Escobar syndrome is a rare, autosomal recessive disorder that affects the musculoskeletal system and the skin. Mutations in the CHRNG and TPM2 genes are associated with this pathology. In this study, we conducted a clinical and genetic investigation of five patients and also explored via in silico and gene expression analysis their phenotypic variability. In detail, we identified a patient with a novel composite heterozygous variant of the CHRNG gene and two recurrent mutations in both CHRNG and TPM2 in the rest of the patients. As for the clinical particularities, we reported a list of modifier genes in a patient suffering from myopathy. Moreover, we identified decreased expression of IGF-1, which could be related to the short stature of Escobar patients, and increased expression of POLG1 specific to patients with TPM2 mutation. Through this study, we identified the genetic spectrum of Escobar syndrome in the Tunisian population, which will allow setting up genetic counseling and prenatal diagnosis for families at risk. In addition, we highlighted relevant biomarkers that could differentiate between patients with different genetic defects.

OBJECTIVE: Muscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes.
METHODS: We constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes.
RESULTS: The results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPARγ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly.
CONCLUSIONS: Overexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.

Escobar syndrome is a milder variant of multiple pterygium syndrome characterized by pterygia, scoliosis, and multiple congenital contractures. It is most frequently due to a genetic variant in CHRNG , which encodes the γ-subunit of the nicotinic acetylcholine receptor. Though the subunit is considered a \"fetal\" form and transitions to the \"adult\" ε-subunit by 33 weeks\' gestation, the pathogenic musculoskeletal effects during fetal development render children with this condition permanently affected. We report a neonate with homozygous CHRNG c.117dupC and discuss some of the downstream clinical effects we observed with this variant.

Multiple pterygium syndrome (MPS) is a genetically heterogeneous rare form of arthrogryposis multiplex congenita characterized by joint contractures and webbing or pterygia, as well as distinctive facial features related to diminished fetal movement. It is divided into prenatally lethal (LMPS, MIM253290) and nonlethal (Escobar variant MPS, MIM 265000) types. Developmental spine deformities are common, may present early and progress rapidly, requiring regular fo llow-up and orthopedic management.
Retrospective chart review and prospective data collection were conducted at three hospital centers. Molecular diagnosis was confirmed with whole exome or whole genome sequencing.
This case series describes the clinical features and scoliosis treatment on 12 patients from 11 unrelated families. A molecular diagnosis was confirmed in seven; two with MYH3 variants and five with CHRNG. Scoliosis was present in all but our youngest patient. The remaining 11 patients spanned the spectrum between mild (curve ≤ 25°) and malignant scoliosis (≥50° curve before 4 years of age); the two patients with MYH3 mutations presented with malignant scoliosis. Bracing and serial spine casting appear to be beneficial for a few years; non-fusion spinal instrumentation may be needed to modulate more severe curves during growth and spontaneous spine fusions may occur in those cases.
Molecular diagnosis and careful monitoring of the spine is needed in children with MPS.

Smoking is considered the leading cause of preventable morbidity and mortality worldwide. Studies have sought to identify predictors of response to smoking cessation treatments. The aim of this study was to analyze a possible association of target gene expression for smoking cessation with varenicline.
We included 74 smokers starting treatment with varenicline. Gene expression analysis was performed through the custom RT² Profiler qPCR array assay, including 17 genes. Times for sample collection were before the start of therapy (T0) and two weeks (T2) and four weeks (T4) after the start of treatment.
For gene expression analysis, we selected 14 patients who had success and 13 patients resistant to varenicline treatment. Success was considered to be when a patient achieved tobacco abstinence until the fourth week of treatment and resistant was when a patient had not stopped smoking as of the fourth week of treatment. We observed a significant difference for CHRNA7 gene expression: in the resistant group, samples from T2 and T4 had lower expression compared with T0 (fold change: 0.38, P = 0.007; fold change: 0.67, P = 0.004; respectively).
This exploratory clinical study, searching for a possible predictor of effectiveness for varenicline, reaffirmed the association of the α7 nAChR subunit for nicotine dependence and smoking therapy effectiveness with varenicline.

