Doc Ophthalmol DOI 10.1007/s10633-015-9497-7
CLINICAL CASE REPORT
Reduced rod electroretinograms in carrier parents of two Japanese siblings with autosomal recessive retinitis pigmentosa associated with PDE6B gene mutations Kazuki Kuniyoshi • Hiroyuki Sakuramoto • Kazutoshi Yoshitake • Kazuho Ikeo • Masaaki Furuno • Kazushige Tsunoda • Shunji Kusaka Yoshikazu Shimomura • Takeshi Iwata
•
Received: 18 March 2014 / Accepted: 25 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose To present the clinical and genetic findings in two siblings with autosomal recessive retinitis pigmentosa (RP) and their non-symptomatic parents. Methods We studied two siblings, a 48-year-old woman and her 44-year-old brother, and their parents. They had general ophthalmic examinations including ophthalmoscopy, perimetry, and electroretinography (ERG). Their whole exomes were analyzed by the next-generation sequence technique. Results The two siblings had night blindness for a long time, and clinical examinations revealed diffuse retinal degeneration with bone spicule pigmentation, constriction of the visual field, and non-recordable ERGs. Their parents were non-symptomatic and had normal fundi; however, their rod ERGs were reduced. Genetic
examination revealed compound heterozygous mutations of I535N and H557Y in the PDE6B gene in the siblings, and the parents were heterozygous carriers of the mutations. Conclusions Heterozygous mutation in the PDE6B gene can cause a reduction in the rod function to different degrees. The retinal function of non-symptomatic carriers of autosomal recessive RP should be evaluated with care.
K. Kuniyoshi (&) H. Sakuramoto Y. Shimomura Department of Ophthalmology, Kinki University Faculty of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589-8511, Japan e-mail: [email protected]
K. Tsunoda Laboratory of Visual Physiology, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
K. Yoshitake K. Ikeo Laboratory for DNA Data Analysis, National Institute of Genetics, Shizuoka, Japan M. Furuno Division of Genomic Technologies, Life Science Accelerator Technology Group, Transcriptome Technology Team, RIKEN Center for Life Science Technologies, Yokohama, Japan
Keywords PDE6B Retinitis pigmentosa Congenital stationary night blindness Electroretinograms Carrier Autosomal recessive Japanese
S. Kusaka Department of Ophthalmology, Sakai Hospital, Kinki University Faculty of Medicine, Osaka, Japan T. Iwata Division of Molecular and Cellular Biology, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
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Introduction Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes a slow, progressive retinal degeneration with night blindness and visual field loss [1, 2]. The onset of the symptoms and signs of the disease can vary from infancy to late adulthood, and the visual acuity is ultimately reduced. The estimated prevalence of RP is 1:2000–1:7000, which is dependent on the ethnicity and country [1, 2]. RP is a complex of various retinal dystrophies and is associated with different kinds of inheritance pattern, e.g., autosomal dominant, autosomal recessive, and X-linked recessive [1, 2]. To date, 48 different causative genes have been discovered to cause autosomal recessive RP [3]. Among them, the PDE6B gene [4] was first reported as causative for autosomal recessive RP by McLaughlin et al. [5] in 1993 (RP40, MIM# 613801) [5], and it accounts for 4–5 % of autosomal recessive RP [5, 6]. Since then, several studies have been published on human RP which showed that the RP was caused by PDE6B mutations [5–20]. The PDE6B gene encodes the b-subunit of rod phosphodiesterase 6 (PDE6) [4]. PDE6 regulates the cytoplasmic level of cyclic guanosine monophosphate (cGMP) in the photoreceptors [21]. PDE6 is activated by light stimulation which then reduces the level of cGMP. This leads to closure of the cGMP-gated Na? and Ca?? channels which then hyperpolarizes the rod plasma membrane [21]. A dysfunction of the bsubunit of PDE6 results in a high concentration of cGMP and Ca?? in rod photoreceptors, and it promote apoptosis of the rod photoreceptors [22]. The phenotype of the PDE6B-associated RP is typical of RP, namely, attenuated retinal vessels, intraretinal bone spicule pigments around mid-periphery to periphery, and waxy-pale optic nerve head [5– 20]. In past reports, the ages of the RP patients at the time of the ophthalmic examinations ranged from 14 to 81 years, and most of them reported night blindness starting in early childhood [5–20]. The ophthalmic examinations showed constricted visual fields, but the central vision can be preserved until the late stages of the disease process. At the early stages, the rod responses of the electroretinograms (ERGs) are nonrecordable and the cone responses are reduced. At the late stages, the cone responses will be extinguished. Cataract is commonly seen in the middle age of RP
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patients. Recent studies reported cystic macular edema which seems to be one of characteristics of the PDE6A&B-associated RP [18–20]. Although no effective therapy is available on human PDE6B-associated RP, attempts of replacing pde6b by lentiviral [23] or adenoassociated virus [24, 25] vectors have been reported in animal models with PDE deficiency. The efficacy of calcium channel blockers is also being examined as a sole treatment [26] or with an adenoassociated virus vector [25] for blocking the apoptosis of photoreceptors. We report the clinical findings of two patients with autosomal recessive RP and their carrier parents who carried missense mutations in the PDE6B gene.
