Exagamglogene Autotemcel for Severe Sickle Cell Disease.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A. METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).

Constitutional Microsatellite Instability, Genotype, and Phenotype Correlations in Constitutional Mismatch Repair Deficiency.

BACKGROUND & AIMS: Constitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsatellite instability (cMSI) is a CMMRD diagnostic hallmark and may associate with cancer risk. We quantified cMSI in a large CMMRD patient cohort to explore genotype-phenotype correlations using novel MSI markers selected for instability in blood. METHODS: Three CMMRD, 1 Lynch syndrome, and 2 control blood samples were genome sequenced to >120× depth. A pilot cohort of 8 CMMRD and 38 control blood samples and a blinded cohort of 56 CMMRD, 8 suspected CMMRD, 40 Lynch syndrome, and 43 control blood samples were amplicon sequenced to 5000× depth. Sample cMSI score was calculated using a published method comparing microsatellite reference allele frequencies with 80 controls. RESULTS: Thirty-two mononucleotide repeats were selected from blood genome and pilot amplicon sequencing data. cMSI scoring using these MSI markers achieved 100% sensitivity (95% CI, 93.6%-100.0%) and specificity (95% CI 97.9%-100.0%), was reproducible, and was superior to an established tumor MSI marker panel. Lower cMSI scores were found in patients with CMMRD with MSH6 deficiency and patients with at least 1 mismatch repair missense variant, and patients with biallelic truncating/copy number variants had higher scores. cMSI score did not correlate with age at first tumor. CONCLUSIONS: We present an inexpensive and scalable cMSI assay that enhances CMMRD detection relative to existing methods. cMSI score is associated with mismatch repair genotype but not phenotype, suggesting it is not a useful predictor of cancer risk.

The German sickle cell disease registry reveals a surprising risk of acute splenic sequestration and an increased transfusion requirement in patients with compound heterozygous sickle cell disease HbS/ß-thalassaemia and no or low HbA expression.

Patients with sickle cell disease (SCD) in Germany exhibit a substantial genetic diversity in the ß-globin genotype. Data collected by the national German SCD registry reflect this diversity and allowed us to analyze the phenotypes associated with different SCD genotypes. Our study focused on 90 patients with HbS/ß-thalassaemia (HbS/ß-thal) and compared these to patients with HbSS and HbSC. Patients with HbS/ß-thal were classified into three groups: HbS/ß0-thal (no HbA), HbS/ß+-thal (HbA < 14%), and HbS/ß++-thal (HbA≥14%). In comparison to HbSS, patients with HbS/ß++-thal had higher Hb-levels, lower hemolytic activity and rarely required red blood cell transfusions. HbS/ß0-thal and HbS/ß+-thal closely resembled each other and are jointly referred to as HbS/ß0/+-thal. Compared to HbSS, patients with HbS/ß0/+-thal experienced a similar frequency of vasoocclusive crises and degree of hemolysis. However, the frequency of red blood cell transfusions (0.6 vs. 0.39/year, p = .0049) and splenic sequestration crises (42.4 vs. 15.5% of patients, p = 3.799e-05) was higher in HbS/ß0/+-thal than in HbSS, but close to zero in HbS/ß++-thal. In conclusion, the level of HbA expression determines the phenotype of HbS/ß+-thal. HbS/ß-thal expressing no or little HbA is hematologically similar to HbSS, but causes a previously unknown high risk of splenic sequestration.

Genetic modifiers of fetal hemoglobin affect the course of sickle cell disease in patients treated with hydroxyurea.

