Nonsense Mutation Research Articles

Powerful QTL mapping and favorable allele mining in an all-in-one population: a case study of heading date.

The multiparent advanced generation intercross (MAGIC) population is characterized with great potentials in power and resolution of quantitative trait locus (QTL) mapping, but single nucleotide polymorphism (SNP)-based GWAS does not fully reach its potential. In this study, a MAGIC population of 1021 lines was developed from four Xian and four Geng varieties from five subgroups of rice. A total of 44000 genes showed functional polymorphisms among eight parents, including frameshift variations or premature stop codon variations, which provides the potential to map almost all genes of the MAGIC population. Principal component analysis results showed that the MAGIC population had a weak population structure. A high-density bin map of 24414 bins was constructed. Segregation distortion occurred in the regions possessing the genes underlying genetic incompatibility and gamete development. SNP-based association analysis and bin-based linkage analysis identified 25 significant loci and 47 QTLs for heading date, including 14 known heading date genes. The mapping resolution of genes is dependent on genetic effects with offset distances of <55kb for major effect genes and <123kb for moderate effect genes. Four causal variants and noncoding structure variants were identified to be associated with heading date. Three to four types of alleles with strong, intermediate, weak, and no genetic effects were identified from eight parents, providing flexibility for the improvement of rice heading date. In most cases, japonica rice carries weak alleles, and indica rice carries strong alleles and nonfunctional alleles. These results confirm that the MAGIC population provides the exceptional opportunity to detect QTLs, and its use is encouraged for mapping genes and mining favorable alleles for breeding.

Analysis of Phenotypes Associated with Deficiency of PAX6 Haplotypes in Chinese Aniridia Families.

To examine the clinical phenotype and genetic deficiencies present in Chinese aniridia families with PAX6 haplotype deficiency. A comprehensive questionnaire and ophthalmological assessments were administered to both affected patients and unaffected relatives. The clinical feature analysis included the evaluation of visual acuity, intraocular pressure, slit-lamp anterior segment examination, fundus photography, and spectral domain optical coherence tomography. To identify the mutation responsible for aniridia, targeted next-generation sequencing was used as a beneficial technique. A total of 4 mutations were identified, consisting of two novel frameshift mutations (c.314delA, p.K105Sfs*33 and c.838_845dup AACACACC, p.S283Tfs*85), along with two recurring nonsense mutations (c.307C>T, p.R103X and c.619A>T, p.K207*). Complete iris absence, macular foveal hypoplasia, and nystagmus were consistent in these PAX6 haplotype-deficient Chinese aniridia families, while corneal lesions, cataracts, and glaucoma exhibited heterogeneity both among the families and within the same family. In our study, two novel PAX6 mutations associated with aniridia were identified in Chinese families, which expanded the phenotypic and genotypic spectrum of PAX6 mutations. We also analyzed the clinical characteristics of PAX6 haplotype deficiency in Chinese aniridia families.

Leukemia Inhibitory Factor Protects against Degeneration of Cone Photoreceptors Caused by RPE65 Deficiency.

Retinal pigment epithelium (RPE) 65 is a key enzyme in the visual cycle involved in the regeneration of 11-cis-retinal. Mutations in the human RPE65 gene cause Leber's congenital amaurosis (LCA), a severe form of an inherited retinal disorder. Animal models carrying Rpe65 mutations develop early-onset retinal degeneration. In particular, the cones degenerate faster than the rods. To date, gene therapy has been used successfully to treat RPE65-associated retinal disorders. However, gene therapy does not completely prevent progressive retinal degeneration in patients, possibly due to the vulnerability of cones in these patients. In the present study, we tested whether leukemia inhibitory factor (LIF), a trophic factor, protects cones in rd12 mice harboring a nonsense mutation in Rpe65. LIF was administered to rd12 mice by intravitreal microinjection. Apoptosis of retinal cells was analyzed by TUNEL assay. The degeneration of cone cells was evaluated by immunostaining of retinal sections and retinal flat-mounts. Signaling proteins regulated by LIF in the retinal and cultured cells were determined by immunoblotting. Intravitreal administration of LIF activated the STAT3 signaling pathway, thereby inhibiting photoreceptor apoptosis and preserving cones in rd12 mice. Niclosamide (NCL), an inhibitor of STAT3 signaling, effectively blocked STAT3 signaling and autophagy in cultured 661W cells treated with LIF. Co-administration of LIF with NCL to rd12 mice abolished the protective effect of LIF, suggesting that STAT3 signaling and autophagy mediate the protection. LIF is a potent factor that protects cones in rd12 mice. This finding implies that LIF can be used in combination with gene therapy to achieve better therapeutic outcomes for patients with RPE65-associated LCA.

