Inherited retinal disorders: a genotype-phenotype correlation in an Indian cohort and the importance of genetic testing and genetic counselling.

PURPOSE: Recent advances in sequencing technologies have enabled radical and rapid progress in the genetic diagnosis of inherited retinal disorders (IRDs). Although the list of gene variations continues to grow, it lacks the genetic etiology of ethnic groups like South Asians. Differences in racial backgrounds and consanguinity add to genetic heterogeneity and phenotypic overlaps. METHODS: This retrospective study includes documented data from the Gen-Eye clinic from years 2014 to 2019. Medical records and pedigrees of 591 IRD patients of Indian origin and genetic reports of 117 probands were reviewed. Genotype-phenotype correlations were performed to classify as correlating, non-correlating and unsolved cases. RESULTS: Among the 591 patients, we observed a higher prevalence of clinically diagnosed retinitis pigmentosa (38.9%) followed by unspecified diagnoses (28.5%). Consanguinity was reported to be high (55.6%) in this cohort. Among the variants identified in 117 probands, 36.4% of variants were pathogenic, 19.2% were likely pathogenic, and 44.4% were of uncertain significance. Among the pathogenic and likely pathogenic variants, autosomal recessive inheritance showed higher prevalence. About 35% (41/117) of cases showed genotype-phenotype correlation. Within the correlating cases, retinitis pigmentosa and Stargardt disease were predominant. Novel variants identified in RP, Stargardt, and LCA are reported here. CONCLUSION: This first-of-a-kind report on an Indian cohort contributes to existing knowledge and expansion of variant databases, presenting relevant and plausible novel variants. Phenotypic overlap and variability lead to a differential diagnosis and hence a clear genotype-phenotype correlation helps in precise clinical confirmation. The study also emphasizes the importance of genetic counselling and testing for personalized vision care in a tertiary eye hospital.

Solar retinopathy-correlation between adaptive optics and spectral domain optical coherence tomography with visual acuity.

BACKGROUND: To study the characteristics of photoreceptors on adaptive optics (AO) in patients of solar retinopathy and its correlation to changes in spectral domain optical coherence tomography (SDOCT) and visual acuity. METHODS AND MATERIAL: This is a cross-sectional observational study of six eyes of five patients with clinically diagnosed solar retinopathy. Five age-matched controls were included for comparison. A flood-illuminated adaptive optics retinal camera (AO; rtx1, Imagine Eyes, Orsay, France) was used to image the photoreceptors and a spectral domain optical coherence tomography (SDOCT; Spectralis™ Heidelberg Engineering, Germany) was used to study the retinal layers in these patients. The corrected distance visual acuity (CDVA) was assessed using Snellen's chart. RESULTS: Defects on the SDOCT were smaller (0.25 to 0.75°) at the fovea compared with AO (more than 1°). The cone mosaic was preserved beyond 1°. The cone count from the foveal center at 0.75, 1, 2, and 3° was decreased in all patients of solar retinopathy (mean value 5352) compared with age-matched controls (mean value 15693). A corresponding increase in spacing (mean value 19.13) compared with controls (mean value 9.34) was also observed. The CDVA ranged from 20/30 to 20/80. There was no statistically significant relationship between cone density, at any of the eccentricities measured, with CDVA or OCT defect (p > 0.05, for all). CONCLUSIONS: Adaptive optics is a sensitive and accurate tool to analyze photoreceptor damage in solar maculopathy. It can be used as an adjunct for better understanding of the pathology and to observe or predict changes in these patients.

Inadvertent triple globe penetration during peribulbar anesthesia.

Inadvertent globe perforation following peribulbar anesthesia can lead to unpleasant experiences if not identified early and managed appropriately. We present the case of a 75-year-old female who came with decreased vision in the left eye (LE) following cataract surgery under peribulbar block. Her visual acuity in the right eye (RE) was 6/24 and LE was 6/75. Fundus examination of LE showed vitreous hemorrhage with localized subretinal hemorrhage along the inferotemporal arcade suggestive of globe perforation. Optical coherence tomography (OCT, Spectralis. Heidelberg imaging, Germany) scan across the perforation site showed subretinal hemorrhage and full-thickness retinal tear. On follow-up, two more focal points of retinal whitening were noted in the inferotemporal equatorial region. The patient was kept under close monitoring, and 4 weeks later, vision improved to 6/9 and barrage laser was done around the perforation sites. Serial OCT scans and close follow-up in iatrogenic globe perforation can result in good visual outcomes.

