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Mutant Fam20c knock-in mice recapitulate both lethal and non-lethal human Raine Syndrome

BMC Molecular and Cell Biology volume 26, Article number: 1 (2025) Cite this article

Abstract

Background

Inactivation or mutations of FAM20C causes human Raine Syndrome, which manifests as lethal osteosclerosis bone dysplasia or non-lethal hypophosphatemia rickets. However, it is only hypophosphatemia rickets that was reported in the mice with Fam20c deletion or mutations. To further investigate the local and global impacts of Fam20c mutation, we constructed a knock-in allele carrying Fam20c mutation (D446N) found in the non-lethal Raine Syndrome. The Fam20cD446N allele replaced the WT Fam20c by 3.6Kb Col1a1-Cre to get the conditional knock-in mice, and by Hprt-cre to get conventional knock-in mice, respectively.

Results

The radiology, serum biochemistry and immunohistochemistry indicated that all conditional and most conventional Fam20cD446N knock-in mice displayed hypophosphatemic rickets with the increased Fgf23 and deceased Dmp1 expression, which survived to adulthood. However, a few conventional Fam20cD446N knock-in mice died before weaning with the osteosclerotic X-ray radiography, though micro-CT assay displayed a reduced mineral density and increased porosity in the osteosclerotic tibia. Our results suggested that hypophosphatemia rickets was the predominant phenotype in both conditional and conventional Fam20c deficient mice, while the lethal osteosclerotic phenotype occasionally took place in the conventional Fam20c mutant mice.

Conclusion

This finding also implicated that the osteosclerotic features resulting from Fam20c deficiency could be a semblance on the basis of rickets, which is most likely triggered by the alterations in the systems other than skeleton.

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Background

Family with sequence similarity 20-member C (FAM20C) encodes the kinase of FAM20C which resides in Golgi apparatus and plays critical roles in secreted pathway [1,2,3,4]. Previous study demonstrated that FAM20C phosphorylates more than 70% of the secreted phosphoproteome by specifically recognizing and phosphorylating the S-X-E (Serine-any-Glutamate) motif [5, 9]. The wide spectrum of FAM20C substrates includes not only the extracellular matrices for skeletogenesis and biomineralization, but also soluble growth factors, adhesion molecules, etc., which is coincided to the ubiquitous FAM20C expression in both mineralized or non-mineralized tissues [6,7,8].

FAM20C deficiency in human causes Raine Syndrome [OMIM: 259775] that displays the controversial clinical manifestations, the lethal osteosclerotic bone dysplasia or the non-lethal hypophosphataemia rickets [9,10,11,12,13]. Through multiple genetic mouse models, hypophosphataemia rickets resulted from Fam20c deficiency have been well documented. Conventional inactivation or conditional abrogation of Fam20c in osteogenic tissues of mice reduced Dmp1 expression, but increased the Fgf23 concentration in circulating blood, both of which were able to independently trigger hypophosphataemia rickets by decreasing serum phosphorus [14]. Further investigation indicated that Fam20c regulates the life-span of Fgf23 by phosphorylating Fgf23 on Ser180 [15]. Lack of the phosphorylation on Ser180 of Fgf23 increased the glycosylation on Thr178, which blocked the cleavage of Fgf23 between Arg179 and Ser180 by Furin. Thus, Fam20c deficiency increases Fgf23 concentration in circulating blood by elongating its half-life, which in turn, promotes the excretion of phosphate and eventually causes hypophosphataemia rickets.

In contrast to hypophosphataemic rickets, the molecular mechanism resulting in the lethal osteosclerotic bone dysplasia in Rain Syndrome remains elusive. Previously, it is assumed that the lethal osteosclerotic bone dysplasia resulted from the enhanced kinase activity of FAM20C by certain point mutations. However, the Fam20c knock-out mice expressing the transgenic Fam20cG374R (a mutation from the lethal osteosclerotic bone dysplasia of human Raine Syndrome) also exhibited the typical features of hypophosphataemic rickets, including the decreased bone mineral density, reduced Dmp1 expression, increased Fgf23 expression and serum Fgf23 concentration, though all the Fam20c knock-out mice expressing Fam20cG374R transgene died in adolescence [16]. Therefore, further investigations are required to unveil the pathogenesis of lethal osteosclerotic bone dysplasia in Rain Syndrome.

