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1.  Medium-Chain Acyl-CoA Dehydrogenase Deficiency in Gene-Targeted Mice  
PLoS Genetics  2005;1(2):e23.
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD−/−) by gene targeting in embryonic stem (ES) cells. The MCAD−/− mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD−/− mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD−/− pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation.
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common inherited disorders of metabolism. This defect in fatty acid oxidation can lead to severe and sometimes fatal disease, especially in young children because they are unable to tolerate a fasting episode. Metabolic complications include very low blood glucose concentrations and generation of toxic by-products. This disorder can result in sudden infant death. Using a process known as gene targeting in mouse embryonic stem cells, the authors have developed a mouse model with the same enzyme deficiency. This mouse model of MCAD deficiency develops many of the same disease characteristics found in affected children. The MCAD-deficient mouse model shows a high rate of newborn loss, intolerance to cold, and the characteristic biochemical changes in the blood, tissues, and urine that are very similar to those found in the human disease counterpart. The MCAD-deficient mouse model will allow researchers to better understand disease mechanisms so that new preventive measures or therapies can be developed.
PMCID: PMC1189074  PMID: 16121256
2.  Sudden unexpected infant death (SUDI) in a newborn due to medium chain acyl CoA dehydrogenase (MCAD) deficiency with an unusual severe genotype 
Medium chain acyl CoA dehydrogenase deficiency (MCAD) is the most common inborn error of fatty acid oxidation. This condition may lead to cellular energy shortage and cause severe clinical events such as hypoketotic hypoglycemia, Reye syndrome and sudden death. MCAD deficiency usually presents around three to six months of life, following catabolic stress as intercurrent infections or prolonged fasting, whilst neonatal-onset of the disease is quite rare. We report the case of an apparently healthy newborn who suddenly died at the third day of life, in which the diagnosis of MCAD deficiency was possible through peri-mortem blood-spot acylcarnitine analysis that showed very high concentrations of octanoylcarnitine. Genetic analysis at the ACADM locus confirmed the biochemical findings by demonstrating the presence in homozygosity of the frame-shift c.244dup1 (p.Trp82LeufsX23) mutation, a severe genotype that may explain the unusual and very early fatal outcome in this newborn. This report confirms that inborn errors of fatty acid oxidation represent one of the genetic causes of sudden unexpected deaths in infancy (SUDI) and underlines the importance to include systematically specific metabolic screening in any neonatal unexpected death.
PMCID: PMC3502270  PMID: 23095120
Medium chain acyl CoA dehydrogenase deficiency; Sudden unexpected deaths in Infancy; Sudden infant death syndrome; Fatty acid oxidation disorders
3.  A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns 
PLoS ONE  2009;4(7):e6449.
Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.
LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.
Principal Findings
VLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.
Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.
PMCID: PMC2715108  PMID: 19649258
4.  Risk stratification by residual enzyme activity after newborn screening for medium-chain acyl-CoA dehyrogenase deficiency: data from a cohort study 
Since the introduction of medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency in population newborn bloodspot screening (NBS) programs, subjects have been identified with variant ACADM (gene encoding MCAD enzyme) genotypes that have never been identified in clinically ascertained patients. It could be hypothesised that residual MCAD enzyme activity can contribute in risk stratification of subjects with variant ACADM genotypes.
We performed a retrospective cohort study of all patients identified upon population NBS for MCAD deficiency in the Netherlands between 2007–2010. Clinical, molecular, and enzymatic data were integrated.
Eighty-four patients from 76 families were identified. Twenty-two percent of the subjects had a variant ACADM genotype. In patients with classical ACADM genotypes, residual MCAD enzyme activity was significantly lower (median 0%, range 0-8%) when compared to subjects with variant ACADM genotypes (range 0-63%; 4 cases with 0%, remainder 20-63%). Patients with (fatal) neonatal presentations before diagnosis displayed residual MCAD enzyme activities <1%. After diagnosis and initiation of treatment, residual MCAD enzyme activities <10% were associated with an increased risk of hypoglycaemia and carnitine supplementation. The prevalence of MCAD deficiency upon screening was 1/8,750 (95% CI 1/7,210–1/11,130).
Determination of residual MCAD enzyme activity improves our understanding of variant ACADM genotypes and may contribute to risk stratification. Subjects with variant ACADM genotypes and residual MCAD enzyme activities <10% should be considered to have the same risks as patients with classical ACADM genotypes. Parental instructions and an emergency regimen will remain principles of the treatment in any type of MCAD deficiency, as the effect of intercurrent illness on residual MCAD enzyme activity remains uncertain. There are, however, arguments in favour of abandoning the general advice to avoid prolonged fasting in subjects with variant ACADM genotypes and >10% residual MCAD enzyme activity.
PMCID: PMC3543239  PMID: 22630369
Population newborn screening; Enzyme; Genotype; Prevalence
5.  Familial Occurrence of Systemic Mast Cell Activation Disease 
PLoS ONE  2013;8(9):e76241.
