Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.

Clinical, ethical and legal considerations in the treatment of newborns with non-ketotic hyperglycinaemia.

Non-ketotic hyperglycinaemia (NKH) is a devastating neurometabolic disorder leading, in its classical form, to early death or severe disability and poor quality of life in survivors. Affected neonates may need ventilatory support during a short period of respiratory depression. The transient dependence on ventilation dictates urgency in decision-making regarding withdrawal of therapy. The occurrence of patients with apparent transient forms of the disease, albeit rare, adds uncertainty to the prediction of clinical outcome and dictates that the current practice of withholding or withdrawing therapy in these neonates be reviewed. Both bioethics and law take the view that treatment decisions should be based on the best interests of the patient. The medical-ethics approach is based on the principles of non-maleficence, beneficence, autonomy and justice. The law relating to withholding or withdrawing life-sustaining treatment is complex and varies between jurisdictions. Physicians treating newborns with NKH need to provide families with accurate and complete information regarding the disease and the relative probability of possible outcomes of the neonatal presentation and to explore the extent to which family members are willing to take part in the decision making process. Cultural and religious attitudes, which may potentially clash with bioethical and juridical principles, need to be considered.

The relation of cerebrospinal fluid and plasma glycine levels in propionic acidaemia, a 'ketotic hyperglycinaemia'.

The characteristic elevation of plasma glycine concentrations observed in propionic acidaemia (PA) and other 'ketotic hyperglycinaemias' has been attributed to secondary inhibition of the hepatic glycine cleavage system (GCS) by accumulating CoA derivatives of branched-chain amino acid metabolites. In nonketotic hyperglycinaemia (NKH), cerebrospinal fluid (CSF) and plasma glycine levels and their ratio are increased due to primary deficiency of central nervous system (CNS) as well as hepatic GCS. Whether the GCS in the CNS is also inhibited in PA is unclear, as there are scant data available on CSF glycine levels in this disorder. We studied the relation of CSF and plasma glycine levels in 6 paired samples from 4 PA patients, including one PA patient with bacterial meningitis who underwent ventriculoperitoneal shunting and multiple CSF analyses (n = 26). In contrast to the CSF glycine levels which were generally elevated in all four PA patients, the CSF/plasma glycine concentration ratios in paired samples were normal (0.016-0.029), with the exception of a single sample (0.132) with extremely high CSF protein concentration (2010 mg/L) during the course of meningitis indicating a disturbed blood-brain barrier. This finding of normal CSF/plasma glycine ratio in PA suggests that the observed elevations of CSF glycine levels are a reflection of the concurrent hyperglycinaemia resulting from secondary inhibition of hepatic GCS, but that brain GCS is not affected, in contrast to the situation in NKH. The neurological sequelae in PA are therefore unlikely to be related to disturbed glycine metabolism.

Investigation of human giardiasis by karyotype analysis.

The patterns of transmission of Giardia lamblia and the potential contribution of strain differences to pathogenicity of infection is poorly understood. We used pulsed field gradient gel electrophoresis (PFGE) to separate chromosome-sized DNA molecules of 22 stocks of G. lamblia isolated from 13 individuals (6 symptomatic, 7 asymptomatic) living in Jerusalem. PGFE gels run under a variety of conditions revealed up to nine ethidium bromide-stained bands per isolate ranging in size from 0.7 to greater than 3 megabasepairs. Relative staining intensities indicated that some bands contained multiple chromosomes. Major differences in the number, size, and intensity of bands allowed a clear differentiation of the karyotypes of isolates from each of the different individuals. This is in contrast to previous studies where the karyotype of different isolates have been strikingly homogeneous. Hybridization of Southern blots with surface antigen, beta-tubulin, and ribosomal RNA genes revealed that these gene families were distributed to different sized chromosomes amongst the different isolates. PFGE thus revealed major differences in the karyotypes of different G. lamblia isolates that were obtained over a short period of time from a relatively confined geographic area. In contrast, karyotypes of isolates established either by direct cultivation of duodenal trophozoites or by excystation of stool cysts from the same individuals were almost identical. Also, isolates from the same individuals obtained over a prolonged period of time revealed only minor differences in their karyotype, suggesting that recurrent infection can be caused by genetically similar organisms. We conclude that chronic giardiasis can result from recurrence of occult infection or reinfection from a common source.

Severe infantile type of carnitine palmitoyltransferase II (CPT II) deficiency due to homozygous R503C mutation.

