Mutations in Hcfc1 and Ronin result in an inborn error of cobalamin metabolism and ribosomopathy

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

Easily Misdiagnosed cblC Deficiency in Adolescents: the Clinical and Metabolic Studies

Purpose: Adolescents are easily attacked by potential inherited metabolic disorders. cblC deficiency is the most common type of methylmalonic aciduria in China. The late-onset patients present with varied non-specific symptoms and usually being misdiagnosed. The purpose of this study is to investigate the clinical features of patients with adolescence-onset cblC deficiency and explore the prevention and control strategies. Methods: Fifty-seven patients (34 males and 23 females) with adolescence-onset cblC deficiency were admitted in our clinic from 2002 to September 2021. The diagnosis was confirmed by metabolic and genetic tests. The clinical and biochemical features, disease triggers, outcome and genotypes-phenotypes correlation were examined.Results: The onset ages ranged from 10 to 25 years old (median age was 12 years). 16 cases (28.0%) presented with symptoms after infection or sports training. 46 patients (80.7%) had neuropsychiatric diseases. 14 patients (24.6%) displayed cardiovascular diseases. Five cases (8.9%) showed pulmonary hypertension. Renal damage was observed in seven cases (12.3%). 23 mutations were identified from the MMACHC gene of 57 patients. 37 patients demonstrated c.482G>A (64.9%) and 16 cases had c.609G>A (26.3%). Among 13 patients that exhibited spastic paraplegia as a main manifestation, 10 patients had c.482G>A (76.9%). Five patients presented with psychotic disorders and spastic paraplegia with c.482G>A. All patients improved after metabolic treatment with cobalamin, L-carnitine, and betaine. 30 school-aged patients returned to school. Two patients were married and had healthy babies.Conclusion: Patients with adolescence-onset cblC deficiency presented with varied neuropsychiatric symptoms or multiple organ damage. Metabolic studies and individualized treatment are keys to improve the outcome of the patients.

Genetic testing is necessary for correct diagnosis and treatment in patients with isolated methylmalonic aciduria: a case report

Background Isolated methylmalonic aciduria can be caused by pathogenic mutations in the gene for methylmalonyl-CoA mutase or in the genes encoding enzymes involved in the intracellular metabolism of cobalamin. Some of these mutations may be cobalamin responsive. The type of methylmalonic aciduria cannot always be assumed from clinical manifestation and the responsiveness to cobalamin has to be assessed for appropriate cobalamin administration, or to avoid unnecessary treatment. The cases presented herein highlight the importance of genetic testing in methylmalonic aciduria cases and the need for standardisation of the in vivo cobalamin-responsiveness assessment. Case presentation We describe two patients who presented in the first week of life with rapid neurological deterioration caused by metabolic acidosis with severe hyperammonaemia requiring extracorporeal elimination in addition to protein restriction, energy support, carnitine, and vitamin B12 treatment. The severity of the clinical symptoms and high methylmalonic acid concentrations in the urine (>30,000 μmol/mmol of creatinine) without hyperhomocysteinaemia in both of our patients suggested isolated methylmalonic aciduria. Based on the neonatal manifestation and the high methylmalonic acid urine levels, we assumed the cobalamin non-responsive form. The in vivo test of responsiveness to cobalamin was performed in both patients. Patient 1 was evaluated as non-responsive; thus, intensive treatment with vitamin B12 was not used. Patient 2 was responsive to cobalamin, but the dose was decreased to 1 mg i.m. every two weeks with daily oral treatment due to non-compliance. Genetic tests revealed bi-allelic mutations in the genes MMAB and MMAA in Patient 1 and 2, respectively. Based on these results, we were able to start intensive treatment with hydroxocobalamin in both patients. After the treatment intensification, there was no acute crisis requiring hospitalisation in Patient 1, and the urine methylmalonic acid levels further decreased in Patient 2. Conclusions Despite carrying out the in vivo test of responsiveness to cobalamin in both patients, only the results of molecular genetic tests led us to the correct diagnosis and enabled intensive treatment with hydroxocobalamin. The combination of the standardized in vivo test of cobalamin responsiveness and genetic testing is needed for accurate diagnosis and appropriate treatment of isolated methylmalonic aciduria.

Insights into energy balance dysregulation from a mouse model of methylmalonic aciduria

Inherited disorders of mitochondrial metabolism, including isolated methylmalonic aciduria (MMAuria), present unique challenges to energetic homeostasis by disrupting energy producing pathways. To better understand global responses to energy shortage, we investigated a hemizygous mouse model of methylmalonyl-CoA mutase (Mmut) type MMAuria. We found Mmut mutant mice to have reduced appetite, energy expenditure and body mass compared to littermate controls, along with a relative reduction in lean mass but increase in fat mass. Brown adipose tissue showed a process of whitening, in line with lower body surface temperature and lesser ability to cope with cold challenge. Mutant mice had dysregulated plasma glucose, delayed glucose clearance and a lesser ability to regulate energy sources when switching from the fed to fasted state, while liver investigations indicated metabolite accumulation and altered expression of peroxisome proliferator-activated receptor and Fgf21-controlled pathways. Together, these indicate hypometabolism, energetic inflexibility and increased stores at the expense of active tissue as energy shortage consequences.

