Choroideremia (CHM) is an incurable progressive chorioretinal dystrophy. Little is known about the natural disease course of visual acuity in the Japanese population. We aimed to investigate the genetic spectrum of the CHM gene and visual acuity outcomes in 24 CHM patients from 16 Japanese families. We measured decimal best-corrected visual acuity (BCVA) at presentation and follow-up, converted to logMAR units for statistical analysis. Sanger and/or whole-exome sequencing were performed to identify pathogenic CHM variants/deletions. The median age at presentation was 37.0 years (range, 5–76 years). The mean follow-up interval was 8.2 years. BCVA of the better-seeing eye at presentation was significantly worsened with increasing age (r = 0.515, p < 0.01), with a high rate of BCVA decline in patients > 40 years old. A Kaplan–Meier survival curve suggested that a BCVA of Snellen equivalent 20/40 at follow-up remains until the fifties. Fourteen pathogenic variants, 6 of which were novel [c.49 + 5G > A, c.116 + 5G > A, p.(Gly176Glu, Glu177Ter), p.Tyr531Ter, an exon 2 deletion, and a 5.0-Mb deletion], were identified in 15 families. No variant was found in one family only. Our BCVA outcome data are useful for predicting visual prognosis and determining the timing of intervention in Japanese patients with CHM variants.
Choroideremia (CHM, OMIM: #303100) is an X-linked recessive disorder that leads to progressive degeneration of the choriocapillaris, retinal pigment epithelium (RPE), and photoreceptors. CHM is caused by sequence variants or deletions in the CHM gene (OMIM: *300390) encoding Rab escort protein 1 (REP1)[
As of now, no treatment is available for CHM, but CHM gene therapy could be a promising treatment option because the only cause of CHM is the loss of functional REP1. In fact, the first human clinical trial for CHM gene supplementation therapy was achieved in 2014 using the adeno-associated virus 2 (AAV2) vector encoding REP1 (NSR-REP1; Nightstar Therapeutics, London, UK) in 6 patients with CHM variants[
To date, only two cohort studies from 1999 have been published of Japanese CHM patients with CHM variants[
Determination of the genetic background is required for upcoming phase 3 clinical trials for NSR-REP1 gene therapy in the Japanese population. Here, we aimed to investigate the genetic spectrum of CHM and visual acuity outcomes in a single-center cohort of Japanese patients with CHM.
In total, 24 CHM patients from 16 unrelated Japanese families were studied (Fig. 1). Clinical findings are summarized in Table 1. All 24 patients were male. The median age at presentation was 37.0 years (range, 5–76 years), whereas the median age at follow-up was 45.5 years (range, 9–78 years). The mean follow-up interval was 8.2 years (range, 0–24 years). Four patients (16.7%) were evaluated once. Eight patients (33.3%) presented before 21 years old. Eleven patients (45.8%) presented after 40 years old.
Graph: Figure 1 Pedigrees of 16 Japanese families with choroideremia. The proband is indicated by an arrow in each family. Affected males are indicated by solid squares and carrier females by a circle with a dot. Unaffected males and females are indicated by open squares and circles. The slash symbol indicates deceased individuals. M: mutation/pathogenic variants.
Clinical findings of 24 patients with choroideremia.
