Haplotype-Resolved Analysis of the Filaggrin Gene Elucidates its Complex Role in Human Adaptation and Disease

The filaggrin gene (FLG) encodes a key epidermal barrier protein, and both copy number variations (CNVs) and loss-of-function (LoF) mutations are major contributors to atopic dermatitis (AD) and allergic diseases. However, the highly repetitive structure of FLG has impeded a complete understanding of its functional landscape. Analyzing 697 haplotype-resolved assemblies from ethnically diverse populations, we provide a comprehensive, nucleotide-level atlas of FLG variation. We resolved the sequences of major FLG alleles—defined by both internal repeat number and nucleotide variation—uncovering sequences with 9–20 repeats (expanding beyond the canonical R10, R11, and R12 allele groups) and delineating eight recurrent alleles (e.g., R11.1, R12.1) with ancestry-specific distributions. We identified tag variants for specific FLG alleles and refined population-specific AD burden estimates by correcting misannotated LoF variants. We detected strong signatures of local adaptation in European and southern African Khoisan-speaking populations. We further identified rs1933064 as a causal regulatory variant within an FLG enhancer and demonstrated that the ancestral A allele reduces enhancer activity and FLG expression. This allele is associated with increased AD and asthma risk but higher serum vitamin D levels, and ancient DNA analysis reveals it has risen in frequency over the past ~10,000 years—consistent with positive selection. Collectively, we present an unprecedented sequence-resolved atlas of FLG variation, revealing the contributions of distinct alleles to AD risk, vitamin D biology, and environmental adaptation. Overall design: HaCaT cells were subjected to lentiviral CRISPR perturbation to generate FLG enhancer knockout cells and compared to control HaCaT cells. Cells were infected with lentiviruses carrying Cas9 (lentiCRISPR v2; Addgene 52961) and sgRNA (pLKO5 backbone), selected with puromycin, expanded, and harvested for RNA extraction. RNA-seq was performed on biological replicates of each condition.