Family Study

This is a prospective observational case series and a genetic laboratory investigation that included review of medical records; clinical, electrophysiological, and radiological examination; and whole exome sequencing. Patients and their unaffected family members were invited to participate in the study. The purpose and the procedure of the study were explained to all participants and all signed an informed letter of consent. Parental consent was obtained for the 12-year-old girl included. The study adhered to the tenets of the Declaration of Helsinki, and the study as well as the letter of consent were approved by the Regional Committee for Medical and Research Ethics, Western Norway (Institutional Review Board [IRB] no. 00001872, ref. no. 2012/329). Approval included permission to obtain data from medical records and to perform clinical and radiological examinations, and genetic analysis.

The family pedigree is shown in Figure 1 . The unaffected parents of the proband (I-1 and I-2) and his siblings underwent a routine ophthalmologic examination while affected family members underwent an extended clinical examination including measurements of visual acuity (Snellen chart), intraocular pressure (IOP) (Goldmann tonometry), axial length (Zeiss IOL Master 500; Carl Zeiss Meditec, Jena, Germany), stereopsis (Lang stereotest, Titmus stereotest), retinal correspondence (Bagolini striated lens test), color vision (Ishihara and Farnsworth D15 color vision tests), and visual field (Octopus 900 perimeter program G2 dyn; Haag-Streit Diagnostics, Koenitz, Switzerland). Fundus photographs and spectral-domain optical coherence tomography (OCT) (Topcon 3D OCT-1000 Mark II; Topcon Corporation, Tokyo, Japan) of the macula and optic disc were obtained. In addition, a detailed neurologic examination was performed. All affected family members underwent cerebral and ocular 3 Tesla magnetic resonance imaging (MRI), testing of VER, pure tone and speech audiometry, and renal ultrasound examination.

Pedigree of the family with high myopia–excavated optic disc anomaly. Affected family members are indicated by solid square/circular symbols. N/N: no MYCBP2 mutation detected, N/M: heterozygous for the MYCPB2 frameshift mutation.

Isolation of DNA, Genotyping, and Exome Sequencing

Blood samples for genotyping were obtained from all family members. Genomic DNA was isolated from blood. Targeted capture and trio exome sequencing were performed at HudsonAlpha Institute for Biotechnology (Huntsville, Alabama, USA). Exome capture was performed using Roche-NimbleGen Sequence Capture EZ Exome v2 kit and paired-end 100nt sequencing on the Illumina HiSeq. The paired-end reads were aligned to the hg19 reference genome using the Burrows-Wheeler Alignment tool (BWA v.0.5.8c), providing between 6.1 and 14.4 gigabases of aligned sequence data for the 3 samples. This resulted in an average of 56X median coverage of the target capture regions with 96% of target bases covered at least 8X. After the alignment, PCR duplicates were removed with Picard v1.90 ( http://broadinstitute.github.io/picard ), and Genome Analysis Toolkit (GATK nightly-2013-07-09-gce5e621) was used for base quality recalibration, realignment around known indels, and multisample variant calling (GATK’s UnifiedGenotyper tool) according to GATK’s Best Practices workflow. Minimum depth of coverage required to consider a variant was set to 8X for total depth and 5X for the variant allele. ANNOVAR was used for variant annotation. Coding and putative splice sites (defined as 2 bps flanking coding exons) were filtered against variants with MAF frequency >0.8% in an in-house database of more than 300 Norwegian exomes and variants present at >0.5% allele frequency in the 1000 Genomes database (phase 1 release v3 called from 20101123 alignment). The de novo mutations were verified by Sanger sequencing. The entire coding region of PAX2 was Sanger sequenced in the proband.

RNA Analysis

Total RNA was purified from blood using the Tempus system (Life Technologies, Carlsbad, California, USA). Reverse transcription and cDNA synthesis were performed using the SuperScript VILO cDNA synthesis kit (Life Technologies). The expression of wild-type and mutant MYCBP2 was detected by Sanger sequencing using primers spanning exon junctions near the mutation of interest.

Ophthalmologic Examination

The ocular findings are illustrated in Figure 2 and the Supplemental Figure (available at AJO.com ), and the MRI findings are shown in Figure 3 . The clinical findings are summarized in the Table .

Fundus photographs (Upper row), optical coherence tomography (OCT) images (Middle row) and visual fields (Lower row) of patients with high myopia–excavated optic disc anomaly. Shown are Patient II-2 (first and second column from left), Patient III-1 (third and fourth column from left), and Patient III-2 (fifth and sixth column from left). Variable optic disc dysplasia is seen with the most pronounced changes in II-2 (Upper row, first and second from left) and the least changes in the right eye of III-2 (Upper row, fifth from left). The latter resembles a vacant disc. A general reduction of sensitivity with deeper scotomas is found on visual field examination. The optic discs are deeply excavated on OCT.

Axial T2-weighted images of the orbits of patients with high myopia–excavated optic disc anomaly. The globe shows an ovoid pattern bilaterally in Patient II-2 (Upper row) and Patient III-1 (Middle row), and unilaterally in Patient III-2 (Lower row).

