Molecular characterization of apparently balanced chromosomal rearrangements by next-generation sequencing in 55 patients with intellectual disability and/or multiple congenital anomalies – ANI

Next-generation sequencing (NGS) is able to determine the sequence of millions of DNA molecules in parallel. Bioinformatics analysis compares each sequence obtained on the reference genome and thus determines the exact position and explores possible variations. Structural variations, such as chromosomal breakpoints, can be characterized by studying the distance and orientation of the sequences.
The breakpoints of patients will be determined by NGS. These results will be verified by conventional Sanger sequencing. A bioinformatics analysis will determine the characteristics of each breakpoint (sequence, repeated elements, genes, regulatory sequences). For each breakpoint, expression study will be performed for the disrupted gene and two adjacent genes in order to examine the functional consequences.

The study of the 6 first patients showed that ABCR are often more complex as they appear. We identified a case of constitutional chromothripsis, a rare type of complex rearrangement, whose mechanisms are still unclear. Among 7 disrupted genes, 2 allowed to explain the phenotype of 2 patients. In 2 patients, breakpoints localization was not possible because they lie in heterochromatine. An alternative analysis should be developed for these cases.

In 2 patients, ABCR disrupted known pathogenic genes, allowing a diagnosis in these cases. It shows the relevance of this strategy in diagnostic. Gene disruption has been reported only once for each gene, to our knowledge. A case of constitutional chromothripsis was identified. It offers the opportunity to better understand the mechanisms of these rare complex rearrangements.

A preliminary study on this project has already been published (Schluth-Bolard et al, J Med Genet, 2013. 50:144-150). The results of the present study will be published and presented to the scientific community, clinicians and patients.

Apparently balanced chromosomal rearrangement (ABCR) associated with an abnormal phenotype is a rare but problematic event. It occurs in 6% of de novo reciprocal translocations and 9% of de novo inversions. Abnormal phenotype, including intellectual disability and / or multiple congenital anomalies (ID/MCA) may be explained either by associated cryptic genomic imbalances detectable by array-CGH or by gene disruption at the breakpoint. However, breakpoint cloning using conventional methods (i.e., fluorescent in situ hybridization (FISH), Southern blot) is often laborious and time consuming and cannot be performed routinely. Without complete investigation of these rearrangements, genetic counseling is a real challenge. Recently, we and others showed that Next-Generation Sequencing (NGS) is a powerful and rapid technique to characterize ABCR breakpoints at the molecular level.
The ANI project (ABCR NGS ID) aims at characterizing at the molecular level ABCR in 55 patients presenting with intellectual disability and/or multiple congenital anomalies (ID/MCA) using NGS. We make the hypothesis that ABCR account for the patient phenotype, either by gene disruption or position effect, since genomic imbalance would have been previously excluded by array-CGH.
The ANI project is a 3-year-long study that will be conducted by a consortium of 21 partners, including 19 french hospital cytogenetics laboratories, a research team (TIGER), and a cellular biotechnology center. Patients will be recruited by each Cytogenetics laboratory. ABCR breakpoints will be molecularly characterized by NGS and a first bio-informatics analysis. The results will be verified by amplification of junction fragments by PCR followed by Sanger sequencing, allowing the localization of breakpoints at the base-pair level. In some complex cases, FISH experiment will be necessary to clarify the results. A second bio-informatics analysis will then determine breakpoints’ characteristics (sequence, repeated elements, gene and regulatory elements). Finally, for each breakpoint, gene expression studies will be performed including the gene disrupted by the breakpoint and two neighboring genes. All these data, together with those already available in the literature and databases will be integrated to determine if the gene could account for the patient’s phenotype, allowing an appropriate genetic counseling.
This project will identify new candidate genes involved in ID and developmental anomalies. It will also contribute to the development and evaluation of NGS as a diagnostic tool for ABCR and ID/MCA. It will also allow unraveling mechanisms and functional consequences of ABCR, in particular in term of position effect.
In conclusion, the ANI project will contribute to the improvement of diagnostic management and genetic counseling of patients with ID/MCA and ABCR. It will also contribute to the understanding of ABCR patho-physiology and to the unraveling of pathway involved in development and brain function, thus improving genetic counseling for ID/MCA patients in general.