The acetylcholine receptor (AChR) is a member of the superfamily of transmitter-gated ion channels having a critical role in controlling electrical signals between nerves and muscle cells. Disruptive mutations in genes encoding different subunits of AChR result in multiple pterygium syndrome (MPS), which can be associated with a severe prenatally lethal presentation. This study aimed to investigate the etiology of lethal MPS (LMPS) in two consanguineous families with a history of miscarriages. DNA was extracted from a tissue sample of two aborted fetuses (probands) from two different families with a history of spontaneous miscarriages. Parental peripheral blood samples were collected for confirmatory analysis and follow-up testing. Whole-exome sequencing (WES) was performed on DNA from the probands. The results were confirmed and segregated by Sanger sequencing. Moreover, protein structure evaluations were accomplished. We identified a homozygous frameshift mutation of c.753_754delCT (p.V253fs*44) and a homozygous missense mutation of c.715C>T (p.Arg239Cys) in the CHRNG gene. Both aborted fetuses had pterygium, severe arthrogryposis, and fetal hydrops with cystic hygroma, being compatible with LMPS. The heterozygous state was confirmed in parents for both CHRNG variants. Likewise, CHRNG mutation was predicted to display the damaging effects by lowering the number of helixes and modifying the surface electrostatic potential. The present study identified rare sequence variants in the CHRNG gene in aborted fetuses from consanguineous couples with recurrent miscarriage history. WES is a comprehensive and cost-effective approach to study heterogeneous diseases including MPS. Such findings can improve our knowledge of MPS databases, particularly for genetic counseling of high-risk families and preimplantation genetic diagnosis.

Background: Escobar syndrome, a nonlethal variant of multiple pterygium syndromes (MPS), is a rare autosomal recessive disorder characterized by pterygia and multiple joint contractures along with other anomalies. Variants in cholinergic receptor nicotinic gamma subunit (CHRNG) have been previously reported in patients with Escobar syndrome. Objective: We studied a consanguineous Pakistani family affected with Escobar syndrome to identify the underlying genetic defect through short tandem repeat (STR) genotyping and direct DNA sequencing. Results: Genotyping with microsatellite markers (D2S427, D2S2344, and D2S206) revealed linkage of the disease phenotype in the family to the CHRNG locus. Using Sanger sequencing, we identified a homozygous nonsense CHRNG variant c.136C>T (p.R46*), predicted to produce a truncated protein that leads to acetylcholine receptor deficiency, causing MPS. The unaffected parents and siblings in the family were heterozygous carriers of this disease-causing variant. Conclusion: We report the identification of a nonsense CHRNG variant in a consanguineous Pakistani family affected with Escobar syndrome.

The Escobar variant of multiple pterygium syndrome (OMIM #265000) is a rare, autosomal recessive disorder associated with mutations in the γ-subunit of the nicotinic acetylcholine receptor (CHRNG). CHRNG is expressed in fetal muscle during motor development and contributes to the formation of neuromuscular junctions (NMJs). Anomalies in NMJ structure and function have not been investigated in patients with Escobar syndrome. We report five patients identified as having Escobar syndrome, from four families. In three families, the same mutation (c.459dupA) was identified in CHRNG. A biopsy from brachioradialis muscle was collected from a patient from one of these families and analyzed for NMJ organization using fluorescence microscopy. Compared to spinalis muscle from control patients with idiopathic scoliosis or cerebral palsy (CP), the patient with Escobar syndrome had a significantly higher degree of acetylcholine receptor present outside acetylcholinesterase and significantly less acetylcholinesterase outside acetylcholine receptors. Given the role of the acetylcholine receptor γ-subunit in fetal neuromuscular signal transduction and in establishing the primary encounter of muscle and motor nerve terminal, the CHRNG mutations described in Escobar syndrome may cause a broader disruption of postsynaptic proteins and result in aberrant development of the NMJ due to impaired prenatal neuromuscular transmission and/or abnormal neuromuscular synaptogenesis.