Biallelic Loss of Function Variants in SENP7 Cause Immunodeficiency with Neurologic and Muscular Phenotypes.

To evaluate a novel candidate disease gene, we engaged international collaborators and identified rare, biallelic, specifically homozygous, loss of function variants in SENP7 in four children from three unrelated families presenting with neurodevelopmental abnormalities, dysmorphism, and immunodeficiency. Their clinical presentations were characterized by hypogammaglobulinemia, intermittent neutropenia, and ultimately death in infancy for all four patients. SENP7 is a sentrin-specific protease involved in posttranslational modification of proteins essential for cell regulation, via a process referred to as deSUMOylation. We propose that deficiency of deSUMOylation may represent a novel mechanism of primary immunodeficiency.

Large-scale mutational analysis identifies UNC93B1 variants that drive TLR-mediated autoimmunity in mice and humans.

Nucleic acid-sensing Toll-like receptors (TLR) 3, 7/8, and 9 are key innate immune sensors whose activities must be tightly regulated to prevent systemic autoimmune or autoinflammatory disease or virus-associated immunopathology. Here, we report a systematic scanning-alanine mutagenesis screen of all cytosolic and luminal residues of the TLR chaperone protein UNC93B1, which identified both negative and positive regulatory regions affecting TLR3, TLR7, and TLR9 responses. We subsequently identified two families harboring heterozygous coding mutations in UNC93B1, UNC93B1+/T93I and UNC93B1+/R336C, both in key negative regulatory regions identified in our screen. These patients presented with cutaneous tumid lupus and juvenile idiopathic arthritis plus neuroinflammatory disease, respectively. Disruption of UNC93B1-mediated regulation by these mutations led to enhanced TLR7/8 responses, and both variants resulted in systemic autoimmune or inflammatory disease when introduced into mice via genome editing. Altogether, our results implicate the UNC93B1-TLR7/8 axis in human monogenic autoimmune diseases and provide a functional resource to assess the impact of yet-to-be-reported UNC93B1 mutations.

Genomic Answers for Kids: Toward more equitable access to genomic testing for rare diseases in rural populations.

Next-generation sequencing has revolutionized the speed of rare disease (RD) diagnoses. While clinical exome and genome sequencing represent an effective tool for many RD diagnoses, there is room to further improve the diagnostic odyssey of many RD patients. One recognizable intervention lies in increasing equitable access to genomic testing. Rural communities represent a significant portion of underserved and underrepresented individuals facing additional barriers to diagnosis and treatment. Primary care providers (PCPs) at local clinics, though sometimes suspicious of a potential benefit of genetic testing for their patients, have significant constraints in pursuing it themselves and rely on referrals to specialists. Yet, these referrals are typically followed by long waitlists and significant delays in clinical assessment, insurance clearance, testing, and initiation of diagnosis-informed care management. Not only is this process time intensive, but it also often requires multiple visits to urban medical centers for which distance may be a significant barrier to rural families. Therefore, providing early, "direct-to-provider" (DTP) local access to unrestrictive genomic testing is likely to help speed up diagnostic times and access to care for RD patients in rural communities. In a pilot study with a PCP clinic in rural Kansas, we observed a minimum 5.5 months shortening of time to diagnosis through the DTP exome sequencing program as compared to rural patients receiving genetic testing through the "traditional" PCP-referral-to-specialist scheme. We share our experience to encourage future partnerships beyond our center. Our efforts represent just one step in fostering greater diversity and equity in genomic studies.

Significance Associated with Phenotype Score Aids in Variant Prioritization for Exome Sequencing Analysis.

Several in silico annotation-based methods have been developed to prioritize variants in exome sequencing analysis. This study introduced a novel metric Significance Associated with Phenotypes (SAP) score, which generates a statistical score by comparing an individual's observed phenotypes against existing gene-phenotype associations. To evaluate the SAP score, a retrospective analysis was performed on 219 exomes. Among them, 82 family-based and 35 singleton exomes had at least one disease-causing variant that explained the patient's clinical features. SAP scores were calculated, and the rank of the disease-causing variant was compared with a known method, Exomiser. Using the SAP score, the known causative variant was ranked in the top 10 retained variants for 94% (77 of 82) of the family-based exomes and in first place for 73% of these cases. For singleton exomes, the SAP score analysis ranked the known pathogenic variants within the top 10 for 80% (28 of 35) of cases. The SAP score, which is independent of detected variants, demonstrates comparable performance with Exomiser, which considers both phenotype and variant-level evidence simultaneously. Among 102 cases with negative results or variants of uncertain significance, SAP score analysis revealed two cases with a potential new diagnosis based on rank. The SAP score, a phenotypic quantitative metric, can be used in conjunction with standard variant filtration and annotation to enhance variant prioritization in exome analysis.

