Epidermolysis bullosa (EB) is a family of rare, incurable, inherited disorders characterized by extreme skin and mucosal fragility. The two most severe forms are dystrophic EB (DEB), caused by genetic variance in type-VII collagen, and junctional EB (JEB), caused by genetic variance in genes encoding basement membrane proteins laminin 332 (α3, β3, γ2), type XVII collagen or integrin α6β4. Despite clinical similarities, there are dramatic differences in the life expectancies of patients with these subtypes. Patients with RDEB survive to early or midadulthood, comparatively patients with JEB rarely survive beyond the first year of life. We aim to explore the disparity in life expectancy using a transcriptomic and an in vivo approach. We performed bulk RNA sequencing on 7 DEB and 13 JEB samples (3 LAMA3, 8 LAMB3 and 2 LAMC2) to identify molecular and cellular differences underpinning these diseases. Principal component analysis and differential gene expression (DEG) analysis was conducted to explore the relatedness between subtypes. Immunohistochemistry was used to validate these findings in mouse models of EB. Three distinct clusters of samples were grouped by condition; one DEB and two JEB clusters separated by LAMA3 and LAMC2 mutations. Pathway and gene ontology (GO) analysis of DEGs indicated an upregulation in cell death, proinflammatory pathways and interleukins (ILs) (IL-1, IL-11, CXCL12 and β-defensins) which stimulate the adaptive immune response. In addition, an abundance of transcriptional factors linked to wound healing (MMP2, COL1A1), collagen remodelling (LOX), fibrosis (TGFβ) and epidermal development (EPPK1, TGM1, FLG and KRT14) were upregulated in patients with JEB compared with those with DEB. These changes have been validated in mouse models of EB at Queen Mary University of London. We have identified and validated significant changes between EB subtypes. The use of pathway and GOterm analysis identified altered pathways and molecular drivers that could be used as a novel approach for therapeutic treatments for EB.

BACKGROUND:Desmosomes are complex cell junction structures that connect intermediate filaments providing strong cell-to-cell adhesion in tissues exposed to mechanical stress.OBJECTIVES:To identify causal variants in individuals with woolly hair and skin fragility of unknown genetic cause.METHODS:This research was conducted using whole-genome sequencing, whole-exome sequencing, clinical phenotyping, haplotype analysis, single-cell RNA sequencing data analysis, immunofluorescence microscopy and transmission electron microscopy.RESULTS:We identified homozygous predicted loss-of-function tuftelin-1 (TUFT1) variants in nine individuals, from three families, with woolly hair and skin fragility. One donor splice-site variant, c.60+1G>A, was present in two families, while a frameshift variant, p.Gln189Asnfs*49, was found in the third family. Haplotype analysis showed the c.60+1G>A substitution to be a founder variant in the Irish population that likely arose approximately 20 generations ago. Human and mouse single-cell RNA sequencing data showed TUFT1 expression to be enriched in the hair dermal sheath and keratinocytes. TUFT1 expression was highly correlated with genes encoding desmosomal components implicated in diseases with phenotypes that overlap with the cohort presented here. Immunofluorescence showed tuftelin-1 to be mainly localized to the peripheral cell membranes of keratinocytes in normal skin. Skin samples from individuals with TUFT1 variants showed markedly reduced immunoreactivity for tuftelin-1, with a loss of the keratinocyte cell membrane labelling. Light microscopy revealed keratinocyte adhesion, mild hyperkeratosis and areas of superficial peeling. Transmission electron microscopy showed panepidermal acantholysis with widening of intercellular spaces throughout the epidermis and desmosomal detachment through the inner plaques.CONCLUSIONS:Biallelic loss-of-function TUFT1 variants cause a new autosomal recessive skin/hair disorder characterized by woolly hair texture and early-onset skin fragility. Tuftelin-1 has a role in desmosomal integrity and function.

