Cone-Rod Degeneration with Sensorineural Hearing Loss

Transcription

1 The American Journal of Human Genetics, Volume 99 Supplemental Data Bi-allelic Truncating Mutations in CEP78, Encoding Centrosomal Protein 78, Cause Cone-Rod Degeneration with Sensorineural Hearing Loss Prasanthi 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, and Dror Sharon

2 SUPPLEMENTAL NOTE: CASE REPORTS Clinical data were collected in two medical centers: Hadassah-Hebrew University Medical Center located in Jerusalem (families MOL0679, MOL0773, and MOL1310) and Tel Aviv Sourasky Medical Center (families TB279 and TB365). At Hadassah-Hebrew University Medical Center- International Society for Clinical Electrophysiology of Vision (ISCEV) standard full-field electroretinography (fferg) was performed using monopolar corneal electrodes (Henkes type; Medical Workshop B.V., Groningen, The Netherlands) and a computerized system (UTAS 3000; LKC, Gaithersburg, MD, USA). Cone responses to 30-Hz flashes of white light were acquired under a background light of 21 cd/m 2. Scotopic responses, including a rod response to a dim blue flash and a mixed cone rod response to a standard white flash, were acquired after minute of dark adaptation. Between 2 and 4 sets of responses were recorded in each condition for reproducibility. All ERG responses were filtered at 0.3 to 500 Hz, and signal averaging was applied. Color fundus photos were obtained using a Zeiss fundus camera (model FF450PLUS; Carl Zeiss Meditec, Dublin, CA, USA). Wide angle color and autofluorescence fundus photos were obtained using an OPTOS system (OPTOS, Dunfermline, Scotland, United Kingdom). At Tel Aviv Sourasky Medical Center ff-erg recordings were conducted according to an expanded protocol of the ISCEV ERG guidelines using an Espion Eᵌ Electrophysiology System (Diagnosys, Lowell, MA, USA) and bipolar Burian-Allen corneal electrodes (Hansen Ophthalmic Development Lab, Coralville, IA, USA). Light-adapted ERG responses were recorded under white background illumination of 30 cd/m 2, using white light stimuli of different energies covering about 3.3 log units ( cd-s/m 2 ) and 30 Hz flicker (3 cds/m 2 ). Following 20 minutes of dark adaptation, scotopically matched dim blue and bright red light stimuli were used to record isolated rod response and cone response. Then, a series of

3 increasing white light stimuli of energy covering 4.6 log units (0.005 to 200 cd-s/m 2 ) were used. Fundus photography was obtained with a fundus camera (FF450 plus fundus camera, ZEISS). Family MOL0679 includes two affected brothers (Figure 1A, III:1 and III:2). The index case (MOL0679 III:1) was known to have visual impairment since the age of 10, including photophobia, poor day vision and relatively preserved night vision. ERG recording performed at 37 years of age revealed nondetectable cone and reduced rod function (Table 1), leading to the clinical diagnosis of CRD. Interestingly, at age 44, peripheral Goldmann visual fields were preserved using the III4e and V4e targets. Visual acuity (VA) at ages 37 and 44 was 0.08 (earlier measurements are not available) and funduscopy at these ages showed mild macular retinal pigment epithelium (RPE) changes, ring of atrophy along the vascular arcades surrounding the macula, and subtle peripheral salt and pepper changes with no bone-spiculelike pigmentation (BSP) (Figure 2D-F; note relatively minimal changes on color images), with more prominent findings evident on fundus autofluorescence (FAF) imaging (Figure 2F). Optical coherence tomography (OCT) shows thinning of the ONL without edema (data not shown). Mild hearing loss appeared only at the age of 45 years. Tinnitus and occasional vertigo were also reported. Audiometry at the age of 47 years revealed mild SNHL at high frequencies, with a notch in the right ear at 3,000 Hz (Figure S1A). MRI and CT were normal. While we presume this hearing loss is associated with his disease, injury related to noise exposure or past head injury cannot be ruled out. His medical history is significant for diabetes mellitus diagnosed at the age of 35 years, hypertension and obesity. His brother (MOL0679 III:2) was found on a screening test to have hearing impairment at the age of 11 years, with SNHL at high frequencies. MRI at the age of 30 years was within normal limits. Began using hearing aids at the age of 38 years, and reported that the hearing level is relatively stable since. Audiometry at the age of 47 years revealed

