Andrew W. Johnson

Guanidinoacetate methyltransferase deficiency: New clinical features

Guanidinoacetate methyltransferase deficiency is a recently described inborn error of creatine biosynthesis that responds to treatment with oral creatine supplementation. The previously reported clinical features consist of developmental arrest and an extrapyramidal movement disorder. We describe a patient who presented with epilepsy, global developmental delay, and a persistently low plasma creatinine level. The diagnosis was established by measuring urinary guanidinoacetate and by demonstrating absence of the creatine/phosphocreatine peak in the patients basal ganglia in 1H magnetic resonance spectroscopy. The clinical and biochemical abnormalities responded to creatine replacement.

Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry

Abstract Objective: To assess the feasibility of screening for cholestatic hepatobiliary disease and extrahepatic biliary atresia by using tandem mass spectrometry to measure conjugated bile acids in dried blood spots obtained from newborn infants at 7-10 days of age for the Guthrie test. Setting: Three tertiary referral clinics and regional neonatal screening laboratories. Design: Unused blood spots from the Guthrie test were retrieved for infants presenting with cholestatic hepatobiliary disease and from the two cards stored on either side of each card from an index child. Concentrations of conjugated bile acids measured by tandem mass spectrometry in the two groups were compared. Main outcome measures: Concentrations of glycodihydroxycholanoates, glycotrihydroxycholanoates, taurodihydroxycholanoates, and taurotrihydroxycholanoates. Receiver operator curves were plotted to determine which parameter (or combination of parameters) would best predict the cases of cholestatic hepatobiliary disease and extrahepatic biliary atresia. The sensitivity and specificity at a selection of cut off values for each bile acid species and for total bile acid concentrations for the detection of the two conditions were calculated. Results: 218 children with cholestatic hepatobiliary disease were eligible for inclusion in the study. Two children without a final diagnosis and five who presented at <14 days of age were excluded. Usable blood spots were obtained from 177 index children and 708 comparison children. Mean concentrations of all four bile acid species were significantly raised in children with cholestatic hepatobiliary disease and extrahepatic biliary atresia compared with the unaffected children (P<0.0001). Of 177 children with cholestatic hepatobiliary disease, 104 (59%) had a total bile acid concentration >33 μmol/l (97.5th centile value for comparison group). Of the 61 with extrahepatic biliary atresia, 47 (77%) had total bile acid concentrations >33 μmol/l Taurotrihydroxycholanoate and total bile acid concentrations were the best predictors of both conditions. For all cholestatic hepatobiliary disease, a cut off level of total bile acid concentration of 30 μmol/l gave a sensitivity of 62% and a specificity of 96%, while the corresponding values for extrahepatic biliary atresia were 79% and 96%. Conclusion: Most children who present with extrahepatic biliary atresia and other forms of cholestatic hepatobiliary disease have significantly raised concentrations of conjugated bile acids as measured by tandem mass spectrometry at the time when samples are taken for the Guthrie test. Unfortunately the separation between the concentrations in these infants and those in the general population is not sufficient to make mass screening for cholestatic hepatobiliary disease a feasible option with this method alone. Key messages The prognosis of cholestatic hepatobiliary disease in infancy, in particular biliary atresia, is improved by early detection Infants destined to present with cholestatic jaundice in the first few months of life have raised concentrations of bile acids in the blood spots obtained at 7-10 days for current neonatal screening programmes Tandem mass spectrometry can be used to detect this marker of neonatal cholestasis Unfortunately there is too much overlap between bile acid concentrations in infants with cholestasis and those in control infants for this to be used as a single screening test for cholestatic hepatobiliary disease in general and biliary atresia Tandem mass spectrometry is a powerful tool for neonatal screening but every potential application must be carefully assessed

Application of magnetic chromatography to the isolation of lysosomes from fibroblasts of patients with lysosomal storage disorders

A method for the purification of lysosomes from fibroblasts has been developed which uses endocytosis of superparamagnetic colloidal iron dextran particles followed by separation of the iron‐containing lysosomes in a magnetic field. This permitted isolation of lysosomes from fibroblasts from patients with infantile sialic acid storage disorder and other lysosomal storage diseases in which a shift in lysosomal density induced by the storage material prevents purification by centrifugation in a Percoll gradient. The magnetic lysosomes isolated from these cells are very similar to those from normal cells as judged by lysosomal marker enzyme activity and 2D‐PAGE analysis of the enriched proteins.

Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry

OBJECTIVE To establish criteria for the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency in the UK population using a method in which carnitine species eluted from blood spots are butylated and analysed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). DESIGN Four groups were studied: (1) 35 children, aged 4 days to 16.2 years, with proven MCAD deficiency (mostly homozygous for the A985G mutation, none receiving carnitine supplements); (2) 2168 control children; (3) 482 neonates; and (4) 15 MCAD heterozygotes. RESULTS All patients with MCAD deficiency had an octanoylcarnitine concentration ([C8–Cn]) > 0.38 μM and no accumulation of carnitine species > C10 or < C6. Among the patients with MCAD deficiency, the [C8–Cn] was significantly lower in children > 10 weeks old and in children with carnitine depletion (free carnitine < 20 μM). Neonatal blood spots from patients with MCAD deficiency had a [C8–Cn] > 1.5 μM, whereas in heterozygotes and other normal neonates the [C8–Cn] was < 1.0 μM. In contrast, the blood spot [C8–Cn] in eight of 27 patients with MCAD deficiency > 10 weeks old fell within the same range as five of 15 MCAD heterozygotes (0.38–1.0 μM). However, the free carnitine concentrations were reduced (< 20 μM) in the patients with MCAD deficiency but normal in the heterozygotes. CONCLUSIONS Criteria for the diagnosis of MCAD deficiency using ESI-MS/MS must take account of age and carnitine depletion. If screening is undertaken at 7–10 days, the number of false positive and negative results should be negligible. Because there have been no instances of death or neurological damage following diagnosis of MCAD deficiency in our patient group, a strong case can be made for neonatal screening for MCAD deficiency in the UK. Key messages Electrospray ionisation tandem mass spectrometry (ESI-MS/MS) of acylcarnitine species is a reliable first line test for the diagnosis of MCAD deficiency Criteria for the diagnosis of MCAD deficiency using ESI-MS/MS must take account of age and carnitine depletion If screening is undertaken at 7–10 days, the number of false positive and negative results should be negligible Because there have been no instances of death or neurological damage following diagnosis of MCAD deficiency in our patient group, a strong case can be made for neonatal screening for MCAD deficiency in the UK

A Method for the Quantitation of Conjugated Bile Acids in Dried Blood Spots Using Electrospray Ionization-Mass Spectrometry

Bile acid concentrations are elevated in the blood of neonates with cholestatic hepatobiliary disorders providing a possible means of screening for treatable conditions including biliary atresia. A method is described for the determination of concentrations of conjugated bile acids in dried blood spots using electrospray ionization mass spectrometry. Bile acids were eluted from the blood spots using methanol containing, as internal standards, the taurine and glycine conjugates of D4-chenodeoxycholic acid and D4-cholic acid. The samples were then reconstituted in acetonitrile/water and injected by autosampler into the electrospray source operating in negative ion mode. Optimal conditions were determined for both single quadrupole and tandem mass spectrometry analysis. Blood spot bile acid profiles were studied in two groups of infants (<1 y), a cholestatic group(conjugated bilirubin >25 μmol/L; n = 49), and a control group(n = 96). The best discrimination between the two groups was provided by measurements of taurodihydroxycholanoates (normal <5 μmol/L; cholestatic group 18-94 μmol/L) and glycodihydroxycholanoates (normal <5μmol/L; cholestatic group 11-66 μmol/L). The method can also be adapted to detect unusual bile acids which are diagnostic of inborn errors of bile acid synthesis and peroxisomal disorders. The method is fast, reliable, reproducible, and relatively cheap; however, much more work is required to determine whether it can be used for mass screening for cholestasis. It will be necessary to show that measurement of bile acid concentrations in blood spots obtained at 7-10 d can be used to detect infants who currently present with jaundice, pale stools, and dark urine during the first 6 mo of life.

Delta 4-3-oxosteroid 5 beta-reductase deficiency: failure of ursodeoxycholic acid treatment and response to chenodeoxycholic acid plus cholic acid.

BACKGROUND--In some infants with liver disease, 3-oxo-delta 4 bile acids are the major bile acids in urine, a phenomenon attributed to reduced activity of the delta 4-3-oxosteroid 5 beta-reductase required for synthesis of chenodeoxycholic acid and cholic acid. These patients form a heterogeneous group. Many have a known cause of hepatic dysfunction and plasma concentrations of chenodeoxycholic acid and cholic acid that are actually greater than those of the 3-oxo-delta 4 bile acids. It is unlikely that these patients have a primary genetic deficiency of the 5 beta-reductase enzyme. AIMS--To document the bile acid profile, clinical phenotype, and response to treatment of an infant with cholestasis, increased plasma concentrations of 3-oxo-delta 4 bile acids, low plasma concentrations of chenodeoxycholic acid and cholic acid, and no other identifiable cause of liver disease. PATIENTS--This infant was compared with normal infants and infants with cholestasis of known cause. METHODS--Analysis of bile acids by liquid secondary ionisation mass spectrometry and gas chromatography-mass spectrometry. RESULTS--The plasma bile acid profile of the patient was unique. She had chronic cholestatic liver disease associated with malabsorption of vitamins D and E and a normal gamma-glutamyltranspeptidase when the transaminases were increased. The liver disease failed to improve with ursodeoxycholic acid but responded to a combination of chenodeoxycholic acid and cholic acid. CONCLUSION--Treatment of primary 5 beta-reductase deficiency requires the use of bile acids that inhibit cholesterol 7 alpha-hydroxylase.

