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Virginie G Peter,1 Sophia C Morandi,1,2 Elio L Herzog,1– 3 Martin S Zinkernagel,1,2 Denise C Zysset-Burri1,2 1Department of Ophthalmology, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; 2Department for BioMedical Research, University of Bern, Bern, Switzerland; 3Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, SwitzerlandCorrespondence: Virginie G Peter, Department of Ophthalmology, Inselspital, Bern University Hospital, University of Bern, Freiburgstrasse 15, Bern, 3010, Switzerland, Email virginie.peter@insel.chAbstract: While pathogens of the eye have been studied for a very long time, the existence of resident microbes on the surface of healthy eyes has gained interest only recently. It appears that commensal microbes are a normal feature of the healthy eye, whose role and properties are currently the subject of extensive research. This review provides an overview of studies that have used 16s rRNA gene sequencing and whole metagenome shotgun sequencing to characterize microbial communities associated with the healthy ocular surface from kingdom to genus level. Bacteria are the primary colonizers of the healthy ocular surface, with three predominant phyla: Proteobacteria, Actinobacteria, and Firmicutes, regardless of the host, environment, and method used. Refining the microbial classification to the genus level reveals a highly variable distribution from one individual and study to another. Factors accounting for this variability are intriguing - it is currently unknown to what extent this is attributable to the individuals and their environment and how much is artifactual. Clearly, it is technically challenging to accurately describe the microorganisms of the ocular surface because their abundance is relatively low, thus, permitting substantial contaminations. More research is needed, including better experimental standards to prevent biases, and the exploration of the ocular surface microbiome’s role in a spectrum of healthy to pathological states. Outcomes from such research include the opportunity for therapeutic interventions targeting the microbiome.Keywords: ocular surface, ocular surface microbiome, 16s rRNA gene sequencing, whole metagenome shotgun sequencing

Background: Palmar/plantar osteochondral disease (POD) of the metacarpal/tarsal condyles is a common pathological finding in racehorses., Objective: To compare diagnoses, imaging details, and measurements of POD lesions between cone-beam computed tomography CT (CBCT), fan-beam CT (FBCT), and low-field magnetic resonance imaging (MRI) using macroscopic pathology as a gold standard., Study Design: Cross-sectional study., Methods: Thirty-five cadaver limbs from 10 horses underwent CBCT, FBCT, MRI, and macroscopic examination. CT and MR images were examined for presence of POD, imaging details of POD, and measurements of POD dimensions and areas. Imaging diagnoses, details, and measurements were compared with macroscopic examination and between modalities., Results: Forty-eight POD lesions were seen over 70 condyles. Compared with macroscopic examination the sensitivity and specificity of diagnosis were 95.8% (CI95 = 88%-99%) and 63.6% (CI95 = 43%-81%) for FBCT, 85.4% (CI95 = 74%-94%) and 81.8% (CI95 = 63%-94%) for CBCT, and 69.0% (CI95 = 54%-82%) and 71.4% (CI95 = 46%-90%) for MRI. Inter-modality agreement on diagnosis was moderate between CBCT and FBCT (κ = 0.56, p < 0.001). POD was identified on CT as hypoattenuating lesions with surrounding hyperattenuation and on MRI as either T1W, T2*W, T2W, and STIR hyperintense lesions or T1W and T2*W heterogeneous hypointense lesions with surrounding hypointensity. Agreement on imaging details between CBCT and FBCT was substantial for subchondral irregularity (κ = 0.61, p < 0.001). Macroscopic POD width strongly correlated with MRI (r = 0.81, p < 0.001) and CBCT (r = 0.79, p < 0.001) and moderately correlated with FBCT (r = 0.69, p < 0.001). Macroscopic POD width was greater than all imaging modality (p < 0.001)., Main Limitations: Effect of motion artefact in live horse imaging could not be assessed., Conclusions: All imaging modalities were able to detect POD lesions, but underestimated lesion size. The CT systems were more sensitive, but the differing patterns of signal intensity may suggest that MRI can detect changes associated with POD pathological status or severity. The image features observed by CBCT and FBCT were similar., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Pain localised to the metacarpophalangeal (MCP) and metatarsophalangeal (MTP) region represents a frequent cause of lameness in sport horses, and standing magnetic resonance imaging (MRI) of these regions is increasingly being acquired. This multicentre retrospective study describes the ranges of abnormalities identified on standing MRI of the MCP/MTP region and compares patterns of abnormalities between forelimbs, hindlimbs and different sports disciplines. In total, 341 MRI reports were reviewed. Subchondral bone plate irregularities, condylar and proximal phalanx pathologies were frequently identified with subchondral bone defect, sclerosis and increased intensity on STIR images often described. Medial pathology was frequently identified in the forelimbs, and more lateral pathology was reported in the hindlimbs, which could potentially reflect differences in the loading patterns. Significant differences in MRI findings were found between different sports activities, with MCP/MTP bone pathology occurring more frequently in MRI reports from race and endurance horses and MCP/MTP soft tissue injuries being reported more frequently in dressage and show-jumping horses, particularly in the suspensory apparatus, including the distal sesamoidean ligaments. The findings of this study identify patterns of MCP/MTP abnormalities detected using standing MRI, with differences seen between forelimbs and hindlimbs and between different sports disciplines.

