Incorporating second-tier genetic screening for multiple acyl-CoA dehydrogenase deficiency

Objective For clinically understanding of glutaric acidemia type Ⅰ (GA-Ⅰ), the relationship of the clinical manifestations and the serum levels of free carnitine (C0), glutarylcarnitine (C5DC), and capryloylcarnitine (C8) as well as the urinary levels of glutaric acid and mevalonolactone of patients with GA-Ⅰ were studied. Methods From December 2007 to April 2016, a total of 62 patients with GA-Ⅰ (GA-Ⅰ group) and 62 healthy children (control group) were included. Tandem mass spectrometry was used to measure the blood levels of C0, C5DC, and C8, while, gas chromatography-mass spectrometry was used to measure the urine levels of glutaric acid and mevalonolactone. Results Total 62 cases, 36 males and 26 females, median age 1.2 years (range from 27 days to 9.3 years) were enrolled. Clinical features included macrocephaly, dyskinesia, seizures, growth and mental retardation, abnormal muscular tension, as well as feeding difficulties. The median levels of C5DC, C5DC/C8, glutaric acid, mevalonolactone were 0.90 μmol/L, 24.25, 936.30 mmol/mol creatinine, and 141.5 mmol/mol creatinine respectively, which were higher than those of the control group (all P<0.01). While the median level of C0 was between the reference value, and the median level of C8 was lower than the control group (0.04 vs 0.10 μmol/L). Cranial magnetic resonance image (MRI) had been carried out in 34 patients. Multiple and symmetrical abnormal signal in white matter and basal ganglia, widening of the sylvian fissure, expansion of cerebro-spinal fluid spaces anterior to the temporal lobes were cardinal signs of GA-Ⅰ. Conclusions Patients with GA-Ⅰ present with complex and variable clinical features, mainly including abnormal increase of head circumference, retarded motor and language development, repeated convulsions and characteristic abnormalities in brain MRI. Significant increase in the blood levels of C5DC and C5DC/C8 and in urine level of glutaricacid and mevalonolactonemay are specific to the diagnosis of GA-Ⅰ. For mild cases, repeated tests and genomic mutation analysis are necessary. (Chin J Endocrinol Metab, 2017, 33: 730-734) Key words: Glutaric acidemia Ⅰ; Glutarylcarnitine; Glutaric acid; Tandem mass spectrometry; Gas chromatography-mass spectrometry

Since 2001, tandem mass spectrometry was introduced for newborn screening by two Taiwan national newborn screening centers: Taipei Institute of Pathology and Chinese Foundation of Health, in Taiwan, respectively. A total of 24 metabolic disorders, including 6 amino acidopathies, 8 organic acid disorders and 9 fatty acid disorders, etc., were screened and the newborns with positive screening result were referred to Taipei Veterans General Hospital for confirmatory diagnosis. Until Dec 2014, a total of 1,391,583 newborns have been screened by these two newborn screening centers. The overall incidence of inborn errors of metabolism identified by tandem mass was approximately 1/6,000. The most common inborn errors were defects of phenylalanine metabolism (phenylketonuria: 1/60,000; 6-pyruvoyltertrahydropterin synthase (PTPS): 1/110,000). Maple syrup urine disease was the second most common amino acidopathy (1/100,000) and, significantly, most MSUD patients (75%) belonged to the Austronesian aboriginal tribes of southern Taiwan. The most frequently detected organic acid disorder was 3-methylcrotonyl-CoA carboxylase deficiency (1/35,000). Glutaric aciduria type 1 and methylmalonic academia were the second most common organic acid disorders (1/100,000 for each disease). The incidence of fatty acid disorder is relatively less than most western countries. Out of them, carnitine transport defect was the most common fatty acid disease (1/100,000). In this report we will present this large population study of tandem mass newborn screening in Chinese population.

