BBS1 Mutations in a Wide Spectrum of Phenotypes Ranging from Nonsyndromic Retinitis Pigmentosa to Bardet-Biedl Syndrome

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD BBS1 Mutations in a Wide Spectrum of Phenotypes Ranging From Nonsyndromic Retinitis Pigmentosa to Bardet-Biedl Syndrome

Alejandro Estrada-Cuzcano, BSc; Robert K. Koenekoop, MD, PhD; Audrey Senechal, PhD; Elfride B. W. De Baere, MD, PhD; Thomy de Ravel, MD, PhD; Sandro Banfi, MD; Susanne Kohl, MD; Carmen Ayuso, MD, PhD; Dror Sharon, PhD; Carel B. Hoyng, MD; Christian P. Hamel, MD, PhD; Bart P. Leroy, MD, PhD; Carmela Ziviello, BS; Irma Lopez, PhD; Alexandre Bazinet, MD; Bernd Wissinger, PhD; Ieva Sliesoraityte, MD; Almudena Avila-Fernandez, PhD; Karin W. Littink, MD, PhD; Enzo M. Vingolo, MD; Sabrina Signorini, MD, PhD; Eyal Banin, MD, PhD; Liliana Mizrahi-Meissonnier, PhD; Eberhard Zrenner, MD; Ulrich Kellner, MD; Rob W. J. Collin, PhD; Anneke I. den Hollander, PhD; Frans P. M. Cremers, PhD; B. Jeroen Klevering, MD, PhD

Objective: To investigate the involvement of the Bardet- 2 BBS1 variants showed the entire clinical spectrum, from Biedl syndrome (BBS) gene BBS1 p.M390R variant in non- nonsyndromic RP to full-blown BBS. In 8 of 14 patients, syndromic autosomal recessive retinitis pigmentosa (RP). visual acuity was significantly reduced. In patients with electroretinographic responses, a rod-cone pattern of pho- Methods: Homozygosity mapping of a patient with iso- toreceptor degeneration was observed. lated RP was followed by BBS1 sequence analysis. We performed restriction fragment length polymorphism analy- Conclusions: Variants in BBS1 are significantly associ- sis of the p.M390R allele in 2007 patients with isolated ated with nonsyndromic autosomal recessive RP and rela- RP or autosomal recessive RP and in 1824 ethnically tively mild forms of BBS. As exemplified in this study by matched controls. Patients with 2 BBS1 variants under- the identification of a homozygous p.M390R variant in went extensive clinical and ophthalmologic assessment. a control individual and in unaffected parents of BBS patients in other studies, cis-ortrans-acting modifiers may Results: In an RP proband who did not fulfill the clini- influence the disease phenotype. cal criteria for BBS, we identified a large homozygous region encompassing the BBS1 gene, which carried the Clinical Relevance: It is important to monitor pa- p.M390R variant. In addition, this variant was detected tients with an early diagnosis of mild BBS phenotypes for homozygously in 10 RP patients and 1 control, com- possible life-threatening conditions. pound heterozygously in 3 patients, and heterozygously in 5 patients and 6 controls. The 14 patients with Arch Ophthalmol. 2012;130(11):1425-1432

ARDET-BIEDL SYNDROME is based on the presence of at least 4 pri- (BBS) (OMIM 209900) is a mary features or a combination of 3 pri- clinically and genetically mary plus at least 2 secondary features.2 The heterogeneous disorder char- clinical phenotypic spectrum of BBS is wide, acterized by a wide spec- and genotype-phenotype correlations have trumB of clinical features. It is considered a not been clearly established.3,4 Some clini- member of the group of conditions collec- cal features overlap those of other ciliopa- tively called ciliopathies.1 Primary clinical thies, such as McKusick-Kaufman (OMIM features include retinitis pigmentosa (RP) 604896), Alstrom (OMIM 203800), or Lau- (OMIM 268000), polydactyly, obesity, rence-Moon (OMIM 245800) syndromes, learning disabilities, hypogonadism in which have been described in families with males, and renal abnormalities. Secondary the BBS6, BBS10,orBBS12 mutation.5-7 The clinical features include speech disorders, transmission of the disease is autosomal re- strabismus, brachydactyly or syndactyly, cessive,8-10 but a digenic inheritance in the developmental delay, polydipsia-polyuria form of triallelism has been reported in some (diabetes insipidus), ataxia, mild spastic- families in which either 3 mutations in 2 BBS ity, diabetes mellitus, dental crowding or genes are required to manifest the pheno- 11,12 Author Affiliations are listed at hypodontia, congenital heart diseases, and type or a third variant may modulate the Author Affil the end of this article. hepatic fibrosis. The clinical diagnosis of BBS expression of the clinical phenotype.13-15 the end of th

