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Use of high-throughput targeted exome sequencing in genetic diagnosis of Chinese family with congenital cataract

Ming-Fu Ma¹, Lian-Bing Li¹, Yun-Qi Pei², Zhi Cheng²

¹Key Laboratory of Birth Defects and Reproductive Health of the National Health and Family Planning Commission (Chongqing Population and Family Planning Science and Technology Research Institute), Chongqing 400020, China

²Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China

Co-first authors: Ming-Fu Ma and Lian-Bing Li

Correspondence to: Zhi Cheng. Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China. zcheng@cqmu.edu.cn

Received: 2015-06-03 Accepted: 2015-07-28

Abstract

AIM: To identify disease-causing mutation in a congenital cataract family using enrichment of targeted genes combined with next-generation sequencing.

METHODS: A total of 371 known genes related to inherited eye diseases of the proband was selected and captured, followed by high-throughput sequencing. The sequencing data were analyzed by established bioinformatics pipeline. Validation was performed by Sanger sequencing.

RESULTS: A recurrent heterozygous non-synonymous mutation c.130G>A (p.V44M) in the GJA3 gene was identified in the proband. The result was confirmed by Sanger sequencing. The mutation showed co-segregation with the disease phenotype in the family but was not detected in unaffected controls.

CONCLUSION: Targeted exome sequencing is a rapid, high-throughput and cost-efficient method for screening known genes and could be applied to the routine gene diagnosis of congenital cataract.

KEYWORDS: genetic diagnosis; targeted exome sequencing; congenital cataract

DOI:10.18240/ijo.2016.05.02

Citation: Ma MF, Li LB, Pei YQ, Cheng Z. Use of high-throughput targeted exome sequencing in genetic diagnosis of Chinese family with congenital cataract. Int J Ophthalmol 2016;9(5):650-654

INTRODUCTION

Congenital cataract is the leading cause of visual impairment and blindness in children. It has an estimated incidence of 1-6 per 10 000 live births and accounts for nearly 10% of irreversible childhood blindness worldwide^[1-2]. For affected children, early diagnosis is important because timely and appropriate intervention can obtain good visual function^[3].

Approximately one-third of congenital cataract cases are believed to be hereditary, and most occur in an autosomal dominant pattern^[4]. To date, more than 40 loci have been mapped in congenital cataracts and 32 genes have been identified^[5]. These genes contain a total of 250 coding fragments (Table 1). Therefore, genetic diagnosis through traditional approaches, such as direct sequencing is time-consuming and costly. A more efficient method to detect the genetic defects is needed.

Table 1 Known causative genes of congenital cataract

No.	Locus	Gene	Coding exons
1	1p32	^1FOXE3	1
2	1p36	EPHA2	18
3	1q21.1	GJA8	2
4	2q33.3	CRYGC	3
5	2q33.3	CRYGD	3
6	3p21.31	FYCO1	22
7	3q22.1	BFSP2	7
8	3q27.3	^1CRYGS	3
9	6p24.2	GCNT2	11
10	7q34	AGK	17
11	8q13.3	EYA1	20
12	9q22.33	TDRD7	17
13	10p13	^1VIM	9
14	10q24.32	PITX3	4
15	11q25	JAM3	9
16	11q22.3-q23.1	CRYAB	3
17	12q13	MIP	4
18	13q12.11	GJA3	3
19	16p13.2	^1TMEM114	4
20	16q21	HSF4	15
21	16q22-q23	MAF	2
22	17q11.2	CRYBA1	6
23	19q13.33	FTL	4
24	19q13.4	LIM2	5
25	20p12.1	BFSP1	13
26	20q11.22	CHMP4B	5
27	21q22.3	CRYAA	4
28	22q11.23	CRYBB2	7
29	22q11.23	CRYBB3	6
30	22q12.1	CRYBB1	6
31	22q12.1	CRYBA4	7
32	Xp22.13	NHS	10
Total			250

¹Genes which were not captured in the current study.

With the progresses on next-generation sequencing and bioinformatics, whole exome sequencing has been proved to be a powerful tool for the genetic diagnosis of both Mendelian and complex diseases^[6-7]. And it has been successfully applied in identifying disease-causing genes and mutations for congenital cataract^[8-9]. However, the large amount of information, subsequent difficult data processing and high cost limit its potential application in clinical practice. In this study, we utilized high-throughput targeted exome sequencing (TES) to study genetic defects in a congenital cataract family and attempt to establish a strategy feasible to genetic diagnosis of congenital cataract patients.

