Marion B. Coulter-Mackie

Pyridoxine dependent epilepsy and antiquitin deficiency Clinical and molecular characteristics and recommendations for diagnosis, treatment and follow-up

Antiquitin (ATQ) deficiency is the main cause of pyridoxine dependent epilepsy characterized by early onset epileptic encephalopathy responsive to large dosages of pyridoxine. Despite seizure control most patients have intellectual disability. Folinic acid responsive seizures (FARS) are genetically identical to ATQ deficiency. ATQ functions as an aldehyde dehydrogenase (ALDH7A1) in the lysine degradation pathway. Its deficiency results in accumulation of α-aminoadipic semialdehyde (AASA), piperideine-6-carboxylate (P6C) and pipecolic acid, which serve as diagnostic markers in urine, plasma, and CSF. To interrupt seizures a dose of 100 mg of pyridoxine-HCl is given intravenously, or orally/enterally with 30 mg/kg/day. First administration may result in respiratory arrest in responders, and thus treatment should be performed with support of respiratory management. To make sure that late and masked response is not missed, treatment with oral/enteral pyridoxine should be continued until ATQ deficiency is excluded by negative biochemical or genetic testing. Long-term treatment dosages vary between 15 and 30 mg/kg/day in infants or up to 200 mg/day in neonates, and 500 mg/day in adults. Oral or enteral pyridoxal phosphate (PLP), up to 30 mg/kg/day can be given alternatively. Prenatal treatment with maternal pyridoxine supplementation possibly improves outcome. PDE is an organic aciduria caused by a deficiency in the catabolic breakdown of lysine. A lysine restricted diet might address the potential toxicity of accumulating αAASA, P6C and pipecolic acid. A multicenter study on long term outcomes is needed to document potential benefits of this additional treatment. The differential diagnosis of pyridoxine or PLP responsive seizure disorders includes PLP-responsive epileptic encephalopathy due to PNPO deficiency, neonatal/infantile hypophosphatasia (TNSALP deficiency), familial hyperphosphatasia (PIGV deficiency), as well as yet unidentified conditions and nutritional vitamin B6 deficiency. Commencing treatment with PLP will not delay treatment in patients with pyridox(am)ine phosphate oxidase (PNPO) deficiency who are responsive to PLP only.

Primary hyperoxaluria type 1: update and additional mutation analysis of the AGXT gene†

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, inherited disorder of glyoxylate metabolism arising from a deficiency of the alanine:glyoxylate aminotransferase (AGT) enzyme, encoded by the AGXT gene. The disease is manifested by excessive endogenous oxalate production, which leads to impaired renal function and associated morbidity. At least 146 mutations have now been described, 50 of which are newly reported here. The mutations, which occur along the length of the AGXT gene, are predominantly single‐nucleotide substitutions (75%), 73 are missense, 19 nonsense, and 18 splice mutations; but 36 major and minor deletions and insertions are also included. There is little association of mutation with ethnicity, the most obvious exception being the p.Ile244Thr mutation, which appears to have North African/Spanish origins. A common, polymorphic variant encoding leucine at codon 11, the so‐called minor allele, has significantly lower catalytic activity in vitro, and has a higher frequency in PH1 compared to the rest of the population. This polymorphism influences enzyme targeting in the presence of the most common Gly170Arg mutation and potentiates the effect of several other pathological sequence variants. This review discusses the spectrum of AGXT mutations and polymorphisms, their clinical significance, and their diagnostic relevance. Hum Mutat 30, 910–917, 2009.

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

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.

