Development of a Targeted Multi-Disorder High-Throughput Sequencing Assay for the Effective Identification of Disease-Causing Variants

<div><p>Background</p><p>While next generation sequencing (NGS) is a useful tool for the identification of genetic variants to aid diagnosis and support therapy decision, high sequencing costs have limited its application within routine clinical care, especially in economically depressed areas. To investigate the utility of a multi-disease NGS based genetic test, we designed a custom sequencing assay targeting over thirty disease-associated areas including cardiac disorders, intellectual disabilities, hearing loss, collagenopathies, muscular dystrophy, Ashkenazi Jewish genetic disorders, and complex Mendelian disorders. We focused on these specific areas based on the interest of our collaborative clinical team, suggesting these diseases being the ones in need for the development of a sequencing-screening assay.</p><p>Results</p><p>We targeted all coding, untranslated regions (UTR) and flanking intronic regions of 650 known disease-associated genes using the Roche-NimbleGen EZ SeqCapV3 capture system and sequenced on the Illumina HiSeq 2500 Rapid Run platform. Eight controls with known variants and one HapMap sample were first sequenced to assess the performance of the panel. Subsequently, as a proof of principle and to explore the possible utility of our test, we analyzed test disease subjects (n = 16). Eight had known Mendelian disorders and eight had complex pediatric diseases. In addition to assess whether copy number variation may be of utility as a companion assay relative to these specific disease areas, we used the Affymetrix Genome-Wide SNP Array 6.0 to analyze the same samples.</p><p>Conclusion</p><p>We identified potentially disease-associated variants: 22 missense, 4 nonsense, 1 frameshift, and 1 splice variants (16 previously identified, 12 novel among dbSNP and 15 novel among NHLBI Exome Variant Server). We found multi-disease targeted high-throughput sequencing to be a cost efficient approach in detecting disease-associated variants to aid diagnosis.</p></div

Cost comparison of target sequencing panel Einstein_v1 versus Whole Exome Sequencing.

<p>* The number of SNVs/InDels identified was based on samples used in the current analysis (n = 2 for WES and matching target sequencing).</p><p>** Based on estimated $1,400/lane 150 bp pair end sequencing on Illumina 2500.</p><p>Cost comparison of target sequencing panel Einstein_v1 versus Whole Exome Sequencing.</p

Custom panel design.

<p>The pie chart illustrates the percent of genes included in the custom design categorized based on specific diseases/abnormalities. Of note the Ashkenazi Jews variant disorders have been kept separate because they represent an ethnic division commonly associated with specific disease and genetic variants.</p

Summary of sequencing coverage and detected variants for test cohort.

<p>* indicates samples that were multiplexed together.</p><p>TG471.002 was added to another lane for logistic reasons.</p><p>Summary of sequencing coverage and detected variants for test cohort.</p

Project pipeline.

<p>Sample quality is conducted in the laboratory (blue box), subsequent library prep and sequencing is conducted in the Epigenomics Shared Facility (pink box). The sequence reads automatically progress into the WASP pipeline for quality control parameters, alignment to reference sequence through BWA and duplicate removal through Picard is performed (yellow boxes). Local Realignment, base quality recalibration, variant discovery and annotation take place via GATK or VarScan2 (somatic) (green boxes). Clinically relevant variants are prioritized through Alamut and visualized through IGV (orange boxes). Validation is then performed by Sanger sequencing and results are visualized through Sequencher 4.0.1 (purple box).</p

Mutational pedigree of TG471.001 and TG472.002, sisters with DCM.

<p>Affected sisters are indicated in black. Associated variants are provided under each family member.</p

Demographic, clinical features, and sequencing results of the sixteen patients in test cohort.

<p>Legend: Ethnicitiy: AA = African American, Cau = Caucasian, His = Hispanic, Isr = Israeli, Ben = Bengali, Mex = Mexican, N/A = Not Available; Clinical Information: DD = Developmental Delay, DF = Dyspmorphic Features, MCA = Multiple Congenital Anomalies, FSGS = Focal segmental glomerulosclerosis, ID = Intellectual Disability, VSD = Ventricular Septal Defect, SD = Speech Delay, PDD = Pervasive Developmental Delay, DCM = Dilated Cardiomyopathy, LQTS = Long QT Syndrome, CPVT = Catecholaminergic Polymorphic Ventricular Tachycardia, SPOH = Severe Postural Orthostatic Hypertension, HCM = Hypertrophic Cardiomyopathy.</p><p>Demographic, clinical features, and sequencing results of the sixteen patients in test cohort.</p

Restriction enzyme digestion to validate the <i>CIB2</i> mutation.

<p>Lanes 2 and 3 on the agarose gel represent the restriction digest of a PCR product that was performed on both affected children. The presence of the c.556C>T mutation abolishes the restriction site and results in a single product of 206bp (Lanes 2 and 3, depict the proband and sibling, respectively). Lanes 4 and 5 contain both parental samples and as a result the there is a PCR product of 206bp representing the mutant allele as well as two additional digested fragments at 124bp and 82bp, which represent the normal allele. Lanes 6 and 7 are restriction enzyme digests from two normal, unrelated individuals, with no PCR product corresponding to the mutant allele of 206bp and only two digested PCR products corresponding to the normal allele. Lane 1 contains the DNA size standard ladder.</p

The p.Arg186Trp mutation does not affect the targeting of CIB2 to the stereocilia tips of vestibular system hair cells.

<p>Gene gun transfection of P3 vestibular system with a CIB2^WT-GFP expression vector shows targeting of CIB2 to the cell body, the cuticular plate (Pseudocolor, *) and also along the length of stereocilia of hair cells (top set of panels). As previously shown, CIB2 also accumulates to the stereocilia tips (Pseudocolor, arrows). The p.Arg186Trp mutation does not affect the localization of CIB2 in the cuticular plate or to the tip of stereocilia (pseudocolor, *, arrows) as shown in the bottom set of panels. Scale bars, 5μm.</p

The C-terminal helix of CIB2 mutation may be destabilized because of steric hindrance.

<p>Molecular models using the Protein Data Bank (PDB) 1XO5 crystal structure of Ca²⁺-CIB1 as a template. A) The backbone ribbon of the C-terminal helix of CIB1 is highlighted in red, and the four Ca²⁺ ions are represented by white spheres. B) The side-chain of the Arg186 residue is represented in white and blue (dash line), and the Trp residue is overlapped in green at position 186.</p