X-linked Retinitis Pigmentosa (XLRP) and Choroideremia Panel

Retinitis pigmentosa (RP) represents a group of hereditary retinal dystrophies with a worldwide prevalence of ~1 in 4000 (Booij 2005). RP is clinically characterized by retinal pigment deposits visible on fundus examination, nyctalopia, followed by progressive degeneration of the photoreceptors, which eventually leads to blindness (van Soest et al. 1999). X-linked RP (XLRP) is one of the most severe forms of RP, with early onset and rapid progression in males. Milder symptoms can also present in females, probably due to random X inactivation (Friedrich et al. 1993).

Choroideremia (CHM) is an X-linked retinal dystrophy characterized by progressive degeneration of the choriocapillaris, retinal pigment epithelium and photoreceptors (Coussa and Traboulsi 2012; MacDonald et al. 2003). This blinding disorder typically affects males with a progression from nyctalopia (night blindness) in the first or second decades of life to peripheral visual field loss. Central vision is preserved until age 40-50 years in most CHM cases. Carrier females are usually asymptomatic, but progressive chorioretinal degenerations can occur.

Nonsyndromic RP is remarkably heterogeneous both clinically and genetically and exhibits autosomal dominant (AD), autosomal recessive (AR) or X-linked (XL) inheritance. At least five different genetic loci have been linked to XLRP: Xp11.2 (RP2), Xp21.1 (RPGR), Xp21.2-21.3 (RP6), Xp22 (RP23 or OFD1) and Xp26-27 (RP24) (RetNet). To date, only the RPGR, RP2 and OFD1 genes have been identified.

RPGR encodes the retinitis pigmentosa GTPase regulator (Meindl et al. 1996), and RP2 encodes a plasma membrane-associated protein. Pathogenic variants in RPGR account for the vast majority (~80%) of all XL RP cases, with RP2 accounting for about 10% of cases, and the other loci likely accounting for the remaining ~10% (Breuer et al. 2002; Sharon et al. 2003). Males hemizygous for RPGR or RP2 pathogenic variants typically display classic features of RP, such as initial night blindness and loss of peripheral vision (Sharon et al. 2003). However, RPGR pathogenic variants have also been detected in patients presenting with cone and cone-rod dystrophy (Yang et al. 2002; Demirci et al. 2002) or atrophic macular degeneration (Ayyagari et al. 2002). Female carriers of RPGR and RP2 pathogenic variants showed variable manifestation of the phenotypes ranging from asymptomatic to severe retinal degeneration similar to affected male patients (Friedrich et al. 1993; Neidhardt et al. 2008).

The RPGR gene encodes several alternatively spliced isoforms, although RP-causing mutations are only found in isoform C (CCDS35229.1). Isoform C consists of 15 coding exons, with exon 15 (usually referred to as ORF15; see Bader et al. 2003 for a detailed description of ORF15) coding for nearly 50% of the protein. Accordingly, pathogenic variants in ORF15 account for ~50% of all XL RP cases (Vervoort et al. 2000; Sharon et al. 2003). Pathogenic variants in RPGR are a mix of missense, nonsense, splicing, small deletions and insertions (mostly frameshift) and larger deletions.

The RP2 gene consists of 5 exons with pathogenic variants found throughout all five exons; no founder pathogenic variants or mutational hotspots have been reported. Most pathogenic variants result in premature protein termination (frameshift, nonsense and splice-site), although a few causative missense variants have also been identified (Mears et al. 1999; Hardcastle et al. 1999). Patients with pathogenic variants in RP2 appear to retain less visual acuity than patients with pathogenic variants in RPGR (Sharon et al. 2003). Thus, knowledge of which XL RP gene is mutated is useful for making the most accurate long-term visual prognosis.

Exome sequencing has identified that OFD1 is associated with XL RP in one pedigree. The deep intronic variant c.935+706A>G (alternatively known as IVS9+706A>G) was identified in all the affected males and the obligate female carriers were heterozygous for this variant. This variant causes the insertion of a cryptic exon producing an aberrant transcript. In vivo expression of this variant suggested that the normal OFD1 transcript is produced, but at reduced level (39%). They speculated that these levels are sufficient for the other tissues, but not for the photoreceptors. OFD1 is localized to the connecting cilium of photoreceptors, and is probably required for primary cilium biogenesis (Webb et al. 2012).

CHM (Rab Escort Protein 1 - REP-1) has 15 coding exons that encode a protein involved in vesicular trafficking (Cremers et al. 1990; Coussa and Traboulsi 2012; MacDonald et al. 2003). CHM is the only gene to date known to be associated with choroideremia. Nearly all CHM pathogenic variants lead to a null allele or truncated nonfunctional REP-1 protein. The major types of CHM defects are frameshifts (30%), nonsense variants (25%), partial and whole gene deletions (20%), and splicing variants (17%) while missense pathogenic variants are very rare (2%) (Human Gene Mutation Database). Gross insertions and complex rearrangements have been also reported but are uncommon.

This test is predicted to detect a causative variant in over 90% of all patients with presumptive XL RP (Sharon et al. 2003; Hong 2005; Breuer et al. 2002), or ~45% of patients with X-linked cone dystrophy (Demirci et al. 2002). The sensitivity for X-linked atrophic macular degeneration is unknown (Ayyagari et al. 2002). The clinical sensitivity for OFD1 is uncertain. So far, an OFD1 causative variant was only reported in one pedigree (Webb et al. 2012). Direct sequencing of the 15 exons and adjacent splice sites detects CHM pathogenic variants in 60%-95% of affected males with Choroideremia (MacDonald et al. 2003).

Gross deletions and duplications have been reported in the RPGR and RP2 genes (Human Gene Mutation Database; Bader 2003; Pelletier et al. 2007; Vervoort et al. 2000). While an exact frequency of gross deletions and duplications is unknown, they are not a common cause of disease. Large deletions that have been reported in OFD1 are associated with Oral-facial-digital syndrome 1 (Human Gene Mutation Database). Large deletions in CHM are relatively common (Human Gene Mutation Database).

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary.

This panel provides 100% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ≥20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

We also sequence the OFD1 intronic c.935+706A>G variant (Webb et al. 2012).

Analysis of RPGR exon 15 (commonly referred to as ORF15) has historically been difficult due to the highly repetitive, purine-rich sequence. When NGS does not achieve full coverage or there is a variant detected by NGS that needs confirmation, then we utilize a specialized chemistry Sanger sequencing.

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).