Eduard Orvisky - Academia.edu (original) (raw)

Papers by Eduard Orvisky

High-molecular-weight hyaluronan : a valuable tool in testing the antioxidative activity of amphiphilic drugs stobadine and vinpocetine

Journal of Pharmaceutical and Biomedical Analysis, 1997

Journal of Pharmaceutical Sciences, 2011

Silicone oil is often used to decrease glide forces in prefilled syringes and cartridges, common ... more Silicone oil is often used to decrease glide forces in prefilled syringes and cartridges, common primary container closures for biopharmaceutical products. Silicone oil has been linked to inducing protein aggregation (Diabet Med 1989;6:278; Diabet Care 1987;10:786-790), leading to patient safety and immunogenicity concerns. Because of the silicone oil application process (Biotech Adv 2007;25:318-324), silicone oil levels tend to vary between individual container closures. Various silicone oil levels were applied to a container closure prior to filling and lyophilization of an albumin and interferon alfa-2b fusion protein (albinterferon alfa-2b). Data demonstrated that high silicone oil levels in combination with intended and stress storage conditions had no impact on protein purity, higher order structure, stability trajectory, or biological activity. Subvisible particulate analysis (1-10 :m range) from active and placebo samples from siliconized glass barrels showed similar particle counts. Increases in solution turbidity readings for both active and placebo samples correlated well with increases in silicone oil levels, suggesting that the particles in solution are related to the presence of silicone oil and not large protein aggregates. Results from this study demonstrate that silicone oil is not always detrimental to proteins; nevertheless, assessing the impact of silicone oil on a product case-by-case basis is still recommended.

Journal of Medical Genetics, 2005

Background: Gaucher disease is classified into neuronopathic and non-neuronopathic forms with wid... more Background: Gaucher disease is classified into neuronopathic and non-neuronopathic forms with wide phenotypic variation among patients sharing the same genotype. While homozygosity for the common L444P allele usually correlates with the neuronopathic forms, how a defined genotype leads to a phenotype remains unknown. Methods: The genetic and epigenetic factors causing phenotypic differences were approached by a clinical association study in 32 children homozygous for the point mutation L444P. Direct sequencing and Southern blots were utilised to establish the genotype and exclude recombinant alleles. Glucocerebrosidase activity was measured in lymphoblast and fibroblast cell lines. Results: Residual enzyme activity was highly variable and did not correlate with the observed clinical course. There was also a wide spectrum of phenotypes. Average age at diagnosis was 15 months, and slowed saccadic eye movements were the most prevalent finding. The most severe systemic complications and highest mortality occurred in splenectomised patients before the advent of enzyme replacement therapy (ERT). On ERT, as morbidity and mortality decreased, developmental and language deficits emerged as a major issue. Some trends related to ethnic background were observed. Conclusion: The wide clinical spectrum observed in the L444P homozygotes implicates the contribution of genetic modifiers in defining the phenotype in Gaucher disease. G aucher disease (MIM 230800, 230900, 2301000), the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GBA; EC 3.2.1.45), results from an abnormal accumulation of the lipid substrate glucocerebroside. This multi-system disease presents with a vast spectrum of phenotypes, including a number of patients who defy categorisation into the classic three types, non-neuronopathic (type 1) and neuronopathic (types 2 and 3), yet represent a part of the phenotypic continuum. 1 2 While it has been suggested that factors such as the environment or modifier genes contribute to the complicated phenotypes associated with Gaucher disease, the impact of these factors still remains elusive. Chronic neuronopathic, or type 3, Gaucher disease commonly presents with diverse neurologic features at any time during infancy and early childhood. The most prevalent neurologic finding is a horizontal supranuclear palsy, often seen in association with severe systemic involvement. 3 4 Other patients have mild systemic manifestations and moderate organomegaly, yet develop a slowly progressive neurological disease, with generalised seizures, myoclonus, ataxia, and/or dementia.

