Chatbot Artificial Intelligence for Genetic Cancer Risk Assessment and Counseling: A Systematic Review and Meta-Analysis.

PURPOSE: Most individuals with a hereditary cancer syndrome are unaware of their genetic status to underutilization of hereditary cancer risk assessment. Chatbots, or programs that use artificial intelligence to simulate conversation, have emerged as a promising tool in health care and, more recently, as a potential tool for genetic cancer risk assessment and counseling. Here, we evaluated the existing literature on the use of chatbots in genetic cancer risk assessment and counseling. METHODS: A systematic review was conducted using key electronic databases to identify studies which use chatbots for genetic cancer risk assessment and counseling. Eligible studies were further subjected to meta-analysis. RESULTS: Seven studies met inclusion criteria, evaluating five distinct chatbots. Three studies evaluated a chatbot that could perform genetic cancer risk assessment, one study evaluated a chatbot that offered patient counseling, and three studies included both functions. The pooled estimated completion rate for the genetic cancer risk assessment was 36.7% (95% CI, 14.8 to 65.9). Two studies included comprehensive patient characteristics, and none involved a comparison group. Chatbots varied as to the involvement of a health care provider in the process of risk assessment and counseling. CONCLUSION: Chatbots have been used to streamline genetic cancer risk assessment and counseling and hold promise for reducing barriers to genetic services. Data regarding user and nonuser characteristics are lacking, as are data regarding comparative effectiveness to usual care. Future research may consider the impact of chatbots on equitable access to genetic services.

Venous Tortuosity in COL4A2-Associated Gould Syndrome.

Mutations in collagen-encoding genes have been linked to numerous systemic diseases. Specifically, pathologic alterations in COL4A2 have been linked to Gould syndrome, a hereditary angiopathy affecting the brain, kidneys, and eyes. However, the ocular phenotype associated with COL4A2-associated disease has yet to be fully characterized. In this report, we describe a novel variant in COL4A2 identified in a 48-year-old woman and her 15-year-old daughter. Funduscopic examination demonstrated significant venous and arteriolar tortuosity. Genetic testing revealed a novel variant, c.2321G>A:p.(Gly774Glu), in COL4A2. This vascular phenotype is similar to the familial retinal arterial tortuosity seen in COL4A2-associated Gould syndrome with additional venous involvement. [Ophthalmic Surg Lasers Imaging Retina 2023;54:536-539.].

Partner and localizer of BRCA2 (PALB2) pathogenic variants and ovarian cancer: A systematic review and meta-analysis.

OBJECTIVE: Approximately 20% of ovarian cancers are due to an underlying germline pathogenic variant. While pathogenic variants in several genes have been well-established in the development of hereditary ovarian cancer (e.g. BRCA1/2, RAD51C, RAD51D, BRIP1, mismatch repair genes), the role of partner and localizer of BRCA2 (PALB2) remains uncertain. We sought to utilize meta-analysis to evaluate the association between PALB2 germline pathogenic variants and ovarian cancer. METHODS: We conducted a systematic review and meta-analysis. We searched key electronic databases to identify studies evaluating multigene panel testing in people with ovarian cancer. Eligible trials were subjected to meta-analysis. RESULTS: Fifty-five studies met inclusion criteria, including 48,194 people with ovarian cancer and information available on germline PALB2 pathogenic variant status. Among people with ovarian cancer and available PALB2 sequencing data, 0.4% [95% CI 0.3-0.4] harbored a germline pathogenic variant in the PALB2 gene. The pooled odds ratio (OR) for carrying a PALB2 pathogenic variant among the ovarian cancer population of 20,474 individuals who underwent germline testing was 2.48 [95% CI 1.57-3.90] relative to 123,883 controls. CONCLUSIONS: Our meta-analysis demonstrates that the pooled OR for harboring a PALB2 germline pathogenic variant among people with ovarian cancer compared to the general population is 2.48 [95% CI 1.57-3.90]. Prospective studies evaluating the role of germline PALB2 pathogenic variants in the development of ovarian cancer are warranted.

Generation of an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Prime Editor.

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.

Generation of CRB1 RP Patient-Derived iPSCs and a CRISPR/Cas9-Mediated Homology-Directed Repair Strategy for the CRB1 c.2480G>T Mutation.

