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A program for high throughput molecular diagnostics of the mutation negative syndromic and nonsyndromic craniosynostoses

Final Report Summary - CRANIOTECHGENE (A program for high throughput molecular diagnostics of the mutation negative syndromic and nonsyndromic craniosynostoses)

Aim and outcomes: The primary aim of this fellowship was to identify novel genes that contribute to non-syndromic craniosynostosis (NSC) using the novel strategies of copy number variation identification and next generation sequencing of patient samples. This aim has been achieved with the identification of two novel genes for NSC.

Main results: The FREM1 gene was identified as a candidate gene for metopic craniosynostosis through fine-mapping studies of the boundaries of a copy number variant on chromosome 9p in families and individuals with this disorder. A total of five alleles of the FREM1 gene were identified to be associated with metopic craniosynostosis; three were de novo copy number variations and a further two de novo and inherited point mutations with reduced penetrance. Mutations in FREM1 may account for up to 7 % of cases of metopic craniosynostosis. Through collaborations with colleagues in Seattle and Melbourne the FREM1 gene was shown to localise to the metopic suture equivalent in mice, and when mutated cause premature fusion and deviation of the posterofrontal suture. This study has been submitted for publication, has passed editorial review and is currently in peer review, and was presented in 2010 as a platform address at the European Society of Human Genetics meeting.

The second gene identified is still undergoing validation studies. It is a member of the Leucine-Rich Repeat (LRR) gene family identified in a four member sibship with sagittal craniosynostosis. DNA was obtained from two members of the sibship and was analysed by whole exome next generation sequencing. The carrier parent in this family is a gonadal mosaic for the mutation. The observation that all four affected member in this family have inherited this mutation in non-mosaic form s prime facie evidence of pathogenicity. Review of the literature indicates that closely related proteins are able to interact with fibroblast growth factor receptors and signal through the MAP-kinase pathway, a commonly involved process in craniosynostosis. Substantial additional work remains to be completed on this study.

The third gene identified during this Marie Curie fellowship was the demonstration that mutations in the SLC29A3 gene can cause a rare inherited form of insulin dependant diabetes mellitus. This was a side project (unrelated to craniosynostosis) using unique clinical resources from Sydney, Australia. This project was very successful and led to the a presentation in the prestigious first plenary session of the European Society of Human Genetics Meeting in Vienna 2009, as well as three publications on aspects of SLC29A3 mutations and their role in susceptibility for insulin dependant diabetes mellitus.

The innovative methods used in the CRANIOTECHGENE project were sequence capture followed by Next generation sequencing (NGS). As part of this project the analytic and clinical validity of the NGS were estimated in DNA samples from patients with autosomal recessive ataxia which showed that with 15-fold coverage it was possible to automatically detect deletions, hetero- and homozygous point mutations for 6/7 mutant alleles, as well as having greater than 99 % accuracy for known SNP variants.