OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD Novel Compound Heterozygous TULP1 Mutations in a Family With Severe Early-Onset

Anneke I. den Hollander, PhD; Janneke J. C. van Lith-Verhoeven, MD, PhD; Maarten L. Arends, BSc; Tim M. Strom, MD, PhD; Frans P. M. Cremers, PhD; Carel B. Hoyng, MD, PhD

Objective: To describe the clinical characteristics and tion analysis of TULP1 identified novel compound het- determine the genetic defect in a Surinamese family with erozygous mutations (p.Arg482Trp and p.Leu504fsX140) autosomal recessive retinitis pigmentosa. in all affected family members.

Methods: Family members underwent blood sampling Conclusions: The affected members of the Surinamese and ophthalmologic examinations. After exclusion of all family have a severe early-onset form of autosomal re- known mutations in all involved in autosomal re- cessive retinitis pigmentosa, which is caused by com- cessive retinitis pigmentosa, a genome-wide linkage scan pound heterozygous mutations in the TULP1 . was performed using 11 555 single-nucleotide polymor- phisms spread throughout the genome. Mutation analysis Clinical Relevance: Clinical and molecular genetic char- of the TULP1 gene was performed by direct sequencing. acterization of autosomal recessive retinitis pigmentosa may help to provide a more accurate prognosis in indi- Results: All affected family members had a severe reti- vidual patients. This study confirms that TULP1 muta- nal dystrophy with a history of nystagmus, low visual tions cause a severe early-onset form of autosomal re- acuity, and nyctalopia since infancy. The scotopic and cessive retinitis pigmentosa. photopic responses were nonrecordable on electroreti- nography. A genome-wide scan suggested linkage to the chromosomal region containing the TULP1 gene. Muta- Arch Ophthalmol. 2007;125(7):932-935

ETINITIS PIGMENTOSA (RP) IS tions have a very severe visual handicap, a heterogeneous group of which may be better described as Leber progressive retinal dystro- congenital amaurosis.3,4,8,9 phies, which has a preva- TULP1 is a member of the tubby-like lence of approximately 1 in (TULP) family. The TULP pro- Author Affiliations: 4000R individuals. Retinitis pigmentosa is teins are characterized by a highly con- Departments of Human characterized by nyctalopia, a progres- served C-terminal tubby domain; their ex- Genetics (Drs den Hollander sive constriction of the visual fields, pig- pression is mainly restricted to neuronal and Cremers, and Mr Arends) ment depositions in the midperipheral tissues. Expression of TULP1 is confined and Ophthalmology (Drs van Lith-Verhoeven and retina, and a gradual reduction of visual to the retina, where it localizes primarily Hoyng), and Nijmegen Centre acuity. The disease can be inherited in an to the inner segments and connecting for Molecular Life Sciences autosomal recessive, autosomal domi- of the photoreceptor cells. TULP1 (Drs den Hollander and nant, or X-linked fashion. Mutations in 19 is involved in protein trafficking and is es- Cremers), Nijmegen Medical genes have been reported to cause auto- sential for the transport of rhodopsin from Centre, Radboud University, somal recessive RP, and 5 loci have been its site of synthesis in the inner segments Nijmegen, the Netherlands; and reported for which the causative gene has through the connecting cilium to the outer Institute of Human Genetics, not yet been identified.1 segments.10,11 German Science Foundation Mutations in the TULP1 gene are found In this study, we describe a Surinamese National Research Center for in approximately 1% to 2% of patients with family with multiple members who were Environment and Health, 2-6 Munich-Neuherberg, and autosomal recessive RP. To date, 14 dif- affected by a severe early-onset form of au- Institute of Human Genetics, ferent mutations have been found in the tosomal recessive RP. The involvement of Technical University, Munich, TULP1 gene.2-8 In comparison with other all known mutations in all known auto- Germany (Dr Strom). forms of RP, patients with TULP1 muta- somal recessive RP genes was excluded.

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 A B

Figure 1. A, Posterior subcapsular cataract in patient II-4. B, Fundus photograph of the left eye of patient II-1 showing diffuse bone spicule pigmentation extending to the macular region, narrowed arterioles, disc pallor, and atrophy of the pigment epithelium.

