DiGeorge Research Articles

Home continuous positive airway pressure therapy in infants: a single-center experience.

There are limited data on indications and outcomes of home continuous positive airway pressure (CPAP) therapy in the first year of life. We aimed to analyze the clinical, demographic, and polysomnographic characteristics of a cohort of children initiated on home CPAP for treatment of sleep-disordered breathing and as respiratory support in the first year of life. Children started on CPAP in the first year of life at the Queensland Children's Hospital were retrospectively evaluated for clinical and demographic parameters, underlying diagnoses, respiratory support, airway surgical intervention, and polysomnography results at baseline and on CPAP. Twenty-nine infants (median age [interquartile range] at CPAP initiation, 182 days [126-265.5 days]) were included. The underlying etiology included Trisomy 21 (n = 6), craniofacial syndromes (n = 5), hypotonia (n = 8; 5 with noncraniofacial syndrome), airway malacia (n = 5), skeletal dysplasia (n = 2), nonsyndromic upper airway obstruction (n = 2), and chronic neonatal lung disease (n = 1). The median (interquartile range) obstructive apnea-hypopnea index was 14 events/h (6.2-31 events/h) at CPAP initiation, which improved on CPAP to 3.4 events/h (1.4-6.4 events/h). The median (interquartile range) transcutaneous CO2 max remained unchanged on CPAP (56.6 mm Hg [49-66.5 mm Hg] pre-CPAP vs 54.9 mm Hg [47-62 mm Hg] on CPAP). Fifteen children needed surgical airway intervention (11 pre-CPAP and 4 post-CPAP). CPAP therapy could be successfully stopped in 9 children, 2 children needed tracheostomy, and 1 child died during the follow-up period. Home CPAP as respiratory support is an effective long-term therapy in infancy, and these patients can be weaned from CPAP therapy even if it was initiated early. Prospective studies with predefined criteria for CPAP initiation and cessation would help ascertain long-term outcomes in this poorly researched group. Joshi SS, Sivapalan D, Leclerc M-J, Kapur N. Home continuous positive airway pressure therapy in infants: a single-center experience. J Clin Sleep Med. 2023;19(3):473-477.

Jacobsen syndrome: a case report and clinical features of a rare genetic syndrome

Objective: Jacobsen syndrome is an infrequent contiguous gene syndrome that involves the deletion of the long arm of chromosome 11. It is mostly accompanied by intellectual disability and other abnormalities. The majority of the patients are hospitalized or lost within the first two years of life. Case(s): We report a case of a fetus at 21 weeks of gestation with Jacobsen syndrome who presented with a conotruncal cardiac defect. Amniocentesis was performed, and karyotype analysis revealed that there was a de novo deletion of chromosome 11. The family decided to terminate the pregnancy. Conclusion: Prenatal diagnosis of Jacobsen syndrome is not always possible, since the characteristic ultrasound findings vary greatly between patients. Additionally, existing symptoms and signs may not always be found with imaging techniques. However, if present, certain ultrasonographic findings should lead clinicians to consider the syndrome. The study aims to present a rare case of Jacobsen syndrome, inform the clinicians, and guide on this syndrome and its possible outcomes.

Prenatal Identification and Confirmation of Jacobsen Syndrome: A Series of Four Cases.

Jacobsen syndrome (JBS) is a rare contiguous gene disorder caused by partial deletion of the distal part of the long arm of chromosome 11. Only a few prenatal cases of JBS have been reported, and data on prenatal ultrasonographic findings are relatively scarce. We analysed four cases of JBS diagnosed prenatally in our centre. All four cases received ultrasound examination in the second trimester. Cardiac defects and intrauterine growth retardation (IUGR) were present in three cases. Ventriculomegaly, shortened femur length and pyelectasis were found in two cases. According to the literature, IUGR, pyelectasis and ventriculomegaly are common prenatal phenotypes of JBS. In addition, cardiac defects, trigonocephaly and shortened femur are also found. Our presentation of these cases provides more ultrasonic information for the prenatal diagnosis of this rare disease. Key Words:Ultrasound, Prenatal diagnosis, Jacobsen syndrome, Chromosomal abnormalities, Fetal malformation.

Abstract 3

Background: Hypoparathyroidism is commonly idiopathic (besides post-surgical). Syndromic forms are mostly sporadic and it is the specific clinical features which help to identify the relevant diagnosis. Many times, these syndromic features are subtle and may be missed in a busy clinical practice. Also, the Ca-P deposition in the brain may lead to overlapping neurological features. Conversely hypoparathyroidism (17 – 60%) may not be part of the initial clinical presentation in these syndromes. Case Report: We report a 26-year male who presented with seizure disorder and hypocalcemia, poorly controlled on anti-epileptics and oral calcium. Apart from seizures, which commenced at the age of 12 days, he had a history of recurrent chest infections and middle ear infections requiring multiple hospitalizations (8 – 10 till date). The patient had a reduced attention span, irritable behaviour, and poor scholastic performance. On examination, the patient had low set ears, carious teeth, high pitched nasal voice, positive Trousseau’s sign, mucopurulent discharge with perforation of the tympanic membrane in both ears. Investigations revealed hypocalcemia (6.3 mg/dl) with hyperphosphatemia (6.4 mg/dl) with inappropriately low iPTH (11.4 pg/ml) levels. Non-contrast CT head showed bilateral calcification at the basal ganglia and thalamus. 2 D echocardiography did not reveal any cardiac abnormality. CT thorax revealed absence of thymus. A deformity was noted at the distal phalynx of both thumbs, which on X-rays was identified as a small bony outgrowth from the distal phalynx of bilateral thumbs. It was actually a preaxialpolydactyly type II (Wassel’s classification). These two pointers led us to do specific genetic testing for DiGeorge syndrome and Next-generation sequencing (NGS) revealed microdeletion at chromosome 22q11.2. Conclusion: The spectrum of DiGeorge syndrome is vast and varied. The characteristic defects including congenital heart disease and velo-facial manifestations may not be present in all. Looking out for subtle clinical pointers to syndromic features may help in directing specific genetic testing to clinch the diagnosis. In our case, the deformed shape of the thumbs led us to explore further leading us to unravelling the diagnosis.

