Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis

Die Knochendichte regulierende genetische Faktoren

Objective: To evaluate factors regulating craniofacial morphologies and bone mass by investigat-ing high bone mass disorders (eg. autosomal recessive osteopetrosis, dysosteosclerosis, cranio-frontonasal syndrome, and osteosclerotic metaphyseal dysplasia) and their genetic defects on a molecular level. Methods: Next Generation Sequencing was performed on patient DNA, bioinformatic filtering was used to decipher new disease-causing mutations in a group of patients. Sanger sequencing was used to confirm the variants. Patient cells – osteoclasts, mesenchymal stem cells, and osteo-blasts – were grown in cell culture. Protein and RNA were extracted with Radioimmunoprecipita-tion assay (RIPA) buffer and Trizol lysis respectively. Immunoblot assays, cDNA sequencing, and reverse transcription-polymerase chain reaction (RT-PCR) were performed to analyze the ef-fect of the mutation on protein- and RNA levels. Osteoclasts were grown, stained for Tartrate-resistant acid phosphatase (TRAP), and visualized by fluorescence microscopy. A resorption assay was performed to illustrate the resorption behavior of mutated osteoclasts. Results: Two novel mutations in the Solute Carrier Family 29 Member 3 (SLC29A3) gene and one new splice-site mutation in the T-Cell Immune Regulator 1 (TCIRG1) gene were found in pa-tients with moderate autosomal-recessive osteopetrosis. Four new mutations in the Ephrin-B1 (EFNB1) gene were depicted in an Indian cohort of non-consanguineous individuals with cranio-frontonasal syndrome. In a patient suffering from osteopetrosis and severe osteonecrosis of the jaws, we described the fourth mutation in the Leucine-Rich Repeat Kinase 1 (LRRK1) gene known to date. LRRK1-mutated osteoclasts showed shallow resorption pits and ineffective bone resorp-tion. Conclusion: We could broaden the spectrum of high bone mass disorders as well as of the disease entity of craniofrontonasal syndrome by the detection of new disease-causing mutations and the description of the resulting molecular effects. In the future, this can facilitate diagnosis and reveal new potential molecular targets for the therapy of osteoporosis.Fragestellung: Analyse von genetischen Einflüssen auf Skelettentwicklung und -homöostase durch Untersuchung der seltenen genetischen Erkrankungen kraniofrontonasale Dysplasie, auto-somal-rezessive Osteopetrose, Dysosteosklerose und osteosklerotische metaphysäre Dysplasie. Darstellung der Auswirkungen der gefundenen genetischen Veränderungen auf zellulärer und mo-lekularer Ebene. Methoden: Patienten-DNA wurde mittels Next Generation Sequencing dechiffriert und bioinfor-matische Filtermethoden wurden verwendet, um neue krankheitsbedingende Mutationen bei Pati-enten mit den verschiedenen Erkrankungen mit erhöhter Knochendichte zu detektieren. Mittels Sanger Sequenzierung wurden die Varianten bestätigt. Patientenzellen – Osteoklasten, mesen-chymale Stammzellen, und Osteoblasten – wurden in der Zellkultur gezüchtet. Protein- und RNA-Extraktionen wurden mit RIPA-Puffer bzw. Trizol durchgeführt. Durch funktionelle Analysen mit Western Blots, cDNA-Sequenzierungen und RT-PCR wurden die Effekte der Mutationen auf Pro-tein-, cDNA- und RNA-Ebene analysiert. Osteoklasten wurden aus perpipheren mononukleären Zellen differenziert, mittels tartartresistenter saurer Phosphatase (TRAP)-Aktivität angefärbt und durch Fluoreszenzmikroskopie visualisiert. Ein Resorptionsassay wurde durchgeführt, um das Resorptionsverhalten von Osteoklasten darzustellen. Ergebnisse: Zwei neue Mutationen in dem für den Nukleosidtransporter 3 kodierenden Solute Carrier Family 29 Member 3 (SLC29A3) Gen und eine neue splice-site Mutation in dem T-Zell-Immunregulator 1 Gen (TCIRG1) wurden bei Patient*innen mit moderater autosomal-rezessiver Osteopetrose gefunden. Bei vier nicht-konsanguinen indischen Patientinnen mit kraniofrontonasa-ler Dysplasie konnten vier neue Mutationen Ephrin B1 Gen (EFNB1) bestätigt werden. In einem unter Osteopetrose und schweren rezidivierenden Kiefernekrosen leidenden Patienten haben wir die vierte weltweit beschriebene Mutation in dem Leucin-rich repeat kinase 1 (LRRK1) Gen nach-gewiesen. LRRK1-mutierte Osteoklasten zeigten abgeschwächtes Resorptionsverhalten, flachere Resorptionspits und eine ineffektive Knochenresorption. Schlussfolgerung: Im Rahmen des Spektrums von Erkrankungen mit erhöhter Knochenmasse und des Krankheitsbildes der kraniofrontonasalen Dysplasie konnten wir neue krankheitsbedin-gende Mutationen finden und die Effekte auf molekularer Ebene darstellen. Zukünftig kann dies die Diagnostik von seltenen Erkrankungen mit erhöhter Knochendichte erleichtern und langfristig neue molekulare Ansatzpunkte für die Osteoporosetherapie aufzeigen