Junctional epidermolysis bullosa (JEB) is a rare autosomal recessive genodermatosis with a broad spectrum of phenotypes. Current genotype-phenotype paradigms are insufficient to accurately predict JEB subtype and characteristics from genotype, particularly for splice site variants, which account for over a fifth of disease-causing variants in JEB. This study evaluated the genetic and clinical findings from a JEB cohort, investigating genotype-phenotype correlations through bioinformatic analyses and comparison with previously reported variants. Eighteen unique variants in LAMB3, LAMA3, LAMC2, or COL17A1 were identified from 17 individuals. Seven had severe JEB, 9 had intermediate JEB, and 1 had laryngo-onycho-cutaneous syndrome. Seven variants were previously unreported. Deep phenotyping was completed for all intermediate JEB cases and demonstrated substantial variation between individuals. Splice site variants underwent analysis with SpliceAI, a state-of-the-art artificial intelligence tool, to predict resultant transcripts. Predicted functional effects included exon skipping and cryptic splice site activation, which provided potential explanations for disease severity and in most cases correlated with laminin-332 immunofluorescence. RT-PCR was performed for 1 case to investigate resultant transcripts produced from the splice site variant. This study expands the JEB genomic and phenotypic landscape. Artificial intelligence tools show potential for predicting the functional effects of splice site variants and may identify candidates for confirmatory laboratory investigation. Investigation of RNA transcripts will help to further elucidate genotype-phenotype correlations for novel variants.

Abstract Over one-fifth of disease-causing mutations in junctional epidermolysis bullosa (JEB) are splice site variants. These affect pre-mRNA splicing through the activation of cryptic splice sites and/or exon skipping, resulting in altered mRNA and translated proteins. Prediction of the precise location of activated cryptic splice sites is vital, as these can potentially alter the reading frame of the resulting transcript. Out-of-frame transcripts contain premature termination complexes (PTCs), which generally result in nonfunctional protein and often correlate with severe JEB in conjunction with other PTC-introducing mutations. SpliceAI is a 32-layer convolutional neural network that examines 10 000 nucleotides of flanking sequence around a variant of interest to predict its effects on RNA splicing. It was used to predict splice site mutation consequences in five patients with JEB with LAMB3 mutations. Genotypes, predicted transcripts and JEB subtypes are summarized in Table 1. SpliceAI predicted PTCs in cases 1 and 2, where transcripts were either out of frame or novel PTCs were introduced following aberrant splicing. An in-frame transcript with no PTC was predicted for case 3, which may explain this individual’s mild phenotype. Reverse transcriptase polymerase chain reaction data were reviewed for cases 4 and 5, and two predicted transcripts were confirmed. Two additional in-frame transcripts not predicted by SpliceAI were found to be produced in low levels. It is likely that these in-frame transcripts generated partially functional protein, alleviating JEB severity to a degree. This study highlights the importance of examining RNA transcripts for accurate genotype–phenotype correlation. In silico tools such as SpliceAI show potential in elucidating mechanisms of severity. They may facilitate selection of cases for further laboratory investigation and can guide which regions of the gene or transcript to sequence for confirmatory analyses. Where RNA analyses were available, SpliceAI correctly predicted two of the resultant transcripts. Further validation of this tool is required with additional cases in this rare disease.Table 1Genotypes, predicted transcripts and JEB subtypes found with SpliceAICaseZygosityMutation (LAMB3)Exon skipping predictionCryptic splice site activation predictionPredicted outcome commentsJEB subtype1Homozygousc.2701+1G>AOOF exon 18 skipping20 nt excluded from exon 18Both outcomes OOFSevere2Heterozygousc.565-2A>G, c.2914C>T (p.R972X)OOF exon 7 skipping117 nt included from intron 6 (including TGA PTC at c.565-60)In-frame cryptic activation with novel PTC introducedSevereOOF exon skipping3Heterozygousc.1705C>T (p.R569X), c.943+2T>COOF exon 9 skipping27 nt included from intron 9In-frame cryptic activationIntermediateOOF exon skipping4Homozygousc.298+5G>COOF exon 4 skipping64 nt included from intron 4Both outcomes OOFIntermediate5Heterozygousc.3119G>A (p.W1040X), c.629-12T>APredicted to be unlikely10 nt included from intron 7OOF cryptic activationIntermediatent, nucleotides; OOF, out of frame.

