Rapid liquid chromatography tandem mass-spectrometry screening method for urinary metabolites of primary hyperoxaluria.

BACKGROUND: The primary hyperoxalurias are inherited disorders of glyoxylate metabolism that lead to overproduction of oxalate, urolithiasis and renal failure. Delays in diagnosis can be costly in terms of preserving renal function. Here we present a rapid liquid chromatography tandem mass-spectrometry screening method for the analysis of metabolites (primary hyperoxaluria metabolites) produced in excess by primary hyperoxaluria patients that include glycolate, glycerate and 2,4-dihydroxyglutarate. METHODS: Assay performance was compared to our existing gas chromatography-mass spectrometry method and clinical utility established by analysis of urine samples from patients with confirmed primary hyperoxalurias (11 PH1, 12 PH2 and 8 PH3) and controls ( n = 12). An additional 67 urine samples from patients with PH3 were used postvalidation to confirm the derived 2,4-dihydroxyglutarate cut-off. RESULTS: Glycolate, glycerate and 2,4-dihydroxyglutarate showed a mean bias of 3.3, -22.8 and 5.7%, respectively, compared to our previously published gas chromatography-mass spectrometry method. The mean total imprecision for glycolate, glycerate and 2,4-dihydroxyglutarate was shown to be 6.4, 10 and 11%, respectively. Clinical assessment confirmed that mean urinary glycolate, glycerate and 2,4-dihydroxyglutarate excretion were significantly elevated in patients with PH1, PH2 and PH3, respectively. The greatest sensitivity and specificity for PH1, PH2 and PH3 was achieved at cut-offs of 193, 100 and 4.9 µmol/mmol for glycolate, glycerate and 2,4-dihydroxyglutarate, respectively. CONCLUSIONS: A rapid screening method for the identification and differentiation of patients with suspected PH1, PH2 and PH3 is presented that allows focussing of genetic testing, saving time, money and, with earlier treatment, potential preservation of renal function for these patients.

A novel point mutation in P450c17 (CYP17) causing combined 17alpha-hydroxylase/17,20-lyase deficiency.

CONTEXT: Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare cause of congenital adrenal hyperplasia and hypogonadism. Novel single amino acid changes in P450c17 provide potentially important insights into key structural domains for enzyme function. OBJECTIVE, DESIGN, AND SETTING: We report a novel missense mutation in P450c17 in a 17-yr-old female presenting with a malignant mixed germ cell tumor with yolk sac elements who demonstrated clinical and biochemical features of combined 17alpha-hydroxylase/17,20-lyase deficiency. METHODS: Quantitative urinary steroid analysis was performed by high resolution gas chromatography. All eight coding exons of CYP17 were PCR amplified and sequenced. The position of arginine at codon 96 was modeled using the CYP17 structure 2c17 (www.rcsb.org). The CYP17 genes were subcloned into pcDNA3, expressed in HEK-293 cells, and chromatographed. PATIENT AND RESULTS: 17alpha-Hydroxylase deficiency was confirmed by marked reductions in urinary and serum cortisol, androgens, and estradiol. Mutational analysis revealed a novel homozygous R96Q missense mutation in P450c17, affecting an amino acid in a key substrate-binding region of the enzyme, leading to complete inactivity. CONCLUSION: The description of a second missense mutation at codon 96 (R96W and R96Q) in the substrate-binding region of P450c17 provides strong evidence for the key role of this amino acid in 17alpha-hydroxylase/17,20-lyase function. An association between a malignant germ cell tumor and 17alpha-hydroxylase deficiency has not been reported previously, although the presence of gonadoblastoma in the ovary of a patient with this condition has recently been described.

A preliminary account of the properties of recombinant human Glyoxylate reductase (GRHPR), LDHA and LDHB with glyoxylate, and their potential roles in its metabolism.

Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.

Two brothers with non-classical 21-hydroxylase deficiency: to treat or not to treat?

