Clinical and Molecular Genetic Analysis with Methylmalonic Acidemia Combined with Homocystinuria.

BACKGROUND: Based on research, c.609G>A (p.W203X) is a universal mutation site for MMACHC in methylmalonic acidemia (MMA) combined with homocystinuria, cblC type (cblC disease), and c.467G>A (p.G156D) mutation in families with such disease have not yet been reported. To conduct clinical and molecular genetic analysis of a family with cblC disease. METHODS: This work followed the Declaration of Helsinki. All testing methods were performed under the informed consent of our children patients' parents. A second-generation cblC family with 5 members, was selected as the research subject, including sick siblings and parents and an older sister with normal phenotype, given newborn screening for acylcarnitine spectrum via liquid chromatography tandem mass spectrometry (LC-MS/MS), and diagnosed through combining urine organic acid with homocysteine detection via gas chromatography-mass spectrometry (GC-MS) with second-generation gene sequencing technology. The peripheral blood of five family members was collected for genomic DNA extraction, and the changes were screened in disease-related MMACHC sequence via PCR and direct DNA sequencing. RESULTS: The family conformed to the autosomal recessive inheritance, the proband and younger sister were cblC patients, diagnosed in February and at 22d given relevant treatment. The proband died, whereas the younger sister received follow-up treatment. Their parents and sister had normal phenotype. In 2 cases, there was compound heterozygous mutation in MMACHC called c.609G>A (p.W203X) nonsense mutation and c.467G>A (p.G156D) missense mutation in exon 4, while the father with normal phenotype had heterozygous mutation c.609G>A in exon 4 coding area. In its protein, the 203rd amino acid changed from tryptophan to a stop codon (p.W203 x). The normal mother and sister had a heterozygous mutation c.467G>A in exon 4 coding area. In its protein, the 156th amino acid changed from glycine to aspartic acid (p.G156D). CONCLUSIONS: The cblC family results from c.609G>A (p.W203X) and c.467G>A (p.G156D) compound heterozygous mutations in MMACHC, which has a pathogenic impact.

METHYLATION-ASSOCIATED PATHWAYS IN MACULAR TELANGIECTASIA TYPE 2 AND OPHTHALMOLOGIC FINDINGS IN PATIENTS WITH GENETIC METHYLATION DISORDERS.

PURPOSE: Serine (Ser) and glycine (Gly) levels were reported to differ between patients with macular telangiectasia type 2 (MacTel) compared with healthy controls. Because they are closely related to methylation metabolism, this report investigates methylation-associated metabolite levels in patients with MacTel and retinal changes in monogenetic methylation disorders. METHODS: Prospective, monocentric study on patients with MacTel and healthy controls underwent a standardized protocol including a blood draw. Methylation-associated metabolite levels in plasma were determined using targeted quantitative metabolomics. Furthermore, patient records of cystathionine beta-synthase, methylenetetrahydrofolate reductase, and methylmalonic aciduria and homocystinuria type C protein (MMACHC) deficiency were screened for reported retinal changes. RESULTS: In total, 29 patients with MacTel and 27 healthy controls were included. Patients with MacTel showed lower plasma Ser ( P = 0.02 and P = 0.01) and Gly ( P = 0.11 and P = 0.11) levels than controls. Principal component analyses revealed that methylation-associated metabolite, especially homocysteine, contributed to a distinct clustering of patients with MacTel. No retinal changes were seen in cystathionine beta-synthase (n = 1) and methylenetetrahydrofolate reductase (n = 2) deficiency, while two patients with MMACHC (n = 4) deficiency displayed extensive macular dystrophy. CONCLUSION: Patients with MacTel show distinct clustering of methylation-associated metabolite compared with controls. Of the three homocystinurias, only MMACHC resulted in macular dystrophy, possibly due to distinct compensatory pathways.

Late-onset methylmalonic acidemia and homocysteinemia (cblC disease): systematic review.

