Functional Characterization of ABCA4 Missense Variants Aids Variant Interpretation and Phenotype Prediction in Patients With ABCA4-Retinal Dystrophies.

Purpose: Biallelic pathogenic variants in the gene encoding the ATP-binding cassette transporter ABCA4 are the leading cause of irreversible vision loss in inherited retinal dystrophies (IRDs). Interpretation of ABCA4 variants is challenging, due to cis-modifying and hypomorphic variants. We have previously detected 10 missense variants of unknown significance (VUS) in patients with suspected ABCA4-retinal dystrophies (ABCA4-RDs) in Norway. In this study, we functionally characterized the VUS to aid interpretation of the variants and to determine if they are associated with the disease. Methods: The ABCA4 VUS were expressed in HEK293T cells and the ABCA4 expression level and ATPase activity were determined and correlated with the patients' phenotype. The functional data further used for reclassification of the VUS following the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Of the 10 VUSs, 2 variants, Cys205Phe and Asn415Thr, were categorized as functionally severe. The age at presentation in the 2 patients carrying these variants was divergent and seemed to be driven by the patients' second pathogenic variants Gly1961Glu and c.5461-10T>C, respectively. Three variants, Val643Gly, Pro799Leu, and Val1433Ile were categorized as functionally moderate, and were found in patients with intermediate/late age at presentation. The remaining five variants were categorized as functionally normal/mild. Based on our data, c.614G>T p.(Cys205Phe), c.1244A>C p.(Asn415Thr), and c.2396C>T p.(Pro799Leu) were reclassified to (likely) pathogenic, while 4 of the functionally normal/mild variants could be reclassified to likely benign. Conclusions: Functional analyses of ABCA4 variants are a helpful tool in variant classification and enable us to better predict the disease severity in patients with ABCA4-RDs.

Diagnosis and management in Rubinstein-Taybi syndrome: first international consensus statement.

Rubinstein-Taybi syndrome (RTS) is an archetypical genetic syndrome that is characterised by intellectual disability, well-defined facial features, distal limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in either of two genes (CREBBP, EP300) which encode for the proteins CBP and p300, which both have a function in transcription regulation and histone acetylation. As a group of international experts and national support groups dedicated to the syndrome, we realised that marked heterogeneity currently exists in clinical and molecular diagnostic approaches and care practices in various parts of the world. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria for types of RTS (RTS1: CREBBP; RTS2: EP300), molecular investigations, long-term management of various particular physical and behavioural issues and care planning. The recommendations as presented here will need to be evaluated for improvements to allow for continued optimisation of diagnostics and care.

Long-Term Follow-Up of Pediatric Excimer Laser-Assisted Penetrating Keratoplasty for Congenital Stromal Corneal Dystrophy.

PURPOSE: The purpose of this study was to highlight characteristic clinical and microscopic findings and report the long-term follow-up of pediatric excimer laser-assisted penetrating keratoplasty (excimer-PKP) for congenital stromal corneal dystrophy (CSCD). METHODS: A 2-year-old Greek child presented with CSCD at our department. Clinical examination showed bilateral flake-like whitish corneal opacities affecting the entire corneal stroma up to the limbus. Genetic testing identified a mutation of the decorin gene (c.962delA). The variant was not present in the parents and represented a de novo mutation. The uncorrected visual acuity was 20/100 in both eyes. Excimer-PKP (8.0/8.1 mm) was performed on the right eye at the age of 2.5 years and on the left eye at the age of 3 years. Postoperatively, alternating occlusion treatment was performed. RESULTS: The light microscopic examination demonstrated a disorganized extracellular matrix of the corneal stroma characterized by a prominent irregular arrangement of stromal collagen lamellae with large interlamellar clefts containing ground substance, highlighted by periodic acid-Schiff- and Alcian blue-positive reaction detecting acid mucopolysaccharides. Electron microscopy showed disorganization and caliber variation of collagen lamellae and thin filaments within an electron-lucent ground substance. The postoperative course was unremarkable. Both grafts remained completely clear 14 years postoperatively. Corneal tomography showed moderate regular astigmatism with normal corneal thickness. The corrected distance visual acuity was 20/25 in both eyes. CONCLUSIONS: Excimer-PKP for CSCD might be associated with excellent long-term results and a good prognosis, particularly when the primary surgery is performed at a very young age. However, this requires close postoperative follow-up examinations by an experienced pediatric ophthalmologist to avoid severe amblyopia.