Mutations in the Crumbs-homologue-1 (CRB1) gene lead to a spectrum of severe inherited retinal diseases, including retinitis pigmentosa (RP). The establishment of a genotype-phenotype correlation in CRB1 patients has been difficult due to the substantial variability and phenotypic overlap between CRB1-associated diseases. This phenotypic modulation may be due to several factors, including genetic modifiers, deep intronic mutations, isoform diversity, and copy number variations. Induced pluripotent stem cell (iPSC)-derived patient retinal organoids are novel tools that can provide sensitive, quantitative, and scalable phenotypic assays. CRB1 RP patient iPSC-derived retinal organoids have shown reproducible phenotypes compared to healthy retinal organoids. However, having genetically defined iPSC isogenic controls that take into account potential phenotypic modulation is crucial. In this study, we generated iPSC from an early-onset CRB1 patient and developed a correction strategy for the c.2480G>T, p.(Gly827Val) CRB1 mutation using CRISPR/Cas9-mediated homology-directed repair.

Do people with hereditary cancer syndromes inform their at-risk relatives? A systematic review and meta-analysis.

Purpose: To evaluate rates of familial disclosure of hereditary cancer syndrome information. Methods: A systematic review and meta-analysis was conducted in accordance with PRISMA guidelines (PROSPERO no.: CRD42020134276). Key electronic databases were searched to identify studies evaluating hereditary cancer syndrome cascade relative disclosure. Eligible studies were subjected to meta-analysis. Results: Thirty-four studies met inclusion criteria. Among 11,711 included relatives, 70% (95% CI 60 - 78%) were informed of their risk of carrying a cancer-associated pathogenic variant; of 2,875 relatives informed of their risk who were evaluated for uptake of cascade testing, 43% (95% CI 27 - 61%) completed testing. Rates of disclosure were higher among female vs male relatives (79% [95% CI 73% - 84%] vs 67% [95% CI 57% - 75%]) and first-degree vs second-degree relatives (83% [95% CI 77% - 88%] vs 58% [95% CI 45 - 69%]). Conclusion: Nearly one-third of at-risk relatives remain uninformed of their risk of carrying a cancer-associated pathogenic variant. Even among those informed, fewer than half subsequently complete genetic testing, representing a critical missed opportunity for precision cancer prevention. Innovation: Five studies evaluating interventions to improve disclosure rates were generally ineffective. Urgent work is needed to elucidate barriers to relative disclosure by probands to develop targeted interventions that can optimize proband-mediated cascade genetic testing rates.

Web-based tool for cancer family history collection: A prospective randomized controlled trial.

OBJECTIVES: Approximately 1% of individuals have a hereditary cancer predisposition syndrome, however, the majority are not aware. Collecting a cancer family history (CFH) can triage patients to receive genetic testing. To rigorously assess different methods of CFH collection, we compared a web-based tool (WBT) to usual care (clinician collects CFH) in a randomized controlled trial. METHODS: New gynecologic oncology patients (seen 9/2019-9/2021) were randomized to one of three arms in a 2:2:1 allocation ratio: 1) usual care clinician CFH collection, 2) WBT completed at home, or 3) WBT completed in office. The WBT generated a cancer-focused pedigree and scores on eight validated cancer risk models. The primary outcome was collection of an adequate CFH (based on established guidelines) with usual care versus the WBT. RESULTS: We enrolled 250 participants (usual care - 110; WBT home - 105; WBT office - 35 [closed early due to COVID-19]). Within WBT arms, 109 (78%) participants completed the tool, with higher completion for office versus home (33 [94%] vs. 76 [72%], P = 0.008). Among participants completing the WBT, 63 (58%) had an adequate CFH versus 5 (5%) for usual care (P < 0.001). Participants completing the WBT were significantly more likely to complete genetic counseling (34 [31%] vs. 15 [14%], P = 0.002) and genetic testing (20 [18%] vs. 9 [8%], P = 0.029). Participant and provider WBT experience was favorable. CONCLUSIONS: WBTs for CFH collection are a promising application of health information technology, resulting in more comprehensive CFH and a significantly greater percentage of participants completing genetic counseling and testing.

Patient perspectives on risk-reducing salpingectomy with delayed oophorectomy for ovarian cancer risk-reduction: A systematic review of the literature.