Patients and methods The patients were two Japanese siblings who were diagnosed with RP (Patients 1 and 2 in Fig. 1) and their non-symptomatic parents (Carriers 1 and 2 in Fig. 1). Twenty-four eyes of 24 age-matched individuals with no visual symptom and normal fundi served as controls. The age of the control group ranged from 71 to 86 years with a mean age of 77.3 ± 4.5 years. The ERG recordings in the normal controls were performed in the Seichokai Fuchu Hospital and Kinki University Hospital. The research protocol was approved by the Ethics Review Board of the Kinki University Faculty of
Fig. 1 Pedigree of family with RP. Proband was Patient 1 (kinki-1016), and family ID# was kinki-F10. Parents of the patients were non-consanguineous
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Medicine in November 2011, and the procedures conformed to the tenets of the Declaration of Helsinki. The genetic studies were performed after obtaining a signed informed consent form from all of the patients and their parents. Clinical studies The ophthalmic examinations including slit-lamp biomicroscopy, ophthalmoscopy, OctopusÒ static automated perimetry, and International Society for Clinical Electrophysiology of Vision (ISCEV)-standard ERGs [27] were performed as clinical studies. The ERGs were recorded with a portable ERGrecording system and were elicited by white lightemitting diodes embedded in a contact lens electrode (LE-4000; TOMEY Corporation, Nagoya, Japan). The clinical data of Patient 1 were obtained in the Department of Ophthalmology, Nishida Clinic (Izumi-Sano City, Osaka, Japan), and those of Patient 2 and Carriers 1 and 2 in the Department of Ophthalmology, Kushimoto Hospital (Kushimoto Town, Wakayama, Japan). Genetic studies Genetic studies were performed using methods described in detail earlier [28]. First, the DNA was extracted from blood samples using the Gentra Puregene Blood Kit (Qiagen, Venlo, Netherlands) and shared with Covaris Ultrasonicator (Covaris, Woburn, MA) in the Division of Molecular and Cellular Biology of the National Institute of Sensory Organs of the National Hospital Organization, Tokyo Medical Center (Tokyo, Japan). The construction of paired-end sequence libraries and exome capture were performed using the Agilent Bravo Automated Liquid Handling Platform with SureSelect XT Human All Exon Kit V4 ? UTRs Kit (Agilent Technologies, Santa Clara, CA). The products were sequenced with the Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA) at RIKEN (Yokohama City, Kanagawa, Japan). The detected mutations were analyzed at the National Institute of Genetics (Shizuoka City, Shizuoka, Japan). The filtered mutations were scored with the PolyPhen software version 2.2.2 [29], which predicts their effect on the structure and function of the protein. The entire exome analysis pipeline is available at Maser [30].
Results Patient 1 (proband; kinki-1016) was a 48-year-old woman who reported that she had night vision difficulties for as long as she can remember. Her parents were not consanguineous (Fig. 1). Her decimal best-corrected visual acuity (BCVA) was 0.7 with ?3.25 diopter sphere (DS) and -2.0 D cylinder (DC) ax 100° in the right eye and 0.6 with ?3.25 DS and -1.5 DC ax 90° in the left eye. Slit-lamp examination showed mild nuclear cataracts in both eyes. Ophthalmoscopy revealed diffuse retinal degeneration with bone spicule pigments in the midperiphery (Fig. 2). The retinal vessels were attenuated although they were detectable even in the periphery. Static perimetry showed constricted visual fields (Fig. 3) although she did not notice any visual field disturbances. Full-field ISCEV-standard ERGs were non-recordable except the flicker ERGs which were severely attenuated (Fig. 4). Genetic studies showed 1,083,241 mutations when her whole-exome sequence was compared with a reference human genome (hs37d5). Among them, 456 mutations remained as candidate mutations after focusing on mutations that could change the amino acid sequence and the exclusion of common mutations by 1000 genomes in the Human Genetic Variation Database [31] and in our in-house database. They were filtered based on the inheritance pattern of the pedigree, and four candidate genes remained. They were VPS13D, ZNF518B, GIPR, and PDE6B. Finally, PDE6B was considered to be the gene responsible for the retinal dystrophy in the siblings, because it was the only gene registered in the RetNet database [3] as causing inherited retinal dystrophies among the four candidate genes. Further genetic analysis revealed a compound heterozygous c.1604T[A and c.1669C[T transition in exon 12 and 13 resulting in substitutions I535N and H557Y in the PDE6B gene. The PolyPhen-2 scores for the four candidate genes were: VPS13D (E372K, 0.66), VPS13D (P1047L, 0.103), VPS13D (T2883S, 0.01), ZNF518B (G732R, 0.002), and ZNF518B (H448R, 0.001), GIPR (G162C, 0.994), GIPR (L226H, 0.999), PDE6B (I535N, 0.999), and PDE6B (H557Y, 0.999).
