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2.
Genet Med ; 21(11): 2442-2452, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31160754

RESUMO

PURPOSE: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

4.
FEMS Microbiol Lett ; 366(9)2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30860574

RESUMO

We assessed the relationship between gut microbiome profile and childhood eczema in 172 subjects (age < 3 years, healthy group N = 123, eczema group N = 49) utilizing 16S rRNA gene sequencing. Lower relative abundance of Bifidobacterium was shown to be associated with childhood eczema. Considering that developmental and environmental factors could modify the state of children's gut microbiome, we divided the samples into four age groups: 0-0.5 years, 0.5-1 years, 1-2 years and 2-3 years for farther analyses. Data revealed significant inter-group differences between healthy and eczema samples in all age groups, and decreased microbial diversity was most significantly found in children with eczema of age 2-3 years old. Decreased abundance of Bifidobacterium was a major finding in eczema groups from 0.5-3 years compared to the age matched healthy controls, but not significant in children younger than 6 month old. Of note, Bifidobacterium operational taxonomic units were identified by Random Forest with highly predictive power of 0.83 (AUC = 0.83) in ROC analysis, which also confirmed its role as a key genus that is associated with eczema. To verify the sequencing results, we performed quantitative polymerase chain reaction of Bifidobacterium and Bacteroides in the same cohort, and in a new eczema cohort (N = 57) for validation. Significantly, lower Bifidobacterium quantities were found in both eczema groups with an age range of 0.5-3 years. These results suggest variations in early gut microbiome are associated with childhood eczema.

6.
Front Physiol ; 9: 795, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30018562

RESUMO

Astragaloside IV (AS-IV), the major pharmacological extract from Astragalus membranaceus Bunge, possesses a variety of biological activities in the cardiovascular systems. Here, we aimed to evaluate preclinical evidence and possible mechanism of AS-IV for animal models of myocardial ischemia/reperfusion (I/R) injury. Studies of AS-IV in animal models with myocardial I/R injury were identified from 6 databases from inception to May, 2018. The methodological quality was assessed by using CAMARADES 10-item checklist. All the data were analyzed using Rev-Man 5.3 software. As a result, 22 studies with 484 animals were identified. The quality score of studies ranged from 3 to 6 points. Meta-analyses showed AS-IV can significantly decrease the myocardial infarct size and left ventricular ejection fraction, and increase shortening fraction compared with control group (P < 0.01). Significant decreasing of cardiac enzymes and cardiac troponin and increasing of decline degree in ST-segment were reported in one study each (P < 0.05). Additionally, the possible mechanisms of AS-IV for myocardial I/R injury are promoting angiogenesis, improving the circulation, antioxidant, anti-inflammatory and anti-apoptosis. Thus, AS-IV is a potential cardioprotective candidate for further clinical trials of myocardial infarction.

7.
Genet Med ; 2018 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-29895857

RESUMO

PURPOSE: To improve the accuracy of matching rare genetic diseases based on patient's phenotypes. METHODS: We introduce new methods to prioritize diagnosis of genetic diseases based on integrated semantic similarity (method 1) and ontological overlap (method 2) between the phenotypes expressed by a patient and phenotypes annotated to known diseases. RESULTS: We evaluated the performance of our methods by two sets of simulated data and one set of patient's data derived from electronic health records. We demonstrated that the two methods achieved significantly improved performance compared with previous methods in correctly prioritizing candidate diseases in all of the three sets. Our methods are freely available as a web application ( https://gddp. RESEARCH: cchmc.org/ ) to aid diagnosis of genetic diseases. CONCLUSION: Our methods can capture the diagnostic information embedded in the phenotype ontology, consider all phenotypes exhibited by a patient, and are more robust than the existing methods when phenotypes are incorrectly or imprecisely specified. These methods can assist the diagnosis of rare genetic diseases and help the interpretation of the results of DNA tests.

