Tandem Mass Spectrometry Newborn Screening Research Articles

Partial Biotinidase Deficiency Revealed Imbalances in Acylcarnitines Profile at Tandem Mass Spectrometry Newborn Screening

Biotinidase (BTD) deficiency is an autosomal recessive inherited neurocutaneous disorder. BTD recycles the vitamin biotin, a coenzyme essential for the function of four biotin-dependent carboxylases, including propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and acetyl-CoA carboxylase. Due to deficient activities of the carboxylases, BTD deficiency is also recognized as late-onset multiple carboxylase deficiency and is associated with secondary alterations in the metabolism of amino acids, carbohydrates, and fatty acids. BTD deficiency can be classified as “profound”, with less than 10% of mean normal activity, and as “partial” with 10–30% of mean normal activity. Newborn screening (NBS) of BTD deficiency is performed in most countries and is able to detect both variants. Moreover, mild metabolic alterations related to carboxylase deficiency in profound BTD deficiency could result and possibly be revealed in the metabolic profile by tandem mass spectrometry (MS/MS) NBS. Here, we report the case of a newborn female infant with an initial suspected BTD deficiency at the NBS test, finally confirmed as a partial variant by molecular testing. Although BTD deficiency was partial, interestingly her metabolic profile at birth and during the follow-up tests revealed, for the first time, alterations in specific acylcarnitines as a possible result of the deficient activity of biotin-dependent carboxylases.

Primary and maternal 3‐methylcrotonyl‐CoA carboxylase deficiency: insights from the Israel newborn screening program

3-Methylcrotonyl-CoA carboxylase deficiency (3MCCD) is an inborn error of leucine catabolism. Tandem mass spectrometry newborn screening (NBS) programs worldwide confirmed 3MCCD to be the most common organic aciduria and a relatively benign disorder with favorable outcome. In addition, several asymptomatic 3MCCD mothers were initially identified following abnormal screening of their healthy babies and were appropriately termed maternal 3MCCD. This is a retrospective study that summarizes all the clinical, biochemical, and genetic data collected by questionnaires of all 3MCCD individuals that were identified by the extended Israeli NBS program since its introduction in 2009 including maternal 3MCCD cases. A total of 36 3MCCD subjects were diagnosed within the 50-month study period; 16 were classified primary and 20 maternal cases. Four additional 3MCCD individuals were identified following sibling screening. All maternal 3MCCD cases were asymptomatic except for one mother who manifested childhood hypotonia. Most of the primary 3MCCD individuals were asymptomatic except for two whose condition was also complicated by severe prematurity. Initial dried blood spot (DBS) free carnitine was significantly lower in neonates born to 3MCCD mothers compared with newborns with primary 3MCCD (p = 0.0009). Most of the mutations identified in the MCCC1 and MCCC2 genes were missense, five of them were novel. Maternal 3MCCD is more common than previously thought and its presence may be initially indicated by low DBS free carnitine levels. Our findings provide additional confirmation of the benign nature of 3MCCD and we suggest to exclude this disorder from NBS programs.

At $1,000, is genomic sequencing clinically useful in newborns?

At $1,000, is genomic sequencing clinically useful in newborns?

