José Paulo Cabral de Vasconcellos

Novel mutation in the MYOC gene in primary open angle glaucoma patients

Editor—Glaucoma is the worlds leading cause of irreversible blindness1 and is characterised by progressive optic disc cupping with corresponding visual field loss. Both intraocular pressure (IOP) and positive family history are risk factors for the development of the disease.2 Juvenile open angle glaucoma (JOAG) is a subtype of open angle glaucoma characterised by an early onset (10 to 35 years of age) and autosomal dominant inheritance with high penetrance,3 a characteristic which has led several authors to investigate affected families in an attempt to identify a gene or genes associated with this condition.4-10 With the use of genetic linkage analysis in families with JOAG, a genetic locus (GLC1A) was recognised on chromosome 1q21-q31.4 The gene associated with GLC1A has been identified and it codifies a 57 kDa protein named trabecular meshwork induced glucocorticoid response protein (TIGR),10also known as myocilin (MYOC).11 The MYOC gene is composed of three exons of 604, 126, and 785 bp, respectively.12 During screening for mutations in the MYOC gene in 25 unrelated Brazilian patients with JOAG, an unreported mutation (Cys433Arg) was detected, present in seven of them. Patients were followed at the Glaucoma Service of the State University of Campinas, Brazil. They underwent an ocular examination, including gonioscopy by Posner lens, applanation tonometry, slit lamp biomicroscopy, optic nerve evaluation, and automated perimetry (Humphrey 630, program 30-2). JOAG was defined as the presence of characteristic bilateral optic nerve damage and visual field loss in the presence of an open angle in subjects younger than 36 years of age. Each patient included in this study came from different families according to interview data. The study was approved by the Ethics Committee of the State University of Campinas. At the time of the ocular examination, the mean age …

Keeping an eye on myocilin: a complex molecule associated with primary open-angle glaucoma susceptibility.

MYOC encodes a secretary glycoprotein of 504 amino acids named myocilin. MYOC is the first gene to be linked to juvenile open-angle glaucoma (JOAG) and some forms of adult-onset primary open-angle glaucoma (POAG). The gene was identified as an up-regulated molecule in cultured trabecular meshwork (TM) cells after treatment with dexamethasone and was originally referred to as trabecular meshwork-inducible glucocorticoid response (TIGR). Elevated intraocular pressure (IOP), due to decreased aqueous outflow, is the strongest known risk factor for POAG. Increasing evidence showed that the modulation of the wild-type (wt) myocilin protein expression is not causative of glaucoma while some misfolded and self-assembly aggregates of mutated myocilin may be associated with POAG in related or unrelated populations. The etiology of the disease remains unclear. Consequently, a better understanding of the molecular mechanisms underlyingPOAG is required to obtain early diagnosis, avoid potential disease progression, and develop new therapeutic strategies. In the present study, we review and discuss the most relevant studies regarding structural characterizations, expressions, molecular interactions, putative functions of MYOC gene and/or its corresponding protein in POAG etiology.

CYP1B1 Gene Analysis in Primary Congenital Glaucoma Brazilian Patients Novel Mutations and Association With Poor Prognosis

PurposeTo determine the spectrum of CYP1B1 gene mutations in Brazilian patients with primary congenital glaucoma, and to correlate the presence of alterations in the CYP1B1 gene sequence with clinical aspects of the disease. Materials and MethodsThirty nonrelated patients with primary congenital glaucoma were studied. Molecular analysis consisted of the codifying region sequencing (exons 2 and 3) and intron/exon boundaries. ResultsCYP1B1 gene mutations were present in 9 (30%) of the 30 patients. The structural changes in the CYP1B1 gene previously described in the literature and observed in our study were Q19X, P437L, A443G, g.4340delG, g.7901_79013delGAGTGCAGGCAGA, g.8182delG, and g.8214_8215delG. Three new mutations were observed: 4635delT, 4523delC, and L378Q, in addition to 3793T→C, R48G, A119S, L432V, D449D, and N453S polymorphisms. Patients carrying CYP1B1 gene mutations needed more surgical procedures to control intraocular pressure, either when both eyes were evaluated (P=0.003) or when the worst eye of the patient was analyzed (P=0.011). In relation to the number of affected eyes, all patients with mutations (n=9/9) developed bilateral glaucoma, whereas 11/21 patients without mutations in the CYP1B1 gene had bilateral glaucoma (P=0.013). ConclusionsIn this group of primary congenital glaucoma patients, a 30% mutation frequency in the CYP1B1 gene was observed. The presence of mutations was associated with a more severe form of the disease, requiring more surgeries for intraocular pressure control and with a higher rate of bilateral cases.

