Hidden Mutations in Cornelia de Lange Syndrome Limitations of Sanger Sequencing in Molecular Diagnostics

Abstract

Cornelia de Lange syndrome (CdLS) is a well-characterized developmental disorder. The genetic cause of CdLS is a mutation in one of five associated genes (NIPBL, SMC1A, SMC3, RAD21, and HDAC8) accounting for about 70% of cases. To improve our current molecular diagnostic and to analyze some of CdLS candidate genes, we developed and established a gene panel approach. Because recent data indicate a high frequency of mosaic NIPBL mutations that were not detected by conventional sequencing approaches of blood DNA, we started to collect buccal mucosa (BM) samples of our patients that were negative for mutations in the known CdLS genes. Here, we report the identification of three mosaic NIPBL mutations by our high-coverage gene panel sequencing approach that were undetected by classical Sanger sequencing analysis of BM DNA. All mutations were confirmed by the use of highly sensitive SNaPshot fragment analysis using DNA from BM, urine, and fibroblast samples. In blood samples, we could not detect the respective mutation. Finally, in fibroblast samples from all three patients, Sanger sequencing could identify all the mutations. Thus, our study highlights the need for highly sensitive technologies in molecular diagnostic of CdLS to improve genetic diagnosis and counseling of patients and their families.

Structure of the cohesin loader Scc2

“…These loops may permit considerable motion of the hook structure as suggested by the conformational variability observed in the Scc2 hook EM images8. In addition, the structure of the Scc2 hook contains a number of conserved buried residues that are mutated in CdLS18202324252627. These mutations result in significant changes in their side-chain chemical properties (Supplementary Fig.…”

“…Our Scc2 structure contains a number of residues that are mutated in CdLS and are conserved between human and yeasts18202324252627 (Supplementary Fig. 1c; Supplementary Fig.…”

Clinical utility gene card for: Cornelia de Lange syndrome

“…Therefore, the simultaneous study of DNA from buccal cells with highly sensitive technologies (next-generation sequencing (NGS)) or Sanger sequencing of DNA from fibroblasts (skin biopsy) is recommended to improve genetic diagnosis and counselling. 21 In patients who test negative for mutations in NIPBL, testing of SMC1A, HDAC8, RAD21 and SMC3 should be individually considered.…”

“…Published studies indicate that analytical sensitivity in blood by Sanger sequencing drops far below 70% when low-level mosaicism is present. 21 Mutations outside the coding exons in promoters, regulatory regions or introns will likely be missed as well.…”

Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration

“…Other characteristics include facial dysmorphism, growth retardation, and upper limb defects. Heterozygous mutations in the cohesin loading factor Nipped-B-like ( NIPBL ) have been identified in 50%–60% of cases and are associated with a more severe clinical presentation, while mutations in cohesin complex subunits SMC1A , SMC3 , and RAD21 and in the SMC3-targeting deacetylase HDAC8 account for a further 10% of mostly mildly affected cases (Braunholz et al., 2015). A genetic cause for the remaining 30% of clinically diagnosed CdLS patients remains unknown.…”