Glutaric aciduria type 1 (GA1) is an organic aciduria inherited in an autosomal recessive pattern, with an occurrence rate of one in 100,000. It is caused by a deficiency of the enzyme glutaryl-CoA dehydrogenase (GCDH), encoded by the GCDH gene on chromosome 19. It is an important enzyme in the catabolism of amino acids such as tryptophan, lysine, and hydroxylysine. Its deficiency leads to the accumulation of organic acids such as glutaric acid and 3-hydroxyglutaric acid, which interfere with cerebral energy metabolism and cause neurological symptoms. Here, we discuss the case of a six-month-old male child who presented withstatus epilepticus following an eight-day history of fever. The child was started on anti-epileptics. Initially, the child was on non-invasive ventilation and was later intubated and taken on a mechanical ventilator. Amagnetic resonance imaging (MRI) scan of the brain was performed, and the findings suggested GA1. The child was started on carnitine after samples were sent for tandem mass spectrometry (TMS) and urine gas chromatography-mass spectrometry (GC/MS), which came out to be positive for GA1. Despite the timely intervention, the child did not survive. Most cases exhibit movement disorders, with many presenting in acute encephalitic crises. Additionally, a significant portion of patients experience an insidious onset of the disease. An MRI of the brain shows widened Sylvian fissures in the majority of cases.Treatment of GA1 includes dietary modifications, including a low-lysine diet and administering carnitine. Early diagnosis and management result in decreased mortality and morbidity, which underscores the need for newborn screening.

Lipid storage myopathies (LSMs) are characterized pathologically by the accumulation of lipid droplets in muscle fibers due to impaired cellular lipid metabolism. The purpose of this study was to determine etiologies and genetic mutations associated with LSMs in ethnic Han Taiwanese. The usefulness of the blood acylcarnitine (AC) profile for diagnosing LSMs in adult patients was also investigated. Nine patients were diagnosed with late-onset LSMs following a review of muscle biopsies and medical records and were recruited retrospectively. Genetic studies were performed to detect mutations in the SLC22A5 for primary carnitine deficiency, PNPLA2 for neutral lipid storage disease with myopathy, ABHD5 for neutral lipid storage disease with ichthyosis, ETFDH for multiple acyl-CoA dehydrogenation deficiency (MADD), and CPT2 for carnitine palmitoyltransferase II deficiency. Blood AC levels were measured by tandem mass spectrometry. The mutation c.250G>A in ETFDH was detected in seven (78%) patients, six of whom were homozygous for the variant. Patients with ETFDH mutations had elevated blood levels of ACs ranging from C8 to C16 species, a pattern consistent with MADD. ETFDH c.250G>A mutation is common in Taiwanese patients with late-onset LSMs. The blood AC profile is a sensitive biochemical marker for diagnosing MADD arising from ETFDH mutations in adults.

Multiple acyl-CoA dehydrogenase deficiency (MADD; also known as glutaric aciduria type II) is an ultra-rare inborn error of metabolism (IEM). The disorder is not included in the Dutch newborn blood spot (NBS) screening program due to a lack of evidence for sufficient health gain upon early detection. Complicating factors concern the limited knowledge on the natural history, disease severity prediction and monitoring of the spectrum of MADD patients, and the absence of systematic evidence of an effective treatment for severely affected patients. MADD is also an exemplary IEM that can escape identification due to nonspecific symptoms and unexpected childhood death. In this thesis, we combined reviews of the literature, with experimental research and studies on clinical outcome in patients. In part I, we present the IEMs that are associated with unexpected death in early childhood, and how their detection through acylcarnitine profile analysis can be improved. We recommend that every child participates in a population NBS program, even after death. In part II, we describe that functional studies in patient fibroblasts can predict the MADD phenotype, we propose a clinical monitoring system, and we describe the efficacy and safety of D,L-3-hydroxybutyrate treatment in severe MADD. The results of this thesis can guide clinicians in their care of MADD patients and their families, and can also support decision-makers in their aims to improve NBS programs and facilitate access to novel treatments for (ultra-)rare diseases.

