Whole Exome Sequencing in Individuals with Idiopathic Clubfoot Reveals a Recurrent Filamin B (FLNB) Deletion.

BACKGROUND: Clubfoot, a congenital deformity that presents as a rigid, inward turning of the foot, affects approximately 1 in 1000 infants and occurs as an isolated birth defect in 80% of patients. Despite its high level of heritability, few causative genes have been identified, and mutations in known genes are only responsible for a small portion of clubfoot heritability. QUESTIONS/PURPOSES: (1) Are any rare gene variants enriched (that is, shared) in unrelated patients with isolated clubfoot? (2) Are there other rare variants in the identified gene (Filamin B) in these patients with clubfoot? METHODS: Whole-exome sequence data were generated from a discovery cohort of 183 unrelated probands with clubfoot and 2492 controls. Variants were filtered with minor allele frequency < 0.02 to identify rare variants as well as small insertions and deletions (indels) resulting in missense variants, nonsense or premature truncation, or in-frame deletions. A candidate deletion was then genotyped in another cohort of 974 unrelated patients with clubfoot (a replication cohort). Other rare variants in the candidate gene were also investigated. A segregation analysis was performed in multigenerational families of individuals with clubfoot to see if the genotypes segregate with phenotypes. Single-variant association analysis was performed using the Fisher two-tailed exact test (exact p values are presented to give an indication of the magnitude of the association). RESULTS: There were no recurrent variants in the known genes causing clubfoot in this study. A three-base pair in-frame codon deletion of Filamin B (FLNB) (p.E1792del, rs1470699812) was identified in 1.6% (3 of 183) of probands with clubfoot in the discovery cohort compared with 0% of controls (0 of 2492) (odds ratio infinity (inf) [95% CI 5.64 to inf]; p = 3.18 x 10-5) and 0.0016% of gnomAD controls (2 of 125,709) (OR 1.01 x 103 [95% CI 117.42 to 1.64 x 104]; p = 3.13 x 10-8). By screening a replication cohort (n = 974 patients), we found two probands with the identical FLNB deletion. In total, the deletion was identified in 0.43% (5 of 1157) of probands with clubfoot compared with 0% of controls and 0.0016% of gnomAD controls (OR 268.5 [95% CI 43.68 to 2.88 x 103]; p = 1.43 x 10-9). The recurrent FLNB p.E1792del variant segregated with clubfoot, with incomplete penetrance in two families. Affected individuals were more likely to be male and have bilateral clubfoot. Although most patients had isolated clubfoot, features consistent with Larsen syndrome, including upper extremity abnormalities such as elbow and thumb hypermobility and wide, flat thumbs, were noted in affected members of one family. We identified 19 additional rare FLNB missense variants located throughout the gene in patients with clubfoot. One of these missense variants, FLNB p.G2397D, exhibited incomplete penetrance in one family. CONCLUSION: A recurrent FLNB E1792 deletion was identified in 0.43% of 1157 isolated patients with clubfoot. Given the absence of any recurrent variants in our discovery phase (n = 183) for any of the known genes causing clubfoot, our findings support that novel and rare missense variants in FLNB in patients with clubfoot, although rare, may be among the most commonly known genetic causes of clubfoot. Patients with FLNB variants often have isolated clubfoot, but they and their family members may be at an increased risk of having additional clinical features consistent with Larsen syndrome. CLINICAL RELEVANCE: Identification of FLNB variants may be useful for determining clubfoot recurrence risk and comorbidities.

Selective sweep for an enhancer involucrin allele identifies skin barrier adaptation out of Africa.

The genetic modules that contribute to human evolution are poorly understood. Here we investigate positive selection in the Epidermal Differentiation Complex locus for skin barrier adaptation in diverse HapMap human populations (CEU, JPT/CHB, and YRI). Using Composite of Multiple Signals and iSAFE, we identify selective sweeps for LCE1A-SMCP and involucrin (IVL) haplotypes associated with human migration out-of-Africa, reaching near fixation in European populations. CEU-IVL is associated with increased IVL expression and a known epidermis-specific enhancer. CRISPR/Cas9 deletion of the orthologous mouse enhancer in vivo reveals a functional requirement for the enhancer to regulate Ivl expression in cis. Reporter assays confirm increased regulatory and additive enhancer effects of CEU-specific polymorphisms identified at predicted IRF1 and NFIC binding sites in the IVL enhancer (rs4845327) and its promoter (rs1854779). Together, our results identify a selective sweep for a cis regulatory module for CEU-IVL, highlighting human skin barrier evolution for increased IVL expression out-of-Africa.

Tiled array-based sequencing identifies enrichment of loss-of-function variants in the highly homologous filaggrin gene in African-American children with severe atopic dermatitis.

Filaggrin (FLG) loss-of-function (LOF) variants are a major risk factor for the common inflammatory skin disease, atopic dermatitis (AD) and are often population-specific. African-American (AA) children are disproportionately affected with AD, often later developing asthma and/or allergic rhinitis and comprise an atopy health disparity group for which the role of FLG LOF is not well known. Discovery of FLG LOF using exome sequencing is challenging given the known difficulties for accurate short-read alignment to FLG's high homology repeat variation. Here, we employed an array-based sequencing approach to tile across each FLG repeat and discover FLG LOF in a well-characterized cohort of AA children with moderate-to-severe AD. Five FLG LOF were identified in 23% of our cohort. Two novel FLG LOF singletons, c.488delG and p.S3101*, were discovered as well as p.R501*, p.R826* and p.S3316* previously reported for AD. p.S3316* (rs149484917) is likely an African ancestral FLG LOF, reported in African individuals in ExAC (Exome Aggregation Consortium), Exome Variant Server (ESP), and 4 African 1000G population databases (ESN, MSL, ASW, and ACB). The proportion of FLG LOF (11.5%) among the total FLG alleles in our cohort was significantly higher in comparisons with FLG LOF reported for African individuals in ExAC (2.5%; P = 4.3 × 10-4 ) and ESP (1.7%; P = 3.5 × 10-5 ) suggesting a disease-enrichment effect for FLG LOF. Our results demonstrate the utility of array-based sequencing in discovering FLG LOF, including novel and population-specific, which are of higher prevalence in our AA severe AD group than previously reported.

Regulation of the dynamic chromatin architecture of the epidermal differentiation complex is mediated by a c-Jun/AP-1-modulated enhancer.

The epidermal differentiation complex (EDC) locus comprises a syntenic and linear cluster of genes whose concomitant expression is a hallmark feature of differentiation in the developing skin epidermis. Many of the EDC proteins are cross-linked together to form the cornified envelope, an essential and discrete unit of the mammalian skin barrier. The mechanism underlying coordinate transcriptional activation of the EDC is unknown. Within the human EDC, we identified an epidermal-specific regulatory enhancer, 923, which responded to the developmental and spatiotemporal cues at the onset of epidermal differentiation in the mouse embryo. Comparative chromosomal conformation capture assays in proliferating and differentiated primary mouse keratinocytes revealed multiple physiologically sensitive chromatin interactions between the 923 enhancer and EDC gene promoters, thus depicting the dynamic chromatin topology of the EDC. We elucidate a mechanistic link between c-Jun/AP-1 and 923, whereby AP-1- and 923-mediated EDC chromatin remodeling are required for functional EDC gene activation. Thus, we identify a critical enhancer/transcription factor axis governing the dynamic regulation of the EDC chromatin architecture and gene expression and provide a framework for future studies toward understanding gene regulation in cutaneous diseases.