The proband (Patient II-2), aged 47 years, is the second child of unrelated, healthy parents and has 3 unaffected siblings. There was no history of similar congenital eye disease in the family. An intermittent convergent squint was noted at the age of 6 months. At age 18 months he was examined by an ophthalmologist, who noted bilateral anomalies of the optic discs interpreted as optic disc coloboma. The anterior segment and the intraocular pressure were normal in both eyes (OU). The earliest refraction reported in his medical journal was at the age of 4 years (−4.50/−6.00, OD/OS respectively). His best-corrected visual acuity (BCVA) at the age of 8 years was 6/18 and 6/9 (Østerberg chart). The refraction remained stable in childhood but later the myopia gradually increased.

When examined for this study, his BCVA was −10.00, −2.50 × 20: 0.32 (OD) and −14.00, −1.50 × 180: 0.63 (OS). The axial lengths were 27.11 mm (OD) and 28.99 mm (OS). Ophthalmoscopy revealed grossly dysplastic optic discs that were deeply excavated on OCT ( Figure 2 ). Pigmentary changes involving the macular region were present ( Supplemental Figure , available at AJO.com ). A general reduction of sensitivity was found on visual field examination, most pronounced in his right eye ( Figure 2 ). There was a moderate red-green color vision defect. VER showed reproducible and clear waveforms with pathologically prolonged latencies bilaterally. On MRI both eyes appeared ovoid and both optic nerves were thin. The optic chiasm was normal ( Figure 3 ).

Patient III-1 is the 16-year-old daughter of Patient II-2. She presented with reduced visual function aged 2 years. At the age of 5, she was found to be myopic, −6.00, −2.00 × 160 (OD) and −3.50 (OS). BCVA was 4/6- (OU). When examined for this study, her BCVA was −16.00, −1.50 × 170: 0.32 (OD) and −11.50, −1.75 × 170: 0.4 (OS). Her axial lengths were 29.27 mm (OD) and 27.27 mm (OS). Dysplastic optic discs that were deeply excavated on OCT were found. There was a general reduction of sensitivity on visual field examination ( Figure 2 ). Color vision was normal. Prolonged latencies were found on VER but were less pronounced than in the proband. Both eyes were ovoid on MRI while the optic nerves, chiasm, and the brain were normal ( Figure 3 ).

Patient III-2 is the 12-year-old daughter of Patient II-2. At the age of 4 she was found to be highly anisometropic (+1.00/−10.00 OD/OS, respectively) with reduced visual function in her left eye. When examined for this study, BCVA was −1.00, −0.50 × 180: 0.8 (OD) and −15.75, −3.50 × 180: 0.05 (OS). Axial lengths were 23.42 mm (OD) and 28.38 mm (OS). Ophthalmoscopy revealed a grossly dysplastic left optic disc with less pronounced changes in the right eye. Here the presence of a central excavation and no central retinal circulation suggested a vacant disc. A general reduction of sensitivity was found in the left eye on visual field examination and a scotoma in the lower temporal quadrant was present in the right eye ( Figure 2 ). Color vision was normal. VER was normal on her right side, but on the left side the examination was suboptimal and difficult to interpret. On MRI, her left eye was ovoid while the right eye appeared normal. The optic nerves, chiasm, and the brain were normal ( Figure 3 ).

General Examination

Apart from the visual disturbances, all patients were healthy, of normal intelligence, and with no dysmorphic features. There were no symptoms or signs of connective tissue involvement like joint hyperextensibility or heart valve abnormalities. Neurologic examination, including assessment of the olfactory nerve, was entirely normal. Audiograms were within the normal range, but in Patient II-2 a shallow depression in the midrange could be consistent with a mild form of hereditary hearing loss. Speech perception was normal in all patients. Patients II-2 and III-1 had small hyperechoic foci near the apex of the papillae of the renal pyramids interpreted to be a normal variant. There were no clinical or biological signs of renal disease. All patients had a stature in the lower normal rank; II-2 was 167 cm (2.5nd percentile), III-1 was 157 cm (10th percentile), and III-2 was 140 cm (3rd percentile). Mid-parental height of the parents of the proband was 163.5 cm.

Genotyping/Exome Sequencing

We performed whole exome sequencing of the proband (II-2) and his parents (I-1, I-2), searching for a de novo mutation. We found 18 263 exonic variants in the proband. Variant filtration against noncoding and common variants yielded 245 rare coding variants that were compared against whole exome data from his parents. This analysis revealed only 2 true de novo mutations, a 5 bp deletion in MYCBP2 (c.5906_5910del; p.Glu1969Valfs*26, NM_015057.4) and a synonymous mutation located 2 bp before an exon-intron boundary in PDCD6 (c.C366T (p.=)). Although our in silico analysis did not suggest that the latter mutation would influence normal splicing, we performed Sanger sequencing of both mutations in the pedigree and only the MYCBP2 frameshift deletion was present in both the proband and his affected children and absent in his parents and siblings. Sanger sequencing of cDNA from peripheral blood mRNA from 2 affected individuals confirmed the presence of the MYCBP2 c.5906_5910del mutation in the expected 50% ratio. The variant is located in exon 40 of the gene and has not been reported in publically available databases including the 1000 Genomes database and dbSNP. Furthermore, the mutation has not been seen in our own in-house database of variants from more than 300 whole exomes of Norwegian individuals.