Massively Parallel Sequencing of the Filaggrin Gene Reveals an Association Between FLG Loss-of-function Mutations and Leprosy

Filaggrin, encoded by the FLG gene, plays a crucial role in the barrier function of epidermis, but the association between FLG loss-of-function mutations and infectious skin diseases has not been systematically studied. FLG coding sequences from 945 patients with leprosy and 916 healthy controls were captured and enriched using an array-based high-throughput system, and subjected to next-generation sequencing. The loss-of-function mutations found were further validated by Sanger sequencing. A total of 21 loss-of-function mutations were found in 945 patients with leprosy, with a carrier rate of 17.53%, while the prevalence of these mutations in 916 healthy controls was 14.77%, which was significantly lower than in patients. Two individual FLG loss-of-function mutations (K4022X and Q1790X) were found to be significantly associated with leprosy. These results suggest a possible role for filaggrin in defending against leprosy pathogens.

T he FLG gene encodes filaggrin, which is the main constituent of keratohyalin granules and is indispens able for proper functioning of the epidermal barrier. Lossoffunction (LoF) mutations in the FLG gene, which cause prestop and frameshift of coding, are the cause of the Mendelian disease ichthyosis vulgaris (IV) (1). A strong association between FLG LoF mutations and atopic dermatitis (AD) has also been shown (2)(3)(4). Based on the speculation that an impaired skin barrier may facilitate the invasion of microbes, the association between FLG mutations and skin infection has been studied in patients with AD, and filaggrin deficiency has been shown to increase patients' susceptibility to bacte rial infection (5). Nevertheless, the association between FLG LoF mutations and infectious skin disease has been little studied. In 2017, the lowfrequency FLG mutation K4022X was shown to increase susceptibility to leprosy, in a genomewide analysis of proteincoding variants in a Chinese population (6), highlighting the involvement of FLG in infectious diseases per se.
Leprosy is a chronic infectious disease caused by Mycobacterium leprae or Mycobacterium lepromatosis, which impairs the skin and peripheral nerves, leading to irreversible disabilities (7). Although infection with the pathogen is needed for the onset of leprosy, genetic factors in the host have long been regarded as a major contributor, with estimated heritability of up to 57% (8)(9)(10)(11)(12). Currently, more than 30 susceptibility genes have been identified as associated with leprosy, most of which are immunerelated genes involved in pathogen recognition and the subsequent immune response (6). However, in the case of FLG, Gene Ontology analysis showed that this gene was not included in any immune networks with other reported leprosyassociated genes, indicating its possible barrier role in defending against infection with Mycobacterium leprae (6). Given that a series of lowfrequency and rare LoF mutations in FLG have been reported in AD and IV (13)(14)(15)(16), the finding of only one leprosyassociated lowfrequency (3.54%) LoF mutation (K4022X) in FLG may be not adequately convincing of the potential role of FLG in leprosy (6).
The aim of the current study was to systematically investigate the association between leprosy and FLG LoF mutations through deep sequencing, and to preliminarily evaluate the effect of barrier deficiency caused by FLG LoF mutations on the onset of infectious skin diseases.

Study subjects and genomic DNA preparation
All patients with leprosy and healthy controls were recruited using uniform criteria from Shandong Provincial Institute of Dermato logy and Venereology, as described previously (6). The study was approved by the institutional review board and all subjects gave written informed consent. Leprosy was diagnosed by at least 2 dermatologists based on clinical manifestations and histopatholo gical examination. Blood samples were taken at the time of patient diagnosis. Genomic DNA was extracted using QuickGene DNA Whole Blood Kit L (AADS Biotec, Neyagawa, Japan) following the manufacturer's instructions.

Statistical analysis
Fisher's exact test was used to compare the allele frequen cies between patients and controls. The odds ratios (ORs) of the significant mutation and the combined FLG LoF muta tion genotype were estimated by logistic regression analysis.

RESULTS
All patients and controls were of Chinese Han ethni city and were enrolled from Shandong province, China. The mean ± standard deviation (SD) age of the 945 patients with leprosy was 63.31 ± 8.52 years (range 23-80 years) and 54.6% (516) of patients were male. The mean ± SD age of the 916 controls was 63.61 ± 11.48 years (range 31-90 years), and 53.60% (491) of controls were male. No sex or age bias was observed between patients and controls.
Regardless of mutation frequency in patients, the distribution of the 21 mutations found across the coding sequence of FLG showed no obvious hot spots. Neverthe less, 17 of 21 mutations (80.95%) were located before the 7 th repeat of exon 3 (Fig. 2).

