Biological variation of methylmalonic acid in urine in spanish population.

BACKGROUND-AIM: Methylmalonic acid (MMA) is currently the best biomarker of functional vitamin B12 deficiency. However, for a correct interpretation of the patient's results it is necessary to know its biological variation (BV). No BV data are available for urine MMA values, as measured by mass spectrometry. Hence, the aim of this study was to estimate the within- and between-person coefficients of variation (CVw, CVg) for MMA in a healthy population, and the associated index of individuality (II), as well as to define quality specifications based on BV and the reference change value (RCV). METHODS: Random urine samples from 34 healthy volunteers were collected over four consecutive weeks. Samples were stored at -80 °C until analysis in a single analytical run. MMA excretion was quantified by tandem liquid chromatography coupled to mass spectrometry (HPLC-MS/MS). Results were normalized to urine creatinine. The coefficients of variation were estimated by CV-ANOVA. Confidence intervals (95 %) were calculated. Quality specifications were defined according to international recommendations. RESULTS: A total of 128 samples were included. The coefficients of variation were CVw = 35.7 % (26.1-45.3) and CVg = 67.7 % (58.3-77.0). The associated II was 0.5 and the RCV was 88.1 %. CONCLUSION: Considering the II obtained, MMA in urine has high individuality, therefore, RCV is better to evaluate serial clinical results. Our results will contribute to a better clinical interpretation of this biomarker and will represent a great aid when defining analytical performance specifications for this magnitude.

Feline hyperammonemia associated with functional cobalamin deficiency: A case report.

Ammonia is a major neurotoxic substance associated with the complex pathogenesis of hepatic encephalopathy. Although several primary and secondary conditions have been reported to cause hyperammonemia, in veterinary medicine this condition is considered primarily associated with hepatic disease or portosystemic shunting. Only a few cases of inherited urea cycle enzyme deficiency and organic acid metabolic disorders have been reported in cats with hyperammonemia. To the best of our knowledge, this is the first report of hyperammonemia in a cat caused by accumulation of methylmalonic acid (MMA) secondary to functional cobalamin deficiency. A 2-year-old spayed female Turkish Angora cat exhibited postprandial depression with a 3-month history of hyperammonemia. Serum protein C and bile acid concentrations were normal. Plasma amino acid analysis revealed a deficiency of urea cycle amino acids. Although the serum cobalamin concentration was markedly high, there was no evidence of inflammatory, hepatic, or renal disease or neoplasia on blood, ultrasonographic, and computed tomographic examination. Gas chromatography-mass spectrometry revealed a high MMA concentration in the urine. Based on the results, functional cobalamin deficiency was diagnosed. Following oral amino acid supplementation and initiation of a low-protein diet, the serum ammonia level returned to normal and the postprandial depression improved. Urea cycle amino acid deficiency secondary to functional cobalamin deficiency presumably caused hyperammonemia due to MMA accumulation in this case.

Hyperammoniémie féline associée à un déficit fonctionnel en cobalamine : rapport de cas. L'ammoniac est une substance neurotoxique majeure associée à la pathogenèse complexe de l'encéphalopathie hépatique. Bien que plusieurs affections primaires et secondaires aient été signalées comme étant à l'origine d'une hyperammoniémie, en médecine vétérinaire, cette affection est considérée comme principalement associée à une maladie hépatique ou à un shunt porto-systémique. Seuls quelques cas de déficit héréditaire en enzymes du cycle de l'urée et de troubles métaboliques des acides organiques ont été signalés chez des chats atteints d'hyperammoniémie. À notre connaissance, il s'agit du premier rapport d'hyperammoniémie chez un chat causée par une accumulation d'acide méthylmalonique (MMA) secondaire à un déficit fonctionnel en cobalamine.Une chatte angora turque stérilisée âgée de 2 ans a présenté une dépression postprandiale avec une histoire d'hyperammoniémie depuis 3 mois. Les concentrations sériques de protéine C et d'acides biliaires étaient normales. L'analyse plasmatique des acides aminés a révélé une déficience en acides aminés du cycle de l'urée. Bien que la concentration sérique de cobalamine ait été nettement élevée, il n'y avait aucun signe de maladie inflammatoire, hépatique ou rénale ou de néoplasie à l'examen sanguin, échographique et tomodensitométrique. La chromatographie en phase gazeuse-spectrométrie de masse a révélé une forte concentration de MMA dans l'urine. Sur la base des résultats, un déficit fonctionnel en cobalamine a été diagnostiqué. Après une supplémentation orale en acides aminés et la mise en place d'un régime pauvre en protéines, le taux sérique d'ammoniac est revenu à la normale et la dépression postprandiale s'est améliorée. Une carence en acides aminés du cycle de l'urée secondaire à une carence en cobalamine fonctionnelle a vraisemblablement causé une hyperammoniémie due à l'accumulation de MMA dans ce cas.(Traduit par Dr Serge Messier).

