Search

AIMS/PURPOSE To investigate Leber congenital amaurosis (LCA) patients' expectations, decision-making processes and gene therapy-related concerns. METHODS Using a qualitative approach, we explored perceptions of gene therapy and clinical trials among individuals with LCA. Young adults with a clinical diagnosis of LCA were recruited through the Ocular Genetics Programme at the Hospital for Sick Children. Semi-structured interviews were conducted with ten patients and analysed following the principles of qualitative description. RESULTS Study participants were aware of ongoing gene therapy research trials and actively sought information regarding advances in ophthalmology and vision restoration. The majority of participants would enrol or were enrolled in a gene-replacement therapy trial, while a minority was ambivalent or would not enrol if provided an opportunity. Participants attributed different values to clinical trials, which influenced their willingness to participate. Intrinsic factors related to coping, adaptation to vision loss and resilience also influenced decision-making. DISCUSSION This study highlights the complex factors involved in gene-therapy-related decision-making and acts as a proponent for adopting patient-centred care strategies when counselling individuals considering gene therapy or clinical trial participation.

Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

Genetic eye diseases (GEDs) are clinically and genetically heterogeneous. Results from molecular genetic testing are redefining the etiology and clinical spectrum of these heritable diseases, helping to explain the observed overlap in clinical phenotypes. Patients and parents benefit from DNA analysis when results confirm a diagnosis or make a diagnosis more specific. Accurate counseling can then be provided regarding the prognosis, presymptomatic diagnosis, life and family-planning issues and whether to anticipate other ocular or systemic medical problems. Patients hope that mutation identification will enable their participation in future clinical trials for treatment. Requests for molecular testing are increasing while those in the visually impaired world and the healthcare professionals who manage them wait to learn long-term results of the first human clinical trials for a GED to be treated with gene therapy, RPE65-type Leber congenital amaurosis. The advantages and disadvantages of molecular testing...

Background: Patients who suffer from ocular genetic diseases have special needs in terms of diagnosis and management of rare entities, low-vision needs, genetic counselling, and psychosocial adjustments that are usually not addressed by an ophthalmologist alone.The Ocular Genetics Program (OGP) at the Hospital for Sick Children,Toronto, was established in 1994 to provide comprehensive, multidisciplinary care of patients with inherited eye disorders.We now assess the benefits of such a program and of integrating research into the care of patients. Methods: We report our experience in developing a multidisciplinary ocular genetics program and the results of a pilot patient satisfaction survey that involved 61 patients. Results: The OGP multidisciplinary aspects are described. Of the 61 patients surveyed, 98% stated that they were satisfied with the OGP; 93%–96% of patients were content with "one day of appointments","understanding of eye problem", and "coordination of ancillary tests such as visual fields test, electrophysiology, and others"; and for 70%–86% of respondents "waiting time to get an appointment", "information received on current research", and "primary health care provider adequately informed" were satisfactory. Interpretation: The OGP is a unique service in Canada, which strives to provide the comprehensive care needed by ocular genetic patients.High patient satisfaction is an indicator of the success of this approach. Long waiting times for appointments and application of laboratory research in clinical care remain challenging.

To investigate psychosocial adjustment to visual loss in patients with retinitis pigmentosa (RP).Cross-sectional study.Thirty-three legally blind patients with RP participated in the study. Information regarding the patients' adjustment to their visual loss was obtained using the Psychological Adjustment to Illness Scale (PAIS-SR). Seven psychosocial domains were tested: health-care orientation, vocational environment, domestic environment, sexual relationships, extended family relationships, social environment, and psychological distress. These scores were compared with the psychosocial adjustment of patients with diabetes.Significantly elevated scores (a high score reflecting poor adjustment) were seen in four out of seven domains. The highest relative score was seen in the health-care orientation domain (65 +/- 14, p0.001), followed by vocational function (61 +/- 11, p0.001), social environment (58 +/- 9, p0.001), and extended family relationships (55 +/- 9, p0.05). The total PAIS score was significantly elevated (58 +/- 8; p0.001).Patients with RP have difficulties in adjusting to their visual loss particularly with respect to health-care orientation, vocational environment, social environment, and extended family relationships. They face more difficulties in these domains than diabetic patients in adjusting to their illness.

