Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Conjunctival lymphangiectasia presenting as pediatric pseudopterygium.

OBJECTIVES: The aim of this study is to report a case of conjunctival lymphangiectasia simulating a pediatric pterygium. METHODS: A 10-year-old girl with Turner syndrome and familial history of pterygia presented because of a conjunctival growing lesion clinically consistent with pterygium in her left eye. Visual acuity (VA) was 20/20 in the right eye and 20/40 in the left eye. Cycloplegic refraction was +1.50sph -0.50cyl×93° and +9.00sph -9.00cyl×180° in the right and left eyes, respectively. RESULTS: Resection of the lesion with amniotic membrane implantation and conjunctival autograft was performed. The histologic examination revealed conjunctival lymphangiectasia. Ten months later, the patient did not show any signs of recurrence. Refraction in the left eye was +0.5sph -1.5cyl×70°, and spontaneous VA was 20/20 in both eyes. CONCLUSIONS: Pediatric pseudopterygium may be the clinical presentation of several ocular surface disorders. Thus, pathologic analysis of this lesion should be considered to determine its exact nature. Surgery lessens the refractive defect and hastens visual recovery.

Microcephaly-lymphedema-chorioretinal dysplasia associated with pachymicrogyria and atrophy of the cerebellar vermis: an integration of brain-ocular migration disorders.

BACKGROUND: Microcephaly-lymphedema-chorioretinal dysplasia (OMIM 152950) is a rare malformative inherited disorder that can be associated with other systemic features. Other ocular and brain anomalies rather than microcephaly and chorioretinal dysplasia have been inconstantly reported in this syndrome. METHODS: We present a case of microcephaly-lymphedema-chorioretinal dysplasia with a dysmorphic facies, hypertonicity in the extremities and neuropsychomotor delay. Ophthalmological examination revealed bilateral nystagmus, microphthalmia, posterior subcapsular cataratacts, extensive chorioretinal dysplasia, optic nerve aplasia, persistent fetal vasculature, and absent retinal vessels. RESULTS: Magnetic resonance revealed pachymicrogyria and discrete atrophy of vermis cerebelosum and confirmed optic nerve hypoplasia. CONCLUSIONS: The developmental alterations observed in the retina of this patient could be analogous to central nervous system anomalies, reflecting a reduction in neural population. Ophthalmic examination of children with microcephaly is warranted.

Cystic peripapillary staphyloma: a report of two cases.

Peripapillary staphyloma is a rare, nonhereditary, congenital anomaly in which an excavation surrounds the optic disk. We present 2 cases of peripapillary staphylomas resembling cystic cavities, both of which caused severe visual impairment. We propose the term cystic peripapillary staphyloma to describe this anomaly. The first case was associated with microphthalmia; the second, with encephalocele and Chiari malformation, an association that has not been previously described. We propose the term "cystic peripapillary staphyloma" to describe this anomaly.

Peripheral primitive neuroectodermal tumour of the orbit.

Peripheral primitive neuroectodermal tumours (pPNETs) are a group of soft-tissue tumours of neuroepithelial origin that arise outside the central and sympathetic nervous system. Orbital location is infrequent, and to the best of the authors' knowledge only 16 cases have been reported in the literature. With this article, the authors report the demographics and clinical characteristics, diagnostic features, differential diagnosis, prognosis and therapeutic options of primary orbital peripheral primitive neuroectodermal tumour, based on their patients and on the cases reported in the literature to date. A differential diagnosis should be made with other small round cell tumours; immunohistochemical and ultrastructural techniques are essential for this purpose. Although bone invasion and extraorbital extension are possible, systemic metastases are uncommon in the cases of orbital pPNETs. Surgery has been the initial treatment in most cases; chemotherapy with or without radiotherapy is considered the best additional treatment. The orbital pPNET could be less aggressive than other forms of pPNETs, since most of the patients reported were alive after the follow-up period (at least 6 months).

Pathogenic validation of unique germline intronic variants of RB1 in retinoblastoma patients using minigenes.

