Major Pathogenic Gene Research Articles

Generation of a human induced pluripotent stem cell line (SMUSHi002-A) from an ALS patient carrying a heterozygous mutation c.1562G > A in the FUS gene

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Affected patients experience gradual loss of their spinal cord and cortical motor neurons with consequent muscle weakness and emaciation, and eventual respiratory failure. The pathogenesis of ALS remains largely unknown although the FUS (sarcoma fusion gene) gene is known to be one of the major pathogenic genes. We have generated an induced pluripotent stem cell line SMUSHi002-A from an ALS patient who carries a heterozygous mutation c.1562G > A in FUS. This cell line will serve as a useful model to investigate disease pathogenesis and develop potential therapeutic approaches for ALS.

Abstract 12562: DYRK1A/PPARG/EGR1 Pathway Elicits Endothelial Dysfunctions and Predispose to Developing Pulmonary Hypertension in Down Syndrome

Introduction: Patients with Down syndrome (DS) have a higher prevalence of pulmonary arterial hypertension (PAH). Pulmonary vascular endothelial dysfunction has been recognized to be one of the predisposing factors to develop PAH, but pathophysiology of pulmonary endothelial cells in DS specific PAH is still unknown. Hypothesis: Endothelial dysfunctions contribute to developing PAH in DS. Methods: Induced pluripotent stem cell-derived vascular endothelial cells (iPSC-ECs) from DS patients were utilized for analyzing cellular and mitochondrial functions. A Cre-loxP mediated isogenic correction line of disomy 21 was established and used as a control. Results: DS-iPSC-ECs showed increased apoptosis, reduced angiogenesis, increased mitochondrial reactive oxygen species, and decreased ATP production, which were all consistent with features of endothelial dysfunctions previously reported in other types of PAH. RNA-seq revealed increased expression of EGR1 in DS-iPSC-ECs, and its inhibition restored cellular and mitochondrial dysfunctions, demonstrated by si EGR1 and pioglitazone treatment. Moreover, we revealed that increased expression of DYRK1a , one of the major pathogenic genes in DS critical region on chromosome 21, led to elevated EGR1 expression thorough PPARG suppression. These experimental results were reinforced by the observations that pulmonary ECs of a DS patient with severe PAH also exhibited increased EGR1. Conclusions: DYRK1A/PPARG/EGR1 pathway in DS is responsible for endothelial dysfunctions and might be a new therapeutic target for DS specific PAH.

Analysis of amelogenesis imperfecta with abnormal tooth eruption caused by FAM83H mutation

Objective: FAM83H is one of the major pathogenic genes of amelogenesis imperfecta (AI). Previous studies focused on the abnormal enamel development and mineralization caused by the mutations in FAM83H. Here we aimed to observe other effects of FAM83H mutations on tooth eruption besides AI through clinical case analysis. Methods: Published AI cases with FAM83H mutations were searched through PubMed database, and the characteristics of tooth eruption of each cases were counted and analyzed. The literature search range was from January 1, 2008 to February 28, 2023, using the keywords FAM83H and amelogenesis imperfecta. The included literature must provide the detailed radiographic imaging or dental eruption information of AI patients, as well as FAM83H gene mutation information. The basic clinical information, tooth phenotypes, and mutations of all the enrolled cases were collected and analyzed in order to find the characteristics of abnormal tooth eruption. Results: Among 45 papers about FAM83H related to AI, twenty meeting the inclusion criteria were selected, involving 50 AI patients carrying FAM83H mutations who had radiographic image data or the detailed description of tooth eruption. A total of 34 abnormal erupted teeth were from 12 patients (12/50, 24%), among which 85% (29/34) had clear eruption path without any eruption obstructions, either embedded (25/34, 74%) or partially erupted (4/34, 12%). Tooth position analysis found that abnormal eruption of canines and second molars accounted for the highest proportion, accounting for 38% (13/34) respectively. Conclusions: The mutations in FAM83H may lead to amelogenesis imperfecta as well as abnormal tooth eruption at specific tooth positions.

Identification of renal cyst cells of type I Nephronophthisis by single-nucleus RNA sequencing.