Patient # Patient ID Family # Age (years) at presentation Chief complaint BCVA at presentation Follow-up interval (years) BCVA at follow up Notes Decimal (RE/LE) LogMAR (RE/LE) Decimal (RE/LE) LogMAR (RE/LE) 1 JU0097 1 35 NB 1.2/– − 0.08/– 18.0 0.9/– 0.05/– 2 JU0141 2 42 NB, VFD 1.0/0.8 0/0.1 20.7 0.6/0.9 0.22/0.05 3 JU0462 2 8 NB 1.2/1.0 − 0.08/0 15.4 1.2/0.9 − 0.08/0.05 4 JU0463 2 10 NB, photophobia 1.2/1.2 − 0.08/− 0.08 9.2 1.2/1.2 − 0.08/− 0.08 5 JU0395 3 21 Photophobia 1.2/1.2 − 0.08/− 0.08 23.8 0.7/1.2 0.15/− 0.08 6 JU0425 4 45 NB 0.8/0.5 0.1/0.3 12.2 0.3/HM 0.52/2.7 7 JU0426 5 49 NB 1.2/1.2 − 0.08/− 0.08 19.8 0.9/0.8 0.05/0.1 8 JU0433 6 58 NB 0.7/1.0 0.15/0 11.7 0.7/0.7 0.15/0.15 9 JU1850 6 68 NB, photophobia 0.4/0.3 0.4/0.52 0 0.4/0.3 0.4/0.52 10 JU0752 7 43 Photophobia 0.9/1.0 0.05/0 8.0 0.2/0.7 0.7/0.15 11 JU1817 7 26 NB 0.02/1.0 1.7/0 24.0 0.08/0.01 1.1/2.3 12 JU1059 8 45 NB, decreased VA 1.2/1.5 − 0.08/− 0.18 6.2 0.9/1.2 0.05/− 0.08 13 JU1248 9 5 NB 1.2/1.0 − 0.08/0 3.9 1.0/1.5 0/− 0.18 14 JU1254 10 26 Distorted vision 1.5/0.5 − 0.18/0.3 5.6 1.2/0.6 − 0.08/0.22 MH in LE 15 JU1457 11 9 Decreased VA 1.2/0.07 − 0.08/1.15 2.6 0.2/0.3 0.7/0.52 CNV in BE 16 JU1458 11 9 No symptom 1.2/1.2 − 0.08/− 0.08 2.6 0.3/1.0 0.52/0 CNV in RE 17 JU1520 11 76 NB, decreased VA HM/0.5 2.7/0.3 1.9 HM/0.6 2.7/0.22 18 JU1681 12 13 Decreased VA 1.5/0.15 − 0.18/0.82 1.5 1.5/0.15 − 0.18/0.82 CNV in LE 19 JU1681b 12 11 No symptom 1.2/1.2 − 0.08/− 0.08 0 1.2/1.2 − 0.08/− 0.08 20 JU1808 13 47 NB 0.6/0.6 0.22/0.22 8.3 0.5/0.6 0.3/0.22 21 JU1877 13 42 NB 1.2/0.8 − 0.08/0.1 0 1.2/0.8 − 0.08/0.1 22 JU1831 14 16 Decreased VA 0.01/1.2 2.30/− 0.08 0.2 0.01/1.2 2.3/-0.08 CNV in RE 23 JU1835 15 39 NB, photophobia 1.2/1.2 − 0.08/− 0.08 0.3 1.2/1.2 − 0.08/− 0.08 24 JU1688 16 46 NB, Decreased VA 0.8/1.2 0.1/− 0.08 0 0.8/1.2 0.1/− 0.08
NB night blindness, VA visual acuity, BCVA best-corrected visual acuity, HM hand motions, RE right eye, LE left eye, BE both eyes, MH macular hole, CNV choroidal neovasscularizetion.
The left eye (LE) of patient 1 had been enucleated due to ocular trauma. Our patients presented with a variety of chief complaints including night blindness only (9/24 patients, 37.5%), night blindness and photophobia (3/24 patients, 12.5%), night blindness and visual field defect (1/24 patient, 4.2%), night blindness and decreased visual acuity (3/24 patients, 12.5%), photophobia (2/24 patients, 8.3%), decreased visual acuity (3/24 patients, 12.5%), distorted vision (1/24 patient, 4.2%), and no symptoms (2/24 patient, 8.3%). The two patients (patients 16 and 19) without any symptoms, whose brothers were diagnosed with CHM, underwent ophthalmic examinations for diagnosis of CHM.
As for visual acuity, there were differences in the logarithm of the minimum angle of resolution (logMAR) acuities at presentation between the right eye (RE) and LE in 7 patients due to sight-threatening retinal conditions such as macular hole (LE of patient 14[
Graph: Figure 2 Scatter plots of the best-corrected visual acuity as a function of age. The best-corrected visual acuity (logMAR equivalent) of the better-seeing eye significantly declined as a function of age (n = 24, r = 0.515, p < 0.01). The segmented linear regression [≤ 40 years old (n = 13, r = − 0.22) and > 40 years old (n = 11, r = 0.512)] indicates that BCVA worsens particularly for > 40 years old.