Variations in phenotype in 21-hydroxylase deficiency (21OHD) have cautioned against initiating treatment in the absence of abnormal clinical features. We report 2 Caucasian brothers with compound heterozygous mutations of the CYP21 gene and mild clinical and biochemical features of late-presenting 21OHD. The index case presented aged 8.5 years with mild genital virilization and bone age advanced by 5 years. Elevated basal and synacthen-stimulated 17-hydroxyprogesterone (17OHP; 22.4 and 246 nmol/l) and androstenedione (10.9 and 19.9 nmol/l) levels confirmed 21OHD. His younger brother was investigated at age 7.3 years, and although examination showed normal pre-pubertal genitalia, basal and synacthen-stimulated 17OHP (32.4 and 281 nmol/l) and androstenedione (6.2 and 9.0 nmol/l) were abnormal, and bone age was advanced by 1.5 years. Because of actual or incipient virilization, both patients were treated with glucocorticoid replacement 8-12 mg/m(2)/day. This decision is discussed in the context of published guidelines for the management of 21OHD.

17-Hydroxylase/17,20 lyase deficiency diagnosed during childhood.

We present a case of familial 17alpha-hydroxylase/17,20 lyase (CYP17) deficiency in which the index case, a 14-year-old XX girl, led to the diagnosis of the condition in a 9-year-old XY sister. No mutations in the CYP 17 gene were found in any of the girls.

Three novel deletions in the alanine:glyoxylate aminotransferase gene of three patients with type 1 hyperoxaluria.

We describe three novel deletions in the human AGT gene in three patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme, alanine glyoxylate aminotransferase (AGT; EC 2.6.1.44). A deletion of 4 nucleotides in the exon 6/intron 6 splice junction (679-IVS6+2delAAgt) is expected to cause missplicing. It would also code for a K227E missense alteration in any mRNA successfully spliced. A 2-bp deletion in exon 11 (1125-1126del CG, cDNA) results in a frameshift. A deletion of at least 5-6 kb, EX1 EX5del, spanned exons 1-5 and contiguous upstream sequence. All three deletions are heterozygous with previously documented missense mutations; the intron 6 deletion with F152I, the exon 11 deletion with G82E, and EX1 EX5del with the common mistargeting mutation, G170R.

Increased nuchal translucency as a prenatal manifestation of congenital adrenal hyperplasia.

We present two cases of pregnant women with a previous history of congenital adrenal hyperplasia. In both cases the only abnormal feature in the initial pregnancy had been increased nuchal translucency at 10-14 weeks of gestation. The fetal karyotype was normal and a diagnosis of congenital adrenal hyperplasia was made after delivery. In their current pregnancies, both fetuses also had increased nuchal translucency and normal fetal karyotype. Diagnosis of 21-hydroxylase deficiency was made prenatally by DNA analysis. These findings in four affected fetuses suggest that congenital adrenal hyperplasia should be added to the list of genetic anomalies associated with an increase in nuchal translucency.

Identification and expression of a cDNA for human glycolate oxidase.

The isolation and expression of a human liver cDNA encoding a 43 kDa protein with glycolate oxidase activity is described. The cDNA (HAO1) is 1128 bp and has a 1113 bp open reading frame. Northern blot analysis detects a 1.8 kb mRNA with expression restricted to the liver. The 370 amino acid protein has 89% sequence similarity to glycolate oxidase from mouse liver and 53% similarity to the spinach and Arabidopsis enzymes. The protein has glycolate oxidase activity in vitro, as measured by the reduction of 2, 6-dichlorophenolindophenol in the presence of flavin mononucleotide and glycolate. The genomic sequence is contained within eight exons and encompasses approximately 57 kb of chromosome 20p12. This paper offers the first full functional description of a gene encoding human glycolate oxidase.

Evidence for digenic inheritance in some cases of Antley-Bixler syndrome?

The Antley-Bixler syndrome has been thought to be caused by an autosomal recessive gene. However, patients with this phenotype have been reported with a new dominant mutation at the FGFR2 locus as well as in the offspring of mothers taking the antifungal agent fluconazole during early pregnancy. In addition to the craniosynostosis and joint ankylosis which are the clinical hallmarks of the condition, many patients, especially females, have genital abnormalities. We now report abnormalities of steroid biogenesis in seven of 16 patients with an Antley-Bixler phenotype. Additionally, we identify FGFR2 mutations in seven of these 16 patients, including one patient with abnormal steroidogenesis. These findings, suggesting that some cases of Antley-Bixler syndrome are the outcome of two distinct genetic events, allow a hypothesis to be formulated under which we may explain all the differing and seemingly contradictory circumstances in which the Antley-Bixler phenotype has been recognised.

