cblC Defect Research Articles

Evaluation of the clinical, biochemical, and molecular spectrum of Cobalamin C (CblC) defect in 33 patients from Pakistan

Background: Cobalamin C is the most common inborn error of intracellular cobalamin metabolism caused by biallelic pathogenic variants in the MMACHC gene, leading to impaired conversion of dietary vitamin B12 into its two metabolically active forms, methylcobalamin and adenosylcobalamin. Biochemical hallmarks are elevated plasma total homocysteine (HCYs) and low methionine accompanied by methylmalonic aciduria. This study aimed to evaluate the clinical, biochemical, and molecular analysis of Pakistani patients with CblC defect. Methods: Medical charts, urine organic acid (UOA) chromatograms, plasma amino acid levels, plasma tHcy and MMACHC gene results of patients presenting at the Biochemical Genetics Clinic, AKUH from 2013–2021 were reviewed. Details were collected on a pre-structured questionnaire. SPSS 22 was used for data analysis. Results: CblC was found in 33 cases (Male:Female 19:14). The median age of symptoms onset and diagnosis were 300 (IQR:135–1800) and 1380 (IQR: 240–2730) days. The most common clinical features were cognitive impairment (n = 29), seizures (n = 23), motor developmental delay (n = 20), hypotonia (n = 17), and sparse/hypopigmented scalp hair (n = 16). The MMACHC gene sequencing revealed homozygous pathogenic variant c.394C > T, (p.Arg132*) in 32 patients, whereas c.609G > A, (p.TRP203*) in one patient whose ancestors had settled in Pakistan from China decades ago. The median age of treatment initiation was 1530 (IQR: 240–2790). The median pre-treatment HCYs levels were 134 (IQR:87.2–155.5) compared to post-treatment levels of 33.3 (IQR: 27.3–44.95) umol/L. Conclusions: Thirty-three cases of CblC defect from a single center underscores a significant number of the disorder within Pakistan. Late diagnosis emphasizes the need for increased clinical awareness and adequate diagnostic facilities.

Improved biochemical and neurodevelopmental profiles with high-dose hydroxocobalamin therapy in cobalamin C defect.

Cobalamin C (Cbl-C) defect causes methylmalonic acidemia, homocystinuria, intellectual disability and visual impairment, despite treatment adherence. While international guidelines recommend parenteral hydroxocobalamin (OH-Cbl) as effective treatment, dose adjustments remain unclear. We assessed OH-Cbl therapy impact on biochemical, neurocognitive and visual outcomes in early-onset Cbl-C patients treated with different OH-Cbl doses over 3 years. Group A (n = 5), diagnosed via newborn screening (NBS), received high-dose OH-Cbl (median 0.55 mg/kg/day); Group B1 (n = 3), NBS-diagnosed, received low-dose OH-Cbl (median 0.09 mg/kg/day); Group B2 (n = 12), diagnosed on clinical bases, received low-dose OH-Cbl (median 0.06 mg/kg/day). Biochemical analyses revealed better values of homocysteine, methionine and methylmalonic acid in Group A compared to Group B1 (p < 0.01, p < 0.05 and p < 0.01, respectively) and B2 (p < 0.001, p < 0.01 and p < 0.001, respectively). Neurodevelopmental assessment showed better outcome in Group A compared to low-dose treated Groups B1 and B2, especially in Developmental Quotient, Hearing and Speech and Performance subscales without significant differences between Group B2 and Group B1. Maculopathy was detected in 100%, 66% and 83% of patients in the three groups, respectively. This study showed that "high-dose" OH-Cbl treatment in NBS-diagnosed children with severe early-onset Cbl-C defect led to a significant improvement in the metabolic profile and in neurocognitive outcome, compared to age-matched patients treated with a "low-dose" regimen. Effects on maculopathy seem unaffected by OH-Cbl dosage. Our findings, although observed in a limited number of patients, may contribute to improve the long-term outcome of Cbl-C patients.