Glucocerebrosidase mutation T369M appears to be another polymorphism

Clinical Genetics, 2003

To the Editor: Over 200 different mutations in the gene encoding glucocerebrosidase (EC 3.2.1.45)... more To the Editor: Over 200 different mutations in the gene encoding glucocerebrosidase (EC 3.2.1.45) have been identified in patients with Gaucher disease (MIM 230800). We recently showed that one such mutation, E326K, was probably a polymorphism, because it was found only in patients who carried another glucocerebrosidase mutation on the same allele and was also identified in normal controls (1). We have now observed that another previously identified mutation, T369M (2), an amino acid substitution of methionine for threonine, may also be a polymorphism. This substitution of a T for a C nucleotide at cDNA position 1223 occurs in the last codon of exon 8 of the glucocerebrosidase gene. The original description of this mutation, by Beutler et al. (2), did not explicitly rule out the existence of an additional mutation on the same allele. There was also no published expression data for this mutation. In routine sequencing of the glucocerebrosidase gene in 200 alleles from individuals without Gaucher disease, two alleles (1%) were found to have the T369M mutation. We were intrigued to find that one patient in our series with type 1 Gaucher disease carried an allele with both R120W and T369M mutations, and, in the literature, a second patient with type 1 Gaucher disease carried D409H and T369M mutations on the same allele (3). Of note, both mutations R120W and D409H occur in the glucocerebrosidase pseudogene sequence, whereas T369M does not. Therefore, we set out to determine the frequency of this alteration in patients and controls. DNA from 126 patients and a total of 210 control individuals was examined for the presence of T369M either by direct sequencing or by restriction digestion. Sequencing of the glucocerebrosidase gene was performed as previously described (4). A 417-bp fragment encompassing exon 8 was amplified using the forward primer 50GTTGCATTCTTCCCGTCACC-30 and the reverse primer 50-CTGGACAGGAAGGGCTTCTG-30. The amplification product was digested for 2 h at 37 C with the enzyme NlaIII (New England Biolabs, Beverly, MA) and electrophoresed on an 8% Tris-bonate polyacrylamide (TBE) gel (Invitrogen, Carlsbad, CA). The T369M mutation generates an additional restriction site in exon 8. In the normal allele, the 417-bp band is cleaved into three fragments of 191, 140 and 86 bp in size. When the T369M mutation is present, the 191-bp band is cut further into 102and 89-bp fragments. The presence of the 102-bp band corresponds to an allele with the T369M mutation (Fig. 1). Among the 126 patients with Gaucher disease, only one was found to have the T369M mutation (Table 1). This was the previously identified patient with genotype N370S/R120WþT369M. The control group included 179 Caucasian individuals. The T369M alteration was identified in five of these subjects. It was also found in one out

Cancer Epidemiology Biomarkers & Prevention, 2006

To examine the association between recreational physical activity and invasive colon adenocarcino... more To examine the association between recreational physical activity and invasive colon adenocarcinoma among women enrolled in a prospective cohort study. Methods: 120,147 CTS participants residing in California and ages 20-84 years with no prior history of colon cancer were included in the analyses. Three hundred nine-five were diagnosed with invasive colon cancer between 1996 and 2002. The relative risks associated with lifetime recreational physical activity were estimated using multivariable Cox proportional hazards regression models. Results: Recreational physical activity was not associated with colon cancer risk in the cohort overall. However, physical activity reduced colon cancer risk among post-menopausal women who had never taken estrogen or combined hormone therapy. Women who reported an average lifetime moderate or strenuous recreational physical activity (from high school through age 54 years) of at least 4 hrs/wk/yr had 48% lower colon cancer risk (relative risk 0.52, 95% confidence interval 0.31-0.85) than women with a lifetime average of less than 0.5hr/wk/yr. Risk was not reduced among postmenopausal women with a history of hormone therapy use. We observed no effect modification by age, smoking status, level of folate intake, or body mass index. Summary: These data suggest that lifetime recreational physical activity protects against colon cancer in post-menopausal women who have not taken hormone therapy. Hormone therapy users benefit from a lower colon cancer risk associated with hormone therapy use, but recreational physical activity does not appear to reduce risk further among these women.

