Hypoxanthine-guanine Phosphoribosyltransferase Gene Research Articles

Genetic Modification of Mouse Bone Marrow by Lentiviral Vector-Mediated Delivery of Hypoxanthine-Guanine Phosphoribosyltransferase Short Hairpin RNA Confers Chemoprotection Against 6-Thioguanine Cytotoxicity

We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection.

DNA-Reactive Protein Monoepoxides Induce Cell Death and Mutagenesis in Mammalian Cells

Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source

BackgroundBioinformatic analysis of the genes coding for the chitinase in Pyrococcus furiosus and Thermococcus kodakarensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase gene. This splits the enzyme into two separate genes, PF1233 and PF1234, in comparison to Thermococcus kodakarensis. Furthermore, our attempts to grow the wild type strain of Pyrococcus furiosus on chitin were negative. From these data we assume that Pyrococcus furiosus is most likely unable to use chitin as a carbon source. The aim of this study was to analyze in vivo if the one nucleotide insertion is responsible for the inability to grow on chitin, using a recently described genetic system for Pyrococcus furiosus.ResultsA marker-less genetic system for Pyrococcus furiosus was developed using simvastatin for positive selection and 6-methylpurine for negative selection. Resistance against simvastatin was achieved by overexpression of the hydroxymethylglutaryl coenzyme A reductase gene. For the resistance to 6-methylpurine the hypoxanthine-guanine phosphoribosyltransferase gene was deleted. This system was used to delete the additional nucleotide at position 1006 in PF1234. The resulting chitinase in the mutant strain was a single subunit enzyme and aligns perfectly to the enzyme from Thermococcus kodakarensis. A detailed analysis of the wild type and the mutant using counted cell numbers as well as ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. An additional mutant strain containing a reduced chitinase version containing just one catalytic and one chitin-binding domain showed diminished growth on chitin in comparison to the mutant containing the single large enzyme.ConclusionsWild type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. The overall high sequence identity of the two chitinases between P. furiosus and T. kodakarensis indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme using programmed +/−1 frameshifting.

Identification of Novel Mutations in the Human HPRT Gene

Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch–Nyhan syndrome (LNS) or Lesch–Nyhan variants (LNVs). We report three novel independent mutations in the coding region of HPRT gene: exon 3: c.141delA, p.D47fs53X; exon 5: c.400G>A, p.E134K; exon 7: c.499A>G, p.R167G from three LNS affected male patients.

Carrier and prenatal diagnosis of Lesch–Nyhan disease due to a defect in HPRT gene expression regulation

Lesch–Nyhan disease (LND) is caused by lack of hypoxanthine–guanine phosphoribosyltransferase (HPRT) activity. Mutations in HPRT1 gene show variability in type and location within the gene, and in certain patients the HPRT coding sequence is normal and the molecular defect cannot be found. These patients presented a decreased HPRT1 expression of unknown cause. This is the first report of a carrier and prenatal diagnosis of LND due to a defect in HPRT gene expression regulation.

High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells

The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

Alternaria toxins: Altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells

Altertoxin II (ATX II) is one of the several mycotoxins produced by Alternaria fungi. It has a perylene quinone structure and is highly mutagenic in Ames Salmonella typhimurium, but its mutagenicity in mammalian cells has not been studied before. Here we report that ATX II is a potent mutagen in cultured Chinese hamster V79 cells, inducing a concentration-dependent increase of mutations at the hypoxanthine guanine phosphoribosyltransferase gene locus at concentrations similar to that of the established mutagen 4-quinoline-N-oxide. Thus, ATX II is at least 50-times more potent as a mutagen than the common Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME). In contrast to AOH and AME, ATX II does not affect the cell cycle of V79 cells. ATX II also causes DNA strand breaks in V79 cells, with a potency again exceeding that of AOH and AME. The high mutagenic and DNA strand breaking activity of ATX II raises the question of whether this Alternaria toxin poses a risk for public health, and warrants studies on the occurrence of ATX II and other perylene quinone-type mycotoxins in food and feed.