Case presentation: a severe case of cobalamin c deficiency presenting with nephrotic syndrome, malignant hypertension and hemolytic anemia

BackgroundThe etiology of nephrotic syndrome can vary, with underlying metabolic diseases being a potential factor. Cobalamin C (cblC) defect is an autosomal recessive inborn error of metabolism caused by mutations in the MMACHC gene, resulting in impaired vitamin B12 processing. While cblC defect typically manifests with hematological and neurological symptoms, renal involvement is increasingly recognized but remains rare.Case PresentationWe describe a 7-month-old male patient presenting with fatigue and edema. His initial laboratory findings showed anemia, thrombocytopenia, hypoalbuminemia and proteinuria. Further examinations reveals hemolysis in peripheral blood smear. During his follow up respiratory distress due to pleural effusion in the right hemithorax was noticed. And fluid leakage to the third spaces supported a diagnosis of nephrotic syndrome. The patient’s condition deteriorated, leading to intensive care admission due to, hypertensive crisis, and respiratory distress. High total plasma homocysteine and low methionine levels raised suspicion of cobalamin metabolism disorders. Genetic testing confirmed biallelic MMACHC gene mutations, establishing the diagnosis of cblC defect. Treatment with hydroxycobalamin, folic acid, and betaine led to remarkable clinical improvement.Discussion/ConclusionThis case underscores the significance of recognizing metabolic disorders like cblC defect in atypical presentations of nephrotic syndrome. Early diagnosis and comprehensive management are vital to prevent irreversible renal damage. While cblC defects are more commonly associated with atypical hemolytic uremic syndrome, this case highlights the importance of considering cobalamin defects in the differential diagnosis of nephrotic syndrome, especially when associated with accompanying findings such as hemolysis. Our case, which has one of the highest homocysteine levels reported in the literature, emphasizes this situation again.

Late-onset cblC defect: clinical, biochemical and molecular analysis

BackgroundcblC defect is the most common type of methylmalonic acidemia in China. Patients with late-onset form (>1 year) are often misdiagnosed due to heterogeneous symptoms. This study aimed to describe clinical characteristics and evaluate long-term outcomes of Chinese patients with late-onset cblC defect.MethodsA total of 85 patients with late-onset cblC defect were enrolled. Clinical data, including manifestations, metabolites, molecular diagnosis, treatment and outcome, were summarized and analyzed.ResultsThe age of onset ranged from 2 to 32.8 years old (median age 8.6 years, mean age 9.4 years). The time between first symptoms and diagnosis ranged from a few days to 20 years (median time 2 months, mean time 20.7 months). Neuropsychiatric symptoms were presented as first symptoms in 68.2% of cases, which were observed frequently in schoolchildren or adolescents. Renal involvement and cardiovascular disease were observed in 20% and 8.2% of cases, respectively, which occurred with the highest prevalence in preschool children. Besides the initial symptoms, the disease progressed in most patients and cognitive decline became the most frequent symptom overall. The levels of propionylcarnitine, propionylcarnitine / acetylcarnitine ratio, methylmalonic acid, methylcitric acid and homocysteine, were decreased remarkably after treatment (P<0.001). Twenty-four different mutations of MMACHC were identified in 78 patients, two of which were novel. The c.482G>A variant was the most frequent mutated allele in this cohort (25%). Except for 16 patients who recovered completely, the remaining patients were still left with varying degrees of sequelae in a long-term follow-up. The available data from 76 cases were analyzed by univariate analysis and multivariate logistic regression analysis, and the results showed that the time from onset to diagnosis (OR = 1.025, P = 0. 024) was independent risk factors for poor outcomes.ConclusionsThe diagnosis of late-onset cblC defect is often delayed due to poor awareness of its various and nonspecific symptoms, thus having an adverse effect on the prognosis. It should be considered in patients with unexplained neuropsychiatric and other conditions such as renal involvement, cardiovascular diseases or even multiple organ damage. The c.482G>A variant shows the highest frequency in these patients. Prompt treatment appears to be beneficial.

Prominent renal complications associated with MMACHC pathogenic variant c.80A > G in Chinese children with cobalamin C deficiency.