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1425

©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 The prevalence of BBS ranges from 1:125 000 to METHODS 1:160 000 in Europe16-18 and 1:65 000 in the Arab popu- 19 lation ; a higher incidence was observed in isolated popu- PATIENTS AND CLINICAL EVALUATION lations of Newfoundland (1:13 000),7 Kuwait (1:17 000),20 21 and the Faroe Islands (1:3700). In Canada, Europe, and Israel, 2007 unrelated patients with To date, 15 genes have been associated with BBS.12,22 isolated or autosomal recessive RP (80 from Ghent, 96 from Most BBS proteins can be divided into 2 groups. One forms Jerusalem, 245 from Madrid, 361 from Montpellier, 343 from a complex called the BBSome (BBS1, BBS2, BBS4, BBS5, Montreal, 143 from Naples, 215 from Nijmegen, and 524 from BBS7, BBS8, and BBS9), which is believed to recruit ␥-glu- Tu¨ bingen) and 1824 ethnically matched individuals serving as tamyl transpeptidase Rab8,23 and the second group com- controls (103 from Ghent, 100 from Jerusalem, 320 from Ma- 24 drid, 56 from Montpellier, 361 from Montreal, 140 from Naples, prises chaperoninlike proteins, including BBS6, BBS10, 233 from Nijmegen, and 511 from Tu¨ bingen) were included and BBS12. Mutations in known BBS genes are detected in the study. The diagnosis of RP was based on ophthalmo- in approximately 75% of families, with BBS1 and BBS10 logic examination that included measurement of best- each accounting for 20% to 25% of families of European corrected visual acuity, Goldmann visual fields, slitlamp bio- descent with BBS1-associated disease.25,26 Two wide- microscopy, dilated indirect ophthalmoscopy, and fundus spread mutations resulting from founder effects have been photography. In addition, full-field flash electroretinography described, p.M390R in BBS18,13,27 and p.C91fsX95 in (ERG), according to the guidelines of the International Soci- 41 BBS10.26 The p.M390R variant is the most common BBS1 ety of Clinical Electrophysiology of Vision, was performed mutation and has been observed8,25 inapproximately 80% when Goldmann visual fields were measurable and indicated a preserved visual field with peripheral limits beyond the peri- of families with BBS1-associated disease. central 10°. A broad definition of RP was used to avoid exclud- Retinitis pigmentosa is the most common inherited ing patients with atypical features: progressive visual loss, night retinal degeneration, with an estimated worldwide preva- blindness, and abnormal ERG findings in a rod-cone pattern lence of 1:4000,28 and is characterized by progressive pho- when recordable. Blood samples were obtained after informed toreceptor dysfunction, followed by photoreceptor cell consent forms were signed. Ethics approval was given to all par- death. The disease is highly heterogeneous and displays ticipating institutions, and the study conformed to the tenets all patterns of inheritance, that is, autosomal recessive, of the Declaration of Helsinki. Participants were evaluated again autosomal dominant, or X-linked. In addition, there are after the identification of the BBS1 gene defects, since they had previously received a diagnosis of nonsyndromic RP. Special some cases with mitochondrial mutations and digenic in- 29,30 attention was given to identifying systemic features associated heritance. Mutations in 36 genes have been identi- with BBS,2 which include the primary features of RP, polydac- fied that cause autosomal recessive nonsyndromic RP, tyly, obesity, learning disabilities, hypogonadism in males, and and mutations in 51 genes cause autosomal recessive syn- renal abnormalities, and the secondary features of speech dis- dromic retinal dystrophy, including BBS and Usher syn- orders, cataract, brachydactyly or syndactyly, developmental drome (RetNet, http://www.sph.uth.tmc.edu/RetNet/). delay, polydipsia/polyuria (diabetes insipidus), ataxia, mild spas- These genes encode proteins involved in the phototrans- ticity, diabetes mellitus, dental crowding or hypodontia, con- duction cascade, vitamin A metabolism, cellular or cy- genital heart diseases, and hepatic fibrosis. Where and when appropriate, patients were clinically reexamined or their rec- toskeletal structure, cell-to-cell signaling or synaptic in- ords were again reviewed. Clinical evaluation included best- teraction, gene expression, phagocytosis, and RNA splicing corrected visual acuity and slitlamp biomicroscopy of the an- 30 factors. Several RP proteins, including approximately terior segment and retina. Additional examinations included half the BBS proteins, are part of the photoreceptor sen- kinetic and/or static perimetry and ERG according to the pro- sory cilium proteome, a large protein complex associ- tocol of the International Society for Clinical Electrophysiol- ated with and constituting the connecting cilium.31 ogy of Vision. Homozygosity mapping has proven to be fruitful to identify defects in known and newly identified genes im- HOMOZYGOSITY MAPPING AND BBS1 plicated in autosomal recessive retinal degenera- MUTATION ANALYSIS tions32-34 and to establish novel genotype-phenotype cor- 35,36 Genomic DNAs were isolated from lymphocytes by standard relations. Using this approach, we identified a patient 42 with RP with a large homozygous region encompassing salting-out procedures. Only the DNA sample of patient 5 was genotyped, using a microarray that contains 500 000 single- the BBS1 gene. The fact that retinal degeneration is con- nucleotide polymorphisms (SNPs) (GeneChip Genome-Wide sistently seen in BBS patients with BBS1 mutations and Human SNP Array 5.0; Affymetrix). Array experiments were previous observations that some mutations in BBS3/ performed according to protocols provided by the manufac- ARL6, BBS8/TTC8, BBS12, and BBS14/CEP290 cause non- turer. The 5.0 array data were genotyped (Genotype Console, syndromic retinal degeneration or retinal degeneration version 2.1; Affymetrix) and, subsequently, regions of homo- with minimal systemic features37-40 prompted us to screen zygosity were identified using commercial software (Partek, ver- BBS1 in this patient and to further test the hypothesis sion 6.1, Partek, Inc). Primers for the amplification of the 17 coding exons and that mutations in BBS1 are also a significant cause of non- 43 syndromic RP. In this study, we analyzed the p.M390R splice junctions of BBS1 were designed by Primer3 and are available on request. Polymerase chain reaction (PCR) prod- BBS1 mutation in 2007 patients with RP. We identified ucts were purified with 96-well filter plates (Multiscreen HTS- p.M390R BBS1 mutation in a homozygous or com- PCR; Millipore) or by gel extraction (Qia-Quick Gel Extrac- pound heterozygous manner in 14 patients with a wide tion Kit; Qiagen). Sequencing was performed with dye terminator clinical spectrum ranging from nonsyndromic RP to clas- chemistry (BigDye Terminator, version 3 on a 3100, 3730, or sic BBS, as well as in 1 putative healthy individual. 3730XL DNA analyzer; Applied Biosystems, Inc).

