OPHTHALMIC MOLECULAR GENETICS Genomewide Homozygosity Mapping and Molecular Analysis of a Candidate Gene Located on 22q13 (-1) in a Previously Undescribed Vitreoretinal Dystrophy

Maike Weigell-Weber, MD; Gian-Marco Sarra, MD; Dieter Kotzot, MD; Lodewijk Sandkuijl, PhD†; Elmar Messmer, MD; Martin Hergersberg, PhD

Objectives: To localize the gene that causes an autoso- meric) to D22S1170 (telomeric). Lod scores were be- mal recessively inherited vitreoretinal dystrophy that has tween 0.017 and 2.36 (theta=0). A mutation analysis of not been described, to our knowledge, and to analyze a the complete coding region of FBLN1, which encodes in- candidate gene mapped to 22q13 (fibulin-1 [FBLN1]). teracting proteins, revealed 4 pre- viously undescribed single nucleotide polymorphisms. Methods: Homozygosity mapping with 500 microsat- ellite markers spread over the whole genome (mean dis- Conclusions: A genomewide homozygosity mapping tance, 7.2 centimorgans [cM]) and mutation analysis of analysis supported the hypothesis that the gene respon- the complete coding region of FBLN1. sible for a unique vitreoretinal dystrophy is located on 22q13. No obviously pathogenic muta- Results: Homozygosity for all analyzed markers was tion was found in the candidate gene, FBLN1. found in the 4 affected siblings in a region on chromo- some 22 encompassing 12 cM from D22S444 (centro- Arch Ophthalmol. 2003;121:1184-1188

EREDITARY vitreoretinal COL11A2 on 6p21.3], Wagner syndrome on dystrophies are poten- 5q13-14, Knobloch syndrome on 21q22.3, tially blinding disorders Goldmann-Favre [enhanced S-cone] syn- characterized by an abnor- drome on 15q23, and multiple vitreoreti- mal vitreous gel, associ- nopathies on 11q13) were excluded by link- atedH retinal changes, and a heterogeneous age analysis. From the Institute of Medical origin. The phenotype is extremely vari- Genetics, University of Zurich able in both syndromic and nonsyndromic See also page 1109 (Drs Weigell-Weber and types. Some of them are associated with axial Hergersberg); Department of myopia of different extents and retinal de- Here, we report on a genomewide ho- Ophthalmology, University tachment. Disease-causing mutations have mozygosity mapping approach that found Hospital Zurich, been identified in various genes that en- a 12-centimorgan (cM) homozygous re- Frauenklinikstr (Drs Sarra and code components of the extracellular ma- gion on chromosome 22q13 containing, Messmer), Zurich, Switzerland; trix (ECM).1 Syndromic types of vitreoreti- among other genes, fibulin-1 (FBLN1). the Institute of Medical Biology nal dystrophies are Knobloch syndrome This attractive candidate gene was fur- and Human Genetics, (Online Mendelian Inheritance in Man University of Innsbruck, ther analyzed for mutations. Innsbruck, Austria (Dr Kotzot); [OMIM] *120328) and the rather heterog- Department of Medical eneous Stickler syndrome (OMIM #108300, METHODS Statistics, Leiden, the OMIM #604841, and OMIM #184840). Netherlands (Dr Sandkuijl). Nonsyndromic types are the Wagner syn- SUBJECTS Dr Sarra is currently affiliated drome (OMIM *143200), Goldmann- with the Department of Favre dystrophy (OMIM #268100), and the The family originated from eastern Switzer- Ophthalmology, Inselspital, allelic enhanced S cone syndrome. In ad- land. Informed consent and medical history Berne, Switzerland; dition, similar ophthalmologic findings were obtained from 3 healthy and 4 affected Dr Messmer, with Stadtspital have been described in families with X- siblings aged 68 to 77 years at the time of in- Triemli, Zurich; and chromosomal, autosomal dominant, and au- vestigation. The eighth sibling as well as both Dr Hergersberg, with Zentrum parents were not affected and died prior to this tosomal recessive inheritance. As reported fu¨r Labormedizin, Aarau, 2 investigation. The clinical data have been re- Switzerland. The authors have by Sarra et al, the chromosomal loci of some ported in detail.2 Briefly, in the 4 affected sib- no relevant financial interest of these disorders (high myopia on 18p11, lings, the onset of visual disturbance started in in this article. 12q21-23, and 7q36, Stickler syndrome early childhood, followed by progressive night †Deceased. [COL1A1 on 12q13, COL11A1 on 1q21, blindness and visual field impairment during

