Deletion Of Exon Research Articles

A novel intronic TCOF1 pathogenic variant in a Chinese family with Treacher Collins syndrome

BackgroundTreacher Collins syndrome (TCS; OMIM 154500) is a craniofacial developmental disorder.MethodsTo investigate the genetic features of a four-generation Chinese family with TCS, clinical examinations, hearing tests, computed tomography, whole-exome sequencing (WES), Sanger sequencing, reverse transcription (RT)-PCR, and the Minigene assay were performed.ResultsThe probands, an 11-year-old male and his cousin exhibited typical clinical manifestations of TCS including conductive hearing loss, downward slanting palpebral fissures, and mandibular hypoplasia. Computed tomography revealed bilateral fusion of the anterior and posterior stapedial crura and malformation of the long crura of the incus. WES of both patients revealed a novel heterozygous intronic variant, i.e., c.4342 + 5_4342 + 8delGTGA (NM_001371623.1) in TCOF1. Minigene expression analysis revealed that the c.4342 + 5_4342 + 8delGTGA variant in TCOF1 caused a partial deletion of exon 24 (c.4115_4342del: p.Gly1373_Arg1448del), which was predicted to yield a truncated protein. The deletion was further confirmed via RT-PCR and sequencing of DNA from proband blood cells. A heterozygous variant in the POLR1C gene (NM_203290; exon6; c.525delG) was found almost co-segregated with the TCOF1 pathogenic variant.ConclusionsIn conclusion, we identified a heterozygous TCOF1 splicing variant c.4342 + 5_4342 + 8delGTGA (splicing) in a Chinese TSC family with ossicular chain malformations and facial anomalies. Our findings broadened the spectrum of TCS variants and will facilitate diagnostics and prognostic predictions.

DEFB126 2-nt Deletion (rs11467417) as a Potential Risk Factor for Chlamydia Trachomatis Infection and Subsequent Infertility in Iranian Men.

Chlamydia trachomatis (CT) is one of the most prevalent sexually transmitted infections, causing genital tract infections and infertility. Defensins have an immunomodulatory function and play an important role in sperm maturation, motility, and fertilization. DEFB126 is present on ejaculated spermatozoa and is essential for them to pass through the female reproductive tract. The purpose of the study was to determine the frequency of the 2-nt deletion of the DEFB126 (rs11467417) in Iranian infertile males with a recurrent history of CT. Semen samples of 1080 subfertile males were investigated. Among patients who had CT-positive results, sperm DNA from 50 symptomatic and 50 asymptomatic patients were collected for the DEFB126 genotype analysis. Additionally, a control group comprising 100 DNA samples from individuals with normal spermogram and testing negative for CT was included in the study. The PCR-sequencing for detecting the 2-nt deletion of the second exon of the DEFB126 was performed. The Chi-squared test comparing all three groups revealed no significant difference across the different genotypes. Moreover, no significant difference between the symptomatic and asymptomatic groups was seen. However, analysis within CT-positive patients and controls demonstrated significant difference between the frequencies of homozygous del/del. The higher frequency of the 2-nt deletion of the DEFB126 in CT- positive patients suggests that the occurrence of mutations in the DEFB-126 may cause the impairment of the antimicrobial activity of the DEFB126 protein and consequently makes individuals more susceptible to infections such as CT.

Neuron type-specific proteomics reveals distinct Shank3 proteoforms in iSPNs and dSPNs lead to striatal synaptopathy in Shank3B–/– mice

Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies. To overcome the limitations associated with bulk measures of synaptic protein abundance, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomes in vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Investigation of Shank3B–/– mice lacking exons 13–16 within the Shank3 gene, reveal distinct Shank3 protein isoform expression in iSPNs and dSPNs. In Shank3B-/- striatum, Shank3E and Shank3NT are expressed by dSPNs but are undetectable in iSPNs. Proteomic analysis indicates significant and selective alterations in the postsynaptic proteome of Shank3B–/– iSPNs. Correspondingly, the deletion of exons 13–16 diminishes dendritic spine density, reduces spine head diameter, and hampers corticostriatal synaptic transmission in iSPNs. Remarkably, reintroducing Shank3E in adult Shank3B–/– iSPNs significantly rectifies the observed dendritic spine morphological and corticostriatal synaptic transmission deficits. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.