CblC deficiency, the most common cobalamin metabolic abnormality, is caused by pathogenic variants in the MMACHC gene. The renal complications of this disease have been described only in a small number of cases. This study aimed to better delineate renal phenotype and genetic characteristics in Chinese children with cblC defect. Children with cblC deficiency who manifested as kidney damage were enrolled. Clinical, renal pathological, and genetic data were reviewed in detail. Seven cases were enrolled. Ages at disease onset ranged from 9 months to 5 years. All patients presented with hematuria and proteinuria, and 2/7 cases presented with nephrotic syndrome. Renal dysfunction was observed in 4/7 cases. Renal biopsy was performed in 5/7 cases, and all of them had renal thrombotic microangiopathy. Macrocytic anemia was detected in all seven patients. Six out of seven cases had hypertension, and 2/7 cases presented with pulmonary hypertension. Two of them had a mild intellectual disability, and one suffered from epilepsy. Increased urine methylmalonic acid and plasma homocysteine were detected in seven cases, while two patients had normal levels of urine methylmalonic acid at the initial evaluation. After diagnosis, all seven cases were treated with hydroxocobalamin IM. Six cases were followed-up for 3-8 years. After treatments, anemia was the first to be recovered, followed by proteinuria. Renal function recovered after 1 year in two cases, whereas patient 2 progressed to stage 2 chronic kidney disease 13 years after onset. While a case presented with end-stage kidney disease because of late diagnosis, one case died 3 months after disease onset due to giving up treatment. Three MMACHC pathogenic variants c.80A > G (8/14), c.609G > A (4/14), and c.658_660delAAG (2/14) were detected in all seven children. MMACHC variant c.80A > G may be associated with prominent renal complications in Chinese cblC patients. Macrocytic anemia and hyperhomocysteinemia are useful clues for patients with hematuria and proteinuria caused by cblC defect. The most frequent renal pathological manifestation is thrombotic microangiopathy. Early diagnosis and treatment resulted in improving renal and hematological signs.

Follow-up study of neuropsychological scores of infant patients with cobalamin C defects and influencing factors of cerebral magnetic resonance imaging characteristics.

The purpose of this study was to investigate whether baseline cerebral magnetic resonance imaging (MRI) characteristics could predict therapeutic responsiveness in patients with cobalamin C (cblC) defects. The cerebral MRI results of 40 patients with cblC defects were evaluated by a neuroradiologist. Neuropsychological scores and imaging data were collected. Neuropsychological tests were performed before and after standardized treatment. Thirty-eight patients initially underwent neuropsychological testing [developmental quotient (DQ)]. CblC defects with cerebellar atrophy, corpus callosum thinning and ventricular dilation had significantly lower DQs than those without (P < 0.05). Through a multivariate linear stepwise regression equation after univariate analysis, ventricular dilation was the most valuable predictor of lower DQs. Thirty-six patients (94.7%) underwent follow-up neuropsychological testing. The pre- and post-treatment DQ values were not significantly different (Z = -1.611, P = 0.107). The post-treatment DQ classification (normal, moderately low, or extremely low) showed nearly no change compared to the pretreatment DQ classification (k = 0.790, P < 0.001). Ventricular dilation, cerebral atrophy and corpus callosum thinning are the main MRI abnormalities of cblC defects, and these manifestations are significantly correlated with delayed development in children. MRI findings can be considered an important tool for determining the severity of cblC defects.

Case report: A late-onset cobalamin C defect first presenting as a depression in a teenager

Background: The cobalamin C (cblC) defect, a common inborn disorder of cobalamin metabolism due to a genetic mutation in MMACHC, can cause combined methylmalonic acid and homocysteine accumulation in blood, urine, or both. In this article, a late-onset case was reported, and the patient first presented with depression identified with the MMACHC gene. We summarized the clinical features of the cblC defect, the relationship between genotype and phenotype, and the clinical experience concerning the diagnosis and treatment of the cblC defect. Case presentation: Initially presented with depression, the 16-year-old female patient showed progressive abnormal gait and bilateral lower limb weakness after 3 months. Blood routine examination suggested severe hyperhomocysteinemia, and screening for urine organic acids found elevated methylmalonic acid. Family gene sequencing showed mutations detected in MMACHC. She had a compound heterozygous mutation, while the c.271dupA (p.R91Kfs∗14) was only detected in her father and the c.482G>A (p.R161Q) was only detected in her mother. Hence, she was diagnosed with a cblC defect and treated with B vitamin supplements. The muscle strength of both lower limbs improved notably. Conclusion: This case indicated that depression could be a presenting sign of cblC-type methylmalonic aciduria and homocysteinemia, and enhanced the genotype-phenotype relationship of the cblC defect, which will contribute to further understanding of this emerging disease.

