In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease.

The radiation-inactivation size of membrane-bound acid beta-glucosidase in cultured skin fibroblasts of four normal individuals, five Gaucher type 1 (non-neuropathic), four Gaucher type 2 (acute neuropathic) and three Gaucher type 3 (sub-acute neuropathic) patients was determined using the radiation-inactivation method. The radiation-inactivation size of the enzyme in the control, Gaucher type 2 and Gaucher type 3 fibroblasts ranged from 94 000 to 128 800, and no statistical significant difference was found in the enzyme size between the normal and Gaucher cells nor among the Gaucher type 2 and type 3 cells. Contrary to the normal, Gaucher type 2 and Gaucher type 3 enzyme, the radiation-inactivation size of membrane-bound acid beta-glucosidase in all of the Gaucher type 1 fibroblasts tested is significantly higher, ranging from 158 400 to 235 300. The size of the control lysosomal enzyme, sphingomyelinase, also determined by the radiation-inactivation method in fibroblasts of normal individuals and patients with the three Gaucher subtypes, was between 70 000 and 74 500 and indistinguishable from each other. Since the molecular weight of acid beta-glucosidase subunit determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was about 60 000 (Pentchev, P.G., Brady, R.O., Hibbert, S.P., Gal, A.E. and Shapiro, C. (1973) J. Biol. Chem. 248, 5256-5261), the above data suggest that: (i) the normal fibroblast enzyme, as well as the Gaucher type 2 and type 3 mutant enzyme, in the membrane-bound form, exists as a dimer; (ii) the underlying biochemical and genetic defect in non-neuropathic (type 1) and neuropathic (type 2 and type 3) Gaucher disease is very different from each other; and (iii) subunit interaction of the mutant enzyme may be present in Gaucher type 1 fibroblasts, resulting in the formation of a higher-molecular-weight aggregate.

Gaucher disease: comparative study of acid phosphatase and glucocerebrosidase in normal and type-1 Gaucher tissues.

Acid phosphatase activity was determined in serum, cultured fibroblasts, and peripheral blood lymphocytes of six splenectomized adult patients with non-neuropathic Gaucher disease in two Canadian families. Elevated levels of serum acid phosphatase activity (520-711% of normal) were found in four patients who also developed orthopedic complications associated with Gaucher disease, including intermittent bone pain, arthritis, collapse of femoral head, and pathological fractures. Serum acid phosphatase activity in two patients who do not have bone involvement were found to be within the normal range. Contrary to the serum enzyme, acid phosphatase activity in lymphocytes and cultured fibroblasts of all of the patients was within the normal range. Deficient glucocerebrosidase (7.5-15.5% of normal) and acid beta-glucosidase (13.8-27.8% of normal) activities were noted in all probands. Similarly, normal levels of fibroblast and lymphocyte acid phosphatase activity were found in Gaucher heterozygotes whose glucocerebrosidase activity was about 50% of normal. Acid polyacrylamide gel electrophoresis and acid phosphatase activity staining of the patients' sera showed that the elevated acid phosphatase is isozyme type 5 osteoclastic origin. The apparent Michaelis constant, Km, of fibroblast glucocerebrosidase for the natural substrate was 0.6 +/- 0.1 mM for controls and 0.6 +/- 0.2 mM for the patients. These data suggest that the assay of serum acid phosphatase activity for the presumptive diagnosis of Gaucher disease is not completely reliable and that the elevated level of serum acid phosphatase in Gaucher disease is most likely a secondary phenomenon which may be indicative of bone involvement in some patients with this disorder. It also demonstrates the clinical heterogeneity of type 1 Gaucher disease, even among full sibs of the same heterozygous parents.

Mutation analysis of Gaucher disease using dot-blood samples on FTA filter paper.

