Functional investigation of a novel ANKRD11 frameshift variant identified in a Chinese family with KBG syndrome.

KBG syndrome is a rare autosomal dominant condition characterized by multisystem developmental disorder, primarily caused by loss-of-function variants in ankyrin repeat domain-containing protein 11 (ANKRD11). Approximately 80 % of ANKRD11 variants associated with KBG syndrome, are frameshift and nonsense variants. Current insight into the pathogenesis of KBG syndrome resulting from ANKRD11 truncating variants remains limited. Here, we presented two members from a non-consanguineous Chinese pedigree both exhibiting characteristics fitting the KBG syndrome-associated phenotypic spectrum. Whole-exome sequencing identified a novel heterozygous frameshift variant in ANKRD11 (NM_013275.6, c.2280_2281delGT, p.Y761Qfs*20) in the proband. Sanger sequencing confirmed that the variant was inherited from her mother and co-segregated with KBG syndrome phenotype. In vitro functional assays revealed that the frameshift variant escaped nonsense-mediated mRNA decay, and resulting in a truncated protein with significantly increased expression levels compared to full-length ANKRD11. Immunofluorescence results demonstrated that truncated protein was predominantly expressed in the nucleus of HEK293 cells, while wild-type ANKRD11 was equally distributed in both the nucleus and cytoplasm. Moreover, the truncated protein significantly reduced CDKN1A/P21-promoter luciferase activity in comparison to wild-type ANKRD11 protein, as well as a remarkably decrease in the endogenous CDKN1A/P21 mRNA level in HEK293 cells. These findings suggest a loss of transcriptional activation function and potentially a dominant-negative mechanism. Overall, our study expands the mutational spectrum of ANKRD11 gene and provides new insights into the pathogenic mechanism of KBG syndrome caused by ANKRD11 truncating variants.

Identification and functional analysis of first heterozygous frameshift mutation in the GHRH gene in a Chinese boy with isolated growth hormone deficiency.

BACKGROUND: Isolated growth hormone deficiency (IGHD) is a rare genetically heterogeneous disorder caused primarily by mutations in GH1 and GH releasing hormone receptor (GHRHR). The aim of this study was to identify the molecular etiology of a Chinese boy with IGHD. METHODS: Whole-exome sequencing, sanger sequencing and bioinformatic analysis were performed to screen for candidate mutations. The impacts of candidate mutation on gene expression, intracellular localization and protein function were further evaluated by in vitro assays. RESULTS: A novel heterozygous frameshift mutation in the GHRH gene (c.91dupC, p.R31Pfs*98) was identified in a Chinese boy clinically diagnosed as having IGHD. The mutation was absent in multiple public databases, and considered as deleterious using in silico prediction, conservative analysis and three-dimensional homology modeling. Furthermore, mRNA and protein expression levels of mutant GHRH were significantly increased than wild-type GHRH (p < 0.05). Moreover, mutant GHRH showed an aberrant accumulation within the cytoplasm, and obviously reduced ability to stimulate GH secretion and cAMP accumulation in human GHRHR-expressing pituitary GH3 cells compared to wild-type GHRH (p < 0.05). CONCLUSION: Our study discovered the first loss-of function mutation of GHRH in a Chinese boy with IGHD and provided new insights on IGHD pathogenesis caused by GHRH haploinsufficiency.