In this study, we constructed Loxp-flanked Fam20CD446N (a mutation from the non-lethal hypophosphatemca rickets of human Raine Syndrome) knock-in allele to exclude the side effects of random insertion of transgenic Fam20c on phenotype. By crossing the mice carrying Fam20cD446N knock-in alleles with Hprt-cre and 3.6 Kb Col1a1-Cre lines, respectively, we generated the conventional and conditional Fam20cD446N knock-in mice which could act as the reliable tools to explore the pathogenesis of Raine Syndrome.

Materials and methods

Ethics statements

All mouse lines were maintained and expanded in the Specific Pathogenic Free System of the Institute of Genome Engineered Animal Models for Human Diseases at Dalian Medical University. All the mouse utilization and procedures in this study were approved by the Institutional Animal Care and Use Committee at Dalian Medical University with the protocol number AEE17038. All the research performance is accorded with the NIH Guide for the Care and Use of Laboratory Animals.

Constructing conditional Fam20C D446N knock-in allele

To construct the mouse Fam20cD446N knock-in allele, the targeting fragment spanning from exons 7 to 10 of mouse Fam20c was subcloned between two loxP sequences in targeting vector. An Frt flanked PKG-Neo cassette was inserted between the exon 10 and the 3’ loxP. The other fragment containing the exon 7–10 sequence fused with an IRES-eGFP element was ligated downstream to the 3’ loxP. The 5’ homologous arm of the targeting vector was 5.4Kb long and subcloned into the vector upstream of the 5’ loxP, while the 3’ homologous arm was 4.1Kb long and subcloned into the vector downstream of the IRES-eGFP element. Finally, a negative selection MC1-HSC-TK cassette was subcloned into the downstream of the 3’ homologous arm in the targeting vector. The final targeting construct was linearized with NotI and electroporated into W4 ES cells. After G418 selection, the genomic DNA was extracted from the ES clones and a 30 PCR screen was performed to screen the targeted clones. The 5’ screen was performed with Fam20c-ScrF2 as the forward primers (5’-TTTCTGTCCTAGGTAAGGGTGA AG-3’) and Lox-ScR2 as backward primer (5’-TGAAGTTCCTATTCTATAACTTCG − 3’) to expand the 5.4Kb fragment. The 3’ screen was performed with the forward primer (eGFP-ScF3:5’-AACGGC CACAATTCAGCGTGTCC-3’) and backward primer (Fam20c-ScrR2: 5’-ACAGCT TCTTGAATTGGGATAAAG-3’) to produce the 4.1 Kb fragment. The 5’ and 3’ screening PCR products were sequenced to confirm the presence of 5’ and 3’ loxP sites, the exons 7–10 containing D446N point mutation and IRES-eGFP element. After the neo cassette was removed from the targeted ES clones by transient transfection of pCAGGS-flpEpuro vector (Addgene, Watertown, MA, USA), two correctly targeted ES cell clones (Clone 5 A and 10 C) were selected and injected into the blastocysts from C57BL/6 mice in the Gene targeting and Transgenic Facility, University of Connecticut Health Center, Farmington, CT, USA. The male chimeras were crossbred with C57BL/6 females to produce F1 agouti offspring. The floxed alleles of F1 agouti mice (Fam20cD446N-flox/+) were genotyped by PCR analyses. With forward (LGF: 5’-CTATCTAAGATGTAGAAGAGGTG-3’) and backward primers (FrtR: 5’-CTCACAGTAATCAGGGTCGAC-3’), the WT allele produced 432 bp segment, and the Fam20cD446N-flox allele gave a 527 bp product.