Identification of potential interferents of methylmalonic acid: A previously unrecognized pitfall in clinical diagnostics and newborn screening.

OBJECTIVES: Determination of methylmalonic acid (MMA) from dried blood spots (DBS) is commonly performed in clinical diagnostics and newborn screening for propionic acidemia (PA) and methylmalonic acidemia. Isobaric compounds of MMA having the same mass can affect diagnostic reliability and quantitative results, which represents a previously unrecognized pitfall in clinical assays for MMA. We set out to identify interfering substances of MMA in DBS, serum and urine samples from confirmed patients with PA and methylmalonic acidemia. METHODS: Techniques included quadrupole time-of-flight high-resolution mass spectrometry (QTOF HR-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC) and tandem mass spectrometry (MS/MS). RESULTS: The five isobaric metabolites detected in DBS, serum and urine from PA and methylmalonic acidemia patients were confirmed as 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-hydroxyisovalerate, 3-hydroxyvalerate and succinate using a series of experiments. An additional unknown substance with low abundance remained unidentified. CONCLUSIONS: The presented results facilitate the diagnostic and quantitative reliability of the MMA determination in clinical assays. Isobaric species should be investigated in assays for MMA to eliminate possible interference in a wide range of conditions including PA, methylmalonic acidemia, a vitamin B12 deficiency, ketosis and lactic acidosis.

Clinical characteristics and genotype analysis of five infants with cblX type of methylmalonic acidemia.

OBJECTIVE: To investigate the clinical and genetic characteristics of infants with cobalamin (cbl) X type of methylmalonic acidemia (MMA). METHODS: The clinical data of 5 infants with cblX type of MMA diagnosed in Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Shanghai Children's Hospital from the year 2016 to 2020 were collected. The levels of blood acylcarnitines were detected by tandem mass spectrometry, the levels of urinary organic acids were detected by gas-chromatography mass spectrometry, the pathogenic genes were detected by whole exon gene sequencing, and the effect of new pathogenic mutations on three-dimensional protein structure was predicted by bioinformatics analysis. RESULTS: Five infants with cblX type were diagnosed, including 4 males and 1 female, and the onset age was 0-6 months. The main clinical manifestations of 4 males were intractable epilepsy, mental and motor retardation, metabolic abnormalities presented mild increase of blood homocysteine level. Among them, 3 cases were accompanied by slight increase of urinary methylmalonic acid, and 1 case was accompanied by increase of blood propionylcarnitine (C3) and C3/acetylcarnitine (C2). Gene detection found that 2 cases carried a same hemizygous mutation c.344C>T (p.A115V) of HCFC1 gene, which was the most reported mutation, and the other 2 cases carried novel pathogenic mutations, c.92G>A (p.R31Q) and c.166G>C (p.V56L). These 3 gene mutations located in the Kelch domain of HCFC1 protein. One female infant carried a benign mutation of c.3731G>T (p.R1244L). Her clinical symptoms were mild, and only the urinary methylmalonic acid was slightly increased. CONCLUSIONS: The clinical manifestations of children with cblX type of MMA are intractable epilepsy, mental and motor retardation, and other serious neurological symptoms. Their metabolic abnormalities present the increase of blood homocysteine with methylmalonic acid (urinary methylmalonic acid or/and blood C3, C3/C2). The clinical and biochemical phenotypes are separated, so the diagnosis should be in combination with the results of gene testing.

