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Ammar A, Bubshait DK, Al Ojan A, Alshari SA, Cyrus C, Alanazi R, Al Ghamdi MA, Keating BJ, Al-Anazi A, Al Qahtani NH, Al-Ali AK. Family-base rare variant association analysis in Saudi Arabian hydrocephalus subjects using whole exome sequencing. J Neurosurg Sci 2024; 68:698-703. [PMID: 37158713 DOI: 10.23736/s0390-5616.23.06010-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
BACKGROUND Hydrocephalus is a highly heterogeneous multifactorial disease that arises from genetic and environmental factors. Familial genetic studies of hydrocephalus have elucidated four robustly associated hydrocephalus associated loci. This study aims to identify potential genetic causation in cases of hydrocephalus, with or without spina bifida and Dandy Walker Syndrome (DWS), using family-based rare variant association analysis of whole exome sequencing. METHODS We performed whole exome sequencing in 143 individuals across 48 families where at least one offspring was affected with hydrocephalus (N.=27), with hydrocephalus with spina bifida (N.=21) and with DWS (N.=3), using Illumina HiSeq 2500 instrument. RESULTS No pathogenic or putative pathogenic single-nucleotide variants were evident in the four known hydrocephalus loci in our subjects. However, after examining 73 known hydrocephalus genes previously identified from literature, we identified three potentially impactful variants from the cohort. Using a gene panel comprising variants in known neural tube defects loci, we identified a total of 1024 potentially deleterious variants, of which 797 were missense variants and 191 were frameshift variants, 36 were stop gain/loss variants. A small portion of our family pedigree analyses yielded putative genetic signals which may be responsible for hydrocephaly elated phenotypes, however the low diagnostic yield may be due to lack of capture of genetic variants in the exonic regions i.e. structural variants may only be evident from whole genome sequencing. CONCLUSIONS We identified three potentially impactful variants from our cohort in 73 known hydrocephalus genes previously identified in literature.
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Affiliation(s)
- Ahmed Ammar
- Department of Neurosurgery, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Dalal K Bubshait
- Department of Pediatric, Generic Unit, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Abdulrazaq Al Ojan
- Department of Neurosurgery, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Shuroq A Alshari
- Department of Neurosurgery, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Cyril Cyrus
- Department of Clinical Biochemistry, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Rawan Alanazi
- Department of Neurosurgery, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Mohammed A Al Ghamdi
- Department of Pediatric, Generic Unit, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Brendan J Keating
- Department of Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
| | - Abdulrahman Al-Anazi
- Department of Neurosurgery, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Noorah H Al Qahtani
- Department of Obstetrics and Gynecology, King Fahd Hospital of the University, Alkhobar College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia
| | - Amein K Al-Ali
- Department of Clinical Biochemistry, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia -
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Sterling NA, Cho SH, Kim S. Entosis implicates a new role for P53 in microcephaly pathogenesis, beyond apoptosis. Bioessays 2024; 46:e2300245. [PMID: 38778437 DOI: 10.1002/bies.202300245] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Revised: 05/02/2024] [Accepted: 05/08/2024] [Indexed: 05/25/2024]
Abstract
Entosis, a form of cell cannibalism, is a newly discovered pathogenic mechanism leading to the development of small brains, termed microcephaly, in which P53 activation was found to play a major role. Microcephaly with entosis, found in Pals1 mutant mice, displays P53 activation that promotes entosis and apoptotic cell death. This previously unappreciated pathogenic mechanism represents a novel cellular dynamic in dividing cortical progenitors which is responsible for cell loss. To date, various recent models of microcephaly have bolstered the importance of P53 activation in cell death leading to microcephaly. P53 activation caused by mitotic delay or DNA damage manifests apoptotic cell death which can be suppressed by P53 removal in these animal models. Such genetic studies attest P53 activation as quality control meant to eliminate genomically unfit cells with minimal involvement in the actual function of microcephaly associated genes. In this review, we summarize the known role of P53 activation in a variety of microcephaly models and introduce a novel mechanism wherein entotic cell cannibalism in neural progenitors is triggered by P53 activation.
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Affiliation(s)
- Noelle A Sterling
- Shriners Hospitals Pediatric Research Center, Department of Neural Sciences, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania, USA
- Biomedical Sciences Graduate Program, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania, USA
| | - Seo-Hee Cho
- Center for Translational Medicine, Department of Medicine, Sydney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Seonhee Kim
- Shriners Hospitals Pediatric Research Center, Department of Neural Sciences, Lewis Katz School of Medicine, Temple University, Philadelphia, Pennsylvania, USA
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3
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Stehle IF, Imventarza JA, Woerz F, Hoffmann F, Boldt K, Beyer T, Quinn PM, Ueffing M. Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina. Life Sci Alliance 2024; 7:e202302440. [PMID: 38570189 PMCID: PMC10992996 DOI: 10.26508/lsa.202302440] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2023] [Revised: 03/26/2024] [Accepted: 03/26/2024] [Indexed: 04/05/2024] Open
Abstract
Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1-CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1-CRB2 interactions. We observed no or a mild loss of CRB1-CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.
