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Drosophila melanogaster (fruit fly) insulin receptor (D‐IR) is highly homologous to the human counterpart. Like the human pathway, D‐IR responds to numerous insulin‐like peptides to activate cellular signals that regulate growth, development, and lipid metabolism in fruit flies. Allelic mutations in the D‐IR kinase domain elevate life expectancy in fruit flies. We developed a robust heterologous expression system to express and purify wild‐type and longevity‐associated mutant D‐IR kinase domains to investigate enzyme kinetics and substrate specificities. D‐IR exhibits remarkable similarities to the human insulin receptor kinase domain but diverges in substrate preferences. We show that longevity‐associated mutations reduce D‐IR catalytic activity. Deletion of the unique kinase insert domain portion or mutations proximal to activating tyrosines do not influence kinase activity, suggesting their potential role in substrate recruitment and downstream signaling. Through biochemical investigations, this study enhances our comprehension of D‐IR's role in Drosophila physiology, complementing genetic studies and expanding our knowledge on the catalytic functions of this conserved signaling pathway. [ABSTRACT FROM AUTHOR]

Ras-related C3 botulinum toxin substrate 2 (RAC2) is a small guanine nucleotide binding molecule that is exclusively expressed in hematopoietic cell lineages as a switcher. Based on in vivo and/or in vitro model experiments, RAC2 plays important roles in different cells through proliferation, secretion, and phagocytosis. It also performs a suppressing function in immunoglobulin (Ig) switching in Rac2-/- animals or cells. Several RAC2 natural mutations have been described in patients with primary immunodeficiency. RAC2 mutations can be classified into loss-of-function inactivating (LoF-I) and gain-of-function activating mutations according to their functional effects. Only two LoF-I mutations on RAC2 have been reported, including a dominant D57N mutation in several cases that exhibit granulocyte function defects and a recessive D56X mutation in cases with common variable immunodeficiency. Regardless of the type of mutation, most of the reported RAC2 mutant cases have shown reduced IgG, IgA, and IgM levels. Herein, we report on a family with three members that suffer from persistent HPV infection, recurrent respiratory infections, bronchiectasis, and autoimmune disease. The immunologic profile suggests that the family was affected by combined immunodeficiency (CID) with increased serum levels of IgG, IgA, and IgE. Exome sequencing identified a de novo RAC2 mutation (c.44G > A/p.G15D) that was co-segregated with the disease in the family. Gene functional experiments identified that such mutation results in reduced guanosine triphosphate binding activity and RAC2 protein expression. In patients' lymphocytes, impaired aggregation and proliferation effects, decreased mitochondrial membrane potential, and increased levels of cell apoptosis were observed, although no functional abnormalities were detected in neutrophils. To our knowledge, this study was the first to identify a LoF-I mutation of RAC2 affecting lymphocyte function that consequently led to CID and increased levels of serum IgG, IgE, and IgA. This study presents a novel subtype of RAC2-related immune disorder. [ABSTRACT FROM AUTHOR]

The CTNNB1 Syndrome is a rare neurodevelopmental disorder associated with developmental delay, intellectual disability, and delayed or absent speech. The aim of the present study is to systematically review the available data on the prevalence of clinical manifestations and to evaluate the correlation between phenotype and genotype in published cases of patients with CTNNB1 Syndrome. Studies were identified by systematic searches of four major databases. Information was collected on patients' genetic mutations, prenatal and neonatal problems, head circumference, muscle tone, EEG and MRI results, dysmorphic features, eye abnormalities, early development, language and comprehension, behavioral characteristics, and additional clinical problems. In addition, the mutations were classified into five groups according to the severity of symptoms. The study showed wide genotypic and phenotypic variability in patients with CTNNB1 Syndrome. The most common moderate-severe phenotype manifested in facial dysmorphisms, microcephaly, various motor disabilities, language and cognitive impairments, and behavioral abnormalities (e.g., autistic-like or aggressive behavior). Nonsense and missense mutations occurring in exons 14 and 15 were classified in the normal clinical outcome category/group because they had presented an otherwise normal phenotype, except for eye abnormalities. A milder phenotype was also observed with missense and nonsense mutations in exon 13. The autosomal dominant CTNNB1 Syndrome encompasses a wide spectrum of clinical features, ranging from normal to severe. While mutations cannot be more generally categorized by location, it is generally observed that the C-terminal protein region (exons 13, 14, 15) correlates with a milder phenotype. [ABSTRACT FROM AUTHOR]

Abstract: Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction. [ABSTRACT FROM AUTHOR]