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 the third to fifth decade of life. Clinical evaluation revealed a peculiar vitreoretinal degeneration characterized by vitreous liq- uefaction and strand formation along with diffuse retinal pig- I-1 I-2 ment epithelium atrophy and pigment clumping of the retina, and high myopia with deep posterior staphyloma. The mean axial length of the eyes was 31.4 mm (range, 28.49-33.41 mm). II-1 II-2 II-3 II-4 II-5 II-6 II-7 II-8 D22S928 2 3 1 31212 12 12 1 3 The depth of the posterior staphylomata was 0.8 mm to 2.7 mm D22S444 2 2 1 21111 11 11 1 2 (mean, 1.75 mm). Visual acuity ranged from no light percep- D22S1153 2 1 2 12222 22 22 2 1 tion to 20/70. Kinetic perimetry revealed severe constrictions D22S532 2 1 1 11111 11 11 1 1 of the isopters, with remaining small central islands. Electro- D22S1141 1 1 1 11111 11 11 1 1 physiologic investigations showed severe retinal damage, sug- D22S1160 21 212222 22 22 21 gestive of a superimposed widespread retinal dystrophy. D22S1149 31 212222 22 22 21 A genealogic analysis of 5 generations revealed no evi- D22S1170 11 212222 22 22 21 dence for parental consanguinity (Manuel Aicher, Genealo- D22S1161 1 2 2 22121 21 21 2 2 gische Forschung, Dietikon, Switzerland). However, the ori- gin of most of the ancestors from a relatively isolated population Figure 1. Pedigree of the family, with solid symbols representing affected individuals. Haplotypes for microsatellite polymorphisms most closely linked and several consanguinities in the different parental pedigrees to fibulin-1 (FBLN1) are depicted below each symbol. The markers showing were indicated. homozygosity in the affected siblings are boxed.