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1426

©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 SCREENING FOR THE BBS1 p.M390R AND in 2006 patients with isolated or autosomal recessive RP MGC1203 C.403CϾT VARIANTS in Canada, Europe, and Israel. We found 8 additional unrelated patients and a sibling pair with a homozygous We developed a restriction enzyme test to screen the patients p.M390R mutation (Table 1). Three patients carried the and controls for the recurrent BBS1 p.M390R mutation be- p.M390R allele combined with a second BBS1 variant in Ͼ cause the c.1169T G variant in exon 12 introduces an AvaII a compound heterozygous state. Two of these muta- recognition site (G/GWCC). The PCR analysis of exon 12 of tions (c.1163TϾC [p.L388P] and c.803GϾC [p.R268P], BBS1 was performed with forward primer 5Ј-CCTGTCTT- GCTTTCCTCTCC-3Ј and reverse primer 5Ј-TCCTCCTCTTTC- detected in patients 2 and 13, respectively) are novel, and CCCAGAAG-3Ј, using 50 ng of DNA and 10 pmol of each primer 1 mutation (c.1130_1134del [p.C377WfsX36] in pa- 27 in a standard 25-µL reaction. The PCR amplification was per- tient 4) was described previously. Alignment of the BBS1 formed (PTC-200 Thermo Cycler; MJ Research) under the fol- amino acid sequences of various orthologs showed that lowing conditions: 3 minutes at 94°C, 35 cycles at 94°C for 30 the substituted amino acids (ie, leucine at position 388 seconds, 58°C for 30 seconds, 72°C for 45 seconds, and a final and arginine at position 268) are highly conserved from extension step at 72°C for 10 minutes. The PCR products were human to C elegans. PolyPhen-2 predicted that p.L388P purified with 96-well filter plates (Multiscreen HTS-PCR; Mil- and p.R268P variants are probably damaging, and SIFT lipore). At that time, 10 µL of the PCR product was digested analysis revealed that neither is tolerated. with2UofAvaII (New England Biolabs) in the appropriate Analysis using restriction fragment length polymor- buffer at 37°C for 2 hours, and the size was discriminated by agarose gel electrophoresis. After detecting the mutations, Sanger phism revealed 5 patients with only 1 p.M390R variant; sequencing was performed to confirm the presence of the subsequent sequence analysis of the 17 BBS1 exons did mutation. not reveal a second allele. In 1824 ethnically matched con- The presence of the MGC1203 c.403CϾT variant was tested trol individuals, we found 6 heterozygous p.M390R car- in all patients with 2 BBS1 variants and in the control indi- riers and 1 individual who was homozygous for this vari- vidual with BBS1 p.M390R in homozygosity. This was done by ant. Because these were anonymous healthy individuals, sequencing PCR products generated using forward primer 5Ј- further clinical assessments could not be performed. ACTGGTGCATAGGCAGATGG-3Ј and reverse primer 5Ј- Because Badano et al14 identified a modifier allele for Ј CATGGACTGGGCCCTACAG-3 . The PCR products were pu- BBS in MGC1203, we tested the presence of the c.403CϾT rified with 96-well filter plates (Multiscreen HTS-PCR; variant in all cases and the 1 control individual with 2 Millipore). Sequencing was performed with dye terminator chemistry (BigDye Terminator, version 3 on a 3100, 3730, or BBS1 alleles. Using restriction fragment length polymor- 3730XL DNA analyzer; Applied Biosystems, Inc). phism analysis, this modifier allele was not detected in any of these cases. IN SILICO ASSESSMENT OF MISSENSE SUBSTITUTIONS CLINICAL FINDINGS

We used PolyPhen-244 (http://genetics.bwh.harvard.edu/pph2 The clinical findings of the 14 patients with BBS1 vari- /index.shtml) and SIFT45 (http://sift.jcvi.org) to predict the func- ants are reported in Table 1. Patients were divided into tional effect of novel missense substitutions identified in this 4 groups. Group A contained the 3 patients who ap- study. To assess the evolutionary conservation, amino acid se- peared to have a nonsyndromic form of RP. In these pa- quences of orthologous BBS1 proteins (Pan troglodites tients, cataract was the only feature that might be re- XP_001171950.1, Sus scrofa NP_001091721, Canis lupus fa- lated to BBS. Group B consisted of 4 patients who may miliaris XP_540830.2, Mus musculus NP_001028300, Xeno- have a nonsyndromic form of RP. These patients showed pus tropicalis NP_001116276.1, Drosophila melanogaster few extraocular features that may be associated with the NP_648080.1, and Caenorhabditis elegans NP_740933.1) were obtained from the NCBI protein database (http://www.ncbi retinal phenotype. Three of these patients were obese, .nlm.nih.gov/protein/) and were aligned with the protein se- and, in 1 patient, routine ultrasound revealed a few mild quence of human BBS1 (NP_078925.3) using Vector NTI soft- renal cysts without renal dysfunction. The 6 patients in ware (version 11; Invitrogen). group C demonstrated definite extraocular features commonly associated with BBS but insufficient to warrant a RESULTS diagnosis of classic BBS. Finally, group D included 1 patient who fulfilled the criteria for the diagnosis of classic BBS after reevaluation. IDENTIFICATION OF BBS1 MUTATIONS The results of the ophthalmologic examinations of the IN RP PATIENTS 14 patients with RP or (mild) BBS are summarized in Table 2. In most patients, night blindness was the first Upon homozygosity mapping, several homozygous re- symptom of retinal degeneration. Early photoreceptor dys- gions were identified in patient 5, who received a diag- function, at or before the age of 10 years, was observed nosis of severe RP, the largest of which measured 19 Mb in 6 of the 14 patients (including 2 siblings, patients 3 and encompassed the BBS1 gene on chromosome 11q13.2. and 7). A visual acuity level of counting fingers or less Sequence analysis of all 17 coding exons of BBS1 re- was observed in 8 patients, and none of the patients dis- vealed the homozygous c.1169TϾG (p.M390R) mis- played visual acuity better than 20/40. Figure 1 (pa- sense mutation in exon 12. To determine whether the tient 4) clearly illustrates the severity of the phenotype p.M390R variant could be a rather frequent cause of non- in these patients. A relatively later age at onset did not syndromic RP, we carried out a restriction fragment length necessarily reflect positively on the chance of encoun- polymorphism analysis for the p.M390R BBS1 mutation tering severe visual loss, as demonstrated by patient 14,

