The co-occurrence of genetic variants in the TYR and OCA2 genes confers susceptibility to albinism.

Although rare genetic conditions are mostly caused by DNA sequence alterations that functionally disrupt individual genes, large-scale studies using genome sequencing have started to unmask additional complexity. Understanding how combinations of variants in different genes shape human phenotypes is expected to provide important insights into the clinical and genetic heterogeneity of rare disorders. Here, we use albinism, an archetypal rare condition associated with hypopigmentation, as an exemplar for the study of genetic interactions. We analyse data from the Genomics England 100,000 Genomes Project alongside a cohort of 1120 individuals with albinism, and investigate the effect of dual heterozygosity for the combination of two established albinism-related variants: TYR:c.1205 G > A (p.Arg402Gln) [rs1126809] and OCA2:c.1327 G > A (p.Val443Ile) [rs74653330]. As each of these changes alone is insufficient to cause disease when present in the heterozygous state, we sought evidence of synergistic effects. We show that, when both variants are present, the probability of receiving a diagnosis of albinism is significantly increased (odds ratio 12.8; 95% confidence interval 6.0 - 24.7; p-value 2.1 ×10-8). Further analyses in an independent cohort, the UK Biobank, support this finding and highlight that heterozygosity for the TYR:c.1205 G > A and OCA2:c.1327 G > A variant combination is associated with statistically significant alterations in visual acuity and central retinal thickness (traits that are considered albinism endophenotypes). The approach discussed in this report opens up new avenues for the investigation of oligogenic patterns in apparently Mendelian disorders.

Using computational approaches to enhance the interpretation of missense variants in the PAX6 gene.

The PAX6 gene encodes a highly-conserved transcription factor involved in eye development. Heterozygous loss-of-function variants in PAX6 can cause a range of ophthalmic disorders including aniridia. A key molecular diagnostic challenge is that many PAX6 missense changes are presently classified as variants of uncertain significance. While computational tools can be used to assess the effect of genetic alterations, the accuracy of their predictions varies. Here, we evaluated and optimised the performance of computational prediction tools in relation to PAX6 missense variants. Through inspection of publicly available resources (including HGMD, ClinVar, LOVD and gnomAD), we identified 241 PAX6 missense variants that were used for model training and evaluation. The performance of ten commonly used computational tools was assessed and a threshold optimization approach was utilized to determine optimal cut-off values. Validation studies were subsequently undertaken using PAX6 variants from a local database. AlphaMissense, SIFT4G and REVEL emerged as the best-performing predictors; the optimized thresholds of these tools were 0.967, 0.025, and 0.772, respectively. Combining the prediction from these top-three tools resulted in lower performance compared to using AlphaMissense alone. Tailoring the use of computational tools by employing optimized thresholds specific to PAX6 can enhance algorithmic performance. Our findings have implications for PAX6 variant interpretation in clinical settings.

Non-syndromic retinal dystrophy associated with biallelic variation of SUMF1 and reduced leukocyte sulfatase activity.

Biallelic variants in SUMF1 are associated with multiple sulfatase deficiency (MSD), a rare lysosomal storage disorder typically diagnosed in early infancy or childhood, marked by severe neurodegeneration and early mortality. We present clinical and molecular characterisation of three unrelated patients aged 13 to 58 years with milder clinical manifestations due to SUMF1 disease variants, including two adult patients presenting with apparent non-syndromic retinal dystrophy. Whole genome sequencing identified biallelic SUMF1 variants in all three patients; Patient 1 homozygous for a complex allele c.[290G>T;293T>A]; p.[(Gly97Val);(Val98Glu)], Patient 2 homozygous for c.866A>G; p.(Tyr289Cys), and Patient 3 compound heterozygous for c.726-1G>C and p.(Tyr289Cys). Electroretinography indicated a rod-cone dystrophy with additional possible inner retinal dysfunction in all three patients. Biochemical studies confirmed reduced, but not absent, sulfatase enzyme activity in the absence of extra-ocular disease (Patient 1) or only mild systemic disease (Patients 2, 3). These cases are suggestive that non-null SUMF1 genotypes can cause an attenuated clinical phenotype, including retinal dystrophy without systemic complications, in adulthood.

