The impact of FBN1 variant types on pregnancy-related aortic dissection in women with Marfan syndrome.

Monoallelic RELA variants resulting in haploinsufficiency (HI) have been linked to recurrent mucocutaneous ulcers and enteritis. Heterozygous RELA dominant-negative (DN) variants often exhibit autoinflammatory phenotypes associated with type I interferonopathy beyond those typically associated with RELA-HI variants. The vast majority of documented cases of autosomal dominant (AD) RelA deficiency are caused by non-missense deleterious variants introducing a premature stop codon. We sought to characterize the clinical manifestations and pathologic significance of RELA variants and to establish the boundary separating RELA-HI and RELA-DN variants to facilitate position-based estimation of the nature of RELA variants. RELA variants were characterized via a nuclear factor-κB reporter assay, immunoblotting, immunoprecipitation, and electrophoretic mobility shift assay in RELA and NFKB1 double knockout cells. Eight patients from 5 families with AD RelA deficiency were identified, and all harbored novel RELA variants. A comprehensive functional study using RELA nonsense variants identified amino acid P290 as the boundary between RELA-HI and RELA-DN variants in non-missense deleterious variants. In patients with RELA-DN variants, corticosteroid preparations were relatively ineffective, leading to increased use of biological drugs, mainly anti-TNF agents. We also identified atypical additional variants, including a missense variant and an in-frame variant, and experimentally confirmed their pathogenicity. The positions of non-missense deleterious variants in RELA allow the estimation of the associated functional changes, facilitating the precise diagnosis of AD RelA deficiency. Conversely, RELA missense variants require functional verification, as their impact cannot be predicted solely from their position.

Heterozygous STAT3 variants acting by dominant negative (DN), gain-of-function (GOF), or haploinsufficiency (HI) mechanisms that have been associated with different inborn errors of immunity. However, the biological impact of STAT3 variants is complex to determine and it generally involves vector-generation, transfection, and multiple iterations for definition. To establish a single, reliable test to functionally characterize putative disease-causing STAT3 variants in patient primary cells. We recruited patients carrying previously published/validated STAT3-DN (n = 13), STAT3-GOF (n = 4), and STAT3-HI (n = 5) variants. We functionally evaluated the IL-10-mediated/STAT3-dependent inhibition ratio of LPS-induced TNF-α production by peripheral blood mononuclear cells (PBMC) using flow cytometry to evaluate the biologic impact of STAT3 variants. Optimization and threshold definition were performed by bootstrapping and k-fold cross-validation of Receiver Operating Characteristic (ROC)-area under the curve (AUC). The inhibition ratio of TNF-α production was significantly diminished in STAT3-DN (-3.44-fold, p < .0001) and STAT3-HI samples (-2.28-fold, p < .0001), while significantly augmented in STAT3-GOF samples (+1.47-fold, p = .002) when compared to healthy controls (HC, n = 16). Optimizing the combinations of IL-10 concentrations discriminated between HC and STAT3-DN (ROC-AUC = 1), and STAT3-GOF (ROC-AUC = 0.89). Further test optimization allowed discrimination between STAT3-HI variants when compared to HC (ROC-AUC = 1) and to STAT3-DN (ROC-AUC = 0.92). Determination of the IL-10 mediated, STAT3-dependent TNFα inhibition ratio in PBMC-derived monocytes by flow cytometry is a sensitive and specific method to functionally assess and discriminate STAT3-DN, -GOF, and -HI variants. This test can be used in research and clinical laboratory settings.

The transcription factor IKAROS, encoded by IKZF1, is crucial for lymphocyte development and differentiation. Germline heterozygous IKZF1 mutations cause B cell immunodeficiency, but also affect T cells. Patients with IKZF1 haploinsufficiency (HI) or dimerization-defective (DD) variants show reduced naive and increased memory T cells, while dominant-negative (DN) mutations result in the opposite phenotype. Gain-of-function patients display variable patterns. To investigate IKAROS's role in shaping the human naive/memory T cell phenotype, we performed IKAROS immunomodulation and knockdown experiments and analyzed early T cell development in an artificial thymic organoid (ATO) system using CD34+ cells from patients with representative IKZF1 variants. IKAROS inhibition by lenalidomide or silencing by small hairpin RNA directly altered expression of HNRNPLL, the master regulator of CD45 isoform splicing that defines CD45RA+/naive and CD45RO+/memory phenotypes. In the ATO system, IKAROS-DN precursor cells were blocked at the CD4-CD8-/double-negative stage and retained a CD45RA+ phenotype, whereas IKAROS-HI cells inefficiently reached the CD4+CD8+/double-positive stage and partially transitioned from CD45RA to CD45RO. Analysis of public gene expression data showed high HNRNPLL expression in double-positive thymic cells, beyond the stages affected by IKZF1 DN and HI mutations. Collectively, these findings indicate that IKAROS regulates early and late T cell development by mechanisms, including HNRNPLL modulation.