Gene therapy for retinal diseases: From genetics to treatment.

The gene therapy approach for retinal disorders has been considered largely over the last decade owing to the favorable outcomes of the US Food and Drug Administration-approved commercial gene therapy, Luxturna. Technological advances in recent years, such as next-generation sequencing, research in molecular pathogenesis of retinal disorders, and precise correlations with their clinical phenotypes, have contributed to the progress of gene therapies for various diseases worldwide, and more recently in India as well. Thus, considerable research is being conducted for the right choice of vectors, transgene engineering, and accessible and cost-effective large-scale vector production. Many retinal disease-specific clinical trials are presently being conducted, thereby necessitating the collation of such information as a ready reference for the scientific and clinical community. In this article, we present an overview of existing gene therapy research, which is derived from an extensive search across PubMed, Google Scholar, and clinicaltrials.gov sources. This contributes to prime the understanding of basic aspects of this cutting-edge technology and information regarding current clinical trials across many different conditions. This information will provide a comprehensive evaluation of therapies in existing use/research for personalized treatment approaches in retinal disorders.

Practice Patterns and Challenges in Managing Inherited Retinal Diseases Across Asia-Pacific: A Survey from the APIED Network.

PURPOSE: The objective of this paper is to shed light on the current landscape of genotyping practices, phenotyping practices and availability of essential vision rehabilitation management for inherited retinal diseases (IRD) in the Asia-Pacific (APAC) Region. METHODS: The 62-item questionnaire was distributed electronically via email. The questions covered five domains: (1) structure of the IRD service and registry/database; (2) genotyping practices; (3) genetic counselling; (4) deep phenotyping practices; (5) low-vision rehabilitation services. RESULTS: The survey was completed by 36 of 45 centres in twelve countries and regions in APAC. Among these centres, 42 % reported managing more than 1000 patients. Notably, 39 % of centres lack an IRD database or registry, and 44 % of centres have tested less than one-quarter of their IRD patients. The majority of centres (67 %) do not have genetic counsellors. While there was consistency in the imaging-based investigations, there was marked heterogeneity for functional testing using electrophysiology and formal perimetry. Only 34 % of centres confirmed the availability of access to low-vision assistive devices. CONCLUSIONS: This study reveals several critical gaps in managing IRDs in the APAC region. These include the lack of IRD database/registry in one-third of centres, a substantial proportion of patients remaining genetically undiagnosed, and limited availability of genetic counsellors. The findings also underscore a need to harmonise investigations for evaluating retinal function and identify areas for improvement in the provision of low-vision rehabilitation services.

Rationale and protocol paper for the Asia Pacific Network for inherited eye diseases.

PURPOSE: There are major gaps in our knowledge of hereditary ocular conditions in the Asia-Pacific population, which comprises approximately 60% of the world's population. Therefore, a concerted regional effort is urgently needed to close this critical knowledge gap and apply precision medicine technology to improve the quality of lives of these patients in the Asia-Pacific region. DESIGN: Multi-national, multi-center collaborative network. METHODS: The Research Standing Committee of the Asia-Pacific Academy of Ophthalmology and the Asia-Pacific Society of Eye Genetics fostered this research collaboration, which brings together renowned institutions and experts for inherited eye diseases in the Asia-Pacific region. The immediate priority of the network will be inherited retinal diseases (IRDs), where there is a lack of detailed characterization of these conditions and in the number of established registries. RESULTS: The network comprises 55 members from 35 centers, spanning 12 countries and regions, including Australia, China, India, Indonesia, Japan, South Korea, Malaysia, Nepal, Philippines, Singapore, Taiwan, and Thailand. The steering committee comprises ophthalmologists with experience in consortia for eye diseases in the Asia-Pacific region, leading ophthalmologists and vision scientists in the field of IRDs internationally, and ophthalmic geneticists. CONCLUSIONS: The Asia Pacific Inherited Eye Disease (APIED) network aims to (1) improve genotyping capabilities and expertise to increase early and accurate genetic diagnosis of IRDs, (2) harmonise deep phenotyping practices and utilization of ontological terms, and (3) establish high-quality, multi-user, federated disease registries that will facilitate patient care, genetic counseling, and research of IRDs regionally and internationally.

