Selected exonic sequencing of the AGXT gene provides a genetic diagnosis in 50% of patients with primary hyperoxaluria type 1

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.447_454delGCTGCTGT, c.449T>C, c.473C>T, c.481G>A, c.481G>T, c.497T>C, c.424-2A>G), and 4 were in exon 7 (c.725insT, c.737G>A, c.757T>C, c.776 + 1G>A). The missense changes were associated with severely decreased AGT catalytic activity and negative immunoreactivity when expressed in vitro. Missense mutation c.26C>A, previously described as a pathological mutation, had activity similar to that of the wild-type enzyme. CONCLUSIONS: Selective exon sequencing can allow a definitive diagnosis in 50% of PH1 patients. The test offers a rapid turnaround time (15 days) with minimal risk to the patient. Demonstration of the expression of missense changes is essential to demonstrate pathogenicity.     

Association of c-fos mRNA expression and excitotoxicity in primary cultures of mouse neocortical and cerebellar neurons

The effect of excitatory amino acids (EAAs) on c-fos mRNA expression was studied in primary cultures of mouse cerebellar granule cells and in neocortical neurons after 2 and 7 days in vitro (div). In cultured granule cells at 2 and 7 div, and in cortical neurons at 2 div, exposure to low levels (≤10 μM) of a variety of EAAs (viz. glutamate [Glu], S-sulpho-L-cysteine [SC], N-methyl-D-aspartate [NMDA], α-amino-3-hydroxy-5-methyl-4-isoxazole [AMPA], and kainate [KA]) resulted in a transient increase in the level of c-fos mRNA which peaked at 30 min but returned to a basal level by 120 min. However, exposure of granule cells (7 div) to high levels (250 μM) of Glu, NMDA, KA, SC and of cortical neurons (7 div) to high levels (250 μM) of Glu, NMDA, KA, SC, or AMPA and to low levels (≤10 μM) of Glu and AMPA resulted in a delay in c-fos mRNA induction but a subsequent, progressive increase that was sustained for at least 240 min. Furthermore, this effect was accompanied by a dose-related increase in the release of the cytosolic enzyme, lactate dehydrogenase, used as an indicator of excitotoxicity. A ratio (Q^240/30) for the steady-state levels of c-fos mRNA after 30 min and 240 min of exposure to EAAs was determined which showed that Q^240/30 >2 correlated reproducibly with excitotoxic cell death, whereas a ratio of ≤1 correlated with a nonexcitotoxic event. In both cell types at 7 div, coadministration of the selective NMDA receptor antagonist, DL(±)-2-amino-5-phosphonopentanoic acid (APV) with cytotoxic levels of Glu 1) protected against EAA-induced neurotoxicity and 2) exhibited a transient c-fos mRNA expression (Q^240/30 values ≈1). In contrast, the AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), provided no protection against excitotoxicity and had no significant effect on the Glu-induced delay in c-fos mRNA expression. These results suggest that the Q^240/30 c-fos mRNA ratio may 1) be used as a predictive index for excitotoxic neuronal death, 2) provide information on the identity of the receptor subtype mediating excitotoxicity in different brain cell types, and 3) aid in establishing the role of excitotoxicity during the development of neurons in vitro. J. Neurosci. Res. 48:533–542, 1997. © 1997 Wiley-Liss Inc.     

Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria

The primary hyperoxalurias, PH1 and PH2, are inherited disorders caused by deficiencies of alanine:glyoxylate aminotransferase and glyoxylate reductase, respectively. Mutations in either of these enzymes leads to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end-stage renal failure and systemic oxalosis. To provide a non-invasive means of accessing kidney cells from individuals with primary hyperoxaluria, we have derived primary cultures of renal proximal tubule cells from the urine of these patients. The cells stain positively for the epithelial markers pan-cytokeratin and zonula occludens 1 and the proximal tubule marker γ-glutamyl transpeptidase. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed glyoxylate reductase at the mRNA level, illustrating their potential value as a source of renal material from these individuals.     

Delayed puberty from partial 17-alpha hydroxylase enzyme deficiency

An 18-year-old woman with primary amenorrhoea and pubertal delay was investigated for mild labile hypertension and secondary hypogonadism. Low renin and normal aldosterone levels combined with evidence of primary adrenal insufficiency suggested partial 17-alpha hydroxylase enzyme deficiency. The diagnosis was confirmed by measurement of 24-hour urine steroid metabolites and whole gene sequencing of CYP17A1 that demonstrated c.160_162delTTC (p.Phe54del) homozygous mutation. Ultrasound showed bilateral small ovaries with multiple cysts. The serum anti-mullerian hormone concentration was unremarkable at 6.6 (normal <12.6 ng/ml) but the outlook for her future ovulatory potential is uncertain. Dexamethasone 0.25 mg pre-bed and hydrocortisone 5 mg on waking normalised her hormonal profile and her blood pressure without side-effects.     

Experimental allergic encephalomyelitis: Modification of optic nerve pathology by antecedent virus infection

Summary Strain 13 guinea pigs, sensitised with spinal cord and Freund's complete adjuvant at 18–21 days of age, developed an experimental allergic encephalomyelitis (EAE) characterised by a non-fatal first attack followed by recovery, then remissions and relapses in the majority of animals. Pretreatment with an i.p. injection of an avirulent strain (A 774) of Semliki forest virus at 1 day of age resulted in an EAE with an accelerated onset and enhanced mortality during the first attack. Unlike the acute phase pathology in unpretreated animals, optic nerve demyelination was observed at 14 days after neuroantigen sensitisation. In sections of optic nerve from virus-treated animals, endothelial cell vesicularisation was seen and the role of this change in disease exacerbation is discussed. Virus treatments of animals older than 1 day of age resulted in a failure to potentiate the disease. Virus inoculated simultaneously with spinal cord alone failed to act as an adjuvent. The mechanism of virus action in modifying the immunopathogenesis of EAE and its relevance to multiple sclerosis is discussed.We thank Multiple Sclerosis Society of Great Britain and Northern Ireland for the financial support of this work