A bioinformatic workflow for analyzing whole genomes in rare Mendelian disease

The vast majority of the human genome (~98%) is non-coding. A symphony of non-coding sequences resides in the genome, interacting with genes and the environment to tune gene expression. Functional non-coding sequences include enhancers, silencers, promoters, non-coding RNA and insulators. Variation in these non-coding sequences can cause disease, yet clinical sequencing in patients with rare Mendelian disease currently focuses mostly on variants in the ~2% of the genome that codes for protein. Indeed, variants in protein-coding genes that can explain a phenotype are identified in less than half of patients with suspected genetic disease by whole exome sequencing (WES). With the dramatic reduction in the cost of whole genome sequencing (WGS), development of algorithms to detect variants longer than 50 bp (structural variants, SVs), and improved annotation of the non-coding genome, it is now possible to interrogate the entire spectrum of genetic variation to identify a pathogenic mutation. A comprehensive pipeline is needed to analyze non-coding variation and structural variation from WGS. In this thesis, I developed and benchmarked a bioinformatics workflow to detect pathogenic non-coding SNVs/indels and pathogenic SVs, and applied this workflow to unsolved patients with rare Mendelian disorders. The pipeline detected ~80-90% of deletions, ~90% of duplications, ~65% inversions, and ~50% of insertions in a simulated genome and the NA12878 genome. The pipeline captured the majority of known pathogenic non-coding single nucleotide variant (SNVs) and insertion deletions (indels), and selectively prioritized a spiked-in known pathogenic non-coding SNV. Several interesting candidate variants were detected in patients, but none could be convincingly implicated as pathogenic. The bioinformatic workflow described in this thesis is complementary to sequencing pipelines that analyze only protein-coding variants from whole genomes. Application of this workflow to larger cohorts of patients with rare Mendelian diseases should identify pathogenic non-coding variants and SVs to increase diagnostic yield of clinical sequencing studies, assist management of genetic diseases, and contribute knowledge of novel pathogenic variants to the scientific community.Science, Faculty ofGraduat

RNA sequencing resolves novel DYNC2H1 variants causing short‐rib thoracic dysplasia type 3: Case report

Abstract Background Intronic variants outside the canonical splice site are challenging to interpret and therefore likely represent an underreported cause of human disease. Autosomal recessive variants in DYNC2H1 are associated with short‐rib thoracic dysplasia 3 with or without polydactyly (SRTD3), a clinically heterogeneous disease generally presenting with short ribs, shortened tubular bones, narrow thorax and acetabular roof anomalies. We describe a case of SRTD3 with compound heterozygous frameshift and intronic variants and highlight the essential role of RNA sequencing (RNA‐Seq) in variant interpretation. Methods Following inconclusive clinical genetic testing identifying a likely pathogenic frameshift variant and an intronic variant of uncertain significance (VUS) in DYNC2H1 in trans, the family enrolled in the Care4Rare Canada research program, where RNA‐Seq studies were performed. Results The proband presented with post‐axial polydactyly of all four limbs, a significantly small chest with a pectus excavatum and anterior flaring of the ribs. RNA‐Seq investigations revealed a novel splice junction as a result of the intronic VUS and significantly decreased DYNC2H1 gene expression in the proband. Conclusion This case demonstrates the diagnostic utility of RNA‐Seq for variant interpretation following inconclusive clinical testing, which can ultimately lead to diagnosis for patients with rare disease

XYalign: Inferring and Correcting for Sex Chromosome Ploidy in Next-Generation Sequencing Data

Sex chromosome aneuploidies are currently estimated to be as common as 1/400 in humans. Atypical ploidy will affect variant calling and measures of genomic variation that are central to most clinical genomic studies. Further, the high degree of similarity between gametologous sequences on the X and Y chromosomes can lead to the misalignment of sequencing reads and substantially affect variant calling. Here we present XYalign, a new tool that (1) quickly infers sex chromosome ploidy in NGS data (DNA and RNA), (2) remaps reads based on the inferred sex chromosome complement of the individual, and (3) outputs quality, depth, and allele-balance metrics across the sex chromosomes