Systemic mast cell activation disease (MCAD) comprises disorders characterized by an enhanced release of mast cell mediators accompanied by accumulation of dysfunctional mast cells. Demonstration of familial clustering would be an important step towards defining the genetic contribution to the risk of systemic MCAD. The present study aimed to quantify familial aggregation for MCAD and to investigate the variability of clinical and molecular findings (e.g. somatic mutations in KIT) among affected family members in three selected pedigrees. Our data suggest that systemic MCAD pedigrees include more systemic MCAD cases than would be expected by chance, i.e., compared with the prevalence of MCAD in the general population. The prevalence of MCAD suspected by symptom self-report in first-degree relatives of patients with MCAD amounted to approximately 46%, compared to prevalence in the general German population of about 17% (p<0.0001). In three families with a high familial loading of MCAD, the subtype of MCAD and the severity of mediator-related symptoms varied between family members. In addition, genetic alterations detected in KIT were variable, and included mutations at position 816 of the amino acid sequence. In conclusion, our data provide evidence for common familial occurrence of MCAD. Our findings observed in the three pedigrees together with recent reports in the literature suggest that, in familial cases (i.e., in the majority of MCAD), mutated disease-related operator and/or regulator genes could be responsible for the development of somatic mutations in KIT and other proteins important for the regulation of mast cell activity. Accordingly, the immunohistochemically different subtypes of MCAD (i.e. mast cell activation syndrome and systemic mastocytosis) should be more accurately regarded as varying presentations of a common generic root process of mast cell dysfunction, than as distinct diseases.
PMCID: PMC3787002  PMID: 24098785
6.  In vitro and in vivo consequences of variant medium-chain acyl-CoA dehydrogenase genotypes 
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of the mitochondrial fatty acid oxidation, caused by mutations in the ACADM gene. Since the introduction of neonatal screening for MCAD deficiency, a subgroup of newborns have been identified with variant ACADM genotypes that had never been identified before in clinically ascertained patients. In vitro residual MCAD enzyme activity has been found to facilitate risk-stratification. In this study we integrated results of in vitro (residual MCAD enzyme activities) and in vivo (clinical fasting tolerance tests, and phenylpropionic acid loading tests) tests in this subgroup of newborns, defining the consequences of variant ACADM genotypes.
Enzyme analyses were performed in leukocytes with: hexanoyl-CoA (C6-CoA) +/− butyryl-CoA (C4-CoA), and phenylpropionyl-CoA (PP-CoA). In vitro studies were performed in 9 subjects with variant ACADM genotypes, in vivo functional tests in 6 of these subjects.
Enzyme analyses with C6-CoA, C6-CoA + C4-CoA, and PP-CoA identified significantly higher residual MCAD enzyme activities in subjects with variant ACADM genotypes when compared to patients with classical ACADM genotypes.
After prolonged fasting (range 15–18.5 hours) no hypoglycaemia was observed. Increasing concentrations of free fatty acids indicated lipolysis, and ketone body concentrations were sufficient for blood glucose concentrations in 5 out of 6 subjects. Phenylpropionic acid loading clearly demonstrated in vivo residual MCAD enzyme activity in all studied subjects.
Subjects with variant ACADM genotypes and residual MCAD enzyme activities >10% display residual MCAD enzyme activities in vitro and in vivo. Our findings support the hypothesis that the guidelines on maximal duration of fasting might be abandoned in subjects with residual MCAD enzyme activities >10% under normal conditions. An emergency regimen and parental instructions remain necessary in all subjects with MCAD deficiency, regardless of residual MCAD enzyme activity.
PMCID: PMC3610156  PMID: 23509891
ACADM; Enzyme; Genotype; Fasting; Phenylpropionic acid
7.  Prostaglandin D2-supplemented “functional eicosanoid testing and typing” assay with peripheral blood leukocytes as a new tool in the diagnosis of systemic mast cell activation disease: an explorative diagnostic study 
Systemic mast cell activation disease (MCAD) is characterized by an enhanced release of mast cell-derived mediators, including eicosanoids, which induce a broad spectrum of clinical symptoms. Accordingly, the diagnostic algorithm of MCAD presupposes the proof of increased mast cell mediator release, but only a few mediators are currently established as routine laboratory parameters. We thus initiated an explorative study to evaluate in vitro typing of individual eicosanoid pattern of peripheral blood leukocytes (PBLs) as a new diagnostic tool in MCAD.
Using the “functional eicosanoid testing and typing” (FET) assay, we investigated the balance (i.e. the complex pattern of formation, release and mutual interaction) of prostaglandin E2 (PGE2) and peptido-leukotrienes (pLT) release from PBLs of 22 MCAD patients and 20 healthy individuals. FET algorithms thereby consider both basal and arachidonic acid (AA)-, acetylsalicylic acid (ASA)-, and substance P (SP)-triggered release of PGE2 and pLT. The FET assay was further supplemented by analyzing prostaglandin D2 (PGD2), as mast cell-specific eicosanoid.
We observed marked PGE2-pLT imbalances for PBLs of MCAD patients, as indicated by a markedly enhanced mean FET value of 1.75 ± 0.356 (range: 1.14–2.36), compared to 0.53 ± 0.119 (range: 0.36-0.75) for healthy individuals. In addition, mean PGD2 release from PBLs of MCAD patients was significantly, 6.6-fold higher than from PBLs of healthy individuals (946 ± 302.2 pg/ml versus 142 ± 47.8 pg/ml; P < 0.001). In contrast to healthy individuals, PGD2 release from PBLs of MCAD patients was markedly triggered by SP (mean: 1896 ± 389.7 pg/ml; P < 0.001), whereas AA and ASA caused individually varying effects on both PGD2 and pLT release.
