Next generation massively parallel sequencing of targeted exomes to identify genetic mutations in primary ciliary dyskinesia: Implications for application to clinical testing

Abstract

PURPOSE Advances in genetic sequencing technology have the potential to enhance testing for genes associated with genetically heterogeneous clinical syndromes, such as primary ciliary dyskinesia (PCD). The objective of this study was to investigate the performance characteristics of exon-capture technology coupled with massively parallel sequencing for clinical diagnostic evaluation. METHODS We performed a pilot study of four individuals with a variety of previously identified PCD mutations. We designed a custom array (NimbleGen) to capture 2089 exons from 79 genes associated with PCD or ciliary function and sequenced the enriched material using the GS FLX Titanium (Roche 454) platform. Bioinformatics analysis was performed in a blinded fashion in an attempt to detect the previously identified mutations and validate the process. RESULTS Three of three substitution mutations and one of three small insertion/deletion mutations were readily identified using this methodology. One small insertion mutation was clearly observed after adjusting the bioinformatics handling of previously described SNPs. This process failed to detect two known mutations: one single nucleotide insertion and a whole exon deletion. Additional retrospective bioinformatics analysis revealed strong sequence-based evidence for the insertion but failed to detect the whole exon deletion. Numerous other variants were also detected, which may represent potential genetic modifiers of the PCD phenotype. CONCLUSIONS We conclude that massively parallel sequencing has considerable potential for both research and clinical diagnostics, but further development is required before widespread adoption in a clinical setting.

Static respiratory cilia associated with mutations in Dnahc11/DNAH11: A mouse model of PCD

“…The prospect that the patient carries mutations in another PCD gene must also be considered with the DNAH11 mutation contributing to a multigenic defect in combination with one or more other PCD locus mutations. A recent report of a PCD patient with only a single frameshift mutation in DNAH11 was suggested to result from digenic inheritance with a DNAH2 (MIM# 603333) variant [Berg et al, 2011], although the authors indicate that further studies are required. However, these human data, combined with the evidence that mutations in the mouse homolog can be associated with static cilia, supports the hypothesis that DNAH11 mutations can cause static cilia, in addition to the previously described association with stiff vibrating/hyperfrequent cilia.…”

“…Mutations causing truncation or frame shift (FS) are shown with a dotted line downstream of the mutation. From the N-terminus mutations are from the following studies c.855-1G>A [Pifferi et al, 2010]; p.Trp1382 * [Pifferi et al, 2010]; lrm3 mouse-50 amino acids missing [Ermakov et al, 2009]; p.Ser2176 * (this study); iv mouse [Supp et al, 1997]; p.Pro2598Leu (this study); p.His2712Arg [Pifferi et al, 2010];p.Arg2852 * [Bartoloni et al, 2002]; p.Arg2907 * (this study); p.Lys3038Thrfs * 13 [Berg et al, 2011]; p.Gly3428Arg [Pifferi et al, 2010]; p.Tyr4128 * [Schwabe et al, 2008]; p.4518 4523delinsQ [Schwabe et al, 2008].…”

“…Two mutations in the dynein heavy chain gene DNAH11 (MIM# 603339) were recently described in a family with PCD associated with hyperkinetic cilia but normal ultrastructure [Schwabe et al, 2008]. Four independent cases with different DNAH11 mutations have also been described [Bartoloni et al, 2002;Berg et al, 2011;Pifferi et al, 2010] although the overall prevalence of DNAH11 mutations in PCD is unknown.…”

Standardizing Nasal Nitric Oxide Measurement as a Test for Primary Ciliary Dyskinesia

“…For nNO validation studies at six (non-UNC) sites, PCD was confirmed by PCD-specific ciliary EM defects and, by the presence of biallelic mutations in PCD genes (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Informed consent was obtained at the University of North Carolina at Chapel Hill and collaborating institutions under the auspices of Committees on the Protection of the Rights of Human Subjects.…”

Identification of a novel Cys146X mutation of SOD1 in familial amyotrophic lateral sclerosis by whole-exome sequencing

“…A clinical implementation of genomics has to be scaled to the new reality of whole-genome analysis. 9 With the advent of next-generation sequencing technologies, the ability to quickly and relatively inexpensively learn the sequence of an individual's entire genome will soon be available to medical practitioners, patients, and consumers. 10,11 In this study of a large FALS family, we opted to apply whole-exome sequencing and, as a consequence, have successfully identified a novel mutation of the superoxide dismutase 1 (SOD1) gene.…”