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

Abstract

Purpose.: To investigate the etiology in a family with autosomal-dominant congenital simple microphthalmia of Chinese origin.

Methods.: A whole-genome scan was performed by using 382 microsatellite DNA markers after the exclusion of reported candidates linked to microphthalmia. Additional fluorescent markers were genotyped for fine mapping. To find out the novel predisposing gene, 14 candidate genes including CRYBA1 and NCOR1 were selected to screen for the mutation by the PCR direct-sequencing method. Genome-wide single-nucleotide polymorphism (SNP) genotyping was performed to find out the pathogenetic copy number variation, as well.

Results.: The most statistically significant linkage results were obtained at D17S1824 (maximum LOD score, 4.97, at recombination fraction 0.00). Haplotype analyses supported the location of the disease-causing gene to a 21.57-cM interval between loci D17S900 and D17S1872 of chromosome 17, region p12-q12. However, no mutation or CNV (copy number variation) was identified to be responsible for the microphthalmia phenotype of this pedigree.

Conclusions.: A novel suggestive linkage locus for congenital microphthalmia was detected in a Chinese family. This linkage region provides a target for susceptibility gene identification.

Microphthalmia (OMIM 309700) is an ocular developmental malformation characterized by unusually small eyes (Online Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/Omim/ National Center for Biotechnology Information [NCBI], Bethesda, MD). The major clinical characteristics include a short axial length (<20 mm), a high degree of hyperopia (+7.00 to +13.00 D), a high lens-to-eye volume ratio, and a high incidence of angle-closure glaucoma after middle age. Some normal adnexal elements and eyelids are usually present. ¹ It is also a common symptom in some other ocular abnormalities. Approximately 80% of microphthalmia cases occur as part of syndromes that include other systemic malformations, especially cardiac defects, facial clefts, microcephaly, and hydrocephaly. ^2,3 The reported prevalence of anophthalmia or microphthalmia at birth is 0.66 of 10,000 around the world and 0.3 of 10,000 in China. ⁴  

Epidemiologic studies have indicated that both heritable and environmental factors cause microphthalmia. Although the precise pathogenesis of microphthalmia is still unknown, studies have demonstrated that it is a genetically heterogeneous disorder. Chromosomal abnormalities may result in syndromic microphthalmia. Studies of different microphthalmia cases and pedigrees have linked it to different chromosomal regions and monogenic causes. Autosomal-dominant microphthalmia pedigrees have been mapped to 2q11–14, ⁵ 3q26.3-q27 (SOX2), ⁶ 11p, ¹ 11p13 (PAX6), 14q22 (OTX2), ⁷ 15q12-q15, ⁸ and 22q11.2-q13.1 (CRYBA4). ⁹ In some families, autosomal recessive microphthalmia has been linked to 2q37.1, ¹⁰ 11q23 (MFRP), ¹¹ 14q24.3 (CHX10), ¹² 14q32, ¹³ and 18q21.3 (RAX). ¹⁴ X-linked anophthalmia pedigrees have been linked to Xp11.4 (BCOR) ¹⁵ and Xq27-q28 (ANOP1). ¹⁶  

Here, we report a linkage and haplotype analysis that indicates a novel locus responsible for microphthalmia. The patients involved in this study were from a congenital simple microphthalmia family of Chinese origin in Shandong Province. The karyotype in the affected family members was normal. Several candidate loci and genes have been excluded in 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, these results may provide more data for further research into the disease. 

Subjects and Methods

Subjects

A five-generation Chinese family from Shandong Province in China that had members with diagnosed microphthalmia was involved in the study (Fig. 1). Thirty-four family members underwent general physical and complete ophthalmic examinations. All family members did not have any other physical anomalies. Nine microphthalmia patients expressed the same full phenotype as previously reported. ^1,18 They were affected by isolated microphthalmia in an autosomal dominant transmission manner in both eyes with onset since birth. Detailed information is available in another publication. ¹⁷  

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. Question mark: genotype not determined.

Peripheral blood samples from 28 individuals including 9 affected family members (containing all patients) and 19 unaffected members were collected for further analysis. All participants gave written informed consent in accordance with the Declaration of Helsinki before they were enrolled in the study. 

