Transcriptome research of human amniocytes identifies hub genes associated with developmental dysplasia in down syndrome.

Trisomy 21, or Down syndrome (DS), is the most frequent human autosomal chromosome aneuploidy, which leads to multiple developmental disorders, especially mental retardation in individuals. The presence of an additional human chromosome 21 (HSA21) could account for the pathological manifestations in DS. In this study, we analyzed the mRNA gene expression profile of DS-derived amniocytes compared with normal amniocytes, aiming to evaluate the relationship between candidate dysregulated HSA21 genes and DS developmental phenotypes. Differentially expressed genes (DEGs) included 1794 upregulated genes and 1411 downregulated genes, which are mainly involved in cell adhesion, inflammation, cell proliferation and thus may play an important role in inducing multiple dysplasia during DS fetal development. Furthermore, STRING protein network studies demonstrated 7 candidate HSA21 genes participated Gene Ontology (GO) terms: cell adhesion and extracellular matrix remodeling (COL6A1, COL6A2, COL18A1, ADAMTS5, JAM2, and POFUT2), inflammation and virus infection response (MX1 and MX2), histone modification and chromatin remodeling (NRIP1), glycerolipid and glycerophospholipid metabolism (AGPAT3), mitochondrial function (ATP5PF and ATP5PO), synaptic vesicle endocytosis (ITSN1 and SYNJ1) and amyloid metabolism (APP). Meanwhile, GSEA enrichment identified several transcription factors and miRNAs, which may target gene expression in the DS group. Our study established connections between dysregulated genes, especially HSA21 genes, and DS-associated phenotypes. The alteration of multiple pathways and biological processes may contribute to DS developmental disorders, providing potential pathogenesis and therapeutic targets for DS.

Integration of ATAC-seq and RNA-seq identifies MX1-mediated AP-1 transcriptional regulation as a therapeutic target for Down syndrome.

BACKGROUND: Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation. METHODS: We performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS. RESULTS: Binding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs. CONCLUSIONS: This study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.

Functional analysis of a novel nonsense variant c.91A>T of the TRAPPC2 gene in a Chinese family with X-linked recessive autosomal spondyloepiphyseal dysplasia tarda.

Spondyloepiphyseal dysplasia tarda (SEDT) is a condition involving late-onset, X-linked recessive skeletal dysplasia caused by mutations in the TRAPPC2 gene. In this paper, we identified a novel nonsense variant in a SEDT pedigree and analyzed the function of the variant in an attempt to explain the new pathogenesis of the TRAPPC2 protein in SEDT. Briefly, DNA and RNA samples from the peripheral blood of SEDT individuals were prepared. The causative variant in the Chinese SEDT family was identified by clinic whole-exome sequencing analysis. Then, we observed the mRNA expression of TRAPPC2 in patients and the mutant TRAPPC2 level in vitro and analyzed the protein stability and subcellular distribution by cell fluorescence and Western blotting. We also investigated the effect of TRAPPC2 knockdown on the expression and secretion of COL2A1 in SW1353 cells or primary human chondrocytes. Herein, we found a nonsense variant, c.91A>T, of the TRAPPC2 gene in the pedigree. TRAPPC2 mRNA expression levels were significantly decreased in the available peripheral blood cell samples of two affected patients. An in vitro study showed that the mutant plasmid exhibited significantly lower mRNA and protein of TRAPPC2, and the mutant protein changed its membrane distribution. TRAPPC2 knockdown resulted in decreased COL2A1 expression and collagen II secretions. Our data indicate that the novel nonsense variant, c.91A>T, of the TRAPPC2 gene is the cause of SEDT in this pedigree. The variant results in a lowered expression of TRAPPC2 and then affects the COL2A1 expression and collagen II secretions, which may explain the mechanism of loss of function of the variant.

Three-way contact analysis characterizes the higher order organization of the Tcra locus.