We report a patient with severe infantile carnitine palmitoyltransferase II (CPT II) deficiency who died at the age of 3 months. Genetic analysis of the CPT2 gene revealed that the patient was homozygous, and her parents were heterozygous, for a R503C missense mutation. Heterozygosity for R503C, without a second mutation, has previously been reported in symptomatic patients from two families, one with the mild adult myopathic form and one with malignant hyperthermia. In contrast, the R503C heterozygous parents of the patient were entirely asymptomatic, suggesting that additional genetic and/or environmental factors must have contributed to the occurrence of symptoms in previously reported carriers. Our findings indicate that the mutation R503C should be added to the handful of mutations associated with the severe phenotype when present in the homozygous state or combined with another severe mutation.

Pica as a presenting symptom in childhood celiac disease.

Persistent pica may be either a cause or a result of iron deficiency. Three children are described with long-standing pica and iron-deficiency anemia and in whom total villous atrophy consistent with celiac disease was found on jejunal biopsy. Additional findings included short stature, delayed bone age, and impaired xylose absorption. A dramatic growth spurt and complete resolution of pica were observed after a gluten-free diet. In these cases pica evidently resulted from iron deficiency secondary to malabsorption. Underlying celiac disease should be considered in children with persistent pica and growth failure even if gastrointestinal disturbances are minimal.

Glycogen storage disease type 1a in Israel: biochemical, clinical, and mutational studies.

Glycogen storage disease type 1a (von Gierke disease, GSD 1a) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The recent cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the characterization of the mutations causing GSD 1a. This, in turn, allows the introduction of a noninvasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. In this study, we report the biochemical and clinical characteristics as well as mutational analyses of 12 Israeli GSD 1a patients of different families, who represent most GSD 1a patients in Israel. The mutations, G6Pase activity, and glycogen content of 7 of these patients were reported previously. The biochemical data and clinical findings of all patients were similar and compatible with those described in other reports. All 9 Jewish patients, as well as one Muslim Arab patient, presented the R83C mutation. Two Muslim Arab patients had the V166G mutation which was not found in other patients' populations. The V166G mutation, which was introduced into the G6Pase cDNA by site-directed mutagenesis following transient expression in COS-1 cells, was shown to cause complete inactivation of the G6Pase. The characterization of all GSD 1a mutations in the Israeli population lends itself to carrier testing in these families as well as to prenatal diagnosis, which was carried out in 2 families. Since all Ashkenzai Jewish patients harbor the same mutation, our study suggests that DNA-based diagnosis may be used as an initial diagnostic step in Ashkenazi Jews suspected of having GSD 1a, thereby avoiding liver biopsy.

Asymptomatic giardiasis in children.

Giardia lamblia infection was identified in 33 of 89 (37%) 3-month-old to 3-yr-old children who were followed with monthly stool examinations for up to 12 months in a day care center. The infection was mainly asymptomatic and usually associated with prolonged carriage of the parasite. There were no significant differences for height and weight achievements and mean hemoglobin values between Giardia-positive and Giardia-negative children. However, Giardia-positive children tended to achieve higher weight and height for age than Giardia-negative children; weight for age was above the 50th percentile in 69% of Giardia-positive vs. 40% of Giardia-negative children (alpha = 0.01). Giardia-positive children tended to have fewer symptoms related to the gastrointestinal and respiratory tracts as recorded by a weekly questionnaire. Lactase deficiency was detected by breath hydrogen testing in 8 of 26 Giardia-positive vs. only 1 of 21 Giardia-negative children (P less than 0.02). Healthy day care children with asymptomatic Giardia infection show no disadvantage and perhaps even an advantage in nutritional status and freedom from other illnesses.

Severe giardiasis in two patients undergoing bone marrow transplantation.

Two patients undergoing bone marrow transplantation developed severe gastrointestinal symptoms, one during the peri-transplantation period and the other several months post-transplantation and following documented intestinal graft-versus-host disease. After detection of Giardia lamblia trophozoites in a stool specimen from one patient and a duodenal aspirate from the other, treatment with metronidazole was administered and all symptoms resolved. Giardiasis should be considered in transplant patients with diarrhea. Negative stool examinations do not rule out this possibility, and consideration should be given to examining a sample of small intestinal contents for Giardia trophozoites.

Protozoan genomes : karyotype analysis, chromosome structure, and chromosome specific libraries.

Protozoa represent a diverse group of single-celled eukaryotes, many of which have parasitic life styles, infecting hundreds of millions of people. Unique aspects of their biology relate to their distinct evolutionary position and their complex life cycles, frequently involving different hosts.

Methylenetetrahydrofolate reductase deficiency: importance of early diagnosis.

