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Research Papers:

Family-based whole exome sequencing of atopic dermatitis complicated with cataracts

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Oncotarget. 2017; 8:59446-59454. https://doi.org/10.18632/oncotarget.19739

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Wenxin Luo, Wangdong Xu, Lin Xia, Dan Xie, Lin Wang, Zaipei Guo, Yue Cheng, Yi Liu and Weimin Li _

Abstract

Wenxin Luo1,*, Wangdong Xu2,*, Lin Xia3, Dan Xie3, Lin Wang4, Zaipei Guo5, Yue Cheng1, Yi Liu2 and Weimin Li1

1Department of Respiratory and Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China

2Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China

3State Key Laboratory of Biotherapy and Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China

4Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China

5Department of Dermatology, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China

*Wenxin Luo and Wangdong Xu contributed equally to this work, and should be considered as the co-first author

Correspondence to:

Weimin Li, email: weimin003@163.com

Keywords: atopic dermatitis, cataracts, mutation

Received: November 07, 2016     Accepted: June 02, 2017     Published: July 31, 2017

ABSTRACT

Background: Atopic dermatitis (AD) is a common skin disorder with elevated prevalence. Cataract induced by AD rarely occurs in adolescent and young adult patients, which is also called atopic cataract. Using whole exome sequencing, we aimed to explore genetic alterations among AD and atopic cataract.

Result: We recruited a 19 year-old Chinese male with AD accompanied with cataracts, his father with AD and his mother without AD or cataract. Through analysis of the exomic sequence of the 3 individuals from the same family, we identified that with respect to AD, there were 162 genes mutated in both this patient and his father but not in his mother. In addition, we found 10 genes mutated in this patient only without in his parents according to cataract.

Conclusion: This research suggests that coinheritance of mutations in these genes may correlate with AD, and the pathogenesis of AD complicated with cataracts was related to genetic factors.


INTRODUCTION

Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease with a worldwide prevalence of 8.7-18.1% in children [1] and 1.5-10.2% in adults [2]. It is characterized by continual itchiness, flares and sleep disturbance, negatively regulating the occupational activities and social relationships of patients, the quality of life of patients and their families [3]. Studies have convinced of a combination of genetic and environmental factors in the pathogenesis of AD. Genetic evidence depicts a complex network comprising epidermal barrier dysfunctions and dysregulation of innate and adaptive immunity in this disease. It has been accepted that mutations in the human filaggrin (FLG) gene are the most significant and well-replicated genetic mutations related to AD. Some other mutations such as SPINK5, SPRR3, and CLDN1 may also correlate with epidermal barriers linked to AD. Genetic variants are able to contribute to the abnormal innate and adaptive responses, such as mutations in IL-1 family cytokines and receptors genes, vitamin D pathway genes, Th2 cytokines genes [4]. A cataract is a clouding of the lens that reduces light transmission to the retina, and it decreases the visual acuity of the bearer. It is one of the severe ocular complications of AD manifested in the eyes. The general classification of cataract includes nuclear, cortical and posterior subcapsular subtypes. Here, we focused on a Chinese male with AD complicated with cataracts via the recently developed whole exome sequencing approach, which has been used to determine the genetic basis of rare diseases.

RESULTS

Clinical description

The patient was a 19 year-old Chinese male who was admitted to our hospital with the chief complaint of relapsing generalized skin rash and blurred vision in August, 2015. His rash began from 10 years ago, accompanied with diffuse red papules all over the body, white desquamation, and skin itchiness. He was diagnosed with AD, and treatment without corticosteroids was not effective. There were persistent skin lesions, with obvious itchiness. His skin became dry and flaking, and some area became hard and thick. Seven months ago, his binocular vision became gradually declined. When admitted, red papules and scratches were displayed on the face, neck, trunk, and four extremities, especially on the face and neck (Figure 1). Both eyelids were hard and thick. The right vision was 0.4, and the left vision was 0.1. Keratic precipitates were negative, but both lens were turbid (Figure 2), of which the right one was more severe than the left. Hemogram analysis revealed eosinophile granulocyte 0.8×109/l (12.1% in WBC), and immunological studies showed that expression of IgE was strongly elevated (>3000.00IU/ml). Other investigations such as expression of complements, immune complex, subpopulation of T cells, anti-double stranded DNA antibody, anti-nuclear antibody were normal. Serum allergen test indicated that combination of willow/poplar/elm, crab, shrimp, combination of dermatophagoides pteronyssinus/dermatophagoides culinae were positive allergens. He has a history of eczema and house dust allergy. Interestingly, his father and grandma were diagnosed with AD, respectively (Figure 3).

