Exome sequencing identified rare recurrent copy number variants and hereditary breast cancer susceptibility.

Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10-7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31-8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62-31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searching for genetic factors underlying hereditary disease predisposition.

Clinical exome sequencing data from patients with inborn errors of immunity: Cohort level diagnostic yield and the benefit of systematic reanalysis.

While next generation sequencing has expanded the scientific understanding of Inborn Errors of Immunity (IEI), the clinical use and re-use of exome sequencing is still emerging. We revisited clinical exome data from 1300 IEI patients using an updated in silico IEI gene panel. Variants were classified and curated through expert review. The molecular diagnostic yield after standard exome analysis was 11.8 %. Through systematic reanalysis, we identified variants of interest in 5.2 % of undiagnosed patients, with 76.7 % being (candidate) disease-causing, providing a (candidate) diagnosis in 15.2 % of our cohort. We find a 1.7 percentage point increase in conclusive molecular diagnoses. We find a high degree of actionability in patients with a genetic diagnosis (76.4 %). Despite the modest absolute diagnostic gain, these data support the benefit of iterative exome reanalysis in IEI patients, conveying the notion that our current understanding of genes and variants involved in IEI is by far not saturated.

Automatized detection of uniparental disomies in a large cohort.

Uniparental disomy (UPD) is the inheritance of both homologues of a chromosome from only one parent. The detection of UPDs in sequencing data is not well established and a common gap in genetic diagnostics. We applied our in-house UPD detection pipeline to evaluate a cohort of 9212 samples, including multigene panels as well as exome sequencing data in a single, duo or trio constellation. We used the results to inform the design of our publicly available web app altAFplotter. UPDs categorized as heterodisomy, whole chromosome or segmental isodisomy were identified and validated with microsatellites, multiplex ligation-dependent probe amplification as well as Sanger sequencing. We detected 14 previously undiagnosed UPDs including nine isodisomies, four segmental isodisomies as well as one heterodisomy on chromosome 22. We characterized eight findings as potentially causative through homozygous pathogenic variants or imprinting disorders. Overall, our study demonstrates the utility of our UPD detection pipeline with our web app, altAFplotter, to reliably identify UPDs. This not only increases the diagnostic yield of cases with growth and metabolic disturbances, as well as developmental delay, but also enhances the understanding of UPDs that may be relevant for recurrence risks and genetic counseling.

Exome variant prioritization in a large cohort of hearing-impaired individuals indicates IKZF2 to be associated with non-syndromic hearing loss and guides future research of unsolved cases.

Although more than 140 genes have been associated with non-syndromic hereditary hearing loss (HL), at least half of the cases remain unexplained in medical genetic testing. One reason is that pathogenic variants are located in 'novel' deafness genes. A variant prioritization approach was used to identify novel (candidate) genes for HL. Exome-wide sequencing data were assessed for subjects with presumed hereditary HL that remained unexplained in medical genetic testing by gene-panel analysis. Cases in group AD had presumed autosomal dominantly inherited HL (n = 124), and in group AR, presumed autosomal recessive HL (n = 337). Variants in known and candidate deafness genes were prioritized based on allele frequencies and predicted effects. Selected variants were tested for their co-segregation with HL. Two cases were solved by variants in recently identified deafness genes (ABHD12, TRRAP). Variant prioritization also revealed potentially causative variants in candidate genes associated with recessive and X-linked HL. Importantly, missense variants in IKZF2 were found to co-segregate with dominantly inherited non-syndromic HL in three families. These variants specifically affected Zn2+-coordinating cysteine or histidine residues of the zinc finger motifs 2 and 3 of the encoded protein Helios. This finding indicates a complex genotype-phenotype correlation for IKZF2 defects, as this gene was previously associated with non-syndromic dysfunction of the immune system and ICHAD syndrome, including HL. The designed strategy for variant prioritization revealed that IKZF2 variants can underlie non-syndromic HL. The large number of candidate genes for HL and variants therein stress the importance of inclusion of family members for variant prioritization.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

Common and rare variants in patients with early onset drusen maculopathy.

