorphic, with about 20% of Caucasians expressing the rarer ALAD2 allele. Previous studies indicated that this polymorphism may be a genetic factor in lead transport, metabolism, and/or distribution. Whole blood lead, serum lead, and ALAD genotype were determined for 381 lead smelter workers, including 70 workers expressing the ALAD2 allele, whose blood lead elevations were observed for more than 20 years of employment. The same employees demonstrated higher serum lead levels. Using a cumulative blood lead index (CBLI) for each worker, based on individual blood lead histories, and in vivo X-ray fluorescence measurements of bone lead to estimate total lead body burden, the slopes of linear relations of bone lead to CBLI were greater for workers homoallelic for ALAD1, indicating more efficient uptake of lead from blood into bone. This effect was most significant in calcaneus bone and for workers hired since 1977 [ALAD1-1: 0.0528 +/- 0.0028 and ALAD1-2 or 2-2: 0.0355 +/- 0.0031 (P < 0.001)]. Decreased transfer of blood lead into bone in individuals expressing the ALAD2 allele contrasted with increased blood lead. Thus the ALAD genotype affected lead metabolism and potentially modified lead delivery to target organs including the brain; however, the ALAD genotype did not significantly affect the net accumulation of lead in bone.

Amino Acid Sequence↗
PubMed1998

Carrier frequency of the Bloom syndrome blmAsh mutation in the Ashkenazi Jewish population.

Bloom syndrome is more common in individuals of Ashkenazi Jewish descent than in any other population, and one particular mutation in the Bloom syndrome gene, blmAsh, is homozygous in nearly all Ashkenazi Jewish persons with Bloom syndrome. We have determined the frequency of blmAsh in 1491 Ashkenazi Jewish persons with no known history of Bloom syndrome and found that 1 in 107 persons was heterozygous. Although not common, genetic screening for Bloom syndrome is feasible in this population.

Adenosine Triphosphatases↗
PubMed1998

Congenital erythropoietic porphyria: prolonged high-level expression and correction of the heme biosynthetic defect by retroviral-mediated gene transfer into porphyric and erythroid cells.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder resulting from the deficient activity of the heme biosynthetic enzyme uroporphyrinogen III synthase (UROS). Severely affected patients are transfusion dependent and have mutilating cutaneous manifestations. Successful bone marrow transplantation has proven curative, providing the rationale for stem cell gene therapy. Toward this goal, two retroviral MFG vectors containing the UROS cDNA were constructed, one with the wild-type sequence (MFG-UROS-wt) and a second with an optimized Kozak consensus sequence (MFG-UROS-K). Following transduction of CEP fibroblasts, the MFG-UROS-wt and MFG-UROS-K vectors increased the endogenous activity without selection to levels that were 18- and 5-fold greater, respectively, than the mean activity in normal fibroblasts. Notably, the MFG-UROS-wt vector expressed UROS activity in CEP fibroblasts at these high levels for over 6 months without cell toxicity. Addition of either delta-aminolevulinic acid (ALA) or ferric chloride did not affect expression of the transduced UROS gene nor did the increased concentrations of uroporphyrin isomers or porphyrin intermediates affect cell viability. Similarly, transduction of CEP lymphoblasts with the MFG-UROS-wt vector without G418 selection increased the endogenous UROS activity by 7-fold or almost 2-fold greater than that in normal lymphoblasts. Transduction of K562 erythroleukemia cells by cocultivation with the MFG-UROS-wt producer cells increased their high endogenous UROS activity by 1.6-fold without selection. Clonally isolated K562 cells expressed UROS for over 4 months at mean levels 4.7-fold greater than the endogenous activity without cell toxicity. Thus, the prolonged, high-level expression of UROS in transduced CEP fibroblasts and lymphoblasts, as well as in transduced K562 erythroid cells, demonstrated that the enzymatic defect in CEP cells could be corrected by retroviral-mediated gene therapy without selection and that the increased intracellular porphyrin intermediates were not toxic to these cells, even when porphyrin production was stimulated by supplemental ALA or iron. These in vitro studies provide the rationale for ex vivo stem cell gene therapy in severely affected patients with CEP.