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Fig. 2 Montage fundus photographs of Patients 1 and 2 and Carrier parents 1 and 2. Patients had typical RP appearance and carriers (parents of the patients) had normal fundi
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Fig. 3 Results of OctopusÒ static automated perimetry. Automated perimetry was performed with G1/Program 32 and Dynamic strategy in OctopusÒ 301, ver. 5.09 (HAAG-STREIT AG, Koeniz, Switzerland). Patients 1 and 2 have constricted visual fields, while Carrier 1 (father) has mild reduction in
sensitivity in the mid-periphery. Rate of false-positive and falsenegative responses during the examination was 0/8 and 1/8 OD, 0/8 and 3/9 OS in Patient 1, 0/8 and 2/8 OD, 0/6 and 1/7 OS in Patient 2, 1/10 and 2/11 OD, 2/11 and 1/12 OS in Carrier 1, and 0/9 and 0/10 OD, 1/9 and 1/10 OS in Carrier 2
Patient 2 (kinki-1030)
-2.25 DC ax 10° in the left eye. Slit-lamp examination revealed intraocular lens implanted in both eyes. Ophthalmoscopy showed diffuse retinal degeneration with numerous bone spicule pigments in the midperiphery (Fig. 2). The retinal vessels were severely attenuated, and the optic discs were waxy-pale in both eyes. Static perimetry showed constricted visual fields
was the 44-year-old younger brother of Patient 1 (Fig. 1). He had night blindness from childhood and noticed visual field disturbances in his mid-thirties. His BCVA was 0.5 with ?0.75 DS and -1.25 DC ax 180° in the right eye and 0.02 with ?1.0 DS with
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Fig. 4 ISCEV-standard full-field ERGs. The amplitudes of the rod ERGs recorded from Carriers 1 and 2 are reduced. Rod ERG; dark-adapted 0.01 ERG, flash ERG; dark-adapted 30
ERG, cone ERG; light-adapted 3.0 ERG, flicker ERG; lightadapted 30-Hz flicker ERG [27]
and reduced sensitivities in the macula (Fig. 3). The ISCEV-standard ERGs were non-recordable in both eyes (Fig. 4). Genetic analysis revealed compound heterozygous mutations of I535N and H557Y substitutions in the PDE6B gene, the same as his sister (Patient 1).
rod b-wave was 98 lV (66 % of lower limit of the normal value) OD and 135 lV (90 %) OS in Carrier 1, and 72 lV (48 %) OD and 85 lV (57 %) OS in Carrier 2 (Table 1). The implicit times in both were not delayed at 114 ms OD and 97 ms OS in Carrier 1, and 87 ms which was 95 % of the lower limit of normal value OD and 84 ms (91 %) OS in Carrier 2. Although Carrier 1 had normal ERGs except for the reduced rod ERGs, Carrier 2 showed reduced flash b-wave resulting in a negative-type ERG (Table 1; Fig. 4). Carrier 2 also had reduced flicker ERG in the left eye (Table 1). Genetic investigations revealed heterozygous mutations in the PDE6B gene, viz., I535N substitution in Carrier 1 (kinki-1031) and H557Y substitution in Carrier 2 (kinki-1032).
Carrier parents (kinki-1031 and 1032) The 82-year-old father (Carrier 1 in Fig. 1) and 72-year-old mother (Carrier 2 in Fig. 1) were investigated both clinically and genetically. They were both non-symptomatic, and their BCVA was 0.8 OD and 0.5 OS in Carrier 1, and 0.8 OD and 1.0 OS in Carrier 2. They had nuclear cataracts, and the fundus examinations were unremarkable except for some aging changes in both eyes (Fig. 2). However, Carrier 1 had a mild reduction in retinal sensitivity by static perimetry (Fig. 3). Both parents had significantly reduced rod ERGs compared with age-matched normal controls (Table 1; Fig. 4). The amplitude of the
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Discussion Patients 1 and 2 had the phenotype of typical RP, and their parents (Carriers 1 and 2) had normal fundi and
Rod ERG in Carriers 1 and 2 showed significantly reduced amplitude. The amplitude is expressed as % of the lower/upper limit of age-matched normal values. One hundred % means within normal value. Rod ERG; dark-adapted 0.01 ERG, flash ERG; dark-adapted 30 ERG, cone ERG; light-adapted 3.0 ERG, flicker ERG; light-adapted 30-Hz flicker ERG [27], SD; standard deviation
75–133 98–172 1.02–1.35 357–515 300–441 149–233 – Normal range
90 (100 %)
67 (90 %)
104 ± 29 135 ± 37 1.19 ± 0.17 436 ± 79 370 ± 70 191 ± 42 77.3 ± 4.5 Mean ± SD Normal controls (n = 24)
139 (100 %)
105 (100 %) 0.85 (84 %)
0.79 (78 %) 340 (95 %)
328 (92 %) 385 (100 %)
429 (100 %) 85 (57 %)
72 (48 %) 72
Left
Right Carrier 2
114 (100 %)
141 (106 %) 190 (110 %)
172 (100 %) 1.2 (100 %)
1.52 (112 %) 628 (122 %)
462 (100 %) 384 (100 %)
413 (100 %) 135 (90 %)
98 (66 %) 82
Left
Left
Right Carrier 1
0
16.0
0 0 – 0 0 0
0
0 –
– 0
0 0
0 0
0
44 Left
Right Patient 2
17.3 0 – 0 0 0 48 Right Patient 1
Table 1 Results of ISCEV-standard ERG
Age (year)
