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ObjectiveGenetics play an important role in systemic lupus erythematosus (SLE) pathogenesis. We calculated the prevalence of rare variants in known monogenic lupus genes among children suspected of monogenic lupus.MethodsWe completed paired-end genome-wide sequencing (whole genome sequencing [WGS] or whole exome sequencing) in patients suspected of monogenic lupus, and focused on 36 monogenic lupus genes. We prioritized rare (minor allele frequency < 1%) exonic, nonsynonymous, and splice variants with predicted pathogenicity classified as deleterious variants (Combined Annotation Dependent Depletion [CADD], PolyPhen2, and Sorting Intolerant From Tolerant [SIFT] scores). Additional filtering restricted to predicted damaging variants by considering reported zygosity. In those with WGS (n = 69), we examined copy number variants (CNVs) > 1 kb in size. We created additive non-HLA and HLA SLE genetic risk scores (GRSs) using common SLE-risk single-nucleotide polymorphisms. We tested the relationship between SLE GRSs and the number of rare variants with multivariate logistic models, adjusted for sex, ancestry, and age of diagnosis.ResultsThe cohort included 71 patients, 80% female, with a mean age at diagnosis of 8.9 (SD 3.2) years. We identified predicted damaging variants in 9 (13%) patients who were significantly younger at diagnosis compared to those without a predicted damaging variant (6.8 [SD 2.1] years vs 9.2 [SD 3.2] years,P= 0.01). We did not identify damaging CNVs. There was no significant association between non-HLA or HLA SLE GRSs and the odds of carrying ≥ 1 rare variant in multivariate analyses.ConclusionIn a cohort of patients with suspected monogenic lupus who underwent genome-wide sequencing, 13% carried rare predicted damaging variants for monogenic lupus. Additional studies are needed to validate our findings.

The factors driving initiation of pathological expansion of tandem repeats remain largely unknown. Here, we assessed theFGF14-SCA27B (GAA)•(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a 5’-flanking 17-bp deletion-insertion in 70.34% of alleles (3,463/4,923). This common sequence variation was present nearly exclusively on alleles with fewer than 30 GAA-pure repeats and was associated with enhanced meiotic stability of the repeat locus.

ObjectiveMacrophage activation syndrome (MAS), a life-threatening complication of systemic lupus erythematosus (SLE), resembles familial hemophagocytic lymphohistiocytosis (HLH), an inherited disorder of hyperinflammation. We compared the proportion of patients with childhood-onset SLE (cSLE) with and without MAS who carried low-frequency HLH nonsynonymous variants.MethodsWe enrolled patients from the Lupus Clinic at SickKids, Toronto. Demographic and clinical features were extracted from the SLE database and ancestry was genetically inferred using multiethnic genotyping array data. Patients with MAS (based on expert diagnosis) underwent either paired-end whole-exome sequencing (WES; read depth: 70-118X) or whole-genome sequencing (WGS). Patients without MAS had WGS (read depth: 37-40X). In 16 HLH genes, we prioritized low-frequency (minor allele frequency [MAF] < 0.05) exonic nonsynonymous variants. We compared the proportion of patients with and without MAS carrying HLH variants (Fisher exact test,P< 0.05). MAFs were compared to an ancestrally matched general population (Trans-Omics for Precision Medicine [TOPMed] and Genome Aggregation Database [gnomAD]).ResultsThe study included 81 patients with cSLE, 19 of whom had MAS. We identified 47 unique low-frequency nonsynonymous HLH variants. There was no difference in the proportion of patients with and without MAS carrying ≥ 1 HLH variants (37% vs 47%,P= 0.44). The MAS cohort did not carry more HLH variants when compared to an ancestrally matched general population.ConclusionIn a single-center multiethnic cSLE cohort, we found no difference in the proportion of patients with MAS carrying nonsynonymous HLH genetic variants compared to patients without MAS. To our knowledge, this is the first study to examine the frequency of HLH genetic variants in relation to MAS among patients with cSLE. Future studies are required to validate our findings.

We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.