A novel compound heterozygous mutation of UFC1 in a patient with neurodevelopmental disorder.

Neurodevelopmental disorders (NDDs) encompass a diverse group of disorders characterized by impaired cognition, behavior, and motor skills. Genetic factor is the leading cause in about 35% of NDDs patients. Mutations of UFC1, an E2 enzyme participating in the post-translational modification of proteins through attachment of ubiquitin-like proteins, were recently reported to be associated with NDDs. However, the UFC1 associated NDDs are rare and the data are scarce, thus making it difficult to identify this disease. This study reported a novel compound heterozygous mutation of UFC1 in a Chinese patient with NDD. Detailed clinical data were recorded. Whole exome sequencing (WES) was performed to determine the genetic cause of the patient. The candidate mutation was verified using Sanger sequencing. WES analysis identified a novel compound heterozygous mutation of UFC1 (c.19C > T, p.Arg7* and c.164G > A, p.Arg55Gln). The nonsense mutation c.19C > T (p.Arg7*) led to a premature truncation of UFC1 and nonsense-mediated RNA decay. Arg55 is highly conserved among orthologues. Molecular modeling predicted that mutation c.164G > A (p.Arg55Gln) may influence the correct folding of UFC1. These two mutations were evaluated as likely pathogenic based on the ACMG guideline. Moreover, neurodevelopmental delay, microcephaly, and epilepsy were confirmed as major phenotypes of UFC1 mutation. This study expands the mutational spectrum of NDDs. We reported the nonsense mutation of UFC1 for the first time. We also confirmed the major phenotypes that may guide clinical identification of UFC1 mutation. Ubiquitination mechanism is highlighted in NDDs pathogenesis.

Genetic Mutations Associated With TNFAIP3 (A20) Haploinsufficiency and Their Impact on Inflammatory Diseases.

TNF-α-induced protein 3 (TNFAIP3), commonly referred to as A20, is an integral part of the ubiquitin-editing complex that significantly influences immune regulation, apoptosis, and the initiation of diverse immune responses. The A20 protein is characterized by an N-terminal ovarian tumor (OTU) domain and a series of seven zinc finger (ZNF) domains. Mutations in the TNFAIP3 gene are implicated in various immune-related diseases, such as Behçet's disease, polyarticular juvenile idiopathic arthritis, autoimmune thyroiditis, autoimmune hepatitis, and rheumatoid arthritis. These mutations can lead to a spectrum of symptoms, including, but not limited to, recurrent fever, ulcers, rashes, musculoskeletal and gastrointestinal dysfunctions, cardiovascular issues, and respiratory infections. The majority of these mutations are either nonsense (STOP codon) or frameshift mutations, which are typically associated with immune dysfunctions. Nonetheless, missense mutations have also been identified as contributors to these conditions. These genetic alterations may interfere with several biological pathways, notably abnormal NF-κB signaling and dysregulated ubiquitination. Currently, there is no definitive treatment for A20 haploinsufficiency; however, therapeutic strategies can alleviate the symptoms in patients. This review delves into the mutations reported in the TNFAIP3 gene, the clinical progression in affected individuals, potential disease mechanisms, and a brief overview of the available pharmacological interventions for A20 haploinsufficiency. Mandatory genetic testing of the TNFAIP3 gene should be performed in patients diagnosed with autoinflammatory disorders to better understand the genetic underpinnings and guide treatment decisions.

Rising Prevalence of Low-Frequency PPM1D Gene Mutations after Second HDCT in Multiple Myeloma.