Enzymatic evaluation of glutaric acidemia type 1 by an in vitro probe assay of acylcarnitine profiling using fibroblasts and electrospray ionization/tandem mass spectrometry (MS/MS)

Genetic and biochemical study in a patient with glutaric acidemia type I.

Molecular analysis of Cypriot patients with Glutaric aciduria type I: Identification of two novel mutations

Background: The present study aims at two tier analysis for diagnosis and confirmation of organic acidemia using simultaneous analysis of dried blood spot (DBS) carnitine profile by tandem mass spectrometry (TMS) and urine metabolic analysis on gas chromatography mass spectrometry (GC/MS). Material and Methods: This cross sectional study included 348 DBS samples and their dried filter paper urine sample came for routine and emergency screening of inborn errors of metabolism (IEM). All DBS samples were anlysed for amino acid and acylcarnitine profile derivatized on TMS. All these cases were simultaneously tested for urine organic acid pattern on GC/MS which involved urease pretreatment, deproteinisation followed by derivatisation. Confirmation of IEM was based on the two tier approach containing the blood and urine metabolic pattern as recommended by panel of American College of Medical Genetics (ACMG). Results: 17 cases were screened positive for IEM analysed separately for amino acid /acylcarnitine profile and urine organic acid profile. However the two tier testing screened and confirmed 8 cases of organic acidemia within 36 hours of receipt of sample which included 2 cases each of maple syrup urine disease (MSUD), glutaric acidemia Type I (GA I), and ?-ketothiolase deficiency (BKT) and one each case of multiple carboxylase deficiency (MCD) and propionic acidemia (PA). Conclusion: The simultaneous two tier metabolome testing for screening of IEM is more comprehensive, cost effective, delivering confirmatory results with significantly reduced turnaround time, thus reducing recall rate by eliminating false-positive results and helps prevent unnecessary anxiety of parents.

Glutaryl carnitine (C5DC) in dried blood spots is used as a biomarker for glutaric aciduria type 1 (GA-1) screening. C5DC, however, is the only screening marker for this condition, and various pathological conditions may interfere with C5DC metabolism. Recently, C5DC elevation has been reported in cases of renal insufficiency. Five patients who were positive for GA-1 on newborn screening with tandem mass spectrometry between September 2012 and March 2015 at Kobe University Hospital were enrolled in this study. GA-1 was not confirmed on urinary organic acids analysis in any of the patients. C5DC decreased immediately in four patients, but one patient, who had high C5DC for at least 4 months, was diagnosed with bilateral renal hypoplasia. In the case of persistently elevated C5DC, renal insufficiency should be considered as a differential diagnosis.

Challenges in translating metabolomic screening into clinical practice

Next generation sequencing as a follow-up test in an expanded newborn screening programme

We identified three different point mutations in the glutaryl-CoA dehydrogenase (GCDH) gene in two unrelated Japanese patients with glutaric aciduria type I (GA-I). One patient was a homozygote for Arg355His and the other a compound heterozygote for Ser305Leu and Met339Val. Arg355His and Met339Val are mutations hitherto undescribed, and all three mutations are predicted to alter the secondary structure of GCDH. Molecular analysis is useful for definite diagnosis and/or prenatal diagnosis of GA-I.

Two brothers with dystonia and slight MRI changes in the basal ganglia had normal urinary glutaric acid excretion, but slightly increased 3-hydroxyglutarate and conjugated glutarate excretions. Both siblings have high residual glutaryl-CoA dehydrogenase activity, and are compound heterozygotes for two mutations - R227P and V400M reported to be disease-causing in patients with glutaric aciduria type I.

Quantification of 3-hydroxyglutaric acid in urine, plasma, cerebrospinal fluid and amniotic fluid by stable-isotope dilution negative chemical ionization gas chromatography–mass spectrometry