Dominant frontonasal dysplasia with ectodermal defects results from increased activity of ALX4

Abstract Frontonasal dysplasia (FND) refers to a group of rare developmental disorders characterized by abnormal morphology of the craniofacial region. We studied a family manifesting with clinical features typical for FND2 including neurobehavioral abnormalities, hypotrichosis, hypodontia, and facial dysmorphism. Whole‐exome sequencing analysis identified a novel heterozygous frameshift insertion in ALX4 (c.985_986insGTGC, p.Pro329Argfs*115), encoding aristaless homeobox 4. This and a previously reported dominant FND2‐causing variant are predicted to result in the formation of a similar abnormally elongated protein tail domain. Using a reporter assay, we showed that the elongated ALX4 displays increased activity. ALX4 negatively regulates the Wnt/β‐catenin pathway and accordingly, patient keratinocytes showed altered expression of genes associated with the WNT/β‐catenin pathway, which in turn may underlie ectodermal manifestations in FND2. In conclusion, dominant FND2 with ectodermal dysplasia results from frameshift variants in ALX4 exerting a gain‐of‐function effect

One year outcome and predictors of treatment outcome in central serous chorioretinopathy: Multimodal imaging based analysis

Purpose: To evaluate the follow up and treatment outcome of central serous chorioretinopathy (CSCR) based on the new multimodal imaging-based classification and identify the predictors for anatomic and visual outcome. Methods: Retrospective, multicentric study on 95 eyes diagnosed with CSCR and a follow up of at least 12 months were included. Eyes with macular neovascularization, atypical CSCR or any other disease were excluded. Results: At the baseline, observation was advised to 70% eyes with simple CSCR whereas photodynamic therapy (PDT) was performed in 49% eyes with complex CSCR. Over the follow up, decrease in CMT was significantly higher in simple CSCR as compared to complex CSCR (P = 0.008) and the recurrences were significantly more in eyes with lower CMT at baseline (P = 0.0002). Median time of resolution of SRF was 3 months and 6 months in simple and complex CSCR respectively (P = 0.09). For the 12 months follow up, the median fluid free period was greater (P = 0.03) while number of interventions performed was lesser in eyes with simple CSCR as compared to complex CSCR (P = 0.006). Multiple regression analysis showed baseline best corrected visual acuity (BCVA) and baseline persistent SRF to be significantly predictive of BCVA and persistent SRF at 12 months (P < 0.0001, 0.04) respectively. Conclusions: Complex CSCR more often required PDT, was associated with shorter fluid free interval and longer time for SRF resolution. Baseline BCVA and persistent SRF were predictive of final visual and anatomical outcome. The new multimodal imaging based classification is helpful in establishing objective criteria for planning treatment approaches for CSCR. © The Author(s) 2021

Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway

SVEP1 plays a crucial role in epidermal differentiation.

SVEP1 is a recently identified multidomain cell adhesion protein, homologous to the mouse polydom protein, which has been shown to mediate cell-cell adhesion in an integrin-dependent manner in osteogenic cells. In this study, we characterized SVEP1 function in the epidermis. SVEP1 was found by qRT-PCR to be ubiquitously expressed in human tissues, including the skin. Confocal microscopy revealed that SVEP1 is normally mostly expressed in the cytoplasm of basal and suprabasal epidermal cells. Downregulation of SVEP1 expression in primary keratinocytes resulted in decreased expression of major epidermal differentiation markers. Similarly, SVEP1 downregulation was associated with disturbed differentiation and marked epidermal acanthosis in three-dimensional skin equivalents. In contrast, the dispase assay failed to demonstrate significant differences in adhesion between keratinocytes expressing normal vs low levels of SVEP1. Homozygous Svep1 knockout mice were embryonic lethal. Thus, to assess the importance of SVEP1 for normal skin homoeostasis in vivo, we downregulated SVEP1 in zebrafish embryos with a Svep1-specific splice morpholino. Scanning electron microscopy revealed a rugged epidermis with perturbed microridge formation in the centre of the keratinocytes of morphant larvae. Transmission electron microscopy analysis demonstrated abnormal epidermal cell-cell adhesion with disadhesion between cells in Svep1-deficient morphant larvae compared to controls. In summary, our results indicate that SVEP1 plays a critical role during epidermal differentiation. Exp Dermatol 2017 May; 26(5):423-430

Effect of <i>Tspear</i> down-regulation on murine hair follicles.