First person – Emily Warren

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Emily Warren is first author on ‘ 17q12 deletion syndrome mouse model shows defects in craniofacial, brain and kidney development, and glucose homeostasis’, published in DMM. Emily conducted the research described in this article while a postdoctoral fellow in Eric M. Morrow's lab at Brown University, Providence, RI, USA. She is now a postdoctoral fellow in the lab of Peter J. McGuire at the National Institutes of Health (NIH) National Human Genome Research Institute (NHGRI), Bethesda, MD, USA, investigating how metabolic dysfunction impacts the brain, particularly in the context of neurodevelopment and neurodegeneration.

17q12 deletion syndrome mouse model shows defects in craniofacial, brain and kidney development, and glucose homeostasis.

17q12 deletion (17q12Del) syndrome is a copy number variant (CNV) disorder associated with neurodevelopmental disorders and renal cysts and diabetes syndrome (RCAD). Using CRISPR/Cas9 genome editing, we generated a mouse model of 17q12Del syndrome on both inbred (C57BL/6N) and outbred (CD-1) genetic backgrounds. On C57BL/6N, the 17q12Del mice had severe head development defects, potentially mediated by haploinsufficiency of Lhx1, a gene within the interval that controls head development. Phenotypes included brain malformations, particularly disruption of the telencephalon and craniofacial defects. On the CD-1 background, the 17q12Del mice survived to adulthood and showed milder craniofacial and brain abnormalities. We report postnatal brain defects using automated magnetic resonance imaging-based morphometry. In addition, we demonstrate renal and blood glucose abnormalities relevant to RCAD. On both genetic backgrounds, we found sex-specific presentations, with male 17q12Del mice exhibiting higher penetrance and more severe phenotypes. Results from these experiments pinpoint specific developmental defects and pathways that guide clinical studies and a mechanistic understanding of the human 17q12Del syndrome. This mouse mutant represents the first and only experimental model to date for the 17q12 CNV disorder. This article has an associated First Person interview with the first author of the paper.

Prenatal Identification and Confirmation of Jacobsen Syndrome: A Series of Four Cases

Prenatal Identification and Confirmation of Jacobsen Syndrome: A Series of Four Cases

Richter transformation in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia can evolve to an aggressive lymphoma-in most of the cases diffuse large B cells lymphoma, rarely Hodgkin lymphoma-and this complication is defined Richter syndrome (RS). Immunogenotypic features that characterize RS include unmutated IgHV status with high prevalence of IgHV4-39/D6-13/J5 sequence; deletion of chromosome 17p or 11q; activation of oncogenes as NOTCH1 and c-MYC; inactivation of onco-suppressors as TP53 and CDKN2A; high expression of CD38 in lymph-nodes. The prognosis of this condition is very poor: patients experience a rapid clinical deterioration with frequent therapeutic failure since the current options include suboptimal strategies as standard chemo-immunotherapy followed by hematopoietic stem cells transplantation or enrollment in clinical trials which investigate the efficacy of target drugs. Understanding the biology of such a heterogeneous condition is crucial to personalize the treatment and improve patient's survival.

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as “giant lamellar bodies” (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.

TBX1 targets the miR-200–ZEB2 axis to induce epithelial differentiation and inhibit stem cell properties

TBX1, which encodes a T-box transcription factor, is considered a candidate gene for DiGeorge syndrome, velocardiofacial syndrome, and conotruncal anomaly face syndrome. Transduction of TBX1 decreases cell proliferation in epithelial cancer cells and Tbx1 ablation induces epithelial proliferation during palatal development. Here, we report that TBX1 regulates stem cell properties and epithelial differentiation through the transcriptional activation of microRNAs. Stable expression of TBX1 induces microRNA-200 (miR-200), whose members repress the epithelial-to-mesenchymal transition and induce epithelial differentiation. TBX1 rescues ZEB2-dependent transcriptional inhibition of the miR-200b/200a/429 cluster, whose promoter region contains conserved overlapping cis-regulatory motifs of the ZEB-binding E-box and TBX-binding element. Consequently, TBX1 activates the expression of both miR-200 and stemness-inhibitor miR-203 to inhibit their common targets, BMI1 and ZEB2. Moreover, Tbx1 ablation affects the differentiation of the palatal epithelium and perturbs the expression of miR-200, miR-203, and their target genes. We propose that TBX1 links stem cell properties and epithelial differentiation by inducing miR-200 and miR-203. Thus, targeting of the ZEB2–miR-200 axis by TBX1 may have potential therapeutic implications in miR-200-associated tumors and cleft palate.

