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Abstract
Background: Mutations in the filaggrin (FLG) gene has been reported to be an indicator of poor prognosis of atopic dermatitis (AD). It has been reported that there is a considerable variation in the mutations detected in the FLG gene in different ethnicities. Aim: To detect the presence of mutations in the FLG gene in pediatric subjects with atopic dermatitis (AD) and to compare the detected mutations with those already reported from different ethnicities. Materials and Methods: Genomic DNA extracted using standard procedure from peripheral venous blood of 30 patient and 15 control samples. Sequence analysis of the FLG gene carried out and detected changes was then cross referenced with those mutations already reported to check for novelty of detected changes. Results: Amino acid changes were detected in 28 of the patient samples and in none of the control samples indicating that changes in the FLG gene were more common in the patient group than the control group (Fishers exact test, P < 0.0001). The most commonly reported mutations R501X and 2282del4 were not detected. Only 5 of the detected 22 amino acid changes H2507Q, L2481S, K2444E, E2398Q, and S2366T have been previously reported and are not clinically significant; however, in one patient a stop codon was detected (S2366STOP). P2238N, R2239W, and V2243L detected in 70% of the samples and S2231E detected in 67% of the patient samples have not been reported so far and their clinical significance is yet to be analyzed. Conclusion: Analyses of mutations already reported showed that the changes detected from this study are novel to Indian traits. While this adds on to the minimal data available from the Indian subcontinent further analyses has to be carried out to analyze the pathogenicity of these detected changes on larger samples sizes.
Keywords: Filaggrin gene, FLG gene mutation, molecular analysis, pediatric atopic dermatitis
Introduction 
The first line of protection the body has from the external environment is the skin. A defective skin barrier is a key factor in the development of chronic inflammatory skin disease.[1]
A highly abundant structural protein that facilitates epidermal differentiation and skin barrier formation is the filament aggregating protein (filaggrin).[1]
Atopic dermatitis (AD) is an itchy, chronic, or chronically relapsing, inflammatory skin condition. Onset of the disease is in infancy and early childhood and is an extremely debilitating inflammatory skin disorder which affects 15-20% of children.[2] The development of AD in infancy and subsequent allergic rhinitis and asthma in later childhood is known as the atopic march. The cause for this progressive atopy is multifactorial and studies have shown strong co-relation with underlying genetic changes and time of onset and severity of AD.[3] Abnormal epidermal barrier function and immunological mechanism are thought to be the major etiological factors for atopic dermatitis. Granting that there is a clear environmental aspect to the pathogenesis of AD, this condition has been found to be inheritable and a number of susceptibility loci have been mapped especially on chromosome 1q21.[4]
The first mutations in the FLG gene were reported in the year 2006 in subjects with ichthyosis vulgaris from Scottish, Irish, and European-American populations. Thereafter, studies were done in different parts of the world on the prevalence of FLG gene mutations is various skin disorders such as atopic dermatitis, eczema, etc., It was observed that the mutations reported from European studies[5] were not observed in studies reported from Ethiopia,[6] Japan,[4] China,[7] Korea,[8] etc., suggesting that there were ethnic variations in the mutations reported.
On the Indian front, while no study has been done on FLG mutations in AD and more specifically in pediatric cases of AD, a study was carried out by Handa et al.,[9] on the presence of mutations in FLG gene in hand eczema.
All this data put together brings to light the fact that while mutations in the FLG gene have been proven to be associated with the atopic march, there is great ethnic variability. In order to develop a successful mutational screening module, the information pertaining to the prevalent FLG mutations for each geographical population has to be mapped. It is therefore imperative to develop a global population genetic map for FLG mutations.[10]
Atopic dermatitis (AD) affects 15-20% of children and is the second most common pediatric skin disease. The mutations in the FLG gene are said to be population specific and no study has been reported on the prevalence of FLG mutations in pediatric subjects with AD from India.
This study aims to detect the presence of mutations in the FLG gene in pediatrics subjects with AD from India and to compare the changes detected with those already reported from different ethnicities.
Materials and Methods This study was an observational and analytical cross-sectional study. It is a pilot study which was carried out at two different centers. Sampling method was non-probability sampling.
