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Functional Studies in Fibroblasts Cultured from Patients with Inborn Errors of Vitamin B12 Metabolism 

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction:
For over 40 years, the Vitamin B12 Laboratory at McGill University has established diagnoses of patients with inborn errors affecting cobalamin (vitamin B12) metabolism using cultured skin fibroblasts submitted by clinicians from around the world. Cobalamin derivatives are required as cofactors for the conversion of methylmalonyl CoA to succinyl CoA (adenosylcobalamin) and for the conversion of homocysteine to methionine (methylcobalamin). Classification of these patients into the appropriate complementation group reflecting the underlying gene affected depends on a variety of studies including propionate and methyltetrahydrofolate incorporation. This functional assessment allows the diagnosis and classification of the patient even when the sequencing results are unclear.





 

Methods:
As a measure of the function of the adenosylcobalamin-dependent pathway, each cell line was incubated with [14C]propionate which via tricarboxylic acid cycle is incorporated into acid insoluble material. As a measure of the function of the methylcobalamin-dependent pathway, each cell line was incubated with [14C]methyltetrahydrofolate and homocysteine which via methionine synthase is incorporated into acid insoluble material. Both procedures were also performed in the presence of 1.5 µmol/L hydroxocobalamin (OHCbl) to gauge potential responsiveness to therapy.  Incorporation values were standardized by Lowry protein assay. The average values and standard deviation for each complementation group were calculated. 



 

Results:
Propionate incorporation (pmol/mg protein/18h) and fold stimulation with added OHCbl [square brackets], methyltetrahydrofolate incorporation (nmol/mg protein/18h) and fold stimulation with added hydroxocobalamin [square brackets] for each complementation group were as follows: Fibroblast control (n= 217) 13.1±4.1 [1.09±0.12], 191±112 [1.68±0.86]; mut0 (n=195) 0.8±0.5 [1.1±0.3]; 241±148 [2.3±1.9]; mut- (n=50) 1.6±1.1 [2.48±1.00]; 214±104 [2.42±1.62]; cblA (n=71) 1.5±0.8 [4.13±1.61]; 179±101 [2.56±2.10]; cblB (n=43) 1.3±1.2 [1.42±0.50]; 184±118 [2.74±2.12]; cblC (n=315) 1.4±1.0 [3.62±1.73]; 32±16 [2.50±1.71]; cblD (n=3) 2.2±1.0 [2.5±0.7]; 29±5 [1.49±0.37]; cblD-Hcy (n=2) 13.1±1.9 [0.98±0.02]; 42±17  [1.30±0.19]; cblD-MMA (n=3) 1.3±0.3 [3.6±0.68]; 163±47 [1.36±0.31]; cblE (n=17) 7.9±2.3 [1.1±0.3]; 23±12 [1.3±0.2]; cblF (n=13) 1.9±1.7 [4.5±1.9]; 42±11 [7.36±3.69]; cblG (n=44) 8.4±3.7 [1.1±0.1]; 31±17 [1.3±0.7]; cblJ (n=2) 1.6±0.8 [3.4±1.8]; 35±12.7 [2.58±0.0]; cblX (n=12) 2.0±1.0 [2.15±0.58]; 41±12 [1.42±0.26].

 

Conclusion:
We have presented a compilation of the values of propionate and methyltetrahydrofolate incorporation that characterize each complementation group of inborn errors of cobalamin metabolism. The response of cells to supplementation with hydroxocobalamin varied among the complementation groups.

 

Agenda

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