INTRODUCTION: Combined methylmalonic acidemia and homocystinuria, cblC type is an inborn error of intracellular cobalamin metabolism and the most common one. The age of onset ranges from prenatal to adult. The disease is characterised by an elevation of methylmalonic acid (MMA) and homocysteine and a decreased production of methionine. The aim is to review existing scientific literature of all late onset cblC patients in terms of clinical symptoms, diagnosis, and outcome. METHODS: A bibliographic database search was undertaken in PubMed (MEDLINE) complemented by a reference list search. We combined search terms regarding cblC disease and late onset. Two review authors performed the study selection, data extraction and quality assessment. RESULTS: Of the sixty-five articles included in this systematic review, we collected a total of 199 patients. The most frequent clinical symptoms were neuropathy/myelopathy, encephalopathy, psychiatric symptoms, thrombotic microangiopathy, seizures, kidney disease, mild to severe pulmonary hypertension with heart failure and thrombotic phenomena. There were different forms of supplementation used in the different studies collected and, within these studies, some patients received several treatments sequentially and/or concomitantly. The general outcome was: 64 patients recovered, 78 patients improved, 4 patients did not improve, or the disease progressed, and 12 patients died. CONCLUSIONS: Most scientific literature regarding the late onset cblC disease comes from case reports and case series. In most cases treatment initiation led to an improvement and even recovery of some patients. The lack of complete recovery underlines the necessity for increased vigilance in unclear clinical symptoms for cblC disease.

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

BACKGROUND: cblC 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: The 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.

Classical homocystinuria presenting with transient basal ganglia pathology and dystonia.

Classical homocystinuria is caused by pathogenic variants in the CBS gene leading to a deficiency of the vitamin B6-dependent enzyme cystathionine beta synthase. The disease is typically associated with high blood homocysteine concentrations. Clinical features include developmental delay/intellectual disability, psychiatric problems, thromboembolism, lens dislocation, and marfanoid habitus. We report on a child with classical homocystinuria presenting with acute episodes of dystonia and symmetrical basal ganglia abnormalities mimicking a mitochondrial disease. After starting treatment with vitamin B6, homocysteine levels rapidly normalized and dystonic episodes did not re-occur. Moreover, brain-imaging findings almost completely disappeared. The case illustrates that homocystinuria should be considered as a treatable differential diagnosis of dystonia.

Combined Newborn Screening Allows Comprehensive Identification also of Attenuated Phenotypes for Methylmalonic Acidurias and Homocystinuria.

Newborn screening (NBS) programs are effective measures of secondary prevention and have been successively extended. We aimed to evaluate NBS for methylmalonic acidurias, propionic acidemia, homocystinuria, remethylation disorders and neonatal vitamin B12 deficiency, and report on the identification of cofactor-responsive disease variants. This evaluation of the previously established combined multiple-tier NBS algorithm is part of the prospective pilot study "NGS2025" from August 2016 to September 2022. In 548,707 newborns, the combined algorithm was applied and led to positive NBS results in 458 of them. Overall, 166 newborns (prevalence 1: 3305) were confirmed (positive predictive value: 0.36); specifically, methylmalonic acidurias (N = 5), propionic acidemia (N = 4), remethylation disorders (N = 4), cystathionine beta-synthase (CBS) deficiency (N = 1) and neonatal vitamin B12 deficiency (N = 153). The majority of the identified newborns were asymptomatic at the time of the first NBS report (total: 161/166, inherited metabolic diseases: 9/14, vitamin B12 deficiency: 153/153). Three individuals were cofactor-responsive (methylmalonic acidurias: 2, CBS deficiency: 1), and could be treated by vitamin B12, vitamin B6 respectively, only. In conclusion, the combined NBS algorithm is technically feasible, allows the identification of attenuated and severe disease courses and can be considered to be evaluated for inclusion in national NBS panels.

Evaluation of the clinical, biochemical, and genetic presentation of neonatal and adult-onset 5,10-methylene tetrahydrofolate reductase (MTHFR) deficiency in patients from Pakistan.