OBJECTIVE: Increasing evidence suggests the fallopian tube as the site of origin of BRCA1/2-associated high-grade ovarian cancers. Several ongoing trials are evaluating salpingectomy with delayed oophorectomy (RRSDO) for ovarian cancer risk reduction and patients are beginning to ask their clinicians about this surgical option. This study sought to systematically review the available literature examining patient preferences regarding RRSDO and risk-reducing salpingo-oophorectomy (RRSO) to provide clinicians with an understanding of patient values, concerns, and priorities surrounding ovarian cancer risk-reducing surgery. METHODS: We conducted a systematic review in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PROSPERO No.: CRD42023400690). We searched key electronic databases to identify studies evaluating acceptance and surgical decision-making regarding RRSO and RRSDO among patients with an increased risk of ovarian cancer. RESULTS: The search yielded 239 results, among which six publications met the systematic review inclusion criteria. Acceptance of RRSDO was evaluated in all studies and ranged from 34% to 71%. Factors positively impacting patients' acceptance of RRSDO included: avoidance of surgical menopause, preservation of fertility, concerns about sexual dysfunction, family history of breast cancer, and avoidance of hormone replacement therapy. Factors limiting this acceptance reported by patients included concerns regarding oncologic safety, surgical timing, and surgical complications. CONCLUSION: To date, few studies have explored patient perspectives surrounding RRSDO. Collectively, the limited data available indicate a high level of acceptance among BRCA1/2 carriers, and provides insight regarding both facilitating and limiting factors associated with patient preferences to better equip clinicians in the counseling and support of their patients.

Clinical and Therapeutic Evaluation of the Ten Most Prevalent CRB1 Mutations.

Mutations in the Crumbs homolog 1 (CRB1) gene lead to severe inherited retinal dystrophies (IRDs), accounting for nearly 80,000 cases worldwide. To date, there is no therapeutic option for patients suffering from CRB1-IRDs. Therefore, it is of great interest to evaluate gene editing strategies capable of correcting CRB1 mutations. A retrospective chart review was conducted on ten patients demonstrating one or two of the top ten most prevalent CRB1 mutations and receiving care at Columbia University Irving Medical Center, New York, NY, USA. Patient phenotypes were consistent with previously published data for individual CRB1 mutations. To identify the optimal gene editing strategy for these ten mutations, base and prime editing designs were evaluated. For base editing, we adopted the use of a near-PAMless Cas9 (SpRY Cas9), whereas for prime editing, we evaluated the canonical NGG and NGA prime editors. We demonstrate that for the correction of c.2843G>A, p.(Cys948Tyr), the most prevalent CRB1 mutation, base editing has the potential to generate harmful bystanders. Prime editing, however, avoids these bystanders, highlighting its future potential to halt CRB1-mediated disease progression. Additional studies investigating prime editing for CRB1-IRDs are needed, as well as a thorough analysis of prime editing's application, efficiency, and safety in the retina.

CRISPR Manipulations in Stem Cell Lines.

Insights into genome engineering in cells have allowed researchers to cultivate and modify cells as organoids that display structural and phenotypic features of human diseases or normal health status. The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. Modified organoids derived from patients' tissue cells are used as models to study diseases and test novel drugs. CRISPR-Cas9 technology has contributed to an explosion of advances that have the ability to edit genomes for the study of monogenic diseases and cancers. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of utmost importance as these cells carry the potential to be differentiated into any cell lineage. We describe recent developments that are broadening our understanding and extend DNA specificity, product selectivity, and fundamental capabilities. Furthermore, fundamental capabilities and remarkable advancements in basic research, biotechnology, and therapeutics development in cell engineering are detailed within this chapter. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter includes detailed protocols for the preparation of reagents to target loci of interest and transfection to genotype single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of ribonucleoprotein (RNP) delivered directly to cells.

Metabolite Extraction from RPE Cells and Retinas Related to Retinitis Pigmentosa.

The application of metabolomics in ophthalmology helps to identify new biomarkers and elucidate disease mechanisms in different eye diseases, as well as aiding in the development of potential treatment options. Extracting metabolites successfully is essential for potential further analysis using mass spectrometry. In this chapter, we describe how to extract metabolites from a variety of sources including (1) cells on a dish, (2) cell culture medium, and (3) tissues in vivo with and without stable isotope tracers. Samples prepared using this protocol are suitable for a range of downstream mass spectrometry analyses and are stable in solvent for weeks at -80 °C.

Generation of Human iPSC-Derived Retinal Organoids for Assessment of AAV-Mediated Gene Delivery.

Human retinal organoids derived from induced pluripotent stem cells (iPSCs) serve as a promising preclinical model for testing the safety and efficacy of viral gene therapy. Retinal organoids recapitulate the stratified multilayered epithelium structure of the developing and maturating human retina. As such, retinal organoids are unique tools to model retinal disease and to test therapeutic interventions toward their amelioration. Here, we describe a method for the generation of human iPSC-derived retinal organoids and how they can be utilized for the assessment of recombinant adeno-associated viral (rAAV)-mediated gene delivery.

Prime Editing for the Installation and Correction of Mutations Causing Inherited Retinal Disease: A Brief Methodology.