Methylmalonic acid analysis using urine filter paper samples to screen for metabolic vitamin B12 deficiency in older adults.

Aim: Methylmalonic acid (MMA) analysis in urine represents a noninvasive approach to screening for vitamin B12 deficiency in older adults. A method allowing the analysis of MMA/creatinine in fasting urine collected on filter paper was developed/validated. Method: Dry urine specimens were eluted using a solution containing internal standards, filtrated and analyzed by ultra-performance LC-MS/MS. Results: The method allowed the chromatographic separation of MMA from succinic acid. Dried urine samples were stable for 86 days at room temperature. The MMA/creatinine ratios measured in urine collected on filter paper were highly correlated with values derived from the corresponding liquid specimens. Conclusion: This robust filter paper method might greatly improve the accessibility and cost-effectiveness of vitamin B12 deficiency screening in older adults.

Delineating the clinical spectrum of isolated methylmalonic acidurias: cblA and mut.

INTRODUCTION: Long-term outcome is postulated to be different in isolated methylmalonic aciduria caused by mutations in the MMAA gene (cblA type) compared with methylmalonyl-CoA mutase deficiency (mut), but case definition was previously difficult. METHOD: Cross-sectional analysis of data from the European Registry and Network for Intoxication type Metabolic Diseases (Chafea no. December 1, 2010). RESULTS: Data from 28 cblA and 95 mut patients in most cases confirmed by mutation analysis (including 4 new mutations for cblA and 19 new mutations for mut). Metabolic crisis is the predominant symptom leading to diagnosis in both groups. Biochemical disturbances during the first crisis were similar in both groups, as well as the age at diagnosis. Z scores of body height and body weight were similar in both groups at birth, but were significantly lower in the mut group at the time of last visit. Glomerular filtration rate was significantly higher in cblA; and as a consequence, chronic renal failure and related complications were significantly less frequent and renal function could be preserved even in older patients. Neurological complications were predominantly found in the mut subgroup. Methylmalonic acidemia (MMA) levels in urine and plasma were significantly lower in cblA. 27/28 cblA patients were reported to be responsive to cobalamin, only 86% of cblA patients were treated with i.m. hydroxocobalamin. In total, 73% of cblA and 98% of mut patients followed a calculated diet with amino acid supplements in 27% (cblA) and 69% (mut). During the study interval, six patients from the mut group died, while all cblA patients survived. CONCLUSION: Although similar at first, cblA patients respond to hydroxocobalamin treatment, subsequently show significantly lower levels of MMA and a milder course than mut patients.

Proteinuria as a presenting sign of combined methylmalonic acidemia and homocysteinemia: case report.

BACKGROUND: Disorders of the metabolism and absorption of vitamin B12 can lead to decrease in activity of methionine synthetase and methylmalonate coenzyme A mutase (MMUT), which results in increased levels of methylmalonic acid and homocysteine in blood and urine. Often, combined methylmalonic acidemia (MMA) and homocysteinemia is misdiagnosed due to a lack of specific symptoms. The clinical manifestations are diverse, but proteinuria as the initial presentation is rare. CASE PRESENTATION: Two cases of MMA with homocysteinemia in children are reported. Proteinuria were a primary presenting symptom, followed by anemia and neurologic symptoms (frequent convulsions and unstable walking, respectively). Screening of amino acids and acyl carnitine in serum showed that the propionyl carnitine:acetylcarnitine ratio increased. Profiling of urinary organic acids by gas chromatography-mass spectrometry revealed high levels of methylmalonic acid. Homocysteine content in blood was increased. Comprehensive genetic analyses of peripheral blood-derived DNA demonstrated heterozygous variants of methylmalonic aciduria type C and homocystinuria (MMACHC) and amnionless (AMN) genes in our two patients, respectively. After active treatment, the clinical manifestations in Case 1 were relieved and urinary protein ceased to be observed; Case 2 had persistent proteinuria and was lost to follow-up. CONCLUSIONS: Analyses of the organic acids in blood and urine suggested MMA combined with homocysteinemia. In such diseases, reports of renal damage are uncommon and proteinuria as the initial presentation is rare. Molecular analysis indicated two different genetic causes. Although the pathologic mechanisms were related to vitamin B12, the severity and prognosis of renal lesions were different. Therefore, gene detection provides new insights into inherited metabolic diseases.