Rod ERG b-wave (lV)
Flash ERG a-wave (lV)
Flash ERG b-wave (lV)
Flash ERG b/a ratio
Cone ERG b-wave (lV)
Flicker ERG (lV)
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were non-symptomatic. However, the rod ERGs of both parents were significantly reduced compared with age-matched normal control. The reduced rod ERGs suggest a mild impairment of the rod system in Carriers 1 and 2 although they did not report night blindness. Although we did not determine the exact cause of the reduction in their rod ERGs, it is notable that past studies [5, 32–35] have also shown rod dysfunction in individuals with heterozygous mutation in the PDE6B gene. McLaughlin et al. [5] reported similar ERG findings in their patients as in our family. Their family had mutations in the PDE6B gene, Q298X and R531X, and the affected members had non-recordable ERGs. The non-symptomatic carriers had reduced and delayed ERGs to dim blue flash and white flicker flashes [5]. They examined the rod function of the carriers in more detail and reported that the light intensity that produced a half-maximal rod response was equal to that in normal controls, even though the maximum rod signal was reduced. This suggested abnormal rods or a reduction in the number of functioning rods in the carriers [5]. The results of other studies [32–35] have shown that the PDE6B gene is causative for autosomal dominant congenital stationary night blindness (CSNB; CSNBAD2, MIM# 163500). Thus, homozygous or compound heterozygous mutations in the PDE6B gene can cause typical RP, and heterozygous mutation in the PDE6B gene can cause retinal dysfunction of various degrees ranging from non-symptomatic mild rod dysfunction to the complete type CSNB. However, we do not know with certainty that the mutations which were found in our family are the cause of the reduction in the rod ERGs in the Carriers 1 and 2 or whether the reduction is common in heterozygous point mutation of the PDE6B gene. Further study is needed. The flash ERGs in Carrier 2 had normal a-wave and reduced b-wave resulting in a negative-type ERG (Fig. 4). The normal a-wave indicates normally functioning photoreceptors that is presumably elicited by normal cone photoreceptors. We did not determine the cause of the negative-type ERG although some ischemic retinal conditions and comorbid systemic diseases and medication for the condition may have caused the reduced flash b-wave in Carrier 2. Saga et al. [12] reported the first Japanese family with PDE6B-associated RP before this report. They
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reported a missense mutation of I535N (c.1604T[A), which is the same as in our pedigree. They also found a heterozygous mutation of I535N in one individual among 90 unrelated healthy Japanese individuals who served as normal controls [12]. Another missense mutation H557Y (c.1669C[T) in our pedigree has been reported in Japan [13] and Korea [18]. Unfortunately, these earlier studies did not report the ERG findings on the heterozygous carrier of I535N or H557Y in the PDE6B gene. Because a few pedigrees were reported from Japan and Korea as PDE6B-associated RP, further studies are needed to confirm that the missense mutations I535N and H557Y in the PDE6B gene is frequently found in eastern Asia. In conclusions, we report the clinical features of two patients with autosomal recessive RP which was caused by missense mutations of I535N and H557Y in the PDE6B gene. Their carrier parents were nonsymptomatic, and their fundi were unremarkable. However, they had reduced rod ERGs. Our findings indicate that non-symptomatic carrier relatives in pedigrees of autosomal recessive RP may have retinal dysfunction of various degrees. Acknowledgments The authors thank Professor Duco Hamasaki of the Bascom Palmer Eye Institute of the University of Miami for critical discussion and final manuscript revisions; Miss Seiko Kawamura, CO, of the Seichokai Fuchu Hospital (Osaka, Japan); and Mr. Tomoaki Nishio of TOMEY Corporation (Nagoya, Japan) for their assistance for ERG examinations. The authors wish to acknowledge RIKEN GeNAS for the sequencing of the exome-enriched libraries. This research was supported by the research grants to T.I., K.T., and K.K. from the Ministry of Health, Labour and Welfare, Japan (13803661), to K.T. and K.K. from the Ministry of Health, Labour and Welfare, Japan (23164001), Y.S from the Ministry of Health, Labour and Welfare, Japan (82259921), S.K. and K.K. from Japan Society for the Promotion of Science, Japan (23592597), and to M.F. from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) for RIKEN Omics Science Center.