Hypotrichosis 12 (HYPT12) is an autosomal dominant, nonsyndromic hypotrichosis, caused by a pathogenic variant in the RPL21 gene encoding ribosomal protein L21, although only two pedigrees harbouring the amino acid substitution, p.Arg32Gln, have been reported previously. We present the case of a 44-year-old White British man with progressive hair loss since the age of 10 months, affecting his scalp, eyebrow, eyelashes and most of his body. Similar hair loss also affected several members of his family, with likely autosomal dominant inheritance. Using whole-exome sequencing, we identified a rare heterozygous missense variant (NM_000982.3:c.127A > G, NP_000973.2:p.Lys43Glu) in RPL21, and subsequent Sanger sequencing confirmed segregation of this variant in affected family members.

This report describes a case of an 18-year-old white British woman with HTSS1, whose phenotype was characterized by the inability to grow long scalp hair. Whole exome sequencing identified a novel pathogenic heterozygous nonsense variant (NM_001264.4: c.484C>T, NP_001255.3: p.Gln162Ter) in CDSN, which encodes corneodesmosin. HTSS1, described in this patient’s case, showed distinct clinical and histopathological features, thereby expanding the genotype–phenotype paradigm of HTSS1.

BACKGROUND:Host specificity among pollinator fig wasps (Agaonidae) depends on host plant specific volatile cues, but fig wasps must also pass through a narrow physical barrier (the ostiole) if they are to pollinate and oviposit. Across South East Asia the dioecious shrub Ficus hirta is associated with at least ten pollinator species allied to Valisia javana. Ficus triloba has a single recorded pollinator, Valisia esquirolianae. Receptive figs of F. hirta are usually much smaller than those of F. triloba, but at a mainland site where F. hirta has atypically large figs we identified both V. esquirolianae and V. javana from both Ficus species using COI and ITS2 sequencing. To investigate whether this host overlap was exceptional we reared fig wasps from the two trees elsewhere and recorded features that may facilitate host transfer between them, including attractant volatiles, reproductive phenology and the sizes of their figs and fig wasps.RESULTS:The two Ficus species were found to support both Valisia species at several of the sites, suggesting that the differences we detected in volatile profiles, ostiole sizes and pollinator head sizes are not strict barriers to host sharing. Valisia javana colonised F. triloba more frequently than V. esquirolianae colonised F. hirta.CONCLUSIONS:This asymmetric sharing of pollinators may reflect the relative abundance of the two species of fig wasps and differences in host reproductive phenology. Asynchronous flowering of individual F. hirta may favor local retention of pollinators, in contrast to the tree-wide synchrony of F. triloba figs, which can generate local shortages of V. esquirolianae. If the pollinator sharing by male figs of F. triloba and F. hirta also occurs in female figs then this could result in gene flow between them.

In the framework of the UK 100 000 Genomes Project, we investigated the genetic origin of a previously undescribed recessive dermatological condition, which we named LIPHAK (LTV1-associated Inflammatory Poikiloderma with Hair abnormalities and Acral Keratoses), in four affected individuals from two UK families of Pakistani and Indian origins, respectively. Our analysis showed that only one gene, LTV1, carried rare biallelic variants that were shared in all affected individuals, and specifically they bore the NM_032860.5:c.503A > G, p.(Asn168Ser) change, found homozygously in all of them. In addition, high-resolution homozygosity mapping revealed the presence of a small 652-kb stretch on chromosome 6, encompassing LTV1, that was haploidentical and common to all affected individuals. The c.503A > G variant was predicted by in silico tools to affect the correct splicing of LTV1's exon 5. Minigene-driven splicing assays in HEK293T cells and in a skin sample from one of the patients confirmed that this variant was indeed responsible for the creation of a new donor splice site, resulting in aberrant splicing and in a premature termination codon in exon 6 of this gene. LTV1 encodes one of the ribosome biogenesis factors that promote the assembly of the small (40S) ribosomal subunit. In yeast, defects in LTV1 alter the export of nascent ribosomal subunits to the cytoplasm; however, the role of this gene in human pathology is unknown to date. Our data suggest that LIPHAK could be a previously unrecognized ribosomopathy.