Efficiency of expanded noninvasive prenatal testing in the detection of fetal subchromosomal microdeletion and microduplication in a cohort of 31,256 single pregnancies

Noninvasive prenatal testing (NIPT) is widely used to screen for common fetal chromosomal aneuploidies. However, the ability of NIPT-Plus to detect copy number variation (CNV) is debatable. Accordingly, we assessed the efficiency of NIPT-Plus to detect clinically significant fetal CNV. We performed a prospective analysis of 31,260 singleton pregnancies, included from June 2017 to December 2020. Cell-free fetal DNA was directly sequenced using the semiconductor sequencing platform for women with high-risk CNV with clinically significant results. Fetal karyotyping and chromosomal microarray analysis (or next-generation sequencing) are recommended for invasive diagnostic procedures. Women at low risk with no other abnormal results continued their pregnancies. We analyzed the expanded NIPT results, diagnostic test results, and follow-up information to evaluate its performance in detecting fetal CNV. Of the 31,260 pregnant women who received NIPT-Plus, 31,256 cases were tested successfully, a high risk of clinically significant CNV was detected in 221 cases (0.71%); 18 women refused further diagnosis; 203 women underwent invasive prenatal diagnosis; and 78 true positive cases and 125 false positive cases, with an overall positive predictive value (PPV) of 38.42% and a false positive rate of 0.40%. For known microdeletion/microduplication syndromes (n = 27), the PPVs were 75% DiGeorge syndrome (DGS), 80% 22q11.22 microduplication, 50% Prader–Willi syndrome, and 50% cri-du-chat. For the remaining clinically significant fetal CNVs (n = 175), the combined PPVs were 46.5% (CNVs > 10 Mb) and 28.57% (CNVs ≤ 10 Mb). NIPT-Plus screening for CNV has certain clinical value. NIPT-Plus yielded relatively high PPVs for 22q11.2 microduplication syndrome and DGS, and low to moderate PPVs for other CNVs.

Construction of an immune-related ceRNA network to screen for potential diagnostic markers for autism spectrum disorder.

Purpose: The diagnosis of autism spectrum disorder (ASD) is reliant on evaluation of patients' behavior. We screened the potential diagnostic and therapeutic targets of ASD through bioinformatics analysis. Methods: Four ASD-related datasets were downloaded from the Gene Expression Omnibus database. The "limma" package was employed to analyze differentially expressed messenger (m)RNAs, long non-coding (lnc)RNAs, and micro (mi)RNAs between ASD patients and healthy volunteers (HVs). We constructed a competing endogenous-RNA (ceRNA) network. Enrichment analyses of key genes were undertaken using the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes database. The ImmucellAI database was used to analyze differences in immune-cell infiltration (ICI) in ASD and HV samples. Synthetic analyses of the ceRNA network and ICI was done to obtain a diagnostic model using LASSO regression analysis. Analyses of receiver operating characteristic (ROC) curves were done for model verification. Results: The ceRNA network comprised 49 lncRNAs, 30 miRNAs, and 236 mRNAs. mRNAs were associated with 41 cellular components, 208 biological processes, 39 molecular functions, and 35 regulatory signaling pathways. Significant differences in the abundance of 10 immune-cell species between ASD patients and HVs were noted. Using the ceRNA network and ICI results, we constructed a diagnostic model comprising five immune cell-associated genes: adenosine triphosphate-binding cassette transporter A1 (ABCA1), DiGeorge syndrome critical region 2 (DGCR2), glucose-fructose oxidoreductase structural domain gene 1 (GFOD1), glutaredoxin (GLRX), and SEC16 homolog A (SEC16A). The diagnostic performance of our model was revealed by an area under the ROC curve of 0.923. Model verification was done using the validation dataset and serum samples of patients. Conclusion: ABCA1, DGCR2, GFOD1, GLRX, and SEC16A could be diagnostic biomarkers and therapeutic targets for ASD.

PTPN1 Deficiency Accelerates the Progression to Myelofibrosis in JAK2V617F-Driven Myeloproliferative Neoplasm