NEUROLOGIC MANIFESTATION OF ORGANIC ACADEMIA

Inborn errors of organic acid metabolism are relatively recently recognized diseases with a wide spectrum of clinical signs and symptoms: ranging from asymptomatic, normal appearing children to death during first few days of life.In my presentation I will try to explain some of the most common clinical presentation of these disorder with stress on neurologic findings. Organic acidemia usually have three clinical manifestations Severe neonatal form, Intermittent late-onset form and chronic progressive form. Recurrent coma, The main feature of these disorders is due to accumulation of toxic metabolites in Central Nervous system with direct effect on the function, while chronic accumulation of these materials may interfere with CNS development or cerebral metabolism leading to developmental delay.Severe neonatal formsFollowing a symptom free interval of a few days from birth, poor sucking and difficult feeding appears in the newborn, followed by unexplained and progressive coma. Seizures may appear during the course of the disease and EEG may show a burst-suppression pattern. During this stage most infants have axial hypotonia with peripheral dystonia, choreoathetosis, episodic opisthotonus and some repetitive bicycling and boxing movements.Associated biochemical abnormalities including metabolic acidosis, ketonuria and hyperammonemia also is usually present. The overall short-term prognosis with recent advances in medical care is improving. But later in life acute intercurrent episodes triggered by a stress often occur, which can be occasionally fatal.bulging fontanelle and cerebral edema may mimic CNS infection in these babies.Intermittent late-onset formsRecurrent attacks of coma or lethargy with ataxia can occur in childhood or even in adolescence or adulthood. These episodes may be frequent, though in between these the child is entirely normal. These attacks are precipitated by conditions that enhance protein catabolism (trauma, infection etc).Sometimes these episodes can lead to death or severe sequel. Seizure disorder is one of these sequels which is generalized in type with myoclonic seizure in infancy and childhood and later tonic-clonic and atypical absence seizures predominate. Also many of the survivors have acute or progressive extra pyramidal syndrome due to bilateral necrosis of basal ganglia.Chronic progressive formsNon specific Developmental delay, hypotonia, muscular weakness, microcephaly and seizures are rarely the only revealing signs in organic acidemia without any acute presentation.Seizures may become refractory to Anti Epileptic Drugs. In addition many asymptomatic or minimally symptomatic infants have been identified during tandem mass spectrometry newborn screening program. Cognitive deterioration associated with movement disorder such as dystonia or chorea may be caused by any form of organic aciduria.

Transcobalamin (TC) deficiency and newborn screening

Dear Editor, We would like to update the readership about transcobalamin (TC) deficiency and newborn screening. In a recent article we described two siblings with TC deficiency (Prasad et al. 2008). The older child was clinically symptomatic with pancytopenia and his sister was diagnosed at birth by mutation analysis. Both children were homozygous for a R399X (c.1195C>T) mutation. Both children are now doing well on oral Vitamin B12 (Cyanocobalamin 10 mg and 5 mg once daily respectively). Propionylcarnitine concentrations were not elevated in the newborn screening cards from the proband and sister, however as indicated in Prasad et al. 2008, the analyses were performed on specimens retrieved from storage after 7 years and 5 years, respectively. The mother has delivered another son who is clinically asymptomatic at age 5 months but had a positive newborn screen for propionic/methylmalonic acidemia with an elevated C3 at 6.59 μM (0.68-3.9) and C3/C200.33. The Ontario provincial newborn screening program considers a result positive for a baby 0.27 and C3 >4.0 μM or the C3/C2>0.23 and C3 >5.5 μM. Hematological parameters showed low hemoglobin at 145 (150–200 g/L) and neutrophils at 4.1 (6–26 )×10*9/L. Platelets were normal at 425 (150–400)×10*9/L and homocysteine was 18.1 (5–12/ L) mildly elevated. Oral Vitamin B12 1 mg daily (Cyanocobalamin) was initiated based on the positive newborn screening results and the diagnosis of TC deficiency was confirmed by mutation analysis. In a paper by Ratschmann et al. 2009, TC deficiency was diagnosed in one of the siblings at birth by measuring TC protein in the umbilical cord however it was not mentioned if newborn screening was done on that affected baby. Our experience indicates that infants with TC deficiency may be identified by C3 elevation on tandem mass spectrometry newborn screening and that TC deficiency should be kept in differential diagnosis when elevated C3 is encountered.

Mountain States Genetics Regional Collaborative Center's Metabolic Newborn Screening Long-Term Follow-Up Study: A collaborative multi-site approach to newborn screening outcomes research

PurposeThis article presents the rationale and design of the Mountain States Genetics Regional Collaborative Center's Metabolic Newborn Screening Long-Term Follow-up Study. MethodsThis study is a collaboration of multi-site metabolic providers throughout the Mountain States region investigating the long-term outcomes of individuals with metabolic conditions detected by newborn screening. ResultsThe Mountain States Genetics Regional Collaborative Center's Metabolic Consortium developed disease-specific care plans that included baseline and follow-up datasets for all metabolic disorders detected by both standard and tandem mass spectrometry newborn screening. ConclusionThese disease-specific care plans are used at multiple metabolic clinics throughout the Mountain States region. The shared datasets consisting of both performance and outcome indicators will be used to explore questions related to the treatment and outcome of these rare metabolic disorders. They will be used to assess the impact of newborn screening by comparing those individuals detected by newborn screening with those individuals diagnosed clinically, therefore allowing the systematic investigation of factors that impact long-term outcome.