The course of sickle cell disease (SCD) is modified by polymorphisms boosting fetal hemoglobin (HbF) synthesis. However, it has remained an open question how these polymorphisms affect patients who are treated with the HbF-inducing drug hydroxyurea/ hydroxycarbamide. The German SCD registry offers the opportunity to answer this question, because >90% of patients are treated according to national guidelines recommending the use of hydroxyurea in all patients above 2 years of age. We analyzed the modifying effect of HbF-related genetic polymorphisms in 417 patients with homozygous SCD >2 years old who received hydroxyurea. HbF levels were correlated with higher total hemoglobin levels, lower rates of hemolysis, a lower frequency of painful crises and of red blood cell transfusions. The minor alleles of the polymorphisms in the γ-globin promoter (rs7482144), BCL11A (rs1427407) and HMIP (rs66650371) were strongly associated with increased HbF levels. However, these associations did not translate into lower frequencies of vaso-occlusive events which did not differ between patients either carrying or not carrying the HMIP and BCL11A polymorphisms. Patients on hydroxyurea carrying the γ-globin promoter polymorphism demonstrated substantially higher hemoglobin levels (P<10-4) but also higher frequencies of painful crises and hospitalizations (P<0.01) when compared to patients without this polymorphism. Taken together, these data indicate that the γ-globin, HMIP and BCL11A polymorphisms correlate with increased HbF in SCD patients on hydroxyurea. While HbF is negatively correlated with the frequency of painful crises and hospitalizations, this was not observed for the presence of known HbF-boosting alleles.

Complete Tracheal Ring Deformity, Recurrent Pneumothoraces and Pleuropulmonary Blastoma in a Child: Coincidence or Common Genetic Cause?

Complete tracheal ring deformity (CTRD) is a rare abnormality of unknown etiology characterized by circumferentially continuous cartilaginous tracheal rings leading to variable degrees of tracheal stenosis with or without additional heart and lung malformations. Pleuropulmonary blastomas (PPB) are rare malignant mesenchymal tumors, which occur almost exclusively in young children. Pathogenic germline DICER1 variants are associated with PPB but also with other tumors like rhabdomyosarcoma or syndromic diseases like GLOW (Global developmental delay, lung cysts, overgrowth and Wilms tumor) syndrome. Here, we report a case with CTRD and recurrent pneumothoraces who additionally developed PPB on the genetic background of a pathogenic DICER1 variant.

High-sensitivity microsatellite instability assessment for the detection of mismatch repair defects in normal tissue of biallelic germline mismatch repair mutation carriers.

INTRODUCTION: Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are hereditary cancer syndromes associated with mismatch repair (MMR) deficiency. Tumours show microsatellite instability (MSI), also reported at low levels in non-neoplastic tissues. Our aim was to evaluate the performance of high-sensitivity MSI (hs-MSI) assessment for the identification of LS and CMMRD in non-neoplastic tissues. MATERIALS AND METHODS: Blood DNA samples from 131 individuals were grouped into three cohorts: baseline (22 controls), training (11 CMMRD, 48 LS and 15 controls) and validation (18 CMMRD and 18 controls). Custom next generation sequencing panel and bioinformatics pipeline were used to detect insertions and deletions in microsatellite markers. An hs-MSI score was calculated representing the percentage of unstable markers. RESULTS: The hs-MSI score was significantly higher in CMMRD blood samples when compared with controls in the training cohort (p<0.001). This finding was confirmed in the validation set, reaching 100% specificity and sensitivity. Higher hs-MSI scores were detected in biallelic MSH2 carriers (n=5) compared with MSH6 carriers (n=15). The hs-MSI analysis did not detect a difference between LS and control blood samples (p=0.564). CONCLUSIONS: The hs-MSI approach is a valuable tool for CMMRD diagnosis, especially in suspected patients harbouring MMR variants of unknown significance or non-detected biallelic germline mutations.

Sickle cell disease in Germany: Results from a national registry.

