Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you when you have a large gene with loss of function mutations?

Abstract

Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of B150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with B90h for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.

SETD2andDNMT3Ascreen in the Sotos-like syndrome French cohort

“…Experiments were performed on the NGS facility of the Cochin hospital, Paris ( Assistance Publique—Hôpitaux de Paris , France), as previously described 15. The custom primers panel targeting the DNMT3A and SETD2 genes was designed using the AmpliSeq Designer (reference IAD62451_192, Life Technologies, Saint-Aubin, France).…”

Dual mTORC1/2 inhibition induces anti-proliferative effect in NF1-associated plexiform neurofibroma and malignant peripheral nerve sheath tumor cells

“…NF1 genotyping was then performed using targeted next generation sequencing allowing NF1 point mutations and copy number variation detection, as previously described [48]. …”

Diagnostic pitfalls for GJB2‐related hearing loss: A novel deletion detected by Array‐CGH analysis in a Japanese patient with congenital profound hearing loss

“…Recently targeted next‐generation sequencing (NGS) has been applied to the high throughput and fast identification of pathological causes 19. In addition, the NGS strategy can be expected to provide an effective method for the identification of multi‐exon deletions or duplications of target genes 20. Therefore, smooth progression to the analysis of gene dosage for confirmation may bring about precise diagnosis and appropriate genetic counseling for GJB2 ‐related HL without the need for Sanger sequencing.…”