Applied Biochemistry and Biotechnology, 1992

ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-... more ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-dimethacrylate and commercially available high-pressure liquid chromatography (HPLC) sorbents Separon HEMA 1000 EL, Separon HEMA 1000 E, and Separon HEMA 1000 EH (Tessek, Prague, CSFR Denmark). Specific, sensitive, and rapid method for determination of immobilized ConA lectin activity was developed, fl-Galactosidase from Aspergilus oryzae oligomannosyl residues was used as specific affinant. After separation of bound and unbound fl-galactosidase, enzyme activity was measured in supernatant and thus immobilized ConA lectin activity was calculated easily. The use of the method for evaluating the properties of immobilized ConA, efficiency of immobilization, specific activity, and thermostability is shown. The method developed could be generalized by using artificially glycosylated enzyme for any lectin.

Proceedings of the 7th annual conference on Genetic and evolutionary computation, 2005

Serum profiling using mass spectrometry is an emerging technology with a great potential to provi... more Serum profiling using mass spectrometry is an emerging technology with a great potential to provide biomarkers for complex diseases such as cancer. However, protein profiles obtained from current mass spectrometric technologies are characterized by their high dimensionality and complex spectra with substantial level of noise. These characteristics have generated challenges in discovery of proteins and protein-profiles that distinguish cancer patients from healthy individuals. This paper proposes a novel machine learning method that combines support vector machines with particle swarm optimization for biomarker discovery. Prior to applying the proposed biomarker selection algorithm, low-level analysis methods are used for smoothing, baseline correction, normalization, and peak detection. The proposed method is applied for biomarker discovery from serum mass spectral profiles of liver cancer patients and controls.

2005 IEEE Symposium on Computational Intelligence in Bioinformatics and Computational Biology, 2005

Mass spectrometric profiles of peptides and proteins obtained by current technologies are charact... more Mass spectrometric profiles of peptides and proteins obtained by current technologies are characterized by complex spectra, high dimensionality, and substantial noise. These characteristics generate challenges in discovery of proteins and protein-profiles that distinguish disease states, e.g. cancer patients from healthy individuals. A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. We present data processing methods and computational intelligence that combines support vector machines (SVM) with particle swarm optimization (PSO) for biomarker selection from MALDI-TOF spectra of enriched serum. SVM classifiers were built for various combinations of m/z windows guided by the PSO algorithm. The method identified mass points that achieved high classification accuracy in distinguishing cancer patients from non-cancer controls. Based on their frequency of occurrence in multiple runs, six m/z windows were selected as candidate biomarkers. These biomarkers yielded 100% sensitivity and 91% specificity in distinguishing liver cancer patients from healthy individuals in an independent dataset. Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.

Biomarker Identification and Rule Extraction from Mass Spectral Serum Profiles

2006 IEEE Symposium on Computational Intelligence and Bioinformatics and Computational Biology, 2006

... The dimension of the spectra was reduced to 23,846 m/z bins. A bin size of 100 ppm was found ... more ... The dimension of the spectra was reduced to 23,846 m/z bins. A bin size of 100 ppm was found adequate. ... REFERENCES [1] Orvisky, E., Drake, SK, Martin, BM, Ressom, H., Varghese, RS, Saha, D., Hortin, GL, Loffredo, CA, and Goldman, R., "Enrichment of low molecular ...

Ant Colony Optimization for Biomarker Identification from MALDI-TOF Mass Spectra

2006 International Conference of the IEEE Engineering in Medicine and Biology Society, 2006

We present a novel method that combines ant colony optimization with support vector machines (ACO... more We present a novel method that combines ant colony optimization with support vector machines (ACO-SVM) to select candidate biomarkers from MALDI-TOF serum profiles of hepatocellular carcinoma (HCC) patients and matched controls. The method identified relevant mass points that achieve high sensitivity and specificity in distinguishing HCC patients from healthy individuals. The results indicate that the MALDI-TOF technology could provide the means to discover novel biomarkers for HCC.

PROTEOMICS, 2006

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins wit... more A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p , 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.