Osteogenesis imperfecta (OI) and Ehlers–Danlos syndrome (EDS) are inherited connective tissue disorders caused by diverse genetic defects, many of which affect collagen biosynthesis. However, the identified genetic variants do not always fully explain the clinical heterogeneity observed in patients, highlighting the need for advanced models and imaging techniques to assess collagen structure and fibroblast behavior at the microscopic level. In this study, we employed 5-week three-dimensional (3D) dermal fibroblast cultures derived from patients with haploinsufficient (HI) and dominant-negative (DN) OI, EDS, and healthy controls. Using label-free higher harmonic generation microscopy (HHGM), we visualized and quantified secreted collagen fibers and fibroblast morphology in situ. We analyzed fibroblast 3D orientation, collagen fiber diameter, collagen amount per cell, and the spatial alignment between fibroblasts and collagen fibers. HI OI fibroblasts secreted significantly less collagen than both control and EDS-derived cells, while EDS samples exhibited thinner collagen fibers compared to controls. Across all groups, collagen fiber orientation was strongly correlated with fibroblast alignment, in line with the role of fibroblasts in matrix organization. In healthy controls and HI OI samples, we observed a depth-dependent, counterclockwise rotation in fibroblast orientation from the culture bottom to the surface—a pattern that was less prominent in DN OI and EDS samples, potentially reflecting altered matrix guidance in diseased tissues. Overall, the quantity and quality of collagen, as well as fibroblast morphology and organization, were markedly altered in the OI and EDS model systems. These alterations may mirror tissue-level manifestations of the diseases, demonstrating the physiological relevance of patient-derived 3D fibroblast models for OI and EDS, as well as the power of harmonic generation microscopy in probing the cellular and extracellular consequences of disease-related gene defects in collagen or its biosynthetic pathways. Extensions of this methodological approach provide a way towards deeper understanding of tissue-level manifestations of collagen dysregulation in connective tissue disorders.

Marfan syndrome (MFS) is an autosomal dominant condition characterised by a wide array of pleiotropic manifestations that affect the cardiovascular, skeletal, ocular and pulmonary systems. This phenotypic diversity arises from the pathogenic variability of the over 3000 identified FBN1 variants. Despite extensive research, correlations between specific FBN1 genotypes and aortic phenotypes remain inconclusive. A comprehensive systematic review and meta-analysis was conducted on data collected from PubMed, Scopus and ScienceDirect up to 1 March 2025. All quantitative studies that reported aortic outcome data and met inclusion criteria were analysed. The primary endpoints assessed were aortic aneurysm, dissection and surgery. Our search strategy identified 17 studies, of which 11 were suitable for meta-analysis. We analysed data from over 6000 adults and conducted genotype-phenotype correlation analyses for six variant classes. Our findings indicate that haploinsufficiency (HI) variants are associated with a 2.5-fold increased risk of developing an aortic presentation compared with dominant negative (DN) variants (pooled RR 2.62; 95% CI 1.90 to 3.61; p<0.001, τ2=0.09, I²=50.4%). Our analysis of the missense cohort revealed a significant positive correlation between substitutions of or by cysteine and adverse aortic events (pooled RR 2.21; 95% CI 1.18 to 4.15; p<0.001, τ2=0.12, I²=76.1%). Subgroup analyses by structural variant classification ranked HI variants as the highest risk, followed by missense and splicing mutations (pooled proportion=0.18 and 0.15). We found significant genotype-aortic phenotype correlations among FBN1 variant classes. Specifically, HI and cysteine-involving variants present the greatest risk and exhibit larger baseline aortic root diameters. Splicing variants, while traditionally grouped under the HI class, demonstrated an aortic risk more comparable to that of missense mutations. In the era of precision medicine, these findings empower clinicians to move beyond one-size-fits-all criteria and tailor monitoring intervals and elective repair decisions according to patient genetic profiles.