Appearance of red papules and scratches in the young patient.

Figure 1: Appearance of red papules and scratches in the young patient.

Anterior subcapsular cataracts and posterior subcapsular cataracts in both lens.

Figure 2: Anterior subcapsular cataracts and posterior subcapsular cataracts in both lens.

Family pedigree of the atopic dermatitis.

Figure 3: Family pedigree of the atopic dermatitis.

Genetic analysis

Due to the rarity of cataract occurred in this young male with AD, we hypothesized that an underlying genetic alteration might be present in this patient. We discussed the genetic relationship between the patient and his parents by whole exome sequencing. In order to discover the candidate mutations of AD, we searched for the genes both mutated in this patient and his father but not in his mother. Results showed that 162 genes were both mutated in this patient and his father but not in his mother (Table 1, Supplementary Table 1).

Table 1: List of 162 genes both mutated in the patient and his father by whole exome sequencing

Chromosome

Position

Gene

SNP

Chromosome

Position

Gene

SNP

chr1

93646190

TMED5

rs185712821

C/T

chr10

75563726

NDST2

NA

C/T

chr1

45271238

PLK3

rs55654497

G/A

chr11

5537592

UBQLNL

rs142657773

G/C

chr1

162569107

UAP1

rs190156359

T/A

chr11

74915493

SLCO2B1

rs192050675

C/A

chr1

214537946

PTPN14

rs200340171

G/A

chr11

78369215

TENM4

rs185503085

C/T

chr1

109260438

FNDC7

NA

T/C

chr11

123988461

VWA5A

rs202202178

A/T

chr1

158533225

OR6P1

NA

C/T

chr11

124266877

OR8B3

rs183842912

A/C

chr1

16907303

NBPF1

rs681623

C/T

chr11

10585620

LYVE1

NA

G/T

chr1

17083872

MST1L

rs11545933

G/A

chr11

118376389

KMT2A

NA

C/T

chr1

17263277

CROCC

rs200228265

G/A

chr11

130060375

ST14

NA

C/T

chr1

181019227

MR1

NA

G/A

chr11

3726498

NUP98

NA

G/A

chr1

232561368

SIPA1L2

NA

C/A

chr11

6661388

DCHS1

rs147698268

G/A

chr1

23637401

HNRNPR

NA

C/T

chr11

71249529

KRTAP5-8

rs200162819

G/A

chr1

248813827

OR2T27

rs1782241

T/C

chr12

6669359

NOP2

rs142370738

G/C

chr1

33237103

KIAA1522

NA

C/T

chr12

7475081

ACSM4

rs7485773

C/T

chr1

57411713

C8B

NA

G/C

chr12

105464439

ALDH1L2

rs199841702

G/C

chr1

86355260

COL24A1

NA

C/G

chr12

109217071

SSH1

rs140582047

T/A

chr1

9780232

PIK3CD

NA

G/A

chr12

110221524

TRPV4

rs55728855

C/T

chr2

90249249

IGKV1D-43

NA

T/C

chr12

112150408

ACAD10

rs145407775

C/T

chr2

113343610

CHCHD5

rs199612227

A/G

chr12

124097777

DDX55

rs117200049

G/A

chr2

209108226

IDH1

rs186787509

T/C

chr12

108956430

ISCU

NA

G/C

chr2

233735070

C2orf82

rs200597442

C/G

chr12

11183661

TAS2R31

NA

C/A

chr2

152484095

NEB

NA

