Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.

We studied two large consanguineous families from Oman with a distinct form of spondyloepiphyseal dysplasia (SED Omani type). By using a genome-wide linkage approach, we were able to map the underlying gene to a 4.5-centimorgan interval on chromosome 10q23. We sequenced candidate genes from the region and identified a missense mutation in the chondroitin 6-O-sulfotransferase (C6ST-1) gene (CHST3) changing an arginine into a glutamine (R304Q) in the well conserved 3 -phosphoadenosine 5 -phosphosulfate binding site. C6ST-1 catalyzes the modifying step of chondroitin sulfate (CS) synthesis by transferring sulfate to the C-6 position of the N-acetylgalactosamine of chondroitin. From the crystal structures of other sulfotransferases, it could be inferred that Arg-304 is essential for the structure of the cosubstrate binding site. We used recombinant C6ST-1 to show that the identified missense mutation completely abolishes C6ST-1 activity. Disaccharide composition analysis of CS chains by anion-exchange HPLC shows that both ⌬HexA-GalNAc(6S) and ⌬HexA(2S)-GalNAc(6S) were significantly reduced in the patient's cells and that ⌬HexA-GalNAc(4S,6S), undetectable in controls, was elevated. Analysis of the patient's urine shows marked undersulfation of CS, in particular reduction in 6-O-sulfated disaccharide and an increase in the nonsulfated unit. Our results indicate that the mutation in CHST3 described here causes a specific but generalized defect of CS chain sulfation resulting in chondrodysplasia with major involvement of the spine.

Tumorigenesis is characterized by alterations of methylation profiles including loss and gain of 5-methylcytosine. Recently, we identified a single CpG, which seemed to be consistently hypomethylated in pilocytic astrocytomas but not in other gliomas. To evaluate its applicability as a biomarker, we examined its methylation status in a large panel of gliomas (n = 97). Methylation-dependent DNA sequence variation may be considered a kind of single nucleotide polymorphism (methylSNP). MethylSNPs can be easily converted into common SNPs of the C/T type by sodium bisulfite treatment of the DNA and afterwards subjected to conventional SNP typing. We adapted SnaPshot trade mark and Pyrosequencing trade mark to determine the methylation of our test CpG in a quantitative manner. The adapted methods, called SNaPmeth and PyroMeth, respectively, gave nearly identical results, however data obtained with PyroMeth showed less scattering. Furthermore, the integrated software for allele frequency determination from Pyrosequencing could be used directly for data analysis while SnaPmeth data had to be exported and processed manually. Although data did not confirm our previous result of a preferential hypomethylation of the tested CpG in pilocytic astrocytomas, we consider quantitative methylSNP analysis by SNaPmeth or PyroMeth a favorable alternative to existing high-throughput methylation assays. It combines single CpG analysis with accurate quantitation and is amenable to high throughput.

Stop mutations are known to disrupt gene function in different ways. They both give rise to truncated polypeptides because of the premature-termination codons (PTCs) and frequently affect the metabolism of the corresponding mRNAs. The analysis of neurofibromin transcripts from different neurofibromatosis type 1 (NF1) patients revealed the skipping of exons containing PTCs. The phenomenon of exon skipping induced by nonsense mutations has been described for other disease genes, including the CFTR (cystic fibrosis transmembrance conductance regulator) gene and the fibrillin gene. We characterized several stop mutations localized within a few base pairs in exons 7 and 37 and noticed complete skipping of either exon in some cases. Because skipping of exon 7 and of exon 37 does not lead to a frameshift, PTCs are avoided in that way. Nuclear-scanning mechanisms for PTCs have been postulated to trigger the removal of the affected exons from the transcript. However, other stop mutations that we found in either NF1 exon did not lead to a skip, although they were localized within the same region. Calculations of minimum-free-energy structures of the respective regions suggest that both changes in the secondary structure of the mRNA and creation or disruption of exonic sequences relevant for the splicing process might in fact cause these different splice phenomena observed in the NF1 gene.

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Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.

We here describe the first example of the replacement of an autosome by two ring chromosomes originating from the missing chromosome, presented in a patient with a single chromosome 18 and two additional ring chromosomes. Detailed fluorescence in situ hybridization (FISH) analysis revealed the chromosome 18 origin of both ring chromosomes and characterized the small and the large ring chromosome as derivatives of the short and long arm of chromosome 18, respectively. The loss of subtelomeric regions of the short and the long arm of chromosome 18 in the ring chromosomes was confirmed by FISH studies. Molecular studies showed the exclusive presence of the paternal alleles for microsatellite markers located distal to the short and long arm loci D18S843 and D18S474, respectively. This indicates the maternal origin of both rings and provides evidence for substantial deletions of the distal parts of both arms of chromosome 18 in the ring chromosomes. The dysmorphic features of the patient can be explained by these deletions in both chromosome arms, as the clinical findings partly overlap with observations in 18p-and 18q-syndrome and are similar to some cases of ring chromosome 18. Centromere misdivision is suggested as one mechanism involved in the formation of the ring chromosomes.