Human Mutation, 2002

Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gauche... more Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gaucher disease. In this study, seven novel missense mutations in the glucocerebrosidase gene (A136E, H162P, K198E, Y205C, F251L, Q350X and I402F) and a splice site mutation (IVS10+2T→A) were identified by direct sequencing of three amplified segments of the glucocerebrosidase gene. Five of the novel mutations were found in patients with neuronopathic forms of Gaucher disease, two of which, K198E and F251L, appear to be associated with type 2 Gaucher disease.

Biochemical and Biophysical Research Communications, 1998

Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previousl... more Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previously isolated and characterized as a single polypeptide chain of 37kDa with one mole of zinc tightly bound to the protein. It exhibited biological activity activating calmodulin dependent bovine brain cAMP phosphodiesterase and was decreased in saliva of patients with loss of taste in whom taste buds showed a specific pathological morphology. Determination of its primary structure by amino acid sequence revealed it was identical with carbonic anhydrase (CA) [EC 4.2.1.1] VI and had two N-linked glycosylation sites. Analysis by reverse phase HPLC and SDS-PAGE before and after deglycosylation confirmed a single peak with molecular weight of the purified protein being 37kDa, the deglycosylated protein, 33kDa. N-linked carbohydrate chains contained N-acetyl glucosamine, galactose, mannose, and fucose interior to di, tri and tetra sialyated termini. By isoelectric focusing five increasingly acidic pI values were determined consistent with addition of sialic acid as the terminal carbohydrate residue on the N-linked glycoforms of the protein. Gustin was found to exhibit CA activity but was inhibited by known CA inhibitors in a different manner than CA I or II. These findings, consistent with analysis of previous investigators, indicate that parotid saliva gustin is CA VI.

Reciprocal and Nonreciprocal Recombination at the Glucocerebrosidase Gene Region: Implications for Complexity in Gaucher Disease

The American Journal of Human Genetics, Mar 31, 2003

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocereb... more Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.

Glucocerebrosidase mutations in subjects with Parkinson's disease

Molecular Genetics and Metabolism

Recent studies showing an association between glucocerebrosidase deficiency and parkinsonism in G... more Recent studies showing an association between glucocerebrosidase deficiency and parkinsonism in Gaucher disease prompted an examination of the glucocerebrosidase gene sequence (GBA) and enzyme activity in brain samples from 57 subjects carrying the diagnosis of Parkinson disease. Alterations in GBA were identified in 12 samples (21%) and were more frequent among the younger subjects. These included eight with mutations (N370S, L444P, K198T, and R329C) and four with probable polymorphisms (T369M and E326K). Our findings suggest that mutations in glucocerebrosidase may be a risk factor for the development of parkinsonism.

2005 IEEE Symposium on Computational Intelligence in Bioinformatics and Computational Biology, 2005

... Fig. 4c shows the binned, normalized, and baseline corrected spectrum. For peak detection, ab... more ... Fig. 4c shows the binned, normalized, and baseline corrected spectrum. For peak detection, abin is identified as a peak if the sign of the intensity's slope changes from positive to negative. ... 10, pp. 860-8, 2004. [19] H. Ressom, RS Varghese, D. Saha, E. Orvisky, L. Goldman, EF ...

Ant Colony Optimization for Biomarker Identification from MALDI-TOF Mass Spectra

2006 International Conference of the IEEE Engineering in Medicine and Biology Society, 2006

We present a novel method that combines ant colony optimization with support vector machines (ACO... more We present a novel method that combines ant colony optimization with support vector machines (ACO-SVM) to select candidate biomarkers from MALDI-TOF serum profiles of hepatocellular carcinoma (HCC) patients and matched controls. The method identified relevant mass points that achieve high sensitivity and specificity in distinguishing HCC patients from healthy individuals. The results indicate that the MALDI-TOF technology could provide the means to discover novel biomarkers for HCC.

PROTEOMICS, 2006

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins wit... more A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p , 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.

Myoclonic Epilepsy in Gaucher Disease: Genotype-Phenotype Insights from a Rare Patient Subgroup

Pediatric Research, 2003

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide s... more Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.