C/G

chr12

12966365

DDX47

NA

G/A

chr2

179466289

TTN

NA

C/T

chr12

48104624

ENDOU

NA

C/T

chr2

187627500

FAM171B

NA

A/G

chr12

52885339

KRT6A

rs199613662

C/T

chr2

233675986

GIGYF2

NA

A/G

chr12

6950473

GNB3

NA

C/T

chr2

73315216

RAB11FIP5

NA

A/T

chr13

103419820

TEX30

rs200314758

T/C

chr2

97877478

ANKRD36

rs10194525

G/A

chr13

96242562

DZIP1

NA

T/G

chr3

7728055

GRM7

rs182447901

C/T

chr14

45432003

FAM179B

rs200775208

C/T

chr3

33644578

CLASP2

rs117166070

C/T

chr14

68241828

ZFYVE26

rs193244014

G/C

chr3

49751251

RNF123

rs117758999

G/A

chr14

105415264

AHNAK2

rs201041268

G/A

chr3

112648174

CD200R1

rs188572017

A/T

chr14

32256995

NUBPL

NA

G/A

chr3

151107788

MED12L

rs199780529

T/C

chr14

70925106

ADAM21

NA

T/C

chr3

124351317

KALRN

NA

G/A

chr15

45456025

DUOX1

rs186783799

G/A

chr3

132319977

CCRL1

NA

G/A

chr15

89402346

ACAN

rs188663484

T/C

chr3

40442466

ENTPD3

rs140869368

G/A

chr16

21994499

UQCRC2

NA

T/A

chr4

42119545

BEND4

rs187366202

G/T

chr16

15761154

NDE1

rs147283674

C/T

chr4

47788868

CORIN

rs186748019

C/A

chr16

55530864

MMP2

rs28730814

G/A

chr4

52948557

SPATA18

rs184617860

C/T

chr16

18849442

SMG1

NA

G/A

chr4

186291928

LRP2BP

NA

C/T

chr16

2287576

DNASE1L2

NA

C/T

chr4

4190576

OTOP1

rs2215642

C/G

chr16

28846489

ATXN2L

NA

T/C

chr5

38451559

EGFLAM

rs140968262

A/G

chr16

30100451

TBX6

rs202193096

G/A

chr5

94814011

TTC37

rs143227096

C/A

chr16

456349

DECR2

NA

C/T

chr5

137722246

KDM3B

rs184734460

C/G

chr16

46637519

SHCBP1

NA

A/G

chr5

178507048

ZNF354C

rs116562180

C/G

chr16

67991689

SLC12A4

NA

G/A

chr5

128442753

ISOC1

NA

G/T

chr16

71163611

HYDIN

NA

T/G

chr5

149357850

SLC26A2

NA

G/T

chr16

71961625

IST1

NA

C/G

chr5

171341357

FBXW11

NA

G/T

chr16

84213027

TAF1C

NA

C/G

chr6

26056145

HIST1H1C

rs79483116

G/A

chr17

76166705

SYNGR2

NA

G/A

chr6

27277365

POM121L2

rs61736085

G/A

chr17

36719794

SRCIN1

rs118094989

C/A

chr6

39847207

DAAM2

rs139876341

A/G

chr17

40714796

COASY

rs200009135

G/C

chr6

43017728

CUL7

rs146808129

C/A

chr17

48916935

WFIKKN2

rs35300894

G/A

chr6

83838955

DOPEY1

rs188246058

A/C

chr17

55918596

MRPS23

rs117734846

C/T

chr6

160485490

IGF2R

rs8191859

G/A

chr17

73096776

SLC16A5

rs116126425

G/A

chr6

119628121

MAN1A1

NA

C/T

chr17

11461158

SHISA6

NA

A/G

chr6

143825320

FUCA2

NA

A/G

chr17

12920199

ELAC2

rs140665334

G/A

chr6

34512160

SPDEF

rs375427681

G/A

chr17

14139300

CDRT15

rs11867613

A/G

chr6

34802049

UHRF1BP1

rs368713702

A/G

chr17

14204942

HS3ST3B1

NA

T/C

chr6

39893446

MOCS1

rs377167949