Purpose: Retinitis pigmentosa (RP) comprises a genetically and clinically heterogeneous group of inherited retinal degenerations, where 20-30% of patients exhibit extra-ocular manifestations (syndromic RP). Understanding the genetic profile of RP has important implications for disease prognosis and genetic counseling. This study aimed to characterize the genetic profile of syndromic RP in Portugal., Methods: Multicenter, retrospective cohort study. Six Portuguese healthcare providers identified patients with a clinical diagnosis of syndromic RP and available genetic testing results. All patients had been previously subjected to a detailed ophthalmologic examination and clinically oriented genetic testing. Genetic variants were classified according to the American College of Medical Genetics and Genomics; only likely pathogenic or pathogenic variants were considered relevant for disease etiology., Results: One hundred and twenty-two patients (53.3% males) from 100 families were included. Usher syndrome was the most frequent diagnosis (62.0%), followed by Bardet-Biedl (19.0%) and Senior-Løken syndromes (7.0%). Deleterious variants were identified in 86/100 families for a diagnostic yield of 86.0% (87.1% for Usher and 94.7% for Bardet-Biedl). A total of 81 genetic variants were identified in 25 different genes, 22 of which are novel. USH2A and MYO7A were responsible for most type II and type I Usher syndrome cases, respectively. BBS1 variants were the cause of Bardet-Biedl syndrome in 52.6% of families. Best-corrected visual acuity (BCVA) records were available at baseline and last visit for 99 patients (198 eyes), with a median follow-up of 62.0 months. The mean BCVA was 56.5 ETDRS letters at baseline (Snellen equivalent ~ 20/80), declining to 44.9 ETDRS letters (Snellen equivalent ~ 20/125) at the last available follow-up (p < 0.001)., Conclusion: This is the first multicenter study depicting the genetic profile of syndromic RP in Portugal, thus contributing toward a better understanding of this heterogeneous disease group. Usher and Bardet-Biedl syndromes were found to be the most common types of syndromic RP in this large Portuguese cohort. A high diagnostic yield was obtained, highlighting current genetic testing capabilities in providing a molecular diagnosis to most affected individuals. This has major implications in determining disease-related prognosis and providing targeted genetic counseling for syndromic RP patients in Portugal., (© 2024. The Author(s).)

