A Novel Locus for Congenital Simple Microphthalmia Family Mapping to 17P12-Q12

Genetics A Novel Locus for Congenital Simple Microphthalmia Family Mapping to 17p12-q12

Zhengmao Hu,1,2,3 Changhong Yu,3,4,5 Jingzhi Li,1 Yiqiang Wang,4 Deyuan Liu,1 Xinying Xiang,1,2 Wei Su,1 Qian Pan,1 Lixin Xie,*,4 and Kun Xia*,1,2

PURPOSE. To investigate the etiology in a family with autosomal- opia (ϩ7.00 to ϩ13.00 D), a high lens-to-eye volume ratio, and dominant congenital simple microphthalmia of Chinese origin. a high incidence of angle-closure glaucoma after middle age. ETHODS Some normal adnexal elements and eyelids are usually pres- M . A whole-genome scan was performed by using 382 1 microsatellite DNA markers after the exclusion of reported ent. It is also a common symptom in some other ocular candidates linked to microphthalmia. Additional fluorescent abnormalities. Approximately 80% of microphthalmia cases markers were genotyped for fine mapping. To find out the occur as part of syndromes that include other systemic malformations, especially cardiac defects, facial clefts, microcephaly, novel predisposing gene, 14 candidate genes including 2,3 CRYBA1 and NCOR1 were selected to screen for the mutation and hydrocephaly. The reported prevalence of anophthalmia or microphthalmia at birth is 0.66 of 10,000 around the by the PCR direct-sequencing method. Genome-wide single- 4 nucleotide polymorphism (SNP) genotyping was performed to world and 0.3 of 10,000 in China. find out the pathogenetic copy number variation, as well. Epidemiologic studies have indicated that both heritable and environmental factors cause microphthalmia. Although the RESULTS. The most statistically significant linkage results were precise pathogenesis of microphthalmia is still unknown, stud- obtained at D17S1824 (maximum LOD score, 4.97, at recom- ies have demonstrated that it is a genetically heterogeneous bination fraction 0.00). Haplotype analyses supported the lo- disorder. Chromosomal abnormalities may result in syndromic cation of the disease-causing gene to a 21.57-cM interval be- microphthalmia. Studies of different microphthalmia cases and tween loci D17S900 and D17S1872 of chromosome 17, region pedigrees have linked it to different chromosomal regions and p12-q12. However, no mutation or CNV (copy number varia- monogenic causes. Autosomal-dominant microphthalmia ped- tion) was identified to be responsible for the microphthalmia igrees have been mapped to 2q11–14,5 3q26.3-q27 (SOX2),6 phenotype of this pedigree. 11p,1 11p13 (PAX6), 14q22 (OTX2),7 15q12-q15,8 and CONCLUSIONS. A novel suggestive linkage locus for congenital 22q11.2-q13.1 (CRYBA4).9 In some families, autosomal reces- microphthalmia was detected in a Chinese family. This link- sive microphthalmia has been linked to 2q37.1,10 11q23 age region provides a target for susceptibility gene (MFRP),11 14q24.3 (CHX10),12 14q32,13 and 18q21.3 (RAX).14 identification. (Invest Ophthalmol Vis Sci. 2011;52: X-linked anophthalmia