High-molecular-weight hyaluronan : a valuable tool in testing the antioxidative activity of amphiphilic drugs stobadine and vinpocetine

Journal of Pharmaceutical and Biomedical Analysis, 1997

Journal of Pharmaceutical Sciences, 2011

Silicone oil is often used to decrease glide forces in prefilled syringes and cartridges, common ... more Silicone oil is often used to decrease glide forces in prefilled syringes and cartridges, common primary container closures for biopharmaceutical products. Silicone oil has been linked to inducing protein aggregation (Diabet Med 1989;6:278; Diabet Care 1987;10:786-790), leading to patient safety and immunogenicity concerns. Because of the silicone oil application process (Biotech Adv 2007;25:318-324), silicone oil levels tend to vary between individual container closures. Various silicone oil levels were applied to a container closure prior to filling and lyophilization of an albumin and interferon alfa-2b fusion protein (albinterferon alfa-2b). Data demonstrated that high silicone oil levels in combination with intended and stress storage conditions had no impact on protein purity, higher order structure, stability trajectory, or biological activity. Subvisible particulate analysis (1-10 :m range) from active and placebo samples from siliconized glass barrels showed similar particle counts. Increases in solution turbidity readings for both active and placebo samples correlated well with increases in silicone oil levels, suggesting that the particles in solution are related to the presence of silicone oil and not large protein aggregates. Results from this study demonstrate that silicone oil is not always detrimental to proteins; nevertheless, assessing the impact of silicone oil on a product case-by-case basis is still recommended.

Journal of Medical Genetics, 2005

Background: Gaucher disease is classified into neuronopathic and non-neuronopathic forms with wid... more Background: Gaucher disease is classified into neuronopathic and non-neuronopathic forms with wide phenotypic variation among patients sharing the same genotype. While homozygosity for the common L444P allele usually correlates with the neuronopathic forms, how a defined genotype leads to a phenotype remains unknown. Methods: The genetic and epigenetic factors causing phenotypic differences were approached by a clinical association study in 32 children homozygous for the point mutation L444P. Direct sequencing and Southern blots were utilised to establish the genotype and exclude recombinant alleles. Glucocerebrosidase activity was measured in lymphoblast and fibroblast cell lines. Results: Residual enzyme activity was highly variable and did not correlate with the observed clinical course. There was also a wide spectrum of phenotypes. Average age at diagnosis was 15 months, and slowed saccadic eye movements were the most prevalent finding. The most severe systemic complications and highest mortality occurred in splenectomised patients before the advent of enzyme replacement therapy (ERT). On ERT, as morbidity and mortality decreased, developmental and language deficits emerged as a major issue. Some trends related to ethnic background were observed. Conclusion: The wide clinical spectrum observed in the L444P homozygotes implicates the contribution of genetic modifiers in defining the phenotype in Gaucher disease. G aucher disease (MIM 230800, 230900, 2301000), the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GBA; EC 3.2.1.45), results from an abnormal accumulation of the lipid substrate glucocerebroside. This multi-system disease presents with a vast spectrum of phenotypes, including a number of patients who defy categorisation into the classic three types, non-neuronopathic (type 1) and neuronopathic (types 2 and 3), yet represent a part of the phenotypic continuum. 1 2 While it has been suggested that factors such as the environment or modifier genes contribute to the complicated phenotypes associated with Gaucher disease, the impact of these factors still remains elusive. Chronic neuronopathic, or type 3, Gaucher disease commonly presents with diverse neurologic features at any time during infancy and early childhood. The most prevalent neurologic finding is a horizontal supranuclear palsy, often seen in association with severe systemic involvement. 3 4 Other patients have mild systemic manifestations and moderate organomegaly, yet develop a slowly progressive neurological disease, with generalised seizures, myoclonus, ataxia, and/or dementia.