A genome-wide linkage suggested linkage to a region dren (third generation) of the 5 affected siblings (second gen- on 6, which contains the TULP1 gene. eration) are affected. The disease-allele frequency was esti- Mutation analysis of the TULP1 gene identified 2 novel mated at 0.001. Haplotypes were constructed with HaploPainter 16 compound heterozygous mutations in all affected indi- V.024 (University of Cologne, Cologne, Germany). viduals. Primers for amplification of the TULP1 coding exons and splice junctions were designed with ExonPrimer (Technical Uni- versity, Munich, Germany; http://ihg.gsf.de/ihg/ExonPrimer METHODS .html) and Primer3 (Whitehead Institute, Cambridge, Massa- chusetts).17 Primer sequences and polymerase chain reaction PATIENTS AND conditions can be requested from the authors. Polymerase chain OPTHALMOLOGICAL EXAMINATIONS reaction products were purified with the QIAquick Gel Extrac- tion Kit (Qiagen, Hilden, Germany). Sequencing was per- Five affected and 5 unaffected members of a Surinamese fam- formed with BigDye Terminator Chemistry, version 3 (Ap- ily with autosomal recessive RP participated in this study. All plied Biosystems, Foster City, California), on a 3730 or 3100 affected family members underwent complete ophthalmo- DNA Analyzer (Applied Biosystems). The p.Arg482Trp mis- logic examinations, including best-corrected projected Snellen sense mutation was analyzed in control individuals by diges- visual acuities, slitlamp biomicroscopy, dilated fundus exami- tion of the exon 14 polymerase chain reaction product with re- nations, Goldmann visual fields, and International Society for striction enzyme HpaII. Clinical Electrophysiology of Vision–standardized electroreti- nography and electrooculography. Informed consent was ob- tained from all participating individuals, consistent with the RESULTS tenets of the Declaration of Helsinki. This study was approved by the institutional review board of the Radboud University Nij- megen Medical Centre, Nijmegen, the Netherlands. CLINICAL EXAMINATION

MOLECULAR ANALYSIS All affected individuals of the Surinamese family had se- vere retinal dystrophy. They had a history of nystag- Standard protocols were used to extract DNA from peripheral 12 mus, low visual acuity, and nyctalopia since infancy. On blood leukocytes. The proband (II-8) of the Surinamese fam- the last examinations (ages, 39-63 years), their visual acu- ily was screened for 505 known mutations and sequence vari- ants in 16 genes known to be involved in autosomal recessive ities ranged from light perception to 1/300 OU. The 3 RP (CERKL, CNGA1, CNGB1, MERTK, PDE6A, PDE6B, PNR, youngest affected family members (II-4, II-8, and II-10) RDH12, RGR, RLBP1, SAG, TULP1, CRB1, RPE65, USH2A, and had myopic refractions, varying from a spherical equiva- USH3A) with a genotyping microarray based on arrayed primer lent of −2.5 to −2.0 diopters. They all had nonrecord- extension technology (AR-RP Chip; Asper Ophthalmics, Tartu, able scotopic and photopic electroretinography re- Estonia).13 Ten members of the Surinamese family underwent sponses and a flat line on electrooculogram. The visual genotyping, with 11 555 single nucleotide polymorphisms spread fields showed a general and central decline in sensitiv- throughout the genome (Affymetrix GeneChip Human Map- ity with progression to a constricted visual field of 10°. ping 10K Array Xba142 2.0; Affymetrix, Santa Clara, Califor- On examination of the anterior segments, posterior sub- nia). Multipoint parametric linkage analysis was performed with capsular cataracts were found (Figure 1A). The oldest Allegro v1.2c (Decode Genetics, Reykjavik, Iceland)14 in the EasyLinkage Plus v4.00b software package (University of Wu¨ rz- affected individual (II-1) already underwent cataract sur- burg, Wu¨ rzburg, Germany)15 using the Decode Genetics ge- gery. Retinal examination revealed narrowed arterioles, netic single nucleotide polymorphism map and the white al- optic disc pallor, atrophy of the pigment epithelium, and lele frequencies. An autosomal recessive mode of inheritance diffuse bone spicule pigmentation extending to the macu- with complete penetrance was assumed, as none of the chil- lar region (Figure 1B).