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Conflict of interest All authors have no commercial interests related to this research. 13.
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CLINICAL CASE REPORT
Reduced rod electroretinograms in carrier parents of two Japanese siblings with autosomal recessive retinitis pigmentosa associated with PDE6B gene mutations Kazuki Kuniyoshi • Hiroyuki Sakuramoto • Kazutoshi Yoshitake • Kazuho Ikeo • Masaaki Furuno • Kazushige Tsunoda • Shunji Kusaka Yoshikazu Shimomura • Takeshi Iwata
•
Received: 18 March 2014 / Accepted: 25 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose To present the clinical and genetic findings in two siblings with autosomal recessive retinitis pigmentosa (RP) and their non-symptomatic parents. Methods We studied two siblings, a 48-year-old woman and her 44-year-old brother, and their parents. They had general ophthalmic examinations including ophthalmoscopy, perimetry, and electroretinography (ERG). Their whole exomes were analyzed by the next-generation sequence technique. Results The two siblings had night blindness for a long time, and clinical examinations revealed diffuse retinal degeneration with bone spicule pigmentation, constriction of the visual field, and non-recordable ERGs. Their parents were non-symptomatic and had normal fundi; however, their rod ERGs were reduced. Genetic
examination revealed compound heterozygous mutations of I535N and H557Y in the PDE6B gene in the siblings, and the parents were heterozygous carriers of the mutations. Conclusions Heterozygous mutation in the PDE6B gene can cause a reduction in the rod function to different degrees. The retinal function of non-symptomatic carriers of autosomal recessive RP should be evaluated with care.
K. Kuniyoshi (&) H. Sakuramoto Y. Shimomura Department of Ophthalmology, Kinki University Faculty of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589-8511, Japan e-mail: [email protected]
K. Tsunoda Laboratory of Visual Physiology, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
K. Yoshitake K. Ikeo Laboratory for DNA Data Analysis, National Institute of Genetics, Shizuoka, Japan M. Furuno Division of Genomic Technologies, Life Science Accelerator Technology Group, Transcriptome Technology Team, RIKEN Center for Life Science Technologies, Yokohama, Japan
Keywords PDE6B Retinitis pigmentosa Congenital stationary night blindness Electroretinograms Carrier Autosomal recessive Japanese
S. Kusaka Department of Ophthalmology, Sakai Hospital, Kinki University Faculty of Medicine, Osaka, Japan T. Iwata Division of Molecular and Cellular Biology, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
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Introduction Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes a slow, progressive retinal degeneration with night blindness and visual field loss [1, 2]. The onset of the symptoms and signs of the disease can vary from infancy to late adulthood, and the visual acuity is ultimately reduced. The estimated prevalence of RP is 1:2000–1:7000, which is dependent on the ethnicity and country [1, 2]. RP is a complex of various retinal dystrophies and is associated with different kinds of inheritance pattern, e.g., autosomal dominant, autosomal recessive, and X-linked recessive [1, 2]. To date, 48 different causative genes have been discovered to cause autosomal recessive RP [3]. Among them, the PDE6B gene [4] was first reported as causative for autosomal recessive RP by McLaughlin et al. [5] in 1993 (RP40, MIM# 613801) [5], and it accounts for 4–5 % of autosomal recessive RP [5, 6]. Since then, several studies have been published on human RP which showed that the RP was caused by PDE6B mutations [5–20]. The PDE6B gene encodes the b-subunit of rod phosphodiesterase 6 (PDE6) [4]. PDE6 regulates the cytoplasmic level of cyclic guanosine monophosphate (cGMP) in the photoreceptors [21]. PDE6 is activated by light stimulation which then reduces the level of cGMP. This leads to closure of the cGMP-gated Na? and Ca?? channels which then hyperpolarizes the rod plasma membrane [21]. A dysfunction of the bsubunit of PDE6 results in a high concentration of cGMP and Ca?? in rod photoreceptors, and it promote apoptosis of the rod photoreceptors [22]. The phenotype of the PDE6B-associated RP is typical of RP, namely, attenuated retinal vessels, intraretinal bone spicule pigments around mid-periphery to periphery, and waxy-pale optic nerve head [5– 20]. In past reports, the ages of the RP patients at the time of the ophthalmic examinations ranged from 14 to 81 years, and most of them reported night blindness starting in early childhood [5–20]. The ophthalmic examinations showed constricted visual fields, but the central vision can be preserved until the late stages of the disease process. At the early stages, the rod responses of the electroretinograms (ERGs) are nonrecordable and the cone responses are reduced. At the late stages, the cone responses will be extinguished. Cataract is commonly seen in the middle age of RP
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patients. Recent studies reported cystic macular edema which seems to be one of characteristics of the PDE6A&B-associated RP [18–20]. Although no effective therapy is available on human PDE6B-associated RP, attempts of replacing pde6b by lentiviral [23] or adenoassociated virus [24, 25] vectors have been reported in animal models with PDE deficiency. The efficacy of calcium channel blockers is also being examined as a sole treatment [26] or with an adenoassociated virus vector [25] for blocking the apoptosis of photoreceptors. We report the clinical findings of two patients with autosomal recessive RP and their carrier parents who carried missense mutations in the PDE6B gene.