Background: Dorsoproximal osteochondral defects commonly affect the proximal phalanx, but information about diagnosis on computed tomography (CT) and magnetic resonance imaging (MRI) is limited., Objectives: To assess CT and MRI diagnoses of osteochondral defects, describe the lesions and compare sensitivity and specificity of the modalities using macroscopic pathology as gold standard., Study Design: Cross-sectional study., Methods: Thirty-five equine cadaver limbs underwent standing cone-beam CT (CBCT), fan-beam CT (FBCT), low-field MRI and pathological examination. CT and MR images were examined for proximal phalanx dorsomedial and dorsolateral eminence osteochondral defects. Defect dimensions were measured. Imaging diagnoses and measurements were compared with macroscopic examination., Results: Fifty-six defects were seen over 70 potential locations. On CBCT and FBCT, osteochondral defects appeared as subchondral irregularity/saucer-shaped defects. On MRI, osteochondral defects were a combination of articular cartilage defect on dorsal images and subchondral flattening/irregularity on sagittal images. Subchondral thickening and osseous short tau inversion recovery hyperintensity were found concurrent with osteochondral defects. Compared with pathological examination, the sensitivity and specificity of diagnosis were 86% (95% confidence interval [95% CI] 75%-93%) and 64% (95% CI 38%-85%) for FBCT; 64% (95% CI 51%-76%) and 71% (95% CI 46%-90%) for CBCT; and 52% (95% CI 39%-65%) and 71% (95% CI 46%-90%) for MRI. Sensitivity of all modalities increased with defect size. Macroscopic defect dimensions were strongly correlated with CBCT (r = 0.76, p < 0.001) and moderately correlated with FBCT and MRI (r = 0.65, p < 0.001). Macroscopic measurements were significantly greater than all imaging modality dimensions (p < 0.001), potentially because macroscopy included articular cartilage pathology., Main Limitations: Influence of motion artefact could not be assessed., Conclusions: Osteochondral defects could be visualised using both CT and MRI with sensitivity increasing with defect size. Diagnostic performance was best using FBCT, followed by CBCT then MRI, but CBCT-measured defect size best correlated with macroscopic examination. MRI provided useful information on fluid signal associated with defects, which could represent active pathology., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research., Competing Interests: Declaration of interests The authors declare that there is no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fissure in the third metacarpal/tarsal parasagittal groove and proximal phalanx sagittal groove is a potential prodromal pathology of fracture; therefore, early identification and characterisation of fissures using non-invasive imaging could be of clinical value. Thirty-three equine cadaver limbs underwent standing cone-beam (CB) computed tomography (CT), fan-beam (FB) CT, low-field magnetic resonance imaging (MRI), and macro/histo-pathological examination. Imaging diagnoses of fissures were compared to microscopic examination. Imaging features of fissures were described. Histopathological findings were scored and compared between locations with and without fissures on CT. Microscopic examination identified 114/291 locations with fissures. The diagnostic sensitivity and specificity were 88.5% and 61.3% for CBCT, 84.1% and 72.3% for FBCT, and 43.6% and 85.2% for MRI. Four types of imaging features of fissures were characterised on CT: (1) CBCT/FBCT hypoattenuating linear defects, (2) CBCT/FBCT striated hypoattenuated lines, (3) CBCT/FBCT subchondral irregularity, and (4) CBCT striated hypoattenuating lines and FBCT subchondral irregularity. Fissures on MRI appeared as subchondral bone hypo-/hyperintense defects. Microscopic scores of subchondral bone sclerosis, microcracks, and collapse were significantly higher in locations with CT-identified fissures. All imaging modalities were able to identify fissures. Fissures identified on CT were associated with histopathology of fatigue injuries.