OBJECTIVES: To study the biochemical, clinical and molecular characteristics of 5,10- methylenetetrahydrofolate reductase (MTHFR) deficiency in Pakistani patients from a single center. METHODS: Medical charts, urine organic acid chromatograms, plasma methionine and Hcys levels, and molecular testing results of MTHFR gene of patients presenting at the Biochemical Genetics Clinic, AKUH from 2016 to 2022 were reviewed. RESULTS: Neonatal MTHFR deficiency was found in five patients. The median (IQR) age of symptom onset and diagnosis were 18 (8.5-22) and 26 (16.5-31) days. The median lag between symptom onset and diagnosis was 8 (4.5-12.5) days. The median age of treatment initiation and duration of treatment were 26 (16.5-49) and 32 (25.5-54) days. The most common clinical features were lethargy, poor feeding, and seizures. The MTHFR gene sequencing revealed homozygous variants p.K510K, p.R567*, and p.R157W. Renal insufficiency manifesting as elevated serum creatinine and responding to betaine therapy was noted in one patient. This has not been previously reported in neonatal MTHFR deficiency and may reflect engagement of alternate pathways of remethylation. Adult onset MTHFR deficiency was found in six patients, with a heterogeneous neurological presentation. The median lag between symptoms onset and diagnosis was 7 (3-11) years. MTHFR gene sequencing revealed homozygous variant p.A195V in five patients from one family and p.G261V in the other. Two of the five reported variants are novel that include p.R157W and p.G261V. CONCLUSIONS: Eleven patients of this rare disorder from a single center indicate the need for clinical awareness and appropriate biochemical evaluation to ensure optimal outcomes.

Adolescent/Adult-Onset Leukodystrophy with MTHFR Deficiency - A Treatable Cause.

Leukodystrophies and genetic leukoencephalopathies comprise a diverse group of neurodegenerative disorders of white matter with a wide age of onset and phenotypic spectrum. Patients with white matter abnormalities detected on magnetic resonance imaging (MRI) often present a diagnostic challenge to both general and specialist neurologists. Patients typically present with a progressive syndrome including various combinations of cognitive impairment, movement disorders, ataxia, and upper motor neuron signs. There are a number of important and treatable acquired causes for this imaging and clinical presentation; one of the causes is hyperhomocystinemia due to 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency. MTHFR deficiency is a genetic disorder that can occur at any age and can be easily detected by increased serum homocysteine levels and it is a treatable cause. Metabolic therapies like betaine were shown to be effective in children and adults to stop the disease progression and sometimes improve neurologic disabilities. Herein, we report a 16-year-old male with gradually progressive spastic paraparesis with history of cerebral venous sinus thrombosis and poor scholastic performance. The patient was diagnosed with MTHFR enzyme deficiency presenting as leukodystrophy with spastic paraparesis, which is treatable on early diagnosis. Treatment with betaine produced a rapid decline of homocysteine and improved the condition.

Contribution of Genetic Test to Early Diagnosis of Methylenetetrahydrofolate Reductase (MTHFR) Deficiency: The Experience of a Reference Center in Southern Italy.

BACKGROUND: the deficiency of 5,10-Methylenetetrahydrofolate reductase (MTHFR) constitutes a rare and severe metabolic disease and is included in most expanded newborn screening (NBS) programs worldwide. Patients with severe MTHFR deficiency develop neurological disorders and premature vascular disease. Timely diagnosis through NBS allows early treatment, resulting in improved outcomes. METHODS: we report the diagnostic yield of genetic testing for MTHFR deficiency diagnosis, in a reference Centre of Southern Italy between 2017 and 2022. MTHFR deficiency was suspected in four newborns showing hypomethioninemia and hyperhomocysteinemia; otherwise, one patient born in pre-screening era showed clinical symptoms and laboratory signs that prompted to perform genetic testing for MTHFR deficiency. RESULTS: molecular analysis of the MTHFR gene revealed a genotype compatible with MTHFR deficiency in two NBS-positive newborns and in the symptomatic patient. This allowed for promptly beginning the adequate metabolic therapy. CONCLUSIONS: our results strongly support the need for genetic testing to quickly support the definitive diagnosis of MTHFR deficiency and start therapy. Furthermore, our study extends knowledge of the molecular epidemiology of MTHFR deficiency by identifying a novel mutation in the MTHFR gene.

