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 malfor- mations, 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 anophthal- mia 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- Investigative Ophthalmology & Visual Science, May 2011, Vol. 52, No. 6 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 3425 Downloaded from iovs.arvojournals.org on 10/01/2021 3426 Hu et al. IOVS, May 2011, Vol. 52, No. 6 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 cosegregat- ing 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.