Report of rapid diagnosis and precise management of MMADHC-related intracellular cobalamin defect

Abstract

Disorders of intracellular cobalamin metabolism are a group of metabolic disorders that lead to varied clinical presentation from intrauterine life to adulthood. We report a male infant with developmental regression, macrocytic anaemia and hyperpigmentation. Exome sequencing identified a homozygous pathogenic variant in the MMADHC gene, known to cause homocystinuria, cblD type (MIM #277410). We describe significant clinical improvement with targeted therapy and emphasise the relevance of genomic testing in accurate management of inherited metabolic disorders.

Background

Disorders of intracellular cobalamin metabolism occur due to a defect in the synthesis of adenosylcobalamin (AdoCbl) or methylcobalamin (MeCbl). Intracellular cobalamin undergoes a series of steps within the cytosol and mitochondria and finally terminates into one of the two pathways in the synthesis of AdoCbl or MeCbl. The most common type of these disorders is type cblC. The incidence of other rare subtypes remains unknown.¹ Based on the biochemical anomaly, these disorders may be classified as disorders with methylmalonic acidaemia, disorders with homocystinuria, and disorders with combined methylmalonic acidaemia and homocystinuria. The cblD type, due to variants in MMADHC, can present as isolated homocystinuria, isolated methylmalonic aciduria (MMA), or a combination of MMA and homocystinuria, depending on which part of the gene is affected.^{1 2} These disorders are potentially treatable with vitamin B₁₂ supplementation. Response to therapy depends on the timing of initiation of therapy, emphasising the role of early diagnosis. We describe a male infant with homocystinuria, cblD type (Mendelian Inheritance in Man (MIM) #277410) who showed significant clinical improvement with hydroxocobalamin (OHCbl) treatment.

Case presentation

A 9-month-old male infant, born to non-consanguineous parents (figure 1A), presented with regression of acquired developmental milestones from 5 months of age. Antenatal and perinatal history was uneventful. At birth, he weighed 3.2 kg (−0.2 SD), with a head circumference of 34.5 cm (mean) and length of 49.5 cm (mean). He had attained head control and partial rollover by 4 months, which were later lost. No language or social milestones were achieved. Around the same time, the parents noted gradual loss and hypopigmentation of scalp hair.

Figure 1.

(A) Pedigree, (B) sparse hypopigmented hair prior to treatment, (C) improved alertness and hair pigmentation, (D) chromatogram showing c.746A>G, p.(Y249C) in exon 8 of MMADHC in homozygous state in the proband and heterozygous state in the parents, and (E and F) sagittal T2 and T2 fluid attenuated inversion recovery sequence (FLAIR) MRI brain images showing cerebral cortical atrophy and absent corpus callosum.

On examination, he had hypopigmented sparse scalp hair. His weight was 7.84 kg (mean), length was 73.5 cm (0.6 SD) and head circumference was 43 cm (−1.58 SD). Posterior plagiocephaly, scalp seborrhoea, generalised hypopigmentation of hair and skin (figure 1B), and hyperpigmented knuckles were noted. He had spontaneous nystagmus, generalised hypotonia and diminished deep tendon reflexes. No hepatosplenomegaly was noted. A complete eye evaluation was performed with no evidence of optic nerve atrophy or maculopathy.

Investigations

Evaluation done prior to referral showed mild anaemia (haemoglobin 10.3 g/dL), total white cell count of 7.5×10⁹/L and platelet count of 739×10⁹L (range: 100–400). He had elevated mean corpuscular volume (99 fL; normal: 72–88 fL). Peripheral smear showed macrocytes. Vitamin B₁₂ level was 424.2 ng/mL (normal: 197–7712 ng/mL). Ammonia, lactate, and renal and liver functions were within normal range. Plasma homocysteine level was not available.

MRI of the brain done at 9 months of age showed mild cerebral atrophy and thin corpus callosum (figure 1E, F). Tandem mass spectroscopy showed normal acylcarnitine profile and methionine levels. Urine gas chromatography-mass spectrometry did not show MMA. Exome sequencing (ES) was performed in view of developmental regression of unknown aetiology. ES identified a known disease-causing variant, c.746A>G, p.(Tyr249Cys), in exon 8 of the MMADHC gene in homozygous state concordant with homocystinuria, cblD type (MIM #277410). This was confirmed by Sanger sequencing (figure 1D). Total plasma homocysteine was 140.98 μmol/L (normal: 5.46–16.2 μmol/L).