G/A

chr17

26823582

SLC13A2

NA

G/A

chr7

75617513

TMEM120A

rs372363121

C/T

chr17

2966032

OR1D5

rs2676564

C/G

chr7

141464509

TAS2R3

NA

T/C

chr17

5036211

USP6

rs201674756

C/T

chr7

144096938

NOBOX

NA

G/A

chr17

74869016

MGAT5B

NA

G/A

chr7

148801869

ZNF425

NA

C/G

chr18

72913819

ZADH2

rs191356988

A/G

chr7

2689594

TTYH3

NA

G/T

chr18

44584631

KATNAL2

NA

C/T

chr7

2962827

CARD11

rs3735133

G/A

chr19

50028070

FCGRT

rs374439544

C/T

chr8

8748876

MFHAS1

rs201875377

C/A

chr19

54743747

LILRA6

rs10403230

C/G

chr8

17928855

ASAH1

rs11538152

G/A

chr19

4504673

PLIN4

rs201143997

G/A

chr8

21766971

DOK2

rs202013016

G/A

chr19

15730502

CYP4F8

rs61746468

C/T

chr8

42711517

RNF170

rs147488061

T/C

chr19

15839677

OR10H2

NA

T/C

chr8

107691450

OXR1

rs200863692

A/G

chr19

18119274

ARRDC2

NA

G/A

chr8

146067346

ZNF7

NA

A/G

chr19

22846981

ZNF492

NA

A/C

chr8

52733228

PCMTD1

rs73592211

G/A

chr19

40743901

AKT2

NA

C/T

chr8

70541824

SULF1

NA

C/T

chr19

43420636

PSG6

rs370759098

G/A

chr9

2719083

KCNV2

rs143382624

G/C

chr19

58370766

ZNF587

rs77577775

G/A

chr9

18776971

ADAMTSL1

rs117558542

G/A

chr20

31685424

BPIFB4

NA

T/C

chr9

19345978

DENND4C

rs145052586

G/A

chr21

33690064

URB1

rs145519835

C/T

chr9

84226764

TLE1

rs141959893

C/T

chr21

37584306

DOPEY2

rs117132686

C/A

chr9

139750000

MAMDC4

rs200545888

T/C

chr21

19666690

TMPRSS15

NA

C/T

chr9

131670227

LRRC8A

NA

C/T

chr22

22673302

IGLV5-52

NA

C/T

chr10

25314128

THNSL1

rs78131600

C/T

chr22

20127408

ZDHHC8

rs200408305

A/G

chr10

63810739

ARID5B

rs201704836

G/A

chr22

46725974

GTSE1

rs188655025

C/G

chr10

128192832

C10orf90

NA

C/T

chrX

2833605

ARSD

rs111939179

C/T

SNP, single nucleotide polymorphism; NA, not available.

We used OMIM database and GeneCards Database to further interpret these genes and found that 4 genes among the 162 genes might have relationship with the predisposition and/or oncogenesis of AD (Figure 4). To find the candidate mutations of atopic cataracts, we searched for the genes only in this patient without in his parents. We found 10 genes mutated in this patient only without in his parents (Table 2, Supplementary Table 2). Intriguingly, we compared these genes in this special patient with the patients those had been diagnosed with cataracts and had genes mutation, so as to discuss whether these 10 genes are belonging to this special kind of disease. After analyzing the available evidence, we found no data that may suggest these genes have been reported to correlate with cataracts. It is possible that these genes may uniquely belong to AD complicated with cataracts.