8.
J Clin Invest ; 128(7): 3071-3087, 2018 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-29889099

RESUMO

Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

9.
Biomed Pharmacother ; 105: 545-552, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29886375

RESUMO

Glycolysis is a metabolic pathway that is enhanced in cancer cells. miR-214 plays an important role in cancer development and can modulate glycolysis. However, whether miR-214 can regulate glycolysis in non-small-cell lung cancer (NSCLC) cells has not yet been investigated. The expression levels of miR-214 in 7 NSCLC cell lines were measured by qRT-PCR. MTT assay was performed to evaluate the cell proliferation. Glucose consumption and lactate production were measured to assess the level of glycolysis. The expression of hexokinase 2 (HK2) and pyruvate kinase isozyme M2 (PKM2) was measured by qRT-PCR and western blot analysis. Luciferase reporter assay was carried out to confirm the target gene of miR-214. The levels of PTEN, p-Akt, Akt, p-mTOR, mTOR, p-S6K, and S6K were assessed by western blot analysis. Results showed that miR-214 levels were significantly increased in the 7 NSCLC cell lines compared with those in the human bronchial epithelial cell line. Down-regulation of miR-214 inhibited cell proliferation, glucose consumption, lactate production, and expression of HK2 and PKM2 in NSCLC cells. We also confirmed that miR-214 directly targeted PTEN and regulated the PTEN/Akt/mTOR pathway. Inhibition of the PTEN/Akt/mTOR pathway attenuated the effect of miR-214 mimics on glucose consumption, lactate production, and expression of HK2 and PKM2 in NSCLC cells. These results demonstrated that miR-214 down-regulation inhibited cell proliferation and glycolysis by down-regulating the expression of HK2 and PKM2 via the PTEN/Akt/mTOR pathway in NSCLC cells. Hence, our findings suggested that miR-214 might serve as a novel therapeutic target for NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Glicólise/genética , Hexoquinase/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Piruvato Quinase/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/antagonistas & inibidores , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais
11.
Int J Hematol ; 108(3): 319-328, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29777376

RESUMO

Epstein-Barr virus (EBV) is associated with several life-threatening diseases, such as lymphoproliferative disease (LPD), particularly in immunocompromised hosts. Some categories of primary immunodeficiency diseases (PIDs) including X-linked lymphoproliferative syndrome (XLP), are characterized by susceptibility and vulnerability to EBV infection. The number of genetically defined PIDs is rapidly increasing, and clinical genetic testing plays an important role in establishing a definitive diagnosis. Whole-exome sequencing is performed for diagnosing rare genetic diseases, but is both expensive and time-consuming. Low-cost, high-throughput gene analysis systems are thus necessary. We developed a comprehensive molecular diagnostic method using a two-step tailed polymerase chain reaction (PCR) and a next-generation sequencing (NGS) platform to detect mutations in 23 candidate genes responsible for XLP or XLP-like diseases. Samples from 19 patients suspected of having EBV-associated LPD were used in this comprehensive molecular diagnosis. Causative gene mutations (involving PRF1 and SH2D1A) were detected in two of the 19 patients studied. This comprehensive diagnosis method effectively detected mutations in all coding exons of 23 genes with sufficient read numbers for each amplicon. This comprehensive molecular diagnostic method using PCR and NGS provides a rapid, accurate, low-cost diagnosis for patients with XLP or XLP-like diseases.


Assuntos
Análise Mutacional de DNA/métodos , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Hospedeiro Imunocomprometido , Lactente , Transtornos Linfoproliferativos/etiologia , Masculino , Pessoa de Meia-Idade , Mutação , Perforina/genética , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Adulto Jovem
12.
Qual Life Res ; 27(7): 1921-1931, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29730851

RESUMO

PURPOSE: The Child Health Utility 9D (CHU9D), a new generic preference-based health-related quality of life (HRQoL) instrument, was developed specifically for the application in cost-effectiveness analyses of treatments and interventions for children and adolescents. The main objective of this study was to examine the psychometric property of the Chinese version of CHU9D (CHU9D-CHN) in a large school-based sample in China. METHODS: Data were collected using a multi-stage sampling method from third-to-ninth-grade students in Shaanxi Province, China. Participants self-completed a hard-copy questionnaire including the CHU9D-CHN instrument, the Pediatric Quality of Life Inventory™ 4.0 Generic Core Scales (PedsQL), information on socio-demographic characteristics and self-reported health status. The psychometric properties of the CHU9D-CHN, including the internal consistency, 2-week test-retest reliability, convergent and known-groups validity were studied. RESULTS: A total of 1912 students participated in the survey. The CHU9D-CHN internal consistency and test-retest reliability were good to excellent with a Cronbach's alpha of 0.77 and an intra-class correlation coefficient of 0.65, respectively. The CHU9D utility scores moderately correlated with the PedsQL total scores (r = .57, P < .001), demonstrating good convergent validity. Difference of the CHU9D utility scores among the different participants with levels of self-reported general health, health services utilisation and left-behind status demonstrated good construct validity. CONCLUSION: The findings demonstrated adequate psychometric performance for the CHU9D-CHN. The CHU9D-CHN was a satisfactory, reliable and valid instrument to measure and value HRQoL for children and adolescents in China.

