Annagiusi Gargiulo

Filamin A Is Mutated in X-Linked Chronic Idiopathic Intestinal Pseudo-Obstruction with Central Nervous System Involvement

We have previously reported that an X-linked recessive form of chronic idiopathic intestinal pseudo-obstruction (CIIPX) maps to Xq28. To select candidate genes for the disease, we analyzed the expression in murine fetal brain and intestine of 56 genes from the critical region. We selected and sequenced seven genes and found that one affected male from a large CIIPX-affected kindred bears a 2-bp deletion in exon 2 of the FLNA gene that is present at the heterozygous state in the carrier females of the family. The frameshift mutation is located between two close methionines at the filamin N terminus and is predicted to produce a protein truncated shortly after the first predicted methionine. Loss-of-function FLNA mutations have been associated with X-linked dominant nodular ventricular heterotopia (PVNH), a central nervous system (CNS) migration defect that presents with seizures in females and lethality in males. Notably, the affected male bearing the FLNA deletion had signs of CNS involvement and potentially has PVNH. To understand how the severe frameshift mutation we found can explain the CIIPX phenotype and its X-linked recessive inheritance, we transiently expressed both the wild- type and mutant filamin in cell culture and found that filamin translation can start from either of the two initial methionines in these conditions. Therefore, translation of a normal shorter filamin can occur in vitro from the second methionine downstream of the 2-bp insertion we found. We confirmed this, demonstrating that the filamin protein is present in the patients lymphoblastoid cell line that shows abnormal cytoskeletal actin organization compared with normal lymphoblasts. We conclude that the filamin N terminal region between the initial two methionines is crucial for proper enteric neuron development.

MicroRNA-Restricted Transgene Expression in the Retina

Background Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.

AAV-mediated tyrosinase gene transfer restores melanogenesis and retinal function in a model of oculo-cutaneous albinism type I (OCA1).

Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.

Molecular and Clinical Characterization of Albinism in a Large Cohort of Italian Patients

PURPOSE The purpose of this study was to identify the molecular basis of albinism in a large cohort of Italian patients showing typical ocular landmarks of the disease and to provide a full characterization of the clinical ophthalmic manifestations. METHODS DNA samples from 45 patients with ocular manifestations of albinism were analyzed by direct sequencing analysis of five genes responsible for albinism: TYR, P, TYRP1, SLC45A2 (MATP), and OA1. All patients studied showed a variable degree of skin and hair hypopigmentation. Eighteen patients with distinct mutations in each gene associated with OCA were evaluated by detailed ophthalmic analysis, optical coherence tomography (OCT), and fundus autofluorescence. RESULTS Disease-causing mutations were identified in more than 95% of analyzed patients with OCA (28/45 [62.2%] cases with two or more mutations; 15/45 [33.3%] cases with one mutation). Thirty-five different mutant alleles were identified of which 15 were novel. Mutations in TYR were the most frequent (73.3%), whereas mutations in P occurred more rarely (13.3%) than previously reported. Novel mutations were also identified in rare loci such as TYRP1 and MATP. Mutations in the OA1 gene were not detected. Clinical assessment revealed that patients with iris and macular pigmentation had significantly higher visual acuity than did severe hypopigmented phenotypes. CONCLUSIONS TYR gene mutations represent a relevant cause of oculocutaneous albinism in Italy, whereas mutations in P present a lower frequency than that found in other populations. Clinical analysis revealed that the severity of the ocular manifestations depends on the degree of retinal pigmentation.

Evaluation of Italian patients with leber congenital amaurosis due to AIPL1 mutations highlights the potential applicability of gene therapy.