BACKGROUND: Limited data on the prevalence and medical care of sickle cell disease (SCD) in Germany are available. Here, we make use of a patient registry to characterize the burden of disease and the treatment modalities for patients with SCD in Germany. PROCEDURE: A nationwide German registry for patients with SCD documents basic data on diagnosis and patient history retrospectively at the time of registration. A prospective annual documentation provides more details on complications and treatment of SCD. For the current analyses, data of 439 patients were available. RESULTS: Most patients had homozygous SCD (HbSS 75.1%, HbS/ß-thalassemia 13.2%, and HbSC 11.3%). The median age at diagnosis was 1.9 years (interquartile range, 0.6-4.4 years), most patients were diagnosed when characteristic symptoms occurred. Sepsis and stroke had affected 3.2% and 4.2% of patients, respectively. During the first year of observation, 48.3% of patients were admitted to a hospital and 10.1% required intensive care. Prophylactic penicillin was prescribed to 95.6% of patients with homozygous SCD or HbS/ß thalassemia below the age of six and hydroxycarbamide to 90.4% of patients above the age of two years. At least one annual transcranial Doppler ultrasound was documented for 74.8% of patients between 2 and 18 years. CONCLUSION: With an estimated number of at least 2000, the prevalence of SCD in Germany remains low. Prospectively, we expect that the quality of care for children with SCD will be further improved by an earlier diagnosis after the anticipated introduction of a newborn screening program for SCD.

A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes.

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.

Newborn screening by tandem mass spectrometry confirms the high prevalence of sickle cell disease among German newborns.

Sickle cell disease (SCD) is a severe inherited blood disorder associated with significant morbidity and mortality in early childhood. Since simple interventions are available to prevent early fatal courses, SCD is a target condition of several national newborn screening (NBS) programs worldwide, but not in Germany. Traditionally, the diagnosis of SCD is made by high-performance liquid chromatography (HPLC), isoelectric focusing (IEF), or capillary electrophoresis (CE), but globally, most NBS programs in place are based on tandem mass spectrometry (MS/MS). Recently, several publications have shown that MS/MS is an appropriate screening technique to detect hemoglobin patterns suggestive of SCD in newborns, too. We have studied dried blood spot samples of 29,079 German newborns by both CE and MS/MS and observed a 100% congruence of test results. Seven babies had hemoglobin patterns characteristic of SCD (1:4154). Our study confirms that (a) the suitability of MS/MS as an adequate substitute for CE in NBS for SCD and (b) the high prevalence of SCD among German newborns. Our results support the thesis that German newborns should be screened for SCD by MS/MS.

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

Cognitive and fine motor deficits in a pediatric sickle cell disease cohort of mixed ethnic origin.

Cerebrovascular disease is an important feature of pediatric sickle cell disease (SCD) and may lead to cognitive and motor impairment. Our cross-sectional study examined the incidence and severity of these impairments in a pediatric cohort without clinical cerebrovascular events from Berlin of mixed ethnic origin. Thirty-two SCD patients (mean age 11.14 years, range 7.0-17.25 years; males 14) were evaluated for full-scale intelligence (IQ) (German version WISC-III), fine motor function (digital writing tablet), and executive function (planning, attention, working memory, and visual-spatial abilities) with the Amsterdam Neuropsychological Tasks (ANT) program and the Tower of London (ToL). Data on clinical risk factors were retrieved from medical records. Full-scale IQ of patients was preserved, whereas performance IQ was significantly reduced (91.19 (SD 12.17) d = 0.7, p = 0.007). SCD patients scored significantly lower than healthy peers when tested for executive and fine motor functions, e.g., planning time in the ToL (6.73 s (SD 3.21) vs. 5.9 s in healthy peers (SD 2.33), d = 0.5, p = <0.001) and frequency on the writing tablet (mean z score -0.79, d = 0.7, p < 0.001). No clinical risk factors were significantly associated with incidence and severity of cognitive and motor deficits. Despite the preservation of full-scale IQ, our SCD cohort of mixed origin exhibited inferior executive abilities and reduced fine motor skills. Our study is limited by the small size of our cohort as well as the lack for control of sociodemographic and socioeconomic factors modulating higher functions but highlights the need for early screening, prevention, and specific interventions for these deficits.

The epidemiology of sickle cell disease in Germany following recent large-scale immigration.

