Disseminated Talaromyces marneffei Infection in a Non-HIV Infant With a Homozygous Private Variant of RELB.

Objective: This study presents a relatively rare case of disseminated Talaromyces marneffei (T. marneffei) infection in an HIV-negative patient. Methods: An 8-month-old girl was hospitalized because of uncontrollable fever and cough for 6 days. Routine laboratory tests, biochemical detection, immunological tests, pathogenic examination, and imaging inspection were performed. Genetic tests of trio whole genome sequencing (Trio-WES), trio copy number sequencing (Trio-CNVseq), and Sanger sequencing were conducted to identify pathogenic variants. In silico analysis of the sequence alignment and structural modeling results was carried out to study the possible pathogenicity of the identified variant. Western blotting was performed to investigate the expression of the identified gene at the protein level. Results: Enhanced CT and MRI scanning demonstrated thymic dysplasia, diffuse pulmonary and liver nodules, and many balloon-like air sacs in both lungs. The white blood cell count, neutrophil count, and neutrophil ratio were normal or elevated. The patient was HIV-negative and bone marrow and blood culture showed T. marneffei infection. Total lymphocyte count, CD3+ T lymphocyte count, CD3+CD4+ T lymphocyte count, CD3+CD8+ T lymphocyte count, and NK cell count decreased, while the number of CD19 positive B cells increased. However, the ratio of CD3+CD4+:CD3+CD8+ T cells increased. Trio-WES identified a homozygous private variant of NM_006509: c.400_c.401insAGC/p.Lys134 delinsLysGln in RELB and Sanger sequencing validated the result. Structural modeling indicated that the variant may be pathogenic. Reverse transcription-polymerase chain reaction and Western blot analysis showed that the expression of RelB in the patient was lower than that in the healthy controls at mRNA and protein levels. Conclusion: This is the first report on disseminated T. marneffei infection in a patient with a homozygous private variant of RELB.

Clinical Utility of Next-Generation Sequencing for Developmental Disorders in the Rehabilitation Department: Experiences from a Single Chinese Center.

This study investigated the clinical and genetic characteristics of developmental disorders (DDs) in children attending a rehabilitation department. A total of 94 children with suspected rare and undiagnosed DDs were included in this study. All patients were subjected to next-generation sequencing by means of proband single whole-exome sequencing (Pro-WES) or trio whole-exome sequencing (Trio-WES). To investigate the copy number variations (CNVs), 63 patients were subjected to the trio strategy, and 17 cases were subjected to the proband single strategy. The patients developed early and suffered from severe symptoms. WES reached a high diagnostic rate (48.7%, 46/94), and de novo (48.3%, 28/58) was the main pathogenic form. Most identified single-nucleotide variations (SNVs)/small insertions and deletions (indels) were found only in one patient. The number of uncertain significant locus in the patients taking Trio-WES was significantly lower than that in patients taking Pro-WES (2.1% vs 2.8%). Compared with hereditary mutations passed from parents, pathogenicity was more obvious in de novo mutations. The diagnostic rate of WES accompanied by CNVseq (57.5%, 46/80) was significantly higher (p = 0.016) than WES alone. Next-generation sequencing exhibited a satisfactory diagnostic rate for DDs patients in the rehabilitation department. Compared with the proband-only model, the family trio strategy should be employed more frequently because it can reduce the number of uncertain significant sites and help to identify de novo pathogenic mutations.

Potential role of intestinal microflora in disease progression among patients with different stages of Hepatitis B.

