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Gill Rumsby

Publications and source records attributed to Gill Rumsby.

18 recordsLinked to original sources
PubMed2006

Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase.

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.

Alcohol Oxidoreductases↗
PubMed2006

Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells.

Failure to detoxify the intermediary metabolite glyoxylate in human hepatocytes underlies the metabolic pathology of two potentially lethal hereditary calcium oxalate kidney stone diseases, PH (primary hyperoxaluria) types 1 and 2. In order to define more clearly the roles of enzymes involved in the metabolism of glyoxylate, we have established singly, doubly and triply transformed CHO (Chinese-hamster ovary) cell lines, expressing all combinations of normal human AGT (alanine:glyoxylate aminotransferase; the enzyme deficient in PH1), GR/HPR (glyoxylate/hydroxypyruvate reductase; the enzyme deficient in PH2), and GO (glycolate oxidase). We have embarked on the preliminary metabolic analysis of these transformants by studying the indirect toxicity of glycolate as a simple measure of the net intracellular production of glyoxylate. Our results show that glycolate is toxic only to those cells expressing GO and that this toxicity is diminished when AGT and/or GR/HPR are expressed in addition to GO. This finding indicates that we have been able to reconstruct the glycolate-->glyoxylate, glyoxylate-->glycine, and glyoxylate-->glycolate metabolic pathways, catalysed by GO, AGT, and GR/HPR respectively, in cells that do not normally express them. These results are compatible with the findings in PH1 and PH2, in which AGT and GR/HPR deficiencies lead to increased oxalate synthesis, due to the failure to detoxify its immediate precursor glyoxylate. These CHO cell transformants have a potential use as a cell-based bioassay for screening small molecules that stabilize AGT or GR/HPR and might have use in the treatment of PH1 or PH2.

Alcohol Oxidoreductases↗
PubMed2006

A de novo mutation in the AGXT gene causing primary hyperoxaluria type 1.

Primary hyperoxaluria type 1 is caused by mutations in the alanine-glyoxylate aminotransferase (AGXT) gene. In cases in which no mutation was identified, linkage analysis can be used to confirm or exclude the diagnosis in other siblings. We present a family in which a sibling of the index case predicted to have primary hyperoxaluria type 1 by means of linkage analysis failed to show hyperoxaluria during the following 7 years, putting the diagnosis into question. Whole-gene sequence analysis identified 2 causative mutations in the index case, of which only 1, c.646A (Gly216Arg), was inherited. The other sequence change, c.33_34insC, was a de novo mutation occurring on the paternal allele. This particular mutation is a relatively common cause of primary hyperoxaluria type 1. It occurs in a run of 8 cytosines and therefore potentially is susceptible to polymerase slippage. This case illustrates 2 important points. First, biochemical confirmation of a genetic diagnosis should always be made in siblings diagnosed by using genetic tests. Second, de novo mutations should be considered as a potential, albeit rare, cause of primary hyperoxaluria type 1.

Child, Preschool↗
PubMed2006

Congenital adrenal hyperplasia in adults: a review of medical, surgical and psychological issues.

Our knowledge of the experience of adults with congenital adrenal hyperplasia (CAH) as they pass through life is only now emerging. In this review we gather medical, surgical and psychological literature pertaining to adults with CAH and consider this alongside practical experience gained from a dedicated adult CAH clinic. There is increasing awareness for the need for multidisciplinary teams who have knowledge of CAH particularly with respect to gynaecological surgery and clinical psychology for women and testicular function in men. Routine management of CAH comprises adjustment of glucocorticoid and mineralocorticoid treatment according to individual needs balancing biochemical markers, compliance and long term risks. Bone density is one such long term concern and is not greatly reduced in most individuals with CAH. More recently, attention has turned to cardiovascular risk factors and catecholamine deficiency in adults with CAH. Women with CAH require access to an experienced gynaecologist, specialised pregnancy care and psychosexual support. The very low fertility rates for women with CAH previously reported appear to be improving with time. Men with CAH are often lost to follow up and therefore miss out on surveillance for hypogonadism either through the effect of adrenal rests of from suppression of gonadotrophins resulting in a high prevalence of oligospermia.

Adrenal Hyperplasia, Congenital↗
PubMed2006

An introduction to PCR techniques.

