Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Autosomal dominant familial acanthosis nigricans caused by a C-terminal nonsense mutation of FGFR3.

FGFR3 encodes a transmembrane receptor tyrosine kinase that has six autophosphorylation sites of tyrosine. Among them, Y770 is a negative regulatory site for the downstream signaling of FGFR3. Constitutive active mutations in FGFR3 are involved in human developmental disorders including familial acanthosis nigricans, an autosomal dominant disorder characterized by general hyperpigmentation with mild acanthosis of the epidermis. Here, we report two unrelated cases of familial acanthosis nigricans with a heterozygous c.2302G>T (p.E768*) mutation in FGFR3 (NM_000142.5). FGFR3 mRNA purified from the skin lesion neither showed aberrant splicing nor nonsense-mediated mRNA decay, indicating that the FGFR3 mutant simply lacked the C-terminal 768-806 amino acids including Y770. While all of the known pathogenic mutations were missense mutations in FGFR3 showing autosomal dominant trait, the c.2302G>T mutation of FGFR3 is a unique autosomal dominant nonsense mutation that causes familial acanthosis nigricans probably via loss of negative regulatory autophosphorylation site of FGFR3.

Mutations in SERPINB7, encoding a member of the serine protease inhibitor superfamily, cause Nagashima-type palmoplantar keratosis.

"Nagashima-type" palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266(∗)) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.

Tubulin polyglutamylation is essential for airway ciliary function through the regulation of beating asymmetry.

Airway epithelial cilia protect the mammalian respiratory system from harmful inhaled materials by providing the force necessary for effective mucociliary clearance. Ciliary beating is asymmetric, composed of clearly distinguished effective and recovery strokes. Neither the importance of nor the essential components responsible for the beating asymmetry has been directly elucidated. We report here that the beating asymmetry is crucial for ciliary function and requires tubulin glutamylation, a unique posttranslational modification that is highly abundant in cilia. WT murine tracheal cilia have an axoneme-intrinsic structural curvature that points in the direction of effective strokes. The axonemal curvature was lost in tracheal cilia from mice with knockout of a tubulin glutamylation-performing enzyme, tubulin tyrosine ligase-like protein 1. Along with the loss of axonemal curvature, the axonemes and tracheal epithelial cilia from these knockout (KO) mice lost beating asymmetry. The loss of beating asymmetry resulted in a reduction of cilia-generated fluid flow in trachea from the KO mice. The KO mice displayed a significant accumulation of mucus in the nasal cavity, and also emitted frequent coughing- or sneezing-like noises. Thus, the beating asymmetry is important for airway ciliary function. Our findings provide evidence that tubulin glutamylation is essential for ciliary function through the regulation of beating asymmetry, and provides insight into the molecular basis underlying the beating asymmetry.

Premature aging syndrome showing random chromosome number instabilities with CDC20 mutation.

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.

Clonal Expansion of Second-Hit Cells with Somatic Recombinations or C>T Transitions Form Porokeratosis in MVD or MVK Mutant Heterozygotes.

Patients with disseminated superficial actinic porokeratosis (DSAP) and linear porokeratosis (LP) exhibit monoallelic germline mutations in genes encoding mevalonate pathway enzymes, such as MVD or MVK. Here, we showed that each skin lesion of DSAP exhibited an individual second hit genetic change in the wild-type allele of the corresponding gene specifically in the epidermis, indicating that a postnatal second hit triggering biallelic deficiency of the gene is required for porokeratosis to develop. Most skin lesions exhibited one of two principal second hits, either somatic homologous recombinations rendering the monoallelic mutation biallelic or C>T transition mutations in the wild-type allele. The second hits differed among DSAP lesions but were identical in those of congenital LP, suggesting that DSAP is attributable to sporadic postnatal second hits and congenital LP to a single second hit in the embryonic period. In the characteristic annular skin lesions of DSAP, the central epidermis featured mostly second hit keratinocytes, and that of the annular ring featured a mixture of such cells and naïve keratinocytes, implying that each lesion reflects the clonal expansion of single second hit keratinocytes. DSAP is therefore a benign intraepidermal neoplasia, which can be included in the genetic tumor disorders explicable by Knudson's two-hit hypothesis.

Collapse of the keratin filament network through the expression of mutant keratin 6c observed in a case of focal plantar keratoderma.

Focal palmoplantar keratoderma (PPK) with severe pain is a hallmark of pachyonychia congenita, a rare autosomal dominant disorder involving PPK and hypertrophic nail dystrophy. Some families present focal PPK with either minimal or no nail changes. Dominant-negative mutations in any of the four identified keratin genes, KRT6A, KRT6B, KRT16 or KRT17, lead to pachyonychia congenita. However, the majority of families with focal PPK showing minimal or no nail changes do not harbor mutations in these genes. Recently, mutations of KRT6C were identified in families with focal PPK alone. Here, we report a 26-year-old Japanese man with focal plantar hyperkeratosis that developed at approximately 10 years of age with no palmar involvement and no nail alterations. We identified a missense KRT6C mutation c.1414G>A resulting in an p.Glu472Lys substitution, as reported in other Japanese patients. When the mutant keratin 6c protein is exogenously expressed in human HaCaT cells, a collapse of the keratin filament network is observed in a dose-dependent manner, suggesting the mutation has a dominant-negative effect on keratin filament network formation. The mutated residue is located at the helix termination motif of keratin 6c. The peptide sequence around this residue is highly conserved among type II, III and IV intermediate filament proteins. Glu to Lys mutations of the equivalent residue have been reported in a variety of inherited diseases, including neurodegenerative diseases, corneal dystrophy and skin disorders, suggesting that this residue is vital to keratin function.

Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS) facilitates surface expression of GluR2-containing AMPA receptors.

Some ubiquitin-like (UBL) domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS) protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP) were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development.

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.

SCRAPPER-dependent ubiquitination of active zone protein RIM1 regulates synaptic vesicle release.

Little is known about how synaptic activity is modulated in the central nervous system. We have identified SCRAPPER, a synapse-localized E3 ubiquitin ligase, which regulates neural transmission. SCRAPPER directly binds and ubiquitinates RIM1, a modulator of presynaptic plasticity. In neurons from Scrapper-knockout (SCR-KO) mice, RIM1 had a longer half-life with significant reduction in ubiquitination, indicating that SCRAPPER is the predominant ubiquitin ligase that mediates RIM1 degradation. As anticipated in a RIM1 degradation defect mutant, SCR-KO mice displayed altered electrophysiological synaptic activity, i.e., increased frequency of miniature excitatory postsynaptic currents. This phenotype of SCR-KO mice was phenocopied by RIM1 overexpression and could be rescued by re-expression of SCRAPPER or knockdown of RIM1. The acute effects of proteasome inhibitors, such as upregulation of RIM1 and the release probability, were blocked by the impairment of SCRAPPER. Thus, SCRAPPER has an essential function in regulating proteasome-mediated degradation of RIM1 required for synaptic tuning.