GENOTYPING Blood samples were obtained from all 7 living siblings after writ- the Applied Biosystems Dye terminator sequencing kit (Foster ten informed consent was obtained. DNA was extracted using stan- City, Calif), followed by sequence analysis on an ABI 3700 cap- dard salting out procedures. For homozygosity mapping, the DNA- illary sequencer (Microsynth, Balgach, Switzerland). RNA was pooling method was applied (affected vs unaffected).3 The mean isolated from EDTA anticoagulated blood using a QIAamp RNA distance between the 500 highly polymorphic markers used was Blood Mini Kit (Qiagen). Reverse transcriptase (RT)-PCR analy- 7.2 cM. Microsatellites chosen for maximum informativeness were sis was performed with the Titan RT-PCR kit (Roche Diagnos- obtained from the Genethon human genetic linkage map4 and from tics, Rotkreuz, Switzerland), using the experimental condi- the Marshfield map.5 Polymerase chain reaction (PCR) primers tions provided by the supplier. The primers used for RT-PCR were from Research Genetics (Huntsville, Ala) or were synthe- were A4F, TCTGCCACTGACACCTCAAG, and A4R, sized with the available sequence information. Polymerase chain GGATTTTGGAGGCAACACAT (nucleotides 2141-2160 and 2321-2344, respectively, of the complementary DNA se- reaction was performed using standard conditions. Products were 9 electrophoretically separated on 6% polyacrylamide sequencing quence of variant A of FBLN1 ). The RT reaction was per- gels and visualized by silver staining.6 Linkage was calculated us- formed at 60°C for 30 minutes, followed by 15 minutes at 95°C. ing the LINKAGE (version 5.1) program package (Laboratory of The denaturation, annealing, and extension temperatures were Statistical Genetics at Rockefeller University, New York, NY), as- 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 sec- suming autosomal recessive inheritance, 100% penetrance in both onds, respectively, for 40 cycles. sexes, and a gene frequency of 0.001. The same assumptions were used for the lod score calculation for the homozygous region with HAPLOTYPE ANALYSIS OF FBLN1 the MAPMAKER/HOMOZ algorithm.7 For this calculation, each marker allele that showed homozygosity in the 4 patients was as- Assays were developed for the 4 single nucleotide polymor- sumed to be the most frequent allele of the corresponding marker. phisms (SNPs) identified during mutation analysis of FBLN1. The allele frequencies of the different SNPs and the haplo- MUTATION ANALYSIS OF FBLN1 types arising from the different allele combinations were ana- lyzed in the DNA of 30 families (parents and at least 1 child) The PCR primers, annealing temperatures, and PCR product not affected by vitreoretinal dystrophy or severe myopia. These sizes for the amplification of all 20 exons of FBLN1, including families had given written informed consent that their anony- the exon-intron boundaries, were derived from the gene struc- mous DNA samples could be used for research. ture as described by Pan et al,8 and are available on request. Heteroduplex analysis of the PCR products was performed by denaturing high-performance liquid chromatography on a Wave RESULTS system (Transgenomic Inc, Omaha, Neb). The column used was a DNAsep Cartridge (Transgenomic Inc). After heteroduplex HOMOZYGOSITY MAPPING formation, DNA fragments were run, with column tempera- tures between 50°C to 68°C. The column temperature was A homozygous region on chromosome 22q13 was iden- changed in incremental steps of 2°C. An elution time of 14 min- tified, which extends from the proximal marker, D22S444, utes was used: the linear acetonitrile–triethylammonium acetate– to the distal marker, D22S1170 (Figure 1). The calcu- buffer gradient ratio went from 43%/57% to 68%/32% during lated lod score for the polymorphic markers that are ho- 10 minutes, with a buffer flow of 0.9 mL/minute, followed by mozygous in this region was between 0.017 and 2.18 (theta a 100% acetonitrile wash for 30 seconds, and in an equilibra- =0). This region contains FBLN1 (base pairs 29/341/ tion step, was reduced to 43%/57% for the last 4 minutes. For 390-29/439/641 on ), which was ana- sequence analysis, PCR products were purified with Quia- lyzed further. The highest possible lod score of 2.18 was quick spin columns (Quiagen, Hilden, Germany). Sequencing found for the microsatellite marker D22S444, which is was done on an ALF Express sequencing automate (Amer- sham Pharmacia Biotech, Uppsala, Sweden), using the respec- located in the largest intron (intron 14) of FBLN1. This tive Cy5-labelled PCR primers as sequencing primers with the lod score was also found for the markers D22S1170 and thermofluorescent-labeled primer cycle sequencing kit (Am- D22S928. The latter polymorphism is located outside the ersham Pharmacia Biotech). Alternatively, the PCR primers were homozygous region and is heterozygous in the 4 af- used for sequencing of the PCR amplification products with fected persons. This is additional evidence that the 4 pa-

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 1. Single-Nucleotide Polymorphisms Identified in the FBLN1 Candidate Gene and Techniques for Their Detection*

Base change 543 + 17CϾG 963CϾT, P318I 2147CϾT 2228CϾG Location Intron 5 Exon 9 Exon 17 Exon 17 Detection technique AcyI Allele-specific amplification HinfI TseI Allele frequency 0.34/0.67 0.58/0.42 0.39/0.61 0.33/0.67

Abbreviation: FBLN1, fibulin-1. *The polymorphisms in exons/introns 5 and 17 were detected by digest of the exon-specific polymerase chain reaction (PCR) products with the indicated restriction enzymes. The PCR primers for the allele-specific amplification of the polymorphism in exon 9 are available on request.

product from the total RNA extracted from the blood of 3 123 4 5 6 7 affected brothers and sisters and from the total RNA ex- tracted from the blood of 3 unaffected sisters (Figure 2).

