Model mice for mild-form glycine encephalopathy: behavioral and biochemical characterizations and efficacy of antagonists for the glycine binding site of N-methyl D-aspartate receptor.

Glycine encephalopathy (GE) is caused by an inherited deficiency of the glycine cleavage system (GCS) and characterized by accumulation of glycine in body fluids and various neurologic symptoms. Coma and convulsions develop in neonates in typical GE while psychomotor retardation and behavioral abnormalities in infancy and childhood are observed in mild GE. Recently, we have established a transgenic mouse line (low-GCS) with reduced GCS activity (29% of wild-type (WT) C57BL/6) and accumulation of glycine in the brain (Stroke, 2007; 38:2157). The purpose of the present study is to characterize behavioral features of the low-GCS mouse as a model of mild GE. Two other transgenic mouse lines were also analyzed: high-GCS mice with elevated GCS activity and low-GCS-2 mice with reduced GCS activity. As compared with controls, low-GCS mice manifested increased seizure susceptibility, aggressiveness and anxiety-like activity, which resembled abnormal behaviors reported in mild GE, whereas high-GCS mice were less sensitive to seizures, hypoactive and less anxious. Antagonists for the glycine-binding site of the N-methyl-D-aspartate receptor significantly ameliorated elevated locomotor activity and seizure susceptibility in the low-GCS mice. Our results suggest the usefulness of low-GCS mice as a mouse model for mild GE and a novel therapeutic strategy.

A novel KCNQ4 one-base deletion in a large pedigree with hearing loss: implication for the genotype-phenotype correlation.

Autosomal-dominant, nonsyndromic hearing impairment is clinically and genetically heterogeneous. We encountered a large Japanese pedigree in which nonsyndromic hearing loss was inherited in an autosomal-dominant fashion. A genome-wide linkage study indicated linkage to the DFNA2 locus on chromosome 1p34. Mutational analysis of KCNQ4 encoding a potassium channel revealed a novel one-base deletion in exon 1, c.211delC, which generated a profoundly truncated protein without transmembrane domains (p.Q71fsX138). Previously, six missense mutations and one 13-base deletion, c.211_223del, had been reported in KCNQ4. Patients with the KCNQ4 missense mutations had younger-onset and more profound hearing loss than patients with the 211_223del mutation. In our current study, 12 individuals with the c.211delC mutation manifested late-onset and pure high-frequency hearing loss. Our results support the genotype-phenotype correlation that the KCNQ4 deletions are associated with later-onset and milder hearing impairment than the missense mutations. The phenotypic difference may be caused by the difference in pathogenic mechanisms: haploinsufficiency in deletions and dominant-negative effect in missense mutations.

Cystathionine beta-synthase is enriched in the brains of Down's patients.

Down's syndrome (DS) or trisomy 21 is the most common genetic cause of mental retardation, and adults with DS develop Alzheimer type of disease (AD). Cystathionine beta-synthase (CBS) is encoded on chromosome 21 and deficiency in its activity causes homocystinuria, the most common inborn error of sulfur amino acid metabolism and characterized by mental retardation and vascular disease. Here, we show that the levels of CBS in DS brains are approximately three times greater than those in the normal individuals. CBS is localized to astrocytes and those surrounding senile plaques in the brains of DS patients with AD. The over-expression of CBS may cause the developmental abnormality in cognition in DS children and that may lead to AD in DS adults.

Mild variant of nonketotic hyperglycinemia with typical neonatal presentations: mutational and in vitro expression analyses in two patients.

In neonatal-onset nonketotic hyperglycinemia, severe psychomotor retardation is the expected uniform outcome. We report two patients with typical neonatal presentation who showed far better developmental outcomes. The in vitro expression analysis of the identified GLDC mutations revealed considerable residual enzyme activity, suggesting prognostic and enzymatic heterogeneity even in neonatal-onset nonketotic hyperglycinemia.

Glycine cleavage system in neurogenic regions.