The deletion of chromosome 20q (del20q) is frequently associated with myeloid malignancies including myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The del20q cytogenetic abnormality was observed most frequently in patients with myelofibrosis (MF). However, the key target genes within 20q involved in the pathogenesis of myelofibrosis has remained elusive. We previously reported that PTPN1 gene encoding protein tyrosine phosphatase non-receptor type 1 is a potential tumor suppressor within 20q in MPN/MF (Jobe et al. Leukemia 2017). The del20q involving PTPN1 deletion was more commonly found in MPN/MF in comparison to other myeloid neoplasms. We also observed significant co-occurrence of del20q involving PTPN1 deletion with JAK2V617F mutation in patients with MF. So, we hypothesize that PTPN1 deficiency may cooperate with JAK2V617F in the pathogenesis of MF. In this study, we investigated the effects of heterozygous and homozygous PTPN1 deletion in JAK2V617F-induced MPN using a conditional PTPN1 knockout allele and a JAK2V617F knock-in mouse model of MPN. We previously reported the generation of a conditional JAK2V617F knock-in mouse. Mice expressing heterozygous JAK2V617F (JAK2VF/+) exhibit a PV-like MPN characterized by increase in red blood cells (RBC), white blood cells (WBC), neutrophils and platelets in the peripheral blood. Heterozygous deletion of PTPN1 significantly increased WBC, neutrophil and platelet counts but reduced RBC and hemoglobin levels in JAK2VF/+ mice. Flow cytometric analysis showed that heterozygous deletion of PTPN1 significantly increased myeloid (Gr-1+Mac-1+) and megakaryocytic (CD41+CD61+) precursors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared with JAK2VF/+ mice. Additionally, deletion of PTPN1 significantly increased hematopoietic stem cells and myeloid progenitors in the BM and spleens of PTPN1-deficient JAK2VF/+ mice compared to JAK2VF/+ mice. Spleen weight was significantly increased in JAK2VF/+ mice, while deletion of PTPN1 further enhanced splenomegaly in PTPN1-deficient JAK2VF/+ mice. Furthermore, deletion of PTPN1 significantly enhanced bone marrow fibrosis in JAK2VF/+mice. Homozygous deletion of PTPN1 resulted in more robust and accelerated development of lethal myelofibrosis in JAK2VF/+ mice. The cooperative effects of JAK2V617F and PTPN1 deficiency in myelofibrotic transformation is associated with increased production of proinflammatory cytokines including IL-1beta, IL-6 and TGF-beta. We also observed enhanced activation of STAT1, STAT3, ERK1/2 and NF-kB in PTPN1-deficient JAK2VF/+ mice bone marrow compared with JAK2VF/+ or WT mice bone marrow. CRISPR/Cas9-mediated deletion of PTPN1 also resulted in enhanced activation of these signaling molecules and increased proliferation in JAK2V617F-positive mouse and human hematopoietic cells. Furthermore, deletion of PTPN1 significantly reduced ruxolitinib sensitivity against JAK2V617F-positive hematopoietic cells, suggesting that del20q involving PTPN1 deletion may cause ruxolitinib resistance in MPN. Overall, this study demonstrates that PTPN1 is an important target in del20q-associated MPN, and deficiency of PTPN1 collaborates with JAK2V617F in the progression to myelofibrosis.

Functional Human iPSC-Derived Thymic Epithelial Progenitor Cells Reconstitute T Cell Development and Function in an In Vivo Model of Thymic Aplasia

The thymus is an essential primary lymphoid organ in which T cells mature, proliferate, and acquire a broad and self-tolerant T cell receptor repertoire. In the thymus, specialized thymic epithelial cells (TECs) select T cells capable of recognizing foreign antigens, and eliminate self-reactive T cells to prevent autoimmunity. In addition, the thymus also contributes to the differentiation of regulatory T cells which confine immune responses to actively preserve central tolerance. During thymic development, the interactions between hematopoietic lymphoid progenitor cells and thymic epithelial progenitor cells (TEPCs) provides indispensable signals to the development of the thymic epithelium. In humans, unlike in mice, the development of the thymus is completed before birth. There is no evidence of thymic stem cells that could contribute to a sustained regeneration of the thymic epithelium throughout life. Accordingly, the thymus has no endogenous capacity to regenerate. The thymus peaks in size during infancy and involutes with age, leading to the weakened immune function of the elderly known as immune senescence. Allogeneic hematopoietic stem cell transplant (HSCT) is a curative strategy for many malignant and non-malignant hematopoietic diseases, however, often at the cost of causing severe thymic dysfunction due to conditioning medications and graft-versus-host disease (GVHD). This can severely impair T cell immune reconstitution post-HSCT leading to life-threatening complications. In addition, genetic forms of congenital athymia (e.g. 22q11 Deletion Syndrome, TBX1, TBX2, PAX1, and CHD7 deficiency) can result in absence of T cell development. Complete congenital athymia is fatal early in life unless immune reconstitution is achieved by allogeneic thymus transplantation. However, the shortage of transplantable donor thymus tissue and lack of HLA-matching between donor and recipient limits the use of allogeneic thymus transplantation to a small number of children with complete congenital athymia. Thus, there is a critical unmet clinical need to provide HLA-matched thymic tissues for a larger group of patients with irreversible thymic injury and severe thymic dysfunction. Creating a regenerative thymus replacement therapy by generating functional TEPCs from induced-pluripotent stem cells (iPSCs) has been a longstanding aspiration of the transplant community. We have taken a novel approach by developing a robust in vitro TEPC differentiation protocol that sequentially induces all stages of human thymic ontogeny including bipotent third (III) pharyngeal pouch endoderm (PPEIII), ventralization of the PPE (PPEv), and TEPC commitment, usually lymphoid dependent. Here, we show that TEPCs from various iPSC-lines (tissue source, donor age and sex) express key markers of thymic epithelial fate and function, including FOXN1, PAX1, PAX9, HLA-DR. To examine their functional capacity, we transplanted iPSC-derived TEPCs under the kidney capsule of humanized (hCD34+ HSCs engrafted) athymic NSG-nude mice (NOD.scid.Il2Rγcnull, Foxn1null knockout allele). We observed robust hCD3+ development (40-60% out of hCD45 population) with CD4+ and CD8+ T cells (±70% and ±25%, respectively) at different stages of maturation (17% CD45RA+ CD45RO-, naïve; 80% CD45RA- CD45RO+, memory). Similar levels of T cells were found in mice that received human fetal thymus transplants, while T cells were not detected in NSG-nude mice without any thymus graft (primary or iPSC-derived). Mice with iPSC-derived thymus grafts had no clinical signs of autoimmunity or GVHD. In vivo matured grafts express levels of FOXN1, PAX1, PAX9, DLL4, PSMB11 and CD205 protein that are comparable to primary thymus tissue grafts. Altogether, our protocol reliably produces functional TEPCs from a diverse set of iPSCs that support T cell differentiation in vivo. We are currently characterizing the transcriptomic and epigenomic profile of iPSC-derived TEPCs on a single cell level before and after transplant. In addition, we are examining in vivo T cell function by analyzing the TCR repertoire and response to T-cell dependent antigens. We foresee that iPSC-derived TEPCs will offer a safe alternative as an HLA-matched thymic replacement therapy that will benefit a large cohort of patients, i.e. patients undergoing HSCT and patients receiving solid organ transplants in addition to patients with congenital thymic aplasia.