Pigs lacking the SRCR5 domain of CD163 protein demonstrate heritable resistance to the PRRS virus and no changes in animal performance from birth to maturity.

Porcine reproductive and respiratory syndrome (PRRS) is one of the world's most persistent viral pig diseases, with a significant economic impact on the pig industry. PRRS affects pigs of all ages, causing late-term abortions and stillbirths in sows, respiratory disease in piglets, and increased susceptibility to secondary bacterial infection with a high mortality rate. PRRS disease is caused by a positive single-stranded RNA PRRS virus (PRRSV), which has a narrow host-cell tropism limited to monocyte-macrophage lineage cells. Several studies demonstrated that the removal of CD163 protein or, as a minimum, its scavenger receptor cysteine-rich domain 5 (SRCR5) precludes the viral genome release, conferring resistance to PRRSV in live animals. Today, very limited information exists about the impact of such edits on animal performance from birth to maturity in pigs. Using CRISPR-Cas9 with dual-guide RNAs and non-homologous end joining (NHEJ), first-generation (E0) pigs were produced with a deletion of exon 7 in the CD163 gene. The selected pigs were bred to produce the next three generations of pigs to establish multiple lines of pigs homozygous for the edited allele, thereby confirming that the CD163 gene with removed exon 7 was stable during multiple breeding cycles. The pigs were evaluated relative to non-edited pigs from birth to maturity, including any potential changes in meat composition and resistance to PRRSV. This study demonstrates that removing the SRCR5 domain from the CD163 protein confers resistance to PRRSV and, relative to unedited pigs, resulted in no detected differences in meat composition and no changes in the growth rate, health, and ability to farrow. Together, these results support the targeted use of gene editing in livestock animals to address significant diseases without adversely impacting the health and well-being of the animals or the food products derived from them.

Characterization of an Aptamer Targeting Neu5Gc, as an Endogenous Pathogenic Factor Derived from Red Meat.

N-glycolylneuraminic acid (Neu5Gc), a sialic acid predominantly found in the non-neurohumoral fluids of hind-mouthed animals, is incapable of synthesizing Neu5Gc due to a deletion in the CMAH exon of the gene encoding human CMP-Neu5Gc hydroxylase. But consumption of animal-derived foods that contain Neu5Gc, such as red meat, can instigate an immune response in humans, as Neu5Gc is recognized as a foreign substance by the human immune system. This recognition leads to the production of anti-Neu5Gc antibodies, subsequently resulting in chronic inflammation. When Neu5Gc is consumed excessively or frequently, it may contribute to the development of heart disease and cancer. This makes Neu5Gc, an endogenous pathogenic factor derived from red meat, a new hot topic in red meat safety research. In this study, aptamers obtained by the magnetic bead SELEX technique were subjected to homology and secondary structure prediction analysis as well as affinity determination. The result indicated that the aptamer 2B.N2A9 exhibited a robust binding affinity, with an affinity constant (Ka) of 1.87 × 108 L/mol. This aptamer demonstrated optimal binding specificity within a pH range of 5.4 to 7.4. Molecular docking analysis further revealed that aptamer 2B.N2A9 formed stable binding interactions with the target Neu5Gc at specific sites, namely G-14, C-15, G-13, G-58, G-60, and C-59. An Enzyme-Linked Oligonucleotide Sorbent Assay (ELOSA) methodology was established to detect the endogenous pathogenic factor Neu5Gc present in red meat. This method demonstrated a limit of detection (LOD) of 0.71 ng/mL, along with an average recovery rate of 92.23%. The aptamer obtained in this study exhibited favorable binding properties to Neu5Gc. The assay was relatively convenient and demonstrated good sensitivity. Further investigation into the distribution of Neu5Gc in various red meats is of public health significance and scientific potential. A practical detection method should be provided to guide red meat diets and ensure the nutrition and safety of meat products.

Long term clinical follow up of four patients with Wolfram syndrome and urodynamic abnormalities.