Mass spectrometry analysis of urinary methylmalonic acid to screen for metabolic vitamin B12 deficiency in older adults.

Aim: Vitamin B12 deficiency is characterized metabolically by increased serum and urine methylmalonic acid (MMA). Urinary MMA/creatinine ratio is suggested for screening for metabolic vitamin B12 deficiency in older populations. Results: A UPLC-MS/MS method for the analysis of urinary MMA and creatinine was developed/validated. A good separation of MMA from succinic acid, its structural isomer, was achieved. Intra- and interday accuracy biases and precision coefficients were all ≤6.3% for MMA and creatinine. Urine and serum samples of 34 individuals of the NuAge Biobank were analyzed for technical comparisons showing that urinary MMA/creatinine ratios by UPLC-MS/MS strongly correlated with GC-MS values, and with serum MMA values. Conclusion: The UPLC-MS/MS method developed is rapid/reliable for the analysis of urinary MMA/creatinine ratios.

Metabolic profiling by reversed-phase/ion-exchange mass spectrometry.

Metabolic profiling is commonly achieved by mass spectrometry (MS) following reversed-phase (RP) and hydrophilic interaction chromatography (HILIC) either performed independently, leading to overlapping datasets, or in a coupled configuration, requiring multiple liquid chromatography (LC) systems. To overcome these limitations, we developed a single, 20-minute chromatographic method using an in-line RP-ion-exchange (IEX) column arrangement and a single LC system. This configuration separates clinically significant polar and non-polar compounds without derivatization or ion-pairing reagents, allowing ionization in both polarities. An in-house library was created with 397 authentic standards, including acylcarnitines, amino acids, bile acids, nucleosides, organic acids, steroid hormones, and vitamins. Analysis of pooled plasma and urine samples revealed 5445 and 4111 ion features, leading to 88 and 82 confirmed metabolite identifications, respectively. Metabolites were detected at clinically relevant concentrations with good precision, and good chromatographic separation was demonstrated for clinically significant isomers including methylmalonic acid and succinic acid, as well as alloisoleucine and isoleucine/leucine. Evaluation of the samples by unsupervised principal component analysis showed excellent analytical quality.

Infantile Tremor Syndrome or a Neurocutaneous Infantile B12 Deficiency (NIB) Syndrome?

OBJECTIVES: To prospectively study the clinical and developmental profile; hematological profile and the B-12 status using multiple parameters in children with Infantile tremor syndrome (ITS). METHODS: In this observational study (NCT02762682) (July 2015 through December 2016) children (and their mothers) with a clinical diagnosis of ITS were evaluated clinically; and development was assessed by CAPUTE scales. A complete blood count (CBC); peripheral blood smear examination; markers of vitamin B12 status (serum B12, homocysteine, folate); acylcarnitines [using Tandem mass spectrometry (TMS)] and urine methylmalonic acid (MMA) [Gas chromatography mass spectrometry (GCMS)] were estimated. A control group of children and their mothers were sampled for comparison. RESULTS: A total of 286 individuals were enrolled for this study. One-hundred-ten children with ITS were screened and 92 (20 with tremors; age 12.7 ± 5 mo, 61 boys) children and their mothers were enrolled. Fifty-one children and their mothers were enrolled as controls. The median clinical linguistic & auditory milestone-developmental quotient (CLAM-DQ) was 32 (IQR 20.6-45.5) and median cognitive adaptive test-developmental quotient (CAT-DQ) was 36.2 (IQR 18.7-49.0). All babies except 9 (ovo-veg) had vegetarian mothers. Head circumference below 2 SD (WHO standards) was seen in 84% and below 3 SD in 58%. The CBC findings were; Hb- 8.3 ± 1.6 g/dl, Thrombocytopenia-29 (32%), mean corpuscular volume (MCV)- 92.2 ± 13.4, MCV- more than 95 fL-38(42%), Red cell distribution width (RDW)- 21.6 ± 6.5, and macrocytes on peripheral smear in 68%. In 89 (97%) out of 92 children had laboratory features of deficient B12 status. Two-thirds of the mothers also had evidence of B12 deficiency. CONCLUSIONS: ITS is, in all likelihood is a consequence of vitamin B12 defeciency. It has a significant impact on head growth and development of affected infants.