Serial ERG monitoring of response to therapy in vitamin A deficiency related night blindness.

Two male patients with known systemic disorders who presented with complaints of nyctalopia underwent a complete ophthalmic examination including electrophysiological evaluation and serum vitamin A (retinol) levels. A significant vitamin A deficiency was detected, supplementation started and repeat electroretinogram (ERG) testing was carried out to monitor the timeline of recovery. Restoration of rod and generalised cone function was rapid within the first week of receiving treatment and near normal recovery was seen after 1 month of supplementation. Serial monitoring of ERG changes in vitamin A deficiency (VAD) associated night blindness plays an important role to demonstrate functional recovery post-treatment. The different effects of VAD on rod and cone function, and their rate of recovery, may reflect differences in the visual cycle between the two photoreceptors. We report the serial ERG changes in VAD related night blindness secondary to intestinal lipofuscinosis and liver cirrhosis in two patients.

Advancing precision medicines for ocular disorders: Diagnostic genomics to tailored therapies.

Successful sequencing of the human genome and evolving functional knowledge of gene products has taken genomic medicine to the forefront, soon combining broadly with traditional diagnostics, therapeutics, and prognostics in patients. Recent years have witnessed an extraordinary leap in our understanding of ocular diseases and their respective genetic underpinnings. As we are entering the age of genomic medicine, rapid advances in genome sequencing, gene delivery, genome surgery, and computational genomics enable an ever-increasing capacity to provide a precise and robust diagnosis of diseases and the development of targeted treatment strategies. Inherited retinal diseases are a major source of blindness around the world where a large number of causative genes have been identified, paving the way for personalized diagnostics in the clinic. Developments in functional genetics and gene transfer techniques has also led to the first FDA approval of gene therapy for LCA, a childhood blindness. Many such retinal diseases are the focus of various clinical trials, making clinical diagnoses of retinal diseases, their underlying genetics and the studies of natural history important. Here, we review methodologies for identifying new genes and variants associated with various ocular disorders and the complexities associated with them. Thereafter we discuss briefly, various retinal diseases and the application of genomic technologies in their diagnosis. We also discuss the strategies, challenges, and potential of gene therapy for the treatment of inherited and acquired retinal diseases. Additionally, we discuss the translational aspects of gene therapy, the important vector types and considerations for human trials that may help advance personalized therapeutics in ophthalmology. Retinal disease research has led the application of precision diagnostics and precision therapies; therefore, this review provides a general understanding of the current status of precision medicine in ophthalmology.

Merits of multicolor imaging for tractional retinal detachment.

PURPOSE: To compare multicolor imaging (MCI) with Optos color fundus photography (OCFP) for the evaluation of morphology and extent of preretinal membranes in diabetic tractional retinal detachments (TRD). METHODS: In this retrospective study, 30 eyes with diabetic TRDs were imaged using the MCI feature of the Heidelberg Spectralis Spectral-domain optical coherence tomography (SD-OCT) and color photo using the Optos Daytona ultra-widefield fundus camera. Two investigators independently graded and determined the agreeability between the two modalities with respect to the extent of the TRD and preretinal membranes on the SD-OCT B-scan images. RESULTS: The MCI provided better visualization of the attachments and traction points of the posterior hyaloid face and preretinal membranes and is comparable to the SD-OCT B-scan images. The inter-rater agreeability rates for OCFP had a Kappa (κ) value of 0.37, while the MCI had a κ value of 0.46. When comparing between images of different wavelengths, grading using infrared reflectance (IR) had a poor agreement (-0.04 ± 0.04) while green reflectance (GR) (0.46 ± 0.32) and blue reflectance (BR) (0.53 ± 0.19) had a moderate agreement. The composite MCI and GR images also had comparatively higher intraclass coefficient when compared to the OCFP (0.25 [-0.09-0.55]) and IR (-0.03 [-0.39-0.34]) images. CONCLUSION: MCI is more sensitive for determining the extent of TRDs and for the detection of secondary membranes when compared to OCFP, thus, aiding in better surgical planning.

Prevalence of primary angle-closure disease in retinitis pigmentosa.

