Various phenotypes of autosomal dominant cone-rod dystrophy with cone-rod homeobox mutation in two Chinese families.

AIM: To present the clinical manifestations of 5 autosomal dominant cone-rod dystrophy (adCORD) patients from two Chinese families with cone-rod homeobox (CRX) mutation (p.R41W), and to explore the clinical heterogeneity of adCORD with CRX mutation (p.R41W). METHODS: Interrogation and ophthalmological examinations were undertaken in all patients and unaffected members. Analysis of clinical features was performed by visual acuity, slit lamp examination, visual field examination, fundoscopy, autofluorescence and spectral domain optical coherence tomography. Targeted next-generation sequencing was applied as a useful tool to identify the causative mutation of CORD genes. RESULTS: A CRX missense mutation c.121C>T was identified in all patients, resulting in an amino acid change from arginine acid to tryptophan (p.R41W). The patients presented with early onset, progressive and different severities with CORD. CONCLUSION: This is the first report of the clinical phenotype of CRX mutation (p.R41W) in Chinese families, and the mutation can lead to a wide range of various retinal phenotypes.

Pineal gland differentiation in mature teratoma: An under-recognized condition and potential pitfalls for overdiagnosis.

We herein report three cases of mature teratomas with pineal gland differentiation, which is a less recognized phenomenon. Case 1 was a 6-year-old male with a neck mass, Case 2 was a 23-year-old female with a retroperitoneal mass, and Case 3 was a 45-year-old female with a retroperitoneal mass. Each case showed the typical macroscopic and histological findings of mature teratoma, such as solid and cystic lesions mainly lined with a mature squamous epithelium. All cases also showed glial differentiation. Small foci of lobulated cell nests were detected in the center of or adjacent to mature glial tissue. Cells had a clear to pale eosinophilic cytoplasm with small round nuclei. Immunohistochemically, cells were positive for synaptophysin, neurofilament protein with a perivascular "club-shaped swelling" pattern, and cone-rod homeobox protein. To the best of our knowledge, this is the first report of pineal gland differentiation arising in mature teratoma, which may be easily overlooked or misdiagnosed as somatic-type tumors, particularly neuroendocrine tumors. To avoid overtreatment, pathologists need to be aware that pineal gland differentiation may occur in mature teratomas.

Mixed germ cell tumor infiltrating the pineal gland without elevated tumor markers: illustrative case.

BACKGROUND: Tumors in the pineal region consist of various histological types, and correct diagnosis from biopsy specimens is sometimes difficult. The authors report the case of a patient with a mixed germ cell tumor infiltrating into the pineal gland despite showing no elevation of tumor markers. OBSERVATIONS: An 18-year-old man complained of headache and nausea and showed disturbance of consciousness. Magnetic resonance imaging showed hydrocephalus associated with a cystic pineal tumor. The patient underwent tumor biopsy followed by endoscopic third ventriculostomy for hydrocephalus in a local hospital. A pineocytoma was diagnosed, and the patient was referred to the authors' hospital for treatment. Concentrations of placental alkaline phosphatase, alpha-fetoprotein (AFP), and beta-human chorionic gonadotropin in cerebrospinal fluid were not elevated. However, the authors' review of the tumor specimen revealed some immature cells infiltrating the pineal gland. These cells were positive for AFP, Sal-like protein 4, and octamer-binding transcription factor 3/4; and the diagnosis was changed to mixed germ cell tumor. Chemoradiotherapy was initiated, followed by surgical removal of the residual tumor. LESSONS: Careful examination of all tumor specimens and immunohistochemical analyses are important for accurate diagnosis of pineal tumors.

Crx Is Posttranscriptionally Regulated by Light Stimulation in Postnatal Rat Retina.

Cone rod homeobox (Crx) plays a key role at the center of a regulatory network that coordinates many pathways in the retina. Its abnormal expression induces retinal disorders. However, the underlying regulatory mechanism of Crx expression is not well defined. Here, we present data that show that the levels of Crx mRNA were inconsistent with that of Crx protein in primary retinal neurocytes cultured in light conditions. Crx protein levels were significantly higher (2.56-fold) in cells cultured in the dark than in cells cultured in light, whereas Crx mRNA was not changed in either type of cell. Moreover, the expression of Crx protein showed a significant light intensity-dependent decrease. Consistently, Crx downstream genes rhodopsin and arrestin also decreased in retinal neurocytes upon light exposure. Furthermore, Crx promoter activity assay performed in primary retinal neurocytes further indicated that light exposure and darkness did not affect its inducibility. In addition, the inconsistency between Crx mRNA and protein expression after light exposure was not observed in 661w cells transfected with plasmid pcDNA3.1-Crx, suggesting that the inconsistency between Crx mRNA and protein induced by light was specific to the endogenous Crx. More importantly, this observation was confirmed in vivo in postnatal day 15 (P15) retinas but not in adult retinas, further implicating that the posttranscriptional regulation mechanism may be involved in Crx expression in the developing retina. Therefore, our study sheds light on the mechanism of Crx expression in postnatal rat retina.