Multiple myeloma (MM) first-line treatment algorithms include immuno-chemotherapy (ICT) induction, high-dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) consolidation, followed by lenalidomide maintenance. After these initial therapies, most patients suffer a disease relapse and require subsequent treatment lines including ICT, additional HDCT and ASCT, or novel immunotherapies. The presence of somatic mutations in peripheral blood cells has been associated with adverse outcomes in a variety of hematological malignancies. Nonsense and frameshift mutations in the PPM1D gene, a frequent driver alteration in clonal hematopoiesis (CH), lead to the gain-of-function of Wip1 phosphatase, which may impair the p53-dependent G1 checkpoint and promote cell proliferation. Here, we determined the presence of PPM1D gene mutations in peripheral blood cells of 75 subsequent myeloma patients in remission after first or second HDCT/ASCT. The prevalence of truncating PPM1D gene mutations emerged at 1.3% after first HDCT/ASCT, and 7.3% after second HDCT/ASCT, with variant allele frequencies (VAF) of 0.01 to 0.05. Clinical outcomes were inferior in the PPM1D-mutated (PPM1Dmut) subset with median progression-free survival (PFS) of 15 vs. 37 months (p = 0.0002) and median overall survival (OS) of 36 vs. 156 months (p = 0.001) for the PPM1Dmut and PPM1Dwt population, respectively. Our data suggest that the occurrence of PPM1D gene mutations in peripheral blood cells correlates with inferior outcomes after ASCT in patients with multiple myeloma.

Truncation mutations of CRYGD gene in congenital cataracts cause protein aggregation by disrupting the structural stability of γD-crystallin

Congenital cataracts, a prevalent cause of blindness in children, are associated with protein aggregation. γD-crystallin, essential for sustaining lens transparency, exists as a monomer and exhibits excellent structural stability. In our cohort, we identified a nonsense mutation (c.451_452insGACT, p.Y151X) in the CRYGD gene. To explore the effect of truncation mutations on the structure of γD-crystallin, we examined the Y151X and T160RfsX8 mutations, both located in the Greek key motif 4 at the cellular and protein level in this study. Both truncation mutations induced protein misfolding and resulted in the formation of insoluble aggregates when overexpressed in HLE B3 and HEK 293T cells. Moreover, heat, UV irradiation, and oxidative stress increased the proportion of aggregates of mutants in the cells. We next purified γD-crystallin to estimate its structural changes. Truncation mutations led to conformational disruption and a concomitant decrease in protein solubility. Molecular dynamics simulations further demonstrated that partial deletion of the conserved domain within the Greek key motif 4 markedly compromised the overall stability of the protein structure. Finally, co-expression of α-crystallins facilitated the proper folding of truncated mutants and mitigated protein aggregation. In summary, the structural integrity of the Greek key motif 4 in γD-crystallin is crucial for overall structural stability.

An Epilepsy-Associated CILK1 Variant Compromises KATNIP Regulation and Impairs Primary Cilia and Hedgehog Signaling.

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling.

Genomic analysis reveals the association of KIT and MITF variants with the white spotting in swamp buffaloes

BackgroundSwamp-type buffaloes with varying degrees of white spotting are found exclusively in Tana Toraja, South Sulawesi, Indonesia, where spotted buffalo bulls are highly valued in accordance with the Torajan customs. The white spotting depigmentation is caused by the absence of melanocytes. However, the genetic variants that cause this phenotype have not been fully characterized. The objective of this study was to identify the genomic regions and variants responsible for this unique coat-color pattern.ResultsGenome-wide association study (GWAS) and selection signature analysis identified MITF as a key gene based on the whole-genome sequencing data of 28 solid and 39 spotted buffaloes, while KIT was also found to be involved in the development of this phenotype by a candidate gene approach. Alternative candidate mutations included, in addition to the previously reported nonsense mutation c.649 C > T (p.Arg217*) and splice donor mutation c.1179 + 2T > A in MITF, a nonsense mutation c.2028T > A (p.Tyr676*) in KIT. All these three mutations were located in the genomic regions that were highly conserved exclusively in Indonesian swamp buffaloes and they accounted largely (95%) for the manifestation of white spotting. Last but not the least, ADAMTS20 and TWIST2 may also contribute to the diversification of this coat-color pattern.ConclusionsThe alternative mutations identified in this study affect, at least partially and independently, the development of melanocytes. The presence and persistence of such mutations may be explained by significant financial and social value of spotted buffaloes used in historical Rambu Solo ceremony in Tana Toraja, Indonesia. Several de novo spontaneous mutations have therefore been favored by traditional breeding for the spotted buffaloes.

A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation.

Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.

