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

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).

Figure 1

Appearance of red papules and scratches in the young patient

Figure 2

Anterior subcapsular cataracts and posterior subcapsular cataracts in both lens

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.

Figure 4

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

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.

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 [9-12]. 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 [22-25], 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).