Background: Nephronophthisis (NPH) is the most common genetic cause of end-stage renal disease (ESRD) in childhood, and NPHP1 is the major pathogenic gene. Cyst formation at the corticomedullary junction is a pathological feature of NPH, but the mechanism underlying cystogenesis is not well understood. The isolation and identification of cystic cell subpopulation could help to identify their origins and provide vital clues to the mechanisms underlying cystogenesis in NPH. Methods: Single-nucleus RNA sequencing (snRNA-seq) was performed to produce an atlas of NPHP1 renal cells. Kidney samples were collected from WT (Nphp1 +/+) mice and NPHP1 (Nphp1 del2-20/del2-20) model mice. Results: A comprehensive atlas of the renal cellular landscape in NPHP1 was generated, consisting of 14 basic renal cell types as well as a subpopulation of DCT cells that was overrepresented in NPHP1 kidneys compared to WT kidneys. GO analysis revealed significant downregulation of genes associated with tubular development and kidney morphogenesis in this subpopulation. Furthermore, the reconstruction of differentiation trajectories of individual cells within this subpopulation confirmed that a specific group of cells in NPHP1 mice become arrested at an early stage of differentiation and proliferate to form cysts. We demonstrate that Niban1 is a specific molecular marker of cystic cells in both mice and human NPHP1. Conclusion: In summary, we report a novel subpopulation of DCT cells, marked by Niban1, that are classified as cystic cells in the NPHP1 mice kidney. These results offer fresh insights into the cellular and molecular basis of cystogenesis in NPH.

Decreased Imiquimod-Induced Psoriasis-Like Skin Inflammation in a Novel MvdF250S/+ Knock-In Mouse Model.

The mevalonate-diphosphate decarboxylase (MVD) gene, a member of the mevalonate pathway, plays a critical role in regulating the biosynthesis of cholesterol, steroid hormones, and non-steroid isoprenoids. Previous studies have suggested that the MVD c.746T > C mutation is a major pathogenic gene of porokeratosis (PK), an autoinflammatory keratinization disease (AIKD) with unclear pathogenesis, few effective treatments, and no suitable animal model. To investigate the function of MvdF250S/+ mutation, we developed a novel MvdF250S/+ mouse model carrying an equivalent point mutation to the most common genetic variation among Chinese PK patients (MVDF249S/+) using CRISPR/Cas9 technology, which exhibited reduced cutaneous expression of Mvd protein. In the absence of external stimulation, MvdF250S/+ mice did not display specific phenotypes. However, upon induction with imiquimod (IMQ), MvdF250S/+ mice exhibited decreased susceptibility to skin acute inflammation compared to wild-type (WT) mice, as evidenced by reduced cutaneous proliferation and lower protein levels of IL-17a and IL-1β. Additionally, after IMQ induction, the MvdF250S/+mice exhibited downregulated collagen generation and upregulated expression of Fabp3 compared to WT mice, whereas no significant changes in the key genes related to cholesterol regulation were found. Furthermore, the MvdF250S/+ mutation activated autophagy. Our findings provided insights into the biological function of MVD in the skin.

The relationship between genotype of familial hypercholesterolemia and the efficacy of PCSK9 inhibitors