Early onset drusen maculopathy (EODM) can lead to advanced macular degeneration at a young age, affecting quality of life. However, the genetic causes of EODM are not well studied. We performed whole genome sequencing in 49 EODM patients. Common genetic variants were analysed by calculating genetic risk scores based on 52 age-related macular generation (AMD)-associated variants, and we analysed rare variants in candidate genes to identify potential deleterious variants that might contribute to EODM development. We demonstrate that the 52 AMD-associated variants contributed to EODM, especially variants located in the complement pathway. Furthermore, we identified 26 rare genetic variants predicted to be pathogenic based on in silico prediction tools or based on reported pathogenicity in literature. These variants are located predominantly in the complement and lipid metabolism pathways. Last, evaluation of 18 genes causing inherited retinal dystrophies that can mimic AMD characteristics, revealed 11 potential deleterious variants in eight EODM patients. However, phenotypic characteristics did not point towards a retinal dystrophy in these patients. In conclusion, this study reports new insights into rare variants that are potentially involved in EODM development, and which are relevant for future studies unravelling the aetiology of EODM.

Presence of Genetic Variants Among Young Men With Severe COVID-19.

Importance: Severe coronavirus disease 2019 (COVID-19) can occur in younger, predominantly male, patients without preexisting medical conditions. Some individuals may have primary immunodeficiencies that predispose to severe infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objective: To explore the presence of genetic variants associated with primary immunodeficiencies among young patients with COVID-19. Design, Setting, and Participants: Case series of pairs of brothers without medical history meeting the selection criteria of young (age <35 years) brother pairs admitted to the intensive care unit (ICU) due to severe COVID-19. Four men from 2 unrelated families were admitted to the ICUs of 4 hospitals in the Netherlands between March 23 and April 12, 2020. The final date of follow-up was May 16, 2020. Available family members were included for genetic variant segregation analysis and as controls for functional experiments. Exposure: Severe COVID-19. Main Outcome and Measures: Results of rapid clinical whole-exome sequencing, performed to identify a potential monogenic cause. Subsequently, basic genetic and immunological tests were performed in primary immune cells isolated from the patients and family members to characterize any immune defects. Results: The 4 male patients had a mean age of 26 years (range, 21-32), with no history of major chronic disease. They were previously well before developing respiratory insufficiency due to severe COVID-19, requiring mechanical ventilation in the ICU. The mean duration of ventilatory support was 10 days (range, 9-11); the mean duration of ICU stay was 13 days (range, 10-16). One patient died. Rapid clinical whole-exome sequencing of the patients and segregation in available family members identified loss-of-function variants of the X-chromosomal TLR7. In members of family 1, a maternally inherited 4-nucleotide deletion was identified (c.2129_2132del; p.[Gln710Argfs*18]); the affected members of family 2 carried a missense variant (c.2383G>T; p.[Val795Phe]). In primary peripheral blood mononuclear cells from the patients, downstream type I interferon (IFN) signaling was transcriptionally downregulated, as measured by significantly decreased mRNA expression of IRF7, IFNB1, and ISG15 on stimulation with the TLR7 agonist imiquimod as compared with family members and controls. The production of IFN-γ, a type II IFN, was decreased in patients in response to stimulation with imiquimod. Conclusions and Relevance: In this case series of 4 young male patients with severe COVID-19, rare putative loss-of-function variants of X-chromosomal TLR7 were identified that were associated with impaired type I and II IFN responses. These preliminary findings provide insights into the pathogenesis of COVID-19.

Whole-Exome Sequencing Identifies Biallelic IDH3A Variants as a Cause of Retinitis Pigmentosa Accompanied by Pseudocoloboma.