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Affiliation(s)
- Isabel F Stehle
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Joel A Imventarza
- Department of Ophthalmology, Vagelos College of Physicians & Surgeons, Columbia University; New York, NY, USA
| | - Franziska Woerz
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Felix Hoffmann
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Karsten Boldt
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Tina Beyer
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Peter Mj Quinn
- Department of Ophthalmology, Vagelos College of Physicians & Surgeons, Columbia University; New York, NY, USA
| | - Marius Ueffing
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
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4
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Möller-Kerutt A, Schönhoff B, Rellmann Y, George B, Braun DA, Pavenstädt H, Weide T. Loss of surface transport is a main cellular pathomechanism of CRB2 variants causing podocytopathies. Life Sci Alliance 2023; 6:6/3/e202201649. [PMID: 36549870 PMCID: PMC9780758 DOI: 10.26508/lsa.202201649] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 12/12/2022] [Accepted: 12/13/2022] [Indexed: 12/24/2022] Open
Abstract
Crumbs2 (CRB2) is a central component of the renal filtration barrier and part of the slit diaphragm, a unique cell contact formed by glomerular podocytes. Some CRB2 variants cause recessive inherited forms of steroid-resistant nephrotic syndrome. However, the disease-causing potential of numerous CRB2 variants remains unknown. Here, we report the establishment of a live-cell imaging-based assay, allowing a quantitative evaluation of the pathogenic potential of so far non-categorized CRB2 variants. Based on in silico data analysis and protein prediction software, putative disease-associated CRB2 missense variants were selected, expressed as CRB2-GFP fusion proteins, and analyzed in reporter cell lines with BFP-labeled plasma membrane. We found that in comparison with PM-localized WT, disease-associated CRB2 variants remained predominantly at the ER. Accumulation at the ER was also present for several non-characterized CRB2 variants and variants in which putative disulfide bridge-forming cysteines were replaced. Strikingly, WT CRB2 retained inside the ER in cells lacking protein disulfide isomerase A3, indicating that posttranslational modification, especially the formation of disulfide bridges, is a crucial step for the CRB2 PM transport.
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Affiliation(s)
- Annika Möller-Kerutt
- University Hospital of Muenster (UKM), Internal Medicine (MedD), Muenster, Germany
| | - Birgit Schönhoff
- University Hospital of Muenster (UKM), Internal Medicine (MedD), Muenster, Germany
| | - Yvonne Rellmann
- Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, Muenster, Germany
| | - Britta George
- University Hospital of Muenster (UKM), Internal Medicine (MedD), Muenster, Germany
| | - Daniela Anne Braun
- University Hospital of Muenster (UKM), Internal Medicine (MedD), Muenster, Germany
| | - Hermann Pavenstädt
- University Hospital of Muenster (UKM), Internal Medicine (MedD), Muenster, Germany
| | - Thomas Weide
- University Hospital of Muenster (UKM), Internal Medicine (MedD), Muenster, Germany
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5
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Tessier A, Roux N, Boutaud L, Lunel E, Hakkakian L, Parisot M, Garfa-Traoré M, Ichkou A, Elkhartoufi N, Bole C, Nitschke P, Amiel J, Martinovic J, Encha-Razavi F, Attié-Bitach T, Thomas S. Bi-allelic variations in CRB2, encoding the crumbs cell polarity complex component 2, lead to non-communicating hydrocephalus due to atresia of the aqueduct of sylvius and central canal of the medulla. Acta Neuropathol Commun 2023; 11:29. [PMID: 36803301 PMCID: PMC9940441 DOI: 10.1186/s40478-023-01519-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2022] [Accepted: 01/23/2023] [Indexed: 02/22/2023] Open
Abstract
Congenital hydrocephalus is a common condition caused by the accumulation of cerebrospinal fluid in the ventricular system. Four major genes are currently known to be causally involved in hydrocephalus, either isolated or as a common clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. Here, we report 3 cases from 2 families with congenital hydrocephalus due to bi-allelic variations in CRB2, a gene previously reported to cause nephrotic syndrome, variably associated with hydrocephalus. While 2 cases presented with renal cysts, one case presented with isolated hydrocephalus. Neurohistopathological analysis allowed us to demonstrate that, contrary to what was previously proposed, the pathological mechanisms underlying hydrocephalus secondary to CRB2 variations are not due to stenosis but to atresia of both Sylvius Aqueduct and central medullar canal. While CRB2 has been largely shown crucial for apico-basal polarity, immunolabelling experiments in our fetal cases showed normal localization and level of PAR complex components (PKCι and PKCζ) as well as of tight (ZO-1) and adherens (β-catenin and N-Cadherin) junction molecules indicating a priori normal apicobasal polarity and cell-cell adhesion of the ventricular epithelium suggesting another pathological mechanism. Interestingly, atresia but not stenosis of Sylvius aqueduct was also described in cases with variations in MPDZ and CCDC88C encoding proteins previously linked functionally to the Crumbs (CRB) polarity complex, and all 3 being more recently involved in apical constriction, a process crucial for the formation of the central medullar canal. Overall, our findings argue for a common mechanism of CRB2, MPDZ and CCDC88C variations that might lead to abnormal apical constriction of the ventricular cells of the neural tube that will form the ependymal cells lining the definitive central canal of the medulla. Our study thus highlights that hydrocephalus related to CRB2, MPDZ and CCDC88C constitutes a separate pathogenic group of congenital non-communicating hydrocephalus with atresia of both Sylvius aqueduct and central canal of the medulla.
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Affiliation(s)
- Aude Tessier
- Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France. .,INSERM UMR 1163, Institut Imagine, Université Paris Cité, Paris, France.