Patients and methods The patients were two Japanese siblings who were diagnosed with RP (Patients 1 and 2 in Fig. 1) and their non-symptomatic parents (Carriers 1 and 2 in Fig. 1). Twenty-four eyes of 24 age-matched individuals with no visual symptom and normal fundi served as controls. The age of the control group ranged from 71 to 86 years with a mean age of 77.3 ± 4.5 years. The ERG recordings in the normal controls were performed in the Seichokai Fuchu Hospital and Kinki University Hospital. The research protocol was approved by the Ethics Review Board of the Kinki University Faculty of
Fig. 1 Pedigree of family with RP. Proband was Patient 1 (kinki-1016), and family ID# was kinki-F10. Parents of the patients were non-consanguineous
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Medicine in November 2011, and the procedures conformed to the tenets of the Declaration of Helsinki. The genetic studies were performed after obtaining a signed informed consent form from all of the patients and their parents. Clinical studies The ophthalmic examinations including slit-lamp biomicroscopy, ophthalmoscopy, OctopusÒ static automated perimetry, and International Society for Clinical Electrophysiology of Vision (ISCEV)-standard ERGs [27] were performed as clinical studies. The ERGs were recorded with a portable ERGrecording system and were elicited by white lightemitting diodes embedded in a contact lens electrode (LE-4000; TOMEY Corporation, Nagoya, Japan). The clinical data of Patient 1 were obtained in the Department of Ophthalmology, Nishida Clinic (Izumi-Sano City, Osaka, Japan), and those of Patient 2 and Carriers 1 and 2 in the Department of Ophthalmology, Kushimoto Hospital (Kushimoto Town, Wakayama, Japan). Genetic studies Genetic studies were performed using methods described in detail earlier [28]. First, the DNA was extracted from blood samples using the Gentra Puregene Blood Kit (Qiagen, Venlo, Netherlands) and shared with Covaris Ultrasonicator (Covaris, Woburn, MA) in the Division of Molecular and Cellular Biology of the National Institute of Sensory Organs of the National Hospital Organization, Tokyo Medical Center (Tokyo, Japan). The construction of paired-end sequence libraries and exome capture were performed using the Agilent Bravo Automated Liquid Handling Platform with SureSelect XT Human All Exon Kit V4 ? UTRs Kit (Agilent Technologies, Santa Clara, CA). The products were sequenced with the Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA) at RIKEN (Yokohama City, Kanagawa, Japan). The detected mutations were analyzed at the National Institute of Genetics (Shizuoka City, Shizuoka, Japan). The filtered mutations were scored with the PolyPhen software version 2.2.2 [29], which predicts their effect on the structure and function of the protein. The entire exome analysis pipeline is available at Maser [30].
Results Patient 1 (proband; kinki-1016) was a 48-year-old woman who reported that she had night vision difficulties for as long as she can remember. Her parents were not consanguineous (Fig. 1). Her decimal best-corrected visual acuity (BCVA) was 0.7 with ?3.25 diopter sphere (DS) and -2.0 D cylinder (DC) ax 100° in the right eye and 0.6 with ?3.25 DS and -1.5 DC ax 90° in the left eye. Slit-lamp examination showed mild nuclear cataracts in both eyes. Ophthalmoscopy revealed diffuse retinal degeneration with bone spicule pigments in the midperiphery (Fig. 2). The retinal vessels were attenuated although they were detectable even in the periphery. Static perimetry showed constricted visual fields (Fig. 3) although she did not notice any visual field disturbances. Full-field ISCEV-standard ERGs were non-recordable except the flicker ERGs which were severely attenuated (Fig. 4). Genetic studies showed 1,083,241 mutations when her whole-exome sequence was compared with a reference human genome (hs37d5). Among them, 456 mutations remained as candidate mutations after focusing on mutations that could change the amino acid sequence and the exclusion of common mutations by 1000 genomes in the Human Genetic Variation Database [31] and in our in-house database. They were filtered based on the inheritance pattern of the pedigree, and four candidate genes remained. They were VPS13D, ZNF518B, GIPR, and PDE6B. Finally, PDE6B was considered to be the gene responsible for the retinal dystrophy in the siblings, because it was the only gene registered in the RetNet database [3] as causing inherited retinal dystrophies among the four candidate genes. Further genetic analysis revealed a compound heterozygous c.1604T[A and c.1669C[T transition in exon 12 and 13 resulting in substitutions I535N and H557Y in the PDE6B gene. The PolyPhen-2 scores for the four candidate genes were: VPS13D (E372K, 0.66), VPS13D (P1047L, 0.103), VPS13D (T2883S, 0.01), ZNF518B (G732R, 0.002), and ZNF518B (H448R, 0.001), GIPR (G162C, 0.994), GIPR (L226H, 0.999), PDE6B (I535N, 0.999), and PDE6B (H557Y, 0.999).
