DataSheet1_Case Report: Decrypting an interchromosomal insertion associated with Marfan’s syndrome: how optical genome mapping emphasizes the morbid burden of copy-neutral variants.pdf

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient’s neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.</p

Additional file 2: Figure S2. of A donor splice site mutation in CISD2 generates multiple truncated, non-functional isoforms in Wolfram syndrome type 2 patients

Chromatograms of the 400 nt-5′-RACE products not subjected to subcloning. (TIFF 155 kb

Genes associated with the phenotype.

<p>Genes associated with the phenotype.</p

Data fusion framework.

<p>Sequencing data are collapsed to calculate their mutational loads using four ROIs, namely genes, pathways, domains and PPIs. This allows studying ROI-phenotype associations along the four correspondent axes. Each element tested for association then becomes a feature for a prediction model. Single ROI types are combined to create data sets. Each data set is split into a training and test set. The training set is used to tune the learning parameters of a RF model and then select the best set of features, while the test set is used to measure the prediction performances.</p

Examples of variants influencing more than one gene.

<p>Protein encoded by gene <i>A</i> interacts with proteins encoded by gene <i>B</i> and gene <i>C</i>. (a) Variant on gene <i>A</i>, <i>V</i>_<i>A</i> contributes to the score both for <i>A</i> and <i>B</i> due to their interaction. In the same way, variants on gene <i>B</i>, <i>V</i>_<i>B</i>, and on gene <i>C</i>, (<i>V</i>_<i>C</i>) both contribute to the scores of genes <i>A</i> <i>B</i> and <i>C</i>. (b) Resulting variant contributions on final PPIs scores.</p

Interconnected, statistically significant ROIs.

<p>Graphical representation of statistically significant ROIs and their overlapping. The direction of the arrow means that an element is included into another. Gene ROIs (light blue) can be part of pathway (green) or PPI (grey) ROIs, while domain ROIs (purple) can be part of gene ROIs.</p

Classification performance for each data set.

<p>Classification performance for each data set.</p

Q-RT PCR results.

<p>Histograms represent the copy numbers ratio of a non-polymorphic probe within Sox9 gene in the two duplicated dogs (dog10 and dog44, red bars) relative to five normal control dogs (blue bars). The data have been normalized against two different reference sequences (Abs17, Bglr2).</p

List of CNVs identified with array-CGH in the seven cases with the indication of their code, type, location and size (CanFam2 assembly).

<p>CNVs were checked for occurrence in the Database of Genomic Copy Number Variants in the dog genome (<a href="http://dogs.genouest.org/LUPA.dir/CNV.html" target="_blank">http://dogs.genouest.org/LUPA.dir/CNV.html</a>) and in several papers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101244#pone.0101244-Chen1" target="_blank">[29]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101244#pone.0101244-Berglund1" target="_blank">[33]</a>.</p

Graphical representation of the SOX9 locus duplications discovered.

<p>The figure shows a 1,6(canFam2 assembly) and magnified views of the two SOX9 duplications detected, by array-CGH, in cases C10 (left) and C44 (right), respectively. The shaded areas indicate a gain in DNA copy number (duplication, average log2 ratios: +0, 5) detected by red dots. Asterisks indicate the 168 bp repeats.</p