ATM inhibition enhance immunotherapy by activating STING signaling and augmenting MHC Class I

Accumulating evidence supports the concept that DNA damage response targeted therapies can improve antitumor immune response by increasing the immunogenicity of tumor cells and improving the tumor immune microenvironment. Ataxia telangiectasia mutated (ATM) is a core component of the DNA repair system. Although the ATM gene has a significant mutation rate in many human cancers, including colorectal, prostate, lung, and breast, it remains understudied compared with other DDR-involved molecules such as PARP and ATR. Here, we found that either gene knockout or drug intervention, ATM inhibition activated the cGAS/STING pathway and augmented MHC class I in CRC cells, and these effects could be amplified by radiation. Furthermore, we found that MHC class I upregulation induced by ATM inhibition is dependent on the activation of the NFκB/IRF1/NLRC5 pathway and independent of STING. Animal experiments have shown increasing infiltration and cytotoxic function of T cells and better survival in ATM-deficient tumors. This work indicated that ATM nonsense mutation predicted the clinical benefits of radiotherapy combined with immune checkpoint blockade for patients with CRC. It also provides a molecular mechanism rationale for ATM-targeted agents for patients with CRC.

Identification of a novel nonsense SLC16A2 gene mutation in an infant with severe neurologic phenotype: A case report.

Allan-Herndon-Dudley syndrome (AHDS) results from a pathogenic variant in the hemizygous subunit of the SLC16A2 gene, which encodes monocarboxylate transporter 8 and follows an X-linked recessive pattern. AHDS manifests as neuropsychomotor developmental delay, intellectual disability, movement disorders, and thyroid hormone abnormalities. It is frequently misdiagnosed as cerebral palsy or hypothyroidism. A 9-month-old male infant exhibited poor head control, hypodynamia, motor retardation, hypertonic limbs, and thyroid abnormalities. Despite levothyroxine supplementation and rehabilitation therapy, no improvements were observed. Whole-exome sequencing identified a novel nonsense mutation in SLC16A2 (c.124G > T, p.E42X), which unequivocally established the diagnosis. AHDS was confirmed. Levothyroxine treatment commenced early in infancy, followed by 3 months of rehabilitation therapy, starting at 5 months of age. The combined administration of levothyroxine and methimazole was initiated at 1 year and 10 months of age, respectively. While improvements were noted in thyroid hormone levels, neurological developmental delays persisted. AHDS should be considered in patients presenting with atypical neurological features and thyroid hormone abnormalities such as elevated triiodothyronine and decreased thyroxine levels. The early utilization of exome sequencing aids in prompt diagnosis. The identified SLC16A2 nonsense mutation correlates with severe neurological phenotypes and adds to the spectrum of genetic variations associated with AHDS.

MRNA-specific readthrough of nonsense codons by antisense oligonucleotides (R-ASOs).

Nonsense mutations account for >10% of human genetic disorders, including cystic fibrosis, Alagille syndrome, and Duchenne muscular dystrophy. A nonsense mutation results in the expression of a truncated protein, and therapeutic strategies aim to restore full-length protein expression. Most strategies under development, including small-molecule aminoglycosides, suppressor tRNAs, or the targeted degradation of termination factors, lack mRNA target selectivity and may poorly differentiate between nonsense and normal stop codons, resulting in off-target translation errors. Here, we demonstrate that antisense oligonucleotides can stimulate readthrough of disease-causing nonsense codons, resulting in high yields of full-length protein in mammalian cellular lysate. Readthrough efficiency depends on the sequence context near the stop codon and on the precise targeting position of an oligonucleotide, whose interaction with mRNA inhibits peptide release to promote readthrough. Readthrough-inducing antisense oligonucleotides (R-ASOs) enhance the potency of non-specific readthrough agents, including aminoglycoside G418 and suppressor tRNA, enabling a path toward target-specific readthrough of nonsense mutations in CFTR, JAG1, DMD, BRCA1 and other mutant genes. Finally, through systematic chemical engineering, we identify heavily modified fully functional R-ASO variants, enabling future therapeutic development.

Intricate MIB1-NOTCH-GATA6 Interactions in Cardiac Valvular and Septal Development.

