Bi-allelic Truncating Mutations in CEP78 , Encoding Centrosomal Protein 78, Cause Cone-Rod Degeneration with Sensorineural Hearing Loss
2016; Elsevier BV; Volume: 99; Issue: 3 Linguagem: Inglês
10.1016/j.ajhg.2016.07.010
ISSN1537-6605
AutoresPrasanthi Namburi, Rinki Ratnapriya, Samer Khateb, Csilla H. Lazar, Yael Kinarty, Alexey Obolensky, Inbar Erdinest, Devorah Marks-Ohana, Eran Pras, Tamar Ben‐Yosef, Hadas Newman, Menachem Gross, Anand Swaroop, Eyal Banin, Dror Sharon,
Tópico(s)Connexins and lens biology
ResumoInherited retinal diseases (IRDs) are a diverse group of genetically and clinically heterogeneous retinal abnormalities. The present study was designed to identify genetic defects in individuals with an uncommon combination of autosomal recessive progressive cone-rod degeneration accompanied by sensorineural hearing loss (arCRD-SNHL). Homozygosity mapping followed by whole-exome sequencing (WES) and founder mutation screening revealed two truncating rare variants (c.893-1G>A and c.534delT) in CEP78, which encodes centrosomal protein 78, in six individuals of Jewish ancestry with CRD and SNHL. RT-PCR analysis of CEP78 in blood leukocytes of affected individuals revealed that the c.893-1G>A mutation causes exon 7 skipping leading to deletion of 65bp, predicted to result in a frameshift and therefore a truncated protein (p.Asp298Valfs∗17). RT-PCR analysis of 17 human tissues demonstrated ubiquitous expression of different CEP78 transcripts. RNA-seq analysis revealed three transcripts in the human retina and relatively higher expression in S-cone-like photoreceptors of Nrl-knockout retina compared to rods. Immunohistochemistry studies in the human retina showed intense labeling of cone inner segments compared to rods. CEP78 was reported previously to interact with c-nap1, encoded by CEP250 that we reported earlier to cause atypical Usher syndrome. We conclude that truncating mutations in CEP78 result in a phenotype involving both the visual and auditory systems but different from typical Usher syndrome. Inherited retinal diseases (IRDs) are a diverse group of genetically and clinically heterogeneous retinal abnormalities. The present study was designed to identify genetic defects in individuals with an uncommon combination of autosomal recessive progressive cone-rod degeneration accompanied by sensorineural hearing loss (arCRD-SNHL). Homozygosity mapping followed by whole-exome sequencing (WES) and founder mutation screening revealed two truncating rare variants (c.893-1G>A and c.534delT) in CEP78, which encodes centrosomal protein 78, in six individuals of Jewish ancestry with CRD and SNHL. RT-PCR analysis of CEP78 in blood leukocytes of affected individuals revealed that the c.893-1G>A mutation causes exon 7 skipping leading to deletion of 65bp, predicted to result in a frameshift and therefore a truncated protein (p.Asp298Valfs∗17). RT-PCR analysis of 17 human tissues demonstrated ubiquitous expression of different CEP78 transcripts. RNA-seq analysis revealed three transcripts in the human retina and relatively higher expression in S-cone-like photoreceptors of Nrl-knockout retina compared to rods. Immunohistochemistry studies in the human retina showed intense labeling of cone inner segments compared to rods. CEP78 was reported previously to interact with c-nap1, encoded by CEP250 that we reported earlier to cause atypical Usher syndrome. We conclude that truncating mutations in CEP78 result in a phenotype involving both the visual and auditory systems but different from typical Usher syndrome. Inherited retinal diseases (IRDs) are a broad and growing group of heterogeneous neurodegenerative disorders1Rivolta C. Sharon D. DeAngelis M.M. Dryja T.P. Retinitis pigmentosa and allied diseases: numerous diseases, genes, and inheritance patterns.Hum. Mol. Genet. 2002; 11: 1219-1227Crossref PubMed Google Scholar, 2Hartong D.T. Berson E.L. Dryja T.P. Retinitis pigmentosa.Lancet. 