The Follow-Up of Chinese Patients in cblC Type Methylmalonic Acidemia Identified Through Expanded Newborn Screening.

Objective: The cblC type of combined methylmalonic acidemia and homocystinuria, an inherited disorder with variable phenotypes, is included in newborn screening (NBS) programs at multiple newborn screening centers in China. The present study aimed to investigate the long-term clinical benefits of screening individual. Methods: A national, retrospective multi-center study of infants with confirmed cblC defect identified by NBS between 2004 and 2020 was conducted. We collected a large cohort of 538 patients and investigated their clinical data in detail, including disease onset, biochemical metabolites, and gene variation, and explored different factors on the prognosis. Results: The long-term outcomes of all patients were evaluated, representing 44.6% for poor outcomes. In our comparison of patients with already occurring clinical signs before treatment to asymptomatic ones, the incidence of intellectual impairment, movement disorders, ocular complications, hydrocephalus, and death were significantly different (p < 0.01). The presence of disease onset [Odd ratio (OR) 12.39, 95% CI 5.15–29.81; p = 0.000], variants of c.609G>A (OR 2.55, 95% CI 1.49–4.35; p = 0.001), and c.567dupT (OR 2.28, 95% CI 1.03–5.05; p = 0.042) were independently associated with poor outcomes, especially for neurodevelopmental deterioration. Conclusion: NBS, avoiding major disease-related events and allowing an earlier treatment initiation, appeared to have protective effects on the prognosis of infants with cblC defect.

Value of amniotic fluid homocysteine assay in prenatal diagnosis of combined methylmalonic acidemia and homocystinuria, cobalamin C type

BackgroundCombined methylmalonic acidemia and homocystinuria, cobalamin C type (cblC defect) is the most common inborn error of cobalamin metabolism, and different approaches have been applied to its prenatal diagnosis. To evaluate the reliability of biochemical method for the prenatal diagnosis of cblC defect, we conducted a retrospective study of our 10-year experience at a single center.Methods248 pregnancies whose probands were diagnosed as cblC defect were referred to our center for prenatal diagnosis from January 2010 to December 2019. Prenatal data of Hcy levels determined by enzymatic cycling assay, acylcarnitine analysis using liquid chromatography tandem mass spectrometry, organic acid analysis using gas chromatography mass spectrometry, and genetic analysis by direct sequencing of 248 at-risk fetuses were retrospectively reviewed.ResultsFor 2.0 and 16.0 μmol/L levels of Hcy AF samples, the relative errors were − 2.5% and 2.8%, respectively. The respective measurement uncertainties were 13.07% and 14.20%. For the 248 at-risk fetuses, 63 fetuses were affected and 185 fetuses were unaffected. Hcy level of 13.20 (6.62–43.30) μmol/L in 63 affected fetuses was significantly higher than that in 185 unaffected fetuses of 2.70 (0.00–5.80) μmol/L, and there was no overlap between the affected and unaffected groups. The diagnostic sensitivity and specificity of Hcy were 100% and 92.05%, respectively. The positive and negative predictive values of the combination of Hcy, propionylcarnitine (C3), ratio of C3 to acetylcarnitine (C2; C3/C2), methylmalonic acid (MMA), and methylcitric acid (MCA) were both 100%. Sixteen fetuses displayed inconclusive genetic results of MMACHC variants, in which seven fetuses were determined to be affected with elevated levels of Hcy, C3, C3/C2 and MMA, and their levels were 18.50 (6.70–43.30) μmol/L, 8.53(5.02–11.91) μmol/L, 0.77 (0.52–0.97), 8.96 (6.55–40.32) mmol/mol Cr, respectively. The remaining nine fetuses were considered unaffected based on a normal amniotic fluid metabolite profile.ConclusionsHcy appears to be another characteristic biomarker for the prenatal diagnosis of cblC defect. The combination of Hcy assay with acylcarnitine and organic acid analysis is a fast, sensitive, and reliable prenatal diagnostic biochemical approach. This approach could overcome the challenge of the lack of genetic analysis for families with at-risk cblC defect fetuses.

