Alanine-glyoxylate Aminotransferase Gene Research Articles

32-year-old diabetic patient with progressive vision loss and crystalline retinopathy.

To report the case of severe bilateral retinal vascular occlusion in a patient with hyperoxalosis and chronic renal failure. Observational case report. Medical and imaging records were retrospectively reviewed. The patient was imaged with ultra-widefield (UWF) fundus photography and fluorescein angiography (UWF-FA), cross sectional and en face spectral-domain optical coherence tomography (SD-OCT), and OCT angiography. A 32-year-old diabetic patient receiving peritoneal dialysis was referred because of severe vision loss. UWF color fundus photography showed diffuse sclerotic retinal vessels and diffuse intraretinal crystals in both eyes. UWF-FA illustrated near-complete retinal vascular occlusion and capillary wipe out in both eyes. SD-OCT demonstrated diffuse inner and middle retina thinning in both eyes and multiple intraretinal hyperreflective foci consistent with crystalline deposits in all retina layers of both eyes. OCT angiography revealed severe capillary and large vessel non-perfusion in the superficial and deep retinal capillary plexus of each eye. The serum oxalate levels were increased at 28 µmol/L (reference range < 2 µmol/L) and genetic testing was positive for a heterozygous mutation of the AGXT (Alanine-Glyoxylate Amino Transferase) gene that causes type 1 autosomal recessive primary hyperoxaluria. A diagnosis of hyperoxalosis causing severe retinal vascular occlusion was rendered. Hyperoxalosis was the result of multiple factors including heterozygous AGXT mutation, chronic renal failure insufficiently treated with peritoneal dialysis, and a diet high in oxalate. This case highlights the importance of ruling out retinal oxalosis in patients on peritoneal dialysis in order to initiate prompt hemodialysis and prevent severe retinal vascular occlusion.

Primary hyperoxaluria: a case series

BackgroundPrimary hyperoxaluria (PH) is a rare genetic disorder characterized by the excessive production and accumulation of oxalate. We present five cases of PH, each exhibiting varying manifestations of the disorder including a case presenting as postpartum kidney failure. Notably, three of these cases involve a previously unreported mutation.Case presentationsWe evaluated five Indian patients who presented with varying manifestations of PH. The first case, a 30 year old woman, presented as post-partum kidney failure and was found to be having oxalate nephropathy precipitated by dietary oxalate overload in the setting of previously undiagnosed PH. Genetic analysis revealed a previously unreported mutation in the alanine-glyoxylate aminotransferase gene. The patient underwent simultaneous kidney liver transplant. The second and third cases, 26 and 28 year old women respectively, were asymptomatic siblings of the first patient, who were diagnosed through screening. The fourth case is a 12 year boy with PH type 1 presenting as nephrolithiasis and rapidly worsening kidney function requiring combined kidney liver kidney transplant. Case 5 is a 6 year old male child with type 2 PH presenting with nephrolithiasis, nephrocalcinosis and normal kidney function. All the patients were born to consanguineous parents.ConclusionsDue to limited clinical suspicion and inadequate diagnostic resources in certain countries with limited resources, it is possible for PH to go undiagnosed. The manifestations of the disease can range from no noticeable symptoms to severe disease. Interestingly, in some individuals with primary hyperoxaluria, the disease may not exhibit any symptoms until it is triggered by a high intake of dietary oxalate.

Generation and characterization of a novel rat model of primary hyperoxaluria type 1 with a nonsense mutation in alanine-glyoxylate aminotransferase gene.