pedigrees have been linked to Xp11.4 3425–3429) DOI:10.1167/iovs.10-6747 (BCOR)15 and Xq27-q28 (ANOP1).16 Here, we report a linkage and haplotype analysis that indi- icrophthalmia (OMIM 309700) is an ocular developmen- cates a novel locus responsible for microphthalmia. The pa- Mtal malformation characterized by unusually small eyes tients involved in this study were from a congenital simple (Online Mendelian Inheritance in Man; http://www.ncbi.nlm. microphthalmia family of Chinese origin in Shandong Prov- nih.gov/Omim/ National Center for Biotechnology Information ince. The karyotype in the affected family members was nor- [NCBI], Bethesda, MD). The major clinical characteristics in- mal. Several candidate loci and genes have been excluded in clude a short axial length (Ͻ20 mm), a high degree of hyper- our previous study.17 To identify the gene responsible for this family, we performed a whole-genome scan analysis and gene screening. Although no novel pathogenic mutation was found, From the 1The State Key Laboratory of Medical Genetics, Central these results may provide more data for further research into South University, Changsha, Hunan, China; 2The School of Biological the disease. Science and Technology, Central South University, Changsha, Hunan, China; the 4State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, China; and the 5College of Medicine, Qingdao University, Qingdao, SUBJECTS AND METHODS China. 3These authors contributed equally to the work presented here Subjects and should therefore be regarded as equivalent authors. Supported by National Natural Science Foundation of China A five-generation Chinese family from Shandong Province in China that Grants 30630062, 81070081, and 81070759. had members with diagnosed microphthalmia was involved in the Submitted for publication October 19, 2010; revised December study (Fig. 1). Thirty-four family members underwent general physical 22, 2010; accepted January 17, 2011. and complete ophthalmic examinations. All family members did not Disclosure: Z. Hu, None; C. Yu, None; J. Li, None; Y. Wang, have any other physical anomalies. Nine microphthalmia patients ex- None; D. Liu, None; X. Xiang, None; W. Su, None; Q. Pan, None; L. pressed the same full phenotype as previously reported.1,18 They were Xie, None; K. Xia, None affected by isolated microphthalmia in an autosomal dominant trans- *Each of the following is a corresponding author: Lixin Xie, Shan- mission manner in both eyes with onset since birth. Detailed informa- dong Eye Institute, 5 Yanerdao Road, Qingdao, 266071, China; 17 [email protected]. tion is available in another publication. Kun Xia, The State Key Laboratory of Medical Genetics, Central South Peripheral blood samples from 28 individuals including 9 affected University, 110 Xiangya Road, Changsha, Hunan, China; family members (containing all patients) and 19 unaffected members [email protected]. were collected for further analysis. All participants gave written in-