Complex trait associations in rare diseases and impacts on Mendelian variant interpretation.

Emerging evidence implicates common genetic variation - aggregated into polygenic scores (PGS) - impacting the onset and phenotypic presentation of rare diseases. In this study, we quantified individual polygenic liability for 1,151 previously published PGS in a cohort of 2,374 probands enrolled in the Genomic Answers for Kids (GA4K) rare disease study, revealing widespread associations between rare disease phenotypes and PGSs for common complex diseases and traits, blood protein levels, and brain and other organ morphological measurements. We observed increased polygenic burden in probands with variants of unknown significance (VUS) compared to unaffected carrier parents. We further observed an enrichment in overlap between diagnostic and candidate rare disease genes and large-effect PGS genes. Overall, our study supports and expands on previous findings of complex trait associations in rare disease phenotypes and provides a framework for identifying novel candidate rare disease genes and in understanding variable penetrance of candidate Mendelian disease variants.

Characterization and visualization of tandem repeats at genome scale.

Tandem repeat (TR) variation is associated with gene expression changes and numerous rare monogenic diseases. Although long-read sequencing provides accurate full-length sequences and methylation of TRs, there is still a need for computational methods to profile TRs across the genome. Here we introduce the Tandem Repeat Genotyping Tool (TRGT) and an accompanying TR database. TRGT determines the consensus sequences and methylation levels of specified TRs from PacBio HiFi sequencing data. It also reports reads that support each repeat allele. These reads can be subsequently visualized with a companion TR visualization tool. Assessing 937,122 TRs, TRGT showed a Mendelian concordance of 98.38%, allowing a single repeat unit difference. In six samples with known repeat expansions, TRGT detected all expansions while also identifying methylation signals and mosaicism and providing finer repeat length resolution than existing methods. Additionally, we released a database with allele sequences and methylation levels for 937,122 TRs across 100 genomes.

Pangenome graphs improve the analysis of structural variants in rare genetic diseases.

Rare DNA alterations that cause heritable diseases are only partially resolvable by clinical next-generation sequencing due to the difficulty of detecting structural variation (SV) in all genomic contexts. Long-read, high fidelity genome sequencing (HiFi-GS) detects SVs with increased sensitivity and enables assembling personal and graph genomes. We leverage standard reference genomes, public assemblies (n = 94) and a large collection of HiFi-GS data from a rare disease program (Genomic Answers for Kids, GA4K, n = 574 assemblies) to build a graph genome representing a unified SV callset in GA4K, identify common variation and prioritize SVs that are more likely to cause genetic disease (MAF < 0.01). Using graphs, we obtain a higher level of reproducibility than the standard reference approach. We observe over 200,000 SV alleles unique to GA4K, including nearly 1000 rare variants that impact coding sequence. With improved specificity for rare SVs, we isolate 30 candidate SVs in phenotypically prioritized genes, including known disease SVs. We isolate a novel diagnostic SV in KMT2E, demonstrating use of personal assemblies coupled with pangenome graphs for rare disease genomics. The community may interrogate our pangenome with additional assemblies to discover new SVs within the allele frequency spectrum relevant to genetic diseases.

Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.

Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort.

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.

Can endoscopic follow-up after acute diverticulitis be rationalised?

BACKGROUND: Acute diverticulitis (AD) is a common cause of presentation to emergency surgical services. Follow-up with endoluminal investigation to exclude colorectal cancer (CRC) remains controversial. Guidelines are increasingly moving to a more restrictive follow-up based on severity of disease and age. The purpose of this observational study was to assess the prevalence of CRC in AD patients and the impact of follow-up on endoscopy services. METHODS: Patients admitted with a diagnosis of AD over a 2-year period were reviewed. The proportion of patients undergoing endoscopic follow-up and the CRC detection rate were recorded. The potential impact of a more conservative approach to follow-up was evaluated. RESULTS: There were 484 patients with AD presenting 546 times (M:F = 198:286; median age = 63 years). 80% of admissions were aged 50 or older. There were 43 emergency interventions in 39 patients (10 percutaneous drain; 33 surgery). The remainder were managed conservatively. 28 patients (5.1%) underwent colonic resection with cancer found in one specimen (3.6%). 287 patients underwent endoluminal follow-up with cancer diagnosed in 3 cases (1.0%). There was no significant difference in the prevalence of CRC between patients requiring emergency surgery and those managed conservatively, or between patients with complicated versus uncomplicated diverticulitis. CONCLUSION: CRC masquerading as acute diverticulitis is rare. The incidence of neoplasia both at endoscopic follow-up and in patients requiring emergency intervention is low. Conservative follow-up strategies appear safe, but their effectiveness in reducing the burden on endoscopy services may be limited by current age-based recommendations. Restricting follow-up to those with complicated AD would reduce the number of patients requiring endoluminal investigation by 70%.

Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing.

Spinal muscular atrophy, a leading cause of early infant death, is caused by bi-allelic mutations of SMN1. Sequence analysis of SMN1 is challenging due to high sequence similarity with its paralog SMN2. Both genes have variable copy numbers across populations. Furthermore, without pedigree information, it is currently not possible to identify silent carriers (2+0) with two copies of SMN1 on one chromosome and zero copies on the other. We developed Paraphase, an informatics method that identifies full-length SMN1 and SMN2 haplotypes, determines the gene copy numbers, and calls phased variants using long-read PacBio HiFi data. The SMN1 and SMN2 copy-number calls by Paraphase are highly concordant with orthogonal methods (99.2% for SMN1 and 100% for SMN2). We applied Paraphase to 438 samples across 5 ethnic populations to conduct a population-wide haplotype analysis of these highly homologous genes. We identified major SMN1 and SMN2 haplogroups and characterized their co-segregation through pedigree-based analyses. We identified two SMN1 haplotypes that form a common two-copy SMN1 allele in African populations. Testing positive for these two haplotypes in an individual with two copies of SMN1 gives a silent carrier risk of 88.5%, which is significantly higher than the currently used marker (1.7%-3.0%). Extending beyond simple copy-number testing, Paraphase can detect pathogenic variants and enable potential haplotype-based screening of silent carriers through statistical phasing of haplotypes into alleles. Future analysis of larger population data will allow identification of more diverse haplotypes and genetic markers for silent carriers.

Insurance denials and diagnostic rates in a pediatric genomic research cohort.

PURPOSE: This study aimed to assess the amount and types of clinical genetic testing denied by insurance and the rate of diagnostic and candidate genetic findings identified through research in patients who faced insurance denials. METHODS: Analysis consisted of review of insurance denials in 801 patients enrolled in a pediatric genomic research repository with either no previous genetic testing or previous negative genetic testing result identified through cross-referencing with insurance prior-authorizations in patient medical records. Patients and denials were also categorized by type of insurance coverage. Diagnostic findings and candidate genetic findings in these groups were determined through review of our internal variant database and patient charts. RESULTS: Of the 801 patients analyzed, 147 had insurance prior-authorization denials on record (18.3%). Exome sequencing and microarray were the most frequently denied genetic tests. Private insurance was significantly more likely to deny testing than public insurance (odds ratio = 2.03 [95% CI = 1.38-2.99] P = .0003). Of the 147 patients with insurance denials, 53.7% had at least 1 diagnostic or candidate finding and 10.9% specifically had a clinically diagnostic finding. Fifty percent of patients with clinically diagnostic results had immediate medical management changes (5.4% of all patients experiencing denials). CONCLUSION: Many patients face a major barrier to genetic testing in the form of lack of insurance coverage. A number of these patients have clinically diagnostic findings with medical management implications that would not have been identified without access to research testing. These findings support re-evaluation of insurance carriers' coverage policies.

Genetic heterogeneity and enrichment of variants in DNA-repair genes in ameloblastoma.

OBJECTIVE: Ameloblastomas are a group of relatively common odontogenic tumors that frequently originate from the dental epithelium. These tumors are aggressive in nature and present as slow-growing painless cortical expansion of the jaw. Histologically, the follicular and plexiform subtypes constitute two-thirds of solid/multicystic ameloblastomas. The objective of this study was to understand the genetic architecture of follicular and plexiform ameloblastomas using deep whole-exome sequencing. METHODS: Archived formalin-fixed paraffin-embedded tissue blocks of follicular (n = 4) and plexiform (n = 6) ameloblastomas were retrieved and genomic DNAs were isolated from the tumor tissue dissected from the formalin-fixed paraffin-embedded block. The exomes were enriched using the Integrated DNA Technologies Exome Research Panel (IDT, Coralville, IA) and paired-end sequencing was completed on an Illumina NovaSeq 6000 with an average output of 20 GB of data resulting in a mean coverage of 400×. Variant analysis was completed using custom-developed software: Rapid Understanding of Nucleotide variant Effect Software and variant integration and knowledge interpretation in genomes. RESULTS: Our analyses focused on examining somatic variants (gnomAD minor allele frequency ≤1%) in genes found on an Food and Drug Administration -approved clinical cancer sequencing panel (FoundationOne®CDx). In follicular tumors, variants (>20% of the reads) were identified in BRAF, KMT2D, and ABL1 genes. In plexiform tumors, variants (>20% of the reads) were identified in ALK, BRAF, KRAS, KMT2D, SMO, KMT2A, and BRCA2 genes. Enrichment analysis showed a significant role of DNA repair genes in the development of these tumors. CONCLUSION: The variants identified in follicular and plexiform ameloblastomas were enriched in DNA-repair genes. The observed genetic heterogeneity in these ameloblastomas may contribute to the aggressive nature and recurrence risk of these tumors.

Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype.

Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation.

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

Clinical Validation of Genome Reference Consortium Human Build 38 in a Laboratory Utilizing Next-Generation Sequencing Technologies.

BACKGROUND: Laboratories utilizing next-generation sequencing align sequence data to a standardized human reference genome (HRG). Several updated versions, or builds, have been released since the original HRG in 2001, including the Genome Reference Consortium Human Build 38 (GRCh38) in 2013. However, most clinical laboratories still use GRCh37, which was released in 2009. We report our laboratory's clinical validation of GRCh38. METHODS: Migration to GRCh38 was validated by comparing the coordinates (lifting over) of 9443 internally curated variants from GRCh37 to GRCh38, globally comparing protein coding sequence variants aligned with GRCh37 vs GRCh38 from 917 exomes, assessing genes with known discrepancies, comparing coverage differences, and establishing the analytic sensitivity and specificity of variant detection using Genome in a Bottle data. RESULTS: Eight discrepancies, due to strand swap or reference base, were observed. Three clinically relevant variants had the GRCh37 alternate allele as the reference allele in GRCh38. A comparison of 88 295 calls between builds identified 8 disease-associated genes with sequence differences: ABO, BNC2, KIZ, NEFL, NR2E3, PTPRQ, SHANK2, and SRD5A2. Discrepancies in coding regions in GRCh37 were resolved in GRCh38. CONCLUSIONS: There were a small number of clinically significant changes between the 2 genome builds. GRCh38 provided improved detection of nucleotide changes due to the resolution of discrepancies present in GRCh37. Implementation of GRCh38 results in more accurate and consistent reporting.

Phenotypic expansion and variable expressivity in individuals with JARID2-related intellectual disability: A case series.

Loss of function variants in JARID2 were recently reported in 16 patients with a neurodevelopmental disorder characterized by delays, intellectual and learning disability, autism, behavioral abnormalities, and dysmorphic features. Most cases were de novo, with only one variant inherited from an affected parent. Here, we present seven additional individuals from five families with pathogenic or likely pathogenic JARID2 variants, confirming this gene-disease association and highlighting palatal abnormalities and heart defects as part of the phenotype. In addition, we report inheritance of JARID2 variants from mildly affected parents, demonstrating the variable expressivity of the disease. We also note the high prevalence of intragenic JARID2 copy number variants, emphasizing the importance of exon-level analysis.

Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes.

PURPOSE: This study aimed to provide comprehensive diagnostic and candidate analyses in a pediatric rare disease cohort through the Genomic Answers for Kids program. METHODS: Extensive analyses of 960 families with suspected genetic disorders included short-read exome sequencing and short-read genome sequencing (srGS); PacBio HiFi long-read genome sequencing (HiFi-GS); variant calling for single nucleotide variants (SNV), structural variant (SV), and repeat variants; and machine-learning variant prioritization. Structured phenotypes, prioritized variants, and pedigrees were stored in PhenoTips database, with data sharing through controlled access the database of Genotypes and Phenotypes. RESULTS: Diagnostic rates ranged from 11% in patients with prior negative genetic testing to 34.5% in naive patients. Incorporating SVs from genome sequencing added up to 13% of new diagnoses in previously unsolved cases. HiFi-GS yielded increased discovery rate with >4-fold more rare coding SVs compared with srGS. Variants and genes of unknown significance remain the most common finding (58% of nondiagnostic cases). CONCLUSION: Computational prioritization is efficient for diagnostic SNVs. Thorough identification of non-SNVs remains challenging and is partly mitigated using HiFi-GS sequencing. Importantly, community research is supported by sharing real-time data to accelerate gene validation and by providing HiFi variant (SNV/SV) resources from >1000 human alleles to facilitate implementation of new sequencing platforms for rare disease diagnoses.