The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer Eα has been implicated in regulating the accessibility of Vα and Jα genes through long-range interactions during rearrangements of the T-cell antigen receptor gene Tcra. However, direct evidence for Eα physically mediating the interaction of Vα and Jα genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multiway contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal Jα genes, Vα genes and Eα in CD4+CD8+ double-positive thymocytes. Notably, the INT2-TEAp loop emerged as a prominent structure likely to be responsible for bringing the proximal Jα genes and the Vα genes into proximity. Moreover, the enhancer Eα utilizes this loop to establish physical proximity with the proximal Vα gene region. This study provides insights into the higher order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination.

Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by regulating the activity of super-enhancers.

CD4+ and CD8+ double-positive (DP) thymocytes play a crucial role in T cell development in the thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCRß. DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, regulatory T cells, or invariant nature kill T cells (iNKT) in response to TCR signaling. Chromatin organizer SATB1 is highly expressed in DP cells and is essential in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing shows that Satb1 deletion changes the cell identity of DP thymocytes and down-regulates genes specifically and highly expressed in DP cells. Super-enhancers regulate the expressions of DP-specific genes, and our Hi-C data show that SATB1 deficiency in thymocytes reduces super-enhancer activity by specifically decreasing interactions among super-enhancers and between super-enhancers and promoters. Our results reveal that SATB1 plays a critical role in thymocyte development to promote the establishment of DP cell identity by globally regulating super-enhancers of DP cells at the chromatin architectural level.

Inhibition of NOS1 promotes the interferon response of melanoma cells.

BACKGROUND: NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown. METHODS: The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer. RESULTS: NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients. CONCLUSION: These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells.

A Novel Variant of the KIF11 Gene, c.2922G>T, Is Associated with Microcephaly by Affecting RNA Splicing.

Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR) is an inherited disorder characterized by severe microcephaly and abnormal facial features. Kinesin family member 11 (KIF11) mutations have been reported closely related to microcephaly in different cases, while the pathogenicity was still unclear. Here, we report a de novo heterozygous mutation in exon 20 of the KIF11 (c.2922G>T; p.Pro974=) from a microcephaly patient through whole-exome sequencing. Further studies identified that this variant affected the normal splicing of KIF11 pre-mRNA, thus leading to the c.2815_2922 deletion of exon 20 through PBMC-derived pre-mRNA splicing assay and minigene experiment. Moreover, c.2815_2922 deletion would produce a shortened KIF11 protein, which may competitively bind to the normal KIF11 protein, suggesting a dominant negative effect mechanism in c.2922G>T mutation-induced MCLMR.

Phase separation of Epstein-Barr virus EBNA2 protein reorganizes chromatin topology for epigenetic regulation.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transactivator of viral and cellular gene expression, which plays a critical role in the Epstein-Barr virus-associated diseases. It was reported that EBNA2 regulates gene expression by reorganizing chromatin and manipulating epigenetics. Recent studies showed that liquid-liquid phase separation plays an essential role in epigenetic and transcriptional regulation. Here we show that EBNA2 reorganized chromatin topology to form accessible chromatin domains (ACDs) of the host genome by phase separation. The N-terminal region of EBNA2, which is necessary for phase separation, is sufficient to induce ACDs. The C-terminal domain of EBNA2 promotes the acetylation of accessible chromatin regions by recruiting histone acetylase p300 to ACDs. According to these observations, we proposed a model of EBNA2 reorganizing chromatin topology for its acetylation through phase separation to explain the mechanism of EBNA2 hijacking the host genome by controlling its epigenetics.

Downregulation by CNNM2 of ATP5MD expression in the 10q24.32 schizophrenia-associated locus involved in impaired ATP production and neurodevelopment.

