The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

A diagnostic approach to syndromic retinal dystrophies with intellectual disability.

Inherited retinal dystrophies are a group of monogenic disorders that, as a whole, contribute significantly to the burden of ocular disease in both pediatric and adult patients. In their syndromic forms, retinal dystrophies can be observed in association with intellectual disability, frequently alongside other systemic manifestations. There are now over 80 genes implicated in syndromic retinal dystrophies with intellectual disability. Identifying and accurately characterizing these disorders allows the clinician to narrow the differential diagnosis, evaluate for relevant associated features, arrive at a timely and accurate diagnosis, and address both sight-threatening ocular manifestations and morbidity-causing systemic manifestations. The co-occurrence of retinal dystrophy and intellectual disability in an individual can be challenging to investigate, diagnose, and counsel given the considerable phenotypic and genotypic heterogeneity that exists within this broad group of disorders. We performed a review of the current literature and propose an algorithm to facilitate the evaluation, and clinical and mechanistic classification, of these individuals.

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

THE NATURAL HISTORY OF FULL-FIELD STIMULUS THRESHOLD DECLINE IN CHOROIDEREMIA.

PURPOSE: To evaluate full-field sensitivity thresholds (FSTs) across a wide range of choroideremia (CHM) disease stages and to determine their applicability as functional endpoints for CHM clinical trials. METHODS: Thirty CHM subjects (60 eyes) and 50 healthy controls (50 eyes) underwent FST testing under dark-adapted conditions to determine rod- and cone-mediated FSTs. Central retinal structure and function were assessed using fundus autofluorescence and microperimetry. Correlation and regression analyses were performed to compare FST responses with the residual area of retinal pigment epithelium in the peri- and parafoveal regions, as well as the mean and highest macular microperimetry sensitivity. RESULTS: All patients with CHM had a baseline of 18 dB elevation in dark-adapted rod FSTs, including the least affected individuals. Further FST sensitivity loss was exponentially associated with decrease in the area of residual peri- and parafoveal retinal pigment epithelium, with precipitous loss of sensitivity noted for fundus autofluorescence areas less than 5 mm. Cone FSTs were comparable with controls, except for advanced stages of CHM. Full-field sensitivity threshold responses showed high correlation with both mean and highest macular microperimetry thresholds (P < 0.001). In some cases of absent macular fundus autofluorescence, the peripheral retina could contribute to detectable rod FST responses but with severely diminished cone-driven responses. CONCLUSION: Full-field sensitivity threshold testing demonstrated a baseline level of rod dysfunction in CHM present in all rod photoreceptors. Further decline in FST responses correlated strongly with the extent of central retina structural and functional loss. Full-field sensitivity threshold allowed quantification of residual rod function in peripheral islands of vision, which cannot be reliably achieved with other conventional tests. As such, the FST can serve as a complimentary tool to guide patient selection and expand the eligibility criteria for current and future CHM clinical trials.

Single-base substitutions in the CHM promoter as a cause of choroideremia.

Although over 150 unique mutations affecting the coding sequence of CHM have been identified in patients with the X-linked chorioretinal disease choroideremia (CHM), no regulatory mutations have been reported, and indeed the promoter has not been defined. Here, we describe two independent families affected by CHM bearing a mutation outside the gene's coding region at position c.-98: C>A and C>T, which segregated with the disease. The male proband of family 1 was found to lack CHM mRNA and its gene product Rab escort protein 1, whereas whole-genome sequencing of an affected male in family 2 excluded the involvement of any other known retinal genes. Both mutations abrogated luciferase activity when inserted into a reporter construct, and by further employing the luciferase reporter system to assay sequences 5' to the gene, we identified the CHM promoter as the region encompassing nucleotides c.-119 to c.-76. These findings suggest that the CHM promoter region should be examined in patients with CHM who lack coding sequence mutations, and reveals, for the first time, features of the gene's regulation.

Reduction of Arachidonate Is Associated With Increase in B-Cell Activation Marker in Infants: A Randomized Trial.

