ABSTRACT
BACKGROUND: Functional T-cell responses are essential for virus clearance and long-term protection after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, whereas certain clinical factors, such as older age and immunocompromise, are associated with worse outcome. OBJECTIVE: We sought to study the breadth and magnitude of T-cell responses in patients with coronavirus disease 2019 (COVID-19) and in individuals with inborn errors of immunity (IEIs) who had received COVID-19 mRNA vaccine. METHODS: Using high-throughput sequencing and bioinformatics tools to characterize the T-cell receptor ß repertoire signatures in 540 individuals after SARS-CoV-2 infection, 31 IEI recipients of COVID-19 mRNA vaccine, and healthy controls, we quantified HLA class I- and class II-restricted SARS-CoV-2-specific responses and also identified several HLA allele-clonotype motif associations in patients with COVID-19, including a subcohort of anti-type 1 interferon (IFN-1)-positive patients. RESULTS: Our analysis revealed that elderly patients with COVID-19 with critical disease manifested lower SARS-CoV-2 T-cell clonotype diversity as well as T-cell responses with reduced magnitude, whereas the SARS-CoV-2-specific clonotypes targeted a broad range of HLA class I- and class II-restricted epitopes across the viral proteome. The presence of anti-IFN-I antibodies was associated with certain HLA alleles. Finally, COVID-19 mRNA immunization induced an increase in the breadth of SARS-CoV-2-specific clonotypes in patients with IEIs, including those who had failed to seroconvert. CONCLUSIONS: Elderly individuals have impaired capacity to develop broad and sustained T-cell responses after SARS-CoV-2 infection. Genetic factors may play a role in the production of anti-IFN-1 antibodies. COVID-19 mRNA vaccines are effective in inducing T-cell responses in patients with IEIs.
Subject(s)
COVID-19 , Immunocompromised Host , SARS-CoV-2 , Humans , COVID-19/immunology , SARS-CoV-2/immunology , Male , Middle Aged , Female , Immunocompromised Host/immunology , Adult , Aged , T-Lymphocytes/immunology , COVID-19 Vaccines/immunology , Immunocompetence/immunologyABSTRACT
In the maintenance phase of Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP)- Berlin-Frankfurt-Muenster (BFM) acute lymphoblastic leukemia (ALL) 2009 protocol, mercaptopurine (MP) is given at the planned dose of 50 mg/m2 /day; however, dose adjustments are routinely performed to target patients' white blood cells to the optimal range of 2,000-3,000 cells/µL. Pediatric patients with ALL (n = 290, age: median (1st-3rd quartile): 4.8 (3.0-8.1) years; boys: 56.9%) were enrolled mainly in 4 medium-large Italian pediatric hospitals; 14.1% of patients relapsed after a median (1st-3rd quartile) follow-up time of 4.43 (3.82-5.46) years from maintenance beginning. MP metabolites (thionucleotide (TGN) and methyl-derivatives (MMPN)) were measured in the erythrocytes of 387 blood samples of 200 patients by high performance liquid chromatography with ultraviolet detection. Single-nucleotide polymorphisms (SNPs; (rs1800462, rs1800460, and rs1142345 in TPMT gene, rs116855232 in NUDT15, rs1127354, rs7270101, rs6051702 in ITPA, and rs2413739 in PACSIN2) were characterized by Taqman SNP genotyping assays. Cox proportional hazard models did not show an impact of TGN levels and variability on relapse. In contrast, after multivariate analysis, relapse hazard ratio (HR) increased in children with ALL of the intermediate risk arm compared with those in standard risk arm (3.44, 95% confidence interval (CI), 1.31-9.05, P = 0.012), and in carriers of the PACSIN2 rs2413739 T allele compared with those with the CC genotype (heterozygotes CT: HR, 2.32, 95% CI, 0.90-5.97, P = 0.081; and homozygous TT: HR, 4.14, 95% CI, 1.54-11.11, P = 0.005). Future studies are needed to confirm the lack of impact of TGN levels and variability on relapse in the AIEOP-BFM ALL trials, and to clarify the mechanism of PACSIN2 rs2413739 on outcome.
