Complete Deletion of Slc52a2 Causes Embryonic Lethality in Mice

Abstract

Riboflavin (vitamin B2) plays an important role in cellular growth and function. Riboflavin transporter 2 (RFVT2) is widely expressed in several tissues, especially in the brain and salivary glands, and plays an important role in the tissue disruption of riboflavin. During the last 10 years, mutations in SLC52A2 have been documented in patients with a rare neurological disorder known as Brown–Vialetto–Van Laere syndrome. However, no suitable animal model of this disease has been reported. Here, we aimed to clarify the physiological role of RFVT2 using Slc52a2-mutant mice. The appearance, body weight, and plasma riboflavin concentration of Slc52a2 heterozygous mutant (Slc52a2+/−) mice were similar to those of wild-type (WT) mice. However, intercrossing between Slc52a2+/− mice failed to generate Slc52a2 homozygous mutant (Slc52a2−/−) mice. This suggested that Slc52a2 gene deficiency results in early embryonic lethality. Our findings suggested that RFVT2 is essential for growth and development, and its deletion may influence embryonic survival.

INTRODUCTION

Riboflavin (vitamin B2) is an indispensable nutrient for cellular growth and function.¹⁾ The active coenzymes, flavin mononucleotide (FMN) and FAD, which are made from riboflavin. Riboflavin deficiency leads to growth impairment, which is causally related to the role of riboflavin in generation of energy from mitochondrial metabolism.²⁾ Human riboflavin transporters RFVT1–3/SLC52A1–3 have been identified.³⁾ RFVT2 predicted to have 10 membrane-spanning domains.⁴⁾ The RFVT2-mediated uptake of riboflavin has been shown to be Na⁺-, Cl⁻-, and pH-independent.^4,5)RFVT2 mRNA is ubiquitously expressed.⁴⁾ It has been suggested that RFVT2 is essential for tissue distribution of water-soluble riboflavin.⁵⁾

Since 2010, several mutations in the SLC52A3 and SLC52A2 genes have been shown to be linked to Brown–Vialetto–Van Laere syndrome (BVVLS).⁶⁾ BVVLS patients with SLC52A3 mutations have a higher frequency of facial weakness and lower blood riboflavin levels.⁷⁾ However, abnormal gait and/or ataxia and optic nerve atrophy appear to be more prevalent features of patients with SLC52A2 mutations.⁷⁾ In addition, improvements in motor abilities, respiratory function and/or cranial nerve deficits upon riboflavin supplementation are observed in 70% patients, with the remaining patients showing stabilization of the current disease stage. The responses to riboflavin supplementation are similar in patients with SLC52A2 and SLC52A3 mutations. It has been suggested that immediate and continuous riboflavin administration may prevent neurological changes.⁷⁾ In previous studies, we have shown that Slc52a3-knockout mice exhibit phenotypes similar to those seen in patients with SLC52A3 mutations, which are associated with riboflavin deficiency.⁸⁾ An analysis of skin fibroblasts from patients with SLC52A2 mutations revealed a significant reduction in electron transport chain complex I and II activity.⁹⁾ However, the pathophysiological mechanism of these symptoms is unclear.

In this study, we aimed to clarify the significance of Rfvt2 in vivo using Slc52a2-mutant mice. The appearance, body weight, and plasma riboflavin concentration of Slc52a2 heterozygous mutant (Slc52a2+/−) mice were not different from those of wild-type (WT) mice. However, intercrossing between Slc52a2+/− mice failed to generate Slc52a2 homozygous mutant (Slc52a2−/−) mice. These results suggested that Rfvt2 deficiency causes embryonic lethality in mice.

