Riboflavin, also known as vitamin B2
, is a water-soluble aromatic substance and has to be absorbed from the food. In humans, at least two riboflavin transporters strongly expressed in small intestine epithelial cells, hRFT1 and hRFT2 encoded by SLC52A1
(GenBank accession no. NM_017986) and SLC52A3
, respectively, cooperatively function in riboflavin uptake and subsequent distribution into the blood (Yao et al 2010
). Uptake into the target cells from the blood might occur via passive diffusion but more likely depends on specific transport systems (Foraker et al 2003
). Inside the cells, riboflavin is converted into its bioactive coenzymes FMN and FAD. The latter functions as an electron acceptor for numerous dehydrogenases involved, e.g., in mitochondrial fatty acid oxidation and branched chain amino acid catabolism. In rats, severe riboflavin deficiency mimics the metabolic signature of a multiple acyl-coA dehydrogenation defect (MADD) (Goodman 1981
). Both, decreased levels of riboflavin and its metabolites and biochemical findings suggestive for mild MADD have recently been reported in hRFT2 mutant BVVLS individuals (Bosch et al 2011
). Plasma acylcarnitines and urine organic acids levels were corrected to normal values upon oral riboflavin supplementation and clinical features such as muscle weakness improved within weeks (Anand et al 2012
; Bosch et al 2011
). In addition to these patients, another study had investigated a patient with a complex neonatal neurological disorder and a biochemical profile suggestive for MADD (Ho et al 2010
). Sequence analysis of SLC52A1
revealed that the mother carried a heterozygous deletion of exons 2 and 3. During pregnancy, this defect led to a haploinsufficiency in riboflavin transport in the mother with subsequent riboflavin deprivation of the infant. Clinical signs and biochemical abnormalities completely resolved after riboflavin treatment.
Here, we report causal mutations in a third member of the riboflavin transporter family. SLC52A2
is located on chromosome 8 and encodes a 445 amino acid protein containing 11 predicted membrane-spanning domains (Fig. ). It has been initially reported to act as a receptor for porcine endogenous retrovirus subgroup A. Based on its amino acid sequence similarity with hRFT1 (86.7 %) and hRFT2 (44.1 %) a physiological function in riboflavin transport has been postulated and was subsequently confirmed by [3
H]riboflavin uptake experiments. The protein was therefore proposed to be renamed hRFT3. Expression analysis had shown that SLC52A2
is highly expressed in human brain and salivary gland, whereas the other two members of the riboflavin transporter family are strongly expressed in the small intestine. These findings suggested a role of hRFT3 in brain riboflavin homeostasis (Yao et al 2010
). While mutant hRFT2 results in decreased plasma levels of riboflavin and its coenzyme forms FMN and FAD (Bosch et al 2011
), riboflavin and its derivates were within normal range in the presented hRFT3-mutant individual. However, slightly elevated acylcarnitine levels as found in patients with decreased levels of FAD argue for impaired riboflavin uptake from the blood into the cell (Fig. ). Of note, clinically relevant disturbance of brain metabolism despite normal serum levels has also been reported for another vitamin, thiamine. Missense mutations in SLC19A3
, coding for human thiamine transporter 2, cause a Wernicke encephalopathy-like disease. Thiamine serum levels were normal and the disorder was responsive to oral high-dose thiamine treatment (Kono et al 2009
Fig. 3 Potential role of hRFT3 in riboflavin metabolism. Riboflavin is taken up from nutrition and passed through into the blood by intestinal epithelial cells. Proposed transporters involved are hRFT1 and hRFT2. Uptake into the target cells might occur to a (more ...)
An oral supplementation with riboflavin (10 mg/kg body weight/day) has been initiated in the SLC52A2-
mutant individual presented in this study and led to a normalization of six out of seven altered acylcarnitine species and moderate clinical improvement. Nevertheless, longer follow up of a larger cohort is necessary to judge the clinical effectiveness of riboflavin in this subtype of BVVLS. Although our in vitro studies indicate a residual function of hRFT3, one has to keep in mind that the gradient from blood to brain is likely to be significantly higher (Nagatsu et al 1967
) than from intestine to epithelial cells or blood. In addition, superfluous riboflavin undergoes renal excretion thus limiting the possible increase in blood riboflavin concentration by supplementation.
In summary, we demonstrate the power of next generation sequencing in the genetic diagnosis of rare metabolic disorders providing the basis for a mechanistic therapy in some cases, like in our patient a high-dose supplementation with riboflavin. The causal nature of the mutations was confirmed by in vitro analysis of transporter activities, thereby substantiating the role of defective riboflavin transport in the pathogenesis of BVVLS. The clinical overlap with motor neuron diseases such as amyotrophic lateral sclerosis bridges the gap to more common disorders and a better understanding of riboflavin metabolism might help to pinpoint the molecular defect in these entities while providing a therapeutic perspective.