Dominant dystrophic epidermolysis bullosa is associated with glycolytically active GATA3+ T helper 2 cells which may contribute to pruritus in lesional skin.

BACKGROUND: Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by trauma-induced blisters and, in some individuals, intense pruritus. Precisely what causes itch in DDEB and optimal ways to reduce it have not been fully determined. OBJECTIVES: To characterize DDEB skin transcriptomes to identify therapeutic targets to reduce pruritus in patients. METHODS: Using bulk RNA sequencing, we evaluated affected and unaffected skin biopsy samples from six patients with DDEB (all with the very itchy pruriginosa subtype) and four healthy individuals. Single-cell transcriptomes of affected (n = 2) and unaffected (n = 1) DDEB skin and healthy skin (n = 2) were obtained. Dupilumab treatment was provided for three patients. RESULTS: The skin bulk transcriptome showed significant enrichment of T helper (Th)1/2 and Th17 pathways in affected DDEB skin compared with nonlesional DDEB skin and healthy skin. Single-cell transcriptomics showed an association of glycolytically active GATA3+ Th2 cells in affected DDEB skin. Treatment with dupilumab in three people with DDEB led to significantly reduced visual analogue scale (VAS) itch scores after 12 weeks (mean VAS 3.83) compared with pretreatment (mean VAS 7.83). Bulk RNAseq and quantitative polymerase chain reaction showed that healthy skin and dupilumab-treated epidermolysis bullosa (EB) pruriginosa skin have similar transcriptomic profiles and reduced Th1/Th2 and Th17 pathway enrichment. CONCLUSIONS: Single-cell RNAseq helps define an enhanced DDEB-associated Th2 profile and rationalizes drug repurposing of anti-Th2 drugs in treating DDEB pruritus.

Dominant dystrophic epidermolysis bullosa (DDEB) is a rare inherited skin disease that causes fragile skin that blisters easily, often triggered by minor injuries. These blisters are accompanied by intense itching, which can be distressing. The underlying cause of DDEB lies in genetic mutations in a gene called COL7A1. This gene encodes 'type VII collagen', a protein crucial for attaching the outer skin layer (epidermis) to the layer beneath (dermis). Although the genetic basis of DDEB is understood, the causes of itch are not known. As well as this, effective treatments for DDEB are lacking, which has driven scientists to explore innovative approaches like repurposing existing drugs. Drug repurposing involves using medications that have already been approved for other health conditions. One such drug is dupilumab, which is used for severe atopic dermatitis (eczema). Dupilumab targets immune cells called Th2 cells, which play a role in inflammation and allergies. While dupilumab has shown promise in relieving DDEB itching, the way it works in this condition is unclear. This study, carried out by a group of researchers in Taiwan, looked at gene expression in DDEB-affected and unaffected skin, and compared it to gene expression in healthy skin samples. We found heightened activity in Th2 immune cells and abnormal gene signals related to itching, similar to atopic dermatitis. These findings support using dupilumab and other anti-inflammatory drugs to alleviate itching in DDEB. Clinical trials will be crucial to evaluate the effectiveness of these drugs for managing DDEB symptoms. This research opens doors for enhanced treatment options and improving the quality of life of people living with DDEB.

Novel SLC5A6 mutations lead to B lymphocyte maturation defects with metabolic abnormality rescuable by biotin replenishment.

We characterized a family diagnosed with immunodeficiency disease presenting with low immunoglobulin levels and skin dyskeratosis. Exome sequencing revealed compound heterozygous missense variants in SLC5A6, the gene encoding a cellular sodium-dependent multivitamin transporter (SMVT) responsible for transporting vitamins, including biotin (vitamin B7). We showed that the biotin deficiency was caused by the SLC5A6 variants resulting in defective B cell differentiation and antibody deficiency. Altered cellular metabolic profiles, including aberrant mitochondrial respiration and reliance on glycolysis, may underlie the failure in plasma cell maturation. Replenishment of biotin improved plasma cell maturation and recovered the antibody producing activity in the patient and in a CRISPR-Cas9 gene-edited mouse model bearing a patient-specific SLC5A6 variant. Our results demonstrate the critical role of metabolic reprogramming in the maturation of plasma cells and nominate SLC5A6 as a causative gene for immunodeficiency that may be treated by biotin replenishment.

