Yosuke Shigematsu

To minimize false-positive cases in newborn screening by tandem mass spectrometry in Japan, practical second-tier liquid chromatography-tandem mass spectrometry analyses have been developed using a multimode ODS column with a single set of mobile phases and different gradient elution programs specific to the analysis of acylcarnitines, acylglycines, amino acids, and organic acids. Most analyses were performed using underivatized samples, except for analysis of methylcitric acid, and careful conditioning of the column was necessary for analyses of organic acids. Our second-tier tests enabled us to measure many metabolites useful for detection of target disorders, including allo-isoleucine, homocysteine, methylmalonic acid, and methylcitric acid. We found that accumulation of 3-hydroxyglutaric acid was specific to glutaric acidemia type I and that the ratio of 3-hydroxyisovaleric acid to 3-hydroxyisovalerylcarnitine was useful to detect newborns of mothers with 3-methylcrotonyl-CoA carboxylase deficiency. Data from the analysis of short-chain acylcarnitine and acylglycine were useful for differential diagnosis in cases positive for C5-OH-acylcarnitine or C5-acylcarnitine.

Neonatal screening (NS) for methylmalonic acidemia uses propionylcarnitine (C3) as a primary index, which is insufficiently sensitive at detecting methylmalonic acidemia caused by defects in the adenosylcobalamin synthesis pathway. Moreover, homocystinuria from cystathionine β-synthase deficiency is screened by detecting hypermethioninemia, but methionine levels decrease in homocystinuria caused by defects in homocysteine remethylation. To establish NS detection of methylmalonic acidemia and homocystinuria of these subtypes, we evaluated the utility of indices (1) C3 ≥ 3.6 μmol/L and C3/acetylcarnitine (C2) ≥ 0.23, (2) C3/methionine ≥ 0.25, and (3) methionine < 10 μmol/L, by retrospectively applying them to NS data of 59,207 newborns. We found positive results in 116 subjects for index (1), 37 for (2), and 15 for (3). Second-tier tests revealed that for index 1, methylmalonate (MMA) was elevated in two cases, and MMA and total homocysteine (tHcy) were elevated in two cases; for index 2 that MMA was elevated in one case; and for index 3 that tHcy was elevated in one case. Though data were anonymized, two cases identified by index 1 had been diagnosed with maternal vitamin B₁₂ deficiency during NS. Methylene tetrahydrofolate reductase deficiency was confirmed for the case identified by index 3, which was examined because an elder sibling was affected by the same disease. Based on these data, a prospective NS study is underway.

Urea cycle disorders (UCDs) are inherited metabolic disorders that present with hyperammonemia, and cause significant mortality and morbidity in infants and children. These disorders are not well reported in the Indian population, due to lack of a thorough study of the clinical and molecular profile. We present data from two major metabolic centres in India, including 123 cases of various UCDs. The majority of them (72/123, 58%) presented in the neonatal period (before 30 days of age) with 88% on or before day 7 of life (classical presentation), and had a high mortality (64/72, 88%). Citrullinemia type 1 was the most common UCD, observed in 61/123 patients. Ornithine transcarbamylase (OTC) deficiency was the next most common, seen in 24 cases. Argininosuccinic aciduria was diagnosed in 20 cases. Deficiencies of arginase, N-acetylglutamate synthase, carbamoyl phosphate synthetase, citrin, and lysinuric protein intolerance were also observed. Molecular genetic analysis revealed two common ASS1 mutations: c.470G > A (p.Arg157His) and c.1168G > A (p.Gly390Arg) (36 of 55 tested patients). In addition, few recurrent point mutations in ASL gene, and a deletion of the whole OTC gene were also noted. A total of 24 novel mutations were observed in the various genes studied. We observed a poor clinical outcome with an overall all time mortality of 63% (70/110 cases with a known follow-up), and disability in 70% (28/40) among the survivors. Prenatal diagnosis was performed in 30 pregnancies in 25 families, including one pre-implantation genetic diagnosis. We report the occurrence of UCDs in India and the spectrum that may be different from the rest of the world. Citrullinemia type 1 was the most common UCD observed in the cohort. Increasing awareness amongst clinicians will improve outcomes through early diagnosis and timely treatment. Genetic diagnosis in the proband will enable prenatal/pre-implantation diagnosis in subsequent pregnancies.