Heterotopic mineralization in equine distal limbs has been considered an incidental finding and little is known about its imaging features. The study aimed to identify heterotopic mineralization and adjacent pathology in the fetlock region with cone-beam (CB) computed tomography (CT), fan-beam (FB) CT, and low-field magnetic resonance imaging (MRI). Images from 12 equine cadaver limbs were examined for heterotopic mineralization and adjacent pathology and verified by macro-examination. Retrospective review of the CBCT/MR images from 2 standing horses was also included. CBCT and FBCT identified twelve mineralization's with homogeneous hyperattenuation: oblique-sesamoidean-ligament (5) without macroscopic abnormality; deep-digital-flexor-tendon (1) and suspensory-branch (6) with macroscopic abnormalities. MRI failed to identify all mineralization's, but detected suspensory branch splitting, and T2 and STIR hyperintensity in 4 suspensory-branches and 3 oblique-sesamoidean-ligaments. Macro-examination found corresponding disruption/splitting and discoloration. All modalities identified 7 ossified fragments showing cortical/trabecular pattern: capsular (1), palmar sagittal ridge (1), proximal phalanx (2) without macroscopic abnormality, and proximal sesamoid bones (3). On MRI, fragments were most identifiable on T1 images. All abaxial avulsions had suspensory-branch splitting on T1 images with T2 and STIR hyperintensity. Macro-examination showed ligament disruption/splitting and discoloration. Suspensory-branch/intersesamoidean ligament mineralization's were identified by CBCT in standing cases; 1 had associated T2 hyperintensity. Both CT systems were generally superior in identifying heterotopic mineralization's than MRI, while MRI provided information on soft tissue pathology related to the lesions, which may be important for management., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Inherited retinal diseases (IRDs) are a group of ocular conditions characterized by an elevated genetic and clinical heterogeneity. They are transmitted almost invariantly as monogenic traits. However, with more than 280 disease genes identified so far, association of clinical phenotypes with genotypes can be very challenging, and molecular diagnosis is essential for genetic counseling and correct management of the disease. In addition, the prevalence and the assortment of IRD mutations are often population-specific. In this work, we examined 230 families from Portugal, with individuals suffering from a variety of IRD diagnostic classes (270 subjects in total). Overall, we identified 157 unique mutations (34 previously unreported) in 57 distinct genes, with a diagnostic rate of 76%. The IRD mutational landscape was, to some extent, different from those reported in other European populations, including Spanish cohorts. For instance, the EYS gene appeared to be the most frequently mutated, with a prevalence of 10% among all IRD cases. This was, in part, due to the presence of a recurrent and seemingly founder mutation involving the deletion of exons 13 and 14 of this gene. Moreover, our analysis highlighted that as many as 51% of our cases had mutations in a homozygous state. To our knowledge, this is the first study assessing a cross-sectional genotype-phenotype landscape of IRDs in Portugal. Our data reveal a rather unique distribution of mutations, possibly shaped by a small number of rare ancestral events that have now become prevalent alleles in patients., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Introduction: Fracture configuration is often more complex than is radiographically appreciable. The objective of this study is to describe the influence of pre-operative computed tomography (CT) for surgical planning in a variety of fracture types. This has not been described in previous studies., Materials and Methods: All cases with pre-operative radiographs, admitted for CT and surgical repair of a suspected limb fracture from January 2010-December 2020 were reviewed. CT was acquired under general anaesthesia in a multi-slice helical scanner; any surgery was then performed immediately. Three diplomates (two surgical; one diagnostic imaging) performed a blinded retrospective review of the radiographs and CT for each horse. A consensus decision was made on any change in surgical plan prior to and after CT review, and cases divided into three categories: CT of major, intermediate or minor relevance, as previously described by Genton et al, 2019., Results: 55 cases were collated. Thoroughbred racehorses predominated. The median age was 3 years. A diverse range of fractures were presented: proximal phalanx (18/55), carpal (17/55), metacarpal/tarsal (11/55), sesamoid (5/55), tarsal (3/55), and middle phalanx (1/55). In 13 of 55 cases (23.6%, 95% CI[12%,35%]) CT was of major relevance. In 21 of 55 cases CT was of intermediate relevance (38.2%, 95% CI[25%,51%]). In 21 of 55 cases CT was of minor relevance (38.2%, 95% CI[25%,51%]). A Fisher's exact test demonstrated no statistical difference in CT relevance between fracture types (p<0.05)., Discussion/conclusions: This study demonstrates that CT has a significant role in surgical planning, and in the majority (61.8%) of cases added additional information or significantly changed the surgical plan. In all cases CT ensured confidence in surgical planning., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Taylor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Pathogenic variants in KCNJ1 3 have been associated with both autosomal dominant Snowflake vitreoretinal degeneration (SVD) and autosomal recessive Leber congenital amaurosis. SVD is characterized by aberrant vitreoretinal interface leading to increased risk of retinal detachment, crystalline retinal snowflake deposits, optic disc abnormalities, early-onset cataract, and cornea guttae. Reduced dark adaptation and reduced scotopic rod b-waves have also been described. We report a novel phenotype associated with the R162W variant in KCNJ13 ., Methods: Four affected members of a Swedish family were included. Three of them were examined with best corrected visual acuity, Goldmann perimetry, full-field-and multifocal electroretinography, optical coherence tomography, fundus color photographs, fundus autofluorescence images, slit lamp inspection, and genetic testing. The fourth subject only managed genetic testing., Results: All subjects carry the pathogenic missense variant; c.484C>T (NM&#95;002242.4), R162W, in KCNJ13 . ERG measurements revealed reduced macular-as well as general retinal function. Two of the subjects had a history of retinal detachment and the two younger subjects demonstrated early onset cataract. They all had structural macular changes and slightly gliotic optic discs., Conclusion: In this family, the R162W variant in KCNJ13 , previously described in association with SVD, causes a somewhat novel phenotype including macular dystrophy and moderate reduction of general retinal function as the main features combined with disc abnormalities, retinal detachment, and presenile cataract that has been described before. In times of up-coming gene-based therapies, it is important to report new genotype-phenotype associations to improve the possibilities to identify future treatment candidates.