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 A 1 2 I Affected individual rs886390 11 11 rs3871466 12 11 rs3906268 11 11 rs763469 22 21 rs3800472 21 22 TULP1 M1 + M2 + rs2007741 11 11 rs708024 12 21 rs2145887 12 22 rs1579028 21 11 rs726108 21 12 rs2050180 1 1 11

1 2 3 4 5 6 7 8 9 10 II

rs886390 1111 111111111111 11 rs3871466 1111 212121211121 11 rs3906268 1111 111111111111 11 rs763469 2222 222221212222 22 rs3800472 2222 221212122212 22 TULP1 M1 M2 M1 M2 M1 M2 + M2 ++++M1 M2 + M2 M1 M2 rs2007741 1111 111111111111 11 rs708024 1212 122221211222 12 rs2145887 1212 122222221222 12 rs1579028 2121 211111112111 21 rs726108 2121 211112122111 22 rs2050180 1 1 11 111111111111 11

B Patient II-8 Exon 14 Control Exon 14

CCCTTAAG G CCCCC/T G G G T C A A CCCTTAAG G CCCC CGG G T C A A FQGVTR>W FQGVTR

Patient II-8 Exon 15 Control Exon 15

GGTTCC GA GGGGGG TTT C CCC GGTTCC GA GGGGGG TT CCCC T C GGGGGGGGGCCT AA CC 11-bp Deletion

G368W F382S R400W R420P C

Human TULP1 360 SRGG E N F I GK L RSN L L GNR F T V F DNGQNPQRGY S T NV A S – – L RQ E L A A V I Y E T NV L G F RGPRRMT V I I PG Chimpanzee Tulp1 360 SRGG E N F I GK L RSN L L GNR F T V F DNGQNPQRGY S T NV A S – – L RQ E L A A V I Y E T NV L G F RGPRRMT V I I PG Canine Tulp1 360 SRGG E N F I GK L RSN L L GNR F T V F DNGQNPHRGGS T DVGS – – L RQ E L A A V I Y E T NV L G F RGPRRMT V I I PG Mouse Tulp1 361 SRGG E N F I GK L RSN L L GNR F T V F DNGQNPQRGGGGDVGS – – L RQ E L A A V V Y E T NV L G F RGPRRMT V I I PG Chicken Tulp1 177 SRGG E N F I GK L RSN LMG T K F T V F DNGANPDR – A S ADWSN – – VRQ E L S A V V Y E T NV L G F KGPRKMT V I I PG Human TUB 322 SRGGDS Y I GK L RSN LMG T K F T V YDNGVNPQK A S S S T L E SG T L RQ E L A A V C Y E T NV L G F KGPRKMS V I V PG Human TULP2 343 SRDGDN F VGK VRSNV F S T K F T I F DNGVNPDR E H L T RN T AR – I RQ E L GA V C Y E PNV L GY L GPRKMT V I L PG Human TULP3 259 SR E G E S Y VGK L RSN LMG T K F T V YDRG I C PMKGRG – L VGA AH T RQ E L A A I S Y E T NV L G F KGPRKMS V I I PG Drosophila king-tubby 283 SRNADG F CGK L RSNV F G T S F T V F DNGNK E S T E S – – – – – – – – PR L D L A V I I YD T N I L G F KGPRNMT V I L PG Caenorhabditis elegans tub-1 241 SR E G E GY C A K VRSNA L G T Q F T V YDSGQNP K K T T N – – – – HA A I RQ E L A A V I Y E T NV L G F KGPRKMT I VMPG

