Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Estimating ON and OFF contributions to the photopic hill: normative data and clinical applications.

BACKGROUND: With progressively brighter stimuli, the amplitude of the b-wave of the human photopic electroretinogram (ERG) first increases to a maximal value (Vmax) and then decreases to finally reach a plateau, a phenomenon known as the photopic hill (PH). A mathematical model combining a Gaussian (G) and a logistic (L) growth function was previously proposed to fit this unusual luminance-response curve, where the G and L functions were suggested to represent, respectively, the OFF and ON retinal pathway contributions to the building of the PH. METHOD: The PHs of patients presenting stationary diseases affecting specifically the ON (3 CSNB-1) or OFF (4 CPCPA) retinal pathways as well as patients affected with retinitis pigmentosa (14 RP) of different stages or etiology were analyzed using this mathematical model and compared to the PHs of a group of 28 normal subjects. RESULTS: The PH of the CSNB-1 patients had a much larger contribution from the G function compared to normal subjects, whereas the opposite was observed for the CPCPA patients. On the other hand, analysis of data from RP patients revealed variable G-L contributions to the building of their PH. CONCLUSION: In this study, we confirm the previous claim that the luminance-response function of the photopic ERG b-wave can be decomposed into a Gaussian function and a logistic growth function representing, respectively, the OFF and ON retinal pathways. Furthermore, our findings suggest that this mathematical decomposition could be useful to further segregate and potentially follow the progression of retinopathies such as RP.

WDR19: an ancient, retrograde, intraflagellar ciliary protein is mutated in autosomal recessive retinitis pigmentosa and in Senior-Loken syndrome.

Autosomal recessive retinitis pigmentosa (arRP) is a clinically and genetically heterogeneous retinal disease that causes blindness. Our purpose was to identify the causal gene, describe the phenotype and delineate the mutation spectrum in a consanguineous Quebec arRP family. We performed Arrayed Primer Extension (APEX) technology to exclude ∼500 arRP mutations in ∼20 genes. Homozygosity mapping [single nucleotide polymorphism (SNP) genotyping] identified 10 novel significant homozygous regions. We performed next generation sequencing and whole exome capture. Sanger sequencing provided cosegregation. We screened another 150 retinitis pigmentosa (RP) and 200 patients with Senior-Løken Syndrome (SLS). We identified a novel missense mutation in WDR19, c.2129T>C which lead to a p.Leu710Ser. We found the same mutation in a second Quebec arRP family. Interestingly, two of seven affected members of the original family developed 'sub-clinical' renal cysts. We hypothesized that more severe WDR19 mutations may lead to severe ciliopathies and found seven WDR19 mutations in five SLS families. We identified a new gene for both arRP and SLS. WDR19 is a ciliary protein associated with the intraflagellar transport machinery. We are currently investigating the full extent of the mutation spectrum. Our findings are crucial in expanding the understanding of childhood blindness and identifying new genes.

GPR98 mutations cause Usher syndrome type 2 in males.

Mutations in the large GPR98 gene underlie Usher syndrome type 2C (USH2C), and all patients described to date have been female. It was speculated that GPR98 mutations cause a more severe, and eventually lethal, phenotype in males. We describe for the first time two male patients with USH2 with novel GPR98 mutations. Clinical characterization of a male patient and his affected sister revealed a typical USH2 phenotype in both. GPR98 may have been excluded from systematic investigation in previous studies, and the proportion of patients with USH2C probably underestimated. GPR98 should be considered in patients with USH2 of both sexes.

A complete, homozygous CRX deletion causing nullizygosity is a new genetic mechanism for Leber congenital amaurosis.

CRX is a transcription factor required for activating the expression of many photoreceptor-neuron genes. CRX may be mutated in three forms of human blindness; Leber congenital amaurosis (LCA), cone-rod degeneration (CRD) and retinitis pigmentosa (RP). The pathogenic mechanism in most cases is likely dominant negative, with gain of function. We report a novel, complete homozygous CRX deletion in LCA. We identified a Lebanese family with 3 affected LCA cases. The proband was sequenced by NGS. Quantitative PCR, array comparative genomic hybridization, and long range PCR were performed. Full eye examinations, OCT and photography were performed. We identified a homozygous 56,000 bp deletion of CRX, which co-segregates and is heterozygous in four parents, who report normal vision. The blind children with LCA manifest severe retinal degeneration, a phenotype typical for CRX and LCA. We hypothesized that a single copy of CRX (haplo-insufficiency) in the causes mild abnormal foveal development, but not LCA. Two parents had significant inner and outer foveal and photoreceptor abnormalities. This is the first reported case of a homozygous, complete CRX deletion. Nullizygosity of CRX thus causes LCA while haplo-insufficiency of CRX causes abnormal foveal development, but not LCA. Our data suggest a new disease mechanism for CRX.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photoreceptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

Knobloch syndrome involving midline scalp defect of the frontal region.