Generating conditional and conventional Fam20c D446N knock-in mice

To generate the conditional Fam20c knock-in mice, the Fam20cD446N-flox/+ mice were first crossbred with 3.6 Kb Col1a1-Cre transgenic mice to gave rise to 3.6 Kb Col1a1-Cre; Fam20cD446N-flox/+ mice, which were mated with the Fam20cD446N-flox/+ mice to get the 3.6 Kb Col1a1-Cre; Fam20cD446N-flox/D446N-flox mice (referred as the conditional Fam20cD446N knock-in mice). When the Fam20cD446N-flox/+ male mice were crossbred with the Hprt-cre female mice (Stock NO. 004302, Jackson Laboratory), the loxP flanked sequence in the conditional Fam20cD446N-flox allele would be eliminated by Cre in the fertilized eggs or blastocysts. Thus, the conditional Fam20cD446N-flox allele would be transformed into the conventional Fam20cD446N-KI allele. We mated the Fam20cD446N-KI males with the Fam20cD446N-KI females to get Fam20cD446N-KI/D446N-KI mice (referred as the conventional Fam20cD446N knock-in mice). The gentyping PCT with the primers of LGF and FrtR produced a 374 bp fragment for the conventional Fam20cD446N knock-in allele, which distinguished from the 527 bp fragment of the conditional Fam20cD446N knock-in allele.

Plain X-ray and micro-CT examination

The X-ray radiography was performed by the inspector of Faxitron MX-20DC12 (Faxitron Bioptics, Tucson, Arizona, USA). The micro-CT scanning was conducted by the SCANCO Medical µCT system with a medium resolution of 7.0 mm slice increment, and the scanning data were analyzed and reconstructed by NRecon v1.6 and CTAn v1.13.8.1 software. The male WT and conditional or conventional Fam20cD446N knock-in mice from a litter were euthanized by carbon dioxide (followed by cervical dislocation), and then, dissected to get ribs, femurs and tibia (including the knee) for X-ray radiography. For the conventional Fam20cD446N knock-in mice die before weaning, we compared them to their littermates with same gender.

Histology and immunohistochemistry

The male P4W WT and conditional or conventional Fam20cD446N knock-in mice from a litter mice were euthanized and dissected for the femurs. The femurs were fixed in 4% PFA overnight, separated from soft tissues, and decalcified in 15% trypsin- ethylenediaminetetraacetic acid (EDTA) for 2 weeks. The decalcified femurs were dehydrated in gradient alcohols and embedded in paraffin for the 10 μm thick slices. Masson staining for the slices was described previously. Immunohistochemical staining was performed as previously described with the antibodies against Fam20c, Fgf23 and Dmp1 [7, 17, 18]. ABC kit and the DAB kit (Vector Laboratories, Burlingame, CA) were applied to color development with the methyl green counter-staining according to the manufacturer’s instructions. The negative controls of immunohistochemistry were conducted by above procedures without primary antibodies.

Serum biochemistry

The sera of male P4W WT and conditional or conventional Fam20cD446N knock-in mice were collected as previous described [14]. Fgf23 concentration were measured with a full-length FGF23 ELISA kit (Kainos Laboratories, Tokyo, Japan), phosphorus concentration with a kit using the phosphomolybdate ascorbic acid method (Stanbio Laboratory, Boerne, TX, USA), and calcium concentration with a colorimetric calcium kit (Stanbio Laboratory, Boerne, TX, USA). All the concentrations of Fgf23, phosphorus and calcium were quantified by a SPECTRA max 250 microplate spectrophotometer (Molecular Device Corporation, Sunnyvale, California, USA).

Statistical analysis

Statistical analysis were performed using a Student t test with SPSS 21.0 software, and stated as the Mean ± SD (standard derivation). P value less than 0.05 was regarded as the significant difference between two individual groups. To eliminate the discrepancy between genders, only the the body weights of the male WT and conditional or conventional Fam20cD446N knock-in mice were collected for statistical assay.