In pulsar astronomy, detecting effective pulsar signals among numerous pulsar candidates is an important research topic. Starting from space X-ray pulsar signals, the two-dimensional autocorrelation profile map (2D-APM) feature modelling method, which utilizes epoch folding of the autocorrelation function of X-ray signals and expands the time-domain information of the periodic axis, is proposed. A uniform setting criterion regarding the time resolution of the periodic axis addresses pulsar signals without any prior information. Compared with the traditional profile, the model has a strong anti-noise ability, a greater abundance of information and consistent characteristics. The new feature is simulated with double Gaussian components, and the characteristic distribution of the model is revealed to be closely related to the distance between the double peaks of the profile. Next, a deep convolutional neural network (DCNN) is built, named Inception-ResNet. According to the order of the peak separation and number of arriving photons, 30 data sets based on the Poisson process are simulated to construct the training set, and the observation data of PSRs B0531+21, B0540-69 and B1509-58 from the Rossi X-ray Timing Explorer (RXTE) are selected to generate the test set. The number of training sets and the number of test sets are 30 000 and 5400, respectively. After achieving convergence stability, more than 99% of the pulsar signals are recognized, and more than 99% of the interference is successfully rejected, which verifies the high degree of agreement between the network and the feature model and the high potential of the proposed method in searching for pulsars.

Polygalae Radix has long been used in China for calming the mind, promoting intelligence, communicating the heart and kidney, eliminating phlegm, and reducing swelling. At present, it is used to treat amnesia, insomnia, and malaise. Modern research has revealed that Polygalae Radix mainly contains triterpenoid saponins, xanthone, oligosaccharide esters, etc., with the activities of improving memory, resisting dementia, protecting the brain, relieving cough, and removing phlegm, as well as sedation and hypnosis. The present study reviews the research progress on chemical composition, pharmacological action, quality control, and metabolism of Polygalae Radix in the past 30 years, to provide a theoretical basis for further research and development.

Background/Objectives Laryngo-onycho-cutaneous syndrome (LOC) is a rare subtype of junctional epidermolysis bullosa (JEB), featuring aberrant granulation tissue formation in the skin, larynx, and eyes. So far, three mutations including the specific (founder) mutation in exon 39 of LAMA3 (c.151dup) have been identified, but sparse data exists regarding the natural history, the genotype-phenotype correlation, and its differentiation from other JEB types. Methods We reviewed our pediatric EB database to identify English children with clinical and genetically diagnosed LOC within the last 15 years. Their demographic, clinical, and laboratory data were examined. We searched three databases for case reports of LOC between January 1986 and November 2020 and extracted clinical and molecular details. Results We identified 6 LOC patients, all female (mean age 5.4 years). Periungual hypergranulation and skin fragility were the earliest presenting signs (0-3 months), followed by laryngeal stenosis, symblepharon (mean onset 10.7 and 11.8 months, respectively), and dental abnormalities. Five children developed anemia at an average of 19.2 months. We identified 22 published studies in English with 31 cases. Conclusions This study delineates the disease course of LOC and highlights the overlap with some forms of JEB. Classical signs/symptoms including anemia appear early in life. Genetic analysis revealed three new LOC-associated variants and underscores the finding that interpretation of skin immunolabeling and molecular diagnostics can be challenging. We provide recommendations on management of this complex syndrome.

