Cholecystokinin receptor-1 mediates the inhibitory effects of exogenous cholecystokinin octapeptide on cellular morphine dependence.

BACKGROUND: Cholecystokinin octapeptide (CCK-8), the most potent endogenous anti-opioid peptide, has been shown to regulate the processes of morphine dependence. In our previous study, we found that exogenous CCK-8 attenuated naloxone induced withdrawal symptoms. To investigate the precise effect of exogenous CCK-8 and the role of cholecystokinin (CCK) 1 and/or 2 receptors in morphine dependence, a SH-SY5Y cell model was employed, in which the µ-opioid receptor, CCK1/2 receptors, and endogenous CCK are co-expressed. RESULTS: Forty-eight hours after treating SH-SY5Y cells with morphine (10 µM), naloxone (10 µM) induced a cAMP overshoot, indicating that cellular morphine dependence had been induced. The CCK receptor and endogenous CCK were up-regulated after chronic morphine exposure. The CCK2 receptor antagonist (LY-288,513) at 1-10 µM inhibited the naloxone-precipitated cAMP overshoot, but the CCK1 receptor antagonist (L-364,718) did not. Interestingly, CCK-8 (0.1-1 µM), a strong CCK receptor agonist, dose-dependently inhibited the naloxone-precipitated cAMP overshoot in SH-SY5Y cells when co-pretreated with morphine. The L-364,718 significantly blocked the inhibitory effect of exogenous CCK-8 on the cAMP overshoot at 1-10 µM, while the LY-288,513 did not. Therefore, the CCK2 receptor appears to be necessary for low concentrations of endogenous CCK to potentiate morphine dependence in SH-SY5Y cells. An additional inhibitory effect of CCK-8 at higher concentrations appears to involve the CCK1 receptor. CONCLUSIONS: This study reveals the difference between exogenous CCK-8 and endogenous CCK effects on the development of morphine dependence, and provides the first evidence for the participation of the CCK1 receptor in the inhibitory effects of exogenous CCK-8 on morphine dependence.

Sequence CLCN1 and SCN4A genes in patients with nondystrophic myotonia in Chinese people.

BACKGROUND: This study aimed to characterize the genetic, pathological, and clinical alterations of 17 patients in China presenting with nondystrophic myotonia (NDM) and to analyze the relationship between genotype and clinical phenotype. METHODS: CLCN1 and SCN4A genes in patients with clinical features and muscle pathology indicative of NDM were sequenced. Furthermore, KCNE3 and CACNA1S genes were assessed in patients with wild-type CLCN1 and SCN4A. RESULTS: Patients may have accompanying atypical myopathy as well as muscle hypertrophy, secondary dystonia, and joint contracture as determined by needle electromyography. All the study participants were administered mexiletine in combination with carbamazepine and showed significant improvements in myotonia symptoms in response to this therapy. CLCN1 gene mutation was detected in 8 cases diagnosed with myotonia congenital using gene screening. The detected mutations included 5 missense, 2 nonsense, 1 deletion, and 2 insertions. Further gene analysis showed 4 mutations in the SCN4A gene in patients diagnosed with paramyotonia congenita. CONCLUSIONS: Myotonia congenita and paramyotonia congenita are the predominant forms of NDM in China. NDM may be best diagnosed using genetic analysis in associated with clinical features.

Genome sequencing identified a novel exonic microdeletion in the RUNX2 gene that causes cleidocranial dysplasia.

BACKGROUND AND AIMS: Cleidocranial dysplasia (CCD) represents a rare autosomal dominant skeletal dysplasia caused by mutations that induce haploinsufficiency in RUNX2, the important transcription factor of osteoblasts related to bone/cartilage development and maintenance. Clavicular hypoplasia, which involves aberrant tooth/craniofacial bone/skeletal formation, is a feature of classic CCD. RUNX2 mutations can be found in approximately 60-70% of patients with CCD, and around ∼10% of these mutations are microdeletions. The present paper describes the radiological and clinical characteristics of a 5-year-old girl who showed representative CCD features, including extra teeth, aplasia of clavicles, sloping shoulders, marked calvarial hypomineralization, and osteoporosis. MATERIALS AND METHODS: We obtained genomic DNA of her family members and performed whole-genome sequencing (WGS) for samples collected from the proband. Quantitative fluorescent PCR (QF-PCR) and specific PCR plus electrophoresis were then performed as validation assays for all participants. In vitro analysis was performed. Luciferase assay for Runx2 transcription activity and evaluation of mRNA levels of Runx2 downstream osteogenic markers were conducted. RESULTS: WGS identified a 11.38-kb microdeletion in RUNX2 comprising 8-9 exons, which was validated by QF-PCR and specific PCR plus electrophoresis. In vitro experiments confirmed the pathogenicity of this variation. CONCLUSION: The present study identified a 11.38-kb microdeletion in RUNX2 that causes CCD. The deletion in the PST domain of RUNX2 reduces its transcription activity and reduces osteogenic marker levels, eventually decreasing the differentiation of osteoblasts. These findings clarify the role of the CCD-related mechanism in the development of CCD and suggest that it is important to consider copy number variation for the suspected familial patients early.

