Array-based sequencing of filaggrin gene for comprehensive detection of disease-associated variants

The filaggrin gene (FLG) is essential for skin differentiation and epidermal barrier formation. FLG loss-of-function (LoF) variants are associated with ichthyosis vulgaris and the major genetic risk factor for developing atopic dermatitis (AD).1, 2, 3 Genetic stratification of patients with AD according to FLG LoF risk is a common practice for both research and clinical studies; however, few studies comprehensively sequence the entire FLG coding region. Most studies that include FLG genotyping have screened for common predominant LoF variants to report allele frequencies after full Sanger sequencing of a smaller batch of test patient samples or previously published data. This strategy potentially results in underreporting of the genetic contribution especially in ethnicities where FLG LoF variants are highly diverse.4 Distinct LoF variants have been reported for most ethnicities studied to date. For example, 2 predominant sequence variants (p.R501X and c.2282del4) make up approximately 80% of the mutation burden in northern Europeans,5 whereas in East Asian ethnicities, a larger FLG LoF mutation spectrum is found with fewer predominating variants.6, 7 However, routinely Sanger sequencing the entire FLG coding region for large cohorts is not always feasible, although desirable as it is essential to correctly stratify patients. To address this, we developed a robust and cost-effective high-throughput PCR-based method for analyzing the entire coding region of FLG using Fluidigm microfluidics technology and next-generation sequencing (NGS). We have applied this method to fully resequence cohorts of Chinese, Malay, and Indian patients with AD from the Singaporean population.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Exome Sequencing and Rare Variant Analysis Reveals Multiple Filaggrin Mutations in Bangladeshi Families with Atopic Eczema and Additional Risk Genes

M.P was supported by a Fellowship from the German Research Foundation (DFG). This work received infrastructure support through the DFG Cluster of Excellence “Inflammation at Interfaces” (grants EXC306 and EXC306/2), and was supported by grants (WE2678/6-1, WE2678/6-2, WE2678/9) from the DFG and the e:Med sysINFLAME grant no. 01ZX1306A from the German Federal Ministry of Education and Research (BMBF). J.E.A.C. and X.F.C.C.W. are funded by A*STAR SPF funding for translational skin research and genetic orphan disease

Induced pluripotent stem cell line heterozygous for p.R501X mutation in filaggrin:KCLi003-A

We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay

The Polyamine Regulator AMD1 Upregulates Spermine Levels to Drive Epidermal Differentiation

Maintaining tissue homeostasis depends on a balance of cell proliferation, differentiation and apoptosis. Within the epidermis the levels of the polyamines putrescine, spermidine and spermine are altered in many different skin conditions yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, AMD1, as a crucial regulator of keratinocyte differentiation. AMD1 protein is upregulated on differentiation and highly expressed in the suprabasal layers of the human epidermis. During keratinocyte differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown/inhibition of AMD1 results in reduced spermine levels and inhibition of keratinocyte differentiation. Supplementing AMD1-knockdown keratinocytes with exogenous spermidine/spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signalling proteins that drive keratinocyte differentiation including KLF4 and ZNF750. These findings demonstrate that human keratinocytes use controlled changes in polyamine levels to modulate gene expression to drive cellular behaviour changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or be used in the treatment of hyper-proliferative skin disorders