14.
Arthritis Rheumatol ; 70(6): 963-970, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29409136

RESUMO

OBJECTIVE: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (JIA) and has pathologic similarity to hemophagocytic lymphohistiocytosis (HLH). Intronic variants in UNC13D are found in patients with familial HLH type 3 (FHLH3), but the role of noncoding variants in MAS is unknown. The objective of this study was to identify deep intronic UNC13D variants in patients with MAS. METHODS: A custom enrichment library was constructed to sequence a genomic region of ~1 Mb flanking UNC13D in 24 patients with systemic JIA, recurrent MAS, and negative results of prior genetic (exon/coding) testing. The functional consequences of intronic variants were assessed using quantitative polymerase chain reaction in patient-derived peripheral blood mononuclear cells (PBMCs), electromobility shift assay, in vitro transcriptional enhancer assays, and natural killer (NK) cell degranulation assays. RESULTS: We evaluated a patient with systemic JIA and recurrent MAS in whom a novel functional intronic variant in UNC13D, c.117+143A>G, was observed. This variant occurred in a proposed regulatory region that drives lymphocyte-specific UNC13D expression and is associated with reduced transcript levels in patient PBMCs. This variant also disrupted NF-κB binding to a functional transcriptional enhancer, leading to reduced enhancer activity in vitro. Partial knockdown of UNC13D expression also led to impaired NK cell degranulation. An additional patient was identified with a previously described UNC13D intronic variant, for a total noncoding variant hit rate of 8.3% (2 of 24). CONCLUSION: These findings highlight the notion that intronic variants in key regulatory regions may be associated with MAS in patients with systemic JIA and support deep sequencing approaches when causative coding variants are not identified.

15.
Mol Cell Endocrinol ; 473: 166-177, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29378236

RESUMO

Germinal heterozygous activating STAT3 mutations represent a novel monogenic defect associated with multi-organ autoimmune disease and, in some cases, severe growth retardation. By using whole-exome sequencing, we identified two novel STAT3 mutations, p.E616del and p.C426R, in two unrelated pediatric patients with IGF-I deficiency and immune dysregulation. The functional analyses showed that both variants were gain-of-function (GOF), although they were not constitutively phosphorylated. They presented differences in their dephosphorylation kinetics and transcriptional activities under interleukin-6 stimulation. Both variants increased their transcriptional activities in response to growth hormone (GH) treatment. Nonetheless, STAT5b transcriptional activity was diminished in the presence of STAT3 GOF variants, suggesting a disruptive role of STAT3 GOF variants in the GH signaling pathway. This study highlights the broad clinical spectrum of patients presenting activating STAT3 mutations and explores the underlying molecular pathway responsible for this condition, suggesting that different mutations may drive increased activity by slightly different mechanisms.

16.
Pediatr Dermatol ; 35(2): 188-197, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29334134

RESUMO

BACKGROUND: Historically, diagnosis of epidermolysis bullosa has required skin biopsies for electron microscopy, direct immunofluorescence to determine which gene(s) to choose for genetic testing, or both. METHODS: To avoid these invasive tests, we developed a high-throughput next-generation sequencing (NGS)-based diagnostic assay called EBSEQ that allows simultaneous detection of mutations in 21 genes with known roles in epidermolysis bullosa pathogenicity. Mutations are confirmed with traditional Sanger sequencing. RESULTS: We present our EBSEQ assay and preliminary studies on the first 43 subjects tested. We identified 11 cases of epidermolysis bullosa simplex, five cases of junctional epidermolysis bullosa, 11 cases of dominant dystrophic epidermolysis bullosa, 15 cases of recessive dystrophic epidermolysis bullosa, and one case that remains without diagnosis. We also found an additional 52 variants of uncertain clinical significance in 17 of the 21 epidermolysis bullosa-associated genes tested. Three of the variants of uncertain clinical significance were also found in three other patients, for a total of 49 unique variants of uncertain clinical significance. We found the clinical sensitivity of the assay to be 75% to 98% and the analytical sensitivity to be 99% in identifying base substitutions and small deletions and duplications. Turnaround time was 3 to 6 weeks. CONCLUSIONS: EBSEQ is a sensitive, relatively rapid, minimally invasive, comprehensive genetic assay for the diagnosis of epidermolysis bullosa.


