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Next-generation sequencing for D47N mutation in Cx50 analysis associated with autosomal dominant congenital cataract in a six-generation Chinese family

  • Chao Shen1,
  • Jingbing Wang1,
  • Xiaotang Wu1,
  • Fuchao Wang1,
  • Yang Liu2,
  • Xiaoying Guo1,
  • Lina Zhang1,
  • Yanfei Cao1,
  • Xiuhua Cao1 and
  • Hongxing Ma1Email author
Contributed equally
BMC OphthalmologyBMC series – open, inclusive and trusted201717:73

DOI: 10.1186/s12886-017-0476-5

Received: 22 September 2016

Accepted: 15 May 2017

Published: 19 May 2017

Abstract

Background

Congenital cataract is the most frequent cause of blindness during infancy or early childhood. To date, more than 40 loci associated with congenital cataract have been identified, including at least 26 genes on different chromosomes associated with inherited cataract. This present study aimed to identify the genetic mutation in a six-generation Chinese family affected with congenital cataract.

Methods

A detailed six-generation Chinese cataract family history and clinical data of the family members were recorded. A total of 27 family members, including 14 affected and 13 unaffected individuals were recruited. Whole exome sequencing was performed to determine the disease-causing mutation. Sanger sequencing was used to confirm the results.

Results

A known missense mutation, c. 139G > A (p. D47N), in Cx50 was identified. This mutation co-segregated with all affected individuals and was not observed in the unaffected family members or in 100 unrelated controls. The homology modeling showed that the structure of the mutant protein was different with that wild-type Cx50.

Conclusions

The missense mutation c.139G > A in GJA8 gene is associated with autosomal dominant congenital cataract in a six-generation Chinese family. The result of this present study provides further evidence that the p. D47N mutation in CX50 is a hot-spot mutation.

Keywords

Congenital cataract GJA8 Whole exome sequencing Next-generation sequencing

Background

Congenital cataract is the most frequent cause of blindness during infancy or early childhood, with an occurrence of 1–15/10,000 live births worldwide [1, 2]. It explains for 10%–30% of childhood blindness [3]. Congenital cataract is characterized by the presence of an opacification of the lens at birth or during babyhood. On the basis of morphology, congenital cataract can be classified into several subtypes, including nuclear, sutural, polar, cortical cataract, etc. [4]. Congenital cataract pathogenesis involves several distinct reasons including gene defects, chromosomal abnormalities, metabolic disorders, and infections during embryogenesis. Approximately half of congenital cataracts are inherited [3]. Though autosomal recessive and X-linked inheritances have been reported, inheritance is mainly autosomal dominant [5]. Up to date, over 40 loci associated with congenital cataract have been confirmed, including no less than 26 genes on different chromosomes related to congenital cataract [6, 7]. Among these mutant genes, the connexin genes and crystallin genes are the most widespread. Briefly, half of the mutations were discovered in the crystalline genes, such as alpha crystallins, beta crystallins and gamma crytallins, and approximately 25% involve mutations in membrane transport genes, such as connexin proteins (Cx43, Cx46, and Cx50) [514].

In current study, we utilized next-generation sequencing of whole exome to investigate genetic defects in a Chinese pedigree with congenital cataract.

Methods

Subject recruitment and DNA sampling

A six-generation Chinese cataract family was examined at the General Hospital of Daqing Oil Field, Heilongjiang province, China. Pedigree medical history was taken directly by interviewing the family members. A total of 27 family members, including 14 affected (III12, IV11, IV28, IV30, IV39, IV72, IV73, V9, V11, V27, V28, VI3, VI9, and VI15) and 13 unaffected individuals (IV40, IV68, IV69, IV70, IV71, IV74, V10, V14, V19, V42, V57, V62, VI13) were recruited (Fig. 1). Ethical approval for current research was obtained from the ethics committee of General Hospital of Daqing Oil Field and the study was conducted according to the Declaration of Helsinki of the World Medical Association. All members recruited in this study underwent ophthalmologic examinations, including slit lamp ophthalmoscopy, biometry, visual acuity, and fundus examination. In addition, 100 unrelated healthy subjects without cataracts were also recruited from General Hospital of Daqing Oil Field.
Fig. 1

Clinical evaluation of a Chinese pedigree with autosomal dominant congenital cataract. a Pedigree of a six-generation Chinese family with autosomal dominant congenital cataracts. The arrow indicates the proband. Squares and circles symbolize males and females, respectively. Black and white denote the status of family members affected or unaffected, respectively, by congenital cataract. b Photo was taken with a surgical microscope

DNA samples were extracted using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) from peripheral blood.