Urgent metabolic service improves survival in long‐chain 3‐hydroxyacyl‐CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a fatty acid oxidation disorder with especially high mortality and uncertain long-term outcome. The aim of the study was to analyze the influence of diagnostic approach on survival in 59 affected children. Referral to a metabolic center was replaced over time by urine/blood testing in centralized metabolic laboratory (selective screening) and by pilot tandem mass spectrometry newborn screening (NBS). Molecular analysis revealed the prevalent mutation in the HADHA gene in all 58 examined cases. Twenty patients died. The number of detections and number of deaths were respectively 9 and 4 (44%) in the patients recognized by differential diagnosis, 28 and 9 (32%) - by selective screening, and 11 and 1 (9%) - by NBS. In 80% of cases the death occurred before or within 3 weeks from the identification. Urgent and active metabolic service remarkably influenced the surviving. The current age of 39 survivors is 0.5 to 23 yrs (mean 7.2 yrs). The disease frequency estimated on the patients number was 1: 115 450, whereas in the pilot NBS - 1: 109 750 (658 492 neonates tested). Interestingly, the phenylalanine level in asymptomatic neonates frequently exceeded the cut-off values. 1) Urgent metabolic intervention decreases mortality of LCHAD-deficient patients, but the prognosis is still uncertain. 2) Emergent metabolic reporting and service are crucial also for the survival of neonates detected by NBS. 3) The nationwide selective screening appeared efficient in LCHADD detection in the country. 4) Transient mild hyperphenylalaninaemia may occur in LCHAD-deficient newborns.

Expanded newborn screening: reducing harm, assessing benefit

Achieving the goals of newborn screening is, as for any screening, a balancing act: getting the maximum benefit from screening while producing the minimum harm. The advent of "expanded" newborn screening, with a large number of disorders detectable using a single test, has also posed problems, not new, but now more obvious. One is the finding of many more cases by screening, the extra cases being largely patients who have attenuated phenotypes and may remain asymptomatic for many years, even throughout life. These may or may not require active management in the short term, but do need lifelong awareness. Additionally, disorders have been included that are now thought benign or largely so. Babies risk being unnecessarily medicalized. Assessing outcome has also proved difficult because of the rarity of some disorders and the impracticality of randomized controlled trials. The requirements for valid studies include the need for case definitions, comparable comparison groups and probably assessment on a whole-population basis. An Australia-wide study of tandem mass spectrometry newborn screening involving 2 million screened and unscreened babies has demonstrated benefits overall to screened patients at age 6years. The study was too small to provide conclusions for individual disorders other than for medium-chain acyl-CoA dehydrogenase deficiency.

Tandem Mass Spectrometry Newborn Screening and Its Impact on Inborn Errors of Metabolism

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Molecular analysis of a presymptomatic case of carnitine palmitoyl transferase І (CPT I) deficiency detected by tandem mass spectrometry newborn screening in Japan

Carnitine palmitoyl transferase І (CPT І) deficiency is a rare disorder of long-chain fatty acid oxidation. It is one of the metabolic diseases detectable by tandem mass spectrometry. We report herein a presymptomatic CPT І deficiency detected in a Japanese female newborn by tandem mass spectrometry newborn screening. A mutation analysis of the CPT1A gene revealed two novel mutations, p.R446X and p.G719D.

Economic Evaluation of Tandem Mass Spectrometry Newborn Screening in Australia

The goal was to investigate the cost-effectiveness of tandem mass spectrometry screening for the detection of inborn metabolic errors in an Australian setting. Cost-effectiveness analysis from the health service perspective was undertaken on the basis of registry data for affected individuals. The intervention group was contrasted with both a contemporaneous group in nonscreening states and a historical cohort. The registry covers all individuals identified in Australia between 1994 and 2002. Main outcome measures were the total net cost of screening, the cost of treatment, life-years saved, and deaths averted. The total net cost of testing was estimated to be A$218 000 per 100 000 infants. Medical costs incurred by the intervention group exceeded those for the control group by A$131 000 per 100 000 infants. The number of life-years saved per 100 000 infants screened was 32.378 life-years per 100 000 infants through an expected mortality rate reduction of 0.738 deaths per 100 000 infants. The cost per death averted was estimated to be A$472 913 and the cost per life-year saved was estimated to be A$10 779, which compare favorably with existing cost-effectiveness standards. This conclusion is particularly robust because conservative assumptions were made throughout, because of data limitations. Sensitivity analyses suggested that this result was relatively robust to adjustment of model parameters. Tandem mass spectrometry screening for conditions caused by rare errors of metabolism is likely to be a cost-effective intervention in Australia.