IC3D Classification of Corneal Dystrophies-Edition 3.

PURPOSE: The International Committee for the Classification of Corneal Dystrophies (IC3D) was created in 2005 to develop a new classification system integrating current information on phenotype, histopathology, and genetic analysis. This update is the third edition of the IC3D nomenclature. METHODS: Peer-reviewed publications from 2014 to 2023 were evaluated. The new information was used to update the anatomic classification and each of the 22 standardized templates including the level of evidence for being a corneal dystrophy [from category 1 (most evidence) to category 4 (least evidence)]. RESULTS: Epithelial recurrent erosion dystrophies now include epithelial recurrent erosion dystrophy, category 1 ( COL17A1 mutations, chromosome 10). Signs and symptoms are similar to Franceschetti corneal dystrophy, dystrophia Smolandiensis, and dystrophia Helsinglandica, category 4. Lisch epithelial corneal dystrophy, previously reported as X-linked, has been discovered to be autosomal dominant ( MCOLN1 mutations, chromosome 19). Classic lattice corneal dystrophy (LCD) results from TGFBI R124C mutation. The LCD variant group has over 80 dystrophies with non-R124C TGFBI mutations, amyloid deposition, and often similar phenotypes to classic LCD. We propose a new nomenclature for specific LCD pathogenic variants by appending the mutation using 1-letter amino acid abbreviations to LCD. Pre-Descemet corneal dystrophies include category 1, autosomal dominant, punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD) ( PRDX3 mutations, chromosome 10). Typically asymptomatic, it can be distinguished phenotypically from pre-Descemet corneal dystrophy, category 4. We include a corneal dystrophy management table. CONCLUSIONS: The IC3D third edition provides a current summary of corneal dystrophy information. The article is available online at https://corneasociety.org/publications/ic3d .

Elevated cholesterol in ATAD3 mutants is a compensatory mechanism that leads to membrane cholesterol aggregation.

Aberrant cholesterol metabolism causes neurological disease and neurodegeneration, and mitochondria have been linked to perturbed cholesterol homeostasis via the study of pathological mutations in the ATAD3 gene cluster. However, whether the cholesterol changes were compensatory or contributory to the disorder was unclear, and the effects on cell membranes and the wider cell were also unknown. Using patient-derived cells, we show that cholesterol perturbation is a conserved feature of pathological ATAD3 variants that is accompanied by an expanded lysosome population containing membrane whorls characteristic of lysosomal storage diseases. Lysosomes are also more numerous in Drosophila neural progenitor cells expressing mutant Atad3, which exhibit abundant membrane-bound cholesterol aggregates, many of which co-localize with lysosomes. By subjecting the Drosophila Atad3 mutant to nutrient restriction and cholesterol supplementation, we show that the mutant displays heightened cholesterol dependence. Collectively, these findings suggest that elevated cholesterol enhances tolerance to pathological ATAD3 variants; however, this comes at the cost of inducing cholesterol aggregation in membranes, which lysosomal clearance only partly mitigates.

Corneal Vascularization Associated With a Novel PDGFRB Variant.

Purpose: The purpose of this study was to identify the genetic cause of aggressive corneal vascularization in otherwise healthy children in one family. Further, to study molecular consequences associated with the identified variant and implications for possible treatment. Methods: Exome sequencing was performed in affected individuals. HeLa cells were transduced with the identified c.1643C>A, p.(Ser548Tyr) variant in the platelet-derived growth factor receptor beta gene (PDGFRB) or wild-type PDGFRB. ELISA and immunoblot analysis were used to detect the phosphorylation levels of PDGFRß and downstream signaling proteins in untreated and ligand-stimulated cells. Sensitivity to various receptor tyrosine kinase inhibitors (TKIs) was determined. Results: A novel c.1643C>A, p.(Ser548Tyr) PDGFRB variant was found in affected family members. HeLa cells transduced with this variant did not have increased baseline levels of phosphorylated PDGFRß. However, upon stimulation with ligand, excessive activation of PDGFRß was observed compared to cells transduced with the wild-type variant. PDGFRß with the p.(Ser548Tyr) amino acid substitution was successfully inhibited with tyrosine kinase inhibitors (axitinib, dasatinib, imatinib, and sunitinib) in vitro. Conclusions: A novel c.1643C>A, p.(Ser548Tyr) PDGFRB variant was found in family members with isolated corneal vascularization. Cells transduced with the newly identified variant showed increased phosphorylation of PDGFRß upon ligand stimulation. This suggests that PDGF-PDGFRß signaling in these patients leads to overactivation of PDGFRß, which could lead to abnormal wound healing of the cornea. The examined TKIs prevented such overactivation, introducing the possibility for targeted treatment in these patients.