A novel dominant CRX mutation causes adult-onset macular dystrophy.

BACKGROUND: To present the ophthalmological findings of a mother and son initially diagnosed with benign concentric annular macular dystrophy (BCAMD) and later discovered to carry a novel nonsense mutation in the cone-rod homeobox (CRX) gene (19q13.3). MATERIALS AND METHODS: Patients received ophthalmic examinations and diagnostic testing. The proband underwent sequencing of 131 retinal dystrophy genes. His mother had targeted testing of the identified sequence variations in CRX and BBS12 (4q27). RESULTS: The proband (Case I) presented at age 35 years for routine care. He had several male and female relatives with adult-onset retinal degeneration. Over 11 years, visual acuity (VA) dropped from 20/20 OU to 20/25 OD and 20/50 OS. Ophthalmoscopy showed bull's eye macular lesions OU. The proband's mother (Case II) presented at age 57 years with unilateral, progressive vision loss. Over 5 years, VA fluctuated 20/80-20/400 OD and remained 20/25 OS. Ophthalmoscopy showed similar findings to Case I. A clinical diagnosis of BCAMD was given because of the characteristic retinal lesion, preserved VA, and presumed autosomal dominant inheritance. Genetic testing identified a novel nonsense mutation in CRX (c.766C>T; p.Gln256Ter) in both patients. The son was also heterozygous for a missense mutation in BBS12 (c.1859A>G; p.Gln620Arg), which was absent in Case II. CONCLUSIONS: CRX mutations are associated with a variety of clinical phenotypes, including an adult-onset macular dystrophy that simulates BCAMD with a bull's eye macular lesion and fairly well preserved VA.

Using Zinc Finger Nuclease Technology to Generate CRX-Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation.

The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone-Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3' UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3' of the CRX gene, generating a CRX-GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX-GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation.

De novo mutations in the cone-rod homeobox gene associated with leber congenital amaurosis in Chinese patients.

BACKGROUND: The cone-rod homeobox (CRX) gene plays an important role in photoreceptor development. Recently, mutant alleles of the CRX gene have been associated with autosomal dominant Leber congenital amaurosis (LCA) and cone-rod dystrophy. The purpose of this study was to analyze the CRX mutations in a cohort of Chinese patients with LCA or early-onset severe retinal dystrophy (EOSRD) and to provide the clinical features of these patients. METHODS: Patients with LCA or EOSRD were enrolled from 2003 to 2012. Detailed ocular examinations including optical coherence tomography (OCT) and standardized electrophysiology were performed. Genomic DNA was isolated with standard methods of genetic diagnosis. All three exons of CRX were amplified with PCR and screened for mutations through direct DNA sequencing. A total of 200 unrelated healthy Chinese subjects were screened to exclude nonpathogenic polymorphisms. Offspring-parent relationship was tested to confirm de novo mutation. RESULTS: A total of 109 probands from 109 unrelated families were selected for mutation screening of the CRX gene. Two individuals with LCA were confirmed to carry de novo CRX mutations c.421delT (p.Ser141Pro fsX46) and c.571delT (p.Tyr191Met fsX3), respectively. The daughter of Case 1 also carried the same CRX mutation (c.421delT) and had LCA symptoms. Pigmentary retinopathy in the peripheral retina and macular atrophy were observed in the two probands. Macular atrophy without normal lamination structure was the retina phenotype under OCT. CONCLUSIONS: Two de novo mutations in CRX were found in Chinese patients with LCA. The CRX mutation might create a dominantly inherited trait.

Identification and characterization of novel alternative splice variants of human SAMD11.

Sterile alpha motif domain-containing 11 (SAMD11) is evolutionarily conserved from zebrafish to human. Mouse Samd11 is predominantly expressed in developing retinal photoreceptors and the adult pineal gland, and its transcription is directly regulated by the cone-rod homeodomain protein Crx. However, there has been little research on human SAMD11. To investigate the function of human SAMD11, we first cloned its coding sequence (CDS) and identified up to 45 novel alternative splice variants (ASVs). Mouse Samd11 ASVs were also identified by aligning the mouse Samd11 expressed sequence tags (ESTs) with the annotated sequence. However, the range of expression and transcriptional regulation of SAMD11 differs between human and mouse. Human SAMD11 was found to be widely expressed in many cell lines and ocular tissues and its transcription was not regulated by CRX, OTX2 or NR2E3 proteins. Furthermore, functional analysis indicated that human SAMD11 could promote cell proliferation slightly. In conclusion, this study elucidated the basic characteristics of human SAMD11 and revealed that, although the occurrence of alternative splicing of SAMD11 was conserved, the function of SAMD11 may vary in different species.