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FIGURE 1. Pedigree of the family studied and haplotypes obtained with 10 microsatellite DNA markers on chromosome 17. Solid symbols: affected individuals; open symbols: unaffected individuals. The sequence of markers is from centromere to telomere. The haplotype cosegregating with the disorder is boxed. Ques- tion mark: genotype not deter- mined.

formed consent in accordance with the Declaration of Helsinki before Multipoint analysis was computed (Genehunter-Modscore, ver. 3.0; they were enrolled in the study. http://linkage.rockefeller.edu/soft/gh/ Rockefeller University, New York, NY). Marker order and map distances were obtained from the Genotyping Marshfield genetic map. A 3-mL peripheral blood sample was taken from each individual after Haplotype Reconstruction informed consent was obtained. Genomic DNA was extracted by using The haplotype was constructed, using a commercial program (Cyrillic the standard phenol-chloroform method. Software, Lake Orion, MI) to define the borders of the cosegregating The whole-genome scan was performed by using 382 fluorescent region and then modifying it by hand. microsatellite markers in the 22 pairs of autosomes, with an average spacing of 10-cM scattering on the human genome (Prism Linkage Mutation Analysis Mapping Set Version 2.0; Applied Biosystems, Inc. [ABI], Foster City, CA). PCR was performed in a 5-␮L volume with 50 ng genomic DNA as After the whole-genome scan, a candidate approach was used to search a template, 0.5 ␮L PCR 10ϫ buffer, 0.1 ␮L dNTP mix (2.5 mM), 0.06 for possible candidate genes. The exons of candidate genes were ␮ ␮ L primers, 0.6 L MgCl2 (15 mM), 0.05 U Taq polymerase (AmpliTaq amplified by PCR, and the primers were designed on computer (Pre- Gold; ABI), and distilled water up to 5 ␮L. Thermal cycling was mier 5.0; Premier Biosoft, Palo Alto, CA). The PCR reaction included 1 performed (GeneAmp 2720; ABI) at 95°C for 12 minutes, then 15 ␮L (50 ng) genomic DNA, 1 ␮L (30 ng) each of the primers, 1 ␮L PCR ϫ cycles of 94°C for 30 seconds, 63°C for 1 minute, degrading 0.5°C per 10 buffer with MgCl2 (Roche Diagnostics, USA, Indianapolis, IN), cycle, and 72°C for 1 minute 50 seconds, followed by 24 cycles of 94°C 0.05 ␮L (5U) Taq polymerase (AmpliTaq Gold; ABI), and 5.85 ␮L for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute 50 seconds, distilled water. Then, PCR products were purified with shrimp alkaline with a final extension of 72°C for 15 minutes. PCR products were phosphatase (Fermentas International, Glen Burnie, MD) and exonu- analyzed on an automated sequencer (model 3100; ABI). A GS400 size clease I (Fermentas International) for 85 minutes at 37°C to remove the standard was used as the internal standard and run in the same lane phosphoryl groups. The samples were then sequenced on an auto- with the markers. Alleles were then analyzed (GeneScan, ver. 3.0 and mated sequencer (model 3100; ABI) in both directions. GenoTyper ver. 3.7; ABI). In the fine mapping phase, additional To explore the possible pathogenetic role of copy number vari- fluorescent markers (D17S799, D17S900, D17S839, D17S261, ation (CNV), we performed genome-wide SNP genotyping D17S1843, D17S740, D17S953, D17S2196, D17S1288, D17S793, (Human660W-Quad BeadChip; Illumina, San Diego, CA). Affected D17S1871, D17S783, D17S1824, D17S1880, D17S1293, D17S1872, individuals III-8 and V-3 were genotyped according to the manufactur- D17S933, D17S92, and D17S1788) were selected from the Marshfield er’s guidelines. To call CNVs, we used the PennCNV algorithm (www. database (http://research.marshfieldclinic.org/ Marshfield Clinic, openbioinformatics.org, an unaffiliated repository of software), which Marshfield, WI). combines multiple sources of information, including log R ratio (LRR) and B allele frequency (BAF) at each SNP marker, along with SNP Linkage Analysis spacing and population frequency of the B allele to generate CNV calls. Two-point LOD scores were calculated by the MLINK program of the LINKAGE package (ver. 5.1; http://linkage.rockefeller.edu/soft/). The RESULTS allele frequencies of markers were assumed to be equal, as were the Linkage Analysis recombination frequencies in males and females. The disease was specified to be an autosomal dominant inheritance with 95% pen- A whole-genome scan study was performed, with markers etrance. We assumed a disease allele frequency of 0.0001 and no sex located in four regions—chromosomes 10, region p13; 11, difference in the recombination rates. region q23.3; and 17, regions p11.2 and p13.1—demonstrated

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TABLE 1. Markers with Two-Point LOD Scores Ͼ1.0 in a Whole-Genome Scan

Recombination Fraction (␪)

␪ Marker Location 0.00 0.01 0.05 0.10 0.20 0.30 0.40 Zmax max

D10S1653 10p13 Ϫ5.37 0.07 1.22 1.49 1.40 1.00 0.48 1.49 0.10 D11S925 11q23 Ϫ6.89 0.62 1.29 1.43 1.26 0.82 0.29 1.43 0.10 D17S1852 17p13 Ϫ2.51 0.80 1.46 1.61 1.43 0.99 0.43 1.61 0.10 D17S1857 17p11 4.72 4.64 4.30 3.86 2.93 1.93 0.86 4.72 0.00