FGF primes angioblast formation by inducing ETV2 and LMO2 via FGFR1/BRAF/MEK/ERK.

It is critical to specify a signal that directly drives the transition that occurs between cell states. However, such inferences are often confounded by indirect intercellular communications or secondary transcriptomic changes due to primary transcription factors. Although FGF is known for its importance during mesoderm-to-endothelium differentiation, its specific role and signaling mechanisms are still unclear due to the confounding factors referenced above. Here, we attempted to minimize the secondary artifacts by manipulating FGF and its downstream mediators with a short incubation time before sampling and protein-synthesis blockage in a low-density angioblastic/endothelial differentiation system. In less than 8 h, FGF started the conversion of KDRlow/PDGFRAlow nascent mesoderm into KDRhigh/PDGFRAlow angioblasts, and the priming by FGF was necessary to endow endothelial formation 72 h later. Further, the angioblastic conversion was mediated by the FGFR1/BRAF/MEK/ERK pathway in mesodermal cells. Finally, two transcription factors, ETV2 and LMO2, were the early direct functional responders downstream of the FGF pathway, and ETV2 alone was enough to complement the absence of FGF. FGF's selective role in mediating the first-step, angioblastic conversion from mesoderm-to-endothelium thus allows for refined control over acquiring and manipulating angioblasts. The noise-minimized differentiation/analysis platform presented here is well-suited for studies on the signaling switches of other mesodermal-lineage fates as well.

Two Cases of Interleukin-7-Deficient Generalized Verrucosis.

We report 2 generalized verrucosis (GV) patients homozygous for a novel mutation in the start codon of IL7. Unlike the previous report in which IL-7 deficiency accompanied CD4 T lymphocytopenia, circulating CD4 T cells were not depleted in one of our patients, suggesting a GV pathogenesis other than poor T-cell development.

Plectin Missense Mutation p.Leu319Pro in the Pathogenesis of Autosomal Recessive Epidermolysis Bullosa Simplex.

is missing (Short communication).

Next-generation sequencing identifies rare variants associated with Noonan syndrome.

Noonan syndrome (NS) is a relatively common genetic disorder, characterized by typical facies, short stature, developmental delay, and cardiac abnormalities. Known causative genes account for 70-80% of clinically diagnosed NS patients, but the genetic basis for the remaining 20-30% of cases is unknown. We performed next-generation sequencing on germ-line DNA from 27 NS patients lacking a mutation in the known NS genes. We identified gain-of-function alleles in Ras-like without CAAX 1 (RIT1) and mitogen-activated protein kinase kinase 1 (MAP2K1) and previously unseen loss-of-function variants in RAS p21 protein activator 2 (RASA2) that are likely to cause NS in these patients. Expression of the mutant RASA2, MAP2K1, or RIT1 alleles in heterologous cells increased RAS-ERK pathway activation, supporting a causative role in NS pathogenesis. Two patients had more than one disease-associated variant. Moreover, the diagnosis of an individual initially thought to have NS was revised to neurofibromatosis type 1 based on an NF1 nonsense mutation detected in this patient. Another patient harbored a missense mutation in NF1 that resulted in decreased protein stability and impaired ability to suppress RAS-ERK activation; however, this patient continues to exhibit a NS-like phenotype. In addition, a nonsense mutation in RPS6KA3 was found in one patient initially diagnosed with NS whose diagnosis was later revised to Coffin-Lowry syndrome. Finally, we identified other potential candidates for new NS genes, as well as potential carrier alleles for unrelated syndromes. Taken together, our data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients.

Cytoplasmic Lipid Droplets Predict Worse Prognosis in Diffuse Large B-Cell Lymphoma: Next-Generation Sequencing Deciphering Lipogenic Genes.

