Decreased apoptotic priming and loss of BCL-2 dependence are functional hallmarks of Richter's syndrome.

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

OBJECTIVE: The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. DESIGN AND METHODS: To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. RESULTS: Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. CONCLUSIONS: This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

Technical Advances in Circulating Cell-Free DNA Detection and Analysis for Personalized Medicine in Patients' Care.

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients' conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients' quality of life.

A molecular circuit linking the BCR to the NAD biosynthetic enzyme NAMPT is an actionable target in Richter syndrome.

ABSTRACT: This works defines, to the best of our knowledge, for the first time a molecular circuit connecting nicotinamide mononucleoside phosphoribosyl transferase (NAMPT) activity to the B-cell receptor (BCR) pathway. Using 4 distinct xenograft models derived from patients with Richter syndrome (RS-PDX), we show that BCR cross-linking results in transcriptional activation of the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme NAMPT, with increased protein expression, in turn, positively affecting global cellular NAD levels and sirtuins activity. NAMPT blockade, by using the novel OT-82 inhibitor in combination with either BTK or PI3K inhibitors (BTKi or PI3Ki), induces rapid and potent apoptotic responses in all 4 models, independently of their mutational profile and the expression of the other NAD biosynthetic enzymes, including nicotinate phosphoribosyltransferase. The connecting link in the circuit is represented by AKT that is both tyrosine- and serine-phosphorylated by PI3K and deacetylated by sirtuin 1 and 2 to obtain full kinase activation. Acetylation (ie, inhibition) of AKT after OT-82 administration was shown by 2-dimensional gel electrophoresis and immunoprecipitation. Consistently, pharmacological inhibition or silencing of sirtuin 1 and 2 impairs AKT activation and induces apoptosis of RS cells in combination with PI3Ki or BTKi. Lastly, treatment of RS-PDX mice with the combination of PI3Ki and OT-82 results in significant inhibition of tumor growth, with evidence of in vivo activation of apoptosis. Collectively, these data highlight a novel application for NAMPT inhibitors in combination with BTKi or PI3Ki in aggressive lymphomas.

Relevance of next generation sequencing (NGS) data re-analysis in the diagnosis of monogenic diseases leading to organ failure.

BACKGROUND: In 2018, our center started a program to offer genetic diagnosis to patients with kidney and liver monogenic rare conditions, potentially eligible for organ transplantation. We exploited a clinical exome sequencing approach, followed by analyses of in silico gene panels tailored to clinical suspicions, obtaining detection rates in line with what reported in literature. However, a percentage of patients remains without a definitive genetic diagnosis. This work aims to evaluate the utility of NGS data re-analysis for those patients with an inconclusive or negative genetic test at the time of first analysis considering that (i) the advance of alignment and variant calling processes progressively improve the detection rate, limiting false positives and false negatives; (ii) gene panels are periodically updated and (iii) variant annotation may change over time. METHODS: 114 patients, recruited between 2018 and 2020, with an inconclusive or negative NGS report at the time of first analysis, were included in the study. Re-alignment and variant calling of previously generated sequencing raw data were performed using the GenomSys Variant Analyzer software. RESULTS: 21 previously not reported potentially causative variants were identified in 20 patients. In most cases (n = 19), causal variants were retrieved out of the re-classification from likely benign to variants of unknown significance (VUS). In one case, the variant was included because of inclusion in the analysis of a newly disease-associated gene, not present in the original gene panel, and in another one due to the improved data alignment process. Whenever possible, variants were validated with Sanger sequencing and family segregation studies. As of now, 16 out of 20 patients have been analyzed and variants confirmed in 8 patients. Specifically, in two pediatric patients, causative variants were de novo mutations while in the others, the variant was present also in other affected relatives. In the remaining patients, variants were present also in non-affected parents, raising questions on their re-classification. CONCLUSIONS: Overall, these data indicate that periodic and systematic re-analysis of negative or inconclusive NGS data reports can lead to new variant identification or reclassification in a small but significant proportion of cases, with benefits for patients' management.

Anti-tumor activity of selinexor in combination with antineoplastic agents in chronic lymphocytic leukemia.