2006; 368: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (2277) Google Scholar that can be either stationary or show progression with age primarily due to photoreceptor degeneration. IRD phenotypes are clinically distinguished by the onset of rod and cone involvement. The most common inherited retinal degeneration, retinitis pigmentosa (RP, MIM 268000) is characterized by a rod > cone pattern of dysfunction, accompanied by early impairment of night vision and progressive constriction of the visual fields, often leading to marked visual loss.3Berson E.L. Retinitis pigmentosa. The Friedenwald Lecture.Invest. Ophthalmol. Vis. Sci. 1993; 34: 1659-1676PubMed Google Scholar Cone-rod degeneration (CRD) is characterized by a cone > rod pattern, often exhibiting macular involvement and accompanied by loss of visual acuity, impairment of color vision, photophobia, and nystagmus in many cases.4Hamel C.P. Cone rod dystrophies.Orphanet J. Rare Dis. 2007; 2: 7Crossref PubMed Scopus (322) Google Scholar The age of onset in CRD is variable, but most individuals demonstrate disease phenotype in the first two decades of life. Globally, RP is estimated to affect approximately 1 in about 3,500 individuals,5Bundey S. Crews S.J. A study of retinitis pigmentosa in the City of Birmingham. II Clinical and genetic heterogeneity.J. Med. Genet. 1984; 21: 421-428Crossref PubMed Scopus (45) Google Scholar, 6Bunker C.H. Berson E.L. Bromley W.C. Hayes R.P. Roderick T.H. Prevalence of retinitis pigmentosa in Maine.Am. J. Ophthalmol. 1984; 97: 357-365Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 7Haim M. Epidemiology of retinitis pigmentosa in Denmark.Acta Ophthalmol. Scand. Suppl. 2002; 233: 1-34Crossref PubMed Scopus (196) Google Scholar, 8Rosenberg T. Epidemiology of hereditary ocular disorders.Dev. Ophthalmol. 2003; 37: 16-33Crossref PubMed Scopus (32) Google Scholar and we recently reported a higher prevalence of nonsyndromic RP in the vicinity of Jerusalem (∼1:2,200).9Sharon D. Banin E. Nonsyndromic retinitis pigmentosa is highly prevalent in the Jerusalem region with a high frequency of founder mutations.Mol. Vis. 2015; 21: 783-792PubMed Google Scholar The prevalence of CRD is much lower estimated as 1 in 40,000.4Hamel C.P. Cone rod dystrophies.Orphanet J. Rare Dis. 2007; 2: 7Crossref PubMed Scopus (322) Google Scholar Both phenotypes are genetically heterogeneous (RetNet), with defects identified in pathways primarily involved in photoreceptor function including phototransduction, transcription and splicing, and intracellular trafficking.10Veleri S. Lazar C.H. Chang B. Sieving P.A. Banin E. Swaroop A. Biology and therapy of inherited retinal degenerative disease: insights from mouse models.Dis. Model. Mech. 2015; 8: 109-129Crossref PubMed Scopus (171) Google Scholar As the photoreceptor outer segment is a modified cilium, mutations in genes associated with cilia biogenesis and transport cause pleiotropic ciliopathies with high penetrance of photoreceptor degeneration.11Valente E.M. Rosti R.O. Gibbs E. Gleeson J.G. Primary cilia in neurodevelopmental disorders.Nat. Rev. Neurol. 2014; 10: 27-36Crossref PubMed Scopus (143) Google Scholar, 12Rachel R.A. Li T. Swaroop A. Photoreceptor sensory cilia and ciliopathies: focus on CEP290, RPGR and their interacting proteins.Cilia. 2012; 1: 22Crossref PubMed Scopus (97) Google Scholar, 13Estrada-Cuzcano A. Roepman R. Cremers F.P. den Hollander A.I. Mans D.A. Non-syndromic retinal ciliopathies: translating gene discovery into therapy.Hum. Mol. Genet. 2012; 21: R111-R124Crossref PubMed Scopus (91) Google Scholar, 14Sorusch N. Wunderlich K. Bauss K. Nagel-Wolfrum K. Wolfrum U. Usher syndrome protein network functions in the retina and their relation to other retinal ciliopathies.Adv. Exp. Med. Biol. 2014; 801: 527-533Crossref PubMed Scopus (25) Google Scholar About 10%–20% of individuals with RP suffer from Usher syndrome (USH) in which rod, followed by cone, degeneration is accompanied by sensorineural hearing loss (SNHL) and varying degrees of vestibular system involvement.2Hartong D.T. Berson E.L. Dryja T.P. Retinitis pigmentosa.Lancet. 2006; 368: 1795-1809Abstract Full Text Full Text PDF PubMed Scopus (2277) Google Scholar, 15Rosenberg T. Haim M. Hauch A.M. Parving A. The prevalence of Usher syndrome and other retinal dystrophy-hearing impairment associations.Clin. Genet. 1997; 51: 314-321Crossref PubMed Scopus (138) Google Scholar, 16Keats B.J. Savas S. Genetic heterogeneity in Usher syndrome.Am. J. Med. Genet. A. 2004; 130A: 13-16Crossref PubMed Scopus (40) Google Scholar, 17Kimberling W.J. Orten D. Pieke-Dahl S. Genetic heterogeneity of Usher syndrome.Adv. Otorhinolaryngol. 