Burkitt lymphoma is characterized by high cell turnover and numerous cytoplasmic vacuoles that are demonstrated to be lipid droplets (LDs) decorated by adipophilin. By contrast, cytoplasmic vacuoles are variably observed in diffuse large B-cell lymphoma (DLBCL) and less well characterized. In this study, we first validated in DLBCL that cytoplasmic vacuoles are indeed LDs by Oil-red-O stain, Bodipy fluorescent stain, and electron microscopy. Second, in a cohort of DLBCL patients (n=52) we showed that LDs in effusional lymphoma cells were associated with a poorer prognosis (P=0.029, log-rank test) and higher International Prognostic Index (IPI) score (94% vs. 66%, P=0.026) than those without. Moreover, using adipophilin as a surrogate marker for LDs, we found in another cohort of biopsy specimen (n=85) that expression of adipophilin by lymphoma cells predicted a poorer prognosis (P=0.007, log-rank test) and higher IPI score (63% vs. 30%, P=0.005). In addition, whole exome sequencing of effusional DLBCL cells showed LD-positive DLBCL shared genetic features with the MCD (MYD88 and CD79B mutations) subtype and highlighted OSBPL10 and CUBN as the most frequently mutated genes involved in lipogenesis. Whole transcriptome analysis by comparing effusional DLBCL cells with versus without LDs showed upregulation of EHHADH, SLC1A1, CD96, INPP4B, and RNF183 relevant for lymphoma lipogenesis and upregulation of epithelial-mesenchymal transition and KRAS signaling pathways. Higher expression of EHHADH and CD96 were validated in LD-positive clinical samples and LD-rich cell lines than LD-poor cells along with the known lipogenic gene, FASN. Our findings highlight the roles of LDs and adipophilin expression in DLBCL, suggest that these markers may predict prognosis and show that lipogenic genes may be potential therapeutic targets.

Syndromic ciliopathy: a taiwanese single-center study.

BACKGROUND: Syndromic ciliopathies are a group of congenital disorders characterized by broad clinical and genetic overlap, including obesity, visual problems, skeletal anomalies, mental retardation, and renal diseases. The hallmark of the pathophysiology among these disorders is defective ciliary functions or formation. Many different genes have been implicated in the pathogenesis of these diseases, but some patients still remain unclear about their genotypes. METHODS: The aim of this study was to identify the genetic causes in patients with syndromic ciliopathy. Patients suspected of or meeting clinical diagnostic criteria for any type of syndromic ciliopathy were recruited at a single diagnostic medical center in Southern Taiwan. Whole exome sequencing (WES) was employed to identify their genotypes and elucidate the mutation spectrum in Taiwanese patients with syndromic ciliopathy. Clinical information was collected at the time of patient enrollment. RESULTS: A total of 14 cases were molecularly diagnosed with syndromic ciliopathy. Among these cases, 10 had Bardet-Biedl syndrome (BBS), comprising eight BBS2 patients and two BBS7 patients. Additionally, two cases were diagnosed with Alström syndrome, one with Oral-facial-digital syndrome type 14, and another with Joubert syndrome type 10. A total of 4 novel variants were identified. A recurrent splice site mutation, BBS2: c.534 + 1G > T, was present in all eight BBS2 patients, suggesting a founder effect. One BBS2 patient with homozygous c.534 + 1G > T mutations carried a third ciliopathic allele, TTC21B: c.264_267dupTAGA, a nonsense mutation resulting in a premature stop codon and protein truncation. CONCLUSIONS: Whole exome sequencing (WES) assists in identifying molecular pathogenic variants in ciliopathic patients, as well as the genetic hotspot mutations in specific populations. It should be considered as the first-line genetic testing for heterogeneous disorders characterized by the involvement of multiple genes and diverse clinical manifestations.

Profibrotic Subsets of SPP1+ Macrophages and POSTN+ Fibroblasts Contribute to Fibrotic Scarring in Acne Keloidalis.