Table 2: List of 10 genes mutated in the patient without in his parents by whole exome sequencing

Chromosome

Position

Gene

SNP

chr12

11183066

TAS2R31

rs138895028

A/T

chr15

22473171

IGHV4OR15-8

NA

A/G

chr17

16068287

NCOR1

rs201932638

A/T

chr19

33490566

RHPN2

rs74582927

T/C

chr1

16890607

NBPF1

rs200783506

G/A

chr22

22730788

IGLV5-45

NA

G/A

chr22

22730800

IGLV5-45

rs114116194

A/C

chr2

90249202

IGKV1D-43

NA

G/A

chr2

90249205

IGKV1D-43

NA

A/C

chr5

140594470

PCDHB13

rs17844610

G/A

chr7

142149078

TRBV5-5

NA

T/G

chr7

142149017

TRBV5-5

NA

G/C

chr7

142149029

TRBV5-5

NA

T/G

chr7

142149030

TRBV5-5

NA

C/G

chr7

142149058

TRBV5-5

NA

T/A

chr7

142149059

TRBV5-5

NA

T/C

chr7

142149060

TRBV5-5

NA

T/C

chr7

142149066

TRBV5-5

NA

A/C

chr7

142149071

TRBV5-5

NA

T/C

chr7

142149072

TRBV5-5

NA

G/A

chr7

142149075

TRBV5-5

NA

A/G

chr7

142149086

TRBV5-5

NA

G/A

chr7

142149092

TRBV5-5

NA

T/A

chr7

142149101

TRBV5-5

rs199978351

A/G

chr9

33385750

AQP7

rs114484742

C/T

SNP, single nucleotide polymorphism; NA, not available.

Gene prediction scores of the four genes and residual variation intolerance score of the genes.

Figure 4: Gene prediction scores of the four genes and residual variation intolerance score of the genes.

DISCUSSION

Here, we presented a rare case of AD with cataract, and familial analysis by whole exome sequencing suggested that the pathogenesis of AD was related to genetic factors. Atopic cataract was firstly described in detail in 1936, where the author demonstrated the association of juvenile cataract with AD in 10 out of 101 AD patients, the mean age was 22 year-old, similar to our findings [5]. From 1940 to 1953, an ophthalmological check in 1,158 AD patients showed typical atopic cataract in 136 patients (11.7%) including 79 cases of visual disturbance [6]. To date, literatures describing cataracts in AD are mainly from Asian populations, including the Japanese population reporting the incidence of atopic cataracts around 10-15% [7], Filipino population [8] and Singapore population without Chinese population. Based on this, it seems that a greater interest may exist in Asians, or the prevalence and significance of this disease is greater in these populations. We firstly reported cataracts in a Chinese patient with AD with cataract. Interestingly, his father and his grandma are also AD patients.

It is known that cataract may develop as a result of aging, metabolic disorders, trauma, or heredity. Location of the cataract in the lens regulates visual acuity. There are two types of cataracts in AD patients in subcapsular region, anterior subcapsular cataracts (ASCs) and posterior subcapsular cataracts (PSCs). The literatures about ASCs or PSCs development in AD patients are inconsistent. Disease onset of ASCs is typically rapid, shieldlike bilateral visual impairment [4, 9], therefore, presentation of ASCs seems to be the “classic” cataract because ASCs in the absence of AD is not common [9]. On the contrary, some investigations showed that PSCs may be more common in AD patients [912]. In a 29 year-old male, AD presented with bilateral ASCs [13]. Histopathologic analysis of the ASCs tissues indicated a fibrous and amorphous mass, most likely extracellular matrix owing to the presence of irregularly arranged bundled strands of fibrils, typical of collagen. Lens epithelial cells (LECs) at the plaque were densely packed and myofibroblast-like and immunoreactive for alpha-smooth muscle actin. Similarly, a 6 year-old African American girl presented with an uncontrolled flare of AD, and her medical history was significant for asthma and allergic rhinitis with a family history of AD [14]. This was in agreement with our study that the male patient’s father and grandmother were also AD. Our results showed that ASCs and PSCs were both existed in the left and right lens of the patient (Figure 2).