FTA((R)) filter papers were used as an effective means of blood cell collection, genomic DNA processing, and delivery. Minute blood samples (<1 microL) were collected onto the filters via a simple lateral prick to the patient's finger, circumventing the need for intravenous blood puncture. Collected samples, which are stable at room temperature for several years, were subsequently sent through the postal system to the diagnostic laboratory, bypassing the stringent requirements of courier delivery. Using this method, we performed restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis on prevalent mutations among Canadian and Chinese Gaucher disease patients. Of the 12 alleles (six patients) analyzed, 42% (5/12) have the N370S mutation and 58% (7/12) the L444P mutation, the two most common alleles found among Jewish and non-Jewish Gaucher disease patients. Uniquely, a Chinese Gaucher disease patient was found to have an N370S mutation. Although the presence of the N370S mutation is regarded as common in other ethnic groups, previous to this report it had not been noted in an individual of Asian descent. PvuII polymorphism analysis showed that the N370S mutation found in the Chinese patient was linked to the Pv1.1(-) polymorphism, as has been previously seen in the Jewish population. The use of FTA((R)) filter paper facilitates access of samples to diagnostic centers, and therefore provides an effective means of performing population-based mutational analysis of Gaucher disease internationally.

Gaucher disease: accurate identification of asymptomatic French-Canadian carrier using nonlabeled authentic sphingolipid substrate N-palmitoyl dihydroglucocerebroside.

Gaucher disease is an autosomal recessive sphingolipidosis associated with deficient glucocerebroside beta-glucosidase activity. It is a panethnic metabolic disorder, but the carrier frequency is particularly high among Ashkenazi Jews (estimated between 1:12-1:25). In order to establish a reliable and convenient biochemical assay method for differentiating asymptomatic Gaucher carriers from normal individuals, glucocerebroside beta-glucosidase activity was determined in peripheral blood lymphocytes and cultured skin fibroblasts of 11 Gaucher obligate heterozygotes using the authentic nonlabeled sphingolipid substrate N-palmitoyl dihydroglucocerebroside and the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGP). The level of lymphocyte beta-glucosidase activity on the glucocerebroside substrate was observed to range from 42-65% of that of the control mean, and there was no overlap of enzyme activity between the Gaucher heterozygotes and controls. However, when the artificial fluorogenic substrate 4MUGP was used, the level of beta-glucosidase activity in 2 of the Gaucher obligate heterozygotes was noted to overlap with that of the control individuals. Contrary to findings in the lymphocytes, cultured skin fibroblasts appear to be a reliable enzyme source for Gaucher carrier detection even when the artificial fluorogenic 4MUGP substrate was used, as the level of beta-glucosidase activity in all of the Gaucher obligate heterozygotes tested was intermediate and distinctly separated from that of the control persons. Using the lymphocyte glucocerebroside beta-glucosidase assay and fibroblast 4MUGP beta-glucosidase assay methods, we identified the carrier status in 3 other relatives and ruled it out in 4 others. These data suggest that nonlabeled glucocerebroside is a reliable and highly specific substrate for either lymphocyte or fibroblast beta-glucosidase activity assay in identifying asymptomatic Gaucher carriers. Use of the 4MUGP substrate for differentiating Gaucher heterozygotes from control persons, on the other hand, should be restricted to the fibroblast enzyme assay method, as considerable overlap of enzyme activity was noted in lymphocytes.

Identification of two novel and four uncommon missense mutations among chinese Gaucher disease patients.

Gaucher disease is the most prevalent lysosomal storage disease. It is panethnic and results from an inherited deficiency of glucocerebrosidase. Most mutations to date have been identified among Jewish and non-Jewish Caucasian patients; mutations in Chinese patients are largely unknown. We have performed nucleotide sequence analysis of PCR-amplified glucocerebrosidase genomic DNA from five unrelated Chinese patients affected with type 1 (non-neuropathic) Gaucher disease. A novel heterozygous C --> T mutation at cDNA nucleotide position 475 (R120W) was detected in a patient who is also heterozygous for a C --> T transition at cDNA nucleotide position 259 (R48W). In a second patient, a novel, heterozygous T --> G transversion at cDNA 226 (F37V) was detected. Mutation 1448 (L444P), the most prevalent mutation among non-Jewish Caucasian Gaucher patients, was found in the heterozygous form in four patients. The mutations in the second Gaucher allele in the other three patients are mutations 254 (G46E), 680 (N188S), and 754 (F213I), which were recently reported in Korean, Arab, and Chinese (Taiwanese) patients. We have developed screening methods that utilize PCR amplification of glucocerebrosidase genomic DNA and Eco571, Nci1, Hinc11, BsaJ1, and Bsr1 restriction endonuclease analyses for the detection of each of these mutations. The population genetics of some of these Gaucher alleles and their implications in genotype/phenotype correlation are discussed.

Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient.

Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomalrecessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed while the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A-->G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase therapy that began 31 months ago has been favorable, though incomplete.