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Fig. 2 Montage fundus photographs of Patients 1 and 2 and Carrier parents 1 and 2. Patients had typical RP appearance and carriers (parents of the patients) had normal fundi
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Fig. 3 Results of OctopusÒ static automated perimetry. Automated perimetry was performed with G1/Program 32 and Dynamic strategy in OctopusÒ 301, ver. 5.09 (HAAG-STREIT AG, Koeniz, Switzerland). Patients 1 and 2 have constricted visual fields, while Carrier 1 (father) has mild reduction in
sensitivity in the mid-periphery. Rate of false-positive and falsenegative responses during the examination was 0/8 and 1/8 OD, 0/8 and 3/9 OS in Patient 1, 0/8 and 2/8 OD, 0/6 and 1/7 OS in Patient 2, 1/10 and 2/11 OD, 2/11 and 1/12 OS in Carrier 1, and 0/9 and 0/10 OD, 1/9 and 1/10 OS in Carrier 2
Patient 2 (kinki-1030)
-2.25 DC ax 10° in the left eye. Slit-lamp examination revealed intraocular lens implanted in both eyes. Ophthalmoscopy showed diffuse retinal degeneration with numerous bone spicule pigments in the midperiphery (Fig. 2). The retinal vessels were severely attenuated, and the optic discs were waxy-pale in both eyes. Static perimetry showed constricted visual fields
was the 44-year-old younger brother of Patient 1 (Fig. 1). He had night blindness from childhood and noticed visual field disturbances in his mid-thirties. His BCVA was 0.5 with ?0.75 DS and -1.25 DC ax 180° in the right eye and 0.02 with ?1.0 DS with
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Fig. 4 ISCEV-standard full-field ERGs. The amplitudes of the rod ERGs recorded from Carriers 1 and 2 are reduced. Rod ERG; dark-adapted 0.01 ERG, flash ERG; dark-adapted 30
ERG, cone ERG; light-adapted 3.0 ERG, flicker ERG; lightadapted 30-Hz flicker ERG [27]
and reduced sensitivities in the macula (Fig. 3). The ISCEV-standard ERGs were non-recordable in both eyes (Fig. 4). Genetic analysis revealed compound heterozygous mutations of I535N and H557Y substitutions in the PDE6B gene, the same as his sister (Patient 1).
rod b-wave was 98 lV (66 % of lower limit of the normal value) OD and 135 lV (90 %) OS in Carrier 1, and 72 lV (48 %) OD and 85 lV (57 %) OS in Carrier 2 (Table 1). The implicit times in both were not delayed at 114 ms OD and 97 ms OS in Carrier 1, and 87 ms which was 95 % of the lower limit of normal value OD and 84 ms (91 %) OS in Carrier 2. Although Carrier 1 had normal ERGs except for the reduced rod ERGs, Carrier 2 showed reduced flash b-wave resulting in a negative-type ERG (Table 1; Fig. 4). Carrier 2 also had reduced flicker ERG in the left eye (Table 1). Genetic investigations revealed heterozygous mutations in the PDE6B gene, viz., I535N substitution in Carrier 1 (kinki-1031) and H557Y substitution in Carrier 2 (kinki-1032).
Carrier parents (kinki-1031 and 1032) The 82-year-old father (Carrier 1 in Fig. 1) and 72-year-old mother (Carrier 2 in Fig. 1) were investigated both clinically and genetically. They were both non-symptomatic, and their BCVA was 0.8 OD and 0.5 OS in Carrier 1, and 0.8 OD and 1.0 OS in Carrier 2. They had nuclear cataracts, and the fundus examinations were unremarkable except for some aging changes in both eyes (Fig. 2). However, Carrier 1 had a mild reduction in retinal sensitivity by static perimetry (Fig. 3). Both parents had significantly reduced rod ERGs compared with age-matched normal controls (Table 1; Fig. 4). The amplitude of the
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Discussion Patients 1 and 2 had the phenotype of typical RP, and their parents (Carriers 1 and 2) had normal fundi and
Rod ERG in Carriers 1 and 2 showed significantly reduced amplitude. The amplitude is expressed as % of the lower/upper limit of age-matched normal values. One hundred % means within normal value. Rod ERG; dark-adapted 0.01 ERG, flash ERG; dark-adapted 30 ERG, cone ERG; light-adapted 3.0 ERG, flicker ERG; light-adapted 30-Hz flicker ERG [27], SD; standard deviation
75–133 98–172 1.02–1.35 357–515 300–441 149–233 – Normal range
90 (100 %)
67 (90 %)
104 ± 29 135 ± 37 1.19 ± 0.17 436 ± 79 370 ± 70 191 ± 42 77.3 ± 4.5 Mean ± SD Normal controls (n = 24)
139 (100 %)
105 (100 %) 0.85 (84 %)
0.79 (78 %) 340 (95 %)
328 (92 %) 385 (100 %)
429 (100 %) 85 (57 %)
72 (48 %) 72
Left
Right Carrier 2
114 (100 %)
141 (106 %) 190 (110 %)
172 (100 %) 1.2 (100 %)
1.52 (112 %) 628 (122 %)
462 (100 %) 384 (100 %)
413 (100 %) 135 (90 %)
98 (66 %) 82
Left
Left
Right Carrier 1
0
16.0
0 0 – 0 0 0
0
0 –
– 0
0 0
0 0
0
44 Left
Right Patient 2
17.3 0 – 0 0 0 48 Right Patient 1
Table 1 Results of ISCEV-standard ERG
Age (year)
Rod ERG b-wave (lV)
Flash ERG a-wave (lV)
Flash ERG b-wave (lV)
Flash ERG b/a ratio
Cone ERG b-wave (lV)