We report on a 4-year-old boy with Knobloch syndrome. He has vitreoretinal degeneration, high myopia, cataract, telecanthus, hypertelorism, and a high-arched palate. He also has a defect of the anterior midline scalp with involvement of the frontal bone as documented by a computed tomography (CT) scan. The brain was normal on CT scan and magnetic resonance imaging. We present a review of the 23 published cases with this syndrome. Our patient illustrates the importance of investigating for underlying ocular and central nervous system pathology whenever midline scalp defects are present.

Ocular findings in a family with Sotos syndrome (cerebral gigantism).

PURPOSE/METHODS: We examined the ocular features in a two-generation family with Sotos syndrome (cerebral gigantism). Sotos syndrome is characterized by excessive growth in prenatal and early life, advanced bone age, and typical facial features. RESULTS/CONCLUSION: One patient had presenile nuclear sclerotic cataracts, megalophthalmos, hypoorbitism, and exotropia. One of her daughters had megalocornea, exophoria, and iris hypoplasia. Her other daughter had megalocornea. The ophthalmologist can play an important role in the diagnosis and treatment of Sotos syndrome.

Clinical features of autosomal dominant congenital nystagmus linked to chromosome 6p12.

PURPOSE: To describe the clinical features of a large pedigree with autosomal dominant congenital nystagmus linked to chromosome 6p12. METHODS: In a prospective evaluation of 54 living family members in a single pedigree, 21 persons were affected with autosomal dominant congenital nystagmus, and clinical examinations were performed on 14. Selected persons underwent further studies, including electroretinography, scanning laser ophthalmoscopy, nerve fiber layer studies, visual evoked potential studies, and eye movement recordings. RESULTS: Among seven affected persons whose parents were able to report whether the nystagmus was present congenitally, onset at birth was noted in two persons and between 3 and 6 months in five persons. Best-corrected binocular Snellen visual acuity ranged from 20/30 to 20/100, with a mode of 20/50. Strabismus was present in 14 examined patients (36%). Eye movement recordings, performed on five persons, included asymmetric pendular (three), asymmetric pendular combined with dual waveform jerk (one), and unidirectional jerk nystagmus (one). CONCLUSIONS: Autosomal dominant congenital nystagmus represents a disorder with variable expressivity. While onset is typically during infancy, it can be noted at birth. Intrafamilial variation in visual acuity, ocular alignment, and nystagmus waveform suggests a role for modifying influences on expression of disease.

A new association of congenital hydrocephalus, albinism, megalocornea, and retinal coloboma in a syndromic child: a clinical and genetic study.

We describe a child with global developmental delay, prominent metopic suture, trigonocephaly, and cryptorchidism whose symptoms resemble the well-known 9p deletion syndrome or 9p monosomy. We also noted congenital hydrocephalus, oculocutaneous albinism, retinal coloboma, and megalocornea, which are not typical features of 9p monosomy. When a new albinism gene was localized to 9p (Chintamaneni et al., Biochem Biophys Res Commun 1991;178:227-235; Murty et al., Genomics 1992;13:227-229), we hypothesized that our patient had the 9p deletion syndrome plus albinism, with the deletion involving the albinism gene. We used FISH probes to test this hypothesis and found that the 9p region was normal, therefore excluding the 9p deletion syndrome. To our knowledge, the association of congenital hydrocephalus, albinism, megalocornea, and retinal coloboma has not been described in the literature. The purpose of this report is to describe this new association of congenital ocular and cerebral anomalies in a syndromic child.

Mutational analysis and clinical correlation in Leber congenital amaurosis.

UNLABELLED: Leber congenital amaurosis (LCA, MIM 204001) is a clinically and genetically heterogeneous retinal disorder characterized by severe visual loss from birth, nystagmus, poor pupillary reflexes, retinal pigmentary or atrophic changes, and a markedly diminished electroretinogram (ERG). PURPOSE: To examine 100 consecutive patients with LCA in order to assess the relative burden of the three known genes involved in LCA, namely retinal guanylyl cyclase (GUCY2D), retinal pigment epithelium protein ( RPE65), and the cone-rod homeobox (CRX), and to define their clinical correlates. METHODS: Mutational analysis and detailed clinical examinations were performed in patients diagnosed with LCA at the Johns Hopkins Center for Hereditary Eye Diseases and the Montreal Children's Hospital. RESULTS: Mutations were identified in 11% of our patients: GUCY2D mutations accounted for 6%, while RPE65 and CRX gene mutations accounted for 3% and 2%, respectively. The clinical presentation was variable; however, the visual evolution in patients with mutations in GUCY2D and CRX remained stable, while individuals with mutations in the RPE65 gene showed progressive visual loss. CONCLUSIONS: This study suggests that molecular diagnosis of Leber congenital amaurosis could provide important information concerning prognosis and course of treatment.