Results

Generation of conditional and conventional Fam20c D446N knock-in mice

To generate the conditional mouse Fam20cD446N knock-in allele, we converted the gat encoding the 446 Aspartate (D) at the Fam20c amino acid sequence into aat encoding Asparagine (N; Fig. 1A). The targeting vector contained the full length DNA from the exons 7 to 10 with the floxing loxP sites, which was followed by the cDNA from exon 7 to 10 carrying the mutation of Fam20cD446N (Fam20cD446N-flox allele; Fig. 1B). Once gene targeting was accomplished, only the full length Fam20c DNA was transcribed into the normal Fam20c mRNA, while the inserted Fam20c cDNA carrying point mutation would not be expressed. In the presence of Cre, the full length DNA from the exons 7 to 10 floxed by loxps was removed to form the recombined Fam20c knock-in allele, which was constituted by the full length DNA from exon 1 to 6, and the cDNA from 7 to 10 containing the mutation of D446N (Fam20cD446N-KI allele; Fig. 1B). The correct targeting events in ES clones were confirmed by PCR screening with the primers eGFP-ScF3 and Fam20c-ScrR2 for a 4.1Kb segment at 3’ arm, and the primers Fam20c-ScrF2 and Lox-ScR2 for a 5.4Kb segment at 5’ arm (Fig. 1B, C). Two correctly targeted ES cell clones were chosen for injection, and both gave chimera through germline transmission (Fig. 1C). Then, the male chimeric mouse was chosen to give agouti F1 mice which were used to generate conditional and conventional Fam20cD446N knock-in mice as described in Materials and methods.

Fig. 1
figure 1

Generating conditional and conventional Fam20cD446N knock-in mice by gene targeting. (A) Sequencing of the targeting vector showed that the gat encoding Aspartate (D) was mutated into aat encoding Asparagine (N). (B) In the targeting construct, the full DNA from exon 7 to 10 and a PGK-neo cassette flanked by Frt sites (orange ovals) were flanked by loxp sites (blue triangles). A cDNA of exon 7–10 carrying the D446N mutation was fused with a IRES-EGFP reporter cassette and followed the loxp flanked sequence. A MC1-HSV-TK cassette was downstream to the 3’-homologous arm (target construct). The PGK-neo cassette was removed from the targeted allele after correct targeting (Neo-removed Fam20c cKI allele). To ensure the correct targeting, a 5.4Kb segment at 5’ arm and a 4.1Kb segment at 3’ arm of the target construct were tested through PCR with the primers eGFP-ScF3 and Fam20c-ScrR2, and the primers Fam20c-ScrF2 and Lox-ScR2, respectively (Fam20c targeted allele). When the loxp-flanking exon 7–10 was removed by Cre, the Fam20c KI allele was formed by recombining the exon 1–7 with the cDNA of exon 7–10 carrying the mutation of D446N. (C) PCR screening for targeted ES clones showed the correct targeting took place in 13 ES clones. Two correctly targeted ES clones (5 A and 10 C) were selected for injection and both got germline transmission

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Rickets/osteomalacia in the conditional and conventional Fam20c D446N knock-in mice

Compared to their WT littermates, both the conditional and conventional Fam20cD446N knock-in mice showed obvious retardation in growth at P2W, which was still remarkable at P4W (Fig. 2A). Statistical assay of the body weights at P4W verified the discrepancy between WT, and the conditional and conventional Fam20cD446N knock-in mice, and further indicated that there was no significant difference in body weight between the conditional and conventional Fam20cD446N knock-in mice (Fig. 2B). As shown in plain X-ray images, both the conditional and conventional Fam20cD446N knock-in mice exhibited a rickets/osteomamalcia characteristics in the skeleton at P4W, including not only the shorter length of femur and tibia, but also the widened growth plates and thinner tibia cortex (Fig. 2C). To P8W, the conditional and conventional Fam20cD446N knock-in mice still showed the fainter reflection in both cortical and spongy bone of the shorted femur and tibia, though the widened growth plates were not so noticeable as those at P4W. Such rickets/osteomamalcia features in the conditional and conventional Fam20cD446N knock-in mice were similar to the 3.6 Kb Col1a1-cre; Fam20cf/f mice (KO) (Fig. 2C).