Primary hyperoxaluria type 1 (PH1) is a severe inherited disorder caused by a genetic defect in alanine-glyoxylate aminotransferase (AGXT), which results in recurrent urolithiasis and renal failure. Animal models that precisely reflect human PH1 phenotypes are lacking. We aimed to develop a novel PH1 rat model and study the mechanisms involved in PH1 deterioration. One cell stage Sprague-Dawley embryos were injected with the CRISPR/Cas9 system to introduce a Q84X mutation in Agxt. Liver tissues were harvested to determine Agxt expression. Urine oxalate, crystals, and electrolyte levels in AgxtQ84X and wild-type (WT) littermates were evaluated. Kidney tissues were used for Pizzolato staining and kidney injury evaluation. Data showed that Agxt mRNA and protein were absent in AgxtQ84X rats. At 4 and 24 wk, AgxtQ84X rats displayed 2.1- and 2.9-fold higher urinary oxalate levels, respectively, compared with WT littermates. As a result, calcium oxalate (CaOx) crystals in urine were revealed in all AgxtQ84X rats but in none of the WT rats. We also observed bladder stones in 36.4% of AgxtQ84X rats, of which 44.4% had renal CaOx deposition. Moreover, the elevated serum urea and creatinine levels indicated the impaired renal function in AgxtQ84X rats. Further investigation revealed significantly increased expression of inflammation-, necroptosis-, and fibrosis-related genes in the kidneys of AgxtQ84X rats with spontaneous renal CaOx deposition, indicating that these pathways are involved in PH1 deterioration. Collectively, these results suggest that this rat model has broad applicability in mechanistic studies and innovative therapeutics development for PH1 and other kidney stone diseases.NEW & NOTEWORTHY Primary hyperoxaluria type 1 is a severe inherited disorder that results in recurrent urolithiasis and renal failure. We generated an alanine-glyoxylate aminotransferase (Agxt)Q84X nonsense mutant rat model that displayed an early onset of hyperoxaluria, spontaneous renal CaOx precipitation, bladder stone, and kidney injuries. Our results suggest an interaction of renal CaOx crystals with the activation of inflammation-, fibrosis-, and necroptosis-related pathways. In all, the AgxtQ84X rat strain has broad applicability in mechanistic studies and the development of innovative therapeutics.

CRISPR/Cas9–mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1

Primary hyperoxaluria type I is caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT), leading to accumulation of glyoxylate and subsequent production of oxalate and urolithiasis. Here, we generated a novel rat model of primary hyperoxaluria type I that carries a D205N mutation in the partially humanized Agxt gene through the CRISPR/Cas9 system. The AgxtD205N mutant rats showed undetectable alanine glyoxylate aminotransferase protein expression, developed hyperoxaluria at 1 month of age and exhibited severe renal calcium oxalate deposition after ethylene glycol challenge. This suggests our novel model is more relevant to the human disease than existing animal models. To test whether this model could be used for the development of innovative therapeutics, SaCas9 targeting hydroxyacid oxidase 1, responsible for metabolizing glycolate into glyoxylate, was delivered via adeno-associated viral vectors into newborn rats with primary hyperoxaluria type 1. This approach generated nearly 30% indels in the Hao1 gene in the liver, leading to 42% lower urine oxalate levels in the treated group than in the control group and preventing the rats with primary hyperoxaluria type 1 from undergoing severe nephrocalcinosis for at least 12 months. Thus, our results demonstrate that this partially humanized AgxtD205N rat strain is a high-performing model of primary hyperoxaluria type 1 for understanding pathology, and the development of novel therapeutics, such as reprogramming of the metabolic pathway through genome editing.

Wheat differential gene expression induced by different races of Puccinia triticina.

Puccinia triticina, the causal agent of wheat leaf rust, causes significant losses in wheat yield and quality each year worldwide. During leaf rust infection, the host plant recognizes numerous molecules, some of which trigger host defenses. Although P. triticina reproduces clonally, there is still variation within the population due to a high mutation frequency, host specificity, and environmental adaptation. This study explores how wheat responds on a gene expression level to different P. triticina races. Six P. triticina races were inoculated onto a susceptible wheat variety and samples were taken at six days post inoculation, just prior to pustule eruption. RNA sequence data identified 63 wheat genes differentially expressed between the six races. A time course, conducted over the first seven days post inoculation, was used to examine the expression pattern of 63 genes during infection. Forty-seven wheat genes were verified to have differential expression. Three common expression patterns were identified. In addition, two genes were associated with race specific gene expression. Differential expression of an ER molecular chaperone gene was associated with races from two different P. triticina lineages. Also, differential expression in an alanine glyoxylate aminotransferase gene was associated with races with virulence shifts for leaf rust resistance genes.