Acne keloidalis is a primary scarring alopecia characterized by longstanding inflammation in the scalp causing keloid-like scar formation and hair loss. Histologically, acne keloidalis is characterized by mixed leukocytic infiltrates in the acute stage followed by a granulomatous reaction and extensive fibrosis in the later stages. To further explore its pathogenesis, bulk RNA sequencing, single-cell RNA sequencing, and spatial transcriptomics were applied to occipital scalp biopsy specimens of lesional and adjacent no-lesional skin in patients with clinically active disease. Unbiased clustering revealed 19 distinct cell populations, including 2 notable populations: POSTN+ fibroblasts with enriched extracellular matrix signatures and SPP1+ myeloid cells with an M2 macrophage phenotype. Cell communication analyses indicated that fibroblasts and myeloid cells communicated by SPP1 signaling networks in lesional skin. A reverse transcriptomics in silico approach identified corticosteroids as possessing the capability to reverse the gene expression signatures of SPP1+ myeloid cells and POSTN+ fibroblasts. Intralesional corticosteroid injection greatly reduced SPP1 and POSTN gene expression as well as acne keloidalis disease activity. Spatial transcriptomics and immunofluorescence staining verified microanatomic specificity of SPP1+ myeloid cells and POSTN+ fibroblasts with disease activity. In summary, the communication between POSTN+ fibroblasts and SPP1+ myeloid cells by SPP1 axis may contribute to the pathogenesis of acne keloidalis.

The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer.

Most susceptibility to colorectal cancer (CRC) is not accounted for by known risk factors. Because MLH1, MSH2 and MSH6 mutations underlie high-penetrance CRC susceptibility in hereditary nonpolyposis colon cancer (HNPCC), we hypothesized that attenuated alleles might also underlie susceptibility to sporadic CRC. We looked for gene variants associated with HNPCC in Israeli probands with familial CRC unstratified with respect to the microsatellite instability (MSI) phenotype. Association studies identified a new MLH1 variant (415G-->C, resulting in the amino acid substitution D132H) in approximately 1.3% of Israeli individuals with CRC self-described as Jewish, Christian and Muslim. MLH1 415C confers clinically significant susceptibility to CRC. In contrast to classic HNPCC, CRCs associated with MLH1 415C usually do not have the MSI defect, which is important for clinical mutation screening. Structural and functional analyses showed that the normal ATPase function of MLH1 is attenuated, but not eliminated, by the MLH1 415G-->C mutation. The new MLH1 variant confers a high risk of CRC and identifies a previously unrecognized mechanism in microsatellite-stable tumors. These studies suggest that variants of mismatch repair proteins with attenuated function may account for a higher proportion of susceptibility to sporadic microsatellite-stable CRC than previously assumed.

Whole-Exome Sequencing Identified Rare Genetic Variants Associated with Undervirilized Genitalia in Taiwanese Pediatric Patients.

Disorders/differences of sex development (DSDs) are a group of rare and phenotypically variable diseases. The underlying genetic causes of most cases of 46XY DSDs remains unknown. Despite the advent of genetic testing, current investigations of the causes of DSDs allow genetic-mechanism identification in about 20-35% of cases. This study aimed primarily to establish a rapid and high-throughput genetic test for undervirilized males with and without additional dysmorphic features. Routine chromosomal and endocrinological investigations were performed as part of DSD evaluation. We applied whole-exome sequencing (WES) complemented with multiplex ligation-dependent probe amplification to seek explainable genetic causes. Integrated computing programs were used to call and predict the functions of genetic variants. We recruited 20 patients and identified the genetic etiologies for 14 (70%) patients. A total of seven of the patients who presented isolated DSD phenotypes were found to have causative variants in the AR, MAP3K1, and FLNA genes. Moreover, the other seven patients presented additional phenotypes beyond undervirilized genitalia. Among them, two patients were compatible with CHARGE syndrome, one with Robinow syndrome, and another three with hypogonadotropic hypogonadism. One patient, who carried a heterozygous FLNA mutation, also harbored a heterozygous PTPN11 mutation and thus presented some phenotypes of Noonan syndrome. We identified several genetic variants (12 nonsense mutations and one microdeletion) that account for syndromic and nonsyndromic DSDs in the Taiwanese population. The identification of these causative genes extended our current understanding of sex development and related congenital disorders.

RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m5C), in the transcriptome remains unclear. Aberrant regulation of 5-methylcytosine turnover in cancer may affect posttranscriptional modifications in coding and noncoding RNAs in disease pathogenesis. Mutations in NSUN2 have been reported as drivers of neurodevelopmental disorders in mice, and upregulated expression of NSUN2 in tumors of the breast, bladder, and pancreas has been reported. In this study, we conducted mRNA whole transcriptomic bisulfite sequencing to categorize NSUN2 target sites in the mRNA of human pancreatic cancer cells. We identified a total of 2829 frequent m5C sites in mRNA from pancreatic cancer cells. A total of 90.9% (2572/2829) of these m5C sites were mapped to annotated genes in autosomes and sex chromosomes X and Y. Immunohistochemistry staining confirmed that the NSUN2 expression was significantly upregulated in cancer lesions in the LSL-KrasG12D/+;Trp53fl/fl;Pdx1-Cre (KPC) spontaneous pancreatic cancer mouse model induced by Pdx1-driven Cre/lox system expressing mutant KrasG12D and p53 deletion. The in vitro phenotypic analysis of NSUN2 knockdown showed mild effects on pancreatic cancer cell 2D/3D growth, morphology and gemcitabine sensitivity in the early phase of tumorigenesis, but cumulative changes after multiple cell doubling passages over time were required for these mutations to accumulate. Syngeneic transplantation of NSUN2-knockdown KPC cells via subcutaneous injection showed decreased stromal fibrosis and restored differentiation of ductal epithelium in vivo. SIGNIFICANCE: Transcriptome-wide mRNA bisulfite sequencing identified candidate m5C sites of mRNAs in human pancreatic cancer cells. NSUN2-mediated m5C mRNA metabolism was observed in a mouse model of pancreatic cancer. NSUN2 regulates cancer progression and epithelial differentiation via mRNA methylation.

Mutational analysis of epidermolysis bullosa in Taiwan by whole-exome sequencing complemented by RNA sequencing: a series of 77 patients.

BACKGROUND: Epidermolysis bullosa (EB) is a heterogeneous group of hereditary skin diseases characterized by skin fragility. Primary data on Taiwanese population remain scarce. METHODS: We gathered clinical information from EB patients at National Cheng Kung University Hospital from January, 2012, to June, 2021. Diagnostic tests including transmission electron microscopy, immunofluorescence studies, and whole-exome sequencing (WES) were performed. The pathogenicity of novel splice-site mutations was determined through reverse transcriptase-PCR of skin mRNA followed by Sanger and/or RNA sequencing. RESULTS: Seventy-seven EB patients from 45 families were included: 19 EB simplex, six junctional EB, and 52 dystrophic EB. Pathogenic variants were identified in 37 of 38 families (97.4%), in which WES was used as a first-line tool for mutational analysis; RNA sequencing determined pathogenic variants in the remaining one family. A total of 60 mutations in EB-related genes were identified, including 22 novel mutations. The mutations involved KRT5, KRT14, PLEC, COL17A1, LAMB3, LAMA3, ITGB4, and COL7A1. Over one-quarter of DEB patients had EB pruriginosa. CONCLUSIONS: The distinct clinical presentation and molecular pathology of EB in Taiwan expand our understanding of this disorder. WES was an effective first-line diagnostic tool for identifying EB-associated variants. RNA sequencing complemented WES when multiple potentially pathogenic splice-site mutations were found.

Novel roles for MLH3 deficiency and TLE6-like amplification in DNA mismatch repair-deficient gastrointestinal tumorigenesis and progression.

