Fumarase Deficiency: A Safe and Potentially Disease Modifying Effect of High Fat/Low Carbohydrate Diet.

Fumarate hydratase deficiency (FHD) caused by biallelic alterations of the FH (fumarate hydratase) gene is a rare disorder of the tricarboxylic acid cycle, classically characterized by encephalopathy, profound psychomotor retardation, seizures, a spectrum of brain abnormalities and early death in childhood. Less common milder phenotypes with moderate cognitive impairment and long-term survival have been reported. In addition, heterozygous mutations of the FH gene are responsible for hereditary leiomyomatosis and renal cell cancer (HLRCC). There is currently no recommended disease modifying treatment for FHD and only isolated reports of unsuccessful dietary modifications. Herein, we describe the safe and possibly disease modifying effect of a high fat, low carbohydrate diet in a 14-year-old female with severe FHD.

Unique presentation of cutis laxa with Leigh-like syndrome due to ECHS1 deficiency.

Clinical finding of cutis laxa, characterized by wrinkled, redundant, sagging, nonelastic skin, is of growing significance due to its occurrence in several different inborn errors of metabolism (IEM). Metabolic cutis laxa results from Menkes syndrome, caused by a defect in the ATPase copper transporting alpha (ATP7A) gene; congenital disorders of glycosylation due to mutations in subunit 7 of the component of oligomeric Golgi (COG7)-congenital disorders of glycosylation (CDG) complex; combined disorder of N- and O-linked glycosylation, due to mutations in ATPase H+ transporting V0 subunit a2 (ATP6VOA2) gene; pyrroline-5-carboxylate reductase 1 deficiency; pyrroline-5-carboxylate synthase deficiency; macrocephaly, alopecia, cutis laxa, and scoliosis (MACS) syndrome, due to Ras and Rab interactor 2 (RIN2) mutations; transaldolase deficiency caused by mutations in the transaldolase 1 (TALDO1) gene; Gerodermia osteodysplastica due to mutations in the golgin, RAB6-interacting (GORAB or SCYL1BP1) gene; and mitogen-activated pathway (MAP) kinase defects, caused by mutations in several genes [protein tyrosine phosphatase, non-receptor-type 11 (PTPN11), RAF, NF, HRas proto-oncogene, GTPase (HRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), MEK1/2, KRAS proto-oncogene, GTPase (KRAS), SOS Ras/Rho guanine nucleotide exchange factor 2 (SOS2), leucine rich repeat scaffold protein (SHOC2), NRAS proto-oncogene, GTPase (NRAS), and Raf-1 proto-oncogene, serine/threonine kinase (RAF1)], which regulate the Ras-MAPK cascade. Here, we further expand the list of inborn errors of metabolism associated with cutis laxa by describing the clinical presentation of a 17-month-old girl with Leigh-like syndrome due to enoyl coenzyme A hydratase, short chain, 1, mitochondria (ECHS1) deficiency, a mitochondrial matrix enzyme that catalyzes the second step of the beta-oxidation spiral of fatty acids and plays an important role in amino acid catabolism, particularly valine.

Utility of next-generation sequencing technologies for the efficient genetic resolution of haematological disorders.

Next-generation sequencing (NGS) has now evolved to be a relatively affordable and efficient means of detecting genetic mutations. Whole genome sequencing (WGS) or whole exome sequencing (WES) offers the opportunity for rapid diagnosis in many paediatric haematological conditions, where phenotypes are variable and either a large number of genes are involved, or the genes are large making sanger sequencing expensive and labour-intensive. NGS offers the potential for gene discovery in patients who do not have mutations in currently known genes. This report shows how WES was used in the diagnosis of six paediatric haematology cases. In four cases (Diamond-Blackfan anaemia, congenital neutropenia (n = 2), and Fanconi anaemia), the diagnosis was suspected based on classical phenotype, and NGS confirmed those suspicions. Mutations in RPS19, ELANE and FANCD2 were found. The final two cases (MYH9 associated macrothrombocytopenia associated with multiple congenital anomalies; atypical juvenile myelomonocytic leukaemia associated with a KRAS mutation) highlight the utility of NGS where the diagnosis is less certain, or where there is an unusual phenotype. We discuss the advantages and limitations of NGS in the setting of these cases, and in haematological conditions more broadly, and discuss where NGS is most efficiently used.

