Case report: vitamin D‐dependent rickets type 1 caused by a novel CYP27B1 mutation

Vitamin D‐dependent rickets type 1 VDDR‐1 is a recessive inherited disorder with impaired activation of vitamin D, caused by mutations in CYP27B1. We present long‐time follow‐up of a case with a novel mutation including high‐resolution peripheral quantitative computed tomography of the bone. Adequate treatment resulted in a normalized phenotype.     

A Novel GJB2 compound heterozygous mutation c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) causes sensorineural hearing loss in a Chinese family

Objective

To investigate whether a novel compound heterozygous mutations c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) in GJB2 result in hearing loss.

Methods

Allele‐specific PCR‐based universal array (ASPUA) screening and sequence analysis were applied to identify these mutations. 3D model was built to perform molecular dynamics (MD) simulation to verify the susceptibility of the mutations. Furthermore, WT‐ and Mut‐GJB2 DNA fragments, containing the mutation of c.257C>G and c.176del16 were respectively cloned and transfected into HEK293 and spiral ganglion neuron cell (SGNs) by lenti‐virus delivery system to indicate the subcellular localization of the WT‐ and Mut‐CX26 protein.

Results

A novel compound heterozygous mutation c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) in GJB2 was identified in a Chinese family, in which 4 siblings with profound hearing loss, but the fifth child is normal. By ASPUA screening and sequencing, a compound heterozygote mutations in GJB2 c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) were identified in these four deaf children, each of the mutated GJB2 gene were inherited from their parents. There is no mutation of GJB2 gene identified in the normal child. Besides, the compound heterozygous mutation GJB2 c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) could lead to the alterations of the subcellular localization of each corresponding mutated CX26 protein and could cause the hearing loss, which has been predicted by MD simulation and verified in both 293T and SGNs cell line.

Conclusion

The c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) compound mutations in GJB2 detected in this study are novel, and which may be associated with hearing loss in this Chinese family.

     

Evolution of the neural sex‐determination system in insects: does fruitless homologue regulate neural sexual dimorphism in basal insects?

In the brain of holometabolous insects such as the fruit fly Drosophila melanogaster, the fruitless gene produces sex‐specific gene products under the control of the sex‐specific splicing cascade and contributes to the formation of the sexually dimorphic circuits. Similar sex‐specific gene products of fruitless homologues have been identified in other holometabolous insects such as mosquitoes and a parasitic wasp, suggesting the fruitless‐dependent neural sex‐determination system is widely conserved amongst holometabolous insects. However, it remains obscure whether the fruitless‐dependent neural sex‐determination system is present in basal hemimetabolous insects. To address this issue, identification, characterization, and expression analyses of the fruitless homologue were conducted in the two‐spotted cricket, Gryllus bimaculatus, as a model hemimetabolous insect. The Gryllus fruitless gene encodes multiple isoforms with a unique zinc finger domain, and does not encode a sex‐specific gene product. The Gryllus Fruitless protein is broadly expressed in the neurones and glial cells in the brain, and there was no prominent sex‐related difference in the expression levels of Gryllus fruitless isoforms. The results suggest that the Gryllus fruitless gene is not involved in the neural sex‐determination in the cricket brain.     

Modular cis‐regulatory logic of yellow gene expression in silkmoth larvae

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis‐regulatory regions of pigmentation genes and have revealed cis‐regulatory modularity. Here, we used well‐developed transgenic techniques in Bombyx mori and demonstrated that cis‐regulatory modularity controls tissue‐specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue‐specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis‐regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans‐regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis‐regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.     

A case‐control study and meta‐analysis reveal the association between COX‐2 G‐765C polymorphism and primary end‐stage hip and knee osteoarthritis

Background

Osteoarthritis (OA) is a popular arthrosis featured as pain, limited joint activity, and deformity. Cyclooxygenase‐2 (COX‐2) has been reported to be up‐regulated in arthritic tissues and is integral to the progression of osteoarthritis (OA). Previous studies showed the COX‐2 promoter G‐765C polymorphism could influence COX‐2 expression. However, the relationship between the variant and OA risk is contrasting.

Methods

We conducted a case‐control study with 196 primary end‐stage hip and knee OA cases and 196 controls in a Chinese Han population. Subsequently, we integrated this case‐control study in a meta‐analysis to acquire greater statistical power. The results from our case‐control study using MassARRAY genotyping technology and binary logistic regression statistical methods.

Results

The variant carriers in the Chinese Han population had a lower primary end‐stage hip and knee OA susceptibility (C vs G: OR = 0.350, 95%CI: 0.154‐0.797, P = .012; GC vs GG: adjusted OR = 0.282, 95%CI: 0.118‐0.676, P = .005). Stratification studies indicated that a higher GC frequency in women decreased not only knee OA susceptibility but also unilateral knee OA risk. The meta‐analysis showed that the variant exhibited a significantly decreased OA risk through comparisons involving allelic, homozygous, heterozygous, and dominant models.