BACKGROUND: Infants who are not breast-fed benefit from formula with both docosahexaenoic acid (C22:6n3) and arachidonic acid (ARA; C20:4n6). The amount of ARA needed to support immune function is unknown. Infants who carry specific fatty acid desaturase (FADS) polymorphisms may require more dietary ARA to maintain adequate ARA status. OBJECTIVE: The aim of the study was to determine whether ARA intake or FADS polymorphisms alter ARA levels of lymphocytes, plasma, and red blood cells in term infants fed infant formula. METHODS: Infants (N = 89) were enrolled in this prospective, double-blind controlled study. Infants were randomized to consume formula containing 17 mg docosahexaenoic acid and 0, 25, or 34 mg ARA/100 kcal for 10 weeks. Fatty acid composition of plasma phosphatidylcholine and phosphatidylethanolamine, total fatty acids of lymphocytes and red blood cells, activation markers of lymphocytes, and polymorphisms in FADS1 and FADS2 were determined. RESULTS: Lymphocyte ARA was higher in the 25-ARA formula group than in the 0- or 34-ARA groups. In plasma, 16:0/20:4 and 18:0/20:4 species of phosphatidylcholine and phosphatidylethanolamine were highest and 16:0/18:2 and 18:0/18:2 were lowest in the 34-ARA formula group. In minor allele carriers of FADS1 and FADS2, plasma ARA content was elevated only at the highest level of ARA consumed. B-cell activation marker CD54 was elevated in infants who consumed formula containing no ARA. CONCLUSIONS: ARA level in plasma is reduced by low ARA consumption and by minor alleles in FADS. Dietary ARA may exert an immunoregulatory role on B-cell activation by decreasing 16:0/18:2 and 18:0/18:2 species of phospholipids. ARA intake from 25 to 34 mg/100 kcal is sufficient to maintain cell ARA level in infants across genotypes.

Choroideremia.

PURPOSE OF REVIEW: Although much has been written to define the phenotype and genotype of choroideremia (CHM), research continues to provide new insights that serve to better understand its pathogenesis and the directions for potential experimental therapies. RECENT FINDINGS: We would like to highlight new findings, expanding the type of disease-causing mutations to include mutations in the CHM promoter that will dramatically influence gene expression. Information derived from careful phenotyping of patients points increasingly to the central role of the retinal pigment epithelium as the key cell layer affected in the degenerative process. Finally, we will review the current initiatives that are testing vector-mediated gene replacement approaches in humans, including our current understanding of the likelihood of success by this approach. SUMMARY: Clinical and basic vision science have benefited greatly by the active engagement of patients with CHM in clinical research studies. The impetus for their involvement in these studies has been generated by the initial results of safety from subretinal injection of and AAV2.REP1 vector in humans. Follow-up studies in the next few years are expected to show if this approach will modify disease progression.

Molecular genetic diagnostic techniques in choroideremia.

PURPOSE: To optimize and streamline molecular genetics techniques in diagnosing choroideremia (CHM). METHODS: PCR primers were designed for exons 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15 of the CHM gene. Each PCR protocol was optimized so that all exons could be amplified with the same component ratio and PCR conditions. Sense and antisense primers were tested for their ability to be used as sequencing primers. Fibroblast cells were cultured, and an immunoblot analysis was performed to detect the presence or absence of Rab escort protein 1 (REP-1) in a suspected CHM patient sample when no mutation was detected with sequencing. Multiplex ligation-dependent probe amplification (MLPA) of the CHM gene was performed and used to detect deletions and duplications in affected males and female carriers. RNA analysis using cDNA was used to detect the presence or absence of the CHM transcript and to search for splice defects. RESULTS: The newly designed PCR primers allow for more efficient PCR preparation and sequencing to detect point mutations in affected males and female carriers. Immunoblot successfully detects the absence of REP-1 in a CHM patient. MLPA identifies deletions and duplications spanning multiple exons in the CHM gene. RNA analysis aids in detecting splice variants. CONCLUSIONS: The development of new molecular biology techniques and ongoing optimization of existing methods allows for an improved integrated approach to confirm CHM diagnosis and carrier status in consideration of patient family history and available patient sample materials. CHM can be confirmed with an immunoblot assay. To detect the molecular cause of CHM, an examination of the genomic DNA or the mRNA must be performed. Presymptomatic carriers with no identifiable fundus signs can be identified only through molecular analysis of genomic DNA or through quantitative assays.