Peripheral Nervous System Involvement in Late-Onset Cobalamin C Disease?

Background: Cobalamin C (cblC) has a fundamental role in both central and peripheral nervous system function at any age. Neurologic manifestations may be the earliest and often the only manifestation of hereditary or acquired cblC defect. Peripheral neuropathy remains a classical but underdiagnosed complication of cblC defect, especially in late-onset cblC disease caused by mutations in the methylmalonic aciduria type C and homocysteinemia (MMACHC) gene. So the clinical, electrophysiological, and pathological characteristics of late-onset cblC disease are not well-known.Methods: A retrospective study of patients with late-onset cblC disease was conducted at our hospital on a 3-year period. The neuropathy was confirmed by the nerve conduction study. Sural biopsies were performed in 2 patients.Results: Eight patients were identified, with a mean onset age of 16.25 ± 6.07 years. All patients had methylmalonic aciduria, homocysteinemia, compound heterozygous MMACHC gene mutations were detected in all patients, and 7/8 patients with c.482G>A mutation. One patient concomitant with homozygote c.665C>T mutation in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. All patients showed limb weakness and cognitive impairment. Five patients had possible sensorimotor axonal polyneuropathy predominantly in the distal lower limbs. Sural biopsies showed loss of myelinated and unmyelinated fibers. Electro microscopy revealed crystalline-like inclusions bodies in Schwann cells and axonal degeneration.Conclusion: Late-onset cblC disease had possible heterogeneous group of distal axonal neuropathy. c.482G>A mutation is a hot spot mutation in late-onset cblC disease.

When to measure plasma homocysteine and how to place it in context: The homocystinurias.

This review presents clinical patterns that should trigger homocysteine measurement in blood, as well as the further diagnostic work-up focused on inborn errors of metabolism and disorders of vitamin B12 (cobalamin) absorption and supply. The numerous conditions (e.g. cardiovascular disease, Alzheimer’s disease) for which mild-to-moderate hyperhomocysteinaemia caused by genetic polymorphisms or acquired reasons is considered a risk factor are beyond the scope of this review.Homocysteine is a sulphur-containing amino acid, which is derived from the amino acid methionine. Homocysteine is either trans-sulphurated to form cystathionine and then cysteine, or re-methylated to methionine. The trans-sulphuration reaction depends on the enzyme cystathionine beta synthase and its cofactor vitamin B6. The re-methylation reaction not only involves the enzymes methionine synthase and methionine synthase reductase but also depends on the cofactor cobalamin and on the provision of methyl groups from the folate cycle. Because the homocysteine–methionine cycle provides for the vast majority of methyl groups in the body, it is central to numerous pathways that depend on methyl group supply, such as creatine synthesis or DNA methylation. Based on this premise, the severity of clinical presentations of inborn errors of metabolism, such as classical homocystinuria or the cobalamin C (cblC) defect, affecting this pathway is unsurprising.

Early neurodevelopmental characterization in children with cobalamin C/defect.

Cobalamin C (cblC) defect is the most common inherited disorder of cobalamin metabolism. Developmental delay, behavioral problems, and maculopathy are common, but they have not been systematically investigated. The aim of this study was to define early neurodevelopment in cblC patients and the possible contribution of different factors, such as mode of diagnosis, age at diagnosis, presence of brain lesions and epilepsy. Children up to the age of 4 years with a visual acuity ≥1/10 were evaluated using the Griffiths' Mental Development Scales. Eighteen children were enrolled (age range 12-48 months). Four were diagnosed by newborn screening (NBS); in the others mean age at diagnosis was 3.5 months (range 0.3-18 months). Eight had seizures: three in the first year, and five after the second year of life. Fourteen had brain lesions on magnetic resonance imaging (MRI). Neurovisual assessment evidenced low visual acuity (<3/10) in 4/18. NBS diagnosed patients had higher general and subquotients neurodevelopmental scores, normal brain MRI, and no epilepsy. The others showed a progressive reduction of the developmental quotient with age and language impairment, which was evident after 24 months of age. Our findings showed a progressive neurodevelopmental deterioration and a specific fall in language development after 24 months in cblC defect. The presence of brain lesions and epilepsy was associated with a worst neurodevelopmental outcome. NBS, avoiding major disease-related events and allowing an earlier treatment initiation, appeared to have a protective effect on the development of brain lesions and to promote a more favorable neurodevelopment.