Mitochondrial dysfunction in the skeletal muscle of a mouse model of Rett syndrome (RTT): implications for the disease phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

Update on transcobalamin deficiency: clinical presentation, treatment and outcome.

Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, and pancytopenia or agammaglobulinemia. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Based on the data presented, current practice appears to favour treatment of individuals with TC deficiency by intramuscular injections of hydroxy- or cyanocobalamin. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. Finally, continuing IM treatment into adulthood appears to be beneficial.

Congenital disorder of glycosylation type Ia: heterogeneity in the clinical presentation from multivisceral failure to hyperinsulinaemic hypoglycaemia as leading symptoms in three infants with phosphomannomutase deficiency.

We describe three patients with congenital disorder of glycosylation (CDG) type Ia, all of whom had persistent hyperinsulinaemic hypoglycaemia responding to diazoxide therapy as a common feature. The first patient, an infant girl, presented with recurrent vomiting, failure to thrive, liver impairment, hypothyroidism and a pericardial effusion. The second patient, also female, had a milder disease with single organ involvement, presenting as isolated hyperinsulinaemic hypoglycaemia, not associated with any cognitive impairment. The third patient, a boy presented with multi-organ manifestations including congenital hypothyroidism, persistent hyperinsulinaemic hypoglycaemia, coagulopathy, olivopontocerebellar hypoplasia and recurrent pancreatitis. All three patients had a type 1 serum transferrin isoform pattern, and were subsequently found to have low phosphomannomutase activity, confirming the diagnosis of CDG type Ia. Our findings emphasize that CDG should be considered as a differential diagnosis in patients with persistent hyperinsulinaemic hypoglycaemia and that it may even occasionally be the leading symptom in CDG Ia.

Potential of AAV vectors in the treatment of metabolic disease.

Inborn errors of metabolism are collectively common, frequently severe and in many instances difficult or impossible to treat. Accordingly, there is a compelling need to explore novel therapeutic modalities, including gene therapy, and examine multiple phenotypes where the risks of experimental therapy are outweighed by potential benefits to trial participants. Among available gene delivery systems recombinant AAV shows special promise for the treatment of metabolic disease given the unprecedented efficiencies achieved in transducing key target tissues, such as liver and muscle, in small animal models. To date over 30 metabolic disease phenotypes have been investigated in small animal studies with complete phenotype correction being achieved in a substantial proportion. Achieving adequately widespread transduction within the central nervous system, however, remains a major challenge, and will be critical to realization of the therapeutic potential of gene therapy for many of the most clinically troubling metabolic disease phenotypes. Despite the relatively low immunogenicity of AAV vectors, immune responses are also emerging as a factor requiring special attention as efforts accelerate toward human clinical translation. Four metabolic disease phenotypes have reached phase I or I/II trials with one, targeting lipoprotein lipase deficiency, showing exciting early evidence of efficacy.

Myelodysplastic syndrome (RARS) with +i(12p) abnormality in a patient 10 months after diagnosis and successful treatment of a mediastinal germ cell tumor (MGCT).

We report on a 21-year-old man with a mediastinal germ cell tumor (MGCT) who developed a myelodysplastic syndrome (MDS) (refractory anemia with ringed sideroblasts, RARS) 10 months after the start of successful treatment with cisplatin, etoposide, ifosfamide, and paclitaxel. A very rare early occurrence of a therapy-related MDS was suspected. Cytogenetic analysis of the bone marrow revealed an aberrant karyotype, showing a deletion in 12p, an isochromosome 5p, as well as gain of an isochromosome 12p. Isochromosome 12p is a specific aberration frequently observed in MGCT. It also was described in patients who developed hematological transformation of a mediastinal germ cell tumor. In this report the association between mediastinal germ cell tumors and hematological malignancies including the possibility of a common genetic origin is discussed.

Coronary arteriographic findings in symptomatic and asymptomatic subjects with coronary artery calcification.