SNP Variants in RET and PAX2 and Their Possible Contribution to the Primary Hyperoxaluria Type 1 Phenotype

Primary hyperoxaluria type 1 (PH1) is a rare genetic kidney disease caused by a deficiency of alanine:glyoxylate aminotransferase (AGT). Genetic heterogeneity of the AGT gene cannot fully account for heterogeneity in the clinical phenotype. This study investigates a possible contribution to the clinical phenotype from SNPs in RET or PAX2 genes associated with reduced nephron number. The frequencies of these SNPs were compared in PH1-affected DNA samples and normal controls, and relative to age of onset in PH1-affected individuals. The frequencies of the risk alleles were higher with early age of onset, although not significantly so. However, homozygosity for the risk alleles of RET and PAX2 was not seen in the late onset group. The overall frequencies of risk alleles and the numbers of homozygotes were significantly higher for PAX2 in PH1 samples versus controls, suggestive of a bias towards more severe clinical phenotypes in the PH1 samples submitted for analysis.

Long-Term Renal Allograft Survival After Posttransplantation Diagnosis of Primary Hyperoxaluria

Primary hyperoxaluria (PH) is a rare metabolic disorder with autosomal recessive inheritance characterized by excessive production and systemic deposition of oxalate. Unfortunately, the diagnosis in some patients is not made until after kidney transplantation. These grafts are nearly always lost unless the patient receives a liver transplant to correct the underlying defect in oxalate metabolism. We describe a patient who developed extensive oxalate deposition within her transplanted kidney necessitating a return to hemodialysis (HD). After treatment with pyridoxine, high-flux dialysis, and substitution of sirolimus for Cyclosporin A, she ceased dialysis and maintained stable allograft function. A 47-year-old woman was admitted for a deceased-donor kidney transplantation in 2006 with kidney failure attributed to reflux nephropathy and a history of traumatic nephrectomy during childhood. She underwent peritoneal dialysis before transitioning to HD for 3 years. Her medical history included painful peripheral neuropathy and small-joint polyarthropathy characterized by subperiosteal erosions. Surgery was uncomplicated with immediate graft function. Immunosuppression consisted of induction with basiliximab and maintenance with prednisolone, mycophenolate mofetil, and cyclosporin. Kidney biopsies performed on days 7 and 11 after transplantation, for an unexplained decline in renal function, showed changes of mild acute tubular necrosis. A further decline in kidney function on day 22 prompted another biopsy that demonstrated extensive deposition of calcium oxalate crystals with associated tubular damage (Fig. 1).FIGURE 1: Kidney biopsy at 3 weeks after transplantation showing extensive oxalate deposition. A, hematoxylin and eosin. Original magnification, ×100. B, under polarized light. Original magnification, ×400.Given the likely diagnosis of PH, daily high-flux HD, pyridoxine (1), dithiazide, magnesium and phosphate supplements, and a low-oxalate diet were commenced (2). Urinary oxalate excretion was elevated at 1.2 mM/day (normal, <0.6 mM/day). Cyclosporin was ceased given the association with hyperoxaluria and hypocitraturia, renal calcium-oxalate deposition (3), and poor kidney transplantation outcomes (4, 5). The serum creatinine stabilized at 150 μM/L after 5 weeks. Rapid and extensive deposition of oxalate within the small joints of the hand resulted in a gross deforming polyarthropathy and, within the bone marrow, led to refractory anemia. The diagnosis of type-1 hyperoxaluria was confirmed by genetic testing where a mutation in the alanine:glyoxylate aminotransferase (AGT) gene (homozygous 508A missense mutation, Gly170Arg) was identified. Subsequent family screening identified several other family