XYalign: Version 1.1.4

<p>The high degree of similarity between gametologous sequences on the sex chromosomes can lead to the misalignment of sequencing reads and substantially affect variant calling. Here we present XYalign, a new tool that (1) quickly infers sex chromosome ploidy in NGS data, (2) remaps reads based on the inferred sex chromosome complement of the individual, and (3) outputs quality, depth, and allele-balance metrics across chromosomes.</p

Aspartylglycosamine is a biomarker for NGLY1-CDDG, a congenital disorder of deglycosylation

BACKGROUND: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently. METHODS: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases. RESULTS: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8-8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of -0.1 (range: -2.1-4.0) in DBS of healthy controls and patients with other diseases. DISCUSSION: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible

Comprehensive whole genome sequence analyses yields novel genetic and structural insights for Intellectual Disability

Background: Intellectual Disability (ID) is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. Whole genome sequencing (WGS) is able to interrogate the entire genome, providing potential to diagnose idiopathic patients. Methods: We conducted WGS on eight children with idiopathic ID and brain structural defects, and their normal parents; carrying out an extensive data analyses, using standard and discovery approaches. Results: We verified de novo pathogenic single nucleotide variants (SNV) in ARID1B c.1595delG and PHF6 c.820C > T, potentially causative de novo two base indels in SQSTM1 c.115_116delinsTA and UPF1 c.1576_1577delinsA, and de novo SNVs in CACNB3 c.1289G > A, and SPRY4 c.508 T > A, of uncertain significance. We report results from a large secondary control study of 2081 exomes probing the pathogenicity of the above genes. We analyzed structural variation by four different algorithms including de novo genome assembly. We confirmed a likely contributory 165 kb de novo heterozygous 1q43 microdeletion missed by clinical microarray. The de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms. We also interrogated regulatory sequence variation for known and hypothesized ID genes and present useful strategies for WGS data analyses for non-coding variation. Conclusion: This study provides an extensive analysis of WGS in the context of ID, providing genetic and structural insights into ID and yielding diagnoses.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofPediatrics, Department ofReviewedFacult

Aspartylglycosamine is a biomarker for NGLY1-CDDG, a congenital disorder of deglycosylation

Background: NGLY1-CDDG is a congenital disorder of deglycosylation caused by a defective peptide:N-glycanase (PNG). To date, all but one of the reported patients have been diagnosed through whole-exome or whole-genome sequencing, as no biochemical marker was available to identify this disease in patients. Recently, a potential urinary biomarker was reported, but the data presented suggest that this marker may be excreted intermittently. Methods: In this study, we performed untargeted direct-infusion high-resolution mass spectrometry metabolomics in seven dried blood spots (DBS) from four recently diagnosed NGLY1-CDDG patients, to test for small-molecule biomarkers, in order to identify a potential diagnostic marker. Results were compared to 125 DBS of healthy controls and to 238 DBS of patients with other diseases. Results: We identified aspartylglycosamine as the only significantly increased compound with a median Z-score of 4.8 (range: 3.8–8.5) in DBS of NGLY1-CDDG patients, compared to a median Z-score of −0.1 (range: −2.1–4.0) in DBS of healthy controls and patients with other diseases. Discussion: The increase of aspartylglycosamine can be explained by lack of function of PNG. PNG catalyzes the cleavage of the proximal N-acetylglucosamine residue of an N-glycan from the asparagine residue of a protein, a step in the degradation of misfolded glycoproteins. PNG deficiency results in a single N-acetylglucosamine residue left attached to the asparagine residue which results in free aspartylglycosamine when the glycoprotein is degraded. Thus, we here identified aspartylglycosamine as the first potential small-molecule biomarker in DBS for NGLY1-CDDG, making a biochemical diagnosis for NGLY1-CDDG potentially feasible

Additional file 5: Supplementary Figures. of Comprehensive whole genome sequence analyses yields novel genetic and structural insights for Intellectual Disability

Figure S1. IGV image and Sanger verification trace files for indel in ARID1B and missense variation in UPF1. Figure S2. UK10K mutation load – counts as per variant annotation type on one patient. Figure S3. Histogram of mutation burden per patient in the UK10K cohort. Figure S4. Pathway interactions showing convergence onto UPP pathway. Figure S5. Plots for CNV distribution for two chromosomes as called by CNAseq. (DOCX 1972 kb