Biallelic variants in Plexin B2 (PLXNB2) cause amelogenesis imperfecta, hearing loss and intellectual disability.

BACKGROUND: Plexins are large transmembrane receptors for the semaphorin family of signalling proteins. Semaphorin-plexin signalling controls cellular interactions that are critical during development as well as in adult life stages. Nine plexin genes have been identified in humans, but despite the apparent importance of plexins in development, only biallelic PLXND1 and PLXNA1 variants have so far been associated with Mendelian genetic disease. METHODS: Eight individuals from six families presented with a recessively inherited variable clinical condition, with core features of amelogenesis imperfecta (AI) and sensorineural hearing loss (SNHL), with variable intellectual disability. Probands were investigated by exome or genome sequencing. Common variants and those unlikely to affect function were excluded. Variants consistent with autosomal recessive inheritance were prioritised. Variant segregation analysis was performed by Sanger sequencing. RNA expression analysis was conducted in C57Bl6 mice. RESULTS: Rare biallelic pathogenic variants in plexin B2 (PLXNB2), a large transmembrane semaphorin receptor protein, were found to segregate with disease in all six families. The variants identified include missense, nonsense, splicing changes and a multiexon deletion. Plxnb2 expression was detected in differentiating ameloblasts. CONCLUSION: We identify rare biallelic pathogenic variants in PLXNB2 as a cause of a new autosomal recessive, phenotypically diverse syndrome with AI and SNHL as core features. Intellectual disability, ocular disease, ear developmental abnormalities and lymphoedema were also present in multiple cases. The variable syndromic human phenotype overlaps with that seen in Plxnb2 knockout mice, and, together with the rarity of human PLXNB2 variants, may explain why pathogenic variants in PLXNB2 have not been reported previously.

Clinical, biochemical and molecular analysis in a cohort of individuals with gyrate atrophy.

BACKGROUND: Gyrate atrophy of the choroid and retina is a rare autosomal recessive metabolic disorder caused by biallelic variants in the OAT gene, encoding the enzyme ornithine δ-aminotransferase. Impaired enzymatic activity leads to systemic hyperornithinaemia, which in turn underlies progressive chorioretinal degeneration. In this study, we describe the clinical and molecular findings in a cohort of individuals with gyrate atrophy. METHODS: Study participants were recruited through a tertiary UK clinical ophthalmic genetic service. All cases had a biochemical and molecular diagnosis of gyrate atrophy. Retrospective phenotypic and biochemical data were collected using electronic healthcare records. RESULTS: 18 affected individuals from 12 families (8 male, 10 female) met the study inclusion criteria. The median age at diagnosis was 8 years (range 10 months - 33 years) and all cases had hyperornithinaemia (median: 800 micromoles/L; range: 458-1244 micromoles/L). Common features at presentation included high myopia (10/18) and nyctalopia (5/18). Ophthalmic findings were present in all study participants who were above the age of 6 years. One third of patients had co-existing macular oedema and two thirds developed pre-senile cataracts. Compliance with dietary modifications was suboptimal in most cases. A subset of participants had extraocular features including a trend towards reduced fat-free mass and developmental delay. CONCLUSIONS: Our findings highlight the importance of multidisciplinary care in families with gyrate atrophy. Secondary ophthalmic complications such as macular oedema and cataract formation are common. Management of affected individuals remains challenging due to the highly restrictive nature of the recommended diet and the limited evidence-base for current strategies.

A multilayered approach to the analysis of genetic data from individuals with suspected albinism.

Albinism is a clinically and genetically heterogeneous group of conditions characterised by visual abnormalities and variable degrees of hypopigmentation. Multiple studies have demonstrated the clinical utility of genetic investigations in individuals with suspected albinism. Despite this, the variation in the provision of genetic testing for albinism remains significant. One key issue is the lack of a standardised approach to the analysis of genomic data from affected individuals. For example, there is variation in how different clinical genetic laboratories approach genotypes that involve incompletely penetrant alleles, including the common, 'hypomorphic' TYR c.1205G>A (p.Arg402Gln) [rs1126809] variant. Here, we discuss the value of genetic testing as a frontline diagnostic tool in individuals with features of albinism and propose a practice pattern for the analysis of genomic data from affected families.