Wolfram syndrome is characterised by insulin-dependent diabetes (IDDM), diabetes insipidus (DI), optic atrophy, sensorineural deafness and neurocognitive disorders. The DIDMOAD acronym has been recently modified to DIDMOAUD suggesting the rising awareness of the prevalence of urinary tract dysfunction (UD). End stage renal disease is the commonest cause of mortality in Wolfram syndrome. We present a case series with main objective of long term follow up in four children having Wolfram syndrome with evaluation of their urodynamic profile. A prospective follow up of four genetically proven children with Wolfram syndrome presenting to a tertiary care pediatric diabetes clinic in Pune, India was conducted. Their clinical, and urodynamic parameters were reviewed. IDDM, in the first decade, was the initial presentation in all the four children (three male and one female). Three children had persistent polyuria and polydipsia despite having optimum glycemic control; hence were diagnosed to have DI and treated with desmopressin. All four patients entered spontaneous puberty. All patients had homozygous mutation in WFS1 gene; three with exon 8 and one with exon 6 novel mutations. These children with symptoms of lower urinary tract malfunction were further evaluated with urodynamic studies; two of them had hypocontractile detrusor and another had sphincter-detrusor dyssynergia. Patients with hypocontractile bladder were taught clean intermittent catheterization and the use of overnight drain. We report a novel homozygous deletion in exon 6 of WFS-1 gene. The importance of evaluation of lower urinary tract malfunction is highlighted by our case series. The final bladder outcome in our cases was a poorly contractile bladder in three patients.

Testing SIPA1L2 as a modifier of CMT1A using mouse models.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a demyelinating peripheral neuropathy caused by the duplication of peripheral myelin protein 22 (PMP22), leading to muscle weakness and loss of sensation in the hands and feet. A recent case-only genome-wide association study of CMT1A patients conducted by the Inherited Neuropathy Consortium identified a strong association between strength of foot dorsiflexion and variants in signal induced proliferation associated 1 like 2 (SIPA1L2), indicating that it may be a genetic modifier of disease. To validate SIPA1L2 as a candidate modifier and to assess its potential as a therapeutic target, we engineered mice with deletion of exon 1 (including the start codon) of the Sipa1l2 gene and crossed them to the C3-PMP22 mouse model of CMT1A. Neuromuscular phenotyping showed that Sipa1l2 deletion in C3-PMP22 mice preserved muscular endurance assayed by inverted wire hang duration and changed femoral nerve axon morphometrics such as myelin thickness. Gene expression changes suggest involvement of Sipa1l2 in cholesterol biosynthesis, a pathway that is also implicated in C3-PMP22 mice. Although Sipa1l2 deletion did impact CMT1A-associated phenotypes, thereby validating a genetic interaction, the overall effect on neuropathy was mild.

Optimized MLPA workflow for spinal muscular atrophy diagnosis: identification of a novel variant, NC_000005.10:g.(70919941_70927324)del in isolated exon 1 of SMN1 gene through long-range PCR

BackgroundSpinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1.MethodsWe analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing.ResultsTwo unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5’ region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1.ConclusionRemarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.

Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency and MECP2 duplication syndrome

To explore the genetic basis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency (OTCD) and MECP2 duplication syndrome. A proband who was admitted to the Neonatal Intensive Care Unit of Gansu Provincial Maternal and Child Health Care Hospital on December 19, 2017 was selected as the study subject. High-throughput sequencing and multiplex ligation-dependent probe amplification (MLPA) were carried out for her pedigree, and short tandem repeat-based linkage analysis and chromosome copy number variation sequencing (CNV-seq) were used for the prenatal diagnosis. The proband, a 3-day-old female, was found to harbor heterozygous deletion of exons 7-9 of the OTC gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PVS1+PM2_Supporting+PP4). The proband was diagnosed with OTCD , which was in keeping with her acute encephalopathy and metabolic abnormalities (manifesting as hyperammonemia, decreased blood citrulline, and increased urine orotic acid). Prenatal diagnosis was carried out for the subsequent pregnancy. The fetus did not harbor the exons 7-9 deletion of the OTC gene, but was found to carry a duplication in Xq28 region (which encompassed the whole region of MECP2 duplication syndrome) and was positive for the SRY sequence. The same duplication was also found in the proband and her mother. Considering the possible existence of X-chromosome inactivation, the proband was diagnosed with two X-linked recessive disorders including OTCD and MECP2 duplication syndrome, and the fetus was determined as a male affected with the MECP2 duplication syndrome. Discoveries of the pathogenic variants underlying the OTCD and MECP2 duplication syndrome have enabled clinical intervention, treatment, genetic counseling and prenatal diagnosis for this pedigree.