Despite recent progress in the understanding of the genetic etiologies of rare diseases (RDs), a significant number remain intractable to diagnostic and discovery efforts. Broad data collection and sharing of information among RD researchers is therefore critical. In 2018, the Care4Rare Canada Consortium launched the project C4R-SOLVE, a subaim of which was to collect, harmonize, and share both retrospective and prospective Canadian clinical and multiomic data. Here, we introduce Genomics4RD, an integrated web-accessible platform to share Canadian phenotypic and multiomic data between researchers, both within Canada and internationally, for the purpose of discovering the mechanisms that cause RDs. Genomics4RD has been designed to standardize data collection and processing, and to help users systematically collect, prioritize, and visualize participant information. Data storage, authorization, and access procedures have been developed in collaboration with policy experts and stakeholders to ensure the trusted and secure access of data by external researchers. The breadth and standardization of data offered by Genomics4RD allows researchers to compare candidate disease genes and variants between participants (i.e., matchmaking) for discovery purposes, while facilitating the development of computational approaches for multiomic data analyses and enabling clinical translation efforts for new genetic technologies in the future.

Genome-wide sequencing (GWS) is a standard of care for diagnosis of suspected genetic disorders, but the proportion of patients found to have pathogenic or likely pathogenic variants ranges from less than 30% to more than 60% in reported studies. It has been suggested that the diagnostic rate can be improved by interpreting genomic variants in the context of each affected individual's full clinical picture and by regular follow-up and reinterpretation of GWS laboratory results. Trio exome sequencing was performed in 415 families and trio genome sequencing in 85 families in the CAUSES study. The variants observed were interpreted by a multidisciplinary team including laboratory geneticists, bioinformaticians, clinical geneticists, genetic counselors, pediatric subspecialists, and the referring physician, and independently by a clinical laboratory using standard American College of Medical Genetics and Genomics (ACMG) criteria. Individuals were followed for an average of 5.1 years after testing, with clinical reassessment and reinterpretation of the GWS results as necessary. The multidisciplinary team established a diagnosis of genetic disease in 43.0% of the families at the time of initial GWS interpretation, and longitudinal follow-up and reinterpretation of GWS results produced new diagnoses in 17.2% of families whose initial GWS interpretation was uninformative or uncertain. Reinterpretation also resulted in rescinding a diagnosis in four families (1.9%). Of the families studied, 33.6% had ACMG pathogenic or likely pathogenic variants related to the clinical indication. Close collaboration among clinical geneticists, genetic counselors, laboratory geneticists, bioinformaticians, and individuals’ primary physicians, with ongoing follow-up, reanalysis, and reinterpretation over time, can improve the clinical value of GWS.

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.

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.

BackgroundMammalian X and Y chromosomes share a common evolutionary origin and retain regions of high sequence similarity. Similar sequence content can confound the mapping of short next-generation sequencing reads to a reference genome. It is therefore possible that the presence of both sex chromosomes in a reference genome can cause technical artifacts in genomic data and affect downstream analyses and applications. Understanding this problem is critical for medical genomics and population genomic inference.ResultsHere, we characterize how sequence homology can affect analyses on the sex chromosomes and present XYalign, a new tool that (1) facilitates the inference of sex chromosome complement from next-generation sequencing data; (2) corrects erroneous read mapping on the sex chromosomes; and (3) tabulates and visualizes important metrics for quality control such as mapping quality, sequencing depth, and allele balance. We find that sequence homology affects read mapping on the sex chromosomes and this has downstream effects on variant calling. However, we show that XYalign can correct mismapping, resulting in more accurate variant calling. We also show how metrics output by XYalign can be used to identify XX and XY individuals across diverse sequencing experiments, including low- and high-coverage whole-genome sequencing, and exome sequencing. Finally, we discuss how the flexibility of the XYalign framework can be leveraged for other uses including the identification of aneuploidy on the autosomes. XYalign is available open source under the GNU General Public License (version 3).ConclusionsSex chromsome sequence homology causes the mismapping of short reads, which in turn affects downstream analyses. XYalign provides a reproducible framework to correct mismapping and improve variant calling on the sex chromsomes.