Glutaric aciduria type I: outcome following detection by newborn screening

Glutaric aciduria type I (GA I), a cerebral organic acidaemia with the potential for severe neurological consequences, can now be detected by tandem mass spectrometry newborn screening. Early detection with implementation of careful management strategies appears to lessen the likelihood of neurological damage. We assessed the outcome in all 10 GA I patients detected in New South Wales during the last decade. Three patients were detected clinically and 7 by newborn screening. Diagnosis was confirmed by detection of significantly elevated urinary 3-hydroxybutyrate and glutarate in urine, isolated elevation of glutarylcarnitine in plasma, typical clinical and MRI findings in several, and mutation analysis or enzyme analysis on cultured skin fibroblasts in 4 cases. The birth frequency was 1:90,000. Following diagnosis, treatment was initiated in all children with oral carnitine (100 mg/kg per day) and a low-protein diet supplemented with a lysine-free, low-tryptophan amino acid formula. Disability was assessed in fields of motor, cognitive and speech development and scored according to Kyllerman. Clinically diagnosed patients were all symptomatic, with severity scores (out of 9) of 3, 5 and 9. Six of seven patients detected by newborn screening are asymptomatic, 4 being aged 2-6 years. One patient had a severe decompensation at 7 months, despite full management advice and treatment, and later died. Our data support previous findings that early diagnosis reduces neurological complications, but show that even with early diagnosis and careful management severe complications may ensue in some.

Non-Ketotic Hyperglycinemia is usually not detectable by tandem mass spectrometry newborn screening

Diagnosis of Non-Ketotic Hyperglycinemia by MSMS newborn screening might benefit patients with post-neonatal presentation. We screened 733,527 babies over eight years, and nine babies were subsequently diagnosed with NKHG. Two had newborn glycine levels above our cut-off and presented within 72 h. The remaining patients could not have been diagnosed by newborn screening without an unacceptably high recall rate. We conclude that babies with NKHG are not usually identifiable by current newborn screening strategies.

Isobutyryl-CoA dehydrogenase deficiency with a novel ACAD8 gene mutation detected by tandem mass spectrometry newborn screening

Isobutyryl-CoA dehydrogenase (IBD) is an enzyme involved in the catabolism of the branched-chain amino acid valine. IBD deficiency is a very rare metabolic disorder, whereby only a few cases have been reported thus far. Recently, we observed a Korean newborn boy with elevated concentration of C4-acylcarnitine detected by tandem mass spectrometry neonatal screening. Subsequent sequencing analysis of ACAD8 gene revealed that he was compound heterozygous for a novel mutation involving the first codon (c.3G>T; Met1Ile) and a previously reported mutation (c.1000C>T; Arg334Cys). To the best of our knowledge, this is the first confirmed case of IBD deficiency based on biochemical and genetic studies not only in Korea but also in Asia.

Variations in IBD (ACAD8) in Children with Elevated C4-Carnitine Detected by Tandem Mass Spectrometry Newborn Screening