2000; 56: 11-18Crossref PubMed Scopus (23) Google Scholar USH is a heterogeneous condition, both at the genetic and clinical levels, with mutations in at least 12 genes (see the databases RetNet and USHbases), historically classified into 3 main clinical sub-types. In recent years, a growing number of families with "atypical USH" were reported, but in all cases the symptoms related to rod photoreceptor involvement preceded cone degeneration.18Bashir R. Fatima A. Naz S. A frameshift mutation in SANS results in atypical Usher syndrome.Clin. Genet. 2010; 78: 601-603Crossref PubMed Scopus (25) Google Scholar, 19Kalay E. de Brouwer A.P. Caylan R. Nabuurs S.B. Wollnik B. Karaguzel A. Heister J.G. Erdol H. Cremers F.P. Cremers C.W. et al.A novel D458V mutation in the SANS PDZ binding motif causes atypical Usher syndrome.J. Mol. Med. 2005; 83: 1025-1032Crossref PubMed Scopus (40) Google Scholar, 20Liu X.Z. Hope C. Walsh J. Newton V. Ke X.M. Liang C.Y. Xu L.R. Zhou J.M. Trump D. Steel K.P. et al.Mutations in the myosin VIIA gene cause a wide phenotypic spectrum, including atypical Usher syndrome.Am. J. Hum. Genet. 1998; 63: 909-912Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Khateb S. Zelinger L. Mizrahi-Meissonnier L. Ayuso C. Koenekoop R.K. Laxer U. Gross M. Banin E. Sharon D. A homozygous nonsense CEP250 mutation combined with a heterozygous nonsense C2orf71 mutation is associated with atypical Usher syndrome.J. Med. Genet. 2014; 51: 460-469Crossref PubMed Scopus (59) Google Scholar The association of SNHL with CRD has not been previously reported. In the present study, we used a combination of homozygosity mapping, whole-exome sequencing (WES), and founder mutation screening to identify the genetic cause of disease in families with the rare combination of CRD and SNHL inherited in an autosomal recessive (AR) mode. The tenets of the Declaration of Helsinki were followed, the study was approved by the Institutional Review Board (IRB), and prior to donation of a blood sample, informed consent was obtained from all participants. In our cohort of over 1,500 Israeli and Palestinian families with IRDs, six affected individuals from five families (Figure 1A) shared the same ethnicity (Jews of Iranian, Iraqi, or Afghani origin) and presented with a unique combination of CRD and SNHL. While some variability was present in the clinical manifestations between subjects (detailed case reports are provided in the Supplemental Note), a characteristic phenotype could be identified. All affected individuals presented with similar cone-dominated symptoms with a variable age of onset (ranging from 10 to 35 years) including photophobia, low visual acuity, impaired color vision, and visual field defects accompanied by SNHL, which also had a variable onset (ranging from 10 to 45 years). Electroretinographic (ERG) testing correlated with the visual symptoms, showing progressive loss of cone, followed by rod, photoreceptor function, in some cases accompanied by a reduced b/a wave ratio in the dark-adapted state (Table 1). Electrooculography (EOG) testing was performed in four affected individuals, and the Arden ratio was found to be reduced (Table 1), probably being secondary to photoreceptor degeneration. Funduscopic findings were characterized by the presence of prominent retinal atrophy along the major vascular arcades surrounding the macula that tended to expand at older ages. In addition, mild macular retinal pigment epithelium (RPE) changes and subtle peripheral salt and pepper changes were evident, with no bone-spicule-like pigmentation (BSP) (Figure 2). Characteristic ring-shaped atrophy was clearly evident on fundus autofluorescence (FAF) imaging, manifesting as a hypo-autofluorescent band along the vascular arcades surrounded by hyper-autofluorescent borders (Figure 2, right panels). In the three individuals who had visual acuity that permitted performance of color vision testing, none of the panels on the Ishihara 38-plate test were identified, and multiple errors with non-specific axes were present using the Farnsworth-Munsell D-15 test. Audiometry tests revealed bilateral SNHL affecting the middle frequencies, high frequencies, or both (Figure S1 and Table 1). One of the individuals, MOL0773 II:1, underwent audiometry at 47 years showing bilateral SNHL with a flat curve, involving all hearing frequencies (Figure S1C). Importantly, none of the affected individuals reported significant abnormalities of the vestibular system (detailed in Supplemental Note).Table 1Clinical Data of