The polymerase chain reaction (PCR) is a method for the enzymatic amplification of DNA and has revolutionised our approach to both diagnostics and research, permitting the amplification of small amounts of genetic material, either derived from genomic (chromosomal DNA) or RNA via reverse transcription to cDNA. This chapter gives a primer of methods for the preparation of materials for PCR and some advice on the requirements for the PCR itself.

Animals↗
PubMed2006

Outcome of bilateral adrenalectomy in congenital adrenal hyperplasia: one unit's experience.

OBJECTIVE: The use of bilateral adrenalectomy in the management of congenital adrenal hyperplasia (CAH) is controversial. We set out to review the outcome of 5 cases of CAH who have undergone adrenalectomy in our unit. DESIGN: A retrospective case note review and subject interview of the experience of adrenalectomy in the setting of a tertiary adult CAH clinic. METHODS: Subjects who had undergone adrenalectomy were reviewed at a routine clinic visit with particular reference to clinical and biochemical outcomes after adrenalectomy. RESULTS: Two subjects underwent surgery for the sole indication of desire for fertility with successful outcome and without subsequent adrenal crises. Three women suffered from the common clinical management problem of unsuppressible hyperandrogenism and worsening obesity. Whilst the outcome of improved appearance and weight loss was achieved in these subjects, all three experienced significant complications including pigmentation and acute episodes of adrenal insufficiency. CONCLUSION: We present a mean follow-up of 4.2 patient years and conclude that this procedure may be suitable for selected women with CAH. Outcomes for those pursuing fertility were positive; however, complications were noted in those women for whom the procedure was performed for symptom control. Accepting that the number of subjects is small, it is clear that more data are required before widely recommending this procedure.

Adrenal Hyperplasia, Congenital↗
PubMed2005

An overview of the role of genotyping in the diagnosis of the primary hyperoxalurias.

The aim of this paper is to give an overview of our current state of knowledge with respect to genotyping for the primary hyperoxalurias and the role of molecular genetics alongside the more traditional biochemical and enzymatic tests for the diagnosis and prognosis of these disorders. The published literature was reviewed to establish the frequency of different mutations and thus the value of testing for a limited number of these mutations in patients with clinical suspicion of primary hyperoxaluria (PH). This approach was compared with whole gene sequencing of the AGXT and GRHPR genes. A limited genetic screen can provide a first line test for PH1 and PH2 in symptomatic patients and can provide a full diagnosis in approximately a third of cases. Molecular genetic analysis is essential for carrier testing and prenatal diagnosis. The value of molecular genetics in prognosis requires a wider evidence base.

Genotype↗
PubMed2005

Intra-familial clinical heterogeneity: absence of genotype-phenotype correlation in primary hyperoxaluria type 1 in Israel.

BACKGROUND/AIMS: Primary hyperoxaluria type 1 (PH1) is caused by the deficiency of the liver enzyme alanine:glyoxylate aminotransferase which results in increased synthesis and excretion of oxalate. The clinical manifestations of PH1 are heterogeneous with respect to the age of onset and rate of progression. The aim of this study was to investigate possible relationships between a given genotype, the biochemical profile and the clinical phenotype. METHODS: We conducted a study of 56 patients from 22 families with PH1 from Israel. The clinical and biochemical data were compiled and the genotype was determined for each family. RESULTS: The prevalent phenotype was of early onset with progression to end-stage renal disease during the first decade of life. Fifteen PH1-causing mutations were detected in 21 families: 10 were first described in this patient population. Marked intra-familial clinical heterogeneity was noted, meaning that there was no correlation between a given genotype and the phenotype. CONCLUSIONS: The clinical course of patients with PH1 is not dictated primarily by its genotype. Other genetic and/or environmental factors play a role in determining the ultimate phenotype.

Child↗
PubMed2004

Molecular aetiology of primary hyperoxaluria and its implications for clinical management.