Isolated subacute combined degeneration in late-onset cobalamin C deficiency in children: Two case reports and literature review.

RATIONALE: Subacute combined degeneration (SCD) is a disease caused by decreased vitamin B12 intake or metabolic disorders. It is more common in the elderly and rarely seen in children. Here, we report 2 pediatric cases of SCD in late-onset cobalamin C (CblC) deficiency. PATIENT CONCERNS: The patients complained of unsteady gait. Their physical examination showed sensory ataxia. Magnetic resonance imaging showed classic manifestations of SCD. The serum vitamin B12 level was normal, but urine methylmalonic acid and serum homocysteine levels were high. DIAGNOSIS: The pathogenic gene was confirmed as MMACHC. The 2 patients each had 2 pathogenic mutations C.482 G>A and C.271dupA and C.365A>T and C.609G>A in this gene. They were diagnosed with combined methylmalonic acidemia and homocysteinemia-CblC subtype. INTERVENTIONS: The patients were treated with methylcobalamin 500 µg intravenous injection daily after being admitted. After the diagnosis, levocarnitine, betaine, and vitamin B12 were added to the treatment. OUTCOMES: Twelve days after treatment, the boy could walk normally, and his tendon reflex and sense of position returned to normal. The abnormal gait seemed to have become permanent in the girl and she walked with her legs raised higher than normal. LESSONS: To the best of our knowledge, this is the first report of 2 cases of isolated SCD in children with late-onset CblC disorder. Doctors should consider that SCD could be an isolated symptom of CblC disorder. The earlier the treatment, the lower the likelihood of sequelae.

Clinical Application of LC-MS/MS in the Follow-Up for Treatment of Children with Methylmalonic Aciduria.

INTRODUCTION: To explore the value of high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the follow-up for treatment of children with methylmalonic aciduria (MMA). METHODS: Methylmalonic acid (MMA), 2-methylcitric acid (MCA) and homocysteine (Hcy) were detected by LC-MS/MS in a total of 1016 samples whose estimated 0.5th and 99.5th percentiles was taken as the reference value. The samples of children with MMA and propionic aciduria (PA) who were followed up in our hospital from January 2017 to August 2018 were collected. Samples of dried blood spots, serum, and urine were taken from each patient on the same day. The concentration of the C3 indicator in the dried blood spots was tested by MS/MS. MMA, MCA, and Hcy in the dried blood spots were quantitatively determined by LC-MS/MS, the concentrations of MMA and MCA in urine filter papers were determined by gas chromatography-mass spectrometry (GC/MS), and the concentration of homocysteine in serum was determined by enzymatic cycling assay. RESULTS: Reference values of MMA, MCA and HCY by LC-MS/MS in the newborn population were determined. The samples from a total of 50 patients were collected, 48 were from children with MMA, and 2 were from children with PA. The first-order equation regression coefficient of MMA in the blood spots and MMA in urine was significant (P < 0.05), r2 = 0.736; the first-order equation regression coefficient of MCA in bthe lood spots and MCA in urine was significant (P < 0.05), r2 = 0.946; the first-order equation regression coefficient of tHcy in bthe lood spots and Hcy in serum was significant (P < 0.05), r2 = 0.771. CONCLUSION: LC-MS/MS can be used for the follow-up of children with MMA after treatment, but it is necessary to establish a reference interval suitable for the local population.

Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry.

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.

Daily oral cyanocobalamin supplementation in Beagles with hereditary cobalamin malabsorption (Imerslund-Gräsbeck syndrome) maintains normal clinical and cellular cobalamin status.

BACKGROUND: Efficacy of PO cobalamin (Cbl) supplementation in dogs with hereditary Cbl malabsorption (Imerslund-Gräsbeck syndrome, IGS) is unknown. OBJECTIVES: To evaluate PO Cbl supplementation in Beagles with IGS previously treated parenterally. We hypothesized that 1 mg cyano-Cbl daily PO would maintain clinical and metabolic remission. ANIMALS: Three client-owned Beagles with IGS and 48 healthy control dogs. METHODS: Prospective study. Daily PO cyanocobalamin (cyano-Cbl; 1 mg) supplementation was monitored for 13 (2 dogs) and 8 months (1 dog). Health status was assessed by owner observations. Methylmalonic acid (MMA)-to-creatinine concentrations were measured using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-TMS) method on urine samples collected monthly. Concurrent measurements of serum MMA concentration (n = 7; UPLC-TMS) were available for 1 dog. RESULTS: All dogs remained in excellent health during PO supplementation. Urine MMA remained consistently low in 2 dogs (median, 2.5 mmol/mol creatinine; range, 1.2-9; healthy dogs [n = 30], median, 2.9 mmol/mol creatinine; range, 1.3-76.5). Urine MMA ranged from 38.9-84.9 mmol/mol creatinine during the first 6 months in 1 dog already known to excrete comparable amounts when supplemented parenterally. Brief antibiotic treatment for an unrelated condition after 6 months resulted in low urine MMA (median, 2.8 mmol/mol creatinine; range, 1.9-4.8) for the next 7 months. All concurrent serum MMA concentrations (median, 651 nmol/L; range, 399-919) before and after month 6 were within the established reference interval (393-1476 nmol/L; n = 48). CONCLUSIONS AND CLINICAL IMPORTANCE: One milligram of cyano-Cbl daily PO appears efficacious for maintaining normal clinical status and normal cellular markers of Cbl metabolism in Beagles with IGS.

Vitamin B12 Deficiency in Children With Infantile Spasms: A Case-Control Study.

There have been few case reports showing association of vitamin B12 deficiency with infantile spasms. We planned this study to see if there was an association of serum vitamin B12 deficiency in children with development of infantile spasms. Cases included children with infantile spasms of ages 6 months to 3 years. The controls were children in the same age group who had global developmental delay but no history of epileptic spasms. Mean serum vitamin B12, serum homocysteine, and urinary methylmalonic acid levels were measured in both groups and compared. Children with infantile spasms had lower mean serum vitamin B12 levels (354.1 pg/mL; standard deviation 234.1 pg/mL) as compared to children with global developmental delay without spasms (466.7 pg/mL; standard deviation 285.5 pg/mL) ( P value < .05). Mean serum homocysteine level (13.9 vs 7.8 µmol/L, P = .02) and mean urinary methylmalonic acid level (68.1 mmol/mol of creatinine vs 26.1 mmol/mol of creatinine, P = .03) were elevated in children with infantile spasms than in controls. Fourteen children (35.0%) with infantile spasms were vitamin B12 deficient compared with 3 (7.50%) controls ( P = .005). Thus, vitamin B12 deficiency may have an association with infantile spasms. More studies are needed before recommending routine measurement of serum B12 levels in children with infantile spasms.

A male case with CDKL5-associated encephalopathy manifesting transient methylmalonic acidemia.