Lack of association between optineurin gene variants T34T, E50K, M98K, 691_692insAG and R545Q and primary open angle glaucoma in Brazilian patients.

Purpose: To verify the frequencies of T34T, E50K, M98K, 691_692insAG, and R545Q variants in the optineurin (OPTN) gene in Brazilian subjects with primary open-angle glaucoma (POAG) and controls. Patients and Methods: Ninety-nine patients with POAG and 100 normal controls were enrolled in this study. The frequency of alterations in the OPTN gene was analyzed by direct sequencing and enzymatic digestion of PCR products. Results: None of the five alterations evaluated was significantly associated with POAG when compared to controls. However, the T34T silent change was present in greater frequency in POAG patients (37.37% vs. 23.00% in controls), while the R545Q change was more prevalent in controls (23.00% vs. 10.10% in POAG). The M98K and 691_692insAG presented with low frequencies in POAG patients (1.01% and 2.02%, respectively) and controls (2.00% and 2.00%, respectively). The E50K substitution was not observed. Conclusion: Our data show no association between the five evaluated variants and POAG in the Brazilian population.

Tratamento clínico do glaucoma em um hospital universitário: custo mensal e impacto na renda familiar

Purpose: To verify the social characteristics and the impact of glaucoma treatment on the familial income of patients followed at a university hospital. Methods: One hundred and forty six glaucomatous patients were interviewed at the Hospital das Clinicas da Universidade de Campinas to evaluate their social economic profile. The questionnaire investigated the occupation, the individual and familial income, as well as the type and frequency of the antiglaucomatous drugs used by each patient. Knowing the monthly cost of antiglaucomatous drugs available in Brazil, we were able to calculate the monthly cost of glaucoma treatment and the percentage of committed familial income. Results: The mean monthly cost of glaucoma treatment was 36.09 ± 31.99 reais, which corresponded to 15.5% of the familial income. Thirty-six (24%) patients had 25 percent or more of the familial income spent on their treatment. Sixty-six (45.2%) patients had difficulty in buying their medications. Factors associated with this difficulty included low familial income (p=0.0001), and high percentage of the income used to buy the drugs (p=0.0002). Conclusion: The cost of glaucoma treatment is high compared to the income of patients treated at a public institution. This population has a low familial income, of which a high percentage is required to acquire antiglaucoma medications. We suggest that these patients may be at risk for low compliance due to economical limitations.

Intraoperative mitomycin-C without conjunctival and Tenon’s capsule touch in primary trabeculectomy

OBJECTIVE To verify whether intraoperative mitomycin C (MMC) without conjunctival and Tenons touch is effective in inhibiting the development of thin, avascular blebs in eyes undergoing primary trabeculectomy. DESIGN Noncomparative, interventional study. PARTICIPANTS Fifteen eyes of 15 consecutive patients undergoing primary trabeculectomy. INTERVENTION All eyes underwent trabeculectomy with intraoperative MMC (0.25 mg/ml for 3 minutes) without either conjunctival or Tenons touch. Patients were examined 1 month, 3 months, 6 months, and 12 months after surgery. Intraocular pressure (IOP) and number of medications were evaluated at each examination. The appearance of the bleb was classified at the last examination into one of three groups: flat and vascularized; elevated but not avascular; or elevated, thin, and avascular. MAIN OUTCOME MEASURES Intraocular pressure, number of antiglaucoma medications, and appearance of the bleb. RESULTS Preoperative mean IOP was 30.57 +/- 10.92 mmHG: Statistically significant IOP reductions were observed 1 month, 3 months, 6 months, and 1 year after surgery (P < 0.01). Twelve months after surgery, the mean IOP was 14.92 +/- 6.53 mmHG: Five eyes (33.3%) showed an IOP less than 15 mmHg without antiglaucoma medication at the 12-month examination. The bleb was considered elevated, thin, and avascular in 12 of 15 eyes (80%) at the end of follow-up. CONCLUSIONS Intraoperative MMC at 0.25 mg/ml for 3 minutes without either conjunctival or Tenons touch was not effective in eliminating the development of thin, avascular blebs in eyes undergoing primary trabeculectomy.

