Three patients in a consanguineous family from southern Morocco were referred to our Department of Medical Genetics at the National Health in Rabat for Corneal Dystrophy. A five-generational pedigree was constructed after a thorough interview of the affected mother (III-2). The available three family members were affected (IV-7, IV-8 and III-2), including two phenotypically unaffected individuals (IV-1, IV-3). An autosomal dominant mode of inheritance was determined (Fig. 1). Clinical features of the patients are given below.
Patient 1 (IV-7) was a 34-year-old woman who complained of recurrent episodes of corneal pain since the age of 18 associated with a decrease in visual acuity. Her vision was 4/10 OD and 6/10 OS. Slit lamp examinations revealed anterior epithelial and stromal corneal dystrophy in the two eyes in the form of spaced microvacuoles by heterogeneous thickening of the epithelium due to thickening of an abnormal subepithelial fibrous layer and poorly individualized anterior opacities, with fuzzy edges (Fig. 2). The rest of the examination of the anterior segment was normal, including a normal iris, a clear lens, and an intraocular pressure of 17 mmHg in the ODG. The optical coherence tomography (OCT) scan showed an unevenness of the epithelial layers by a homogeneous confluent layer of hyperreflected deposits with a serrated anterior border taking the sawtooth appearance, replacing the Bowman layer and reaching the anterior stroma (Fig. 3 (A, B, C, D)). It is thicker and becomes thinner on the periphery and disappears towards the limb. The pachymetry is 512 μm in OD and 523 μm in OS.
Patient 2 (IV-8) is a 44-year-old woman who presents episodes of recurrent keratitis with a progressive decrease in visual acuity since 20 years of age. Visual acuity in OD shows that she counts the fingers at 3 m/OS at 2/10 (irremovable). Examination with the slit lamp showed anterior epithelial and stromal corneal dystrophy in the form of microvacuoles in the OD and OS groups, especially in the periphery, with heterogeneous thickening of the epithelium, which was more pronounced in the centre, giving a large central opacity with fuzzy edges in both eyes (Fig. 4 (A, B)). She had a clear lens; ocular tone at 15 mmHg in OD 14 mmHg in OS, and a normal fundus. OCT of the cornea showed unevenness of the anterior epithelial and stromal layers with thicker hyperreflective deposits and a clear central opacity in both eyes (Fig. 5 (A, B, C, D)), as well as pachymetry at 538 μm in OD and 543 μm in OS.
Patient 3 (III-2) is a 72-year-old woman with bilateral osteoarthritis, and she reports episodes of recurrent corneal pain. For her visual acuity, in far vision OD, she could barely see the movement of the fingers/OS (she counted the fingers at 3 m). The slit lamp examination showed central and paracentric yellowish and gelatinous central and paracentric deposition in the right eye associated with a corneal opacity deeper than the previous one and affecting the epithelial layers of Bowman's membrane and the anterior stroma in the form of an epithelial fibrous layer, which covered almost the entire corneal surface (Fig. 6). The rest of the examination is hampered by the very important dystrophy of the right eye (i.e., ineclairable fundus). In the left eye, slit lamp examination showed CD involving the epithelial layers of Bowman's membrane and the anterior stroma in the form of heterogeneous thickening at the centre, associated with opacity with fuzzy boundaries and a corticonuclear cataract (Fig. 6). In the fundus, pupillary glow with flattened retina was noted. OCT of the cornea showed thin patches (blue arrows) of microvacuoles in the right eye associated with a significant loss of epithelial cells with disorganization of the epithelial layers, the absence of Bowman's membrane, and an anterior intrastromal bubble (white arrows). The pachymetry is at 586 μm (Fig. 7). In the left eye, OCT showed irregularity of the corneal surface with disorganization of the epithelial layers, discontinuous Bowman’s membrane, and anterior stromal reshaping, especially in the centre (Fig. 7). The process of corneal transplantation in this patient is ongoing.
