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.

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 0.8×10 9 /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).

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).
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.

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 SNP, single nucleotide polymorphism; NA, not available.
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][10][11][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 myofibroblastlike 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][23][24][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 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.

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.

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

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

Figure 3 :
Figure 3: Family pedigree of the atopic dermatitis.

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