Genome-wide association studies (GWAS) have accelerated the discovery of numerous genetic variants associated with schizophrenia. However, most risk variants show a small effect size (odds ratio (OR) <1.2), suggesting that more functional risk variants remain to be identified. Here, we employed region-based multi-marker analysis of genomic annotation (MAGMA) to identify additional risk loci containing variants with large OR value from Psychiatry Genomics Consortium (PGC2) schizophrenia GWAS data and then employed summary-data-based mendelian randomization (SMR) to prioritize schizophrenia susceptibility genes. The top-ranked susceptibility gene ATP5MD, encoding an ATP synthase membrane subunit, is observed to be downregulated in schizophrenia by the risk allele of CNNM2-rs1926032 in the schizophrenia-associated 10q24.32 locus. The Atp5md knockout (KO) in mice was associated with abnormal startle reflex and gait, and ATP5MD knockdown (KD) in human induced pluripotent stem cell-derived neurons disrupted the neural development and mitochondrial respiration and ATP production. Moreover, CNNM2-rs1926032 KO could induce downregulation of ATP5MD expression and disruptions of mitochondrial respiration and ATP production. This study constitutes an important mechanistic component that links schizophrenia-associated CNNM2 regions to disruption in energy adenosine system modulation and neuronal function by long-distance chromatin domain downregulation of ATP5MD. This pathogenic mechanism provides therapeutic implications for schizophrenia.

METTL3-Mediated N6-Methyladenosine Modification Is Involved in the Dysregulation of NRIP1 Expression in Down Syndrome.

Down syndrome (DS) is a common genetic condition in which a person is born with an extra copy of chromosome 21. Intellectual disability is the most common characteristic of DS. N6-methyladenosine (m6A) is a common RNA modification that is implicated in many biological processes. It is highly enriched within the brain and plays an essential role in human brain development. However, the mRNA m6A modification in the fetal brain of DS has not been explored. Here, we report m6A mRNA profiles and mRNA expression profiles of fetal brain cortex tissue from DSs and controls. We observed that the m6A modification in DS brain tissues was reduced genome-wide, which may be due to decreased the m6A methyltransferase like 3 (METTL3) protein expression. The nuclear receptor-interacting protein 1 (NRIP1/RIP140) is coded by a highly conserved chromosome 21 (Hsa21) gene. Overexpression of NRIP1 is associated with mitochondrial dysfunction in DS. The NRIP1 mRNA increased in fetal brain tissues of DS, whereas the m6A modification of the NRIP1 mRNA significantly decreased. METTL3 knockdown reduced the m6A modification of NRIP1 mRNA and increased its expression, and an increase in NRIP1 m6A modification and a decrease in its expression were observed in METTL3-overexpressed cells. The Luciferase reporter assay confirmed that METTL3 regulates NRIP1 expression in an m6A-dependent manner. The decay rate of NRIP1 mRNA was significantly reduced in METTL3-knockdown cells but increased in METTL3-overexpressed cells. We proposed that the m6A modification of NRIP1 mRNA in DS fetal brain tissue is reduced, reducing its transcript degradation rate, resulting in abnormally increased expression of NRIP1, at least partially, in the DS brain. It provides a new mechanism for the molecular pathology of DS and leads to a new insight that may become therapeutically relevant.

The role of S-nitrosylation of PFKM in regulation of glycolysis in ovarian cancer cells.

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.

Intravitreal Injection of an Exosome-Associated Adeno-Associated Viral Vector Enhances Retinoschisin 1 Gene Transduction in the Mouse Retina.

To investigate whether exosome-associated adeno-associated virus (AAV) retinoschisin 1 (RS1) vector improved the transduction efficiency of RS1 in the mouse retina. pAAV2-RS1-ZsGreen plasmid was constructed by homologous recombination. Exosome-associated AAV vectors containing human RS1 gene (exosome-associated AAV [exo-AAV]2-RS1-ZsGreen) were isolated from producer cells' supernatant, and confirmed by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. In vitro, HEK-293T cells were transduced with AAV2-RS1-ZsGreen and exo-AAV2-RS1-ZsGreen. In vivo, 1 µL of AAV2-RS1-ZsGreen or 1 µL exo-AAV2-RS1-ZsGreen (2 × 108 genome copies/µL) was injected intravitreally into the C57BL/6J mouse eyes. Phosphate buffer saline was injected as controls. The mRNA and the protein expression in the retina were detected. Exo-AAV2-RS1-ZsGreen possessed lipid bilayers, a saucer-like structures and an average of 120 nm particle size. The expression of RS1 and ZsGreen in exo-AAV2-RS1-ZsGreen group were 7.6 times and 5.7 times that of AAV2-RS1-ZsGreen group in HEK-293T cells, respectively. Furthermore, RS1 protein expression increased by 11.8 times in HEK-293T cells. Intravitreal injection of exo-AAV significantly increased the transduction efficiency of RS1 than AAV. Exo-AAV may be a powerful gene delivery system for gene therapy of X-link retinoschisis as well as other inherited retina degenerations.