PURPOSE: To identify the genetic cause of and describe the phenotype in 4 families with autosomal recessive retinitis pigmentosa (arRP) that can be associated with pseudocoloboma. DESIGN: Case series. PARTICIPANTS: Seven patients from 4 unrelated families with arRP, among whom 3 patients had bilateral early-onset macular pseudocoloboma. METHODS: We performed homozygosity mapping and whole-exome sequencing in 5 probands and 2 unaffected family members from 4 unrelated families. Subsequently, Sanger sequencing and segregation analysis were performed in additional family members. We reviewed the medical history of individuals carrying IDH3A variants and performed additional ophthalmic examinations, including full-field electroretinography, fundus photography, fundus autofluorescence imaging, and optical coherence tomography. MAIN OUTCOME MEASURES: IDH3A variants, age at diagnosis, visual acuity, fundus appearance, visual field, and full-field electroretinography, fundus autofluorescence, and optical coherence tomography findings. RESULTS: We identified 7 different variants in IDH3A in 4 unrelated families, that is, 5 missense, 1 nonsense, and 1 frameshift variant. All participants showed symptoms early in life, ranging from night blindness to decreased visual acuity, and were diagnosed between the ages of 1 and 11 years. Four participants with biallelic IDH3A variants displayed a typical arRP phenotype and 3 participants were diagnosed with arRP and pseudocoloboma of the macula. CONCLUSIONS: IDH3A variants were identified as a novel cause of typical arRP in some individuals associated with macular pseudocoloboma. We observed both phenotypes in 2 siblings carrying the same compound heterozygous variants, which could be explained by variable disease expression and warrants caution when making assertions about genotype-phenotype correlations.

Whole genome sequencing identifies elusive variants in genetically unsolved Italian inherited retinal disease patients.

Inherited retinal diseases (IRDs) are a group of rare monogenic diseases with high genetic heterogeneity (pathogenic variants identified in over 280 causative genes). The genetic diagnostic rate for IRDs is around 60%, mainly thanks to the routine application of next-generation sequencing (NGS) approaches such as extensive gene panels or whole exome analyses. Whole-genome sequencing (WGS) has been reported to improve this diagnostic rate by revealing elusive variants, such as structural variants (SVs) and deep intronic variants (DIVs). We performed WGS on 33 unsolved cases with suspected autosomal recessive IRD, aiming to identify causative genetic variants in non-coding regions or to detect SVs that were unexplored in the initial screening. Most of the selected cases (30 of 33, 90.9%) carried monoallelic pathogenic variants in genes associated with their clinical presentation, hence we first analyzed the non-coding regions of these candidate genes. Whenever additional pathogenic variants were not identified with this approach, we extended the search for SVs and DIVs to all IRD-associated genes. Overall, we identified the missing causative variants in 11 patients (11 of 33, 33.3%). These included three DIVs in ABCA4, CEP290 and RPGRIP1; one non-canonical splice site (NCSS) variant in PROM1 and three SVs (large deletions) in EYS, PCDH15 and USH2A. For the previously unreported DIV in CEP290 and for the NCCS variant in PROM1, we confirmed the effect on splicing by reverse transcription (RT)-PCR on patient-derived RNA. This study demonstrates the power and clinical utility of WGS as an all-in-one test to identify disease-causing variants missed by standard NGS diagnostic methodologies.

Early detection of active Human CytomegaloVirus (hCMV) infection in pregnant women using data generated for noninvasive fetal aneuploidy testing.