BACKGROUND: The epidemiology of sickle cell disease (SCD) in Germany is currently changing fundamentally with ongoing immigration. Here, we address the challenges resulting from the increased frequency, that is, the morbidity, and mortality of SCD in this population. PROCEDURE: The number of immigrants with SCD was estimated based on the data of the German central registry of migrants (2007-2015) and published epidemiologic data. Additional data analysis was based on nationwide aggregated data from the diagnosis-related groups' (DRG) statistics of the German Federal Statistical Office. RESULTS: The total number of patients with SCD among migrants was estimated at 2,016 in 2007 and 3,216 in 2015, thus showing a 60% increase, which was particularly remarkable during 2014 and 2015. The countries of origin included those of West sub-Saharan Africa, followed by Syria, and other countries of the Middle East. In parallel, the number of SCD inpatient treatments increased from 780 in 2002 to 1,340 in 2015. Between 2012 and 2014, 42 patients with SCD died in hospital, mostly at an age of less than 5 years (n = 7) or over 30 years (n = 29). CONCLUSION: More than 3,000 patients with SCD are estimated to live among the immigrant population in Germany. In addition, the number of SCD patients of German nationality is not known. The increasing number of inpatient treatments and the death of young children from SCD indicate the need for a general newborn screening program and an increased awareness of this disease among medical practitioners in a country in which SCD used to be rare.

Guido Fanconi (1892-1979): a jack of all trades.

In 1927 the Swiss paediatrician Guido Fanconi described a family in which three boys had physical birth defects and died of a condition that resembled pernicious anaemia. In the 1960, inspired by Fanconi's theoretical considerations, it was shown that the disorder is based on an underlying chromosomal instability and is associated with a predisposition to bone marrow failure and cancer. As the 80th anniversary of the first description of Fanconi anaemia approaches, we were motivated to pay tribute to Guido Fanconi as an outstanding figure in European medicine and to honour his contribution to cancer research.

Gene Therapy in Transfusion-Dependent Non-ß0/ß0 Genotype ß-Thalassemia: First Real-World Experience of Beti-cel.

Gene addition and editing strategies for transfusion-dependent ß-thalassemia have gained momentum as potentially curative treatment options, with studies showcasing their efficacy and safety. We report the first real-world application of betibeglogene autotemcel (beti-cel; ZYNTEGLO™) during its period of active license in Europe from January 2020 to March 2022 for patients aged ≥ 12 years without a ß0/ß0 genotype and without a human leukocyte antigen (HLA)-matched sibling donor, before beti-cel marketing authorization was withdrawn by its holder due to non-safety reasons. Among 15 screened patients, 4 opted out for fertility and safety concerns, 2 were excluded because of marked hepatic siderosis, and 1 had apheresis collection failure. Eight patients received beti-cel post busulfan myeloablative conditioning, all achieving transfusion independence within 8 to 59 days with posttreatment hemoglobin levels ranging from 11.3 to 19.3 g/dL. No deaths occurred, but acute toxicity mirrored busulfan's known effects. Posttreatment platelet management faced challenges due to HLA-antibodies in 3 patients. Monitoring up to Month 24 revealed pituitary-gonadal endocrine dysfunction in all 3 female and in 2 of 5 male patients. Additionally, we observed unexpected posttreatment sequelae: 1 patient developed polycythemia that could not be explained by known genetic or acquired mechanisms, 1 patient developed posttreatment depression and anxiety prohibiting her from returning to work, and 1 patient developed fatigue severely compromising both quality of life and work capacity. This real-world experience corroborates beti-cel's efficacy and safety and provides information on adverse events observed during real-world use of the therapy.

Sickle cell disease landscape and challenges in the EU: the ERN-EuroBloodNet perspective.

Sickle cell disease is a hereditary multiorgan disease that is considered rare in the EU. In 2017, the Rare Diseases Plan was implemented within the EU and 24 European Reference Networks (ERNs) were created, including the ERN on Rare Haematological Diseases (ERN-EuroBloodNet), dedicated to rare haematological diseases. This EU initiative has made it possible to accentuate existing collaborations and create new ones. The project also made it possible to list all the needs of people with rare haematological diseases not yet covered health-care providers in the EU to allow optimised care of individuals with rare pathologies, including sickle cell disease. This Viewpoint is the result of joint work within 12 EU member states (ie, Belgium, Cyprus, Denmark, France, Germany, Greece, Ireland, Italy, Portugal, Spain, Sweden, and The Netherlands), all members of the ERN-EuroBloodNet. We describe the role of the ERN-EuroBloodNet to improve the overall approach to and the management of individuals with sickle cell disease in the EU through specific on the pooling of expertise, knowledge, and best practices; the development of training and education programmes; the strategy for systematic gathering and standardisation of clinical data; and its reuse in clinical research. Epidemiology and research strategies from ongoing implementation of the Rare Anaemia Disorders European Epidemiological Platform is depicted.