Inherited retinal diseases (IRDs) encompass a large heterogeneous group of rare blinding disorders whose etiology originates from mutations in the 280 genes identified to date. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems represent a promising avenue for the treatment of IRDs, as exemplified by FDA clinical trial approval of EDIT-101 (AGN-151587), which removes a deep intronic variant in the CEP290 gene that causes Leber congenital amaurosis (LCA) type 10. Prime editing is a novel double-strand break (DSB) independent CRISPR/Cas system which has the potential to correct all 12 possible transition and transversion mutations in addition to small deletions and insertions. Here, as a proof-of-concept study, we describe a methodology using prime editing for the in vitro installation and correction of the classical Pde6brd10 c.1678C > T (p.Arg560Cys) mutation which causes autosomal recessive retinitis pigmentosa (RP) in mice.

Culture of Human Retinal Explants for Ex Vivo Assessment of AAV Gene Delivery.

Due to the clinically established safety and efficacy profile of recombinant adeno-associated viral (rAAV) vectors, they are considered the "go to" vector for retinal gene therapy. Design of a rAAV-mediated gene therapy focuses on cell tropism, high transduction efficiency, and high transgene expression levels to achieve the lowest therapeutic treatment dosage and avoid toxicity. Human retinal explants are a clinically relevant model system for exploring these aspects of rAAV-mediated gene delivery. In this chapter, we describe an ex vivo human retinal explant culture protocol to evaluate transgene expression in order to determine the selectivity and efficacy of rAAV vectors for human retinal gene therapy.

What happens in the long term: Uptake of cancer surveillance and prevention strategies among at-risk relatives with pathogenic variants detected via cascade testing.

BACKGROUND: Cascade genetic testing for hereditary cancer syndromes offers affected relatives the opportunity to pursue cancer screening and risk-reducing surgery and thus reduces morbidity and mortality. The purpose of this study was to measure the long-term utilization of targeted cancer prevention and quality of life among at-risk relatives offered clinician-facilitated cascade genetic testing. METHODS: In a pilot study, at-risk relatives of patients with a hereditary cancer syndrome were contacted directly by the clinical team and offered telephone genetic counseling and genetic testing via an at-home, mailed saliva kit. Two-year follow-up results evaluating the use of targeted cancer prevention strategies and the quality of life for enrolled relatives were reported. Quality-of-life was measured with validated surveys, and scores were compared to the time of initial contact by the Wilcoxon signed-rank test. RESULTS: Ninety-five at-risk relatives were enrolled in the initial pilot study, and 72 (76%) participated in the 2-year follow-up; 57 of these (79%) had completed genetic testing. Twenty-five of those 57 relatives (44%) were found to harbor an inherited pathogenic variant. Guideline-based cancer surveillance was recommended to 18 relatives; 13 (72%) completed at least one recommended screening, and six (33%) completed all recommended screenings. Risk-reducing surgery was recommended to 10 relatives; four (40%) completed a total of eight procedures. Quality-of-life surveys demonstrated low levels of anxiety, depression, distress, and uncertainty. CONCLUSIONS: The 2-year follow-up of the original pilot study revealed that clinician-facilitated cascade testing resulted in genetically targeted cancer screening and prevention with preserved quality of life. These results, to be confirmed by larger randomized controlled trials, suggest that medical systems should consider supporting clinician-facilitated cascade testing programs.

Foveolar thickness as potential standardized structural outcome measurement in studies of Bietti crystalline dystrophy.

Bietti crystalline dystrophy (BCD) is an ultra-rare orphan disorder that can lead to blindness. Because of the variable rates of progression of the disease, it is necessary to identify suitable outcome measurements for tracking progression in BCD. A retrospective analysis of patients with a clinical and genetic diagnosis of BCD was conducted. Four measurements of spectral domain-optical coherence tomography were compared to patients' best corrected visual acuity. We observed that patients with higher measurements of foveolar thickness, choroidal thickness in the foveolar region, ellipsoid zone band length and the outer nuclear layer + area, had on average better visual acuity. Future studies are needed to validate the structural-functional correlations we observed in BCD and to propose a sensitive and clinically meaningful outcome measurement for tracking this rare, variable disease.

Multimodal imaging reveals retinoschisis masquerading as retinal detachment in patients with choroideremia.