| | - Nathalie Roux
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France
| | - Lucile Boutaud
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France ,grid.508487.60000 0004 7885 7602INSERM UMR 1163, Institut Imagine, Université Paris Cité, Paris, France
| | - Elodie Lunel
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France
| | - Leila Hakkakian
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France
| | - Mélanie Parisot
- grid.7429.80000000121866389Genomics Core Facility, Institut Imagine-Structure Fédérative de Recherche Necker, INSERM U1163 et INSERM US24/CNRS UAR3633, Paris Descartes Sorbonne Paris Cite University, Paris, France
| | - Meriem Garfa-Traoré
- grid.462420.6Cell Imaging Platform, INSERM-US24-CNRS UMS 3633 Structure Fédérative de Recherche Necker, Paris University, 75015 Paris, France
| | - Amale Ichkou
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France
| | - Nadia Elkhartoufi
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France
| | - Christine Bole
- grid.7429.80000000121866389Genomics Core Facility, Institut Imagine-Structure Fédérative de Recherche Necker, INSERM U1163 et INSERM US24/CNRS UAR3633, Paris Descartes Sorbonne Paris Cite University, Paris, France
| | - Patrick Nitschke
- grid.462336.6Bioinformatics Platform, Institut Imagine, Paris, France
| | - Jeanne Amiel
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France ,grid.508487.60000 0004 7885 7602INSERM UMR 1163, Institut Imagine, Université Paris Cité, Paris, France
| | - Jelena Martinovic
- grid.413738.a0000 0000 9454 4367Unité de Foetopathologie, AP-HP, Hôpital Antoine Béclère, Groupe Hospitalo-Universitaire Paris Saclay, Clamart, France
| | - Férechté Encha-Razavi
- grid.412134.10000 0004 0593 9113Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France
| | - Tania Attié-Bitach
- Service de Médecine Génomique des Maladies Rares, Hôpital Universitaire Necker-Enfants Malades, Paris, France. .,INSERM UMR 1163, Institut Imagine, Université Paris Cité, Paris, France.
| | - Sophie Thomas
- INSERM UMR 1163, Institut Imagine, Université Paris Cité, Paris, France.
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Exome Sequencing Revealed a Novel Splice Site Variant in the CRB2 Gene Underlying Nephrotic Syndrome. MEDICINA (KAUNAS, LITHUANIA) 2022; 58:medicina58121784. [PMID: 36556986 PMCID: PMC9781877 DOI: 10.3390/medicina58121784] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Revised: 11/29/2022] [Accepted: 11/30/2022] [Indexed: 12/11/2022]
Abstract
Background and Objectives: Nephrotic syndrome (NS) is a kidney disease where the patient has a classic triad of signs and symptoms including hypercholesterolemia, hypoalbuminemia, proteinuria (>3.5 g/24 h), and peripheral edema. In case of NS, the damaged nephrons (structural and functional unit of the kidney) filter unwanted blood contents to make urine. Thus, the urine contains unwanted proteins (proteinuria) and blood cells (hematuria), while the bloodstream lacks enough protein albumin (hypoalbuminemia). Nephrotic syndrome is divided into two types, primary NS, and secondary NS. Primary NS, also known as primary glomerulonephrosis, is the result of a glomerular disease that is limited to the kidney, while secondary NS is a condition that affects the kidney and other parts of the body. The main causes of primary NS are minimal change disease, membranous glomerulonephritis, and focal segmental glomerulosclerosis. In the present study we recruited a family segregating primary NS with the aim to identify the underlying genetic etiology. Such type of study is important in children because it allows counseling of other family members who may be at risk of developing NS, predicts risk of recurrent disease phenotypes after kidney transplant, and predicts response to immunosuppressive therapy. Materials and Methods: All affected individuals were clinically evaluated. Clinical examination, results of laboratory tests, and biopsy investigations led us to the diagnosis. The next-generation sequencing technique (whole-exome sequencing) followed by Sanger sequencing identified a novel homozygous splice site variant (NM_173689.7: c.941-3C>T) in the CRB2 gene. The variant was present in a homozygous state in the affected individuals, while in a heterozygous state in phenotypically normal parents. Results: The study expanded the spectrum of the mutations in the gene CRB2 responsible for causing NS. Conclusions: In addition, the study will also help in genetic counseling, carrier testing, and prenatal and/or postnatal early diagnosis of the disease in the affected family.
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Lu J, Guo YN, Dong LQ. Crumbs homolog 2 mutation in two siblings with steroid-resistant nephrotic syndrome: Two case reports. World J Clin Cases 2021; 9:3056-3062. [PMID: 33969091 PMCID: PMC8080757 DOI: 10.12998/wjcc.v9.i13.3056] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 02/22/2021] [Accepted: 03/11/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Crumbs homolog 2 (CRB2) is a recently discovered gene that is closely related to the maintenance of normal polarity in podocytes; mutations can directly lead to steroid-resistant nephrotic syndrome (SRNS). However, the characteristics of nephrotic syndrome (NS) caused by CRB2 mutations have not been described.
CASE SUMMARY We report a novel compound heterozygous mutation of the CRB2 gene in two siblings with SRNS. The two siblings had edema, proteinuria, hypoproteinemia and hyperlipidemia. Both their father and mother had normal phenotypes (no history of NS). Whole exon sequencing (WES) of the family showed a novel compound heterozygous mutation, c.2290 (exon 8) C > T and c.3613 (exon 12) G > A. Glucocorticoid therapy (methylprednisolone pulse therapy or oral prednisone) and immunosuppressive agents (tacrolimus) had no effect. During a 3-year follow-up after genetic diagnosis by WES, proteinuria persisted, but the patient was healthy.
CONCLUSION CRB2 mutations related to SRNS often occur in exons 7, 10, and 12. Clinical manifestations of SRNS caused by CRB2 mutations are often less severe than in other forms of SRNS.
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Affiliation(s)
- Jing Lu
- Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
| | - Yan-Nan Guo
- Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
| | - Li-Qun Dong
- Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
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8
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Genetic and preimplantation diagnosis of cystic kidney disease with ventriculomegaly. J Hum Genet 2020; 65:455-459. [PMID: 32051522 DOI: 10.1038/s10038-020-0731-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Revised: 01/10/2020] [Accepted: 01/22/2020] [Indexed: 11/09/2022]
Abstract
Ventriculomegaly with cystic kidney disease (VMCKD) is a rare and severe disorder characterized by cerebral ventriculomegaly, greatly elevated maternal serum alpha-fetoprotein (MSAFP) or amniotic fluid alpha-fetoprotein (AFAFP) levels and kidney disease similar to Finnish congenital nephrosis. Recessive mutations in the CRB2 (NM_173689) gene have been shown to cause the syndrome. Here, we described a nonconsanguineous Chinese family with two fetuses affected with VMCKD. A novel compound heterozygous mutation was identified in the CRB2 gene with co-segregation. One mutation [c.1960G>C (p.A654P)] was inherited from the father, while another mutation [c.3078_c.3093delGGCGCGGCCCCGGCCC (p.L1026Lfs*110)] was inherited from the mother. Preimplantation genetic testing for monogenic disease (PGT-M) was performed for the carrier couple with full informed consent and successfully blocked the inheritance of the disease. Our study has important implications on molecular diagnosis and genetic counseling for VMCKD and extends the mutation spectrum in CRB2 gene.