The relation of coronary artery calcification with the presence of symptoms of coronary artery disease and its angiographic severity is not clear. We studied 37 apparently healthy, asymptomatic subjects that were found by digital cinefluoroscopy to have coronary calcium and compared to age- and sex-matched group of patients with coronary calcium and symptomatic coronary artery disease. Normal coronary arteries and non-obstructive lesions only were found in 12/37 (32.4%) and 11/37 (29.7%) asymptomatic subjects vs. 1/37 (2.7%) and 2/37 (5.4%) patients; P<0.001 and P<0.012, respectively. Obstructive lesions were more rare in asymptomatic subjects than in patients, 14/37 (37.8%) vs. 34/37 (91.9%) (P<0.0001), as well as total occlusions, 2/37 (5.4%) vs. 10/37 (27%) (P<0.024). Median worst lesion stenosis was 30% in asymptomatic subjects and 95% in patients (P<0.0001). In asymptomatic usual cardiovascular risk subjects, coronary calcium detection by digital cinefluoroscopy is accompanied by a relatively high probability of obstructive disease, although less severe angiographically than in age- and sex-matched catheterized patients with symptomatic coronary artery disease.

Flow cytometric evaluation of defects of the mitochondrial respiratory chain.

Cultured human skin fibroblasts from 12 patients with a variety of mitochondrial respiratory chain defects were examined for their capacity to oxidize dihydrorhodamine-123 to the fluorescent molecule rhodamine-123 using a flow cytometer. We found that cells from patients with functional defects in respiratory chain enzymes were less able to oxidize dihydrorhodamine-123 than those of healthy controls. Ten of the cell strains had reduced activity in at least one of the respiratory chain complexes and also showed significantly reduced fluorescence when compared to the mean of eight normal control cell strains. One patient had mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (with the A3243G mutation) and reduced respiratory chain activities in muscle and liver. Molecular analysis did not show the mutation in cultured skin fibroblasts, and had correspondingly normal fluorescence. The 12th cell strain showed reduced fluorescence but did not reach statistical significance. This strategy could be of use in helping direct further investigations in patients, and in studying the biochemical pathogenesis of mitochondrial DNA mutations in cybrid studies.

Surfactant protein B deficiency: clinical, histological and molecular evaluation.

Congenital alveolar proteinosis due to surfactant protein B deficiency is an inherited disease which results in severe respiratory failure in term infants soon after birth. The pathophysiologic basis of this disease is now known to be an inability to synthesise adequate quantities of normally functioning surfactant protein B. We report a male infant with fatal respiratory failure of neonatal onset, and histopathological features typical of those seen in congenital alveolar proteinosis. Molecular analysis of genomic DNA revealed two mutations, the 'common' 121ins2 mutation in exon 4, and a novel 2bp frameshift mutation in exon 5. We believe this is the first Australian case of surfactant protein B deficiency confirmed by molecular analysis.

Respiratory chain complex III [correction of complex] in deficiency with pruritus: a novel vitamin responsive clinical feature.

We report a child with an isolated complex III respiratory chain deficiency and global developmental delay who had severe pruritus with elevated plasma bile acid levels. A liver biopsy showed micronodular cirrhosis, and enzymologic evaluation demonstrated an isolated complex III deficiency in both liver and muscle. His pruritus improved and serum bile acid levels decreased after treatment with menadione and vitamin C.

Non-invasive estimation of pulmonary arterial hypertension in chronic obstructive pulmonary disease.

The feasibility and reliability of the combination of several noninvasive methods using a multivariate method of analysis to predict pulmonary artery hypertension (PAH) is evaluated in 20 patients with chronic obstructive pulmonary disease. These methods comprised arterial blood gases (Pao2, Paco2), pulmonary functional parameters (FEV1), echo-Doppler parameters (tricuspid regurgitation jets, acceleration time on pulmonary valve), computed tomography measurements (transhilar distance, hilar thoracic index, and measurement of the descending branch of the right pulmonary artery to the lower lobe). A multiple stepwise regression analysis (including one Doppler parameter, two parameters of arterial blood gases, and one functional parameter) revealed a coefficient of determination (R2) equal to 0.954 for mean pulmonary artery pressure (MPAP) with a standard error of estimate (S.E.E.) of 5.25 mmHg. A stepwise regression analysis including computed tomography and radiographic parameters revealed an R2 equal to 0.970 for PAP with a S.E.E. of 4.26 mmHg. Logistical regression analysis classified correctly 80% of patients with PAH using noninvasive methods such as the diameter of the main pulmonary artery and the diameter of the left pulmonary arterial branch calculated by computed tomography. Not only the presence of PAH but also the level of MPAP can be estimated by the combination of multiple stepwise and logistical regression analyses.

