Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene.

Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low-visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α-subunit of the G-protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease-causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients carry just a single heterozygous variant. In addition to our previous study on GNAT2-ACHM, we also present detailed clinical data of these patients.

Where are the missing gene defects in inherited retinal disorders? Intronic and synonymous variants contribute at least to 4% of CACNA1F-mediated inherited retinal disorders.

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB.

Abnormal differentiation of B cells and megakaryocytes in patients with Roifman syndrome.

BACKGROUND: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome. OBJECTIVE: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. METHODS: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. RESULTS: The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor-dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. CONCLUSION: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome.

Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs(∗)57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.

Blau syndrome: cross-sectional data from a multicentre study of clinical, radiological and functional outcomes.

OBJECTIVE: To report baseline articular, functional and ocular findings of the first international prospective cohort study of Blau syndrome (BS). METHODS: Three-year, multicentre, observational study on articular, functional (HAQ, Childhood HAQ and VAS global and pain), ophthalmological, therapeutic and radiological data in BS patients. RESULTS: Baseline data on the first 31 recruited patients (12 females and 19 males) from 18 centres in 11 countries are presented. Of the 31 patients, 11 carried the p.R334W NOD2 mutation, 9 the p.R334Q and 11 various other NOD2 missense mutations; 20 patients were sporadic and 11 from five BS pedigrees. Median disease duration was 12.8 years (1.1-57). Arthritis, documented in all but one patient, was oligoarticular in 7, polyarticular in 23. The median active joint count was 21. Functional capacity was normal in 41%, mildly impaired in 31% and moderate-severe in 28% of patients. The most frequently involved joints at presentation were wrists, ankles, knees and PIPs. On radiographs, a symmetrical non-erosive arthropathy was shown. Previously unknown dysplastic bony changes were found in two-thirds of patients. Ocular disease was documented in 25 of 31 patients, with vitreous inflammation in 64% and moderate-severe visual loss in 33%. Expanded manifestations (visceral, vascular) beyond the classic clinical triad were seen in 52%. CONCLUSION: BS is associated with severe ocular and articular morbidity. Visceral involvement is common and may be life-threatening. Bone dysplastic changes may show diagnostic value and suggest a previously unknown role of NOD2 in bone morphogenesis. BS is resistant to current drugs, suggesting the need for novel targeted therapies.

Spectral sensitivity measurements reveal partial success in restoring missing rod function with gene therapy.

Restored rod visual function after gene therapy can be established unequivocally by demonstrating that, after dark adaptation, spectral sensitivity has the shape characteristic of rods and that this shape collapses to a cone-like shape before rods have recovered after an intense bleach. We used these tests to assess retinal function in eight young adults and children with early-onset severe retinal dystrophy from Phase II of a clinical gene-therapy trial for RPE65 deficiency that involved the subretinal delivery of a recombinant adeno-associated viral vector carrying RPE65. We found substantial improvements in rod sensitivity in two participants: dark-adapted spectral sensitivity was rod-like after treatment and was cone-like before rods had recovered after a bleach. After 40 min of dark adaptation, one participant showed up to 1,000-fold sensitivity improvements 4 months after treatment and the second up to 100-fold improvements 6 months after treatment. The dark-adapted spectral sensitivities of the other six participants remained cone-like and showed little improvement in sensitivity.

Identity-by-descent-guided mutation analysis and exome sequencing in consanguineous families reveals unusual clinical and molecular findings in retinal dystrophy.