Therapy-Related Myelodysplastic Syndromes with Isolated Del(5q): Comparative Analysis of Phenotype and Long-Term Survival

Background: Myelodysplastic syndromes (MDS) with chromosome 5q deletion [MDS-del(5q)] is a subcategory of MDS recognized by both the International Consensus (ICC) and World Health Organization (WHO) classification systems. Diagnostic criteria according to ICC-2022 include i) presence of del(5q) alone or with only one other additional cytogenetic abnormality, other than -7/del(7q), ii) absence of multi-hit TP53 mutation, iii) presence of ≥1 cytopenias, iv) presence of bone marrow myeloid dysplasia in ≥1 lineages, v) absence of ≥5% bone marrow blasts, and vi) absence of ≥2% circulating blasts (Arber et al. Blood 2022). We used WHO-2016 diagnostic criteria to identify Mayo Clinic patients with MDS-del(5q) and confirmed a study population that also met ICC-2022 criteria. The main objective of the current study was to describe the phenotype and long-term survival of patients with therapy-related MDS-del(5q) and compare the findings with a cohort of patients with primary MDS-del(5q), sourced from the same database. Methods: The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patients with MDS-del(5q) who were seen at the Mayo Clinic. Diagnosis of MDS-del(5q) was retrofitted to comply with ICC-2022 criteria, with the exception that patients with additional cytogenetic abnormalities were excluded from the current report (Arber et al. Blood 2022). Most patients were treated with lenalidomide, often given at the recommended dose of 10 mg/day x 21-28 days, with dose adjustements at physician discretion. Erythroid response criteria included transfusion independence for at least 8 weeks for transfusion dependent patients and an increase in hemoglobin by 1.5 g/dl lasting for at least 8 weeks, in transfusion independent patients. Transfusion dependency was assigned to those patients who had received at least 3 units of packed red cells in the 8 weeks prior to diagnosis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results A total of 93 patients (median age 73 years; range 24-89; females 63%) were included in the current study, including primary (n=79) and therapy-related (n=14) MDS-del(5q). Cancer/treatment history for the latter group of patients included prostate cancer/radiotherapy (n=5), thyroid cancer/radiotherapy (n=2), breast cancer/radiotherapy (n=2), bladder cancer/chemotherapy (n=2), colon cancer/chemotherapy (n=1), lung cancer/chemotherapy (n=1), and non-Hodgkin's lymphoma/chemotherapy/radiotherapy (n=1). Median (range) time from radiotherapy/chemotherapy and recognition of MDS-del(5q) was 16 years (1.9-30). Comparison of presenting features for therapy-related vs primary MDS-del(5q) revealed the former to be associated with older age (median 77 vs 72 years; p=0.002), male gender (72% vs 30%; p=0.003), and lesser likelihood of transfusion-dependent anemia, at time of initial diagnosis (28% vs 68%; p=0.03). The two groups were otherwise similar in their leukocyte and platelet counts and mutational landscape, including prevalence of TP53 mutations (25% vs 13%, respectively). All 79 patients with primary vs 10 of the 14 with therapy-related disease received lenalidomide therapy with erythroid response rates of 62% vs 80% (p=0.2) and complete/partial cytogenetic response rates of 13%/10% vs 0/0, respectively (p=0.05); the latter information was available in a subset of 64 patients. After a median follow up of 4.8 years, 38 (41%) deaths and 16 (17%) leukemic transformations were documented; the corresponding figures for therapy related/primary disease were 50%/39% and 14%/18%, respectively (p=0.4 and 0.7). Figure 1 depicts similar (p=0.6) survival data for therapy-related vs primary MDS-del(5q), with corresponding median survival (3-year survival) rates of 6.3 years (60%) vs 7.7 years (63%). Conclusions: The current study describes the circumstances surrounding the development of therapy-related MDS-del(5q) and highlights differences in age and gender distribution, compared to primary MDS-del(5q). The two groups were otherwise similar in their response to lenalidomide therapy, overall survival, and leukemic transformation rates while their similarity in mutation distribution requires validation with higher number of informative cases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TP53 Mutation Status Defines a Distinct Clinicopathological Entity of Therapy-Related Myeloid Neoplasm, Characterized By Genomic Instability and Extremely Poor Outcome