Differential diagnosis

The presence of macrocytic anaemia and hyperpigmentation with neuroregression could indicate vitamin B₁₂ deficiency or a defect in the cobalamin transport or an intracellular metabolic pathway defect. A normal vitamin B₁₂ level points to cobalamin pathway or transport defect. The presence or absence of MMA and elevated plasma homocysteine are important biochemical clues to further differentiate these disorders into subtypes.

Treatment

In view of a clinical diagnosis of macrocytic anaemia, the child was given a single dose of cyanocobalamin at 9 months of age. Subsequently, after molecular diagnosis at 1 year of age, he was started on OHCbl at a dose of 1 mg (0.3 mg/kg) once a day intramuscularly. Folic acid was supplemented. Total plasma homocysteine done at this time was 140.98 μmol/L (normal: 5.46–16.2 μmol/L).

Outcome and follow-up

On follow-up after 3 months, he had regained head control and social smile. He was alert and showed improvement in interaction. His hair showed mildly increased pigmentation. During the second follow-up visit after 15 days of parenteral OHCbl, his social interaction improved further and his nystagmus resolved. Macrocytosis had also resolved. After 6 months of parenteral OHCbl treatment, the child was able to roll over, can sit with support and speak monosyllables. The levels of plasma homocysteine reduced to 18.74 μmol/L. The frequency of OHCbl administration was reduced to twice a week. After this initial improvement, he has not attained any milestones even by 2 years of age. A repeat plasma homocysteine was 21.04 μmol/L. In view of high homocysteine levels and lack of clinical improvement, a daily dose of OHCbl was reinitiated. He was also started on oral betaine. He will be closely followed up for neurodevelopmental outcome.

Discussion

Disorders of cobalamin metabolism are a group of disorders (termed as cblA-cblG, cblJ and cblX) that result from disruption in the intracellular processing of cobalamin. In this group of disorders, the biochemical phenotype is determined by whether the defect lies in AdoCbl (cofactor for methylmalonyl-CoA mutase) or in MeCbl (cofactor for methionine synthase).

The cblD type of defect results from variations in the MMADHC gene and can result into three distinct biochemical phenotypes: isolated homocystinuria, isolated MMA or a combination depending on the type and location of the variant. While truncating variants towards the N-terminus (exons 3 and 4) lead to AdoCbl deficiency, missense variants towards the C-terminus (exons 6 and 8) cause MeCbl deficiency. Combined AdoCbl and MeCbl deficiency occurs when truncating variants occur in exons 5, 7 and 8.¹

cblD is a rare disorder, and to the best of our knowledge a total of 26 cases of cblD have been reported in the literature to date. Of these, eight are of isolated homocystinuria type, nine are isolated MMA and the remaining nine are of combined type. Clinical features are diverse, ranging from global developmental delay, seizures, hypotonia, poor feeding, megaloblastic anaemia, small head size, repeated stereotypic movements and reduced social interaction, poor feeding and lethargy, muscle weakness, ataxic gait, nystagmus, dehydration, vomiting and ketotic coma.^2–10 The present proband presented with global developmental delay, nystagmus and megaloblastic anaemia. Other clinical findings in our patient which are not reported in the literature include hypopigmented hair and hyperpigmented knuckles. MRI in patients with cblD shows cortical atrophy, thin corpus callosum and hypomyelination.^{3 8–10} Our patient showed cortical atrophy and thin corpus callosum, but hypomyelination was not seen.

Biochemical testing to look for MMA, total plasma homocysteine and methionine levels will help in identifying the biochemical phenotypes. Serum vitamin B₁₂ levels should be assessed in all suspected individuals to rule out vitamin B₁₂ deficiency. Eye evaluation is required to look for evidence of macular/retinal disease. Due to clinical, biochemical and genetic heterogeneity, it is cost-effective to consider comprehensive genomic testing such as ES for all individuals with suspected intracellular cobalamin defects.