Purpose: To report three cases of retinoschisis in patients with intermediate to advanced choroideremia. Observations: Three patients were referred for evaluation of retinal detachment in the context of an inherited retinal degenerative disease. In all three cases, patients carried variants in the CHM gene suspected to be pathogenic and exhibited the characteristic findings of choroideremia, including pigment clumping and chorioretinal atrophy with scleral exposure and prominent choroidal vessels. Interestingly, these patients were also found to have areas of typical retinoschisis and cystoid degeneration located in the outer plexiform layer of the mid periphery or macula. Retinoschisis was confirmed by spectral domain optical coherence tomography (SD-OCT). Conclusions/Importance: This paper draws attention to the occurrence of retinoschisis in patients with choroideremia. OCT can be used to confirm the presence of retinoschisis rather than retinal detachment, as the clinical exam findings that distinguish the two conditions are not helpful in the setting of advanced chorioretinal atrophy. Although it remains unclear whether patients with choroideremia as a group are at increased risk of retinoschisis, it is possible that abnormal vesicular traffic in the RPE and photoreceptors could contribute to abnormalities in cell adhesion and the extracellular matrix. As gene therapy by subretinal injection of adeno-associated virus becomes the standard of care to slow down or arrest retinal degeneration in choroideremia, it will be critical to carefully screen these patients for retinoschisis prior to surgical intervention and to incorporate any such findings into surgical planning.

Proteomic analysis of autoimmune retinopathy implicates NrCAM as a potential biomarker.

Purpose: To identify vitreous molecular biomarkers associated with autoimmune retinopathy (AIR). Design: Case-control study. Participants: We analyzed six eyes from four patients diagnosed with AIR and eight comparative controls diagnosed with idiopathic macular holes and epiretinal membranes. Methods: Vitreous biopsies were collected from the participants and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or multiplex ELISA. Outcome Measures: Protein expression changes were evaluated by 1-way ANOVA (significant p-value <0.05), hierarchical clustering, and pathway analysis to identify candidate protein biomarkers. Results: There were 16 significantly upregulated and 17 significantly downregulated proteins in the vitreous of three AIR patients compared to controls. The most significantly upregulated proteins included lysozyme C (LYSC), zinc-alpha-2-glycoprotein (ZA2G), complement factor D (CFAD), transforming growth factor-beta induced protein (BGH3), beta-crystallin B2, and alpha-crystallin A chain. The most significantly downregulated proteins included disco-interacting protein 2 homolog (DIP2C), retbindin (RTBDN), and amyloid beta precursor like protein 2 (APLP2). Pathway analysis revealed that vascular endothelial growth factor (VEGF) signaling was a top represented pathway in the vitreous of AIR patients compared to controls. In comparison to a different cohort of three AIR patients analyzed by multiplex ELISA, a commonly differentially expressed protein was neuronal cell adhesion molecule (NrCAM) with p-values of 0.027 in the LC-MS/MS dataset and 0.035 in the ELISA dataset. Conclusion: Protein biomarkers such as NrCAM in the vitreous may eventually help diagnose AIR.

Photoreceptor Manifestations of Primary Mitochondrial Optic Nerve Disorders.

Purpose: To compare the manifestations of photoreceptors (PRs) in three hereditary optic neuropathies affected by primary mitochondrial dysfunction and discuss whether the retinal ganglion cells (RGCs) or the PRs are preferentially affected. Methods: A retrospective analysis of patients with genetically confirmed diagnoses of optic neuropathies associated with mitochondrial dysfunction was performed. This cohort included Leber's hereditary optic neuropathy (LHON), autosomal dominant optic atrophy type 1 (OPA1), and optic atrophy type 13 (OPA13). Patient chart evaluations included clinical characteristics, best-corrected visual acuity (BCVA), fundus photography, spectral-domain optical coherence tomography (SD-OCT), electroretinogram (ERG), and visual evoked potential data. Results: This analysis included seven patients with LHON, six with OPA1, and one with OPA13 from a tertiary medical center. Thirteen of the 14 individuals were male. The average BCVA at diagnosis was 20/285 and 20/500 in the right and left eyes, respectively. Five of the seven patients with LHON, and three of the six patients with OPA1 also showed a mild amplitude reduction or delayed latency on light-adapted ERG and 30-Hz flicker responses; however, SD-OCT imaging did not show correlated PR abnormalities. Notably, a 7-year follow-up of a patient with OPA13 revealed degeneration of RGCs prior to the degeneration of PRs. Follow-up data also demonstrated continuous loss of cone outer segment tips on SD-OCT imaging. Conclusions: RGCs are, in general, affected by mitochondrial dysfunction, whereas variable PR dysfunction exists in patients with LHON and OPA1, especially with respect to the cone responses. Involvement of PRs is particularly evident in OPA13 after RGC degenerations.