MIR204 n.37C>T variant as a cause of chorioretinal dystrophy variably associated with iris coloboma, early-onset cataracts and congenital glaucoma.

Four members of a three-generation Czech family with early-onset chorioretinal dystrophy were shown to be heterozygous carriers of the n.37C>T in MIR204. The identification of this previously reported pathogenic variant confirms the existence of a distinct clinical entity caused by a sequence change in MIR204. Chorioretinal dystrophy was variably associated with iris coloboma, congenital glaucoma, and premature cataracts extending the phenotypic range of the condition. In silico analysis of the n.37C>T variant revealed 713 novel targets. Additionally, four family members were shown to be affected by albinism resulting from biallelic pathogenic OCA2 variants. Haplotype analysis excluded relatedness with the original family reported to harbour the n.37C>T variant in MIR204. Identification of a second independent family confirms the existence of a distinct MIR204-associated clinical entity and suggests that the phenotype may also involve congenital glaucoma.

Causal factors in primary open angle glaucoma: a phenome-wide Mendelian randomisation study.

Primary open angle glaucoma (POAG) is a chronic, adult-onset optic neuropathy associated with characteristic optic disc and/or visual field changes. With a view to identifying modifiable risk factors for this common neurodegenerative condition, we performed a 'phenome-wide' univariable Mendelian randomisation (MR) study that involved analysing the relationship between 9661 traits and POAG. Utilised analytical approaches included weighted mode based estimation, the weighted median method, the MR Egger method and the inverse variance weighted (IVW) approach. Eleven traits related to POAG risk were identified including: serum levels of the angiopoietin-1 receptor (OR [odds ratio] = 1.11, IVW p = 2.34E-06) and the cadherin 5 protein (OR = 1.06, IVW p = 1.31E-06); intraocular pressure (OR = 2.46-3.79, IVW p = 8.94E-44-3.00E-27); diabetes (OR = 5.17, beta = 1.64, IVW p = 9.68E-04); and waist circumference (OR = 0.79, IVW p = 1.66E-05). Future research focussing on the effects of adiposity, cadherin 5 and angiopoietin-1 receptor on POAG development and progression is expected to provide key insights that might inform the provision of lifestyle modification advice and/or the development of novel therapies.

Cav1.4 congenital stationary night blindness is associated with an increased rate of proteasomal degradation.

Pathogenic, generally loss-of-function, variants in CACNA1F, encoding the Cav1.4α1 calcium channel, underlie congenital stationary night blindness type 2 (CSNB2), a rare inherited retinal disorder associated with visual disability. To establish the underlying pathomechanism, we investigated 10 clinically derived CACNA1F missense variants located across pore-forming domains, connecting loops, and the carboxy-tail domain of the Cav1.4α subunit. Homology modeling showed that all variants cause steric clashes; informatics analysis correctly predicted pathogenicity for 7/10 variants. In vitro analyses demonstrated that all variants cause a decrease in current, global expression, and protein stability and act through a loss-of-function mechanism and suggested that the mutant Cav1.4α proteins were degraded by the proteasome. We showed that the reduced current for these variants could be significantly increased through treatment with clinical proteasome inhibitors. In addition to facilitating clinical interpretation, these studies suggest that proteasomal inhibition represents an avenue of potential therapeutic intervention for CSNB2.

The Role of Genetic Testing in Children Requiring Surgery for Ectopia Lentis.

Non-traumatic ectopia lentis can be isolated or herald an underlying multisystemic disorder. Technological advances have revolutionized genetic testing for many ophthalmic disorders, and this study aims to provide insights into the clinical utility of genetic analysis in paediatric ectopia lentis. Children that underwent lens extraction for ectopia lentis between 2013 and 2017 were identified, and gene panel testing findings and surgical outcomes were collected. Overall, 10/11 cases received a probable molecular diagnosis. Genetic variants were identified in four genes: FBN1 (associated with Marfan syndrome and cardiovascular complications; n = 6), ADAMTSL4 (associated with non-syndromic ectopia lentis; n = 2), LTBP2 (n = 1) and ASPH (n = 1). Parents appeared unaffected in 6/11 cases; the initial presentation of all six of these children was to an ophthalmologist, and only 2/6 had FBN1 variants. Notably, 4/11 cases required surgery before the age of 4 years, and only one of these children carried an FBN1 variant. In summary, in this retrospective cohort study, panel-based genetic testing pointed to a molecular diagnosis in >90% of paediatric ectopia lentis cases requiring surgery. In a subset of study participants, genetic analysis revealed changes in genes that have not been linked to extraocular manifestations and highlighted that extensive systemic investigations were not required in these individuals. We propose the introduction of genetic testing early in the diagnostic pathway in children with ectopia lentis.