The Exactis trial (NCT00984048), assessed plasma circulating-tumor DNA (ctDNA) mutations and whether they correlated with their relative levels in metastatic tumor biopsies in 50 mCRC enrolled patients undergoing first-line treatment. Exactis is a Canadian National Centre of Excellence in Personalized Medicine and clinical trials. Two-hundred and twenty-two plasma ctDNA samples collected at baseline, on treatment, and at time of clinical resistance (median 4 samples per patient) were sequenced and analyzed using the Contextual Genomics FOLLOW ITTM assay with QUALITY NEXUSTM bioinformatics analytical pipeline. This panel assesses hotspot mutations and frequently mutated regions in 30 commonly mutated cancer genes. Patient overall objective response was based on RECIST v1.0 criteria. We detected ctDNA mutations in at least one plasma timepoint in 92% (46/50) of patients and in multiple timepoints in 76% (38/50), including mutations in KRAS or NRAS (52%), PIK3CA (26%), BRAF (6%), and TP53 (74%). In 3 of the 4 patients with no detectable mutations, exome sequencing of the tumor revealed no mutations covered by the ctDNA assay. The other had both a KRAS and PIK3CA mutation in the tumor data; both were present in the FOLLOW ITTM sequencing data but were below the clinically validated threshold for the assay. Of interest, 8 out of 26 patients with ctDNA KRAS mutations detected (31%) had a G12D or G12C variant, which may have implications for new targeted therapeutics. We also observed a trend toward enrichment of KRAS/NRAS mutations in nonresponder (NR) compared to responder (R) patients (16/23 NR versus 10/23 R, p=0.07, Chi-square test). Furthermore, the occurrence of PIK3CA alone or in combination with KRAS mutations was significantly higher in the NR patients (10/23 NR versus 3/23 R p=0.02; and 8/23 NR versus 1/23 R p=0.0092, Chi-square test). Within the NR patient population, we found that 17 out of 23 (74%) patients harbored detectable ctDNA mutations preceding progression detection based on CT scan imaging, highlighting the potential of liquid biopsies to monitor disease progression in mCRC. Our study showed that the FOLLOW ITTM assay is capable of detecting mutations in CRC driver genes using liquid biopsy within a clinical trial setting. The identification of plasma ctDNA mutations in this context could allow for re-evaluation and potential change in management before clinical or CT scan detected relapse has occurred. In addition, the detection of ctDNA PIK3CA and KRAS mutations enrichment in the NR population warrants further validation and investigation for its clinical utility for patient stratification. Citation Format: Melissa K. McConechy, Suzan McNamara, Mathilde Couetoux du Tertre, Karen Gambaro, Maud Marques, Salem Malikic, Ka Mun Nip, Sonal Brahmbhatt, Adrian Kense, Kevin Tam, Rosalia Aguirre Hernandez, Ruth Miller, Madeline Couse, Jas Khattra, David G. Huntsman, Gerald Batist. Sequential ctDNA analysis detected preclinical relapse in patients with metastatic colorectal cancer from the Exactis trial (NCT00984048) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A07.

Mammalian X and Y chromosomes share a common evolutionary origin and retain regions of high sequence similarity. This sequence homology can cause the mismapping of short sequencing reads derived from the sex chromosomes and affect variant calling and other downstream analyses. Understanding and correcting this problem is critical for medical genomics and population genomic inference. Here, we characterize how sequence homology can affect analyses on the sex chromosomes and present XYalign, a new tool that: (1) facilitates the inference of sex chromosome complement from next-generation sequencing data; (2) corrects erroneous read mapping on the sex chromosomes; and (3) tabulates and visualizes important metrics for quality control such as mapping quality, sequencing depth, and allele balance. We show how these metrics can be used to identify XX and XY individuals across diverse sequencing experiments, including low and high coverage whole genome sequencing, and exome sequencing. We also show that XYalign corrects mismapped reads on the sex chromosomes, resulting in more accurate variant calling. Finally, we discuss how the flexibility of the XYalign framework can be leveraged for other use cases including the identification of aneuploidy on the autosomes. XYalign is available open source under the GNU General Public License (version 3).

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3671-0) contains supplementary material, which is available to authorized users.