I459L R482W K489R F491L

Human TULP1 428 MS A E N E RV P I RPRN – – – – – A SDG L L VRWQNK T L E S L I E L HNK P P VWNDDSGS Y T L N F QGRV T QA S V KN F Q Chimpanzee Tulp1 428 MS A E N E RV P I RPRN – – – – – A SDG L L VRWQNK T L E S L I E L HNK P P VWNDDSGS Y T L N F QGRV T QA S V KN F Q Canine Tulp1 428 MN T DN E RV P I RPRN – – – – – A SDG L L VRWQNK T L E S L I E L HNK P P VWN E DSGS Y T L N F QGRV T QA S V KN F Q Mouse Tulp1 429 MNSDN E RV P I RPRN – – – – – A SDG L L VRWQNK T L E S L I E L HNK P P I WN E DSGS Y T L N F QGRV T QA S V KN F Q Chicken Tulp1 244 MNSDN E RV P I RPRN – – – – – DNDG L LMRWQNKNMDN I I E L HNK A P VWND E T QS Y V L N F HGRV T HA S V KN F Q Human TUB 392 MNMVH E RV S I RPRN – – – – – E H E T L L ARWQNKN T E S I I E L QNK T P VWNDD T QS Y V L N F HGRV T QA S V KN F Q Human TULP2 412 T NSQNQR I NVQP L N – – – – – E Q E S L L SRYQRGDKQG L L L L HNK T P SWDK E NGV Y T L N F HGRV T RA S V KN F Q Human TULP3 328 MT L NHKQ I P YQPQN – – – – – NHDS L L SRWQNR TME N L V E L HNK A P VWNSD T QS Y V L N F RGRV T QA S V KN F Q Drosophila king-tubby 345 MT E DDQRV K I S S ADP – – – – KQQG I L D LWKMKNMDN I V E L HNK T P VWND E T QS Y V L N F HGRV T QA S V KN F Q Caenorhabditis elegans tub-1 307 I E P P T E NRP A VRC P VRP I QDKH T L L E RYR L ND L DS L K I L SNK S P VWND E T QS Y V L N F HGRV T QA S V KN F Q

Figure 2. Molecular analysis of the genetic defect in a Surinamese family with autosomal recessive retinitis pigmentosa. A, Pedigree structure and haplotype analysis at chromosome 6p21. Mutations in the TULP1 gene segregate with the disease in the family. Mutation 1 (M1), p.Leu504fsX140; mutation 2 (M2), p.Arg482Trp. Black bars indicate uninformative single nucleotide polymorphism alleles. B, Genomic sequence of the TULP1 gene in proband II-8 and a control individual. The proband carries a heterozygous missense mutation p.Arg482Trp in exon 14 and a heterozygous 11– (bp) deletion in exon 15. C, Alignment of a part of the C-terminal tubby domain of TULP1 orthologs and TULP family members and position of all missense mutations identified in the TULP1 gene to date, including the novel p.Arg482Trp mutation. Identical amino acids are indicated in black boxes, conserved residues in gray boxes. positions are shown after mutation names.

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 MOLECULAR ANALYSES Submitted for Publication: November 2, 2006; final revi- sion received December 21, 2006; accepted December 28, The proband (II-8) of the family was analyzed with the 2006. autosomal recessive RP mutation chip13 and did not carry Correspondence: Anneke I. den Hollander, PhD, De- any mutations in the genes known to be involved in au- partment of Human Genetics, Radboud University, Nij- tosomal recessive RP. Genome-wide linkage analysis re- megen Medical Centre, PO Box 9101, 6500 HB Nijme- vealed 2 regions with a maximum multipoint logarithm gen, the Netherlands ([email protected]). of the odds score of 2.9. Haplotype analysis confirmed Financial Disclosure: None reported. that the single nucleotide polymorphism alleles at both Funding/Support: This work was supported by grant of these regions completely segregated with the disease. 916.56.160 from the Netherlands Organisation for Sci- One region is located on chromosome 5p15 between single entific Research (Dr den Hollander) and grant 543 from nucleotide polymorphisms rs4487467 and rs2008011, the British Retinitis Pigmentosa Society (Drs den Hol- spans 6.6 megabase pairs (Mb) of genomic DNA, and con- lander and Cremers). tains 11 genes. The second region is located on chromo- Additional Contributions: Arjan P. de Brouwer, PhD, some 6p21 between single nucleotide polymorphisms provided helpful discussions, and Mrs Christel Beumer rs3871466 and rs726108, spans 7.8 Mb of genomic DNA, and Mrs Saskia van der Velde-Visser provided technical and contains more than 150 genes, including the TULP1 assistance. gene (Figure 2A). 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