5.3 Molecular Mechanisms Underlying the Contrastin...
The contrasting phenotypic consequences of CEL-HYB1 andCEL-HYB2 imply very different effects on gene expression and/or protein function.In this regard，we noted that whereas CELHYB2 variants harbour a premature stop codon within the second last exon，CEL-HYB1 variants harbour a premature stop codon within the last exon（Fig.6.1a）.We therefore surmised that CEL-HYB2 mRNAs could be subject to significant degradation by nonsense-mediated mRNA decay（NMD）；by contrast，this may not be the case for CEL-HYB1 because mRNAs that harbour a stop codon in the final exon usually escape degradation by NMD（Karam et al.2013）.To test these postulates，we amplified the full-length genomic CEL-HYB2a and CELHYB1 sequences by means of polymerase chain reaction（PCR）from their corresponding carriers and cloned the resulting PCR products into the pcDNA3.1/V5-His-TOPO vector.Reverse transcription polymerase chain reaction（RT-PCR）analyses of mRNAs from subsequently transfected HEK293T cells indicated lower CEL-HYB2a mRNA expression as compared to CEL-HYB1（Fig.6.1b）.Further quantitative RT-PCR analyses demonstrated that the mRNA expression of CEL-HYB2a accounted for only 60%of that of CEL-HYB1（Fig.6.1c）.Finally，we tested whether the mRNA expression level of CEL-HYB2a could be increased by treatment of the transfected cells with cycloheximide，a known NMD inhibitor（Pereverzev et al.2015）and found this to be the case（Fig.6.1d）.Consequently，we concluded that mRNA expression from the CEL-HYB2a allele is significantly reduced by NMD.We believe that this conclusion should apply to CEL-HYB2b due to its high sequence similarity with CELHYB2a（refer to Fig.6.1a）.(https://www.daowen.com)
Finally，it is important to mention that pancreatic exocrine function has been found to be normal in Cel-knockout mice（Vesterhus et al.2010）.This finding，taken together with the significant degradation of CEL-HYB2a mRNA by NMD，was held to account for the apparent lack of any association between CEL-HYB2a and chronic pancreatitis（Zou et al.2016c）.