Functional characterization of all-trans retinoic acid-induced differentiation factor (ATRAID).

All-trans retinoic acid-induced differentiation (ATRAID) factor was first identified in HL60 cells. Several mRNA isoforms exist, but the respective proteins have not been fully characterized. In transfected cells expressing Myc-Flag-tagged ATRAID Isoform (Iso) A, B, and C, Iso C was found to be expressed at high levels, Iso A was found to be expressed at low levels due to rapid degradation, and the predicted protein expressed from Iso B was not detected. Iso C was present mainly in an N-glycosylated form. In subcellular fractionation experiments, Iso C localized to the membranous and nuclear fractions, while immunofluorescence analysis revealed that Iso C is located close to the plasma membrane, mainly in cytoplasmic vesicles and in the Golgi area. We confirm that Iso C colocalizes to some extent with endosomal/lysosomal markers LAMP1 and LAMP2. Furthermore, we show that ATRAID co-localizes with RAB11, a GTPase associated with recycling endosomes and implicated in regulating vesicular trafficking.

A Pellino-2 variant is associated with constitutive NLRP3 inflammasome activation in a family with ocular pterygium-digital keloid dysplasia.

Ocular pterygium-digital keloid dysplasia (OPDKD) is a rare hereditary disease characterized by corneal ingrowth of vascularized conjunctival tissue early in life. Later, patients develop keloids on fingers and toes but are otherwise healthy. In a recently described family with OPDKD, we report the presence of a de novo c.770C > T, p.(Thr257Ile) variant in PELI2 in the affected individual. PELI2 encodes for the E3 ubiquitin ligase Pellino-2. In transgenic U87MG cells overexpressing Pellino-2 with the p.(Thr257Ile) amino acid substitution, constitutive activation of the NLRP3 inflammasome was observed. However, the Thr257Ile variant did not affect Pellino-2 intracellular localization, its binding to known interaction partners, nor its stability. Our findings indicate that constitutive autoactivation of the NLRP3 inflammasome contributes to the development of PELI2-associated OPDKD.

Clinical and molecular response to dasatinib in an adult patient with Penttinen syndrome.

Penttinen type of premature aging syndrome is an autosomal-dominant disorder that can be caused by the c.1994T>A pVal665Ala pathogenic variant in platelet-derived growth factor receptor-B (PDGFRB). Imatinib, a receptor tyrosine kinase (RTK) inhibitor, has been used in Penttinen syndrome (PS) patients with good results. A 21-year-old male presented shortly after birth with a prematurely aged appearance with distinctive facial features and cutaneous atrophy with hypertrophic scar-like lesions. Generalized brachydactyly with acro-osteolysis was observed. Flexion contractures limited his daily activities. Cognitive impairment was not present. Genetic testing found a heterozygous variant c.1994T>A pVal665Ala in exon 14 of PDGFRB. A diagnosis of PS was made and imatinib treatment was started with partial response. After lack of further improvement, in vitro molecular studies with imatinib and dasatinib showed that the Val665Ala variant had greater sensitivity to dasatinib than imatinib. This was seen examining levels of P-PDGFRB directly and on downstream ligands P-AKT and P-STAT. Improved clinical response was observed after treatment with dasatinib. We report a new case of PS with clinical and molecular response to dasatinib after incomplete response to imatinib. Our work provides further molecular and clinical evidence of RTK inhibitors' efficacy in this rare disorder.

Pellino-2 in nonimmune cells: novel interaction partners and intracellular localization.

Pellino-2 is an E3 ubiquitin ligase that mediates intracellular signaling in innate immune pathways. Most studies of endogenous Pellino-2 have been performed in macrophages, but none in nonimmune cells. Using yeast two-hybrid screening and co-immunoprecipitation, we identified six novel interaction partners of Pellino-2, with various localizations: insulin receptor substrate 1, NIMA-related kinase 9, tumor necrosis factor receptor-associated factor 7, cyclin-F, roundabout homolog 1, and disheveled homolog 2. Pellino-2 showed cytoplasmic localization in a wide range of nonimmune cells under physiological potassium concentrations. Treatment with the potassium ionophore nigericin resulted in nuclear localization of Pellino-2, which was reversed by the potassium channel blocker tetraethylammonium. Live-cell imaging revealed intracellular migration of GFP-tagged Pellino-2. In summary, Pellino-2 interacts with proteins at different cellular locations, taking part in dynamic processes that change its intracellular localization influenced by potassium efflux.