13q12.3 microdeletion syndrome is a rare cause of syndromic intellectual disability. Identification and genetic characterization of patients with 13q12.3 microdeletion syndrome continues to expand the phenotypic spectrum associated with it. Previous studies identified four genes within the approximately 300 Kb minimal critical region including two candidate protein coding genes: KATNAL1 and HMGB1. To date, no patients carrying a sequence‐level variant or a single gene deletion in HMGB1 or KATNAL1 have been described. Here we report six patients with loss‐of‐function variants involving HMGB1 and who had phenotypic features similar to the previously described 13q12.3 microdeletion syndrome cases. Common features included developmental delay, language delay, microcephaly, obesity and dysmorphic features. In silico analyses suggest that HMGB1 is likely to be intolerant to loss‐of‐function, and previous in vitro data are in line with the role of HMGB1 in neurodevelopment. These results strongly suggest that haploinsufficiency of the HMGB1 gene may play a critical role in the pathogenesis of the 13q12.3 microdeletion syndrome. [ABSTRACT FROM AUTHOR]

Arthrogryposis describes the presence of multiple joint‐contractures. Clinical severity of this phenotype is variable, and more than 400 causative genes have been proposed. Among these, ERGIC1 is a recently reported candidate encoding a putative transmembrane protein of the ER‐Golgi interface. Two homozygous missense variants have been reported in patients with relatively mild non‐syndromic arthrogryposis. In a consanguineous family with two affected siblings presenting congenital arthrogryposis and some facial dysmorphism we performed prenatal array‐CGH, postnatal targeted exome and genome sequencing. Genome sequencing identified a homozygous 22.6 Kb deletion encompassing the promoter and first exon of ERGIC1. mRNA quantification showed the complete absence of ERGIC1 expression in the two affected siblings and a decrease in heterozygous parents. Our observations validate the pathogenic role of ERGIC1 in congenital arthrogryposis and demonstrate that complete loss of function causes a relatively mild phenotype. These findings will contribute to improve genetic counseling of ERGIC1 mutations. [ABSTRACT FROM AUTHOR]

We report a patient with thrombocytopenia from a Japanese family with hemophilia A spanning four generations. Various etiologies of thrombocytopenia, including genetic, immunological, and hematopoietic abnormalities, determine the prognosis for this disease. In this study, we identified a novel heterozygous mutation in a gene encoding cytochrome c, somatic (CYCS, MIM123970) using whole exome sequencing. This variant (c.301_303del:p.Lys101del) is located in the α‐helix of the cytochrome c (CYCS) C‐terminal domain. In silico structural analysis suggested that this mutation results in protein folding instability. CYCS is one of the key factors regulating the intrinsic apoptotic pathway and the mitochondrial respiratory chain. Using the yeast model system, we clearly demonstrated that this one amino acid deletion (in‐frame) resulted in significantly reduced cytochrome c protein expression and functional defects in the mitochondrial respiratory chain, indicating that the loss of function of cytochrome c underlies thrombocytopenia. The clinical features of known CYCS variants have been reported to be confined to mild or asymptomatic thrombocytopenia, as was observed for the patient in our study. This study clearly demonstrates that thrombocytopenia can result from CYCS loss‐of‐function variants. [ABSTRACT FROM AUTHOR]

Mutations within STXBP1 have been associated with a range of neurodevelopmental disorders implicating the pleotropic impact of this gene. Although the frequency of de novo mutations within STXBP1 for selective cohorts with early onset epileptic encephalopathy is more than 1%, there is no evidence for a hotspot within the gene. In this study, we analyzed the genomic context of de novo STXBP1 mutations to examine whether certain motifs indicated a greater risk of mutation. Through a comprehensive context analysis of 136 de novo/rare mutation (SNV/Indels) sites in this gene, strikingly 26.92% of all SNV mutations occurred within 5bp upstream or downstream of a 'GTA' motif (P < 0.0005). This implies a genomic context modulated mutagenesis. Moreover, 51.85% (14 out of 27) of the 'GTA' mutations are splicing compared to 14.70% (20 out of 136) of all reported mutations within STXBP1. We also noted that 11 of these 14 'GTA' associated mutations are de novo in origin. Our analysis provides strong evidence of DNA motif modulated mutagenesis for STXBP1 de novo splicing mutations. [ABSTRACT FROM AUTHOR]

Introduction: We describe a 6-year-old girl with a T118M PMP22 mutation and heterozygous deletion of PMP22 on chromosome 17 (17p11.2-p12) resulting in a severe sensorimotor polyneuropathy.Methods: This study is a case report in which the relevant mutations are described.Results: Foot pain, cavovarus feet, tibialis anterior atrophy, absent reflexes, and inability to walk were found when the patient was age 6 years. Nerve conduction studies showed evidence of a sensorimotor polyneuropathy and compressive mononeuropathies of bilateral median nerves at the wrist and ulnar nerves at the elbow. Genetic testing revealed deletion of a PMP22 allele and T118M PMP22 mutation in the remaining allele.Conclusions: The severe sensorimotor polyneuropathy and hereditary neuropathy with liability to pressure palsies (HNPP) in this patient was likely a consequence of both decreased expression of PMP22 causing features consistent with HNPP and unopposed expression of the T118M mutant form of PMP22 that is relatively benign in the heterozygous state. The T118M mutant form of PMP22 can be disease-modifying in the appropriate circumstances. [ABSTRACT FROM AUTHOR]