Graph: Figure 3 Kaplan–Meier survival curve. The Kaplan–Meier survival curve for a decimal BCVA equal of 0.5 (Snellen equivalent 20/40) or worse in the worse-seeing eye (n = 24) at follow up demonstrated that the median age of survival was 57.0 years.
Among 22 patients assessed by optical coherence tomography (OCT), 4 patients were excluded for unclear foveal ellipsoid zone (EZ) bilaterally (patients 6, 8, 20) and bilateral CNV (patient 15). We measured central foveal thickness (CFT) and the EZ width. We found no correlation (n = 18, r = − 0.13) between the CFT of the better-seeing eye and age at follow up (Fig. 4). On the other hand, there was a significant negative correlation (n = 18, r = − 0.798, p < 0.01) between the EZ width of the better-seeing eye and age at follow up (Fig. 4).
Graph: Figure 4 Scatter plots of optical coherence tomography findings of the better-seeing eye and age at follow up. No correlation between the central foveal thickness and age is found (n = 18, r = − 0.13). On the other hand, there is a significant negative correlation between the ellipsoid zone width and age (n = 18, r = − 0.798, p < 0.01).
Fundus autofluorescence (FAF) images were shown from the three representative patients in Fig. 5. The preserved autofluorescence (PAF) area was measured from better-seeing eye of patients 22, 14 and 10 when they were 16 years, 27 years and 52 years of age, respectively. The PAF area in patient 10 was smaller than that in patients 14 and 22 (Fig. 5).
Graph: Figure 5 Fundus autofluorescence images. Fundus autofluorescence images are shown from the three representative patients. The preserved autofluorescence (PAF) area is measured from better-seeing eye of patients 22 (A), 14 (B) and 10 (C) when they are 16 years, 27 years and 52 years of age, respectively. The PAF area in patient 10 is smaller than that in patients 22 and 14 (D).
A total of 14 different pathogenic variants including large deletions of CHM were identified in 16 families, but no pathogenic variant or deletion was found only in one family (family 6) (Fig. 1 and Table 2). Eight variants [c.315_318del (p.Ser105ArgfsTer20), c.616dupA (p.Thr206AsnfsTer17), c.646delA (p.Thr216LeufsTer16), c.757C > T (p.Arg253Ter), c.820-2A > T (splice site variant), c.1034C > G (p.Ser345Ter), c.1079delA (p.Asn360ThrfsTer49), and c.1649 T > C (p.Leu550Pro)] have been previously reported as causes of CHM[
Genetic findings of 24 patients with choroideremia.
Patient # Patient ID Family # Nucleotide change Protein change HGMD ClinVar/GnomAD ACMG References 1 JU0097 1 c.1593 T > A p.Tyr531Ter ND ND Pathogenic (PVS1,PM2,PP3) This study 2 JU0141 2 c.820-2A > T (Splicing change) ND ND Pathogenic (PVS1,PM2,PP3) Described 3 JU0462 2 4 JU0463 2 5 JU0395 3 c.1079delA p.Asn360ThrfsTer49 CD137317 ND Pathogenic (PVS1,PM2,PP3) Described 6 JU0425 4 c.527G > A, c.(529G > T; 530A > G) p.Gly176Glu, p.Glu177Ter ND ND Likely pathogenic (PVS1,PM2) This study 7 JU0426 5 Exon 2 deletion (Gross deletion) ND Not applicable Not applicable This study 8 JU0433 6 9 JU1850 6 10 JU0752 7 c.1034C > G p.Ser345Ter CM983733 ND Pathogenic (PVS1,PM2,PP3) Described 11 JU1817 7 12 JU1059 8 c.315_318del p.Ser105ArgfsTer20 CD983792 ND Pathogenic (PVS1,PM2,PP3,PP5) Described 13 JU1248 9 c.646delA p.Thr216LeufsTer16 ND ND Likely pathogenic (PVS1,PM2) Described 14 JU1254 10 c.757C > T p.Arg253Ter CM994349 ND Pathogenic (PVS1,PM2,PP3,PP5) Described 15 JU1457 11 c.616dupA p.Thr206AsnfsTer17 CI1515670 ND Likely pathogenic (PVS1,PM2) Described 16 JU1458 11 17 JU1520 11 18 JU1681 12 c.1649 T > C p.Leu550Pro CM093648 ND Uncertain significance (PM2,PP3,PP5,BP1) Described 19 JU1681b 12 Not determined 20 JU1808 13 c.616dupA p.Thr206AsnfsTer17 CI1515670 ND Likely pathogenic (PVS1,PM2) Described 21 JU1877 13 22 JU1831 14 c.49 + 5G > A (Splicing change) ND Likely pathogenic/ND Uncertain significance (PM2,PP5,BP5) This study 23 JU1835 15 Deletion of 5.0 Mb (Gross deletion) ND Not applicable Not applicable This study 24 JU1688 16 c.116 + 5G > A (Splicing change) ND ND Uncertain significance (PM2,BP4) This study