members with the same AGT gene mutation. A protocol kidney biopsy at 3 months showed acute T-cell–mediated rejection (Grade IB) and persistent oxalate deposition. The rejection resolved after three doses of methylprednisolone and addition of sirolimus. Eight months after transplantation, she had a prolonged hospital admission with an ischemic toe requiring amputation, Clostridium difficile colitis, and extensive deep-vein thrombosis. A rise in serum creatinine prompted a biopsy that demonstrated T-cell–mediated rejection (Grade IA) and extensive oxalate deposition. Because of her multiple medical problems, the rejection episode was not treated. HD was recommenced and immunosuppression (prednisolone, mycophenolate mofetil, and low-dose sirolimus; levels 4–6 ng/mL) continued to preserve residual graft function and aid in oxalate clearance. While on HD, she maintained a daily urine output of 1.5 to 2 L/day. After 18 months of HD, the patient’s predialysis serum creatinine began to decline. HD was slowly weaned, and she now maintains a serum creatinine level of 175 μmol/L 5 years later. PH type 1 is caused by a deficiency of the liver-specific peroxisomal enzyme AGT, and PH type 2 is caused by a deficiency of the cytosolic/mitochondrial enzyme glyoxylate/hydroxypyruvate reductase (2). PH type 3 has only recently been described and is caused by the loss of function of the mitochondrial 4-hydroxy-2-oxoglutarate aldolase enzyme (6). Clinical presentation of PH is heterogenous, often leading to delayed diagnosis. Prognosis and treatment options vary according to the type of PH (reviewed in Hoppe [7]). Liver transplantation with or without kidney transplantation is the only cure for PH type 1. Transplantation outcomes in children and adults with PH demonstrate better kidney graft survival in patients with combined liver-kidney transplantation (8, 9) than in those with kidney transplantation alone. However, in patients with PH type 1 with the homozygous G170R mutation of alanine-glyoxalate aminotransferase and who are responsive to pyridoxine treatment, kidney transplantation alone may be considered (10). Late diagnosis of PH is not uncommon, and it is important to secure the genetic mutation involved as soon as possible. Our patient has a mutation that responded to pyridoxine treatment (11). This case also illustrates that it is possible to preserve kidney allograft function in some patients with PH. Furthermore, replacing Cyclosporin A with a mammalian target of rapamycin inhibitor may reduce oxalate deposition and contribute to improved graft outcomes (12). Veena Roberts 1 Sid V. Rajakumar1 Sandra Crikis2 Karen M. Dwyer1 Prudence A. Hill3 David J. Goodman1 1Department of Nephrology St Vincent’s Hospital Melbourne Melbourne, Australia 2Department of Nephrology Western Health Fitzroy, Australia 3Department of Anatomical Pathology St Vincent’s Hospital Melbourne Melbourne, Australia

Differential expression of liver and kidney proteins in a mouse model for primary hyperoxaluria type I

Mutations in the alanine-glyoxylate aminotransferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. A deeper understanding of the changes in the metabolic pathways secondary to the lack of AGXT expression is needed in order to explore substrate depletion as a therapeutic strategy to limit oxalate production in primary hyperoxaluria type I. We have developed an Agxt knockout (AgxtKO) mouse that reproduces some key features of primary hyperoxaluria type I. To improve our understanding of the metabolic adjustments subsequent to AGXT deficiency, we performed a proteomic analysis of the changes in expression levels of various subcellular fractions of liver and kidney metabolism linked to the lack of AGXT. In this article, we report specific changes in the liver and kidney proteome of AgxtKO mice that point to significant variations in gluconeogenesis, glycolysis and fatty acid pathways.

