Background Inborn errors of immunity (IEI) and inherited bone marrow failure syndromes (IBMFS) are associated with an increased lifetime cancer risk. However, the role of most IEI/IBMFS in childhood cancer predisposition remains largely unexplored. This study investigated the potential contribution of germline variants in IEI/IBMFS-associated genes to pediatric hematological malignancy development. Methods We analyzed 151 children with leukemia or lymphoma for germline (likely) pathogenic variants in 541 IEI/IBMFS-associated genes. None had features indicative of IEI/IBMFS prior to their cancer diagnosis. Results Six patients (4 %) had monoallelic (likely) pathogenic variants in autosomal dominant genes (TNFRSF13B, MPL, AIRE, NLRP12). Thus far, these genes have no proven association with childhood cancer predisposition. The carrier frequency of monoallelic (likely) pathogenic variants in recessive genes was 33 %, considered to align with general population data. Twelve patients (8 %) carried variants in genes involved in DNA repair or chromosomal stability, half of which had been identified in previous work by our group or were known before the cancer diagnosis. Conclusion This study assessed for the first time in an unbiased and comprehensive manner the role of IEI/IBMFS in childhood hematological malignancy predisposition. Although the overall yield of this exploratory study was limited, our findings support the importance of research on childhood cancer predisposition at the intersection of hematological malignancies and IEI/IBMFS. We identified several variants in both dominant and recessive genes of which it would be interesting to investigate their causality. However, for now, sequencing of IEI/IBMFS-associated genes should be restricted to research context or in case of clinical suspicion.

Background Germline data have become widely available in paediatric oncology since the introduction of paired tumour-germline sequencing. To guide best practice in cancer predisposition syndrome (CPS) diagnostics, we aimed to assess the diagnostic yield of extensive germline analysis compared with clinical selection-based genetic testing among all children with cancer. Methods In this prospective diagnostic study, all children (aged 0-19 years) with newly diagnosed neoplasms treated in the Netherlands national centre, the Princess M & aacute;xima Center for Pediatric Oncology (Utrecht, Netherlands), between June 1, 2020, and July 31, 2022, were offered two approaches to identify CPSs. In a phenotype-driven approach, paediatric oncologists used the McGill Interactive Pediatric OncoGenetic Guidelines tool to select children for referral to a clinical geneticist, and for genetic testing. In a phenotype-agnostic approach, CPS gene panel sequencing (143 genes) was offered to all children. In children declining the research CPS gene panel, 49 CPS genes were still analysed as part of routine diagnostics by the pathologist. Children with a causative CPS identified before neoplasm diagnosis were excluded. The primary objective was to compare the number and type of patients diagnosed with a CPS between the two approaches. Findings 1052 children were eligible for this study, of whom 733 (70%) completed both the phenotype-driven approach and received phenotype-agnostic CPS gene panel sequencing (143 genes n=600; 49 genes n=133). In 53 children, a CPS was identified: 14 (26%) were diagnosed by the phenotype-driven approach only, 22 (42%) by CPS gene sequencing only, and 17 (32%) by both approaches. In 27 (51%) of the 53 children, the identified CPS was considered causative for the child's neoplasm. Only one (4%) of the 27 causative CPSs was missed by the phenotype-driven approach and was identified solely by phenotype-agnostic CPS gene sequencing. In 26 (49%) children, a CPS with uncertain causality was identified, including 14 adult-onset CPSs. The CPSs with uncertain causality were mainly detected by the phenotype-agnostic approach (21 [81%] of 26). Interpretation Phenotype-driven genetic testing and phenotype-agnostic CPS gene panel sequencing were complementary. The phenotype-driven approach identified the most causative CPSs. CPS gene panel sequencing identified additional CPSs, many of those with uncertain causality, but some with clinical utility. We advise clinical evaluation for CPSs in all children with neoplasms. Phenotype-agnostic testing of all CPS genes is preferably conducted only in research settings and should be paired with counseling. Copyright (c) 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.