Despite recent relevant therapeutic progresses, chronic lymphocytic leukemia (CLL) remains an incurable disease. Selinexor, an oral inhibitor of the nuclear export protein XPO1, is active as single agent in different hematologic malignancies, including CLL. The purpose of this study was to evaluate the anti-tumor effects of selinexor, used in combination with chemotherapy drugs (i.e. fludarabine and bendamustine) or with the PI3Kδ inhibitor idelalisib in CLL. Our results showed a significant decrease in CLL cell viability after treatment with selinexor-containing drug combinations compared to each single compound, with demonstration of synergistic cytotoxic effects. Interestingly, this drug synergism was exerted also in the presence of the protective effect of stromal cells. From the molecular standpoint, the synergistic cytotoxic activity of selinexor plus idelalisib was associated with increased regulatory effects of this drug combination on the tumor suppressors FOXO3A and IkBα compared to each single compound. Finally, selinexor was also effective in potentiating the in vivo anti-tumor effects of the PI3Kδ inhibitor in mice treated with the drug combination compared to single agents. Our data provide preclinical evidence of the synergism and potential efficacy of a combination treatment targeting XPO1 and PI3Kδ in CLL.

The humoral and cellular response to mRNA SARS-CoV-2 vaccine is influenced by HLA polymorphisms.

Host genetic variability contributes to susceptibility to SARS-CoV-2 infection and COVID-19 evolution and the role of HLA system has not clearly emerged, suggesting the involvement of other factors. Studying response to vaccination with Spyke protein mRNA represents an ideal model to highlight whether the humoral or cellular responses are influenced by HLA. Four hundred and sixteen workers, vaccinated with Comirnaty beginning 2021, were selected within the Azienda Ospedaliera Universitaria "Città della Salute e della Scienza di Torino." The humoral response was determined with the LIAISON® kit, while the analysis of the cellular response was performed with the Quantiferon SARS-CoV-2 assay, for the S1 (receptor-binding domain; Ag1) and S1 and S2 (Ag2) subunits of the Spyke protein. Six HLA loci were typed by next-generation sequencing. Associations between HLA and vaccine response were performed with univariate and multivariate analyses. An association was found between A*03:01, B*40:02 and DPB1*06:01 and high antibody concentration and between A*24:02, B*08:01 and C*07:01 and low humoral responses. The haplotype HLA-A*01:01 ~ B1*08:01 ~ C*07:01 ~ DRB1*03:01 ~ DQB1*02:01 conferred an increased risk of low humoral response. Considering cellular responses, 50% of the vaccinated subjects responded against Ag1 and 59% against Ag2. Carriers of DRB1*15:01 displayed a higher cellular response both to Ag1 and Ag2 compared to the rest of the cohort. Similarly, DRB1*13:02 predisposed to a robust cellular response to Ag1 and Ag2, while DRB1*11:04 showed an opposite trend. Cellular and humoral responses to Comirnaty are influenced by HLA. Humoral response is mainly associated to class I alleles, with A*03:01, previously associated to protection against severe COVID-19, and response to vaccination, standing out. Cellular response predominantly involves class II alleles, with DRB1*15:01 and DPB1*13:01 prevailing. Affinity analysis for Spyke peptides is generally in line with the association results.

The role of genetic testing in the diagnostic workflow of pediatric patients with kidney diseases: the experience of a single institution.

PURPOSE: Inherited kidney diseases are among the leading causes of kidney failure in children, resulting in increased mortality, high healthcare costs and need for organ transplantation. Next-generation sequencing technologies can help in the diagnosis of rare monogenic conditions, allowing for optimized medical management and therapeutic choices. METHODS: Clinical exome sequencing (CES) was performed on a cohort of 191 pediatric patients from a single institution, followed by Sanger sequencing to confirm identified variants and for family segregation studies. RESULTS: All patients had a clinical diagnosis of kidney disease: the main disease categories were glomerular diseases (32.5%), ciliopathies (20.4%), CAKUT (17.8%), nephrolithiasis (11.5%) and tubular disease (10.5%). 7.3% of patients presented with other conditions. A conclusive genetic test, based on CES and Sanger validation, was obtained in 37.1% of patients. The highest detection rate was obtained for ciliopathies (74.4%), followed by nephrolithiasis (45.5%), tubular diseases (45%), while most glomerular diseases and CAKUT remained undiagnosed. CONCLUSIONS: Results indicate that genetic testing consistently used in the diagnostic workflow of children with chronic kidney disease can (i) confirm clinical diagnosis, (ii) provide early diagnosis in the case of inherited conditions, (iii) find the genetic cause of previously unrecognized diseases and (iv) tailor transplantation programs.

