Francis Y.M. Choy

Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris

The lysosomal hydrolase glucocerebrosidase catalyzes the penultimate step in the breakdown of membrane glycosphingolipids. An inherited deficiency in this enzyme leads to the onset of Gaucher disease, the most common lysosomal storage disorder. Exogenous sources of this protein are required for biochemical and biophysical investigations and enzyme replacement therapy of Gaucher disease. Heterologous expression of glucocerebrosidase has been successful in mammalian and insect cell lines and although its use in enzyme replacement therapy of Gaucher disease has proven efficacious, current production levels limit the availability of the enzyme. Initial attempts to express human glucocerebrosidase using the methylotrophic yeast Pichia pastoris had limited success, despite significant levels of transcription. Using fragments of the glucocerebrosidase cDNA fused to the luciferase cDNA as a translational read-through reporter, the impact of synonymous codon usage bias on protein expression in P. pastoris was examined. A table of preferred codons was determined for P. pastoris and the codon usage of a 186-bp fragment of the glucocerebrosidase gene was optimized to that of the P. pastoris preferred set. A second construct with altered G+C content but no codon optimization was created for comparison. While the native glucocerebrosidase coding region limited luciferase activity to baseline levels, the codon optimized and G+C altered constructs increased luciferase activity 10.6- and 7.5-fold, respectively. Optimized G+C content, regardless of corresponding codon optimization, appears to be the major contributor to increased translational efficiency in this heterologous expression host.

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 patients 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 and Cancer: Concept and Controversy

Gaucher disease is an inherited disorder caused by a deficiency in the lysosomal hydrolase glucocerebrosidase. There is a wide spectrum of clinical presentations, with the most common features being hepatosplenomegaly, skeletal disease, and cytopenia. Gaucher disease has been classified into three broad phenotypes based upon the presence or absence of neurological involvement: Type 1 (nonneuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). The two main treatment options include enzyme replacement therapy and substrate reduction therapy. Recently, discussion has escalated around the association of Gaucher disease and cancer, with conflicting reports as to whether Gaucher patients have an increased risk of malignancy. In this review, we present both the concept and controversy surrounding the association of Gaucher disease with cancer.

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 and the synucleinopathies: refining the relationship

Gaucher disease (OMIM 230800, 230900, 231000), the most common lysosomal storage disorder, is due to a deficiency in the enzyme glucocerebrosidase. Gaucher patients display a wide spectrum of clinical presentation, with hepatosplenomegaly, haematological changes, and orthopaedic complications being the predominant symptoms. Gaucher disease is classified into three broad phenotypes based upon the presence or absence of neurological involvement: Type 1 (non-neuronopathic), Type 2 (acute neuronopathic), and Type 3 (subacute neuronopathic). Nearly 300 mutations have been identified in Gaucher patients, with the majority being missense mutations. Though studies of genotype-to-phenotype correlations have revealed significant heterogeneity, some consistent patterns have emerged to inform prognostic and therapeutic decisions. Recent research has highlighted a potential role for Gaucher disease in other comorbidities such as cancer and Parkinsons Disease. In this review, we will examine the potential relationship between Gaucher disease and the synucleinopathies, a group of neurodegenerative disorders characterized by the development of intracellular aggregates of α-synuclein. Possible mechanisms of interaction will be discussed.

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.

Perinatal lethal Gaucher's disease without prenatal complications.

To the Editor: Gaucher’s disease (GD, OMIM 230800, 230900 and 231000) is the most prevalent sphingolipidosis with a frequency of 1 in 50,000–100,000 live births in Caucasians and 1 in 850 in Ashkenazi Jews (1). GD results from an inherited deficiency of the lysosomal enzyme glucocerebrosidase (GBA) (E.C. 3.2.1.45) (2). More than 200 mutations in the Gba gene are known to date. Three clinical forms (types 1, 2, and 3) of GD have been described. The perinatal lethal form of Gaucher’s disease (PLGD, a type 2 variant) presents with symptoms at or shortly after birth that include rapid neurodegeneration, hepatosplenomegaly, ichthyosis, and on occasion, non-immune hydrops fetalis. Here, we present the clinical and molecular analysis of an infant with PLGD and a brief review on 30 reported cases. The female patient was the second child born to non-consanguineous Caucasian parents at 39 weeks of gestation after an uncomplicated pregnancy. The infant had fetal decelerations 2 h prior to delivery and was delivered by vacuum extraction. The infant exhibited spontaneous tremors with baseline hypotonia. She had a very weak cry and weak respiratory effort that required mechanical ventilation. The initial Apgar scores were 1, 5, and 7 at 1, 5, and 10 min with initial pH of 7.2. Miosis, lethargy and hypotonia led to an initial diagnosis of Sarnat and Sarnat stage 2 perinatal asphyxia. Detailed physical examination identified an infant encased in a glistening translucent covering resembling ichthyosis. There was no evidence of ectropion or limb contractures; however, cobbling of the ears was notable. Abdominal examination showed hepatomegaly at 3 cm without splenomegaly. In the subsequent 24–48 h, seizures and multisystem organ involvementwerenoted (i.e. hematuria, elevated creatinine, elevated liver enzymes and thrombocytopenia). The infant was extubated to room air on her fifth day of life without residual cardiorespiratory issues. The hematuria resolved and creatinine levels returned to baseline, however, liver enzymes remained elevated and thrombocytopenia persisted, requiring multiple transfusions. Abdominal examinations showed a progressively enlarging liver and splenomegaly. Physical examination at 2 weeks of age was remarkable for the following findings: hypertension (.95th percentile for age), scleral icterus, generalized jaundice, marked abdominal distension with caput medusae, hepatomegaly of 8–10 cm and splenomegaly of 5 cm. There was resolution of the ichthyosis, but the hands were held in a flexed position with evidence of contractures. Abdominal ultrasound and hepatobiliary iminodiacetic acid scans showed a markedly enlarged liver and spleen with a combined volume filling at least three-fourth of the abdominal cavity. The diagnosis of PLGD was evaluated and confirmed biochemically, as shown by deficient leukocyte GBA activity at 0.9 nanomole/h/ mg protein (normal: 5.6–12.5 nanomole/h/mg protein). The infant expired from cardiorespiratory failure at 2 months of age. Gba genomic DNA was amplified from a dot blood sample collected on FTA filter paper (Fitzco Inc., Maple Plain, MN) using a two-step nested polymerase chain reaction method and resolved by agarose gel electrophoresis (3). DNA sequence analysis was performed on a CEQGenetic Analysis System Capillary Electrophoresis DNA Sequencer (Beckman Coulter, Mississauga, ON) using the CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter). A novel C/T missense and heterozygous mutation of paternal origin was identified by DNA sequence analysis in exon 8 (cDNA position 1112), resulting in proline to leucine substitution at GBA polypeptide position 332 (P332L). Residue P332 lies in the loop between a-helix 6 and bbarrel 7 in the catalytic domain of GBA (4). The presence of proline, an alpha-imino acid with an established role in protein secondary structure, between the two catalytic moieties (E235, the acid/ base catalyst and E340, the nucleophile) may be important for correct positioning of the two