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

Phenome-wide Mendelian randomisation analysis identifies causal factors for age-related macular degeneration.

Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the industrialised world and is projected to affect >280 million people worldwide by 2040. Aiming to identify causal factors and potential therapeutic targets for this common condition, we designed and undertook a phenome-wide Mendelian randomisation (MR) study. Methods: We evaluated the effect of 4591 exposure traits on early AMD using univariable MR. Statistically significant results were explored further using: validation in an advanced AMD cohort; MR Bayesian model averaging (MR-BMA); and multivariable MR. Results: Overall, 44 traits were found to be putatively causal for early AMD in univariable analysis. Serum proteins that were found to have significant relationships with AMD included S100-A5 (odds ratio [OR] = 1.07, p-value = 6.80E-06), cathepsin F (OR = 1.10, p-value = 7.16E-05), and serine palmitoyltransferase 2 (OR = 0.86, p-value = 1.00E-03). Univariable MR analysis also supported roles for complement and immune cell traits. Although numerous lipid traits were found to be significantly related to AMD, MR-BMA suggested a driving causal role for serum sphingomyelin (marginal inclusion probability [MIP] = 0.76; model-averaged causal estimate [MACE] = 0.29). Conclusions: The results of this MR study support several putative causal factors for AMD and highlight avenues for future translational research. Funding: This project was funded by the Wellcome Trust (224643/Z/21/Z; 200990/Z/16/Z); the University of Manchester's Wellcome Institutional Strategic Support Fund (Wellcome ISSF) grant (204796/Z/16/Z); the UK National Institute for Health Research (NIHR) Academic Clinical Fellow and Clinical Lecturer Programmes; Retina UK and Fight for Sight (GR586); the Australian National Health and Medical Research Council (NHMRC) (1150144).

Retinal gene therapy in RPE-65 gene mediated inherited retinal dystrophy.

BACKGROUND: Voretigene neparvovec (VN) is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. We illustrate, both the benefits and pitfalls associated with ocular gene therapy in the same patient. METHODS: Two eyes of one patient with bi-allelic RPE65 mutations have been treated with VN. The clinical examinations included visual acuity (VA, in normal and low luminance), colour vision, contrast sensitivity, International Society for Clinical Electrophysiology of Vision (ISCEV) standard retinal electrophysiology and dark-adapted full-field stimulus threshold (FST), Goldmann VF analysis and imaging studies, including optical coherence tomography (OCT) and autofluorescence. These were performed at baseline, 2-weeks, 3 and 6-months, 1 and 2-years follow-up. RESULTS: The first eye showed improvement in rod photoreceptor function with increased peripheral and low luminance vision (baseline VA: 0.9 logMAR and 2-years post-operative VA: 0.7 logMAR). The second eye, whilst showing increased light sensitivity, suffered a drop in central vision (at 2-weeks) with loss of foveal photoreceptors as shown by the loss of ellipsoid zone on OCT scan (baseline VA: 0.6, 2-year post-operative VA: 1.2). FST improvements were maintained in both eyes indicating a sustained efficacy of VN with little waning of its effect. CONCLUSIONS: We present a previously unreported adverse complication of subretinal VN therapy in bi-allelic RPE65, indicating a probable immune response in treatment of the second eye, resulting in loss of foveal photoreceptors. This case-series highlights the potential and pitfalls of retinal gene therapy in the same patient. The immune responses of the body to a 'foreign vector', remains a challenge.

Late-Onset Autosomal Dominant Macular Degeneration Caused by Deletion of the CRX Gene.

PURPOSE: To characterize the phenotype observed in a case series with macular disease and determine the cause. DESIGN: Multicenter case series. PARTICIPANTS: Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. METHODS: Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. MAIN OUTCOME MEASURES: Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. RESULTS: All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. CONCLUSIONS: Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.