Primary hyperoxaluria in a compound heterozygote infant

Primary hyperoxaluria type 1 is a rare disorder caused by a defect in the hepatic metabolism of glyoxylate. Cases presenting in infancy are very uncommon and often have a severe course leading to early end-stage renal failure. We treated a case of early presentation of primary hyperoxaluria type 1 and reviewed the relevant literature. A 4-month-old female infant was admitted to our hospital because of acute renal failure and nephrocalcinosis. Mutational analysis of alanine-glyoxylate aminotransferase gene revealed compound heterozygosity in the infant, confirming the development of primary hyperoxaluria type 1. A few weeks later, the condition of the infant worsened during an interdialytic period and died. Interest of this case is based on the coexistence of two mutations of alanine-glyoxylate aminotransferase gene recently reported, and it confirms the severe course of the disease when it presents in infancy. It also highlights the importance of the association of nephrocalcinosis and urolithiasis as key diagnostic manifestations of primary hyperoxaluria type 1.

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer

Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt(-/-) mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria.

Consequences of missense mutations for dimerization and turnover of alanine:glyoxylate aminotransferase: Study of a spectrum of mutations

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme, deficiency of which results in primary hyperoxaluria type 1 (PH1). More than 65 PH1-related mutations are now documented in the AGT gene ( AGXT), of which about 50% are missense. We have generated a spectrum of 15 missense changes including the most common PH1 mutation, G170R, and expressed them on the appropriate background of the major or minor allele, in an Escherichia coli overexpression system and in a rabbit reticulocyte transcription/translation system. We have investigated their effects on enzyme activity, dimerization, aggregation, and turnover. The effect of pyridoxal phosphate (PLP) on dimerization and stability was also investigated. Although all 15 mutant AGTs were expressed as intact proteins in E. coli, only three: G41R and G41V on the major allele, and the common mutation G170R, resulted in significant amounts of enzymatic activity. Dimerization failure was a frequent observation (13/15) except for G41V and D183N. Dimerization was poor with S187F but was substantially improved with PLP. Proteasome-mediated protein degradation was observed for all the mutations except G41R on the major allele, G41V, D183N, G170R, and S218L. Increases in the stability of the mutant enzymes in the presence of PLP were small; however, G41R on the minor allele showed a direct relationship between its half life and the concentration of PLP. The minor allele AGT product and many of the mutants were subject to a limited non-proteasomal proteolytic cleavage when ATP was depleted.

Hepatic alanine-glyoxylate aminotransferase activity and oxalate metabolism in vitamin B6 deficient rats.

Urinary oxalate has an important role in the formation of calcium oxalate stone and approximately 50% to 60% of urinary oxalate is derived from the endogenous metabolism of glyoxylate. Glyoxylate is enzymatically converted to glycine by alanine-glyoxylate aminotransferase in the liver and vitamin B6 has a key role as a coenzyme. Therefore, we evaluated hepatic alanine-glyoxylate aminotransferase activity and oxalate metabolism in vitamin B6 deficient rats. A total of 12 male Wistar rats were fed a control or a vitamin B6 deficient diet. After 4 weeks creatinine, oxalate, glycolate, glycine and citrate in the urine, and alanine-glyoxylate aminotransferase activity and its mRNA level in the liver were measured. Urinary oxalate-to-creatinine and glycolate-to-creatinine ratios were significantly higher in vitamin B6 deficient rats than in control rats but urinary glycine-to-creatinine and citrate-to-creatinine ratios were significantly lower. Hepatic alanine-glyoxylate aminotransferase activity and its mRNA level were significantly lower in vitamin B6 deficient rats than in control rats. Vitamin B6 deficiency not only decreased alanine-glyoxylate aminotransferase activity, but also down-regulated alanine-glyoxylate aminotransferase gene expression by hepatocytes and led to hyperoxaluria and hyperglycolic aciduria secondary to impaired metabolism of oxalate precursors. Hyperoxaluria with hypocitruria may also contribute to calcium oxalate stone formation in vitamin B6 deficiency.