4 bilateral down-slopping SNHL at middle and high frequencies (Figure S1B). Tinnitus and vertigo were not reported. Visual impairment was first noticed at age 15, with difficulty particularly under high levels of ambient light and good function at night. His VA at the age of 30 was 0.15, but he reported severe deterioration in his forties and at the age of 47 only light perception with partial projection was measured in BE. At the age of 33, photopic and scotopic ERG responses were nondetectable. At the age of 30 the fundus was reported as normal, and at age 47 small anterior capsular cataracts, hypermetropic optic discs, mild macular RPE changes and atrophy along the vascular arcades were apparent. The peripheral retina showed mild salt and pepper changes, without BSPs or overt pigmentary changes (Figure 2J-L). Changes on FAF imaging (Figure 2L) were more prominent than in his brother (Figure 2F). His general health is good, with hypercholesterolemia and benign prostatic hyperplasia (BHP). The four remaining affected individuals were isolate cases (Figure 1A): MOL0773 II:1 underwent repeated electroretinography (ERG) tests at ages 29, 33, 43, and 48 showing progressive loss of cone, followed by rod, photoreceptor function (Table 1). Her VA is very low (Table 1) and she suffered from photophobia and mild cataract with preserved night vision. Fundoscopic changes are minimal but changes can be seen by FAF (Figure 2M-O). She reported hearing impairment since the age of 42 years, without tinnitus or vertigo. Disequilibrium was reported by the affected individuals. Audiometry at 47 year showed bilateral flat curve (Figure S1C) indicating SNHL at low, medium, and high frequencies. Individual MOL1310 II:1 reported mild hearing loss and visual impairment since the age of 35 years. Visual symptoms included photophobia, difficulties in night vision, and constricted visual fields. Ocular examination revealed quite anterior segment with mild central posterior sub-capsular cataract in both eyes. Fundus examination showed atrophic

5 macular changes, very mild peripheral salt and pepper pigmentation with no BSPs, and attenuated blood vessels. Individual TB279 II:1 reported non-progressive hearing loss since the age of 20 years as well as tinnitus in the right ear. He underwent audiometry testing at the age of 48 years which showed bilateral down-slopping SNHL affecting the middle and high frequencies (Figure S1D). He is not using hearing aids. He reported central progressive visual loss and photophobia since age 30. At 47 years of age, he had nystagmus and BCVA of in both eyes. ERG responses were consistent with severe generalized cone-rod dysfunction, accompanied by an electronegative pattern (reduced b/a ratio) and a reduced Arden ratio on electrooculography (EOG) (Table 1). Funduscopy showed pale optic discs, epiretinal membranes (ERM) in both eyes with a lamellar hole in the right eye, and atrophic changes along the vascular arcades. FAF imaging revealed hypo-autofluorescence along the vascular arcades with a macular hyper-autofluorescent ring (Figure 2G-I). Individual TB365 II:1 reported hearing loss since the age of 10 years and had photophobia and progressive deterioration of central vision since age 28. Audiometry at the age of 39 years showed bilateral down-slopping SNHL affecting the middle and high frequencies (Figure S1E). He is using hearing aids. Tinnitus and dis-equilibrium were reported. ERG responses, recorded at 37 years of age, were consistent with generalized conerod dysfunction, accompanied by an electronegative pattern (Table 1). At the age of 38 years, his BCVA was 0.13 in the RE, and 0.1 in the LE. Funduscopy showed pale optic discs, mild macular RPE changes and subtle atrophic changes along the vascular arcades with no BSPs. FAF imaging revealed hypo-autofluorescence at the vascular arcades with a subtle macular hyper-autofluorescent ring (Figure 2A-C).

6 Figure S1- Audiometry of five individuals with bi-allelic CEP78 mutations. (A) MOL0679 III:1 (age 45 years), (B) MOL0679 III:2 (47 years), (C) MOL0773 II:1 (47 years), (D) TB279 II:1 (48 years), (E) TB365 II:1 (39 years).

7 Figure S2- Homozygosity mapping of MOL0679 III:2. Homozygosity mapping of MOL679 III:2 was performed using whole genome single nucleotide polymorphism (SNP) microarray (Affymetrix 6.0) and analyzed using the HomozygosityMapper server (Berlin, Germany [03/2016]): the x-axis represents the chromosome number and y-axis represents the threshold for the Homozygosity score as a ratio over the maximum score.