Correlation of Clinical Fibrillar Layer Detection and Corneal Thickness in Advanced Fuchs Endothelial Corneal Dystrophy

Central subendothelial geographic deposits are formed as a fibrillar layer (FL) in advanced Fuchs endothelial corneal dystrophy (FECD). Previous studies demonstrated a significant decrease in corneal endothelial cell (CEC) density and an increase in focal corneal backscatter in the FL area. The present study investigated the association of the FL with edema formation and its localization. Patients (n = 96) presenting for Descemet membrane endothelial keratoplasty (DMEK) for advanced FECD were included. Slit-lamp biomicroscopy with FECD grading was followed by Scheimpflug imaging with en face backscatter analysis and pachymetric analysis. FL dimensions were measured, and correlation with pachymetric values was performed. An FL was detected in 74% of all eyes (n = 71). Pachymetric values in FL-positive versus FL-negative eyes were for corneal thickness at the apex (ACT) 614 ± 52 µm and 575 ± 46 µm (p = 0.001), for peripheral corneal thickness at 1 mm (PCT1mm) 616 ± 50 µm and 580 ± 44 µm (p = 0.002), for PCT2mm 625 ± 48 µm and 599 ± 41 µm (p = 0.017), for PCT3mm 651 ± 46 µm and 635 ± 40 µm (p = 0.128) and for PCT4mm 695 ± 52 µm and 686 ± 43 µm (p = 0.435), respectively. Correlation analysis indicated a weak correlation for the FL maximum vertical caliper diameter with ACT and PCT1mm values but no further relevant correlations. In FL-positive eyes, increased focal corneal backscatter and increased corneal thickness showed primarily central and inferotemporal localization. In conclusion, Scheimpflug imaging shows an association of the FL with increased corneal thickness in advanced FECD and shows localization of the FL and increased corneal thickness in the central and inferotemporal region. This may provide important information for progression assessment and therapeutic decision making in FECD patients in the future

Scheimpflug Backscatter Imaging of the Fibrillar Layer in Fuchs Endothelial Corneal Dystrophy

Purpose: A central collagen-rich subendothelial fibrillar layer (FL) correlates with areas of accentuated loss of corneal endothelial cells in advanced Fuchs endothelial corneal dystrophy (FECD). The present study sought to investigate whether the FL may be visualized by en face Scheimpflug backscatter imaging in vivo. Design: Retrospective analysis of a prospective observational case series. Methods: A total of 34 eyes (34 subjects) undergoing Descemet membrane endothelial keratoplasty (DMEK) surgery with preoperative high-quality Scheimpflug backscatter imaging data were included. The Descemet endothelium complex (DEC) was retrieved during DMEK surgery, and immunofluorescence staining was performed for collagens I, III, and IV. The FL morphology in en face Scheimpflug backscatter and immunofluorescence imaging was compared and agreement of FL parameters was analyzed using intraclass correlation coefficients (ICC) and Bland-Altman plots. Results: Scheimpflug backscatter imaging delineated the FL in 26 eyes and was FL negative in 8 eyes with deviation compared to immunofluorescence in 1 case and good agreement of morphology characteristics. Horizontical caliper diameter +/- SD was 4.84 +/- 0.85 mm, vertical caliper diameter was 3.92 +/- 0.78 mm, maximum caliper diameter was 5.12 +/- 0.82 mm, and surface area was 12.43 +/- 4.74 mm(2). Compared to immunofluorescence imaging, mean difference (95% limits of agreement) and intraclass correlation coefficients were for horizontal caliper diameter 0.13 mm (-0.81 to 1.1 mm) and 0.88, vertical caliper diameter 0.23 mm (-0.76 to 1.2 mm) and 0.81, maximum caliper diameter 0.06 mm (-1.1 to 1.2 mm) and 0.86, and surface area 1.4 mm(2) (-3.9 to 6.7 mm(2)) and 0.85. Conclusions: Scheimpflug backscatter imaging facilitates visualization of the FL in advanced FECD eyes, offering the potential to identify particularly diseased areas of the FECD endothelium in vivo. ((C) 2021 Elsevier Inc. All rights reserved.