Mutations in the X-linked gene CDKL5 cause early-onset epileptic encephalopathy and severe developmental delay. Because this disorder predominantly affects females, the full clinical spectrum of male patients remains elusive. We herein report a 16-year-old boy, who suffered from intractable seizures 20 days after birth. Serial electroencephalograms detected recurrent focal epileptiform discharges from age 4 months, which evolved to hypsarrhythmia later in infancy. Mass-spectrometric analyses revealed increase in urinary excretion of methylmalonic acid without perturbed concentrations of propionic acid, homocystein and methionine. Whole-exome sequencing identified a de novo, truncating mutation in CDKL5 (NM_003159.2:c.419dupA, p.Asn140Lysfs*8). Targeted sequencing excluded concomitant mutations in methylmalonic academia-associated genes. No methylmalonic acidemia has been reported in children with CDKL5 disorder. Extensive analyses on organic acid metabolism for males with CDKL5 mutations will gain more insight into their biochemical profiles in infancy.

Prospective long-term evaluation of parenteral hydroxocobalamin supplementation in juvenile beagles with selective intestinal cobalamin malabsorption (Imerslund-Gräsbeck syndrome).

BACKGROUND: Prospective studies on maintenance treatment for Beagles with hereditary selective cobalamin (Cbl) malabsorption (Imerslund-Gräsbeck syndrome, IGS) are lacking. In our experience, measurement of methylmalonic acid (MMA), a Cbl-dependent metabolite, seems more helpful to monitor Cbl status as compared with serum Cbl concentrations. OBJECTIVES: To evaluate a standardized Cbl supplementation scheme in Beagles with IGS. We hypothesized that a single parenteral dose of 1 mg hydroxocobalamin (OH-Cbl) would maintain clinical and metabolic remission for up to 2 months. ANIMALS: Six client-owned juvenile Beagles with genetically confirmed IGS and 28 healthy control dogs. METHODS: Prospective study. Monthly IM OH-Cbl (1 mg) supplementation was done over a median of 9 months (range, 6-13) in 6 dogs, followed by bimonthly (every 2 months) injections in 5 dogs over a median of 6 months (range, 3-10). Health status was assessed by routine clinical examinations at injection time points and owner observations. Voided urine samples were collected immediately before OH-Cbl injections for measurement of MMA-to-creatinine concentrations using a gas-liquid chromatography-tandem mass spectrometry (GC-MS) method. RESULTS: All dogs were clinically healthy while receiving monthly and bimonthly OH-Cbl supplementation. Urinary MMA results in healthy dogs ranged from 1.3 to 76.5 mmol/mol creatinine (median, 2.9). Median urinary MMA concentrations did not differ between dogs with IGS receiving monthly (n = 49; 5.3 mmol/mol creatinine; range, 2.3-50.4) and bimonthly (n = 31; 5.3 mmol/mol creatinine; range, 1.6-50) injections. CONCLUSIONS AND CLINICAL IMPORTANCE: A maintenance parenteral dose of 1 mg OH-Cbl monthly or bimonthly appears adequate in Beagles with IGS monitored by metabolic testing.

Methylmalonyl-CoA Epimerase Deficiency Mimicking Propionic Aciduria.

Methylmalonyl-CoA epimerase (MCE) converts d-methylmalonyl-CoA epimer to l-methylmalonyl-CoA epimer in the propionyl-CoA to succinyl-CoA pathway. Only seven cases of MCE deficiency have been described. In two cases, MCE deficiency was combined with sepiapterin reductase deficiency. The reported clinical pictures of isolated MCE are variable, with two asymptomatic patients and two other patients presenting with metabolic acidosis attacks. For combined MCE and sepiapterin reductase deficiency, the clinical picture is dominated by neurologic alterations. We report isolated MCE deficiency in a boy who presented at five years of age with acute metabolic acidosis. Metabolic investigations were consistent with propionic aciduria (PA). Unexpectedly, propionyl-CoA carboxylase activity was within the reference range. Afterward, apparently intermittent and mild excretion of methylmalonic acid (MMA) was discovered. Methylmalonic pathway gene set analysis using the next-generation sequencing approach allowed identification of the common homozygous nonsense pathogenic variant (c.139C > T-p.Arg47*) in the methylmalonyl-CoA epimerase gene (MCEE). Additional cases of MCE deficiency may help provide better insight regarding the clinical impact of this rare condition. MCE deficiency could be considered a cause of mild and intermittent increases in methylmalonic acid.