Stanley H. Korman

Mutations in the Fatty Acid 2-Hydroxylase Gene Are Associated with Leukodystrophy with Spastic Paraparesis and Dystonia

Myelination is a complex, developmentally regulated process whereby myelin proteins and lipids are coordinately expressed by myelinating glial cells. Homozygosity mapping in nine patients with childhood onset spasticity, dystonia, cognitive dysfunction, and periventricular white matter disease revealed inactivating mutations in the FA2H gene. FA2H encodes the enzyme fatty acid 2-hydroxylase that catalyzes the 2-hydroxylation of myelin galactolipids, galactosylceramide, and its sulfated form, sulfatide. To our knowledge, this is the first identified deficiency of a lipid component of myelin and the clinical phenotype underscores the importance of the 2-hydroxylation of galactolipids for myelin maturation. In patients with autosomal-recessive unclassified leukodystrophy or complex spastic paraparesis, sequence analysis of the FA2H gene is warranted.

IDH2 Mutations in Patients with d-2-Hydroxyglutaric Aciduria

A mutation that changes the specificity of an enzyme in human cancer is also found in an inherited metabolic disorder. Heterozygous somatic mutations in the genes encoding isocitrate dehydrogenase-1 and -2 (IDH1 and IDH2) were recently discovered in human neoplastic disorders. These mutations disable the enzymes’ normal ability to convert isocitrate to 2-ketoglutarate (2-KG) and confer on the enzymes a new function: the ability to convert 2-KG to d-2-hydroxyglutarate (D-2-HG). We have detected heterozygous germline mutations in IDH2 that alter enzyme residue Arg140 in 15 unrelated patients with d-2-hydroxyglutaric aciduria (D-2-HGA), a rare neurometabolic disorder characterized by supraphysiological levels of D-2-HG. These findings provide additional impetus for investigating the role of D-2-HG in the pathophysiology of metabolic disease and cancer.

An overview of L‐2‐hydroxyglutarate dehydrogenase gene (L2HGDH) variants: a genotype–phenotype study

L‐2‐Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2‐hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT‐PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty‐five novel mutations as well as 35 reported mutations and 14 nondisease‐related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype–genotype relationship. Hum Mutat 30:1–11, 2010.

Phenotype and genotype variation in primary carnitine deficiency

Purpose: Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the carnitine transporter gene SLC22A5. The objective of this study was to extend mutational analysis to four additional families with this disorder and determine whether recurrent mutations could be found.Methods: The SLC22A5 gene encoding the OCTN2 carnitine transporter was sequenced, and the missense mutations identified were expressed in Chinese hamster ovary (CHO) cells.Results: DNA sequencing revealed four novel mutations (Y4X; dup 254–264, 133X; R19P; R399Q). Alleles introducing premature STOP codons reduced the levels of OCTN2 mRNA. Carnitine transport in CHO cells expressing the R19P and R399Q mutations was reduced to < 5% of normal. The 133X mutation was found in two unrelated European families. Two patients within the same family, both homozygous for the same mutation (R399Q) had completely different clinical presentation.Conclusions: Heterogeneous mutations in the SLC22A5 gene cause primary carnitine deficiency. Different presentations are observed even in children with identical mutations.

Elevated plasma citrulline and arginine due to consumption of Citrullus vulgaris (watermelon)

SummaryA 19-month-old girl with developmental delay was found to have moderately elevated plasma citrulline and mildly elevated plasma arginine concentrations. Dietary history revealed that she consumed large quantities of watermelon (Citrullus vulgaris), a fruit containing high free citrulline and arginine concentrations. In order to determine whether the patient’s high watermelon intake could account for her elevated plasma citrulline and arginine concentrations, we studied the response of plasma citrulline and arginine to ingestion of watermelon in six healthy adult volunteers. All developed markedly elevated plasma citrulline (mean maximum 593 μmol/L, range 386–1069) and moderately elevated plasma arginine (mean maximum 199 μmol/L, range 128–251). Physicians and laboratory personnel performing metabolic investigations should be aware of watermelon-induced citrullinaemia. Its hallmarks are elevated plasma citrulline, and to a lesser extent arginine, in the absence of orotic or arginosuccinic aciduria or hyperammonaemia. This phenomenon has implications for the management of patients with urea cycle and related disorders.

Pitfalls in the diagnosis of glycine encephalopathy (non-ketotic hyperglycinemia).