Genome-wide association studies and experimental mouse models implicate the MIB1 and GATA6 genes in congenital heart disease (CHD). Their close physical proximity and conserved synteny suggest that these two genes might be involved in analogous cardiac developmental processes. Heterozygous Gata6 loss-of-function mutations alone or humanized Mib1 mutations in a NOTCH1-sensitized genetic background cause bicuspid aortic valve (BAV) and a membranous ventricular septal defect (VSD), consistent with MIB1 and NOTCH1 functioning in the same pathway. To determine if MIB1-NOTCH and GATA6 interact in valvular and septal development, we generated compound heterozygote mice carrying different Mib1 missense (Mib1K735R and Mib1V943F) or nonsense (Mib1R530X) mutations with the Gata6STOP/+ heterozygous null mutation. Combining Mib1R530X/+ or Mib1K735R/+ with Gata6STOP/+ does not affect Gata6STOP/+ single mutant phenotypes. In contrast, combining Mib1V943F/+ with Gata6STOP/+ decreases the incidence of BAV and VSD by 50%, suggesting a suppressive effect of Mib1V943F/+ on Gata6STOP/+. Transcriptomic and functional analyses revealed that while the EMT pathway term is depleted in the Gata6STOP/+ mutant, introducing the Mib1V943F variant robustly enriches this term, consistent with the Mib1V943F/+ phenotypic suppression of Gata6STOP/+. Interestingly, combined Notch1 and Gata6 insufficiency led to a nearly fully penetrant VSD but did not affect the BAV phenotype, underscoring the complex functional relationship between MIB1, NOTCH, and GATA6 in valvular and septal development.

Sulfotransferase 4A1 Coding Sequence and Protein Structure Are Highly Conserved in Vertebrates.

Cytosolic sulfotransferases (SULTs) are Phase 2 drug-metabolizing enzymes that catalyze the conjugation of sulfonate to endogenous and xenobiotic compounds, increasing their hydrophilicity and excretion from cells. To date, 13 human SULTs have been identified and classified into five families. SULT4A1 mRNA encodes two variants: (1) the wild type, encoding a 284 amino acid, ~33 kDa protein, and (2) an alternative spliced variant resulting from a 126 bp insert between exon 6 and 7, which introduces a premature stop codon that enhances nonsense-mediated decay. SULT4A1 is classified as an SULT based on sequence and structural similarities, including PAPS-domains, active-site His, and the dimerization domain; however, the catalytic pocket lid 'Loop 3' size is not conserved. SULT4A1 is uniquely expressed in the brain and localized in the cytosol and mitochondria. SULT4A1 is highly conserved, with rare intronic polymorphisms that have no outward manifestations. However, the SULT4A1 haplotype is correlated with Phelan-McDermid syndrome and schizophrenia. SULT4A1 knockdown revealed potential SULT4A1 functions in photoreceptor signaling and knockout mice display hampered neuronal development and behavior. Mouse and yeast models revealed that SULT4A1 protects the mitochondria from endogenously and exogenously induced oxidative stress and stimulates cell division, promoting dendritic spines' formation and synaptic transmission. To date, no physiological enzymatic activity has been associated with SULT4A1.

Regulatory mechanism of cold-inducible diapause in Caenorhabditis elegans

Temperature is a critical environmental cue that controls the development and lifespan of many animal species; however, mechanisms underlying low-temperature adaptation are poorly understood. Here, we describe cold-inducible diapause (CID), another type of diapause induced by low temperatures in Caenorhabditis elegans. A premature stop codon in heat shock factor 1 (hsf-1) triggers entry into CID at 9 °C, whereas wild-type animals enter CID at 4 °C. Furthermore, both wild-type and hsf-1(sy441) mutant animals undergoing CID can survive for weeks, and resume growth at 20 °C. Using epistasis analysis, we demonstrate that neural signalling pathways, namely tyraminergic and neuromedin U signalling, regulate entry into CID of the hsf-1 mutant. Overexpression of anti-ageing genes, such as hsf-1, XBP1/xbp-1, FOXO/daf-16, Nrf2/skn-1, and TFEB/hlh-30, also inhibits CID entry of the hsf-1 mutant. Based on these findings, we hypothesise that regulators of the hsf-1 mutant CID may impact longevity, and successfully isolate 16 long-lived mutants among 49 non-CID mutants via genetic screening. Furthermore, we demonstrate that the nonsense mutation of MED23/sur-2 prevents CID entry of the hsf-1(sy441) mutant and extends lifespan. Thus, CID is a powerful model to investigate neural networks involving cold acclimation and to explore new ageing mechanisms.