Gaucher disease: functional expression of the normal glucocerebrosidase and Gaucher T1366G and G1604A alleles in Baculovirus-transfected Spodoptera frugiperda cells.

Gaucher disease is an inherited sphingolipidosis resulting from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1 as non-neuronopathic, type 2 as acute neuronopathic, and type 3 as subacute neuronopathic. We recently identified a rare mutation (G-->A at glucocerebrosidase cDNA nucleotide position 1604) [Choy et al., 1994a, Am J Med Genet 51:156-160] and a novel mutation (T-->G at glucocerebrosidase cDNA nucleotide position 1366) in two type 1 Gaucher patients by sequence analysis of the entire glucocerebrosidase coding region [Choy et al., 1994a, 1994b, Hum Mol Genet 3:821-823]. To demonstrate that these are deleterious and not neutral mutations, we cloned the full-length glucocerebrosidase cDNA of patients and of a normal control in the plasmid vector pAcUW1, recombined the human gene into the Baculovirus genome downstream of its polyhedron p10 promoter, and expressed the inserted gene in cultured cells of Spodoptera frugiperda transfected by recombinant Baculovirus. The levels of residual glucocerebrosidase activity determined in transfected cells with the Gaucher G1604A and T1366G alleles are 6.9% and 2.9% of that expressed by the normal allele (normal = 352.0 nmol/hr/mg protein or 100%). By comparison, the enzyme-specific activity expressed in transfected cells by 2 known Gaucher alleles, A1226G and T1448C, that are prevalent in type 1 and type 2 Gaucher disease are 23.4% and 3.3% of normal. No endogeneous glucocerebrosidase activity was detected in cultured cells transfected by either the wild-type Baculovirus or Baculovirus with the pAcUW1 plasmid vector without the glucocerebrosidase cDNA insert. These findings show that the Baculovirus expression system in cultured Spodoptera frugiperda cells is a suitable system for the functional expression and characterization of the normal and mutant glucocerebrosidase alleles. Moreover, the use of this expression system demonstrates that the G1604A and T1366G mutations are both deleterious mutations resulting in profoundly deficient glucocerebrosidase activity and subsequent Gaucher disease.

Molecular analysis of Gaucher disease in a Vietnamese-Czechoslovak patient with high residual glucocerebrosidase activity.

A Vietnamese-Czechoslovak type 1 Gaucher disease patient with mild hematological complications was found to have approximately 20% of the normal level of fibroblast glucocerebrosidase activity. Using primers that recognize exon 9 sequences of the glucocerebrosidase structural gene absent in the pseudogene, genomic DNA sequences flanking exons 9 and 10 of the glucocerebrosidase structural gene were amplified by the polymerase chain reaction. Allele-specific oligonucleotide hybridization to amplified genomic DNA sequence of exons 9 and 10 showed an A----G transition in exon 9 that resulted in the 370Ser----370Asp substitution in one of the alleles. In the other allele, a T----C transition in exon 10 resulted in the 444Leu----444Pro substitution, creating a NciI cleavage site. The heterozygote status of the patient's parents was confirmed biochemically by the detection of intermediate levels (42-55% of normal) of fibroblast glucocerebrosidase activity. Allele-specific oligonucleotide hybridization to amplified parental genomic DNA showed that the exon 9 mutation was present in the Czechoslovak father, whereas the exon 10 mutation was inherited from the patient's Vietnamese mother. This is the first report of the exon 10 mutation in a person of Vietnamese origin.

DNA analysis of an uncommon missense mutation in a Gaucher disease patient of Jewish-Polish-Russian descent.

Gaucher disease is the most frequent lysosomal lipid storage disease. It results from deficient glucocerebrosidase activity and is transmitted as an autosomal recessive trait. Three clinical forms of Gaucher disease have been described: type 1, non-neuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. We have sequenced the full length cDNA of the glucocerebrosidase gene and identified an uncommon mutation in nucleotide position 1604 (genomic DNA nucleotide position 6683) from a Gaucher disease patient of Jewish-Polish-Russian descent with type 1 Gaucher disease. It is a G-->A transition in exon 11 that results in 496Arg-->496His of glucocerebrosidase. This missense mutation is present in the heterozygous form and creates a new cleavage site for the endonuclease HphI. We have developed a simple method to detect the presence of this mutation by using HphI restriction fragment length polymorphism analysis of glucocerebrosidase genomic DNA or cDNA. The mutation in the other Gaucher allele of this patient is an A-->G transition at cDNA nucleotide position 1226 which creates an XhoI cleavage site after PCR mismatch amplification. The presence of this mutation was also confirmed by sequence analysis. Based on previous reports that mutation 1226 is present only in type 1 Gaucher disease and the observation that there is no neurological involvement in this patient, we conclude that our patient with the 1226/1604 genotype is diagnosed as having type 1 Gaucher disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular analysis of Gaucher disease: screening of patients in the Montreal/Quebec region.

