We report here the molecular analysis of CANT1 and CHST3 in 38 DD cases. As previously reported, we identified CANT1 mutations in all DD type 1. We also identified CANT1 mutation in one Kim variant and in one DD case without any accessory center, but with major finger dislocations, epiphyseal interphalangeal anomalies, and thumb digitalization, expanding the phenotypic spectrum of hand anomalies observed with CANT1 mutations.
Up till now, 28 patients have been reported with CANT1
mutations (literature and our present series [Faden et al., 2010
; Furuichi et al., 2011
; Huber et al., 2009
; Laccone et al., 2011
]). Among them, 20 patients had characteristic hand anomalies, namely, extra ossification centers or bifid distal phalanx of the thumb with or without additional anomalies such as hypoplasic or low inserted triphalangeal thumbs and phalangeal dislocations. Apart from our case with an homozygous CANT1
splice site mutation and atypical hand anomalies (thumb digitalization and major phalangeal dislocations but without extra ossification center), only one case has been reported so far with “normal” hands, but no clinical or radiological data were presented [Furuichi et al., 2011
]. Finally, six patients were classified as Kim variants characterized by recognizable hand anomaly, including five patients from Japan and Korea and one Turkish patient currently reported [Kim et al., 2010
]. While Kim variant was first described in Japan/Korean population sharing a common haplotype and supporting a founder effect in this population, our findings support the existence of Kim variant in other population.
Twenty-four distinct CANT1
mutations have been reported so far located throughout the gene and including eleven nonsense mutations, eleven missense mutations, one large deletion in 5′UTR and one splice site mutation (current publication [Faden et al., 2010
; Furuichi et al., 2011
; Huber et al., 2009
; Laccone et al., 2011
]). The arginine 300 substitution has been identified in 6/28 unrelated DD type 1 patients (p.Arg300Cys [3/6], p.Arg300His [(3/6]). The valine 226 substitution has been identified in five patients with a Kim variant phenotype from Japan/Korea. No other obvious correlation between genotype and phenotype could be established.
DD type 2 patients represented two-thirds of our cohort. All were characterized by normal hands, apart from advanced carpal ossification. CHST3
mutations have been reported in spondyloepiphyseal dysplasia with congenital joint dislocations and in one case of DD type 2 [Hermanns et al., 2008
]. Our DD type 2 case harbored an homozygous p.Leu259Pro mutation previously reported in spondyloepiphyseal dysplasia with congenital joint dislocations. However, some clinical features were atypical for DD, such as camptodactyly and elbow limitation, and were more suggestive of the clinical spectrum reported in CHST3 conditions.
The clinical overlap observed in DD and DTDST/CHST3 conditions, which are due to generalized undersulfation and a lack of 6-O-sulfation of the GAG chains, respectively [Hermanns et al., 2008
; Rossi et al., 1998
], support the involvement of CANT1 in GAG biosynthesis. Proteoglycan synthesis was within normal value in basal medium; however, in this culture condition, comparisons are difficult and it has been previously reported that proteoglycan synthesis varies greatly among cultures depending on tissue source of the cells, the in vitro age of the culture and as yet unidentified factors [Harper et al., 1987
]. It is well known that β-d
-xyloside acts as an artificial chain initiator in the Golgi and maximally stimulates chondroitin/dermatan sulfate synthesis, relieving the rate limitation normally exerted by core-protein supply [Robinson et al., 1975
]. Thus, by treating cultures with β-d
-xyloside we tested the cell ability to synthesize GAGs under condition of markedly stimulated GAG synthesis and we clearly found a significant reduction of proteoglycan synthesis in DD fibroblasts in the presence of β-d
The same defect enhancement has been observed in other disorders of proteoglycan metabolism, namely, the DTDST family of disorders and CHST3 disorders resulting in generalized undersulfation and a lack of 6-O-sulfation of the GAG chains, respectively. Indeed, the sulfation defect was enhanced when fibroblasts were incubated with xyloside suggesting that this condition might mimic the cartilage situation because it is thought that chondrocytes synthesize higher amounts of proteoglycans than any other tissue.
It has been suggested that CANT1
encodes a uridin diphosphate (UDP) nucleotidase putatively needed for proteoglycan synthesis and involved in vesicular trafficking in Golgi apparatus by calcium release through inositol 1,4,5-triphosphate receptor activation [Huber et al., 2009
]. Our results support the hypothesis that CANT1
in the ER/Golgi compartment might play a role in proteoglycan synthesis through the hydrolysis of UDP a product of glycosyl transferase reactions. Thanks to UDP removal, glycosyltransferase reactions are not inhibited and uridin monophosphate (UMP) is exchanged with cytosolic UDP sugars through an antiporter exchanger (). Thus, functional impairment of CANT1
would result in increased Golgi UDP level causing feedback inhibition of glycosyltranferase activities and reduced transport of UDP sugars in the Golgi compartment, affecting, overall, GAG synthesis. In this condition, reduced GAG synthesis occurs when cells are pressed to synthesize high amount of proteoglycans as is the case during incubation with β-d
-xyloside in vitro or, in vivo, in tissues with high proteoglycan content (i.e., cartilage). Interestingly, reduced hydrodynamic size of GAG chains was also detected, suggesting a reduced elongation rate of GAG chains, even if enhanced oligosaccharide degradation might not be excluded. Finally, our finding of normal hyaluronic acid synthesis, which occurs on the plasma membrane through hyaluronan synthases [Vigetti et al., 2009
] further support the involvement of CANT1
in the ER/Golgi compartment
Figure 5 Schematic representation of the suggested role of CANT1 in proteoglycan metabolism. UDP sugars are transported into the lumen of the Golgi apparatus where sugars are transferred by specific glycosyltransferases to the growing GAG chains. UDP, the other (more ...)
We conclude that CANT1 is the major gene responsible for DD and expand the spectrum of hand anomalies observed in this disorder. We also demonstrated the role of CANT1 in the rate of proteoglycan synthesis. Ongoing studies will hopefully lead to identify other disease gene(s) responsible for DD type 2 presumably involved in proteoglycan synthesis.