Precise identification of the pathogenic character of germline mutations in the retinoblastoma gene (RB1) is fundamental to provide genetic counselling to patients at risk of developing retinoblastoma. In contrast to bona fide oncogenic RB1 mutations like nonsense or frameshift mutations, and those affecting invariant dinucleotides at splice sites, intronic variants affecting less conserved splice motifs require additional analysis to ascertain whether splicing is altered. Although the frequency of these variations is low, their impact on genetic counselling is high, since they are usually associated with low penetrance phenotypes and unaffected carriers. In this work, we used minigene assays to study infrequent germline intronic variations for which functional data were not available. Using this approach, the aberrant splicing and the resulting oncogenic nature of three intronic RB1 mutations was established (c.501-15T>G, c.719-9C>G, c.2326-8T>A). Conversely, the intronic variant c.1961-12T>C was categorized by minigene assay as a very infrequent neutral polymorphism. To our knowledge this is the first report describing the use of minigene constructs to study the oncogenic character of intronic RB1 variants detected during mutational screening and show the utility of this approach to ascertain the oncogenic nature of unique RB1 intronic variants for which no previous functional and clinical data are available. Minigene assay can be especially useful when lymphocyte RNA is not available for study, or when aberrant mRNA can not be detected as a consequence of nonsense mediated decay. Since RB1 minigene are time-consuming assays, owing to the genomic organization of the RB1 gene, it should be welcome the design of new expression vectors that make this type of studies more straightforward.

Pars planitis in children: epidemiologic, clinical, and therapeutic characteristics.

PURPOSE: To report the demographics and clinical characteristics, therapy logarithm, and prognosis of children with pars planitis. PATIENTS AND METHODS: The medical records were reviewed of all patients diagnosed with pars planitis between June 1995 and December 2005 in the Department of Pediatric Ophthalmology at Hospital Universitario La Paz, Madrid, Spain. A retrospective, descriptive, and longitudinal study of 30 eyes in 16 children was performed. RESULTS: Pars planitis was bilateral in 87.5% and more frequent in males (68.8%). Average age at onset was 9.2 years. The main ophthalmologic findings recorded were snowballs (96.7%) and vitritis (93.3%). Cataract formation was the most prevalent complication (36.7%). Mean initial and final best-corrected visual acuities were 0.640 and 0.840, respectively. Periocular corticosteroids were used in 33.3% of cases and cryotherapy or laser photocoagulation in 16.7%. Complications requiring surgical management occurred in 4 eyes (13.3%). CONCLUSION: Pars planitis treated with adequate medical and surgical procedures has a good prognosis in most cases.

[Molecular diagnosis of retinoblastoma: molecular epidemiology and genetic counseling]. / Diagnóstico molecular del retinoblastoma: epidemiología molecular y consejo genético.

BACKGROUND AND OBJECTIVE: Retinoblastoma, a prototype of hereditary cancer, is the most common intraocular tumor in children and a potential cause of blindness from therapeutic eye ablation, second tumors in germ line mutation carriers, and even death when untreated. The molecular scanning of RB1 in search of germ line mutations in 213 retinoblastoma patients from Spain, Cuba, Colombia and Serbia, has led to the detection of 106 mutations whose knowledge is important for genetic counselling and characterization of phenotypic-genotypic relations. PATIENTS AND METHOD: Mutational study (PCR-sequentiation and microsatellites analysis) in patients with retinoblastoma, from Spain, Cuba, Colombia and Serbia. RESULTS: 45% of mutations, including most of the frame shift (FS), missense (MS) and splicing (SP), were new, while all nonsense mutations (NS) corresponded to hypermutable sites in RB1. Germ line mutations were found in 22% of unilateral sporadic patients. The incidence of SP plus MS mutations in this group of patients was greater (p = 0.018) than in bilateral patients. The frequency of SP mutations was higher (p = 0.0003) in Spain and France than in Germany and United Kingdom, while the incidence of NS mutations was lower (p = 0.0006). SP mutations were associated with the low penetrance phenotype and were also overrepresented (p = 0.018) in patients with delayed retinoblastoma onset. CONCLUSIONS: Mutational scanning of unilateral patients is important for genetic counselling and may help decipher the molecular mechanisms leading to low penetrance or expressivity. The functional characterization of mutations associated with low-penetrance or expressivity phenotypes and the molecular classification of tumors using multiple expression profiling is important for a better understanding of the retinoblastoma pathogenesis.

Diagnóstico molecular del retinoblastoma: epidemiología molecular y consejo genético / Molecular diagnosis of retinoblastoma: molecular epidemiology and genetic counseling