We used a machine learning approach to analyze the within-gene distribution of missense variants observed in hereditary conditions and cancer. When applied to 840 genes from the ClinVar database, this approach detected a significant non-random distribution of pathogenic and benign variants in 387 (46%) and 172 (20%) genes, respectively, revealing that variant clustering is widespread across the human exome. This clustering likely occurs as a consequence of mechanisms shaping pathogenicity at the protein level, as illustrated by the overlap of some clusters with known functional domains. We then took advantage of these findings to develop a pathogenicity predictor, MutScore, that integrates qualitative features of DNA substitutions with the new additional information derived from this positional clustering. Using a random forest approach, MutScore was able to identify pathogenic missense mutations with very high accuracy, outperforming existing predictive tools, especially for variants associated with autosomal-dominant disease and cancer. Thus, the within-gene clustering of pathogenic and benign DNA changes is an important and previously underappreciated feature of the human exome, which can be harnessed to improve the prediction of pathogenicity and disambiguation of DNA variants of uncertain significance., Competing Interests: Declaration of interests D.N.C. and P.D.S. acknowledge QIAGEN Inc. for their financial support through a License Agreement with Cardiff University. The other authors do not declare any conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Inherited retinal dystrophies (IRDs) constitute one of the most heterogeneous groups of Mendelian human disorders. Using autozygome-guided next-generation sequencing methods in 17 consanguineous pedigrees of Iranian descent with isolated or syndromic IRD, we identified 17 distinct genomic variants in 11 previously-reported disease genes. Consistent with a recessive inheritance pattern, as suggested by pedigrees, variants discovered in our study were exclusively bi-allelic and mostly in a homozygous state (in 15 families out of 17, or 88%). Out of the 17 variants identified, 5 (29%) were never reported before. Interestingly, two mutations (GUCY2D:c.564dup, p.Ala189ArgfsTer130 and TULP1:c.1199G > A, p.Arg400Gln) were also identified in four separate pedigrees (two pedigrees each). In addition to expanding the mutational spectrum of IRDs, our findings confirm that the traditional practice of endogamy in the Iranian population is a prime cause for the appearance of IRDs., (© 2021. The Author(s).)

We studied a family in which the first-born child, a girl, had developmental delay, facial dysmorphism, and agenesis of the corpus callosum (ACC). The subsequent pregnancy was interrupted as the fetus was found to be also affected by ACC. Both cases were heterozygous for two KDM5B variants predicting p (Ala635Thr) and p (Ser1155Ala fs Ter4) that were shown to be in trans . KDM5B variants have been previously associated with moderate to severe developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), and dysmorphism in a few individuals, but the pathogenetic mechanisms are not clear yet as patients with both monoallelic and biallelic variants have been observed. Interestingly, one individual has previously been reported with ACC and severe ID in association with biallelic KDM5B variants. Together with the observations in this family, this suggests that agenesis of the corpus callosum may be part of the phenotypic spectrum associated with KDM5B variants and that the KDM5B gene should be included in gene panels to clarify the etiology of ACC both in the prenatal and postnatal setting.