Arrhythmogenic right ventricular cardiomyopathy (ARVC), with skin manifestations, has been associated with mutations in JUP encoding plakoglobin. Genotype–phenotype correlations regarding the penetrance of cardiac involvement, and age of onset have not been well established. We examined a cohort of 362 families with skin fragility to screen for genetic mutations with next-generation sequencing-based methods. In two unrelated families, a previously unreported biallelic mutation, JUP: c.201delC; p.Ser68Alafs*92, was disclosed. The consequences of this mutation were determined by expression profiling both at tissue and ultrastructural levels, and the patients were evaluated by cardiac and cutaneous work-up. Whole-transcriptome sequencing by RNA-Seq revealed JUP as the most down-regulated gene among 21 skin fragility-associated genes. Immunofluorescence showed the lack of plakoglobin in the epidermis. Two probands, 2.5 and 22-year-old, with the same homozygous mutation, allowed us to study the cross-sectional progression of cardiac involvements in relation to age. The older patient had anterior T wave inversions, prolonged terminal activation duration (TAD), and RV enlargement by echocardiogram, and together with JUP mutation met definite ARVC diagnosis. The younger patient had no evidence of cardiac disease, but met possible ARVC diagnosis with one major criterion (the JUP mutation). In conclusion, we identified the same biallelic homozygous JUP mutation in two unrelated families with skin fragility, but cardiac findings highlighted age-dependent penetrance of ARVC. Thus, young, phenotypically normal patients with biallelic JUP mutations should be monitored for development of ARVC.

X-ray pulsar signals commonly have a very low amplitude and suffer from high observation noise and statistical correlation interference, which seriously degrade the signal detection performance given finite observation times. Here, noise refers to the uncertainty distribution of the photon Poisson statistics, and interference refers to time-dependent broadband or narrowband signals, which may originate from some artificial interference source or other pulsars. To address the above problems, this paper proposes a feature representation method for pulsar signal detection based on high-order cyclostationarity theory. First, we establish a photon signal model of an X-ray detector based on the cyclostationary characteristics of pulsar signals, considering period jitter, correlation interference, and Gaussian noise in the pulsar emission process. By introducing cyclic statistics, we use a direct estimation method for the high-order spectrum, including a nonuniform sampling strategy, to accurately extract signal features and effectively suppress correlation interference and noise. Finally, we use simulation data and Rossi X-ray Timing Explorer observation data to verify the proposed method. The results show that the proposed method is a promising feature modeling solution that is superior to traditional one-dimensional spectra and epoch folding in counteracting interference and noise. While such feature modeling is particularly useful for applications such as pulsar navigation, we believe that this method has general promise for a wide range of pulsar-related research.

Background Epidermolysis bullosa with pyloric atresia (EB-PA), also known as Carmi syndrome, is an uncommon, autosomal recessive genodermatosis that typically affects the skin and gastrointestinal tract. EB-PA is caused by homozygous or compound heterozygous mutations in the integrin alpha 6 ( ITGA6 ) gene on chromosome 2q31.1 or in the integrin beta 4 ( ITGB4 ) gene on 17q25.1. Case presentation A male premature infant was born with aplasia cutis, atresia of the pylorus, and bilateral hydronephrosis. His clinical and imaging findings were compatible with EB-PA. A novel, small deletion of the last two bases in exon 6 and the first two nucleotides of intron 6 (c.565_566+2del) in ITGB4 gene was identified. Conclusion EB-PA-aplasia cutis congenita is known to be a non-treatable condition with a poor prognosis as the reported case. The novel mutation reported in this patient may lead to the lethal form of this disease. Identification of underlying genetic abnormality is critical to give genetic counseling.

Next-generation sequencing (NGS) is helpful in diagnosing complex genetic disorders and phenotypes, particularly when more than one overlapping condition is present. From a large cohort of 362 families with clinical manifestations of skin and mucosal fragility, referred by several major medical centers, one patient was found by NGS to have two overlapping heritable skin diseases, recessive dystrophic epidermolysis bullosa (RDEB; COL7A1 mutations) and acrodermatitis enteropathica (AE; SLC39A4 mutations). The pathogenicity of the variants was studied at gene expression as well as ultrastructural and tissue levels. Although there is no specific treatment for RDEB except avoiding trauma, supplementation with oral zinc (3 mg center dot kg(-1)center dot day(-1)) for the AE resulted in rapid amelioration of the skin findings. This case demonstrates the power of NGS in identifying two genetically unlinked diseases that led to effective treatment with major clinical benefits as an example of genomics-guided treatment.