Individuals with Bi-allelic CEP78 MutationsIndividual Number (Age)aThe age at the time of first ERG testing is written in parenthesis in the Individual Number column. When additional clinical tests were performed at other ages, the age of testing is provided in parenthesis in the appropriate column. All ages are in years.GenotypeOnset of Visual Impairment (Age)Best Corrected Visual Acuity (Age)Refraction (Age)Full-Field ERG ResultsbFull-field electroretinography (ERG) results include the following details: light-adapted (LA) cone flicker amplitude (normal 60–144 μV) and implicit time (IT, normal 27–33 ms); dark-adapted (DA) rod response b-wave amplitude (normal range 200–500 μV); dark-adapted mixed cone-rod a- and b-wave amplitudes (normal a-wave 90–350 μV, normal b-wave 380–630 μV).,cTel Aviv Sourasky Medical Center ERG normal ranges (applicable to individuals TB279 II:1 and TB365 II:1): LA cone flicker amplitude (65–190 μV) and implicit time (IT, ms, normal 24–30); DA rod response b-wave amplitude (normal 60–250 μV); Dark-adapted mixed cone-rod a- and b-wave amplitudes (normal a-wave 80–340 μV, normal b-wave 260–600 μV).EOG (%)dElectro-oculography (EOG) results are provided as the Arden ratio (light peak/dark trough) in percent (normal > 185%). (Age)Hearing Impairment30 Hz Cone Flicker, AMP/IT (Age)Mixed Cone-Rod Response (Age)Rod Response (Age)Onset (Age)FrequenciesMOL0679 III:1 (37)c.893–1G>A / c.893–1G>A100.08−3.00NDa = 172, b = 175110–––0.08 (44)–ND (44)a = 50, b = 43 (44)47 (44)122 (44)45highMOL0679 III:2 (30)c.893–1G>A / c.893–1G>A100.15–––––––LP (47)−0.25 (47)NDNDNDNA11medium and highMOL0773 II:1 (29)c.893–1G>A / c.893–1G>A200.08−2.50NAa = 127, b = 287180135––––25/42 (33)a = 91, b = 229 (33)75 (33)–––0.03 (43)–15/43 (43)a = 65, b = 94 (43)ND (43)–42bilateral flat curveHM (48)−1.25 (48)ND (48)ND (48)ND (48)131 (47)––MOL1310 II:1 (38)c.534delT / c.534delT350.4−8.25NANANANA36NATB279 II:1 (47)c.534delT / c.534delT300.03planoNDa = 41, b = 31411020medium and highTB365 II:1 (38)c.893–1G>A / c.534delT280.12−2.5020/38a = 134, b = 9149810medium and highND, nondetectable; NA, not available. Data represent average between both eyes. Best-corrected visual acuity is given as a decimal ratio; if numbers on ETDRS chart could not be identified even at a distance of 1 meter, residual vision described as hand movements (HM) or light perception (LP). Refraction is given as mean spherical equivalent of both eyes in diopters.a The age at the time of first ERG testing is written in parenthesis in the Individual Number column. When additional clinical tests were performed at other ages, the age of testing is provided in parenthesis in the appropriate column. All ages are in years.b Full-field electroretinography (ERG) results include the following details: light-adapted (LA) cone flicker amplitude (normal 60–144 μV) and implicit time (IT, normal 27–33 ms); dark-adapted (DA) rod response b-wave amplitude (normal range 200–500 μV); dark-adapted mixed cone-rod a- and b-wave amplitudes (normal a-wave 90–350 μV, normal b-wave 380–630 μV).c Tel Aviv Sourasky Medical Center ERG normal ranges (applicable to individuals TB279 II:1 and TB365 II:1): LA cone flicker amplitude (65–190 μV) and implicit time (IT, ms, normal 24–30); DA rod response b-wave amplitude (normal 60–250 μV); Dark-adapted mixed cone-rod a- and b-wave amplitudes (normal a-wave 80–340 μV, normal b-wave 260–600 μV).d Electro-oculography (EOG) results are provided as the Arden ratio (light peak/dark trough) in percent (normal > 185%). Open table in a new tab Figure 2Imaging of Individuals with Bi-allelic CEP78 Variants Ordered by AgeShow full caption(A–C) TB365 II:1 (38 years).(D–F) MOL0679 III:1 (44 years), (G–I) TB279 II:1 (47 years), (J–L) MOL0679 III:2 (47 years), and (M–O) MOL0773 II:1 (48 years). Color fundus photos of the posterior pole (left panels) and Optos wide-angle images (middle panels) show mild macular RPE changes, ring of atrophy along the vascular arcades surrounding the macula, and subtle peripheral salt and pepper changes with no BSPs. The findings are more clearly evident on fundus auto fluorescence (FAF) imaging (right panels) showing hypo-autofluorescence along the vascular arcades with a macular hyper-autofluorescent ring.