SUBJECTS AND METHODS

Subjects The proband, a 7-year-old boy, was diagnosed with bilateral cataracts at Affiliated Hospital of Chongqing Population and Family Planning Science and Technology Research Institute. A family history revealed six members in three generations. Five family members participated in the study. The research protocol was approved by the ethics committee of Chongqing Population and Family Planning Science and Technology Research Institute. All participants from the family provided their written consent for participation in the research. And they didn't receive any stipend. The study was conducted according to the principles in the Declaration of Helsinki. Peripheral blood samples were collected from all study participants.

Targeted Capture Preparation and Next-generation Sequencing Genomic DNA was extracted from whole blood using TIANamp Blood DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). The DNA was quantified with Nanodrop 2000 (Thermo Fisher Scientific, MA, USA). A minimum of 3 μg DNA was used for the indexed Illumina libraries according to manufacturer’s protocol. The final library size 350-450 bp including adapter sequences was selected. The coding exons and flanking regions of 371 genes related to inherited eye diseases were selected and captured using a GenCap custom enrichment kit^[10]. The methods used for DNA target capture, enrichment and elution followed previously described protocols ^[11-13]. The eluted DNA was finally amplified as follows: 98℃ for 30s; 98℃ for 25s, 65℃ for 30s, 72℃ for 30s (15 cycles); 72℃ for 5min. The polymerase chain reaction (PCR) product was purified using SPRI beads (Beckman Coulter, CA, USA) according to manufacturer’s protocol. The purified product was sequenced on Illumina Solexa HiSeq 2000 sequencer (Illumina, CA, USA).

Bioinformatics Analysis After sequencing, the Solexa QA package and the cutadapt program (http://code.google.com/p/cutadapt/) were used to filtering out the low quality reads and adaptor sequences^[14]. The SOAPaligner program was used to align the clean read sequences to the human reference genome (hg19)^[15].

The PCR duplicates were removed by the Picard software and single nucleotide polymorphisms (SNPs) were identified using the SOAPsnp program^[15-16]. Subsequently, reads were realigned to the reference genome using the Burrows-Wheeler alignment program, and insertions or deletions (InDels) were identified with the Genome Analysis Toolkit^[17-18]. The identified SNPs and InDels were annotated using the Exome-assistant program (http://122.228.158.106/exomeassistant). MagicViewer was used to view the short read alignment and validate the candidate SNPs and InDels^[19]. Finally, nonsynonymous variants were evaluated by four algorithms, SIFT, PolyPhen_2, MutationTaster and GERP++, to determine pathogenicity.

Expanded Validation Sanger sequencing was used to confirm the potential pathogenic variants detected by TES. Primers were used to amplify coding exons containing the candidate variants and their flanking regions (GJA3-F: 5’-CGGTGTTCATGAGCATTTTC-3’ and GJA3-R: 5’-CTCTTCAGCTGCTCCTCCTC-3’). The PCR products were sequenced with the ABI3730 Automated Sequencer (Applied Biosystems, CA, USA). DNA samples from all participating members of the family were analyzed. The sequencing results were analyzed using Chromas software and compared with the reference sequences in the NCBI database.

RESULTS

Clinical Evaluation Five family members of a three-generation Chinese family with a history of cataracts participated in the study (three affected and two unaffected individuals; Figure 1). All patients in this family had bilateral cataracts. The proband, a 7-year-old boy，had been diagnosed with bilateral cataracts at the age of 6mo. His grandfather (I:1) and mother (II:1) also had poor vision in their childhood. The boy’s best corrected visual acuity was 0.2/0.4. There was no family history of other ocular or systemic abnormalities. To date, all of the affected individuals have had cataract surgery.

Figure 1 Pedigree of family with congenital cataract Circles represent females, while squares indicate males. Affected individuals are denoted by black symbols. The arrow points to the proband. The phenotypes of proband’s brother is unknown while the study was carried on.