Corneal Infantile Myofibromatosis Caused by Novel Activating Imatinib-Responsive Variants in PDGFRB

Purpose: To investigate the genetic cause, clinical characteristics, and potential therapeutic targets of infantile corneal myofibromatosis. Design: Case series with genetic and functional in vitro analyses. Participants: Four individuals from 2 unrelated families with clinical signs of corneal myofibromatosis were investigated. Methods: Exome-based panel sequencing for platelet-derived growth factor receptor beta gene (PDGFRB) and notch homolog protein 3 gene (NOTCH3) was performed in the respective index patients. One clinically affected member of each family was tested for the pathogenic variant detected in the respective index by Sanger sequencing. Immunohistochemical staining on excised corneal tissue was conducted. Functional analysis of the individual PDGFRB variants was performed in vitro by luciferase reporter assays on transfected porcine aortic endothelial cells using tyrosine kinase inhibitors. Protein expression analysis of mutated PDGFRB was analyzed by Western blot. Main outcome measures: Sequencing data, immunohistochemical stainings, functional analysis of PDGFRB variants, and protein expression analysis. Results: We identified 2 novel, heterozygous gain-of-function variants in PDGFRB in 4 individuals from 2 unrelated families with corneal myofibromatosis. Immunohistochemistry demonstrated positivity for alpha-smooth muscle actin and β-catenin, a low proliferation rate in Ki-67 (< 5%), marginal positivity for Desmin, and negative staining for Caldesmon and CD34. In all patients, recurrence of disease occurred after corneal surgery. When transfected in cultured cells, the PDGFRB variants conferred a constitutive activity to the receptor in the absence of its ligand and were sensitive to the tyrosine kinase inhibitor imatinib. The variants can both be classified as likely pathogenic regarding the American College of Medical Genetics and Genomics classification criteria. Conclusions: We describe 4 cases of corneal myofibromatosis caused by novel PDGFRB variants with autosomal dominant transmission. Imatinib sensitivity in vitro suggests perspectives for targeted therapy preventing recurrences in the future

Corneal Infantile Myofibromatosis Caused by Novel Activating Imatinib-Responsive Variants in PDGFRB

Purpose: To investigate the genetic cause, clinical characteristics, and potential therapeutic targets of infantile corneal myofibromatosis. Design: Case series with genetic and functional in vitro analyses. Participants: Four individuals from 2 unrelated families with clinical signs of corneal myofibromatosis were investigated. Methods: Exome-based panel sequencing for platelet-derived growth factor receptor beta gene (PDGFRB) and notch homolog protein 3 gene (NOTCH3) was performed in the respective index patients. One clinically affected member of each family was tested for the pathogenic variant detected in the respective index by Sanger sequencing. Immunohistochemical staining on excised corneal tissue was conducted. Functional analysis of the individual PDGFRB variants was performed in vitro by luciferase reporter assays on transfected porcine aortic endothelial cells using tyrosine kinase inhibitors. Protein expression analysis of mutated PDGFRB was analyzed by Western blot. Main Outcome Measures: Sequencing data, immunohistochemical stainings, functional analysis of PDGFRB variants, and protein expression analysis. Results: We identified 2 novel, heterozygous gain-of-function variants in PDGFRB in 4 individuals from 2 unrelated families with corneal myofibromatosis. Immunohistochemistry demonstrated positivity for alpha-smooth muscle actin and β-catenin, a low proliferation rate in Ki-67 (< 5%), marginal positivity for Desmin, and negative staining for Caldesmon and CD34. In all patients, recurrence of disease occurred after corneal surgery. When transfected in cultured cells, the PDGFRB variants conferred a constitutive activity to the receptor in the absence of its ligand and were sensitive to the tyrosine kinase inhibitor imatinib. The variants can both be classified as likely pathogenic regarding the American College of Medical Genetics and Genomics classification criteria. Conclusions: We describe 4 cases of corneal myofibromatosis caused by novel PDGFRB variants with autosomal dominant transmission. Imatinib sensitivity in vitro suggests perspectives for targeted therapy preventing recurrences in the future. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article