КЛИНИЧЕСКИЙ СЛУЧАЙ БОЛЕЗНИ КУГЕЛЬБЕРГА-ВЕЛАНДЕРА (СПИНАЛЬНОЙ МЫШЕЧНОЙ АТРОФИИ III ТИПА)

Background. Differential diagnosis of neuromuscular diseases is one of the most difficult areas in neurology. Molecular genetic research in these patients is of particular importance. The article presents a clinical case of 5q spinal muscular atrophy type III (Kugelberg – Welander disease), confirmed by molecular genetic testing. Description of the clinical case. The article presents the clinical observation of a patient with an initial diagnosis of Erb – Roth myopathy. A genetic study was carried out. The analysis of the presence of exon 7 of the SMN1/SMN2 genes showed the absence of a signal corresponding to exon 7 of the SMN1 gene. A search for deletions in the SMN1 gene showed the homozygous deletion of exons 7–8 of the SMN1 gene in the proband. Homozygous deletion of exons 7–8 of the SMN1 gene causes 5q proximal spinal muscular atrophy. As a result of determining the num-ber of copies of the SMN1 and SMN2 genes, 0 copies of exons 7–8 of the SMN1 gene and 4 copies of exons 7–8 of the SMN2 gene were registered, which confirms the diagnosis of 5q spinal muscular atrophy type III. Conclusion. Adult patients suffering from neuromuscular diseases are recommended to undergo confirmatory DNA diagnostics to prescribe pathogenetic treatment, which increases their chances of survival. In young people with a clinical diagnosis of neuromuscular disease, genetic testing is required to clarify the diagnosis and further treatment tactics.

Taking a color photo: A homozygous 25-bp deletion in Bace2 may cause brown-and-white coat color in giant pandas

Brown-and-white giant pandas (hereafter brown pandas) are distinct coat color mutants found exclusively in the Qinling Mountains, Shaanxi, China. However, its genetic mechanism has remained unclear since their discovery in 1985. Here, we identified the genetic basis for this coat color variation using a combination of field ecological data, population genomic data, and a CRISPR-Cas9 knockout mouse model. We de novo assembled a long-read-based giant panda genome and resequenced the genomes of 35 giant pandas, including two brown pandas and two family trios associated with a brown panda. We identified a homozygous 25-bp deletion in the first exon of Bace2, a gene encoding amyloid precursor protein cleaving enzyme, as the most likely genetic basis for brown-and-white coat color. This deletion was further validated using PCR and Sanger sequencing of another 192 black giant pandas and CRISPR-Cas9 edited knockout mice. Our investigation revealed that this mutation reduced the number and size of melanosomes of the hairs in knockout mice and possibly in the brown panda, further leading to the hypopigmentation. These findings provide unique insights into the genetic basis of coat color variation in wild animals.

Complex analysis of the national Hereditary angioedema cohort in Slovakia – Identification of 12 novel variants in SERPING1 gene

BackgroundHereditary angioedema (HAE) is a rare autosomal dominant genetic disease characterised by acute episodes of non-pruritic skin and submucosal swelling caused by increase in vascular permeability. ObjectiveHere we present the first complex analysis of the National HAE Slovakian cohort with the detection of 12 previously un-published genetic variants in SERPING1 gene. MethodsIn patients diagnosed with hereditary angioedema caused by deficiency or dysfunction of C1 inhibitor (C1–INH-HAE) based on clinical manifestation and complement measurements, SERPING1 gene was tested by DNA sequencing (Sanger sequencing/massive parallel sequencing) and/or multiplex ligation-dependent probe amplification for detection of large rearrangements. ResultsThe Slovakian national cohort consisted of 132 living patients with confirmed HAE. We identified 51 index cases (32 families, 19 sporadic patients/112 adults, 20 children). One hundred seventeen patients had HAE caused by deficiency of C1 inhibitor (C1–INH-HAE-1) and 15 patients had HAE caused by dysfunction of C1 inhibitor (C1–INH-HAE-2). The prevalence of HAE in Slovakia has recently been calculated to 1:41 280 which is higher than average calculated prevalence. The estimated incidence was 1:1360 000. Molecular-genetic testing of the SERPING1 gene found 22 unique causal variants in 26 index cases, including 12 previously undescribed and unreported. ConclusionThe first complex report about epidemiology and genetics of the Slovakian national HAE cohort expands the knowledge of the C1–INH-HAE genetics. Twelve novel causal variants were present in the half of the index cases. A higher percentage of inframe variants comparing to other studies was observed. Heterozygous deletion of exon 3 found in a large C1–INH-HAE-1 family probably causes the dysregulation of the splicing isoforms balance and leads to the decrease of full-length C1–INH level.

Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years.

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases.

Effect of deletion of the protein kinase PRKD1 on development of the mouse embryonic heart.

Congenital heart disease (CHD) is the most common congenital anomaly, with an overall incidence of approximately 1% in the United Kingdom. Exome sequencing in large CHD cohorts has been performed to provide insights into the genetic aetiology of CHD. This includes a study of 1891 probands by our group in collaboration with others, which identified three novel genes-CDK13, PRKD1, and CHD4, in patients with syndromic CHD. PRKD1 encodes a serine/threonine protein kinase, which is important in a variety of fundamental cellular functions. Individuals with a heterozygous mutation in PRKD1 may have facial dysmorphism, ectodermal dysplasia and may have CHDs such as pulmonary stenosis, atrioventricular septal defects, coarctation of the aorta and bicuspid aortic valve. To obtain a greater appreciation for the role that this essential protein kinase plays in cardiogenesis and CHD, we have analysed a Prkd1 transgenic mouse model (Prkd1em1) carrying deletion of exon 2, causing loss of function. High-resolution episcopic microscopy affords detailed morphological 3D analysis of the developing heart and provides evidence for an essential role of Prkd1 in both normal cardiac development and CHD. We show that homozygous deletion of Prkd1 is associated with complex forms of CHD such as atrioventricular septal defects, and bicuspid aortic and pulmonary valves, and is lethal. Even in heterozygotes, cardiac differences occur. However, given that 97% of Prkd1 heterozygous mice display normal heart development, it is likely that one normal allele is sufficient, with the defects seen most likely to represent sporadic events. Moreover, mRNA and protein expression levels were investigated by RT-qPCR and western immunoblotting, respectively. A significant reduction in Prkd1 mRNA levels was seen in homozygotes, but not heterozygotes, compared to WT littermates. While a trend towards lower PRKD1 protein expression was seen in the heterozygotes, the difference was only significant in the homozygotes. There was no compensation by the related Prkd2 and Prkd3 at transcript level, as evidenced by RT-qPCR. Overall, we demonstrate a vital role of Prkd1 in heart development and the aetiology of CHD.

Characterizing Homozygous Variants in Bardet-Biedl Syndrome-Associated Genes Within Iranian Families: Unveiling a Founder Variant in BBS2, c.471G>A.

Bardet-Biedl syndrome (BBS) is a rare inherited ciliopathy disorder characterized by a broad spectrum of clinical symptoms such as retinal dystrophy, obesity, polydactyly, genitourinary and kidney anomalies, learning disability, and hypogonadism. The understanding of the variants involved in BBS-causing genes remains incomplete, highlighting the need for further research to develop a molecular diagnostic strategy for this syndrome. Singleton whole-exome sequencing (WES) was performed on sixteen patients. Our study revealed (1) nine patients carried eight homozygous pathogenic variants with four of them being novel (2) Specifically, a synonymous splicing variant (c.471G > A) in BBS2 gene in six patients with Baloch ethnicity. The identification of runs of homozygosity (ROH) calling was performed using the BCFtools/RoH software on WES data of patients harboring c.471G > A variant. The presence of shared homozygous regions containing the identified variant was confirmed in these patients. In-silico analysis predicted the effect of the c.471G > A variants on BBS2 mRNA splicing. This variant results in disrupted wild-type donor site and intron retention in the mature mRNA. (3) And a deletion of exons 14 to 17 in the BBS1 gene was identified in one patient by Copy-Number Variation (CNV) analysis using the ExomeDepth pipeline. Our results identified the founder variant c.471G > A in the BBS2 gene in the Baloch ethnicity of the Iranian population. This finding can guide the diagnostic approach of this syndrome in future studies.