Presumed photic retinopathy after cataract surgery: an angiographic study.

To ascertain the prevalence rate of presumed photic retinopathy after cataract surgery, 276 fluorescein angiograms of 147 patients were reviewed. The diagnosis was based on strict angiographic criteria. Lesions consistent with healed photic retinopathy were noted in four patients (prevalence rate of 3%). This finding is discussed and comparisons with the literature are made.

Leber's congenital amaurosis with anterior keratoconus in Pakistani families is caused by the Trp278X mutation in the AIPL1 gene on 17p.

BACKGROUND: Leber's congenital amaurosis (LCA) represents the earliest and severest form of retinal dystrophy leading to congenital blindness. A total of 20% of children attending blind schools have this disease. LCA has a multigenic basis and is proving central to our understanding of the development of the retina. We describe the clinical and molecular genetic features of four inbred pedigrees from neighbouring remote villages in northern Pakistan, in which some of the affected members have concurrent keratoconus. METHODS: History-taking and physical and eye examinations were performed in the field. Venipuncture, DNA extraction, studies of linkage to known LCA genes, automated sequencing and polymorphism analyses for haplotype assessments were done. RESULTS: We examined 12 affected and 15 unaffected family members. By history, there were an additional nine blind people in the four pedigrees. In each pedigree a consanguineous marriage was evident. We found a homozygous nonsense mutation in the AIPL1 gene, which replaces a tryptophan with a stop codon (Trp278X). The phenotype is severe and variable, despite the common molecular genetic etiology in each family. Affected patients had hand motion to no light perception vision and fundus findings ranging from maculopathy to diffuse pigmentary retinopathy. Three affected members had definite keratoconus, and two were suspects based on mild cone formation in the cornea of at least one eye. INTERPRETATION: We have identified four Pakistani families with a severe form of LCA that is associated with severe keratoconus in some affected members. The molecular etiology in all four families is a homozygous nonsense mutation, Trp278X, in the photoreceptor-pineal gene AIPL1. To our knowledge, this is one of the first phenotype-genotype correlations of AIPL1-associated LCA.

Evidence supportive of a functional discrimination between photopic oscillatory potentials as revealed with cone and rod mediated retinopathies.

We report on a family where four of the eleven children presented with reduced visual acuities, a red-green deficit at the Farnsworth-Munsel FM 100-hue test, normal appearing fundi and unexpected electroretinographic findings. Light- (photopic) and dark- (scotopic) adapted electroretinograms (ERG) and oscillatory potentials (OPs) were obtained following an accepted standard protocol. The b-wave of their photopic ERG was significantly more attenuated than the a-wave due to the specific abolition of OP4, while the amplitudes of OP2 and OP3 were within the normal range, giving to the b-wave a truncated appearance reminiscent of that seen in congenital stationary night blindness (CSNB) with myopia. Interestingly in the latter condition, which is believed to result from an ON-retinal pathway anomaly, it is OP2 and OP3 which are specifically abolished while OP4 is of normal amplitude thus resulting in an OP response pattern which complements that seen with our patients. Also of interest is the fact that, in our patients, the amplitude of the dark-adapted OP2 was, on average, 240% larger than that measured in light-adaptation while, in normal, a non-significant 14% increase is noted; a finding which is in keeping with other studies reporting supernormal scotopic ERGs in some forms of cone dystrophies. Based on the photopic OP response pattern, our patients represent the electrophysiological complement of patients affected with CSNB. Interestingly their symptoms are also complementary, a finding which could support a functional discrimination between the photopic OPs.

A gene for autosomal dominant congenital nystagmus localizes to 6p12.

Congenital nystagmus is an idiopathic disorder characterized by bilateral ocular oscillations usually manifest during infancy. Vision is typically decreased due to slippage of images across the fovea. As such, visual acuity correlates with nystagmus intensity, which is the amplitude and frequency of eye movements at a given position of gaze. X-linked, autosomal dominant, and autosomal recessive pedigrees have been described, but no mapping studies have been published. We recently described a large pedigree with autosomal dominant congenital nystagmus. A genome-wide search resulted in six markers on 6p linked by two-point analysis at theta = 0 (D6S459, D6S452, D6S465, FTHP1, D6S257, D6S430). Haplotype analysis localizes the gene for autosomal dominant congenital motor nystagmus to an 18-cM region between D6S271 and D6S455.