Pseudoxanthoma elasticum and retinitis pigmentosa in a patient with a novel mutation in the ABCC6 gene.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is an autosomal recessive condition caused by mutations in the ABCC6 gene. Ocular features include angioid streaks, peau d'orange fundus, and drusen. We report a novel ABCC6 mutation causing PXE in a patient with a mixed phenotype of PXE and retinitis pigmentosa (RP). CASE: A 37-year-old female presented with decreased peripheral vision and nyctalopia. Ocular imaging revealed angioid streaks emanating from the optic nerve as well as peripheral pigmentary changes and bone spicules. Genetic testing revealed two mutations in ABCC6 in trans. No other mutation was identified. CONCLUSION: We present a rare case with ocular findings of PXE and RP in a patient with a novel ABCC6 mutation. The patient presented both with peripheral pigmentary changes and angioid streaks. Further investigation into this novel mutation would be beneficial to determine if the mutation is involved in the RP phenotype.

A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.

An internet-based health survey on the co-morbidities of choroideremia patients.

PURPOSE: To examine the prevalence of systemic and ocular disease among choroideremia patients and carriers. METHODS: A cross-sectional analysis was performed on responses from affected males with choroideremia, female carriers, and unaffected brothers to an Internet-based survey made available from September 2009 to November 2010. Affected males were classified into two groups, those with or without functional vision. Carrier females were classified into those with and without symptoms. Comparisons were made between these groups. RESULTS: There was a higher prevalence of dry eye in our respondents than the North American population. The prevalence of dry eye, cataract, hypertension, diabetes, psychological problems and hypercholesterolemia were higher in choroideremia males without functional vision compared to those with functional vision. Likewise, statin intake was more prevalent among the affected males without functional vision than those with functional vision. After age adjustment, any differences between the two subgroups of male patients (with and without functional vision) were not significant. CONCLUSION: Age plays an important role in determining the onset of severe visual impairment with loss of functional vision in male subjects affected by choroideremia. Although Internet surveys have limitations such as the use of self-reported diagnoses and the possibility that the responses may not be representative of the population as a whole, this study shows that such surveys can provide data quickly and easily, and for rare diseases such as choroideremia, with relatively large numbers of responses.

Breaking bad news in ophthalmology: a pilot skills workshop for residents.

OBJECTIVE: To design and implement a formal skills workshop for ophthalmology residents to practice breaking bad news. METHODS: A 2-session workshop was developed for 7 ophthalmology residents at the University of Alberta based on a workshop published by Ohio State University. Residents discussed the SPIKES protocol for breaking bad news, practiced mock cases with standardized patients, and listened to shared experiences from patients who had received ocular diagnoses. RESULTS: All the residents (n = 6; p = 0.03) at the University of Alberta reported an increase in confidence in 3 measures of an encounter in which they had to break bad news, one of which shared the significant improvement reported by the Ohio State group (n = 9; p = 0.01): setting realistic expectations without destroying hope. Standardized patients discussed their satisfaction with their case training and suggested the provision of eye models or printouts to enhance the realism in the examination rooms. The University of Alberta workshop results replicated those from Ohio State in that the SPIKES lecture and standardized patient session were ranked highly in efficacy (median, 4 of 5). The University of Alberta panel discussion was ranked lower than at Ohio State University, which may have resulted from 1 of 2 patient guest speakers being unexpectedly unable to attend. CONCLUSION: The pilot Breaking Bad News Workshop was well received overall and may serve to inform future incorporation of soft skills development in a formal residency curriculum.

Upward saccadic intrusions as the presenting feature for incomplete congenital stationary night blindness.