K+ regulates relocation of Pellino-2 to the site of NLRP3 inflammasome activation in macrophages.

Pellino proteins are E3 ubiquitin ligases involved in the innate immune system. Recently, Pellino-2 was reported to modulate the activation of the mouse Nlrp3 inflammasome. We examined the intracellular localization of human Pellino-2 in THP1-derived macrophages during activation with LPS and ATP. We observed that Pellino-2 changed intracellular localization and colocalized with the inflammasome proteins NLRP3 and ASC late in the assembly of the inflammasome. Colocalization with NLRP3 and ASC was also seen in cells maintained in potassium-free medium. The colocalization and inflammasome activation were abrogated by several potassium channel inhibitors, supporting a role for potassium efflux in modulating intracellular localization of Pellino-2. The data suggest that Pellino-2 is essential for mediating the effect of potassium efflux on inflammasome activation.

Temperature-dependent autoactivation associated with clinical variability of PDGFRB Asn666 substitutions.

Ocular pterygium-digital keloid dysplasia (OPDKD) presents in childhood with ingrowth of vascularized connective tissue on the cornea leading to severely reduced vision. Later the patients develop keloids on digits but are otherwise healthy. The overgrowth in OPDKD affects body parts that typically have lower temperature than 37°C. We present evidence that OPDKD is associated with a temperature sensitive, activating substitution, p.(Asn666Tyr), in PDGFRB. Phosphorylation levels of PDGFRB and downstream targets were higher in OPDKD fibroblasts at 37°C but were further greatly increased at the average corneal temperature of 32°C. This suggests that the substitution cause significant constitutive autoactivation mainly at lower temperature. In contrast, a different substitution in the same codon, p.(Asn666Ser), is associated with Penttinen type of premature aging syndrome. This devastating condition is characterized by widespread tissue degeneration, including pronounced chronic ulcers and osteolytic resorption in distal limbs. In Penttinen syndrome fibroblasts, equal and high levels of phosphorylated PDGFRB was present at both 32°C and 37°C. This indicates that this substitution causes severe constitutive autoactivation of PDGFRB regardless of temperature. In line with this, most downstream targets were not affected by lower temperature. However, STAT1, important for tissue wasting, did show further increased phosphorylation at 32°C. Temperature-dependent autoactivation offers an explanation to the strikingly different clinical outcomes of substitutions in the Asn666 codon of PDGFRB.

Clinical features and molecular genetics of patients with ABCA4-retinal dystrophies.

PURPOSE: Pathogenic variations in the ABCA4 gene are a leading cause of vision loss in patients with inherited retinal diseases. ABCA4-retinal dystrophies are clinically heterogeneous, presenting with mild to severe degeneration of the retina. The purpose of this study was to clinically and genetically characterize patients with ABCA4-retinal dystrophies in Norway and describe phenotype-genotype associations. METHODS: ABCA4 variants were detected in 111 patients with inherited retinal disease undergoing diagnostic genetic testing over a period of 12 years. In patients where only a single ABCA4 variant was found, whole-gene ABCA4 sequencing was performed and intronic variants were investigated by mRNA analyses in fibroblasts. Medical journals were used to obtain a clinical description and ultrawidefield autofluorescence images were used to analyse retinal degeneration patterns. RESULTS: The genetic diagnostic yield was 89%. The intronic splice variant c.5461-10T>C was the most prevalent disease-causing variant (27%). Whole-gene ABCA4 sequencing detected two novel intronic variants (c.6729+81G>T and c.6817-679C>A) that we showed affected mRNA splicing. Peripheral retinal degeneration was identified in 33% of patients and was associated with genotypes that included severe loss of function variants. By contrast, peripheral degeneration was not found in patients with a disease duration over 20 years and genotypes including p.(Asn1868lle), c.4253+43G>A or p.(Gly1961Glu) in trans with a loss of function variant. CONCLUSION: This study demonstrates the clinical and genetic heterogeneity of ABCA4-retinal dystrophies in Norway. Further, the study presents novel variants and increases our knowledge on phenotype-genotype associations and the presence of peripheral retinal degeneration in ABCA4-retinal dystrophy patients.