MRI-assessed diaphragmatic function can predict frequent acute exacerbation of COPD: a prospective observational study based on telehealth-based monitoring system.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) have considerably high mortality and re-hospitalisation rate. Diaphragmatic dysfunction (DD) is common in COPD patients. However, whether diaphragmatic dysfunction is related to acute exacerbation is yet to be elucidated. This study aimed to evaluate the diaphragm function by magnetic resonance imaging (MRI) in COPD patients and assess whether the impact of DD may help predict AECOPD. METHODS: 20 healthy adult volunteers and 80 COPD patients were enrolled. The diaphragms function parameters were accessed by MRI. Patients were guided to start self-management by the Telehealth-based monitoring system following the enrolment. Events of acute exacerbation of COPD were recorded by the system and confirmed by healthcare providers. Binary univariate and multivariate logistic regression analyses were performed to investigate the factors associated with the frequency of AECOPD. Receiver operating characteristic (ROC) curves were further used to assess the value of prediction indexes. RESULTS: Fifty-nine COPD patients completed a one-year follow-up based on the Telehealth-based monitoring system. The clinical outcomes showed that the diaphragm function parameters at the end of maximal breathing were lower in the COPD group than in the healthy control group (P < 0.05). ANOVA showed significant differences among Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages for diaphragm function parameters, including chest wall motion, lung area, upper-lower diameter, and the diaphragm thickening fraction at the end of maximal breathing (P < 0.05). Moreover, significant differences in diaphragm function parameters were observed between patients with infrequent AECOPD (n = 28) and frequent AECOPD (n = 31) based on the frequency of AECOPD (P < 0.05). The diaphragm thickening fraction and the chest wall motion were associated with AECOPD after adjusting for age, sex, BMI, and lung functions, and the combination of predictions showed better accuracy in predicting the frequency of AECOPD. CONCLUSIONS: In COPD patients, diaphragm function parameters correlate with the severity of airflow limitation. The diaphragm thickening fraction and the chest wall motion were associated with the frequency of AECOPD and can predict it.

R158Q and G212S, novel pathogenic compound heterozygous variants in SLC12A3 of Gitelman syndrome.

The dysfunction of Na+-Cl- cotransporter (NCC) caused by mutations in solute carrier family12, member 3 gene (SLC12A3) primarily causes Gitelman syndrome (GS). In identifying the pathogenicity of R158Q and G212S variants of SLC12A3, we evaluated the pathogenicity by bioinformatic, expression, and localization analysis of two variants from a patient in our cohort. The prediction of mutant protein showed that p.R158Q and p.G212S could alter protein's three-dimensional structure. Western blot showed a decrease of mutant Ncc. Immunofluorescence of the two mutations revealed a diffuse positive staining below the plasma membrane. Meanwhile, we conducted a compound heterozygous model-Ncc R156Q/G210S mice corresponding to human NCC R158Q/G212S. NccR156Q/G210S mice clearly exhibited typical GS features, including hypokalemia, hypomagnesemia, and increased fractional excretion of K+ and Mg2+ with a normal blood pressure level, which made NccR156Q/G210S mice an optimal mouse model for further study of GS. A dramatic decrease and abnormal localization of the mutant Ncc in distal convoluted tubules contributed to the phenotype. The hydrochlorothiazide test showed a loss of function of mutant Ncc in NccR156Q/G210S mice. These findings indicated that R158Q and G212S variants of SLC12A3 were pathogenic variants of GS.

Identification and Functional Characterization of a Novel Variant in the SEMA3A Gene in a Chinese Family with Kallmann Syndrome.

Background: Kallmann syndrome (KS) is a rare genetic disease characterized by the reproductive system and olfactory dysplasia due to the defective migration of gonadotropin-releasing hormone (GnRH) neurons. However, this disorder is clinically heterogeneous and the genotype-phenotype relationship has not been determined. Objective: The present study aimed to identify the variant causing KS in a Chinese family and evaluate the functional consequences and phenotypes associated with the novel variant. Methods: A Chinese family with KS was screened for pathogenic variants by whole-exome sequencing (WES). Bioinformatic analysis was performed to predict the consequences of the identified variant. The expression of the mutant protein was examined in vitro. Results: A novel heterozygous variant (NM_006080.2 : c.814G > T) in SEMA3A was identified in the patient and his father, which caused the substitution of aspartic acid with tyrosine in codon 272. It was predicted to result in pathogenic significance with a high damaging score and seriously affect protein structure by bioinformatic analysis. In vitro experiments revealed this variant could significantly decrease the expression of SEMA3A. Furthermore, it may cause the disease by failing to induce the phosphorylation of focal adhesion kinase (FAK) in GnRH neurons. Conclusion: Identification and functional characterization of this novel variant in the SEMA3A gene in a Chinese family with Kallmann syndrome extend the genetic variant spectrum of SEMA3A and provide more data about the heterogeneity of KS, which may provide further insights into the diagnosis of KS and help patients get additional data in genetic counseling and timely treatment.

