Methylmalonic Acidemia with Novel MUT Gene Mutations

A 5-year-old boy presented with recurrent episodes of fever, feeding problems, lethargy, from the age of 11 months, and poor weight gain. He was admitted and evaluated for metabolic causes and diagnosed as having methylmalonic acidemia (MMA). He was treated with vit B12 and carnitine supplements and has been on follow-up for the last 3 years. Mutation analysis by next generation sequencing (NGS), supplemented with Sanger sequencing, revealed two novel variants in the MUT gene responsible for MMA in exon 5 and exon 3, respectively. Recently he developed dystonic movements including orofacial dyskinesia. With advent of NGS, judicious use of NGS with Sanger sequencing can help identify causative possibly pathogenic mutations.


Case Presentation
e child presented for the rst time at the age of months, with complaints of fever, vomiting, poor feeding, and lethargy. On examination he had pallor and tachypnea and was drowsy. Further evaluation was suggestive of high anion-gap metabolic acidosis with ketonuria (urine ketones-+) with normal electrolytes, blood sugar ( mg/dl), vitamin B , and homocysteine. Plasma ammonia was units, and plasma lactate was . units. TMS was normal but urine GC-MS revealed elevated -OH propionic acid [ . retention time (RT)] as well as elevated methyl malonic acid [ . RT, Suppl Figure 1, in Supplementary Material available online at https://doi.org/ .
/ / ]. Since then this child was on low protein diet, carnitine, biotin, thiamine, and vitamin B injections. Child was therea er admitted on multiple occasions ( times) with acute decompensation and managed as per protocol. Mutational analysis was sent for MMA which showed single heterozygous missense variant c.
A>G (p.Arg Gly) in exon of MUT gene (genomic coordinates: chr : ) as a variant of uncertain signi cance. Chromosomal microarray analysis done did not reveal any major deletion or duplication which could disrupt the gene. Since exon and exon were not adequately covered by NGS, further evaluation by Sanger sequencing for targeted exons was done and a nd mutation in exon c. G>A (p.=) was identi ed. e variants were found to be damaging on SIFT database score (Suppl data). ey were also predicted to be deleterious on Polyphen-and Mutation Taster and not found in the ExAC database. MRI brain done at the age of years was showing multifocal cystic encephalomalacic changes with surrounding gliosis in deep white matter predominantly in frontoparietal regions ( Figure ). In latest admission child was found to have fresh neurological ndings in the form of perioral tremors, generalised hypertonia, and generalised dystonia with clonus with exaggerated deep tendon re exes. He was treated with intravenous dextrose and sodium bicarbonate and was continued on carnitine and injection of vitamin B . Plasma ammonia was units and lactate level was . units. MRI brain was repeated and revealed bilateral basal ganglia hyperintensities suggestive of metabolic stroke. A er the subsidence of acute crisis he was discharged on carnitine, injection of vitamin B , and trihexyphenidyl. Parents were counseled regarding prognosis and for prenatal diagnosis next pregnancy.

Discussion
MMA presents with lethargy, acidosis, hypoglycemia/ hyperglycemia, ketosis, and recurrent episodes. MMA due to MUT gene mutations usually led to severe phenotype, and around -% of cases are due to new mutations [ , ]. ere can be missense or nonmutations, deletions, insertions, and so on leading to clinical phenotype. e advent of NGS technology has enabled better characterization of mutations in several populations. However, Sanger sequencing remains useful adjunct in molecular testing of these cases. Sometimes in NGS, due to incomplete coverage of the exons, Sanger sequencing is required to nd mutations, if there is strong clinical suspicion. By careful use of both techniques, we could nd the two variants responsible for the clinical condition. In a Saudi study on patients of MMA, nonsense, missense, and frameshi mutations were detected across the MUT gene [ ]. Another study in Chinese patients identi ed recurrent mutations and novel mutations [ ]. A previous Indian study in patients of clinically diagnosed MMA identi ed one novel exon mutation in MUT gene with predicted pathogenicity. Here, we identi ed two novel variants, one in exon and another in exon of the MUT gene. Both were labelled as variants of unknown signi cance (VUS). e exon variant is a synonymous variant, and a di erent nucleotide change c. G>C (p.Lys Asn) has been reported earlier in ClinVar. Some synonymous variants can also a ect the splicing or protein function and lead to clinical phenotypes. e identi ed exon variant is new, but another close variant c. G>A (p.Arg Lys) has been reported in ClinVar. e variants were found to be deleterious on bioinformatic analysis and were not found in ExAC database. Both variants identi ed in present case possibly explain the phenotype of MMA in the child.
MUT-related MMA has poor prognosis in most cases. Specialised diet and supplements may not improve outcomes, even if diagnosed early. Early recognition and appropriate treatment of acute crises are necessary. Metabolic stroke can sometimes occur in the absence of acute metabolic decompensation, so meticulous neurological examination at each visit is useful. e options for therapy include early liver transplantation [ ] and possibly gene therapy in the future. Genetic counseling and prenatal diagnosis help these families in making reproductive decisions.

Conflicts of Interest
e authors declare that they have no con icts of interest.