A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax

Abstract

BackgroundPrimary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletions and duplications of the mutations. These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory. In addition, as a genetically heterogeneous disorder, PSP may be caused by mutations in multiple genes include FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 genes. For differential diagnosis, these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive.MethodsForty PSP patients were divided into 2 groups. Nineteen patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group, 21 random PSP patients without any genetic screening were included in blinded sample group. 7 PSP genes including FLCN, FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system, sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method.ResultsWe demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data. Noteworthy, by our in-house copy number analysis of the sequence data, we could not only detect intragenic deletions, but also determine approximate deletion junctions simultaneously.ConclusionsNGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients, as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs, small indels, large intragenic deletions and determining deletion junctions in PSP-related genes.

Clinical and genetic characteristics of chinese patients with Birt-Hogg-Dubé syndrome

“…2Mutation spectrum of the FLCN gene responsible for BHD syndrome. Top of this figure: mutations identified in this study; Bottom of this figure: mutations identified in other studies as reviewed in Schmidt et al [11], Furuya et al [14], Zhang et al [24], Rossing et al [25]. Definitions of abbreviations: FS = frameshift; MS = missense; NS = nonsense; AAΔ, amino acid deletion inframe; pMet1?…”

Childhood pneumothorax in Birt‐Hogg‐Dubé syndrome: A cohort study and review of the literature

“…In line with the interfamilial variability seen in BHD it is well recognized that BHD families exist in which SP is the sole manifestation (Gunji et al., ; Painter, Tapanainen, Somer, Tukiainen, & Aittomaki, ). Based on molecular genetic screening studies of persons with primary SP, it has been suggested that BHD accounts for up to 10% of all SP in the adult population (Johannesma et al., ; Ren et al., ; Zhang et al., ). There are only few previous reports of pediatric pneumothorax in BHD and similar studies in pediatric populations have not been published.…”

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next‐generation sequencing

“…This is likely to produce results similar to those observed using equimolar pooling without the additional cost of individual library assessment using a Bioanalyzer. Lack of coverage uniformity across regions using the HaloPlex target enrichment system has been previously reported by others . The impact of uneven coverage can be minimized by ensuring that the DoC reaches the required minimum for the regions with the lowest coverage, although this may limit the number of samples that can be multiplexed.…”