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ND, nondetectable; NA, not available. Data represent average between both eyes. Best-corrected visual acuity is given as a decimal ratio; if numbers on ETDRS chart could not be identified even at a distance of 1 meter, residual vision described as hand movements (HM) or light perception (LP). Refraction is given as mean spherical equivalent of both eyes in diopters. (A–C) TB365 II:1 (38 years). (D–F) MOL0679 III:1 (44 years), (G–I) TB279 II:1 (47 years), (J–L) MOL0679 III:2 (47 years), and (M–O) MOL0773 II:1 (48 years). Color fundus photos of the posterior pole (left panels) and Optos wide-angle images (middle panels) show mild macular RPE changes, ring of atrophy along the vascular arcades surrounding the macula, and subtle peripheral salt and pepper changes with no BSPs. The findings are more clearly evident on fundus auto fluorescence (FAF) imaging (right panels) showing hypo-autofluorescence along the vascular arcades with a macular hyper-autofluorescent ring. Aiming to identify the cause of disease in family MOL0679, in which the parents are distantly related, we performed homozygosity mapping on genomic DNA sample of the index case (III:2; Figure S2). Two large homozygous regions passed the analysis criteria: a very large region of 65.6 Mb on chromosome 9 (ranging from 19.8 to 85.4 Mb in reference sequence hg19) flanked by SNP markers rs1857437 and rs728695, and a smaller region of 34 Mb on chromosome 19 (15.3 to 49.1 Mb) flanked by markers rs2366267 and rs1073653. These regions harbor 628 and 744 genes, respectively, but do not harbor any known IRD-associated gene. We therefore performed WES analysis on the DNA sample of MOL0679 III:1 that revealed 30,379 sequence variants. Because compound heterozygous mutations can be identified in consanguineous families, we initially inspected a set of all 226 known IRD-associated genes22Beryozkin A. Shevah E. Kimchi A. Mizrahi-Meissonnier L. Khateb S. Ratnapriya R. Lazar C.H. Blumenfeld A. Ben-Yosef T. Hemo Y. et al.Whole Exome Sequencing Reveals Mutations in Known Retinal Disease Genes in 33 out of 68 Israeli Families with Inherited Retinopathies.Sci. Rep. 2015; 5: 13187Crossref PubMed Scopus (52) Google Scholar and did not identify any putative pathogenic disease-causing mutations. Analysis of known polymorphic markers in the two above-mentioned homozygous regions showed that the two siblings shared only a single large homozygous region on chromosome 9 (spanning 16 Mb, ranging from 69.4 to 85.4 Mb [Figure 1B], flanked by SNP markers rs369854466 and rs728695). We then performed detailed analysis of this homozygous region and identified 15 homozygous variants (Table S1), three of which were rare variants (minor allele frequency [MAF] < 0.005) that are predicted to affect the amino-acid sequence of the corresponding gene. Two of the variants were missense that are predicted to be benign by the online prediction tools (PolyPhen 2, SIFT, and MutationTaster). The remaining single variant was a homozygous transition (c.893-1G>A, Figure 1D) in the acceptor site of exon 7 of CEP78 (Genbank: NM_032171.1 [Figures 1C and 1D]) with coverage of 43X that was verified by Sanger sequencing (Figure 1E) and co-segregated with the phenotype in the family. Exon 7 includes 65 nucleotides and skipping of this exon is expected to result in a frameshift mutation (c.893_957del; p.Asp298Valfs∗17). The sequence variant was not found in public databases (including the Exome Aggregation Consortium [ExAC] Server [Cambridge, MA, 03/2016]) and in an in-house database of 408 Israeli exomes. Aiming to study whether c.893-1G>A is a recurrent mutation, we screened the index cases of the four remaining families that had a similar phenotype (MOL0773, MOL1310, TB279, and TB365, Figure 1A) for this mutation. Two index cases harbored the rare variant: MOL0773 II:1 was homozygous and TB365 II:1 was heterozygous. Sequencing analysis of the remaining exons in TB365 II:1 revealed an additional variant: a heterozygous deletion of thymidine in exon 4 (c.534delT), which is expected to cause a frameshift (p.Lys179Argfs∗10). The two remaining index cases, MOL1310 II:1 and TB279 II:1, were found to be homozygous for c.534delT. In addition, we sequenced both exons 4 and 7, as well as flanking intronic sequences in 249 Eastern Jewish index cases with IRDs, but none of them carried either mutation or additional possible CEP78 disease-causing mutation. Because both variants have been identified in individuals