Genetic prescreening of a candidate for laser refractive surgery identifies risk for inadequate tissue response: a case report.

BACKGROUND: Inadequate response to corneal laser refractive surgery, e.g., ectatic corneal diseases, may not be identified by conventional examinations, hence creating therapeutic uncertainty. Herein we demonstrate the application of genetic prescreening to augment preassessment for corneal laser refractive surgery and highlight the ability to prevent the possibility of enrolling a subject at risk for developing ectatic corneal diseases. CASE PRESENTATION: Preoperative tests were performed alongside deoxyribonucleic acid (DNA) sequencing of 75 genes specific to the structure and health of the eye of a 44-year-old Caucasian male candidate for corneal laser refractive surgery. The patient had no medical, family, or psychosocial history, nor symptoms that could lead to suspect any corneal abnormalities, and conventional preoperative tests confirmed that no corneal abnormalities were present. The sequencing results uncovered rare DNA variants within the ADGRV1, PTK2, ZNF469, and KRT15 genes. These variants were considered potential risk factors for inadequate response in the patient post corneal laser refractive surgery. Subsequent reevaluation with three different last-generation corneal tomographers identified in the left eye a "warning" for a deformity of the posterior profile of the cornea. CONCLUSIONS: Genetic prescreening identifies potential risk of inadequate response to corneal laser refractive surgery where current technologies in use may lead to a hazardous predictive diagnostic uncertainty.

Evaluation of the IgG antibody response to SARS CoV-2 infection and performance of a lateral flow immunoassay: cross-sectional and longitudinal analysis over 11 months.

OBJECTIVE: To evaluate the dynamics and longevity of the humoral immune response to SARS-CoV-2 infection and assess the performance of professional use of the UK-RTC AbC-19 Rapid Test lateral flow immunoassay (LFIA) for the target condition of SARS-CoV-2 spike protein IgG antibodies. DESIGN: Nationwide serological study. SETTING: Northern Ireland, UK, May 2020-February 2021. PARTICIPANTS: Plasma samples were collected from a diverse cohort of individuals from the general public (n=279), Northern Ireland healthcare workers (n=195), pre-pandemic blood donations and research studies (n=223) and through a convalescent plasma programme (n=183). Plasma donors (n=101) were followed with sequential samples over 11 months post-symptom onset. MAIN OUTCOME MEASURES: SARS-CoV-2 antibody levels in plasma samples using Roche Elecsys Anti-SARS-CoV-2 IgG/IgA/IgM, Abbott SARS-CoV-2 IgG and EuroImmun IgG SARS-CoV-2 ELISA immunoassays over time. UK-RTC AbC-19 LFIA sensitivity and specificity, estimated using a three-reference standard system to establish a characterised panel of 330 positive and 488 negative SARS-CoV-2 IgG samples. RESULTS: We detected persistence of SARS-CoV-2 IgG antibodies for up to 10 months post-infection, across a minimum of two laboratory immunoassays. On the known positive cohort, the UK-RTC AbC-19 LFIA showed a sensitivity of 97.58% (95.28% to 98.95%) and on known negatives, showed specificity of 99.59% (98.53 % to 99.95%). CONCLUSIONS: Through comprehensive analysis of a cohort of pre-pandemic and pandemic individuals, we show detectable levels of IgG antibodies, lasting over 46 weeks when assessed by EuroImmun ELISA, providing insight to antibody levels at later time points post-infection. We show good laboratory validation performance metrics for the AbC-19 rapid test for SARS-CoV-2 spike protein IgG antibody detection in a laboratory-based setting.

Gene Editing for Corneal Stromal Regeneration.

CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.

Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor ß-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.

Biochemical mechanisms of aggregation in TGFBI-linked corneal dystrophies.

Transforming growth factor-ß-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-ß-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.

Evaluation of TGFBI corneal dystrophy and molecular diagnostic testing.

To date, 70 different TGFBI mutations that cause epithelial-stromal corneal dystrophies have been described. At present one commercially available test examines for the five most common of these mutations: R124H, R124C, R124L, R555W, and R555Q. To expand the capability of identifying the causative mutation in the remaining cases, 57 mutations would need to be added. The aim of this study was to obtain a better understanding of the worldwide distribution and population differences of TGFBI mutations and to assess which mutations could be included or excluded from any potential assay. A total of 184 published papers in Human Gene Mutation Database (HGMD) and PubMed from 34 countries worldwide reporting over 1600 corneal dystrophy cases were reviewed. Global data from 600,000 samples using the commercially available test were analyzed. Case studies by University College of London (UCL), Moorfield's Corneal Dystrophy Study data and 19 samples from patients with clinical abnormality or uncertainty for which the current test detected no mutation were used to predict an achievable detection rate. Data from the literature search showed no difference in the spectrum and frequency of each mutation in different populations or geographical locations. According to our analysis, an increase to the worldwide detection rate in all populations from 75 to 90% could be achieved by the addition of six mutations-H626R, A546D, H572R, G623D, R124S, and M502V-to the currently available test and that may be beneficial for LASIK pre-screening worldwide.

TGFBI Gene Mutation Analysis of Clinically Diagnosed Granular Corneal Dystrophy Patients Prior to PTK: A Pilot Study from Eastern China.

This study investigated the TGFBI gene mutation types in outpatients clinically diagnosed with granular corneal dystrophy (GCD) prior to phototherapeutic keratectomy (PTK), also calculated the mutation rate of subjects with normal corneas, but positive family history. Clinical GCD outpatients and consanguineous family members were enrolled in this study. Among total 42 subjects: 24 patients from 23 unrelated families had typical signs of GCD on corneas; 5 patients from 5 unrelated families had atypical signs; 13 subjects from 11 unrelated families had no corneal signs but positive family history. Using Avellino gene test kit, the TGFBI mutation detection was performed on DNA samples from all subjects. 36 subjects were detected to carry heterozygous TGFBI gene mutations. Among 24 clinical GCD patients, the proportion of R124H, R555Q, R124L, R555W and R124C were 37.5%, 16.7%, 25.0%, 20.8% and 0%, respectively, and 2 patients had been diagnosed with GCD according to the opacities thriving after LASIK (R124H) and PRK (R555W). The mutation rate of 13 subjects having no signs but positive family history was 69.2%. R124H mutation is the most prominent mutation type among GCD outpatients in Eastern China. It is recommended to conduct gene detection for patients with positive family history prior to refractive surgeries.