Molecular Vision 2023; 29:25-30
Received 31 July 2022 | Accepted 28 April 2023 | Published 29 April 2023
1Stein Eye Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Department of Ophthalmology, Malayan Ophthalmologic Center, Yerevan, Armenia; 3Case Western Reserve University School of Medicine; 4Hoopes Vision Research Center, Draper, UT; 5John A. Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT
Correspondence to: Anthony J. Aldave, Department of Ophthalmology, Stein Eye Institute 200 Stein Plaza, UCLA, Los Angeles CA 90095-7003; Phone: (310) 206-7202; email: firstname.lastname@example.org
Purpose: Pre-Descemet corneal dystrophy (PDCD) with X-linked ichthyosis (XLI) is associated with mutations in or deletions of the steroid sulfatase gene (STS). As only three cases of genetically confirmed PDCD associated with XLI have been reported, we sought to expand our understanding of the genetic basis of PDCD by screening STS in two previously unreported families.
Materials and Methods: The affected individuals underwent cutaneous and slit-lamp examinations. Saliva samples collected from each affected individual served as a source of DNA for the amplification of the 10 coding exons of STS and flanking DNA markers.
Results: The slit-lamp examination of three affected men (two of whom were brothers) from two families revealed bilateral punctate posterior corneal stromal opacities anterior to the Descemet membrane. Cutaneous examination demonstrated dry, coarse, scaly ichthyotic changes characteristic of XLI in all individuals. Genetic examination of the STS locus on the X chromosome in Case 1 revealed a deletion that spanned across DNA markers DXS1130–DXS237, which includes all the coding exons (exons 1–10) of STS. Genetic screening of Cases 2 and 3 revealed a partial deletion of the STS locus involving exons 1–7 and flanking DNA marker DXS1130 on the X chromosome.
Conclusions: PDCD with XLI may be associated with either partial or complete deletion of STS. Despite the identification of point mutations, partial deletion, and complete deletion of STS in different affected families reported to date, there was no apparent difference in the affected phenotype between the families, suggesting that the identified variants likely all resulted in loss of function of steroid sulfatase.
With an estimated prevalence of 1:1500–1:6000 male individuals worldwide, X-linked ichthyosis (XLI) is an inherited skin disorder with a typical onset soon after birth that initially presents with large, thin, translucent scales, which are replaced later in life by polygonal, dark lesions restricted to the extremities, trunk, and neck [1–4]. Located on chromosome Xp22.3 and spanning an approximately 135 kb genomic region with 10 exons, the steroid sulfatase (STS) gene encodes a membrane-bound enzyme called steroid sulfatase. STS mutations are believed to cause XLI by impairing steroid sulfatase activity, ultimately leading to the retention of hyperkeratosis and impaired skin permeability [5,6]. Approximately 90% of reported XLI patients who underwent genetic screening demonstrated deletions of the entire STS gene and flanking sequences, while point mutations or partial deletions accounted for the remaining 10% of cases . Larger deletions involving contiguous genes may result in syndromic conditions that involve other tissues and organs, resulting in cryptorchidism, corneal opacification, and neurologic deficits . Nonsyndromic ocular disorders associated with XLI, in addition to corneal opacification, include recurrent corneal epithelial erosions, corneal ulcers, posterior embryotoxon, and deuteranopsia .
Pre-Descemet corneal dystrophy (PDCD) is associated with multiple small, gray, deep stromal punctate opacities; in mild cases, opacities can be seen not only immediately anterior to the Descemet membrane but also involving the posterior stroma . Approximately 25% of female carriers of XLI will demonstrate the characteristic pre-Descemet opacities of PDCD, which are thought to be due to the accumulation of cholesterol sulfate anterior to the Descemet membrane and serve to distinguish male individuals with XLI from other types of ichthyosis, as well as to identify female carriers .
Similar to the majority of individuals with XLI without corneal manifestations, the majority of individuals with both XLI and PDCD have demonstrated deletions spanning the entire STS gene locus [11–13]. While partial deletions of STS have been reported nearly two dozen times in association with XLI without PDCD, a partial deletion of STS has never been reported previously in association with PDCD. Here, we report three individuals from two previously unreported families with PDCD and XLI associated with deletions in the STS locus, including the first report of PDCD associated with a partial STS deletion.
This study followed the guidelines established by the Declaration of Helsinki for the treatment of all subjects, and all research performed was compliant with HIPAA guidelines. Informed written consent was obtained from all participants and was approved by the Institutional Review Board at the University of California, Los Angeles (UCLA IRB#11–000020). Ophthalmologic evaluation of the affected individuals was performed using slit-lamp biomicroscopy. The diagnosis of PDCD was based on the presence of punctate opacities in the posterior corneal stroma immediately anterior to the Descemet membrane in both eyes.
After informed consent was obtained from all participating individuals, saliva samples were collected using an Oragene Saliva Collection Kit (DNA Genotek, Inc., Ottawa, CA). Genomic DNA was then isolated using the Oragene PrepIT-L2P Kit (DNA Genotek, Inc.). Previously published primers were used to amplify exons 1–10 of STS (NG_021472.2) and DNA markers that flank STS [14,15]. Each PCR was performed in a 25-μl reaction mixture consisting of 50 ng of genomic DNA template, 12.5 μL of GoTaq® DNA Polymerase (Promega, Madison, WI), 10 μM forward and reverse primer (Integrated DNA Technologies, Coralville, IA), and 10.3 μl of nuclease free water. Amplification reactions were performed under the following conditions: denaturation at 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for STS exons 1–10 and 58 °C for STS flanking markers for 30 s, elongation at 72 °C for 35 s, and a final extension step at 72 °C for 5 min. The PCR-amplified sequences used in this study are arranged on the X chromosome as follows: telomere-DXS89-DXS996-DXS1139-DXS1130-STS exons 1–10-DXS1131+-DXS1133-DXS237-DXS1132-DXF22S1-DXS278-DXS1134-centromere. All PCR-amplified products were separated on a 1.25% (wt/vol) agarose gel with ethidium bromide and imaged using the INGenius3 system (Syngene, Frederick, MD).
