ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2012) 29 P1442

Are ''in silico'' predictions reliable regarding splice-site mutations? - Studies in the aryl hydrocarbon receptor-interacting protein (AIP)

F. Martucci1,2, G. Trivellin2, B. Khoo2, S. Owusu-Antwi2, K. Stals3, A. Kumar2, S. Ellard3, A. Grossman2, P. Bouloux2 & M. Korbonits2

1Universita’ degli Studi Milano, Milano, Italy; 2Queen Mary’s School of Medicine, London, UK; 3Royal Devon and Exeter NHS Foundation Trust, Exeter, UK.

Background: It is often difficult to define the clinical relevance of a novel gene variant. In silico analyses of variants located close to exon–intron-junctions are utilised to predict the result of these basepair changes. We have previously identified two splice-site variants in AIP and confirmed the predicted changes for c.249G>T, p.G83AfsX15 and c.807C>T. We identified the c.469-2A>G heterozygous variant located at the end of intron-3 in a childhood-onset acromegaly patient. This change has been previously described and was suggested to result in the loss of exon-four based on in silico analysis.

Methods: RNA has been extracted from our patient’s peripheral blood sample and it was amplified using RT-PCR with primers located on exon-2 and exon-6. After electrophoresis DNA bands were extracted from agarose gel and sequenced. In silico prediction was carried out by the ALAMUT software (

Results: The agarose gel revealed two bands in the patient sample and a single band in the control sample. The common band showed the expected wild-type sequence. The additional lower-size band revealed the loss of 84-nucleotides at the 5′ end of exon-4, while the rest of exon-4 was normal. This change is not predicted to result in a frameshift.

Conclusion: The disruption of intron-3 acceptor splice-site resulted in the appearance of a cryptic splice-acceptor site in the middle of exon-4. The resulting RNA is predicted to yield a shorter protein missing 27 amino acids in the second alpha-helix of the first tetratricopeptide repeat motif of the AIP molecule. This variant has been identified in our childhood-onset acromegaly patient and previously in an adult-onset case, but has not been reported in normal subjects, except for family members of the above patients. We suggest that experimental confirmation of in silico predictions of gene variants is helpful for appropriate genetic counselling and clinical management of these families.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.