The new in-vitro FET assay, supplemented with analysis of PGD2, demonstrated that the individual patterns of eicosanoid release from PBLs can unambiguously distinguish MCAD patients from healthy individuals. Notably, in our analyses, the FET value and both basal and triggered PGD2 levels were not significantly affected by MCAD-specific medication. Thus, this approach may serve as an in-vitro diagnostic tool to estimate mast cell activity and to support individualized therapeutic decision processes for patients suffering from MCAD.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0213-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4283146  PMID: 25113638
Systemic mast cell activation disease; Eicosanoids; PGE2; PGD2; Peptido-leukotrienes; Functional eicosanoid testing and typing; Peripheral blood leukocytes; Substance P; Acetylsalicylic acid; Arachidonic acid
8.  Outcome of medium chain acyl-CoA dehydrogenase deficiency after diagnosis 
Archives of Disease in Childhood  1999;80(5):459-462.
BACKGROUND—Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inborn error of fatty acid metabolism. Undiagnosed, it has a mortality rate of 20-25%. Neonatal screening for the disorder is now possible but it is not known whether this would alter the prognosis.
OBJECTIVE—To investigate the outcome of MCAD deficiency after the diagnosis has been established.
METHOD—All patients with a proved diagnosis of MCAD deficiency attending one centre in a four year period were reviewed.
RESULTS—Forty one patients were identified. Follow up was for a median of 6.7 years (range, 9 months to 14 years). Nearly half of the patients were admitted to hospital with symptoms characteristic of MCAD deficiency before the correct diagnosis was made. After diagnosis, two patients were admitted to hospital with severe encephalopathy but there were no additional deaths or appreciable morbidity. There was a high incidence (about one fifth) of previous sibling deaths among the cohort.
CONCLUSIONS—Undiagnosed, MCAD deficiency results in considerable mortality and morbidity. However, current management improves outcome, supporting the view that the disorder should be included in newborn screening programmes.

PMCID: PMC1717923  PMID: 10208954
9.  Evidence for involvement of medium chain acyl-CoA dehydrogenase in the metabolism of phenylbutyrate 
Molecular genetics and metabolism  2012;107(4):684-689.
Sodium phenylbutyrate is used for treating urea cycle disorders, providing an alternative for ammonia excretion. Following conversion to its CoA ester, phenylbutyryl-CoA is postulated to undergo one round of β-oxidation to phenylacetyl-CoA, the active metabolite. Molecular modeling suggests that medium chain acyl-CoA dehydrogenase (MCAD; EC, a key enzyme in straight chain fatty acid β-oxidation, could utilize phenylbutyryl-CoA as substrate. Moreover, phenylpropionyl-CoA has been shown to be a substrate for MCAD and its intermediates accumulate in patients with MCAD deficiency. We have examined the involvement of MCAD and other acyl-CoA dehydrogenases (ACADs) in the metabolism of phenylbutyryl-CoA. Anaerobic titration of purified recombinant human MCAD with phenylbutyryl-CoA caused changes in the MCAD spectrum that are similar to those induced by octanoyl-CoA, its bona fide substrate, and unique to the development of the charge transfer ternary complex. The calculated apparent dissociation constant (KD app) for these substrates was 2.16 μM and 0.12 μM, respectively. The MCAD reductive and oxidative half reactions were monitored using the electron transfer flavoprotein (ETF) fluorescence reduction assay. The catalytic efficiency and the Km for phenylbutyryl-CoA were 0.2 mM−1· sec−1 and 5.3 μM compared to 4.0 mM−1· sec−1 and 2.8 μM for octanoyl-CoA. Extracts of wild type and MCAD-deficient lymphoblast cells were tested for the ability to reduce ETF using phenylbutyryl-CoA as substrate. While ETF reduction activity was detected in extracts of wild type cells, it was undetectable in extracts of cells deficient in MCAD. The results are consistent with MCAD playing a key role in phenylbutyrate metabolism.