Introduction of Universal Newborn Screening for Sickle Cell Disease in Germany-A Brief Narrative Review.

Sickle cell disease (SCD) is a severe non-malignant disorder of hemoglobin and is inherited in an autosomal-recessive manner [...].

Benefits of a Disease Management Program for Sickle Cell Disease in Germany 2011-2019: The Increased Use of Hydroxyurea Correlates with a Reduced Frequency of Acute Chest Syndrome.

Sickle Cell Disease (SCD) is the most common monogenic disorder globally but qualifies as a rare disease in Germany. In 2012, the German Society for Paediatric Oncology and Haematology (GPOH) mandated a consortium of five university hospitals to develop a disease management program for patients with SCD. Besides other activities, this consortium issued treatment guidelines for SCD that strongly favour the use of hydroxyurea and propagated these guidelines in physician and patient education events. In order to quantify the effect of these recommendations, we made use of claims data that were collected by the research institute (WIdO) of the major German insurance company, the Allgemeine Ortskrankenkasse (AOK), and of publicly accessible data collected by the Federal Statistical Office (Statistisches Bundesamt, Destatis). While the number of patients with SCD in Germany increased from approximately 2200 in 2011 to approximately 3200 in 2019, important components of the recently issued treatment guidelines have been largely implemented. Specifically, the use of hydroxyurea has more than doubled, resulting in a proportion of approximately 44% of all patients with SCD being treated with hydroxyurea in 2019. In strong negative correlation with the use of hydroxyurea, the frequency of acute chest syndromes decreased. Similarly, the proportion of patients who required analgesics and hospitals admissions declined. In sum, these data demonstrate an association between the dissemination of treatment guidelines and changes in clinical practice. The close temporal relationship between the increased use of hydroxyurea and the reduction in the incidence of acute chest syndrome in a representative population-based analysis implies that these changes in clinical practice contributed to an improvement in key measures of disease activity.

Thalassemias: An Overview.

Thalassemia syndromes are among the most serious and common genetic conditions. They are indigenous in a wide but specific geographical area. However, through migration they are spreading across regions not previously affected. Thalassemias are caused by mutations in the α (HBA1/HBA2) and ß globin (HBB) genes and are usually inherited in an autosomal recessive manner. The corresponding proteins form the adult hemoglobin molecule (HbA) which is a heterotetramer of two α and two ß globin chains. Thalassemia-causing mutations lead to an imbalanced globin chain production and consecutively to impaired erythropoiesis. The severity of the disease is largely determined by the degree of chain imbalance. In the worst case, survival is dependent on regular blood transfusions, which in turn cause transfusional iron overload and secondary multi-organ damage due to iron toxicity. A vigorous monitoring and treatment regime is required, even for the milder syndromes. Thalassemias are a major public health issue in many populations which many health authorities fail to address. Even though comprehensive care has resulted in long-term survival and good quality of life, poor access to essential components of management results in complications which increase the cost of treatment and lead to poor outcomes. These requirements are not recognized by measures such as the Global Burden of Disease project, which ranks thalassemia very low in terms of disability-adjusted life years (DALYs), and fails to consider that it ranks highly in the one to four-year-old age group, making it an important contributor to under-5 mortality. Thalassemia does not fulfil the criteria to be accepted as a target disease for neonatal screening. Nevertheless, depending on the screening methodology, severe cases of thalassemia will be detected in most neonatal screening programs for sickle cell disease. This is very valuable because: (1) it helps to prepare the affected families for having a sick child and (2) it is an important measure of secondary prevention.