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Quinn PM, Mulder AA, Henrique Alves C, Desrosiers M, de Vries SI, Klooster J, Dalkara D, Koster AJ, Jost CR, Wijnholds J. Loss of CRB2 in Müller glial cells modifies a CRB1-associated retinitis pigmentosa phenotype into a Leber congenital amaurosis phenotype. Hum Mol Genet 2019; 28:105-123. [PMID: 30239717 DOI: 10.1093/hmg/ddy337] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2018] [Accepted: 09/17/2018] [Indexed: 11/14/2022] Open
Abstract
Variations in the human Crumbs homolog-1 (CRB1) gene lead to an array of retinal dystrophies including early onset of retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) in children. To investigate the physiological roles of CRB1 and CRB2 in retinal Müller glial cells (MGCs), we analysed mouse retinas lacking both proteins in MGC. The peripheral retina showed a faster progression of dystrophy than the central retina. The central retina showed retinal folds, disruptions at the outer limiting membrane, protrusion of photoreceptor nuclei into the inner and outer segment layers and ingression of photoreceptor nuclei into the photoreceptor synaptic layer. The peripheral retina showed a complete loss of the photoreceptor synapse layer, intermingling of photoreceptor nuclei within the inner nuclear layer and ectopic photoreceptor cells in the ganglion cell layer. Electroretinography showed severe attenuation of the scotopic a-wave at 1 month of age with responses below detection levels at 3 months of age. The double knockout mouse retinas mimicked a phenotype equivalent to a clinical LCA phenotype due to loss of CRB1. Localization of CRB1 and CRB2 in non-human primate (NHP) retinas was analyzed at the ultrastructural level. We found that NHP CRB1 and CRB2 proteins localized to the subapical region adjacent to adherens junctions at the outer limiting membrane in MGC and photoreceptors. Our data suggest that loss of CRB2 in MGC aggravates the CRB1-associated RP-like phenotype towards an LCA-like phenotype.
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Affiliation(s)
- Peter M Quinn
- Department of Ophthalmology, Leiden University Medical Center, RC Leiden, The Netherlands
| | - Aat A Mulder
- Department of Cell & Chemical Biology, Leiden University Medical Center (LUMC), RC Leiden, The Netherlands
| | - C Henrique Alves
- Department of Ophthalmology, Leiden University Medical Center, RC Leiden, The Netherlands
| | - Mélissa Desrosiers
- Department of Therapeutics, Institut de la Vision, Sorbonne Universités, UPMC Univ Paris, UMR_S INSERM, CNRS, UMR, Paris, France
| | - Sharon I de Vries
- Department of Axonal Signaling, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), BA Amsterdam, The Netherlands
| | - Jan Klooster
- Department of Retina Signal Processing, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences (KNAW), BA Amsterdam, The Netherlands
| | - Deniz Dalkara
- Department of Therapeutics, Institut de la Vision, Sorbonne Universités, UPMC Univ Paris, UMR_S INSERM, CNRS, UMR, Paris, France
| | - Abraham J Koster
- Department of Cell & Chemical Biology, Leiden University Medical Center (LUMC), RC Leiden, The Netherlands
| | - Carolina R Jost
- Department of Cell & Chemical Biology, Leiden University Medical Center (LUMC), RC Leiden, The Netherlands
| | - Jan Wijnholds
- Department of Ophthalmology, Leiden University Medical Center, RC Leiden, The Netherlands.,The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, BA Amsterdam, The Netherlands
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10
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Date P, Ackermann P, Furey C, Fink IB, Jonas S, Khokha MK, Kahle KT, Deniz E. Visualizing flow in an intact CSF network using optical coherence tomography: implications for human congenital hydrocephalus. Sci Rep 2019; 9:6196. [PMID: 30996265 PMCID: PMC6470164 DOI: 10.1038/s41598-019-42549-4] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 04/02/2019] [Indexed: 12/30/2022] Open
Abstract
Cerebrospinal fluid (CSF) flow in the brain ventricles is critical for brain development. Altered CSF flow dynamics have been implicated in congenital hydrocephalus (CH) characterized by the potentially lethal expansion of cerebral ventricles if not treated. CH is the most common neurosurgical indication in children effecting 1 per 1000 infants. Current treatment modalities are limited to antiquated brain surgery techniques, mostly because of our poor understanding of the CH pathophysiology. We lack model systems where the interplay between ependymal cilia, embryonic CSF flow dynamics and brain development can be analyzed in depth. This is in part due to the poor accessibility of the vertebrate ventricular system to in vivo investigation. Here, we show that the genetically tractable frog Xenopus tropicalis, paired with optical coherence tomography imaging, provides new insights into CSF flow dynamics and role of ciliary dysfunction in hydrocephalus pathogenesis. We can visualize CSF flow within the multi-chambered ventricular system and detect multiple distinct polarized CSF flow fields. Using CRISPR/Cas9 gene editing, we modeled human L1CAM and CRB2 mediated aqueductal stenosis. We propose that our high-throughput platform can prove invaluable for testing candidate human CH genes to understand CH pathophysiology.