Pathogenic variants in INPP5E cause Joubert syndrome (JBTS), a ciliopathy with retinal involvement. However, despite sporadic cases in large cohort sequencing studies, a clear association with non-syndromic inherited retinal degenerations (IRDs) has not been made. We validate this association by reporting 16 non-syndromic IRD patients from ten families with bi-allelic mutations in INPP5E. Additional two patients showed early onset IRD with limited JBTS features. Detailed phenotypic description for all probands is presented. We report 14 rare INPP5E variants, 12 of which have not been reported in previous studies. We present tertiary protein modeling and analyze all INPP5E variants for deleteriousness and phenotypic correlation. We observe that the combined impact of INPP5E variants in JBTS and non-syndromic IRD patients does not reveal a clear genotype-phenotype correlation, suggesting the involvement of genetic modifiers. Our study cements the wide phenotypic spectrum of INPP5E disease, adding proof that sequence defects in this gene can lead to early-onset non-syndromic IRD.

Four individuals from two families presented with a multisystemic condition of suspected genetic origin that was diagnosed only after genome analysis. The main phenotypic features were immune system dysregulation (severe immunodeficiency with autoimmunity) and intellectual disability. The four individuals were found to be homozygous for a 4.4 Kb deletion removing exons 20-23 (NM&#95;003291.4) of the TPP2 gene, predicting a frameshift with premature termination of the protein. The deletion was located on a shared chromosome 13 haplotype indicating a Swiss founder mutation. Tripeptidyl peptidase 2 (TPP2) is a protease involved in HLA/antigen complex processing and amino acid homeostasis. Biallelic variants in TPP2 have been described in 10 individuals with variable features including immune deficiency, autoimmune cytopenias, and intellectual disability or chronic sterile brain inflammation mimicking multiple sclerosis. Our observations further delineate this severe condition not yet included in the OMIM catalog. Timely recognition of TPP2 deficiency is crucial since (1) immune surveillance is needed and hematopoietic stem cell transplantation may be necessary, and (2) for provision of genetic counselling. Additionally, enzyme replacement therapy, as already established for TPP1 deficiency, might be an option in the future., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In murine and canine animal models, mutations in the Arylsulfatase G gene (ARSG) cause a particular lysosomal storage disorder characterized by neurological phenotypes. Recently, two variants in the same gene were found to be associated with an atypical form of Usher syndrome in humans, leading to visual and auditory impairment without the involvement of the central nervous system. In this study, we identified three novel pathogenic variants in ARSG, which segregated recessively with the disease in two families from Portugal. The probands were affected with retinitis pigmentosa and sensorineural hearing loss, generally with an onset of symptoms in their fourth decade of life. Functional experiments showed that these pathogenic variants abolish the sulfatase activity of the Arylsulfatase G enzyme and impede the appropriate lysosomal localization of the protein product, which appears to be retained in the endoplasmic reticulum. Our data enable to definitely confirm that different biallelic variants in ARSG cause a specific deaf-blindness syndrome, by abolishing the activity of the enzyme it encodes., (© 2020 Wiley Periodicals LLC.)

Homozygosity mapping is a powerful method for identifying mutations in patients with recessive conditions, especially in consanguineous families or isolated populations. Historically, it has been used in conjunction with genotypes from highly polymorphic markers, such as DNA microsatellites or common SNPs. Traditional software performs rather poorly with data from Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS), which are now extensively used in medical genetics. We develop AutoMap, a tool that is both web-based or downloadable, to allow performing homozygosity mapping directly on VCF (Variant Call Format) calls from WES or WGS projects. Following a training step on WES data from 26 consanguineous families and a validation procedure on a matched cohort, our method shows higher overall performances when compared with eight existing tools. Most importantly, when tested on real cases with negative molecular diagnosis from an internal set, AutoMap detects three gene-disease and multiple variant-disease associations that were previously unrecognized, projecting clear benefits for both molecular diagnosis and research activities in medical genetics.