with a shared origin, we used a set of 21 SNP markers flanking CEP78 to study whether c.893-1G>A and c.534delT are founder mutations (Table S2). Haplotype analysis revealed a shared and distinct haplotype for each mutation, covering homozygous regions of 6.3 and 11.3 Mb respectively, indicating that indeed, as expected, these are founder mutations in Jews originated from Iraq, Iran, and Afghanistan. To study the effect of the c.893-1G>A mutation on splicing, we employed RT-PCR using RNA from blood leukocytes to amplify a fragment of CEP78 mRNA. The primers we used are located in exons 6 and 9 (Table S3) and produced an expected 290 bp fragment in controls (Figure 3A) and a shorter 225 bp fragment in individuals who are homozygous for the mutation (Figure 3A, lane 1). Sanger sequencing of the mutant band revealed skipping of exon 7 leading to deletion of 65 nucleotides (Figure 3A, left panel). RT-PCR analysis of all available affected individuals and family members did not reveal significant differences in the abundance of the WT and the two mutant alleles (Figure S3), indicating that in leukocytes, short mutant CEP78 proteins that are likely to result in no protein function, are expected to be produced. CEP78 includes 17 coding exons in the human genome spanning a genomic region of 31 Kb. To study the expression of CEP78 in the human retina, we analyzed RNA-seq data of 11 individuals and identified three different protein-coding transcripts (Figures 1C and 3B). Analysis of retinal transcripts deposited in public databases show that exon 15 is an alternatively-spliced exon. In addition, we show here using RNA-seq analysis that transcripts including exon 17 (CEP78-001 and 003) are also expressed in the human retina (Figure 3B). All three transcripts show a similar level of expression with CEP78-003 the less common one (Figure 3B). Subsequent semiquantitative analysis of the distribution of CEP78 transcripts in various human tissues revealed a ubiquitous and variable expression pattern (Figure 3C, upper two panels, and Figures S4A and S4B). In addition, the analysis revealed similar expression levels of transcripts including and lacking exon 15 in most studied tissues (Figure 3C, middle panel, and Figures S4B and S4D) with a relatively high expression level in the thymus. To study Cep78 expression in the mouse retina, we used RT-PCR (followed by verification with Sanger sequencing) at different age points (embryonic day 14 [E14], postnatal day 0 [P0], and postnatal day 30 [P30]) in the WT mouse retina (Figures 3D, S4E, and Table S3). Cep78 expression was evident at all time points with more intense amplification at P30 (Figures 3D and S4E). In addition, RT-PCR analysis of the inner ear shows robust amplification of Cep78, which is similar to that obtained in the retina (Figure S4E). To gain more insight on the expression level in specific retinal cells, we used RNA-seq to analyze the expression levels in flow-sorted photoreceptors of wild-type versus the Nrl-KO retina in mice (Figure 3E).23Mears A.J. Kondo M. Swain P.K. Takada Y. Bush R.A. Saunders T.L. Sieving P.A. Swaroop A. Nrl is required for rod photoreceptor development.Nat. Genet. 2001; 29: 447-452Crossref PubMed Scopus (710) Google Scholar We observed a higher Cep78 expression level in cones. In all studied affected individuals, we observed a retinal phenotype showing initial and more pronounced cone dysfunction, which is followed and accompanied by less prominent rod involvement. This is reflected both in the symptoms experienced by the affected individuals (including decreased visual acuity, increased sensitivity to light and impaired color vision), in the funduscopic findings described above that mainly involve the posterior pole, and further substantiated by the ERG results that show a cone more than rod pattern of injury. Aiming to study the immunolocalization of CEP78 in the human retina, we performed immunohistochemistry studies with an anti-CEP78 antibody in sections of normal human (Figure 4) and mouse (Figure S5) retinas. The analysis revealed immunolocalization of CEP78 in many retinal cell types (Figure 4A compared to DAPI staining in Figure 4B). A similar immunolabeling retinal pattern was reported previously for FAM161A.24Di Gioia S.A. Letteboer S.J. Kostic C. Bandah-Rozenfeld D. Hetterschijt L. Sharon D. Arsenijevic Y. Roepman R. Rivolta C. FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies.Hum. Mol. Genet. 