The immunomodulatory molecule TIGIT is expressed by chronic lymphocytic leukemia cells and contributes to anergy.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory checkpoint receptor that negatively regulates Tcell responses. CD226 competes with TIGIT for binding to the CD155 ligand, delivering a positive signal to the T cell. Here we studied the expression of TIGIT and CD226 in a cohort of 115 patients with chronic lymphocytic leukemia (CLL) and report expression of TIGIT and CD226 by leukemic cells. By devising a TIGIT/CD226 ratio, we showed that CLL cells favoring TIGIT over CD226 are typical of a more indolent disease, while those favoring CD226 are characterized by a shorter time to first treatment and shorter progression-free survival after first treatment. TIGIT expression was inversely correlated to the B-cell receptor (BCR) signaling capacity, as determined by studying BTK phosphorylation, cell proliferation and interleukin- 10 production. In CLL cells treated with ibrutinib, in which surface IgM and BCR signaling capacity are temporarily increased, TIGIT expression was downmodulated, in line with data indicating transient recovery from anergy. Lastly, cells from patients with Richter syndrome were characterized by high levels of CD226, with low to undetectable TIGIT, in keeping with their high proliferative drive. Together, these data suggest that TIGIT contributes to CLL anergy by downregulating BCR signaling, identifying novel and actionable molecular circuits regulating anergy and modulating CLL cell functions.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

Macrophage- and BCR-derived but not TLR-derived signals support the growth of CLL and Richter syndrome murine models in vivo.

A large amount of circumstantial evidence has accumulated suggesting that Toll-like receptor (TLR) signals are involved in driving chronic lymphocytic leukemia (CLL) cell proliferation, but direct in vivo evidence for this is still lacking. We have now further addressed this possibility by pharmacologically inhibiting or genetically inactivating the TLR pathway in murine CLL and human Richter syndrome (RS) patient-derived xenograft (PDX) cells. Surprisingly, we show that pharmacologic inhibition of TLR signaling by treatment with an IRAK1/4 inhibitor delays the growth of the transplanted malignant cells in recipient mice, but genetic inactivation of the same pathway by CRISPR/Cas9-mediated disruption of IRAK4 or its proximal adaptor MyD88 has no effect. We further show that treatment with the IRAK1/4 inhibitor results in depletion of macrophages and demonstrate that these cells can support the survival and enhance the proliferation of both murine Eµ-TCL1 leukemia and human RS cells. We also show that genetic disruption of the B-cell receptor (BCR) by CRISPR/Cas9 editing of the immunoglobulin M constant region gene inhibits the growth of human RS-PDX cells in vivo, consistent with our previous finding with murine Eµ-TCL1 leukemia cells. Finally, we show that genetic disruption of IRAK4 does not result in negative selection of human CLL cell lines xenografted in immunodeficient mice. The obtained data suggest that TLR signals are unlikely to represent a major driver of CLL/RS cell proliferation and provide further evidence that signals from macrophages and the BCR promote the growth and survival of CLL and RS cells in vivo.

Validation of a Simple, Rapid, and Cost-Effective Method for Acute Rejection Monitoring in Lung Transplant Recipients.

Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

Lift the curtain on long non-coding RNAs in hematological malignancies: Pathogenic elements and potential targets.