Glucocerebrosidase mutation T369M appears to be another polymorphism

Clinical Genetics, 2003

To the Editor: Over 200 different mutations in the gene encoding glucocerebrosidase (EC 3.2.1.45)... more To the Editor: Over 200 different mutations in the gene encoding glucocerebrosidase (EC 3.2.1.45) have been identified in patients with Gaucher disease (MIM 230800). We recently showed that one such mutation, E326K, was probably a polymorphism, because it was found only in patients who carried another glucocerebrosidase mutation on the same allele and was also identified in normal controls (1). We have now observed that another previously identified mutation, T369M (2), an amino acid substitution of methionine for threonine, may also be a polymorphism. This substitution of a T for a C nucleotide at cDNA position 1223 occurs in the last codon of exon 8 of the glucocerebrosidase gene. The original description of this mutation, by Beutler et al. (2), did not explicitly rule out the existence of an additional mutation on the same allele. There was also no published expression data for this mutation. In routine sequencing of the glucocerebrosidase gene in 200 alleles from individuals without Gaucher disease, two alleles (1%) were found to have the T369M mutation. We were intrigued to find that one patient in our series with type 1 Gaucher disease carried an allele with both R120W and T369M mutations, and, in the literature, a second patient with type 1 Gaucher disease carried D409H and T369M mutations on the same allele (3). Of note, both mutations R120W and D409H occur in the glucocerebrosidase pseudogene sequence, whereas T369M does not. Therefore, we set out to determine the frequency of this alteration in patients and controls. DNA from 126 patients and a total of 210 control individuals was examined for the presence of T369M either by direct sequencing or by restriction digestion. Sequencing of the glucocerebrosidase gene was performed as previously described (4). A 417-bp fragment encompassing exon 8 was amplified using the forward primer 50GTTGCATTCTTCCCGTCACC-30 and the reverse primer 50-CTGGACAGGAAGGGCTTCTG-30. The amplification product was digested for 2 h at 37 C with the enzyme NlaIII (New England Biolabs, Beverly, MA) and electrophoresed on an 8% Tris-bonate polyacrylamide (TBE) gel (Invitrogen, Carlsbad, CA). The T369M mutation generates an additional restriction site in exon 8. In the normal allele, the 417-bp band is cleaved into three fragments of 191, 140 and 86 bp in size. When the T369M mutation is present, the 191-bp band is cut further into 102and 89-bp fragments. The presence of the 102-bp band corresponds to an allele with the T369M mutation (Fig. 1). Among the 126 patients with Gaucher disease, only one was found to have the T369M mutation (Table 1). This was the previously identified patient with genotype N370S/R120WþT369M. The control group included 179 Caucasian individuals. The T369M alteration was identified in five of these subjects. It was also found in one out

Cancer Epidemiology Biomarkers & Prevention, 2006

To examine the association between recreational physical activity and invasive colon adenocarcino... more To examine the association between recreational physical activity and invasive colon adenocarcinoma among women enrolled in a prospective cohort study. Methods: 120,147 CTS participants residing in California and ages 20-84 years with no prior history of colon cancer were included in the analyses. Three hundred nine-five were diagnosed with invasive colon cancer between 1996 and 2002. The relative risks associated with lifetime recreational physical activity were estimated using multivariable Cox proportional hazards regression models. Results: Recreational physical activity was not associated with colon cancer risk in the cohort overall. However, physical activity reduced colon cancer risk among post-menopausal women who had never taken estrogen or combined hormone therapy. Women who reported an average lifetime moderate or strenuous recreational physical activity (from high school through age 54 years) of at least 4 hrs/wk/yr had 48% lower colon cancer risk (relative risk 0.52, 95% confidence interval 0.31-0.85) than women with a lifetime average of less than 0.5hr/wk/yr. Risk was not reduced among postmenopausal women with a history of hormone therapy use. We observed no effect modification by age, smoking status, level of folate intake, or body mass index. Summary: These data suggest that lifetime recreational physical activity protects against colon cancer in post-menopausal women who have not taken hormone therapy. Hormone therapy users benefit from a lower colon cancer risk associated with hormone therapy use, but recreational physical activity does not appear to reduce risk further among these women.