Non-ketotic hyperglycinemia (NKH), also termed glycine encephalopathy (MIMa 605899), is an autosomal recessive inborn error of glycine degradation which leads to severe neurological symptoms and profound psychomotor disability. In NKH, glycine accumulates in all body fluids and tissues, including the CNS. The biochemical hallmark of NKH is increased glycine concentration in the plasma and to an even greater extent in the CSF, leading to an elevation of the CSF:plasma glycine ratio (C:PGR) to above 0.08 (normal <0.04). The fundamental defect is in the glycine cleavage system (GCS), a multienzyme complex located in the inner mitochondrial membrane of the liver, kidney, brain, and placenta. It consists of four individual protein components termed P (a pyridoxal phosphate-dependent glycine decarboxylase), H (a lipoic acid-containing hydrogen carrier protein), T (a tetrahydrofolate-dependent protein), and L (a lipoamide dehydrogenase). In more than 80% of patients the defect is in the P protein (MIM 238300), but defects in the T (MIM 238310) and H (MIM 238330) proteins have also been described. The pathogenesis of NKH is related to the properties of glycine as an excitatory neurotransmitter acting via the N-methyl-Daspartate receptor in the cortex and an inhibitory neurotransmitter in the brainstem and spinal cord.1–3 Classically, NKH presents in the early neonatal period with progressive lethargy, hypotonia, myoclonic jerks, hiccups, and apnea, usually leading to total unresponsiveness, coma, and death unless the patient is supported through this stage with mechanical ventilation. Survivors almost invariably display profound neurological disability and intractable seizures. In a minority of NKH cases the presentation is atypical with a later onset and features including seizures, developmental delay and/or regression, hyperactivity, spastic diplegia, spinocerebellar degeneration, optic atrophy, vertical gaze palsy, ataxia, chorea, and pulmonary hypertension.4–20 Atypical cases are more likely to have milder elevations of glycine concentrations and C:PGR with residual GCS activity. A handful of transient NKH cases have been reported21–26 with neonatal onset and characteristic EEG and biochemical abnormalities which, however, return to normal by 2 months of age, usually with complete clinical resolution. Such cases have been attributed to delayed maturation of the hepatic and cerebral GCS. NKH is generally considered to be a rare disease, but relatively higher incidences have been reported in Northern Finland,27 British Columbia,28,29 and Israel.30–32 In our own laboratory, one of four in Israel performing amino acid analyses, we have diagnosed 15 new unrelated cases of NKH in the past 3 years. Ideally, the diagnosis of NKH should be confirmed by demonstrating deficient GCS activity using the [1-14C] glycine decarboxylation assay, together with assay of the individual components of the complex.33 This necessitates a liver biopsy, as NKH activity is not expressed in fibroblasts nor untransformed lymphocytes. An open liver biopsy is necessary in order to obtain the required amount of tissue for the complete evaluation. In most cases, the treating physician and/or family are unwilling to perform this procedure, particularly in a critically ill infant. Furthermore, the assay is available in only a few centres worldwide necessitating transport of a limited and crucial sample under stringent conditions. Enzymatic diagnosis of NKH due to P-protein deficiency using Epstein-Barr virus transformed lymphoblasts has been reported.34 However, the normal GCS activity in lymphoblasts is low, and some other laboratories have been unable to reproduce these results consistently.35 Furthermore, lymphoblast GCS activity may be normal despite unequivocally deficient hepatic GCS activity.17 At present, molecular diagnosis of NKH by mutation analysis is not a practicable alternative for most sporadic cases. With the exception of a common mutation in the P-protein gene among Finnish patients36 representing a founder effect, there have been few reports of recurring mutations in the Por T-protein genes in unrelated patients.37–39 Thus, A nntation

TMEM70 mutations are a common cause of nuclear encoded ATP synthase assembly defect: further delineation of a new syndrome

Background The TMEM70 gene defect was recently identified as a novel cause of autosomal recessive ATP synthase deficiency. Most of the 28 patients with TMEM70 disorder reported to date display a distinctive phenotype characterised by neonatal onset of severe muscular hypotonia hypertrophic cardiomyopathy, facial dysmorphism, profound lactic acidosis, and 3-methylglutaconic aciduria. Almost all share a common Roma descent and are homozygous for a single founder splice site mutation. Methods Six new patients from four separate families, with clinical and biochemical diagnosis of ATP synthase deficiency, were studied. TMEM70 sequence analysis of the three exons and their flanking splice junction consensus sequences was performed in all patients. In addition their clinical phenotype and disease course was strictly studied. Results Four novel deleterious homozygous TMEM70 mutations were identified. The previously described clinical spectrum was expanded to include infantile onset cataract, early onset gastrointestinal dysfunction and congenital hypertonia with multiple contractures resembling arthrogryposis. The first characterisation of fetal presentation of the syndrome is also provided, featuring significant intrauterine growth retardation, severe oligohydramnios, fetal hypotonia, and myocardial wall thickening. Conclusions The current report corroborates the previously described unique phenotype of TMEM70 deficiency. The study identifies TMEM70 gene defect as a pan-ethnic disorder and further redefines it as the most common cause of nuclear-origin ATP synthase deficiency.

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. Ann Neurol 2004;56:139–143

Evidence for genetic heterogeneity in D-2-hydroxyglutaric aciduria†

We performed molecular, enzyme, and metabolic studies in 50 patients with D‐2‐hydroxyglutaric aciduria (D‐2‐HGA) who accumulated D‐2‐hydroxyglutarate (D‐2‐HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D‐2‐hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D‐2‐hydroxyglutarate dehydrogenase (D‐2‐HGDH). Enzyme assay of D‐2‐HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D‐2‐HGA whose enzyme activity was normal did not have mutations. Significantly lower D‐2‐HG concentrations in body fluids were observed in mutation‐positive D‐2‐HGA patients than in mutation‐negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D‐2‐HG. Accordingly, we suggest a new classification: D‐2‐HGA Type I associates with D‐2‐HGDH deficiency, whereas idiopathic D‐2‐HGA manifests with normal D‐2‐HGDH activity and higher D‐2‐HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D‐2‐HGA patients with diverse genetic loci will be revealed in future studies. Hum Mutat 31:1–5, 2010.

Treatment from birth of nonketotic hyperglycinemia due to a novel GLDC mutation

To determine whether the devastating outcome of neonatal‐onset glycine encephalopathy (NKH) could be improved by instituting treatment immediately at birth rather than after symptoms are already well established.