BACKGROUND: The prognostic and therapeutic impact of TP53-mutated (TP53mut) myeloid neoplasm (MN) is an area of intense interest, with the 5th edition of the World Health Organization, the International Consensus Classification, and the European LeukemiaNet guidelines, all recommending major changes MN classification based on TP53mut. However, studies driving these changes predominantly included de novo MDS (Bernard et al. Nat Med 2020), AML and high risk MDS treated with intensive chemotherapy (Grob et al. Blood 2022), or MN with complex karyotype (CK, Weinberg et al. Blood Adv 2022). Therefore, the significance of these findings as they relate to t-MN is not known. METHODS: We analyzed therapy-related myeloid neoplasm (t-MN) (n=377) patients evaluated at Mayo Clinic, Rochester and South Australian MDS Registry with clinical, integrated genomic profiling, and long-term follow-up to characterize the genomic profile and outcome of TP53mut t-MN. RESULTS: Our cohort of 377 t-MN included t-MDS (n=245, 65%) and t-AML (n=132, 35%), with a median age at t-MN diagnosis being 65.7 years. 184 TP53mut at variant allele frequency (VAF) ≥2% were detected in 131 (34.74%) patients. Median overall survival (OS) of TP53mut VAF <10% (n=14) was similar to wild-type TP53 (TP53wt), hence further analysis was restricted to TP53mut with VAF >10% (n=111). TP53mut patients without VAF (n=6) were not included for further analysis. Genomic instability was highly evident in TP53mut t-MN compared to TP53wt t-MN. CK, monosomal karyotype (MK), chromosome 5q deletion, and marker chromosomes were enriched in TP53mut t-MN. In contrast, driver oncogenic gene mutations such as IDH2, SRSF2, RAS, and TET2 were less frequent in TP53mut. TP53mut also dictated the clinical presentation, longer latency (interval between primary cancer and t-MN diagnosis), and inferior OS compared to TP53wt (8.2 vs. 19.4 months; P <0.001). On multivariate Cox regression analyses, TP53mut was associated with inferior OS (HR 1.43 95% CI 1.01-2; P =0.03) independent of CK and bone marrow blast %. A significantly higher proportion of CK patients had TP53mut compared to non-CK patients (74.4% vs. 5.4%, P <0.001). Even within the CK cohort, TP53mut was associated with structural genomic instability, with a higher proportion of TP53mut harbouring MK (P =0.013), marker chromosome (P =0.033), deletion 5q (P <0.001), abnormal chromosome 17 (P =0.001), and other structural chromosomal aberrancies compared to TP53wt-CK. In contrast to the predominance of structural chromosomal aberrancies in the TP53mut-CK cohort, mutations driving MN were sparse in TP53mut-CK compared to TP53wt-CK. Finally, TP53mut-CK was associated with significantly poor survival compared to the TP53wt-CK (8.2 vs. 17 months; P <0.001). Though bone marrow (BM) % predicted poor outcome in the whole t-MN cohort, it failed to predict outcome in TP53mut t-MN patients. We compared the clinical and molecular profiling of TP53mut t-MDS (<5%, 5-9%, 10-19% blasts) and t-AML (≥20% blasts) at t-MN diagnosis. Genomic instability, TP53mut allelic status, number of TP53mut, and the clone size were similar across all four cohorts. Remarkably, the survival of TP53mut patients were equally dismal irrespective of blast percentage (P=0.2, Figure 1A). Together, these findings suggest that TP53mut t-MN represent a homogenous group irrespective of BM blast percentage. Integrating the mutation, conventional cytogenetic, copy number variation, FISH, and SNP array analysis, TP53mut were classified as single- or multi-hit (Grob et al. Blood 2022). In total, 31 (25.2%) of the 123 patients with TP53mut (n=111) and/or loss of TP53 (n=12) locus were considered single-hit, and 92 (74.8%) were considered as multi-hit. In contrast to prior reports, we did not observe enrichment of genomic instability, or differences in degree of cytopenia, bone marrow blast % and t-MN phenotype in multi- vs single-hit TP53mut patients. Importantly, no OS differences were observed when TP53mut were stratified by type of TP53mut (truncation vs. missense), site of missense mutation (hot- vs. non-hot spot) and single- vs multi-hit (Figure 1B). CONCLUSION: The classification of t-MN based on TP53mut status is clinically and biological relevant, irrespective of blast % or single- or multiple-hit status. TP53mut t-MN is a molecularly defined unique group enriched for genetic instability and is associated with extremely poor outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Molecular Pathophysiology of Ineffective Erythropoiesis at Single Cell Resolution