MATERIALS AND METHODS

Animals

All animal studies were conducted in accordance with the Guidelines for Animal Experiments of Kyoto University. Embryos with an Slc52a2 mutation (C57BL/6-Slc52a2^{tm1(KOMP)Vlcg}) were purchased from the Knockout Mouse Project (KOMP) Repository.¹⁰⁾ The targeting vector is described in Fig. 1A. To determine mouse genotypes, genomic DNA was isolated from tail biopsies using the GeneAmp^® PCR System 9700 (Applied Biosystems, Foster City, CA, U.S.A.), and PCR analysis was performed using the TaKaRa Ex Taq^® Hot Start Version reaction mix (TaKaRa Bio, Shiga, Japan). The primer sets were as follows: a forward primer, 5′-CCA GAC CCT AAG GCC CAT CAG-3′, and a reverse primer, 5′-CAG CAC GCC ATT GGT CAG AG-3′, for detecting the wild-type alleles and a forward primer, 5′-GGT AAA CTG GCT CGG ATT AGG G-3′, and a reverse primer, 5′-TTG ACT GTA GCG GCT GAT GTT G-3′, for detecting mutant alleles. PCR cycling conditions were as follows: 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. Heterozygous mice (8 weeks old) were mated overnight and vaginal plugs were examined the following morning. Plug detection was considered to correspond to day 0.5 of pregnancy. Embryos at E10.5, pups at postnatal day 0, and adult mice older than 8 weeks were used for subsequent experiments. The mice were housed under a 12-h light/dark cycle in a temperature-controlled environment, and were given water ad libitum and a standard chow diet (F-2; Funabashi Farm, Funabashi, Japan) before being used in experiments. All protocols were approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University (Permission No. MedKyo20121).

Fig. 1. Targeted Disruption of Slc52a2

(A) Diagram of Slc52a2 mutant construct. Exons are indicated by white closed boxes. Primers used for PCR are depicted as arrowheads. (B) Slc52a2 mRNA expression in brains from 8-week-old WT and Slc52a2+/− mice. Each bar represents the mean ± standard error of the mean (S.E.M.). (WT, n = 6; Slc52a2+/−, n = 22 from three litters). **** p < 0.0001, compared to WT.

Real-Time PCR

Total RNA was isolated from brains dissected at 8 weeks of age, using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and was then reverse transcribed. TaqMan Gene Expression assays were obtained from Life Technologies (Slc52a2, Mm01205717_g1; Carlsbad, CA, U.S.A.). Real-time PCR was performed to determine the mRNA expression level of Slc52a2 as described previously.⁴⁾

Measurement of Riboflavin

We collected samples of blood and tissue from 16-week-old mice. The concentrations of riboflavin in blood and tissue samples were measured by HPLC (LC-10ADVP; Shimadzu, Kyoto, Japan) according to a previously reported method.¹¹⁾

Statistical Analysis

Statistics were performed using GraphPad Prism (version 7; GraphPad Software, Inc., La Jolla, CA, U.S.A.). All values are expressed as the mean ± standard error of the mean (S.E.M.), and the differences were analyzed for significance using an unpaired Welch’s t-test. Multiple comparisons were performed using Bonferroni’s two-tailed test, after a one-way ANOVA. The significance was shown based on the p-value (**** p < 0.0001).

RESULTS

Targeted Disruption of the Slc52a2 Gene

The mouse Slc52a2 gene was deleted from exon 2 to 5, and was integrated with a trapping cassette. PCR analysis confirmed the targeted Slc52a2 allele in genomic DNA isolated from tail biopsies of the offspring (Fig. 1A). Furthermore, real-time PCR analysis demonstrated that Slc52a2 mRNA levels in the brain were significantly lower in Slc52a2+/− mice than in WT mice (Fig. 1B).

Genotyping of newborn pups from Slc52a2+/− parents revealed that intercrossing between heterozygotes only produced Slc52a2+/− and WT mice, and failed to generate Slc52a2−/− mutant mice (Fig. 2A). The same result was observed in embryos at E10.5 (Fig. 2B).

Fig. 2. Genotypic Analysis of Slc52a2-Mutant Mice

(A) Genotype distributions after birth (n = 204 from 72 litters). The white bar represents the number of surviving mice and the black bar represents the number of mice that died immediately after birth. (B) Genotype distributions in fetal mice at E10.5 (n = 20 from three litters).