Base Sequence↗
PubMed1998

Murine alpha-N-acetylgalactosaminidase: isolation and expression of a full-length cDNA and genomic organization: further evidence of an alpha-galactosidase gene family.

Recent characterization of the human sequences encoding two lysosomal hydrolases, alpha-galactosidase A (alpha-Gal A) and alpha-N-acetylgalactosaminidase (alpha-GalNAc) revealed that these two enzymes with distinct enzymatic activities shared about 50% overall amino acid identity and that their genomic sequences had a conserved common gene structure. These findings suggested that these genes, which are located on different chromosomes, arose by duplication and divergence from a common ancestral gene. To further compare this alpha-galactosidase gene family, the murine alpha-GalNAc cDNA and genomic sequences were isolated and characterized. The full-length cDNA contained an open-reading frame of 1245 bp encoding a 415 amino acid polypeptide and had 5' and 3' untranslated regions of 94 and 333 bp, respectively. The coding region had 81% nucleotide and 81.9% amino acid identities with those of the corresponding human alpha-GalNAc sequence. Northern analysis revealed a single transcript of approximately 1.9 kb. The functional integrity of the cDNA was demonstrated by transient expression in COS-1 cells. The murine alpha-GalNAc genomic sequence spanned approximately 9 kb and was identical in structure with the human alpha-GalNAc gene with eight introns interrupting the coding sequence at identical positions. In addition, the deduced amino acid sequence of the murine alpha-GalNAc gene was highly homologous with alpha-GalNAc and alpha-Gal A genes from other species providing further support for a common evolutionary ancestor of the alpha-galactosidase gene family. The availability of the murine gene will permit additional evolutionary comparisons, structure/function analyses, and the generation of mice with alpha-GalNAc deficiency by gene targeting.

Amino Acid Sequence↗
PubMed1998

Molecular genetics of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of the cytosolic enzyme, uroporphyrinogen III synthase (URO-synthase). The accumulation of the nonphysiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I, leads to the clinical manifestations of CEP. Disease severity in unrelated patients is markedly heterogeneous, ranging from fetal demise or severe transfusion dependency throughout life to milder adult cases with only cutaneous photosensitivity. To date, 18 mutations causing CEP have been described in the URO-synthase gene, including single base substitutions, insertions and deletions, and splicing defects. Most mutations have been identified in one or a few unrelated families with the exception of C73R, L4F, and T228M which occurred in about 33%, 8%, and 7% of the mutant alleles studied, respectively. Prokaryotic expression of the mutant URO-synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for severe to milder phenotypes of this clinically heterogeneous disease. As successful bone marrow transplantation in severely affected patients has proven curative, current efforts are underway to develop hematopoietic stem cell gene therapy for CEP.

Humans↗
PubMed1998

Paternal uniparental disomy for chromosome 1 revealed by molecular analysis of a patient with pycnodysostosis.

Molecular analysis of a patient affected by the autosomal recessive skeletal dysplasia, pycnodysostosis (cathepsin K deficiency; MIM 265800), revealed homozygosity for a novel missense mutation (A277V). Since the A277V mutation was carried by the patient's father but not by his mother, who had two normal cathepsin K alleles, paternal uniparental disomy was suspected. Karyotyping of the patient and of both parents was normal, and high-resolution cytogenetic analyses of chromosome 1, to which cathepsin K is mapped, revealed no abnormalities. Evaluation of polymorphic DNA markers spanning chromosome 1 demonstrated that the patient had inherited two paternal chromosome 1 homologues, whereas alleles for markers from other chromosomes were inherited in a Mendelian fashion. The patient was homoallelic for informative markers mapping near the chromosome 1 centromere, but he was heteroallelic for markers near both telomeres, establishing that the paternal uniparental disomy with partial isodisomy was caused by a meiosis II nondisjunction event. Phenotypically, the patient had normal birth height and weight, had normal psychomotor development at age 7 years, and had only the usual features of pycnodysostosis. This patient represents the first case of paternal uniparental disomy of chromosome 1 and provides conclusive evidence that paternally derived genes on human chromosome 1 are not imprinted.

Adult↗
PubMed1998

Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles.

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Alleles↗