CHRAT Vacation Studentship – Summer 2013
Student:
Beverley Wilson
Supervisor(s):
Dr Miriam Schmidts and Dr Hannah Mitchison
Title of Project:
Candidate gene analysis to identify genes causing unsolved cases of isolated situs
inversus.
Aims of Project:
To screen suitable cohorts of patients for mutations in the LEFTY1, LEFTY2, CCDC11
and CFC1 genes.
Brief description of project and results. Please also comment on the value of your experience:
This project involved a cohort of 26 individuals all diagnosed with isolated situs inversus without any other
symptoms. Several genes are known to cause isolated situs inversus and there are many more good
candidates. The cohort had already undergone next generation sequence analysis, whereby an Illumina gene
panel was screened for mutations in an external collaboration between this lab and the UK10K project. This
gene panel, however, was not entirely comprehensive so known situs inversus genes and good candidates
need to be excluded in order to proceed to whole exome sequencing to determine the gene defect.
Limited patient DNA was first amplified using isothermal Whole Genome Amplification. Primers were
designed to amplify the open reading frame of four candidate genes, and these were then used to perform
PCR-based Sanger sequencing on the cohort. The resulting chromatograms were analysed by Sequencher
software, allowing the comparison of the patient sequence to the reference to identify all genetic variants.
Genetic variants were then analysed using Ensembl, dbSNP and Polyphen-2 for their likely causative nature,
by examining their frequency in controls and their predicted functional effect.
The results of the sequencing revealed six suspected changes. In the LEFTY1 gene two coding missense
changes were found c.178G>A (p.Arg33Gln) and c.949A>C (p.Ile317Leu), when these two changes were
analysed using Polyphen-2 they came up as benign changes and are therefore unlikely to be pathogenic. One
coding missense was also found in the LEFTY2 gene [c.1203C>T (p.Pro284Leu)], once again this was analysed
using Polyphen-2 and also came up as a benign change. A synonymous nucleotide change was found in
CCDC11 [c.175A>G (P.Lys19Lys)]. However since this mutation does not result in an amino acid change it is
unlikely that it is pathogenic. An additional genetic variant was found in CCDC11 c.997G>A, that had a
frequency of approximately 20% in the normal Caucasian population using information available from
Ensemble. This provides strong evidence suggesting this change is a polymorphism. Finally a missense
change was found in exon 5 of the CFC1 gene [c.433G>A (p.Ala145Thr)], and once again analysis using
Polyphen-2 revealed this change was likely to be benign and therefore unlikely to be pathogenic.
My time at the Institute of Child Health this summer has been an invaluable experience. I have learnt so
much including various techniques and the analysis of data. The correct practices of working in a laboratory
to avoid contamination, ensure personal safety and set up controls to obtain accurate data were invaluable
lessons that I can carry forward into future lab work. Everyone I worked with was very friendly and overall it
has given me a real insight into what research entails, it being both challenging and rewarding. I am very
grateful to have been given this opportunity and it has driven me to follow a path in research after
completing my university studies.