The Human Genome Project in 2001 has opened the Pandora's box on the complexity of DNA structure and transcriptional regulation. Only a small fraction of the 3 billion bases is part of the protein-coding genes, while approximately 98.5% is represented by non-coding sequences. Besides the classical messenger, ribosomal and transfer RNAs, the "cellular RNA world" is made of short and long non-coding RNAs (lncRNAs) that play regulatory or structural roles, shifting the balance of pathogenic gene variations from coding to non-coding genome. LncRNAs are 200 and 100,000 nucleotide long molecules, not translated into protein, highly heterogeneous in terms of expression within the cells, showing tissue and stage specificity. They are emerging as modifiers of epigenetic, transcription, and translation processes, and can be implicated in the pathogenesis of cancers. In this review, we will focus on B, T and NK hematological malignancies, with the aim of presenting an update on lncRNAs landscape and on their role as potential oncogenes or oncosuppressors. Moreover, we will talk over the bi-directional crosstalk between lncRNAs and epigenetics since these modifications can impact on lncRNAs expression, and, in turn, non-coding transcripts can regulate chromatin organization and transcriptional processes. Finally, we will point the attention on their use as potential biomarkers for diagnostic and prognostic purposes, and possibly as attractive targets in a translational perspective, opening for novel therapeutic options.

Novel Approaches for the Treatment of Patients with Richter's Syndrome.

OPINION STATEMENT: In the last 10-15 years, the way to treat cancers has dramatically changed towards precision medicine approaches. These treatment options are mainly based on selective targeting against signaling pathways critical for or detrimentally activated in cancer cells in cancer cells, as well as exploiting molecules that are specifically expressed on neoplastic cells, also known as tumor-associated antigens. These considerations hold true also in the hematological field where a plethora of novel targeted agents have reached patients' bedside, significantly improving clinical responses. Chronic lymphocytic leukemia (CLL) is an example of how targeted therapies, such as BTK, PI3K, or Bcl-2 inhibitors as well as anti-CD20 antibodies, have improved patients' management, even when adopted as frontline treatment. However, these advancements do not apply to Richter's syndrome (RS), the transformation of CLL into a very aggressive and fatal lymphoma, occurring in 2-10% of patients. RS is usually a fast-growing lymphoma of the diffuse large B cell or the Hodgkin's variant, with a dismal prognosis. Despite advancements in depicting and understanding the genetic background of RS and its pathogenesis, no significant clinical results have been registered. In the last couple of years, several studies have started to investigate the impact of novel drugs or drug combinations and some of them have opened for clinical trials, currently in phase I or II, whose results will be soon available. This review will present an overview of current and most recent therapeutic options in RS, discussing also how results coming from xenograft models may help in designing and identifying novel treatment opportunities to overcome the lack of effective therapies.

Adult-onset CblC deficiency: a challenging diagnosis involving different adult clinical specialists.

BACKGROUND: Methylmalonic aciduria and homocystinuria, CblC type (OMIM #277400) is the most common disorder of cobalamin intracellular metabolism, an autosomal recessive disease, whose biochemical hallmarks are hyperhomocysteinemia, methylmalonic aciduria and low plasma methionine. Despite being a well-recognized disease for pediatricians, there is scarce awareness of its adult presentation. A thorough analysis and discussion of cobalamin C defect presentation in adult patients has never been extensively performed. This article reviews the published data and adds a new case of the latest onset of symptoms ever described for the disease. RESULTS: We present the emblematic case of a 45-year-old male, describing the diagnostic odyssey he ventured through to get to the appropriate treatment and molecular diagnosis. Furthermore, available clinical, biochemical and molecular data from 22 reports on cases and case series were collected, resulting in 45 adult-onset CblC cases, including our own. We describe the onset of the disease in adulthood, encompassing neurological, psychiatric, renal, ophthalmic and thromboembolic symptoms. In all cases treatment with intramuscular hydroxycobalamin was effective in reversing symptoms. From a molecular point of view adult patients are usually compound heterozygous carriers of a truncating and a non-truncating variant in the MMACHC gene. CONCLUSION: Adult onset CblC disease is a rare disorder whose diagnosis can be delayed due to poor awareness regarding its presenting insidious symptoms and biochemical hallmarks. To avoid misdiagnosis, we suggest that adult onset CblC deficiency is acknowledged as a separate entity from pediatric late onset cases, and that the disease is considered in the differential diagnosis in adult patients with atypical hemolytic uremic syndromes and/or slow unexplained decline in renal function and/or idiopathic neuropathies, spinal cord degenerations, ataxias and/or recurrent thrombosis and/or visual field defects, maculopathy and optic disc atrophy. Plasma homocysteine measurement should be the first line for differential diagnosis when the disease is suspected. To further aid diagnosis, it is important that genes belonging to the intracellular cobalamin pathway are included within gene panels routinely tested for atypical hemolytic uremic syndrome and chronic kidney disorders.