Data on cardiac lncRNA STX18-AS1 expression in developing human hearts and function during in vitro hESC-cardiomyocyte differentiation.

This article presents data concerning STX18-AS1, a long noncoding RNA gene identified from a Genome-wide association study of Atrial Septal Defect (ASD). The data describes its expression patterns in human tissues and functions in regulating cardiomyocyte differentiation in vitro. STX18-AS1 is a lncRNA with a higher abundance in developing tissues, including hearts. Its transcription distribution within the embryonic hearts during key heart septation stages supports STX18-AS1's association with risk SNPs for ASD. The CRISPR stem cell pool in which STX18-AS1 was knocked down, showed reduced CM differentiation efficiency and lower expression of key cardiac transcriptional factors. This indicated its regulative role in supporting the lineage specification from cardiac mesoderm into cardiac progenitors and cardiomyocytes. These data can benefit the understanding of human embryonic heart developmental biology, and the time-course changes of cardiac transcriptional factors during in vitro cardiomyocyte differentiation from human embryonic stem cells.

Assessing the Pathogenicity of In-Frame CACNA1F Indel Variants Using Structural Modeling.

Small in-frame insertion-deletion (indel) variants are a common form of genomic variation whose impact on rare disease phenotypes has been understudied. The prediction of the pathogenicity of such variants remains challenging. X-linked incomplete congenital stationary night blindness type 2 (CSNB2) is a nonprogressive, inherited retinal disorder caused by variants in CACNA1F, encoding the Cav1.4α1 channel protein. Here, structural analysis was used through homology modeling to interpret 10 disease-correlated and 10 putatively benign CACNA1F in-frame indel variants. CSNB2-correlated changes were found to be more highly conserved compared with putative benign variants. Notably, all 10 disease-correlated variants but none of the benign changes were within modeled regions of the protein. Structural analysis revealed that disease-correlated variants are predicted to destabilize the structure and function of the Cav1.4α1 channel protein. Overall, the use of structural information to interpret the consequences of in-frame indel variants provides an important adjunct that can improve the diagnosis for individuals with CSNB2.

Generation of a human induced pluripotent stem cell line carrying the TYR c.575C>A (p.Ser192Tyr) and c.1205G>A (p.Arg402Gln) variants in homozygous state using CRISPR-Cas9 genome editing.

TYR encodes tyrosinase, the enzyme catalysing the first steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). The TYR c.575C>A (p.Ser192Tyr) [rs1042602] and c.1205G>A (p.Arg402Gln) [rs1126809] variants are prevalent genetic changes that have been associated with multiple pigmentation traits. Notably, individuals who are homozygous for these two missense variants are predisposed to having albinism. Here we used CRISPR-Cas9 technology to generate an induced pluripotent stem cell (iPSC) line (WTSIi253-A-2) that carries both c.575C>A and c.1205G>A in homozygous state. The line expresses pluripotency markers and exhibits multi-lineage differentiation potential, providing a useful in vitro model for investigating albinism pathogenesis.

The contribution of common regulatory and protein-coding TYR variants to the genetic architecture of albinism.

Genetic diseases have been historically segregated into rare Mendelian disorders and common complex conditions. Large-scale studies using genome sequencing are eroding this distinction and are gradually unmasking the underlying complexity of human traits. Here, we analysed data from the Genomics England 100,000 Genomes Project and from a cohort of 1313 individuals with albinism aiming to gain insights into the genetic architecture of this archetypal rare disorder. We investigated the contribution of protein-coding and regulatory variants both rare and common. We focused on TYR, the gene encoding tyrosinase, and found that a high-frequency promoter variant, TYR c.-301C>T [rs4547091], modulates the penetrance of a prevalent, albinism-associated missense change, TYR c.1205G>A (p.Arg402Gln) [rs1126809]. We also found that homozygosity for a haplotype formed by three common, functionally-relevant variants, TYR c.[-301C;575C>A;1205G>A], is associated with a high probability of receiving an albinism diagnosis (OR>82). This genotype is also associated with reduced visual acuity and with increased central retinal thickness in UK Biobank participants. Finally, we report how the combined analysis of rare and common variants can increase diagnostic yield and can help inform genetic counselling in families with albinism.