Lesch–Nyhan Variant Syndrome: Real-Time RT-PCR for mRNA Quantification in Variable Presentation in Three Affected Family Members

Inherited mutations of hypoxanthine guanine phosphoribosyltransferase (HPRT) give rise to Lesch–Nyhan syndrome (LNS) or variants (LNV). We report molecular insights from real-time RT-PCR for HPRT mRNA quantification into the mechanism by which a single mutation located in exon 7 of the HPRT gene: c.500G>T, p.R167M, led to different clinical phenotypes from three male LNV-affected patients in the same family manifesting parallel differences in enzymatic activities. This approach can be applied for understanding genotype-phenotype correlations for other human genetic diseases.

Epigallocatechin-3-gallate decreases UVA-induced HPRT mutations in human skin fibroblasts accompanied by increased rates of senescence and apoptosis.

Our study was designed to determine the protective effect of epigallocatechin-3-gallate (EGCG) on cultured human skin fibroblasts (HSFs) from multiple ultraviolet A (UVA) irradiation-induced hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant colony formation and its underlying mechanisms. In our study, the mutation frequency of the HPRT gene was examined by mutagenesis assay. Cell senescence was determined by histochemical staining of senescence-associated β-galactosidase. The apoptosis rate was detected by flow cytometry. EGCG decreased the UVA-induced HPRT gene mutation frequency by 47.85%. However, EGCG further increased the number of senescent cells by 38.92% and the apoptosis rate by 56.92% in HSFs. The photo-protective effect of EGCG on multiple UVA-exposed HSFs is related to a significant reduction in UVA-induced HPRT mutant cells. This may be caused by the induction of damaged cells to proceed to senescence and apoptosis.

Susceptibility of Human Lymphocytes to 2-Nitrofluorene-Induced Gene Mutation is Strongly Dependent on Metabolic Genotypes

Genetic polymorphisms in drug metabolism contribute largely to adverse drug reactions in susceptible individuals due to excess production of reactive metabolites and active oxygen species. We conducted an in vitro study that clearly demonstrates that functional impact of common polymorphisms in the metabolic genes, such as N-acetyltransferase 2 (NAT2), can be easily reflected in mutant induction in the gene coding for hypoxanthineguanine phosphoribosyl transferase (HPRT) in T-lymphocytes isolated from human peripheral blood. NAT2 is involved in the metabolic activation of 2-nitrofluorene (2-NF) to the known aromatic amide carcinogen N-acetyl- 2-aminofluorene. Subsequent deactivation through glutathione conjugation involves glutathione S-transferase M1 (GSTM1). We obtained a clear dose-related increase in the HPRT mutant frequency after treating mitogen-stimulated lymphocytes isolated from a normal blood donor with 2-NF (up to 5 fold at 400 μg/mL, 24h exposure), while no HPRT mutant induction was observed using cells from another blood donor. The susceptible cells turned out to have the NAT2 rapid and GSTM1 null genotype combination (capable of activation, with insufficient deactivation), while the resistant cells had the NAT2 slow and GSTM1 positive combination. Although the contribution of GSTM1 genotype is less clear, our finding suggests that functional polymorphisms in key metabolic genes do affect induction of gene mutations at the HPRT locus and at least the NAT2 genotype plays a critical role in determining the susceptibility of human cells to genotoxicity of 2-NF. Further, human peripheral T-lymphocytes and the in vitro HPRT gene mutation assay can be utilized to study the functional impact of common genetic polymorphisms in drug metabolism and to identify risk genotypes susceptible for drug toxicity and somatic mutations.

Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Mutations in the Asian Population

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch– Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout (Kelley–Seegmiller syndrome). The marked heterogeneity of HPRT deficiency is well known, with more than 300 mutations at the HPRT gene locus having been reported (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exons 1 to 9). We have identified mutations in Asian families with patients manifesting different clinical phenotypes, including rare cases of female subjects, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the polymerase chain reaction technique coupled with direct sequencing. We developed suitable methods to detect the mutations identified from respective families with HPRT deficiency. Then, prenatal genetic diagnoses in HPRT-deficient families were carried out using both mRNA and genomic DNA from chorionic villi or amniotic fluid cells. As shown here in the heterogeneity of HPRT mutations, the spectrum of 70 mutations identified in the Asian population fits the four main conclusions that emerged previously from worldwide analysis.

Insight into the roles of hypoxanthine and thydimine on cultivating antibody-producing CHO cells: cell growth, antibody production and long-term stability

The potential of hypoxanthine and thymidine (H&T) to promote growth of CHO cells and production of monoclonal antibody (mAb) was explored in this study. It was demonstrated that H&T stimulated the initial cell growth and enhanced volumetric production of anti-human CD20 mAb by 22%, mainly through the elevated integrated viable cell concentration (IVCC). The moderate alteration in cell cycle distribution might partially account for the increased cell growth. Subsequent long-term stability studies indicated that H&T did not accelerate decay kinetics in mAb productivity. Specifically, cells under both nucleic acids-replete (H&T supplementation) and nucleic acids-hungry (methotrexate treatment) culture conditions showed similar stable mAb production during the first 2 months, followed by a gradual decline in the specific production rate (q(mAb)) with a 40% drop at the fourth month. In addition, the decreased transcript level of intracellular heavy chain (HC) of anti-human CD20 mAb correlated well with the decreased q(mAb). Furthermore, genomic mutation rate regarding the loss-of-function occurrence of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was determined, showing that H&T repressed the HPRT spontaneous mutation rate while methotrexate (MTX) provoked the mutation rate. Collectively, our data illustrated that H&T as potential medium additives promoted both initial cell growth and volumetric production of mAb, while not affecting the long-term stability of antibody-producing CHO cells.

The role of human cytochrome P4503A4 in biotransformation of tissue-specific derivatives of 7H-dibenzo[ c,g]carbazole

The environmental pollutant 7H-dibenzo[ c,g]carbazole (DBC) and its derivative, 5,9-dimethylDBC (DiMeDBC), produced significant and dose-dependent levels of micronuclei followed by a substantial increase in the frequency of apoptotic cells in the V79MZh3A4 cell line stably expressing the human cytochrome P450 (hCYP) 3A4. In contrast, neither micronuclei nor apoptosis were found in cells exposed to the sarcomagenic carcinogen, N-methylDBC ( N-MeDBC). A slight but significant level of gene mutations and DNA adducts detected in V79MZh3A4 cells treated with N-MeDBC, only at the highest concentration (30 μM), revealed that this sarcomagenic carcinogen was also metabolized by hCYP3A4. Surprisingly, DBC increased the frequency of 6-thioguanine resistant (6-TG r) mutations only at the highest concentration (30 μM), while DiMeDBC failed to increase the frequency of these mutations. The resistance to 6-thioguanine is caused by the mutations in the hypoxanthine-guanine phosphoribosyltransferase ( Hprt) gene. The molecular analysis of the coding region of Hprt gene showed a deletion of the entire exon 8 in DiMeDBC-induced 6-TG r mutants, while no changes in the nucleotide sequences were identified in 6-TG r mutants produced by DBC and N-MeDBC. Based on our results, we suggest that hCYP3A4 is involved in the metabolism of DBC and its tissue-specific derivatives. While hCYP3A4 probably plays an important role in biotransformation of the liver carcinogens, DBC and DiMeDBC, it might only have a marginal function in N-MeDBC metabolism.

