NGS in Neurodevelopmental Disorders: from small variants to syndromes

Date: 7 November 2016

Venue: UAntwerp, Campus Drie Eiken, Building S, Auditorium S1 - Universiteitsplein 1 - 2610 Wilrijk (Antwerp) (route: UAntwerpen, Campus Drie Eiken)

Time: 4:00 PM - 6:00 PM

PhD candidate: Céline Helsmoortel

Principal investigator: Frank Kooy

Co-principal investigator: Geert Vandeweyer

Short description: PhD defence Céline Helsmoortel - Department of Biomedical Sciences


The introduction of Next Generation Sequencing, also called massive parallel sequencing, enabled the genome-wide detection of genetic variants with a resolution of up to 1 basepair. For the genetics of neurodevelopmental disorders, including intellectual disability, autism spectrum disorders and other clinical abnormalities, this technical revolution paved the way for novel diagnostics.

Despite the fact that patients with severe NDDs do not reproduce, the inci- dence of neurodevelopmental disorders remains stable in the general popula- tion. Therefore it was hypothesised that neurodevelopmental disorders are a consequence of de novo mutations. This paradigm for the genetics of NDDs was just published when I started this PhD research.

As a pilot study we sequenced the exomes of 10 patients along with their parents. Using trio-based filtering we were able to diagnose two patients with mutations in known disease genes immediately, one with Kleefstra and one with Costello syndrome. For one other patient, we identified a 4bp frameshift deletion in the ADNP gene. ADNP is a transcription factor involved in the nBAF chromatin remodeling complex, a complex from which other proteins were already previously linked to NDDs. In the context of a large international collaboration we identified ten more patients with a mutation in the same gene, suggesting alterations in this gene are one of the most frequent causes of syndromic ASD.

In a follow-up study we sequenced eight more families. In one patient we identified exactly the same PACS1 mutation as previously published and sub- sequently contributed to the expansion of the associated phenotype. Addition- ally, by discovering mutations in the DDX3X gene in two unrelated patients, we contributed to the definition of this novel syndrome. In the remaining patients suggestively relevant candidate mutations were identified. However, more research is needed to fully establish their clinical consequences. Taken together our diagnostic yield from these whole exome sequencing (WES) stud- ies is 6/18 (30%) for patients in which all previous genetic testing returned negative. Based on the outcome of these studies, WES has been implemented in the diagnostics of the Center of Medical Genetics in Antwerp.

The experimental workflow of NGS experiments are complex and may last for several weeks. As a consequence, sample swaps may occur. Therefore we designed an assay to confirm the identity of the patients and detect such even- tual sample swaps. We opted to develop a multiplex HRM assay, genotyping 21 SNPs independently, a setting which is applicable to most genetic labs. Our procedure is in line with the European guidelines for NGS testing, that recommended implementation of such a test in NGS workflows.

Moving forward, after applying whole genome sequencing (WGS), we identified a compound heterozygous mutation in the HTR7 gene, causing severe developmental delay and autism in a dizygotic twin. WGS may be the standard for genetic testing in the future, but to date it is still too expensive to run routinely.

In the second part of this thesis we elaborated more on the characterisation of deleterious mutations in the ADNP gene as a novel NDD cause, namely the Helsmoortel-Van der Aa syndrome. Since the initial publication(s), mutations in 57 more patients were identified, bringing the total number of patients known to us to date to 68. It seems likely that more will be identified thanks to the growing number of patients being included in NGS research and diagnostics worldwide. All patients carry deleterious mutations in the ADNP gene and have similar phenotypes (amongst others autism spectrum disorder, intellectual disability and specific facial dysmorphic features). By applying genome-wide expression micro-arrays and qPCR on a subset of patients we gained further insight on the effect of the mutations on gene expression.

In conclusion, NGS was proven to be a very powerful technology to identify (novel) causes of neurodevelopmental disorders.