PMCID: PMC3504130  PMID: 23141465
Buphenyl; Phenybutyrate; hyperammonemia; urea-cycle disorders; medium chain acyl-CoA dehydrogenase
10.  Hypoglycaemia related to inherited metabolic diseases in adults 
In non-diabetic adult patients, hypoglycaemia may be related to drugs, critical illness, cortisol or glucagon insufficiency, non-islet cell tumour, insulinoma, or it may be surreptitious. Nevertheless, some hypoglycaemic episodes remain unexplained, and inborn errors of metabolism (IEM) should be considered, particularly in cases of multisystemic involvement. In children, IEM are considered a differential diagnosis in cases of hypoglycaemia. In adulthood, IEM-related hypoglycaemia can persist in a previously diagnosed childhood disease. Hypoglycaemia may sometimes be a presenting sign of the IEM. Short stature, hepatomegaly, hypogonadism, dysmorphia or muscular symptoms are signs suggestive of IEM-related hypoglycaemia. In both adults and children, hypoglycaemia can be clinically classified according to its timing. Postprandial hypoglycaemia can be an indicator of either endogenous hyperinsulinism linked to non-insulinoma pancreatogenic hypoglycaemia syndrome (NIPHS, unknown incidence in adults) or very rarely, inherited fructose intolerance. Glucokinase-activating mutations (one family) are the only genetic disorder responsible for NIPH in adults that has been clearly identified so far. Exercise-induced hyperinsulinism is linked to an activating mutation of the monocarboxylate transporter 1 (one family). Fasting hypoglycaemia may be caused by IEM that were already diagnosed in childhood and persist into adulthood: glycogen storage disease (GSD) type I, III, 0, VI and IX; glucose transporter 2 deficiency; fatty acid oxidation; ketogenesis disorders; and gluconeogenesis disorders. Fasting hypoglycaemia in adulthood can also be a rare presenting sign of an IEM, especially in GSD type III, fatty acid oxidation [medium-chain acyl-CoA dehydrogenase (MCAD), ketogenesis disorders (3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) lyase deficiency, and gluconeogenesis disorders (fructose-1,6-biphosphatase deficiency)].
PMCID: PMC3458880  PMID: 22587661
Inborn errors of metabolism; Hypoglycaemia; Non-insulinoma pancreatogenic hypoglycaemia syndrome; Glycogen storage disease; Fatty acid oxidation disorder; Gluconeogenesis
11.  Clinical Relevance and Discriminatory Value of Elevated Liver Aminotransferase Levels for Dengue Severity 
Elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is prominent in acute dengue illness. The World Health Organization (WHO) 2009 dengue guidelines defined AST or ALT≥1000 units/liter (U/L) as a criterion for severe dengue. We aimed to assess the clinical relevance and discriminatory value of AST or ALT for dengue hemorrhagic fever (DHF) and severe dengue.
Methodology/Principal Findings
We retrospectively studied and classified polymerase chain reaction positive dengue patients from 2006 to 2008 treated at Tan Tock Seng Hospital, Singapore according to WHO 1997 and 2009 criteria for dengue severity. Of 690 dengue patients, 31% had DHF and 24% severe dengue. Elevated AST and ALT occurred in 86% and 46%, respectively. Seven had AST or ALT≥1000 U/L. None had acute liver failure but one patient died. Median AST and ALT values were significantly higher with increasing dengue severity by both WHO 1997 and 2009 criteria. However, they were poorly discriminatory between non-severe and severe dengue (e.g., AST area under the receiver operating characteristic [ROC] curve = 0.62; 95% confidence interval [CI]: 0.57–0.67) and between dengue fever (DF) and DHF (AST area under the ROC curve = 0.56; 95% CI: 0.52–0.61). There was significant overlap in AST and ALT values among patients with dengue with or without warning signs and severe dengue, and between those with DF and DHF.
Although aminotransferase levels increased in conjunction with dengue severity, AST or ALT values did not discriminate between DF and DHF or non-severe and severe dengue.
Author Summary
Dengue is a global public health problem, as the incidence of the disease has reached hyperendemic proportions in recent decades. Infection with dengue can cause acute, febrile illness or severe disease, which can lead to plasma leakage, bleeding, and organ impairment. One of the most prominent clinical characteristics of dengue patients is increased aspartate and alanine aminotransferase liver enzyme levels. The significance of this is uncertain, as it is transient in the majority of cases, and most patients recover uneventfully without liver damage. In this study, we characterized this phenomenon in the context of dengue severity and found that, although liver enzyme levels increased concurrently with dengue severity, they could not sufficiently discriminate between dengue fever and dengue hemorrhagic fever or between non-severe and severe dengue. Therefore clinicians may need to use other parameters to distinguish dengue severity in patients during early illness.
PMCID: PMC3367991  PMID: 22679523
12.  Direct Comparison of the BD Phoenix System with the MicroScan WalkAway System for Identification and Antimicrobial Susceptibility Testing of Enterobacteriaceae and Nonfermentative Gram-Negative Organisms▿  
Journal of Clinical Microbiology  2008;46(7):2327-2333.