The anemias of myelodysplastic syndrome (MDS) and Diamond Blackfan anemia (DBA) are generally macrocytic, always reflect ineffective erythropoiesis, and yet result from diverse genetic mutations. To delineate the shared mechanisms that lead to the death of maturing erythroid cells, we studied single marrow cells from MDS-5q patients, DBA patients, and normal individuals with CITE-seq (antibody barcoding of cell surface proteins) and RNA sequencing. After testing several approaches, we opted to align transcriptomes with SCVelo pseudotime analysis, which includes incompletely processed mRNAs, since these are highly prevalent and regulated during erythropoiesis. We identified 38,296 erythroid lineage cells then plotted CITE-seq protein quantitation data versus the velocity pseudotime to identify BFU-E through orthochromatic erythroblasts based upon the sequential CD117, CD36, CD71, and GlyA expression levels, using well-established criteria. The analysis identified two distinct transcriptional trajectories. These diverge during the CFU-E stage, following upregulation of the transferrin receptor, CD71, and initiation of iron import, and right after upregulation of ALAS2, the first and rate-limiting step of heme synthesis. The normal (healthy) trajectory (trajectory A) contains cells at all stages, including the late stages of erythroid maturation, and there is high expression of mitochondrial oxidative metabolism and erythroid differentiation pathway genes. In contrast, the second trajectory (trajectory B) ends abruptly near the start of terminal erythroid differentiation as defined by cell surface proteins, and highly expresses gene pathways associated with excess heme and with apoptosis. There is loss of mitochondrial gene expression, premature loss of nuclear gene expression (increased % mitochondrial genes), and elevated levels of BAX and other apoptosis markers, implying that this is a death pathway. The significant increase in expression of genes in the heme-responsive dataset defined by Liao et al (Cell Rep. 2020. 31:107832) in trajectory B cells along with the pathway analysis, suggests that DBA and MDS-5q erythroid cells died from heme toxicity, a finding supportive of our earlier work (Yang et al. Sci Transl Med 2016. 8:338ra67). Cells along each trajectory were present in all samples but at different frequencies: 57±4% of MDS-5q, 73±2% of DBA, and 24±12% of normal cells followed trajectory B. That 24±12% of normal cells followed trajectory B was an unexpected finding, and indicated that heme might regulate the ineffective vs. effective differentiation of normal, as well as DBA and MDS-5q cells. As a corollary, drugs or interventions decreasing heme synthesis might modulate trajectory choice and be therapeutically beneficial. Also notably, fate decisions occur well before cell death, allowing time for therapeutic intervention. In addition, since we can independently track the fate and transcriptomes of cells with an intact chromosome 5q vs. cells with chromosome 5q deletion, these experiments provide insight into why anemia occurs early in MDS-5q (i.e., at presentation) often only 50-75% of the marrow is replaced by neoplastic cells. The analysis revealed both 5q- and 5q+ cells show early upregulation of HIF1A targets, ROS response, cell cycle regulation and spliceosome pathways and down regulation of erythroid differentiation and mTOR signaling pathways, indicating both 5q- and 5q+ cells share common pathologies. Additionally, the 5q- cells upregulate p53 pathways. The fact that the 5q+ cells are not normal implies that the presence of neoplastic 5q- cells influence the erythroid differentiation of the 5q+ cells. That they too have transcriptomic findings attributable to excessive heme may suggest a cell-extrinsic role for heme toxicity, which has led us to study heme processing by the central macrophage of erythroblastic islands (EBIs). Initial studies suggest EBI macrophage dysfunction contributes to the poor differentiation of 5q+ cells. This abstract includes data available on the medRxiv preprint server. https://doi.org/10.1101/2022.08.01.22278133

Characterization of Inborn Errors of Immunity in Recurrent Autoimmune Cytopenias in Children: A Report from Central California Cohort

Abstract Autoimmune cytopenias are characterized by autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), and/or autoimmune neutropenia. Evans syndrome (ES) is a condition characterized by simultaneous or sequential development of AIHA and ITP and/or immune neutropenia. Inborn errors of immunity driven multilineage cytopenias including ES requires further characterization to identify therapeutic targets. We evaluated our cohort of children with immune cytopenias to characterize mutations in inborn disorders of immunity and response to targeted therapy. To this end, we retrospectively reviewed the medical records of children between the ages of 0-21 years who presented to our hematology clinic with recurrent immune cytopenia between the years of 2018-2021. Rheumatological diseases, lymphoproliferative disorders, bone marrow failure syndromes, infections, drugs, toxins and metabolic disorders were excluded in the study. Children presenting with isolated idiopathic thrombocytopenic purpura or neutropenia were also excluded from the study. We utilized lymphocyte immunophenotyping and next generation sequencing involving 474 known genes (Invitae: Test code-08100) causing primary immunodeficiency in our pediatric patients with recurrent AIHA or multilineage immune cytopenias. Of the 59 patients with AIHA and multilineage immune cytopenias, 18 (30.5%) had recurrence of cytopenias after initial diagnosis. The age ranged from 1-20 years and 33% (n=6) were of the female gender (Table 1). AIHA (n=5, 28 %) and ES (n=5, 28%) were the most common presentation in patients with recurrent cytopenias. Autoimmune pancytopenia (n=4, 22.2%) was another common presentation. One child progressed to autoimmune pancytopenia after being diagnosed with AIHA. Pathogenic mutations in inborn errors of immunity were identified in 38.8% of the children. No mutations or variants were identified in 5 children (28%). Majority of patients received immunomodulatory treatments (Table 2) which resulted in remission (n=12, 66.6%). Immune cytopenias associated with Di-George syndrome were responsive to sirolimus with both children attaining remission. There was a death in the cohort related to the underlying condition. In conclusion, inborn errors of immunity were identified in recurrent immune cytopenias. Targeted therapies helped to achieve durable remission in children with specific mutations. Larger studies are warranted to understand the long-term therapeutic response in these children. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NOTCH1 Signaling Is Dysregulated in CLL By the Deubiquitinase USP28 That Is Recurrently Affected By Del(11q) and Can be Therapeutically Targeted

Active NOTCH1 signaling is present in approximately half of chronic lymphocytic leukemia (CLL) patients and was shown to be a marker for disease outcome. The NOTCH1 signaling pathway can be aberrantly activated through mutations in the NOTCH1 gene itself or co-factors such as FBXW7, resulting in protein stabilization of cleaved NOTCH1 (NICD). However, 50% of CLL cases with NOTCH1 activation do not harbor alterations in these genes, suggesting additional mechanisms for NOTCH1 hyperactivation. Since NICD can be stabilized due to defects in the ubiquitination/degradation machinery, we aimed to elucidate the role of the on chromosome 11q23 located deubiquitinase USP28 in the regulation of NOTCH1 activity and as a novel therapeutic target for NOTCH1-dependent CLL patients. First, we evaluated USP28 expression levels in a previously published cohort of 285 untreated CLL patients characterized via SNP array analysis and gene expression profiling (Edelmann et al, Blood 2012; Bloehdorn et al, Nat Commun 2021). The presence of monoallelic USP28 deletion was identified in 90% of CLL patients harboring a heterozygous deletion of chromosome 11q (del(11q); n=96), resulting in a significant decrease of USP28 RNA expression in comparison to non-del(11q) cases (n=199, P<0.0001). In non-del(11q) cases, USP28 expression levels were variable and assessment of protein levels of USP28 and its target proteins by western blotting in primary CLL cells (n=8) revealed a correlation between high protein levels of USP28 and remarkably high levels of NICD and other USP28 targets such as c-MYC or c-JUN. In addition, interaction of USP28 with NICD was shown by immunoprecipitation assays. In parallel, we implemented the CRISPR/Cas9 system to generate HG3 CLL-derived cell lines harboring USP28 biallelic deletion (HG3-USP28KO, n=5), monoallelic 11q deletion (HG3-del(11q), 17 Mb size including both ATM and USP28 genes, n=3), biallelic ATM truncation (HG3-ATMKO, n=3) or the combination of both events (HG3-del(11q) ATMKO, n=3). Overexpression of USP28 in HG3WT and HG3-USP28KO cells increased NOTCH1 activity measured by luciferase reporter assays (P<0.05, P<0.001, respectively). In contrast, in HG3-del(11q) cells decreased USP28 RNA and protein expression resulted in reduced NOTCH1 activity compared to HG3WT cells (P<0.05). Notably, HG3-del(11q) ATMKO cell lines showed even lower levels of NOTCH1 activity (P<0.01). Given that ATM phosphorylates USP28 (Zhang et al, Cell 2006), we hypothesized that in CLL cells ATM regulates USP28 activity and thereby also downstream NOTCH1 signaling. Indeed, NOTCH1 activity was reduced in HG3-ATMKO cells (P<0.01). However, USP28 overexpression in HG3-ATMKO cells did not lead to increased NOTCH1 signaling activity as observed in HG3WT cells. Moreover, pharmacological ATM inhibition (5 µM of KU-55933) also resulted in reduced NOTCH1 activity in HG3WT cells (P<0.0001) and reduced NICD levels in primary CLL cells (n=12; P<0.05). We finally explored the impact of the small molecule USP28 inhibitor AZ1 (alone or in combination with ibrutinib or venetoclax) on NICD protein levels and viability of primary CLL cells (n=15). AZ1 treatment (10 µM) resulted in decreased NICD levels, independent of mutations within NOTCH1 or FBXW7 (P<0.01). ATP quantification and 7-AAD staining revealed that increasing doses of AZ1 significantly reduced viability of NOTCH1/FBXW7-MUT primary cells (n=5) as well as of NOTCH1/FBXW7-WT cells (n=6) expressing high protein levels of USP28 and NICD (P<0.01; P<0.05, respectively). In contrast, AZ1 treatment did not affect viability of CLL patient cells expressing low NICD levels (n=4; P=0.56) Furthermore, AZ1 combined with venetoclax potentiated the reduction of cell viability especially in NOTCH1/FBXW7-MUT or patient cells expressing high USP28 and NICD levels (P<0.001; P<0.05). We propose that high levels of the deubiquitinase USP28 are responsible for hyperactivation of NOTCH1 signaling in CLL. This hypothesis is further confirmed in del(11q) CLL cells, where USP28 heterozygous deletion resulted in low NOTCH1 activity. In addition, our data reveal that USP28 activity is regulated by ATM, uncovering a novel mechanism of NOTCH1 modulation via the ATM-USP28-NOTCH1 axis (Figure 1). Finally, we demonstrate that USP28 inhibition by AZ1 suppresses USP28-mediated NOTCH1 activation and could represent a novel therapeutic strategy in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hypoparathyroidism and late-onset hypogonadism in an adult male with familial 22q11.2 deletion syndrome: a case report with 3-year follow-up and review of the literature

Background22q11.2 deletion syndrome (DiGeorge syndrome) is associated with multiple organ dysfunctions such as cardiac defects, immunodeficiency, and hypoplasia of parathyroid glands. Moreover, the phenotype of 22q11.2 DS has clinical variability and heterogeneity.Case presentationIn this report, we present the case of a 35-year-old patient with a past medical history that included recurrent infections, mild learning difficulties in childhood, pediatric obesity, and cataract. He was admitted to the endocrinology department for the management of hypogonadism and hypocalcemia. During the 3-year follow-up, the patient gradually developed primary hypoparathyroidism, hypogonadism, chronic renal failure, and heart failure, and his medical condition deteriorated. Meanwhile, in order to improve clinicians’ awareness of the endocrine manifestations of adult 22q11.2 DS and reduce missed diagnoses, we reviewed 28 case reports of adult 22q11.2 DS to analyze the clinical characteristics.DiscussionHere, we report the case of a young man diagnosed with 22q11.2 DS presented a rare combination of multiple endocrine disorders. This is the first time that a patient with 22q11.2DS had late-onset hypogonadism caused by primary testicular failure combined with decreased pituitary gonadotropin reserve in a patient with 22q11.2DS.