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Usher syndrome-associated retinitis pigmentosa (RP) causes progressive retinal degeneration, which has no cure. The pathomechanism of Usher type 1B (USH1B)-RP caused by MYO7A mutation remains elusive because of the lack of faithful animal models and limited knowledge of MYO7A function. Here, we analyzed 3D retinal organoids generated from USH1B patient-derived induced pluripotent stem cells. Increased differential gene expression occurred over time without excessive photoreceptor cell death in USH1B organoids compared with controls. Dysregulated genes were enriched first for mitochondrial functions and then proteasomal ubiquitin-dependent protein catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression in rod photoreceptor and Müller glial cells corresponding to upregulation of stress responses in NRL

Purpose To develop a comprehensive next-generation sequencing panel assay that screens genes known to cause developmental eye disorders and inherited eye disease and to evaluate its diagnostic yield in a pediatric cohort with malformations of the globe, anterior segment anomalies, childhood glaucoma, or a combination thereof. Design Evaluation of diagnostic test. Participants Two hundred seventy-seven children, 0 to 16 years of age, diagnosed with nonsyndromic or syndromic developmental eye defects without a genetic diagnosis. Methods We developed a new oculome panel using a custom-designed Agilent SureSelect QXT target capture method (Agilent Technologies, Santa Clara, CA) to capture and perform parallel high-throughput sequencing analysis of 429 genes associated with eye disorders. Bidirectional Sanger sequencing confirmed suspected pathogenic variants. Main Outcome Measures Collated clinical details and oculome molecular genetic results. Results The oculome design covers 429 known eye disease genes; these are subdivided into 5 overlapping virtual subpanels for anterior segment developmental anomalies including glaucoma (ASDA; 59 genes), microphthalmia–anophthalmia–coloboma (MAC; 86 genes), congenital cataracts and lens-associated conditions (70 genes), retinal dystrophies (RET; 235 genes), and albinism (15 genes), as well as additional genes implicated in optic atrophy and complex strabismus (10 genes). Panel development and testing included analyzing 277 clinical samples and 3 positive control samples using Illumina sequencing platforms; more than 30× read depth was achieved for 99.5% of the targeted 1.77-Mb region. Bioinformatics analysis performed using a pipeline based on Freebayes and ExomeDepth to identify coding sequence and copy number variants, respectively, resulted in a definitive diagnosis in 68 of 277 samples, with variability in diagnostic yield between phenotypic subgroups: MAC, 8.2% (8 of 98 cases solved); ASDA, 24.8% (28 of 113 cases solved); other or syndromic, 37.5% (3 of 8 cases solved); RET, 42.8% (21 of 49 cases solved); and congenital cataracts and lens-associated conditions, 88.9% (8 of 9 cases solved). Conclusions The oculome test diagnoses a comprehensive range of genetic conditions affecting the development of the eye, potentially replacing protracted and costly multidisciplinary assessments and allowing for faster targeted management. The oculome enabled molecular diagnosis of a significant number of cases in our sample cohort of varied ocular birth defects.

Mutations in more than 250 genes are implicated in inherited retinal dystrophy; the encoded proteins are involved in a broad spectrum of pathways. The presence of unsolved families after highly parallel sequencing strategies suggests that further genes remain to be identified. Whole-exome and -genome sequencing studies employed here in large cohorts of affected individuals revealed biallelic mutations in ARHGEF18 in three such individuals. ARHGEF18 encodes ARHGEF18, a guanine nucleotide exchange factor that activates RHOA, a small GTPase protein that is a key component of tight junctions and adherens junctions. This biological pathway is known to be important for retinal development and function, as mutation of CRB1, encoding another component, causes retinal dystrophy. The retinal structure in individuals with ARHGEF18 mutations resembled that seen in subjects with CRB1 mutations. Five mutations were found on six alleles in the three individuals: c.808A>G (p.Thr270Ala), c.1617+5G>A (p.Asp540Glyfs ∗ 63), c.1996C>T (p.Arg666 ∗ ), c.2632G>T (p.Glu878 ∗ ), and c.2738_2761del (p.Arg913_Glu920del). Functional tests suggest that each disease genotype might retain some ARHGEF18 activity, such that the phenotype described here is not the consequence of nullizygosity. In particular, the p.Thr270Ala missense variant affects a highly conserved residue in the DBL homology domain, which is required for the interaction and activation of RHOA. Previously, knock-out of Arhgef18 in the medaka fish has been shown to cause larval lethality which is preceded by retinal defects that resemble those seen in zebrafish Crumbs complex knock-outs. The findings described here emphasize the peculiar sensitivity of the retina to perturbations of this pathway, which is highlighted as a target for potential therapeutic strategies.