AN FBLN1 HAPLOTYPE SEGREGATING WITH THE DISEASE PHENOTYPE

The SNPs identified in FBLN1 were used to determine II-5 II-3 II-6 II-2 II-7 II-1 the frequency of the different haplotypes of this gene in the DNA of 30 unrelated families (both parents and at Affected Not Affected least 1 child). Genotyping of these families allowed the Figure 2. Results of reverse-transcriptase polymerase chain reaction identification of 10 of the 16 possible haplotypes in these (RT-PCR) analysis of the FBLN1 (fibulin-1) transcript. In lane 1, there is a families (Table 2). The homozygous haplotype 16 iden- 100–base-pair ladder, and in lane 2 to lane 7, the products of the RT-PCR tified in the 4 patients was also found in 1 control sub- analysis performed with total RNA isolated from whole blood of 3 affected (II-3, II-5, II-6) and 3 unaffected (II-1, II-2, II-7) siblings were loaded. No ject with normal vision. It is therefore unlikely that the difference was detectable. observed haplotype per se is responsible for the de- scribed phenotype of the eye.

tients inherited the same ancestral chromosomal region COMMENT from both parents. A statistical calculation using the MAPMAKER/HOMOZ algorithm yielded a lod score of We report on a genomewide homozygosity mapping ap- 2.36 under the assumption that there was a hidden con- proach and analysis of a candidate gene located on 22q13 sanguinity in the family at least 6 generations ago, and (FBLN1) in a nonsyndromic and previously unde- that the markers are homozygous for the most frequent scribed vitreoretinal dystrophy. This vitreoretinal dys- allele of the particular marker. In addition to the 4 af- trophy is characterized by excessive early-onset axial myo- fected patients, 2 unaffected sisters were included in this pia with deep and sharply demarcated posterior calculation. staphylomata, pronounced vitreous liquefaction, reti- nal degeneration with diffuse retinal pigment epithe- MUTATION ANALYSIS OF FBLN1 lium atrophy, and premature cataract formation. As de- scribed in Sarra et al,2 the clinical picture and the striking Denaturing high-performance liquid chromatography analy- electrophysiologic and visual field defects suggested the sis of the PCR products of the 20 exons of FBLN1 was fol- presence of a primary vitreoretinal dystrophy rather than lowed by sequence analysis. No pathogenetic mutation was just late sequelae of a pathologic myopia. detected, but 4 SNPs were identified (Table 1). One of The rarity of the disease phenotype in siblings, and the SNPs (963CϾT) codes for a proline-leucine amino acid the origin of their parents from a relatively isolated popu- exchange at amino acid position 318 in exon 9. The 3 other lation in neighboring villages, led us to the hypothesis of SNPs were located in intron 5 (543+17CϾG) and in the a hidden consanguinity. By a genomewide homozygosity 3Ј untranslated region of exon 17 (2147CϾT and mapping approach with more than 500 polymorphic mark- 2218CϾG). The SNPs were designated following the rec- ers, homozygosity for a 12-cM region located on 22q13 ommendations of Antonarakis et al,10 beginning with the was found. The maximal lod score of 2.18 was identified initiation codon of the FBLN1 transcript. The 4 affected sib- for the markers D22S1170 and D22S444. Both markers lings were homozygous for 1 allele of all SNPs. To deter- were either heterozygous or homozygous for other alle- mine the pathogenic significance of the identified base les in the unaffected siblings (Figure 1). The affected per- changes, their incidence in a control population was de- sons were heterozygous for the marker D22S928, which termined. Both alleles of all 4 SNPs were found in the con- was adjacent to the homozygous region and which also trol population in significant numbers and also in the ho- had a lod score of 2.18 due to an identical genotype in the mozygous state, thereby excluding the possibility that 1 of 4 affected siblings and different genotypes in the 3 non- the 4 SNPs is a mutation causing the observed phenotype. affected siblings. Recently, evidence has been published Reverse-transcriptase–PCR analysis with a primer pair spe- that homozygous or autozygous genomic regions can oc- cific for the 3Ј untranslated sequence of variant A of the cur with a higher then expected percentage, either through FBLN1 gene resulted in roughly the same amount of PCR underestimation of the extent of inbreeding or by