two-point LOD scores Ͼ1.0 (Table 1). The maximum LOD cM on chromosome 17, region p12-q12, bounded proximally score of 4.72 (␪ ϭ 0) was obtained at D17S1857. by locus D17S900 and distally by locus D17S1872. Other surrounding markers were genotyped for fine mapping. Among these microsatellite markers, D17S740 failed to Mutation Screening amplify, and four markers—D17S839, D17S953, D17S2196, and D17S1788—were excluded because of low heterozygos- According to the data from the UCSC Genome Bioinformatics ity. Finally, 15 markers remained: D17S799, D17S900, Site (http://www.genome.ucsc.edu/University of California, D17S261, D17S1843, D17S1857, D17S1288, D17S793, Santa Cruz) using the NCBI RNA reference sequence collection D17S1871, D17S783, D17S1824, D17S1880, D17S1293, (RefSeq; www.ncbi.nlm.nih.gov/locuslink/refseq), the miR- D17S1872, D17S933, and D17S927. The two-point LOD Base sequence database (http://www.mirbase.org/ University scores of these markers were calculated. A maximum LOD of Manchester, Manchester, UK) and the snoRNABase (http:// score of 4.97 at recombination 0.00 was obtained at D17S1824 www.snorna.biotoul.fr/coordinates.php), 153 protein-coding on chromosome 17, region q12. Seven other markers genes, 9 miRNAs, and 13 snoRNAs were identified in the (D17S900, D17S261, D17S1843, D17S1857, D17S1288, interval between D17S900 and D17S1872. Then, 14 candidate D17S783, and D17S1880) around D17S1824 also obtained a genes were selected for mutation screening: UNC119, LOD score Ͼ3.0 at recombination fraction 0.00 (Table 2), CRYBA1, RPL23A, NCOR1, COPS3, ALDOC, C17orf39, MED9, which is suggestive of linkage to microphthalmia. NLK, FLII, NUFIP2, CCL8, PROCA1, and SPAG5. These genes Multipoint linkage analysis resulted in a LOD score Ͼ3.0 in are thought to be likely to share similarities with known mi- the region between D17S900 and D17S1880. The highest crophthalmia genes, or else to be expressed specifically in multipoint LOD score, 4.1, was obtained between D17S261 eyes, or their function is thought to be associated with eyes. and D17S1871 (Fig. 2). The 14 candidate genes were screened in individual IV-8. The coding regions and intron/exon splicing regions of these 14 Haplotype Analysis candidate genes were sequenced, but no pathogenic mutation was found (Table 3). Ten microsatellite markers for fine mapping were used to Through CNV analysis, 265 CNVs were found in affected construct the haplotypes (Fig. 1). The informative recombina- individual III-8, and 239 CNVs were found in affected individ- tion event was present in normal individual III-3 between ual V-3. However, no possible pathogenic CNV was identified markers D17S900 and D17S1843, placing the disease-causing in the region. gene centromeric to the marker D17S900. Similarly, recombination events between loci D17S1293 and D17S1872 oc- curred in affected individuals III-11, IV-8, and V-3, indicating DISCUSSION that the disease gene is telomeric to locus D17S1293.In addition, haplotype analysis showed that a cosegregated hap- This is the second Chinese background, congenital simple lotype expanding from D17S1843 to D17S1293 was inherited microphthalmia pedigree reported after Li et al.5 reported one by all nine affected members in the family. Thus, in this family, in 2008 and linked a novel locus to this pedigree. A whole- the disease gene lies within a region of approximately 21.57 genome scan and precise localization were performed, and a

TABLE 2. Two-Point LOD Scores between Microphthalmia and Markers of 17p12-q12

؍ LOD Score at ␪

Marker cM 0.00 0.01 0.05 0.1 0.2 0.3 0.4

D17S799 31.96 Ϫ4.45 0.12 0.65 0.72 0.58 0.33 0.12 D17S900 36.14 3.30 3.30 3.21 2.99 2.36 1.57 0.69 D17S261 41.12 3.39 3.32 3.06 2.71 2.00 1.24 0.46 D17S1843 41.12 3.44 3.38 3.11 2.76 2.02 1.21 0.39 D17S1857 43.90 4.72 4.64 4.30 3.86 2.93 1.93 0.86 D17S1288 45.93 4.91 4.82 4.48 4.04 3.09 2.05 0.91 D17S793 47.00 2.46 2.46 2.41 2.23 1.71 1.08 0.40 D17S783 47.00 4.43 4.36 4.05 3.65 2.80 1.86 0.82 D17S1871 48.07 2.61 2.57 2.38 2.14 1.64 1.11 0.54 D17S1824 49.67 4.97 4.89 4.54 4.10 3.14 2.09 0.94 D17S1880 53.41 4.77 4.69 4.34 3.90 2.96 1.95 0.88 D17S1293 56.48 2.36 2.31 2.14 1.92 1.47 0.99 0.49 D17S1872 57.71 Ϫ7.91 2.12 2.53 2.45 1.95 1.25 0.48 D17S933 57.71 2.41 2.36 2.19 1.97 1.51 1.03 0.51 D17S927 58.25 Ϫ7.50 2.51 2.88 2.77 2.20 1.44 0.59