A large deletion in TSC2 causes tuberous sclerosis complex by dysregulating PI3K/AKT/mTOR signaling pathway

Background/aimTuberous sclerosis complex (TSC) is a multi-system syndrome caused by loss-of-function mutation in TSC1 or TSC2. Most TSC patients present with cardiac rhabdomyoma or cortical tubers during fetal life, and the symptoms are not uniform as their age. The gene products of TSC1/2 are components of the TSC protein complex and are important role in the PI3K/AKT/mTOR (PAM) signaling pathway. Based on three members of a family with variable expressivity, the purpose of this study was to clarify the clinical features of TSC in different age groups and to analyze the genetic characteristics of TSC2 gene. MethodsClinical exome sequencing and co-segregation were used to identify a three-generation family with four affected individuals. HEK-293T cell model was constructed for subsequent experiments. Quantitative RT-PCR, western blotting, and subcellular localization were used to analyze the expression effect of TSC2 mutation. CCK-8 assay, wound healing assay, and cell cycle analysis were used to analyze the function effect of TSC2 mutation. ResultWe identified a TSC family with heterozygous deletion of exon 4 in TSC2 by clinical exon sequencing. Sanger sequencing indicated that the affected individuals have 2541-bp deletion that encompassed exon 4 and adjacent introns. Deletion of exon 4 decreased the TSC2 mRNA and protein levels in HEK-293T cells, and activated the PI3K/AKT/mTOR pathway, thereby altering the cell cycle and promoting cell proliferation and migration. ConclusionWe confirmed the pathogenicity of the large deletion in TSC2 in a three- generations family.. Deletion of exon 4 of TSC2 affected cell proliferation, migration, and cell cycle via abnormal activation of the PAM pathway. This study evaluated the pathogenic effect of deletion of exon 4 of TSC2 and investigated the underlying mechanism.

NAIP Gene Deletion and SMN2 Copy Number as Molecular Tools in Predicting the Severity of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is one of the most prevalent autosomal recessive illnesses with type I being the most severe type. Genomic alterations including survival motor neuron (SMN) copy number as well as deletions in SMN and Neuronal Apoptosis Inhibitory Protein (NAIP) are greatly implicated in the emergence of SMA. However, the association of such alterations with the severity of the disease is yet to be investigated. This study was directed to elucidate the molecular assessment of NAIP and SMN genomic alterations as a useful tool in predicting the severity of SMA among patients. This study included 65 SMA pediatric patients (30 type I and 35 type II) and 65 healthy controls. RFLP-PCR was employed to determine the genetic polymorphisms of the SMN1, SMN2, and NAIP genes. In addition, qRT-PCR was used to identify the expression of the SMN1 and SMN2 genes, and serum levels of creatine kinase were measured using a colorimetric method. DNA sequencing was performed on some samples to detect any single nucleotide polymorphisms in SMN1, SMN2, and NAIP genes. All SMA patients had a homozygous deficiency of SMN1 exon 7. The homozygous deficiency of SMN1 exons 7 and 8, with the deletion of NAIP exon 5 was found among the majority of Type I patients. In contrast, patients with the less severe condition (type II) had SMN1 exons 7 and 8 deleted but did not have any deletions in NAIP, additionally; 65.7% of patients had multiple copies of SMN2. Analysis of NAIP deletion alongside assessing SMN2 copy number might enhance the effectiveness of the diagnosis that can predict severity among Spinal Muscular Atrophy patients.

Deletion of exons 2 and 3 from Actb and cell immortalization lead to widespread, β-actin independent alterations in gene expression associated with cell cycle control

The cytoplasmic actin proteins, β- and γ-actin, are 99% identical but thought to perform non-redundant functions. The nucleotide coding regions of cytoplasmic actin genes, Actb and Actg1, are 89% identical. Knockout (KO) of Actb by Cre-mediated deletion of first coding exons 2 and 3 in mice is embryonic lethal and fibroblasts derived from KO embryos (MEFs) fail to proliferate. In contrast, Actg1 KO MEFs display with a much milder defect in cell proliferation and Actg1 KO mice are viable, but present with increased perinatal lethality. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the β-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry reveal largescale changes to the transcriptome, proteome, and phosphoproteome in cells lacking Actb but not those only lacking β-actin protein. Pathway analysis of genes and proteins differentially expressed upon Actb KO suggest widespread dysregulation of genes involved in the cell cycle that may explain the severe defect in proliferation.

Expanding the genetic and phenotypic spectrum of congenital myasthenic syndrome: new homozygous VAMP1 splicing variants in 2 novel individuals.

