To investigate the diverse ocular manifestations and identify the causative mutation in a large family with autosomal dominant anterior segment dysgenesis accompanied in some individuals by cerebral vascular disease.
Retrospective observational case series and laboratory investigation.
Forty-five family members from 4 generations underwent ophthalmic examination. Molecular genetic investigation included analysis with single nucleotide polymorphism (SNP) markers and DNA sequencing. Whole exome sequencing was performed in 1 individual.
A broad range of ocular manifestations was observed. Typical cases presented with corneal clouding, anterior synechiae, and iris hypoplasia. Posterior embryotoxon, corectopia, and early cataract development were also seen. One obligate carrier and several other family members had minor ocular anomalies, thus confounding the scoring of affected and unaffected individuals. Cerebral hemorrhages had occurred in 4 individuals, in 3 at birth or during the first year of life. Seven patients with corneal clouding were considered “definitely affected” for linkage studies. Haplotype mapping revealed that they shared a 14 cM region in the terminal part of chromosome 13q that included the locus for COL4A1 . The affected family members were heterozygous for a novel COL4A1 sequence variant c.4881C>G (p.Asn1627Lys) predicted to be damaging and not found among 185 local blood donors. Exome sequencing showed that this variant was the only one in the candidate region not found in dbSNP.
Among the family members shown to carry the novel COL4A1 mutation, heterogenous presentations of anterior segment dysgenesis was seen. Testing family members for this mutation also made a definite diagnosis possible in patients with a clinical presentation difficult to classify. In families where anterior segment dysgenesis occurs together with cerebral hemorrhages, genetic analysis of COL4A1 should be considered.
Anterior segment dysgenesis is a term used to denote the presence of developmental anomalies that affect one or several of the structures of the anterior part of the eye. Included are the Axenfeld-Rieger group of disorders (Axenfeld-Rieger anomaly, iris hypoplasia, and iridogoniodysgenesis), Peters anomaly, and aniridia. Anomalies affecting other organs are found in Axenfeld-Rieger syndrome, iridogoniodysgenesis syndrome, and Peters Plus disease. There is a considerable overlap between the different entities, and their presentation may vary widely between affected family members. Anterior segment dysgenesis may also be part of several multisystem disorders, such as Alagille syndrome, Wolf-Hirschhorn syndrome, SHORT syndrome, and Abruzzo-Erikson syndrome.
Mutations in more than 12 genes have been associated with anterior segment dysgenesis. Most single gene disorders show autosomal dominant inheritance. In mice, dysfunction of the collagen IV alpha gene ( Col4a1 ) was found to be associated with severe congenital anomalies in the eye, brain, and kidney. In humans, COL4A1 mutations were first shown to cause autosomal dominant porencephaly, cerebral hemorrhages, and microangiopathy including retinal arteriolar tortuosity. Later, the HANAC syndrome was described, consisting of hereditary angiopathy, nephropathy (renal cysts, hematuria), aneurysms, and muscle cramps. Recently, mutations in COL4A1/Col4a1 have been associated with an autosomal dominant form of muscle-eye-brain disease (MEB) and Walker-Warburg Syndrome (WWS), both in humans and in mice. Anterior segment dysgenesis associated with mutations in COL4A1 has been reported in 2 French families. In the affected individuals, congenital cataract was the most frequent finding, followed by iris anomalies (hypoplasia and corectopia), microcornea with peripheral corneal opacities, and elevated intraocular pressure.
Type IV collagens are basement membrane proteins that are widely expressed. COL4A1 and COL4A2 are the most abundant ones. COL4A1 is not required for the formation of basement membranes, but is essential for their structural integrity and function. In the eye, presence of COL4A1 has been demonstrated in the basement membranes of the conjunctiva, corneal epithelium, corneal endothelium, trabecular meshwork, Schlemm canal, and choroid body, as well as in the lens, retinal inner limiting membrane, Bruch membrane, and vascular basement membranes. Misfolded COL4A1 may thus disrupt the integrity of basement membranes in most parts of the eye. In addition, at least in mice, it may also contribute to cataract development.