Although the pathogenesis of AD complicated with cataract has not been clearly elucidated, severe lesions of AD located over the face may be a critical factor in the development of atopic cataracts. In addition, AD complicated with cataracts may correlate with prolonged usage of corticosteroids and repetitive periorbital scratching [11]. Physical examination of the present patient showed a scratch on the face, neck, trunk, and four extremities, especially on the face and neck, suggesting that AD complicated with cataract in this patient may correlate with scratching. However, several studies reported that the presence of cataracts (both ASCs and PSCs) were not correlated with the disease onset, severity, or duration of AD [15, 16], and the clinical features of AD patients who developed cataracts were similar to the patients who did not have. It is notable that cataract was seen in some patients with only mild facial involvement [16, 17]. On the other hand, systemic corticosteroids are known to cause ocular complications. It is reported that incidence of cataract is dose and treatment duration dependent, where the patients received the equivalent of prednisone, 10 to 15 mg/d for at least 1 year displayed the greatest risk [18]. However, Niwa, et al discussed the incidence of cataract among 3 groups of AD patients [11]. The patients were treated with topical corticosteroids, or treated with both topical and systemic corticosteroids, or corticosteroid-naive patients, respectively. The authors found no difference among these groups. Interestingly, there are 37 patients developed cataract, by which 86% showed posterior cataract [11]. This finding was similar to our study, where the patient had no history of corticosteroids. Tatham, et al reported two boys about 10 year-old diagnosed with widespread AD of the face, neck, trunk and limbs. After diagnosis and treatment with topical steroids for 2 years, both of them complained of gradual onset of blurred vision in both eyes, ophtalmic testing found PSCs in these patients, suggesting that AD and topical corticosteroids may be associated with cataracts in children [19]. Together, whether usage of corticosteroids and scratching may be susceptible factors to AD complicated with cataract is still needed to be clarified in the future with large scales of patients.

Genetic epidemiologic studies on monozygotic twins [20], and genetic association studies indicated a genetic susceptibility for AD [21]. In the present study, four genes including CORIN, CARD11, MMP2, DNASE1L, which were previously reported to be risk factors for AD [2225], were also mutated in this patient and his father. CARD11 encodes CARMA1, an essential scaffold protein for lymphocyte activation via T cell receptor and B cell receptor signaling [26]. CARMA1 plays important roles in T cell differentiation, regulation of JunB, GATA3 and the subsequent generation of Th2 cell specific cytokines [27]. Mice that are homozygous for the mutation affecting CARMA1 showed gradual development of AD with high level of serum IgE [28]. Li, et al [29] showed that chronic loss of epidermal caspase-8 recapitulates many aspects of AD, such as a spongiotic phenotype whereby intercellular adhesion between epidermal keratinocytes is disrupted, adversely affecting tissue architecture and function. However, subcutaneous injection of matrix metalloproteinase-2 (MMP2) inhibitor strongly down-regulated the intercellular space found in the suprabasal layers of the epidermis [29]. Suppression of MMP2 also restored full length E-cadherin to normal levels and significantly decreased the amount of the cleaved E-cadherin C-terminal fragments product. Transepidermal water loss through the epidermis from caspase-8 conditional knockout mice treated with the MMP2 inhibitor was strongly reduced relative to controls, suggesting that suppression of MMP2 is able to abrogate the effect of caspase-8 knockout induced AD. In a whole exome sequencing study of early-onset AD from a Korean population, Heo, et al discussed family-specific candidate genetic variants from three separate families, and validated the possible genes in 112 AD patients and 61 controls. Results showed that three variants of the COL6A6 gene appeared in all three families and were in close proximity to AD related loci on chromosome 3q21 [30]. The homozygous frequency for the rs16830494 minor allele (AA) and the rs59021909 (TT) allele and the rs200963433 heterozygous (CT) frequency were all higher in AD cases compared to controls, suggesting that COL6A6 variants may be risk factors for AD.