Analysis by matrix assisted laser desorption/ ionisation‐time of flight mass spectrometry of the post‐translational modifications of α1‐antitrypsin isoforms separated by two‐ dimensional polyacrylamide gel electrophoresis

The state of protein glycosylation in terms of occupation of potential N‐linked glycosylation sites (macroheterogeneity) and type of glycosylation at that site (microheterogeneity) is important when investigating the consequences of aberrant glycosylation in the pathophysiology of disease. Protocols have been developed to permit characterisation of the site‐specific glycosylation of individual isoforms of glycoproteins after separation by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and analysis of the peptide mixture by peptide mass fingerprinting using matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry (MALDI‐TOF). High resolution of the individual isoforms of α1‐ antitrypsin was achieved by using narrow range (4.5–5.5) pI strips. The individual isoforms were then subjected to sequential digestion with a recombinant N‐glycanase followed by a protease. Using this strategy it was possible not only to increase the coverage of the amino acid sequence but also to monitor the occupancy of all three putative N‐linked glycosylation sites. Glycans were enzymatically released from α1‐antitrypsin which had been separated in gels formed with a low percentage of bis‐acrylamide cross‐linker and analysed. Profiles of the N‐linked glycans of the individual isoforms of α1‐antitrypsin were obtained by MALDI‐TOF.

Congenital disorders of glycosylation type I leads to altered processing of N-linked glycans, as well as underglycosylation

The N-linked glycans on transferrin and alpha(1)-antitrypsin from patients with congenital disorders of glycosylation type I have increased fucosylation and branching relative to normal controls. The elevated levels of monofucosylated biantennary glycans are probably due to increased alpha-(1-->6) fucosylation. The presence of bi- and trifucosylated triantennary and tetra-antennary glycans indicated that peripheral alpha-(1-->3), as well as core alpha-(1-->6), fucosylation is increased. Altered processing was observed on both the fully and underglycosylated glycoforms.

A case of the carbohydrate-deficient glycoprotein syndrome type 1 (CDGS type 1) with normal phosphomannomutase activity

The carbohydrate-deficient glycoprotein syndrome (CDGS) is a group of disorders characterized biochemically by abnormal glycosylation of serum and cellular glycoproteins. It has been classified into four forms on the basis of the isoelectric focusing pattern of serum transferrin and differences in clinical presentation. A deficiency of phosphomannomutase (PMM) has been reported in most patients with type 1. Seven of our eight CDGS patients, classified clinically as type 1, were shown to have a deficiency of phosphomannomutase in their fibroblast or lymphoblastoid cells (0.04-0.2 nmol/min per mg, compared with a control range of 1.0-2.1 nmol/min per mg). The eighth patient, who had many clinical features of the severe neonatal form of CDGS type 1, but lacked definite signs of CNS and ocular involvement, had a normal phosphomannomutase activity in his fibroblasts. There were approximately equal amounts of disialo- and tetrasialotransferrin and only a trace amount of asialotransferrin in the serum and ascitic fluid of this patient. The disialo- and tetrasialotransferrin isoforms were purified by ion-exchange chromatography and analysed by SDS-PAGE. The disialotransferrin had a lower molecular mass than the tetrasialotransferrin, consistent with the absence of an N-linked glycan. The N-linked glycans released enzymically from both isoforms consisted exclusively of disialylated biantennary chains, suggesting that disialotransferrin results from underglycosylation, as in the PMM-deficient CDGS type 1 patients. It is concluded that the clinical and biochemical phenotype in CDGS type 1 can result from more than one basic defect.

Ataxia associated with increased plasma concentrations of pristanic acid, phytanic acid and C27 bile acids but normal fibroblast branched-chain fatty acid oxidation.

SummaryInvestigations of peroxisomal function were undertaken in an 8-year-old girl who developed motor difficulties at the age of 3.5 years and went on to develop a progressive ataxia and dysarthria. There were no other neurological abnormalities and she was of normal intelligence. Analysis of plasma very long-chain fatty acids revealed a normal C26 concentration and normal C24/C22 and C26/C22 ratios. Analysis of branched-chain fatty acids showed an elevated plasma phytanic acid concentration of 60 µmol/L (normal<15) and a considerably elevated pristanic acid concentration of 50 µmol/L (normal<2). Plasma concentrations of the C27 bile acids 3α,7α-dihydroxycholestanoic acid (DHCA) and 3α,7α,12α-trihydroxycholestanoic acid (THCA) and of the C29-dicarboxylic acid were also increased. We postulated that these results might be due to deficiency of the peroxisomal branched-chain acyl-CoA oxidase, but when oxidation of branched-chain fatty acids was studied in cultured skin fibroblasts it was found to be normal. Alternative explanations for the accumulation of branched-chain substrates for peroxisomalβ-oxidation are discussed. Treatment with a low-phytanic acid diet arrested the progression of the ataxia and led to a slight improvement.