Implications of Genotype and Enzyme Phenotype in Pyridoxine Response of Patients with Type I Primary Hyperoxaluria

Mutations in AGXT, a locus mapped to 2q37.3, cause deficiency of liver-specific alanine:glyoxylate aminotransferase (AGT), the metabolic error in type 1 primary hyperoxaluria (PH1). Genetic analysis of 55 unrelated probands with PH1 from the Mayo Clinic Hyperoxaluria Center, to date the largest with availability of complete sequencing across the entire AGXT coding region and documented hepatic AGT deficiency, suggests that a molecular diagnosis (identification of two disease alleles) is feasible in 96% of patients. Unique to this PH1 population was the higher frequency of G170R, the most common AGXT mutation, accounting for 37% of alleles, and detection of a new 3 end deletion (Ex 11_3UTR del). A described frameshift mutation (c.33_34insC) occurred with the next highest frequency (11%), followed by F152I and G156R (frequencies of 6.3 and 4.5%, respectively), both surpassing the frequency (2.7%) of I244T, the previously reported third most common pathogenic change. These sequencing data indicate that AGXT is even more variable than formerly believed, with 28 new variants (21 mutations and seven polymorphisms) detected, with highest frequencies on exons 1, 4, and 7. When limited to these three exons, molecular analysis sensitivity was 77%, compared with 98% for whole-gene sequencing. These are the first data in support of comprehensive AGXT analysis for the diagnosis of PH1, obviating a liver biopsy in most well-characterized patients. Also reported here is previously unavailable evidence for the pathogenic basis of all AGXT missense variants, including evolutionary conservation data in a multisequence alignment and use of a normal control population.

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“…Because the haplotype analysis in this patient suggested a potential partial c Previously published in a smaller series (37).…”

Section: Resultsmentioning

confidence: 78%

Comprehensive Mutation Screening in 55 Probands with Type 1 Primary Hyperoxaluria Shows Feasibility of a Gene-Based Diagnosis

Monico

Rossetti

Schwanz

et al. 2007

Journal of the American Society of Nephrology

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“…Pyridoxine, which is broken down to form PLP in cells, has long been known to improve the outcome in patients with certain forms of PH1 (1,12), but it has been unclear how this effect is mediated. Such treatment improves the disease in certain patients with residual AGT enzymatic activity but has no effect on patients that lack enzyme activity, leading researchers to hypothesize that PLP may be acting to enhance protein folding or stability.…”

Section: Discussionmentioning

confidence: 99%

“…Molecules that provide even a modest increase in protein stability may therapeutically benefit PH1 patients with partially functional AGT variants. One chemical that has been used in pharmacological treatment of PH1 is pyridoxine, which appears to improve outcome for certain subtypes of PH1 (1,12) through an unknown mechanism. Pyridoxine is broken down in the cell to form pyridoxal 5Ј-phosphate (PLP), a cofactor for AGT.…”

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confidence: 99%

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Hopper

Pittman

Fitzgerald

et al. 2008

Journal of Biological Chemistry

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Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.Deficiencies in the enzyme alanine:glyoxylate aminotransferase (AGT) 3 cause primary hyperoxaluria type I (PH1), a severe autosomal recessive kidney stone disease (1, 2). In humans, AGT is responsible for conversion of glyoxylate to glycine in the liver. Without functional AGT, glyoxylate builds up and is converted to calcium oxalate, which is deposited in the kidneys and can lead to kidney stones and renal failure. In many patients, deficiency of AGT results from one or two amino acid changes that decrease the stability of this enzyme. As a result, AGT may be degraded, become improperly localized, or form nonfunctional aggregates (1, 2).Over 50 different mutations of AGT and two polymorphic variants have been identified (1, 3). The two allelic forms consist of a "wild-type" major allele, AGTma, and a minor allele, AGTmi. The minor allele is present in ϳ20% of European and North American populations and contains P11L and I340M substitutions in the amino acid sequence and a 74-bp duplication in intron 1 (4, 5). The P11L and I340M substitutions, particularly P11L, have several biochemical effects, including decreasing catalytic activity and slowing the dimerization rate, but by themselves are not disease-causing (4, 5). The minor allele of AGT is delete...

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“…We detected an additional 6 missense changes that have previously been reported, although not expressed in vitro. These mutations are c.26CϾA (13,14 ) and c.106CϾT (15 ) in exon 1, c.466GϾA (16 ) and c.518GϾA (17 ) in exon 4, and c.697CϾT and c.698GϾA (17 ) in exon 7.…”

Section: Construction and Analysis Of Mutantsmentioning

confidence: 99%

Selected Exonic Sequencing of the AGXT Gene Provides a Genetic Diagnosis in 50% of Patients with Primary Hyperoxaluria Type 1

Williams

Rumsby²

2007

Clinical Chemistry

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Background: Definitive diagnosis of primary hyperoxaluria type 1 (PH1) requires analysis of alanine:glyoxylate aminotransferase (AGT) activity in the liver. We have previously shown that targeted screening for the 3 most common mutations in the AGXT gene (c.33_ 34insC, c.508G>A, and c.731T>C) can provide a molecular diagnosis in 34.5% of PH1 patients, eliminating the need for a liver biopsy. Having reviewed the distribution of all AGXT mutations, we have evaluated a diagnostic strategy that uses selected exon sequencing for the molecular diagnosis of PH1. Methods: We sequenced exons 1, 4, and 7 for 300 biopsy-confirmed PH1 patients and expressed the identified missense mutations in vitro. Results: Our identification of at least 1 mutation in 224 patients (75%) and 2 mutations in 149 patients increased the diagnostic sensitivity to 50%. We detected 29 kinds of sequence changes, 15 of which were novel. Four of these mutations were in exon 1 (c.2_3delinsAT, c.30_ 32delCC, c.122G>A, c.126delG), 7 were in exon 4 (c

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Implications of Genotype and Enzyme Phenotype in Pyridoxine Response of Patients with Type I Primary Hyperoxaluria

Cited by 83 publications

References 23 publications

Comprehensive Mutation Screening in 55 Probands with Type 1 Primary Hyperoxaluria Shows Feasibility of a Gene-Based Diagnosis

Comprehensive Mutation Screening in 55 Probands with Type 1 Primary Hyperoxaluria Shows Feasibility of a Gene-Based Diagnosis

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Selected Exonic Sequencing of the AGXT Gene Provides a Genetic Diagnosis in 50% of Patients with Primary Hyperoxaluria Type 1

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