Fundamento y objetivo: El retinoblastoma, prototipo de cáncer hereditario, puede causar ceguera, por enucleación terapéutica, segundos tumores en pacientes con mutación germinal e incluso muerte si no se trata. El diagnóstico molecular de 213 pacientes a lo largo de 5 años ha conducido a la detección de 106 mutaciones que se analizan desde la perspectiva de la epidemiología molecular y consejo genético. Pacientes y método: Estudio mutacional (reacción en cadena de la polimerasa, secuenciación y análisis de microsatélites) en pacientes con retinoblastoma procedentes de España, Cuba, Colombia y Serbia. Resultados: Un 45% de las mutaciones analizadas son nuevas y corresponden a mutaciones de tipo de corrimiento de pauta de lectura, cambio de aminoácido o procesado del ácido ribonucleico. Todas las mutaciones sin sentido corresponden a sitios de alta mutabilidad. La tasa de detección de mutaciones en pacientes unilaterales esporádicos es alta (22%). En este grupo de pacientes se detecta una mayor incidencia (p = 0,018) de mutaciones de cambio de aminoácido y procesamiento. España y Francia muestran una incidencia mayor de mutaciones del procesado (p = 0,0003) que Alemania y el Reino Unido, países en los que predominan las mutaciones sin sentido (p = 0,0006). Las mutaciones del procesado se asocian al fenotipo de baja penetración y retraso en la aparición de tumores (p = 0,018). Conclusiones: La incidencia de mutaciones germinales en pacientes unilaterales y las relaciones fenotipo/genotipo analizadas indican la necesidad del consejo genético basado en el diagnóstico molecular temprano. La mejora de las técnicas diagnósticas, la caracterización funcional de mutaciones asociadas a baja penetrancia o expresividad y el estudio del transcriptoma de los tumores son objetivos necesarios para definir mejor la patogenia del retinoblastoma

Background and objective: Retinoblastoma, a prototype of hereditary cancer, is the most common intraocular tumor in children and a potential cause of blindness from therapeutic eye ablation, second tumors in germ line mutation carriers, and even death when untreated. The molecular scanning of RB1 in search of germ line mutations in 213 retinoblastoma patients from Spain, Cuba, Colombia and Serbia, has led to the detection of 106 mutations whose knowledge is important for genetic counselling and characterization of phenotypic-genotypic relations. Patients and method: Mutational study (PCR-sequentiation and microsatellites analysis) in patients with retinoblastoma, from Spain, Cuba, Colombia and Serbia. Results: 45% of mutations, including most of the frame shift (FS), missense (MS) and splicing (SP), were new, while all nonsense mutations (NS) corresponded to hypermutable sites in RB1. Germ line mutations were found in 22% of unilateral sporadic patients. The incidence of SP plus MS mutations in this group of patients was greater (p = 0.018) than in bilateral patients. The frequency of SP mutations was higher (p = 0.0003) in Spain and France than in Germany and United Kingdom, while the incidence of NS mutations was lower (p = 0.0006). SP mutations were associated with the low penetrance phenotype and were also overrepresented (p = 0.018) in patients with delayed retinoblastoma onset. Conclusions: Mutational scanning of unilateral patients is important for genetic counselling and may help decipher the molecular mechanisms leading to low penetrance or expressivity. The functional characterization of mutations associated with low-penetrance or expressivity phenotypes and the molecular classification of tumors using multiple expression profiling is important for a better understanding of the retinoblastoma pathogenesis

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients.

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.

Imaging of orbital disorders in pediatric patients.

The spectrum of orbital lesions occurring in childhood is wide, including a variety of both benign and malignant disorders. Although physical examination and fundoscopy may aid in establishing the diagnosis of retro-ocular lesions, imaging remains a critical step in the evaluation of the pediatric orbit. Ultrasonography, CT, and MR imaging are the primary modalities for the evaluation of the diseased orbit, and careful observation of the characteristic radiological features usually leads to correct diagnosis; however, some of the lesions look very similar and are difficult to differentiate from each other. The purpose of this article is to review the common and unusual entities that may involve the pediatric orbit, to describe the radiological features, and to evaluate the efficacy of US, CT, and MRI in the diagnosis and management of these conditions.

Apolipoprotein D expression in retinoblastoma.

The presence of apolipoprotein D (apoD) synthesis in brain suggests that this protein could play a major role in mediating neuronal degeneration and regeneration in the central nervous system (CNS). For instance, apoD is overexpressed in neural tissues in Niemann-Pick's type C disease, after acute CNS injury and cerebral ischemia, Alzheimer's disease and by a number of different tumors such as breast, prostate, ovarian and endometrial carcinomas. Recent data have raised the possibility that apoD expression could be a marker of cellular differentiation and growth arrest. By immunohistochemistry, we evaluated the tumoral expression of apoD in retinoblastoma, a tumor that results from malignant transformation of primitive retinal cells before final differentiation, and analyzed the possible relationship with clinicopathological parameters such as: age, sex, histological type, staging, local invasion, metastasis, preoperative or postoperative treatment and bilateral tumors. A total of eleven retinoblastomas (55%) showed apoD-positive immunostaining, but no significant correlation was found between apoD expression and patient or tumor characteristics.