Targeted Exome Sequencing and Co-segregation Analysis Identified Causative Mutation The genomic DNA of the proband was subjected to TES. Three hundred and seventy-one genes related to inherited eye diseases were analyzed including 28 genes involved in congenital cataract. The average sequencing depth on the targeted regions was 215.45. And the sample had 95.30% coverage of the targeted regions. Meanwhile, 89.40% and 81.50% targeted exons are covered with at least 4 and 10 reads, respectively (Table 2). A total of 23 variants were identified in the 28 known cataract genes. After excluding variants reported in HapMap 28 and the SNPs release of the 1000 Genome Project with minor allele frequency >0.05, a previously reported heterozygous non-synonymous mutation c.130G>A (p.V44M) was discovered in exon 2 of GJA3 (Figure 2). It was predicted to be damaging by bioinformatic software tools. The mutation was further confirmed in the proband by Sanger sequencing. It was also found in two other affected family members but was absent in unaffected family members (Figure 3). The mutation was not detected in the 100 unrelated controls either^[20]. So, it co-segregated with the congenital cataract phenotype. Additionally, a heterozygous non-synonymous mutation c.605A>G (p.E202G) in GCNT2 was also detected in the proband.

Table 2 Data summary of the targeted exome sequencing

Sample	Proband
Raw data (Mb)	418.5
Clean data (Mb)	415.78
Aligned	99.8
Initial bases on target	951791
Base covered on target	907495
Coverage of target region	95.30%
Total effective yield (Mb)	332.63
Effective sequence on target (Mb)	205.06
Fraction of effective bases on target	61.60%
Average sequencing depth on target	215.45
Fraction of target covered with at least 4×	89.40%
Fraction of target covered with at least 10×	81.50%
Fraction of target covered with at least 20×	72.90%
Duplication rate (%)	17.4741

Figure 2 GJA3 mutation identified by TES in the proband.

Figure 3 DNA sequences of GJA3 in affected and unaffected individuals A: The DNA sequence chromatograms showing the heterozygous c.130G>A transition that replaces valine by methionine at codon 44 in an affected individual; B: The DNA sequence chromatograms of an unaffected individual.

DISCUSSION

Congenital cataract is clinically and genetically highly heterogeneous. It is known that different mutations in the same gene can cause similar phenotypes, while the same mutation in a single gene can lead to different cataract patterns within the families^[21]. Together with the fact that a huge number of coding exons exist in known genes, traditional screening of each region is impractical for genetic diagnosis and testing in clinical practice. In this study, we used enrichment of targeted genes in combination with high-throughput sequencing to screen mutations in a Chinese pedigree with congenital cataract. And we successfully identified a previously reported recurrent disease-causing mutation. The results were validated by Sanger sequencing. It demonstrated the robustness and potential application of this approach in clinical genetic diagnosis of congenital cataract.

At present, whole exome sequencing has emerged as a useful new method in genetic diagnosis of congenital cataract^[8-9]. Although its cost is now falling, it’s still too high for clinical practice. In the current study, our approach only captured and analyzed the exons of targeted genes. The cost was saved at least 50% compared with whole exome sequencing. And a large amount of work in data analysis was saved simultaneously. Furthermore, the sequencing depth was increased as a result of the decreased targeted region. Therefore, our approach is more feasible than whole exome sequencing in clinical practice.

GJA3, which encodes Connexin 46, is mainly expressed in lens fiber cells and plays a key role in maintaining normal lens transparency. Knock-out of GJA3 in mice leads to different degree of cataracts depending on genetic background^[22-23]. To date, more than 20 mutations of GJA3 have been reported to cause congenital cataract^[24]. Utilizing TES, we identified a heterozygous mutation of GJA3 (p.V44M) in a Chinese family. The same mutation had been reported in a Han Chinese family and in a Caucasian American family^[20,25]. It indicated that the V44 might be a mutation hotspot.

Through TES, a heterozygous mutation of GCNT2 (p.E202G) was also detected in the proband. But it could not be the causative mutation of the family because the GCNT2 had been demonstrated to associate with autosomal recessive cataract ^[26-27]. It indicated that TES could provide complete information of genetic defects which might be undetectable by traditional methods.

In summary, we diagnosed congenital cataract genetically using enrichment of targeted genes combined with next-generation sequencing and proved that it is a rapid, high-throughput and cost-efficient method. This strategy can be also used to genetically diagnose of other monogenic diseases.

ACKNOWLEDGEMENTS

Foundations: Supported by the National Natural Science Foundation of China (No.81300463; No.81130051); Fundamental Research Funds for Non-profit Public Scientific Research Institutions of Chongqing (No.2012CSTC-jbby-01704); Natural Science Foundation Project of CQ CSTC (No.cstc2013jcyjA10086); Promotion Program for Young and Middle-aged Teacher in Scientific Research of Basic Medicine College, Chongqing Medical University (No.JC201306).

Conflicts of Interest: Ma MF, None; Li LB, None; Pei YQ, None; Cheng Z, None.

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