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1427

Primary Features Secondary Features No. of BBS Features Patient No./Sex Country Ocular Extraocular Ocular Extraocular Primary Secondary Diagnosis Allele 1/Allele 2 Group A 1/M NL RP None Cataract None 1 1 Nonsyndromic RP M390R/M390R 2/M FR RP None Cataract None 1 1 Nonsyndromic RP M390R/L388P 3/Fa IT RP None Cataract None 1 1 Nonsyndromic RP M390R/M390R Group B 4/M NL RP Mild renal cysts without None None 1 1 Possibly M390R/C377WfsX36 renal dysfunction nonsyndromic RP 5/M CA RP Obesity; not truncal Cataract None 2 1 Possibly M390R/M390R nonsyndromic RP 6/M FR RP Obesity None None 2 0 Possibly M390R/M390R nonsyndromic RP 7/Ma IT RP Obesity Cataract None 2 0 Possibly M390R/M390R nonsyndromic RP Group C 8/Mb BE RP None None Light spasm and 1 1 Mild BBS M390R/M390R clubfoot 9/F GE RP Learning difficulties None Speech disorder 2 1 Mild BBS M390R/M390R 10/Fc FR RP Obesity Cataract None 2 1 Mild BBS M390R/M390R 11/M IT RP Nephronophthisis Cataract None 2 1 Mild BBS M390R/M390R 12/F FR RP Obesity/learning Cataract None 3 1 Mild BBS M390R/M390R difficulties 13/F NL RP Obesity/polydactyly Cataract None 3 1 Mild BBS M390R/R268P Group D 14/M GE RP Obesity/learning None Polyuria-polydipsia/ 3 2 BBS M390R/M390R difficulties diabetes insipidus/congenital heart disease

Abbreviations: BBS, Bardet-Biedl syndrome; BE, Belgium; CA, Canada; FR, France; GE, Germany; IT, Italy; NL, the Netherlands; RP, retinitis pigmentosa. a Patients 3 and 7 are siblings. b Sister with polydactyly. c Sister with polydactyly and RP.

who experienced visual field loss at age 35 years, but at controls (P = .02, Fisher exact test). Similarly, the BBS3/ age 54 years, her visual acuity was reduced to light per- ARL6 variant p.A89V and the BBS12 variant p.S701X have ception. In most patients there was extensive loss of the been implicated in both nonsyndromic RP and BBS.40,47 visual field. Four patients could not see the largest tar- A splice mutation affecting a retina-specific exon of BBS8/ get or showed only a modest temporal region of remain- TTC8 also causes nonsyndromic RP.48 In other stud- ing sensitivity; 6 patients demonstrated severe visual field ies,49-51 considerable variations of systemic features have constriction. When ERG responses could be elicited, a been described in patients with BBS. rod-cone pattern of photoreceptor degeneration was ob- The phenotype of the BBS1-associated retinal dystro- served. In 10 of 13 patients, the ERG became nonrecord- phy in our patient group was more severe than that re- able during the course of their disease. ported by Azari and coworkers.52 Eight of the 14 patients in the current study had visual acuity of counting COMMENT fingers or lower, whereas Azari and coworkers observed only 1 patient with visual acuity below 20/200 in their Mutations in the BBS1 gene have been associated with 10 patients with principally classic BBS. In addition, the BBS,27 and in one study46 they were associated with RP age at onset was relatively early in our patients and nys- and variable mild systemic features. For some BBS fami- tagmus was present in 3 patients. For the most part, the lies, a digenic-triallelic inheritance model has been pro- patients in the current study showed classic RP with a posed in which interactions were suggested between BBS1 relatively homogeneous clinical presentation. In view of and BBS2 or BBS1 and BBS6,13-15 although this has not the early age at onset, low visual acuity, and extensive been replicated in other studies.8-10 We found that the BBS1 visual field loss, the photoreceptor dystrophy of the 14 variant p.M390R is significantly associated with nonsyn- patients in this study should be considered severe. dromic RP or mild BBS because we identified it in 21 of The disease spectrum of the patients in this study is 4014 alleles in our probands vs 8 of 3648 alleles in the broad, and any attempt at organization is arbitrary at best.

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1428

Visual Acuity Patient First Age at Anterior Scotopic/ No. Symptoms Examination, y OD OS Segment Ophthalmoscopy Perimetry Photopic ERG Group A 1 Night blindness 33 CF CF Polar subcapsular Typical RP with Ring scotoma at At age 12 y: atage3y,VA cataract symmetric atrophy in age 12 y; later in severely loss at age the posterior pole life, central disturbed in 12-15 y islands, 15° rod-cone pattern; later, NR/NR 2 Night blindness 31 20/50 20/63 Moderate cortical Punctate pigment Central islands, 10° NR/NR at age 18 y cataract deposits and peripheral bone spicules; preserved maculae 3a Nystagmus and 55 LP LP Unknown Typical RP with marked Modest temporal NR/NR hemeralopia peripheral region of at age 1 y pigmentation remaining sensitivity Group B 4 Night blindness 43 LP LP Posterior cortical Extensive atrophy, Patient could not NR/NR at age 8 y opacity striking macular observe targets atrophy, attenuated vessels and irregular midperipheral pigmentations (Figure 1) 5 Night blindness 43 HM HM Pseudophakia Severe maculopathy, Patient could not NR/NR at age 18 y leopard spots, diffuse observe targets pigmentation 6 Night blindness 19 20/50 20/63 No abnormalities Typical RP with atrophy Central islands, 5° Not performed at age 10 y at the fovea 7a Nystagmus and 55 LP LP Pseudophakia Typical RP with macular Modest temporal NR/NR hemeralopia involvement region of at age 1 y remaining sensitivity Group C 8 Night blindness 16 20/80 20/160 Unknown Typical RP Severely constricted Severely decreased at age 12 y (30°) cone response/rod NR 9 Night blindness 43 20/200 20/200 Posterior cortical RP with diffuse pigment Relative central NR/NR at age 5 y opacity clumping and scotoma of 60°, depigmentation with relative and absolute regions 10 VA loss at age 41 HM HM Pseudophakia at Profound macular Large central Severely decreased 23 y, age 41 y atrophy, bone scotoma of 40° rods more than moderate spicules, and cones night attenuated vessels blindness 11 Nystagmus 21 LP LP Unknown Typical RP with macular Patient could not NR/NR since infancy involvement observe targets 12 VA loss at age 36 20/63 20/63 Subcapsular Typical RP, normal Severely constricted Severely decreased 10 y cataract aspect of macula (30°) cone response/rod NR 13 Night blindness 38 20/50 20/40 Cortical cataract Typical RP with severe Severely constricted NR/NR at age 18 y atrophy (30-35°) Group D 14 Visual field loss 54 LP LP Cataract RP without Severely constricted NR/NR at age 35 y pigmentation, severe (20°-45° OU) atrophy central and in midperiphery