Exome sequencing

Ten patients (III12, IV11, IV28, IV30, IV73, V9, V27, VI3, VI9 and VI15) and one unaffected member of the family (IV40) were selected for exome sequencing. The whole exome-enriched library was built using NimbleGen SeqCap EZ Exome 64 Mb solution-based SeqCap EZ capture reagents, and solution hybridization exome capture was conducted in according with the manufacturer’s protocol. Exome sequencing was taken by using an Illumina HiSeq2000 sequencer.

Short-read alignment, variant calling and annotation

Low quality reads and PCR duplicates with >5 unknown bases were eliminated [15], for insertion/deletion (indel) and single nucleotide polymorphism (SNP), respectively. Aligning between read and the National Center for Biotechnology Information human reference genome (hg 19) were performed by sequencing reads were aligned to using Burrows-Wheeler Aligner (BWA) [15] and Short Oligonucleotide Analysis Package (SOAP3) tools [16]. Indels were validated according to the alignment result with the Genome Analysis Toolkit (GATK), and SNP calling was performed with Short Oligonucleotide Analysis Package (SOAPsnp). Variants were annotated using ANNOVAR tool.

Validation of mutation by Sanger sequencing

Sanger sequencing was used to validate the variants identified by exome sequencing. Specific primers were designed by Primer Premier 3.0 software for the target region. Genomic DNA from participants and 100 normal controls was analyzed.

Genomic DNA samples were amplified with the forward primer (5′- GCAGATCATCTTCGTCTCCA-3′) and the reverse primer(5′- GGCCACAGACAACATGAACA-3′). The following program was used: 95 °C for 3 min (1 cycle); 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s (30 cycles); 72 °C for 10 min (1 cycle).

Bioinformatics analysis

The effects of wild-type amino acid sequences with the p. D47N mutant of Cx50 on the secondary structure were performed using Antheprot 2000 software (version 6.6.5, IBCP, Lypn, France). The solved structure of gap junction protein beta 2(Cx26) was taken as template (Protein Data Bank No.2ZW3). The model structure of homomeric wild-type and the mutant of GJA8 were modelled by Swiss-Model Server [17]. In addition, the possible functional effect of the amino acid change was predicted by PolyPhen-2 and SIFT.

Results

Clinical evaluations

Among 171 members in this six-generation Chinese family, affected individuals account for 23.39% (Fig. 1). All affect individuals in the pedigree had bilateral cataracts. Autosomal dominant inheritance mode of the congenital cataract was ascertained by the presence of affected individuals in each generation of the family, and male-to-male transmission. The proband’s son (VI 9) had been diagnosed with cataracts when he was 15 months old. Slit-lamp examination of his left eye showed perinuclear cataract.

Identification of Cx50 mutation

Whole exome sequencing was performed on genomic DNA from nine patients of congenital cataract family (III12, IV11, IV28, IV30, IV73, V9, V27, VI3, VI9 and VI15) and one unaffected individual (IV40) though next-generation sequencing technology. As demonstrated in Table 1, we obtained at least 64.06 million reads that mapped to targeted exome regions; more than 99.49% of the target region was covered. The mean depth of the target exome region was 180.98×, 191.56×, 191.23×, 155.43×, 184.67×, 197.75×, 203.48×, 160.48×, 167.92×, 155.12× and 187.92×, respectively. The raw Indel/SNP sequencing data are shown in Table 2. To help identify candidate mutations, untranslated regions, variants falling within intergenic, synonymous substitutions, intronic were excluded. Then the remaining variants were filtered out in at least four public genetic variant databases, including 1000 Genomes, dbSNP, HapMap and YH. Variants with an allele frequency > 0.5% were rejected. Variants shared by 10 patients and absent from 1 unaffected individual were analyzed.
Table 1