DNA mismatch repair suppresses gastrointestinal tumorgenesis. Four mammalian E. coli MutL homologues heterodimerize to form three distinct complexes: MLH1/PMS2, MLH1/MLH3, and MLH1/PMS1. To understand the mechanistic contributions of MLH3 and PMS2 in gastrointestinal tumor suppression, we generated Mlh3(-/-);Apc(1638N) and Mlh3(-/-);Pms2(-/-);Apc(1638N) (MPA) mice. Mlh3 nullizygosity significantly increased Apc frameshift mutations and tumor multiplicity. Combined Mlh3;Pms2 nullizygosity further increased Apc base-substitution mutations. The spectrum of MPA tumor mutations was distinct from that observed in Mlh1(-/-);Apc(1638N) mice, implicating the first potential role for MLH1/PMS1 in tumor suppression. Because Mlh3;Pms2 deficiency also increased gastrointestinal tumor progression, we used array-CGH to identify a recurrent tumor amplicon. This amplicon contained a previously uncharacterized Transducin enhancer of Split (Tle) family gene, Tle6-like. Expression of Tle6-like, or the similar human TLE6D splice isoform in colon cancer cells increased cell proliferation, colony-formation, cell migration, and xenograft tumorgenicity. Tle6-like;TLE6D directly interact with the gastrointestinal tumor suppressor RUNX3 and antagonize RUNX3 target transactivation. TLE6D is recurrently overexpressed in human colorectal cancers and TLE6D expression correlates with RUNX3 expression. Collectively, these findings provide important insights into the molecular mechanisms of individual MutL homologue tumor suppression and demonstrate an association between TLE mediated antagonism of RUNX3 and accelerated human colorectal cancer progression.

New Structural and Single Nucleotide Mutations in Type I and Type II Collagens in Taiwanese Children With Type I and Type II Collagenopathies.

Collagenopathy is a rare genetic condition characterized by abnormality in either collagen structure or metabolism. Variations in its clinical presentations highlight diversity in the genetic causes and potential existence of concurrent mutations. Through whole exome sequencing (WES) complemented with multiplex ligation-dependent probe amplification, we identified the genetic etiologies for six cases with osteogenesis imperfecta (OI) in COL1A1 (p.T1298N, p.Q1280Pfs∗51, and p.G557Vfs∗23) and COL1A2 (c.1-1677_133-441del) as well as three cases with spondyloepiphyseal dysplasia congenita in COL2A1 (p.G1041S, p.G654S, and p.G441A). Co-occurrence of COL1A1 and WNT1 mutations was found in a patient with a mild OI phenotype but severe osteoporosis. These findings extended the pathogenic variant spectrum of COL1A1, COL1A2, and COL2A1 for type I and type II collagenopathies. Although WES provides a fast and accurate method to identify the genetic causes in most of the patients with type I and type II collagenopathies, its limitation of detecting CNVs because of variable capturing uniformity should be kept in mind when interpreting the results. Taken together, we demonstrate that multiple genetic characterizing technologies can provide an accurate and efficient molecular diagnostic of new genetic variants in disease-causing genes that are compatible with clinical phenotypes.

NOX2-Deficient Neutrophils Facilitate Joint Inflammation Through Higher Pro-Inflammatory and Weakened Immune Checkpoint Activities.

Immune-mediated arthritis is an important chronic inflammatory disease of joints causing debilitating morbidity in affected patients. The mechanisms underlying immune-mediated arthritis have been intensively investigated, however the cellular and molecular factors contributing to the joint inflammation in different redox conditions have not been clearly elucidated. Previous research showed that phagocyte-produced reactive oxygen species (ROS) plays an anti-inflammatory role in K/BxN serum-transfer arthritis and NOX2-deficient mice tend to have more severe arthritis. Although many leukocytes play critical roles in the development of immune-mediated arthritis, the role of neutrophils, which are the main producers of ROS in inflammation, is still controversial. We hence assessed the immunomodulatory function of neutrophils from arthritic joints of NOX2-deficient and wild type mice in this study. We found more neutrophils accumulation in NOX2-deficient inflamed joints. RNA-sequencing and quantitative PCR revealed significantly increased expression of acute inflammation genes including IL1b, Cxcl2, Cxcl3, Cxcl10 and Mmp3 in activated neutrophils from the inflamed joints of NOX2-deficient mice. Moreover, gene set enrichment analysis (GSEA) showed enriched gene signatures in type I and II IFN responses, IL-6-JAK-STAT3 signaling pathway and TNF-α signaling pathway via NF-κB in NOX2-deficient neutrophils. In addition, we found that NOX2-deficient neutrophils expressed lower levels of PD-L1 and were less suppressive than WT neutrophils. Moreover, treatment of PD-L1-Fc decreased cytokine expression and ameliorated the severity of inflammatory arthritis. Our results suggest that NOX2-derived ROS is critical for regulating the function and gene expression in arthritic neutrophils. Both the strong pro-inflammatory and weakened anti-inflammatory functions of neutrophils due to abnormal redox regulation may be targets of treatment for immune-mediated arthritis.

