IMPORTANCESevere 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 PARTICIPANTSCase 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 MEASURESResults 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. RESULTSThe 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 RELEVANCEIn 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.

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Leber congenital amaurosis (LCA) represents the most severe form of inherited retinal dystrophies with an onset during the first year of life. Currently, 21 genes are known to be associated with LCA and recurrent mutations have been observed in AIPL1, CEP290, CRB1 and GUCY2D. In addition, sequence analysis of LRAT and RPE65 may be important in view of treatments that are emerging for patients carrying variants in these genes. Screening of the aforementioned variants and genes was performed in 64 Danish LCA probands. Upon the identification of heterozygous variants, Sanger sequencing was performed of the relevant genes to identify the second allele. In combination with prior arrayed primer extension analysis, this led to the identification of two variants in 42 of 86 cases (49%). Remarkably, biallelic RPE65 variants were identified in 16% of the cases, and one novel variant, p.(D110G), was found in seven RPE65 alleles. We also collected all previously published RPE65 variants, identified in 914 alleles of 539 patients with LCA or early-onset retinitis pigmentosa, and deposited them in the RPE65 Leiden Open Variation Database (LOVD). The in silico pathogenicity assessment of the missense and noncanonical splice site variants, as well as an analysis of their frequency in~60 000 control individuals, rendered 864 of the alleles to affect function or probably affect function. This comprehensive database can now be used to select patients eligible for gene augmentation or retinoid supplementation therapies.

Bietti's crystalline dystrophy (BCD) is a rare, autosomal recessive retinal degenerative disease associated with mutations in CYP4V2. In this study, we describe the genetic and clinical findings in 19 unrelated BCD patients recruited from five international retinal dystrophy clinics. Patients underwent ophthalmic examinations and were screened for CYP4V2 mutations by Sanger sequencing and quantitative polymerase chain reaction (qPCR) copy number variation screening. Eight CYP4V2 mutations were found in 10/19 patients, including three patients in whom only monoallelic mutations were detected. Four novel mutations were identified: c.604G>A; p.(Glu202Lys), c.242C>G; p.(Thr81Arg), c.604+4A>G; p.(?), and c.1249dup; p.(Thr417Asnfs*2). In addition, we identified a heterozygous paternally inherited genomic deletion of at least 3.8 Mb, encompassing the complete CYP4V2 gene and several other genes, which is novel. Clinically, patients demonstrated phenotypic variability, predominantly showing choroidal sclerosis, attenuated vessels, and crystalline deposits of varying degrees of severity. To our knowledge, our study reports the first heterozygous CYP4V2 deletion and hence a novel mutational mechanism underlying BCD. Our results emphasize the importance of copy number screening in BCD. Finally, the identification of CYP4V2-negative patients with indistinguishable phenotypes from CYP4V2-positive patients might suggest the presence of mutations outside the coding regions of CYP4V2, or locus heterogeneity, which is unreported so far.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10−5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10−4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

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PURPOSE. To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1). METHODS. Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253þ43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769-784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs). RESULTS. Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253þ43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253þ43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253þ43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253þ43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e.,~30% of the c.769-784C>T allele alone). CONCLUSIONS. Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene.

Inherited retinal diseases (IRDs) are a major cause of visual impairment. These clinically heterogeneous disorders are caused by pathogenic variants in more than 270 genes. As 30–40% of cases remain genetically unexplained following conventional genetic testing, we aimed to obtain a genetic diagnosis in an IRD cohort in which the genetic cause was not found using whole-exome sequencing or targeted capture sequencing. We performed whole-genome sequencing (WGS) to identify causative variants in 100 unresolved cases. After initial prioritization, we performed an in-depth interrogation of all noncoding and structural variants in genes when one candidate variant was detected. In addition, functional analysis of putative splice-altering variants was performed using in vitro splice assays. We identified the genetic cause of the disease in 24 patients. Causative coding variants were observed in genes such as ATXN7, CEP78, EYS, FAM161A, and HGSNAT. Gene disrupting structural variants were also detected in ATXN7, PRPF31, and RPGRIP1. In 14 monoallelic cases, we prioritized candidate noncanonical splice sites or deep-intronic variants that were predicted to disrupt the splicing process based on in silico analyses. Of these, seven cases were resolved as they carried pathogenic splice defects. WGS is a powerful tool to identify causative variants residing outside coding regions or heterozygous structural variants. This approach was most efficient in cases with a distinct clinical diagnosis. In addition, in vitro splice assays provide important evidence of the pathogenicity of rare variants.