We describe the case of a 9-month-old boy presenting with isolated intermittent vertical eye movements most in keeping with upward saccadic pulses, a form of saccadic intrusions. Full-field electroretinogram was consistent with a generalized retinal dystrophy, and genetic testing revealed a hemizygous pathogenic mutation in the CACNA1F gene, confirming the diagnosis of incomplete congenital stationary night blindness (iCSNB). This case describes vertical saccadic pulses as the sole presenting sign of a retinal dystrophy.

AAV2-Mediated Gene Therapy for Choroideremia: 5-Year Results and Alternate Anti-sense Oligonucleotide Therapy.

PURPOSE: To assess the long-term safety and efficacy of AAV2-REP1 in choroideremia (CHM) patients, and to test a potential antisense oligonucleotide therapy for CHM. DESIGN: Extended, prospective phase 1/2 clinical trial and laboratory investigation. METHODS: Five patients who received a single subfoveal injection of AAV2-REP1 were studied. The long-term safety was evaluated by ophthalmic examination, spectral domain optical coherence tomography, and fundus autofluorescence (FAF) for up to 5 years. Functional and structural changes were determined by different test modalities. Four antisense oligonucleotides (ASOs) were designed to treat the CHM c.1245-521A>G mutation, which was present in 2 patients within this trial. RESULTS: Subject P3 experienced a localized intraretinal immune response that resulted in a significant loss of preserved retinal pigment epithelium (RPE). P4 experienced an exacerbation of peripheral retinoschisis. P2 had a constant ≥15-letter best-corrected visual acuity (BCVA) gain in the treated eye, whereas P5 had ≥15-letter BCVA improvement once in the untreated eye. The preserved FAF areas declined more rapidly in the treated eyes compared to the untreated eyes (P = .043). A customized 25-mer ASO recovered 83.2% to 95.0% of the normal RNA and 57.5% of the normal protein in fibroblasts from 2 trial patients. CONCLUSIONS: Intraretinal inflammation triggered by AAV2-REP1 subretinal injection stabilized after 2 years but resulted in permanent damage to the retinal structure. Long-term progression of the disease was seen in both treated and untreated eyes, casting doubt as to the effectiveness of this approach in late-stage CHM. Alternative approaches such as ASO may have a therapeutic effect in a subgroup of CHM patients.

Overcoming the Challenges to Clinical Development of X-Linked Retinitis Pigmentosa Therapies: Proceedings of an Expert Panel.

X-linked retinitis pigmentosa (XLRP) is a rare inherited retinal disease manifesting as impaired night vision and peripheral vision loss that progresses to legal blindness. Although several trials of ocular gene therapy for XLRP have been conducted or are in progress, there is currently no approved treatment. In July 2022, the Foundation Fighting Blindness convened an expert panel to examine relevant research and make recommendations for overcoming the challenges and capitalizing on the opportunities in conducting clinical trials of RPGR-targeted therapy for XLRP. Data presented concerned RPGR structure and mutation types known to cause XLRP, RPGR mutation-associated retinal phenotype diversity, patterns in genotype/phenotype relationships, disease onset and progression from natural history studies, and the various functional and structural tests used to monitor disease progression. Panel recommendations include considerations, such as genetic screening and other factors that can impact clinical trial inclusion criteria, the influence of age on defining and stratifying participant cohorts, the importance of conducting natural history studies early in clinical development programs, and the merits and drawbacks of available tests for measuring treatment outcomes. We recognize the need to work with regulators to adopt clinically meaningful end points that would best determine the efficacy of a trial. Given the promise of RPGR-targeted gene therapy for XLRP and the difficulties encountered in phase III clinical trials to date, we hope these recommendations will help speed progress to finding a cure. Translational Relevance: Examination of relevant data and recommendations for the successful clinical development of gene therapies for RPGR-associated XLRP.

Absence of NR2E1 mutations in patients with aniridia.

