Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PurposeGenetic 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.MethodsWe 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.ResultsWGS 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.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

OTX2 mutations cause autosomal dominant pattern dystrophy of the retinal pigment epithelium.

PURPOSE: To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment epithelium (RPE) in two families. METHODS AND RESULTS: Two families with autosomal-dominant PD were identified. Eight members of family 1 (five affected) were subjected to whole-genome SNP genotyping; multipoint genome-wide linkage analysis identified 7 regions of potential linkage, and genotyping four additional individuals from family 1 resulted in a maximum logarithm of odds score of 2.09 observed across four chromosomal regions. Exome sequencing of two affected family 1 members identified 15 shared non-synonymous rare coding sequence variants within the linked regions; candidate genes were prioritised and further analysed. Sanger sequencing confirmed a novel heterozygous missense variant (E79K) in orthodenticle homeobox 2 (OTX2) that segregated with the disease phenotype. Family 2 with PD (two affected) harboured the same missense variant in OTX2. A shared haplotype of 19.68 cM encompassing OTX2 was identified between affected individuals in the two families. Within the two families, all except one affected demonstrated distinct 'patterns' at the macula. In vivo structural retinal imaging showed discrete areas of RPE-photoreceptor separation at the macula in all cases. Electroretinogram testing showed generalised photoreceptor degeneration in three cases. Mild developmental anomalies were observed, including optic nerve head dysplasia (four cases), microcornea (one case) and Rathke's cleft cyst (one case); pituitary hormone levels were normal. CONCLUSIONS: This is the first report implicating OTX2 to underlie PD. The retinal disease resembles conditional mice models that show slow photoreceptor degeneration secondary to loss of Otx2 function in the adult RPE.

Gene therapy: perspectives from young adults with Leber's congenital amaurosis.

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.

Genetic counseling and genetic testing in ophthalmology.

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.

Psychosocial adjustment to visual loss in patients with retinitis pigmentosa.

PURPOSE: To investigate psychosocial adjustment to visual loss in patients with retinitis pigmentosa (RP). DESIGN: Cross-sectional study. METHODS: 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. RESULTS: 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, p < 0.001), followed by vocational function (61 +/- 11, p < 0.001), social environment (58 +/- 9, p < 0.001), and extended family relationships (55 +/- 9, p < 0.05). The total PAIS score was significantly elevated (58 +/- 8; p < 0.001). CONCLUSIONS: 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.

Ocular Genetics Program: multidisciplinary care of patients with ocular genetic eye disease.

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.

Eight previously unidentified mutations found in the OA1 ocular albinism gene.

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.

Utility of molecular testing for related retinal dystrophies.

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.

Mutational analysis of the OA1 gene in ocular albinism.

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.

Exome sequencing identifies NMNAT1 mutations as a cause of Leber congenital amaurosis.

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.507G>A, p.Trp169*) and missense (c.769G>A, 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.

Management and outcome of unilateral retinoblastoma.

BACKGROUND: 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. METHODS: We retrospectively reviewed the presentation, age at diagnosis, classification, genetic status, treatment, and long-term outcome of 130 patients with unilateral retinoblastoma (1988-2008). RESULTS: 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 (p < 0.001) 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. CONCLUSIONS: 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.

Unique insertional translocation in a childhood Wilms' tumor survivor detected when his daughter developed bilateral retinoblastoma.

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%.

Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma.

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 <3 wk and decreased direct costs by one-third. The multistep method reported here detected 89% (199/224) of mutations in bilaterally affected probands and both mutant alleles in 84% (112/134) of tumors from unilaterally affected probands. For 23 of 27 exons and the promoter region, QM-PCR was a highly accurate measure of deletions and insertions (accuracy 95%). By revealing those family members who did not carry the mutation found in the related proband, molecular analysis enabled 97 at-risk children from 20 representative families to avoid 313 surveillance examinations under anesthetic and 852 clinic visits. The average savings in direct costs from clinical examinations avoided by children in these families substantially exceeded the cost of molecular testing. Moreover, health care savings continue to accrue, as children in succeeding generations avoid unnecessary repeated anaesthetics and examinations.