Purpose To evaluate the phenotypic variability and natural history of ocular disease in a cohort of 28 individuals with MYO7A -related disease. Mutations in the MYO7A gene are the most common cause of Usher syndrome type 1, characterized by profound congenital deafness, vestibular arreflexia, and progressive retinal degeneration. Design Retrospective case series. Participants Twenty-eight patients from 26 families (age range, 3–65 years; median, 32) with 2 likely disease-causing variants in MYO7A . Methods Clinical investigations included fundus photography, optical coherence tomography, fundus autofluorescence (FAF) imaging, and audiologic and vestibular assessments. Longitudinal visual acuity and FAF data (over a 3-year period) were available for 20 and 10 study subjects, respectively. Main Outcome Measures Clinical, structural, and functional characteristics. Results All patients with MYO7A mutations presented with features consistent with Usher type 1. The median visual acuity for the cohort was 0.39 logarithm of the minimum angle of resolution (logMAR; range, 0.0–2.7) and visual acuity in logMAR correlated with age (Spearman's rank correlation coefficient, r = 0.71; P 0.0001). Survival analysis revealed that acuity ≤0.22 logMAR was maintained in 50% of studied subjects until age 33.9; legal blindness based on loss of acuity (≥1.00 logMAR) or loss of field (≤20°) was reached at a median age of 40.6 years. Three distinct patterns were observed on FAF imaging: 13 of 22 patients tested had relatively preserved foveal autofluorescence surrounded by a ring of high density, 4 of 22 had increased signal in the fovea with no obvious hyperautofluorescent ring, and 5 of 22 had widespread hypoautofluorescence corresponding to retinal pigment epithelial atrophy. Despite a number of cases presenting with a milder phenotype, there seemed to be no obvious genotype–phenotype correlation. Conclusions MYO7A -related ocular disease is variable. Central vision typically remains preserved at least until the third decade of life, with 50% of affected individuals reaching legal blindness by 40 years of age. Distinct phenotypic subsets were identified on FAF imaging. A specific allele, previously reported in nonsyndromic deafness, may be associated with a mild retinopathy.

Purpose To determine the prevalence of CYP1B1 mutations in a cohort of patients with congenital corneal opacification (CCO), infantile glaucoma, or both and to describe a developmental CCO associated with CYP1B1 mutation that may explain von Hippel's original description of an internal ulcer. Design Retrospective genotyping of a cohort of patients with infantile glaucoma and CCO. Participants Thirty-three patients with CCO, infantile glaucoma, or both. Methods All patients underwent a full clinical evaluation with or without examination under anesthetic including anterior segment photography, ultrasound biomicroscopy (for CCO patients; n=22), and histopathologic analysis in patients in whom penetrating keratoplasty (PK) was performed (n = 10). Patient DNA and DNA from 50 normal control individuals who had undergone a full ophthalmologic examination were screened for CYP1B1 mutations. Main Outcome Measures Classification of the developmental corneal opacity phenotype in infantile glaucoma patients with CYP1B1 mutations. Results Nine distinct pathogenic recessive CYP1B1 mutations were found in 11 patients from 6 unrelated families, including 1 patient with an entire deletion of the CYP1B1 gene. Two of these patients, including the patient with the deletion, had isolated infantile congenital glaucoma with no other abnormalities. No CYP1B1 mutations were found in another 13 patients (7 of whom underwent PK in at least 1 eye) who had CCO with iridocorneal or keratolenticular adhesions (Peters' anomaly types I and II, respectively). Eight further children with CYP1B1 mutations who had CCO from birth and glaucoma underwent successful glaucoma treatment but had persistent diffuse CCO without iridocorneal or keratolenticular adhesions. Three of these underwent bilateral PK, and the histologic results were not consistent with any hitherto recognized congenital corneal dystrophy and showed abnormalities of the central corneal endothelium. Conclusions Both severe CCO and isolated infantile glaucoma are associated with CYP1B1 mutations. The severe CCO phenotype reported herein often requires PK and has typical histopathologic changes. The mutations associated with this phenotype have not been reported previously. This phenotype may explain the patient described by Von Hippel in 1897. Financial Disclosure(s) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