Prospective evaluation of pregnancy outcome in an Italian woman with late-onset combined homocystinuria and methylmalonic aciduria

BackgroundCobalamin metabolism disorders are rare, inherited diseases which cause megaloblastic anaemia and other clinical manifestations. Early diagnosis of these conditions is essential, in order to allow appropriate treatment as early as possible.Case presentationHere we report the case of a patient who was apparently healthy until the age of 20, when she presented with impaired renal function and normocytic anaemia. At the age of 34, when her first pregnancy resulted in an intrauterine death of a morphologically normal growth-restricted foetus, she was diagnosed with homocystinuria and methylmalonic aciduria due to cyanocobalamin C (cblC) defect, which was confirmed by molecular investigation. Consequently, hydroxocobalamin was administered to correct homocysteine plasma levels. This treatment was efficacious in lowering homocysteine plasma levels and restored anaemia and renal function. During a second pregnancy, the patient was also administered a prophylactic dose of low molecular -weight heparin. The pregnancy concluded with a full-term delivery of a healthy male.ConclusionsThis case emphasises the importance of awareness and appropriate management of rare metabolic diseases during pregnancy. We suggest that women with late-onset cblC defect can have a positive pregnancy outcome if this metabolic disease is treated adequately.

Molecular genetic characterization of cblC defects in 126 pedigrees and prenatal genetic diagnosis of pedigrees with combined methylmalonic aciduria and homocystinuria

BackgroundWe sought to analyse MMACHC variants among 126 pedigrees with cobalamin (cbl) C deficiency and combined methylmalonic aciduria and homocystinuria by Sanger sequencing, characterize the spectrum of MMACHC gene variants, and perform prenatal genetic diagnosis by chorionic villus sampling among these pedigrees.MethodsPeripheral blood was collected from 126 probands and their parents who visited the Genetic Counseling Clinic at our hospital between January 2014 and December 2017, and DNA was extracted from the blood. Then, we amplified the coding sequence and splicing regions of the MMACHC gene by PCR, and the PCR products were further sequenced to detect the variants in each pedigree. In 62 families, pregnant women were subjected to chorionic villus sampling for prenatal genetic diagnosis.ResultsIn total, 31 distinct variants were detected in the 126 pedigrees, and the most frequent variants were c.609G > A (p.Trp203Ter), c.658_660delAAG (p.Lys220del), c.567dupT (p.Ile190Tyrfs*13) and c.80A > G (p.Gln27Arg). Two of these variants have not been previously reported in the literature. One variant [c.463_465delGGG (p.Gly155del)] is a small-scale deletion, and the other variant [c.637G>T(p.Glu213Ter)] is a nonsense mutation. Among the 62 pedigrees who received a prenatal diagnosis, 16 foetuses were normal, 34 foetuses were carriers of heterozygous variants, and the remaining 12 foetuses harboured compound heterozygous variants or homozygous variants. Couples whose foetuses were normal or carriers continued the pregnancy, whereas couples whose foetuses harboured compound heterozygous variants or homozygous variants decided to terminate the pregnancy. The follow-up results were consistent with the prenatal diagnosis.ConclusionsTwo novel MMACHC variants were identified, and prenatal genetic diagnosis is an accurate and convenient method that helps avoid the delivery of combined methylmalonic aciduria and homocystinuria patients.