Fig. 2
figure 2

The skeletal characteristics of the conditional and conventional Fam20cD446N knock-in mice. (A) The gross views of the P2W WT, conditional and conventional Fam20cD446N knock-in mice (left) and the P4W WT, conditional and conventional Fam20cD446N knock-in mice (middle and right). (B) The statistical analysis on the body weights of P4W WT (17.14 ± 1.30 g), conditional (13.04 ± 1.57 g) and conventional Fam20cD446N knock-in (12.38 ± 1.20 g) mice. (**p < 0.01,*** p < 0.001). (C) Comparison of the plain X-ray examination on the femurs between P4W WT and conditional Fam20cD446N knock-in mice (most left); between P4W WT and conventional Fam20cD446N knock-in mice (the second image from left), between P8W WT and conditional Fam20cD446N knock-in mice (the second image from right), and among P8W WT, conditional Fam20c knock-out (KO) and conventional Fam20cD446N knock-in mice (most right). (Arrowheads delineated growth plates and arrows pointed to tibia cortex)

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Both the conditional and conventional Fam20c D446N knock-in mice suffered from hypophosphatemic rickets

Histological analysis with Masson staining confirmed the rickets/osteomamalcia characteristics in the P4W conditional and conventional Fam20cD446N knock-in mice. Although the gross views seemed no discrepancy (Fig. 3A-C), the growth plates in the femoral heads of the conditional and conventional Fam20cD446N knock-in mice displayed the less proliferating chondrogenic cells and the enlongated columns of hypertrophyic chondrocytes compared to the WT controls (Fig. 3A’-C’). Consistently, the cortex in the femoral diaphysis of the conditional Fam20cD446N knock-in mice was more porous than WT counterparts (Fig. 3A’’, B’’). More evidently, the cortex in the conventional Fam20cD446N knock-in femoral diaphysis was much more porous even compared to the counterpart in the conditional Fam20cD446N knock-in mice (Fig. 3B’’, C’’).

Fig. 3
figure 3

The skeletal characteristics of the conditional and conventional Fam20cD446N knock-in mice. (A, A’, A’’) Masson staining of the P4W WT femoral metaphysis (A), femur heads (A’) and femoral cortex (A’’). (B, B’, B’’) Masson staining of the P4W conditional Fam20cD446N knock-in femoral metaphysis (B), femur heads (B’) and cortex (B’’). (C, C’, C’’) Masson staining of the P4W conventional Fam20cD446N knock-in femoral metaphysis (C), femur heads (C’) and cortex (C’’). (D, D’, D’’) Immunohistochemical staining with antibody against Fam20C in the tibiae of the P4W WT (D), conditional (D’) and conventional Fam20cD446N knock-in mice (D’’). (E, E’, E’’) Immunohistochemical staining with antibody against Fgf23 in the tibiae of the P4W WT (E), conditional (E’) and conventional Fam20cD446N knock-in mice (E’’). (F, F’, F’’) Immunohistochemical staining with antibody against DMP1 in the tibiae of the P4W WT (F), conditional (F’) and conventional Fam20cD446N knock-in mice (F’’). (The yellow parentheses indicated the width of growth plates in femurs; Scale bar is 200 μm)

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To further explore the rickets/osteomamalcia phenotype, we performed the serum biochemistrical assays of the P4W conditional and conventional Fam20cD446N knock-in mice. There were greatly elevated Fgf23 concentration, as well as reduced serum phosphorus and relative normal calcium concentrations in both the conditional and conventional Fam20cD446N knock-in mice (Table 1). Worthy of notice, the serum Fgf23 concentrations in the conditional and conventional Fam20cD446N knock-in mice were significantly higher than not only the WT mice, but also the KO mice (Table 1).