ABSTRACT
Background: PTX3 is an important mediator of inflammation and innate immunity. We aimed at assessing its prognostic value in a large cohort of patients hospitalized with COVID-19. Methods: Levels of PTX3 were measured in 152 patients hospitalized with COVID-19 at San Gerardo Hospital (Monza, Italy) since March 2020. Cox regression was used to identify predictors of time from admission to in-hospital death or mechanical ventilation. Crude incidences of death were compared between patients with PTX3 levels higher or lower than the best cut-off estimated with the Maximally Selected Rank Statistics Method. Results: Upon admission, 22% of the patients required no oxygen, 46% low-flow oxygen, 30% high-flow nasal cannula or CPAP-helmet and 3% MV. Median level of PTX3 was 21.7 (IQR: 13.5-58.23) ng/ml. In-hospital mortality was 25% (38 deaths); 13 patients (8.6%) underwent MV. PTX3 was associated with risk of death (per 10 ng/ml, HR 1.08; 95%CI 1.04-1.11; P<0.001) and death/MV (HR 1.04; 95%CI 1.01-1.07; P=0.011), independently of other predictors of in-hospital mortality, including age, Charlson Comorbidity Index, D-dimer and C-reactive protein (CRP). Patients with PTX3 levels above the optimal cut-off of 39.32 ng/ml had significantly higher mortality than the others (55% vs 8%, P<0.001). Higher PTX3 plasma levels were found in 14 patients with subsequent thrombotic complications (median [IQR]: 51.4 [24.6-94.4] versus 21 [13.4-55.2]; P=0.049). Conclusions: High PTX3 levels in patients hospitalized with COVID-19 are associated with a worse outcome. The evaluation of this marker could be useful in prognostic stratification and identification of patients who could benefit from immunomodulant therapy.
Subject(s)
COVID-19 , Thrombosis , Humans , Hospital Mortality , Serum Amyloid P-Component/metabolism , Thrombosis/etiology , Intubation, IntratrachealABSTRACT
BACKGROUND: l-Asparaginase (ASP) plays a crucial role in the treatment of childhood acute lymphoblastic leukemia (ALL). Currently, different ASP products are available in the market, including both native and pegylated drugs. Several biogeneric Escherichia coli ASP (GEN-ASP) products have been developed in response to shortages and expensiveness of the native E. coli ASP innovator compounds, but some concerns have been raised about their quality. Recently, a number of generic pegylated ASP products (GEN-PEG-ASP) have been marketed to substitute for the innovator product (PEG-ASP). METHODS: Clinical courses and serum asparaginase activity (SAA) levels were monitored in 12 children with ALL, who were treated in our institution with two doses of a GEN-PEG-ASP product, given IV at 2500 IU/m2 during the remission induction phase. Results were compared with those obtained in a reference cohort of 35 patients treated in our institution, who received the innovator PEG-ASP product at same dosage and within the same chemotherapy background. RESULTS: Compared to the reference cohort treated with PEG-ASP, SAA levels were significantly lower in the 12 patients receiving GEN-PEG-ASP (p < .0001); a higher proportion of ASP-associated hypersensitivity reactions (2/12 vs. 0/35; p = .061) and silent inactivation (3/12 vs. 0/35; p = .014) were observed in comparison with the reference cohort. CONCLUSIONS: Our results highlighted different pharmacological profiles and different rates of hypersensitivity reactions and silent inactivation in the GEN-PEG-ASP cohort compared to those treated with the innovator product. Our findings suggest that a rigorous clinical attention and a thorough pharmacological monitoring are advisable in patients treated with GEN-PEG-ASP products.