ND not described. HGMD: the Human Gene Mutation Database (https://www.hgmd.org); ClinVar: https://
We performed whole exome sequencing (WES) for patient 7 (family 5) due to the failure of polymerase chain reaction amplification of exon 2. However, all exons except for exon 2 were amplified for Sanger sequencing. The IGV demonstrated a deletion of approximately 34.2 kb containing exon 2, which was determined by comparison with two controls (Fig. 6). The presence of exon 1 and exons 3–15 was confirmed, consistent with the results of Sanger sequencing analysis. WES was also performed in patient 23 (family 15), demonstrating an extremely large deletion of probably 5.0 mb encompassing the entire CHM gene (Supplementary Figure S1) and other genes (TGIF2LX, CPXCR1, KLHL4, DACH2, MIR1321, POF1B, SATL1, UBE2DNL) in the vicinity of CHM.
Graph: Figure 6 Whole-exome sequencing data of patient 7 and two controls. The Integrative Genomics Viewer visualization of exons 1–4 in the CHM gene indicated a deletion of approximately 34.2 kb containing exon 2 in patient 7.
In patients 8 and 9 (family 6), we performed whole genome sequencing (WGS) because no pathogenic CHM variant was found in both Sanger sequencing and WES. In addition, no pathogenic variant was found in both RP2 and RPGR genes, which are responsible for X-linked retinitis pigmentosa. IGV revealed no decrease in read depth of any exons or introns in either patient (Supplementary Figure S2), indicating no obvious deletion region in the CHM gene. In addition, no rare variant was found in the promoter region. As a result, we could not find any pathogenic variant or deletion in the CHM gene, even though five male CHM patients were found in family 6.
In this study, we investigated the genetic spectrum of CHM, visual acuity outcomes and OCT findings in a single-center cohort of 24 Japanese patients (from 16 families) with CHM. Genetic analysis identified 14 different CHM variants or deletions, 6 of which were novel. The visual acuity outcomes revealed that BCVA significantly worsened with increasing age, and the Kaplan–Meier survival analysis suggested that a BCVA of 0.5 (Snellen equivalent 20/40) or better remains preserved until the fifties. Also, we found a significant negative correlation between the EZ width and age.
Previous studies have revealed that the majority of CHM patients harbor loss-of-function/null-type variants in the CHM gene[
Visual acuity is the most important parameter for the assessment of visual function. As for the relationship between BCVA and age, a cross-sectional study of 120 CHM patients, collected from 24 studies and/or case reports, showed that BCVA decreases very slowly until 50 years of age[
An OCT study of 61 eyes from 39 CHM patients has revealed that the central retinal thickness was within normal limits until the 40 s, followed by significant thinning between 40 and 60 years of age[
A previous study of CHM genotype and phenotype correlations found no significant difference in terms of visual acuity by variants involving the C-terminus and those occurring upstream[
Until recently, no treatment was available for CHM. The first-in-human phase1/2 gene therapy trial was achieved in 2014 in which 6 CHM patients underwent vitrectomy accompanied with subfoveal injection of AAV2 containing CHM cDNA[
Our study had a few limitations, including a relatively small sample size (24 patients) and recruitment from a single center. Nevertheless, we found a variety of genetic variations: 14 different CHM variants in 15 families. In addition, our inclusion criteria might have had selection bias because some elderly CHM patients with total blindness, who cannot come to the hospital due to various reasons, might be excluded. Further studies with a larger sample size from multiple centers would give more strength to our visual acuity outcomes and OCT findings in disease progression.
