Diagnostic application of a capture based <scp>NGS</scp> test for the concurrent detection of variants in sequence and copy number as well as <scp>LOH</scp>

Vetro, Annalisa; Goidin, Didier; Lesende, Iván; Limongelli, Ivan; Ranzani, Guglielmina Nadia; Novara, Francesca; Bonaglia, María Clara; Rinaldi, Berardo; Franchi, F; Manolakos, Emmanouil; Lonardo, Fortunato; Scarano, Francesca; Scarano, Gioacchino; Costantino, Lucy; Tedeschi, Silvana; Giglio, Sabrina; Zuffardi, Orsetta

doi:10.1111/cge.13060

Cited by 13 publications

(6 citation statements)

References 41 publications

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“…Case 25 (class A, Online Resource 2: Figure S9). The karyotype of this female child (case 8 in Vetro et al 2018), affected by severe neurodevelopmental delay, showed a simple unbalanced translocation der(2)t(2;14)(q37.2;q24.3) in 80% of peripheral blood cells and an apparently normal karyotype in the remaining ones. Array CGH and subtelomeric FISH analysis performed in both blood and fibroblasts revealed a terminal 2q deletion in 100% of the cells, while the 14q duplication was detected in about 70% of blood cells and 30% of fibroblasts.…”

Section: Chromosome Mosaicismmentioning

confidence: 87%

De novo unbalanced translocations have a complex history/aetiology

et al. 2018

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We investigated 52 cases of de novo unbalanced translocations, consisting in a terminally deleted or inverted-duplicated deleted (inv-dup del) 46th chromosome to which the distal portion of another chromosome or its opposite end was transposed. Array CGH, whole-genome sequencing, qPCR, FISH, and trio genotyping were applied. A biparental origin of the deletion and duplication was detected in 6 cases, whereas in 46, both imbalances have the same parental origin. Moreover, the duplicated region was of maternal origin in more than half of the cases, with 25% of them showing two maternal and one paternal haplotype. In all these cases, maternal age was increased. These findings indicate that the primary driver for the occurrence of the de novo unbalanced translocations is a maternal meiotic non-disjunction, followed by partial trisomy rescue of the supernumerary chromosome present in the trisomic zygote. In contrast, asymmetric breakage of a dicentric chromosome, originated either at the meiosis or postzygotically, in which the two resulting chromosomes, one being deleted and the other one inv-dup del, are repaired by telomere capture, appears at the basis of all inv-dup del translocations. Notably, this mechanism also fits with the origin of some simple translocations in which the duplicated region was of paternal origin. In all cases, the signature at the translocation junctions was that of non-homologous end joining (NHEJ) rather than nonallelic homologous recombination (NAHR). Our data imply that there is no risk of recurrence in the following pregnancies for any of the de novo unbalanced translocations we discuss here.

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Section: Chromosome Mosaicismmentioning

confidence: 87%

De novo unbalanced translocations have a complex history/aetiology

et al. 2018

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“…Furthermore, several studies are ongoing in order to evaluate the benefits that are provided by this technology in routine diagnostics, above all in ovarian cancer BRCA1/2 assessment [ 13 , 14 , 15 , 16 , 17 ]. NGS platforms are able to provide reliable qualitative data: nevertheless, one of the main challenges regards its ability to precisely identify quantitative changes, like gain or loss in the genes investigated [ 18 , 19 ]. Allele status evaluation is becoming a powerful marker of genome instability, being one of the defected targeted by PARP-1 inhibitors.…”