<p>(a) Tspear is expressed in mouse hair follicles (HFs) in the hair matrix keratinocytes, outer root sheath, inner root sheath, hair shaft and the infundibulum (scale bar = 50 μm); (b) Back skin tissue strips from <i>K14-H2B-GFP</i> mice were transfected with <i>Tspear</i> siRNA or control siRNA. RNA was extracted from transfected HFs and <i>Tspear</i> RNA expression levels were assessed by qRT-PCR. Results were normalized to <i>Gapdh</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; **p<0.01); (c-f) Z stacks optical sections of <i>K14-H2B-GFP</i> mouse HFs (c) obtained 24h following transfection with control siRNA (d) or <i>Tspear</i> siRNA (e) were used to calculate average hair bulb diameter. Three measurements were done for each HF in the bulb and proximal hair shaft (c, dashed white lines) and an average diameter was calculated accordingly. Epithelial nuclei are marked with <i>GFP</i> (scale bars = 100 μm). Data was pooled from three independent experiments (F, two sided t-test; **p<0.01); (g-i) Melanin content was assessed by quantitative Masson-Fontana histochemistry in <i>Tspear</i> siRNA treated HFs (h) compared to control (g). Data was pooled from two independent experiments (I, two sided t-test; ***p<0.001) (scale bars = 50 μm); (j-o) Apoptosis was assessed by the TUNEL assay (TUNEL, green; DAPI, blue) at the hair bulb (j-l) and infundibular (m-o) compartments of HFs downregulated for <i>Tspear</i> (k,n) compared to control siRNA treated HFs (j,m) (scale bars = 50 μm). Average number of TUNEL-positive cells in hair follicles in the respective compartments. Data were pooled from two independent experiments (l,o, two sided t-test; ***p<0.001) (scale bars = 50 μm). White dotted lines delineate the outer epidermal surface; (p) RNA was extracted from <i>Tspear</i> siRNA and control siRNA transfected HFs and <i>Notch1</i> RNA expression level was assessed by qRT-PCR. Results were normalized to <i>Rplp0</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; *p<0.05). E—epidermis; INF–Infundibulum; D—dermis; DP—dermal papilla; IRS—inner root sheath; ORS—outer root sheath; HM—hair matrix; HS—hair shaft; TUNEL—terminal deoxynucleotidyl transferase dUTP nick end labeling.</p

Effect of <i>Tspear</i> down-regulation on murine hair follicles.