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 Table 2. Sixteen Possible Haplotypes of 4 Different Intragenic SNPs in FBLN1 and Frequency of Their Occurrence in 30 Families (Parents and 1 Child)*

Haplotype 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Intron 5 CϾG CCCCCCCCGGGGGGGG Exon 9 CϾT CCCCTTTTCCCCTTTT Exon 17 CϾT CCTTCCTTCCTTCCTT Exon 17 CϾG CGCGCGCGCGCGCGCG No. of haplotypes 5 . . . 1 8 1 ...... 5 7 . . . 2 15 1 ...... 11

Abbreviations: SNP, single-nucleotide polymorphism; FBLN1, fibulin-1; and ellipses, not found. *The identified number of haplotypes is determined by the number of informative families. Haplotype 16 was identified in the affected probands.

chance.11,12 On one hand, this allows the interpretation that C>G C>T C>T, C>G homozygosity is possible in a family despite lack of evi- dence for parental consanguinity due to a relatively small and inbreeding population. The genealogic analysis dis- ACB covered 3 consanguinities between ancestors of the pro- 283 5 9 12 1415 16 17 18 19 20 band’s father. On the other hand, it has to be mentioned that the observed association between the phenotype and D Variant the homozygous region can result from chance only. How- ever, no other polymorphic marker used in the genome Figure 3. Scheme of the fibulin-1 gene (FBLN1) with all exons according to scan showed the same allele distribution pattern, ie, ho- the description of Pan et al8 and the 4 protein variants FBLN1 A-D due to mozygosity in the 4 affected siblings and different geno- different splicing sites. types in the 3 unaffected siblings. It is therefore unlikely that the occurrence and distribution of these homozy- identified in the gene that encodes the epidermal growth gous segments is a chance event resulting from a gener- factor–containing fibulin-like extracellular matrix pro- ally low genetic heterogeneity in this family. If we as- tein 1 (EFEMP1), formerly designated Sl-5, or FBLN3.14 sume that parental consanguinity exists, their closest (2) The FBLN1 protein interacts with a number of ECM connection must be at least 6 generations ago (a result of proteins, including , nidogen, aggrecan, and the genealogic investigation). A statistical analysis with .1 In particular, FBLN1 interacts with ␣-1(XVIII) MAPMAKER/HOMOZ, assuming the existence of such a collagen, which was mutated in several families with Knob- distant connection, yielded an overall lod score of 2.36 for loch syndrome.15,16 (3) The 2 ECM proteins, aggrecan and linkage to this same region. This lod score represents a very versican, to which FBLN1 binds, are expressed in the sclera conservative estimate of the evidence for linkage because with dynamic expression patterns during growth and ag- a conservative estimate for the frequency of the marker ing.1,17 Because the versican gene is a candidate gene for alleles for which the patients are homozygous was used: Wagner disease, it is tempting to speculate that the simi- for each marker, the frequency of the most frequent allele lar phenotype of the affected family members described was specified. here with Wagner syndrome on one side and Knobloch The homozygous region on chromosome 22 in the syndrome on the other side is the consequence of a func- 4 affected persons contains at least 11 genes predicted tional connection between the protein products of the by computer searches, and 16 genes identified both by genes that mutated in these diseases.18 (4) A further mem- bioinformatic analysis and by transcript analysis. From ber of the fibulin gene family, FBLN4 (also designated these 27 genes, FBLN1 is the most interesting candidate EFEMP2), was recently identified in the multiple reti- gene. FBLN1 encodes a calcium-binding ECM protein and nopathy critical region on 11q13 and considered as a is characterized by a complex splicing pattern, resulting candidate gene for the different vitreoretinopathies in 4 protein variants, FBLN1 A-D, which differ in their mapped to this region. Other members of the fibulin gene carboxy termini (Figure 3).1 These variants differ in func- family in addition to FBLN1 are FBLN2 on 3p25, and tion and tissue expression.1,8,13 FBLN1 expression has been EVEC/DANCE/FBLN5 on 14q32.1.19 detected in a wide range of tissues in a complex temporal- Apart from 4 SNPs, no mutation was identified in spatial pattern, including the fetal eye and adult retinal FBLN1 in the present study. The distribution of the SNP pigment epithelium sheets. It can therefore be con- haplotypes was determined in 30 families, resulting in the cluded that FBLN1 is expressed in the tissues of the fam- identification of the same haplotype in a homozygous state ily described here (Unigene database). Recently, disrup- in one other person without an obvious ophthalmologic tion of FBLN1 was reported in a family with a t(12;22) phenotype. Therefore, yet undiscovered mutations in associated with a complex type of synpolydactyly.13 This FBLN1 or in other genes in the homozygous region may translocation specifically interferes with the synthesis of play a role in susceptibility for vitreoretinal dystrophy with variant FBLN1-D. No ocular symptoms were reported. high myopia. It is also possible that homozygosity of an FBLN1 is a particularly interesting candidate gene FBLN1 allele with tissue-specific splicing activity results for the following reasons: (1) in the allelic retinopa- in an abnormal tissue-specific protein. This phenom- thies, Malattia leventinese and Doyne honeycomb reti- enon was found in the RPGR (retinitis pigmentosa gua- nal dystrophy (OMIM #126600), a mutation has been nosin triphosphate hydrolase [GTPase] regulator) gene,