In summary, we investigated the genetic spectrum of CHM, visual acuity outcomes and OCT findings in a single-center cohort of 24 Japanese CHM patients. Genetic analysis using Sanger sequencing and/or WES identified 6 novel variants/deletions in the CHM gene. The majority of CHM variants (14/15, 93%) were predicted to be loss-of-function/null-type variants in our Japanese families. The visual acuity outcomes revealed that BCVA significantly worsened with increasing age, especially in patients > 40 years old. The Kaplan–Meier survival analysis suggested that a BCVA of Snellen equivalent 20/40 or better remains preserved until the fifties. This was the largest cohort study to investigate visual acuity as a function of age in Japanese patients with CHM variants/deletions. Our results will be pivotal clinical data for upcoming phase 3 clinical trials of AAV2 gene therapy using NSR-REP1 to determine participants' eligibility as inclusion criteria for visual acuity.
A single-center cohort of 24 CHM patients from 16 Japanese families was included in this study (Fig. 1). The medical records from The Jikei University Hospital were retrospectively reviewed including age, gender, chief complaint, visual acuity and OCT. The clinical diagnosis of CHM was made between 2002 and 2020, based on the characteristic ophthalmoscopic appearance, X-linked recessive transmission, and each carrier's ophthalmoscopy findings (when possible). We measured the BCVA at a 5-m distance using Landolt C charts at presentation and follow-up. Horizontal cross-setional retinal images (6.0 mm) were evaluated using spectral domain OCT (Cirrus HD-OCT, Carl Zeiss Meditec AG, Dublin, CA, USA) from all patients, except for patients 7 and 19, at follow-up visit. We measured the CFT and EZ width using a ruler within the OCT device. The CFT was difined as the distance between the vitreoretinal interface and the inner surface of the retinal pigment epithelium at the fovea, whereas the EZ width was difined as the horizontal linear distance between two locations. The Spectralis HRA2 (Heidelberg Engineering, Heidelberg, Germany) was used to obtain FAF images from patients 10, 14 and 22 as representative cases. The PAF area (mm
Genomic DNA from leucocytes in venous blood samples was extracted using a Gentra Puregene Blood kit (Qiagen, Hilden, Germany) from the probands/patients (depicted by arrows in Fig. 1) and their family members. The exonic regions of the CHM gene were analyzed by Sanger sequencing[
The Database used in the study are publicly available.
The mean, median, and range (min and max values) were used for assessment. Decimal BCVA was converted to the logMAR units for statistical analysis. BCVA of hand motions was converted to 2.7 logMAR units[
The Institutional Review Boards of The Jikei University School of Medicine (approval number: 24-231 6997), National Hospital Organization Tokyo Medical Center (approval number: R14-050) and Nippon Medical School (approval number: 27-02) approved the protocol for this study. The protocol adhered to the tenets of the Declaration of Helsinki, with informed consent obtained from participants and/or their legal guardians.
The authors would like to thank the patients and their families for participating in this study. This research was supported by grants from Practical Research Project for Rare/Intractable Diseases (15ek0109072h0003, 16ek0109072h0003, 17ek0109282s0001, 18ek0109282h0002, 19ek0109282h0003 to T.I.) and from the Japan Agency for Medical Research and Development (AMED), Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17K11434 to T.H.), Japanese Retinitis Pigmentosa Society (JRPS) Research Grant 2019 to T.H. and The Jikei University Research Fund to T.H.
S.K. (Shuhei Kameya), D.K., S.K. (Sachiko Kikuchi),K.Y. and T.I. performed the molecular genetic analyses. T.H., T.I. and K.M. interpreted the data and wrote the manuscript. T.H., S.K. (Shuhei Kameya), A.M. (Atsushi Mizota), A.M. (Akira Murakami) and T.N. assisted with data interpretation. T.H. designed and supervised the study. All authors have read and approved the final manuscript.
The authors declare no competing interests.
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By Takaaki Hayashi; Shuhei Kameya; Kei Mizobuchi; Daiki Kubota; Sachiko Kikuchi; Kazutoshi Yoshitake; Atsushi Mizota; Akira Murakami; Takeshi Iwata and Tadashi Nakano
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