Favorable course of previously undiagnosed Methylmalonic Aciduria with Homocystinuria (cblC type) presenting with pulmonary hypertension and aHUS in a young child: a case report

BackgroundCobalamin C (cblC) defect is the most common inborn error of Vitamin B12 metabolism often causing severe neurological, renal, gastrointestinal and hematological symptoms. Onset with pulmonary hypertension (PAH) and atypical hemolytic-uremic syndrome (aHUS) is rare.Case presentationWe describe the case of a 2-years old child, previously in good health, admitted to the hospital with severe respiratory symptoms, rapid worsening of clinical conditions, O2 desaturation and palmo-plantar edema. The patient showed PAH and laboratory findings compatible with aHUS. cblC defect, an inborn error of metabolism, was identified as the cause of all the symptoms described (cardiac, respiratory and renal involvement). Results of neonatal screening for inborn errors of metabolism had been negative.Administration of IM OHCbl (intramuscular hydroxocobalamin), oral betaine and symptomatic treatment with diuretics and anti-hypertensive systemic and pulmonary drugs induced dramatic improvement of both cardiac and systemic symptoms.ConclusionsIn this case of cblC defect the metabolic treatment completely reverted symptoms of aHUS and PAH. The course was favorable, and the prognosis is what we foresee for the future.

Heterogeneous phenotypes, genotypes, treatment and prevention of 1 003 patients with methylmalonic acidemia in the mainland of China

Objective: To analyz the current situation of the diagnosis, treatment and prevention of methylmalonic acidemia, the phenotypes, biochemical features and genotypes of the patients in the mainland of China, were investigated. Methods: Tottally 1 003 patients of methylmalonic acidemia from 26 provinces and municipalities of the mainland of China were enrolled. The clinical data, biochemical features and gene mutations were studied. Blood aminoacids and acylcarnitines, urine organic acids, and plasma total homocysteine were determined for the biochemical diagnosis. Gene analyses were performed for the genetic study of 661 patients. The patients were treated with individual intervention and long-term follow up. Prenatal diagnoses were carried out for 165 fetuses of the families. Results: Among 1 003 patients (580 boys and 423 girls), 296 cases (29.5%) had isolated methylmalonic acidemia; 707 cases (70.5%) had combined homocysteinemia; 59 patients (5.9%) were detected by newborn screening; 944 patients (94.1%) had the onset at the ages from several minutes after birth to 25 years and diagnosed at 3 days to 25 years of age. The main clinical presentations were psychomotor retardation and metabolic crisis. Multi-organ damage, including hematological abnormalities, pulmonary hypertension, kidney damage, were found. MMACHC, MUT, MMAA, MMAB, HCFC1, SUCLG1, SUCLA2 mutations were found in 631 patients (96.6%) out of 661 patients who accepted gene analysis. MMACHC mutations were detected in 460 patients (94.7%) out of 486 cases of methylmalonic acidemia combined with homocysteinemia. MUT mutations were found in 158 (90.3%) out of 169 cases of isolated methylmalonic acidemia. The development of 59 patients detected by newborn screening were normal; 918 cases (97.2%) were diagnosed after onset accepted the treatment. Forty-five of them completely recovered with normal development. Twenty-six patients (2.7%) died; 873 (92.5%) patients had mild to severe psychomotor retardation. Methylmalonic acidemia were found in 35 out of 165 fetuses by metabolites assay of amniotic fluid and amniocytes gene analysis. Conclusion: Combined methylmalonic acidemia and homocysteinemia is the common type of methylmalonic acidemia in the mainland of China. CblC defect due to MMACHC mutations is the most common type of methylmalonic acidemia combined with homocysteinemia. MUT gene mutations are frequent in the patients with isolated methylmalonic acidemia. Newborn screening is key for the early diagnosis and the better outcome. Combined diagnosis of biochemical assays and gene analysis are reliable for the prenatal diagnosis of methylmalonic acidemia.

Prenatal diagnosis of methylmalonic acidemia using test of homocysteine and mass spectrometry in amniotic fluid