Biochemical and genetic diagnosis of the primary hyperoxalurias: a review.

BACKGROUND AND PURPOSE: The primary hyperoxalurias are a group of inherited disorders of endogenous oxalate overproduction. Diagnosis of the two best-characterized disorders, primary hyperoxaluria (PH) Types 1 and 2, is achieved by sequential measurement of alanine:glyoxylate aminotransferase and glyoxylate reductase enzyme activity in a single needle liver biopsy. While genetic analysis of PH2 is still at a relatively early stage, the AGXT gene defective in the Type 1 disorder is well characterized, and a number of mutations have been identified. METHODS: To determine whether mutation analysis could replace enzymatic analysis for the diagnosis of PH1, DNA samples from 127 consecutive unrelated patients in whom there was a high clinical suspicion of primary hyperoxaluria were analyzed for the presence of the G630A and T853C mutations, which together account for approximately 34% of the mutant alleles in our patient cohort. RESULTS AND CONCLUSIONS: The sensitivity of mutation detection was 47% in those patients with enzymologically confirmed Type 1 disease, showing that mutation analysis cannot effectively replace enzymology at the present time. However, there is little doubt of the value of genetic methods (mutation and linkage analysis) for diagnosing PH1 (and eventually PH2) in other family members and for prenatal diagnosis and carrier testing.

Recent developments in our understanding of primary hyperoxaluria type 2.

Hydroxypyruvate reductase (HPR) has been partially purified from human liver and can be separated into at least two forms by chromatofocusing; these forms therefore differ in their pI values. Both forms, one with a pI of >7.2 (peak A) and the other with a pI between pH 6.5 and 5.5 (peak B), use NADPH as a cofactor. However, only peak B was able to reduce hydroxypyruvate and glyoxylate, with a Km of 2.3 mM for the latter substrate. Peak A coeluted with lactate dehydrogenase and could represent lactate dehydrogenase (which is known to reduce hydroxypyruvate) alone or a mixture of proteins with HPR activity. The Km for hydroxypyruvate of the enzyme(s) in peak A (8 mM) was 80 times greater than that of peak B (0.1 mM), suggesting that the HPR enzyme contained in peak B may be more important physiologically, where the hydroxypyruvate concentrations are in the micromolar range. The data presented provide a biochemical explanation for the previously observed differences in the tissue distribution of HPR and glyoxylate reductase activities in human subjects and support the claim that diagnoses of primary hyperoxaluria type 2 should be made by measurement of glyoxylate reductase activity in the liver.

Treatment of achondroplasia with growth hormone: six years of experience.

We describe the effects of recombinant hGH (r-hGH) therapy for up to 6 y on stature and body proportions of 35 children with achondroplasia (Ach). Consecutive height (Ht) measurements were plotted on disease-specific Ach growth curves, but age and sex SD scores (SDS) of Ht, sitting Ht, subischial leg length, and Ht velocity were made with respect to Tanner normal standards. r-hGH was administered by daily subcutaneous injections at a median (range) dose of 30 (15.8-40) U/m2 per week [0.06 (0.04-0.08) mg.kg(-1).24 h(-1)]. Patients were treated for 3 (1-6) y from age 2.25 (1.2-9.3) y. Before treatment, Ht SDS was -4.6 (-6.5 to -3.24). Treatment caused a significant increase in Ht SDS year to year until y 4 (ANOVA F = 46.94; p < 0.01) that was subsequently sustained with no significant further change (y 5 and 6 versus y 4, p > 0.05). When the response to r-hGH was also expressed as a change in Ht velocity, there was a significant increase in the first year of therapy that was maintained over subsequent treatment years (ANOVA = 4.28, p = 0.001). Age was the most important variable accounting for the first-year response in Ht SDS (r2 = 0.41, p < 0.001), and dose of r-hGH did not influence this. Increments in sitting Ht SDS were greater than subischial leg length SDS (F = 26.25, p < 0.001; F = 9.04, p < 0.001, respectively). r-hGH treatment improved the Ht position of Ach children relative to their normal and Ach peers without obvious side effects. A young age at initiation of therapy prevented the characteristic Ht deficit from accumulating. The greater increase in spinal Ht accentuated the existing disproportion. The addition of later surgical leg lengthening could offer the possibility of proportionate adult stature just within the normal range.

Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase.

The isolation and expression of a human liver cDNA encoding a 40-kDa protein with glyoxylate and hydroxypyruvate reductase activities is described. The cDNA (GLXR) is 1235 bp and consists of a predicted open reading frame of 987 bp with a 225-bp 3'-untranslated region. The 328-amino acid protein has partial sequence similarity to hydroxypyruvate and glyoxylate reductases from a variety of plant and microbial species.

Isoelectric focusing of native urinary uromodulin (Tamm-Horsfall protein) shows no physicochemical differences between stone formers and non-stone formers.

Recent studies have suggested the occurrence of an abnormal form of uromodulin in stone formers which may be related to a reduced sialic acid content of the protein in these patients. Previous attempts to demonstrate these differences have required extensive sample processing prior to analysis, which may in itself alter the nature and behaviour of uromodulin. By employing a sensitive detection system of enhanced chemiluminescence on Western blots following isoelectric focusing we have been able to study the physicochemical properties of uromodulin in whole unprocessed urine from 50 idiopathic calcium stone formers and 15 non-stone formers. Uromodulin, desialated in vitro with either acid or neuraminidase was also analysed using the same system. All urine samples analysed from stone formers and non-stone formers showed a single band of pI 3.5 after isoelectric focusing. Desialated uromodulin showed a series of bands ranging from pI 4.0 to 5.1 reflecting different amounts of sialic acid removed. We conclude there are no charge-related differences in native uromodulin between stone formers and non-stone formers, in particular none relating to the sialic acid content of the protein.

Characterization of polymorphisms at the 11 beta-hydroxylase (CYP11B1) locus.

Four sequence variants in the 11 beta-hydroxylase (CYP11B1) gene are reported. One of the sequence changes occurs in exon 1 and is in linkage disequilibrium with a second variant in intron 3. The other two changes occur at adjacent nucleotides in intron 1. The finding of easily demonstrable, intragenic variants will be beneficial to the study of the role of the CYP11B1 and the adjacent aldosterone synthase (CYP11B2) gene in hypertensive disease.

Genotype-phenotype analysis in late onset 21-hydroxylase deficiency in comparison to the classical forms.

OBJECTIVE: To establish the type and frequency of mutations causing late onset 21-hydroxylase deficiency and associated clinical and biochemical phenotypes and to compare these findings to those from heterozygotes and homozygotes for classical 21-hydroxylase deficiency. DESIGN: Analysis of the 21-hydroxylase genes by DNA amplification and mutation detection. Measurement of serum 17-hydroxyprogesterone following stimulation with adrenocorticotrophic hormone. PATIENTS: Fifteen patients with late onset congenital adrenal hyperplasia, 18 obligate heterozygotes for classical congenital adrenal hyperplasia, 95 patients with classical 21-hydroxylase deficiency. MEASUREMENTS: 17-hydroxyprogesterone following adrenal stimulation with adrenocorticotrophic hormone; frequency of 10 common mutations in the 21-hydroxylase gene. RESULTS: Of alleles from patients with late onset CAH 46% carried V281L and 10% P30L mutations compared to 2.6% and 1.1% respectively of alleles from patients with the classical disease where the most common mutations are a splice site mutation in intron 2 (34%), deletions (25%) and I172N (15%). Serum 17-hydroxyprogesterone following Synacthen stimulation reliably differentiated the late onset patients from carriers for the classical disease, whereas there was overlap of the basal 17-hydroxyprogesterone concentration between these two conditions. CONCLUSIONS: Normal basal serum 17-hydroxyprogesterone levels cannot exclude late onset CAH, although response to adrenocorticotrophic stimulation clearly distinguished this disorder from carriers of the classical disease. The frequency of mutations causing less severe enzyme deficiency was higher in the late onset patients than in a group of classical patients, although other genes or environmental pressures may determine the age of onset of disease.