Reproducibility of water drinking test performed at different times of the day

PURPOSE To evaluate the reproducibility of water drinking test (WDT) performed at different times of the day, in primary open angle glaucoma (POAG) patients and normal individuals. METHODS Fifteen patients with POAG and 30 normal individuals underwent three WDTs at different times of the day (7 AM, 12 PM, and 5 PM) on 3 different days. Test results in POAG patients and normal individuals were compared. Agreement and correlation of intraocular pressure (IOP) baseline levels, peak levels, and IOP change (peak IOP--baseline IOP) on tests performed at different times were evaluated. Only right eye measurements were analyzed. RESULTS Mean baseline IOP, peak IOP and IOP change were significantly higher in POAG patients than in normal individuals, at all time intervals (p<0.05). The Bland-Altman analysis demonstrated limits of agreement for IOP peak levels and IOP changes larger than the clinically acceptable (>3 mmHg), even though Pearsons test revealed good correlation among the results. CONCLUSION The mean IOP peak and mean IOP change observed during WDT are significantly higher in POAG patients than in control individuals. Low levels of agreement among WDTs performed at different times of the day suggest a poor reproducibility of WDT, which may limit its applicability for the diagnosis and follow-up of glaucoma.

New mutation in the myocilin gene segregates with juvenile-onset open-angle glaucoma in a Brazilian family.

Mutations in the myocilin gene (MYOC) account for most cases of autosomal dominant juvenile-onset open-angle glaucoma (JOAG), an earlier and more severe form of POAG. We accessed seven members of a Brazilian JOAG family by clinical and molecular investigation. Four out of seven family members were diagnosed with JOAG. All of these patients presented high intraocular pressure and two of them were bilaterally blind. The disease onset varied from 20 to 30years old. There was a nine-year-old family member who had not yet manifested the disease, although he was also a carrier of the mutation. Ophthalmologic examination included: evaluation of the visual field and optic disc, intraocular pressure measurement, and gonioscopy. The three exons and intron/exon junctions of the MYOC gene were screened for mutations through direct sequencing of PCR-amplified DNA fragments. Mutation screening revealed an in-frame mutation in the third exon of the MYOC gene: an insertion of six nucleotides between the cDNA positions 1187 and 1188 (c.1187_1188insCCCAGA, p.D395_E396insDP). This mutation presented an autosomal dominant pattern of inheritance, segregating with the disease in four family members for three generations, and it was absent in 60 normal controls. We also performed a computational structure modeling of olfactomedin-like domain of myocilin protein and conducted in silico analysis to predict the structural changes in the myocilin protein due to the presence of the mutation. These findings may be important for future diagnosis of other presymptomatic family members, as well as for the increase of the panel of MYOC mutations and their effects on phenotype.

Intra- and interobserver reproducibility of Bruch's membrane opening minimum rim width measurements with spectral domain optical coherence tomography

To investigate the reproducibility of Bruchs membrane opening minimum rim width (BMO‐MRW) and retinal nerve fibre layer thickness (RNFLT) measurements using spectral domain optical coherence tomography (SD‐OCT). Additionally, to investigate the reproducibility of BMO area measurements and fovea to BMO centre (FoBMO) angle.

Allogeneic Mesenchymal Stem Cell Transplantation in Dogs with Keratoconjunctivitis Sicca

Keratoconjunctivitis sicca (KCS) is a dysfunction in tear production associated with clinical signs, which include conjunctival hyperemia, ocular discharge, discomfort, pain, and, eventually, corneal vascularization and pigmentation. Immunosuppressive drugs are routinely administrated for long periods to treat KCS but with side effects and limited results. Evaluation of the clinical benefits of intralacrimal transplantation of allogeneic mesenchymal stem cells (MSCs) in dogs with mild-moderate and severe KCS was done. A total of 24 eyes with KCS from 15 dogs of different breeds were enrolled in the present study. A single transplantation of MSCs (1 × 106) directly into lacrimal glands (dorsal and third eyelid) was performed. The Schirmer tear tests (STTs) and ocular surface improvements were used to assess short- and long-term effects of these cells. The STTs were carried out on day 0 (before MSCs transplantation) and on days 7, 14, 21, and 28, as well as 6 and 12 months after MSC transplantation. Our data demonstrate that allogeneic MSC transplantation in KCS dogs is safe since no adverse effects were observed immediately after transplantation and in short- and long-term follow-ups. A statistically significant increase in the STT and ocular surface improvements was found in all eyes studied. In all the eyes with mild-moderate KCS, STT values reverted to those of healthy eyes, while in eyes with severe KCS, although complete reversion was not found, there was improvement in tear production and in other clinical signs. Our study shows that a single dose of a low number of MSCs can be used to treat KCS in dogs. In contrast to immunosuppressive drug use, MSC transplantation has an effect over a long period (up to 12 months), even after a single administration, and does not require daily drug administration.