Multi‑omics analysis of Del20q reveals PHF20 haploinsufficiency and an HDAC2-centered epigenetic circuit in myeloid neoplasms

X-linked disorders caused by skewed X chromosome inactivation (XCI) result in phenotypic heterogeneity, which is rarely reported. XCI testing is not widely used in clinical cases, making risk assessment for carriers of X-linked unbalanced structural abnormalities challenging. We present genetic data from an asymptomatic female with a de novo 6.31Mb deletion on Xp11.23-p11.22, identified through CMA-array analysis. The deletion includes 101 OMIM genes, 11 haplo-insufficient (HI) genes, and 4 escape genes. An androgen receptor (AR) methylation assay showed a 100% skewed XCI pattern silencing the abnormal X-chromosome. RNA-seq analysis revealed up-regulation of escape genes within the deletion at the transcriptional level. The absence of a severe clinical phenotype, aside from infertility, in this female was most likely attributed to the extremely skewed XCI and the compensatory up-regulation of XCI escape genes. Our data indicate that XCI can modify the phenotype in female carriers of heterozygous X-linked deletion and provide valuable information about the analysis of XCI pattern in risk assessment of this kind cases, especially precious fetuses.

We investigated the corneal biomechanical properties and their genotype-phenotype correlation correlations in patients with Marfan syndrome (MFS) and ectopia lentis (EL). Patients with MFS with EL underwent panel-based next-generation sequencing in this retrospective cohort study. The FBN1 genotypes were categorized into the dominant-negative (DN) group and the haploinsufficiency (HI) group. The DN variants were further subclassified based on the affected residues and their locations. Corneal biomechanical parameters were measured using dynamic Scheimpflug-based biomechanical analysis (CorVis ST). The correlations between corneal biomechanical properties and FBN1 genotype or nongenetic factors were analyzed. The differences between patients with MFS and normal control were also evaluated after matching confounding factors. One hundred one consecutive MFS probands participated in this study, with a median age of 6 years. Patients with HI and DN variants affecting critical residues, namely the DN (-Cys + CaB) variants, exhibited significantly higher deformation amplitude ratios (P = 0.029) and lower stress-strain index values (P = 0.007) compared with those in the DN (others) group, indicating lower corneal stiffness in the former group. DN variants in the FUN-EGF3 region were associated with lower deformation amplitude ratios (P = 0.011) and higher stress-strain index values (P = 0.002), whereas those in the DN-CD region exhibited the opposite pattern. Compromised corneal stiffness was significantly associated with HI and DN (-Cys + CaB) variants (b = -0.184; P = 0.01) and variants located outside the FUN-EGF3 region (b = 0.256; P = 0.001), after adjusting for confounding factors. Compared with matched controls, patients with MFS demonstrated significantly higher deformation amplitude ratios (P = 0.023), further confirming decreased corneal stiffness in this population. The FBN1 genotype impacts the corneal biomechanical properties of patients with MFS and EL. Corneal biomechanics provide a novel platform to study the genotype-phenotype correlation of MFS.

Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor, based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes, with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.

P219: Linkage disequilibrium between SLC12A3 and HYDIN pathogenic variants in the Old Order Amish community

P218: Investigating racial differences and genotype-phenotype variation in Marfan syndrome: A retrospective analysis of genetic and clinical features

Genomic Landscape of Germline <i>RUNX1</i> variants in Adult Myeloid Neoplasia According to Functional Impact

Elucidating the Somatic Genetic Rescue Underlying Del(20q) Myeloid Neoplasms

RationalePathogenic (P)/likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), which is characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis.ObjectivesInvestigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants.MethodsA retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts.ResultsIndividuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0–47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0–54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines.ConclusionDistinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.

Background: Immunodeficiency 70 is a rare disease and there are only very few cases in the literature. Case History: Young boy in his early 20’s from Indian subcontinent presented with recurrent episodes of fever, oral ulcers, loss of weight for past 3 years. Four episodes (july 2019, april 2020, APRIL 2021, Jan 2022) of fever, each high grade associated with chills, rigor, nausea, myalgia, throat pain, holo-cranial headache, painful oral ulcers over the tongue and painless over lips. Initial episode of fever was associated with leucopenia (3200) and thrombocytopenia (122000), acute kidney injury (creatinie-3.0) and pyelonephritis, transaminitis. He received 2 weeks of parenteral antibiotics and improved. Subsequent episodes of fever is not associated with any localizing symptoms or signs. No history Suggestive of connective tissue disease. Family history was non contributory. Physical examination: He was febrile (101 degree F), pulse rate 100/minute, respiratory rate 18/minute, tongue ulceration in lateral border, no lymphadenopathy, no organomegaly, normal sytemic examination. Evaluation: The patient was evaluated for chronic infections, hematalogical malignancy, Behcet’s disease, auto inflammatory syndromes like Haplo insufficiency of A20. His complete blood count, electrolytes, renal function test, liver function test urine microscopy, blood culture were normal. CRP was elevated (58 mg/l). Tzanck smear from buccal mucosa was negative. CT thorax and abdomen showed no significant lymphadenopathy, organomegaly, evidence of infections or malignancy. In view of oral ulcers &amp; fever Behcet’s disease was considered and HLA b51 was sent and came negative. Exome genome sequencing was done to diagnose immunodeficiency or autoinflammatory syndromes which came positive for immunodeficiency 70. Treatment: He was treated with colchicine and fever subsided and leucopenia improved Discussion: Immunodeficiency-70 (IMD70) is an autosomal dominant immunologic disorder characterized by severe cutaneous warts on the hands, feet, and face, suggesting increased susceptibility to human papillomavirus (HPV) infection. Affected individuals may also have recurrent bacterial infections like pneumonia, boils, sinusitis, as well as feature of autoinflammation, such as colitis, celiac disease, and retinal vasculitis. Laboratory studies show decreased CD4+ T cells and decreased CD19+ B cells; hypogammaglobulinemia, combined T cell and B cell immunodeficiency characterized by decreased CD4+ T cells, decreased CD19+ B cells, recurrent bacterial infections, and severe cutaneous warts on the hands, feet, and face that has material basis in heterozygous mutation in IVNS1ABP on chromosome 1q25.3.