Section: Discussionmentioning

confidence: 99%

A Whole Germline BRCA2 Gene Deletion: How to Learn from CNV In Silico Analysis

Scaglione¹,

Concolino

Bonis

et al. 2018

IJMS

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BRCA1/2 screening in Hereditary Breast and Ovarian Syndrome (HBOC) is an essential step for effective patients’ management. Next-Generation Sequencing (NGS) can rapidly provide high throughput and reliable information about the qualitative and quantitative status of tumor-associated genes. Straightforwardly, bioinformatics methods play a key role in molecular diagnostics pipelines. BRCA1/2 genes were evaluated with our NGS workflow, coupled with Multiplex Amplicon Quantification (MAQ) and Multiplex Ligation-dependent Probe Amplification (MLPA) assays. Variant calling was performed on Amplicon Suite, while Copy Number Variant (CNV) prediction by in house and commercial CNV tools, before confirmatory MAQ/MLPA testing. The germline profile of BRCA genes revealed a unique HBOC pattern. Although variant calling analysis pinpointed heterozygote and homozygote polymorphisms on BRCA1 and BRCA2, respectively, the CNV predicted by our script suggested two conflicting interpretations: BRCA1 duplication and/or BRCA2 deletion. Our commercial software reported a BRCA1 duplication, in contrast with variant calling results. Finally, the MAQ/MLPA assays assessed a whole BRCA2 copy loss. In silico CNV analysis is a time and cost-saving procedure to powerfully identify possible Large Rearrangements using robust and efficient NGS pipelines. Our layout shows as bioinformatics algorithms alone cannot completely and correctly identify whole BRCA1/2 deletions/duplications. In particular, the complete deletion of an entire gene, like in our case, cannot be solved without alternative strategies as MLPA/MAQ. These findings support the crucial role of bioinformatics in deciphering pitfalls within NGS data analysis.

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“…To test for any possible constitutional or mosaic mutation that could explain the polymicrogyria, proband’s DNA from both blood and saliva was analysed by OneSeq protocol, at an average coverage of 194.3x and 134.7x, respectively, on an Illumina HiSeq2500, as previously described. 15 After reads alignment, variant calling, annotation and filtering for possible artefacts was done as previously reported. 16 …”

Section: Molecular Studiesmentioning

confidence: 99%

Chromothripsis and ring chromosome 22: a paradigm of genomic complexity in the Phelan-McDermid syndrome (22q13 deletion syndrome)

et al. 2018

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IntroductionPhelan-McDermid syndrome (PMS) is caused by SHANK3 haploinsufficiency. Its wide phenotypic variation is attributed partly to the type and size of 22q13 genomic lesion (deletion, unbalanced translocation, ring chromosome), partly to additional undefined factors. We investigated a child with severe global neurodevelopmental delay (NDD) compatible with her distal 22q13 deletion, complicated by bilateral perisylvian polymicrogyria (BPP) and urticarial rashes, unreported in PMS.MethodsFollowing the cytogenetic and array-comparative genomic hybridization (CGH) detection of a r(22) with SHANK3 deletion and two upstream duplications, whole-genome sequencing (WGS) in blood and whole-exome sequencing (WES) in blood and saliva were performed to highlight potential chromothripsis/chromoanagenesis events and any possible BPP-associated variants, even in low-level mosaicism.ResultsWGS confirmed the deletion and highlighted inversion and displaced order of eight fragments, three of them duplicated. The microhomology-mediated insertion of partial Alu-elements at one breakpoint junction disrupted the topological associating domain joining NFAM1 to the transcriptional coregulator TCF20. WES failed to detect BPP-associated variants.ConclusionsAlthough we were unable to highlight the molecular basis of BPP, our data suggest that SHANK3 haploinsufficiency and TCF20 misregulation, both associated with intellectual disability, contributed to the patient’s NDD, while NFAM1 interruption likely caused her skin rashes, as previously reported. We provide the first example of chromoanasynthesis in a constitutional ring chromosome and reinforce the growing evidence that chromosomal rearrangements may be more complex than estimated by conventional diagnostic approaches and affect the phenotype by global alteration of the topological chromatin organisation rather than simply by deletion or duplication of dosage-sensitive genes.

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Diagnostic application of a capture based NGS test for the concurrent detection of variants in sequence and copy number as well as LOH

Cited by 13 publications

References 41 publications

De novo unbalanced translocations have a complex history/aetiology

De novo unbalanced translocations have a complex history/aetiology

A Whole Germline BRCA2 Gene Deletion: How to Learn from CNV In Silico Analysis

Chromothripsis and ring chromosome 22: a paradigm of genomic complexity in the Phelan-McDermid syndrome (22q13 deletion syndrome)

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