Matsuda, et al [7] discovered that -56 T allele in the IFNGR1 promoter was significantly associated with an increased risk of ocular AD, especially of atopic cataracts. In our study, the whole exome sequencing revealed the -56 CT genotype in both the patient and his father, which contained -56 T allele, whereas his mother harbored -56 CC genotype. The IFNGR1 gene promoter construct that contained the -56 T allele showed higher transcriptional activity in LECs than did the construct with the -56 C allele after stimulation with IFN-γ, and there was higher IFNGR1 expression in the LECs in atopic than in senile cataracts [7], indicating that the -56 T allele in the IFNGR1 promoter leads to elevated IFNGR1 transcriptional activity and represents a genetic risk factor for atopic cataracts. Hori, et al [25] investigated the role of PAI-1, IFN-γ downstream molecule in the pathogenesis of atopic cataracts. They found that the IFN-γ, PAI-1 and TGF-β1 were involved in the pathophysiology of atopic cataracts.

According to the OMIM database and GeneCards Database, we found 4 genes including CARD11, PIK3CD, LILRB3, C8B, may correlate with the pathogenesis of AD. Among them, CARD11 had been reported to have relation with AD [24]. Phenolyzer were used to examine the association of these candidate genes with AD and we found that PIK3CD, LILRB3, C8B were in the same biosystem with CARD11 in the record of NCBI's Biosystem. According to the result of residual variation intolerance score (RVIS) [31], CARD11 had a RVIS score of-1.39 and a percentile of 4.33%, showing that it was amongst the 4.33% most intolerant of human gene (FDR = 1.87×10-6), and PIK3CD, the 2.72% most intolerant human gene (FDR = 8.11×10-6), had a score of -1.66, while LILRB3 and C8B with positive scores had more common functional variation. The normalized RVIS of CARD11 and PIK3CD was approximated to 1, indicating that these two genes were considered as “intolerant”. PIK3CD had a HI score of 0.607 [32], suggesting that haploinsufficiency of the PIK3CD gene may associate with the pathogenesis of AD (Figure 4).

In conclusion, this is the first report of familial AD with cataracts, and the family-based whole exome sequencing found that 162 genes were both mutated in the young patient and his father, while 10 genes were only mutated in the young patient of AD complicated with cataracts. Further studies with large scale need to discuss the functional role of these genes in AD, especially in AD complicated with cataracts.

MATERIALS AND METHODS

Subjects

There was a 19 year-old young male with AD accompanied with cataracts. His father was AD patient, while his mother was not AD or cataracts patient. All of them were recruited in this study. The grandma was also AD, because of impossibility, the grandma was not included. Patients were collected from the Department of Dermatology of the West China Hospital Sichuan University. AD patients met the diagnostic criteria of Hanifin and Rajka [33]. Data about demographic and clinical features were collected from hospital records or by questionnaire and reviewed by experienced physicians. All subjects gave their written consent to participate before study. This study was approved by the ethics committee of the Sichuan University.

DNA extraction and whole exome sequencing

EDTA anti-coagulated venous blood (10ml) was collected from the young male and his parents. The genomic DNA was extracted using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s protocol. Whole exome enrichment was performed using Agilent SureSelect Human All Exon Kit 50M (Agilent Technologies, Santa Clara, CA, USA) and sequenced with the Illumina HiSeq 4000 System (HiSeq® 3000/4000 SBS Kit).

Sequence alignment, variant calling, and annotation

The sequenced reads were aligned to the hg19 human reference genome sequence using BWA aln and BWA sampe, and removed PCR duplicates with PICARD. Variations were called by GATK HaplotypeCaller with default parameters, after calling genotyping were jointed together by GATK CombineGVCFs/GenotypeGVCFs. Variants were retained considering reads depth DP>= 8, MQ >=20. Beyond that, variants were annotated by ANNOVAR, filtered by position (non-synonymous or gain/loss of stops), VAF < 0.005 (1000 genome project (2012) and HAPMAP), potential damaging effect (variants that were predicted as damaging variants by at least 2 databases, including SIFT, PolyPhen2 HDIV, PolyPhen2 HVAR, LRT, MutationTaste, MutationAssessor, FATHMM, GERP++, PhyloP and SiPhy).

CONFLICTS OF INTEREST

The authors report no declarations of interest.

GRANT SUPPORT

This work was supported by grants from the National Natural Science Foundation of China (81372504 and 81241068).

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