Next-generation sequencing-based clinical diagnosis of choroideremia and comprehensive mutational and clinical analyses

Choroideremia (OMIM 303100) is a rare, X-linked, recessive dystrophy that leads to the progressive degeneration of the retinal pigment epithelium (RPE), photoreceptors in the retina, and the choriocapillaris (CC) [1, 2]. The estimated prevalence is 1 in 50,000–100,000 individuals [3, 4]. Female carriers are affected with varying degrees of severity, but they are generally asymptomatic [5, 6]. Affected male patients suffer from nyctalopia, the progressive loss of the peripheral visual field and a reduction in the central visual field. Central visual acuity usually begins declining from the 30’s (4th decade) [7, 8]. Typically, patchy areas of chorioretinal degeneration occur in the mid periphery of the fundus, and these areas of degeneration proceed centripetally, eventually leading to the exposure of the underlying white sclera [2, 3, 9]. Therefore, choroideremia is often misdiagnosed as retinitis pigmentosa (RP) or other retinal dystrophies, such as Usher syndrome (OMIM 276900), gyrate atrophy (OMIM 258870) and Leber congenital amaurosis [10].

Choroideremia is caused by mutations in the CHM gene (OMIM, 300390), which encodes the geranylgeranyl transferase Rab escort protein-1 (REP-1). REP-1 deficiency affects intracellular vesicular trafficking, resulting in cellular dysfunction and premature cell death [11, 12]. CHM is located on chromosome Xq21.2, contains 15 exons, and encodes a 654-amino acid protein [13, 14]. CHM is the only gene known to be associated with choroideremia. To date, more than 280 mutations in the CHM gene have been reported to be associated with choroideremia, most of which are point mutations that directly introduce premature stop codons [12, 15]. However, the etiology and pathology of retinal degeneration caused by CHM mutations remains poorly characterized, and no phenotype-genotype correlations have been identified [2, 16, 17]. Gene therapy is a promising treatment option for choroideremia because it is caused by mutations in a single gene and the effective treatment window is relatively long; therefore, the impaired functions of the cells may be able to be reversed using gene-replacement therapy before the cells are irreversibly damaged [1, 18, 19]. Encouraging results from a recent phase 2 clinical trial showed that high-dose subfoveal gene therapy using an adeno-associated virus (AAV) expressing REP-1 (AAV2-REP-1) has the potential to maintain, and in some cases improve, the best-corrected visual acuity (BCVA) of patients with choroideremia [19].

With the development of gene sequencing technologies and the advent of gene therapy, genetic testing is becoming increasing attractive for patients with inherited retinal diseases [24, 25]. Genetic testing might be particularly vital for patients with choroideremia because choroideremia is often misdiagnosed as RP [10]. In addition, clinical trials using gene therapy to treat choroideremia have shown that gene therapy represents a promising treatment prospect [19, 26]. An accurate genetic diagnosis will provide support for a clinical diagnosis, modify future disease risks, and provide assistance for clinicians working in the field of retinal genetics. In addition, gene sequencing helps to identify suitable candidates for future choroideremia gene therapy trials. In the present study, seven patients who were previously diagnosed with RP received corrective and accurate diagnoses of choroideremia based on NGS.

Six of the seven variants identified in this study were novel. All of the variants were deleterious mutations, and no missense mutations were identified. This result was similar to the findings reported in other countries, such as America [27], Europe [22], and Canada [28]. This finding implied that most missense mutations in CHM exert little or no effect on the protein structure and function. However, deleterious mutations exert substantial effects on the structure of the encoded protein, potentially leading to a loss of GGTase function and resulting in the insufficient transfer of geranylgeranylpyrophosphate groups onto Rab proteins, inducing CHM [14, 29]. These results not only expand the mutational spectrum of CHM but also suggest an important feature of CHM mutations in this cohort with relevant guiding value for genetic counseling. These results also are important in guiding the selection of sequencing methods used for patients with choroideremia. Various sequencing methods have been used to identify CHM mutations, with Sanger sequencing and NGS representing the most widely used methods [30‐32]. However, mutations in the CHM gene are often not identified. One possible reason is that copy number variations and structural variations are important pathogenic mutations observed in patients with choroideremia and should be considered during routine genetic sequencing, particularly when small mutation analyses produce negative results. Moreover, no hotspots or hot regions were detected among the CHM mutations, and these results have important value for guiding genetic counseling.

Clinically, choroideremia is often misdiagnosed as RP. Approximately 6% of individuals diagnosed with RP might instead have choroideremia [33, 34], while approximately one-quarter of patients with a clinical diagnosis of choroideremia may actually have other diseases, including RP [34]. In the present study, we analyzed and reported the clinical characteristics of patients with choroideremia, and we summarized the detailed differences between choroideremia and RP based on a multimodal imaging analysis of the phenotypes among the six patients. First, unlike RP, patients with choroideremia do not show ‘bone spicules’ pigmentation; instead, they show pigment hypertrophy and clumping. However, the specific types of RP, such as pigment-free or advanced stage RP, can be difficult to identify in patients. Second, the characteristic appearance of patients with choroideremia is a whitish fundus with large areas of profound atrophy observed in the choroid and the retina, and large choroidal vessels and vortex veins are obvious. However, in the early stages of disease, the differentiation between RP and choroideremia may be difficult, as the choroid degeneration may only become apparent during later stages [35]. Third, most of the patients with choroideremia examined in this study presented with better visual acuity than patients with RP; however, this finding cannot be generalized because the vision prognoses among patients with different types of RP vary substantially [36, 37]. Fourth, the majority of patients with choroideremia present with retinal tubulations in the outer layer of the retina. Although the underlying pathogenesis remains unclear, researchers have postulated that these tubulations may represent the tubular rearrangement of degenerating photoreceptors [38‐40]. These tubulations are present around areas of the surviving retina and are thought to represent areas that retain some visual function [10, 41]. Therefore, due to the high level of phenotypic heterogeneity among patients with choroideremia, the clinical analysis described above should only serve as a reference. The “gold standard” for diagnosing choroideremia must be based on a genetic diagnosis. Moreover, CHM should be included as a candidate gene during gene sequencing of patients suspected of having RP.

The limitations of this study include the small number of patients, and more patients with diverse pathological phenotypes and longer follow-up periods are necessary.