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FIGURE 2. Multipoint LOD scores between the disease and markers of 17, region p12–q12. Marker order and map distances were obtained from the Marshﬁeld genetic map.

significantly positive two-point LOD score was obtained with a has been localized to the photoreceptor synapses in the outer maximum 4.97 for marker D17S1824 at a recombination frac- plexiform layer of the retina and has been suggested to play a tion of 0.00. Subsequent haplotype analysis showed that a role in the mechanism of photoreceptor neurotransmitter re- cosegregated haplotype expanding from D17S1843 to lease through the synaptic vesicle cycle.19 NCOR1 encodes a D17S1293 was inherited by all nine affected members in this protein that mediates ligand-independent transcription repres- family. sion of thyroid hormone and retinoic acid receptors by pro- A total of 153 genes have been mapped to this interval moting chromatin condensation and preventing access of the defined by loci D17S900 and D17S1872. Fourteen candidate transcription machinery. ALDOC, COPS3, SPAG5, PROCA1, genes, including UNC119, CRYBA1, RPL23A, NCOR1, COPS3, NLK, RPL23A, and MED9 are at high levels in eyes. Especially, ALDOC, C17orf39, MED9, NLK, FLII, NUFIP2, CCL8, RPL23A, PROCA, and SPAG5 are expressed at high levels in PROCA1, and SPAG5, were screened on the basis of their high fetal eyes, lens, eye anterior segment, optic nerve, and retina, expression and essential function in eyes. Especially, the among other ocular components. The coding regions and in- CRYBA1 protein is the structural constituent of eye lens crys- tron/exon splicing region of these 14 candidate genes were tallins. The mutation in the CRYBA4 gene, which is in the same sequenced, but no pathogenic mutation was found. However, protein family as CRYBA1, is attributed to complex microph- the possibility could not be completely ruled out, because we thalmia in association with genetic cataracts.9 UNC119, which did not screen the control regions (promoter, 5Ј and 3Ј un- is specifically expressed in the photoreceptors in the retina, translated regions [UTRs]) of these genes, the possibility of a

TABLE 3. The Results of Candidate Gene Screening and Variation Detection

Genes Base Change Codon Change SNP ID

ALDOC No variation CCL8 NM_005623.2:c.194ϩ139AϾG rs3138036 NM_005623.2:c.205AϾC LysϾGln rs1133763 NM_005623.2:c.195–77TϾC rs3138037 COPS3 NM_003653.3:c.185–3CϾT rs4985761 NM_003653.3:c.573CϾT IleϾIle rs3182911 CRYBA1 No variation C17orf39 No variation FLII NM_002018.2:c.1596ϩ69AϾG rs2071242 NM_002018.2:c.2191–77GϾC rs35738574 NM_002018.2:c.3051ϩ54TϾC rs2856289 MED9 NM_018019.2:c.243GϾA ProϾPro rs11553995 NCOR1 NM_006311.3:c.1082ϩ64TϾA rs11078333 NM_006311.3:c.1173ϩ75TϾA rs2285579 NM_006311.3:c.4153–5TϾA rs12942295 NM_006311.3:c.5101ϩ41AϾG rs2285583 NM_006311.3:c.5882–38TϾC rs2157991 NM_006311.3:c.6679ϩ67CϾT rs1079533 NLK No variation NUFIP2 No variation PROCA1 No variation RPL23A No variation SPAG5 No variation UNC119 No variation

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