Ethical clearance was obtained from the Institutional Ethics Committee prior to initiation of the project. Consent and assent forms were prepared both in English and Kannada. Written consent was obtained from parents, as the subjects/controls included in the study are minors. In addition, consent/assent forms were signed by children aged 7 years and above. Thorough clinical history was obtained from the subjects prior to inclusion in either group.
Patients and Healthy controls Selection Criteria:
Patients: Patients with atopic dermatitis (AD) visiting department of Dermatology and Pediatrics, East Point College of Medical Sciences and Research Centre, Bangalore.
Inclusion Criteria:
Children with clinical AD based on Hanifin and Rajka criteria.Age of 0-18 yearsPatients between 8-18 years also need to give their consent/assent.Exclusion Criteria:
1. Children with immunodeficiency and other co-morbidities.
Healthy Controls: Patients without clinical AD, bronchial asthma, allergic rhinitis and without any immunocompromised condition from department of Pediatrics and Dermatology, East Point College of Medical Sciences and Research Centre, Bangalore.
Inclusion Criteria:
Children without clinical AD, consent/assent.Age of 0-18 years
Patients between 8-18 years also need to give their consent/assent.Exclusion Criteria:
1. Children with conditions under the atopic march.
Sample Collection: 3-4 ml of peripheral venous blood was collected aseptically in EDTA vacutainers from 30 patients and 15 controls and was then transported under strict aseptic conditions, maintaining cold chain to the laboratory.
Sample Processing: Genomic DNA was extracted from peripheral venous blood using standard procedures QIAamp DNA Blood midi kit (Qiagen, Inc., Valencia, CA). The FLG gene has three exons. While exon 2 is the initiation codon, the major part of the protein is encoded by the much larger and complex exon 3 which carries approximately 10-12 very similar tandem repeats, thus making the FLG gene unique in its own way. The presence of so many repeats and the ensuing structural complexity of the gene could also be the reason why many studies have not studied the entire coding region of this gene but just explored the already reported mutations in their population of study, which could result in novel mutations not being identified. Therefore, to analyze the entire coding region of the FLG gene, sequence analysis of the third exon of FLG was carried out by PCR amplification of 11 overlapping fragments amplified by 11 sequence specific primer pairs.[11] For each sample, 11 PCR reactions were performed and PCR products were run on Agarose gel. The products were then purified and subjected to Sanger sequencing and were run on ABI 3130xl automated sequencer (Applied Biosystems). The sequence data obtained was assembled to get complete exon 3 sequence and analyzed further for mutations at nucleotide level. The nucleotide sequence of exon 3 was converted to amino acid sequence and analyzed for mutations. Detected changes were then cross referenced with those mutations already reported in the Human Gene Mutation Database (HGMD) and clinvar (https://www.ncbi.nlm.nih.gov/clinvar/?term=FLG%5Bgene%5D &redir=gene; clinical variants database of NCBI) to check for novelty and clinical significance of detected changes.
Statistical analysis
Fisher's exact test was used to analyze the frequency distribution of the genetic variations in the two groups studied. Statistical analysis was done using the GraphPad QuickCalcs Website: http://www.graphpad.com/quickcalcs/ConfInterval1.cfm (Accessed March and April 2022) and also using GraphPad Prism 9.4.1 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com. A P value < 0.05 was considered to be statistically significant.
Results Mean age of children in the patient group was 9.43 ± 5.03 years while that of the children in the control group was 8.86 ± 5.71 years. Genomic DNA was extracted from all 45 samples collected (30 patient samples and 15 control samples), amplified, and sequenced successfully. While changes in the DNA sequence was not detected in any of the control samples, changes were detected in the patient samples.
Of the 30 patient samples that were collected, 28 of them were missense mutations, i.e., a single base pair substitution in the DNA sequence resulted in a change in the amino acid [Table 1]. The changes have been detected in repeat regions 6 and 7 of the FLG gene.
Table 1: Amino acid changes detected in the FLG gene in patients with ADSingle nucleotide polymorphisms leading to 22 different amino acid changes were detected and samples had different permutation and combinations of these changes and it was noted that some patients were found to have the same set of amino acid changes; however, a successful correlation of these changes with the clinical features could not be established [Table 2] and [Table 3]. P2238N, R2239W, and V2243L amino acid changes were the most common changes detected followed by S2231E amino acid change in the patient samples.
No changes were detected in any of the 15 control samples that were collected, indicating that changes in the FLG gene are more common in patients with atopic dermatitis (Fishers exact test; P < 0.0001).