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Affiliation(s)
- Priya Date
- Pediatric Genomics Discovery Program, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
- Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
| | - Pascal Ackermann
- Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
- Department of Medical Informatics, Uniklinik RWTH Aachen, Pauwelsstr 30, 52074, Aachen, Germany
| | - Charuta Furey
- Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
- Department of Neurosurgery and Cellular & Molecular Physiology, and Centers for Mendelian Genomics, 333 Cedar Street, New Haven, CT, 06510, USA
| | - Ina Berenice Fink
- Department of Medical Informatics, Uniklinik RWTH Aachen, Pauwelsstr 30, 52074, Aachen, Germany
| | - Stephan Jonas
- Department of Informatics, Technical University of Munich, Munich, Germany
| | - Mustafa K Khokha
- Pediatric Genomics Discovery Program, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
- Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
- Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA
| | - Kristopher T Kahle
- Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA.
- Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA.
- Department of Neurosurgery and Cellular & Molecular Physiology, and Centers for Mendelian Genomics, 333 Cedar Street, New Haven, CT, 06510, USA.
| | - Engin Deniz
- Pediatric Genomics Discovery Program, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA.
- Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, 06510, USA.
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11
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Jiménez-Amilburu V, Stainier DYR. The transmembrane protein Crb2a regulates cardiomyocyte apicobasal polarity and adhesion in zebrafish. Development 2019; 146:dev.171207. [DOI: 10.1242/dev.171207] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Accepted: 04/08/2019] [Indexed: 12/21/2022]
Abstract
Tissue morphogenesis requires changes in cell-cell adhesion as well as in cell shape and polarity. Cardiac trabeculation is a morphogenetic process essential to form a functional ventricular wall. Here we show that zebrafish hearts lacking Crb2a, a component of the Crumbs polarity complex, display compact wall integrity defects and fail to form trabeculae. Crb2a localization is very dynamic at a time when other cardiomyocyte junctional proteins also relocalize. Before the initiation of cardiomyocyte delamination to form the trabecular layer, Crb2a is expressed in all ventricular cardiomyocytes and colocalizes with the junctional protein ZO-1. Subsequently, Crb2a becomes localized all along the apical membrane of compact layer cardiomyocytes and is downregulated in the delaminating cardiomyocytes. We show that blood flow and Nrg/ErbB2 signaling regulate Crb2a localization dynamics. crb2a−/− display a multilayered wall with polarized cardiomyocytes, a unique phenotype. Our data further indicate that Crb2a regulates cardiac trabeculation by controlling the localization of tight and adherens junction proteins in cardiomyocytes. Importantly, transplantation data show that Crb2a controls CM behavior in a cell-autonomous manner in the sense that crb2a−/− cardiomyocytes transplanted into wild-type animals were always found in the trabecular layer. Altogether, our study reveals a critical role for Crb2a during cardiac development.
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Affiliation(s)
- Vanesa Jiménez-Amilburu
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany
| | - Didier Y. R. Stainier
- Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany
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12
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Chen X, Jiang C, Yang D, Sun R, Wang M, Sun H, Xu M, Zhou L, Chen M, Xie P, Yan B, Liu Q, Zhao C. CRB2 mutation causes autosomal recessive retinitis pigmentosa. Exp Eye Res 2018; 180:164-173. [PMID: 30593785 DOI: 10.1016/j.exer.2018.12.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Revised: 12/21/2018] [Accepted: 12/21/2018] [Indexed: 01/29/2023]
Abstract
Retinitis pigmentosa (RP), the most common form of inherited retinal dystrophies, exhibits significant genetic heterogeneity. The crumbs homolog 2 (CRB2) protein, together with CRB1 and CRB3, belongs to the Crumbs family. Given that CRB1 mutations account for 4% of RP cases, the role of CRB2 mutations in RP etiology has long been hypothesized but never confirmed. Herein, we report the identification of CRB2 as a novel RP causative gene in a Chinese consanguineous family and have analyzed its pathogenic effects. Comprehensive ophthalmic and systemic evaluations confirmed the clinical diagnosis of the two patients in this family as RP. WES revealed a homozygous missense mutation, CRB2 p.R1249G, to segregate the RP phenotype, which was highly conserved among multiple species. In vitro cellular study revealed that this mutation not only interrupted the stability of the transcribed CRB2 mRNA and the encoded CRB2 protein, but also interfered with the wild type CRB2 mRNA/protein and decreased their expression. This mutation was also shown to trigger epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, thus impairing regular RPE phagocytosis and induce RPE degeneration and apoptosis. Thus, we conclude that CRB2 p.R1249G mutation causes RP via accelerating EMT, dysfunction and loss of RPE cells, and establish CRB2 as a novel Crumbs family member associated with non-syndromic RP. We provide important hints for understanding of CRB2 defects and retinopathy, and for the involvement of EMT of RPE cells in RP pathogenesis.
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Affiliation(s)
- Xue Chen
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China; Department of Ophthalmology and Vision Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, 200023, China; Key Laboratory of Myopia of State Health Ministry (Fudan University) and Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, 200023, China
| | - Chao Jiang
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Daidi Yang
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Ruxu Sun
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Min Wang
- Department of Ophthalmology and Vision Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, 200023, China; Key Laboratory of Myopia of State Health Ministry (Fudan University) and Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, 200023, China
| | - Hong Sun
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Min Xu
- Department of Ophthalmology, Northern Jiangsu People's Hospital, Yangzhou, 211406, China
| | - Luyin Zhou
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Mingkang Chen
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Ping Xie
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
| | - Biao Yan
- Department of Ophthalmology and Vision Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, 200023, China; Key Laboratory of Myopia of State Health Ministry (Fudan University) and Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, 200023, China
| | - Qinghuai Liu
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
| | - Chen Zhao
- Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China; Department of Ophthalmology and Vision Science, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, 200023, China; Key Laboratory of Myopia of State Health Ministry (Fudan University) and Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, 200023, China.
