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Desbuquois dysplasia is a severe condition characterized by short stature, joint laxity, scoliosis, and advanced carpal ossification with a delta phalanx. Studying nine Desbuquois families, we identified seven distinct mutations in the Calcium-Activated Nucleotidase 1 gene (CANT1), which encodes a soluble UDP-preferring nucleotidase belonging to the apyrase family. Among the seven mutations, four were nonsense mutations (Del 5' UTR and exon 1, p.P245RfsX3, p.S303AfsX20, and p.W125X), and three were missense mutations (p.R300C, p.R300H, and p.P299L) responsible for the change of conserved amino acids located in the seventh nucleotidase conserved region (NRC). The arginine substitution at position 300 was identified in five out of nine families. The specific function of CANT1 is as yet unknown, but its substrates are involved in several major signaling functions, including Ca2+ release, through activation of pyrimidinergic signaling. Importantly, using RT-PCR analysis, we observed a specific expression in chondrocytes. We also found electron-dense material within distended rough endoplasmic reticulum in the fibroblasts of Desbuquois patients. Our findings demonstrate the specific involvement of a nucleotidase in the endochondral ossification process.

Unlabelled: COL4A3/COL4A4 mutations: From familial hematuria to autosomal-dominant or recessive Alport syndrome., Background: Mutations of the type IV collagen COL4A5 gene cause X-linked Alport syndrome (ATS). Mutations of COL4A3 and COL4A4 have been reported both in autosomal-recessive and autosomal-dominant ATS, as well as in benign familial hematuria (BFH). In the latter conditions, however, clinical features are less defined, few mutations have been reported, and other genes and non-genetic factors may be involved., Methods: We analyzed 36 ATS patients for COL4A3 and COL4A4 mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Sporadic patients who had tested negative for COL4A5 mutations were included with typical cases of autosomal recessive ATS to secure a better definition of the phenotype spectrum., Results: We identified seven previously undescribed COL4A3 mutations: in two genetic compounds and three heterozygotes, and one in COL4A4. In agreement with the literature, some of the mutations of compound heterozygotes were associated with microhematuria in healthy heterozygous relatives. The mutations of heterozygous patients are likely dominant, since no change was identified in the second allele even by sequencing, and they are predicted to result in shortened or abnormal chains with a possible dominant-negative effect. In addition, both genes showed rare variants of unclear pathogenicity, and common polymorphisms that are shared in part with other populations., Conclusions: This study extends the mutation spectrum of COL4A3 and COL4A4 genes, and suggests a possible relationship between production of abnormal COL IV chains and dominant expression of a continuous spectrum of phenotypes, from ATS to BFH.

A total of 108 patients affected by Alport's syndrome, taken from 97 families, were enrolled in a genetic and ultrastructural study. Sixty-four families (75 patients) were X-linked, seven autosomal recessive, two autosomal dominant, five uninterpretable, and 19 sporadic. The ultrastructural features were consistent with Alport's syndrome in 66, doubtful in 20, and not significant for Alport's syndrome in 22 patients in the X-linked, sporadic, and genetically uninterpretable groups (without significant differences), as well as in the autosomal group. Mutations of the COL4A5 gene were present in 36 patients in the first three groups, without significant differences. More severe mutations were more frequently present in patients with an ultrastructural pattern consistent with Alport's syndrome. Nevertheless, there seems to be no strict correlation between mutation and ultrastructure, because a major rearrangement was found in a patient with no significant lesions, and different morphologic patterns were detected in patients Belonging to the same family. Immunohistochemical investigation into 24 patients for alpha (IV) chains showed that both alpha 3(IV) and alpha 5(IV) were lacking in the glomerular basement membrane of 13 patients (five with mutations) and were expressed in another six (three with mutations and one in the autosomal group). On the contrary, in this study the retained expression of alpha 3(IV) chain was found, despite the lack of alpha 5(IV) in the glomerular basement membrane of five patients (two with mutation). These different patterns could be related to both the type and severity of the COL4A5 mutations. All of the ultrastructural patterns were identified in all three immunohistochemical groups. Ultrastructural features and alpha 5(IV) chain production, even if an expression of a genetic mutation, do not strictly correlate. The combined use of analysis of collagen expression and electron microscopy made it possible to diagnose Alport's syndrome in 92% of the cohort, and therefore this approach is advisable. A multidisciplinary approach is recommended in the study of Alport's syndrome in an attempt to achieve a better diagnostic definition of and insight into the pathogenetic mechanisms.