Genotype and phenotype in hypochondroplasia.

Mutations in the tyrosine kinase domain of fibroblast growth factor receptor gene (FGFR3) have been described in some cases of hypochondroplasia (Hch). We screened 65 children with Hch diagnosed by clinical and radiologic criteria for 2 previously described mutations, C1620A and C1620C in FGFR3; 28 (43%) of 65 patients were heterozygous for the C1620A transversion resulting in lysine to asparagine substitution at codon 540 in the tyrosine kinase domain of FGFR3. The height, sitting height, and subischial leg length of these children and of 18 children with achondroplasia were analyzed at presentation, and SD scores were calculated. For comparison of growth data the patients were divided into three groups: group 1, achondroplasia defined by radiology and the presence of the G1138A mutation in the transmembrane domain of FGFR3; group 2, Hch with C1620A mutation; and group 3, Hch with no mutation identified so far. Height, sitting height, and subischial leg length SD scores were analyzed as group mean data by analysis of variance with the Student Neuman-Keuls test after testing for multiple contrasts were performed. All three groups were significantly compromised in height, although the children with achondroplasia were much shorter with significant reduction in subischial leg length. The same pattern was evident in group 2, with additional shortening of the back, the third group was proportionately short. Children with the common C1620A mutation met all of the criteria for the diagnosis of Hch with a severe phenotype that resembled achondroplasia and disproportionate short stature in early childhood. However, a substantial number of patients with proportionate short stature presented at an older age with the same radiologic characteristics and failure of the puberty growth spurt. The genetic basis of this milder phenotype not yet known.

Carrier status for steroid 21-hydroxylase deficiency is only one factor in the variable phenotype of acne.

OBJECTIVE: Previous endocrine studies of women with acne have produced diverse results. This study was designed to seek evidence, from endocrine and genetic studies, for impaired steroid biosynthesis in patients with acne. DESIGN: Adrenal stimulation tests with synthetic adrenocorticotrophic hormone (ACTH) were performed. MEASUREMENTS: Steroid hormones were measured basally and 30 minutes after ACTH. The results were correlated with analysis of the steroid 21-hydroxylase gene (CYP21). PATIENTS: Fifty-one consecutive female patients (mean age 27.1 years) referred with acne. RESULTS: The median plasma 17-hydroxyprogesterone (17-OHP) before and 30 minutes after ACTH were 2.5 nmol/l (range 1.1-8.2) and 7.3 (2.1-17.8) nmol/l which were significantly above normal female controls (n = 11, mean age 25.6 years) at 1.5 (0.9-4.2) and 4.6 (2.6-8.4) nmol/l. Eighteen of 51 acne patients showed an abnormal 17-OHP response. The 21-hydroxylase gene (CYP21) was examined for major deletions and for three common point mutations in 31 of the patients (14 with exaggerated 17-OHP response). One patient had a deletion of CYP21 on one allele consistent with carrier status for the classical congenital adrenal hyperplasia (CAH). Five patients, one of whom had a normal 17-OHP response to Synacthen, were heterozygous for the val 281 leu mutation in exon 7 of the CYP21 and were therefore carriers for a mutation associated with late-onset CAH. One patient with a raised 17-OHP response was homozygous for the splice site mutation in intron 2 and one patient with a normal 17-OHP response was heterozygous for the mutation. None of the patients had the ile 172 asn mutation. Eight of the 31 acne patients who had CYP21 gene analysis were carriers for mutations in the 21-hydroxylase gene but only six would have been detected by an abnormal response of 17-OHP on stimulation. CONCLUSION: Although alterations of the CYP21 gene were more common in acne than in controls there is a poor correlation between these events and raised steroids and acne. Factors other than mild impairment of CYP21 contribute to the variability of the clinical phenotype in hyperandrogenic states including acne.