Genotyping

A 3-mL peripheral blood sample was taken from each individual after informed consent was obtained. Genomic DNA was extracted by using the standard phenol-chloroform method. 

The whole-genome scan was performed by using 382 fluorescent microsatellite markers in the 22 pairs of autosomes, with an average spacing of 10-cM scattering on the human genome (Prism Linkage Mapping Set Version 2.0; Applied Biosystems, Inc. [ABI], Foster City, CA). PCR was performed in a 5-μL volume with 50 ng genomic DNA as a template, 0.5 μL PCR 10× buffer, 0.1 μL dNTP mix (2.5 mM), 0.06 μL primers, 0.6 μL MgCl₂ (15 mM), 0.05 U Taq polymerase (AmpliTaq Gold; ABI), and distilled water up to 5 μL. Thermal cycling was performed (GeneAmp 2720; ABI) at 95°C for 12 minutes, then 15 cycles of 94°C for 30 seconds, 63°C for 1 minute, degrading 0.5°C per cycle, and 72°C for 1 minute 50 seconds, followed by 24 cycles of 94°C for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute 50 seconds, with a final extension of 72°C for 15 minutes. PCR products were analyzed on an automated sequencer (model 3100; ABI). A GS400 size standard was used as the internal standard and run in the same lane with the markers. Alleles were then analyzed (GeneScan, ver. 3.0 and GenoTyper ver. 3.7; ABI). In the fine mapping phase, additional fluorescent markers (D17S799, D17S900, D17S839, D17S261, D17S1843, D17S740, D17S953, D17S2196, D17S1288, D17S793, D17S1871, D17S783, D17S1824, D17S1880, D17S1293, D17S1872, D17S933, D17S92, and D17S1788) were selected from the Marshfield database (http://research.marshfieldclinic.org/ Marshfield Clinic, Marshfield, WI). 

Linkage Analysis

Two-point LOD scores were calculated by the MLINK program of the LINKAGE package (ver. 5.1; http://linkage.rockefeller.edu/soft/). The allele frequencies of markers were assumed to be equal, as were the recombination frequencies in males and females. The disease was specified to be an autosomal dominant inheritance with 95% penetrance. We assumed a disease allele frequency of 0.0001 and no sex difference in the recombination rates. 

Multipoint analysis was computed (Genehunter-Modscore, ver. 3.0; http://linkage.rockefeller.edu/soft/gh/ Rockefeller University, New York, NY). Marker order and map distances were obtained from the Marshfield genetic map. 

Haplotype Reconstruction

The haplotype was constructed, using a commercial program (Cyrillic Software, Lake Orion, MI) to define the borders of the cosegregating region and then modifying it by hand. 

Mutation Analysis

After the whole-genome scan, a candidate approach was used to search for possible candidate genes. The exons of candidate genes were amplified by PCR, and the primers were designed on computer (Premier 5.0; Premier Biosoft, Palo Alto, CA). The PCR reaction included 1 μL (50 ng) genomic DNA, 1 μL (30 ng) each of the primers, 1 μL PCR 10× buffer with MgCl₂ (Roche Diagnostics, USA, Indianapolis, IN), 0.05 μL (5U) Taq polymerase (AmpliTaq Gold; ABI), and 5.85 μL distilled water. Then, PCR products were purified with shrimp alkaline phosphatase (Fermentas International, Glen Burnie, MD) and exonuclease I (Fermentas International) for 85 minutes at 37°C to remove the phosphoryl groups. The samples were then sequenced on an automated sequencer (model 3100; ABI) in both directions. 

To explore the possible pathogenetic role of copy number variation (CNV), we performed genome-wide SNP genotyping (Human660W-Quad BeadChip; Illumina, San Diego, CA). Affected individuals III-8 and V-3 were genotyped according to the manufacturer's guidelines. To call CNVs, we used the PennCNV algorithm (www.openbioinformatics.org, an unaffiliated repository of software), which combines multiple sources of information, including log R ratio (LRR) and B allele frequency (BAF) at each SNP marker, along with SNP spacing and population frequency of the B allele to generate CNV calls. 