Gaucher disease, the most prevalent lysosomal storage disease, is an autosomal recessive sphingolipidosis resulting from deficient glucocerebrosidase activity. Genomic DNA of the structural gene of glucocerebrosidase from normal individuals and fifteen unrelated patients with the three clinical forms of Gaucher disease from the Montreal/Quebec region were amplified by the polymerase chain reaction technique. Allele-specific oligonucleotide dot blot hybridization and restriction fragment length polymorphism were used to screen for five of the mutations [mutations 120, 370, 415, 444 (Nci), and 463] in exons 5, 9, and 10 of glucocerebrosidase gene. It was noted that all of the patients had at least one of the known mutant alleles. However, 9 patients (9/15 = 60%) had an unknown allele. Mutation 370 in exon 9 was present in the heteroallelic form in eight out of the nine patients with type 1 Gaucher disease, but was present in none of the six patients with type 2 or type 3 Gaucher disease. The Nci mutation in exon 10 was present in the heteroallelic form in three patients with type 1 Gaucher disease and in either the heteroallelic or homoallelic form in all of the six patients with type 2 or type 3 Gaucher disease. The 415/Nci mutations were found in a mildly affected 29-year-old patient with type 1 Gaucher disease, as well as in an infant with the type 2 form. These findings demonstrate the clinical and molecular genetic heterogeneities of Gaucher disease, the presence of unknown Gaucher allele(s) in most (60%) of the patients surveyed, and the occasional inexplicable lack of phenotype-genotype correlation among some patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of the Interact Short Form as a tool to evaluate emotion of people with profound intellectual disabilities.

BACKGROUND: One of the essential purposes of intervention programmes for people with profound intellectual disabilities (ID) is to enhance the desirable mood and behaviour and decrease the undesirable ones through stabilizing their emotion. There is lack of validated instrument to offer a comprehensive measure that covers the mood and behaviour, both desirable and undesirable, appropriate for people with profound ID. METHOD: This study aimed to examine the validity and reliability of the Interact Short Form for evaluating the mood and behaviour of people with profound ID, and at the same time, review their emotional profile using the Interact Short Form. Both content validity using expert panel review and construct validity by means of factor analysis were investigated. A total of 75 people with profound ID were recruited. Inter-rater reliability was tested. The results of the Interact Short Form were described to reflect the emotional profile of this group of participants. RESULTS: Using the results of expert panel review and those from factor analysis, we found three subscales representing the mood and behaviour of people with profound ID. They were: 'emotional expression', 'interests towards tasks' and 'behaviours to environment'. All three subscales were found to be internally consistent (alpha = 0.71-0.88). The Interact Short Form- People with profound ID version also showed good inter-rater reliability (mean = 0.72). The results of the Interact Short Form showed that this group of participants had fairly stable emotion under the structured setting and activities in the residential institutions where data were collected. CONCLUSIONS: The Interact Short Form- People with profound ID version serves as a helpful tool for both clinical and research use in assessing the mood and behaviour of people with profound ID in a simple, comprehensive and systematic way.

Intrafamilial clinical variability of type 1 Gaucher disease in a French-Canadian family.