Retinoblastoma and Wilms' tumor are rare childhood embryonic tumors associated with loss or inactivation of tumor suppressor genes, RB1 located within 13q14, and WT1 located within 11p13. Interchromosomal insertional translocations occur rarely, and such rearrangements within RB1 or WT1, even rarer. We report a unique family in which an insertional translocation of a chromosomal segment that included band 13q14 inserted into 11p13 caused childhood Wilms' tumor in the father, and whose child developed bilateral retinoblastoma. This is the first case of an insertional translocation that caused both tumors. This insertional translocation had significant consequences for genetic counseling and in utero diagnosis. The estimated risk for an offspring of this father to develop Wilms' tumor is up to 50%, to develop retinoblastoma up to 25%, to have neither tumor 25%, and to have both tumors 0%.

Glaucoma is a leading cause of irreversible blindness in Canada. Congenital glaucoma usually manifests during the first years of life and is characterised by severe visual loss and autosomal recessive inheritance. Two disease loci, on chromosomes 1p36 and 2p21, have been associated with various forms of congenital glaucoma. A branch of a large six generation family from a consanguineous Amish community in south western Ontario was affected with congenital glaucoma and was studied by linkage and mutational analysis to identify the glaucoma related genetic defects. Linkage analysis using the MLINK component of the LINKAGE package (v 5.1) showed evidence of linkage to the 2p21 region (Zmax=3.34, θ=0, D2S1348 and D2S1346). Mutational analysis of the primary candidate gene, CYP1B1, was done by direct cycle sequencing, dideoxy fingerprinting analysis, and fragment analysis. Two different disease causing mutations in exon 3, 1410del13 and 1505G→A, both segregated with the disease phenotype. The two different combinations of these alleles appeared to result in a variable expressivity of the phenotype. The compound heterozygote appeared to have a milder phenotype when compared to the homozygotes for the 13 bp deletion. The congenital glaucoma phenotype for this large inbred Amish family is the result of mutations in CYP1B1 (2p21). The molecular information derived from this study will be used to help identify carriers of the CYP1B1 mutation in this community and optimise the management of those at risk of developing glaucoma. Keywords: congenital glaucoma; CYP1B1; gene; genetic counselling

When pondering the term boot camp, one often envisions the rigorous physical training soldiers endure prior to military service. Boot camps challenge individuals to exceed limits that may never have been tested and develop physical, mental, and emotional stamina to prepare for future arduous tasks. The Sisters of the Academy (SOTA) Research Boot Camp exemplifies such an environment, featuring strict training for the “academic battlegrounds” of doctoral attainment and the professoriate. It seeks to develop the intellectual muscles of women who have enlisted to pursue the doctorate and academic careers, stretch them beyond their zones of familiarity, and validate commitment to their research interests. Black women have made phenomenal strides in their quests for higher education, but have yet to reach demographic parity in the ivory tower (Evans, 2007). As such, the camp trains its participants for active duty in the academy, from the dissertation writing stage, toward tenure, and beyond.

Carrier status determination for Duchenne and Becker muscular dystrophies (D/BMD), disorders caused by mutations in the dystrophin gene at Xp21, is complicated by a number of factors. These include a high mutation rate and a 5-10% recombination frequency across the dystrophin gene. For these reasons, linkage analysis frequently gives an inconclusive result, and a direct mutation detection method for females at risk is desirable. Because 65% of the mutations that cause D/BMD are deletions of one or more exons of the dystrophin gene, diagnosis in most affected males is relatively easy using multiplex polymerase chain reaction (PCR) analysis. However, deletion analysis in females is more difficult because of the interference of the normal X chromosome in the deletion assay. We have developed a quantitative PCR-based analysis designated computer-assisted laser densitometry (CALD), which uses the automated fluorescent fragment analysis application of the Applied Biosystems (Foster City, California) automated sequencer. This method has proved to be 100% accurate in retrospective blind studies analysing a total of 351 samples. Subsequent analysis of more than 800 women from more than 400 D/BMD families has shown that a highly accurate carrier risk can be given in more than 90% of cases.

Leber congenital amaurosis (LCA) is an autosomal recessive retinal dystrophy that manifests with genetic heterogeneity. We sequenced the exome of an individual with LCA and identified nonsense (c.507GA, p.Trp169*) and missense (c.769GA, p.Glu257Lys) mutations in NMNAT1, which encodes an enzyme in the nicotinamide adenine dinucleotide (NAD) biosynthesis pathway implicated in protection against axonal degeneration. We also found NMNAT1 mutations in ten other individuals with LCA, all of whom carry the p.Glu257Lys variant.

Purpose of review To introduce the issues specific to the genetic counseling profession for genetic eye diseases. Recent findings To discuss current issues in ocular genetic counseling including the use of a focused ophthalmology pedigree, informed consent in the blind population, genetic testing trends and psychosocial issues. Summary Introduce the time-consuming issues to be addressed in genetic counseling for genetic eye disease patients.