8 Figure S3- RT-PCR analysis of CEP78 in leukocytes. (A-B) RT-PCR products of different individuals using primers CEP78-CDS-E3F and CEP78- CDS-E5R (see Table S3) amplifying a 180 bp amplicon (exons 3 through 5) were run on an agarose gel in the following order: WT individuals (lanes 1 and 2), individuals who are homozygous for c.893-1g>a (lane 3- MOL0679 III:1; lane 4- MOL0679 III:2; lane 5- MOL0773 II:1), heterozygotes for c.893-1g>a (lane 6- MOL0773 III:2, lane 7- TB365 I:2), compound heterozygote (lane 8- TB365 II:1), heterozygous for c.534delt (lane 9- TB365 I:1), homozygous for c.534delt (lane 10- TB279 II:1), and a negative control (lane 11). M- represents a Kb marker. (C-D) RT-PCR of the same samples using primers PGM1- CDS-6&7F and PGM1-CDS-10R (see Table S3) of the housekeeping gene PGM1.

9 Figure S4- Semi-quantitative RT-PCR analysis of CEP78. (A-C) Expression level of CEP78 versus PGM1 (MIM: ) in different human tissues (Clontech; cat #636643, lot No A) using two sets of primers amplifying exons 6 through 9 and exons 14 through 16. Lane 1: bone marrow, 2: brain (whole), 3: fetal brain, 4: fetal liver, 5: heart, 6: kidney, 7: liver, 8: lung, 9: placenta, 10: prostate, 11: spleen, 12: thymus, 13: uterus, 14: small intestine, 15: spinal cord, 16: stomach, 17: retina, 18: negative control and M: kb DNA ladder. PGM1 servers as a housekeeping control. (D) A high resolution image of an agarose gel showing the presence of two different transcripts in the human retina (lane 1) and thymus (lane 3) using primers CEP78-CDS-E14-F and CEP78- CDS-E16-R. Lane 2 is a negative control. The upper band (438 bp) includes exon 15 and the lower band (390 bp) lacks this exon, as verified by Sanger sequencing. A faint intermediate band is evident due to heteroduplex PCR products including both transcripts. (E) Expression of Cep78 in the mouse retina and inner ear. Lane 1- mouse retina age E14, 2- mouse retina age P0, 3- mouse retina age P30, 4- mouse inner ear age P30, 5- negative control. Pgk1 servers as a housekeeping control.

10 Figure S5- CEP78 immunolocalization in the mouse retina using immunohistochemical (IHC) analysis. Low (A) and high (B)-magnification images (20X and 40X respectively) of control retina showing double staining with anti-cep78 (green) and anti-s-opsin (red) antibodies. Blue staining represents DAPI labeling. GCL- ganglion cell layer; IPL- inner plexiform layer; INL- inner nuclear layer; OPL- outer plexiform layer; ONL- outer nuclear layer; IS- inner segments of photoreceptors; OS- outer segments of photoreceptors; RPE- retinal pigment epithelium layer. Yellow bars show the scale (panel A- 100 m, panel B- 25 m).

11 Figure S6- Evolutionary analysis of CEP78. Schematic representation of the CEP78 is presented in the upper panels: a yellow box represents the Leucine-rich repeats (LRR) region, a gray box represents a coiled-coil domain. The exon boundaries are shown below. An amino acid sliding window (length of 30 amino acids) comparing the human protein sequence to selected orthologs (macaca, dog, mouse, chicken, frog and zebrafish) is shown. X-axis: amino acid number; Y-axis: percentage of the

12 mean amino acid identity in a 30 amino acid window. The lower left graph represents a summary of the data in mammalian sequences (red) versus all eight sequences (blue), with a cumulative sliding window analysis of data points that are above the average percentage of amino acid identity for each studied sequence (chimp 99%, macaca 97%, dog 87%, cow 86%, rat 77%, mouse 77%, chicken 62%, frog 57%, zebrafish 50%). Amino acid sequences of CEP78 orthologs were extracted from the Homologene database at NCBI and aligned using the phylogeny server (at Marseille, France [03/2016]) multiple alignment tool. (B) A phylogenetic tree of CEP78 using the neighbor joining procedure with the Drosophila willistoni Dwil\GK14449 sequence serving as an outgroup. (C) A comparison of amino acid identity levels (comparing the human protein sequence to its ortholog in each of the eight species) of 14 USH-associated proteins as well as all 55 known ARRP proteins. The results obtained for CEP78 are marked in red.