Coverage statistics with next-generation sequencing in ten patients with autosomal dominant congenital cataract and one unaffected member of family

Sample

III12

IV11

IV28

IV30

IV73

V9

V27

VI3

VI9

VI15

IV40

Total base mapped (G)

11.6

12.31

12.15

9.72

11.92

12.35

13.16

9.65

10.37

11.51

11.84

Region of target kit

64,558,893

64,326,610

64,326,610

64,326,610

64,558,893

64,558,893

64,558,893

64,326,610

64,326,610

64,326,610

64,326,610

Region of covered on target

64,226,731

64,090,887

64,126,819

64,109,790

64,332,815

64,368,416

64,322,956

64,060,274

64,112,774

64,117,790

64,141,226

Coverage of target region (%)

99.49

99.63

99.69

99.66

99.65

99.7

99.63

99.59

99.67

99.68

99.71

Effective bases on target (G)

11.68

12.32

12.3

10

11.92

12.77

13.14

10.32

10.8

9.98

12.09

Average sequencing depth on target region

180.98

191.56

191.23

155.43

184.67

197.75

203.48

160.48

167.92

155.12

187.92

Target coverage with at least 5× (%)

98.72

98.93

99.02

98.93

98.98

99.11

99.01

98.78

98.96

98.92

99.05

Target coverage with at least 10× (%)

98.07

98.32

98.42

98.19

98.37

98.54

98.47

98.09

98.32

98.24

98.45

Target coverage with at least 20× (%)

96.99

97.25

97.31

96.57

97.21

97.42

97.54

96.83

97.17

96.92

97.42

Flank region coverage with at least 5× (%)

22.26

18.67

18.83

18

18.1

17.42

17.67

17.1

16.92

17.07

16.61

Flank region coverage with at least 10× (%)

17.82

14.06

13.87

13.12

13.48

12.06

12.01

13.14

13.22

11.6

11.37

Flank region coverage with at least 20× (%)

15.16

11.79

11.58

10.7

11.18

9.94

9.88

10.88

11.02

9.34

9.34

Exome coverage with at least 5× (%)

98.1

98.3

98.4

98.3

98.4

98.6

98.4

98

98.3

98.3

98.4

Exome coverage with at least 5× (%)

97.2

97.4

97.6

97.4

97.6

97.8

97.6

97

97.4

97.3

97.5

Exome coverage with at least 5× (%)