Abbreviations: CF, counting fingers; ERG, electroretinography; HM, hand motion; LP, light perception; NR, nonrecordable; RP, retinitis pigmentosa; VA, visual acuity. a Patients 3 and 7 are siblings.

Cataract is commonly found in up to 50% of patients with of other primary or secondary signs, we believed that the RP and, in many patients, cataract surgery is performed condition in the 3 patients in group A should be diag- at a relative early age.53-55 Consequently, in the absence nosed as nonsyndromic RP, despite the presence of the

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1429

Unaffected RP/Mild BBS BBS v1 v1 v1 BBS1 BBS1 BBS1 Cis -acting +++ ++ +/– v2 v2 v2 BBS1 BBS1 BBS1

B BBS v1 BBS1 ++ v2 Cis- + trans-acting BBS1

v3 Modifier

Figure 2. Cis- and trans-acting Bardet-Biedl syndrome (BBS) gene BBS1 expression regulator model. A, Enhancer/promoter variants (cis-acting) may modulate the messenger RNA expression levels of BBS1 and thereby determine the penetrance or expression of disease. Unaffected individuals carry hypomorphic variants (v1 and v2, such as the p.M390R variant) on 1 or 2 highly (ϩϩϩ) expressed BBS1 alleles; moderately (ϩϩ) expressed BBS1 alleles result in nonsyndromic retinitis pigmentosa (RP) or mild BBS, and low (ϩ/−) expressed BBS1 alleles result in BBS. B, A trans-acting third variant (v3) present in an interactor of BBS1 or a transcription factor Figure 1. The right eye of patient 4 at age 33 years. This color fundus negatively regulating the expression of BBS1 may contribute to the photograph illustrates the severity of the retinitis pigmentosa phenotype in phenotype. many of the patients in this study. In addition to the midperipheral bone spicules and attenuated vessels, there is widespread chorioretinal atrophy. The atrophy is especially severe at the posterior pole. The visual acuity in this ronophthisis genes and not, to our knowledge, with BBS1 patient was only light perception; electroretinographic responses were mutations. The observation that BBS1 mutations may cause absent. milder BBS phenotypes has been reported in a case history of 2 brothers.46 The danger of a misdiagnosis in pa- secondary feature cataract. The same line of reasoning tients with milder BBS phenotypes is important in view of more or less holds with the primary feature of obesity in the potentially severe consequences of life-threatening con- the patients in group B. Obesity is a rather nonspecific ditions associated with BBS1 mutations, as well as the ac- finding in current Western society and perhaps even more curacy of genetic counseling. so in patients with retinal degeneration who are prone The presence of a presumed unaffected individual with to physical inactivity.56 In addition to RP, the patients in 2 p.M390R variants in our study is not unprecedented; group B had few primary and/or secondary features that Badano and coworkers13 reported on 2 families with 2 could be associated with BBS but may be coincidental. p.M390R alleles in BBS patients and their unaffected fa- The isolated presence of moderate obesity, especially with- thers. The differential penetrance of BBS1 alleles in one out truncal distribution and in combination with paren- of these families was subsequently explained by the de- tal obesity, is not necessarily an indication of extraocu- tection of a heterozygous trans-acting epistatic allele lar abnormalities due to BBS1 mutations, so that (MGC1203; c.430CϾT) in the patient with BBS but not nonsyndromic RP is at least a possibility. Following this in the unaffected father.14 The MGC1203 gene encodes approach, 3 of our 14 patients (group A) exhibited non- a pericentriolar protein that interacts with BBS1, BBS2, syndromic RP and 4 patients (group B) demonstrated a BBS4, BBS5, BBS6, BBS7, and BBS8 and colocalizes with form of RP that may be nonsyndromic. BBS1 and BBS4. The MGC1203 variant c.430CϾT en- Despite the inclusion criterion of autosomal recessive hances the use of a cryptic splice acceptor site, causing and/or isolated RP, 7 patients showed syndromic features the introduction of a premature stop codon and the re- that seem to be associated with the retinal phenotype. In duction of MGC1203 mRNA levels. An alternative ex- one patient (group D), a previously undiagnosed classic form planation for the variable phenotypes associated with BBS1 of BBS was discovered. The extra digits were removed shortly alleles could be cis-acting regulators of expression. We after birth in this patient, emphasizing the importance of did not find this modifier in our case and control pa- obtaining a complete history in patients with RP. In the re- tients with 2 BBS1 alleles. Different enhancer or pro- maining 6 patients (group C), extraocular features were moter variants may result in higher messenger RNA ex- present, but these abnormalities were insufficient to war- pression levels of the BBS1 p.M390R alleles in unaffected rant the diagnosis of classic BBS. Extraocular features in individuals, which may compensate for the reduced ac- these 6 patients were highly divergent, making correct iden- tivity of the p.M390R-carrying BBS1 protein (Figure 2A, tification of this milder form of BBS challenging. In pa- cis-acting modifier). Alternatively, as shown in Figure 2B, tient 11, RP was associated with nephronophthisis, a com- mild BBS or BBS can also be caused by a combination of bination known as the Senior-Løken syndrome. This 2 BBS1 variants and a negatively acting trans-acting modi- syndrome has been associated with mutations in the neph- fier, such as the MGC1203 variant c.430CϾT.