<p>(a) Tspear is expressed in mouse hair follicles (HFs) in the hair matrix keratinocytes, outer root sheath, inner root sheath, hair shaft and the infundibulum (scale bar = 50 μm); (b) Back skin tissue strips from <i>K14-H2B-GFP</i> mice were transfected with <i>Tspear</i> siRNA or control siRNA. RNA was extracted from transfected HFs and <i>Tspear</i> RNA expression levels were assessed by qRT-PCR. Results were normalized to <i>Gapdh</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; **p<0.01); (c-f) Z stacks optical sections of <i>K14-H2B-GFP</i> mouse HFs (c) obtained 24h following transfection with control siRNA (d) or <i>Tspear</i> siRNA (e) were used to calculate average hair bulb diameter. Three measurements were done for each HF in the bulb and proximal hair shaft (c, dashed white lines) and an average diameter was calculated accordingly. Epithelial nuclei are marked with <i>GFP</i> (scale bars = 100 μm). Data was pooled from three independent experiments (F, two sided t-test; **p<0.01); (g-i) Melanin content was assessed by quantitative Masson-Fontana histochemistry in <i>Tspear</i> siRNA treated HFs (h) compared to control (g). Data was pooled from two independent experiments (I, two sided t-test; ***p<0.001) (scale bars = 50 μm); (j-o) Apoptosis was assessed by the TUNEL assay (TUNEL, green; DAPI, blue) at the hair bulb (j-l) and infundibular (m-o) compartments of HFs downregulated for <i>Tspear</i> (k,n) compared to control siRNA treated HFs (j,m) (scale bars = 50 μm). Average number of TUNEL-positive cells in hair follicles in the respective compartments. Data were pooled from two independent experiments (l,o, two sided t-test; ***p<0.001) (scale bars = 50 μm). White dotted lines delineate the outer epidermal surface; (p) RNA was extracted from <i>Tspear</i> siRNA and control siRNA transfected HFs and <i>Notch1</i> RNA expression level was assessed by qRT-PCR. Results were normalized to <i>Rplp0</i> levels and are expressed as expression levels relative to control samples. Data were pooled from three independent experiments (two sided t-test; *p<0.05). E—epidermis; INF–Infundibulum; D—dermis; DP—dermal papilla; IRS—inner root sheath; ORS—outer root sheath; HM—hair matrix; HS—hair shaft; TUNEL—terminal deoxynucleotidyl transferase dUTP nick end labeling.</p

Expression analysis.

<p>(a) A comparative analysis of gene expression profiles of primary keratinocytes transfected with <i>TSPEAR</i> specific siRNA or control siRNA (left panel) revealed a number of differentially expressed genes including <i>NOTCH1</i> and additional genes encoding elements of the <i>NOTCH1</i> regulatory network and/or known to be involved in hair and tooth development. Pathway analysis (IPA software, see details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006369#sec007" target="_blank">Materials and Methods</a>, right panel) revealed that <i>TSPEAR</i> down-regulation affects a <i>NOTCH</i>-associated regulatory network; (b) Gene expression following siRNA-mediated <i>TSPEAR</i> down-regulation was assessed using qRT-PCR. Results are expressed as percentage of gene expression in cells transfected with <i>TSPEAR</i>-specific siRNA relative to gene expression in siRNA control-transfected cells ± standard error (two sided t-test; *p<0.05, **p<0.01). Results are normalized to <i>GAPDH</i> RNA levels; (c,d) NOTCH1 expression was assessed by immunostaining (c) in skin biopsies obtained from an affected individual (IV-4, family A; right panel) and from a healthy individual (left panel). Immunostaining was significantly reduced in affected vs. normal skin (d) (scale bars = 25 μm; (e) HaCaT cells were co-transfected with a NOTCH1-responsive luciferase reporter gene and <i>TSPEAR</i>-specific siRNA or control siRNA. Luciferase activity was measured after 48 hours and normalized to Renilla luciferase. Results represent the mean of three independent experiments ± standard error (two sided t-test; ***p<0.001).</p

Clinical and pathological features.

<p>(a) Family pedigrees are presented in the upper panels. Black symbols denote affected individuals. PCR-RFLP assays, performed as detailed in materials and methods, were used in each family to confirm co-segregation of the mutation with the disease phenotype (lower panels). Mutation c.1728delC is associated with the presence of a 108 bp fragment in families A and B, while mutation c.454_457delCTGG results in a 514 and 256 bp fragments in family B; in addition, both mutations c.1852T>A and c.1915G>A are associated with the presence of a 180 bp fragment in family C; (b-d) Clinical features displayed by the patients include (b) hypodontia with conical teeth, (c) anterior scalp hypotrichosis and (d) follicular accentuation; (e) A skin biopsy obtained from scalp skin of individual IV-4 of family A and stained for hematoxylin and eosin, demonstrates paucity of rudimentary hair follicles; (f-g) Scanning electron microscopy (SEM) analysis of hair shafts obtained from the scalp demonstrates flattened and partially absent cuticular scales (arrows) in the patient hair (f) as compared with a healthy individual (g) (scale bar = 100 μm).</p