Results

Linkage Analysis

A whole-genome scan study was performed, with markers located in four regions—chromosomes 10, region p13; 11, region q23.3; and 17, regions p11.2 and p13.1—demonstrated two-point LOD scores >1.0 (Table 1). The maximum LOD score of 4.72 (θ = 0) was obtained at D17S1857. 

Table 1.

 

Markers with Two-Point LOD Scores >1.0 in a Whole-Genome Scan

Marker	Location	Recombination Fraction (θ)
		0.00	0.01	0.05	0.10	0.20	0.30	0.40	Zmax	θmax
D10S1653	10p13	−5.37	0.07	1.22	1.49	1.40	1.00	0.48	1.49	0.10
D11S925	11q23	−6.89	0.62	1.29	1.43	1.26	0.82	0.29	1.43	0.10
D17S1852	17p13	−2.51	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

Other surrounding markers were genotyped for fine mapping. Among these microsatellite markers, D17S740 failed to amplify, and four markers—D17S839, D17S953, D17S2196, and D17S1788—were excluded because of low heterozygosity. Finally, 15 markers remained: D17S799, D17S900, D17S261, D17S1843, D17S1857, D17S1288, D17S793, D17S1871, D17S783, D17S1824, D17S1880, D17S1293, D17S1872, D17S933, and D17S927. The two-point LOD scores of these markers were calculated. A maximum LOD score of 4.97 at recombination 0.00 was obtained at D17S1824 on chromosome 17, region q12. Seven other markers (D17S900, D17S261, D17S1843, D17S1857, D17S1288, D17S783, and D17S1880) around D17S1824 also obtained a LOD score >3.0 at recombination fraction 0.00 (Table 2), which is suggestive of linkage to microphthalmia. 

Table 2.

 

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

Marker	cM	LOD Score at θ =
		0.00	0.01	0.05	0.1	0.2	0.3	0.4
D17S799	31.96	−4.45	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	−7.91	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	−7.50	2.51	2.88	2.77	2.20	1.44	0.59

Multipoint linkage analysis resulted in a LOD score >3.0 in the region between D17S900 and D17S1880. The highest multipoint LOD score, 4.1, was obtained between D17S261 and D17S1871 (Fig. 2). 

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.

Haplotype Analysis

Ten microsatellite markers for fine mapping were used to construct the haplotypes (Fig. 1). The informative recombination event was present in normal individual III-3 between markers D17S900 and D17S1843, placing the disease-causing gene centromeric to the marker D17S900. Similarly, recombination events between loci D17S1293 and D17S1872 occurred in affected individuals III-11, IV-8, and V-3, indicating that the disease gene is telomeric to locus D17S1293. In addition, haplotype analysis showed that a cosegregated haplotype expanding from D17S1843 to D17S1293 was inherited by all nine affected members in the family. Thus, in this family, the disease gene lies within a region of approximately 21.57 cM on chromosome 17, region p12-q12, bounded proximally by locus D17S900 and distally by locus D17S1872. 

Mutation Screening

According to the data from the UCSC Genome Bioinformatics Site (http://www.genome.ucsc.edu/University of California, Santa Cruz) using the NCBI RNA reference sequence collection (RefSeq; www.ncbi.nlm.nih.gov/locuslink/refseq), the miRBase sequence database (http://www.mirbase.org/ University of Manchester, Manchester, UK) and the snoRNABase (http://www.snorna.biotoul.fr/coordinates.php), 153 protein-coding genes, 9 miRNAs, and 13 snoRNAs were identified in the interval between D17S900 and D17S1872. Then, 14 candidate genes were selected for mutation screening: UNC119, CRYBA1, RPL23A, NCOR1, COPS3, ALDOC, C17orf39, MED9, NLK, FLII, NUFIP2, CCL8, PROCA1, and SPAG5. These genes are thought to be likely to share similarities with known microphthalmia genes, or else to be expressed specifically in eyes, or their function is thought to be associated with eyes. The 14 candidate genes were screened in individual IV-8. The coding regions and intron/exon splicing regions of these 14 candidate genes were sequenced, but no pathogenic mutation was found (Table 3). 

