Identification and characterization of 40 novel hydroxymethylbilane synthase mutations that cause acute intermittent porphyria
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Acute intermittent porphyria (AIP), an autosomal dominant disorder due to the half-normal activity of hydroxymethylbilane synthase (HMBS), is characterized by acute neurovisceral attacks that are precipitated by factors that induce heme biosynthesis. Molecular diagnosis is the most sensitive and specific diagnostic test for AIP, and importantly, it permits the identification of asymptomatic family members for genetic counseling and avoidance of precipitating factors. Here, we report the identification of 40 novel HMBS mutations, including 11 missense, four nonsense, 16 small insertions or deletions, eight consensus splice site mutations, and a complex insertion-deletion mutation in unrelated individuals with AIP. Prokaryotic expression of the missense mutations demonstrated that all mutants had ≤5% of expressed wildtype activity, except for c.1039G>C (p.A347P), which had 51% residual HMBS activity but was markedly thermolabile. Of note, the mutation c.612G>T (p.Q204H) altered the last nucleotide of exon 10, which resulted in an alternative HMBS transcript with an in-frame nine base-pair deletion at the 3'-terminus of exon 10 (encoding protein Q204HΔ3). When expressed, Q204HΔ3 and an in-frame three base-pair deletion (c.639_641delTGC) had no detectable HMBS activity. Western blot analyses and mapping of the missense mutations on the human HMBS crystal structure revealed that mutations near the active site or at the dimerization interface resulted in stably expressed proteins, while most that altered surface residues resulted in unstable proteins, presumably due to improper protein folding. These studies identified novel pathogenic HMBS mutations and expanded the molecular heterogeneity of AIP.
We thank Vendenii Zaikov for his excellent technical assistance.
This work was funded in part by Career Development Awards K01 DK087971 (to M.Y.) and K23 DK095946 (to M.B.) from the National Institutes of Health (NIH) and a cooperative grant (U54 Dk083909) for the Porphyrias Consortium, which is a part of the NIH Rare Diseases Research Network (RDCRN) and supported through collaboration between the NIH Office of Rare Diseases Research (ORDR) at the National Center for Advancing Translational Science (NCATS) and National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. All authors confirm independence from the sponsors and that the content of the article has not been influenced by the sponsors.
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Conflict of interest
Manisha Balwani, Robert Desnick and John Phillips are consultants for Alnylam Pharmaceuticals and Recordati Rare Diseases. Manisha Balwani receives clinical trial grants and Robert Desnick has received research grants from Alnylam Pharmaceuticals and Recordati Rare Diseases. Robert Desnick and Makiko Yasuda are inventors of intellectual property licensed to Alnylam Pharmaceuticals. Brenden Chen, Constanza Solis-Villa, Angelika Erwin, and Irina Nazrenko declare that they have no conflict of interest.
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