Purpose: To determine the prevalence of primary angle-closure disease (PACD) in patients with retinitis pigmentosa (RP). Methods: This was a retrospective review of the electronic medical records of all RP patients over the age of 10 years attending the Genetics Eye Clinic of a tertiary-care hospital during a 7-year period. Information regarding age, gender, vision, refraction, lens, intraocular pressure (IOP), type of RP, and inheritance pattern using pedigree charts for all patients were obtained. Patients with a shallow anterior chamber, high IOP, or glaucomatous optic discs were referred to the glaucoma department where they underwent additional IOP measurements, a gonioscopy, and disc evaluation by a glaucoma specialist. The prevalence of PACD was determined. Results: A total of 618 RP patients were examined during the study period, of which 95.1% had typical RP. The prevalence of primary angle-closure suspects was 2.9%, primary angle closure was 0.65%, and primary angle-closure glaucoma (PACG) was 2.27%. In contrast, the prevalence of primary open-angle glaucoma was 1.29%. The prevalence of PACG in those older than 40 years was 3.8% (95% confidence interval: 1.6-6.0). Conclusion: The prevalence of PACG in RP patients over 40 years was higher than that found in the general population of a similar age (3.8% vs. 0.8%). In our cohort of RP patients, 5.9% had PACD. Hence, gonioscopy is warranted in all RP patients to identify this condition and treat it appropriately.

Foveal cyst: Unknown etiology and unexplained response to treatment.

BACKGROUND: Foveal cysts have been associated with vitreous traction due to a taut posterior hyaloid. These eyes may progress to become a full-thickness macular hole, remain stable, or resolve after a posterior vitreous detachment. A foveal cyst in an eye with a complete posterior vitreous detachment and no other obvious pathology is unusual. CASE PRESENTATION: A 21-year-old male presented with blurred vision in the left eye of 1-month duration. On examination his left-eye vision was 20/60, and anterior segment examination was normal. Fundus examination showed internal limiting membrane folds with a suspected lamellar macular hole. A spectral domain optical coherence scan showed a thin dome-like cyst in the sub foveal region involving the outer retinal layer with a detached posterior hyaloid face. A trial of intravitreal dexamethasone implant was given, following which there was improvement in vision and resolution of the foveal cyst. At the third-month follow-up, the patient presented with a stellate pattern of hyporeflectivity in the macular area both on spectral domain optical coherence scan and multicolor imaging with no change in visual acuity. CONCLUSION: The presence of a foveal cyst in a young male without any risk factors is peculiar, and the rapid response to intravitreal dexamethasone implant raises questions about the pathogenesis. The stellate pattern of hyporeflectivity on spectral domain optical coherence scan imaging with an intraretinal "filling defect"-like picture on fluorescein angiography suggests a possible ongoing ischemic pathology.

Morphological features of focal choroidal excavation and its association with macular pathology in Asian Indian eyes.

Purpose: To study the characteristics of focal choroidal excavation (FCE) in Indian eyes based on spectral-domain optical coherence tomography (SD-OCT) findings and their association with macular pathologies. Methods: Retrospective study of 26 patients diagnosed with FCE. All patients' clinical and imaging data were reviewed. Results: There were eight females and 18 males aged between 24 and 85 years. FCE was noted in 31 eyes of 26 patients - unilateral in 21 and bilateral in 5. The conforming type was noted in 13 and nonconforming in 19 eyes. The location was extrafoveal in 16 and subfoveal in 15 eyes. The morphology was bowl-shaped in 24, cone-shaped in six eyes, and mixed type in one eye. Associated pathologies were central serous chorioretinopathy in nine eyes, choroidal neovascular membrane in seven eyes, Stargardt's disease in three eyes, Best disease in four eyes, other retinal dystrophies in two eyes, polypoidal choroidal vasculopathy and moderate non-proliferative diabetic retinopathy, each in one eye. The mean FCE width was 1667.2 ± 817.7 µ, mean depth was 95.7 ± 46.4 µ, and the mean choroidal thickness under the FCE was 234.8 ± 85.9 µ. No abnormal choroidal tissue was found under any FCE. Conclusion: FCE is a relatively common entity and frequently associated with macular pathologies. The presence of an FCE did not alter the course or management of these conditions.

Posterior segment manifestations and imaging features post-COVID-19.