Subject(s)
Antineoplastic Agents , Asparaginase , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Asparaginase/adverse effects , Asparaginase/therapeutic use , Child , Escherichia coli , Humans , Polyethylene Glycols/adverse effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Remission InductionABSTRACT
Understanding the cause of sex disparities in COVID-19 outcomes is a major challenge. We investigate sex hormone levels and their association with outcomes in COVID-19 patients, stratified by sex and age. This observational, retrospective, cohort study included 138 patients aged 18 years or older with COVID-19, hospitalized in Italy between February 1 and May 30, 2020. The association between sex hormones (testosterone, estradiol, progesterone, dehydroepiandrosterone) and outcomes (ARDS, severe COVID-19, in-hospital mortality) was explored in 120 patients aged 50 years and over. STROBE checklist was followed. The median age was 73.5 years [IQR 61, 82]; 55.8% were male. In older males, testosterone was lower if ARDS and severe COVID-19 were reported than if not (3.6 vs. 5.3 nmol/L, p =0.0378 and 3.7 vs. 8.5 nmol/L, p =0.0011, respectively). Deceased males had lower testosterone (2.4 vs. 4.8 nmol/L, p =0.0536) and higher estradiol than survivors (40 vs. 24 pg/mL, p = 0.0006). Testosterone was negatively associated with ARDS (OR 0.849 [95% CI 0.734, 0.982]), severe COVID-19 (OR 0.691 [95% CI 0.546, 0.874]), and in-hospital mortality (OR 0.742 [95% CI 0.566, 0.972]), regardless of potential confounders, though confirmed only in the regression model on males. Higher estradiol was associated with a higher probability of death (OR 1.051 [95% CI 1.018, 1.084]), confirmed in both sex models. In males, higher testosterone seems to be protective against any considered outcome. Higher estradiol was associated with a higher probability of death in both sexes.
Subject(s)
COVID-19/blood , Gonadal Steroid Hormones/blood , Sex Characteristics , Aged , Aged, 80 and over , Cohort Studies , Female , Hospital Mortality , Hospitalization , Humans , Italy , Male , Middle Aged , Retrospective Studies , Risk Factors , SARS-CoV-2ABSTRACT
Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, which negatively affects T-cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T-cell responses limit disease severity. Understanding how cDCs cope with SARS-CoV-2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures, with the upregulation of IFN-stimulated genes and IL-6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti-apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS-CoV-2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T-cell activation.
Subject(s)
COVID-19/immunology , Dendritic Cells/immunology , Lymphocyte Activation , SARS-CoV-2/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , HumansABSTRACT
Following the spread of the SARS-CoV-2 infection and coronavirus disease 2019 (COVID-19) to a global pandemic, concerns have arisen for the disease impact in at-risk populations, especially in immunocompromised hosts. On the other hand, clinical studies have clarified that the COVID-19 clinical burden is mostly due to over-inflammation and immune-mediated multiorgan injury. This has led to downsizing the role of immunosuppression as a determinant of outcome, and early reports confirm the hypothesis that patients undergoing immunosuppressive treatments do not have an increased risk of severe COVID-19 with respect to the general population. Intriguingly, SARS-CoV-2 natural reservoirs, such as bats and mice, have evolved mechanisms of tolerance involving selection of genes optimizing viral clearance through interferon type I and III responses and also dampening inflammasome response and cytokine expression. Children exhibit resistance to COVID-19 severe manifestations, and age-related features in innate and adaptive response possibly explaining this difference are discussed. A competent recognition by the innate immune system and controlled pro-inflammatory signaling seem to be the pillars of an effective response and the premise for pathogen clearance in SARS-CoV-2 infection. Immunosuppression-if not associated with other elements of fragility-do not represent per se an obstacle to this competent/tolerant phenotype in children. Several reports confirm that children receiving immunosuppressive medications have similar clinical involvement and outcomes as the pediatric general population, indicating that maintenance treatments should not be interrupted in suspect or confirmed SARS-CoV-2 infection.
ABSTRACT
Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.