Cisplatin binding sites on human albumin.

Reactions of cisplatin (cis-[PtCl2(NH3)2]) with albumin are thought to play an important role in the metabolism of this anticancer drug. They are investigated here via (i) labeling of cisplatin with 15N and use of two-dimensional 1H,15N NMR spectroscopy, (ii) comparison of natural human serum albumin with recombinant human albumin (higher homogeneity and SH content), (iii) chemical modification of Cys, Met, and His residues, (iv) reactions of bound platinum with thiourea, and (v) gel filtration chromatography. In contrast to previous reports, it is shown that the major sulfur-containing binding site involves Met and not Cys-34, and also a N ligand, in the form of an S,N macrochelate. Additional monofunctional adducts involving other Met residues and Cys-34 are also observed. During the later stages of reactions of cisplatin with albumin, release of NH3 occurs due to the strong trans influence of Met sulfur, which weakens the Pt-NH3 bonds, and protein cross-linking is observed. The consequences of these findings for the biological activity of cisplatin-albumin complexes are discussed.

First prenatal diagnosis of the carnitine transporter defect.

We report the first attempt at prenatal diagnosis of the carnitine transporter defect in a fetus at high risk of having the disorder. Analysis of cultured CVS after prolonged culture predicted that the fetus was not affected but might be heterozygous for the carnitine transporter defect, but chromosome 15 satellite DNA markers showed no paternal contribution, suggesting that the CVS cells assayed were of predominantly maternal origin. Subsequent assay of cultured amniocytes predicted that the fetus would be affected, and this was confirmed in the newborn period. We conclude that prenatal diagnosis of the carnitine transporter defect is possible, but where results depend on extended culture of CVS, molecular studies should be performed to confirm genetic contributions from both parents.

Repolarization abnormalities with prolonged hyperventilation in apparently healthy subjects: incidence, mechanisms and affecting factors.

Brief hyperventilation is occasionally accompanied by repolarization abnormalities in subjects without apparent heart disease. However, the effect of prolonged hyperventilation on repolarization abnormalities is not clearly defined. We analysed the repolarization abnormalities induced by prolonged hyperventilation, correlated with hyperventilation induced haemodynamic changes and exercise test results and assessed the effect on them of age, gender, smoking and hypertension. Prolonged hyperventilation (overbreathing at least 30 respirations/min for 5 min and 10 min recovery) was performed in 474 healthy volunteers (269 men, 205 women) 42.6 +/- 13.5 years old. The electrocardiogram was analysed for transient ST depression or T-wave inversion. Repolarization abnormalities were observed in 72 subjects, 47 men (17.5%) and 25 women (12.2%). Age, gender, smoking and hypertension did not influence the overall incidence of repolarization abnormalities. ST depression was more frequently observed in women (4.4 vs 0.7%, P < 0.01), while T wave inversion from negative to positive was more frequent in men (8.9 vs 2.4%, P < 0.006). All subjects with ST depression were non-smokers (4.1% of the non-smokers, P < 0.003 compared to smokers). Repolarization abnormalities occurred usually (80.5%), but not exclusively, within the first hyperventilation minute. Subjects with repolarization abnormalities developed higher heart rate change with hyperventilation than those without repolarization abnormalities (29.2 +/- 13.1 vs 24.2 +/- 12.7, P < 0.002). A positive exercise test was observed in 45.4% of subjects with hyperventilation induced ST depression but only in 13.1% of subjects with T-wave changes, P < 0.02. Repolarization abnormalities are not uncommon during prolonged hyperventilation. Hear rate change affects the occurrence of repolarization abnormalities, while gender and smoking influence the type of repolarization abnormalities. ST depression but not T-wave inversion during hyperventilation is associated with a positive exercise test. Increased understanding about hyperventilation-induced repolarization abnormalities could affect use of the method in the clinic, either as a provocation test or as a complement to the interpretation of the exercise test.

Coronary artery calcium detected by digital fluoroscopy and risk factors in healthy subjects.

Coronary artery calcium detected by digital fluoroscopy is closely associated with known risk factors of coronary artery disease in asymptomatic low-risk populations. Even in the absence of significant luminal narrowing, this may not be an innocent finding, and subjects with coronary calcium may be at greater risk for developing obstruction and clinical disease.