Hypoparathyroidism-retardation-dysmorphism syndrome—Clinical insights from a large longitudinal cohort in a single medical center

BackgroundHypoparathyroidism, retardation, and dysmorphism (HRD) Syndrome is a rare disease composed of hypoparathyroidism, retardation of both growth and development, and distinctive dysmorphic features. Here, we describe the long-term morbidity and mortality in a large cohort of HRD patients and suggest recommendations for follow up and treatment.MethodsMedical records of 63 HRD syndrome patients who were followed at Soroka Medical Center during 1989–2019 were reviewed retrospectively. Information regarding demographics, medical complications, laboratory findings, and imaging studies was collected.ResultsThe mortality rate was 52%. The main causes of death were infectious diseases including pneumonia, septic shock, and meningitis. Multiple comorbidities were found including brain anomalies in 90% of examined patients (basal ganglia calcifications, tightening of corpus callosum, Chiari malformation, hydrocephalous, and brain atrophy), seizures in 62%, nephrocalcinosis and/or nephrolithiasis in 47%, multiple eye anomalies were recorded in 40%, bowel obstructions in 9.5%, and variable expression of both conductive and senso-neural hearing loss was documented in 9.5%.ConclusionHRD is a severe multisystem disease. Active surveillance is indicated to prevent and treat complications associated with this rare syndrome

Epidermolytic Ichthyosis Sine Epidermolysis

Epidermolytic ichthyosis (EI) is a rare disorder of cornification caused by mutations in KRT1 and KRT10, encoding two suprabasal epidermal keratins. Because of the variable clinical features and severity of the disease, histopathology is often required to correctly direct the molecular analysis. EI is characterized by hyperkeratosis and vacuolar degeneration of the upper epidermis, also known as epidermolytic hyperkeratosis, hence the name of the disease. In the current report, the authors describe members of 2 families presenting with clinical features consistent with EI. The patients were shown to carry classical mutations in KRT1 or KRT10, but did not display epidermolytic changes on histology. These observations underscore the need to remain aware of the limitations of pathological features when considering a diagnosis of EI

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

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

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

Mutation analysis.

<p>(a) Direct sequencing of <i>TSPEAR</i> revealed a homozygous missense transversion c.1726G>T and a homozygous c.1728delC deletion in family A patients; heterozygous c.454_457delCTGG, c.1726G>T and c.1728delC mutations in family B patient; and heterozygous c.1852T>A and c.1915G>A missense mutations in family C patient. Wildtype sequences are given below the mutant sequence for comparison; (b) The predicted consequences of the 4 mutations are depicted along a schematic representation of the TSPEAR protein structure with its different domains.</p