Germline investigation in rare families with multiple affected individuals and large cohorts of pediatric patients with acute lymphoblastic leukemia (ALL) has resulted in the discovery of a growing number of leukemia predisposing genes [1, 2].A rare leukemia predisposition syndrome is caused by germline mutations in PAX5.Paired box 5 (PAX5) encodes for a paired box domain transcriptional factor essential for B-cell development [3].Approximately 30% of the pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor a somatic heterozygous loss-of-function alteration in PAX5 [4].Three germline missense variants in PAX5 have been described in seven families with a high incidence of BCP-ALL [5][6][7][8][9].The first germline PAX5 missense variant c.547G>A (p.Gly183Ser) was identified in four unrelated families [5,6,9].Functional testing showed that the PAX5 p.Gly183Ser variant has significantly reduced activity and results in deregulation of target genes, although the effect was milder than non-functional PAX5 mutants [5].A germline PAX5 missense variant affecting the same hotspot, c.547G>C (p.Gly183Arg), has been reported in one family [9].Two unrelated families were found to carry a germline c.113G>A (p.Arg38His) variant [7,8].Functional studies showed that PAX5 p.Arg38His is also a hypomorphic variant resulting in incomplete B-cell differentiation and is sufficient to predispose to leukemia [8].In all families, the susceptibility to BCP-ALL is inherited in an autosomal dominant pattern with incomplete penetrance.In the leukemia of the affected family members a second somatic alteration was detected in PAX5, either by loss of heterozygosity (LOH) or a second somatic mutation.We report a novel germline PAX5 alteration, a deletion including exon 6, in a boy who developed t(1;19)(q23;p13) (TCF3::PBX1) rearrangement-positive BCP-ALL at the age of 5. Somatic PAX5 aberrations are detected in ~20% of the cases with TCF3::PBX1 BCP-ALL [10].The patient was stratified in the standard risk group of the Dutch Childhood Oncology Group (DCOG) ALL11 treatment protocol and completed treatment without severe complications.He has been in follow up for three years.His father was diagnosed with acute undifferentiated leukemia at the age of 9 months.Cytogenetic testing of the leukemia of the father was not performed at time of diagnosis.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

PURPOSE:Wilms tumor (WT) is associated with (epi)genetic predisposing factors affecting a growing number of WT predisposing genes and loci, including those causing Beckwith-Wiedemann spectrum (BWSp) or WT1-related syndromes. To guide genetic counseling and testing, we need insight into the prevalence of WT predisposing (epi)genetic factors. PATIENTS AND METHODS:All children diagnosed with WT in the Netherlands between 2015 and 2020 were referred to a clinical geneticist. Phenotypic data, disease characteristics, and diagnostic test results were collected. If no genetic predisposition was identified by targeted diagnostic testing, germline (trio-)whole-exome sequencing and BWSp testing on normal kidney-derived DNA were offered. RESULTS:A total of 126 cases were analyzed of 128 identified patients. (Epi)genetic predisposing factors were present in 42 of 126 patients (33.3%) on the basis of a molecular diagnosis in blood-derived DNA (n = 26), normal kidney-derived DNA (n = 12), or solely a clinical diagnosis of BWSp (n = 4). Constitutional, heterozygous DIS3L2 variants were identified as a recurrent predisposing factor in five patients (4%), with a second somatic hit in 4 of 5 tumors. Twenty patients (16%) were diagnosed with BWSp while four additional patients without BWSp features harbored chromosome 11p15 methylation defects in normal kidney tissue. Remaining findings included WT1-related syndromes (n = 10), Fanconi anemia (n = 1), neurofibromatosis type 1 (n = 1), and a pathogenic REST variant (n = 1). In addition, (likely) pathogenic variants in adult-onset cancer predisposition genes (BRCA2, PMS2, CHEK2, and MUTYH) were identified in 5 of 56 (8.9%) patients with available whole-exome sequencing data. Several candidate WT predisposition genes were identified, which require further validation. CONCLUSION:(Epi)genetic WT predisposing factors, including mosaic aberrations and recurrent heterozygous DIS3L2 variants, were present in at least 33.3% of patients with WT. On the basis of these results, we encourage standard genetic testing after counseling by a clinical geneticist.

In a subset of pediatric cancers, a germline cancer predisposition is highly suspected based on clinical and pathological findings, but genetic evidence is lacking, which hampers genetic counseling and predictive testing in the families involved. We describe a family with two siblings born from healthy parents who were both neonatally diagnosed with atypical teratoid rhabdoid tumor (ATRT). This rare and aggressive pediatric tumor is associated with biallelic inactivation of SMARCB1, and in 30% of the cases, a predisposing germline mutation is involved. Whereas the tumors of both siblings showed loss of expression of SMARCB1 and acquired homozygosity of the locus, whole exome and whole genome sequencing failed to identify germline or somatic SMARCB1 pathogenic mutations. We therefore hypothesized that the insertion of a pathogenic repeat‐rich structure might hamper its detection, and we performed optical genome mapping (OGM) as an alternative strategy to identify structural variation in this locus. Using this approach, an insertion of ~2.8 kb within intron 2 of SMARCB1 was detected. Long‐range PCR covering this region remained unsuccessful, but PacBio HiFi genome sequencing identified this insertion to be a SINE‐VNTR‐Alu, subfamily E (SVA‐E) retrotransposon element, which was present in a mosaic state in the mother. This SVA‐E insertion disrupts correct splicing of the gene, resulting in loss of a functional allele. This case demonstrates the power of OGM and long‐read sequencing to identify genomic variations in high‐risk cancer‐predisposing genes that are refractory to detection with standard techniques, thereby completing the clinical and molecular diagnosis of such complex cases and greatly improving counseling and surveillance of the families involved. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