The role of chromatin organizer Satb1 in shaping TCR repertoire in adult thymus.

T cells recognize the universe of foreign antigens with a diverse repertoire of T cell receptors generated by V(D)J recombination. Special AT-rich binding protein 1 (Satb1) is a chromatin organizer that plays an essential role in T cell development. Previous study has shown that Satb1 regulates the re-induction of recombinase Rag1 and Rag2 in CD4+CD8+ thymocytes, affecting the secondary rearrangement of the Tcra gene. Here, we detected the repertoires of four TCR genes, Tcrd, Tcrg, Tcrb, and Tcra, in the adult thymus, and explored the role of the Satb1 in shaping the TCR repertoires. We observed a strong bias in the V and J gene usages of the Tcrd and Tcrg repertoires in WT and Satb1-deleted thymocytes. Satb1 deletion had few effects on the V(D)J rearrangement and repertoire of the Tcrg, Tcrd, and Tcrb genes. The Tcra repertoire was severely impaired in Satb1-deleted thymocytes, while the primary rearrangement was relatively normal. We also found the CDR3 length of TCRα chain was significantly longer in Satb1-deleted thymocytes, which can be explained by the strong bias of the proximal Jα usage. Our results showed that Satb1 plays an essential role in shaping TCR repertoires in αß T cells.

Epicardium-Derived Tbx18+ CDCs Transplantation Improve Heart Function in Infarcted Mice.

Cardiosphere-derived cells (CDCs) constitute a cardiac stem cell pool, a promising therapeutics in treating myocardial infarction (MI). However, the cell source of CDCs remains unclear. In this study, we isolated CDCs directly from adult mouse heart epicardium named primary epicardium-derived CDCs (pECDCs), which showed a different expression profile compared with primary epicardial cells (pEpiCs). Interestingly, pECDCs highly expressed T-box transcription factor 18 (Tbx18) and showed multipotent differentiation ability in vitro. Human telomerase reverse transcriptase (hTERT) transduction could inhibit aging-induced pECDCs apoptosis and differentiation, thus keeping a better proliferation capacity. Furthermore, immortalized epicardium CDCs (iECDCs) transplantation extensively promote cardiogenesis in the infracted mouse heart. This study demonstrated epicardium-derived CDCs that may derive from Tbx18+ EpiCs, which possess the therapeutic potential to be applied to cardiac repair and regeneration and suggest a new kind of CDCs with identified origination that may be followed in the developing and injured heart.

Case Report: Twin Pregnancy Gives Birth to a Girl with Partial Trisomy 21 Mosaicism after in vitro Fertilization and Embryo Transfer.

Objective: To report a rare case in which an IVF-ET twin pregnancy gave birth to a partial trisomy 21 chimera girl. Design: Case report. Setting: University hospital. Patient: A girl with partial trisomy 21 mosaicism after in vitro fertilization and embryo transfer. Interventions: In vitro fertilization (IVF) and embryo transfer (ET). Main Outcome Measure: Karyotype analysis, Copy Number Variation sequencing (CNV-seq), stLFR-WGS, and Short Tandem Repeat (STR) analysis. Results: Being assisted with IVF and EF technology, the couple successfully gave birth to twin sisters at 37 weeks of gestational age. The NonInvasive Prenatal Testing (NIPT) and Nuchal Translucency (NT) examination showed no detectable genetic abnormalities during pregnancy. However, the younger infant displayed growth retardation and feeding difficulties after birth, which was not observed in her twin sister. Further genetic counseling and diagnosis suggested that she is a Chimera with complex partial trisomy 21. The stLFR-WGS assay showed multiple CNV variations in Chr21 and STR analysis confirmed the paternal origin of the additional fragments. Conclusion: It is rare for IVF-ET-assisted twin pregnancy to give birth to a girl with a complex combination of abnormal Chr21, which might result from paternal chromosome rearrangement during meiosis and mitosis.