The Phoenix automated microbiology system (BD Diagnostics, Sparks, MD) is designed for the rapid identification (ID) and antimicrobial susceptibility testing (AST) of clinically significant human bacterial pathogens. We evaluated the performance of the Phoenix instrument in comparison with that of the MicroScan WalkAway system (Dade Behring, West Sacramento, CA) in the ID and AST of gram-negative clinical strains and challenge isolates of Enterobacteriaceae (n = 150) and nonfermentative gram-negative bacilli (NFGNB; 45 clinical isolates and 8 challenge isolates). ID discrepancies were resolved with the API 20E and API 20NE conventional biochemical ID systems (bioMerieux, Durham, NC). The standard disk diffusion method was used to resolve discordant AST results. The overall percentages of agreement between the Phoenix ID results and the MicroScan results at the genus and species levels for clinical isolates of Enterobacteriaceae were 98.7 and 97.7%, respectively; following resolution with conventional biochemical testing, the accuracy of the Phoenix system was determined to be 100%. For NFGNB, the levels of agreement were 100 and 97.7%, respectively. Both systems incorrectly identified the majority of the uncommon nonfermentative nonpseudomonal challenge isolates recovered from cystic fibrosis patients; these isolates are not included in the databases of the respective systems. For AST of Enterobacteriaceae, the rate of complete agreement between the Phoenix results and the MicroScan results was 97%; the rates of very major, major, and minor errors were 0.3, 0.2, and 2.7%, respectively. For NFGNB, the rate of complete agreement between the Phoenix results and the MicroScan results was 89.1%; the rates of very major, major, and minor errors were 0, 0.5, and 7.7%, respectively. Following the confirmatory testing of nine clinical isolates initially screened by the MicroScan system as possible extended-spectrum-β-lactamase (ESBL)-producing organisms (seven Klebsiella pneumoniae isolates and two Escherichia coli isolates), complete agreement was achieved for eight isolates (one ESBL positive and seven negative); one false positive was obtained with the Phoenix instrument. The MicroScan system correctly detected the 10 ESBL challenge isolates, versus the 6 detected by the Phoenix system. Overall, there was good correlation between the Phoenix instrument and the MicroScan system for the ID and AST of Enterobacteriaceae and common NFGNB. The Phoenix system is a reliable method for the ID and AST of the majority of clinical strains encountered in the clinical microbiology laboratory. Until additional performance data are available, results for all Klebsiella pneumoniae or Klebsiella oxytoca and E. coli isolates screened and confirmed as ESBL producers by any automated system should be confirmed by alternate methods prior to the release of final results.
PMCID: PMC2446888  PMID: 18495856
13.  Screening for medium chain acyl-CoA dehydrogenase deficiency using electrospray ionisation tandem mass spectrometry 
Archives of Disease in Childhood  1998;79(2):109-115.
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) 2168control 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.

PMCID: PMC1717662  PMID: 9797589
14.  Evaluation of newborn screening for medium chain acyl-CoA dehydrogenase deficiency in 275 000 babies 
OBJECTIVE—To evaluate newborn screening by tandem mass spectrometry for detection of medium chain acyl-CoA dehydrogenase (MCAD) deficiency, a fatty acid oxidation disorder with significant mortality in undiagnosed patients.
DESIGN—The following were studied: (a) 13 clinically detected MCAD deficient subjects, most homozygous for the common A985G mutation, whose newborn screening sample was available; (b) 275 653 consecutive neonates undergoing routine newborn screening. Screened infants with blood octanoylcarnitine levels ⩾ 1 µmol/l were analysed for the A985G mutation, had analysis of plasma and repeat blood spot acylcarnitines and urinary organic acids, and had fibroblast fatty acid oxidation or acylcarnitine studies.
RESULT—Twelve of the 13 patients later diagnosed clinically had newborn octanoylcarnitine levels > 2.3 µmol/l. Twenty three screened babies had initial octanoylcarnitine levels ⩾ 1 µmol/l. Eleven of 12 babies with persistent abnormalities had metabolite and/or enzyme studies indicating MCAD deficiency. Only four were homozygous for the A985G mutation, the remainder carrying one copy.
CONCLUSIONS—Most patients with symptomatic MCAD deficiency could be detected by newborn screening. Infants actually detected had a lower frequency of A985G alleles than clinically diagnosed cases and may have a lower risk of becoming symptomatic.

PMCID: PMC1721303  PMID: 11517203
15.  Isolated middle cerebral artery dissection: a systematic review 
Acute stroke can be missed in the emergency department, particularly in younger patients and in those with more vague symptoms such as headache or dizziness. Cervicocephalic dissections are one group of etiologies for acute stroke in the young. While cervicocephalic dissections are not uncommon in clinical practice, isolated middle cerebral artery dissection (MCAD) has been rarely reported as a cause for stroke. We sought to review the clinical implications and pathophysiology of an isolated MCAD. We searched the medical literature for isolated MCAD in clinical stroke patients using MEDLINE, HighWire, and Google Scholar databases from 1966 to 2013 using the keywords ‘middle cerebral artery dissection,’ ‘intracerebral artery dissection,’ and ‘middle cerebral artery dissection stroke.’ We reviewed cases to learn various characteristics of isolated MCAD. A total of 61 cases (62.3% male, mean age 44.16 ± 19.17 years) were reviewed from 54 publications. Most cases were reported from Asian countries (78.7%). Ischemic strokes were more common than hemorrhagic strokes (68.9%). Digital subtraction angiography was the most common imaging modality used to diagnose isolated MCAD (75.4%). Surgery was the preferred form of therapeutic intervention (39.3%). Males (n = 27/48, p = 0.0008) and those who presented with only ischemic syndromes (n = 22/48, p = 0.0009) had significantly higher rates of favorable outcome. Isolated MCAD is a rare disease that can contribute to the stroke burden of young patients. Further studies are needed to better characterize optimal treatment strategies and define outcomes for this rare condition.
PMCID: PMC4272800  PMID: 25593617
Dissection; Intracranial; Middle cerebral artery; Stroke
16.  Genome-wide association study identifies genetic variants in GOT1 determining serum aspartate aminotransferase levels 
Journal of human genetics  2011;56(11):801-805.