LINC00152 expression in normal and Chronic Lymphocytic Leukemia B cells.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.

A novel COLEC10 mutation in a child with 3MC syndrome.

3MC syndrome is an autosomal recessive disorder encompassing four rare disorders previously known as the Malpuech, Michels, Mingarelli and Carnevale syndromes. They are characterized by a variable spectrum of abnormalities, including facial dysmorphisms, along with genital, limb and vesico-renal anomalies. The syndrome was originally attributed to mutations in MASP1 and COLEC11, which code for proteins involved in the lectin complement pathway. More recently, mutations in COLEC10, a third gene coding for collectin CL-L1, were identified in a limited number of patients with 3MC syndrome. Here we describe a 4-years-old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, sacral dimple and horseshoe kidney. Initial genetic analysis was based on clinical exome sequencing, where only MASP1 and COLEC11 genes are present, without evidence of pathogenic variants. Sanger sequencing of COLEC10 identified the homozygous frameshift variant c.807_810delCTGT; p.Cys270Serfs*33, which results in the loss of the natural stop codon. The resulting protein is 24 amino acids longer and lacks a conserved cysteine residue (Cys270), which could affect protein folding. Segregation studies confirmed that both parents were carriers for the variant: interestingly they originate from the same area of Apulia in southern Italy. Plasma levels of CL-L1 in the patient and her parents were within normal range, suggesting that this variant does not modify transcription or secretion. However, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells migrate significantly less in response to the mutant protein compared to the wild-type one.

The frequency of rare and monogenic diseases in pediatric organ transplant recipients in Italy.

BACKGROUND: Rare diseases are chronic and life-threatening disorders affecting < 1 person every 2,000. For most of them, clinical symptoms and signs can be observed at birth or childhood. Approximately 80% of all rare diseases have a genetic background and most of them are monogenic conditions. In addition, while the majority of these diseases is still incurable, early diagnosis and specific treatment can improve patients' quality of life. Transplantation is among the therapeutic options and represents the definitive treatment for end-stage organ failure, both in children and adults. The aim of this paper was to analyze, in a large cohort of Italian patients, the main rare genetic diseases that led to organ transplantation, specifically pointing the attention on the pediatric cohort. RESULTS: To the purpose of our analysis, we considered heart, lung, liver and kidney transplants included in the Transplant Registry (TR) of the Italian National Transplantation Center in the 2002-2019 timeframe. Overall, 49,404 recipients were enrolled in the cohort, 5.1% of whom in the pediatric age. For 40,909 (82.8%) transplant recipients, a disease diagnosis was available, of which 38,615 in the adult cohort, while 8,495 patients (17.2%) were undiagnosed. There were 128 disease categories, and of these, 117 were listed in the main rare disease databases. In the pediatric cohort, 2,294 (5.6%) patients had a disease diagnosis: of the 2,126 (92.7%) patients affected by a rare disease, 1,402 (61.1%) presented with a monogenic condition. As expected, the frequencies of pathologies leading to organ failure were different between the pediatric and the adult cohort. Moreover, the pediatric group was characterized, compared to the adult one, by an overall better survival of the graft at ten years after transplant, with the only exception of lung transplants. When comparing survival considering rare vs non-rare diseases or rare and monogenic vs rare non-monogenic conditions, no differences were highlighted for kidney and lung transplants, while rare diseases had a better survival in liver as opposed to heart transplants. CONCLUSIONS: This work represents the first national survey analyzing the main genetic causes and frequencies of rare and/or monogenic diseases leading to organ failure and requiring transplantation both in adults and children.