Whole-exome sequencing facilitates the differential diagnosis of Ehlers-Danlos syndrome (EDS).

Ehlers-Danlos syndromes (EDSs) are a group of rare monogenic conditions with strong heterogeneity and can be caused by 20 genes associating with the essence of the extracellular matrix (ECM). This study enrolled three cases with various subtypes of EDS. Clinical evaluation and genetic testing with whole-exome sequencing (WES) were performed. The clinical manifestations of all three patients were thoroughly monitored; and three de novo diagnostic variants, namely COL5A1: NM_001278074.1: c.4609-2A>C, COL3A1: NM_000090.3: c.3554G>T(p.Gly1185Val), and COL1A1: NM_000088.3: c.545G>T(p.Gly182Val) were identified from them, respectively. The findings in this study expanded the mutation spectrum of EDS and strengthened the efficiency of WES in the differential diagnosis on disorders with overlapping phenotypes and various pathogenesis.

Identification of novel mutations of the CLCN1 gene for myotonia congenital in China.

OBJECTIVES: The identification of disease-specific genetic and electrophysiological patterns for myotonia congenital (MC) could help clinicians apply in the findings of genetic studies to improve diagnosis. We examined the molecular, clinical, and histopathological characteristics of eight patients with MC. METHODS: Optimization PCR was used to exclude myotonic dystrophies and the CLCN1 gene was sequenced in patients having clinical and electrophysiological features indicative of MC. RESULTS: Genetic screening identified nine CLCN1 mutations among the eight patients, including two missense, three nonsense, two insertion, and two deletion mutations. The patients showed typical myotonia and muscle hypertrophy. In contrast to the previous studies, secondary dystonia, joint contracture, and abnormal cardiac activity were also observed. Patients with novel mutations did not show any new muscle pathology compared with established mutations. Disscussion: Molecular genetics analysis offers an accurate method for diagnosing MC. The results of this analysis should be considered alongside clinical and electrophysiological characteristics. In this study, novel mutations in CLCN1 were detected, and the spectrum of CLCN1 mutations known to be associated with MC was expanded.

Optimization PCR for Detection CTG/CCTG-Repeat Expansions in the Diagnosis of Myotonic Dystrophies.

CONTEXT: Myotonic dystrophies (DMs) are a group of autosomal dominant neuromuscular disorders which are caused by large CTG/CCTG-repeat expansions in untranslated regions of DMPK/ZNF9 gene. The "phenotypic overlap" in DMs creates complication in distinguishing patients with DM1 from patients with DM2 and underscores the need for these patients to undergo genetic test; therefore, detection and accurate sizing of the CTG/CCTG-repeat expansions are necessary. Templates with long CTG/CCTG tandem repeats are difficult to amplify by convention PCR. AIMS: The aim of our study was to develop an efficient, economic amplification method which based on combination of primer design, modified annealing, and extension conditions in PCR amplification. SETTINGS AND DESIGN: We detected and analyzed the CTG-repeat expansions in patients having clinical, electrophysiological, and muscle pathology features indicative of DMs by optimization PCR. If no CTG-repeat expansions were detected, we subsequently analyzed the CCTG-repeat expansions in the remaining patients. RESULTS: 42 participants included 25 DMs patients and 17 family members. 22 patients showed CTG-repeat expansions, the CTG-repeat ranged from 53 to 683 and the average was 535; 3 patients showed CCTG-repeat expansions, the CCTG-repeat ranged from 400 to 450 and the average was 416. CONCLUSIONS: Molecular genetic tests are essential for DMs diagnosis; Optimization PCR under the optimal conditions of primer design, modified annealing, and extension conditions can be used for efficient PCR in DMs diagnosis; Optimization PCR can greatly improve the positive detection of DMs, provide an economic, accurate, and rapid method for routine diagnostic use.