Nonsense CD247 mutations show dominant-negative features in T-cell receptor expression and function

BackgroundThe invariant TCRζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its three immunoreceptor tyrosine‐based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. ObjectiveTo analyze and model the potential impact on TCR expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. MethodsJurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with wild-type CD247 or mutations ablating one (Q142X), two (Q101X), or three (Q70X) ITAMs. ResultsThree patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2+3, respectively. Both mutations were associated with low surface CD3 expression, normal intracellular CD247 levels using a transmembrane-specific antibody but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO could not restore normal surface CD3 expression (only 60%, 22% and 10%, respectively), whereas in WT they reduced it (to 39%, 19% and 9% of normal levels), and both effects were ITAM number dependent. All six transfectants showed reduced CD69 induction (25-50%), indicating that they were unable to signal downstream properly neither isolated nor associated with wild-type CD247. ConclusionOur results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

F8 variants and their genotype-phenotype correlations in Thai patients with haemophilia A: a nationwide multicentre study

AimsAnalysis of the F8 gene helps predict the risk of developing factor VIII (FVIII) inhibitors and the depth of phenotype in haemophilia A (HA) patients. Since data in Southeast Asian...

Application of whole exome sequencing technology in the diagnosis of hereditary eye diseases

Objective: To evaluate the application of whole exome sequencing (WES) in the diagnosis of hereditary eye diseases. Methods: A total of 24 patients who came to the Obstetrics and Gynecology Hospital of Fudan University for reproductive genetic counseling from December 2020 to December 2023 with the main complaint of congenital eye disorders were included in this study. All cases had no known infections or exposure to known teratogenic drugs, karyotype and chromosome microarray analysis (CMA) abnormalities. Genomic DNA was extracted from the peripheral blood of the probands and their family members and tested for WES. Among them, three individual WES and 21 Trio WES were performed. Potential pathogenic sites were screened and analyzed by Sanger sequencing. For RPGRIP1:c.1611+26G>A site, minigene vector was constructed and RT-qPCR was performed to detect the effect of mRNA splicing. Results: A total of 24 families were collected in this study, of which 20 yielded positive results, achieving a diagnosis rate of 83.3% (20/24). The results involved 21 genes and identified 30 distinct variants, 19 of which were new variants reported. Prenatal diagnostic analysis of family 3 revealed that the fetus carried a c.6970G>T heterozygous nonsense mutation in the PRPF8 gene. The results of RT-PCR with the minigene vector at the non-classical splice site in family 24 indicated that the transcription product of the mutant plasmid was partially retained 104 bp in intron 12, resulting in a p.Glu538Valfs*12 alteration of the protein. Conclusions: The high detection rate of WES in the diagnosis of hereditary eye diseases further supports the advantages of its application as an important molecular detection tool for determining the etiology of hereditary eye diseases.

Four novel mutations identified in the COL4A3, COL4A4 and COL4A5 genes in 10 families with Alport syndrome

BackgroundAlport syndrome (AS) is an inherited nephropathy caused by mutations in the type IV collagen genes. It is clinically characterized by damage to the eyes, ears and kidneys. Diagnosis of AS is hampered by its atypical clinical picture, particularly when the typical features, include persistent hematuria and microscopic changes in the glomerular basement membrane (GBM), are the only clinical manifestations in the patient.MethodsWe screened 10 families with suspected AS using whole exome sequencing (WES) and analyzed the harmfulness, conservation, and protein structure changes of mutated genes. In further, we performed in vitro functional analysis of two missense mutations in the COL4A5 gene (c.2359G > C, p.G787R and c.2605G > A, p.G869R).ResultsWe identified 11 pathogenic variants in the type IV collagen genes (COL4A3, COL4A4 and COL4A5). These pathogenic variants include eight missense mutations, two nonsense mutations and one frameshift mutation. Notably, Family 2 had digenic mutations in the COL4A3 (p.G1170A) and UMOD genes (p.M229K). Family 3 had a digenic missense mutation (p.G997E) in COL4A3 and a frameshift mutation (p.P502L fs*151) in COL4A4. To our knowledge, four of the 11 mutations are novel mutations. In addition, we found that COL4A5 mutation relation mRNA levels were significantly decreased in HEK 293 T cell compared to control, while the cellular localization remained the same.ConclusionsOur research expands the spectrum of COL4A3-5 pathogenic variants, which is helpful for clinical and scientific research.