Two-thirds of retinoblastoma cases are unilateral, with most presenting with an advanced stage of disease. Primary enucleation is usually the preferred treatment. Conservative therapy may save less involved eyes.We retrospectively reviewed the presentation, age at diagnosis, classification, genetic status, treatment, and long-term outcome of 130 patients with unilateral retinoblastoma (1988-2008).The mean age at presentation was 26 months. Based on retinoblastoma gene (RB1) status in tumors, germ-line status was defined in 92% of patients; 13% had a germ-line mutation. The primary treatment of 106 patients was enucleation. Severe disease at presentation (International Intraocular Retinoblastoma Classification [IIRC] group E) was significantly (p0.001) associated with adverse histopathological risk factors. Of the 16 patients who underwent eye-conserving therapy, treatment was successful in 9 (IIRC group A, 1; B, 5; C, 3). Two patients with a pertinent family history were diagnosed early and were treated solely with focal therapy. Three patients retained vision of 6/18 or better in the treated eye (median follow-up, 33 months; range, 2-120 months). Seven patients (IIRC group: B, 2; C, 4; D, 1) eventually underwent enucleation. One patient died of metastases following delayed parental consent for enucleation and refusal of prophylactic chemotherapy for high-risk histopathologic features.Chemotherapy/focal therapy can save selected eyes, but primary enucleation is preferred for advanced unilateral retinoblastoma. "Conservative" treatment is an option when there is good potential for useful vision without prolonged, costly therapy with potential side effects. Simple enucleation reduces the risk of masking high-risk pathology and promotes early return to normal life.

RESUME Background: The purpose of this study was to describe our experience with the clinical effects of molecular genetic testing for retinitis pigmentosa (RP) and related retinal dystrophies. Methods: Chart review of 303 consecutive patients with retinal dystrophies was done when blood was sent for molecular genetic testing between 1993 and 2001. Phenotype information was retrieved for patients with identified mutations. The yield of positive and clinically useful results was assessed. Results: Participants comprised 35 patients with Leber congenital amaurosis, 18 with Usher syndrome, and 250 with isolated RP or other retinal dystrophies. Of these 303 participants, 203 (67%) received positive or negative results of molecular testing for an average of 2.7 genes. Positive results were available in 19 patients after an average time interval of 38 ± 22 months (median 33 months, range 1-89 months). No results were received for 84 (28%) patients. In 16 (5%) cases, patients received partial results. Only 19 (6%) patients were found to have sequence changes in RHO, RDS, CRB1, or USH2A, 2 of which were thought to be disease-causing. Only 2 sequence changes were previously documented mutations, but several other novel changes were suspected to be disease-causing mutations also. Interpretation: Molecular testing was helpful only in the minority of cases, largely because of a lack of availability, as well as the complexity of the molecular genetics of RP. Improvements in funding, infrastructure, and molecular knowledge will be necessary to improve the transformation of molecular genetic testing into a clinically relevant bedside tool.

Background Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. Methods The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. Results We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. Conclusion The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.

Ocular albinism type 1 (OA1) is an X-linked disorder, mainly characterized by a severe reduction in visual acuity, foveal hypoplasia, nystagmus, hypopigmentation of the retina, the presence of macromelanosomes in the skin and eyes, and the misrouting of optic pathways, resulting in the loss of stereoscopic vision. We screened the OA1 gene for mutations in three unrelated Canadian and French families and in two isolated patients with OA1. We found three different missense mutations and two different nonsense mutations, three of which were novel. To date, 41 mutations (including missense mutations, insertions, and deletions) have been reported in the OA1 gene. Mutation and polymorphism data for this gene are available from the international albinism center albinism database website: http://www.cbc.umn.edu/tad/oa1map.htm.