2012; 21: 5174-5184Crossref PubMed Scopus (35) Google Scholar Intense staining of cone photoreceptor inner segments can be observed, while only weak labeling of the base of rod inner segments is evident (Figure 4C). These results are in-line with the observed retinal phenotype in which the cones are more affected than rods. IHC in the mouse retina (Figure S5) also revealed immunolocalization in various retinal cell-types including photoreceptor inner segments. CEP78 orthologs can be found in mammals, amphibians, aves, fish, and insects. To gain insight into protein regions that are preserved along evolution, we performed a sliding window analysis calculating the mean amino-acid identity at a 30 amino-acid interval, comparing the human protein sequence to selected orthologs (Figure S6A). CEP78 contains a leucine-rich repeat (LRR) region, which is composed of 20–29 residue sequence motifs that are present in many proteins that participate in protein-protein interactions and have different functions and cellular locations. A limited level of amino-acid identity was found in the C terminus, while three regions show a relatively high degree of preservation in all studied species: amino acids 178–287, which cover most of the LRR region, a sharp peak at amino acids 409–413, and a region between amino acids 511 and 566 (Figure S6A). Phylogeny analysis revealed, as expected, closely related sequences among mammalians and clear orthologous sequences in other species (Figure S6B). To compare the level of amino-acid preservation between CEP78 and 69 other IRD proteins (14 USH-related proteins and 55 arRP-related proteins listed in Table S4), we calculated the amino-acid identity levels between each human protein sequence and its orthologs in eight species (Figure S6C). The analysis indicates that CEP78 (red circle in Figure S6C) has an average level of preservation in most species along evolution. Centrosomal protein 78kDa (CEP78) was previously shown to be localized to the human centrosome,25Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1051) Google Scholar which is the major microtubule-organizing center of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity, and motility. The mother centriole determines the position of the flagella and cilia and becomes the basal body of these organelles.26Nigg E.A. Raff J.W. Centrioles, centrosomes, and cilia in health and disease.Cell. 2009; 139: 663-678Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar The centrosome has also a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes during cell division. CEP78 was reported previously to interact with another centrosomal protein, C-Nap1, encoded by CEP250 (MIM 609689).27Fogeron M.L. Müller H. Schade S. Dreher F. Lehmann V. Kühnel A. Scholz A.K. Kashofer K. Zerck A. Fauler B. et al.LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells.Nat. Commun. 2013; 4: 1531Crossref PubMed Scopus (47) Google Scholar Interestingly, we reported previously that a null CEP250 mutation causes atypical USH, characterized by early-onset SNHL, and a relatively mild RP phenotype.21Khateb S. Zelinger L. Mizrahi-Meissonnier L. Ayuso C. Koenekoop R.K. Laxer U. Gross M. Banin E. Sharon D. A homozygous nonsense CEP250 mutation combined with a heterozygous nonsense C2orf71 mutation is associated with atypical Usher syndrome.J. Med. Genet. 2014; 51: 460-469Crossref PubMed Scopus (59) Google Scholar Based on its cellular localization and interacting proteins, CEP78 is considered a ciliary protein.28Brekman A. Walters M.S. Tilley A.E. Crystal R.G. FOXJ1 prevents cilia growth inhibition by cigarette smoke in human airway epithelium in vitro.Am. J. Respir. Cell Mol. Biol. 2014; 51: 688-700Crossref PubMed Scopus (55) Google Scholar Depletion of human CEP78 affects primary cilium assembly in RPE1 cells and decreased the proportion of ciliated cells up to 2-fold.29Azimzadeh J. Wong M.L. Downhour D.M. Sánchez Alvarado A. Marshall W.F. Centrosome loss in the evolution of planarians.Science. 