Probiotics-Containing Yogurt Ingestion and H. pylori Eradication Can Restore Fecal Faecalibacterium prausnitzii Dysbiosis in H. pylori-Infected Children.

This study investigated the compositional differences in fecal microbiota between children with and without H. pylori infection and tested whether probiotics-containing yogurt and bacterial eradication improve H. pylori-related dysbiosis. Ten H. pylori-infected children and 10 controls ingested probiotics-containing yogurt for 4 weeks. Ten-day triple therapy plus yogurt was given to the infected children on the 4th week. Fecal samples were collected at enrollment, after yogurt ingestion, and 4 weeks after successful H. pylori eradication for cytokines and microbiota analysis using ELISA and metagenomic sequencing of the V4 region of the 16S rRNA gene, respectively. The results showed H. pylori-infected children had significantly higher levels of fecal TGF-ß1 than those who were not infected. Eight of 295 significantly altered OTUs in the H. pylori-infected children were identified. Among them, the abundance of F. prausnitzii was significantly lower in the H. pylori-infected children, and then increased after yogurt ingestion and successful bacterial eradication. We further confirmed probiotics promoted F. prausnitzii growth in vitro and in ex vivo using real-time PCR. Moreover, F. prausnitzii supernatant significantly ameliorated lipopolysaccharide-induced IL-8 in HT-29 cells. In conclusions, Probiotics-containing yogurt ingestion and H. pylori eradication can restore the decrease of fecal F. prausnitzii in H. pylori-infected children.

A novel pathogenic mutation on Interleukin-7 receptor leading to severe combined immunodeficiency identified with newborn screening and whole exome sequencing.

BACKGROUND: Patients with severe combined immunodeficiency (SCID), which is caused by genetic defects in immune-related genes involved in the development or activation of the adaptive immune system, often died in infancy due to severe infections before definite molecular diagnosis could be made. Although recent improvement in early diagnosis has been achieved by newborn screening, the genetic basis of many of the patients is still unknown. METHODS: Here we performed whole exome sequencing (WES) to investigate the underlying genetic causes of SCID in a proband identified with newborn screening. Inheritance of the mutation was confirmed with targeted sequencing of the parents. Homozygosity mapping from the WES was used to investigate the consanguinity of the parents. Immunoblotting was used to confirm the loss of expression of the mutant protein. RESULTS: A novel homozygous frameshift mutation of IL7R was identified through WES. Both parents are carriers for this 1-bp deletion. HLA typing and exome-wide homozygous stretch mapping suggested that the parents are consanguineous. Immunoblotting showed no expression of IL7Rα isoform in the whole blood sample of the proband. The proband received peripheral blood stem cell transplantation and her general condition became stable. Our results suggest that IL7R is essential for T cell development but dispensable for the development of certain human NK cells B cells and suggest that WES can be a useful tool for precise genetic diagnosis of SCID following newborn screening in the index patient without the need to screen other members of the whole family.

Rare Compound Heterozygous Frameshift Mutations in ALMS1 Gene Identified Through Exome Sequencing in a Taiwanese Patient With Alström Syndrome.

Alström syndrome (AS) is a rare autosomal recessive disorder that shares clinical features with other ciliopathy-related diseases. Genetic mutation analysis is often required in making differential diagnosis but usually costly in time and effort using conventional Sanger sequencing. Herein we describe a Taiwanese patient presenting cone-rod dystrophy and early-onset obesity that progressed to diabetes mellitus with marked insulin resistance during adolescence. Whole exome sequencing of the patient's genomic DNA identified a novel frameshift mutation in exons 15 (c.10290_10291delTA, p.Lys3431Serfs*10) and a rare mutation in 16 (c.10823_10824delAG, p.Arg3609Alafs*6) of ALMS1 gene. The compound heterozygous mutations were predicted to render truncated proteins. This report highlighted the clinical utility of exome sequencing and extended the knowledge of mutation spectrum in AS patients.