We report a large Chinese family with X-linked postlingual nonsyndromic hearing impairment in which the critical linkage interval spans a genetic distance of 5.41 cM and a physical distance of 15.1 Mb that overlaps the DFN2 locus. Mutation screening of the PRPS1 gene in this family and in the three previously reported DFN2 families identified four different missense mutations in PRPS1. These mutations result in a loss of phosphoribosyl pyrophosphate (PRPP) synthetase 1 activity, as was shown in silico by structural analysis and was shown in vitro by enzymatic activity assays in erythrocytes and fibroblasts from patients. By in situ hybridization, we demonstrate expression of Prps1 in murine vestibular and cochlea hair cells, with continuous expression in hair cells and postnatal expression in the spiral ganglion. Being the second identified gene associated with X-linked nonsyndromic deafness, PRPS1 will be a good candidate gene for genetic testing for X-linked nonsyndromic hearing loss.

Coenzyme Q(10) is a mobile lipophilic electron carrier located in the inner mitochondrial membrane. Defects of coenzyme Q(10) biosynthesis represent one of the few treatable mitochondrial diseases. We genotyped a patient with primary coenzyme Q(10) deficiency who presented with neonatal lactic acidosis and later developed multisytem disease including intractable seizures, global developmental delay, hypertrophic cardiomyopathy, and renal tubular dysfunction. Cultured skin fibroblasts from the patient had a coenzyme Q(10) biosynthetic rate of 11% of normal controls and accumulated an abnormal metabolite that we believe to be a biosynthetic intermediate. In view of the rarity of coenzyme Q(10) deficiency, we hypothesized that the disease-causing gene might lie in a region of ancestral homozygosity by descent. Data from an Illumina HumanHap550 array were analyzed with BeadStudio software. Sixteen regions of homozygosity1.5 Mb were identified in the affected infant. Two of these regions included the loci of two of 16 candidate genes implicated in human coenzyme Q(10) biosynthesis. Sequence analysis demonstrated a homozygous stop mutation affecting a highly conserved residue of COQ9, leading to the truncation of 75 amino acids. Site-directed mutagenesis targeting the equivalent residue in the yeast Saccharomyces cerevisiae abolished respiratory growth.

We report a patient with relatively mild Leigh syndrome and mitochondrial respiratory chain complex II deficiency caused by a homozygous G555E mutation in the nuclear encoded flavoprotein subunit of succinate dehydrogenase. This mutation has previously been reported in a lethal-infantile presentation of complex II deficiency. Such marked phenotypic heterogeneity, although typical of heteroplasmic mutations in the mitochondrial genome, is unusual for nuclear mutations. Comparable activities and stability of mitochondrial respiratory chain enzymes were demonstrated in both patients, so other reasons for the phenotypic variability are considered.

GJB2 mutations account for approximately 50% of recessive non-syndromic deafness, with 35delG being the most prevalent. Homozygous 35delG mutations cause pre-lingual, non-progressive hearing loss that is detected on newborn hearing screening programmes. We present a sibling pair with homozygous 35delG mutations, who passed hearing tests in early infancy and developed progressive sensorineural hearing loss, one requiring a cochlear implant. These cases illustrate that deafness due to such mutations may have a late onset and consequently be missed on neonatal screening programmes and they may present an argument to consider neonatal screening for GJB2 mutations in order to aid early intervention.

Piebaldism is an autosomal dominant disorder, characterized by congenital leukoderma typically on the abdomen, knees, and forehead. Mice models for human piebaldism are the W dominant white spotting, the steel mice and the mice that are mutated for the slug gene. Human genes for these mice models were investigated in this study. Genomic DNAs of peripheral leukocytes were prepared from twenty-two patients with piebaldism. PCR-direct sequencing or screening by SSCP and subsequent sequencing of all of the exons and flanking introns of KIT, SCF (stem cell factor), and SLUG genes disclosed six pathological mutations only in KIT. Among them five were novel: 358delG, IVS3-2A>G, Q346X, H650L, and D792Y. His650 is located in the strand connecting the helix αC and the downstream β-sheet of the N-lobe of the active KIT kinase domain, the crystal fine structure of which was determined recently. Asp792 is situated in the center of the kinase active site of interdomain cleft between the N-lobe and the C-lobe. No pathological mutations were found in SCF or SLUG in our screening system. Analysis of mutations in the KIT kinase domain may offer an insight into the detailed functional role of this fascinating protein.