The isobutyryl-CoA dehydrogenase (IBD) enzyme is involved in the degradation of valine. IBD deficiency was first reported in 1998 and subsequent genetic investigations identified acyl-CoA dehydrogenase (ACAD) 8, now IBD, as the gene responsible for IBD deficiency. Only three individuals homozygous or compound heterozygous for variations in the IBD gene have been reported. We present IBD deficiency in an additional four newborns with elevated C(4)-carnitine identified by tandem mass spectrometry (MS/MS) screening in Denmark and the United States. Three showed urinary excretions of isobutyryl-glycine, and in vitro probe analysis of fibroblasts from two newborns indicated enzymatic IBD defect. Molecular genetic analysis revealed seven new rare variations in the IBD gene (c.348C>A, c.400G>T, c.409G>A, c.455T>C, c.958G>A, c.1000C>T and c.1154G>A). Furthermore, sequence analysis of the short-chain acyl-CoA dehydrogenase (SCAD) gene revealed heterozygosity for the prevalent c.625G>A susceptibility variation in all newborns and in the first reported IBD patient. Functional studies in isolated mitochondria demonstrated that the IBD variations present in the Danish newborn (c.409G>A and c.958G>A) together with a previously published IBD variation (c.905G>A) disturbed protein folding and reduced the levels of correctly folded IBD tetramers. Accordingly, low/no IBD residual enzyme activity was detectable when the variant IBD proteins were overexpressed in Chang cells.

The tandem mass spectrometry newborn screening experience in North Carolina: 1997–2005

North Carolina (NC) was the first US state to initiate universal tandem mass spectrometry (MS/MS) newborn screening. This began as a statewide pilot project in 1997 to determine the incidence and feasibility of screening for fatty acid oxidation, organic acid and selected amino acid disorders. The MS/MS analyses were done by a commercial laboratory and all follow-up and confirmatory testing was performed through the NC Newborn Screening (NBS) Program. In April 1999, the NC NBS Laboratory began the MS/MS analyses in-house. Between 28 July 1997 and 28 July 2005, 944,078 infants were screened and 219 diagnoses were confirmed on newborns with elevated screening results, for an overall incidence of 1:4,300. Ninety-nine infants were identified with fatty acid oxidation disorders, 58 with organic acidaemias and 62 with aminoacidopathies. Medium-chain acyl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase deficiency and disorders of phenylalanine metabolism were the most common disorders detected. Identification of affected infants has allowed retrospective testing of other family members, resulting in an additional 16 diagnoses. Seven neonates died from complications of their metabolic disorders/prematurity despite timely MS/MS screening. In addition, there were six infants who were not identified by elevated NBS results but who presented with symptoms later in infancy. The NC MS/MS NBS Program uses a two-tier system, categorizing results as either 'borderline' or 'diagnostic' elevated, for both the cutoffs and follow-up protocol. Infants with an initial borderline result had only a repeat screen. Infants with a diagnostic or two borderline results were referred for confirmatory testing. The positive predictive value of the NC MS/MS NBS for those infants requiring confirmatory testing was 53% for 2003 and 2004. The success of the NC MS/MS NBS Program in identifying infants with metabolic disorders was dependent on a comprehensive follow-up protocol integrating the public health laboratory and the academic metabolic centres.

Falsely elevated C4‐carnitine as expression of glutamate formiminotransferase deficiency in tandem mass spectrometry newborn screening

Journal of Mass SpectrometryVolume 41, Issue 2 p. 263-265 JMS Letter Falsely elevated C4-carnitine as expression of glutamate formiminotransferase deficiency in tandem mass spectrometry newborn screening Sabrina Malvagia, Sabrina Malvagia Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorGiancarlo la Marca, Giancarlo la Marca Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorBruno Casetta, Bruno Casetta Applied Biosystems, Monza, ItalySearch for more papers by this authorSerena Gasperini, Serena Gasperini Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorElisabetta Pasquini, Elisabetta Pasquini Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorMaria Alice Donati, Maria Alice Donati Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorEnrico Zammarchi, Corresponding Author Enrico Zammarchi enrico.zammarchi@unifi.it Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalyDepartment of Pediatrics, Meyer Children's Hospital, Via Luca Giordano 13-50132 Florence, Italy.===Search for more papers by this author Sabrina Malvagia, Sabrina Malvagia Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorGiancarlo la Marca, Giancarlo la Marca Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorBruno Casetta, Bruno Casetta Applied Biosystems, Monza, ItalySearch for more papers by this authorSerena Gasperini, Serena Gasperini Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorElisabetta Pasquini, Elisabetta Pasquini Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorMaria Alice Donati, Maria Alice Donati Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalySearch for more papers by this authorEnrico Zammarchi, Corresponding Author Enrico Zammarchi enrico.zammarchi@unifi.it Department of Pediatrics, University of Florence, Meyer Children's Hospital. Via Luca Giordano 13-50132 Firenze, ItalyDepartment of Pediatrics, Meyer Children's Hospital, Via Luca Giordano 13-50132 Florence, Italy.===Search for more papers by this author First published: 18 January 2006 https://doi.org/10.1002/jms.964Citations: 26Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume41, Issue2February 2006Pages 263-265 RelatedInformation