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13
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Fan J, Fu R, Ren F, He J, Wang S, Gou M. A case report of CRB2 mutation identified in a Chinese boy with focal segmental glomerulosclerosis. Medicine (Baltimore) 2018; 97:e12362. [PMID: 30212996 PMCID: PMC6156060 DOI: 10.1097/md.0000000000012362] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
RATIONALE Focal segmental glomerulosclerosis (FSGS) is a common disease resulting in end-stage renal disease. The incidence of FSGS is increasing in Western countries. The clinical manifestations include proteinuria, hypoproteinemia, oedema, and hypertension. Single-gene heritable mutations are considered to be the source of FSGS pathogenicity according to recent in-depth studies on the pathogenesis. Here, we first reported the case of a Chinese boy whose histology presented with FSGS caused by a compound heterozygous mutation. PATIENT CONCERNS A 7-year-old Chinese boy was repeatedly admitted to our hospital for fever, cough, and proteinuria since he was 1.6 years old. DIAGNOSES FSGS was identified by renal biopsy. Whole exome sequencing (WES) showed that a novel mutation of crumbs homolog 2 (CRB2) was identified in a Chinese boy with FSGS. INTERVENTIONS Patient was treated with low-dose corticosteroid and mycophenolate mofetil for maintenance therapy. OUTCOMES At last follow-up, protein (+∼++) was observed in his urinalysis. LESSONS We identified a novel mutation of CRB2 in a Chinese boy with FSGS that had never been described in a previous report. These findings suggested that mutations in recessive disease genes are more frequent among early-onset disease.
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14
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Dolón JF, Paniagua AE, Valle V, Segurado A, Arévalo R, Velasco A, Lillo C. Expression and localization of the polarity protein CRB2 in adult mouse brain: a comparison with the CRB1 rd8 mutant mouse model. Sci Rep 2018; 8:11652. [PMID: 30076417 PMCID: PMC6076319 DOI: 10.1038/s41598-018-30210-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Accepted: 07/24/2018] [Indexed: 11/09/2022] Open
Abstract
Acquisition of cell polarization is essential for the performance of crucial functions, like a successful secretion and appropriate cell signaling in many tissues, and it depends on the correct functioning of polarity proteins, including the Crumbs complex. The CRB proteins, CRB1, CRB2 and CRB3, identified in mammals, are expressed in epithelial-derived tissues like brain, kidney and retina. CRB2 has a ubiquitous expression and has been detected in embryonic brain tissue; but currently there is no data regarding its localization in the adult brain. In our study, we characterized the presence of CRB2 in adult mice brain, where it is particularly enriched in cortex, hippocampus, hypothalamus and cerebellum. Double immunofluorescence analysis confirmed that CRB2 is a neuron-specific protein, present in both soma and projections where colocalizes with certain populations of exocytic and endocytic vesicles and with other members of the Crumbs complex. Finally, in the cortex of CRB1rd8 mutant mice that contain a mutation in the Crb1 gene generating a truncated CRB1 protein, there is an abnormal increase in the expression levels of the CRB2 protein which suggests a possible compensatory mechanism for the malfunction of CRB1 in this mutant background.
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Affiliation(s)
- Jorge F Dolón
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain
| | - Antonio E Paniagua
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain.,Department of Ophthalmology and Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Vicente Valle
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain
| | - Alicia Segurado
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain
| | - Rosario Arévalo
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain
| | - Almudena Velasco
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain
| | - Concepción Lillo
- Institute of Neurosciences of Castilla y León, IBSAL, Cell Biology and Pathology, University of Salamanca, 37007, Salamanca, Spain.
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15
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Watanabe S, Aizawa T, Tsukaguchi H, Tsugawa K, Tsuruga K, Shono A, Nozu K, Iijima K, Joh K, Tanaka H. Long-term clinicopathologic observation in a case of steroid-resistant nephrotic syndrome caused by a novel Crumbs homolog 2 mutation. Nephrology (Carlton) 2018; 23:697-702. [PMID: 29473663 DOI: 10.1111/nep.13244] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/19/2018] [Indexed: 01/14/2023]
Abstract
Recent advances in high-throughput sequencing for clinical genetic testing have revealed novel disease-causing genes, such as Crumbs homolog 2 (CRB2) for early-onset steroid-resistant nephrotic syndrome (SRNS). We report the long-term clinicopathologic observation of a Japanese female patient with SRNS caused by a newly identified compound heterozygous mutation of CRB2 (p.Arg628Cys and p.Gly839Trp located in the 10th and 11th epidermal growth factor-like domains, respectively). She was initially examined during a mass urinary screening for 3.5-year-old children in Japan. Although she developed long-standing SRNS without any extrarenal clinical signs thereafter, her renal function was well-preserved over the next 17 years. In total, six sequential renal biopsy specimens revealed histologic alterations ranging from minor glomerular abnormalities to advanced focal segmental glomerulosclerosis (FSGS). A genetic analysis for SRNS performed at 19 years of age revealed a newly identified compound heterozygous mutation in CRB2. Glomerular CRB2 immunoreactivity in biopsy specimens from the patient was scanty, whereas intense expression was observed in those from patients with idiopathic FSGS or in controls. To our knowledge, this is the first report regarding a long-term outcome in a case of SRNS due to an identified CRB2 mutation. Although the phenotype of CRB2 mutation-related syndrome is now expanding, we believe that this case might provide a novel clinicopathologic aspect of this syndrome.