Methylenetetrahydrofolate reductase deficiency is the most common inborn error of folate metabolism and should be suspected when homocystinuria is combined with hypomethioninemia. The main clinical findings are neurologic signs such as severe developmental delay, marked hypotonia, seizures, microcephaly, apnea, and coma. Most patients present in early life. The infantile form is severe, with rapid deterioration leading to death usually within 1 year. Treatment with betaine has been shown to be efficient in lowering homocysteine concentrations and returning methionine to normal, but the clinical response is variable. We report two brothers with methylenetetrahydrofolate reductase deficiency: the first was undiagnosed and died at 8 months of age from neurologic deterioration and apnea, while his brother, who was treated with betaine from the age of 4 months, is now 3 years old and has developmental delay.

Carnitine-deficient myopathy as a presentation of tyrosinemia type I.

Carnitine deficiency secondary to renal Fanconi's tubulopathy has been described in only a few inborn errors of metabolism: cystinosis, galactosemia, and Fanconi-Bieckel syndrome. We report a 27-month-old infant who presented with a sudden change in gait owing to proximal muscle weakness. The laboratory evaluation showed carnitine deficiency associated with Fanconi's tubulopathy. Eventually, tyrosinemia type I was diagnosed. Carnitine deficiency can contribute to the clinical picture of hepatorenal tyrosinemia and should therefore be evaluated and treated.

Reversible deafness caused by biotinidase deficiency.

We present a child with complete biotinidase deficiency who developed bilateral sensorineural deafness without a response to a maximal stimulus of 90 dB in brainstem acoustic-evoked response. After treatment with 20 mg biotin daily, a repeated brainstem acoustic-evoked response demonstrated an improved hearing threshold of 65 dB, and the child began to talk. The case is a rare example of reversible hearing loss caused by to biotinidase deficiency and highlights the need for immediate replacement therapy once the diagnosis is established.

TAT gene mutation analysis in three Palestinian kindreds with oculocutaneous tyrosinaemia type II; characterization of a silent exonic transversion that causes complete missplicing by exon 11 skipping.

Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon-intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5' donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.

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

A 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 micromol/L, range 386-1069) and moderately elevated plasma arginine (mean maximum 199 micromol/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.

Mild glycine encephalopathy (NKH) in a large kindred due to a silent exonic GLDC splice mutation.

BACKGROUND: Classic neonatal-onset glycine encephalopathy (GE) is devastating and life threatening. Milder, later onset variants have been reported but were usually sporadic and incompletely defined. OBJECTIVE: To determine the clinical and biochemical phenotype and molecular basis of mild GE in nine children from a consanguineous Israeli Bedouin kindred. METHODS: Genomic DNA was screened for GLDC, AMT, and GCSH gene mutations. GLDC expression in lymphoblasts was studied by Northern blot and reverse transcriptase PCR analysis. RESULTS: Clinical features included hypotonia, abnormal movements, convulsions, and moderate mental retardation with relative sparing of gross motor function, activities of daily living skills, and receptive language. Aggression and irritability were prominent. CSF-to-plasma glycine ratio was mildly to moderately elevated. All nine patients were homozygous and their parents heterozygous for a novel, translationally silent GLDC exon 22 transversion c.2607C>A. Lymphoblast GLDC mRNA levels were considerably reduced. Three aberrantly spliced cDNA species were identified: exon 22 and exon 22 to 23 skipping, and insertion of an 87-base pair cryptic exon. Homozygosity for c.2607C>A was also identified in an unrelated but haplotypically identical patient with an unusually favorable outcome despite severe neonatal-onset GE. Mutation analysis enabled prenatal diagnosis of three unaffected and one affected pregnancies. CONCLUSIONS: The mutation in this kindred led to missplicing and reduced GLDC (glycine decarboxylase) expression. The 4 to 6% of normally spliced GLDC mRNA in the patients may account for their relatively favorable clinical outcome compared with patients with classic glycine encephalopathy.

The duodenal string test. A simple multipurpose diagnostic tool in clinical pediatrics.

The duodenal string test capsule is a cheap and simple device used for sampling the contents of the upper gastrointestinal tract. Its major applications in pediatrics are in diagnosis of enteric parasitic infestations, confirmation of contaminated small-bowel syndrome, diagnosis of Salmonella infection, and assessment of neonatal cholestasis. Pediatricians should be aware of this invaluable diagnostic aid.

Thrombocytopenic purpura during the incubation of hepatitis B.

A 6-year-old girl presented with acute thrombocytopenic purpura. Hepatitis B surface antigenaemia was present at the time of this illness, and 8 weeks later she developed acute icteric hepatitis B. Screening for hepatitis B virus should be considered in children with apparently idiopathic thrombocytopenic purpura.