Congenital glaucoma refers to a genetically heterogeneous group of distinctive clinical diseases characterised by increased intraocular pressure most often associated with increased corneal diameter, corneal oedema, and consequent visual impairment. Primary congenital glaucoma (PCG) is associated with a primary angle defect, whereas secondary congenital glaucoma is associated with a more generalised developmental anomaly of the anterior segment such as seen in Peters' anomaly. The inheritance of PCG is usually autosomal recessive. Peters' anomaly consists of corneal opacity, defects in the posterior structures of the cornea, and iridocorneal and/or keratolenticular adhesions, and it most frequently occurs sporadically.1Over 50% of subjects develop glaucoma in childhood. Numerous aetiologies have been proposed including chromosomal abnormalities, teratogens,1 and mutations in the eye developmental genes PAX6 2-4 and PITX2 .5 However, large subsets of Peters' anomaly cases are without molecular characterisation. Primary congenital glaucoma has been linked to chromosomes 1p36 (GLC3B) and 2p21 (GLC3A), but only the GLC3A gene has been identified. This gene, CYP1B1 , encodes a broadly expressed cytochrome P450 enzyme (P4501B1) whose natural substrate is unknown.6 7 It is proposed to be the major gene for disease causing mutations in primary congenital glaucoma.6-11 A variety of chain terminating and missense CYP1B1 mutations have been described.6-9 We report two novel mutations in CYP1B1 in a patient who had Peters' anomaly with secondary congenital glaucoma. The subject, a male of Native Indian (Mohawk)/French Canadian background, presented with a history of bilateral cloudy corneas and tearing since birth. Examination at 3 weeks of age showed bilateral corneal oedema with central corneal opacities, superficial pannus (corneal vascularisation), and iridocorneal adhesions with a well formed anterior chamber (fig 1, above). Iris was present for …

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.

Type I oculocutaneous albinism (OCA) is an autosomal recessive disorder caused by the reduction or the absence of tyrosinase (TYR) activity in melanocytes of the skin, hair and eyes. Here we report an analysis of 45 patients with OCA. We found five novel mutations in the tyrosinase gene involved in the pathogenesis of oculocutaneous albinism type IA or type IB (OCA-1A/B) in five unrelated patients. Three mutations are missense mutations (G109R, P205T and H256Y) and two are nucleotide deletions (336-337delCA and 678-680delAGG). One patient is homozygous for the previously known V275F mutation but has an extremely mild OCA phenotype and has no eye features typical of OCA. In several patients we discovered only one or even no mutation in the coding sequence of the TYR gene. Thus, this disease may also result from mutations in non coding regions of the gene or in another gene involved in the biosynthesis of melanin. Hum Mutat 17:352, 2001. © 2001 Wiley-Liss, Inc.

The multiple copies of the human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters that map to the middle of the short arms of chromosomes 13, 14, 15, 21, and 22. Concerted evolution of the gene family is thought to be mediated by interchromosomal recombination between rDNA repeat units, but such events would also result in conservation of the sequences distal to the rDNA on these five pairs of chromosomes. To test this possibility, a DNA fragment spanning the junction between rDNA and distal flanking sequence has been cloned and characterized. Restriction maps, sequence data, and gene mapping studies demonstrate that (i) the rRNA genes are transcribed in a telomere-to-centromere direction, (ii) the 5' end of the cluster and the adjacent non-rDNA sequences are conserved on the five pairs of chromosomes, and (iii) the 5' end of the cluster is positioned about 3.7 kb upstream from the transcription initiation site of the first repeat unit. The data support a model of concerted evolution by interchromosomal recombination.

Timely molecular diagnosis of RB1 mutations enables earlier treatment, lower risk, and better health outcomes for patients with retinoblastoma; empowers families to make informed family-planning decisions; and costs less than conventional surveillance. However, complexity has hindered clinical implementation of molecular diagnosis. The majority of RB1 mutations are unique and distributed throughout the RB1 gene, with no real hot spots. We devised a sensitive and efficient strategy to identify RB1 mutations that combines quantitative multiplex polymerase chain reaction (QM-PCR), double-exon sequencing, and promoter-targeted methylation-sensitive PCR. Optimization of test order by stochastic dynamic programming and the development of allele-specific PCR for four recurrent point mutations decreased the estimated turnaround time to

We report the identification and characterization of a family of repeated restriction fragments whose molecular organization is apparently specific to the human X chromosome. This fragment, identified as an ethidium bromide-staining 2.0 kilobase (kb) band in BamHI-digested DNA from a Chinese hamster-human somatic cell hybrid containing a human X chromosome, has been cloned into pBR325 and characterized. The 2.0 kb repeated family has been assigned to the Xp11 leads to Xq12 region on the X by Southern blot analysis of somatic cell hybrids and is predominantly arranged in tandem clusters of up to seven 2.0 kb monomers. Homologous DNA sequences, not organized as 2.0 kb BamHI fragments, are found elsewhere on the X chromosome and on at least some autosomes, but are not found on the Y chromosome. From a dosing experiment using various amounts of the cloned repeat, we estimate that there are 5,000-7,500 copies of the 2.0 kb BamHI repeat per haploid genome. Since the vast majority, if not all, of these are confined to the X chromosome, this repeated DNA family must account for 5-10% of all X chromosome DNA and must constitute the major sequence component of the pericentromeric region of the X.