Conjunctival melanoma (CJM) is a rare but potentially lethal and highly-recurrent cancer of the eye. Similar to cutaneous melanoma (CM), it originates from melanocytes. Unlike CM, however, CJM is relatively poorly characterized from a genomic point of view. To fill this knowledge gap and gain insight into the genomic nature of CJM, we performed whole-exome (WES) or whole-genome sequencing (WGS) of tumor-normal tissue pairs in 14 affected individuals, as well as RNA sequencing in a subset of 11 tumor tissues. Our results show that, similarly to CM, CJM is also characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions. This, as well as the presence of a UV light-induced mutational signature, are clear signs of the role of sunlight in CJM tumorigenesis. In addition, the genomic classification of CM proposed by TCGA seems to be well-applicable to CJM, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, NF1, RAS, and triple wild-type. In line with these results, transcriptomic analyses revealed similarities with CM as well, namely the presence of a transcriptomic subtype enriched for immune genes and a subtype enriched for genes associated with keratins and epithelial functions. Finally, in seven tumors we detected somatic mutations in ACSS3, a possible new candidate oncogene. Transfected conjunctival melanoma cells overexpressing mutant ACSS3 showed higher proliferative activity, supporting the direct involvement of this gene in the tumorigenesis of CJM. Altogether, our results provide the first unbiased and complete genomic and transcriptomic classification of CJM., Competing Interests: The authors have declared that no competing interests exist.

Dystrophic epidermolysis bullosa is a major form of epidermolysis bullosa and may be inherited as an autosomal dominant or recessive trait, with associated mutations in the COL7A1 gene. Here, we describe a consanguineous Pakistani family with four affected individuals suffering from recessive dystrophic epidermolysis bullosa. Exome sequencing of the proband's DNA revealed a homozygous missense variant (c.8038G>A:p.Gly2680Ser) in COL7A1 which cosegregated with disease in the family. The emergence of this particular glycine substitution in patients from diverse ethnic backgrounds such as China, United Kingdom, Poland, Iran, and Pakistan indicates that this variant most likely constitutes a recurrent mutational hotspot in the COL7A1 gene, rather than a germline mutation present at low levels in the general population.

Variants in more than 271 different genes have been linked to hereditary retinal diseases, making comprehensive genomic approaches mandatory for accurate diagnosis. We explored the genetic landscape of retinal disorders in consanguineous families from North-Western Pakistan, harboring a population of approximately 35 million inhabitants that remains relatively isolated and highly inbred (~50% consanguinity). We leveraged on the high degree of consanguinity by applying genome-wide high-density single-nucleotide polymorphism (SNP) genotyping followed by targeted Sanger sequencing of candidate gene(s) lying inside autozygous intervals. In addition, we performed whole-exome sequencing (WES) on at least one proband per family. We identified 7 known and 4 novel variants in a total of 10 genes ( ABCA4 , BBS2 , CNGA1 , CNGA3 , CNGB3 , MKKS , NMNAT1 , PDE6B , RPE65 , and TULP1 ) previously known to cause inherited retinal diseases. In spite of all families being consanguineous, compound heterozygosity was detected in one family. All homozygous pathogenic variants resided in autozygous intervals ≥2.0 Mb in size. Putative founder variants were observed in the ABCA4 (NM&#95;000350.2:c.214G>A; p.Gly72Arg; ten families) and NMNAT1 genes (NM&#95;022787.3:c.25G>A; p.Val9Met; two families). We conclude that geographic isolation and sociocultural tradition of intrafamilial mating in North-Western Pakistan favor both the clinical manifestation of rare "generic" variants and the prevalence of founder mutations., Competing Interests: The authors declare no conflict of interest.