We carried out a genome-wide association study of serum aspartate aminotransferase (AST) activity in 866 Amish participants of the Heredity and Phenotype Intervention Heart Study and identified significant association of AST activity with a cluster of single nucleotide polymorphisms located on chromosome 10q24.1 (peak association was rs17109512; P=2.80E-14), in the vicinity of GOT1, the gene encoding cytosolic AST (cAST). Sequencing of GOT1 revealed an in-frame deletion of three nucleic acids encoding asparagine at position 389 c.1165_1167delAAC (p.Asn389del) in the gene. Deletion carriers had significantly lower AST activity levels compared with homozygotes for the common allele (mean±s.d.: 10.0±2.8 versus 18.8±5.2 U l−1; P=2.80E-14). Further genotyping of the deletion in other Amish samples (n=1932) identified an additional 20 carriers (minor allele frequency (MAF)=0.0052). The deletion was not detected in 647 outbred Caucasians. Asn at codon 389 is conserved among known mammalian cASTs. In vitro transient transfection of wild-type and mutant cAST indicated that mutant cAST protein was barely detectable in the cells. Furthermore, even after correction for cAST expression, mutant cAST had markedly diminished enzymatic activity. Remarkably, we did not find any association between the deletion and metabolic traits including serum fasting glucose or insulin, fasting and post-meal lipids, inflammatory markers, or sub-clinical markers of cardiovascular disease. In conclusion, we discovered a rare in-frame deletion in GOT1 gene, which inactivates cAST enzyme in the Old Order Amish. This finding will help us to understand structure and function of the enzyme and would be useful for predicting serum AST levels.
PMCID: PMC3608855  PMID: 21900944
Amish; AST; gene; GOT1; mutation
17.  The Domain-Specific and Temperature-Dependent Protein Misfolding Phenotype of Variant Medium-Chain acyl-CoA Dehydrogenase 
PLoS ONE  2014;9(4):e93852.
The implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-CoA dehydrogenase deficiency (MCADD) caused by mutations in the ACADM gene. However, the disease is still potentially fatal. Missense induced MCADD is a protein misfolding disease with a molecular loss-of-function phenotype. Here we established a comprehensive experimental setup to analyze the structural consequences of eight ACADM missense mutations (p.Ala52Val, p.Tyr67His, p.Tyr158His, p.Arg206Cys, p.Asp266Gly, p.Lys329Glu, p.Arg334Lys, p.Arg413Ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. With fever being the crucial risk factor for metabolic decompensation of patients with MCADD, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. Based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in MCADD depends on the structural region that is affected by missense-induced conformational changes with the central β-domain being particularly prone to structural derangement and destabilization. Since systematic classification of conformational derangements induced by ACADM mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.Lys329Glu (K304E), the classical severe mutation, and p.Tyr67His (Y42H), discussed to be mild. Experiments assessing the impact of thermal stress revealed that mutations in the ACADM gene lower the temperature threshold at which MCAD loss-of-function occurs. Consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the MCAD enzyme explaining the life-threatening clinical courses observed during fever episodes. Early and aggressive antipyretic treatment thus may be life-saving in patients suffering from MCADD.
PMCID: PMC3981736  PMID: 24718418
18.  Molecular basis of medium chain acyl-coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation. 
Journal of Clinical Investigation  1990;86(3):1000-1003.
We sequenced polymerase chain reaction (PCR)-amplified variant medium chain acyl-CoA dehydrogenase (MCAD) cDNAs in cultured fibroblasts from three MCAD-deficient patients. In all three patients, an A to G transition was identified at position 985 of the coding region. Since no appropriate restriction sites for detecting this point mutation were found, we devised a PCR method that amplifies an 87-bp fragment from position 955. In the 5' primer encompassing positions 955 to 984, A-981 was artificially substituted with C. With the presence of C-981 and G-985, an Nco I restriction site is introduced in the mutant copies. When cDNA or genomic DNA from fibroblasts of nine MCAD-deficient patients were tested with this method, the copies from all of them completely cleaved into two shorter fragments by Nco I, indicating their homozygosity for the A----G-985 transition. In contrast, the copies from all eight controls remained intact. Thus, this A----G-985 transition is the single prevalent mutation causing MCAD deficiency, a highly unusual feature for any genetic disorder. The PCR/Nco I digestion method is suitable for the diagnosis of MCAD deficiency.
PMCID: PMC296821  PMID: 2394825
19.  Rapid detection of medium chain acyl-CoA dehydrogenase gene mutations by non-radioactive, single strand conformation polymorphism minigels. 
Journal of Medical Genetics  1994;31(7):551-554.
Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inherited metabolic disorder affecting fatty acid beta oxidation. Identification of carriers is important since the disease can be fatal and is readily treatable once diagnosed. Twelve molecular defects have been identified in the MCAD gene; however, a single highly prevalent mutation, A985G, accounts for > 90% of mutant alleles in the white population. In order to facilitate the molecular diagnosis of MCAD deficiency, oligonucleotide primers were designed to amplify the exon regions encompassing the 12 mutations enzymatically, and PCR products were then screened with a single strand conformation polymorphism (SSCP) based method. Minigels were used allowing much faster run times, and silver staining was used after gel electrophoresis to eliminate the need for radioisotopic labelling strategies. Our non-radioactive, minigel SSCP approach showed that normals can be readily distinguished from heterozygotes and homozygotes for all three of the 12 known MCAD mutations which were detected in our sampling of 48 persons. In addition, each band pattern is characteristic for a specific mutation, including those mapping in the same PCR product like A985G and T1124C. When necessary, the molecular defect was confirmed using either restriction enzyme digestion of PCR products or by direct DNA sequence analysis or both. This rapid, non-radioactive approach can become routine for molecular diagnosis of MCAD deficiency and other genetic disorders.
PMCID: PMC1049978  PMID: 7966191
20.  Rhabdomyolysis and acute encephalopathy in late onset medium chain acyl-CoA dehydrogenase deficiency. 
A previously asymptomatic 30 year old man presented with rhabdomyolysis, muscle weakness, and acute encephalopathy after strenuous exertion in the cold without adequate food intake. Serum and muscle carnitine concentrations were decreased. Urinary excretion of carnitine and glycine esters and biochemical examination of skeletal muscle and fibroblasts led to the diagnosis of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. A point mutation at nucleotide position 985 of the coding region of the MCAD gene was found. The MCAD protein was synthesised in the patient's fibroblasts at a normal rate, but was unstable. In general, patients in whom the 985 point mutation has been established show much more severe clinical symptoms and other symptoms than those seen in this patient. The relation of the 985 point mutation and the residual MACD activity to the symptoms is not as straightforward as previously thought.
PMCID: PMC1073319  PMID: 7876853
21.  Functional Effects of Different Medium-Chain Acyl-CoA Dehydrogenase Genotypes and Identification of Asymptomatic Variants 
PLoS ONE  2012;7(9):e45110.
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the ACADM gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%–60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new ACADM genotypes associated with asymptomatic disease variants.
PMCID: PMC3444485  PMID: 23028790
22.  Regulatory role of mitochondria in oxidative stress and atherosclerosis 
World Journal of Cardiology  2010;2(6):150-159.
Mitochondrial physiology and biogenesis play a crucial role in the initiation and progression of cardiovascular disease following oxidative stress-induced damage such as atherosclerosis (AST). Dysfunctional mitochondria caused by an increase in mitochondrial reactive oxygen species (ROS) production, accumulation of mitochondrial DNA damage, and respiratory chain deficiency induces death of endothelial/smooth muscle cells and favors plaque formation/rupture via the regulation of mitochondrial biogenesis-related genes such as peroxisome proliferator-activated receptor γ coactivator (PGC-1), although more detailed mechanisms still need further study. Based on the effect of healthy mitochondria produced by mitochondrial biogenesis on decreasing ROS-mediated cell death and the recent finding that the regulation of PGC-1 involves mitochondrial fusion-related protein (mitofusin), we thus infer the regulatory role of mitochondrial fusion/fission balance in AST pathophysiology. In this review, the first section discusses the possible association between AST-inducing factors and the molecular regulatory mechanisms of mitochondrial biogenesis and dynamics, and explains the role of mitochondria-dependent regulation in cell apoptosis during AST development. Furthermore, nitric oxide has the Janus-faced effect by protecting vascular damage caused by AST while being a reactive nitrogen species (RNS) which act together with ROS to damage cells. Therefore, in the second section we discuss mitochondrial ATP-sensitive K+ channels, which regulate mitochondrial ion transport to maintain mitochondrial physiology, involved in the regulation of ROS/RNS production and their influence on AST/cardiovascular diseases (CVD). Through this review, we can further appreciate the multi-regulatory functions of the mitochondria involved in AST development. The understanding of these related mechanisms will benefit drug development in treating AST/CVD through targeted biofunctions of mitochondria.
PMCID: PMC2999054  PMID: 21160733
Apoptosis; Atherosclerosis; ATP-sensitive K+ channels; Free radical; Mitochondrial biogenesis
23.  Metabolic syndrome is linked to a mild elevation in liver aminotransferases in diabetic patients with undetectable non-alcoholic fatty liver disease by ultrasound 
Despite ongoing findings on the relationship between elevated levels of alanine and aspartate aminotransferases (ALT and AST) and metabolic syndrome (MetS), this association in diabetic patients without a known cause for liver enzymes elevation other than diabetes, per se, remains unclear. In this study, we aimed to assess the relationship between circulating liver enzymes and MetS in a relatively large sample of patients with diabetes.
A total of 670 diabetic patients, without known causes of hepatocellular injury, were enrolled. Patients with ultrasonographic signs of fatty liver disease were not included. Fasting blood samples were obtained and biochemical characteristics were measured. MetS was defined according to the international diabetes federation criteria.
Serum ALT and AST were significantly higher in patients with MetS (p < 0.001). High waist circumference and low HDL-cholesterol were significantly associated with elevated ALT (OR = 2.56 and 2.0, respectively) and AST (OR = 2.23 and 2.21, respectively). ALT and AST were significantly associated with MetS (OR = 2.17 and 2.31, respectively). These associations remained significant after multiple adjustments for age, sex, BMI, diabetes duration, HbA1c and medications. There was a significant (p < 0.01) positive association between the number of the MetS features and the level of ALT or AST.
