Background
Primary hyperoxaluria type 1 (PH1; MIM# 259900) is an autosomal recessive disorder of glyoxylate metabolism, leading to the overproduction of endogenous oxalate; patients present with urolithiasis and/or nephrocalcinosis [
1,
2]. The disease is caused by mutations in the
AGXT gene (MIM#604285), which encodes the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the transamination of glyoxylate to glycine [
3,
4]. Approximately 50% of patients who present with PH1 in childhood will have end-stage renal failure by the age of 15 years. PH1 is a condition that leads to systemic oxalosis with oxalate precipitation in the eye, heart, and bones and that results in significant morbidity and mortality [
5,
6]. The incidence of PH1 is estimated at 1 in 120,000 live births with prevalence ranging from 1.05/10
6 to 2.9/10
6 in France, Switzerland, and the Netherlands [
5,
7‐
9]. Excessive oxalate excretion is an indicator of this disease; however, the test is not specific for PH1 and may have misleading results because oxalate excretion may be reduced during renal failure [
2,
10,
11]. Thus, more sophisticated tests, including genetic analysis and/or enzymology, are required for diagnosis [
12‐
14]. Although a few sporadic cases of PH1 have been reported in mainland China, mutational analysis of
AGXT was not performed in these cases. We identified 2 unrelated cases of PH1 in mainland China. We analysed the clinical features, detected
AGXT mutations in their families, and compared our cases with other ethnic or regional patients previously reported by other authors. We hope that the presentation of rare cases will contribute to understanding the spectrum of the disease by aiding its clinical identification and pathogenetic understanding.
Discussion
PH1 is a severe autosomal recessive inherited disorder of glyoxylate metabolism caused by mutations in the
AGXT gene on chromosome 2q37.3 that encodes the liver-specific PLP-dependent enzyme AGT [
4]. More than 150 different pathogenic mutations, including nonsense, frameshift, and missense mutations, in the
AGXT gene that cause PH1 have been identified to date (
http://www.hgmd.cf.ac.uk/ac/gene.php?gene=AGXT). While nonsense and frameshifts are null mutations that lead to the complete loss of the gene product, the most common type of
AGXT mutations are single amino-acid substitutions that lead to the synthesis of an aberrant gene product [
6]. These mutations are found throughout the entire gene and cause a wide spectrum of clinical severity.
The four most common
AGXT mutations in PH1 in European and North American patients are p.G170R, p.F152I, p.I244T and c.33
-34insC [
19,
20]. The p.S205P mutation is a PH1-specific mutation in Japanese patients [
21]. In the present study, mutational analysis of the
AGXT gene in two Chinese families with PH1 revealed two patients who had compound heterozygous mutations. One patient had p.S81X and p.S275delinsRAfs mutations, and the other had p.M1T and p.I202N mutations. These four mutations are different from the four most common
AGXT mutations in European and North American patients, which are also different from the PH1-specific mutation (p.S205P) in Japanese patients. The p.S275delinsRAfs and p.M1T mutations have been previously reported [
22,
23]. The p.M1T mutation was first reported in another Chinese study from Hong Kong in 2004 [
23]. This mutation has not been reported in other populations to date. The other two mutations, p.S81X and p.I202N, are novel. The p.S81X mutation is a nonsense mutation that leads to a truncated AGT, and this mutation was speculated to be a pathogenic mutation. p.I202N is another missense mutation and is located on exon 6. Exon 6 spans the PLP co-factor binding site consensus sequence (amino acids 201–221) common to aminotransferases and is critical to the catalytic site [
24]. Crystallisation studies confirmed that the lysine at codon 209 in exon 6 is the actual site of the Schiff base with PLP [
25]. The alignment of AGT proteins from several species revealed that the isoleucine at codon 202 is 100% conserved across all analysed species (Figure
2d) and constitute one of the amino acids in the sequence of a highly conserved region of AGT protein. Several clinical reports identified nine mutations also located in this highly conserved sequence motif [
6,
21,
26‐
29]. Given the predictions of the
in silico PolyPhen-2 and SIFT analyses and the fact that the mutation was not identified in 100 healthy controls, the missense p.I202N mutation identified in patient 1 in this highly conserved sequence motif is hypothesised to be a damaging mutation. Therefore, the two novel mutations, p.S81X and p.I202N, were not identified in 100 healthy controls and are considered to be disease-causing mutations.
AGXT gene mutations result in deficiency and/or mistargeting of hepatic AGT, which leads to metabolic overproduction of oxalate and glycolate. The excess oxalate is excreted in the urine but is of low solubility and precipitates as a calcium salt, resulting in urolithiasis, nephrocalcinosis, and progressive renal insufficiency [
11,
30]. Both patients in this study presented with haematuria, back pain, and recurrent urolithiasis. Radiographic screening revealed multiple urolithiasis in both patients. Stone analysis revealed the main component of the stones to be calcium oxalate monohydrate (>95%). The patient with p.S81X and p.S275delinsRAfs mutations, presenting at the age of 3, did not respond to treatment with pyridoxine and progressed rapidly to end-stage renal disease (ESRD) at the age of 10.6. This patient displayed serious clinical features given that she had compound truncation mutations, which led to hepatic AGT deficiency. The patient with the p.M1T and p.I202N missense mutations presented at the age of 5 and received conservative treatment, including a high fluid intake, oral potassium citrate, and oral pyridoxine. Currently, her renal function is normal. PH1 may present at any age [
4]. The presentation varies from infantile nephrocalcinosis and failure to thrive as a result of renal impairment to recurrent or occasional stone formation [
30]. Patients should undergo metabolic screening for PH1 at the presentation of a first kidney stone (in a child), in recurrent or familial stone disease (at any age), or if nephrocalcinosis is detected [
10,
31]. Stone analysis may reveal characteristic morphology and whether the stone contains > 95% calcium oxalate monohydrate, which often presents with a particular morphology [
32]. PH1 should be considered in any patient with renal failure of unknown cause, particularly in the presence of nephrocalcinosis or severe stone burden, because PH1 accounts for 1 to 2% of cases of paediatric ESRD according to registries from Europe, the United States, and Japan [
33]. Radiographic screening of the kidneys may elucidate stones and/or medullary or diffuse nephrocalcinosis [
30]. Although hepatic biopsy is the gold standard for PH1 diagnosis, it is invasive, and facilities for the estimation of enzyme activity are not available in many countries, especially developing countries. Hence, molecular diagnosis using direct sequencing of the whole
AGXT gene is recommended. Therefore, the gold standard method of diagnosis—liver-biopsy-proven enzyme deficiency or enzyme mislocalisation—is now exclusively performed in patients for whom clinical suspicion is still evident but mutation analysis did not result in a precise diagnosis [
2].
This is the first mutational analysis of PH1 performed in mainland China. Because facilities for enzyme estimation of liver biopsies are not available in most centres, there is a need to define clinical features and family histories of consanguinity. We believe that the findings of nephrocalcinosis, recurrent urolithiasis, and progressive renal failure in patients with severe stone burden are sufficient to clinically diagnose PH1. Detection of mutations in the AGXT gene may confirm the diagnosis of PH1.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HX and YA conceptualised the study. G-mL performed the experiment. QS and X-yF obtained funding. HX, G-mL, Y-nG, QS, X-yF, LS, and H-mL acquired the data. G-mL analysed the data. HX, YA, G-mL, and Y-nG contributed to data interpretation and manuscript preparation. All authors read and approved the final manuscript.