Advantage of Whole Exome Sequencing over Allele-specific and Targeted Segment Sequencing, in Detection of Novel <i>TULP1</i> Mutation in Leber Congenital Amaurosis

<div><p></p><p><i>Background</i>: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.</p><p><i>Materials and Methods</i>: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.</p><p><i>Results</i>: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C > T, p.Arg361*) in <i>TULP1</i>, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.</p><p><i>Conclusions</i>: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.</p></div

Detection of Chromosomal Breakpoints in Patients with Developmental Delay and Speech Disorders

<div><p>Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide <i>(GNAQ),</i> RNA-binding protein, fox-1 homolog <i>(RBFOX3),</i> unc-5 homolog D (<i>C.elegans) (UNC5D</i>), transmembrane protein 47 (<i>TMEM47</i>), and X-linked inhibitor of apoptosis (<i>XIAP</i>). Among them, <i>XIAP</i> is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).</p></div

List of SVs obtained after filtration of four patients.

a<p>Discordantly mapped PETs (dPETs) which connect the same two genomic regions are clustered together.</p>b<p>dPET clusters which has passed quality filters to remove sequencing artefacts provided the list of predicted SVs.</p>c<p>Approximately 95% of SVs overlapped with SVs found in normal population (see Methods).</p

Patient CD5 with translocation t(9;17).

<p>A) The pedigree of patient CD5 is indicated. The translocation is transmitted to his two sons (CD21 and CD22). B) Translocation between chromosome 9 and 17 were validated by Sanger sequencing in three translocation carriers. The reference sequence is indicated, showing the fusion of two genes at the genomic level: the first five exons of <i>GNAQ</i> fused to exon 3–14 of <i>RBFOX3</i> and the first two exons of <i>RBFOX3</i> fused to exon 6–7 of <i>GNAQ.</i> C) mRNA expression of <i>GNAQ</i> and <i>RBFOX3</i> showed high expression in fetal brain, adult brain and cerebellum in human tissue panel.</p

Screening of CNVs in cases/controls from published and public datasets.

<p>The total number of cases in Cooper et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852-Cooper1" target="_blank">[28]</a> is 15,767 cases, and for DECIPHER is ∼17,000 cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852-Swaminathan1" target="_blank">[29]</a>. The total number of controls in Cooper et al. is 8329 controls and for 1000 Genome SV Release set is 185 controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852-Mills1" target="_blank">[22]</a>.</p

Patient CD10 with translocation t(6;8).

<p>A) The pedigree of patient CD10 is indicated. The familial translocation is inherited from asymptomatic carrier mother and shared with his affected sister (CD11). B) Sanger sequencing analysis refined the chromosomal breakpoint regions and revealed a loss of 11 bp on chromosome 6 and 8 bp on chromosome 8, with a microhomology of 3 bp between the paired breakpoints. C) <i>UNC5D</i> mRNA expression in human tissue panel showed high expression in the fetal brain, adult brain and cerebellum compared to other tissues. D) The translocation breakpoint is located at intron 1 of <i>UNC5D</i> indicated by the black arrow, encompasses 15 CNVs cases described in the DECIPHER.</p

Patient CD8 with a complex chromosomal inversion.

<p>A) Karyogram of normal chromosome X compared to der(X) in patient CD8. B) FISH validation of 10 SVs shown in Table S5 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090852#pone.0090852.s001" target="_blank">Supporting Information S1</a>) with the respective FISH probes: Hybridization of RP1-296G17-Biot (SV15) and RP1-315-Dig (SV16) were localized on the centromere of the patient’s metaphase. Probes for SV17 and SV21 on Xp21 (RP11-330K13-Biot) and Xq25 (W12-499N23-Dig), respectively resulted in a split signal between Xp21 and the centromeric region in the patient’s chromosome. Further FISH analysis was performed by using probe RP11-762M23-Biot on Xq11.1 (SV22) that was found to localize on the upper chromosomal arm. Probe RP11-655E22 on Xp11.2 was localized on the lower arm of derivative chromosome X. C) Reconstructed derivative chromosome X for patient CD8. Normal human chromosome X according to ISCN 2009 with the arrow orientation from <i>a</i> to <i>d</i> and the proposed mechanism of sequential double inversion in patient CD8. Based on our FISH analysis, an inversion occurred first between Xp21 and Xq25, changing the orientation of p and q arm with a shift of the centromere position towards the lower q-arm shown by inverted red arrow <i>b</i> and <i>c</i>. This was followed by the second inversion that occurred between Xq11.1 and Xq25, altering the orientation of the q-arm (inverted green arrow <i>c</i>). D) Expression of <i>TMEM47</i> in human tissue panel assessed by qRT-PCR. E) Expression analysis of four disrupted genes in patient CD8 assessed by qRT-PCR.</p

Validated breakpoints and disrupted candidate genes from DNA-PET sequencing in four patients.

<p>Validated breakpoints and disrupted candidate genes from DNA-PET sequencing in four patients.</p