We used single nucleotide polymorphism (SNP) microarrays to investigate the cause of a symptomatic epilepsy syndrome in a group of seven distantly related Old Order Mennonite children. Autozygosity mapping was inconclusive, but closer inspection of the data followed by formal SNP copy number analyses showed that all affected patients had homozygous deletions of a single SNP (rs721575) and their parents were hemizygous for this marker. The deleted SNP marked a larger deletion encompassing exons 9-13 of LYK5, which encodes STE20-related adaptor protein, a pseudokinase necessary for proper localization and function of serine/threonine kinase 11 (a.k.a. LKB1). Homozygous LYK5 deletions were associated with polyhydramnios, preterm labour and distinctive craniofacial features. Affected children had large heads, infantile-onset intractable multifocal seizures and severe psychomotor retardation. We designated this condition PMSE syndrome (polyhydramnios, megalencephaly and symptomatic epilepsy). Thirty-eight percent (N = 16) of affected children died during childhood (ages 7 months to 6 years) from medical complications of the disorder, which included status epilepticus, congestive heart failure due to atrial septal defect and hypernatremic dehydration due to diabetes insipidus. A single post-mortem neuropathological study revealed megalencephaly, ventriculomegaly, cytomegaly and extensive vacuolization and astrocytosis of white matter. There was abundant anti-phospho-ribosomal S6 labelling of large cells within the frontal cortex, basal ganglia, hippocampus and spinal cord, consistent with constitutive activation of the mammalian target of rapamycin (mTOR) signalling pathway in brain.

Despite early diagnosis, one-third of Amish infants with glutaryl-CoA dehydrogenase deficiency (GA1) develop striatal lesions that leave them permanently disabled. To better understand mechanisms of striatal degeneration, we retrospectively studied imaging results from 25 Amish GA1 patients homozygous for 1296C>T mutations in GCDH. Asymptomatic infants had reduced glucose tracer uptake and increased blood volume throughout gray matter, which may signify a predisposition to brain injury. Nine children (36%) developed striatal lesions: three had sudden motor regression during infancy whereas six had insidious motor delay associated with striatal lesions of undetermined onset. Acute striatal necrosis consisted of three stages: (1) an acute stage, within 24 h of motor regression, characterized by cytotoxic oedema within the basal ganglia, cerebral oligemia, and rapid transit of blood throughout gray matter; (2) a sub-acute stage, 4–5 days after the onset of clinical signs, characterized by reduced striatal perfusion and glucose uptake, and supervening vasogenic oedema; and (3) a chronic stage of striatal atrophy. Apparent diffusion coefficient maps revealed that at least two of the six patients with insidious motor delay suffered striatal injuries before or shortly after birth, followed by latent periods of several months before disability was apparent. Thus, acute and insidious presentations may occur by similar mechanisms, and differ only with regard to the timing of injury. Intravenous fluid and dextrose therapy for illnesses during the first 2 years of life was the only intervention that was clearly neuroprotective in this cohort (odds ratio for brain injury = 0.04, 95% confidence interval = 0.01–0.34; P < 0.001).

Over a four-year period, we collected clinical and biochemical data from five Amish children who were homozygous for missense mutations in 5,10-methylenetetrahydrofolate reductase (MTHFR c.1129C>T). The four oldest patients had irreversible brain damage prior to diagnosis. The youngest child, diagnosed and started on betaine therapy as a newborn, is healthy at her present age of three years. We compared biochemical data among four groups: 16 control subjects, eight heterozygous parents, and five affected children (for the latter group, both before and during treatment with betaine anhydrous). Plasma amino acid concentrations were used to estimate changes in cerebral methionine uptake resulting from betaine therapy. In all affected children, treatment with betaine (534+/-222 mg/kg/day) increased plasma S-adenosylmethionine, improved markers of tissue methyltransferase activity, and resulted in a threefold increase of calculated brain methionine uptake. Betaine therapy did not normalize plasma total homocysteine, nor did it correct cerebral 5-methyltetrahydrofolate deficiency. We conclude that when the 5-methyltetrahydrofolate content of brain tissue is low, dietary betaine sufficient to increase brain methionine uptake may compensate for impaired cerebral methionine recycling. To effectively support the metabolic requirements of rapid brain growth, a large dose of betaine should be started early in life.

Genetic disorders of homocysteine(Hcy) metabolism or a high-methionine diet lead toelevations of plasma Hcy levels. In humans, severegenetic hyperhomocysteinemia results in prematuredeath from vascular complications whereas dietaryhyperhomocysteinemia is often used to induce athero-sclerosis in animal models. Hcy is mistakenly selectedin place of methionine by methionyl-tRNA synthetaseduring protein biosynthesis, which results in the forma-tion of Hcy-thiolactone and initiates a pathophysiolog-ical pathway that has been implicated in human vasculardisease. However, whether genetic deficiencies in Hcymetabolism or a high-methionine diet affect Hcy-thio-lactone levels in mammals has been unknown. Here weshow that plasma Hcy-thiolactone is elevated 59-foldand 72-fold in human patients with hyperhomocysteine-mia secondary to mutations in methylenetetrahydrofo-late reductase and cystathionine -synthase genes, re-spectively. We also show that mice, like humans,eliminate Hcy-thiolactone by urinary excretion; in con-trast to humans, however, mice also eliminate signifi-cant amounts of plasma total Hcy (38%) by urinaryexcretion. In mice, hyperhomocysteinemia secondaryto a high-methionine diet leads to 3.7-fold and 25-foldincreases in plasma and urinary Hcy-thiolactone levels,respectively. Thus, we conclude that hyperhomocys-teinemia leads to significant increases in the athero-genic metabolite Hcy-thiolactone in humans and mice.