95.9

96.2

96.4

95.9

96.4

96.6

96.5

95.6

96.1

95.9

96.3

Table 2

Variations identified by whole exome sequencing

Mutation type

III12

IV11

IV28

IV30

IV73

V9

V27

VI3

VI9

VI15

IV40

Indel analysis

 Total

15,930

15,690

15,489

13,623

15,448

15,678

18,613

14,028

14,873

14,520

15,657

 1000genome and dbsnp

6813

6707

6678

6153

6663

6561

8340

6123

6324

6375

6586

 1000genome specific

151

134

128

132

131

143

170

154

129

128

122

 dbSNP specific

4846

4570

4462

3853

4544

4448

5594

4036

4406

4324

4559

 dbSNP rate

73.19%

71.87%

71.92%

73.45%

72.55%

70.22%

74.86%

72.42%

72.14%

73.68%

71.18%

 Novel

4120

4279

4221

3485

4110

4526

4509

3715

4014

3693

4390

 Homozygous

4857

4935

4803

4405

4612

4448

3181

4304

4534

4601

4686

 Heterozygous

11,073

10,755

10,686

9218

10,836

11,230

15,432

9724

10,339

9919

10,971

 Frameshift

374

413

394

394

406

458

423

392

417

387

397

 Non-frameshift Insertion

158

180

189

153

181

208

195

173

189

164

169

 Non-frameshift Deletion

61

62

63

67

66

81

83

68

66

72

60

 Non-frameshift codon substitution plus Insertion

61

77

61

58

73

80

88

70

75

55

84

 Non-frameshift codon substitution plus Deletion

28

28

35

25

33

34

30

38

25

23

26

 Stopgain

4

14

10

4

9

5

7

9

9

10

12

 Stoploss

1

1

0

0

1

2

2

1

1

1

1

 Startloss

0

1

0

0

2

0

2

1

0

1

1

 Exonic

689

777

754

702

772

869

832

754

782

715

751

 Splicing

62

58

57

59

62

60

60

63

61

57

66

 NcRNA

238

229

240

235

233

249

259

239

223

222

248

 UTR5

178

194

188

180

186

198

216

174

172

174

203

 UTR3

1530

1510

1427

1276

1498

1471

1797

1372

1414

1382

1519

 Intronic

11,915

11,636

11,562

10,061

11,403

11,572

13,936

10,245

10,997

10,788

11,579

 Upstream

283

280

307

266

304

293

338

242

279

239

284

 Downstream

733

710

683

603

740

708

846

663

682

688

738

 Intergenic

302

296

271

241

250

258

329

276

263

255

269

SNP analysis

 Total

134,311

134,225

136,378

129,878

134,039

133,761

166,869

127,698

130,216

131,224

134,002

 1000genome and dbsnp

121,404

120,889

122,334

116,489

120,656

119,805

152,022

114,903

117,222

117,467

119,890

 1000genome specific

443

456

451

450

466

500

503

473

473

443

436

 dbSNP specific

4979

5030

5142

4963

5008

5188

5533

4875

5051

5034

5008

 dbSNP rate

94.10%

93.81%

93.47%

93.51%

93.75%

93.45%

94.42%

93.80%

93.90%

93.35%

93.21%

 Novel

7485

7850

8451

7976

7909

8268

8811

7447

7470

8280

8668

 Homozygous

51,982

53,569

52,947

51,462

51,793

50,013

32,944

48,638

50,529

51,424

51,815

 Heterozygous

82,329

80,656

83,431

78,416

82,246

83,748

133,925

79,060

79,687

79,800

82,187

 Synonymous

11,043

11,075

11,209

10,961

10,967

11,123

14,116

11,169

11,048

11,104

11,215

 Missense

10,750

10,857

10,991

10,820

10,768

11,029

13,713

10,878

10,892

10,768

10,992

 Stopgain

100

113

117

110

102

109

139

113

111

117

113

 Stoploss

30

33

31

30

28

35

34

33

31

35

27

 Startgain

506

509

496

468

524

491

639

466

487

495

503

 Startloss

30

29

26

27

30

30

36

24

29

30

32

 Exonic

21,979

22,126

22,390

21,970

21,916

22,346

28,067

22,241

22,131

22,075

22,402

 Splicing

159

162

166

149

150

156

191

168

157

157

158

 NcRNA

3252

3279

3410

3233

3381

3329

3809

3283

3281

3245

3284

 UTR5

1981

2028

2080

1993

2015

2096

2498

1908

1948

1968

2061

 UTR3

7707

7707

7821

7485

7825

7652

9778

7461

7527

7610

7820

 Intronic

89,844

89,674

91,108

86,065

89,445

88,742

111,479

83,963

86,127

87,172

89,093

 Upstream

2248

2299

2339

2195

2262

2344

2743

2040

2170

2152

2237

 Downstream

4596

4483

4540

4325

4523

4471

5408

4202

4364

4429

4516

 Intergenic

2545

2467

2524

2463

2522

2625

2896

2432

2511

2416

2431

 SIFT

1859

1905

1934

1819

1833

1939

2556

1918

1866

1892

1904

After filtering and samples comparison, one heterozygous change was confirmed in all affected individuals in congenital cataract family, G > A, at position 139 (c.139 G > A) in exon 2 of GJA8 (Cx50). This change led to the substitution of aspartic acid by asparagine at position 47 (p. D47N). This mutation was further confirmed by Sanger sequencing (Fig. 2). The D47N substitution co-segregated with all 14 affected individuals, while it was not found in the unaffected family members or in the 100 healthy controls.
Fig. 2