Subject(s)
COVID-19/immunology , COVID-19/mortality , Adrenal Cortex Hormones/therapeutic use , Adult , Aged , Anti-Bacterial Agents/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Antiviral Agents/therapeutic use , Azithromycin/therapeutic use , Biomarkers , COVID-19/genetics , COVID-19/therapy , Calgranulin B/genetics , Calgranulin B/immunology , Case-Control Studies , Chemokine CCL2/genetics , Chemokine CCL2/immunology , Chemokine CXCL9/genetics , Chemokine CXCL9/immunology , Enzyme Inhibitors/therapeutic use , Female , Ferritins/genetics , Ferritins/immunology , Gene Expression Profiling , Humans , Hydroxychloroquine/therapeutic use , Immunologic Factors/therapeutic use , Interferon Type I/genetics , Interferon Type I/immunology , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-1 Receptor-Like 1 Protein/genetics , Interleukin-1 Receptor-Like 1 Protein/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-15/genetics , Interleukin-15/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Lactoferrin/genetics , Lactoferrin/immunology , Lipocalin-2/genetics , Lipocalin-2/immunology , Male , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/immunology , Middle Aged , Multivariate Analysis , NF-kappa B/genetics , NF-kappa B/immunologyABSTRACT
T cells are involved in the early identification and clearance of viral infections and also support the development of antibodies by B cells. This central role for T cells makes them a desirable target for assessing the immune response to SARS-CoV-2 infection. Here, we combined two high-throughput immune profiling methods to create a quantitative picture of the T-cell response to SARS-CoV-2. First, at the individual level, we deeply characterized 3 acutely infected and 58 recovered COVID-19 subjects by experimentally mapping their CD8 T-cell response through antigen stimulation to 545 Human Leukocyte Antigen (HLA) class I presented viral peptides (class II data in a forthcoming study). Then, at the population level, we performed T-cell repertoire sequencing on 1,815 samples (from 1,521 COVID-19 subjects) as well as 3,500 controls to identify shared "public" T-cell receptors (TCRs) associated with SARS-CoV-2 infection from both CD8 and CD4 T cells. Collectively, our data reveal that CD8 T-cell responses are often driven by a few immunodominant, HLA-restricted epitopes. As expected, the T-cell response to SARS-CoV-2 peaks about one to two weeks after infection and is detectable for at least several months after recovery. As an application of these data, we trained a classifier to diagnose SARS-CoV-2 infection based solely on TCR sequencing from blood samples, and observed, at 99.8% specificity, high early sensitivity soon after diagnosis (Day 3-7 = 85.1% [95% CI = 79.9-89.7]; Day 8-14 = 94.8% [90.7-98.4]) as well as lasting sensitivity after recovery (Day 29+/convalescent = 95.4% [92.1-98.3]). These results demonstrate an approach to reliably assess the adaptive immune response both soon after viral antigenic exposure (before antibodies are typically detectable) as well as at later time points. This blood-based molecular approach to characterizing the cellular immune response has applications in clinical diagnostics as well as in vaccine development and monitoring.
ABSTRACT
Cohesin complex components exert fundamental roles in animal cells, both canonical in cell cycle and non-canonical in gene expression regulation. Germline mutations in genes coding for cohesins result in developmental disorders named cohesinopaties, of which Cornelia de Lange syndrome (CdLS) is the best-known entity. However, a basic description of mammalian expression pattern of cohesins in a physiologic condition is still needed. Hence, we report a detailed analysis of expression in murine and human tissues of cohesin genes defective in CdLS. Using both quantitative and qualitative methods in fetal and adult tissues, cohesin genes were found to be ubiquitously and differentially expressed in human tissues. In particular, abundant expression was observed in hematopoietic and central nervous system organs. Findings of the present study indicate tissues which should be particularly sensitive to mutations, germline and/or somatic, in cohesin genes. Hence, this expression analysis in physiological conditions may represent a first core reference for cohesinopathies.