Conclusion

Our findings suggest that the COX‐2 G‐765C polymorphism exerts a protective effect against primary end‐stage knee osteoarthritis in a female Chinese Han population.

     

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9‐mediated mutagenesis of the multiple edematous wings gene induces muscle weakness and flightlessness in Bactrocera dorsalis (Diptera: Tephritidae)

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system is a versatile, efficient and heritable gene editing tool that can be useful for genome engineering. Bactrocera dorsalis (Hendel) is a major pest of agriculture that causes great economic losses. We used the B. dorsalis multiple edematous wings (Bdmew) gene as the target gene to explore the effectiveness of CRISPR/Cas9 for B. dorsalis genome manipulation. We studied the physiological functions of the Bdmew gene, particularly those related to muscle development. Site‐specific genome editing was feasible using direct microinjection of specific guide RNA and the Cas9‐plasmid into B. dorsalis embryos. Mutation frequencies ranged from 12.1 to 30.2% in the injected generation. Mosaic G0, with the mew mutation, was heritable to the next generation. The G1 displayed a series of defective phenotypes including muscle weakness, flightlessness, failure to eclose, wing folds and unbalanced movement. These results demonstrated that CRISPR/Cas9 can act as a highly specific, efficient, heritable tool for genome manipulation in B. dorsalis and this has significance for gene function research and genetic control of pests. The Bdmew gene possesses key functions in muscle development of B. dorsalis. Bdmew mutations cause a series of serious defects by interfering with muscle development and may provide a means for controlling B. dorsalis via a gene‐based method such as gene drive.     

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing

Background

KRAS mutations are the key indicator for EGFR monoclonal antibody‐targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision‐making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next‐generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations.

Methods

We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS.

Results

We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V‐1, G12V‐2, and G12V‐3 were 52.01%, 82.06%, and 17.29%, respectively.

Conclusion

The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.

     

QT length during methadone maintenance treatment: gene × dose interaction

Methadone is known to be a risk factor for sudden death by enlarging ECG QT corrected (QTc) interval. For other medical conditions, QTc lengthening has been described as the result of interactions between pharmacological treatments and genetic factors. Former heroin‐dependent subjects under methadone maintenance treatment in remission for at last 3 months were recruited. We studied the association between QTc length (Bazett formula) and 126 SNPs located on five genes (KCNE1, KCNQ1, KCNH2, NOS1AP and SCN5A) previously associated with drug‐induced QT prolongation. Both SNP‐based and gene‐based approaches were used, and we tested also the interaction of the top SNP with methadone dosage to predict the QTc length. In our sample of 154 patients, current methadone daily dose was associated with QTc length (r_Pearson = 0.26; P = 10^?3). Only one SNP, rs11911509 on KCNE1, remained significantly associated with QT length after correction for multiple testing (P = 3.84 × 10^?4; p_corrected = 0.049). Using a gene‐based approach, KCNE1 was also significantly associated with QTc length (p_empirical = 0.02). We found a significant interaction between methadone dosage and rs11911509 minor allele count (allele A vs. C; P = 0.01). Stratified analysis revealed that the correlation between QTc length and methadone dosage was restricted only to AA carriers of this top SNP. Patients’ genetic background should be taken into account in the case of clinically relevant QT enlargement during methadone maintenance treatment.     

HLA‐DQB1*03 genotype and perioperative blood transfusion are not conducive to the prognosis of patients with gastric cancer

Background

Gastric cancer (GC) is a disease associated with a higher incidence and mortality, and some host genetic polymorphisms have been reported as potential factors contributing to the development of GC. In view of this, the study was conducted to investigate the effects of HLA‐DQB1 gene polymorphisms and perioperative blood transfusion on prognosis of patients with gastric cancer (GC).

Methods

A total of 142 patients with GC (case group) and 150 healthy controls (control group) were enrolled. Relationship between HLA‐DQB1 gene polymorphisms, perioperative blood transfusion, and clinical pathological parameters of patients with GC after operation was analyzed. Kaplan‐Meier curve was applied for analyzing survival rate of patients with GC, and Cox multivariate regression analysis for prognostic factors of patients with GC.

Results

The frequency of HLA‐DQB1*03 gene was increased in patients with GC. Patients with GC with HLA‐DQB1*03 genotype had higher number of tumor size >6 cm, deeper depth of infiltration, higher LNM rate, and later stage of disease. Patients with HLA‐DQB1*03 genotype had lower survival rate compared with other genotypes. Anemia before operation, depth of infiltration in T3 stage and T4 stage, LNM in N1 stage and N2 stage, and HLA‐DQB1*03 genotype were regarded as independent risk factors for patients with GC.