Table 1 Serum concentration of Fgf23, pi and calcium in WT, KO, conditional and conventional Fam20cD446N knock-in mice
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Immunohistochemical staining was preformed to verify the construction of knock-in mice and the consequence of serum biochemistry. The immuno-staining of Fam20c showed no difference among the tibiae of P4W WT, conditional and conventional Fam20cD446N knock-in mice (Fig. 3D, D’, D’’). Consistent with the consequence of serum biochemistry, the Fgf23 staining in the conditional and conventional Fam20cD446N knock-in tibiae was obvious higher than that in WT tibia (Fig. 3E, E’, E’’). In contrast, the conventional Fam20cD446N knock-in tibia was almost devoid of Dmp1 staining (Fig. 3F’’), while the Dmp1 staining in the conditional Fam20cD446N knock-in tibia was much fainter than that in WT controls (Fig. 3F, F’). Supplementary material 1 showed the negative controls of WT, conditional and conventional Fam20cD446N knock-in long bones, in which only secondary antibody and color reagent were applied for color development without the primary antibodies. All of these data indicated that both the conditional and conventional Fam20cD446N knock-in mice suffered from hypophosphatemic rickets.

A few conventional Fam20c D446N knock-in mice died before weaning showed osteosclertic features

Although all the conditional and most conventional Fam20cD446N knock-in mice could survive to adult and suffered from the non-lethal hypophosphatemic rickets, we indeed found several conventional Fam20cD446N knock-in mice (less than 5% of the conventional Fam20cD446N knock-in pups) died before weaning (P1-3 W). These died mice also exhibited growth retardation (Fig. 4A), and plain X-ray examination showed the higher reflection in the cranial base (Fig. 4B), ribs (Fig. 4C) and femurs (Fig. 4D) compared to the WT counterparts. Micro-CT 3D reconstruction showed the much more porous cortex, and the less but thickened trabecula in the osteosclerotic conventional Fam20cD446N knock-in pups, even compared with the conditional and rickets/osteomalacia conventional Fam20cD446N knock-in pups (Fig. 4E). Moreover, the bone mineral density of the osteosclerotic conventional Fam20cD446N knock-in pup was even lower than that in both conditional and rickets/osteomalacia conventional Fam20cD446N knock-in mice (Table 2). Histological assay indicated that although thicker than that of WT, conditional and the survived conventional Fam20cD446N knock-in pups, the tibia cortex of the lethal osteosclerotic conventional Fam20cD446N knock-in pup was much more porous (Fig. 4F), implicating the osteosclerotic features most likely a semblance in radiography. Thus, the increased X-ray reflection, especially in the ribs and femurs of conventional Fam20cD446N knock-in mice seemed result from the more extensive deposition of mineral fibres, as apposed of the increased bone mineral density.

Fig. 4
figure 4

The plain X-ray features of the conventional Fam20cD446N knock-in mice died before weaning. (A) The plain X-ray images of the P18d WT, conditional Fam20cD446N knock-in mice and the died conventional Fam20cD446N knock-in mice. (B-D) There were occasional death (less than 5%) from P1W to weaning in all conventional Fam20cD446N knock-in mice. The increased X-ray reflection was detected in the cranial bases (B), ribs (C) and femurs (D) of the occasionally died conventional Fam20cD446N knock-in mice. (Red arrowheads pointed to cranial bases, yellow arrowheads to ribs, blue arrowheads to cortex in tibiae and green arrowheads to tail vertebrates) (E) The reconstructed cortex and trabeculae in tibiae from P24d WT, conditional Fam20cD446N knock-in mice, as well as the survived and the died conventional Fam20cD446N knock-in mice. (F) The masson staining of the tibiae from P24d WT, conditional Fam20cD446N knock-in mice, as well as the survived and the died conventional Fam20cD446N knock-in mice. (Green arrowheads pointed to the cortex in tibiae; Scale bar is 200 μm)

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Table 2 Quantatative micro-CT analyses of the tibia from P3w WT, conditional, osteosclerotic and rickets/osteomalacia conventional Fam20cD446N knock-in mice
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Discussion

