Biochemical and molecular predictors for prognosis in nonketotic hyperglycinemia.

OBJECTIVE: Nonketotic hyperglycinemia is a neurometabolic disorder characterized by intellectual disability, seizures, and spasticity. Patients with attenuated nonketotic hyperglycinemia make variable developmental progress. Predictive factors have not been systematically assessed. METHODS: We reviewed 124 patients stratified by developmental outcome for biochemical and molecular predictive factors. Missense mutations were expressed to quantify residual activity using a new assay. RESULTS: Patients with severe nonketotic hyperglycinemia required multiple anticonvulsants, whereas patients with developmental quotient (DQ) > 30 did not require anticonvulsants. Brain malformations occurred mainly in patients with severe nonketotic hyperglycinemia (71%) but rarely in patients with attenuated nonketotic hyperglycinemia (7.5%). Neonatal presentation did not correlate with outcome, but age at onset ≥ 4 months was associated with attenuated nonketotic hyperglycinemia. Cerebrospinal fluid (CSF) glycine levels and CSF:plasma glycine ratio correlated inversely with DQ; CSF glycine > 230 µM indicated severe outcome and CSF:plasma glycine ratio ≤ 0.08 predicted attenuated outcome. The glycine index correlated strongly with outcome. Molecular analysis identified 99% of mutant alleles, including 96 novel mutations. Mutations near the active cleft of the P-protein maintained stable protein levels. Presence of 1 mutation with residual activity was necessary but not sufficient for attenuated outcome; 2 such mutations conferred best outcome. Divergent outcomes for the same genotype indicate a contribution of other genetic or nongenetic factors. INTERPRETATION: Accurate prediction of outcome is possible in most patients. A combination of 4 factors available neonatally predicted 78% of severe and 49% of attenuated patients, and a score based on mutation severity predicted outcome with 70% sensitivity and 97% specificity.

Intron retention and frameshift mutations result in severe pyruvate carboxylase deficiency in two male siblings.

This paper describes the molecular characterization of two male siblings displaying the complex (Type B) form of pyruvate carboxylase (PC) deficiency in which severe neonatal lactic acidosis and redox abnormalities results in death within the first few weeks of life. The two male siblings were found to be compound heterozygous for a TAGG deletion at the exon15/intron15 splice site (IVS15+2-5delTAGG) and a dinucleotide deletion in exon 16 (2491-2492delGT) of the PC gene. We also demonstrate through RT-PCR and sequencing of aberrant transcripts that the IVS15+2-5delTAGG results in the retention of intron 15 during pre-mRNA splicing. In addition, both deletions are predicted to result in a frameshift to generate a premature termination codon such that the encoded mRNA could be subject to nonsense mediated decay.

A genomic approach to mutation analysis of holocarboxylase synthetase gene in three Chinese patients with late-onset holocarboxylase synthetase deficiency.

OBJECTIVE: Multiple carboxylase deficiency (MCD, MIM:253270) is a common organic aciduria and caused by deficiency of either biotinidase or holocarboxylase synthetase (HLCS; EC 6.3.4.10). Patients commonly present during early infancy with acute metabolic derangements and severe metabolic acidosis. Recently, a late onset form of HLCS deficiency was also described. The different phenotypes (early and late presenting) may be related to a spectrum of mutations in HLCS gene. Applications of mutation analysis in HLCS had been limited previously by the requirement of cDNA from living tissue for study. We described here a genomic approach for molecular diagnosis of HLCS deficiency which we have used to detect mutations in Chinese patients who had the late-onset form of HLCS deficiency. In addition, a fibroblast cell line with MCD from Coriell Cell repositories was also studied. DESIGN AND METHODS: Three Chinese patients with late onset HLCS deficiency were studied. The genomic sequence of HLCS was retrieved and newly designed primers were used to cover all coding sequences of the gene. PCR products were analyzed by direct sequencing. Population allelic frequencies of mutations detected were determined by genotyping of control samples by restriction fragment length polymorphism. RESULTS: We found a recurrent mutation, R508W, in the three unrelated Chinese patients. Two were homozygous for this mutation. The other patient was a compound heterozygote of R508W and a novel mutation, D634N. The results suggest that R508W may be an important and relatively prevalent disease-causing mutation in Chinese MCD patients. A fibroblast cell-line from an African patient revealed an additional novel mutation, R565X and a known mutation, V550M. CONCLUSION: R508W is a recurrent mutation in Chinese MCD patients which is associated with the late onset phenotype. This new genomic approach for mutation analysis of HLCS gene provides new opportunities in studies of MCD.