We report the cases of two Spanish pediatric patients with hypotonia, muscle weakness and feeding difficulties at birth. Whole-exome sequencing (WES) uncovered two new homozygous VAMP1 (Vesicle Associated Membrane Protein 1) splicing variants, NM_014231.5:c.129+5 G > A in the boy patient (P1) and c.341-24_341-16delinsAGAAAA in the girl patient (P2). This gene encodes the vesicle-associated membrane protein 1 (VAMP1) that is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. VAMP1 has a highly variable C-terminus generated by alternative splicing that gives rise to three main isoforms (A, B and D), being VAMP1A the only isoform expressed in the nervous system. In order to assess the pathogenicity of these variants, expression experiments of RNA for VAMP1 were carried out. The c.129+5 G > A and c.341-24_341-16delinsAGAAAA variants induced aberrant splicing events resulting in the deletion of exon 2 (r.5_131del; p.Ser2TrpfsTer7) in the three isoforms in the first case, and the retention of the last 14 nucleotides of the 3' of intron 4 (r.340_341ins341-14_341-1; p.Ile114AsnfsTer77) in the VAMP1A isoform in the second case. Pathogenic VAMP1 variants have been associated with autosomal dominant spastic ataxia 1 (SPAX1) and with autosomal recessive presynaptic congenital myasthenic syndrome (CMS). Our patients share the clinical manifestations of CMS patients with two important differences: they do not show the typical electrophysiological pattern that suggests pathology of pre-synaptic neuromuscular junction, and their muscular biopsies present hypertrophic fibers type 1. In conclusion, our data expand both genetic and phenotypic spectrum associated with VAMP1 variants.

Cytopathological characteristics of SMARCA4-deficient thoracic undifferentiated tumors in serous effusion

Objective: To investigate the clinicopathological characteristics of SMARCA4-deficient thoracic undifferentiated tumors, and the diagnostic value of the cells in serous effusion. Methods: Eleven cases of SMARCA4-deficient tumor were collected from the Affiliated Hospital of Hebei University, China from January 2018 to July 2023, which were diagnosed using cell block of serous effusion. The clinical, histopathological, immunohistochemical and molecular genetic features were reviewed, along with related literature. Results: All the 11 patients were males with ages ranging from 54 to 77 years (median 64 years). Nine patients were smokers and two had an unknown smoking history. Most of them complained of cough and dyspnea with pleural effusion. The primary tumor sites included lung (9 cases), thoracic wall (1 case), and mediastinum (1 case), while 3 patients had a history of lung surgery. Histologically, tumor cells were large and pleomorphic, with increased nuclear-cytoplasmic ratio. They also showed round nuclei, conspicuous nucleoli, and basophilic cytoplasm in serous effusion. Immunohistochemically, tumor cells in all cases were negative for SMARCA4/BRG1, CKpan and CK7, but positive for SMARCB1/INI1. Some of the cases were positive for CD34 (7/11), synaptophysin (4/11) and SALL4 (2/11). Histologically, monotonous tumor cells formed solid sheets or anastomosing islands with poor cell adhesion and rhabdoid morphology. Brisk mitotic figures were accompanied by large areas of necrosis. Some cases focally exhibited syncytia, and some had bright cytoplasm and vesicular chromatin. The immunohistochemical profiles in the tumor tissues were consistent with those of cytology. Six cases were negative for PD-L1 (22c3). Among the 6 cases analyzed by targeted next generation sequencing, concurrent SMARCA4 and TP53 mutations were detected in all 6 cases. Some of the 6 tumors showed mutations of STK11, CDKN2A, and MET, and amplification of ERBB2, exon deletion of BRCA2, etc. Follow-up information was available in all cases and ranged from 2 to 24 months. The patients showed metastases to various sites, including lymph node, liver, kidney, adrenal gland, brain, bone and other sites. Four patients died of the tumor. The survival time of 4 patients who underwent radical resection or radiofrequency ablation was more than 13 months. Conclusions: SMARCA4-deficient thoracic sarcoma is a rare but highly aggressive tumor with dismal prognosis and rhabdomyoid features. It is difficult to diagnose this disease using only serous effusion samples. This tumor thus warrants careful consideration. Accurate diagnosis can greatly improve early diagnosis and treatment of these tumors.