Novel Mutations in the Human HPRT Gene

Inherited mutation of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT), gives rise to Lesch-Nyhan Syndrome (LNS) or HPRT-related gout. Here, we report five novel independent mutations in the coding region of the HPRT gene from five unrelated male patients manifesting different clinical phenotypes associated with LNS: exon 2: c.133A > G, p.45R > G; c.35A > C, p.12D > A; c.88delG; exon 7: c.530A > T, p.177D > V; and c.318 + 1G > C: IVS3 + 1G > C splice site mutation.

Urine alkalinization may be enough for the treatment of bilateral renal pelvis stones associated with Lesch–Nyhan syndrome

Lesch-Nyhan syndrome is a rare sex-linked disorder of purine metabolism that is caused by a mutation in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene which causes marked hyperuricemia and hyperuricosuria, with signs of gouty arthritis and uric acid stone disease in early childhood. We report a case of renal pelvis calculi which was dissolved within 10 days of urine alkalinization and hydration.

A study on the relationship between TCTA tetranucleotide polymorphism of the HPRT gene and primary hyperuricemia

We examined polymorphism of the TCTA tetranucleotide sequence in the 3rd intron of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the Han population of Ningxia Province in China. We also looked for a possible relationship between STR polymorphism in the 3rd intron of the HPRT gene and primary hyperuricemia. We used Chelex-100 to extract DNA, then PCR, PAGE and silver staining for allele genotyping and DNA sequencing to obtain the distribution of the alleles. We found, for the first time, that there is high STR polymorphism in the 3rd intron of the HPRT gene. We detected 5 STR alleles in this intron in the Han population of Ningxia Province, with 15 genotypes in females; significant differences were observed in the distribution of alleles and genotypes between control and patient groups for both males and females. Alleles of the TCTA repeat in the 3rd intron of the HPRT gene were found to be associated with primary hyperuricemia; consequently, these alleles may be considered risk factors for primary hyperuricemia.

Knockdown of HPRT Enables Selection of Genetically Modified Human Hematopoietic Progenitor Cells.

AbstractAbstract 3772Genetic modification of autologous hematopoietic stem cells (HSC) has the potential for effective treatment of a wide variety of inherited blood disorders. However, HSC gene therapy has shown limited clinical efficacy (with notable exceptions), in part because of the small proportion of engrafted genetically corrected HSCs. The use of drug-resistance genes to enable selection for transduced HSCs has been explored, but with limited success. Previous studies from our laboratory have indicated that murine HSC can be selected with 6-Thioguanine (6TG), a relatively non-toxic drug used in the treatment of leukemias, after knocking down the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT), an enzyme that metabolizes 6TG to its active state. We sought to determine if these findings can be translated to human hematopoietic cells. In the present study, we transduced human myeloid (Molm13, MV4-11) and lymphoid cell lines (Reh) with lentiviral vectors expressing shRNA constructs targeting HPRT or a non-targeted control sequence (Ctrl). Two of the most promising constructs directed against HPRT (491 and 50) were studied in more detail to determine which is most effective. Cells were selected in puromycin and cell lysates analyzed for HPRT gene expression. Reverse-transcription, real-time PCR (RT qPCR) and western blotting demonstrated that construct 491 was most efficient in knocking down HPRT in human hematopoietic cell lines compared to construct 50 (and Ctrl). To determine whether knockdown of HPRT provided resistance to 6TG, cells were cultured in the absence or presence of different doses of 6TG and live cell concentrations were determined. While Ctrl transduced cells decreased in a dose dependent manner after 72h of 6TG treatment, cells transduced with constructs 491 and 50 were relatively resistant to 6TG. IC50 values for construct 491 were significantly higher (114μM for Molm13 and 46μM for Reh cell lines) than construct 50 (1μM for Molm13 and 10μM for Reh) in comparison to control transduced cells (0.4μM for Molm13 and 3.5μM for Reh). We assessed cell death in human hematopoietic cell lines by annexin V staining after exposure to 6TG at 48 and 72h. As expected, control transduced cells died of apoptosis upon 6TG treatment, while 491 and 50 transduced cells were resistant. Furthermore, 491 transduced cells were more resistant to apoptosis than 50 transduced cells. Based on these results, construct 491 was used to transduce human CD34+ progenitor cells isolated from umbilical cord blood along with control shRNA. Transduction efficiency varied from 25–35% as determined by %GFP expression by flow cytometry. Sorted GFP+ cells showed reduced expression of HPRT in 491 transduced cells compared to controls, as measured by RT qPCR. Similar to the effects in cell lines, in vitro proliferation of control transduced CD34+ cells diminished in response to increasing 6TG concentrations. There was an increase in the percentage of GFP+ cells in 6TG treated 491 transduced cells compared to untreated controls in a dose dependent fashion, indicating a selective advantage conferred to 491 transduced cells in the presence of 6TG. Importantly, 491 transduced cells continued to proliferate despite treatment with 6TG. Like 6TG, cisplatin requires mismatch repair (MMR) for cytotoxicity. To determine if HPRT knockdown had off-target effects impairing MMR, transduced cells were also treated with cisplatin. Both control and 491 transduced cells stopped proliferating in the presence of cisplatin indicating that MMR remained intact. These data indicate that human hematopoietic progenitor cells can be selected in vitro by knock-down of HPRT and treatment with 6TG. Xenografts of Ctrl and 491 transduced human CD34+ cord blood cells have been generated and are being treated with 6TG to determine if human cells can be selected with 6TG in vivo. Disclosures:Off Label Use: Off label use of 6-thioguanine will be suggested.