Isolated peripheral neuropathy in atypical metachromatic leukodystrophy: a recurrent mutation.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a genetic neurodegenerative disorder resulting from a deficiency of arylsulfatase A. Late onset forms are relatively rare. Central nervous system (CNS) involvement is characteristic at all ages. METHODS: A patient in her late 40s with peripheral neuropathy was assessed by EEG, evoked potentials, CT and nerve conduction studies. Nerve and muscle biopsy samples were investigated by electron microscopy. Arylsulfatase A activity in leukocytes and excreted cerebroside sulfate were determined. The arylsulfatase A gene was investigated for mutations using polymerase chain reaction (PCR) and DNA sequencing. The identified mutation was expressed transiently in African green monkey kidney (COS) cells to determine the effect of the mutation on arylsulfatase A activity. RESULTS: Central nervous system functions were normal. Nerve conduction velocities were decreased. Sural nerve biopsy showed inclusions typical of MLD. Arylsulfatase A was less than 5% of normal. A homozygous mutation thr286pro was identified in the arylsulfatase A gene and demonstrated to be deleterious through transient expression studies. CONCLUSIONS: Our patient has a progressive peripheral neuropathy but has apparently intact CNS function at her present age of 57 years. Biochemical, physiological and pathological findings are consistent with a diagnosis of MLD. A homozygous mutation, thr286pro, found in her arylsulfatase A gene, decreased enzyme activity to a level consistent with a late onset form of MLD.

Mutation-based diagnostic testing for primary hyperoxaluria type 1: survey of results.

OBJECTIVES: To test for specific mutations in the alanine:glyoxylate aminotransferase (AGT) gene, in order to diagnose primary hyperoxaluria type 1 (PH1). DESIGN AND METHODS: Samples of liver and/or DNA from 81 patients were submitted to our laboratory for diagnostic testing for PH1. Using a panel of selected mutations, DNA was examined in 64 cases, of which 36 had the diagnosis of PH1 confirmed by liver AGT assay. DNA sequencing was employed if mutation testing revealed only one mutation. RESULTS: Identification of 100% of the mutations in the AGT-confirmed samples led to the development of a focused testing panel currently involving 4 common mutations, 7 mutations recurring at lower frequency and 5 with apparent ethnic associations. CONCLUSIONS: This mutation panel alone would have identified the two causative mutations in 64% of the PH1 samples.

The AGT gene in Africa: a distinctive minor allele haplotype, a polymorphism (V326I), and a novel PH1 mutation (A112D) in Black Africans.

We describe a novel missense mutation (A112D) and polymorphism (V326I) in the human AGT gene in two black African 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). V326I was found in DNA from normal control Blacks with an allele frequency of 3%. Expression studies confirmed that A112D reduced AGT enzyme activity by 95% while V326I had no effect. Both A112D and V326I were homozygous in both patients and lie on a variant of the minor allele of the AGT gene. This variant haplotype, Mi(A), includes an intron 1 duplication and intron 4 VNTR (38 repeat) but lacks the P11L and I340M normally associated with the minor allele in Caucasians. Among the South African Blacks tested, the Mi(A) haplotype had an allele frequency of 12% compared to 3 % for the Caucasian-type minor allele haplotype.

Molecular genetic and potential biochemical characteristics of patients with T-protein deficiency as a cause of glycine encephalopathy (NKH).

A defect in the P-protein component of the glycine cleavage system has been the most frequent abnormality found in patients with glycine encephalopathy (NKH). In a retrospective study of a more specific group of NKH patients, however, we found that >50% had T-protein mutations. The patients studied had one or more of the following unusual biochemical findings: residual glycine cleavage system activity in liver assayed by the standard method or a newly developed micromethod, residual glycine cleavage system activity in lymphoblasts, and/or increased amniotic fluid glycine/serine ratio with a normal amniotic fluid glycine level in prenatal diagnosis. The selected patients had a much higher incidence of T-protein defects than expected in the general NKH patient population. We report, here, three novel mutations and five polymorphisms in the T-protein gene, PCR/restriction enzyme methods for one mutation (R296H) and two polymorphisms (E211K and R318R), and an estimation of their frequency in normal controls. The co-occurrence of the polymorphism E211K with the mutation R320H in patients with a severe phenotype is discussed.

Novel mutations in the P-protein (glycine decarboxylase) gene in patients with glycine encephalopathy (non-ketotic hyperglycinemia).

Eight novel mutations were found in the P-protein (glycine decarboxylase) gene (GLDC) of the glycine cleavage system (EC 2.1.1.10) by screening five exons of the gene in patients with glycine encephalopathy (NKH). The mutations identified were of eight single base changes: a one-base deletion 1054del A, a splice site mutation IVS18-2A-->G and six amino acid substitutions A283P, A313P, P329T, R410K, P700A, and G762R.