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Affiliation(s)
- Shojiro Watanabe
- Department of Pediatrics, Hirosaki University Hospital, Hirosaki, Japan
| | - Tomomi Aizawa
- Department of Pediatrics, Hirosaki University Hospital, Hirosaki, Japan
| | | | - Koji Tsugawa
- Department of Pediatrics, Hirosaki University Hospital, Hirosaki, Japan
| | - Kazushi Tsuruga
- Department of Pediatrics, Hirosaki University Hospital, Hirosaki, Japan
| | - Akemi Shono
- Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Kandai Nozu
- Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Kazumoto Iijima
- Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan
| | - Kensuke Joh
- Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan
| | - Hiroshi Tanaka
- Department of Pediatrics, Hirosaki University Hospital, Hirosaki, Japan
- Department of School Health Science, Hirosaki University Faculty of Education, Hirosaki, Japan
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16
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Richer J, Laberge AM. Secondary findings from next-generation sequencing: what does actionable in childhood really mean? Genet Med 2018; 21:124-132. [DOI: 10.1038/s41436-018-0034-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2017] [Accepted: 03/26/2018] [Indexed: 01/20/2023] Open
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17
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Khan KN, Robson A, Mahroo OAR, Arno G, Inglehearn CF, Armengol M, Waseem N, Holder GE, Carss KJ, Raymond LF, Webster AR, Moore AT, McKibbin M, van Genderen MM, Poulter JA, Michaelides M. A clinical and molecular characterisation of CRB1-associated maculopathy. Eur J Hum Genet 2018; 26:687-694. [PMID: 29391521 PMCID: PMC5945653 DOI: 10.1038/s41431-017-0082-2] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2017] [Revised: 11/14/2017] [Accepted: 12/05/2017] [Indexed: 12/22/2022] Open
Abstract
To date, over 150 disease-associated variants in CRB1 have been described, resulting in a range of retinal disease phenotypes including Leber congenital amaurosis and retinitis pigmentosa. Despite this, no genotype-phenotype correlations are currently recognised. We performed a retrospective review of electronic patient records to identify patients with macular dystrophy due to bi-allelic variants in CRB1. In total, seven unrelated individuals were identified. The median age at presentation was 21 years, with a median acuity of 0.55 decimalised Snellen units (IQR = 0.43). The follow-up period ranged from 0 to 19 years (median = 2.0 years), with a median final decimalised Snellen acuity of 0.65 (IQR = 0.70). Fundoscopy revealed only a subtly altered foveal reflex, which evolved into a bull's-eye pattern of outer retinal atrophy. Optical coherence tomography identified structural changes-intraretinal cysts in the early stages of disease, and later outer retinal atrophy. Genetic testing revealed that one rare allele (c.498_506del, p.(Ile167_Gly169del)) was present in all patients, with one patient being homozygous for the variant and six being heterozygous. In trans with this, one variant recurred twice (p.(Cys896Ter)), while the four remaining alleles were each observed once (p.(Pro1381Thr), p.(Ser478ProfsTer24), p.(Cys195Phe) and p.(Arg764Cys)). These findings show that the rare CRB1 variant, c.498_506del, is strongly associated with localised retinal dysfunction. The clinical findings are much milder than those observed with bi-allelic, loss-of-function variants in CRB1, suggesting this in-frame deletion acts as a hypomorphic allele. This is the most prevalent disease-causing CRB1 variant identified in the non-Asian population to date.
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Affiliation(s)
- Kamron N Khan
- University College London Institute of Ophthalmology, University College London, London, UK.
- Inherited Eye Disease Service, Moorfields Eye Hospital, London, UK.
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK.
- Department of Ophthalmology, St. James's University Teaching Hospital, Leeds, UK.
| | - Anthony Robson
- Department of Electrophysiology, Moorfields Eye Hospital, London, UK
| | - Omar A R Mahroo
- University College London Institute of Ophthalmology, University College London, London, UK
- Inherited Eye Disease Service, Moorfields Eye Hospital, London, UK
| | - Gavin Arno
- University College London Institute of Ophthalmology, University College London, London, UK
| | - Chris F Inglehearn
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK
| | - Monica Armengol
- Inherited Eye Disease Service, Moorfields Eye Hospital, London, UK
| | - Naushin Waseem
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK
| | - Graham E Holder
- Department of Electrophysiology, Moorfields Eye Hospital, London, UK
| | - Keren J Carss
- NIHR BioResource - Rare Diseases, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK
- Department of Haematology, NHS Blood and Transplant Centre, University of Cambridge, Cambridge, CB2 0PT, UK
- Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK
| | - Lucy F Raymond
- NIHR BioResource - Rare Diseases, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge, UK
- Department of Haematology, NHS Blood and Transplant Centre, University of Cambridge, Cambridge, CB2 0PT, UK
- Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, CB2 0XY, UK
| | - Andrew R Webster
- University College London Institute of Ophthalmology, University College London, London, UK
- Inherited Eye Disease Service, Moorfields Eye Hospital, London, UK
| | - Anthony T Moore
- University College London Institute of Ophthalmology, University College London, London, UK
- Inherited Eye Disease Service, Moorfields Eye Hospital, London, UK
- Ophthalmology Department, University of California San Francisco Medical School, San Francisco, CA, USA
| | - Martin McKibbin
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK
- Department of Ophthalmology, St. James's University Teaching Hospital, Leeds, UK
| | - Maria M van Genderen
- Bartiméus Diagnostic Centre for Complex Visual Disorders, Zeist, The Netherlands
- Department of Ophthalmology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - James A Poulter
- Section of Ophthalmology and Neuroscience, Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK
| | - Michel Michaelides
- University College London Institute of Ophthalmology, University College London, London, UK
- Inherited Eye Disease Service, Moorfields Eye Hospital, London, UK
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18
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Udagawa T, Jo T, Yanagihara T, Shimizu A, Mitsui J, Tsuji S, Morishita S, Onai R, Miura K, Kanda S, Kajiho Y, Tsurumi H, Oka A, Hattori M, Harita Y. Altered expression of Crb2 in podocytes expands a variation of CRB2 mutations in steroid-resistant nephrotic syndrome. Pediatr Nephrol 2017; 32:801-809. [PMID: 27942854 DOI: 10.1007/s00467-016-3549-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2016] [Revised: 11/02/2016] [Accepted: 11/08/2016] [Indexed: 10/20/2022]
Abstract
BACKGROUND Steroid-resistant nephrotic syndrome (SRNS) is a genetically heterogeneous disorder for which more than 25 single-gene hereditary causes have been identified. METHODS Whole exome sequencing was performed in a 3-year-old girl with SRNS. We analyzed the expression of Crb2 and slit diaphragm molecules in the patient's glomeruli, and compared it with that of controls or other nephrotic patients. RESULTS Whole-exome analysis identified novel compound heterozygous mutations in exons 10 and 12 of CRB2 (p.Trp1086ArgfsX64 and p.Asn1184Thr, each from different parents; Asn1184 within extracellular 15th EGF repeat domain). Renal pathology showed focal segmental glomerulosclerosis with effaced podocyte foot processes in a small area, with significantly decreased Crb2 expression. Molecules critical for slit diaphragm were well-expressed in this patient's podocytes. Crb2 expression was not altered in the other patients with congenital nephrotic syndrome with NPHS1 mutations. CONCLUSIONS These findings demonstrate that Crb2 abnormalities caused by these mutations are the mechanism of steroid-resistant NS. Although CRB2 mutations previously found in SRNS patients have been clustered within the extracellular tenth EGF-like domain of this protein, the present results expand the variation of CRB2 mutations that cause SRNS.