All changes detected were cross referenced with reported data available from websites mentioned earlier. Of the 22 amino acid changes, only 5 of them, have been previously reported [Table 4]. Of the previously reported mutations, 40% of the patients had the S2366T mutation which was reported as benign, however, in one patient a STOP codon was detected, i.e. S2366STOP. Two mutations (R3435C and G2226V) were heterozygous. Other mutations were homozygous (Table 1). Hardy-Weinberg Equilibrium was not tested for these observed mutations.
Table 4: Previously reported changes also detected in this study; percentage of patients carrying the reported changes Discussion This is one of the first studies from Asia to have sequenced the entire coding region of the FLG gene, albeit on a small sample size. The sequencing of the entire coding region of the FLG gene in pediatric patients with AD brought to light the presence of 22 new missense mutations. Of these, 5 of them H2507Q, L2481S, K2444E, E2398Q, and S2366T have been previously reported and were reported to be not clinically significant, while the remaining 17 changes have not been reported so far. The S2366STOP variant was detected in one patient. The presence of a STOP codon indicates a signal to terminate protein synthesis, and therefore, this change could be pathogenic. However, further analysis using techniques such as nanopore sequencing is required to confirm the impact of this change. The P2238N, R2239W, and V2243L mutations, located in the repeat region 6 and 7 of the coding region of the FLG gene, were the most commonly detected (70%), followed by S2231E mutation (67%) also located in the repeat region 6 and 7 of the coding region of the FLG gene.
It has been reported that there is a great ethnic variation in the type of mutations present in the FLG gene. Most of the studies reported so far have been done either on adult AD or conditions such as ichtyosis vulgaris. The most common mutation reported in the FLG gene, R501X and 2282del4 was not detected in this study. Other novel mutations reported from Korea, Japan, and other Asian countries were not detected as well [Table 5]. To, the best of our knowledge 17 amino acid changes detected in this study have not been reported by any other study so far in adults or children. One reason for this could be that very few studies have analyzed the entire coding region of the FLG gene.
[18]This study brings to light the presence of changes in the FLG gene in pediatric patients with AD from the Indian population. The pathogenicity of these newly discovered mutations on the manifestation of pediatric atopic dermatitis needs to be explored and is beyond the scope of this study. A more meaningful correlation maybe obtained if a significantly more number of cases as well as controls can be studied and analyzed. However, the data obtained sheds light on the possibility of finding a possible association between changes in the FLG gene and atopic dermatitis since no changes were detected in the control group. Keeping in mind the vast ethnic differences reported in the presence of mutations in the FLG gene in atopic dermatitis, the study data adds on to the knowledge pool of the unexplored area of pediatric atopic dermatitis in India. Analyses of mutations already reported showed that the changes detected from this study are novel to Indian traits. While this adds on to the minimal data available from the Indian subcontinent further analyses has to be carried out to analyze the pathogenicity of these detected changes on larger samples sizes.
Limitations
The present study has few limitations. Firstly, the sample size of patients was less for studying the association of the diseased cases (Phenotype) with genotype. We observed several missense mutations which could not correlated to specific diseased conditions of the patients. Secondly, the study was confined to one geographic area. Taking into consideration, the vast diversity of the country and the population, a meaningful conclusion can be drawn from the multicentric study and relating to clinical conditions. Thirdly, the inclusion criteria were based on history of the patients or control and clinical examination. Patients and controls were not examined for autoimmune disease. Skin inflammation caused by aberrant innate immune responses characterizes atopic dermatitis, and a complex dysregulation of innate and adaptive immunity is the cause of autoimmune disorders. Both autoimmune disorders and atopic dermatitis may have similar immunologic mechanisms.
Conclusion In conclusion, the study reveals the existence of several variations at nucleotide and amino acid level in the coding region of FLG gene among Indian population, the clinical significance of which remains unexplored. Finding patients with FLG mutations may make it easier to focus novel treatments strategies. Early life intervention can potentially stop the “atopic march,” lowering the prevalence of asthma and allergic rhinitis.
Financial support and sponsorship
This study was supported financially by the Rajiv Gandhi University of Health Sciences, Bangalore, under the advanced research projects 2019-20 scheme.
Conflicts of interest
There are no conflicts of interest.
References 
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