Conclusion

These results demonstrate that HLA‐DQB1*03 genotype and perioperative blood transfusion are not conducive to the prognosis of patients with GC, which could provide a reference for the treatment of GC.

     

Multiple functions of miR‐8‐3p in the development and metamorphosis of the red flour beetle,Tribolium castaneum

The microRNA miR‐8‐3p is conserved among insects and closely involved in development and immunity, but its functions in vivo are unexplored in the red flour beetle, Tribolium castaneum. Here, we show that miR‐8‐3p was highly expressed in late larva and early adult stages, as determined by quantitative real‐time PCR. It was enriched in the fat body and cuticle in late larval tissues and abundant in the head and cuticle in early adult tissues, indicating this microRNA plays important roles during T. castaneum development. Specific inhibition of miR‐8‐3p in late larvae led to metamorphosis defects in the development of wings, eyes, legs and embryo. Moreover, a series of genes related to organism development were identified as miR‐8‐3p targets by computational prediction and microRNA–messenger RNA interaction validation, including Wingless, Eyg, Fpps and Sema‐1a. These genes were critical for the regulation of the larva‐to‐adult transition. Eyg, as a functional target of miR‐8‐3p, participates in eye development, which was further confirmed by luciferase assay and loss‐of‐function analyses. In brief, miR‐8‐3p is broadly involved in the development of wings, eyes and legs through its target genes and has extensive regulatory roles during T. castaneum development.     

MicroRNA miR‐252 targets mbt to control the developmental growth of Drosophila

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone‐responsive miRNA, miR‐252, is normally upregulated during the pupal and adult stages of Drosophila development. Here, we found that overexpression of miR‐252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decrease in larval size. Furthermore, miR‐252 overexpression led to a delayed larval‐to‐pupal transition with defective anterior spiracle eversion, as well as a decrease in adult size and mass. Conversely, adult flies lacking miR‐252 showed an increase in mass compared with control flies. We found that miR‐252 directly targeted mbt, encoding a p21‐activated kinase, to repress its expression. Notably, co‐overexpression of mbt rescued the developmental and growth defects associated with miR‐252 overexpression, indicating that mbt is a biologically relevant target of miR‐252. Overall, our data support a role for the ecdysone/miR‐252/mbt regulatory axis in growth control during Drosophila development.     

Genotypic effect of a mutation of the MYBPC3 gene and two phenotypes with different patterns of inheritance

Background

MYBPC3 mutations have been described in dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). A mutation, c.3373G>A, has been reported to cause autosomal recessive form of HCM. Here, we report that this mutation can cause autosomal dominant form of DCM.

Methods

Next‐generation sequencing using targeted panel of a total of 23 candidate genes and following Sanger sequencing was applied to detect causal mutations of DCM. Computational analyses were also performed using available software tools. In silico structural and functional analyses including protein modeling and prediction were done for the mutated MYBPC3 protein.

Results and Conclusion

Targeted sequencing showed one variant c.3373G>A (p.Val1125Met) in the studied family following autosomal dominant inheritance. Computational programs predicted a high score of pathogenicity. Secondary structure of the region surrounding p.Val1125 was changed to a shortened beta‐strand based on prediction of I‐TASSER and Phyre2 servers with high confidence value for the mutation. cMyBP‐C protein was modeled to 3dmkA. Our findings suggest that one single mutation of MYBPC3 may have different effects on the cellular mechanisms based of its zygosity. Various factors might be considered for explaining this phenomenon. This gene may have an important role in Iranian DCM and HCM patients.

     

An αIIb mutation in patients with Glanzmann thrombasthenia located in the N‐terminus of blade 1 of the β‐propeller (Asn2Asp) disrupts a calcium binding site in blade 6

Summary. Background: Studies of Glanzmann thrombasthenia (GT)‐causing mutations has generated invaluable information on the formation and function of integrin αIIbβ₃. Objective: To characterize the mutation in four siblings of an Israeli Arab family affected by GT, and to analyze the relationships between the mutant protein structure and its function using artificial mutations. Methods and Results: Sequencing disclosed a new A97G transversion in the αIIb gene predicting Asn2Asp substitution at blade 1 of the β‐propeller. Alignment with other integrin α subunits revealed that Asn2 is highly conserved. No surface expression of αIIbβ₃ was found in patients’ platelets and baby hamster kidney (BHK) cells transfected with mutated αIIb and WT β₃. Although the αIIbβ₃ was formed, the mutation impaired its intracellular trafficking. Molecular dynamics simulations and modeling of the αIIbβ₃ crystal indicated that the Asn2Asp mutation disrupts a hydrogen bond between Asn2 and Leu366 of a calcium binding domain in blade 6, thereby impairing calcium binding that is essential for intracellular trafficking of αIIbβ₃. Substitution of Asn2 to uncharged Ala or Gln partially decreased αIIbβ₃ surface expression, while substitution by negatively or positively charged residues completely abolished surface expression. Unlike αIIbβ₃, αVβ₃ harboring the Asn2Asp mutation was surface expressed by transfected BHK cells, which is consistent with the known lower sensitivity of αVβ₃ to calcium chelation compared with αIIbβ₃. Conclusion: The new GT causing mutation highlights the importance of calcium binding domains in the β‐propeller for intracellular trafficking of αIIbβ₃. The mechanism by which the mutation exerts its deleterious effect was elucidated by molecular dynamics.     