In the present report we describe the findings in a large family with heterogenous presentation of anterior segment dysgenesis accompanied in some individuals by cerebral vascular disease. The family was first reported in 1981. Ocular anomalies were seen in a large proportion of family members. Examination with single nucleotide polymorphism (SNP) markers in a subset of 7 definitely affected individuals revealed that they shared a 14 cM region in the terminal part of chromosome 13q encompassing the COL4A1 locus. DNA sequencing revealed a novel sequence variant in COL4A1 , c.4881C>G, likely to be the cause of the disorder.
Family members were invited to participate and signed a letter of informed consent. This retrospective study consisted of a review of hospital records including data from routine clinical chemistry analyses and computed tomography (CT) and magnetic resonance imaging (MRI) scans, a general medical and ophthalmologic examination, and a genetic laboratory investigation. The ophthalmologic examination included Snellen visual acuity measurements, slit-lamp examination, gonioscopy, Goldmann tonometry, indirect ophthalmoscopy, and anterior segment and fundus photography. The purpose and the procedures of the study were explained to all participants, and all signed a letter of informed consent. The study adhered to the tenets of the Declaration of Helsinki, and the study as well as the letter of consent were approved prospectively by the Regional Committee for Medical and Health Research Ethics, Western Norway (IRB#00001872) (ref. 2010/1552). Approval included permission to obtain clinical, radiological, and laboratory data, and to perform the genetic analysis.
Linkage Studies With SNP Markers
Genomic DNA was isolated from whole blood using the nucleic acid extractor model 341 (Applied Biosystems [ABI], Foster City, California, USA). A genome-wide SNP scan was performed in a subset of definitely affected individuals using the Affymetrix 250K chip (Affymetrix, Santa Clara, California, USA) and search for regions of shared identity by descent was performed using the PLINK software.
DNA Sequencing and Mutation Detection
DNA was amplified by polymerase chain reaction (PCR) using standard procedures. After PCR amplification, the PCR products were treated with SAP/exonuclease I (Amersham, Chalfont St. Giles, United Kingdom) and sequenced using the PRISM BigDye Terminator kit (ver1.1) and an ABI 3730 Genetic Analyzer (ABI). DNA sequences were analyzed by SeqScape software (ABI). DNA from 185 healthy local blood donors was used as controls for the sequence variants identified.
Whole exome capture using Roche-Nimblegen’s SeqCap EZ Exome v2 and sequencing on the Illumina HiSeq was performed as described previously to a median coverage of 78×. Variant analysis was restricted to the chromosome 13 linkage interval.
Forty-five individuals were included in the study. A partial pedigree is shown in Figure 1 . The ocular malformations and their severity varied considerably between the family members examined. Corneal clouding was present in 7 individuals ( Table 1 , Figure 2 ). Most had bilateral involvement, but in 2 mainly the left eye was affected ( Figure 2 ). The clouding was located in the periphery of the cornea, except in III-1, IV-4, and V-1. In III-1 and IV-4, both peripheral and central clouding was seen in one eye, while the opposite cornea was clear except for a posterior embryotoxon ( Figure 2 ). Photographs taken of individual IV-4 at the age of 9 and 31 years ( Figure 2 ) indicate that corneal clouding may progress over time. The most severe involvement was seen in V-1, where both corneas were opaque at birth ( Figure 2 ). He underwent penetrating keratoplasties between the age of 5 and 7 years, twice in the right eye and once in the left. Extensive clouding of the transplants occurred within the first year after surgery, and both corneas became completely opaque, resulting in a visual acuity of light perception in both eyes.
|Patient||Corneal Clouding||Embryotoxon Posterior||Anterior Synechiae||Iris Hypoplasia||Cataract <45 y||Fundus Anomalies||Spastic Hemiparesis/Paraparesis||Cognitive Impairment||Epilepsy||Cerebellar Signs|
a Three groups of family members are listed. In the first group (III-1 to V-1) all have corneal clouding and were, therefore, classified as definitely affected. In the next group, IV-1 and V2 had clear corneas with minimal ocular anomalies. However, both had neurological disease. In the third group, II-6 to IV-3 all had minor ocular anomalies, but no neurological disease. One individual, III-11, is not included in the table. She had no ocular anomalies and a neurologic disease different from that seen in other individuals.