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1430

©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 In conclusion, by analyzing a very large cohort of pa- Hollander and Cremers); grant BR-GE-0510-0489-RAD tients with isolated or autosomal recessive RP, we iden- from the Foundation Fighting Blindness US (Dr den Hol- tified BBS1 variants in individuals with a wide clinical lander); the Algemene Nederlandse Vereniging ter spectrum, ranging from nonsyndromic RP (mild BBS) to Voorkoming van Blindheid, Gelderse Blinden Stichting, classic BBS. Landelijke Stichting voor Blinden en Slechtzienden, Retina Nederland, Stichting Oogfonds Nederland, Stichting Submitted for Publication: June 16, 2011; final revi- Wetenschappelijk Onderzoek het Oogziekenhuis, the Rot- sion received April 23, 2012; accepted June 6, 2012. terdamse Vereniging Blindenbelangen, the Stichting AF Author Affiliations: Departments of Human Genetics (Mr Deutman Researchfonds Oogheelkunde (Drs den Hol- Estrada-Cuzcano and Drs Littink, den Hollander, Col- lander and Cremers); the Foundation Fighting Blind- lin, and Cremers) and Ophthalmology (Drs Hoyng, Col- ness Canada, Canadian Institute of Health Research, Fond lin, den Hollander, and Klevering), Radboud University de la recherche en sante´ du Que´bec, Reseau Vision, and Nijmegen Medical Centre, Nijmegen Center of Molecu- National Intitutes of Health (Dr Koenekoop); Organiza- lar Life Sciences (Mr Estrada-Cuzcano and Drs Collin, cioń Nacional de Ciegos, FIS PS09/00459, and CIBERER den Hollander, and Cremers), Radboud University Ni- (ISCIII) (Dr Ayuso); the Italian Telethon Foundation, the jmegen, Nijmegen, the Netherlands; McGill Ocular Ge- Italian Ministry of Health (Dr Banfi); and grant KAN netics Laboratory, Montreal Children’s Hospital, McGill 1524611N from the Research Foundation–Flanders/ University Health Centre, Montreal, Quebec, Canada FWO (Drs De Baere and Leroy). Drs De Baere and Leroy (Drs Koenekoop, Lopez, and Bazinet); Institut National are Senior Clinical Investigators of the Research Foun- de la Sante´ et de la Recherche Me´dicale (INSERM) U583, dation–Flanders, Belgium, and are further supported by Institute for Neurosciences of Montpellier, Montpellier, Research Foundation–Flanders grants 3E000203, France (Dr Senechal); Centre for Medical Genetics (Drs De 31509107, and 31518209 (Dr De Baere) and OZP Baere and Leroy), Department of Ophthalmology (Dr Le- 3G004306 (Drs Banin and Leroy). roy), Ghent University Hospital and Ghent University, Ghent, Belgium; Department of Clinical Genetics, Cen- ter for Human Genetics, University Hospital Leuven, Leu- REFERENCES ven, Belgium (Dr de Ravel); Telethon Institute of Genet- ics and Medicine (Dr Banfi and Ms Ziviello), and Medical 1. Badano JL, Mitsuma N, Beales PL, Katsanis N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 2006;7:125- Genetics, Department of General Pathology, Second Uni- 148. versity of Naples (Dr Banfi), Naples, Italy; Molecular Ge- 2. Beales PL, Elcioglu N, Woolf AS, Parker D, Flinter FA. New criteria for improved netics Laboratory, University Eye Hospital, Tu¨ bingen, Ger- diagnosis of Bardet-Biedl syndrome: results of a population survey. J Med Genet. many (Drs Kohl and Wissinger); Genetics Department, 1999;36(6):437-446. 3. Deveault C, Billingsley G, Duncan JL, et al. BBS genotype-phenotype assess- Instituto de Investigacioń Sanitaria (IIS)–Fundacioń Jime- ment of a multiethnic patient cohort calls for a revision of the disease definition. nez Dıáz, Centro de Investigacioń Biome´dica en Red de Hum Mutat. 2011;32(6):610-619. Enfermedades Raras (CIBERER) Unit, Madrid, Spain 4. Janssen S, Ramaswami G, Davis EE, et al. Mutation analysis in Bardet-Biedl syn- (Drs Ayuso and Avila-Fernandez); Department of Oph- drome by DNA pooling and massively parallel resequencing in 105 individuals. thalmology, Hadassah-Hebrew University Medical Cen- Hum Genet. 2011;129(1):79-90. 5. Billingsley G, Bin J, Fieggen KJ, et al. Mutations in chaperonin-like BBS genes ter, Jerusalem, Israel (Drs Sharon, Banin, and Mizrahi- are a major contributor to disease development in a multiethnic Bardet-Biedl syn- Meissonnier); Department of Ophthalmology, Centre de drome patient population. J Med Genet. 2010;47(7):453-463. Reference Maladies Sensorielles Genetiques, Hopital Gui 6. Moore SJ, Green JS, Fan YL, et al. Clinical and genetic epidemiology of Bardet- de Chauliac, and School of Medicine, Montpellier Uni- Biedl syndrome in Newfoundland: a 22-year prospective, population-based, cohort study. Am J Med Genet A. 2005;132(4):352-360. versity 1, Montpellier, France (Dr Hamel); Unit of Patho- 7. Schaefer E, Durand M, Stoetzel C, et al. Molecular diagnosis reveals genetic hetero- physiology of Vision, Institute for Ophthalmic Re- geneity for the overlapping MKKS and BBS phenotypes. Eur J Med Genet. 2011; search, Centre for Ophthalmology, University Tu¨ bingen, 54(2):157-160. Tu¨ bingen (Drs Sliesoraityte and Zrenner); Department 8. Mykytyn K, Nishimura DY, Searby CC, et al. Evaluation of complex inheritance of Ophthalmology, A. Fiorini Hospital, Polo Pontino, Uni- involving the most common Bardet-Biedl syndrome locus (BBS1). Am J Hum Genet. 2003;72(2):429-437. versity of Rome “La Sapienza,” Rome, Italy (Dr Vin- 9. Smaoui N, Chaabouni M, Sergeev YV, et al. Screening of the eight BBS genes in golo); Unit of Child Neurology and Psychiatry, Istituto Tunisian families: no evidence of triallelism. Invest Ophthalmol Vis Sci. 2006; Ricerca e Cura a Carattere Scientifico, C. Mondino In- 47(8):3487-3495. stitute of Neurology, University di Pavia, Pavia, Italy 10. Hichri H, Stoetzel C, Laurier V, et al. Testing for triallelism: analysis of six BBS genes in a Bardet-Biedl syndrome family cohort. Eur J Hum Genet. 2005;13 (Dr Signorini); and Center of Rare Retinal Disease, Au- (5):607-616. genZentrum Siegburg, Siegburg, Germany (Dr Kell- 11. Katsanis N, Ansley SJ, Badano JL, et al. Triallelic inheritance in Bardet-Biedl syn- ner). drome, a mendelian recessive disorder. Science. 2001;293(5538):2256-2259. Correspondence: B. Jeroen Klevering, MD, PhD, Depart- 12. Zaghloul NA, Katsanis N. Mechanistic insights into Bardet-Biedl syndrome, a model ment of Ophthalmology, Radboud University Nijmegen ciliopathy. J Clin Invest. 2009;119(3):428-437. 13. Badano JL, Kim JC, Hoskins BE, et al. Heterozygous mutations in BBS1, BBS2 Medical Centre, PO Box 9101, 6500 HB, Nijmegen, the and BBS6 have a potential epistatic effect on Bardet-Biedl patients with two mu- Netherlands ([email protected]). tations at a second BBS locus. Hum Mol Genet. 2003;12(14):1651-1659. Author Contributions: Drs Cremers and Klevering con- 14. Badano JL, Leitch CC, Ansley SJ, et al. Dissection of epistasis in oligogenic Bardet- tributed equally to this manuscript. Biedl syndrome. Nature. 2006;439(7074):326-330. 15. Khanna H, Davis EE, Murga-Zamalloa CA, et al. A common allele in RPGRIP1L is Conflict of Interest Disclosures: None reported. a modifier of retinal degeneration in ciliopathies. Nat Genet. 2009;41(6):739- Funding/Support: These studies were supported by the 745. Radboud University Nijmegen Medical Centre (Drs den 16. Beales PL, Warner AM, Hitman GA, Thakker R, Flinter FA. Bardet-Biedl syn-