13 Table S1- A list of variants identified in the shared homozygous region on chromosome 9 in family MOL0679 Chromosome position Gene Name Gene region ANKRD20A4 exonic ANKRD20A4 exonic CBWD3 exonic MAMDC2 exonic TRPM3 exonic TRPM3 exonic C9orf57 exonic ANXA1 exonic PRUNE2 exonic PRUNE2 exonic PRUNE2 exonic PRUNE2 exonic Transcript #: cdna, protein variant name NM_ : c.1117c>g, p.(q373e) NM_ : c.2088a>t, p.(q696h) NM_ : c.840a>c, p.(e280d) NM_ : c.881g>a, p.(s294n) NM_ : c.5009g>a, p.(r1670q) NM_ : c.4023g>a, p.(s1341s) NM_ : c.204a>g, p.(k68k) NM_ : c.t198c:p.(i66i) NM_ : c.8058g>a, p.(l2686l) NM_ : c.5914a>g, p.(t1972a) NM_ : c.1632c>g, p.(t544t) NM_ : c.1575t>c, p.(s525s) MAF value SIFT score Polyphen2 score Mutation Taster score T B P 0 T B P T B P T B P D (LC) PD P NA NA P NA NA p NA NA p NA NA P T B P NA NA P 0.79 NA NA P

14 CEP78 splicing TLE1 exonic TLE1 exonic NM_ : c.893-1g>a, p.(d298vfs*17) NM_ : c.1689c>t, p.(t563t) NM_ : c.354a>g, p.(e118e) 0 NA NA NA NA NA P NA NA P MAF - minor allele frequency, NA not available D- deleterious, T- tolerated, D (LC)- deleterious with low confidence, PD- probably damaging, B- benign, P- polymorphism. Home pages: SIFT- Polyphen2- Mutationtasrer-

15 Table S2: SNP makers used for haplotype reconstruction SNP # Chr. 9 Position (bp) Alleles (major/m inor) MAF MOL 0679 III:1 MOL 0679 III:2 MOL 0773 II:1 MOL 1310 II:1 TB279 II:1 TB365 II:1 rs A/T 0 A/A A/A A/A A/A A/A A/A rs G/C C/C C/C G/G C/C G/G C/G rs T/C 0.06 C/C C/C T/T T/T C/C C/T rs C/T T/T T/T C/C C/C C/C C/T rs A/G G/G G/G G/G A/A A/A A/G rs A/G G/G G/G A/A A/A A/A A/G rs T/C C/C C/C T/T T/T T/T C/T rs C/T 0.31 C/C C/C T/T C/C C/C C/C rs C/T 0.33 C/C C/C C/C C/C C/C C/C rs A/C 0.37 A/A A/A A/A C/C C/C A/C rs C/T 0.42 T/T T/T T/T C/C C/C T/C rs C/G 0.46 C/C C/C C/C G/G G/G C/G rs G/T 0 T/T T/T T/T T/T T/T T/T rs A/C 0.35 A/A A/A A/A A/A A/A A/A c.534delt T/- 0 T/T T/T T/T -/- -/- -/T c.893-1g>a G/A 0 A/A A/A A/A G/G G/G A/G rs C/T T/T T/T T/T C/C C/C C/T rs A/G A/A A/A A/A G/G A/G A/G rs C/A 0.32 A/C A/A A/C C/C A/C A/C rs /TTT /TTT -/- -/TTT -/TTT -/TTT TTT/T TT rs G/A A/A A/A A/A A/A A/A A/A rs C/T C/T C/C C/T T/T T/T T/T rs T/C C/C C/T C/T C/C C/C C/C Chromosome positions are based on hg19 assembly of the human genome. MAF- minor allele frequency (based on ExAC). Shared homozygous regions are colored in light blue (for the c.893-1g>a haplotype) or pink (for the c.534delt haplotype).

