Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in ,, or , leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. (laminin β3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin β3-null keratinocytes transfected with expression vectors encoding eight different nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin β3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin β3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6β4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Purpose of review Over the past decade many previously poorly understood vascular malformation disorders have been linked to somatic activating mutations in PIK3CA, which regulates cell survival and growth via activation of the mTOR1-AKT pathway. The goal of this article is to describe and provide an update on the clinical features, complications, and management strategies for the PIK3CA-related overgrowth spectrum (PROS). Recent findings PROS encompasses a heterogenous group of disorders with complications related to the tissues harboring the mutation. Vascular malformation syndromes, such as Klippel–Trenaunay syndrome and Congenital Lipomatous Overgrowth Vascular malformations Epidermal nevi and Skeletal abnormalities, have an increased risk of thromboembolic complications, which is accentuated postprocedurally. Asymmetric overgrowth, particularly of limbs, results in a high rate of orthopedic complications. Hypoglycemia screening in the neonatal period and ongoing monitoring for growth failure is recommended in megalencephaly capillary malformation due to its association with multiple endocrinopathies. Recently, sirolimus, an mTOR1 inhibitor, has shown promise in vascular anomalies and now PROS. PIK3CA direct inhibitor, Alpelisib (BYL719), was recently trialed with significant clinical benefit. Summary As the pathogenesis of these conditions is better elucidated and targeted treatments are developed, recognizing the clinical features, comorbidities, and evolving therapeutic landscape across the PROS spectrum becomes more crucial for optimization of care.

Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3 , LAMB3 , or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients’ skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients’ blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.

Hypoxia-inducible factor-1 (HIF-1), a master transcriptional factor for protecting cells from hypoxia, plays a critical role in spermatogenesis and tumorigenesis. For the past two decades, numerous small molecule inhibitors that block mRNA synthesis, protein translation, or DNA binding of HIF-1α have entered clinical trials. To date, few have advanced to FDA approval for clinical applications due to limited efficacy at their toxicity-tolerable dosages. New windows for developing effective and safe therapeutics require better understanding of the specific mechanism of action. The finding that a chaperone-defective mutant heat shock protein-90-alpha (Hsp90α) blocks spermatogenesis, a known hypoxia-driven process in mouse testis prompted us to focus on the role of Hsp90α in HIF-1α. Here we demonstrate that Hsp90α gene knockout causes a dramatic reduction of the high steady-state level of HIF-1α in the testis, blocking sperm production and causing infertility of the mice. In HIF-1α-dependent tumor cells, we found that Hsp90α forms protein complexes with hypoxia-elevated HIF-1α and Hsp90α knockout prevents hypoxia-induced HIF-1α accumulation. In contrast, downregulation of Hsp90β had little effect on hypoxia-induced accumulation of HIF-1α. Instead, Hsp90β protects signaling molecules responsible for cellular homeostasis from assault by 17-AAG (17-N-allylamino-17-demethoxygeldanamycin), a general ATPase inhibitor of both Hsp90α and Hsp90β. Since targeting Hsp90β gene is lethal in both cultured cells and in mice, our new finding explains the toxicity of the previous inhibitor trials and identifies the specific binding of Hsp90α to HIF-1α as a new therapeutic window for developing safer and more effective treatment of male infertility and cancer.

IMPORTANCE Junctional epidermolysis bullosa (JEB) is an incurable blistering skin disorder with high infant mortality often caused by nonsense variants in the genes that encode laminin 332.OBJECTIVE To evaluate the safety and outcomes following intravenous gentamicin readthrough therapy and subsequent laminin 332 expression in patients with JEB. DESIGN, SETTING, AND PARTICIPANTSThis open-label, pilot nonrandomized clinical trial assessed 1 course of low-or high-dose intravenous gentamicin, including follow-up at 30 and 90 days after treatment. Five pediatric patients with JEB (2 with intermediate JEB and 3 with severe JEB) and confirmed nonsense variants in LAMA3 or LAMB3 in 1 or 2 alleles and decreased expression of laminin 332 at the dermal-epidermal junction of their skin participated in the study, which was performed at a single institution in collaboration with physicians and home infusion services near the patients from