BACKGROUND: Prenatal hCMV infections can lead to severe embryopathy and neurological sequelae in neonates. Screening during pregnancy is not recommended by global societies, as there is no effective therapy. Recently, several groups showed that maternal-fetal hCMV transmission can be strongly reduced by administering anti-viral agents early in pregnancy. This calls for a screening method to identify at risk pregnancies at an appropriate gestational age, with the possibility for large-scale enrolment. Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidy screening early in pregnancy is already implemented in many countries and performed on a large-scale basis. We investigated the use of whole genome cell-free DNA (cfDNA) sequencing data, generated for the purpose of NIPT, as (pre-)screening tool to identify women with active hCMV-infections, eligible for therapy. METHODS: Coded raw sequencing NIPT data from 204,818 pregnant women from three testing laboratories were analyzed for the presence of hCMV-cfDNA. Samples were stratified by cfDNA-hCMV load. For validation and interpretation, diagnostic hCMV-qPCR and serology testing were performed on a subset of cfDNA-hCMV-positive (n = 112) and -negative (n = 127) samples. FINDINGS: In 1930 samples (0.94%) hCMV fragments were detected. Validation by hCMV-qPCR showed that samples with high cfDNA-hCMV load tested positive and cfDNA-hCMV-negative samples tested negative. In 32/112 cfDNA-hCMV-positive samples (28.6%) the serological profile suggested a recent primary infection: this was more likely in samples with high cfDNA-hCMV load (78.6%) than in samples with low cfDNA-hCMV load (11.0%). In none of the cfDNA-hCMV-negative samples serology was indicative of a recent primary infection. INTERPRETATION: Our study shows that large-scale (pre-)screening for both genetic fetal aberrations and active maternal hCMV infections during pregnancy can be combined in one cfDNA sequencing test, performed on a single blood sample, drawn in the first trimester of pregnancy. FUNDING: This work was partly funded by the Prenatal Screening Foundation Nijmegen, the Netherlands.

Lipopeptide-mediated Cas9 RNP delivery: A promising broad therapeutic strategy for safely removing deep-intronic variants in ABCA4.

Deep-intronic (DI) variants represent approximately 10%-12% of disease-causing genetic defects in ABCA4-associated Stargardt disease (STGD1). Although many of these DI variants are amenable to antisense oligonucleotide-based splicing-modulation therapy, no treatment is currently available. These molecules are mostly variant specific, limiting their applicability to a broader patient population. In this study, we investigated the therapeutic potential of the CRISPR-Cas9 system combined with the amphipathic lipopeptide C18:1-LAH5 for intracellular delivery to correct splicing defects caused by different DI variants within the same intron. The combination of these components facilitated efficient editing of two target introns (introns 30 and 36) of ABCA4 in which several recurrent DI variants are found. The partial removal of these introns did not affect ABCA4 splicing or its expression levels when assessed in two different human cellular models: fibroblasts and induced pluripotent stem cell-derived photoreceptor precursor cells (PPCs). Furthermore, the DNA editing in STGD1 patient-derived PPCs led to a ∼50% reduction of the pseudoexon-containing transcripts resulting from the c.4539+2001G>A variant in intron 30. Overall, we provide proof-of-concept evidence of the use of C18:1-LAH5 as a delivery system for therapeutic genome editing for ABCA4-associated DI variants, offering new opportunities for clinical translation.

Identification of novel 3D-genome altering and complex structural variants underlying retinitis pigmentosa type 17 through a multistep and high-throughput approach.

Introduction: Autosomal dominant retinitis pigmentosa type 17 (adRP, type RP17) is caused by complex structural variants (SVs) affecting a locus on chromosome 17 (chr17q22). The SVs disrupt the 3D regulatory landscape by altering the topologically associating domain (TAD) structure of the locus, creating novel TAD structures (neo-TADs) and ectopic enhancer-gene contacts. Currently, screening for RP17-associated SVs is not included in routine diagnostics given the complexity of the variants and a lack of cost-effective detection methods. The aim of this study was to accurately detect novel RP17-SVs by establishing a systematic and efficient workflow. Methods: Genetically unexplained probands diagnosed with adRP (n = 509) from an international cohort were screened using a smMIPs or genomic qPCR-based approach tailored for the RP17 locus. Suspected copy number changes were validated using high-density SNP-array genotyping, and SV breakpoint characterization was performed by mutation-specific breakpoint PCR, genome sequencing and, if required, optical genome mapping. In silico modeling of novel SVs was performed to predict the formation of neo-TADs and whether ectopic contacts between the retinal enhancers and the GDPD1-promoter could be formed. Results: Using this workflow, potential RP17-SVs were detected in eight probands of which seven were confirmed. Two novel SVs were identified that are predicted to cause TAD rearrangement and retinal enhancer-GDPD1 contact, one from Germany (DE-SV9) and three with the same SV from the United States (US-SV10). Previously reported RP17-SVs were also identified in three Australian probands, one with UK-SV2 and two with SA-SV3. Discussion: In summary, we describe a validated multi-step pipeline for reliable and efficient RP17-SV discovery and expand the range of disease-associated SVs. Based on these data, RP17-SVs can be considered a frequent cause of adRP which warrants the inclusion of RP17-screening as a standard diagnostic test for this disease.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Unravelling undiagnosed rare disease cases by HiFi long-read genome sequencing.

Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

Exome sequencing identifies novel genes and variants in patients with Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a complex genetic disease characterized by the absence of ganglion cells in the intestines, leading to a functional obstruction in infants. At least 24 genes have been identified for the pathogenesis of HSCR. They contributed to approximately 72% of HSCR cases. We aimed to elucidate further the genetic basis of HSCR in Indonesia using the whole-exome sequencing (WES) approach. METHODS: WES was performed in 39 sporadic non-syndromic HSCR patients and 16 non-HSCR subjects as controls. Variants presented in controls were excluded, followed by in silico prediction tools and population allele frequency databases to select rare variants. We determined the minor allele frequency (MAF) using gnomAD (MAF <0.1%). RESULTS: We involved 24 (61.5%) males and 15 (38.5%) females. Most patients (62%) had short-segment aganglionosis and underwent the Duhamel procedure (41%). We identified several candidate novel variants in HSCR-related genes, including UBR4, GDNF, and ECE1. Moreover, we also identified some novel candidate genes, including a possible compound heterozygous variant in the MUTYH gene: the first variant, a known protein-truncating variant associated with colorectal cancer (CRC), p.Glu452Ter and the second variant is novel, p.Ala39Val. Moreover, the type of variants was not associated with the aganglionosis type. CONCLUSIONS: We identified several novel genes and variants, including the variant associated with CRC, that might contribute to the pathogenesis of HSCR. No genotype-phenotype associations were noted. Our study further confirms the complex network involved in enteric nervous system development and HSCR pathogenesis. LEVEL OF EVIDENCE: Level III.

CRISPR-Cas9 correction of a nonsense mutation in LCA5 rescues lebercilin expression and localization in human retinal organoids.

Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.

Identification of Rare Variants Involved in High Myopia Unraveled by Whole Genome Sequencing.

Purpose: Myopia (nearsightedness) is a condition in which a refractive error (RE) affects vision. Although common variants explain part of the genetic predisposition (18%), most of the estimated 70% heritability is missing. Here, we investigate the contribution of rare genetic variation because this might explain more of the missing heritability in the more severe forms of myopia. In particular, high myopia can lead to blindness and has a tremendous impact on a patient and at the societal level. The exact molecular mechanisms behind this condition are not yet completely unraveled, but whole genome sequencing (WGS) studies have the potential to identify novel (rare) disease genes, explaining the high heritability. Design: Cross-sectional study performed in the Netherlands. Participants: We investigated 159 European patients with high myopia (RE > -10 diopters). Methods: We performed WGS using a stepwise filtering approach and burden analysis. The contribution of common variants was calculated as a genetic risk score (GRS). Main Outcome Measures: Rare variant burden, GRS. Results: In 25% (n = 40) of these patients, there was a high (> 75th percentile) contribution of common predisposing variants; that is, these participants had higher GRSs. In 7 of the remaining 119 patients (6%), deleterious variants in genes associated with known (ocular) disorders, such as retinal dystrophy disease (prominin 1 [PROM1]) or ocular development (ATP binding cassette subfamily B member 6 [ABCB6], TGFB induced factor homeobox 1 [TGIF1]), were identified. Furthermore, without using a gene panel, we identified a high burden of rare variants in 8 novel genes associated with myopia. The genes heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) (proportion in study population vs. the Genome Aggregation Database (GnomAD) 0.14 vs. 0.03, P = 4.22E-17), RNA binding motif protein 20 (RBM20) (0.15 vs. 0.06, P = 4.98E-05), and MAP7 domain containing 1 (MAP7D1) (0.19 vs. 0.06, P = 1.16E-10) were involved in the Wnt signaling cascade, melatonin degradation, and ocular development and showed most biologically plausible associations. Conclusions: We found different contributions of common and rare variants in low and high grade myopia. Using WGS, we identified some interesting candidate genes that could explain the high myopia phenotype in some patients. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Patient-derived glioblastoma organoids reflect tumor heterogeneity and treatment sensitivity.