Genotype-Phenotype Relationship and Follow-up Analysis of a Chinese Cohort With Osteogenesis Imperfecta.

OBJECTIVE: To evaluate the genotype-phenotype relationship and the effect of treatment on the clinical course of osteogenesis imperfecta (OI). METHODS: We established a Chinese hospitalized cohort with OI and followed them up for an average of 6 years. All patients were confirmed as having OI using whole-exome sequencing. We analyzed the genotype-phenotype relationship based on different types, pathogenic mechanisms, and gene inheritance patterns of OI. Additionally, we assessed whether there was a difference in treatment efficacy based on genotype. RESULTS: One hundred sixteen mutations in 6 pathogenic genes (COL1A1, COL1A2, IFITM5, SERPINF1, FKBP10, and WNT1) were identified in 116 patients with type I, III, IV, V, VI, XI, or XV OI. Compared with patients with COL1A1 mutations, patients with COL1A2 mutations were younger at the time of the first fracture, whereas other phenotypes were similar. When 3 groups (helical, haploinsufficiency, and non-collagen I gene mutations) were compared, patients with helical mutations were the shortest and most prone to dentinogenesis imperfecta. Patients with haploinsufficiency mutations were the oldest at the time of the first fracture. Moreover, patients with non-collagen I gene mutations were least susceptible to blue sclerae and had the highest fracture frequency. Furthermore, there were some minor phenotypic differences among non-collagen I gene mutations. Interestingly, pamidronate achieved excellent results in the treatment of patients with OI, and the treatment effect appeared to be unrelated to their genotypes. CONCLUSION: Our findings indicated a genotype-phenotype relationship and a similar effect of pamidronate treatment in patients with OI, which could provide a basis for guiding clinical treatment and predicting OI prognosis.

A home-based pulmonary rehabilitation mHealth system to enhance the exercise capacity of patients with COPD: development and evaluation.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) experience deficits in exercise capacity and physical activity as their disease progresses. Pulmonary rehabilitation (PR) can enhance exercise capacity of patients and it is crucial for patients to maintain a lifestyle which is long-term physically active. This study aimed to develop a home-based rehabilitation mHealth system incorporating behavior change techniques (BCTs) for COPD patients, and evaluate its technology acceptance and feasibility. METHODS: Guided by the medical research council (MRC) framework the process of this study was divided into four steps. In the first step, the prescription was constructed. The second step was to formulate specific intervention functions based on the behavior change wheel theory. Subsequently, in the third step we conducted iterative system development. And in the last step two pilot studies were performed, the first was for the improvement of system functions and the second was to explore potential clinical benefits and validate the acceptance and usability of the system. RESULTS: A total of 17 participants were enrolled, among them 12 COPD participants completed the 12-week study. For the clinical outcomes, Six-Minute Walk Test (6MWT) showed significant difference (P = .023) over time with an improvement exceeded the minimal clinically important difference (MCID). Change in respiratory symptom (CAT score) was statistically different (P = .031) with a greater decrease of - 3. The mMRC levels reduced overall and showed significant difference. The overall compliance of this study reached 82.20% (± 1.68%). The results of questionnaire and interviews indicated good technology acceptance and functional usability. The participants were satisfied with the mHealth-based intervention. CONCLUSIONS: This study developed a home-based PR mHealth system for COPD patients. We showed that the home-based PR mHealth system incorporating BCTs is a feasible and acceptable intervention for COPD patients, and COPD patients can benefit from the intervention delivered by the system. The proposed system played an important auxiliary role in offering exercise prescription according to the characteristics of patients. It provided means and tools for further individuation of exercise prescription in the future.