Common use of pivalate‐generating antibiotics in newborns in Japan and low cutoff value of C5‐acylcarnitine (C5) to detect mild forms of isovaleric acidemia (IVA) led to 1,065 positive results from IVA screening among 146,000 newborns tested by tandem mass spectrometry over the last 3 years. Using our method to determine isovalerylglycine (IVG) levels in dried blood spots (DBS) as a second‐tier test with IVG cutoff value of 0.5 nmol/ml in DBS, one patient with severe IVA was identified, and no recall of the second DBS was needed. Retrospective analysis revealed that most patients with moderate to severe forms of IVA have decreased free‐carnitine levels shortly after birth and higher levels of IVG than those of C5, which suggests that this method is useful in evaluating the severity of IVA. Another second‐tier test, to measure methylmalonic acid (MMA) levels in DBS by gas chromatography/mass spectrometry (GC/MS), has been developed to overcome difficulties in screening methylmalonic aciduria (MMAU) and propionic acidemia. Methanol extract from DBS was dried and derivatized using N‐methyl‐N‐(tert‐butyldimethylsilyl)‐trifluoroacetamide. GC/MS was performed using splitless injection, electron‐impact ionization, and selected ion monitoring for data recording. MMAU patients had much higher DBS concentrations of MMA (24.2–321.9 nmol/ml) than control newborns (0.34 ± 0.11 nmol/ml). MMA measurement in DBS was thought to provide useful information about the severity of MMAU, as MMAU patients with high levels of MMA had decreased levels of free carnitine and mildly increased levels of propionylcarnitine.

The introduction of tandem mass spectrometry (MS/MS) has made it possible to screen for very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. To confirm the diagnosis in cases with an abnormal profile of blood acylcarnitines, we developed a new enzymatic assay method for determining dehydrogenase activity toward palmitoyl-CoA (C16:0) in lymphocytes. Using this method, the production of 2-hexadecenoyl-CoA (C16:1) by crude cell lysates can be directly quantified using high performance liquid chromatography (HPLC). We applied the assay to 7 myopathic patients, 7 hypoglycemic patients, and 2 presymptomatic newborns with elevated levels of tetradecenoylcarnitine (C14:1 AC) in blood, and found impaired VLCAD activity in all of the 7 myopathic patients and both of the 2 newborns. All of the 7 hypoglycemic patients had normal level of the enzyme activity. Results of the ACADVL gene analysis were in consistent with the enzymatic diagnosis. These results suggest that MS/MS-based screening for VLCAD deficiency using blood C14:1 AC as the indicator may show a considerably high false-positive rate in selective screening of symptomatic patients. Our practical enzymatic assay can be a useful test for the accurate diagnosis of VLCAD deficiency cases screened by MS/MS.

We experienced a 24-year-old male patient with myalgia and myoglobuinuria followed by severe exercise from childhood. In 18 years old, he had severe myaglia after a long time-night trip by bus. He was diagnosed as acute renal failure induced by rhabdomyolysis and treated with hemodialysis. In 24 years old, he was admitted to our hospital because of repeated rhabdomyolysis. We performed muscle biopsy from right quadriceps femoris, however histological and immunohistochemistological studies were normal. Ischemic forearm exercise test showed the elevation of lactic acid in serum. Therefore, we performed the analysis of acylcarnitine in serum, and the measurement of enzyme in beta-oxidation in muscle and white blood cells. These showed the lack of very-long-chain-acyl coA dehydrogenase (VLCAD) activity. He was diagnosed as skeletal muscle type VLCAD deficiency. Under the guidance of high carbohydrate and low fat diet, creatine kinase was controlled around 400 IU/l. VLCAD deficiency is important to make a differential diagnosis of young cases with recurrent elevation of creatine kinase.