Posterior embryotoxon is a consistent feature of the Axenfeld-Rieger group of anomalies. An irregular Schwalbe line was seen in one or both eyes of all individuals with corneal clouding. Anterior synechiae extending to the Schwalbe line were present in 5 of these individuals ( Table 1 ). Individuals II-6, III-2, III-8, and V-2 had clear corneas with a small posterior embryotoxon in the temporal part of both eyes. Angle anomalies were infrequent, although in the most severely affected individuals corneal opacities or anterior synechiae prevented detailed examination. None of the family members had elevated intraocular pressure.
Iris hypoplasia was observed in 9 individuals. In 6 of these, the iris appeared dark, with coarse stromal fibers. In III-8, III-9, and IV-1 a slight circumferential or sectorial involvement was seen, resulting in visualization of all or part of the sphincter pupillae muscle. Mild corectopia was seen in III-8, III-9, III-10, and IV-8. A slightly irregular pupil was also seen in III-5, but the iris color was unremarkable and the sphincter pupillae muscle was not visible. Polycoria was not detected in any family members.
Three individuals had cataract surgery before the age of 45 years. Fundus abnormalities were seen in III-1, III-5, III-6, and IV-3. They consisted of irregular vessels around the optic disc (III-1) ( Figure 3 ), unilateral increased excavation of the optic disc (III-5), preretinal fibrosis (III-6), and a narrow avascular zone of the macula (IV-3). Visual acuity was normal in all eyes except those with corneal opacities, where it ranged from 0.8 to light perception.
A slight dysphonia was present in III-9 and III-12. Individual III-12 also suffered from congenital sensorineural hearing loss and a cardiomyopathy of unknown origin. Supraventricular tachycardia was seen in III-10.
When this family was first enrolled for studies, cerebral involvement was observed in 4 individuals. Cerebral hemorrhages occurring either at birth or within the first year of life were seen in III-10, V-1, and V-2, causing spastic paraparesis (III-10 and V-1), hemiparesis (V-2), and impaired psychomotor development. Slightly impaired psychomotor development was also present in individual IV-4. During the next decade, signs of cerebral involvement developed in III-9, III-11, and IV-1 ( Table 1 ). Individual III-9 suffered from several intracerebral hemorrhages, IV-1 had an ischemic cerebellar stroke followed by an episode of optic neuritis, and III-11 died from a neurologic disease of unknown origin, classified as encephalomyelitis. Individuals V-1 and V-2 suffered from epilepsy.
Brain CT and/or MRI scans were available for re-examination for 8 individuals. Five of them showed extensive central white matter changes consistent with the presence of leukoencephalopathy ( Figure 4 ), 1 showed mild white matter lesions, and 2 (14 and 19 years old) were normal. Signs of previous hemorrhage, such as deposits of hemosiderin, calcification, and areas of parenchyma loss, were seen in 5 individuals ( Figure 4 ) ( Table 2 ). Contrast series were available for 2 individuals. One showed no enhancement. The other (III-11) showed extensive contrast enhancement, predominantly in the brain stem and basal ganglia.
|Patient||Leukoencephalopathy||Intracerebral Hemorrhages||Calcifications||Wide Ventricles||Cerebellar Atrophy||Cerebellar Infarct||Contrast Enhancement|
a Porencephalic cysts. Small porencephalic cysts secondary to intracerebral hemorrhages were also seen in III-9 and V-1.
From the clinical examination, 7 individuals were scored as definitely affected by the same disorder based on the presence of corneal clouding ( Figure 1 ). Analysis of a genome-wide scan with SNP markers in these individuals showed that they shared a 14 cM area at the terminal part of chromosome 13q ranging from SNP markers at 110, 173, 324-115, 045, 259 (GRCh37/hg19). This region included the COL4A1 locus now known to be associated with anterior segment dysgenesis. DNA sequencing revealed a novel sequence variant, c.4881C>G, present in these 7 individuals considered certainly affected ( Figure 5 ), but not in a panel of 185 blood donors (370 chromosomes). The same sequence variant was also present in IV-1 and V-2, but not in any of the other sampled family members ( Figure 1 ).
To rule out the possibility of another causal variant present in the linkage region, whole exome sequencing was performed on a sample from Patient III-1. The 2 main candidate genes in the region, COL4A1 and COL4A2 , were highly covered (mean 78× coverage, all 100 exons were covered at >10× mean coverage). The c.4881C>G mutation was the only exonic variant in the 14 cM linkage interval not present in dbSNP ( Supplemental Table , available at AJO.com , contains a complete list of all variants discovered in the region).