Addressing the puzzling role of amidated gastrin17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-κB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-κB activation and decreased expression of the antiapoptotic NF-κB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT–PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFα)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-κB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFα and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFα- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-κB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.Oncogene (2008) 27, 1122–1134; doi:10.1038/sj.onc.1210728; published online 20 August 2007 [ABSTRACT FROM AUTHOR]

Summary: Mutations in the TECTA gene result in sensorineural non-syndromic hearing impairment. TECTA-related deafness can be inherited autosomal dominantly (designated as DFNA8/12) or autosomal recessively (as DFNB21). The α-tectorin protein, which is encoded by the TECTA gene, is one of the major components of the tectorial membrane in the inner ear. Six mutations in the TECTA gene have already been reported in families segregating autosomal recessive non-syndromic hearing impairment. In this study, seventy-five Iranian families segregating autosomal recessive non-syndromic hearing impairment were analyzed for homozygosity at the DFNB21 locus by genotyping two short tandem repeat markers closely linked to the TECTA gene. Allelic segregation consistent with possible linkage to the DFNB21 locus was found in 1/75 families studied. By sequencing all 23 coding exons of TECTA, a 16bp deletion (c.6203-6218del16) in exon 21, leading to a frameshift, segregating with the hearing loss was found. All 3 affected individuals of this family have moderate-to-severe hearing loss across all frequencies, which is more pronounced in the mid frequencies. This new mutation, as well as the six previously reported mutations in the TECTA gene, is inactivating. All of these mutations lead to an easily recognized audiometric profile of moderate to severe hearing impairment as presented by the family in this study too. The TECTA autosomal recessive non-syndromic deafness phenotype differs from the typical profound deafness phenotype that is seen in most families segregating autosomal recessive non-syndromic deafness. On the basis of the recognizable phenotype, we recommend mutation screening of TECTA in families with this hearing phenotype. [Copyright &y& Elsevier]

Simple Summary: Two deleterious RAD50 loss-of-function germline mutations were identified from the blood DNA of a cohort of 3289 Henan individuals by next-generation sequencing. These rare loss-of-function RAD50 variants were associated with a substantial increased risk of familial esophageal squamous cell carcinoma in high-risk Northern China. A functional study suggested that the RAD50 mutations may affect DNA repair and cell survival upon replication stress. Our preliminary functional study provided novel insight and the potential clinical implication that patients with heterozygous RAD50L1264F and RAD50Q672X status may have a potential synthetic lethal therapeutic option with CHK1 inhibitors. Further study is warranted for validation of the implicated genetic susceptibility role of the RAD50 Zinc Hook mutants. Unbiased whole-exome sequencing approaches in familial esophageal squamous cell carcinoma (ESCC) initially prioritized RAD50 as a candidate cancer predisposition gene. The combined study with 3289 Henan individuals from Northern China identified two pathogenic RAD50 protein truncation variants, p.Q672X and a recurrent p.K722fs variant at the zinc hook domain significantly conferring increased familial ESCC risk. Effects of ~10-fold higher familial ESCC risk were observed, when compared to East Asians from the gnomAD database. Functional characterization suggested that the RAD50Q672X mutation contributes a dominant-negative effect in DNA repair of double-stranded breaks. Overexpression of the RAD50Q672X and RAD50L1264F missense mutation also sensitized cell death upon replication stress stimuli induced by formaldehyde treatment and the CHK1 inhibitor, AZD7762. Our study suggested the novel insight of the potential for synthetic lethal therapeutic options for RAD50Q672X and the East-Asian-specific RAD50L1264F variants and CHK1 inhibitors. Our study also suggested the association of RAD50 LOF variants in the zinc hook domain with a higher risk of familial ESCC in Chinese. [ABSTRACT FROM AUTHOR]

Background: Although the genetic spectrum of human colorectal cancer (CRC) is mainly characterized by APC, KRAS and TP53 mutations, driver genes in tumor initiation have not been conclusively demonstrated. In this study, we aimed to identify novel markers for CRC. Methods: We performed exome analysis of sporadic colorectal cancer (sCRC) coding regions to screen loss of function (LoF) mutation genes, and carried out systems-level approaches to confirm top rank gene in this study. Results: We identified loss of BMP5 is an early event in CRC. Deep sequencing identified BMP5 was mutated in 7.7% (8/104) of sCRC samples, with 37.5% truncating mutation frequency. Notably, BMP5 negative expression and its prognostic value is uniquely significant in sCRC but not in other tumor types. Furthermore, BMP5 expression was positively correlated with E-cadherin in CRC patients and its dysregulation play a vital role in epithelial-mesenchymal transition (EMT), thus triggering tumor initiation and development. RNA sequencing identified, independent of BMP/Smads pathway, BMP5 signaled though Jak-Stat pathways to inhibit the activation of oncogene EPSTI1. Conclusions: Our result support a novel concept that the importance of BMP5 in sCRC. The tumor suppressor role of BMP5 highlights its crucial role in CRC initiation and development. [ABSTRACT FROM AUTHOR]