Complete or partial deletions of chromosome 7 (-7/del7q) belong to the most frequent chromosomal abnormalities in myeloid neoplasm (MN) and are associated with a poor prognosis. The disease biology of -7/del7q and the genes responsible for the leukemogenic properties have not been completely elucidated. Chromosomal deletions may create clonal vulnerabilities due to haploinsufficient (HI) genes contained in the deleted regions. Therefore, HI genes are potential targets of synthetic lethal strategies. Through the most comprehensive multimodal analysis of more than 600 -7/del7q MN samples, we elucidated the disease biology and qualified a list of most consistently deleted and HI genes. Among them, 27 potentially synthetic lethal target genes were identified with the following properties: (i) unaffected genes by hemizygous/homozygous LOF mutations; (ii) prenatal lethality in knockout mice; and (iii) vulnerability of leukemia cells by CRISPR and shRNA knockout screens. In -7/del7q cells, we also identified 26 up or down-regulated genes mapping on other chromosomes as downstream pathways or compensation mechanisms. Our findings shed light on the pathogenesis of -7/del7q MNs, while 27 potential synthetic lethal target genes and 26 differential expressed genes allow for a therapeutic window of -7/del7q.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies

IntroductionOsteogenesis Imperfecta is a rare genetic connective tissue disorder, characterized by skeletal dysplasia and fragile bones. Currently only two mouse models have been reported for haploinsufficient (HI) mild Osteogenesis Imperfecta (OI); the Col1a1+/Mov13 (Mov13) and the Col1a1+/-365 mouse model. The Mov13 mice were created by random insertion of the Mouse Moloney leukemia virus in the first intron of the Col1a1 gene, preventing the initiation of transcription. Since the development of the Mov13 mice almost four decades ago and its basic phenotypic characterization in the 90s, there have not been many further studies. We aimed to extensively characterize the Mov13 mouse model in order to critically evaluate its possible use for preclinical studies of HI OI.MethodsBone tissue from ten heterozygous Mov13 and ten wild-type littermates (WT) C57BL/6J mice (50% males per group) was analyzed at eight weeks of age with bone histomorphometry, micro computed tomography (microCT), 3-point bending, gene expression of different collagens, as well as serum markers of bone turnoverResultsThe Mov13 mouse presented a lower bone strength and impaired material properties based on our results of 3-point bending and microCT analysis respectively. In contrast, no significant differences were found for all histomorphometric parameters. In addition, no significant differences in Col1a1 bone expression were present, but there was a significant lower P1NP concentration, a bone formation marker, measured in serum. Furthermore, bone tissue of Mov13 mice presented significantly higher expression of collagens (Col1a2, Col5a1 and Col5a2), and bone metabolism markers (Bglap, Fgf23, Smad7, Edn1 and Eln) compared to WT. Finally, we measured a significantly lower Col1a1 expression in heart and skin tissue and also determined a higher expression of other collagens in the heart tissue.ConclusionAlthough we did not detect a significant reduction in Col1a1 expression in the bone tissue, a change in bone structure and reduction in bone strength was noted. Regrettably, the variability of the bone phenotype and the appearance of severe lymphoma in adult Mov13 mice, does not favor their use for the testing of new long-term drug studies. As such, a new HI OI type 1 mouse model is urgently needed.

Non-missense variants of KCNH2 show better outcomes in type 2 long QT syndrome