Background: Treatment resistance and tumor relapse are the primary causes of mortality in glioblastoma (GBM), with intratumoral heterogeneity playing a significant role. Patient-derived cancer organoids have emerged as a promising model capable of recapitulating tumor heterogeneity. Our objective was to develop patient-derived GBM organoids (PGO) to investigate treatment response and resistance. Methods: GBM samples were used to generate PGOs and analyzed using whole-exome sequencing (WES) and single-cell karyotype sequencing. PGOs were subjected to temozolomide (TMZ) to assess viability. Bulk RNA sequencing was performed before and after TMZ. Results: WES analysis on individual PGOs cultured for 3 time points (1-3 months) showed a high inter-organoid correlation and retention of genetic variants (range 92.3%-97.7%). Most variants were retained in the PGO compared to the tumor (range 58%-90%) and exhibited similar copy number variations. Single-cell karyotype sequencing demonstrated preservation of genetic heterogeneity. Single-cell multiplex immunofluorescence showed maintenance of cellular states. TMZ treatment of PGOs showed a differential response, which largely corresponded with MGMT promoter methylation. Differentially expressed genes before and after TMZ revealed an upregulation of the JNK kinase pathway. Notably, the combination treatment of a JNK kinase inhibitor and TMZ demonstrated a synergistic effect. Conclusions: Overall, these findings demonstrate the robustness of PGOs in retaining the genetic and phenotypic heterogeneity in culture and the application of measuring clinically relevant drug responses. These data show that PGOs have the potential to be further developed into avatars for personalized adaptive treatment selection and actionable drug target discovery and as a platform to study GBM biology.

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Identification of a novel nonsense mutation in RP1 that causes autosomal recessive retinitis pigmentosa in an Indonesian family.

PURPOSE: The purpose of this study was to identify the underlying molecular genetic defect in an Indonesian family with three affected individuals who had received a diagnosis of retinitis pigmentosa (RP). METHODS: Clinical evaluation of the family members included measuring visual acuity and fundoscopy, and assessing visual field and color vision. Genomic DNA of the three affected individuals was analyzed with Illumina 700k single nucleotide polymorphism (SNP) arrays, and homozygous regions were identified using PLINK software. Mutation analysis was performed with sequence analysis of the retinitis pigmentosa 1 (RP1) gene that resided in one of the homozygous regions. The frequency of the identified mutation in the Indonesian population was determined with TaqI restriction fragment length polymorphism analysis. RESULTS: A novel homozygous nonsense mutation in exon 4 of the RP1 gene, c.1012C>T (p.R338*), was identified in the proband and her two affected sisters. Unaffected family members either carried two wild-type alleles or were heterozygous carriers of the mutation. The mutation was not present in 184 Indonesian control samples. CONCLUSIONS: Most of the previously reported RP1 mutations are inherited in an autosomal dominant mode, and appear to cluster in exon 4. Here, we identified a novel homozygous p.R338* mutation in exon 4 of RP1, and speculate on the mutational mechanisms of different RP1 mutations underlying dominant and recessive RP.