Comparison between IV immune globulin (IVIG) and anti‐D globulin for treatment of immune thrombocytopenia: a randomized open‐label study

To compare the effect of IV immune globulin (IVIG) and anti‐D globulin (anti‐D) for treatment of immune thrombocytopenia (ITP) in children. A randomized, open‐label, single‐center clinical trial was carried out in Amir‐Kabir Hospital (Arak, Iran). The study was performed on 60 children with acute and chronic ITP, aged from 1 to 15 years. Patients were randomly assigned (1:1) to 50 μg/kg anti‐D or 1 g/kg IVIG. Platelet counting was performed at baseline and at 3, 7, and 14 days after treatment termination. Safety assessment was performed in all patients. Anti‐D caused a quicker response on the 3rd day of treatment (P < 0.001). Both drugs caused a significant rise in number of platelets on the 7th and the 14th day of treatment. Compared to IVIG, except a significant drop in hemoglobin concentration (P < 0.001), anti‐D had lower rate of side effects including fever (P < 0.05), allergy (P < 0.01), and headache (P < 0.001). Our results showed that anti‐D was associated with rapid rise of platelets compared to IVIG. In addition, anti‐D treatment had acceptable safety profile.     

Phenotypic variation of autosomal recessive pseudohypoaldosteronism type I: a case in point

We present a 27‐month‐old male infant with pseudohypoaldosteronism, with two novel α‐subunits, epithelial sodium channel (ENaC) mutations. Despite the presence of the ENaC in the lungs, kidneys, and exocrine glands, he continues to only have renal and exocrine involvement, stressing differential effects of the mutation in each organ.     

Homozygous F5 deep‐intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet‐rich plasma

Summary. Background: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet‐rich plasma (PRP). Therefore, we hypothesized that FV‐deficient patients with severe bleeding manifestations may lack platelet FV. Objectives: To characterize a FV‐deficient patient with a severe bleeding diathesis. Patients/Methods: We performed FV mutation screening and functional studies in a 31‐year‐old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. Results: The proband was homozygous for a deep‐intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo‐exon with an in‐frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband’s plasma or platelets with a prothrombinase‐based assay. Moreover, no thrombin generation was observed in PRP triggered with 1–50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband’s whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished. Conclusions: This is the first report of a pathogenic deep‐intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.     

NOTCH4 is a possible novel susceptibility gene for dilated cardiomyopathy in the Chinese population: A case‐control study

Background

The incidence of dilated cardiomyopathy (DCM) has increased in recent years, and many studies have sought to further improve the general understanding of this condition. Previous studies have demonstrated that some single nucleotide polymorphisms (SNPs) associated with systemic lupus erythematosus also affect susceptibility to DCM, suggesting that immune‐related diseases may share similar genetic susceptibility. Recent large‐scale and genome‐wide association studies have identified NCR3, NOTCH4, CYP1A2, ITGA1, OPRM1, ST8SIA2, and LINC00704 as genetic risk factors associated with cardiac manifestations of neonatal lupus. Here, we aimed to determine whether these SNPs conferred susceptibility to DCM in the Chinese Han population.

Methods

We investigated the relationship between these polymorphisms and DCM risk in 273 patients with DCM and 548 healthy controls. Genotyping was performed using MassArray iPLEX system.

Results

Logistic regression analysis indicated that the T allele of rs3134942 in NOTCH4 gene increased the risk of DCM by 61% compared with the G allele (P_a = 6.57 × 10⁻³). The SNP rs3134942 was also significantly associated with increased DCM risk in the additive (P_a = 6.57 × 10⁻³) and dominant models (P_a = 1.01 × 10⁻²). Additionally, rs2472299 in CYP1A2 gene showed suggestive association with reduced risk of DCM in the dominant model (P_a = 4.24 × 10⁻²) and was correlated with smoking status in patients with DCM (P_a = 1.56 × 10⁻²).

Conclusions

Our findings suggested that rs3134942 in NOTCH4 may be involved in DCM risk. Further, studies in larger and ethnically diverse populations are required to confirm the results reported in this study.