PURPOSE To evaluate the suitability of gene delivery-based approaches as potential treatment of Leber congenital amaurosis 4 (LCA4) due to AIPL1 mutations. METHODS Genomic DNA from patients was analyzed using a microarray chip and direct sequencing. A detailed clinical evaluation including fundus autofluorescence (FAF) and optical coherence tomography (OCT) was performed in patients with AIPL1 mutations. Aipl1 null mice and porcine eyes were subretinally injected with adeno-associated viral (AAV) vectors harboring the human AIPL1 coding sequence. RESULTS We identified 10 LCA4 patients with mutations in AIPL1. The p.W278X sequence variation was the one most frequently found. Clinical assessment revealed common features including diffuse retinal dystrophies and maculopathy. However, optical coherence tomography showed partially retained photoreceptors in extramacular regions at all ages. The fundus autofluorescence was elicitable at the posterior pole and absent in the fovea. AAV-mediated gene transfer in Aipl1 -/- mice was associated with restoration of AIPL1 and βPDE expression in photoreceptors and protection from degeneration. Administration of a clinically relevant dose of AAV2/8-AIPL1 to the preclinical large porcine retina resulted in high level of AIPL1 photoreceptor expression in the absence of toxicity. CONCLUSIONS Using advanced imaging diagnostics we showed that maculopathy is a main feature of LCA4. We identified retinal areas at the posterior pole with surviving photoreceptors present even in adult LCA4 patients, which could be the target of gene therapy. The possible use of gene therapy for LCA4 is additionally supported by the protection from photoreceptor degeneration observed in Aipl 1-/- mice and by the high levels of photoreceptor transduction in the absence of toxicity observed after AAV2/8 delivery to the large porcine retina.

Immunohistochemical and immunochemical characterization of the distribution of leptin-like proteins in the gastroenteric tract of two teleosts (Dicentrarchus labrax and Carassius auratus L.) with different feeding habits.

Leptin is a modulator of food intake and energy homeostasis both in mammals and in some species of nonmammals vertebrates. In this study, we reported for the first time, using an immunohistochemical and immunochemical approach, the presence and distribution of immunoreactivity to leptin‐like protein in the gastroenteric tract of Dicentrarchus labrax (bass) and Carassius auratus (goldfish), two teleostean species with different feeding and different adaptative morphological organization of the gastroenteric tract. Bass stomach showed intense immunoreactivity to leptin‐like protein in all regions, with immunoreactive cells located at the base of the mucosal plicae and at the apical margin of the gastric crypts. Immunoreactive fibers and neuronal cells were observed close to vascular structures in the pyloric region. In bass and goldfish intestine, rare immunoreactive cells were observed along the mucosal epithelium mostly at the base or the apex of intestinal folds in the proximal and medium intestine; numerous immunoreactive nerve fibers in the circular and longitudinal layers of the tunica muscolaris as well as in the myenteric plexus were observed. Western blot analysis recognized a ∼15 kDa signal with a similar expression pattern for goldfish and sea bass. Our results could contribute to confirm the evolutive conservation of leptin‐like proteins and their probably precocious functional diversification in fish. Microsc. Res. Tech., 2011.

The Role of Optical Coherence Tomography in an Atypical Case of Oculocutaneous Albinism: A Case Report

Background: Oculocutaneous albinism is a group of autosomal recessive disorders featuring hypopigmentation of the hair, skin and eyes. Ocular signs associated with the disease are nystagmus, decreased visual acuity, hypopigmentation of the retina, foveal hypoplasia, translucency of the iris, macular transparency, photophobia and abnormal decussation of nerve fibers at the chiasm. Case Report: An 8-year-old Caucasian girl presented to our clinic ‘Referral Center for Hereditary Retinopathies’ of the Second University of Naples with a diagnosis of Stargardt disease and a progressive reduction in visual acuity in both eyes. She underwent a complete ophthalmic examination including standard electroretinography and optical coherence tomography (OCT). A molecular analysis was also performed. Best-corrected visual acuity was 20/30 in the right eye and 20/40 in the left eye. Biomicroscopy of the anterior segment revealed a transparent cornea, in situ and transparent lens and normally pigmented iris. A mild diffuse depigmentation and macular dystrophy were observed at fundus examination. Standard electroretinography showed normal scotopic and photopic responses. OCT revealed high reflectivity across the fovea without depression. The typical OCT pattern led us to direct the molecular analysis towards the genes involved in oculocutaneous albinism. The molecular analysis identified mutations in the TYR gene. Conclusion: In this case, the role of OCT was crucial in guiding the molecular analysis for the diagnosis of albinism. OCT is therefore instrumental in similar cases that do not present typical characteristics of a disease. The case also proves the relevance of molecular analysis to confirm clinical diagnoses in hereditary retinal diseases.