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1431

©2012 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/25/2021 drome: a molecular and phenotypic study of 18 families. J Med Genet. 1997; 36. Khan MI, Kersten FFM, Azam M, et al. CLRN1 mutations cause nonsyndromic 34(2):92-98. retinitis pigmentosa. Ophthalmology. 2011;118(7):1444-1448. 17. Haim M. Prevalence of retinitis pigmentosa and allied disorders in Denmark, III: 37. Abu Safieh L, Aldahmesh MA, Shamseldin H, et al. Clinical and molecular char- hereditary pattern. Acta Ophthalmol (Copenh). 1992;70(5):615-624. acterisation of Bardet-Biedl syndrome in consanguineous populations: the power 18. Klein D, Ammann F. The syndrome of Laurence-Moon-Bardet-Biedl and allied of homozygosity mapping. J Med Genet. 2010;47(4):236-241. diseases in Switzerland: clinical, genetic and epidemiological studies. J Neurol 38. Aldahmesh MA, Safieh LA, Alkuraya H, et al. Molecular characterization of reti- Sci. 1969;9(3):479-513. nitis pigmentosa in Saudi Arabia. Mol Vis. 2009;15:2464-2469. 19. Farag TI, Teebi AS. Bardet-Biedl and Laurence-Moon syndromes in a mixed Arab 39. den Hollander AI, Koenekoop RKK, Yzer S, et al. Mutations in the CEP290 (NPHP6) population. Clin Genet. 1988;33(2):78-82. gene are a frequent cause of Leber congenital amaurosis. Am J Hum Genet. 2006; 20. Teebi AS. Autosomal recessive disorders among Arabs: an overview from Kuwait. 79(3):556-561. J Med Genet. 1994;31(3):224-233. 40. Pawlik B, Mir A, Iqbal H, et al. A novel familial BBS12 mutation associated with 21. Hjortshøj TD, Grønskov K, Brøndum-Nielsen K, Rosenberg T. A novel founder a mild phenotype: implications for clinical and molecular diagnostic strategies. BBS1 mutation explains a unique high prevalence of Bardet-Biedl syndrome in Mol Syndromol. 2010;1(1):27-34. the Faroe Islands. Br J Ophthalmol. 2009;93(3):409-413. 41. Marmor MF, Fulton AB, Holder GE, Miyake Y, Brigell M, Bach M; International 22. Kim SK, Shindo A, Park TJ, et al. Planar cell polarity acts through septins to con- Society for Clinical Electrophysiology of Vision. ISCEV standard for full-field clini- trol collective cell movement and ciliogenesis. Science. 2010;329(5997):1337- cal electroretinography (2008 update). Doc Ophthalmol. 2009;118(1):69-77. 1340. 42. Aldred MA, Teague PW, Jay M, et al. Retinitis pigmentosa families showing ap- 23. Nachury MV, Loktev AV, Zhang Q, et al. A core complex of BBS proteins coop- parent X linked inheritance but unlinked to the RP2 or RP3 loci. J Med Genet. erates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 2007; 1994;31(11):848-852. 129(6):1201-1213. 43. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist 24. Stoetzel C, Muller J, Laurier V, et al. Identification of a novel BBS gene (BBS12) programmers. Methods Mol Biol. 2000;132:365-386. highlights the major role of a vertebrate-specific branch of chaperonin-related 44. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting proteins in Bardet-Biedl syndrome. Am J Hum Genet. 2007;80(1):1-11. damaging missense mutations. Nat Methods. 2010;7(4):248-249. 25. Beales PL, Badano JL, Ross AJ, et al. Genetic interaction of BBS1 mutations with 45. Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res. alleles at other BBS loci can result in non-mendelian Bardet-Biedl syndrome. Am 2001;11(5):863-874. J Hum Genet. 2003;72(5):1187-1199. 46. Cannon PS, Clayton-Smith J, Beales PL, Lloyd IC. Bardet-Biedl syndrome: an 26. Stoetzel C, Laurier V, Davis EE, et al. BBS10 encodes a vertebrate-specific chap- atypical phenotype in brothers with a proven BBS1 mutation. Ophthalmic Genet. eronin-like protein and is a major BBS locus. Nat Genet. 