2012; 335: 461-463Crossref PubMed Scopus (118) Google Scholar Unlike all cases of retinal degeneration associated with SNHL (e.g., Usher syndrome) that were thus far reported, in which the retinal phenotype was typical RP, the individuals we describe here manifest ocular findings that are compatible with cone-rod degeneration. In line with the retinal phenotype, our immunohistochemistry analysis in the human retina shows more pronounced expression of CEP78 in cones. In summary, our results show that bi-allelic null variants in CEP78 cause a syndrome that is characterized by CRD with SNHL, with relatively late onset of both ocular and hearing impairment. The funduscopic findings are characteristic, showing atrophy along the major vascular arcades that are best visualized using FAF imaging. To the best of our knowledge, this combined retinal and auditory phenotype has not been previously reported. The phenotype we describe here does not fall within the standard classification of Usher syndrome, and it should be noted that a relatively large number of affected individuals have been reported to suffer from "atypical" Usher syndrome with both retinal and hearing features that do not fit the three established Usher subtypes.18Bashir R. Fatima A. Naz S. A frameshift mutation in SANS results in atypical Usher syndrome.Clin. Genet. 2010; 78: 601-603Crossref PubMed Scopus (25) Google Scholar, 19Kalay E. de Brouwer A.P. Caylan R. Nabuurs S.B. Wollnik B. Karaguzel A. Heister J.G. Erdol H. Cremers F.P. Cremers C.W. et al.A novel D458V mutation in the SANS PDZ binding motif causes atypical Usher syndrome.J. Mol. Med. 2005; 83: 1025-1032Crossref PubMed Scopus (40) Google Scholar, 20Liu X.Z. Hope C. Walsh J. Newton V. Ke X.M. Liang C.Y. Xu L.R. Zhou J.M. Trump D. Steel K.P. et al.Mutations in the myosin VIIA gene cause a wide phenotypic spectrum, including atypical Usher syndrome.Am. J. Hum. Genet. 1998; 63: 909-912Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Khateb S. Zelinger L. Mizrahi-Meissonnier L. Ayuso C. Koenekoop R.K. Laxer U. Gross M. Banin E. Sharon D. A homozygous nonsense CEP250 mutation combined with a heterozygous nonsense C2orf71 mutation is associated with atypical Usher syndrome.J. Med. Genet. 2014; 51: 460-469Crossref PubMed Scopus (59) Google Scholar The results presented here further expand the spectrum of syndromes involving the auditory and visual systems that are due to defects in ciliary and/or centrosomal proteins. The authors thank all affected individuals and family members for their participation in this study. We are grateful to Lilach Binyamini and Shelly Stika for assistance in recruitment and clinical evaluation of the individuals and Alaa Abu-Diab for technical assistance. Whole-exome analysis utilized the computational resources of the NIH HPC Biowulf cluster. We thank Matthew Brooks, Jung-Woong Kim, Hyunjin Yang, and Ashley Walton for help with RNA-seq analysis. This research was supported by the United States – Israel Binational Science Foundation (grant number 2011202, to D.S. and A.S.), Foundation Fighting Blindness USA (BR-GE-0214-0639 to D.S., E.B., E.P., H.N., and T.B.-Y.), Israel Science Foundation (#2154/15 to S.K.), Chief Scientist office of the Israeli Ministry of Health and the Lirot association (#300009177 to S.K.), the Yedidut Research Grant (to E.B.), and the Intramural Research Program of the National Eye Institute, National Institutes of Health (to A.S.). The accession numbers for the c.893-1G>A and c.534delT variants reported in this paper were deposited in the Leiden open variation database (LOVD) with respective IDs 0000119346 and 0000119347. Download .pdf (2.07 MB) Help with pdf files Document S1. Figures S1–S6, Tables S1–S4, and Supplemental Experimental Procedures ExAC Browser, http://exac.broadinstitute.org/HomozygosityMapper software, http://www.homozygositymapper.org/LOVD, http://www.lovd.nl/3.0/homeHPC @ NIH, http://hpc.nih.govOMIM, http://www.omim.org/RetNet – Retinal Information Network, https://sph.uth.edu/retnet/home.htmUSHbases, https://grenada.lumc.nl/LOVD2/Usher_montpellier/USHbases.html Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia DefectsNikopoulos et al.The American Journal of Human GeneticsSeptember 01, 2016In BriefCone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. Full-Text PDF Open ArchiveBi-allelic Truncating Mutations in CEP78, Encoding Centrosomal Protein 78, Cause Cone-Rod Degeneration with Sensorineural Hearing LossNamburi et al.The American Journal of Human GeneticsNovember 03, 2016In Brief(The American Journal of Human Genetics 99, 777–784; September 1, 2016) Full-Text PDF Open Archive
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