Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study

Abstract

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.

Genotype and Phenotype Landscape of 283 Japanese Patients with Tuberous Sclerosis Complex

“…Heteroduplex analysis and mismatch cleavage analysis can be performed simultaneously, and since it is more sensitive than Sanger sequencing, it is also useful for detecting mosaic mutations. The remaining 95 patients were analyzed using our newly developed method, called combined long amplicon sequencing (CoLAS) [ 19 , 20 ]. CoLAS is a targeted DNA and RNA sequencing, using long-range PCR-based NGS to detect diverse mutations in TSC1 and TSC2 .…”

“…Since then, we have continued to perform genetic tests of TSC for clinical purposes, receiving patient specimens from hospitals all over Japan. We have also developed a new analysis method that can adapt to various types of mutations using a single platform of next generation sequencing (NGS), named combined long amplicon sequencing (CoLAS) [ 19 , 20 ]. In this study, we analyzed the phenotype and genotype details of 283 TSC patients from 260 families, to clarify the overall picture of TSC in Japan.…”

Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies

“…Additionally, vLAS enables the detection of the breakpoint sequences caused by large intragenic deletion [ 10 ]. In cases where the gene exhibits mRNA expression in blood, splicing abnormalities due to deep intron variants can also be detected using long-range reverse transcribed PCR (RT-PCR) libraries [ 10 , 11 ].…”

Molecular Modelling Hurdle in the Next-Generation Sequencing Era

“…The incorporation of NGS into clinical diagnostics has uncovered imminent needs in terms of clarifying the huge amount of uncertainty that arises from large-scale sequencing studies. Aside from disease-specific strategies, which allow more detailed and complete designs to reach a molecular diagnosis [14][15][16], generic solutions when confronting any kind of rare disease are difficult to achieve. Predicting the clinical significance of a change at the DNA level is a challenging task due to the myriad of structures/processes that could be affected.…”