Newborn screening for medium‐chain acyl‐CoA dehydrogenase deficiency: A global perspective

As judged by tandem mass spectrometry blood spot screening, the incidence of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is 1:14 600 (CI 95%: 1:13 500-1:15 900) in almost 8.2 million newborns worldwide and is 2- to-3 fold higher than that identified in the same populations after clinical presentation. In mass-screened newborn populations, the 985A>G (K329E) mutation accounts for 54-90% of disease alleles, with homozygotes representing about 47-80% of MCAD deficiency cases. Worldwide, octanoylcarnitine levels are an effective primary screen for MCAD deficiency in newborns. Newborns homozygous for the 985A < G mutation have higher octanoylcarnitine levels than do those compound heterozygous for 985A < G and those with other genotypes. Time of sampling after birth also significantly affects octanoylcarnitine levels in MCAD-deficient newborns. Tandem mass spectrometry newborn blood spot screening for MCAD deficiency is accurate and effective, reduces morbidity and mortality, and merits expansion to other populations worldwide.

Medium-chain acyl-CoA dehydrogenase deficiency: Genotype–biochemical phenotype correlations

The fatty acid oxidation disorder most commonly identified by tandem mass spectrometry newborn screening is the potentially fatal medium-chain acyl-CoA dehydrogenase deficiency (MCAD). In clinically presenting cases, 80% are homozygous for the common mutation, c.985A > G and 18% heterozygous. We screened 592,785 babies and identified 34 with MCAD, 17 homozygous for c.985A > G, 14 with one copy, and 3 with no copy. We sequenced the exons of 19 patients, the 17 carrying one or no copy of c.985A > G, and two with marginal findings, and examined correlations between groups of mutations and biochemical markers. We found two known or putative pathogenic mutations in 18 of the 19 patients. Two mutations appeared more than once: c.199T > C, not recorded in clinically presenting cases ( n = 4), and c.583G > A ( n = 2). Patients homozygous for c.985A > G had the highest levels of neonatal octanoylcarnitine, plasma octanoylcarnitine when asymptomatic, and urinary acylglycines. Compound heterozygotes of c.985A > G and other mutations had intermediate levels, and those without c.985A > G, or heterozygous for that and c.199T > C had the lowest levels of these analytes. There was overlap in all values. The c.985A > G and c.583G > A mutations appear to have functional effects towards the severe end of the spectrum, and the c.199T > C mutation a smaller effect, as has been previously postulated. If these results are confirmed and extended, this could influence the advice given to parents of babies with MCAD detected by newborn screening, and make management more specific. In the meantime, all MCAD patients identified by newborn screening have, by definition, a functional defect and require careful clinical management.

Enzymatic diagnosis of medium-chain acyl-CoA dehydrogenase deficiency by detecting 2-octenoyl-CoA production using high-performance liquid chromatography: A practical confirmatory test for tandem mass spectrometry newborn screening in Japan

Many of the previously described enzymatic assay methods for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency have been dependent upon the measurement of radioisotope-labeled co-products or reduction of electron acceptors. We have developed a direct assay method to detect 2-enoyl-CoA production using high-performance liquid chromatography (HPLC). Crude cell lysate prepared from lymphocytes were incubated with n-octanoyl-CoA and ferrocenium hexafluorophosphate. The detection of 2-octenoyl-CoA was significantly reproducible. We applied the assay to samples from four infants suspected to have MCAD deficiency by tandem mass spectrometry (MS/MS) newborn screening conducted in the Hiroshima area of Japan. Three of them were proved to have pathologically reduced residual enzyme activities, although they were associated with various clinical and biochemical phenotypes. In addition, another symptomatic Japanese patient and her presymptomatic sibling who were detected by MS/MS selective screening were successfully diagnosed by our enzymatic assay. These results indicate that the method can be a useful confirmatory test for MS/MS screening of MCAD deficiency.