2006;38(5):521- 2008;29(3):128-132. 524. 47. Pretorius PR, Aldahmesh MA, Alkuraya FS, Sheffield VC, Slusarski DC. Func- 27. Mykytyn K, Nishimura DY, Searby CC, et al. Identification of the gene (BBS1) tional analysis of BBS3 A89V that results in non-syndromic retinal degeneration. most commonly involved in Bardet-Biedl syndrome, a complex human obesity Hum Mol Genet. 2011;20(8):1625-1632. syndrome. Nat Genet. 2002;31(4):435-438. 48. Riazuddin SA, Iqbal M, Wang Y, et al. A splice-site mutation in a retina-specific 28. Haim M. The epidemiology of retinitis pigmentosa in Denmark. Acta Ophthalmol exon of BBS8 causes nonsyndromic retinitis pigmentosa. Am J Hum Genet. 2010; Scand. 2002;80(suppl 233):1-34. doi:10.1046/j.1395-3907.2002.00001.x. 86(5):805-812. 29. Daiger SP, Bowne SJ, Sullivan LS. Perspective on genes and mutations causing 49. Bruford EA, Riise R, Teague PW, et al. Linkage mapping in 29 Bardet-Biedl syn- retinitis pigmentosa. Arch Ophthalmol. 2007;125(2):151-158. drome families confirms loci in chromosomal regions 11q13, 15q22.3-q23, and 30. Berger W, Kloeckener-Gruissem B, Neidhardt J. The molecular basis of human 16q21. Genomics. 1997;41(1):93-99. retinal and vitreoretinal diseases. Prog Retin Eye Res. 2010;29(5):335-375. 50. Carmi R, Elbedour K, Stone EM, Sheffield VC. Phenotypic differences among pa- 31. Liu Q, Tan G, Levenkova N, et al. The proteome of the mouse photoreceptor sen- tients with Bardet-Biedl syndrome linked to three different chromosome loci. Am sory cilium complex. Mol Cell Proteomics. 2007;6(8):1299-1317. J Med Genet. 1995;59(2):199-203. 32. Collin RWJ, Littink KW, Klevering BJ, et al. Identification ofa2Mbhuman or- 51. Riise R, Andre´asson S, Borgastro¨m MK, et al. Intrafamilial variation of the phe- tholog of Drosophila eyes shut/spacemaker that is mutated in patients with reti- notype in Bardet-Biedl syndrome. Br J Ophthalmol. 1997;81(5):378-385. nitis pigmentosa. Am J Hum Genet. 2008;83(5):594-603. 52. Azari AA, Aleman TS, Cideciyan AV, et al. Retinal disease expression in Bardet- 33. Collin RWJ, van den Born LI, Klevering BJ, et al. High-resolution homozygosity Biedl syndrome-1 (BBS1) is a spectrum from maculopathy to retina-wide mapping is a powerful tool to detect novel mutations causative of autosomal re- degeneration. Invest Ophthalmol Vis Sci. 2006;47(11):5004-5010. cessive RP in the Dutch population. Invest Ophthalmol Vis Sci. 2011;52(5): 53. Pruett RC. Retinitis pigmentosa: clinical observations and correlations. Trans Am 2227-2239. Ophthalmol Soc. 1983;81:693-735. 34. Bandah-Rozenfeld D, Collin RWJ, Banin E, et al. Mutations in IMPG2, encoding 54. Hamel C. Retinitis pigmentosa. Orphanet J Rare Dis. 2006;1:40. interphotoreceptor matrix proteoglycan 2, cause autosomal-recessive retinitis doi:10.1186/1750-1172-1-40. pigmentosa. Am J Hum Genet. 2010;87(2):199-208. 55. Lee SH, Yu HG, Seo JM, et al. Hereditary and clinical features of retinitis pig- 35. Estrada-Cuzcano A, Koenekoop RK, Coppieters F, et al. IQCB1 mutations in pa- mentosa in Koreans. J Korean Med Sci. 2010;25(6):918-923. tients with Leber congenital amaurosis. Invest Ophthalmol Vis Sci. 2011;52 56. Pietila¨inen KH, Kaprio J, Borg P, et al. Physical inactivity and obesity: a vicious (2):834-839. circle. Obesity (Silver Spring). 2008;16(2):409-414.

ARCH OPHTHALMOL / VOL 130 (NO. 11), NOV 2012 WWW.ARCHOPHTHALMOL.COM 1432