The mutation in Cx50 was confirmed with Sanger sequencing. a a heterozygous mutation c.139 G > A was identified in all affected participants. b Sequence of unaffected individual. c Sequence of control. The amino acid reading-frame is indicated, GAT encodes Asp (D), and AAT encodes Asn (N)

Bioinformatics analysis

The potential structure and function impact of the D47N mutation was predicted to affect protein function with a score of 0.00, and could probably be damaging with a score of 1.0 by SIFT and PolyPhen-2, respectively. As shown in Fig. 3, the secondary structure of mutant Cx50 protein was different with wild type. The results stated clearly that the wild-type sheet in COOH- terminal portion is likely missing in the D47N mutant. Took the structure of Cx-26 as template, the model structure of the mutant Cx50 have distinct changes (Fig. 4). There are additional helix (red arrow) and shortened sheet (green arrow) in the D47N mutant.
Fig. 3

Comparison of the secondary structure of wild type and mutant. The red line indicates the position of 139 G > A

Fig. 4

Structure homology modeling and comparison of muant protein and wild type Cx 50. a Wild type Cx50. b Mutant protein Cx50

Discussion

In the current study, we confirmed a missense mutation c. 139 G > A in Cx50 (GJA8) in a six-generation Chinese pedigree with congenital cataract. This mutation resulted in an asparagine substitution for aspartic at amino acid residue 47 (D47N).

Cataracts are defined as opacification of the normally transparent crystalline lens, and are the leading cause of vision loss in the world. Congenital cataract is a type of cataract that emerges at birth or during early childhood [5, 18]. The abnormality of lens can interfere with normal development of eyes [5, 19]. Congenital cataracts can be inherited or familial, either as an isolated lens phenotype or as part of a genetic/metabolic disorder, commonly with full penetrance and autosomal dominant transmission [19]. Genetic factors play an important role in congenital cataract [20]. Gene mutations that affecting the lens development during embryonic period are considered to be the main cause [18]. Up to now, more than 39 genes and loci have been confirmed to be involved in the formation of isolate cataract [21, 22], including crystallins, such as α-, β-, γ-crystallins (e.g., CRYAA, CRYBB1, CRYBB2, CRYGD), membrane transport and channel proteins, such as α-connexins (GJA3, GJA8).

Intercellular gap junction channels provide pathways for metabolic and electrical coupling between cells in different tissues, and they are permeable to ions and small solutes, such as ions (K+, Ca2+), nutrients and small metabolites [23]. Gap junction channels consist of connexin protein subunits. Connexin proteins also known as gap junction proteins have four transmembrane domins with two extracellular loops (E1 and E2) and three intracellular regions (the NH2-terminus, a cytoplasmic loop and the COOH-terminus) [24]. Three isoforms of the connexin gene family- Cx43 (GJA1), Cx46 (GJA3) and Cx50 (GJA8) are abundantly expressed in the vertebrate lens.

Cx50 is an important protein and play an important role during lens growth, maturation of lens fiber cells, and lens transparency [25]. Cx50 comprises two exons with exon-2 coding for the entire 433 amino acid residues of gap junction protein α8 (GJA8). Up to date, at least 32 mutations in Cx50 have been identified to contribute to cataract. Of the 32 coding mutations, 29 result in missense substitutions that are involved in autosomal dominant cataract, and two are frameshift mutation associated with autosomal recessive cataract [6]. The majority of missense substitution are situated in the N-terminal half of the protein, which also contains the conserved connexin domain (amino acids 3–109) [6]. Three types of mutation: D47N, D47H and D47Y indicate that the amino acid at position 47 in GJA8 is a mutational hot spot [2628]. Functional findings showed that D47N mutant expressed in Xenopus oocyte pairs could not form functional gap junction channels. Moreover, co-expression of Cx50D47N with wild-type Cx50 did not inhibit the activity of wild-type Cx50 [29]. The similar behavior was also observed in the mouse Cx50D47A, a mutation underlying the cataracts in the No2 mouse [30]. D47N and D47A mutants were loss-of-function mutants. Cellular level studies showed that the mutation of Cx50 prevented its localization to the plasma membrane. And this may lead to a capacity deficiency of Connexin 50, triggering a complex sequence of events, such as disruption of transmembrane ion gradients, loss of membrane potential, decreased cell growth and subsequent decreased metabolic activity [25, 31]. Cx50 is critical for ball-and-socket structures, actin distribution and fiber cell morphology. Cx50 gap junctional communication through ball-and-socket is important for lens development, especially during rapid, early fiber cell growth [32].