Assessment of the cytotoxicity and genotoxicity of haloacetic acids using microplate-based cytotoxicity test and CHO/ HGPRT gene mutation assay

Haloacetic acids (HAAs) are the second most prevalent class of disinfection byproducts found in drinking water. The implications of HAAs presence in drinking water are a public health concern due to their potential mutagenic and carcinogenic effects. In the present study, we examined the cytotoxic and genotoxic effects of six common HAAs using a microplate-based cytotoxicity test and a hypoxanthine–guanine phosphoribosyltransferase ( HGPRT) gene mutation assay in Chinese hamster ovary K1 (CHO-K1) cells. We found that their chronic cytotoxicities (72 h exposure) to CHO-K1 cells varied, and we ranked their levels of toxicity in the following descending order: iodoacetic acid (IA) > bromoacetic acid (BA) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA). The toxicity of IA is 1040-fold of that of TCA. All HAAs except TCA were shown to be mutagenic to CHO-K1 cells in the HGPRT gene mutation assay. The mutagenic potency was compared and ranked as follows: IA > DBA > BA > CA > DCA > TCA. There was a statistically significant correlation between cytotoxicity and mutagenicity of the HAAs in CHO-K1 cells. The microplate-based cytotoxicity assay and HGPRT gene mutation assay were suitable methods to monitor the cytotoxicity and genotoxicity of HAAs, particularly for comparing the toxic intensities quantitatively.

Attenuated variants of Lesch-Nyhan disease: the case of King James VI/I.

Sir, We read with interest the paper by Jinnah et al . (2010) in the March 2010 issue of Brain , describing the clinical characteristics of a large series of patients with an attenuated variant of Lesch–Nyhan disease. All of the patients reported had either a deficiency of hypoxanthine–guanine phosphoribosyltransferase (HPRT) or a mutation in the HPRT gene, and all had evidence of overproduction of uric acid; but neurological involvement was highly variable and none exhibited the self injurious behaviour that is diagnostic of the full-blown condition. The existence of a clinically variable phenotype highlights the importance of considering the condition in the differential diagnosis of atypical neurological presentations in adults as well as children, particularly when they are associated with raised levels of serum and/or urinary uric acid. It also opens new possibilities for retrospective diagnosis of historical figures …

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity.