Unexpected elevation in valproic acid concentration and agranulocytosis in a patient with short-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an autosomal recessive metabolic disorder or condition of fatty acid ß-oxidation, caused by mutations in the gene encoding SCAD (ACADS). We report an infant with SCAD deficiency who unexpectedly exhibited an extremely high blood concentration of valproic acid (VPA) and agranulocytosis. CASE REPORT: An 8-month-old girl was diagnosed with West syndrome (infantile spasms), and VPA was administered at the standard level of 25 mg/kg/day. However, the blood concentration of VPA rose unexpectedly to 230 µg/mL (two- to three-fold higher than the expected value), and continued to remain relatively high even after the dosage was reduced (7 mg/kg/day, blood concentration of 88 µg/mL). Furthermore, she presented with a high-grade fever with agranulocytosis (neutrophil 231/µL). The abnormal pharmacokinetics and toxicity of VPA raised the suspicion of possible inborn errors of metabolism in the fatty acid ß-oxidation pathway. Blood tandem mass spectrometry revealed a transient elevation of C4, and urine gas chromatography-mass spectrometry revealed a continuous elevation of ethylmalonate. Finally, gene analysis revealed compound heterozygous mutations, c.625G > A (p.G209S) and c.1031A > G (p.E344G), in ACADS. CONCLUSION: VPA should be avoided if a patient is suspected to have inborn errors of ß-oxidation including SCAD deficiency.

The minor C-allele of the rs2014355 variant in ACADS gene is associated with exercise-induced increase in HDL cholesterol levels in Taiwanese adults.

ABSTRACT: We investigated the association between high-density lipoprotein cholesterol (HDL-C) and rs2014355 variant in the gene, short-chain acyl-coenzyme A dehydrogenase (ACADS) based on exercise habits.Data collected between 2008 and 2015 for individuals aged 30 to 70 years were available in the Taiwan Biobank (TWB) database. Backward stepwise linear regression was used to evaluate the associations of rs2014355 and exercise with HDL-C levels.We analyzed data of 5515 physically active and 4169 inactive biobank participants. The HDL-C concentrations were higher in the exercise compared to no exercise group (beta value, ß = 1.79856; P < .0001). We observed that the test for interaction was significant for the ACADS rs2014355 variant and exercise (P for interaction =.0412). Multivariate analyses showed significant association between TC+CC genotype and HDL-C in the exercise (ß = 1.09785; P value = .0146) compared to the no-exercise group (ß = -0.03754, P = .9154).In summary, the association between HDL-C and exercise differed significantly with respect to ACADS rs2014355 genotypes. Compared to the TT genotype, the TC+CC genotype together with exercise was associated with higher levels of HDL-C.

Medium-chain acyl-coenzyme A dehydrogenase deficiency: Six cases in the Chinese population.

BACKGROUND: Medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD) is a rare autosomal recessive disorder that affects the degradation of medium-chain fatty acids. Few cases of MCADD have been documented to date in mainland China. METHODS: Medium-chain acyl-coenzyme A dehydrogenase deficiency was diagnosed in six patients (three girls and three boys) from six unrelated Chinese families at ages ranging from 10 days to 3 years old. The diagnosis was confirmed by the identification of a primary biomarker of serum octanoyl-carnitine (C8) and genetic pathogenic mutations. RESULTS: Only two patients were admitted because of vomiting, diarrhea, myasthenia, and coma; the other four patients were diagnosed via the newborn screening process. Six mutations were found in acyl-CoA dehydrogenase medium chain (ACADM). One mutation (c.727C>T) was novel and the others (c.158G>A, c.387+1delG, c.449_452del, c.1045C>T, and c.1085G>A) have been previously reported. CONCLUSIONS: Six Chinese cases of MCADD were identified. One novel mutation was found. c.449_452del and c.1085G>A were common mutations in this study.

Selective Screening of Fatty Acids Oxidation Defects and Organic Acidemias by Liquid Chromatography/tandem Mass Spectrometry Acylcarnitine Analysis in Brazilian Patients.

BACKGROUND: Inborn errors of metabolism (IEM) are diseases which can lead to accumulation of toxic metabolites in the organism. AIM OF THE STUDY: To investigate, by selective screening, mitochondrial fatty acid oxidation defects (FAOD) and organic acidemias in Brazilian individuals with clinical suspicion of IEM. METHODS: A total of 7,268 individuals, from different regions of Brazil, had whole blood samples impregnated on filter paper which were submitted to the acylcarnitines analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) at the Medical Genetics Service of Hospital de Clínicas de Porto Alegre, Brazil, during July 2008-July 2016. RESULTS: Our results showed that 68 patients (0.93%) were diagnosed with FAOD (19 cases) and organic acidemias (49 cases). The most prevalent FAOD was multiple acyl CoA dehydrogenase deficiency (MADD), whereas glutaric type I and 3-OH-3-methylglutaric acidemias were the most frequent disorders of organic acid metabolism. Neurologic symptoms and metabolic acidosis were the most common clinical and laboratory features, whereas the average age of the patients at diagnosis was 2.3 years. CONCLUSIONS: Results demonstrated a high incidence of glutaric acidemia type I and 3-OH-3- methylglutaric acidemia in Brazil and an unexpectedly low incidence of FAOD, particularly medium-chain acyl-CoA dehydrogenase deficiency (MCADD).

Medium-chain acyl-CoA dehydrogenase deficiency: Two novel ACADM mutations identified in a retrospective screening.

Objective The aim of this study was to determine whether an expanded newborn screening programme, which is not yet available in Slovenia, would have detected the first two patients with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in the country. Two novel ACADM mutations are also described. Methods Both patients were diagnosed clinically; follow-up involved analysis of organic acids in urine, acylcarnitines in dried blood spots, and genetic analysis of ACADM. Cut-off values of acylcarnitines in newborns were established using analysis of 10,000 newborns in a pilot screening study. Results In both patients, analysis of the organic acids in urine showed a possible ß-oxidation defect, while the specific elevation of acylcarnitines confirmed MCAD deficiency. Subsequent genetic analysis confirmed the diagnosis; both patients were compound heterozygotes, each with one novel mutation (c.861 + 2T > C and c.527_533del). The results from a retrospective analysis of newborn screening cards clearly showed major elevations of MCAD-specific acylcarnitines in the patients. Conclusions An expanded newborn screening programme would be beneficial because it would have detected MCAD deficiency in both patients before the development of clinical signs. Our study also provides one of the first descriptions of ACADM mutations in Southeast Europe.

[Clinical, biochemical and gene mutation characteristics of short chain acyl-coenzyme A dehydrogenase deficiency by neonatal screening].

Objective: To investigate the incidence, clinical, biochemical and gene mutation characteristics of short chain acyl-coenzyme A dehydrogenase deficiency (SCADD). Method: From January, 2009 to October, 2015, a retrospective analysis of the urine organic acids and acyl-coenzyme A dehydrogenase (ACADS) gene mutation characteristics of patients diagnosed as SCADD by newborn screening using tandem mass spectrometry in Department of Genetics and Metabolism (Newborn screening Center of Zhejiang Province), Children's Hospital, Zhejiang University School of Medicine. Dietary guidance, life management and supplementation of L-carnitine were conducted, and growth and intelligence development were observed during follow-up among the SCADD patients. Result: A total of 1 430 024 neonates, seventeen cases were diagnosed with SCADD with an incidence of 1/84 117. All patients had no clinical symptoms, and intelligence and physical development were normal. Blood butylacyl-carnitine (C4) levels and the ratios increased, C4 0.713.14 µmol/L(reference value 0.03-0.48 µmol/L), C4/C2 0.07-0.23(reference value 0.01-0.04), C4/C3 0.65-2.04(reference value 0.05-0.39). Thirteen with increased urinary ethyl malonic acid (9.30-90.99 mg/g creatinine (reference value 0-6.20 mg/g creatinine )), one patient was accompanied by increased methyl succinic acid (12.33 mg/g creatinine(reference value 0-6.40 mg/g creatinine)), one subject with increased acetylglycine (3.52 mg/g creatinine(reference value 0-0.70 mg/g creatinine)). A total of 13 known mutations were detected in the ACADS gene, 1 homozygous mutation (c.1031A>G), the others are compound heterozygous mutations. One frameshift mutation (c.508_509delGC) and 12 missense mutations were detected. Common mutation were c. 1031A>G(35.3%), c. 164C>T(20.6%) and c. 991G>A(11.8%). SCADD in newborn screening program had no clinical symptoms and normal growth development after 8-42 months follow-up. Conclusion: Cases with SCADD had no clinical symptoms with an incidence of 1/84117. The c. 164C>T and c. 1031A>G may be the common mutations.

Screening of MCAD deficiency in Japan: 16years' experience of enzymatic and genetic evaluation.

BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a representative disorder of fatty acid oxidation and is one of the most prevalent inborn errors of metabolism among Caucasian populations. In Japan, however, it was as late as 2000 when the first patient was found, and enzymatic and genetic evaluation of MCAD deficiency began. METHODS: We measured octanoyl-CoA dehydrogenase activity in lymphocytes of symptomatic children and newborn screening (NBS)-positive subjects who showed elevated levels of C8-acylcarnitine in blood. The results were further confirmed by direct sequencing of the ACADM gene. RESULTS: The disease was diagnosed in 9 out of 18 symptomatic children. The affected patients showed residual activities from 0% to 3% of the normal average value, except for one patient with 10% activity. Concerning 50 NBS-positive subjects, 18 with enzymatic activities around 10% or lower and 14 with activities ranging from 13% to 30% were judged to be affected patients, and biallelic variants were detected in most of the cases tested. Newborns with higher enzymatic activities were estimated to be heterozygous carriers or healthy subjects, though biallelic variants were detected in 5 of them. Genetic analysis detected 22 kinds of variant alleles. The most prevalent was c.449_452delCTGA (p.T150Rfs), which was followed by c.50G>A (p.R17H), c.1085G>A (p.G362E), c.157C>T (p.R53C), and c.843A>T (p.R281S); these five variants accounted for approximately 60% of all the alleles examined. CONCLUSION: Our study has revealed the unique genetic backgrounds of MCAD deficiency among Japanese, based on the largest series of non-Caucasian cases. A continuous spectrum of severity was also observed in our series of NBS-positive cases, suggesting that it is essential for every nation and ethnic group to accumulate its own information on gene variants, together with their enzymatic evaluation, in order to establish an efficient NBS system for MCAD deficiency.

Quantitative acylcarnitine determination by UHPLC-MS/MS--Going beyond tandem MS acylcarnitine "profiles".

Tandem MS "profiling" of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.

A case report of short-chain acyl-CoA dehydrogenase deficiency (SCADD).

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare inherited mitochondrial fatty acid oxidation disorder associated with variations in the ACADS (Acyl-CoA dehydrogenase, C-2 to C-3 short chain) gene. SCADD has highly variable biochemical, genetic and clinical characteristics. Phenotypes vary from fatal metabolic decompensation to asymptomatic individuals. SUBJECT AND METHODS: A Romani boy presented at 3 days after birth with hypoglycaemia, hypotonia and respiratory pauses with brief generalized seizures. Afterwards the failure to thrive and developmental delay were present. Organic acids analysis with gas chromatography-mass spectrometry (GS/MS) in urine and acylcarnitines analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) in dried blood spot were measured. Deoxyribonucleic acid (DNA) was isolated from blood and polymerase chain reactions (PCRs) were performed for all exons. Sequence analysis of all exons and flanking intron sequences of ACADS gene was performed. RESULTS: Organic acids analysis revealed increased concentration of ethylmalonic acid. Acylcarnitines analysis showed increase of butyrylcarnitine, C4-carnitine. C4-carnitine was 3.5 times above the reference range (<0.68 µmol/L). Confirmation analysis for organic acids and acylcarnitine profile was performed on the second independent sample and showed the same pattern of increased metabolites. Sequence analysis revealed 3-bp deletion at position 310-312 in homozygous state (c.310_312delGAG). Mutation was previously described as pathogenic in heterozygous state, while it is in homozygous state in our patient. CONCLUSIONS: In our case clinical features of a patient, biochemical parameters and genetic data were consistent and showed definitely SCAD deficiency.

Oxidized phosphatidylcholines suggest oxidative stress in patients with medium-chain acyl-CoA dehydrogenase deficiency.

Inborn errors of metabolism encompass a large group of diseases caused by enzyme deficiencies and are therefore amenable to metabolomics investigations. Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is a defect in ß-oxidation of fatty acids, and is one of the most well understood disorders. We report here the use of liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics and targeted flow injection analysis-tandem mass spectrometry (FIA-TMS) that lead to discovery of novel compounds of oxidative stress. Dry blood spots of controls (n=25) and patient samples (n=25) were extracted by methanol/water (1/1, v/v) and these supernatants were analyzed by LC-MS method with detection by an Orbitrap Elite MS. Data were processed by XCMS and CAMERA followed by dimension reduction methods. Patients were clearly distinguished from controls in PCA. S-plot derived from OPLS-DA indicated that medium-chain acylcarnitines (octanoyl, decenoyl and decanoyl carnitines) as well as three phosphatidylcholines (PC(16:0,9:0(COOH))), PC(18:0,5:0(COOH)) and PC(16:0,8:0(COOH)) were important metabolites for differentiation between patients and healthy controls. In order to biologically validate these discriminatory molecules as indicators for oxidative stress, a second cohort of individuals were analyzed, including MCADD (n=25) and control (n=250) samples. These were measured by a modified newborn screening method using FIA-TMS (API 4000) in MRM mode. Calculated p-values for PC(16:0,9:0(COOH)), PC(18:0,5:0(COOH)) and PC(16:0,8:0(COOH)) were 1.927×10(-14), 2.391×10(-15) and 3.354×10(-15) respectively. These elevated oxidized phospholipids indeed show an increased presence of oxidative stress in MCADD patients as one of the pathophysiological mechanisms of the disease.

Evidencia del polimorfismo 511C>T en el gen de la acil-CoA deshidrogenasade cadena corta en Colombia / Evidence of 511C>T polymorphism in the short chain acyl-CoA dehydrogenase gene in Colombia / Evidência do polimorfismo 511C>T no gene da acil-CoA desidrogenase de cadeia curta na Colômbia

La acil-CoA deshidrogenasa de cadena corta (SCAD) cataliza la reacción inicial de la ß-oxidación de los ácidos grasos de cadena corta. La deficiencia hereditaria de SCAD ha sido reportada y han sido descritos pocos casos de la misma. El presente estudio pretendió determinar la posible presencia del polimorfismo 511C>T en Caldas (Colombia), debido a que las variantes 625G>A y 511C>T en el gen de la acil-CoA deshidrogenasa de cadena corta están presentes en el 14% de algunas poblaciones estudiadas, causando algunas veces su deficiencia. El presente estudio es descriptivo. Muestras de sangre de 300 voluntarios fueron estudiadas para el polimorfismo 511C>T mediante la técnica de polimorfismo de conformación de la cadena simple, utilizando ADN amplificado por reacción en cadena de la polimerasa. Los resultados fueron confirmados por secuenciación. El polimorfismo fue identificado en tres personas aparentemente sanas. Existe evidencia de la presencia del polimorfismo 511C>T en el gen de la acil-CoA en Colombia, lo que significa que algunas personas de esta población pueden tener riesgo de sufrir su deficiencia.

Short-chain acyl-CoA dehydrogenase (SCAD) catalyzes the initial reaction in short-chain fatty acid ß-oxidation. Hereditary SCAD deficiency has been reported and only few cases of this disorder have been described. The present study was conducted to determine the possible presence of the 511C>T variation in the short-chain acyl-CoA dehydrogenase gene in Caldas (Colombia), as the 625G>A and 511C>T variations are present in 14% of some studied populations causing its deficiency on some occasions. The present study is descriptive, blood samples of three hundred adult volunteers were tested for 511C>T polymorphism, analysing the polymerase chain reaction amplified cDNA, using a single-stranded conformation polymorphism assay. The results were confirmed by direct bidirectional cycle sequencing using DNA from the positive patients. The polymorphism was identified and confirmed in three healthy persons. This is evidence of the presence of 511C>T polymorphism in the short chain acyl-coA dehydrogenase gene in Colombia, which means that some people in these populations can be at risk of suffering SCAD deficiency.

A acil-CoA desidrogenase de cadeia curta (SCAD) catalisa a reação inicial da b-oxidação dos ácidos graxos de cadeia curta. Foi reportada a deficiência hereditária de SCAD e poucos casos da deficiência foram descritos. O presente trabalho quis determinar a possível presença do polimorfismo 511C>T em Caldas (Colômbia), devido a que as variantes 625G>A e 511C>T no gene da acil-CoA desidrogenase de cadeia curta estão presentes em 14% de algumas populações estudadas, produzindo algumas vezes sua deficiência. O presente estudo é descritivo. Amostras de sangue de 300 voluntários foram analisadas para o polimorfismo 511C>T através da técnica de polimorfismo de conformação da cadeia simples, utilizando DNA amplificado por reação em cadeia da polimerase. Os resultados foram confirmados por sequenciamento. O polimorfismo foi identificado em três pessoas aparentemente saudáveis. Existe evidência da presença do polimorfismo 511C>T no gene da acil-CoA na Colômbia, o que significa que algumas pessoas desta população correm o risco de sofrer sua deficiência.

Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin.

Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of l-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10years of expanded newborn screening using MS/MS in Wisconsin (2001-2011), 97 infants had elevated C5 values (≥0.44µmol/L), of whom five were Caucasian infants confirmed to have IVA. Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n=1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentration ≥0.44µmol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8-2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4-24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut-off would need to be as low as 0.20µmol/L to detect all infants homozygous for the ACADSB c.1165 A>G mutation. However, lowering the screening cut-off to 0.20 would also result in five "false positive" (non-homozygous) screening results in the Hmong population for every c.1165 A>G homozygote detected. Increasing the cut-off to 0.60µmol/L and requiring elevated C5/C2 (acetylcarnitine) and C5/C3 (propionylcarnitine) ratios to flag a screen as abnormal would reduce the number of infants screening positive, but would still result in an estimated 5 infants with SBCADD per year who would require follow-up and additional biochemical testing to distinguish between SBCADD and IVA diagnoses. Further research is needed to determine the clinical outcomes of SBCADD detected on newborn screening and the c.1165 A>G mutation before knowing whether the optimal screening cut-off would minimize true positives or false negatives for SBCADD associated with this mutation.

Normal rates of whole-body fat oxidation and gluconeogenesis after overnight fasting and moderate-intensity exercise in patients with medium-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Impairments in gluconeogenesis have been implicated in the pathophysiology of fasting hypoglycemia in medium-chain acyl-CoA dehydrogenase deficiency. However, whole body glucose and fat metabolism have never been studied in vivo. METHODS: Stable isotope methodology was applied to compare fat and glucose metabolism between four adult patients with MCADD and four matched controls both at rest and during 1.5 h of moderate-intensity exercise. Additionally, intramyocellular lipid and glycogen content and intramyocellular acylcarnitines were assessed in muscle biopsies collected prior to and immediately after cessation of exercise. RESULTS: At rest, plasma FFA turnover was significantly higher in patients with MCADD, whereas the plasma FFA concentrations did not differ between patients and controls. Blood glucose kinetics did not differ between groups both at rest and during exercise. Palmitate and FFA turnover, total fat and carbohydrate oxidation rates, the use of muscle glycogen and muscle derived triglycerides during exercise did not differ between patients and controls. Plasma FFA oxidation rates were significantly lower in patients at the latter stages of exercise. Free carnitine levels in muscle were lower in patients, whereas no differences were detected in muscle acetylcarnitine levels. CONCLUSIONS: Whole-body or skeletal muscle glucose and fat metabolism were not impaired in adult patients with MCADD. This implies that MCADD is not rate limiting for energy production under the conditions studied. In addition, patients with MCADD have a higher FFA turnover rate after overnight fasting, which may stimulate ectopic lipid deposition and, as such, make them more susceptible for developing insulin resistance.

Sequencing from dried blood spots in infants with "false positive" newborn screen for MCAD deficiency.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Macro-AST: misleading finding in an adolescent with MCAD-deficiency.

BACKGROUND: MCAD-deficiency is the most common inborn error of fatty acid oxidation now included in many newborn screening programms using MS/MS. During prolonged catabolic episodes, patients may suffer from metabolic decompensation with dysfunction of liver, skeletal- and heart muscle as well as brain. In anabolism, neither clinical symptoms nor biochemical signs of organ dysfunction occur. CASE PRESENTATION: We report a female patient with MCAD-deficiency in whom at the age of 11 years isolated AST-elevation was found without any clinical or biochemical signs of organ dysfunction. We showed by polyethylene glycol precipitation that macro-AST formation was responsible for this biochemical finding. AST was probably complexed with immunoglobulins possibly related to an allergic disposition. Macro-AST formation is not a special feature of MCAD-deficiency but rather a non-specific, coincidental finding which also occurs in healthy individuals. The general practitioner consulted by the patient before coming to our outpatient clinic for inborn errors of metabolism was worried that isolated AST-elevation indicated cell damage in MCAD-deficiency. He ordered further diagnostic tests like ultrasound, ECG and echocardiography without any pathology. CONCLUSION: In isolated AST-elevation, macro-AST has to be considered in order to avoid unnecessary, costly and invasive evaluation. This is not only true for healthy persons but for patients with chronic diseases like MCAD as well.

[Expanded newborn screening in the Region of Murcia, Spain. Three-years experience]. / Cribado neonatal ampliado en la Región de Murcia. Experiencia de tres años.

BACKGROUND AND OBJECTIVE: The early detection of inborn errors of metabolism by mass spectrometry allows expanding the traditional neonatal screening of phenylketonuria and congenital hypothyroidism to test for aminoacidopathies, fatty acid oxidation disorders and organic acid metabolic disorders. Cystic fibrosis and biotinidase deficiency screening is implemented in the Region of Murcia. The aim of the study is to describe our experience in the expanded neonatal screening and to define the prevalence of each of the metabolic disorders early detected. PATIENTS AND METHODS: Since March 2007 until October 2010, a total of 71,595 neonates were screened with this expanded program by mass spectrometry, fluoroimmunoassay or colorimetric methods. RESULTS: Thirty-eight patients (prevalence 1:1,884) were diagnosed of inborn errors of metabolism by mass spectrometry, 13 patients of cystic fibrosis (prevalence 1:5,507), 38 of congenital hypothyroidism (prevalence 1:1,884) and one of biotinidase deficiency. To date, the global frequency of inborn errors of metabolism is estimated to be 1:804. The positive predictive value for the results obtained by mass spectrometry was 20.25%. Two false negative patients were not identified (cystic fibrosis and methylmalonic aciduria patients) and 6 non neonatal patients were detected through expanded neonatal screening. CONCLUSIONS: Our data support the necessity of unifying the set of metabolic diseases to be screened in all Regions of Spain for early detection of a defined panel of inborn errors of metabolism and to provide every newborn the same opportunities to be early diagnosed.

Analysis of plasma ghrelin in patients with medium-chain acyl-CoA dehydrogenase deficiency and glutaric aciduria type II.

OBJECTIVE: Ghrelin requires a fatty acid modification for binding to the GH secretagogue receptor. Acylation of the Ser3 residue of ghrelin is essential for its biological activities. We hypothesized that acyl-CoA is the fatty acid substrate for ghrelin acylation. Because serum octanoyl-CoA levels are altered by fatty acid oxidation disorders, we examined circulating ghrelin levels in affected patients. MATERIALS AND METHODS: Blood levels of acyl (A) and des-acyl (D) forms of ghrelin and acylcarnitine of patients with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and glutaric aciduria type II (GA2) were measured. RESULTS: Plasma acyl ghrelin levels and A/D ratios increased in patients with MCAD deficiency or GA2 when compared with normal subjects. Reverse-phase HPLC confirmed that n-octanoylated ghrelin levels were elevated in these patients. CONCLUSION: Changing serum medium-chain acylcarnitine levels may affect circulating acyl ghrelin levels, suggesting that acyl-CoA is the substrate for ghrelin acylation.

Sudden death in medium chain acyl-coenzyme a dehydrogenase deficiency (MCADD) despite newborn screening.

INTRODUCTION: Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is the most frequent of the fatty acid oxidation disorders (FAOD), a group caused by defects in the mitochondrial B-oxidation of fatty acids. Fatty acid oxidation is critical in supplying energy during periods when glucose is limited or when energy needs are increased beyond the availability of glucose. In MCADD, this energy shortage can result in acute metabolic episodes or sudden death. The prevention of sudden death from MCADD served as the primary impetus to expand newborn screening. However, we have experienced sudden death in four children with MCADD despite their detection by newborn screening. The purpose of this report is to alert others to the danger of sudden death in MCADD even when it is detected by newborn screening, to identify the clinical symptoms that precede sudden death, and to examine the relationship between the newborn screening result and the risk for sudden death. METHODS: We describe these children and their metabolic findings with emphasis on their newborn screening octanoylcarnitine (C8) level, the primary marker for newborn detection of MCADD. We also performed a literature search of cases of sudden death in MCADD in which the clinical status preceding death is described. RESULTS: The newborn screening C8 levels in our four cases were markedly elevated, ranging from 8.4 to 24.8micromol/L (cut off<0.8micromol/L). Only two of the children were homozygous for the common c.985A>G MCAD mutation; the other two were heterozygous for this mutation. Similarly, among the eight reported cases which included MCAD genotypes, five were homozygous for the c.985A>G mutation, while two were heterozygous and one was homozygous for a splice site mutation. Vomiting 12-24h before sudden death was present in all four of our cases, and the review of reported cases of sudden death in MCADD disclosed vomiting as a frequent symptom. CONCLUSION: We suggest that in MCADD (1) a newborn screening C8 level of 6micromol/L or greater represents particular risk of sudden death; (2) that MCAD genotypes other than homozygosity for the c.985A>G mutation are also associated with sudden death; (3) that vomiting is a frequent symptom preceding sudden death; and (4) social support and medical follow-up of these families are crucial in reducing the occurrence of sudden death.

Medium-chain acyl-CoA dehydrogenase deficiency in Saudi Arabia: incidence, genotype, and preventive implications.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD), caused by mutated ACADM gene, is a potentially fatal fatty acid oxidation defect. Detection of MCADD is now part of tandem mass spectrometry (MS-MS)-based newborn screening programs worldwide. To date, more than 67 mutations have been reported to cause MCADD with a single allele, c.985A>G, being the most common in patients of northwestern European descent. In Saudi Arabia, the Newborn Screening Program, officially launched in 2005, screens for 16 disorders including MCADD. Over a period of 3 years, 237,812 newborns were screened; 13 were identified to have MCADD giving an incidence of 1:18,293. Since the introduction of MS-MS to our institution, however, a total of 30 patients were detected to have MCADD. These cases were either newborns, at high-risk family members, or clinically suspected. The C8-carnitine levels (median 3.31, range 0.81-16.33 µM) were clearly diagnostic in all analyzed samples. Sequencing ACADM in 20 DBS revealed two novel mutations: c.362C>T (p.T121I) and c.347G>A (p.C116Y) substitutions, neither of which were detected in 300 chromosomes from controls. Eighteen (90%) patients were homozygous for the T121I mutation and two (10%) were compound heterozygous (T121I/C116Y). Our molecular data lend further support to MS-MS biochemical screening for MCADD and provide evidence for the relatively high incidence of MCADD in the Arab population. The identification of a founder mutation for MCADD has important implications for the preventive screening programs not only in Saudi Arabia but potentially also in other countries in the region.