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Affiliation(s)
- Tomohiro Udagawa
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Tohaku Jo
- Department of Pediatrics, Nippon Medical School, Tokyo, Japan
| | | | - Akira Shimizu
- Department of Analytic Human Pathology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan
| | - Jun Mitsui
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Shoji Tsuji
- Department of Neurology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
| | - Shinichi Morishita
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan
| | - Reiko Onai
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Kenichiro Miura
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Shoichiro Kanda
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Yuko Kajiho
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Haruko Tsurumi
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Akira Oka
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan
| | - Motoshi Hattori
- Department of Pediatric Nephrology, Tokyo Women's Medical University, Sinjuku-ku, Tokyo, Japan
| | - Yutaka Harita
- Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.
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19
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Quinn PM, Pellissier LP, Wijnholds J. The CRB1 Complex: Following the Trail of Crumbs to a Feasible Gene Therapy Strategy. Front Neurosci 2017; 11:175. [PMID: 28424578 PMCID: PMC5380682 DOI: 10.3389/fnins.2017.00175] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Accepted: 03/16/2017] [Indexed: 12/24/2022] Open
Abstract
Once considered science fiction, gene therapy is rapidly becoming scientific reality, targeting a growing number of the approximately 250 genes linked to hereditary retinal disorders such as retinitis pigmentosa and Leber's congenital amaurosis. Powerful new technologies have emerged, leading to the development of humanized models for testing and screening these therapies, bringing us closer to the goal of personalized medicine. These tools include the ability to differentiate human induced pluripotent stem cells (iPSCs) to create a “retina-in-a-dish” model and the self-formed ectodermal autonomous multi-zone, which can mimic whole eye development. In addition, highly specific gene-editing tools are now available, including the CRISPR/Cas9 system and the recently developed homology-independent targeted integration approach, which allows gene editing in non-dividing cells. Variants in the CRB1 gene have long been associated with retinopathies, and more recently the CRB2 gene has also been shown to have possible clinical relevance with respect to retinopathies. In this review, we discuss the role of the CRB protein complex in patients with retinopathy. In addition, we discuss new opportunities provided by stem cells and gene-editing tools, and we provide insight into how the retinal therapeutic pipeline can be improved. Finally, we discuss the current state of adeno-associated virus-mediated gene therapy and how it can be applied to treat retinopathies associated with mutations in CRB1.
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Affiliation(s)
- Peter M Quinn
- Department of Ophthalmology, Leiden University Medical CenterLeiden, Netherlands
| | - Lucie P Pellissier
- Unité Physiologie de la Reproduction et des Comportements, INRA UMR85, Centre National de la Recherche Scientifique UMR-7247, Institut Français du Cheval et de l'Équitation, Université François RabelaisNouzilly, France
| | - Jan Wijnholds
- Department of Ophthalmology, Leiden University Medical CenterLeiden, Netherlands.,Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and SciencesAmsterdam, Netherlands
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20
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Slavotinek AM. The Family of Crumbs Genes and Human Disease. Mol Syndromol 2016; 7:274-281. [PMID: 27867342 DOI: 10.1159/000448109] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/19/2016] [Indexed: 11/19/2022] Open
Abstract
The family of vertebrate Crumbs proteins, homologous to Drosophila Crumbs (Crb), share large extracellular domains with epidermal growth factor-like repeats and laminin-globular domains, a single transmembrane domain, and a short intracellular C-terminus containing a single membrane proximal 4.1/ezrin/radixin/moesin-binding domain and PSD-95/Discs large/ZO-1-binding motifs. There are 3 Crb genes in humans - Crumbs homolog-1 (CRB1), Crumbs homolog-2 (CRB2), and Crumbs homolog-3 (CRB3). Bilallelic loss-of-function mutations in CRB1 cause visual impairment, with Leber's congenital amaurosis and retinitis pigmentosa, whereas CRB2 mutations are associated with raised maternal serum and amniotic fluid alpha feto-protein levels, ventriculomegaly/hydrocephalus, and renal disease, ranging from focal segmental glomerulosclerosis to congenital Finnish nephrosis. CRB3 has not yet been associated with human disease. In this review, we summarize the phenotypic findings associated with deleterious sequence variants in CRB1 and CRB2. We discuss the mutational spectrum, animal models of loss of function for both genes and speculate on the likely mechanisms of disease.
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Affiliation(s)
- Anne M Slavotinek
- Department of Pediatrics, UCSF School of Medicine, University of California San Francisco, San Francisco, Calif., USA
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