Applied Biochemistry and Biotechnology, 1992

ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-... more ConA was immobilized on an epoxy-activated copolymer of 2-hydroxyethyl-methacrylate and ethylene-dimethacrylate and commercially available high-pressure liquid chromatography (HPLC) sorbents Separon HEMA 1000 EL, Separon HEMA 1000 E, and Separon HEMA 1000 EH (Tessek, Prague, CSFR Denmark). Specific, sensitive, and rapid method for determination of immobilized ConA lectin activity was developed, fl-Galactosidase from Aspergilus oryzae oligomannosyl residues was used as specific affinant. After separation of bound and unbound fl-galactosidase, enzyme activity was measured in supernatant and thus immobilized ConA lectin activity was calculated easily. The use of the method for evaluating the properties of immobilized ConA, efficiency of immobilization, specific activity, and thermostability is shown. The method developed could be generalized by using artificially glycosylated enzyme for any lectin.

Proceedings of the 7th annual conference on Genetic and evolutionary computation, 2005

Serum profiling using mass spectrometry is an emerging technology with a great potential to provi... more Serum profiling using mass spectrometry is an emerging technology with a great potential to provide biomarkers for complex diseases such as cancer. However, protein profiles obtained from current mass spectrometric technologies are characterized by their high dimensionality and complex spectra with substantial level of noise. These characteristics have generated challenges in discovery of proteins and protein-profiles that distinguish cancer patients from healthy individuals. This paper proposes a novel machine learning method that combines support vector machines with particle swarm optimization for biomarker discovery. Prior to applying the proposed biomarker selection algorithm, low-level analysis methods are used for smoothing, baseline correction, normalization, and peak detection. The proposed method is applied for biomarker discovery from serum mass spectral profiles of liver cancer patients and controls.

2005 IEEE Symposium on Computational Intelligence in Bioinformatics and Computational Biology, 2005

Mass spectrometric profiles of peptides and proteins obtained by current technologies are charact... more Mass spectrometric profiles of peptides and proteins obtained by current technologies are characterized by complex spectra, high dimensionality, and substantial noise. These characteristics generate challenges in discovery of proteins and protein-profiles that distinguish disease states, e.g. cancer patients from healthy individuals. A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. We present data processing methods and computational intelligence that combines support vector machines (SVM) with particle swarm optimization (PSO) for biomarker selection from MALDI-TOF spectra of enriched serum. SVM classifiers were built for various combinations of m/z windows guided by the PSO algorithm. The method identified mass points that achieved high classification accuracy in distinguishing cancer patients from non-cancer controls. Based on their frequency of occurrence in multiple runs, six m/z windows were selected as candidate biomarkers. These biomarkers yielded 100% sensitivity and 91% specificity in distinguishing liver cancer patients from healthy individuals in an independent dataset. Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.

Biomarker Identification and Rule Extraction from Mass Spectral Serum Profiles

2006 IEEE Symposium on Computational Intelligence and Bioinformatics and Computational Biology, 2006

... The dimension of the spectra was reduced to 23,846 m/z bins. A bin size of 100 ppm was found ... more ... The dimension of the spectra was reduced to 23,846 m/z bins. A bin size of 100 ppm was found adequate. ... REFERENCES [1] Orvisky, E., Drake, SK, Martin, BM, Ressom, H., Varghese, RS, Saha, D., Hortin, GL, Loffredo, CA, and Goldman, R., "Enrichment of low molecular ...

Ant Colony Optimization for Biomarker Identification from MALDI-TOF Mass Spectra

2006 International Conference of the IEEE Engineering in Medicine and Biology Society, 2006

We present a novel method that combines ant colony optimization with support vector machines (ACO... more We present a novel method that combines ant colony optimization with support vector machines (ACO-SVM) to select candidate biomarkers from MALDI-TOF serum profiles of hepatocellular carcinoma (HCC) patients and matched controls. The method identified relevant mass points that achieve high sensitivity and specificity in distinguishing HCC patients from healthy individuals. The results indicate that the MALDI-TOF technology could provide the means to discover novel biomarkers for HCC.

PROTEOMICS, 2006

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins wit... more A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p , 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.

