Messenger RNA transcripts of the hepatocyte nuclear factor-1alpha gene containing premature termination codons are...

Area of Science:

• Genetics
• Molecular Biology
• Endocrinology

Background:

• Mutations in the hepatocyte nuclear factor-1alpha (HNF-1a) gene are a cause of maturity-onset diabetes of the young (MODY).
• A significant proportion of these mutations lead to premature termination codons (PTCs) in mRNA transcripts.
• The nonsense-mediated decay (NMD) pathway is known to degrade transcripts with PTCs in many genes.

Purpose of the Study:

• To investigate whether PTC-containing mutant transcripts of the HNF-1alpha gene are targeted for degradation by the NMD pathway.
• To determine the mechanism by which HNF-1alpha mutations cause MODY.

Main Methods:

• Development of a novel quantitative RT-PCR assay.
• Quantification of ectopically expressed mutant HNF-1alpha transcripts relative to normal transcripts in lymphoblastoid cell lines using a coding single nucleotide polymorphism (cSNP) as a marker.
• Assessment of transcript levels with and without the translation inhibitor cycloheximide.

Main Results:

• Nonsense mutations (R171X, I414G415ATCG-->CCA, P291fsinsC) showed significantly reduced mutant mRNA expression (40%, <0.01%, and 6% of normal, respectively).
• Transcript levels were restored by cycloheximide, confirming NMD involvement.
• Missense mutations (G207D, R229P) did not elicit NMD, though R229P showed moderate RNA instability.

Conclusions:

• This study provides the first evidence that HNF-1alpha PTC mutations are subject to NMD.
• NMD-induced reduction in protein levels implies that dominant-negative activity is unlikely for these mutations.
• Haploinsufficiency is likely the predominant mechanism underlying HNF-1alpha mutations causing MODY.