A novel 5 bp homozygous deletion mutation in ASPH gene associates with Traboulsi syndrome

Traboulsi syndrome is a rare genetic disorder, inherited in an autosomal recessive fashion. It is characterized by facial dysmorphism, lens dislocation, anterior-segment abnormalities, and spontaneous filtering blebs. In 1995, Traboulsi and co-workers reported six members of a consanguineous family from Druze sect in Lebanon with dysmorphic facial features (crowded teeth, large beaked nose, flat cheeks, retracted chin and antimongoloid slanting of palpebral fissures), subluxation of crystalline lenses, iridocorneal adhesions, and spontaneous filtering blebs(1). Subsequently, another Lebanese family and an isolated Lebanese patient were reported with similar clinical phenotype (2,3). This necessitated an entry in Online Mendelian Inheritance In Man (OMIM 601552). The molecular cause of Traboulsi syndrome was first studied through autozygosity mapping and exome sequencing, which revealed two homozygous mutations in exon 22 and 25 of ASPH gene(4). Recently, a homozygous novel variant in exon 2 of ASPH gene has also been found to be associated with Traboulsi syndrome (5). ASPH gene is located at the q arm of chromosome no.8 (8q12.3), which is a widely expressed type II membrane protein involved in calcium homeostasis. It encodes Aspartyl/Asparaginyl beta-hydroxylase that specifically hydroxylate aspartic acid or asparagine residues in the epidermal growth factor (EGF)domain containing proteins using iron as a cofactor(6). Genetically engineered knockout mice of ASPH gene showed abolished beta hydroxylation activity in EGF containing proteins(7). The EGF specific unusual posttranslational modification has significant developmental consequences A 35-year-old male presented with complaints of progressive decrease in vision in the right eye since childhood. There was no prior history of ocular trauma or incisional surgery. On examination, he was found to have a long face, anti-mongoloid slanting of palpebral fissures, prominent beaked nose, retrognathia and malar hypoplasia (Figure 1A and B). His best-corrected visual acuity was 6/24 in right eye (RE) and 6/9 in the left eye (LE). Slit lamp examination of the RE showed a diffuse elevated bleb near the limbus in the superonasal quadrant, shallow anterior chamber, and dislocated microspherophakic lens in the anterior chamber. Left eye showed clear cornea, deep anterior chamber, normal iris pattern and microspherophakic lens (Figure 1C and D). His intraocular pressure (IOP) was 6 and 14 mm Hg in the RE and LE, respectively. Gonioscopy and ultrasound biomicroscopy of the RE showed scleral thinning with iris strands bridging the area (Figure 1E and F). Fundus examination revealed a normal disc and retina. Rest of the physical examination was unremarkable. Laboratory investigations including serum homocysteine and urine lysine were within normal limits. Electrocardiogram and echocardiogram were normal. Based on these findings we ruled out other causes of ectopia lentis and diagnosed our patient with Traboulsi syndrome. We have examined the parents and children of the proband, they were normal without ocular or facial anomalies. Peripheral blood samples were collected from the proband and his parents. DNA was extracted by modified salting-out method (8) and quantified with Nano spectrophotometer (Nano Drop Technologies, Inc Wilmington). Polymerase chain reaction was performed for all 25 exons of ASPH gene in the proband. The PCR products were treated with ExoSAP-IT® reagent to remove the unincorporated primers and DNTP’s with zero sample loss. Cyclic PCR was performed using the forward primer of exon 22 and exon 25. Big dye termination chemistry method was used for sequencing (3130 Genetic Analyzer; Applied Biosystems, CA). The sequencing results were analyzed by Finch TV version 1.5 and BLAST. Sanger sequencing results revealed mutations in exon 22 of ASPH gene in proband which could be associated with the disease phenotype. To understand the genetic basis of inheritance, an exon 22 of ASPH gene was amplified (449 bp) from proband’s parents and a control sample, further it was subjected to sanger sequencing. The chromatogram of proband showed a 5 bp homozygous deletion mutation (c.1869_1873 del GGACT Ref [NM_004318.3]) with an altered reading frame in the coding region of exon 22 and the sequencing results of proband’s parents also displayed a heterozygous peak with predicted frameshift mutation in comparison to the control chromatogram representing a normal peak with intact coding region of ASPH exon 22 (c.1869_1873 GGACT Ref [NM_004318.3]). N Blast analysis