Although characterized by a broader spectrum of disorders [13, 19], the key manifestations of Raine Syndrome was the skeletal defects: the sclerotic bone dysplasia or hypophosphatemia rickets [10, 20]. As the causative gene of Raine Syndrome, how the deficiency of FAM20C resulted in the heterogeneous, even the controversial clinical manifestations in skeleton, attracted the interests of researchers. Initially, the heterogeneous skeletal defects were speculated to result from the altered kinase activity of the FAM20C mutants because the lethal cases of Raine Syndrome contributed more point mutations in the conserved C-terminal kinase domain (CCD), while most point mutations in the non-lethal cases resided out of the CCD [2, 21]. However, the ex vivo study indicated that the kinase activity of the FAM20C mutants in both the lethal and non-lethal Raine subjects was reduced compared to WT FAM20C, and deletion or splicing mutations of FAM20C were detected in both the lethal and non-lethal cases of Raine Syndrome [9, 21]. Thus, the controversial skeletal manifestations in Raine Syndrome seems not determined by certain FAM20C mutations.

In our latest study, we constructed the 3.6 Kb Col1a1-cre; Fam20cf/f; Fam20cG374R-Tg and 3.6 Kb Col1a1-cre; Fam20cf/f; Fam20cD446N-Tg mice to mimic the mutations from lethal and non-lethal Raine Syndrome cases, respectively [16]. The increased porosity in cortex, the decreased trabecular amounts, the elevated serum Fgf23 and reduced phosphorus suggested that both mutant mice also underwent hypophosphatemia rickets as 3.6 Kb Col1a1-cre; Fam20cf/f mice did. Furthermore, the mutant Fam20c from lethal and non-lethal Raine Syndrome showed no discrepancy in elevating Fgf23 expression in both the in vivo and ex vivo conditions. These results indicated that the mutant Fam20c from both the lethal and non-lethal cases primarily led to the hypophosphatemia rickets/osteomalacia, instead of osteosclerosis [16].

In our present study, we generated the conditional knock-in allele of mutant Fam20c to prevent the side effects of random insertion of transgene. Taking the advantage of the conditional knock-in allele, we generated the conventional knock-in mice by Hprt-cre to mimic human Raine Syndrome, and the conditional knock-in mice by 3.6Kb Col1a1-cre to explore the role of Fam20c in bone development and mineralization. It was found that hypophosphatemia rickets/osteomalacia was the predominant phenotype of the conditional and conventional Fam20cD446N knock-in mice, which is consistent with the consequence from the transgenic Fam20c mutant mice. Unexpectedly, a few conventional Fam20cD446N knock-in mice exhibited the osteosclerotic features. Clinically, the co-existence of osteomalacia and osteosclerotic manifestation was found not only in the subject of non-lethal Raine Syndrome [22], but also in the cases of X-linked and autosomal recessive hypophosphatemic rickets [23,24,25], in which most of these osteosclerosis were identified by X-ray radiography and lacked histological and quantitative evidence. However, the micro-CT assay indicated a reduced bone mineral density and severer porosity in the long bone of the osteosclerotic conventional Fam20cD446N knock-in pup, supporting the assumption that FAM20C deficiency primarily causes hypophosphatemia rickets/osteomalacia. Moreover, our finding implicated that the osteosclerotic feature was most likely a semblance resulting from the extended deposition of mineral fibers, as apposed of a real osteosclerosis.

Because of the limited case of osteosclerotic conventional Fam20cD446N knock-in mice, we could not exclude the osteosclerosis in Raine Syndrome. Recently, by transfecting a series of FAM20C mutants reported in lethal cases of Rain syndrome into human osteosarcoma cell lines, Koike et al. reported that the mutant FAM20C was incapable of interacting with chondroitin 4-O-sulfotransferase-1, which reduced the ratio of 4-sulfation/6-sulfation of chondroitin, and in turn, increased the amounts of mineral nodules. They further found that the balance between 4-sulfation and 6-sulfation of chondroitin played a critical role in maintaining bone mineral density, and 6-sulfation was able to enhance osteogenic differentiation ex vivo and biomineralization in vivo [26]. Since neither WT nor mutant FAM20C impacted the 4-sulfation/6-sulfation of chondroitin directly, how FAM20C regulates the biomineralization remains to be elucidated. We speculate that the metabolism and/or endo/paracrine altered by FAM20C deficiency most likely trigger osteosclerotic features, because of the ubiquitous expression of FAM20C and a series of substrates including endo/paracrine factors [15, 27]. Furthermore, we speculate that on the basis of hypophosphatemia rickets/osteomalacia, the various metabolic and/or endo/paracrine factors, combined with more complicated environmental influence, results in osteosclerotic dysplasia in lethal Raine Syndrome. The recent case of Rained Syndrome reported the gradually decreased osteosclerosis, subperiosteal osteoid formation and the emerging bone-in-a-bone appearance in medullary space in a subject with the increasing age supported the speculation [28].