Combined Malonic and Methylmalonic Aciduria Due to ACSF3 Variants Results in Benign Clinical Course in Three Chinese Patients

Introduction: Combined malonic and methylmalonic aciduria (CMAMMA) is a rare metabolic disease caused by biallelic variants in ACSF3 gene. The clinical phenotype is highly heterogeneous in this disorder, ranging from asymptomatic to severe symptoms. No cases with CMAMMA were reported in China.Materials and Methods: In this study, three Chinese pediatric patients were diagnosed with CMAMMA unexpectedly while being treated for other ailments. To better characterize CMAMMA in a Chinese population, we made a multidimensional analysis with detailed clinical phenotype, semi-quantitative detection of urine organic acid, and analysis of ACSF3 gene variants.Results: The clinical presentation of these patients is quite different; their main complaints were anemia, jaundice, or abnormal urine test, respectively. They showed no symptoms of the classic methylmalonic academia, but urine organic acid analysis showed elevated malonic acid and methylmalonic acid in all the patients repeatedly. Variants were found at four sites in ACSF3 gene. Patient 1 carried the compound heterogeneous variant c.689G> A (p.Trp230*)/c.1456G> A (p.Ala486Thr). A compound heterozygous variant c.473C> T (p.Pro158Leu)/c.1456G> A (p.Ala486Thr) was identified in patient 2. Patient 3 harbored a novel homozygous variant c.1447A> G (p.Lys483Glu).Conclusions: Three Chinese patients were diagnosed with CMAMMA caused by ACSF3 variants. Their clinical course revealed that CMAMMA can be a benign condition that does not affect individual growth and development, but severe clinical phenotype may appear when other triggers exist. This study systematically elaborates CMAMMA in a Chinese population for the first time, broadens the spectrum of gene variant, and provides a strong basis for the etiological study of this disorder.

Spectrum and characterization of bi-allelic variants in MMAB causing cblB-type methylmalonic aciduria

Pathogenic variants in MMAB cause cblB-type methylmalonic aciduria, an autosomal-recessive disorder of propionate metabolism. MMAB encodes ATP:cobalamin adenosyltransferase, using ATP and cob(I)alamin to create 5’-deoxyadenosylcobalamin (AdoCbl), the cofactor of methylmalonyl-CoA mutase (MMUT). We identified bi-allelic disease-causing variants in MMAB in 97 individuals with cblB-type methylmalonic aciduria, including 33 different and 16 novel variants. Missense changes accounted for the most frequent pathogenic alleles (p.(Arg186Trp), N = 57; p.(Arg191Trp), N = 19); while c.700C > T (p.(Arg234*)) was the most frequently identified truncating variant (N = 14). In fibroblasts from 76 affected individuals, the ratio of propionate incorporation in the presence and absence of hydroxocobalamin (PI ratio) was associated to clinical cobalamin responsiveness and later disease onset. We found p.(Arg234*) to be associated with cobalamin responsiveness in vitro, and clinically with later onset; p.(Arg186Trp) and p.(Arg191Trp) showed no clear cobalamin responsiveness and early onset. Mapping these and novel variants onto the MMAB structure revealed their potential to affect ATP and AdoCbl binding. Follow-up biochemical characterization of recombinant MMAB identified its three active sites to be equivalent for ATP binding, determined by fluorescence spectroscopy (Kd = 21 µM) and isothermal calorimetry (Kd = 14 µM), but function as two non-equivalent AdoCbl binding sites (Kd1 = 0.55 μM; Kd2 = 8.4 μM). Ejection of AdoCbl was activated by ATP (Ka = 24 µM), which was sensitized by the presence of MMUT (Ka = 13 µM). This study expands the landscape of pathogenic MMAB variants, provides association of in vitro and clinical responsiveness, and facilitates insight into MMAB function, enabling better disease understanding.

PRDX1 gene-related epi-cblC disease is a common type of inborn error of cobalamin metabolism with mono- or bi-allelic MMACHC epimutations

Background The role of epigenetics in inborn errors of metabolism (IEMs) is poorly investigated. Epigenetic changes can contribute to clinical heterogeneity of affected patients but could also be underestimated determining factors in the occurrence of IEMs. An epigenetic cause of IEMs has been recently described for the autosomal recessive methylmalonic aciduria and homocystinuria, cblC type (cblC disease), and it has been named epi-cblC. Epi-cblC has been reported in association with compound heterozygosity for a genetic variant and an epimutation at the MMACHC locus, which is secondary to a splicing variant (c.515-1G > T or c.515-2A > T) at the adjacent PRDX1 gene. Both these variants cause aberrant antisense transcription and cis-hypermethylation of the MMACHC gene promotor with subsequent silencing. Until now, only nine epi-cblC patients have been reported. Methods We report clinical/biochemical assessment, MMACHC/PRDX1 gene sequencing and genome-wide DNA methylation profiling in 11 cblC patients who had an inconclusive MMACHC gene testing. We also compare clinical phenotype of epi-cblC patients with that of canonical cblC patients. Results All patients turned out to have the epi-cblC disease. One patient had a bi-allelic MMACHC epimutation due to the homozygous PRDX1:c.515-1G > T variant transmitted by both parents. We found that the bi-allelic epimutation produces the complete silencing of MMACHC in the patient’s fibroblasts. The remaining ten patients had a mono-allelic MMACHC epimutation, due to the heterozygous PRDX1:c.515-1G > T, in association with a mono-allelic MMACHC genetic variant. Epi-cblC disease has accounted for about 13% of cblC cases diagnosed by newborn screening in the Tuscany and Umbria regions since November 2001. Comparative analysis showed that clinical phenotype of epi-cblC patients is similar to that of canonical cblC patients. Conclusions We provide evidence that epi-cblC is an underestimated cause of inborn errors of cobalamin metabolism and describe the first instance of epi-cblC due to a bi-allelic MMACHC epimutation. MMACHC epimutation/PRDX1 mutation analyses should be part of routine genetic testing for all patients presenting with a metabolic phenotype that combines methylmalonic aciduria and homocystinuria.