In diabetic patients without ultrasonographic evidence of fatty liver, elevated aminotransferases are independently associated with MetS. Despite negative ultrasound results in diabetic patients with MetS, the serum level of liver aminotransferases may be elevated and should be more thoroughly monitored.
PMCID: PMC2987914  PMID: 21047423
24.  Protein misfolding is the molecular mechanism underlying MCADD identified in newborn screening 
Human Molecular Genetics  2009;18(9):1612-1623.
Newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) revealed a higher birth prevalence and genotypic variability than previously estimated, including numerous novel missense mutations in the ACADM gene. On average, these mutations are associated with milder biochemical phenotypes raising the question about their pathogenic relevance. In this study, we analyzed the impact of 10 ACADM mutations identified in NBS (A27V, Y42H, Y133H, R181C, R223G, D241G, K304E, R309K, I331T and R388S) on conformation, stability and enzyme kinetics of the corresponding proteins. Partial to total rescue of aggregation by co-overexpression of GroESL indicated protein misfolding. This was confirmed by accelerated thermal unfolding in all variants, as well as decreased proteolytic stability and accelerated thermal inactivation in most variants. Catalytic function varied from high residual activity to markedly decreased activity or substrate affinity. Mutations mapping to the β-domain of the protein predisposed to severe destabilization. In silico structural analyses of the affected amino acid residues revealed involvement in functionally relevant networks. Taken together, our results substantiate the hypothesis of protein misfolding with loss-of-function being the common molecular basis in MCADD. Moreover, considerable structural alterations in all analyzed variants do not support the view that novel mutations found in NBS bear a lower risk of metabolic decompensation than that associated with mutations detected in clinically ascertained patients. Finally, the detailed insight into how ACADM missense mutations induce loss of MCAD function may provide guidance for risk assessment and counseling of patients, and in future may assist delineation of novel pharmacological strategies.
PMCID: PMC2667288  PMID: 19224950
25.  Sequencing from Dried Blood Spots in Infants with “False Positive” Newborn Screen for MCAD Deficiency 
Newborn screening (NBS) for medium chain acyl-CoA dehydrogenase deficiency (MCADD), one of the most common disorders identified, uses measurement of octanoylcarnitine (C8) from dried blood spots. In the state of Ohio, as in many places, primary care providers, with or without consultation from a metabolic specialist, may perform “confirmatory testing”, with the final diagnostic decision returned to the state. Confirmatory testing may involve measurement of metabolites, enzyme analysis, mutation screening, or sequencing. We now report sequencing results for infants said to have “false positive” NBS results for MCAD deficiency, or who died before confirmatory testing could be performed.
Dried blood spots (DBS) were obtained from all 18 available NBS cards identified as “false positive” by NBS for the 3 year period after screening began in Ohio in 2003 (N=20, thus 2 had no DBS available), and from all 6 infants with abnormal screens who died before confirmatory testing could be obtained. DNA extracted from DBS was screened for the common c.985A>G mutation in exon 11 of the ACADM gene, using a specific restriction digest method, followed by sequencing of the 12 exons, intron-exon junctions, and several hundred base pairs of the 5′ untranslated region.
The NBS cut-off value for C8 used was 0.7 μmol/L. Sequencing of ACADM in six neonates with elevated C8 on NBS who died before confirmatory testing was obtained did not identify any significant variants in the coding region of the gene, suggesting that MCADD was not a contributing factor in these deaths. The mean C8 for the 18 surviving infants labeled as “False Positives” was 0.90 (95%CI 0.77-1.15), much lower than the mean value for confirmed cases. Ten of the 18 were premature births weighing <1200 g, the rest were normal sized and full term. Eight infants, mostly full term with appropriate birth weight, were heterozygous for the common c.985A>G mutation; one of those also has a novel sequence change identified in exon 9 that predicts a PRO to LEU change at residue 258 of the protein. Both the phase and any possible clinical significance of the variant are unknown, but several lines of evidence suggest that it could lead to protein malfunction. That child had an NBS C8 of 2.2, more than double the mean for the False Positive group. Unfortunately, the study design did not provide clinical outcome data, but the child is not known to have presented clinically by age 7 years.
These results suggest that sequencing of ACADM from dried blood spots can be one useful follow-up tool to provide accurate genetic counseling in the situation of an infant with elevated C8 on NBS who dies before confirmatory testing is obtained. Of surviving neonates, there appear to be two populations of infants with false positive NBS C8 values: 1) term AGA infants who are heterozygous for the common c.985A>G mutation, and, 2) premature infants, regardless of carrier status. The finding of two sequence variants in an infant reported to the state as not affected suggests the possibility that some infants with two mutations may be reported as normal at follow-up. State registries may wish to consider asking that metabolic specialists, who are most familiar with the variability of these rare disorders, be involved in the final diagnostic evaluation. Finally, providers may wish to consider ACADM sequencing, or other diagnostic testing, as part of the confirmatory evaluation for infants with NBS C8 concentrations that are significantly above the cut-off value, even if plasma and urine metabolites are not strikingly increased.
PMCID: PMC3676896  PMID: 23151387
Medium-chain acyl-CoA dehydrogenase deficiency; fatty acid oxidation disorders; newborn screening; dried blood spots

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