Multiplexing Homocysteine into First-Tier Newborn Screening Mass Spectrometry Assays Using Selective Thiol Derivatization.

BACKGROUND: Classical homocystinuria (HCU) results from deficient cystathionine ß-synthase activity, causing elevated levels of Met and homocysteine (Hcy). Newborn screening (NBS) aims to identify HCU in pre-symptomatic newborns by assessing Met concentrations in first-tier screening. However, unlike Hcy, Met testing leads to a high number of false-positive and -negative results. Therefore, screening for Hcy directly in first-tier screening would be a better biomarker for use in NBS. METHODS: Dried blood spot (DBS) quality control and residual clinical specimens were used in analyses. Several reducing and maleimide reagents were investigated to aid in quantification of total Hcy (tHcy). The assay which was developed and validated was performed by flow injection analysis-tandem mass spectrometry (FIA-MS/MS). RESULTS: Interferents of tHcy measurement were identified, so selective derivatization of Hcy was employed. Using N-ethylmaleimide (NEM) to selectively derivatize Hcy allowed interferent-free quantification of tHcy by FIA-MS/MS in first-tier NBS. The combination of tris(2-carboxyethyl)phosphine (TCEP) and NEM yielded significantly less matrix effects compared to dithiothreitol (DTT) and NEM. Analysis of clinical specimens demonstrated that the method could distinguish between HCU-positive, presumptive normal newborns, and newborns receiving total parenteral nutrition. CONCLUSIONS: Here we present the first known validated method capable of screening tHcy in DBS during FIA-MS/S first-tier NBS.

Elevated homocysteine levels: What inborn errors of metabolism might we be missing?

Elevated total plasma homocysteine (hyperhomocysteinemia) is a marker of cardiovascular, thrombotic, and neuropsychological disease. It has multiple causes, including the common nutritional vitamin B12 or folate deficiency. However, some rare but treatable, inborn errors of metabolism (IEM) characterized by hyperhomocysteinemia can be missed due to variable presentations and the lack of awareness. The aim of this study is to identify undiagnosed IEM in adults with significantly elevated homocysteine using key existing clinical data points, then IEM specific treatment can be offered to improve outcome. We conducted a retrospective study with data mining and chart review of patients with plasma total homocysteine >30 µmol/L over a two-year period. We offer biochemical and genetic testing to patients with significant hyperhomocysteinemia without a clear explanation to diagnose IEM. We identified 22 subjects with significant hyperhomocysteinemia but no clear explanation. Subsequently, we offered genetic testing to seven patients and diagnosed one patient with classic homocystinuria due to cystathionine beta-synthase deficiency. With treatment, she lowered her plasma homocysteine and improved her health. This study stresses the importance of a thorough investigation of hyperhomocysteinemia in adults to identify rare but treatable IEM. We propose a metabolic evaluation algorithm for elevated homocysteine levels.

Angiopathy and Stroke Associated with Homocystinuria in Childhood.

A previously healthy 13 year-old boy presented with acute-onset headaches, aphasia and right-sided hemiparesis. Imaging showed cerebral ischemic infarction due to bilateral carotid occlusion, and investigation for stroke etiology diagnosed homocystinuria. Homocystinuria is an autosomal recessive condition that affects the metabolism of the amino acid methionine due to an enzyme deficiency. This disorder involves multiple organs systems, and complications include thromboembolic events, ectopia lentis, mental retardation, and skeletal abnormalities. The early diagnosis and treatment of hyperhomocystinemia can significantly improve outcomes. Therefore, metabolic screening for homocystinuria is strongly recommended for children presenting with stroke.

A teenager with combined methylmalonic aciduria and homocystinuria (CblC type) presenting with neurological symptoms and congenital heart diseases: a case report.