A tyrosine kinase-activating variant Asn666Ser in PDGFRB causes a progeria-like condition in the severe end of Penttinen syndrome.

Missense variants located to the "molecular brake" in the tyrosine kinase hinge region of platelet-derived growth factor receptor-ß, encoded by PFGFRB, can cause Penttinen-type (Val665Ala) and Penttinen-like (Asn666His) premature ageing syndromes, as well as infantile myofibromatosis (Asn666Lys and Pro660Thr). We have found the same de novo PDGFRB c.1997A>G p.(Asn666Ser) variants in two patients with lipodystrophy, acro-osteolysis and severely reduced vision due to corneal neovascularisation, reminiscent of a severe form of Penttinen syndrome with more pronounced connective tissue destruction. In line with this phenotype, patient skin fibroblasts were prone to apoptosis. Both in patient fibroblasts and stably transduced HeLa and HEK293 cells, autophosphorylation of PDGFRß was observed, as well as increased phosphorylation of downstream signalling proteins such as STAT1, PLCγ1, PTPN11/SHP2-Tyr580 and AKT. Phosphorylation of MAPK3 (ERK1) and PTPN11/SHP2-Tyr542 appeared unaffected. This suggests that this missense change not only weakens tyrosine kinase autoinhibition, but also influences substrate binding, as both PTPN11 tyrosines (Tyr542 and Tyr580) usually are phosphorylated upon PDGFR activation. Imatinib was a strong inhibitor of phosphorylation of all these targets, suggesting an option for precision medicine based treatment.

Recurrent, Activating Variants in the Receptor Tyrosine Kinase DDR2 Cause Warburg-Cinotti Syndrome.

We have investigated a distinct disorder with progressive corneal neovascularization, keloid formation, chronic skin ulcers, wasting of subcutaneous tissue, flexion contractures of the fingers, and acro-osteolysis. In six affected individuals from four families, we found one of two recurrent variants in discoidin domain receptor tyrosine kinase 2 (DDR2): c.1829T>C (p.Leu610Pro) or c.2219A>G (p.Tyr740Cys). DDR2 encodes a collagen-responsive receptor tyrosine kinase that regulates connective-tissue formation. In three of the families, affected individuals comprise singleton adult individuals, and parental samples were not available for verification of the de novo occurrence of the DDR2 variants. In the fourth family, a mother and two of her children were affected, and the c.2219A>G missense variant was proven to be de novo in the mother. Phosphorylation of DDR2 was increased in fibroblasts from affected individuals, suggesting reduced receptor autoinhibition and ligand-independent kinase activation. Evidence for activation of other growth-regulatory signaling pathways was not found. Finally, we found that the protein kinase inhibitor dasatinib prevented DDR2 autophosphorylation in fibroblasts, suggesting an approach to treatment. We propose this progressive, fibrotic condition should be designated as Warburg-Cinotti syndrome.

Mutations in MAPKBP1 Cause Juvenile or Late-Onset Cilia-Independent Nephronophthisis.

Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.

The intronic ABCA4 c.5461-10T>C variant, frequently seen in patients with Stargardt disease, causes splice defects and reduced ABCA4 protein level.

PURPOSE: Despite being the third most common ABCA4 variant observed in patients with Stargardt disease, the functional effect of the intronic ABCA4 variant c.5461-10T>C is unknown. The purpose of this study was to investigate the molecular effect of this variant. METHODS: Fibroblast samples from patients carrying the ABCA4 variant c.5461-10T>C were analysed by isolating total RNA, followed by real-time polymerase chain reaction (RT-PCR) using specific primers spanning the variant. For detection of ABCA4 protein, fibroblast samples were lysed and analysed by SDS-PAGE followed by immunoblotting using a monoclonal ABCA4 antibody. RESULTS: The ABCA4 variant c.5461-10T>C causes a splicing defect resulting in the reduction of full-length mRNA in fibroblasts from patients and the presence of alternatively spliced mRNAs where exon 39-40 is skipped. A reduced level of full-length ABCA4 protein is observed compared to controls not carrying the variant. CONCLUSIONS: This study describes the functional effect and the molecular mechanism of the pathogenic ABCA4 variant c.5461-10T>C. The variant is functionally important as it leads to splicing defects and a reduced level of ABCA4 protein.