Objective To explore the value of the combination of homocysteine analysis, liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC/MS) in the prenatal diagnosis of combined methylmalonic acidemia and homocystinuria (cblC defect) in amniotic fluid. Methods This is a retrospective study of 187 cases of pregnancies that came to our hospital for prenatal diagnosis between 2014/01-2017/03, among which 78 cases′ probands were cblC defect patients and 109 cases′ probands were not organic academia patients(control group). Amniotic fluid samples from pregnant women were obtained at 16-24 weeks of gestation. Propionylcarnitine (C3) and acetylcarnitine (C2) were measured by LC-MS/MS, methylmalonic acid and methylcitric acid were analyzed by GC/MS, and homocysteine was determined by fluorescence polarization immunoassay. Some pregnancies received MMACHC gene sequencing with cultured cells from amniotic fluid. Data were analyzed using Mann-Whitney U and Kruskal-Wallis H tests. Results Among those 78 pregnant women whose probands were diagnosed to be cblC defect, 24 cases were diagnosed to be cblC defect (positive group) and 54 pregnant women were diagnosed to be negative (negative group). In positive group, levels of homocysteine, C3, C3/C2, methylmalonic acid and methylcitric acid were all significantly higher than their normal reference ranges, negative group and control group (P values are 0.00). Cases that were diagnosed to be cblC defect by MMACHC gene sequencing were all turned out to be positive in the tests of the above metabolites in amniotic fluid. Cases with negative results of the metabolites were all excluded to be cblC defect by gene sequencing. Besides, 2 cases of pregnancies were diagnosed to be positive by homocysteine and mass spectrometric analysis while only one mutation were detected by gene sequencing. Conclusions The combination of homocysteine, LC-MS/MS and GC/MS analysis in amniotic fluid turns out to be reliable for prenatal diagnosis of cblC defect, which may further cover the defect of prenatal diagnosis of those pregnancies whose probands′ gene mutation is unknown. (Chin J Lab Med, 2017, 40: 880-884) Key words: Amino acid metabolism, inborn errors; Prenatal diagnosis; Amniotic fluid; Homocysteine; Chromatography, liquid; Tandem mass spectrometry; Gas chromatography-mass spectrometry

Molecular picture of cobalamin C/D defects before and after newborn screening era.

Objective Birth prevalence of Cobalamin (Cbl) C or D defects in Portugal is an estimated 1:85,000, one of the highest worldwide. We compared the genotype/phenotype of patients identified with CblC or CblD before and after the implementation of expanded newborn screening. Methods Twenty-five Portuguese CblC/D patients, 14 symptomatic and 11 identified through screening, were diagnosed using gas chromatography or tandem mass spectrometry. Molecular characterization was performed through the study of MMACHC and MMADHC genes. Results The most common MMACHC mutation, c.271dupA, was present in 100% of MMACHC alleles of all CblC screened patients, in contrast with the 61% identified before expanded newborn screening. All studied cases (except one, who presented a CblD deficiency) presented a CblC defect. More CblC late-onset patients were diagnosed before the introduction of newborn screening than in the post newborn screening era, probably because some early onset patients died without a definitive diagnosis. Conclusion The molecular data found in this cohort contribute to the improvement of screening and diagnosis of Cbl defects and would enable a confirmatory diagnosis of these patients, reducing the need for complex, costly, laborious, and time-consuming biochemical/enzymatic tests.

Pathobiological Insights into Neurological Involvement in Cobalamin C Deficiency

Cobalamin C (Cbl-C) defects are inherited autosomal recessive disorders of vitamin B 12 (or cyanocobalamin [CNCbl]) metabolism. These defects are caused by mutations in the methylmalonic aciduria and homocystinuria Cbl-C type ( MMACHC ; MIM # 609831) gene located on chromosome 1p34.1, which catalyzes the reductive decyanation of CNCbl, thus impairing the biosynthesis of 5′-deoxyadenosylcobalamin, adenosylcobalamin, and methylcobalamin. This impairment results in methylmalonic acidemia [MMA; MIM # 277400] combined with hyperhomocysteinemia and hypomethioninemia . Clinically, Cbl-C defects are characterized by a constellation of systemic signs and symptoms, including neurological, cognitive, psychiatric, and thromboembolic events. Retinal phenotypes, including maculopathy, pigmentary retinopathy, and optic atrophy, are common in the early-onset form of the disease, but are rare in its adult-onset counterpart. Administration of hydroxocobalamin (OHCbl), betaine, and folinic acid represents main therapeutic approaches. No proven efficacy has been demonstrated for carnitine and dietary protein restrictions. Although early introduction of OHCbl is crucial, no standardized protocols regarding dose adjustment exist. Despite these measures, the long-term outcome is unsatisfactory especially in patients with early onset, who experience frequent progression of their neurological and ocular impairment. The unfavorable outcome suggests that a better understanding of the pathophysiology of the disease is needed to improve treatment protocols and to develop new therapeutic approaches.

Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines.

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency.