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Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura

Abstract

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening systemic illness of abrupt onset and unknown cause. Proteolysis of the blood-clotting protein von Willebrand factor (VWF) observed in normal plasma is decreased in TTP patients. However, the identity of the responsible protease and its role in the pathophysiology of TTP remain unknown. We performed genome-wide linkage analysis in four pedigrees of humans with congenital TTP and mapped the responsible genetic locus to chromosome 9q34. A predicted gene in the identifed interval corresponds to a segment of a much larger transcript, identifying a new member of the ADAMTS family of zinc metalloproteinase genes (ADAMTS13). Analysis of patients' genomic DNA identified 12 mutations in the ADAMTS13 gene, accounting for 14 of the 15 disease alleles studied. We show that deficiency of ADAMTS13 is the molecular mechanism responsible for TTP, and suggest that physiologic proteolysis of VWF and/or other ADAMTS13 substrates is required for normal vascular homeostasis.

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Figure 1: Pedigrees used for linkage analysis.
Figure 2: Plasma VWF-cleaving protease levels.
Figure 3: Identification of the ADAMTS13 gene.
Figure 4: Northern and RT-PCR analysis of ADAMTS13.
Figure 5: ADAMTS13 mutations in TTP patients.

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Acknowledgements

We thank S. J. Weiss for comments on the manuscript; R. L. Nagel, I. I. Sussman, T.-P. Lee, J. Ott and J. E. Sadler for advice and encouragement; and A. Li and S. K. Uniacke for technical assistance. This work was supported in part by grants from the National Institutes of Health to H.-M.T., D.G., T.F. and W.C.N.; D.G. is an investigator of the Howard Hughes Medical Institute.

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Table 2: Single nucleotide polymorphisms
Table 3: New STS markers
Table 4: Primers used for the amplification of ADAMTS13 exons and intron/exon boundaries

Figure 6: Splice donor mutation in family G. a, Sequence trace and schematic showing the intron 13 donor splice site mutation identified in patient G. The exon-intron boundary is indicated on the sequence chromatogram on the left, and the ADAMTS13 intron 13 donor splice site is shown schematically on the right, compared to the consensus splice donor sequence. The patient is seen to be heterozygous for a G® A substitution at the conserved G at +5 of the splice donor consensus. b, Ethidium-bromide stained gel of RT-PCR products from normal control and patient RNA. Control RNA and RNA from patient CII-1 both generate the same wild-type RT-PCR product, consistent with complete splicing of intron 13 (confirmed by direct sequence analysis). In contrast, RT-PCR performed on mRNA from proband G yields the same wild-type band and 2 additional larger products. The wild-type band and additional larger bands from this patient were excised and subjected to direct sequence analysis. This revealed a 69 bp insertion, utilizing an alternate donor splice site in intron 13 (+70 nucleotides), in the larger of the two aberrant bands. The smaller aberrant product was identified as heteroduplex (data not shown). Analysis of a SNP in exon 12 from the wild-type and aberrant splice product, as well as from genomic DNA of this patient is shown at the right. The PCR product from genomic DNA demonstrates that the patient is heterozygous for the 1342C>G SNP. However, sequence analysis of the upper RT-PCR band (middle chromatogram panel) shows only the G SNP allele, whereas direct sequencing of the lower band (bottom chromatogram) shows only the C allele. These data indicate that the upper, aberrantly spliced PCR product is derived entirely from the G allele and the normally spliced lower band from the C allele, demonstrating that little or no normally spliced product is generated from the mutant allele.

Figure 7

(JPG 122 KB)

Additional ADAMTS13 mutations. a, Sequence chromatograms corresponding to the reverse strand of sequenced PCR products in the vicinity of the 3769-3770insT mutation in family C. The first panel shows the sequence of a wild-type control individual. Sequence analysis of total PCR product from the patient (middle panel) shows overlapping peaks starting with the insertion. The PCR product was cloned and individual colonies were subjected to sequence analysis. The sequence of a single colony bearing the T insertion (A on reverse strand shown) is presented in the third panel. The arrows indicate the position of the insertion. b, Missense mutations in families B, C, D and E are shown with corresponding wild-type control sequence chromatograms. The arrows indicate the positions of the mutations.

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Levy, G., Nichols, W., Lian, E. et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413, 488–494 (2001). https://doi.org/10.1038/35097008

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