Subject(s)
Cell Cycle Proteins/genetics , Central Nervous System/metabolism , Chromosomal Proteins, Non-Histone/genetics , De Lange Syndrome/genetics , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Animals , Central Nervous System/embryology , Central Nervous System/growth & development , Chondroitin Sulfate Proteoglycans/genetics , DNA-Binding Proteins , Gene Expression Profiling , Genetic Predisposition to Disease/genetics , Histone Deacetylases/genetics , Humans , Mice , Mutation , Nuclear Proteins/genetics , Phosphoproteins/genetics , Proteins/genetics , Repressor Proteins/genetics , CohesinsABSTRACT
BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare genetic disorder affecting the neurodevelopment, gastrointestinal, musculoskeletal systems. CdLS is caused by mutations within NIPBL, SMC1A, SMC3, RAD21, and HDAC8 genes. These genes codify for the "cohesin complex" playing a role in chromatid adhesion, DNA repair and gene expression regulation. The aim of this study was to investigate retinoic acid (RA) signaling pathway, a master developmental regulator, in CdLS cells. METHODS: Skin biopsies from CdLS patients and healthy controls were cultured and derived primary fibroblast cells were treated with RA or dimethyl sulfoxide (vehicle). After RA treatment, cells were harvested and RNA was isolated for quantitative real-time polymerase chain reaction experiments. RESULTS: We analyzed several components of RA metabolism in a human cell line of kidney fibroblasts (293T), in addition to fibroblasts collected from both NIPBL-mutated patients and healthy donors, with or without RA treatment. In all cases, ADH and RALDH1 gene expression was not affected by RA treatment, while CRABP1 was induced. CRABP2 was dramatically upregulated upon RA treatment in healthy donors but not in CdLS patients cells. CONCLUSION: We investigated if CdLS alterations are associated to perturbation of RA signaling. Cells derived from CdLS patients do not respond to RA signaling as efficiently as healthy controls. RA pathway alterations suggest a possible underlying mechanism for several cellular and developmental abnormalities associated with cohesin function. Birth Defects Research 109:1268-1276, 2017. © 2017 Wiley Periodicals, Inc.
Subject(s)
De Lange Syndrome/genetics , De Lange Syndrome/metabolism , Tretinoin/metabolism , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase 1 Family , Cell Cycle Proteins/genetics , Cell Line , Chromosomal Proteins, Non-Histone/genetics , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression , Genes, cdc , HEK293 Cells , Humans , Mutation , Neurophysins/genetics , Neurophysins/metabolism , Protein Precursors/genetics , Protein Precursors/metabolism , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinal Dehydrogenase , Signal Transduction , Skin/metabolism , Skin/pathology , Vasopressins/genetics , Vasopressins/metabolism , CohesinsABSTRACT
Cornelia de Lange syndrome (CdLS) is a rare genetic condition related to mutation of various cohesion complex related genes. Its natural history is quite well characterized as regard pediatric age. Relatively little information is available regarding the evolution of the disease in young-adult age. In medical literature, only one specific study has been published on this topic. We report on our experience on 73 Italian CdLS patients (40 males and 33 females) with and age range from 15 to 49 years. Our results confirm the previous study indicating that gastroesophageal reflux disease (GERD) is the main medical problem of these patients in childhood and young-adult age. Other medical features that should be considered in the medical follow-up are tendency to overweight/frank obesity, constipation, discrepancy of limbs' length, epilepsy, hearing, and visual problems. Behavioral problems are particularly frequent as well. For this reason, every source of hidden pain should be actively searched for in evaluating a patient showing such a disorder. Finally, recommendations for medical follow-up in adult age are discussed. © 2016 Wiley Periodicals, Inc.
Subject(s)
De Lange Syndrome/complications , De Lange Syndrome/pathology , Adolescent , Adult , Epilepsy , Female , Gastroesophageal Reflux , Hearing Disorders , Humans , Italy/epidemiology , Limb Deformities, Congenital , Male , Mental Disorders , Middle Aged , Obesity , Vision Disorders , Young AdultABSTRACT
Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as "cohesinopathies" that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A-mutated patient-derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis, and specifically down-regulates Ccnd1 levels. The same down-regulation of cohesin targets is observed in SMC1A-mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesins in neural development in vertebrates.
Subject(s)
Apoptosis/physiology , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Cyclin D1/metabolism , De Lange Syndrome/metabolism , Transcription Factors/metabolism , Zebrafish/embryology , Animals , Apoptosis/genetics , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , De Lange Syndrome/genetics , Down-Regulation , Humans , Mice , Mutation/genetics , Transcription Factors/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Cornelia de Lange syndrome (CdLS) is a complex genetic disease with skeletal involvement mostly related to upper limb malformations. We report on three males with clinical and molecular diagnoses of CdLS. Besides typical CdLS features, all showed different cervical spine malformations. To the best of our knowledge, this is an unusual malformation in the CdLS phenotypic spectrum.