The primary hyperoxalurias type 1 (PH1) and type 2 (PH2) are autosomal recessive calcium oxalate kidney stone diseases caused by deficiencies of the metabolic enzymes alanine:glyoxylate aminotransferase (AGT) and glyoxylate/hydroxypyruvate reductase (GR/HPR), respectively. Over 50 mutations have been identified in the AGXT gene (encoding AGT) in PH1, associated with a wide variety of effects on AGT, including loss of catalytic activity, aggregation, accelerated degradation, and peroxisome-to-mitochondrion mistargeting. Some of these mutations segregate and interact synergistically with a common polymorphism. Over a dozen mutations have been found in the GRHPR gene (encoding GR/HPR) in PH2, all associated with complete loss of glyoxylate reductase enzyme activity and immunoreactive protein. The crystal structure of human AGT, but not human GR/HPR, has been solved, allowing the effects of many of the mutations in PH1 to be rationalised in structural terms. Detailed analysis of the molecular aetiology of PH1 and PH2 has led to significant improvements in all aspects of their clinical management. Enzyme replacement therapy by liver transplantation can provide a metabolic cure for PH1, but it has yet to be tried for PH2. New treatments that aim to counter the effects of specific mutations on the properties of the enzymes could be feasible in the not-too-distant future.

Alcohol Oxidoreductases↗
PubMed2004

Evaluation of mutation screening as a first line test for the diagnosis of the primary hyperoxalurias.

BACKGROUND: A definitive diagnosis of primary hyperoxaluria type 1 (PH1) and primary hyperoxaluria type 2 (PH2) requires the measurement of alanine:glyoxylate aminotransferase (AGT) and glyoxylate reductase (GR) activities, respectively, in a liver biopsy. We have evaluated a molecular genetic approach for the diagnosis of these autosomal-recessive diseases. METHODS: Polymerase chain reaction (PCR) was used to detect three common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) and one, c.103delG, in the GRHPR gene in DNA samples from 365 unrelated individuals referred for diagnosis of PH1 and/or PH2 by liver enzyme analysis. RESULTS: One or more of these mutations was found in 183 (68.8%) biopsy proven cases of PH1 and PH2 with a test negative predictive value of 62% and 2%, respectively. 102 (34.1%) patients were homozygous or compound heterozygous, making a molecular diagnosis possible. Age of onset and presenting features were similar in patients homozygous for any of the four mutations. Of the AGXT homozygotes, only the c.508G>A mutant was associated with significant AGT catalytic activity and in two of these activity was in the low normal range, possibly reflecting variation in mitochondrial content of the biopsy as this particular mutation is associated with mitochondrial mistargeting. CONCLUSION: Limited mutation analysis can provide a useful first line test for PH1 and PH2 in patients in whom primary hyperoxaluria is suspected and in whom secondary causes have been excluded. Those patients in whom a single mutation, or no mutation, is found can then be selectively targeted for liver biopsy.

Age Factors↗
PubMed2003

Molecular analysis of the glyoxylate reductase (GRHPR) gene and description of mutations underlying primary hyperoxaluria type 2.

Primary hyperoxaluria type 2, an inherited autosomal recessive disorder of endogenous oxalate overproduction, is caused by mutations in the GRHPR gene encoding the glyoxylate/hydroxypyruvate reductase enzyme. The GRHPR genes from nineteen unrelated patients with PH2 were analysed for mutations using a combination of PCR-SSCP and sequence analysis of genomic and cDNA. Eleven mutations were identified, seven of which are novel. The mutations included five point mutations: c.84-2A>G, c.295C>T (R99X), c.494G>A (G165D), and c.904C>T (R302C) as well as six minor deletions: c.103delG, c.375delG, c.403_405+2 delAAGT, c.540delT, c.608_609delCT and a more complex mutation in intron 1: c.84-13_c.84-12del; c.84-8_c.84-5del. Aberrant transcripts were demonstrated in hepatic mRNA as a result of the c.403_405+2 delAAGT and c.84-2A>G mutations. In addition, a splice variant lacking 28 bp of exon 1 was expressed in a number of tissues but is of unknown function. Two polymorphisms, c.579A>G in exon 6 and a (CT)(n) microsatellite in intron 8 were identified. Expression studies showed that the G165D and R302C mutants had glyoxylate reductase activity 1.5 and 5.6% respectively of the wild type protein. Both mutant proteins were unstable on purification. Although there is wide expression of the GRHPR mRNA demonstrated by northern blot analysis, our study shows that GRHPR protein distribution is predominantly hepatic and concludes that PH2, like the related type 1 disease, is primarily a disorder affecting hepatic glyoxylate metabolism.

Alcohol Oxidoreductases↗
PubMed2002

Primary hyperoxaluria type 2 in children.