Integrative Analysis Extracts a Core ceRNA Network of the Fetal Hippocampus With Down Syndrome.

Accumulating evidence suggests that circular RNAs (circRNAs)-miRNA-mRNA ceRNA regulatory network-may play an important role in neurological disorders, such as Alzheimer's disease (AD). Interestingly, neuropathological changes that closely resemble AD have been found in nearly all Down syndrome (DS) cases > 35 years. However, few studies have reported circRNA transcriptional profiling in DS cases, which is caused by a chromosomal aberration of trisomy 21. Here, we characterized the expression profiles of circRNAs in the fetal hippocampus of DS patients (n = 8) and controls (n = 6) by using microarray. MiRNA, mRNA expression profiling of DS from our previous study and scRNA-seq data describing normal fetal hippocampus development (GEO) were also integrated into the analysis. The similarity between circRNAs/genes with traits/cell-types was calculated by weighted correlation network analysis (WGCNA). miRanda and miRWalk2 were used to predict ceRNA network interactions. We identified a total of 7,078 significantly differentially expressed (DE) circRNAs, including 2,637 upregulated and 4,441 downregulated genes, respectively. WGCNA obtained 15 hub circRNAs and 6 modules with cell type-specific expression patterns among scRNA-seq data. Finally, a core ceRNA network was constructed by 14 hub circRNAs, 17 DE miRNA targets and 245 DE mRNA targets with a cell type-specific expression pattern annotation. Known functional molecules in DS or neurodegeneration (e.g., miR-138, OLIG1, and TPM2) were also included in this network. Our findings are the first to delineate the landscape of circRNAs in DS and the first to effectively integrate ceRNA regulation with scRNA-seq data. These data may provide a valuable resource for further research on the molecular mechanisms or therapeutic targets underlying DS neuropathy.

A novel c.2326G>A KIT pathogenic variant in piebaldism.

INTRODUCTION: Piebaldism is a rare autosomal dominant disorder characterized by congenital patchy depigmentation of the scalp, forehead, trunk, and limbs. The KIT gene is the mainly causative gene to this disease. But how KIT is involved in piebaldism remains unclear. METHODS: Whole exome sequencing was used to explore the genetic cause of a familial case of piebaldism. Sanger sequencing was used to validate the variant. To further examine the variant's pathogenicity, the wild type and the mutated KIT plasmids were constructed and transfected into HEK293T cells. Next STAT5 expression, a signaling target of KIT, was detected by western blotting to explore the potential molecular mechanism of the variant in piebaldism. Based on the classification of the given variant, prenatal diagnosis was further performed in this family. RESULTS: A novel pathogenic variant of KIT c.2326G>A (NM_000222.2) was identified in this family. The phosphorylation of STAT5 was reduced in the mutant KIT transfected cells compared to the wild type after stem cell factor (SCF) treatment, indicating that the KIT signaling was dysfunctional and supported that the variant was a pathogenic one. Prenatal diagnosis results indicated that the fetus exhibited the same genotype as the proband. CONCLUSION: We identified a novel KIT pathogenic variant in the patient with piebaldism to expand the variation spectrum of KIT. The functional study indicated that the mutant KIT was dysfunctional in KIT signaling. The pathogenic variant identification enriches the knowledge about the genotype/phenotype correlation and could serve as the basis for genetic counseling and prenatal diagnosis.

The Ig heavy chain protein but not its message controls early B cell development.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.