Therefore, in our further exploration, we will accumulate more cases of the osteosclerotic conventional Fam20cD446N knock-in mice to address the osteosclerotic characteristics. Additionally, we will examine the ratio of 4-sulfation/6-sulfation of chondroitin, as well as the activities of chondroitin 4-O-sulfotransferase-1 and chondroitin 6-O- sulfotransferase-1, in the conventional Fam20cD446N knock-in mice with the lethal osteosclerotic features, to verify the novel mechanism of FAM20C/Fam20c in biomineralization.

Conclusion

We reported the non-lethal hypophosphatemia rickets/osteomalacia as the predominant phenotype of the conditional and conventional Fam20cD446N knock-in mice, confirming our previous conclusion. We also found a few lethal ostelsclertotic cases in the conventional Fam20cD446N knock-in mice, which recapitulated the lethal osteosclerotic bone dysplasia of human Raine Syndrome. However, the long bone of the lethal ostelsclerotic conventional Fam20cD446N knock-in mice was in lower bone mineral density and more porous, implicating that the osteosclerotic features in Raine Syndrome most likely a semblance of extended mineral deposition, instead of a real osteosclerosis.

Data availability

Data is provided within the manuscript or supplementary information files. All the data and materials of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We sincerely thank Dr. Jianjun Hao in Division of Orthodontics, Department of Craniofacial Sciences, School of Dental Medicine, University of Connecticut, Farmington, USA for his instruction and efforts in generating the mouse carrying Fam20cD446N knock-in allele.

Funding

This research was funded by the Natural Science Foundation of Heilongjiang Province (grant no. H2017030), the Fund of Harbin Science and Technology Bureau (grant no. 2022ZCZJNS055) and the Fund of the First Affiliated Hospital of Harbin Medical University (grant no. 2016B020).

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Author notes

  1. Mengnan Chen, Dongmei Sun and Sui-Pok Yee contributed equally to this work.

Authors and Affiliations

  1. Department of Stomatology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, China

    Mengnan Chen, Dongmei Sun, Zhaoyang Yuan, Li Lin, Bing Cui & Peihong Liu

  2. Department of Cell Biology, Health Science Center, University of Connecticut, Farmington, 06030, USA

    Siu-Pok Yee

  3. Department of Prosthodontics, School of Stomatology, Dalian Medical University, Dalian, 116044, China

    Yi Wang

  4. Department of Oral Pathology, School of Stomatology, Dalian Medical University, Dalian, 116044, China

    Chao Liu

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  1. Mengnan Chen
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Contributions

C.L. and P.L.: conception, design of the research and writing of the manuscript; S.Y.: generation of the mouse carrying Fam20cD446N knock-in allele; M.C. and D.S.: acquisition of histological data, analysis and interpretation of the data, edition of figures and tables; Z.Y.: acquisition of serm biochemistry; L.L.: X-ray plain film and micro-CT scanning; B.C.: Statistical assay; Y.W: maintaining, expanding and identifying the Fam20cD446N knock-in mice. All authors have read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Chao Liu or Peihong Liu.

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All the experiment in this study were conducted in accordance to the guideline and regulation in the animal study protocol approved by the Ethics Committee of Dalian Medical University (Protocol number: AEE17038).

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Chen, M., Sun, D., Yee, SP. et al. Mutant Fam20c knock-in mice recapitulate both lethal and non-lethal human Raine Syndrome. BMC Mol and Cell Biol 26, 1 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12860-024-00526-4

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BMC Molecular and Cell Biology

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