PURPOSE: Autosomal recessive retinal dystrophies are clinically and genetically heterogeneous, which hampers molecular diagnosis. We evaluated identity-by-descent-guided Sanger sequencing or whole-exome sequencing in 26 families with nonsyndromic (19) or syndromic (7) autosomal recessive retinal dystrophies to identify disease-causing mutations. METHODS: Patients underwent genome-wide identity-by-descent mapping followed by Sanger sequencing (16) or whole-exome sequencing (10). Whole-exome sequencing data were filtered against identity-by-descent regions and known retinal dystrophy genes. The medical history was reviewed in mutation-positive families. RESULTS: We identified mutations in 14 known retinal dystrophy genes in 20/26 (77%) families: ABCA4, CERKL, CLN3, CNNM4, C2orf71, IQCB1, LRAT, MERTK, NMNAT1, PCDH15, PDE6B, RDH12, RPGRIP1, and USH2A. Whole-exome sequencing in single individuals revealed mutations in either the largest or smaller identity-by-descent regions, and a compound heterozygous genotype in NMNAT1. Moreover, a novel deletion was found in PCDH15. In addition, we identified mutations in CLN3, CNNM4, and IQCB1 in patients initially diagnosed with nonsyndromic retinal dystrophies. CONCLUSION: Our study emphasized that identity-by-descent-guided mutation analysis and/or whole-exome sequencing are powerful tools for the molecular diagnosis of retinal dystrophy. Our approach uncovered unusual molecular findings and unmasked syndromic retinal dystrophies, guiding future medical management. Finally, elucidating ABCA4, LRAT, and MERTK mutations offers potential gene-specific therapeutic perspectives.

Sclerocornea in a patient with van den Ende-Gupta syndrome homozygous for a SCARF2 microdeletion.

Van den Ende-Gupta Syndrome (VDEGS) is an autosomal recessive disorder characterized by blepharophimosis, distinctive nose, hypoplastic maxilla, and skeletal abnormalities. Using homozygosity mapping in four VDEGS patients from three consanguineous families, Anastacio et al. [Anastacio et al. (2010); Am J Hum Genet 87:553-559] identified homozygous mutations in SCARF2, located at 22q11.2. Bedeschi et al. [2010] described a VDEGS patient with sclerocornea and cataracts with compound heterozygosity for the common 22q11.2 microdeletion and a hemizygous SCARF2 mutation. Because sclerocornea had been described in DiGeorge-velo-cardio-facial syndrome but not in VDEGS, they suggested that the ocular abnormalities were caused by the 22q11.2 microdeletion. We report on a 23-year-old male who presented with bilateral sclerocornea and the VDGEGS phenotype who was subsequently found to be homozygous for a 17 bp deletion in exon 4 of SCARF2. The occurrence of bilateral sclerocornea in our patient together with that of Bedeschi et al., suggests that the full VDEGS phenotype may include sclerocornea resulting from homozygosity or compound heterozygosity for loss of function variants in SCARF2.

Conradi-Hünermann-Happle syndrome: a novel heterozygous missense mutation, c.204G>T (p.W68C).

Conradi-Hünermann-Happle syndrome (X-linked dominant chondrodysplasia punctata, CDPX2 [Online Mendelian Inheritance in Man 302960]) is a rare genodermatosis that presents with blaschkolinear ichthyosis, cicatricial alopecia, chondrodysplasia punctata, asymmetric shortening of the bones, and cataracts. In this case report we describe a child presenting with a patterned alopecia in which supplementary signs and clinical examination of the mother led to the suspicion of Conradi-Hünermann-Happle syndrome. Mutation analysis revealed a heterozygous novel missense mutation, c.204G>T (p.W68C), in exon 2.

Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome.

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

Ophthalmological assessment of children with neurofibromatosis type 1.

UNLABELLED: Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder, caused by mutations in the NF1 gene, located on chromosome band 17q11.2. In 1988, the National Institutes of Health created specific criteria for the diagnosis of NF1. Four cardinal criteria are assessed through ophthalmological screening: Lisch nodules, optic pathway glioma, a distinctive osseous lesion (sphenoid dysplasia), and the (orbital) plexiform neurofibroma. NF1 patients are prone to the development of central and peripheral nervous system tumors. Especially young children are at risk for growing optic pathway gliomas that can threaten their sight. From an early age, children with NF1 undergo regular ophthalmological examinations. Little is known about the natural progress of these clinical features and the guidelines for screening and follow-up are controversial. Several questions remain unanswered. CONCLUSION: Most of these questions could be solved by better understanding of the natural history of optic pathway gliomas. There is a tendency towards using vision as a primary objective in clinical treatment trials; this way we can evaluate new treatment strategies and focus specifically on visual evolution so we will be able to select even more carefully which patient would benefit treatment. For future clinical trials, a standardized visual acuity assessment protocol is therefore mandatory.