Expression of two green fluorescent protein variants in citrate-buffered media in Pichia pastoris.

The Aequorea victoria green fluorescent protein (GFP) has become one of the most frequently employed molecular reporters. A variety of mutational methods have created a large number of GFP variants with differing pH sensitivity [1], differing spectra, and optimized expression [2]. Many of the recent mutational approaches have focused on species-specific codon optimization. First established in 1980 [3], the idea that organisms do not display a random pattern of synonymous codon usage has been validated by the large amount of recent genome sequencing projects [4]. A substantial number of studies have examined the effects of varying codon bias in heterologous protein expression systems under the assumption that disparate patterns of codon bias in the transgene and the expression host will have a significant impact on the levels of recombinant protein produced. It has been suggested that the human codon-optimized enhanced green fluorescent protein (EGFP; Clontech, Palo Alto, CA) is unsuitable for expression in yeast host systems due to such preferred codon discrepancy. However, several studies have demonstrated successful expression in yeast, mainly in Saccharomyces cerevisiae [5–7]. Therefore, we compared the expression of two GFP variants, red-shifted GFP (RSGFP from pRSGFP-C1; Clontech), and EGFP, in the methylotrophic yeast Pichia pastoris. Unlike EGFP, RSGFP is not human codon-optimized; however, like EGFP, RSGFP does have an excitation wavelength shifted toward the red end of the spectrum. Both variants also exhibit greater fluorescence intensity than wildtype GFP: EGFP fluoresces 35 times greater and RSGFP fluoresces 4–6 times greater. We chose to examine the expression of both variants in citrate-buffered rather than phosphate-buffered media to determine whether either variant could serve as a fusion partner with proteins reported to exhibit inhibition in the presence of phosphate. Glucocerebrosidase (acid b-glucosidase, EC 3.2.1.45) activity, for example, has been suggested to be inhibited by phosphate ions [8]. Based on the codon bias theory, we expected to see moderate amounts of RSGFP and minimal amounts of EGFP produced. To create the RSGFP and EGFP constructs, the variant cDNAs were PCR-amplified with the following primers: 50-TAGAATTCCCGGTCGCCACCATG-30 and50-TGTTACAGGGCCCGCGGTTCAGTCGAC-30 for RSGFP; 50-AACGGTCGAATTCATGGTGAGCA AGGG-30 and 50-TATGATCTGAATTGCCGGCCGC TTTACTT-30 for EGFP. PCR amplification was performed with an initial denaturation at 94 C for 2.5min, followed by 30 cycles of 94 C for 1min, 60 C (for RSGFP) or 56 C (for EGFP) for 1.5min, and 72 C for 1.5min. Reactions were carried out with 1ll of template (pRSGFP-C1 or pEGFP-N1 vector), 5ll of 10 PCR buffer, 5ll of 25mM MgCl2, 5ll of 2.5mM dNTPs, and 4 ll forward and reverse primers in a total volume of 50 ll. These amplified cDNAs were digested with EcoR1 and ligated into the vector pPICZaA (Invitrogen, San Diego, CA) prior to electroporation into bacterial cells. Transformantswere selected by resistance toZeocin (0.1mg/ml; Invitrogen) on low-salt Luria–Bertani plates (pH 7.5). Clones were confirmed by PCR amplification and sequenced to ensure the fidelity of the PCR and cloning steps. True positive pPICZaA–RSGFP and pPICZaA– EGFP clones were isolated, purified, and linearized at the BstX1 restriction site prior to electroporation into GS115 P. pastoris cells according to the Pichia Expression Manual protocol (Invitrogen). Transformants were Analytical Biochemistry 311 (2002) 193–195

CASE REPORT: Novel Point Mutation (W184R) in Neonatal Type 2 Gaucher Disease

ABSTRACT 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.