16 Table S3: Primers sequences used in the current study Primer Name Region Species Oligonucleotide Sequence 5'-3' Product Size (bp) CEP78-E1-F GACTCAGCGTTCTTGGGTG Exon1 Human CEP78-E1-R AAAACCCACTTCGCCGC 376 CEP78-E2&3-F AAAGGACATGGCTCATTTCTATG Exon2 & 3 Human CEP78-E2&3-R AAGGTACAGACAGGAATTAACATGG 527 CEP78-E4-F TCAAGACTGACTAGGTTGTATATGCTC Exon4 Human CEP78-E4-R TCAAAATATTAACGTGTGGCAATC 231 CEP78-E5-F TGGTGTGTGTGTAAAGCTCTG Exon5 Human CEP78-E5-R CTTCGGACAACATTACTGTGC 368 CEP78-E6-F CGGAGATAGTGCCACTGC Exon6 Human CEP78-E6-R ACAAAAGTGTAAGATTAGGGAAGG 351 CEP78-E7-F GGGAATAGCCACTCATTGGG Exon7 Human CEP78-E7-R AGGCTGGGCAGTACAGTGAG 202 CEP78-E8-F GGTCCCAGAACTGCTGATTG Exon8 Human CEP78-E8-R GACCTCATAATATGCACGGAGTC 359 CEP78-E9-F CATGTGCCTATTCATCTGTAGACC Exon9 Human CEP78-E9-R CCAAAATAAAGAATTTTGAATCCC 265 CEP78-E10-F TTATTACCTAGTGTTAACTGGTGGTG Exon10 Human CEP78-E10-R ATCTTCCACCCTTCCCATTC 172 CEP78-E11-F CACTTCAGAAAGATCACTTTGATTAG Exon11 Human CEP78-E11-R GAATTCCCATGCAAGCTGTC 319 CEP78-E12-F TTCACTTCTGGCTCTGTCCC Exon12 Human CEP78-E12-R TGACCATTTGCAATACACTATATTTTC 309 CEP78-E13-F GATCCAGGTATGTATTTAAGCTGTTAG Exon13 Human CEP78-E13-R TACAGGTGCAAACCACCAAG 670 CEP78-E14-F GGTTTGGGGAAATGTAAGCTG Exon14 Human CEP78-E14-R AGATGCTTCTGGTCCCTGAG 364 CEP78-E15-F CTTGGCAACTGACGACTTGA Exon15 Human CEP78-E15-R CAGAATGTTCTAGTGGACTGGATTT 845 CEP78-E16-F Exon 16 Human GGGGTCCTTTCCAAGACTGA 444

17 CEP78-E16-R GTCAGTTGCCAAGACCAGTC CEP78-CDS-E3F Exon3 TTTGGTGCACCTGTCTCTTG Human CEP78-CDS-E5R Exon5 TCATGCCTTCTCATGGTCTG 180 CEP78-CDS-E6-F Exon6 CACCAATGAAGGAGCAAAGG Human CEP78-CDS-E9-R Exon9 CAGGTTTCTTTGTAGCCAATCC 290 CEP78-CDS-E14-F Exon14 CCACAATGGCTGGGATAGAT 390 Human CEP78-CDS- 16-R Exon 16 TGCTATTTCTGGACAACTCCTC 438 Cep78-CDS-E2-F Exon2 GCTTAAGTGTATCAGGGGTGCT Mouse Cep78-CDS-E5-R Exon5 CCTGGTCACCAATGAGTGTG 387 PGM1-CDS-6&7F Exon 6&7 TGGTGCTCTGGACCGGGTGG Human PGM1-CDS-10R Exon 10 GCACTCCCAGTGCCGCTCAG 516 Pgk1-E3F Exon3 GATCAAGGCTGCTGTTCCA Mouse Pgk1-E5R Exon 5 GCAGTCCCAAAAGCATCATT 390 Primer sequences are based on following accession numbers: CEP78- NM_ and NM_ , PGM1- NM_ , Cep78 - NM_ , Pgk1- NM_

18 Table S4: A list of genes used for Figure S6C Phenotype Gene Names Total arush ABHD12, CDH23, CEP250, CIB2, CLRN1, DFNB31, GPR98, 14 HARS, MYO7A, PCDH15, USH1C, USH1G, USH2A, PDZD7 arrp ABCA4, ARL6, ARL2BP, BBS1, BBS2, BEST1, C2orf71, C8orf37, CERKL, CLRN1, CNGA1, CNGB1, CRB1, CYP4V2, DHDDS, DHX38, EMC1, EYS, FAM161A, GPR125, HGSNAT, IDH3B, IFT140, IFT172, IMPG2, KIAA1549, KIZ, LRAT,MAK, MERTK, MVK,NEK2, NEUROD1, NR2E3, NRL, PDE6A, PDE6B, PDE6G, PRCD, PROM1, RBP3, RGR, RHO, RLBP1, RP1, RP1L1, RPE65, SAG, SLC7A14, SPATA7, TTC8, TULP1, USH2A, ZNF408, ZNF513 55

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