Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg↓) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for ≈20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg159↓- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor α1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription–PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A → G substitution at nucleotide −2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G → A substitution at nucleotide −1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far—the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

Fibroblast growth factor 2 can replace ectodermal signaling for feather development.

The initiation and morphogenesis of cutaneous appendages depend on a series of reciprocal signaling events between the epithelium and mesenchyme of the embryonic skin. In the development of feather germs, early dermal signals induce the formation of epidermal placodes that in turn signal the mesoderm to form dermal condensations immediately beneath them. We find a spatially and temporally restricted pattern of transcription for the genes that encode fibroblast growth factor (FGF) 2 and FGF receptor (FGFR) 1 in developing feather germs of the chicken embryo. FGF-2 expression is restricted to the epidermal placodes, whereas FGFR-1 expression is limited to the dermal condensations. Transcription of these genes could not be detected in skins of scaleless (sc/sc) embryos that fail to develop feathers as a result of an ectodermal defect. Treatment of sc/sc skins with FGF-2 results in the formation of feathers at the site of application of the growth factor and the induced feathers express FGFR-1 in their dermal condensations. Thus, we have established FGF-2 as an epidermal signal in early feather germ formation. The observation that FGF-2 can rescue the mutant phenotype of sc/sc embryos suggests that FGF-2 either is, or is downstream from, the signal that the sc/sc mutant ectoderm fails to generate.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure.

Myotonic dystrophy is caused by an expansion of a CTG triplet repeat sequence in the 3' noncoding region of a protein kinase gene, yet the mechanism by which the triplet repeat expansion causes disease remains unknown. This report demonstrates that a DNase I hypersensitive site is positioned 3' of the triplet repeat in the wild-type allele in both fibroblasts and skeletal muscle cells. In three unrelated individuals with myotonic dystrophy that have large expansions of the triplet repeat, the allele with the triplet repeat expansion exhibited both overall DNase I resistance and inaccessibility of nucleases to the adjacent hypersensitive site. These results indicate that the triplet repeat expansion alters the adjacent chromatin structure, establishing a region of condensed chromatin, and suggests a molecular mechanism for myotonic dystrophy.

Deep Anterior Lamellar Keratoplasty versus Penetrating Keratoplasty for Macular Corneal Dystrophy: A Randomized Trial

Purpose: To compare outcomes of big-bubble deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PK) for macular corneal dystrophy. Design: Prospective, randomized, interventional case series. Methods: Setting: Single hospital. Patients: Eighty-two eyes of 54 patients requiring keratoplasty for the treatment of macular corneal dystrophy without endothelial involvement were included. Main outcome measures: Operative complications, uncorrected visual acuity, best-corrected visual acuity, contrast sensitivity function, higher-order aberrations, and endothelial cell density were evaluated. Results: The DALK and PK group consisted of 35 and 41 eyes, respectively. Best-corrected visual acuity after surgery was 20/40 or better 68.5% and 70.7% of the eyes in the DALK and PK groups, respectively (P > .05). No statistically significant differences between groups were found in contrast sensitivity function with and without glare for any spatial frequency (P > .05). Significantly higher levels of higher-order aberrations were found in the DALK group (P < .01). In both groups, a progressive and statistically significant reduction in endothelial cell density was found (P < .01). At the last follow-up, the mean endothelial cell loss was 18.1% and 26.9% in DALK and PK groups, respectively (P = .03). Graft rejection episodes were seen in 5 eyes (12.1%) in the PK group, and regrafting was necessary in 3 eyes (7.3%). Recurrence of the disease was documented in 5.7% and 4.8% of the eyes in the DALK and PK groups, respectively. Conclusions: Deep anterior lamellar keratoplasty with the big-bubble technique provided comparable visual and optical results as PK and resulted in less endothelial damage, as well as eliminating endothelial rejection in macular corneal dystrophy. Deep anterior lamellar keratoplasty surgery is a viable option for macular corneal dystrophy without endothelial involvement.

Function and Regulation of Cytochrome P450 4V2 and the Implications in Bietti’s Crystalline Dystrophy

Thesis (Ph.D.)--University of Washington, 2016-06

Sleep-related breathing disorder in Duchenne muscular dystrophy: Disease spectrum in the paediatric population

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease with death usually occurring because of respiratory failure. Signs of early respiratory insufficiency are usually first detectable in sleep. Objective: To study the presentation of sleep-related breathing disorder (SRBD) in patients with DMD. Method:> A retrospective review of patients with DMD attending a tertiary paediatric sleep disorder clinic over a 5-year period. Symptoms, lung function and polysomnographic indices were reviewed. Results: A total of 34 patients with DMD were referred for respiratory assessment (1-15 years). Twenty-two (64%) reported sleep-related symptomatology. Forced vital capacity (FVC) was between 12 and 107% predicted (n = 29). Thirty-two progressed to have polysomnography of which 15 were normal studies (median age: 10 years) and 10 (31%) were diagnostic of obstructive sleep apnoea (OSA) (median age: 8 years). A total of 11 patients (32%) showed hypoventilation (median age: 13 years) during the 5-year period and non-invasive ventilation (NIV) was offered to them. The median FVC of this group was 27% predicted. There was a significant improvement in the apnoea/hypopnoea index (AHI) (mean difference = 11.31, 95% CI = 5.91-16.70, P = 0.001) following the institution of NIV. Conclusions: The prevalence of SRBD in DMD is significant. There is a bimodal presentation of SRBD, with OSA found in the first decade and hypoventilation more commonly seen at the beginning of the second decade. Polysomnography is recommended in children with symptoms of OSA, or at the stage of becoming wheelchair-bound. In patients with the early stages of respiratory failure, assessment with polysomnography-identified sleep hypoventilation and assisted in initiating NIV.

Distinct roles for interleukin-12p40 and tumour necrosis factor in resistance to oral candidiasis defined by gene-targeting

Cell-mediated immunity is important for anti-Candida host defence in mucosal tissues. In this study we used cytokine-specific gene knockout mice to investigate the requirement for T helper type 1 (Th1) and Th2 cytokines in recovery from oral candidiasis. Knockout mice used in this study included interleukin-4 (IL-4), IL-10, IL-12p40, interferon-gamma (IFN-gamma), and tumour necrosis factor (TNF). The mice were challenged either orally or systemically with Candida albicans yeasts, and levels of colonization were determined. IL-12p40 knockout mice developed chronic oropharyngeal candidiasis, but were not more susceptible to systemic challenge. On the other hand, TNF knockout mice displayed increased susceptibility to both oral and systemic challenge, but only in the acute stages of infection. TNF apparently has a protective effect in the acute stages of both oral and systemic candidiasis, whereas IL-12p40 is essential for recovery from oral but not systemic candidiasis. The role of IL-12p40, and its relation to T-cell-mediated responses remain to be determined.