We recruited a 21-year old female patient along with her 58-year old unaffected father, 55-year old unaffected mother, 41-year old unaffected brother and 30-year old unaffected sister (Figure 1A) at the Bangalore West Lions Superspeciality Eye Hospital, Bangalore in April 1999 for decreased vision in the right eye for two years. The family belongs to the city of Bangalore, state of Karnataka, India. On examination, in the right eye there was no perception of light. She improved to 6/9, N8 vision in the left eye with a correction of -10 D (lenticular myopia). The anterior segment examination showed the right eye had exotropia and a fixed dilated pupil. In the left eye, the pupil reacted normally. There was microspherophakia (Figure 1B) with iridodonesis and phacodonesis in both eyes. Gonioscopy showed grade 2 angles in the right eye with peripheral anterior synechiae and grade 2 angles with no synechiae in the left eye. Intraocular pressure (IOP) was 23 mmHg in the right eye and 16 mmHg in the left eye. Fundus evaluation with indirect ophthalmoscopy showed glaucomatous optic atrophy in the right eye and glaucomatous cupping (CD 0.6-0.7) in the left eye. The axial length was 22.5 mm and 21.65 mm in the right eye and the left eye, respectively. She had short stature (131 cm), brachydactyly, and the absence of joint stiffness (Figure 1C-G). There was no cardiac abnormality in the patient. These features suggested that the patient was affected with WMS.
Her father (II:1), mother (III:1), unaffected brother (IV:1), and unaffected sister (IV:2) were 150, 140.2, 158.50, and 155.5 cm tall, respectively. None of her parents and siblings had any ocular, joint, and cardiac problems, except her father had undergone cataract surgery for both eyes at the age of 48. The parents of the patient are uncle and niece.
The patient was advised and had undergone Nd:Yag peripheral iridotomy in the left eye and was put on Glucomol (timolol maleate, Allergan, Bangalore, India) eye drops for the left eye. The patient was followed up regularly for IOP and fundus evaluation. Her IOP was 40 mmHg and 34 mmHg in the right eye and the left eye, respectively, in August 2000. Since the IOP was not controlled by maximum medical management, transscleral cyclophotocoagulation was performed for the right eye to decrease the pressure and control the pain. In April 2001, trabeculectomy was performed in the left eye. The IOP was under control at 10 mmHg in subsequent follow-ups. The patient's vision decreased in the left eye due to cataract changes, and the patient underwent lens aspiration with scleral-fixated intraocular lens implantation in July 2009. She has been on a regular follow-up schedule for vision, IOP, and fundus since then. A retinal hole was detected in her left eye in June 2013 for which a prophylactic laser procedure was performed. There is no history of retinal detachment. On her last visit in December 2013, her unaided vision in the left eye was 6/9 with IOP of 12 mmHg in both eyes and stable retina in the left eye.
This study was approved by an Institution Review Board (an Ethics Committee) and that the study adhered to the tenets of Declaration of Helsinki and the ARVO statement on human subjects. Three to five ml of blood was drawn from each individual in a sodium-EDTA Vacutainer tube (Beckton-Dickinson, Franklin Lakes, NJ) for DNA isolation, using the Wizard genomic DNA purification kit (Promega, Madison, WI). Since the cost of sequencing 101 coding exons of four WMS and WMS-like syndrome genes (viz., ADAMTS10, ADAMTS17, LTBP2, and FBN1) was high, we used whole exome sequencing (WES) on patient IV:3 to find the genetic cause of WMS in this family. For WES, 1 mug of total genomic DNA from the patient was randomly fragmented, size selected to 350-400 bp products, and ligated to adapters on both ends. The resulting library was then amplified and sequenced on an Illumina HiSeq 2000 sequencer (Illumina Inc., San Diego, CA) and run to 45.90X coverage. The resulting reads with an average read length of 101 base pairs were mapped to the human genome (UCSC human genome hg19 build) with the Burrows-Wheeler Aligner (BWA) program. SNPs and coding indels were detected with the SAMTOOLS program. All genetic variants were then compared to those in dbSNP132, exome variant server (EVS), the 1000 Genomes project, and in-house WES databases to determine if they were known or novel. The pathogenicity of the variants was tested with PolyPhen-2, Mutation Taster, and SIFT bioinformatics tools. The domain structure of the ADAMTS17 protein (GenBank accession number NP_620688.2) was identified using the Pfam program.
Specific coding regions of the ADAMTS17, calcium/calmodulin-dependent protein kinase ID (CAMK1D; gene ID 57118, OMIM 607511), pyridine nucleotide-disulphide oxidoreductase domain 2 (PYROXD2; gene ID 84795), and signal-induced proliferation-associated 1 like 3 (SIPA1L3; gene ID 23094) genes were amplified using gene-specific primers to confirm the variants identified with WES. Primer pairs were designed using gene sequences from the UCSC Bioinformatics site (http//genome.ucsc.edu/). The sequences and PCR conditions of the primers are provided in Table 1. Variants were confirmed by sequencing the PCR products from the patient on an ABIprism A370-automated sequencer (PE Biosystems, Foster City, CA). PCR was performed using a standard laboratory procedure. Once a mutation (variant) was identified, we then examined 50 ethnically matched controls for the presence of the mutation, using restriction fragment length polymorphism (RFLP) analysis or Sanger sequencing.
To determine the effect of a splice-site mutation on the splicing of the ADAMTS17 transcript (NM_139057.2), total RNA was isolated from peripheral blood samples using the Tri-Reagent BD (Sigma-Aldrich, St. Louis, MO). Total RNA was then converted into the first-strand cDNA using a RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, Burlington, Canada). Primers specific to exons 4 (5'-ACG CCG ACA TGG TGC AGT ACC AC-3') and 8 (5'-GGC GAT GGT AAA GGC CAA ATT GAG) were used in the reverse transcription (RT)-PCR analysis, and the amplicons were visualized in a 1.5% agarose gel with ethidium bromide.