HIV TAT variants differentially influence the production of glucocerebrosidase in Sf9 cells

Gaucher disease, the most common lysosomal storage disorder, is currently treated with enzyme replacement therapy. This approach, however, is ineffective in altering the progression of neurodegeneration in type 2 and type 3 patients due to the difficulty of transferring the recombinant enzyme across the blood-brain barrier. Human immunodeficiency virus type 1 trans-activating transcriptional activator protein (HIV TAT) contains a protein transduction domain that can be added to a fusion protein partner to allow for transport of the partner across membranes. Consequently, we examined the creation, production, and secretion of fusion constructs containing glucocerebrosidase and either wild-type TAT or modified TAT in Sf9 cells. All three constructs exhibited successful expression, with wild-type TAT chimeras showing lower levels of expression than modified TAT chimeras.

Heterologous expression and characterization of a rare Gaucher disease mutation (c.481C > T) from a Canadian aboriginal population using archival tissue samples.

Gaucher disease is an inherited sphingolipidosis resulting from deleterious mutations in the glucocerebrosidase gene. Through direct sequence analysis of genomic DNA from whole blood, fibroblast cultures, and formalin-fixed archival tissue samples, we have identified a rare homozygous C > T transition at cDNA nucleotide 481 of the glucocerebrosidase gene that results in a proline to serine amino acid substitution (p.P122S) in an aboriginal family of Cree descent in northern Alberta, Canada. A 13-month-old boy (JB) presented with severe visceral Gaucher disease and was treated with enzyme replacement. Currently, at 11 years he is developmentally delayed, with oculomotor apraxia. A cousin (MS) had previously died at age 7 from complications of severe Gaucher disease, before enzyme replacement therapy was available. She was also developmentally delayed. Heterologous expression of this allele using a baculovirus expression system revealed 19.2% of normal enzyme activity on the artificial substrate 4-methylumbelliferyl beta-d-glucopyranoside (4MUGP). Genotype/phenotype correlation is complicated by incomplete clinical details, enzyme replacement therapy, and the difficulty in excluding other genetic and environmental causes of developmental delay. However the development of oculomotor apraxia in JB suggests a Type 3 Gaucher phenotype. The only previous report of this mutation was also from a member of the Cree Nation, who has had a rather similar clinical course. A protocol is described for the isolation of genomic DNA from formalin-fixed bone marrow aspirate archival specimens obtained from the deceased for subsequent PCR-based sequence analysis and mutation detection. This technique will be applicable to the screening of this and other populations for the frequency of known Gaucher mutations where traditional DNA sources are unavailable.

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.

Novel point mutation (W184R) in neonatal type 2 Gaucher disease.

Gaucher disease is the most prevalent inherited sphingolipidosis and results from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1, or non-neuronopathic; type 2, or acute neuronopathic; and type 3, or subacute neuronopathic. We have identified a novel mutation in a patient of Russian-British descent who died of type 2 Gaucher disease a few hours after birth. A heterozygous T --> C transition mutation in exon 6, cDNA nucleotide position 667, results in the substitution of tryptophan by arginine at amino acid residue 184 (W184R) of glucocerebrosidase. This mutation creates a new cleavage site for the restriction endonuclease Hinf1. We developed a method that utilizes Hinf1 restriction endonuclease analysis to confirm the presence of the mutation and test family members. The second mutation identified in the other glucocerebrosidase allele of the patient is mutation L444P, a severe mutation frequent in type 2 and 3 Gaucher disease. Since the patient died very shortly after birth, we postulate that the W184R/L444P genotype may result in little or no detectable glucocerebrosidase activity and thus a poor prognosis.

A novel complex allele and two new point mutations in type 2 (acute neuronopathic) Gaucher disease.

Gaucher disease, the most prevalent inherited sphingolipidosis, is characterized by lipid laden histiocytes in the spleen, liver and bone marrow sinusoids of affected individuals. It results from deleterious mutations in the functional gene of glucocerebrosidase (acid beta-glucosidase, EC. 3.2.1.45) 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. In this report, we describe the identification and characterization of three novel mutations from two patients who died with type 2 Gaucher disease. Two heterozygous missense point mutations, one at cDNA nucleotide 238A (E41L) and the other at cDNA nucleotide 508T (R131C) were identified, both in the context of a cDNA nucleotide 1448C (L444P) mutation in the second allele. One of these L444P mutations was identified as a novel complex allele resulting from a crossover involving the glucocerebrosidase functional gene and pseudogene beginning between genomic nucleotides 5689 and 5723 and extending through the rest of the coding sequence. Based on the recent identification and sequence analysis of the metaxin gene and pseudogene contiguous with the glucocerebrosidase pseudogene and functional gene respectively, we have developed a PCR-based method for the analysis of the origin and extent of this recombination.

A novel mutation (V191G) in a German-British type 1 Gaucher disease patient. Mutations in brief no. 131. Online.

Gaucher disease results from mutations in the glucocerebrosidase gene located on human chromosome 1q21. Three clinical forms of Gaucher disease have been described: type 1, nonneuropathic; type 2, acute neuropathic; and type 3, subacute neuropathic. We have identified a novel mutation in a German-British patient with type 1 Gaucher disease which results in V191G of the glucocerebrosidase polypeptide. Because the mutation abolishes a HphI cleavage site, its presence was confirmed by HphI RFLP analysis of PCR-amplified genomic DNA. In the second allele of the patient, the mutation identified was g.5841A G(N370S). Sequence analysis of the remainder of the coding region of the gene as well as the exon-intron boundaries showed identity to normal controls. Because mutation N370S has so far been found only in type 1 Gaucher disease and postulated to result in mild clinical presentation, and since the clinical course of this patient has been relatively mild with minimal skeletal involvement, we speculate that the V191G/N370S genotype may also result in good prognosis.

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.

Gaucher disease: molecular screening of the glucocerebrosidase 1601G and 1601A alleles in Victoria, British Columbia, Canada.

Gaucher disease is the most prevalent lysosomal storage disease and it results from inherited deficient glucocerebrosidase activity. The glucocerebrosidase gene from normal people was sequenced by several laboratories and it was noted that a G or A nucleotide may be present at cDNA position 1601, resulting in 495arginine or 495histidine in the glucocerebrosidase polypeptide. In order to rule out the possibility of cloning error and to elucidate the genetic status of the two genotypes and their distribution in the population, we have developed a convenient and reliable method for the molecular screening of the 1601G and 1601A genotypes in the population. This method uses PCR amplification of glucocerebrosidase genomic DNA in blood samples, followed by BsaHI restriction fragment length polymorphism analysis. Out of the 256 subjects without Gaucher disease and 15 Gaucher patients surveyed, the 1601G genotype was present in the homozygous form in all of the asymptomatic subjects and 14 Gaucher patients. In one Gaucher patient who was diagnosed as having type 1 (non-neuropathic) Gaucher disease with the A1226G/T1366G mutations, the heterozygous 1601G/A genotype was detected. These findings indicate that the 1601G genotype which encodes 495arginine of the glucocerebrosidase polypeptide is not a cloning error. Instead, it constitutes the normal as well as predominant genotype in the population in the municipality of Greater Victoria, British Columbia. The 1601A genotype, on the other hand, appears to be quite infrequent in this population. The availability of our restriction enzyme based method has allowed the screening and frequency determination of these two alleles in other populations.

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.

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)