DNA extraction protocol impacts ocular surface microbiome profile.

Purpose: The aim of this study is to provide a reference frame to allow the comparison and interpretation of currently published studies on 16S ribosomal ribonucleic acid amplicon sequencing of ocular microbiome samples using different DNA extraction protocols. Alongside, the quantitative and qualitative yield and the reproducibility of different protocols has been assessed. Methods: Both eyes of 7 eligible volunteers were sampled. Five commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome and 2 host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration. The V3-V4 region of the 16S rRNA gene was targeted using Illumina MiSeq sequencing. The DADA2 pipeline in R was used to perform the bio-informatic processing and taxonomical assignment was done using the SILVA v132 database. The Vegdist function was used to calculate Bray-Curtis distances and the Galaxy web application was used to identify potential metagenomic biomarkers via linear discriminant analysis Effect Size (LEfSe). The R package Decontam was applied to control for potential contaminants. Results: Samples analysed with PowerSoil, RNeasy and NucleoSpin had the highest DNA yield. The host DNA depletion kits showed a very low microbial DNA yield; and these samples were pooled per kit before sequencing. Despite pooling, 1 of both failed to construct a library.Looking at the beta-diversity, clear microbial compositional differences - dependent on the extraction protocol used - were observed and remained present after decontamination. Eighteen genera were consistently retrieved from the ocular surface of every volunteer by all non-pooled extraction kits and a comprehensive list of differentially abundant bacteria per extraction method was generated using LefSe analysis. Conclusion: High-quality papers have been published in the field of the ocular surface microbiome but consensus on the importance of the extraction protocol used are lacking. Potential contaminants and discriminative genera per extraction protocol used, were introduced and a reference frame was built to facilitate both the interpretation of currently published papers and to ease future choice - making based on the research question at hand.

Two siblings with Bosch-Boonstra-Schaaf optic atrophy syndrome due to parental gonadal mosaicism.

Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS, OMIM 615722) is a rare autosomal dominant disorder characterized by intellectual disability, optic atrophy, cortical visual impairment, mild facial dysmorphism, hypotonia, hearing problems, attention deficit and a thin corpus callosum. The gene underlying this disorder is NR2F1 located on chromosome 5q15 which encodes for a nuclear receptor protein. Mutations and deletions have been identified in patients. Here we report on a brother and a sister carrying a pathogenic nonsense NR2F1 variant. The patients have a mild phenotype showing optic atrophy, mild intellectual disability, dysmorphic features and thin corpus callosum. This correlates with previously described milder phenotypes in patients with mutations in this domain. The variant was not identified in the parental genome indicating most likely a gonadal mosaicism. Gonadal mosaicism has not yet been reported in Bosch-Boonstra-Schaaf Optic Atrophy Syndrome.

Unilateral Melanoma-Associated Retinopathy Case Report.

In this report, we present a case of unilateral melanoma-associated retinopathy in a 72-year-old woman. The patient's main symptoms were decreased vision and positive dysphotopsia. Unilateral electronegative electroretinogram (ERG) was suggestive for melanoma retinopathy. PET-CT discovered metastatic disease, 3 years after the initial melanoma. A prompt treatment with corticosteroids was started, followed by immunotherapy. The central and peripheral vision of the patient improved, and the ERG showed normalization of the responses. This case highlights the importance of early recognition and individualized treatment strategies for melanoma-associated retinopathy.

Variability in expression of a familial 2.79 Mb microdeletion in chromosome 14q22.1-22.2.