Profound Neonatal Hypoglycemia and Lactic Acidosis Caused by Pyridoxine-Dependent Epilepsy

Pyridoxine-dependent epilepsy (PDE) was first described in 1954. The ALDH7A1 gene mutations resulting in α-aminoadipic semialdehyde dehydrogenase deficiency as a cause of PDE was identified only in 2005. Neonatal epileptic encephalopathy is the presenting feature in >50% of patients with classic PDE. We report the case of a 13-month-old girl with profound neonatal hypoglycemia (0.6 mmol/L; reference range >2.4), lactic acidosis (11 mmol/L; reference range <2), and bilateral symmetrical temporal lobe hemorrhages and thalamic changes on cranial MRI. She developed multifocal and myoclonic seizures refractory to multiple antiepileptic drugs that responded to pyridoxine. The diagnosis of α-aminoadipic semialdehyde dehydrogenase deficiency was confirmed based on the elevated urinary α-aminoadipic semialdehyde excretion, compound heterozygosity for a known splice mutation c.834G>A (p.Val278Val), and a novel putative pathogenic missense mutation c.1192G>C (p.Gly398Arg) in the ALDH7A1 gene. She has been seizure-free since 1.5 months of age on treatment with pyridoxine alone. She has motor delay and central hypotonia but normal language and social development at the age of 13 months. This case is the first description of a patient with PDE due to mutations in the ALDH7A1 gene who presented with profound neonatal hypoglycemia and lactic acidosis masquerading as a neonatal-onset gluconeogenesis defect. PDE should be included in the differential diagnosis of hypoglycemia and lactic acidosis in addition to medically refractory neonatal seizures.

A protocol for detection of mitochondrial DNA deletions: characterization of a novel deletion

OBJECTIVES To develop a protocol capable of identifying deletions in mitochondrial DNA and use it to identify the breakpoints of a mtDNA deletion in a patient with chronic progressive external ophthalmoplegia (CPEO). DESIGN AND METHODS Deletions in mtDNA were identified by a combination of long range PCR and Southern blotting. The precise breakpoints were determined by automated DNA sequencing. RESULTS A series of DNA samples from patients with suspected mitochondrial disease was subjected to a protocol, which combines long range PCR and Southern blotting. We found a unique deletion in a patient with CPEO and we identified the precise location of this deletion through DNA sequencing. CONCLUSIONS Long range PCR has the advantages of speed, minimal samples requirements, and sensitivity. Southern blotting is better able to evaluate heteroplasmy and detect duplications. We suggest a protocol that enables us to identify precisely the breakpoints in a unique mutation of mtDNA in a patient with CPEO.

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.

Variable onset of metachromatic leukodystrophy in a Vietnamese family.

We report two siblings with metachromatic leukodystrophy, one who presented at 7 years of age (juvenile onset) and his sister who presented at 22 years of age (adult onset). They are compound heterozygotes for two novel mutations in the arylsufatase A gene (ARSA). The responsible mutations in this Vietnamese family consist of a missense mutation with 5% enzyme activity (R143G) and a nonsense mutation (W318ter), from which no enzyme activity would be expected. These mutations in the ARSA gene have not been previously reported and may be useful when diagnosing metachromatic leukodystrophy in other affected Vietnamese individuals. The variability in presentation suggests that the genotype alone is not sufficient to determine the onset and course of the disease and modifying genetic and perhaps nongenetic factors likely contribute.

Preliminary Evidence for Ethnic Differences in Primary Hyperoxaluria Type 1 Genotype

Background: Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of peroxisomal alanine:glyoxylate aminotransferase (AGT). In about one third of patients, enzymatically active AGT is synthesized but is mistargeted to mitochondria. There are more than 50 mutations identified in the gene for AGT. Four mutations, G170R, 33_34insC, F152I and I244T account for more than 50% of PH1 alleles. The question arose whether there are ethnic differences in PH1 genotype. Methods: The published data on mutations in the AGT gene were examined with respect to recurrences and geographic or ethnic association. The mutations that have been found in at least 2 unrelated individuals were considered. Results: Two common mutations, G170R and 33_34insC showed no obvious ethnic associations and have been found in a variety of populations. A third common PH1 mutation, I244T, has a strong association with people from a Spanish or North African background. A particularly high frequency among Canary Islands PH1 patients suggests a probable founder effect. Between these two extremes are a number of mutations that recur at low frequency within certain ethnic groups. Conclusions: Ethnic associations of PH1 genotypes span a spectrum ranging from limited recurrences confined to a population group, to a probable founder effect.