Human Mutation, 2002

Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gauche... more Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gaucher disease. In this study, seven novel missense mutations in the glucocerebrosidase gene (A136E, H162P, K198E, Y205C, F251L, Q350X and I402F) and a splice site mutation (IVS10+2T→A) were identified by direct sequencing of three amplified segments of the glucocerebrosidase gene. Five of the novel mutations were found in patients with neuronopathic forms of Gaucher disease, two of which, K198E and F251L, appear to be associated with type 2 Gaucher disease.

Biochemical and Biophysical Research Communications, 1998

Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previousl... more Gustin, a zinc-metalloprotein constituting about 3% of human parotid saliva protein was previously isolated and characterized as a single polypeptide chain of 37kDa with one mole of zinc tightly bound to the protein. It exhibited biological activity activating calmodulin dependent bovine brain cAMP phosphodiesterase and was decreased in saliva of patients with loss of taste in whom taste buds showed a specific pathological morphology. Determination of its primary structure by amino acid sequence revealed it was identical with carbonic anhydrase (CA) [EC 4.2.1.1] VI and had two N-linked glycosylation sites. Analysis by reverse phase HPLC and SDS-PAGE before and after deglycosylation confirmed a single peak with molecular weight of the purified protein being 37kDa, the deglycosylated protein, 33kDa. N-linked carbohydrate chains contained N-acetyl glucosamine, galactose, mannose, and fucose interior to di, tri and tetra sialyated termini. By isoelectric focusing five increasingly acidic pI values were determined consistent with addition of sialic acid as the terminal carbohydrate residue on the N-linked glycoforms of the protein. Gustin was found to exhibit CA activity but was inhibited by known CA inhibitors in a different manner than CA I or II. These findings, consistent with analysis of previous investigators, indicate that parotid saliva gustin is CA VI.

Reciprocal and Nonreciprocal Recombination at the Glucocerebrosidase Gene Region: Implications for Complexity in Gaucher Disease

The American Journal of Human Genetics, Mar 31, 2003

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocereb... more Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.

Glucocerebrosidase mutations in subjects with Parkinson's disease

Molecular Genetics and Metabolism

Recent studies showing an association between glucocerebrosidase deficiency and parkinsonism in G... more Recent studies showing an association between glucocerebrosidase deficiency and parkinsonism in Gaucher disease prompted an examination of the glucocerebrosidase gene sequence (GBA) and enzyme activity in brain samples from 57 subjects carrying the diagnosis of Parkinson disease. Alterations in GBA were identified in 12 samples (21%) and were more frequent among the younger subjects. These included eight with mutations (N370S, L444P, K198T, and R329C) and four with probable polymorphisms (T369M and E326K). Our findings suggest that mutations in glucocerebrosidase may be a risk factor for the development of parkinsonism.

2005 IEEE Symposium on Computational Intelligence in Bioinformatics and Computational Biology, 2005

... Fig. 4c shows the binned, normalized, and baseline corrected spectrum. For peak detection, ab... more ... Fig. 4c shows the binned, normalized, and baseline corrected spectrum. For peak detection, abin is identified as a peak if the sign of the intensity's slope changes from positive to negative. ... 10, pp. 860-8, 2004. [19] H. Ressom, RS Varghese, D. Saha, E. Orvisky, L. Goldman, EF ...

Ant Colony Optimization for Biomarker Identification from MALDI-TOF Mass Spectra

2006 International Conference of the IEEE Engineering in Medicine and Biology Society, 2006

We present a novel method that combines ant colony optimization with support vector machines (ACO... more We present a novel method that combines ant colony optimization with support vector machines (ACO-SVM) to select candidate biomarkers from MALDI-TOF serum profiles of hepatocellular carcinoma (HCC) patients and matched controls. The method identified relevant mass points that achieve high sensitivity and specificity in distinguishing HCC patients from healthy individuals. The results indicate that the MALDI-TOF technology could provide the means to discover novel biomarkers for HCC.

PROTEOMICS, 2006

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins wit... more A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p , 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.

Myoclonic Epilepsy in Gaucher Disease: Genotype-Phenotype Insights from a Rare Patient Subgroup

Pediatric Research, 2003

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide s... more Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.