Flicker ERG (lV)
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were non-symptomatic. However, the rod ERGs of both parents were significantly reduced compared with age-matched normal control. The reduced rod ERGs suggest a mild impairment of the rod system in Carriers 1 and 2 although they did not report night blindness. Although we did not determine the exact cause of the reduction in their rod ERGs, it is notable that past studies [5, 32–35] have also shown rod dysfunction in individuals with heterozygous mutation in the PDE6B gene. McLaughlin et al. [5] reported similar ERG findings in their patients as in our family. Their family had mutations in the PDE6B gene, Q298X and R531X, and the affected members had non-recordable ERGs. The non-symptomatic carriers had reduced and delayed ERGs to dim blue flash and white flicker flashes [5]. They examined the rod function of the carriers in more detail and reported that the light intensity that produced a half-maximal rod response was equal to that in normal controls, even though the maximum rod signal was reduced. This suggested abnormal rods or a reduction in the number of functioning rods in the carriers [5]. The results of other studies [32–35] have shown that the PDE6B gene is causative for autosomal dominant congenital stationary night blindness (CSNB; CSNBAD2, MIM# 163500). Thus, homozygous or compound heterozygous mutations in the PDE6B gene can cause typical RP, and heterozygous mutation in the PDE6B gene can cause retinal dysfunction of various degrees ranging from non-symptomatic mild rod dysfunction to the complete type CSNB. However, we do not know with certainty that the mutations which were found in our family are the cause of the reduction in the rod ERGs in the Carriers 1 and 2 or whether the reduction is common in heterozygous point mutation of the PDE6B gene. Further study is needed. The flash ERGs in Carrier 2 had normal a-wave and reduced b-wave resulting in a negative-type ERG (Fig. 4). The normal a-wave indicates normally functioning photoreceptors that is presumably elicited by normal cone photoreceptors. We did not determine the cause of the negative-type ERG although some ischemic retinal conditions and comorbid systemic diseases and medication for the condition may have caused the reduced flash b-wave in Carrier 2. Saga et al. [12] reported the first Japanese family with PDE6B-associated RP before this report. They
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reported a missense mutation of I535N (c.1604T[A), which is the same as in our pedigree. They also found a heterozygous mutation of I535N in one individual among 90 unrelated healthy Japanese individuals who served as normal controls [12]. Another missense mutation H557Y (c.1669C[T) in our pedigree has been reported in Japan [13] and Korea [18]. Unfortunately, these earlier studies did not report the ERG findings on the heterozygous carrier of I535N or H557Y in the PDE6B gene. Because a few pedigrees were reported from Japan and Korea as PDE6B-associated RP, further studies are needed to confirm that the missense mutations I535N and H557Y in the PDE6B gene is frequently found in eastern Asia. In conclusions, we report the clinical features of two patients with autosomal recessive RP which was caused by missense mutations of I535N and H557Y in the PDE6B gene. Their carrier parents were nonsymptomatic, and their fundi were unremarkable. However, they had reduced rod ERGs. Our findings indicate that non-symptomatic carrier relatives in pedigrees of autosomal recessive RP may have retinal dysfunction of various degrees. Acknowledgments The authors thank Professor Duco Hamasaki of the Bascom Palmer Eye Institute of the University of Miami for critical discussion and final manuscript revisions; Miss Seiko Kawamura, CO, of the Seichokai Fuchu Hospital (Osaka, Japan); and Mr. Tomoaki Nishio of TOMEY Corporation (Nagoya, Japan) for their assistance for ERG examinations. The authors wish to acknowledge RIKEN GeNAS for the sequencing of the exome-enriched libraries. This research was supported by the research grants to T.I., K.T., and K.K. from the Ministry of Health, Labour and Welfare, Japan (13803661), to K.T. and K.K. from the Ministry of Health, Labour and Welfare, Japan (23164001), Y.S from the Ministry of Health, Labour and Welfare, Japan (82259921), S.K. and K.K. from Japan Society for the Promotion of Science, Japan (23592597), and to M.F. from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) for RIKEN Omics Science Center.
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Conflict of interest All authors have no commercial interests related to this research. 13.
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