Deletions in chromosome 14q22-23 have been associated with variable manifestations including malformations of the eye, limbs, palate, and brain, and with developmental and growth delay. Haploinsufficiency of BMP4, OTX2 and possibly SIX6 are thought to contribute to the phenotype. We present a three generation family with four individuals carrying a 2.79 Mb microdeletion 14q22.1-22.2 encompassing BMP4 but not OTX2 nor SIX6. The highly variable manifestations in this family range from multiple congenital malformations with Robin sequence, microphthalmia, postaxial polydactyly, and developmental delay in the index patient to cleft uvula, growth delay, and mild developmental delay in her sister. The adults have a normal intelligence, postaxial polydactyly, and short stature or early cataract. Genotype-phenotype correlations suggest that the severity of eye manifestations in 14q22 deletions are influenced by the size of the deletion, but the marked intrafamilial variability observed in this family, as well as in familial BMP4 or OTX2 intragenic mutations points to additional modifiers outside this region.

A child with bilateral proptosis: a sign of acute myeloid leukemia?

A 5-year-old girl from Rwanda was referred to our pediatric oncology department with severe bilateral proptosis and a bloody, necrotic chemosis of the conjunctiva and infraorbital tissues. Furthermore, she presented with a left facial paresis, hemorrhagic gingiva, epistaxis, and bloody tears. There was a 3-month history of pancytopenia. Investigations that included a complete blood count, peripheral blood smear, and bone marrow aspirate, were conclusive for the diagnosis of acute myeloid leukemia.

Integrity of the inferior longitudinal fasciculus and impaired object recognition in children: a diffusion tensor imaging study.

AIM: In this study, we explored the integrity of the inferior longitudinal fasciculus (ILF) by means of diffusion tensor imaging tractography in children with visual perceptual impairment, and more specifically, object recognition deficits, compared with typically developing children. METHODS: Eleven individuals (nine males, two females; mean age 7 y 8 mo; range 3 y 5 mo-13 y) were assessed with the L94 visual perceptual battery after assessment of performance age. In all participants, an ophthalmological evaluation was carried out. Diffusion tensor imaging tractography of the ILF was performed. The mean fractional anisotropy was determined for every child and compared with data for 11 age- and sex-matched typically developing children. RESULTS: The mean fractional anisotropy value in the left ILF was consistently lower in the study participants than in the comparison group. The five children with L94 impairment showed a significantly lower ILF fractional anisotropy on the left as well as on the right side. Furthermore, the decrease in ILF fractional anisotropy was correlated with the number of impaired subtests. INTERPRETATION: The results suggest an association between ILF integrity loss and object recognition deficits. Moreover, the severity of clinical impairment is reflected in the degree of ILF integrity loss. Therefore, the ILF plays a potential role in object recognition.

Educational paper: Retinopathy of prematurity.

UNLABELLED: Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease affecting the premature infant with an incompletely vascularized retina. The spectrum of ophthalmological findings in ROP exists from minimal sequelae, which do not affect vision, to bilateral retinal detachment and total blindness. With the increased survival of very small infants, retinopathy of prematurity has become one of the leading causes of childhood blindness. Over the past two decades, major advances have been made in understanding the pathogenesis of ROP, to a large extent as a result of changes in clinical risk factors (oxygen and non-oxygen related) and characteristics observed in ROP cases. This article provides a literature review on the evolution in clinical characteristics, classification and treatment modalities and indications of ROP. Special attention is hereby paid to the neonatal factors influencing the development of ROP and to the necessity for everyone caring for premature babies to have a well-defined screening and treatment protocol for ROP. Such screening protocol needs to be based on a unit-specific ROP risk profile and, consequently, may vary between different European regions. CONCLUSION: Retinopathy of prematurity is an important cause of ocular morbidity and blindness in children. With better understanding of the pathogenesis, screening and treatment guidelines have changed over time and are unit specific.