The primary hyperoxalurias (PH1 and PH2) are rare defects of oxalate overproduction. There are only 24 reported cases of PH2, which is characterized by raised urine oxalate and L-glycerate. We describe 13 previously unreported children with PH2, representing the largest single-centre cohort in the world. DNA samples were tested for a common mutation and four other documented mutations in the gene encoding the enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Two of the five kindred showed homozygosity for two different mutations in the GRHPR gene. The genetic defect was not identified in the other three families. The median age at diagnosis of PH2 was 1.7 years. Five children presented with nephrolithiasis between 0.8 and 9 years. Haematuria was common, but urinary tract infection and nephrocalcinosis were not. All had normal renal function at diagnosis, and only 1 patient had a significant decline in glomerular filtration rate. We conclude that all children with nephrolithiasis secondary to hyperoxaluria should have urinary glycerate measured, as PH2 may be more prevalent than currently estimated. DNA mutational analysis may be useful in supporting the diagnosis.

Adolescent↗
PubMed2002

Potential mechanisms of marked hyperoxaluria not due to primary hyperoxaluria I or II.

BACKGROUND: Hyperoxaluria may be idiopathic, secondary, or due to primary hyperoxaluria (PH). Hepatic alanine:glyoxylate aminotransferase (AGT) or glyoxylate/hydroxypyruvate reductase (GR/HPR) deficiency causes PHI or PHII, respectively. Hepatic glycolate oxidase (GO) is a candidate enzyme for a third form of inherited hyperoxaluria. METHODS: Six children were identified with marked hyperoxaluria, urolithiasis, and normal hepatic AGT (N = 5) and GR/HPR (N = 4). HPR was below normal and GR not measured in one. Of an affected sibling pair, only one underwent biopsy. GO mutation screening was performed, and dietary oxalate (Diet(ox)), enteric oxalate absorption (EOA) measured using [13C2] oxalate, renal clearance (GFR), fractional oxalate excretion (FE(ox)) in the children, and urine oxalate in first-degree relatives (FDR) to understand the etiology of the hyperoxaluria. RESULTS: Mean presenting age was 19.2 months and urine oxalate 1.3 +/- 0.5 mmol/1.73 m2/24 h (mean +/- SD). Two GO sequence changes (T754C, IVS3 - 49 C>G) were detected which were not linked to the hyperoxaluria. Diet(ox) was 42 +/- 31 mg/day. EOA was 9.4 +/- 3.6%, compared with 7.6 +/- 1.2% in age-matched controls (P = 0.33). GFR was 90 +/- 19 mL/min/1.73 m2 and FE(ox) 4.2 +/- 1.4. Aside from the two brothers, hyperoxaluria was not found in FDR. CONCLUSIONS: These patients illustrate a novel form of hyperoxaluria and urolithiasis, without excess Diet(ox), enteric hyper-absorption, or hepatic AGT, GR/HPR deficiency. Alterations in pathways of oxalate synthesis, in liver or kidney, or in renal tubular oxalate handling are possible explanations. The affected sibling pair suggests an inherited basis.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Genetic heterogeneity in primary hyperoxaluria type 1: impact on diagnosis.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disease characterized by progressive kidney failure due to renal deposition of calcium oxalate. The disease is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT) which catalyzes the conversion of glyoxylate to glycine. When AGT is absent, glyoxylate is converted to oxalate which forms insoluble calcium salts that accumulate in the kidney and other organs. In the most common phenotype there is a unique phenomenon wherein AGT is mis-targeted to the mitochondria instead of the peroxisomes. The diagnosis of PH1 is complicated by heterogeneity of clinical presentation, course of the disease, biochemical markers, AGT enzymatic activity and genotype. More than 50 mutations and polymorphisms have been reported in the AGT gene; three common mutations accounting for almost 50% of PH1 alleles. The mutations are of all types, with missense making up the largest fraction. There are some mutations with apparent ethnic associations and at least one that appears to be pan-ethnic. Although correlations can in some cases be made between biochemical phenotype and genotype, correlation with clinical phenotype is complicated by the involvement of other genetic and non-genetic factors that affect disease severity. A number of polymorphisms have been described in the AGT gene some of which cause missense changes and, in some cases, alter enzyme activity. As DNA testing becomes more commonly used for diagnosis it is important to correlate observed sequence changes with previously documented changes as an aid to assessing their potential significance.

Genetic Heterogeneity↗