After obtaining written consent from all participants involved in the study, we collected blood samples from family members. Blood samples were collected from five members of the family. Genomic DNA was extracted from whole peripheral blood by using the QIAamp DNA Blood Mini Kit (Qiagen Valencia, CA) strictly following the manufacturer’s protocol. Whole-exome sequencing (WES) was performed for the three patients; 500 ng of fragmented DNA was obtained by enzymatic fragmentation with the Kapa Hyper Plus Kit (KapaBiosystems Inc. Wilmington, MA, USA) and was amplified according to the manufacturer instructions and subjected to enrichment with SeqCap EZ Human Exome v3.0 Roche Nimblegen (Roche, Basel, Switzerland).
The Illumina HiSeq 2500 system was used to sequence 64 enriched megabases in fast-running double-ended mode (2x100 bp). bcl2fastq v1.8.4 (Illumina) was used to convert the original data (bcl file) into a fastq file. The sequence was analysed according to GATK best practice recommendations; BWA-MEM was used for mapping, and GATK (haplotype calling program) was used for variant calling. Variant Studio (Illumina) was used for annotation and filtering steps.
Candidate variants were selected using an autosomal dominant mode of inheritance according to the following criteria: i) heterozygous variants, ii) nonsynonymous variants, iii) variants predicted as pathogenic or likely pathogenic, iv) variants with a minor allele frequency (MAF) of <0.01 were selected from the 1000 Genomes Project (http://www.1000genomes.org/) and ESP6500 exome project (https://evs.gs.washington.edu/EVS/) and v) segregation analysis. Candidate variants in favour of clinical manifestations and passing the criteria were validated by Sanger sequencing.
The established variants were cross-checked with the 1000 Genomes database (http://www.1000genomes.org/) with the Exome Variant Server (http://evs.gs.washington.edu/EVS/), HGMD (http://www.biobase-international. com/product/hgmd) and with the ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/).
To confirm the mutation detected by exome sequencing, standard PCR was carried out to amplify exon 10 of the TGFBI gene by using the TGFBI_F:5’-GACCAGGCTAATTACCATTCTTG-3’ and TGFBI_R:5’-TGAGATATGTCCTGGAGCCC-3’ primer pair. Amplification products were electrophoresed on a 1% agarose gel. Sanger sequencing was performed with dye terminator chemistry (ABI Prism BigDye v3.1) and run on an automated sequencer using the 3130 Genetic Analyzer (Thermo Fisher Scientific). The results obtained were aligned with the reference genome (GRCh37/hg19) and then analysed by DNA variant analysis software (Mutation Surveyor® software).
The transmission form of this family was consistent with autosomal dominant inheritance (Fig. 1). Clinical examinations demonstrated bilaterally multiple superficial, epithelial and stromal anterior granular opacities in different stages of severity among three patients of this family. The three patients with the same mutation shared a mixed phenotype with a superficial form of granular corneal dystrophy (GCD) type 1 and TBCD patterns.
Whole-Exome Sequencing and Variant Validation: WES was performed on three family members (IV-7, IV-8 and III-2). The analysis of the data of the three patients shows 30887 variants in 19712 genes. After filtering, this number was reduced to a single allelic variant in the TGFBI gene. The filtering of variants is illustrated in Fig. 8. Only a heterozygous mutation (c.1772C>A; p.Ser591Tyr) in the TGFBI gene, identified in these three patients, was proposed as the potential pathogenic mutation within this family (Fig. 9 A).
Bioinformatics analysis using Polyphen-2 and SIFT suggested that this mutation was probably damaging. A high degree of conservation of this amino acid was demonstrated by a score of 5.52 calculated by Genomic Evolutionary Rate Profiling (GERP) http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html. Comparative amino acid sequence alignment of TGFBI across different species revealed that this mutation occurred at highly conserved positions (Fig. 9 B) (https://www.ensembl.org/Homo_sapiens/Tools/Blast). The mutation was confirmed by Sanger sequencing in the affected sister and mother and two unaffected brothers.
Furthermore, the variant was not present in the GnomAD browser (accessed July 2020) or in our in-house WES database of 100 unrelated individuals of Moroccan ethnicity who had undergone WES for diseases other than corneal dystrophy.
The genomic and clinical data both supported a diagnosis of Thiel-Behnke corneal dystrophy in this family.