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.194+139A>G		rs3138036
	NM_005623.2:c.205A>C	Lys>Gln	rs1133763
	NM_005623.2:c.195–77T>C		rs3138037
COPS3	NM_003653.3:c.185–3C>T		rs4985761
	NM_003653.3:c.573C>T	Ile>Ile	rs3182911
CRYBA1	No variation
C17orf39	No variation
FLII	NM_002018.2:c.1596+69A>G		rs2071242
	NM_002018.2:c.2191–77G>C		rs35738574
	NM_002018.2:c.3051+54T>C		rs2856289
MED9	NM_018019.2:c.243G>A	Pro>Pro	rs11553995
NCOR1	NM_006311.3:c.1082+64T>A		rs11078333
	NM_006311.3:c.1173+75T>A		rs2285579
	NM_006311.3:c.4153–5T>A		rs12942295
	NM_006311.3:c.5101+41A>G		rs2285583
	NM_006311.3:c.5882–38T>C		rs2157991
	NM_006311.3:c.6679+67C>T		rs1079533
NLK	No variation
NUFIP2	No variation
PROCA1	No variation
RPL23A	No variation
SPAG5	No variation
UNC119	No variation

Through CNV analysis, 265 CNVs were found in affected individual III-8, and 239 CNVs were found in affected individual V-3. However, no possible pathogenic CNV was identified in the region. 

Discussion

This is the second Chinese background, congenital simple microphthalmia pedigree reported after Li et al. ⁵ reported one in 2008 and linked a novel locus to this pedigree. A whole-genome scan and precise localization were performed, and a significantly positive two-point LOD score was obtained with a maximum 4.97 for marker D17S1824 at a recombination fraction of 0.00. Subsequent haplotype analysis showed that a cosegregated haplotype expanding from D17S1843 to D17S1293 was inherited by all nine affected members in this family. 

A total of 153 genes have been mapped to this interval defined by loci D17S900 and D17S1872. Fourteen candidate genes, including UNC119, CRYBA1, RPL23A, NCOR1, COPS3, ALDOC, C17orf39, MED9, NLK, FLII, NUFIP2, CCL8, PROCA1, and SPAG5, were screened on the basis of their high expression and essential function in eyes. Especially, the CRYBA1 protein is the structural constituent of eye lens crystallins. The mutation in the CRYBA4 gene, which is in the same protein family as CRYBA1, is attributed to complex microphthalmia in association with genetic cataracts. ⁹ UNC119, which is specifically expressed in the photoreceptors in the retina, has been localized to the photoreceptor synapses in the outer plexiform layer of the retina and has been suggested to play a role in the mechanism of photoreceptor neurotransmitter release through the synaptic vesicle cycle. ¹⁹ NCOR1 encodes a protein that mediates ligand-independent transcription repression of thyroid hormone and retinoic acid receptors by promoting chromatin condensation and preventing access of the transcription machinery. ALDOC, COPS3, SPAG5, PROCA1, NLK, RPL23A, and MED9 are at high levels in eyes. Especially, RPL23A, PROCA, and SPAG5 are expressed at high levels in fetal eyes, lens, eye anterior segment, optic nerve, and retina, among other ocular components. The coding regions and intron/exon splicing region of these 14 candidate genes were sequenced, but no pathogenic mutation was found. However, the possibility could not be completely ruled out, because we did not screen the control regions (promoter, 5′ and 3′ untranslated regions [UTRs]) of these genes, the possibility of a functional defect in introns has not yet been ruled out. In addition, because the information on genes in this area is limited, the pathogenic gene may not have been identified. With the updated genome and expression studies, however, new eye-related genes will be found that could be our new candidate genes. 

In conclusion, the novel localization for an autosomal dominant congenital simple microphthalmia pedigree has been mapped to chromosome 17, region p12-q12. Some new genes related to mammalian eye development may be identified from this Chinese family in the future. Our screening is still being undertaken with the hope of identifying the disease gene itself. Further, high-throughput sequencing technology for screening the area will also be taken into account. 

Footnotes

 Supported by National Natural Science Foundation of China Grants 30630062, 81070081, and 81070759.

Footnotes

 Disclosure: Z. Hu, None; C. Yu, None; J. Li, None; Y. Wang, None; D. Liu, None; X. Xiang, None; W. Su, None; Q. Pan, None; L. Xie, None; K. Xia, None

The authors thank the family members for their understanding and participation in this study and Cong Wang for assistance in the DNA sequencing.