DISCUSSION
By employing an arraybased highthroughput deep se quencing method, the coding sequence of the filaggrin gene in 945 patients with leprosy and 916 controls was finely mapped. The overall carrier rate of 21 FLG LoF mutations was found to be significantly higher in patients with leprosy than in healthy controls. Two individual mutations (K4022X and Q1790X) were shown to be significantly associated with leprosy. It is well known that the FLG LoF mutations, which can cause prestop and frameshift of coding leading to a deficiency of filaggrin, can be directly treated as causal variants without further functional analysis (14). Thus, our findings are the first, to our knowledge, to indicate a role of the FLG gene in the pathogenesis of leprosy.
FLG sequencing remains a challenge because exon 3 of FLG is very large (>12 kb) and highly repetitive (2), which makes direct sequencing of FLG using the Sanger sequencing method difficult, costly and time consuming. Hence, most previous studies focused only on the genotyping of known predominant LoF mutations in patients, and few studies of the sequencing of the en tire FLG gene were reported in large cohorts. In recent years, efforts have been made to sequence FLG using targetregion enrichment based on the nextgeneration sequencing (NGS) method. In 2017, a probe capture enrichmentbased NGS method (Roche NimbleGen) was first used to sequence FLG in 54 Chinese Han IV patients (13). Another probe capture systembased NGS method (Agilent SureDesign, Agilent Technologies) was also developed and used for FLG sequencing in 741 children with AD (15,19). In addition to these 2 probe capturebased enrichment methods, a highthroughput PCR enrichment method (Access Array, Fluidigm), fol lowed by NGS, was also developed and used to sequence FLG in 334 Singaporean patients with IV or AD (16). Those studies not only confirmed the known FLG LoF mutations, but also found several novel LoF mutations. Herein, using the highthroughput PCR enrichment method, NGS of the FLG coding sequence was perfor med in 945 patients with leprosy and 916 healthy con trols. In total, we identified 21 FLG LoF mutations and, among them, 9 were newly reported, highlighting again the efficacy of the NGS method in FLG sequencing.
Although M. leprae, the pathogenic agent of leprosy, was discovered more than 100 years ago, the transmis sion patterns of M. leprae are still not fully understood (20). The most widely accepted transmission pathway is the upper respiratory tract (21), but several studies have reported M. leprae infection following trauma (20), such as tattoos (22)(23)(24), injury with a glass bangle (25) and knee injury (26), which suggests the possibility of infection through impaired skin. A previous study found that FLG LoF mutations could increase the risk of recurrent skin bacterial infections in patients with AD (5). Moreover, knockdown of FLG was shown to increase the colonization of Staphylococcus aureus in an epidermal model (27). The current study found that the carrier rate of overall FLG LoF mutations and 2 indivi dual mutations were significantly higher in patients with leprosy than in healthy controls, indicating the possible pathogenic role of the FLG gene in leprosy, which sug gested that an impaired skin barrier may facilitate the transmission of M. leprae through direct skin contact.
In addition to barrier function, filaggrin has also been demonstrated to have immune functions. Filag grin deficiencies were reported to cause immunological dysregulation, such as increased CD11c expression in Langerhans cells, increased systemic Th17 response and decreased expression of IFNγ in keratinocytes (28)(29)(30). Considering that Langerhans cells and keratinocytes play roles in the pathogenesis of leprosy, and that Th17 cells are also an important T cell subset involved in the immune response to leprosy (31,32), it is possible that immune dysregulation caused by FLG LoF mutations also contribute to the pathogenesis of leprosy. Further research, using animal or human epidermis models, will be needed to confirm this proposal.
Similar to other studies on FLG in AD or IV, our studies have focused only on the variants that cause prestop or frameshift of coding, and other types of mutations, such as missense mutations, were not included (13,15,16). This was based on the conventional opinion that truncated filaggrin proteins cannot function normally, as with the homozygous R501X, which has been shown to cause the loss of keratohyalin granules in the epidermis granular layer (1). In the current study, all 21 mutations could cause stopgain and frameshift of coding, genera ting truncated protein. In particular, 17 of 21 (80.95%) LoF mutations located before the seventh repeat of exon 3 could cause at least a 40% loss in exon 3, with severe effects on filaggrin function. Another limitation of this study is that the pvalue (p = 0.041) was not corrected for multiple tests, which made it necessary to verify our discovery in other populations and samples with larger sample size.
In summary, a comprehensive analysis of FLG and leprosy using NGS sequencing was performed in 945 patients with leprosy and 916 healthy controls, revealing a significant association between FLG LoF mutations and leprosy. Further investigations will be required to reveal the potential barrier and immunological roles of filaggrin in leprosy pathogenesis.