We describe a case of a boy with neurodevelopmental delay and a diffuse large B-cell lymphoma (DLBCL) in whom we discovered a germline de novo 2p16.3 deletion including MSH6 and part of the FBXO11 gene. A causative role for MSH6 in cancer development was excluded based on tumor characteristics. The constitutional FBXO11 deletion explains the neurodevelopmental delay in the patient. The FBXO11 protein is involved in BCL-6 ubiquitination and BCL-6 is required for the germinal center reaction resulting in B cell differentiation. Somatic loss of function alterations of FBXO11 result in BCL-6 overexpression which is a known driver in DLBCL. We therefore consider that a causative relationship between the germline FBXO11 deletion and the development of DLBCL in this boy is conceivable.

Minimal residual disease (MRD) diagnostics are implemented in most clinical protocols for patients with acute lymphoblastic leukaemia (ALL) and are mostly performed using rearranged immunoglobulin (IG) and/or T-cell receptor (TR) gene rearrangements as molecular polymerase chain reaction targets. Unfortunately, in 5-10% of patients no or no sensitive IG/TR targets are available, and patients therefore cannot be stratified appropriately. In the present study, we used fusion genes and genomic deletions as alternative MRD targets in these patients, which retrospectively revealed appropriate MDR stratification in 79% of patients with no (sensitive) IG/TR target, and a different risk group stratification in more than half of the cases.

Allele specific expression (ASE) concerns divergent expression quantity of alternative alleles and is measured by RNA sequencing. Multiple studies show that ASE plays a role in hereditary diseases by modulating penetrance or phenotype severity. However, genome diagnostics is based on DNA sequencing and therefore neglects gene expression regulation such as ASE. To take advantage of ASE in absence of RNA sequencing, it must be predicted using only DNA variation. We have constructed ASE models from BIOS (n = 3432) and GTEx (n = 369) that predict ASE using DNA features. These models are highly reproducible and comprise many different feature types, highlighting the complex regulation that underlies ASE. We applied the BIOS-trained model to population variants in three genes in which ASE plays a clinically relevant role: BRCA2, RET and NF1. This resulted in predicted ASE effects for 27 variants, of which 10 were known pathogenic variants. We demonstrated that ASE can be predicted from DNA features using machine learning. Future efforts may improve sensitivity and translate these models into a new type of genome diagnostic tool that prioritizes candidate pathogenic variants or regulators thereof for follow-up validation by RNA sequencing. All used code and machine learning models are available at GitHub and Zenodo.

Background and Aims: The low-density lipoprotein receptor (LDLR) in the liver is the major determinant of LDL-cholesterol levels in humans. The discovery of novel genes that regulate the activity of LDLR could lead to the identification of pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease.

Background One of the growing problems in genome diagnostics is the increasing number of variants that get identified through genetic testing but for which it is unknown what the significance for the disease is (Variants of Unknown Significance - VUS) 1,2 . When these variants are observed in patients, clinicians need to be able to determine their relevance for causing the patient’s disease. Here we investigated whether allele-specific expression (ASE) can be used to prioritize disease-relevant VUS and therefore assist diagnostics. In order to do so, we conducted ASE analysis in RNA-seq data from 3,818 blood samples (part of the the Dutch BIOS biobank consortium), to ascertain how VUS affect gene expression. We compared the effect of VUS variants to variants that are predicted to have a high impact, and variants that are predicted to be pathogenic but are either recessive or autosomal-dominant with low penetrance. Results For immune and haematological disorders, we observed that 24.7% of known pathogenic variants from ClinVar show allelic imbalance in blood, as compared to 6.6% of known benign variants with matching allele frequencies. However, for other types of disorders, ASE information from blood did not distinguish (likely) pathogenic variants from benign variants. Unexpectedly, we identified 5 genes ( ALOX5, COMT, PRPF8, PSTPIP1 and SH3BP2 ) in which seven population-based samples had a predicted high impact, autosomal-dominant variant. For these genes the imbalanced expression of the major allele compensates for the lower expression of the minor allele. Conclusions Our analysis in a large population-based gene expression cohort reveals examples of high impact, autosomal-dominant variants that are compensated for by imbalanced expression. Additionally, we observed that ASE analyses in blood are informative for predicting pathogenic variants that are associated with immune and haematological conditions. We have made all our ASE results, including many ASE calls for rare variants (MAF < 1%), available at https://molgenis15.gcc.rug.nl/ .