Background: To report the posterior segment (uvea and retinal) manifestations and imaging characteristics of eyes of patients with and after coronavirus disease 2019 (COVID-19). Methods: We searched the PubMed/MEDLINE database to identify relevant articles using the following search terms: COVID-19, SARS-CoV-2, retina, uvea, optic nerve, retinal findings, posterior segment manifestations, and endophthalmitis. Articles published from December 1, 2019, to May 30, 2021, and indexed in PubMed/ MEDLINE were screened. Results: For the purpose of this review, we included clinical features of 26 case reports and 8 case series. The posterior segment manifestations reported included cotton wool spots, retinal hemorrhages, central serous retinopathy, papillophlebitis, optic neuritis, panuveitis, multifocal retinitis, necrotizing retinitis, central retinal artery/vein occlusion, and Purtschner like retinopathy. In this review, we have also included optical coherence tomography angiography (OCTA) features that have been described in COVID-19 patients with pneumonia. Conclusions: COVID-19 patients can experience uveo-retinal manifestations even after recovery. These patients, even if asymptomatic for eye symptoms, should undergo an eye evaluation to rule out posterior segment involvement. OCTA performed in these patients revealed microvascular changes in the superficial and deep retinal plexuses. Some of these patients may require anticoagulant or antiplatelet therapy.

"Old wine in a new bottle" - post COVID-19 infection, central serous chorioretinopathy and the steroids.

INTRODUCTION: Corona virus disease (COVID-19) pandemic can cause myriad of ocular manifestations. We report a case of unilateral multi focal central serous retinopathy, post COVID-19 infection in an Asian Indian female. CASE PRESENTATION: A 42-year-old female presented to us with unilateral blurring, in the right eye (OD), 12 days after COVID-19 infection. She had fever, chills, shortness of breath and cough with tiredness and was COVID- RT PCR positive. She was administered intravenous and oral antibiotics with injection heparin/remdesivir, during her 7 day stay at the hospital. She was also on steroid inhalers. She had no systemic history of note. On ocular evaluation, her corrected distance visual acuity was 20/40 in OD and 20/20 in left eye (OS). Anterior segment was normal. Anterior vitreous was clear. Fundus examination of the OD showed central serous retinopathy (CSCR) with OS being normal. CONCLUSION: CSCR can occur post COVID-19 due to steroid administration and physicians administering it should be aware of this and refer the patients to an ophthalmologist earlier.

Study of retinal structural-functional relationship in choroideremia using fundus autofluorescence and optical coherence tomography.

OBJECTIVE: The objective of this study was to assess the structural-functional relationship in choroideremia (CHM) patients using optical coherence tomography (OCT) and autofluorescence (AF) images. METHODS: In this study, 53 eyes of 28 CHM patients were included. Demographic, ocular and clinical fundus features were recorded. Fundus AF and OCT images were analysed. Patients were classified into two groups based on AF features: group 1, CHM patients where the foveal island was present and group 2, CHM patients where the foveal island was absent. Inner and outer retinal layer thicknesses, retinal pigment epithelium (RPE) and subfoveal choroidal thickness (SFCT) were measured and correlated with visual acuity (VA). RESULTS: There were 26 eyes in group 1 and 27 eyes in group 2. Mean age in groups 1 and 2 were 51.7 ± 13.4 and 63.6 ± 11.6 years, respectively. Age (p = 0.001) and VA (p < 0.001) between the two groups were significantly different. The retinal and SFCT showed significant differences that were analysed for each eye between the two groups. Reduced VA was noted with increasing age (r = 0.483; p ≤ 0.001), thin total retina (r = -0.378; p = 0.005), inner (r = -0.512; p < 0.001), outer (r = -0.59; p < 0.001) retinal thicknesses and thin RPE (r = -0.653; p < 0.001). Multivariate analysis showed RPE thickness (p = 0.001) as the most important index that affected VA. CONCLUSION: RPE thinning contributes to poor VA in patients with advanced CHM. Further studies are needed to evaluate the role of retinal thickness and SFCT and its relationship to VA.

Macular tractional retinal detachment: A rare complication of blunt trauma.