Some limitations of this study should be addressed. First, we did not collect all of pedigree samples, especially the affected individuals in the congenital cataract family. Secondly, we did not perform more experiments, such as cell function experiment of D47N mutant and animal model experiments. Both of these limit our knowledge of more information of the D47N mutant. Nonetheless, advantages in our study should also be acknowledged. Exome sequencing and next-generation sequencing provide a rational approach to screen all candidate genes for inherited cataract or other inherited disease. In addition, exome sequencing and next-generation sequencing are suitable for molecular diagnosis of hereditary diseases. Our finding supports the enormous potential of exome sequencing in molecular diagnosis of single gene disease.

Conclusions

In conclusion, the present research confirmed a recurrent mutation, c.139 G > A (p.D47N) in Cx50 in a six-generation Chinese family with autosomal dominant congenital cataract. This result provided further evidence for Cx50 in association with congenital cataract, and the amino acid at position 47 is a mutational hot-spot. The function of D47N mutation needs to be further certificated in animal mode. In addition, exome sequencing and next-generation sequencing are suitable for molecular diagnosis of hereditary diseases.

Abbreviations

CRYAA

Crystallin Alpha A

CRYAB

Crystallin Alpha B

CRYBA1

Crystallin Beta A1

CRYBA3

Crystallin Beta A3

CRYBA4

Crystallin Beta A4

CRYBB1

Crystallin Beta B1

CRYBB2

Crystallin Beta B2

CRYBB3

Crystallin Beta B3

CRYGA

Crystallin Gamma A

CRYGC

Crystallin Gamma C

CRYGD

Crystallin Gamma D

CRYGS

Crystallin Gamma S

Cx43

Connexin43

Cx46

Connexin46

Cx50

Connexin50

GJA1: 

Gap Junction Protein Alpha 1

GJA3: 

Gap Junction Protein Alpha 3

GJA8: 

Gap Junction Protein Alpha 8

Declarations

Acknowledgments

We thank all participants including healthy control peoples, patients and their families in this study. We also thank all the people who helped us to complete the research successfully.

Consent for publication

Informed written consent was obtained from all participants of the family. If the participants are children (under 16 years of age), the informed consent was signed by their parents.

Funding

This work was supported by a grant from Daqing Oil Field Innovation Fund (No.2015018).

Availability of data and materials

The datasets in the current study are available from the corresponding author on reasonable request.

Authors’ contributions

CS, HM and XC conceived and designed the experiments; CS, JW, XW, and FW performed the experiments; CS, JW, XG, YC, YL, and LZ analyzed the data; HM contributed reagents/materials/analysis tools; CS wrote the paper; All authors have read and approved the final manuscript.

Competing interests

The authors announce that they have no affiliations with or involvement in any organization or entity with any financial interest, or non-financial interest in the materials or subject matter discussed in this paper.

Ethics approval and consent to participate

This research was approved by the ethics committee of General Hospital of Daqing Oil Field and was conducted according to the Declaration of Helsinki of the World Medical Association. Informed written consent was obtained from the participants or their legal guardians (if the participant was underage).

Publisher’s Note

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Authors’ Affiliations

(1)
Department of Clinical Diagnosis, General Hospital of Daqing Oil Field
(2)
Department of Ophthalmology, General Hospital of Daqing Oil Field

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