Preliminary Evidence for Ethnic Differences in Primary Hyperoxaluria Type 1 Genotype

Background: Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of peroxisomal alanine:glyoxylate aminotransferase (AGT). In about one third of patients, enzymatically active AGT is synthesized but is mistargeted to mitochondria. There are more than 50 mutations identified in the gene for AGT. Four mutations, G170R, 33_34insC, F152I and I244T account for more than 50% of PH1 alleles. The question arose whether there are ethnic differences in PH1 genotype. Methods: The published data on mutations in the AGT gene were examined with respect to recurrences and geographic or ethnic association. The mutations that have been found in at least 2 unrelated individuals were considered. Results: Two common mutations, G170R and 33_34insC showed no obvious ethnic associations and have been found in a variety of populations. A third common PH1 mutation, I244T, has a strong association with people from a Spanish or North African background. A particularly high frequency among Canary Islands PH1 patients suggests a probable founder effect. Between these two extremes are a number of mutations that recur at low frequency within certain ethnic groups. Conclusions: Ethnic associations of PH1 genotypes span a spectrum ranging from limited recurrences confined to a population group, to a probable founder effect.

Hepatic Alanine-glyoxylate Aminotransferase Activity and Oxalate Metabolism in Vitamin B6 Deficient Rats

Hepatic Alanine-glyoxylate Aminotransferase Activity and Oxalate Metabolism in Vitamin B6 Deficient Rats

Molecular Basis for the Dual Mitochondrial and Cytosolic Localization of Alanine:Glyoxylate Aminotransferase in Amphibian Liver Cells

To gain further insights into the molecular basis of the evolution of alanine:glyoxylate aminotransferase (AGT) intracellular targeting in vertebrates, we have studied the molecular basis of its dual mitochondrial and cytosolic distribution in amphibian liver cells. The AGT gene in Xenopus laevis encodes a polypeptide of 415 amino acids, which includes a 24-residue N-terminal mitochondrial targeting sequence (MTS), at either end of which are located two in-frame potential translation start sites. This MTS is necessary to target Xenopus AGT and sufficient to target a green fluorescent fusion protein to mitochondria in transfected COS cells. The C-terminal tripeptide (KKM), despite being similar to the nonconsensus type 1 peroxisomal targeting sequence in human AGT (KKL), was unable to target Xenopus AGT or human AGT to peroxisomes. The Xenopus AGT gene produces two types of transcript. The longer form encodes a polypeptide that contains the MTS and is targeted to mitochondria. The shorter form encodes a polypeptide that does not contain the MTS and remains in the cytosol. These results are discussed not only in terms of the molecular evolution of AGT targeting but also in terms of the ancillary requirements for the peroxisomal targeting of human AGT.

Molecular adaptation of alanine:glyoxylate aminotransferase targeting in primates.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is targeted to different organelles (mitochondria and/or peroxisomes) in different species. Possibly under the influence of dietary selection pressure, the subcellular distribution of AGT has changed on at least eight occasions during the evolution of mammals. AGT targeting is dependent on the variable use of two alternative transcription and translation initiation sites which determine whether or not the region encoding the N-terminal mitochondrial targeting sequence is contained within the open reading frame. In the present study, we sequenced the 5' region of the AGT gene, including both ancestral translation start sites, for 11 anthropoid primates and compared the results with data already available for two others. We show that while the more 3' of the two translation start sites is maintained in all species, the more 5' site has been lost in six species (five of seven catarrhines and one of six platyrrhines). In addition, the remaining two catarrhines, which have maintained the 5' translation start site, are predicted to have lost mitochondrial targeting by a different mechanism, possibly loss of the more 5' transcription start site. Analysis of the relative frequencies of nonsynonymous and synonymous mutations in the region encoding the extant or ancestral mitochondrial targeting sequences led us to suggest that there has been recent strong positive selection pressure to lose, or decrease the efficiency of, mitochondrial AGT targeting in several anthropoid lineages, and that the loss of mitochondrial targeting in this group of mammals is likely to have occurred on at least four, and possibly five, separate occasions.

Primary hyperoxaluria type 1

Primary hyperoxaluria type 1