BACKGROUND: Increasing evidence demonstrate that the gut microbiota is involved in the pathogenesis of liver diseases, and faecal microbiota transplantation is considered to be a promising new treatment option. However, there are no reports on the intestinal flora of asymptomatic HBV carriers using next-generation sequencing. This study intends to investigate the potential role of the intestinal microflora in predicting the progression of Hepatitis B patients in different non-cancerous stages. RESULTS: A total of 266 patients with different stages of Hepatitis B and 31 healthy controls were included in this study. Some of the subjects (217 cases) underwent 16S rRNA gene sequencing. Compared with the control group (CK), the α diversity of patients in Group A (HBV carrier) slightly increased, while that of patients in the other three groups decreased. Each group of patients, especially those in Group C (cirrhosis) and Group D (acute-on-chronic liver failure), could be separated from the CK using weighted UniFrac PCoA and ANOSIM. LEfSe revealed that 40 taxa belonging to three phyla had an LDA larger than 4. In addition to the comparison between Group B (chronic Hepatitis B) and Group C, the specific flora and potential taxonomic function were also identified. Different microbial communities were found to be highly correlated with clinical indicators and the Child-Pugh scores. Changes in the microbial community were highly related to the alternations of host metabolism, which in turn, was related to the development of Hepatitis B. Our analysis identified a total of 47 strains with potential biomarker functions at all levels except for the phylum level. CONCLUSIONS: Faecal microbiota transplantation of some potential beneficial bacteria can change with the occurrence of disease, and HBV carriers might be the most suitable donors.

Asymptomatic COVID-19 infection in late pregnancy indicated no vertical transmission.

This study is to investigate the clinical characteristics of late pregnancy with asymptomatic 2019 novel coronavirus disease (COVID-19) infection, evaluate the outcome of maternal and fetal prognosis, and identify the evidence of intrauterine vertical transmission. A 22-years-old pregnant woman with asymptomatic COVID-19 infection who was admitted to our hospital on 11 February 2020 was enrolled in this study. Clinical data including laboratory test results and chest computed tomography (CT) scanning were collected and reviewed. Diagnosis of late pregnancy with asymptomatic COVID-19 infection was made. Lumbar anesthesia for cesarean section was performed and a female baby was delivered uneventfully, with the Apgar score of 9 to 10 points. Three times of COVID-19 nucleic acid test for the baby was negative after delivery. The puerpera returned to normal after the operation and two times of throat swab COVID-19 nucleic acid test were all negative after antiviral therapy. We reported an asymptomatic COVID-19 pregnant woman with detailed clinical information and our result indicated that for late pregnant women with asymptomatic COVID-19 infection, there might be no intrauterine infection caused by vertical transmission.

IDDCA syndrome in a Chinese infant due to GNB5 biallelic mutations.

Herein, we present a Chinese infant with an early-onset intellectual developmental disorder with cardiac arrhythmia syndrome. A 6-month-old boy visited our hospital because of convulsions and paroxysmal cyanosis for 1 day. Mental development analysis showed that the patient had a neurodevelopmental delay. Frequent seizures occurred, and ECG monitoring demonstrated severe cardiac arrhythmia. Whole-exome sequencing showed that the infant had two compound heterozygous variants, NM_016194:c.458G>A/p.Cys153Tyr and NM_016194:c.1032C>A/p.Tyr344*, in GNB5. The first variant was inherited from his mother, while the other one was a de novo variant. Haplotype analysis indicated that the de novo variant was located in the paternal chromosome. Structural modeling indicated that both mutations could influence the interaction of GNB5 with its binding protein. Our study expanded the known genetic and phenotypic spectrum of GNB5-associated diseases, by presenting a Chinese male infant with IDDCA.

Biallelic INTS1 Mutations Cause a Rare Neurodevelopmental Disorder in Two Chinese Siblings.

This study presents two Chinese siblings with a rare neurodevelopmental disorder (NDD) caused by biallelic INTS1 mutations and investigates the clinical features of this disease by means of in silico analysis. Two siblings, an 11-year-old brother and a 5-year-old sister, visited our hospital due to physical retardation and profound intellectual disability. Whole-exome sequencing (WES) was performed for the girl, and Sanger sequencing was used to validate the identified variants. Phenotype correlation analysis and in silico genetic interaction network analysis were performed to investigate genes that could lead to diseases similar to the rare disease in the patients. Growth retardation, distinct intellectual disability, hypertelorism, mild cataract, uneven teeth, abnormal palmar and plantar creases, and dubious genitalia were noted in the sister. No neurological features related to neuropathy were found. The brother showed features and growth delay similar to his sister. Heterozygous novel variants of c.1645A>G,p.Met549Val and c.5881C>T,p.Gln1961* in INTS1 were considered a candidate etiology. Sanger sequencing demonstrated that the variants were inherited from the grandfather and (maternal) grandmother. Phenotype correlation analysis revealed that CTDP1 mutation-induced congenital cataracts-facial dysmorphism-neuropathy (CCFDN) mostly overlapped with the performance of our patients. In silico analysis of the genetic interaction network showed that INTS1 is highly associated with INTS8 and CTDP1. Our study further validated that biallelic INTS1 mutations could bring about the onset of a novel neurodevelopmental disorder.

Diagnostic Yields of Trio-WES Accompanied by CNVseq for Rare Neurodevelopmental Disorders.

OBJECTIVE: This study is to investigate the diagnostic yield of the combination of trio whole exome sequencing (Trio-WES) and copy number variation sequencing (CNVseq) for rare neurodevelopmental disorders (NDDs). METHODS: Clinical data from consecutive pediatric patients who were diagnosed with rare NDDs that were suspected to be monogenic disorders, who were admitted to our hospital from April 2017 to March 2019, and who underwent next generation sequencing (NGS) were extracted from the medical records. Patients for whom Trio-WES and CNVseq data were available were enrolled in this study. Sanger sequencing was applied for the validation of the variants identified by Trio-WES. Sequence alignment and structural modeling were conducted for analyzing the possibility of the variants in the onset of the NDDs. RESULTS: In total, 54 patients were enrolled in this study, with the median age of 15 (8-26) months. A total of 242 phenotypic abnormalities belonging to 20 different systems were identified in the cohort. Twenty-four patients were diagnosed by Trio-WES, eight patients were diagnosed by CNVseq, and one case was identified by both WES and CNVseq. Compared with Trio-WES, the diagnosis rate of Trio-WES accompanied by CNVseq was significantly higher (P = 0.016). Trio-WES identified 36 variants in 26 different genes, among which 27 variants were novel. CNVseq detected four duplications and eight deletions, ranging from 310 kb to 23.27 Mb. Our case examples demonstrated the high heterogeneity of NDDs and showed the challenges of rare NDDs for physicians. CONCLUSION: The significantly higher diagnosis rate of Trio-WES accompanied by CNVseq makes this strategy a potential alternative to the most widely used approaches for pediatric children with rare and undiagnosed NDDs.

The combination of whole-exome sequencing and copy number variation sequencing enables the diagnosis of rare neurological disorders.

This retrospective study aims to investigate the diagnostic yields of multiple strategies of next-generation sequencing (NGS) for children with rare neurological disorders (NDs). A total of 220 pediatric patients with NDs who visited our hospital between Jan 2017 and Dec 2018 and had undergone NGS were included. Most patients were 5 years old or younger, and the number of patients visiting the hospital decreased with age. Seizures were the most common symptom in this cohort. The positive rates for targeted NGS panels (Panel), whole-exome sequencing (WES), and copy number variation sequencing (CNVseq) were 26.5% (9/34), 36.6% (63/172), and 16.7% (22/132), respectively. The positive rate for patients undergoing a combination of WES and CNVseq (WES + CNVseq) was 47.8% (54/113), which was significantly better than the positive rate for patients who underwent WES alone (32.7%, 37/113). A total of 83 variants were found in 42 genes, and SCN1A was the most frequently mutanted gene. Twenty-four CNVs were identified in 22 patients: two CNVs were inherited from the mother; 12 CNVs were de novo; and the CNV origins could not be determined in 10 patients. WES + CNVseq may potentially be the mostly effective NGS approach for diagnosis of rare NDs in pediatric patients.

Mitochondrial metabolism is inhibited by the HIF1α-MYC-PGC-1ß axis in BRAF V600E thyroid cancer.

BRAF V600E is the most common mutation identified in thyroid cancers. However, the relationship between BRAF V600E and metabolic reprogramming in thyroid cancer is unclear. Here, we investigate the mechanism of metabolic reprogramming in BRAF V600E thyroid cancer by constructing BRAF V600E-overexpressing and BRAF-knockdown thyroid cell lines for use in mitochondrial respiration and glycolysis experiments. Western blot and RT-qPCR were performed to measure the level of metabolism-related proteins, and various approaches were used to investigate transcriptional regulation. In thyroid cancer cells, the overexpression of BRAF V600E inhibited OXPHOS gene expression and mitochondrial respiration but enhanced aerobic glycolysis. Clinical thyroid cancer samples carrying the BRAF V600E mutation had suppressed levels of PGC-1ß but increased expression of HIF1α. Our results show that BRAF V600E reduced mitochondrial respiration by decreasing the expression of PGC-1ß. In addition, HIF1α, which is a target of BRAF V600E, was found to regulate the expression of PGC-1ß via MYC. Furthermore, glycolysis-related enzymes, such as LDHA and PKM2, were upregulated in BRAF V600E mutant thyroid cancer specimens, thereby promoting glycolysis. MEK1/2 inhibitor treatment enhanced the specific dependence of BRAF V600E mutant thyroid cancer on mitochondrial respiration. These results indicate that in thyroid cancer, the BRAF V600E mutation alters the HIF1α-MYC-PGC-1ß axis, causing mitochondrial respiration to be inhibited and aerobic glycolysis to be enhanced.

Compound heterozygous variants in MOGS inducing congenital disorders of glycosylation (CDG) IIb.

This study is to present two Chinese siblings who were diagnosed with congenital disorders of glycosylation (CDG) IIb because of mannosyl-oligosaccharide glucosidase (MOGS) deficiency. The siblings visited our hospital due to "pulmonary infection". Facial dysmorphism including long eyelashes, blepharophimosis, depressed nasal bridge, and high palate was noted. Head MRI of the elder sister showed increased signals on T1W1, bilateral frontal gyrus stenosis, and thin corpus callosum. Both cases presented progressive hepatomegaly and elevated hepatic enzymes. Low immunoglobulin was discovered in the siblings. Compound heterozygous variants of NM_006302:c.1239_1267dup,p.Asp414Leufs*17, c.544 G > A,p.Gly182Arg, and c.1698C > A,p.Asp566Glu in MOGS were identified. Structural modeling demonstrated that the mutations were pathogenic to MOGS. Our study enriched the genetic and phenotypic spectrum of MOGS-CDG, and for children with facial dysmorphism, postnatal dyspnea, seizures, motor developmental delay, hypotonia, and immunological or gastrointestinal dysfunction, this disease should be highly suspected.

Whole-exome Sequencing Helps the Diagnosis and Treatment in Children with Neurodevelopmental Delay Accompanied Unexplained Dyspnea.

Neurodevelopmental delay accompanied unexplained dyspnea is a highly lethal disease in clinic. This study is to investigate the performance characteristics of trio whole exome sequencing (Trio-WES) in a pediatric setting by presenting our patient cohort and displaying the diagnostic yield. A total of 31 pediatric patients showing neurodevelopmental delay accompanied unexplained dyspnea were admitted to our hospital and referred for molecular genetic testing using Trio-WES. Eight genes namely MMACHC, G6PC, G6PT, ETFDH, OTC, NDUFAF5, SLC22A5, and MAGEL2 were suspected to be responsible for the onset of the clinical symptoms and 6 variants were novel. Standard interpretation according to ACMG guideline showed that the variants were pathogenic. Finally, diagnosis of methylmalonic aciduria and homocystinuria, glycogen storage disease, ornithine transcarbamylase deficiency, glutaric acidemia II, mitochondrial complex 1 deficiency, carnitine deficiency, and Schaaf-Yang syndrome was made in 12 out of the 31 patients. Trio-WES is an effective means for molecular diagnosis of infantile neurodevelopmental delay accompanied unexplained dyspnea. As for molecular etiology identification, when routine potential monogenetic inheritance patterns including de novo, autosomal recessive, autosomal dominant, and X-linked recessive inheritance analysis is negative, physicians should take into account imprinted genes.

SREBP2 contributes to cisplatin resistance in ovarian cancer cells.

This study is to investigate transcription factors involved in cisplatin resistance in ovarian cancer cells. The transcriptome of cisplatin resistant and sensitive A2780 epithelial ovarian cancer cells was obtained from GSE15372. Ovarian transcriptome data GSE62944 was downloaded from TCGA and applied for transcription regulatory network analysis. The analysis results were confirmed using quantitative polymerase chain reaction. The roles of SREBP2 in cisplatin-resistant cells were investigated by RNA inference and cell viability analysis. Transcription regulatory network analysis found that 12 transcription factors and their targets were involved in cisplatin resistant in A2780 cells. Among these factors, the targets of EZH2 and SREBP2 revealed by Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining were also enriched in differentially expressed genes between cisplatin resistant and cisplatin sensitive cells. Their targets were enriched mainly in cell cycle and cholesterol metabolic process, respectively. Bioinformatic analysis illustrated three known targets of SREBP2, namely LDLR, FDFT1, and HMGCR were increased in A2780-resistant cell lines. Additionally, the three genes and SREBP2 were also elevated in live cells after cisplatin treatment via quantitative polymerase chain reaction. Importantly, RNA inference of SREBP2 in A2780 cell line resulted in a decrease of cell viability after cisplatin treatment. SREBP2 played important roles in cisplatin resistance and cholesterol metabolic process might be a novel target for cancer therapy. Impact statement Transcriptome of cisplatin resistant and sensitive A2780 epithelial ovarian cancer cells was obtained from GSE15372 and TCGA. Twelve transcription factors and their targets were involved in cisplatin resistant. Among these factors, the targets of EZH2 and SREBP2 revealed by Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining were also enriched in differentially expressed genes. Their targets were enriched mainly in cell cycle and cholesterol metabolic process. Three targets of SREBP2, namely LDLR, FDFT1, and HMGCR were increased in A2780-resistant cell lines and were found elevated in live cells after cisplatin treatment via qPCR. RNAi of SREBP2 in A2780 cell line resulted in a decrease of cell viability after cisplatin treatment. SREBP2 played important roles in cisplatin resistance and might be a novel target for cancer therapy.

De Novo Mutation of Paternal IGF2 Gene Causing Silver-Russell Syndrome in a Sporadic Patient.

Silver-Russell syndrome (SRS) is a rare, but well-recognized disease characterized by growth disorder. To date, there are two reports arguing IGF2 mutation for the onset of SRS. Herein, we present another sporadic case harboring IGF2 mutation. The male proband was the first and only child of a non-consanguineous Chinese couple. He was small for gestational age, with relative macrocephaly at birth. Severe feeding difficulties, low feeding, and growth retardation were revealed during neonatal period. At 4.5 years old, obvious body asymmetry was noted. Whole exome sequencing identified a novel de novo c.101G > A (p.Gly34Asp, NM_000612) variant in IGF2 and Sanger sequencing validated the variant. Amplification refractory mutation system polymerase chain reaction demonstrated that the IGF2 variant was on the paternal allele. Alignment shows the variant is evolutionarily conserved. Structural modeling argues that the variant site might be important for the binding of IGF2 to its receptor. Our study provides further evidence that IGF2 mutation may be another mechanism of SRS, and we consider that IGF2 should be included in a disease specific gene panel in case it is designed for SRS routine diagnostics.

Structural studies of the periplasmic portion of the diguanylate cyclase CdgH from Vibrio cholerae.

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger involved in bacterial signal transduction and produced by diguanylate cyclases (DGCs) generally containing highly variable periplasmic signal-recognition domains. CdgH is a DGC enzyme that regulates rugosity associated phenotypes in Vibrio cholerae. CdgH has two N-terminal tandem periplasmic substrate-binding (PBPb) domains for its signal recognition; however, the role of the tandem PBPb domains remains unclear. Here, we reported the crystal structure of the periplasmic portion of CdgH, which indicated that both tandem PBPb domains consist of typical interlobe ligand-binding architecture. Unexpectedly, the PBPb-I domain binds an L-arginine which apparently has been co-purified from the E. coli expression system, whereas the PBPb-II domain is in an unliganded open state. Structural comparison with other amino acid-binding proteins indicated that despite similar ligand-binding pockets, the PBPb-I domain possesses two ligand-binding residues (E122 and Y148) not conserved in homologs and involved in hydrophilic and hydrophobic interactions with L-arginine. Isothermal titration calorimetry indicated that the PBPb-I is primarily an L-arginine/L-lysine/L-ornithine-binding domain, whereas the PBPb-II domain exhibits a preference for L-glutamine and L-histidine. Remarkably, we found that the periplasmic portion of CdgH forms a stable dimer in solution and L-arginine binding would cause conformational changes of the dimer.

Novel NFU1 Variants Induced MMDS Behaved as Special Leukodystrophy in Chinese Sufferers.

Multiple mitochondrial dysfunctions syndrome (MMDS) is an autosomal recessive disorder of systemic energy metabolism. This study is to present the diagnosis of two MMDS Chinese sufferers. Physical and auxiliary examination was performed. Next generation sequencing (NGS) was conducted to identify candidate causal genes and Sanger sequencing was adopted to validate the variants detected. Fluorescence quantitative polymerase chain reaction (FQ-PCR) amplification was carried out to testify allelic loss existence. Structural investigation was performed to study the possibility of the candidate variants for disease onset. Physical examination showed that the children were with neurological impairment. Auxiliary examination demonstrated energy metabolism disturbance and abnormal brain signals. NGS found that the probands had homozygous mutation of c.545 + 5G > A and compound heterozygous variants of exon 4 deletion and c.721G > T in NFU1, respectively. NFU1 was considered as candidate molecular etiology and indicating that the kids were with MMDS. Sanger sequencing confirmed the variants. FQ-PCR amplification characterized that patient 1 had a de novo allele mutation while patient 2 inherited from his parents. Structural investigation demonstrated that the variants were possible for MMDS occurrence. This is the first report of patients diagnosed as MMDS with novel mutation types from the Asia-Pacific region.

Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system.

YfiBNR is a recently identified bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiB(L43P) complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB(L43P) adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiB(L43P) shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiB(L43P)-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

Crystal structures of YfiR from Pseudomonas aeruginosa in two redox states.

YfiBNR is a recently identified c-di-GMP regulatory system involved in bacterial biofilm formation. The periplasmic protein YfiR inhibits the diguanylate cyclase activity of the inner membrane protein YfiN, whereas YfiB in the outer membrane can release this inhibition by sequestration of YfiR. In addition, this system may respond to anoxic conditions via YfiR, although the detailed mechanism is still unknown. Here we report crystal structures of Pseudomonas aeruginosa YfiR in the absence and presence of oxidative glutathione. Our structures reveal the overall folding of YfiR for the first time and demonstrate that YfiR exist as a dimer. Comparison of the two structures in different redox states revealed a broken/formation of one disulfide bond (Cys71-Cys110) and local conformational change around the other one (Cys145-Cys152). Mutagenesis studies indicated that Cys145-Cys152 plays an important role in maintaining the correct folding of YfiR.