Riboflavin Homeostasis and Phenotypic Analysis in Slc52a2+/− Mice

Macroscopically, the appearance of Slc52a2+/− pups and adults were not different from WT mice (Fig. 3A), and the body weights of Slc52a2+/− and WT mice were similar within 3 weeks of birth (Fig. 3B).

Fig. 3. Phenotypic Analysis of Slc52a2+/− Mice

(A) Gross appearance of whole bodies of WT and Slc52a2+/− mice at postnatal day 0 and 10 weeks. (B) Changes in body weight of WT and Slc52a2+/− mice up to 3 weeks after birth. (WT, n = 5; Slc52a2+/−, n = 7).

We measured riboflavin concentration in plasma (Fig. 4A) and tissues, including the upper and lower small intestine, liver, kidney, lung, heart, muscle, and brain in 16-week-old WT and Slc52a2+/− mice (Fig. 4B). No differences in plasma or tissue riboflavin concentrations were observed between Slc52a2+/− and WT mice.

Fig. 4. Riboflavin Levels in Plasma and Tissues from WT and Slc52a2+/− Mice

Plasma and tissue samples were obtained from 16-week-old WT and Slc52a2+/− mice. Riboflavin levels in the plasma (A) and tissues (B) were measured by HPLC. Each bar represents the mean ± S.E.M. (WT, n = 6; Slc52a2+/−, n = 5).

DISCUSSION

In this study, we attempted to produce Slc52a2-mutant mice as a pathological model of SLC52A2-mutant BVVLS. However, Slc52a2−/− mice were not observed among newborn pups or E10.5 embryos, including those that died due to maternal neglect. RFVT2 is widely expressed in tissues throughout the body. Therefore, the complete deletion of Slc52a2 expression resulted in embryonic lethality in the early stages of embryonic development.

In in vitro functional analyses, SLC52A2 mutations p.G306R and p.L312P show a moderate, but significant, decrease in transport activity.¹²⁾ These mutations have been detected in 30 BVVLS patients (Table 1). Except for one patient, previous studies have shown that BVVLS patients with SLC52A2 mutations have one allele that encodes functional RFVT2.^12,13) A previously described patient with mutations in p.L123P and p.L339P is thought to have survived due to the retention of a low level of RFVT2 activity. Taken together, these data suggested that RFVT2 is essential for embryonic cell survival in vivo, and complete deletion may lead to embryonic lethality.

Table 1. Gene Mutations in Patients with SLC52A2-Mutant BVVLS

Gene mutation	G306R	G306R	L312P	G306R	G306R	L312P	G306R	L312P	L123P
	×	×	×	×	×	×	×	×	×
	G306R	L312P	L312P	L339P	R284D	L339P	Y305C	W31S	L339P
Number	17	1	1	4	2	2	1	1	1

Modified from Haack et al.,¹²⁾ Foley et al.,¹³⁾ and O’Callaghan et al.⁷⁾ In in vitro functional analyses, the mutations with a white background showed a moderate decrease in function, while the mutations with a gray background showed almost complete loss of function.

Phenotypic analysis showed no difference between WT and Slc52a2+/− mice. In addition, the riboflavin concentrations in plasma and tissues were unchanged compared with those in WT mice. These results revealed that Slc52a2+/− mice show normal growth, which is consistent with the results reported for Slc52a3+/− mice.⁸⁾ In clinical reports, parents or sibling with heterozygous mutation are healthy, suggesting an autosomal recessive mode of inheritance.¹⁴⁾ Therefore, the Slc52a2+/− mouse phenotype may mimic the phenotype of parents of BVVLS patients.

When the Slc52a2 gene was completely deleted by homologous recombination with a long-chain sequence, Slc52a2−/− mice were not generated. Creating a single-nucleotide polymorphism animal model, in which some Rfvt2 function is retained, may be an alternative method for producing a pathological model of RFVT2-mutant BVVLS.

In conclusion, RFVT2 is an essential transporter for growth and development, and its deletion may influence embryonic survival.

Acknowledgments

This study was supported in part by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid for Scientific Research [C] to AY [24590190, 15K08095]).

Conflict of Interest

The authors declare no conflict of interest.

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