Objective: This study intends to explore the difference in the efficacy of PCSK9 inhibitors in patients with different FH phenotypes by analyzing the level of blood lipids before and after treatment with PCSK9 inhibitors in patients with familial hypercholesterolemia (FH) with different allele grades. Methods: Patients with FH phenotype, who admitted to Beijing Anzhen Hospital from January 2019 to October 2020, were enrolled. Age, sex and other clinical information were collected from enrolled, and the pathogenic genes were detected by the second generation sequencing technique. The patients were divided into five groups according to the number of alleles involved and the degree of gene damage: single allele-null mutation group, single allele-defect mutation group, multi-allele-null mutation group, multi-allele-defect mutation group and no major pathogenic gene mutation group. The results of blood lipids were collected before medication, 4-6 weeks of intensive statin treatment and one month after combined treatment with PCSK9 inhibitor (PCSK9i). The LDL-C level were compared among groups. ASCVD risk stratification was performed in all patients, and the proportion of LDL-C level reaching the corresponding risk stratification target value of each genotype group after treatment was analyzed. Results: A total of 66 patients with FH phenotype were included, including 47 males (71.2%) and 19 females (28.8%),the mean age was(43.1±13.4 years). There were 7 cases in single allele-null mutation group (10.6%), 25 cases in single allele-defect mutation group (37.9%), 8 cases in multi-allele-null mutation group (12.1%), 18 cases in multi-allele-defect mutation group (27.3%) and 8 cases in no major pathogenic mutation group (12.1%). The degree of LDL-C reduction post combined PCSK9 inhibitor therapy was as follows: single allele mutation group>no major pathogenic mutation group>multi-allele mutation group, general distribution was in the range of 0-90.0%. Two groups of single allele mutation and no major pathogenic mutation group>50.0%>multi-allele mutation group. Under the combined treatment of PCSK9 inhibitors, the further decrease of LDL-C was in the order of single allele mutation group>non-major pathogenic mutant group>multi-allele mutation group. The efficacy of combined therapy on reducing LDL-C at 1 month after treatment decreased with the increase of baseline LDL-C level (r = 0.46, P<0.001) in patients with FH phenotype. In addition, the further decrease of LDL-C level post high-intensity statin therapy combined with PCSK9 inhibitors decreased with the increase of baseline LDL-C levels (r = 0.40, P<0.001). The degree of LDL-C decrease was high and stable by statin combined with PCSK9 inhibitor therapy in single allele mutation group. In the single allele-defect mutant group, the decrease of LDL-C increased with the increase of baseline LDL-C level post intensive statin treatment and combined PCSK9 inhibitor treatment ((r=0.54, P=0.009); r=0.45,P=0.030), and the further decrease of LDL-C level decreased with the increase of baseline LDL-C level in single allele-defect mutant group post combined therapy with PCSK9 inhibitor (r=0.43, P=0.040). The decrease of LDL-C in patients with the multi-allele mutation group varied with different pathogenic gene loci and combinations post combined therapy with PCSK9 inhibitor. There was no significant difference in the level of blood lipids between the group without major pathogenic gene mutation and the group with single allele mutation before and after treatment. The percentage of patients achieving LDL-C goals with different genotypes of phenotypic FH were as follows: single allele mutation group (86.7%), non-major pathogenic mutant group (75.0%) and multi-allele mutation grou (<5.0%). Conclusions: All patients with different FH phenotypes could benefit from the intensive lipid-lowering therapy with statins and PCSK9 inhibitors, however, there are significant differences in the efficacy of lowering LDL-C in Chinese patients with FH phenotype with different molecular etiologies. Therefore, the pathogenic gene analysis may suggest the lipid-lowering effect of PCSK9 inhibitors in patients with FH.

Long Noncoding RNA lnc-TSSK2-8 Activates Canonical Wnt/β-Catenin Signaling Through Small Heat Shock Proteins HSPA6 and CRYAB.

Congenital heart defects (CHDs) are the most common birth defects worldwide. 22q11.2 deletion syndrome is the most common microdeletion disorder that has been frequently associated with conotruncal malformations. By now, the dosage-sensitive gene TBX1 has been adopted as the major pathogenic gene responsible for 22q11.2 deletion, which is regulated by canonical Wnt/β-catenin signaling pathway in heart outflow tract development. Here, we report the long noncoding RNA (lncRNA) lnc-TSSK2-8, which is encompassed in the 22q11.2 region, that can activate canonical Wnt/β-catenin signaling by protecting β-catenin from degradation, which could result from decreased ubiquitination. Such effects were mediated by two short heat shock proteins HSPA6 and α-β-crystallin (CRYAB), whose expression was regulated by lnc-TSSK2-8 through a competing endogenous RNA (ceRNA) mechanism. In clinical practice, the pathogenesis of copy number variation (CNV) was always attributed to haploinsufficiency of protein-coding genes. Here, we report that the 22q11.2 lncRNA lnc-TSSK2-8 significantly activated canonical Wnt/β-catenin signaling, which has major roles in cardiac outflow tract development and should act upstream of TBX1. Our results suggested that lncRNAs should contribute to the etiology of CNV-related CHD.

The Predicted Key Molecules, Functions, and Pathways That Bridge Mild Cognitive Impairment (MCI) and Alzheimer's Disease (AD).

To elucidate the key molecules, functions, and pathways that bridge mild cognitive impairment (MCI) and Alzheimer's disease (AD), we investigated open gene expression data sets. Differential gene expression profiles were analyzed and combined with potential MCI- and AD-related gene expression profiles in public databases. Then, weighted gene co-expression network analysis was performed to identify the gene co-expression modules. One module was significantly negatively associated with MCI samples, in which gene ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that these genes were related to cytosolic ribosome, ribosomal structure, oxidative phosphorylation, AD, and metabolic pathway. The other two modules correlated significantly with AD samples, in which functional and pathway enrichment analysis revealed strong relationships of these genes with cytoplasmic ribosome, protein binding, AD, cancer, and apoptosis. In addition, we regarded the core genes in the module network closely related to MCI and AD as bridge genes and submitted them to protein interaction network analysis to screen for major pathogenic genes according to the connectivity information. Among them, small nuclear ribonucleoprotein D2 polypeptide (SNRPD2), ribosomal protein S3a (RPS3A), S100 calcium binding protein A8 (S100A8), small nuclear ribonucleoprotein polypeptide G (SNRPG), U6 snRNA-associated Sm-like protein LSm3 (LSM3), ribosomal protein S27a (RPS27A), and ATP synthase F1 subunit gamma (ATP5C1) were not only major pathogenic genes of MCI, but also bridge genes. In addition, SNRPD2, RPS3A, S100A8, SNRPG, LSM3, thioredoxin (TXN), proteasome 20S subunit alpha 4 (PSMA4), annexin A1 (ANXA1), DnaJ heat shock protein family member A1 (DNAJA1), and prefoldin subunit 5 (PFDN5) were not only major pathogenic genes of AD, but also bridge genes. Next, we screened for differentially expressed microRNAs (miRNAs) to predict the miRNAs and transcription factors related the MCI and AD modules, respectively. The significance score of miRNAs in each module was calculated using a hypergeometric test to obtain the miRNApivot-Module interaction pair. Thirty-four bridge regulators were analyzed, among which hsa-miR-519d-3p was recognized as the bridge regulator between MCI and AD. Our study contributed to a better understanding of the pathogenic mechanisms of MCI and AD, and might lead to the development of a new strategy for clinical diagnosis and treatment.

Mutation screening of the USH2A gene reveals two novel pathogenic variants in Chinese patients causing simplex usher syndrome 2

BackgroundUsher syndrome (USH) is the most prevalent cause of the human genetic deafness and blindness. USH type II (USH2) is the most common form of USH, and USH2A is the major pathogenic gene for USH2. For expanding the spectrum of USH2A mutations and further revealing the role of USH2A in USH2, we performed the USH2A gene variant screening in Chinese patients with USH2.MethodsGenomic DNA was extracted from peripheral blood of unrelated Chinese USH2 patients, we designed specific primers for amplifying the coding region (exons 2–72) of the USH2A gene. Sanger sequencing was used to study alleles. Silico prediction tools were used to predict the pathogenicity of the variants identified in these patients.ResultsFive heterozygous pathogenic variants were detected in four patients. Two patients were found to have two-mutations and two patients only have one. Two novel variants c.4217C > A (p.Ser1406X) and c.11780A > G (p.Asp3927Gly)) were predicted deleterious by computer prediction algorithms. In addition, three reported mutations (c.8559-2A > G, c.8232G > C and c.11389 + 3A > T) were also found in this study.ConclusionsWe identified five heterozygous pathogenic variants in the USH2A gene in Chinese patients diagnosed with Usher syndrome type 2, two of which were not reported. It expands the spectrum of USH2A variants in USH.

Excessive Startle with Novel GLRA1 Mutations in 4 Chinese Patients and a Literature Review of GLRA1-Related Hyperekplexia.

Background and PurposeHyperekplexia (HPX), a rare neurogenetic disorder, is classically characterized by neonatal hypertonia, exaggerated startle response provoked by the sudden external stimuli and followed by a shortly general stiffness. Glycine receptor alpha 1 (GLRA1) is the major pathogenic gene of the disease. We described the clinical manifestations of genetically confirmed HPX patients and made a literature review of GLRA1-related HPX to improve the early recognition and prompt the management of the disorder.MethodsExtensive clinical evaluations were analyzed in 4 Chinese HPX patients from two unrelated families. Next generation sequencing was conducted in the probands. Sanger sequence and segregation analysis were applied to confirm the findings.ResultsAll four patients including 3 males and 1 female presented with excessive startle reflex, a cautious gait and recurrent falls. Moreover, startle episodes were dramatically improved with the treatment of clonazepam in all cases. Exome sequencing revealed 2 homozygous GLRA1 mutations in the patients. The mutation c.1286T>A p.I429N has been previously reported, while c.754delC p.L252* is novel.ConclusionsHPX is a treatable disease, and clonazepam is the drug of choice. By studying and reviewing the disorder, we summarized the phenotype, expanded the genotype spectrum, and discussed the possible pathogenic mechanisms to enhance the understanding and recognition of the disease. Early awareness of the disease is crucial to the prompt and proper administration, as well as the genetic counseling.

Pathogenic genes and clinical therapeutic strategies for Treacher Collins syndrome

Treacher Collins syndrome is a congenital craniofacial malformation with autosomal dominant inheritance as the main genetic pattern. In this condition, the biosynthesis of ribosomes in neural crest cells and neuroepithelial cells is blocked and the number of neural crest cells that migrate to the craniofacial region decreases, causing first and second branchial arch dysplasia. Definite causative genes include treacle ribosome biogenesis factor 1 (tcof1), RNA polymerase Ⅰ and Ⅲ subunit C (polr1c), and RNA polymerase Ⅰ and Ⅲ subunit D (polr1d). This paper provides a review of research of three major patho-genic genes, pathogenesis, phenotypic research, prevention, and treatment of the syndrome.

Rapid and Reliable Detection of Nonsyndromic Hearing Loss Mutations by Multicolor Melting Curve Analysis

Hearing loss is a common birth defect worldwide. The GJB2, SLC26A4, MT-RNR1 and MT-TS1 genes have been reported as major pathogenic genes in nonsyndromic hearing loss. Early genetic screening is recommended to minimize the incidence of hearing loss. We hereby described a multicolor melting curve analysis (MMCA)-based assay for simultaneous detection of 12 prevalent nonsyndromic hearing loss-related mutations. The three-reaction assay could process 30 samples within 2.5 h in a single run on a 96-well thermocycler. Allelic types of each mutation could be reproducibly obtained from 10 pg ~100 ng genomic DNA per reaction. For the mitochondrial mutations, 10% ~ 20% heteroplasmic mutations could be detected. A comparison study using 501 clinical samples showed that the MMCA assay had 100% concordance with both SNaPshot minisequencing and Sanger sequencing. We concluded that the MMCA assay is a rapid, convenient and cost-effective method for detecting the common mutations, and can be expectedly a reliable tool in preliminary screening of nonsyndromic hearing loss in the Chinese Han population.

Characterization and survival of environmental Escherichia coli O26 isolates in ground beef and environmental samples.

In addition to Escherichia coli O157:H7, shiga toxin-producing E. coli (STEC) O26 was added to the zero-tolerance adulterant list together with other 5 non-O157 STEC serogroups in 2012. Four farm O26 isolates were used in this study; they were obtained from a on-farm survey study conducted in Alabama. The presence of 3 major pathogenic genes (stx1, stx2, and eaeA) was determined through multiplex polymerase chain reaction (PCR). Two major pathogenic gene profiles were observed: 3 of the farm isolates contain only the eaeA gene whereas 1 farm isolate has both the eaeA and the stx1 genes. No significant difference was seen among the 4 farm isolates in the antibiotic resistance tests. To test their survival in ground beef and environmental samples, 2 inoculums were prepared and inoculated at various concentrations into samples of ground beef, bovine feces, bedding materials, and trough water. One inoculum was made of 3 farm isolates containing only the eaeA gene and another inoculum contained the isolate with both the eaeA and stx1 genes. Inoculated beef samples were stored at 4 °C for 10 d and the inoculated environmental samples were stored at ambient temperature for 30 d. Results showed that virulence gene profiles do not have an impact on O26's ability to survive in ground beef and in environment (P > 0.05). The inoculation levels, sample types as well as the storage times are the major factors that impact O26 survival (P < 0.05).

ATP1A3 mutations and genotype-phenotype correlation of alternating hemiplegia of childhood in Chinese patients.

Alternating hemiplegia of childhood (AHC) is a rare and severe neurological disorder. ATP1A3 was recently identified as the causative gene. Here we report the first genetic study in Chinese AHC cohort. We performed whole-exome sequencing on three trios and three unrelated patients, and screened additional 41 typical cases and 100 controls by PCR-Sanger sequencing. ATP1A3 mutations were detected in 95.7% of typical AHC patients. At least 93.3% were de novo. Four late onset, atypical AHC patients were also mutation positive, suggesting the need for testing ATP1A3 mutations in atypical cases. Totally, 13 novel missense mutations (T370N, G706R, L770R, T771N, T771I, S772R, L802P, D805H, M806K, P808L, I810N, L839P and G893R) were identified in our study. By homology modeling of the mutant protein structures and calculation of an extensive list of molecular features, we identified two statistically significant molecular features, solvent accessibility and distance to metal ion, that distinguished disease-associated mutations from neutral variants. A logistic regression classifier achieved 92.9% accuracy by the average of 100 times of five-fold cross validations. Genotype-phenotype correlation analysis showed that patients with epilepsy were more likely to carry E815K mutation. In summary, ATP1A3 is the major pathogenic gene of AHC in Chinese patients; mutations have distinctive molecular features that discriminate them from neutral variants and are correlated with phenotypes.

Mutation analysis of the SCN1A gene in severe myoclonic epilepsy of infancy

To investigate the mutations of the sodium channel alpha 1 subunit gene SCN1A in severe myoclonic epilepsy of infancy (SMEI) patients and analyze its inheritance. Twenty-three patients consistent with the diagnosis of SMEI were selected for SCN1A mutation analysis. Genomic DNA was extracted from peripheral blood lymphocytes of these patients and their parents. All the twenty-six exons of the SCN1A gene were amplified by PCR and sequenced. In the 23 SMEI patients, 17 mutations were identified in 17 unrelated SMEI patients. The SCN1A mutation rate was 73.9% (17/23). The mutations included 8 missense mutations (F90S, I91T, A239T, W952G, T1210K, V1335M, V1390M and G1433E), 3 nonsense mutations (R612X, W768X and W1408X), 3 deletion mutations (A395fsX400, L556fsX557 and V1778fsX1800), 1 insertion mutation (Y1241fsX1270), 1 splice-site mutation (IVS10+3 A to G) and 1 synonymous mutation (K1492K), of which 47.1% (8/17) were truncation mutations. Thirteen mutations (F90S, I91T, T1210K, V1335M, G1433E, R612X, W768X, A395fsX400, L556fsX557, V1778fsX1800, Y1241fsX1270, IVS10+3A to G and K1492K) have not been reported previously. Except for F90S, L556fsX557 and V1778fsX1800, the other 14 mutations were de novo. SCN1A is a major pathogenic gene for SMEI. About a half of the SCN1A mutations in SMEI cause truncation. There were no hotspots of SCN1A mutations in SMEI patients, and most mutations were de novo.

&lt;I&gt;Listeria monocytogenes&lt;/I&gt;が分離された胸腹部大動脈瘤被覆破裂の1症例

We are reporting a case of sealed rupture of thoracoabdominal aortic aneurysm associated with the isolation of Listeria monocytogenes. The patient was a 75-year-old man with previous history of hypertension that had not required medication for the 3 years prior to hospital admission. He was admitted due to chest pain, but he was afebrile. There were no clinical findings indicating infection, although CRP was slightly elevated. During his clinical course, a sealed rupture of a thoracoabdominal aortic aneurysm was found and replaced with an artificial artery. After surgery, he was treated for 2 weeks with sultamicillin. He was discharged from hospital on the 43rd postoperative day. No bacteria were observed after microscopic examination of gram stained samples from the thrombus that was present in the sealed rupture of the aneurysm. However, a L. monocytogenes strain isolated from the thrombus only after enrichment culturing by HK medium at 37 degrees C for 4 days. By histopathology, there was a slight cellular infiltration of lymphocytes and neutrophils at the aperture of the aneurysm. Although L. monocytogenes strains possess major pathogenic genes, such as prfA, hlyA, plcA, plcB, mpl, inlA, inlB and actA that can be identified by PCR, none of the evidence indicated that this case was a mycotic aortic aneurysm due to L. monocytogenes.