Combined methylmalonic acidemia and homocystinuria, is a rare autosomal recessive disorder due to defective intracellular cobalamin metabolism. We report an 18-year-old Chinese male who presented with hypermyotonia, seizures, and congenital heart diseases. Mutation analysis revealed c.365A>T and c.482 G>A mutations in the MMACHC gene, diagnosed with methylmalonic aciduria and homocystinuria (CblC type). After treatment with vitamin B12, L-carnitine, betaine, and folate, which resulted in an improvement in his clinical symptoms and laboratory values. This case emphasizes that inborn errors of metabolism should be considered for a teenager presenting with challenging or neurologic symptoms, especially when combined with unexplained heart diseases.

Late-onset cblC deficiency around puberty: a retrospective study of the clinical characteristics, diagnosis, and treatment.

BACKGROUND: cblC deficiency is the most common type of methylmalonic aciduria in China. Late-onset patients present with various non-specific symptoms and are usually misdiagnosed. The purpose of this study is to investigate the clinical features of patients with late-onset cblC deficiency and explore diagnosis and management strategies around puberty. RESULTS: This study included 56 patients (35 males and 21 females) with late-onset cblC deficiency who were admitted to our clinic between 2002 and September 2021. The diagnosis was confirmed by metabolic and genetic tests. The clinical and biochemical features, disease triggers, outcome, and associated genetic variants were examined. The onset age ranged from 10 to 20 years (median age, 12 years). Fifteen patients (26.8%) presented with symptoms after infection or sports training. Further, 46 patients (82.1%) had neuropsychiatric diseases; 11 patients (19.6%), cardiovascular diseases; and 6 patients (10.7%), pulmonary hypertension. Renal damage was observed in 6 cases (10.7%). Genetic analysis revealed 21 variants of the MMACHC gene in the 56 patients. The top five common variants detected in 112 alleles were c.482G > A (36.6%), c.609G > A (16.1%), c.658_660delAAG (9.8%), c.80A > G (8.0%), and c.567dupT (6.3%). Thirty-nine patients carried the c.482G > A variant. Among 13 patients who exhibited spastic paraplegia as the main manifestation, 11 patients carried c.482G > A variants. Six patients who presented with psychotic disorders and spastic paraplegia had compound heterozygotic c.482G > A and other variants. All the patients showed improvement after metabolic treatment with cobalamin, L-carnitine, and betaine, and 30 school-aged patients returned to school. Two female patients got married and had healthy babies. CONCLUSIONS: Patients with late-onset cblC deficiency present with a wide variety of neuropsychiatric symptoms and other presentations, including multiple organ damage. As a result, cb1C deficiency can easily be misdiagnosed as other conditions. Metabolic and genetic studies are important for accurate diagnosis, and metabolic treatment with cobalamin, L-carnitine, and betaine appears to be beneficial.

Delayed diagnosis of homocystinuria presenting with coronavirus disease 2019 in a 17-year-old boy.

Homocystinuria is a treatable autosomal recessive inherited disorder. This condition may cause life-threatening complications such as thromboembolic events. Coronavirus disease 2019 (COVID-19) is associated with an increased risk of venous thromboembolic events. Here, we report a case of late diagnosis of homocystinuria presenting with deep venous thrombosis and COVID-19. This study highlights a sustained high index of suspicion for homocystinuria to prevent severe thromboembolic complications.

Homocystinuria and ocular complications - A review.

Homocystinuria is a rare metabolic inborn disorder caused due to dysfunctional cystathionine ß-synthase (CBS) enzyme activity, thus resulting in elevated levels of methionine and homocysteine in the blood and urine. The timely recognition of this rare metabolic disorder and prompt methionine-restricted diet are crucial in lessening the systemic consequences. The recalcitrant cases have a higher risk for cardiovascular diseases, neurodegenerative diseases, neural tube defects, and other severe clinical complications. This review aims to present the ophthalmic spectrum of homocystinuria and its molecular basis, the disease management, as well as the current and potential treatment approaches with a greater emphasis on preventive strategies.