Assuntos
Epidermólise Bolhosa/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Epidermólise Bolhosa/diagnóstico , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação , Ohio , Pele/patologia , Adulto Jovem
17.
Hum Mutat ; 39(3): 389-393, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29288557

RESUMO

Pyruvate kinase deficiency (PKD) is the most frequent red blood cell enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Over 250 PKLR-gene mutations have been described, including missense/nonsense, splicing and regulatory mutations, small insertions, small and gross deletions, causing PKD and hemolytic anemia of variable severity. Alu retrotransposons are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. Alu insertions have been associated with a number of human diseases either by disrupting a coding region or a splice signal. Here, we report on two unrelated Middle Eastern patients, both born from consanguineous parents, with transfusion-dependent hemolytic anemia, where sequence analysis revealed a homozygous insertion of AluYb9 within exon 6 of the PKLR gene, causing precipitous decrease of PKLR RNA levels. This Alu element insertion consists a previously unrecognized mechanism underlying pathogenesis of PKD.

19.
Clin Exp Pharmacol Physiol ; 45(2): 198-204, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28963785

RESUMO

It is well-documented that nicotine, the main active ingredient in cigarettes, results in endothelial cell injury in numerous diseases. However, whether nicotine plays a crucial role in endothelial cell injury in diabetes and the exact molecular mechanism that mediates this process have not been fully elucidated. The current study aimed to investigate the effects of nicotine on endothelial cell injury in diabetes and the specific molecular mechanism by which it plays a role. Human umbilical vein endothelial cells (HUVECs) were incubated in HG/HF media and treated with nicotine, PYR-41 (a selective ubiquitin E1 inhibitor), Akt-overexpressing adenovirus, or TTC3 and MUL1 shRNA adenovirus. Cell viability was subsequently detected by the CCK8 assay, and apoptosis was examined by caspase-3 cleavage and activity analysis. Compared to the HG/HF incubated group, nicotine incubation significantly decreased cell survival and increased apoptosis. Moreover, nicotine induced Akt degradation via UPS, and Akt overexpression blocked nicotine-induced apoptosis in HUVECs cultured in HG/HF media. Furthermore, the TTC3 and MUL1 shRNA adenovirus dramatically decreased the Akt ubiquitination and apoptosis induced by nicotine. These results indicate that nicotine-induced Akt ubiquitination and degradation occurs through TTC3 and MUL1 and results in a dramatic increase in apoptosis in HUVECs cultured in HG/HF media.

20.
Haematologica ; 102(11): 1956-1968, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28860338

RESUMO

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome characterized by overwhelming immune activation. A steroid and chemotherapy-based regimen remains as the first-line of therapy but it has substantial morbidity. Thus, novel, less toxic therapy for HLH is urgently needed. Although differences exist between familial HLH (FHL) and secondary HLH (sHLH), they have many common features. Using bioinformatic analysis with FHL and systemic juvenile idiopathic arthritis, which is associated with sHLH, we identified a common hypoxia-inducible factor 1A (HIF1A) signature. Furthermore, HIF1A protein levels were found to be elevated in the lymphocytic choriomeningitis virus infected Prf1-/- mouse FHL model and the CpG oligodeoxynucleotide-treated mouse sHLH model. To determine the role of HIF1A in HLH, a transgenic mouse with an inducible expression of HIF1A/ARNT proteins in hematopoietic cells was generated, which caused lethal HLH-like phenotypes: severe anemia, thrombocytopenia, splenomegaly, and multi-organ failure upon HIF1A induction. Mechanistically, these mice show type 1 polarized macrophages and dysregulated natural killler cells. The HLH-like phenotypes in this mouse model are independent of both adaptive immunity and interferon-γ, suggesting that HIF1A is downstream of immune activation in HLH. In conclusion, our data reveal that HIF1A signaling is a critical mediator for HLH and could be a novel therapeutic target for this syndrome.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfo-Histiocitose Hemofagocítica/metabolismo , Transdução de Sinais , Imunidade Adaptativa , Animais , Biomarcadores , Linhagem Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfo-Histiocitose Hemofagocítica/genética , Linfo-Histiocitose Hemofagocítica/mortalidade , Linfo-Histiocitose Hemofagocítica/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma
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