Lipotoxicity suppresses the synthesis of growth hormone in pituitary somatotrophs via endoplasmic reticulum stress.

Lipotoxicity has been shown to cause dysfunction of many organs and tissues. However, it is unclear whether lipotoxicity is harmful to the somatotrophs, a kind of cell that synthesize growth hormone (GH) in the pituitary. In this study, we performed an epidemiological study, serum levels of triglyceride (TG) and GH showed a negative correlation, even after adjustment for potential confounders. In an animal study, male Sprague-Dawley rats were fed a high-fat diet (HFD) or a control diet for 28 weeks. HFD rats showed impaired GH synthesis, resulting in a decrease in circulating GH levels. The expression of pituitary Pit-1, a key transcription factor of GH, was inhibited. We found that the inositol-requiring enzyme 1α (IRE1α) pathway of endoplasmic reticulum (ER) stress was triggered in HFD rat pituitary glands and palmitic acid-treated GH3 cells, respectively. On the contrary, applying 4-phenyl butyric acid (4-PBA) to alleviate ER stress or 4µ8c to specifically block the IRE1α pathway attenuated the impairment of both Pit-1 and GH expression. In conclusion, we demonstrated that lipotoxicity directly inhibits the synthesis of GH, probably by reducing Pit-1 expression. The IRE1α signaling pathway of ER stress may play an important role in this process.

Using Mobile Health Technology to Deliver a Community-Based Closed-Loop Management System for Chronic Obstructive Pulmonary Disease Patients in Remote Areas of China: Development and Prospective Observational Study.

BACKGROUND: Mobile health (mHealth) technology is an increasingly recognized and effective method for disease management and has the potential to intervene in pulmonary function, exacerbation risk, and psychological status of patients with chronic obstructive pulmonary disease (COPD). OBJECTIVE: This study aimed to investigate the feasibility of an mHealth-based COPD management system designed for Chinese remote areas with many potential COPD patients but limited medical resources. METHODS: The system was implemented based on a tailored closed-loop care pathway that breaks the heavy management tasks into detailed pieces to be quantified and executed by computers. Low-cost COPD evaluation and questionnaire-based psychological intervention are the 2 main characteristics of the pathway. A 6-month prospective observational study at the community level was performed to evaluate the effect of the system. Primary outcomes included changes in peak expiratory flow values, quality of life measured using the COPD assessment test scale, and psychological condition. Acute exacerbations, compliance, and adverse events were also measured during the study. Compliance was defined as the ratio of the actual frequency of self-monitoring records to the prescribed number. RESULTS: A total of 56 patients was enrolled; 39 patients completed the 6-month study. There was no significant difference in the mean peak expiratory flow value before and after the 6-month period (366.1, SD 106.7 versus 313.1, SD 116.6; P=.11). Psychological condition significantly improved after 6 months, especially for depression, as measured using the Patient Health Questionnaire-9 scale (median 6.0, IQR 3.0-9.0 versus median 4.0, IQR 0.0-6.0; P=.001). The COPD assessment test score after 6 months of intervention was also lower than that at the baseline, and the difference was significant (median 4.0, IQR 1.0-6.0 versus median 3.0, IQR 0.0-6.0; P=.003). The median overall compliance was 91.1% (IQR 67%-100%). In terms of acute exacerbation, 110 exacerbations were detected and confirmed by health care providers (per 6 months, median 2.0, IQR 1.0-5.0). Moreover, 72 adverse events occurred during the study, including 1 death, 19 hospitalizations, and 52 clinic visits due to persistent respiratory symptoms. CONCLUSIONS: We designed and validated a feasible mHealth-based method to manage COPD in remote Chinese areas with limited medical resources. The proposed closed-loop care pathway was effective at the community level. Proper education and frequent communication with health care providers may encourage patients' acceptance and use of smartphones to support COPD self-management. In addition, WeChat might play an important role in improving patient compliance and psychological distress. Further research might explore the effect of such systems on a larger scale and at a higher evidence level.