Purpose: We observed four individuals in two unrelated but consanguineous families from Portugal and Brazil affected by early-onset retinal degeneration, sensorineural hearing loss, microcephaly, intellectual disability, and skeletal dysplasia with scoliosis and short stature. The phenotype precisely matched that of an individual of Azorean descent published in 1986 by Liberfarb and coworkers., Methods: Patients underwent specialized clinical examinations (including ophthalmological, audiological, orthopedic, radiological, and developmental assessment). Exome and targeted sequencing was performed on selected individuals. Minigene constructs were assessed by quantitative polymerase chain reaction (qPCR) and Sanger sequencing., Results: Affected individuals shared a 3.36-Mb region of autozygosity on chromosome 22q12.2, including a 10-bp deletion (NM&#95;014338.3:c.904-12&#95;904-3delCTATCACCAC), immediately upstream of the last exon of the PISD (phosphatidylserine decarboxylase) gene. Sequencing of PISD from paraffin-embedded tissue from the 1986 case revealed the identical homozygous variant. In HEK293T cells, this variant led to aberrant splicing of PISD transcripts., Conclusion: We have identified the genetic etiology of the Liberfarb syndrome, affecting brain, eye, ear, bone, and connective tissue. Our work documents the migration of a rare Portuguese founder variant to two continents and highlights the link between phospholipid metabolism and bone formation, sensory defects, and cerebral development, while raising the possibility of therapeutic phospholipid replacement.

Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy., Competing Interests: The authors have declared that no competing interests exist.

Background: Inherited retinal degenerations (IRDs) encompass a wide spectrum of genetic ocular diseases characterized by considerable genetic and clinical heterogeneity., Methods: Complete ophthalmic examination and next-generation sequencing., Results: We describe a patient with no family history of vision loss, who at the age of 28 years developed visual impairment consistent with a severe form of retinitis pigmentosa. Genetic testing by means of whole exome sequencing identified a homozygous variant in the gene IDH3A. To date, only three papers have reported mutations in IDH3A, in families with early-onset retinal degeneration with or without the presence of macular pseudocoloboma., Conclusion: This study highlights the importance of including this rarely-mutated gene in the molecular diagnostic set-ups for IRDs, and further delineates the phenotypic spectrum elicited by mutations in IDH3A.

Reasons for performing study: To investigate the racing performance of Thoroughbred horses with osseous cyst-like lesions (OCLLs) in the distal phalanx causing lameness and treated conservatively. Objectives: To assess horses' ability to race and perform after radiographic identification of OCLL in the distal phalanx of Thoroughbred horses with lameness at the time of detection and undergoing conservative treatment. Study Design: Retrospective case control study. Methods: The clinical database of one equine clinic was reviewed in a 10-year period for Thoroughbreds showing lameness localized to the foot and a radiographic diagnosis of OCLL in the distal phalanx. Sex, age at time of detection of the OCLL, degree of lameness, affected limb, and treatment were recorded. Successful performance of horses was assessed by racing at least once after detection of the OCLL and maximum racing performance rating (RPR). Radiographic features such as size, location, sclerotic rim of the OCLL and irregularity of the articular surface of the distal phalanx were compared to successful performance using univariable statistical analysis. Successful performance of horses with OCLL was compared to a control group of maternal siblings by parametric testing. Results: Twenty-two horses met the inclusion criteria. Thirteen horses raced after the detection of OCLLs. Eight did not race, one case had not yet reached racing age, resulting in 62% (13/21) of racing age racing at least once. The number of successfully performing horses with an OCCL was significantly lower compared to their maternal siblings [ p = 0.03, Odds ratio (OR) = 0.30]. If horses with OCLL in the distal phalanx raced, their RPR was similar to their maternal siblings. No significant association was found between radiographic features of OCLLs and successful performance, but OCLLs in the left forelimb carried a more favorable outcome for racing ( p = 0.02, OR = 2.33 95%CI 1.27, 4.27) compared to OCLLs in any other limb. Conclusions: Horses with lameness and an OCLL in the distal phalanx managed conservatively are less likely to race when compared to their maternal siblings. If horses with OCLLs in the distal phalanx are able to race, their performance, measured as RPR, was comparable to their maternal siblings. Due to the small numbers in this study the results should be interpreted carefully.