Glucocerebroside beta-glucosidase (glucocerebrosidase) activity was determined from peripheral blood lymphocytes and cultured skin fibroblasts of eight full sibs in a French-Canadian family at risk for Gaucher disease, an autosomal recessive sphingolipidosis resulting from deficient glucocerebrosidase activity. The diagnosis of type 1, non-neuronopathic Gaucher disease was made in all of the five affected sibs on the basis of deficient (7.5 to 15.5% of control mean) glucocerebrosidase activity and absence of neurological involvement. Normal levels of enzyme activity were found in two of the three asymptomatic sibs. The third asymptomatic sib had an intermediate level (about 50% of control mean) of fibroblast and lymphocyte glucocerebrosidase activity, indicating that he is a carrier. Considerable clinical heterogeneity was noted among the five affected sibs. One patient is mildly affected and so far has not developed any orthopaedic complications associated with Gaucher disease. His haematological complications were also reversed after splenectomy 24 years ago. In contrast to this mild presentation, the patient's splenectomised sister has been very anaemic and thrombocytopenic. There have been severe orthopaedic complications associated with Gaucher disease, including vertebral compression, avascular necrosis, and pathological fracture of the long bones. The clinical picture of the other three affected sibs appeared to vary between the two extremes. Although the asymptomatic parents of the patients died many years ago, their heterozygous status with respect to Gaucher disease can be deduced by the presence of Gaucher homozygotes, normal homozygotes, and one heterozygote among their eight offspring. Present findings suggest that the clinical variability of type 1 Gaucher disease may be attributed to variable expressions of the same Gaucher mutant alleles, in addition to the presence of multiple mutant alleles that are widely disseminated in the population.

Purification of human placental glucocerebrosidase using a two-step high-performance hydrophobic and gel permeation column chromatography method.

Glucocerebrosidase was purified from human placenta approximately 10,600-fold to apparent homogeneity with an overall yield of 37% using cholate extraction, ammonium sulfate fractionation, butanol delipidation, and a two-step high-performance hydrophobic and gel permeation column chromatography method. A Phenyl-5PW (21.5 X 150 mm) column was used in the first step. Approximately one litre of delipidated and dialysed extract containing 3.7 X 10(6) units of enzyme activity from 1 kg of placental tissue was processed by the column at a flow rate of 5 ml/min. Glucocerebrosidase was eluted using a linear cholate gradient (2-3%). There was a 50-fold purification and 89% recovery. The run was completed in about 7 h. In the second step, the concentrated enzyme preparation from the phenyl column was run through two Bio-Sil TSK 250 gel permeation columns (21.5 X 600 mm) connected in series at a flow rate of 1.5 ml/min. A symmetrical peak of glucocerebrosidase activity (Ve = 253 ml) which had constant specific activity (47,000 units/h/mg protein) was noted. There was a 17-fold purification and 80% recovery in this run which was completed in 4 h. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and protein staining with silver compounds of the purified preparation revealed the presence of one band of Mr 68,000.

Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography.

Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)

Gaucher disease: the effects of phosphatidylserine on glucocerebrosidase from normal and Gaucher fibroblasts.

Glucocerebroside beta-glucosidase (glucocerebrosidase) activity was assayed from cultured fibroblasts of normal individuals, and patients with type 1 (non-neuropathic), type 2 (acute neuropathic), and type 3 (subacute neuropathic) form of Gaucher disease. Residual glucocerebrosidase activity of patients was 8.9 to 17.4% of normal controls, and there was no clear correlation between the level of residual enzyme activity and the different clinical subtypes of the disease. When membrane-bound glucocerebrosidase activity was assayed in the presence of crude brain lipid extracts or purified phosphatidylserine, enzyme from both the normal and type 1 Gaucher fibroblasts was stimulated dramatically (35-60% by crude extracts, 85-90% by phosphatidylserine). This stimulation was not observed with fibroblast glucocerebrosidase of an infantile type 2 and two juvenile type 3 Gaucher patients. The presence of inhibitors of glucocerebrosidase in these type 2 and type 3 Gaucher cells was not detected. Contrary to the mutant enzyme from these Gaucher fibroblasts, glucocerebrosidase from fibroblasts of two adult type 3 Gaucher patients with cerebral involvement was stimulated substantially (72-85%) by phosphatidylserine. When membrane-bound glucocerebrosidase from fibroblasts of the infantile type 2 and juvenile type 3 patients was solubilized with sodium cholate (1% w/v) and delipidated, the phospholipid stimulation of enzyme activity was restored. These findings suggest that considerable clinical and biochemical heterogeneity exists among patients with neuropathic Gaucher disease and that phosphatidylserine activation cannot be used as a reliable indicator in predicting future onset of neurodegeneration in Gaucher patients. The possibility of an aberrant binding of mutant glucocerebrosidase to the lysosomal membrane in juvenile type 3 form of Gaucher disease is discussed.