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/28/2021 which is often mutated at a retina-specific exon in RP3. Schweizerischer Fonds zur Verhu¨tung und Beka¨mpfung der Mutations were therefore not detectable in DNA and RNA Blindheit, Zurich, and the Schweizerischen Nationalfonds from other tissues.20 This hypothesis is also in agreement (No. 31-59267.99), Berne, Switzerland. with the finding that the homozygous knockout null mu- We thank Prof Andreas Huber, MD, for support, and Ms tation of FBLN1 was found to be perinatally lethal in the I. Moro for technical help. We thank the family members for -/- mouse, and that a t(12;22) disrupting the variant their participation and continued interest in this study. FBLN1-D is associated with a complex type of synpoly- Corresponding author and reprints: Martin Hergers- dactyly.13,21 This indicates that the described ophthalmo- berg, PhD, Zentrum fu¨r Labormedizin, Kantonsspital Aarau, logic disease cannot result from a null mutation. Planned CH-5001 Aarau, Switzerland (e-mail: martin.hergersberg experiments will aim at the quantification of the tran- @ksa.ch). script levels of FBLN1 in patients and probands to test the idea that a subtle difference in the transcription rate of the REFERENCES allele causes the ophthalmologic phenotype. Sertie et al16 discussed the idea that yet to be identi- 1. Aspberg A, Adam S, Kostka G, Timpl R, Heinegard D. Fibulin-1 is a ligand for the fied alleles of COL18A1 encoding the ␣1(XVIII) collagen C-type lectin domains of aggrecan and versican. J Biol Chem. 1999;274:20444- 20449. could be susceptibility alleles for high myopia. In light of 2. 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