Currently, out of the 28 variants in MMADHC, 12 are missense, 10 are frameshift and 6 are nonsense variants.^2–10 Additionally, seven frameshift, two nonsense, one missense and three splice site variants are reported in the ClinVar database. The missense variant found in our case, c.746A>G, in exon 8 of MMADHC was classified as pathogenic (PS3, PM2, PP3, PP4, PP5) as per the standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. The variant was found to segregate in the parents, and both parents are heterozygous carriers. This appears to be a recurrent variant, and to date this variant has been reported in five patients.^2–4

Currently, no guidelines exist on the treatment and outcome of cblD and guidelines for the more common cblC are used. Parenteral OHCbl at 0.3 mg/kg by intravenous or intramuscular route is recommended in the management of cblC defect.¹¹ However, there is low evidence and limited experience in using OHCbl in other types of defects. Although it has been shown that OHCbl is more effective than cyanocobalamin in the treatment of cblC defect,¹² our patient did show improvement with the initial dose of parenteral cyanocobalamin. Reports of management and outcome are available for 10 cases in the literature. On starting treatment with OHCbl, developmental milestones were regained in 7 of 9 cases, drop in plasma homocysteine/MMA levels was noted in all 10 cases, and resolution of macrocytic anaemia was noted in 4 cases. Of the 10 patients, 7 reported residual neurodevelopmental deficit, either in learning, attention and developmental delay.3 5 7–10 A similar response was noted in our case. Although nystagmus is known to be less responsive to treatment,² it resolved in our patient.

Clinical follow-up is advised once or twice a month during infancy to monitor for growth, nutrition, feeding and development. During these visits, monitoring for plasma amino acids and homocysteine levels should be done, which serve as a marker to adjust the dose and frequency of OHCbl. Once homocysteine level reaches below 50 μmol/L, OHCbl can be given on alternate days or even once a week. The frequency of monitoring for toddlers and older children is recommended once every 6 months to monitor growth, nutrition and dose adjustment.¹ Betaine is an oral formulation and can be added at a dose of 250 mg/kg/day. These children should be monitored for thromboembolic complications, and aspirin may be considered in children with evidence of thrombosis. Fasting, protein-restricted diet and nitrous oxide anaesthesia can exacerbate symptoms and must be avoided. Parents must be educated on sick day rules when OHCbl doses should not be withheld. Targeted molecular testing is recommended for all asymptomatic siblings of affected children. For newborns, if prenatal testing was not done, molecular testing must be performed within the first week of life as early initiation of therapy is known to result in better clinical outcomes.^{1 13} Genetic counselling regarding reproductive options for parents, including prenatal diagnosis, should be offered to all couples with affected children as the chance of recurrence of this condition is 25% in every pregnancy.

Patient’s perspective.

We were distressed when we noted delay in development in our child. We have visited multiple hospitals till now and multiple tests and scans have been done. After genetic testing, when a definitive diagnosis was available, we were suggested treatment that could possibly halt my child’s disease or even reverse it. We were relieved as we saw a ray of hope and immediately began treatment. It used to pain us when we had to give intramuscular injections everyday to him, but when we noted improvement in his condition, we accepted it. Our son gradually regained his milestones and it was exciting to see him grow. His interaction improved and we are hoping he will be like any other normal child. During our second visit, our concern about his developmental delay had partly subsided. We wanted to know if this condition could recur in our future children. When we were counselled about the 25% risk of recurrence and availability of prenatal testing, we believe, we have enough confidence and can plan our children when required.

Learning points.

• Remethylation disorders should be suspected in patients with neurodevelopmental abnormalities and peripheral blood picture suggestive of megaloblastic anaemia or pancytopaenia.

• Exome sequencing aids in the diagnosis and initiation of appropriate therapy in inherited metabolic disease.

• Cyanocobalamin can be used as initial treatment; however, parenteral hydroxocobalamin is the preferred treatment.

• Early initiation of appropriate therapy is crucial for clinical improvement in patients with cobalamin metabolism disorders.