Traumatic rhegmatogenous retinal detachment after blunt ocular trauma is a known entity. A tractional macular detachment occurring posttrauma without a retinal break is a unique presentation. A 25-year-old gentleman after blunt ocular trauma with a ball presented a week later with a vision of 20/800, large subretinal bleed and resolving vitreous hemorrhage in the right eye. Three weeks later, an extensive glial proliferation at the posterior pole and macular tractional retinal detachment was noted with the worsening of visual acuity. A vitrectomy, membrane peeling, and silicone oil tamponade with a subsequent silicone oil removal at 6 months stabilized the macula, and vision improved to 20/120. A tractional macular detachment post blunt trauma is rare albeit an interesting occurrence, which can be effectively managed with vitrectomy and allied procedures.

Multimodal imaging of an idiopathic florid vascularised epiretinal membrane: Course, treatment, and outcome.

Idiopathic vascular epiretinal membrane is an extremely rare entity and the pathogenesis and clinical course is not clearly understood. A 53-year-old hypertensive female patient presented with complaints of altered vision in the right eye. On examination, her vision was 20/30 and fundoscopy showed a vascularized epiretinal membrane (ERM), which was confirmed on spectral-domain optical coherence tomography. No primary cause was found after investigations. The symptoms and ERM showed slow progression over the next three years with a visual acuity of 20/60. She underwent surgery for removal of the ERM, which was subjected to histopathological evaluation. This is a unique case of a florid proliferative vascularisation of an ERM in the absence of any identifiable cause, which had a good visual outcome following surgery.

Multimodal imaging in retinoschisis.

Retinoschisis is characterized by abnormal splitting of neurosensory retina. We demonstrate imaging of retinoschisis with multiple modalities.

Quantifying microstructural changes in retinitis pigmentosa using spectral domain - optical coherence tomography.

BACKGROUND: Most patients of established retinitis pigmentosa (RP) have subnormal peripheral vision and heavily rely on central vision for their daily activities. Central visual acuity is dependent on photoreceptor survival at the macula. Identification of structural changes that precede visual loss is essential. The aim of this study was to correlate the Spectral Domain-Optical Coherence Tomography (SD-OCT) characteristics with visual acuity in patients with typical RP. METHODS: This was a retrospective, observational case series of 224 eyes of 113 RP patients conducted a tertiary eye care center. SD-OCT imaging was done for all eyes. Central retinal thickness (CRT), photoreceptor outer segment length (PROS), foveal outer segment pigment epithelial thickness (FOSPET) and ellipsoid zone (EZ) extent were measured. A new variable, FOSPET-PROS ratio (FPR), obtained by dividing FOSPET by PROS is defined and correlated to corrected distance visual acuity (CDVA) in logMAR using linear regression. RESULTS: Out of 113 patients, 71 were males and 42 females. Mean age of the patients was 35.4 ± 15.1 years. Mean CDVA was 0.33 ± 0.25 logMAR with no difference between the genders. Mean CRT (218.74 ± 83.5 µm) and FPR (1.63 ± 0.22) significantly correlated to CDVA with a correlation coefficient of r = - 0.139 (p = 0.048) and r = 0.842 (p = 0.0001), respectively. FOSPET (mean = 71.15 ± 13.8 µm) and PROS (mean = 44.85 ± 12.5 µm) did not show a significant correlation to CDVA, independent of FPR. CONCLUSIONS: Retinal microstructural changes on SD-OCT, especially the FPR, can be used as a surrogate marker to monitor disease progression in the central retina in degenerative diseases like RP.

Quantification of Ocular Biomechanics In Ocular Manifestations of Systemic Autoimmune Diseases.

Purpose: To quantify biomechanical change associated with autoimmune diseases using Corvis ST deformation data. Methods: Cross-sectional, observational, case control study of 76 patients with systemic autoimmune disease and 21 control subjects. All patients underwent detailed ophthalmic examination with Corvis-ST (Oculus Optikgerate Gmbh, Germany) measurements for biomechanical properties of the eye. Corneal deformation and also deformation caused by the extracorneal tissue were recorded. Using a mathematical formula, the three parameters, namely, corneal stiffness (kc), extracorneal tissue stiffness (kg), and extracorneal tissue viscosity (µg), were defined. Results: The biomechanical analysis showed that kc and µg were significantly lower in patients with systemic autoimmune disease with eye manifestations than control group. However statistically, it was significant in rheumatoid arthritis (RA) patients only. Conclusion: The new parameters, namely, kc, kg, and µg, can provide a sensitive marker of the ocular activity of the collagen vascular diseases.