A 43-year-old man who presented to the Hoopes Vision Research Center for a refractive surgery consultation had an uncorrected visual acuity of 20/200 in the right eye and 20/250 in the left eye, with a corrected distance visual acuity (CDVA) of 20/20 in each eye after correcting for myopic astigmatism. Slit-lamp biomicroscopy was significant only for numerous tiny, gray-white opacities in the posterior corneal stroma anterior to the Descemet membrane, primarily involving the central and midperipheral cornea bilaterally (Figure 1A). The corneal endothelium was normal in both eyes, and the central corneal thickness measured 475 μm OD and 487 μm OS. The remainder of the ocular examination was unremarkable. The patient was noted to have dry, coarse skin that was characteristic of ichthyosis, with excessive skin flaking predominantly over the forearms and lower extremities (Figure 2A). The patient reported having excessive flaking and scaling since early childhood, which he self-treated with topical moisturizers, but he had not seen a dermatologist and had not been diagnosed with ichthyosis. He had no immediate family history of XLI or other dermatologic disorders.
A 34-year-old man presented to the Malayan Ophthalmologic Center in Yerevan, Armenia, for ophthalmic evaluation. His uncorrected visual acuities measured 20/20 in each eye. Slit-lamp examination demonstrated diffuse, punctate, gray posterior stromal corneal opacities diffusely distributed across the cornea in each eye (Figure 1B). The remaining ocular examination was unremarkable. External examination revealed excessive flaking, scaling, and thickening of the skin over his hands, bilateral lower extremities, and lower back (Figure 2B). The patient was first noted to have abnormal skin findings at age 5 and was subsequently diagnosed with XLI at age 18. His family history was significant for his maternal grandfather and brother having XLI.
A 33-year-old man, the brother of the Case 2 patient, also presented to the Malayan Ophthalmologic Center for ophthalmic evaluation. The patient’s uncorrected visual acuities measured 20/100 OD and 20/25 OS, improving to 20/20 OU with refraction. Slit-lamp biomicroscopy demonstrated numerous tiny gray-white opacities in the posterior stroma bilaterally, with an otherwise unremarkable ocular exam (Figure 1C). Cutaneous examination revealed a dry, scaly appearance of the skin consistent with XLI, which had been diagnosed at age 18 (Figure 2C).
PCR amplification of STS exons 1–10 in Case 1 failed to produce any amplicons, while each of the 10 exons was amplified in two unrelated, healthy controls with the same primers and PCR conditions (Figure 3A), indicating the deletion of all STS exons in Case 1. To identify the extent of the X chromosome deletion in Case 1, PCR amplification of flanking DNA markers was performed, which demonstrated that the boundaries of the ~1.2 Mb deletion were DNA markers DXS1139 and DXS1132 (Figure 3B). Genetic screening of the STS locus in Cases 2 and 3 both failed to produce amplicons for STS exons 1–7, while producing amplicons for exons 8–10, indicating a partial deletion of STS (Figure 3A). PCR amplification of flanking markers demonstrated that the boundaries of the ~0.7 Mb deletion in both Cases 2 and 3 were DNA markers DXS1139 and DXS1131+ (Figure 3B).
PDCD is characterized by punctate corneal opacities anterior to the Descemet membrane, with histopathological studies being rare due to the opacities not affecting visual acuity. The corneal stromal deposits in PDCD usually arise in early adulthood, and previous studies have not shown a correlation between the number of corneal deposits and age [12,16]. Histopathologic studies of the corneal button from an individual with PDCD with associated XLI showed electron dense, polymorphic material in the pre-Descemetic, posterior corneal stroma, suggestive of cholesterol sulfate deposits . In vivo confocal microscopic studies of an individual with genetically confirmed PDCD associated with XLI demonstrated an abundance of hyperreflective aggregates in the cell bodies of posterior stromal keratocytes, likely representative of focal accumulations of cholesterol sulfate, while nongenetically confirmed cases of PDCD similarly showed both intracellular and extracellular hyperreflective particles in mid and deep stromal keratocytes [13,18–20].
In the majority of individuals with XLI with or without PDCD, the genetic basis is a complete deletion of STS, likely as a result of recombination due to homologous sequences and low copy repeats flanking STS [15,21]. To our knowledge, this is the first report of PDCD associated with XLI associated with a partial deletion of STS. The corneal and cutaneous features in the individuals with a partial STS deletion were indistinguishable from those of the individual with the complete STS deletion and from previously reported individuals with XLI and PDCD [11–13], with development in early childhood in all cases. Previous studies examining individuals with isolated XLI secondary to STS mutations and partial and complete STS deletions have also failed to show a genotype–phenotype correlation [7,22–24]. Therefore, the partial deletion of STS that we report in two individuals with PDCD likely results in a complete loss of expression of steroid sulfatase, similar to the complete deletion of STS.
Support was provided by Walton Li Chair in Cornea and Uveitis (A.J.A), National Eye Institute Grant P30EY000331 (core grant), the Stotter Revocable Trust (SEI Cornea Division), UCLA Stein Eye Medical Student Research Program (D.W.), and unrestricted grant from Research to Prevent Blindness to Stein Eye Institute.