The glycine cleavage system (GCS) is the essential enzyme complex for degrading glycine and supplying 5,10-methylenetetrahydrofolate for DNA synthesis. Inherited deficiency of this system causes nonketotic hyperglycinemia, characterized by severe neurological symptoms and frequent association of brain malformations. Although high levels of glycine have been considered to cause the above-mentioned problems, the detailed pathogenesis of this disease is still unknown. Here we show that GCS is abundantly expressed in rat embryonic neural stem/progenitor cells in the neuroepithelium, and this expression is transmitted to the radial glia-astrocyte lineage, with prominence in postnatal neurogenic regions. These data indicate that GCS plays important roles in neurogenesis, and suggest that disturbance of neurogenesis induced by deficiency of GCS may be the main pathogenesis of nonketotic hyperglycinemia.

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.

Persistent NKH with transient or absent symptoms and a homozygous GLDC mutation.

Three of four nonketotic hyperglycinemia patients homozygous for a novel GLDC mutation (A802V) were treated by assisted respiration and/or sodium benzoate with or without ketamine and had transient neonatal or absent symptoms and normal developmental outcome, despite persisting biochemical evidence of nonketotic hyperglycinemia. This exceptional outcome may be related to the high residual activity of the mutant protein (32% of wild type) and therapeutic intervention during a critical period of heightened brain exposure and sensitivity to glycine.

Heterozygous GLDC and GCSH gene mutations in transient neonatal hyperglycinemia.

Transient neonatal hyperglycinemia is clinically or biochemically indistinguishable from nonketotic hyperglycinemia at onset. In the case of transient neonatal hyperglycinemia, the elevated plasma and cerebrospinal fluid glycine levels are normalized within 2 to 8 weeks. To elucidate the pathogenesis of transient neonatal hyperglycinemia, we studied three patients by screening mutations in the genes that encode three components of the glycine cleavage system. Heterozygous mutations were identified in all of the three patients, suggesting that transient neonatal hyperglycinemia develops in some heterozygous carriers for nonketotic hyperglycinemia.

Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness.

Hereditary deafness affects about 1 in 2000 children and mutations in the GJB2 gene are the major cause in various ethnic groups. GJB2 encodes connexin26, a putative channel component in cochlear gap junction. However, the pathogenesis of hearing loss caused by the GJB2 mutations remains obscure. The generation of a mouse model to study the function of connexin26 during hearing has been hampered by the fact that Gjb2 knockout mice are embryonic lethal. To establish viable model mice we generated transgenic mice expressing a mutant connexin26 with R75W mutation that was identified in a deaf family with autosomal-dominant inheritance. The previous expression analysis revealed that the R75W connexin26 inhibited the gap channel function of the co-expressed normal connexin26 in a dominant-negative fashion. We established two lines of transgenic mice that showed severe to profound hearing loss, deformity of supporting cells, failure in the formation of the tunnel of Corti and degeneration of sensory hair cells. Despite robust expression of the transgene, no obvious structural change was observed in the stria vascularis or spiral ligament that is rich in connexin26 and generates the endolymph. The high resting potential in cochlear endolymph essential for hair cell excitation was normally sustained. These results suggest that the GJB2 mutation disturbs homeostasis of cortilymph, an extracellular space surrounding the sensory hair cells, due to impaired K(+) transport by supporting cells, resulting in degradation of the organ of Corti, rather than affecting endolymph homeostasis in mice and probably in humans.

Genetic testing of glycogen storage disease type Ib in Japan: five novel G6PT1 mutations and a rapid detection method for a prevalent mutation W118R.

We devised a simple method using a TaqMan fluorogenic probe for detection of a prevalent G6PT1 mutation W118R among Japanese patients with glycogen storage disease type Ib. The W118R mutation was detected in three of six newly diagnosed Japanese patients. The W118R-negative alleles were screened for causative mutations by sequencing analysis, revealing five novel mutations. The genetic tests using the simple TaqMan method coupled with sequencing analysis would facilitate the early diagnosis of this disorder.