PURPOSE: Nuclear receptor 2E1 (NR2E1) is a transcription factor with many roles during eye development and thus may be responsible for the occurrence of certain congenital eye disorders in humans. To test this hypothesis, we screened NR2E1 for candidate mutations in patients with aniridia and other congenital ocular malformations (anterior segment dysgenesis, congenital optic nerve malformation, and microphthalmia). METHODS: The NR2E1 coding region, 5' and 3' untranslated regions (UTRs), exon flanking regions including consensus splice sites, and six evolutionarily conserved non-coding candidate regulatory regions were analyzed by sequencing 58 probands with aniridia of whom 42 were negative for PAX6 mutations. Nineteen probands with anterior segment dysgenesis, one proband with optic nerve malformation, and two probands with microphthalmia were also sequenced. The control population comprised 376 healthy individuals. All sequences were analyzed against the GenBank sequence AL078596.8 for NR2E1. In addition, the coding region and flanking intronic sequences of FOXE3, FOXC1, PITX2, CYP1B1, PAX6, and B3GALTL were sequenced in one patient and his relatives. RESULTS: Sequencing analysis showed 17 NR2E1 variants including two novel rare non-coding variants (g.-1507G>A, g.14258C>T), and one novel rare coding variant (p.Arg274Gly). The latter was present in a male diagnosed with Peters' anomaly who subsequently was found to have a known causative mutation for Peters' plus syndrome in B3GALTL (c.660+1G>A). In addition, the NR2E1 novel rare variant Arg274Gly was present in the unaffected mother of the patient but absent in 746 control chromosomes. CONCLUSIONS: We eliminated a major role for NR2E1 regulatory and coding mutations in aniridia and found a novel rare coding variant in NR2E1. In addition, we found no coding region variation in the control population for NR2E1, which further supports its previously reported high level of conservation and low genetic diversity. Future NR2E1 studies in ocular disease groups such as those involving retinal and optic nerve abnormalities should be undertaken to determine whether NR2E1 plays a role in these conditions.

Lessons learned from research on choroideremia.

Having devoted over 35 years of my professional life to various projects on choroideremia (CHM), I began to reflect on the many lessons that I learned along the way. One of the most important is: we should pay careful attention to possible, unintended psychological harm in clinical research. This lesson was learned early and then reinforced when I engaged CHM patients in an investigator-sponsored Phase IB clinical trial of ocular gene therapy for choroideremia. My second lesson came from the trial itself in that preliminary data may not be sufficient to predict the risks to patients in a clinical trial. In the significant push to begin a gene therapy trial for CHM patients, writing grants, recruiting personnel, interacting with regulatory authorities, acquiring research equipment to test outcome measures, I missed a third lesson. There is significant bias when the principal investigator of an investigator-sponsored clinical trial is also the treating physician in the trial. Ideally, those two roles should be kept separate. Finally, having completed the clinical trial, I learned that gene replacement with an AAV vector may not be the only genetic therapy for CHM; an antisense oligonucleotide therapy may be possible in select cases.

Zebrafish and inherited photoreceptor disease: Models and insights.

Photoreceptor dysfunctions and degenerative diseases are significant causes of vision loss in patients, with few effective treatments available. Targeted interventions to prevent or reverse photoreceptor-related vision loss are not possible without a thorough understanding of the underlying mechanism leading to disease, which is exceedingly difficult to accomplish in the human system. Cone diseases are particularly challenging to model, as some popular genetically modifiable model animals are nocturnal with a rod-dominant visual system and cones that have dissimilarities to human cones. As a result, cone diseases, which affect visual acuity, colour perception, and central vision in patients, are generally poorly understood in terms of pathology and mechanism. Zebrafish (Danio rerio) provide the opportunity to model photoreceptor diseases in a diurnal vertebrate with a cone-rich retina which develops many macular degeneration-like pathologies. Zebrafish undergo external development, allowing early-onset retinal diseases to be detected and studied, and many ophthalmic tools are available for zebrafish visual assessment during development and adulthood. There are numerous zebrafish models of photoreceptor disease, spanning the various types of photoreceptor disease (developmental, rod, cone, and mixed photoreceptor diseases) and genetic/molecular cause. In this review, we explore the features of zebrafish that make them uniquely poised to model cone diseases, summarize the established zebrafish models of inherited photoreceptor disease, and discuss how disease in these models compares to the human presentation, where applicable. Further, we highlight the contributions of these zebrafish models to our understanding of photoreceptor biology and disease, and discuss future directions for utilising and investigating these diverse models.