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.

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 major allele of the alanine:glyoxylate aminotransferase gene: nine novel mutations and polymorphisms associated with primary hyperoxaluria type 1.

We describe nine novel mutations and polymorphisms occurring on the major allele of the human alanine:glyoxylate aminotransferase gene in 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). The PH1 mutations include two small frameshift mutations, 327delG and 117_118insCA, a large deletion spanning exon 9 and portions of the flanking introns, a splice junction mutation, IVS6+5G>C, and two missense mutations, G161R and S218L. Expression studies of the two missense mutations indicated very little enzymatic activity associated with either of them. Three polymorphisms in the coding sequence were also identified, I279T, A280V, and T235T. Expression studies of I279T and A280V suggested essentially normal AGT activity. I279T, found in two cases, was located on a 33_34insC allele. A280V and T235T were both located on the same allele as IVS6+5G>C. We have also identified recurrences of previously reported rare mutations, 33delC, IVS7-1G>C, and IVS4-1G>A. Five of the six novel PH1 mutations occurred in a compound heterozygous state with either of two common PH1 mutations, G170R or 33_34insC. S218L was apparently homozygous in two individuals. These findings contribute to our overall picture of heterogeneity of mutations in PH1 and the AGT major allele.

The major allele of the alanine:glyoxylate aminotransferase gene: seven novel mutations causing primary hyperoxaluria type 1.

We describe 7 novel mutations occurring on the major allele of the human AGT gene in 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). These mutations include 3 small deletions, 570delG, 744delC, and 983_988del, two splice junction mutations, IVS7-1G-->C and IVS8+1G-->T, and two nonsense mutations, R111X and W251X. We have also identified recurrences of previously identified reported mutations, 679-(IVS6+2)delAAgt, IVS8-3C-->G and 33insC. Deletion mutation 679-(IVS6+2)delAAgt has now been identified in a second Chinese patient and may be specific to that population. In contrast, 33insC has been found in patients of varying ethnic and racial backgrounds; a single vs multiple origin for this mutation is thus an intriguing question. It also appears to occur at a high frequency on the major allele. Five of the novel mutations were detected in patients who were compound heterozygotes for one of the common mis-targeting mutation, G170R or F152I, while the other two mutations occurred in the same patient.

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.

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.