Update on surveillance for Wilms tumor and hepatoblastoma in Beckwith-Wiedemann Syndrome and other predisposition syndromes.

Wilms tumors are commonly associated with predisposition syndromes many, but not all, of which include overgrowth. Several of these syndromes also include a risk of other embryonal malignancies - particularly hepatoblastoma. Guidelines for surveillance in this population were published in 2017 and recently members of the AACR Pediatric Cancer Working Group met to update those guidelines with a review of more recently published evidence and risk estimates. This perspective serves to update pediatric oncologists, geneticists, radiologists, counselors and other healthcare professionals on revised diagnostic criteria, review previously published surveillance guidelines and harmonize updated surveillance recommendations in the North American and Australian context for patients with overgrowth syndromes and other syndromes associated with Wilms tumor predisposition.

Update on Pediatric Cancer Surveillance Recommendations for Patients with Neurofibromatosis Type 1, Noonan Syndrome, CBL Syndrome, Costello Syndrome, and Related RASopathies.

Neurofibromatosis type 1 (NF1), Noonan syndrome and related syndromes, grouped as the RASopathies, result from dysregulation of the RAS-MAPK pathway and demonstrate varied multisystemic clinical phenotypes. Together the RASopathies are among the more prevalent genetic cancer predisposition syndromes and require nuanced clinical management. When compared to the general population, children with RASopathies are at significantly increased risk of benign and malignant neoplasms. In the last decade, clinical trials have shown that targeted therapies can improve outcomes for low-grade and benign neoplastic lesions but have their own challenges, highlighting the multi-disciplinary care needed for such individuals, specifically those with NF1. This perspective, which originated from the 2023 AACR Childhood Cancer Predisposition Workshop, serves to update pediatric oncologists, neurologists, geneticists, counselors, and other healthcare professionals on revised diagnostic criteria, review previously published surveillance guidelines, and harmonize updated surveillance recommendations for patients with NF1 or RASopathies.

Sirolimus for vascular anomalies associated with PTEN hamartoma tumor syndrome.

Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is a rare condition associated with vascular anomalies and increased tumor risk. Sirolimus, an mTOR inhibitor used for managing vascular anomalies is underexplored in PHTS. A single-institution retrospective review of children with PHTS and vascular anomalies treated with sirolimus identified seven patients. Median age at sirolimus initiation was 10 years. After a median 2.5-year follow-up, six of seven patients (86%) showed significant clinical improvement. No significant adverse effects were observed, except mild buccal ulcers and acne. This study supports sirolimus as an effective and safe treatment for vascular anomalies in a small group of children with PHTS.

Neuroblastoma Predisposition and Surveillance-An Update from the 2023 AACR Childhood Cancer Predisposition Workshop.

Genetic predisposition to neuroblastoma (NB) is relatively rare. Only 1% to 2% of patients have a family history of NB, 3% to 4% of cases present with bilateral or multifocal primary tumors, and occasional patients have syndromes that are associated with increased NB risk. Previously, a germline pathogenic variant (GPV) in PHOX2B was associated with Hirschsprung disease and congenital central hypoventilation syndrome. Recently, certain GPVs were shown to be responsible for congenital central hypoventilation syndrome and NB predisposition. Also, several groups determined that activating GPVs in ALK accounted for a substantial number of familial NB. Finally, there are additional genes and cancer predisposition syndromes in which NB occurs with greater frequency or that have been associated with NB based on genome-wide association studies. We review the evidence for all these genes and whether there is sufficient evidence to warrant surveillance. We review recommended surveillance for hereditary patients with NB, including minor updates to surveillance recommendations that were published previously in 2017.

Update on Cancer Predisposition Syndromes and Surveillance Guidelines for Childhood Brain Tumors.

Tumors of the central nervous system (CNS) comprise the second most common group of neoplasms in childhood. The incidence of germline predisposition among children with brain tumors continues to grow as our knowledge on disease etiology increases. Some children with brain tumors may present with nonmalignant phenotypic features of specific syndromes (e.g., nevoid basal cell carcinoma syndrome, neurofibromatosis type 1 and type 2, DICER1 syndrome, and constitutional mismatch-repair deficiency), while others may present with a strong family history of cancer (e.g., Li-Fraumeni syndrome) or with a rare tumor commonly found in the context of germline predisposition (e.g., rhabdoid tumor predisposition syndrome). Approximately 50% of patients with a brain tumor may be the first in a family identified to have a predisposition. The past decade has witnessed a rapid expansion in our molecular understanding of CNS tumors. A significant proportion of CNS tumors are now well characterized and known to harbor specific genetic changes that can be found in the germline. Additional novel predisposition syndromes are also being described. Identification of these germline syndromes in individual patients has not only enabled cascade testing of family members and early tumor surveillance but also increasingly affected cancer management in those patients. Therefore, the AACR Cancer Predisposition Working Group chose to highlight these advances in CNS tumor predisposition and summarize and/or generate surveillance recommendations for established and more recently emerging pediatric brain tumor predisposition syndromes.

A homozygous stop codon in HORMAD2 in a patient with recurrent digynic triploid miscarriage.

BACKGROUND: Recurrent miscarriage (RM) affects 1% to 5% of couples trying to conceive. Despite extensive clinical and laboratory testing, half of the RM cases remain unexplained. We report the genetic analysis of a couple with eight miscarriages and the search for their potential genetic etiology. METHODS: Short tandem repeat (STR) markers, single nucleotide polymorphic (SNP) microarray, and human DNA methylation microarray were used to analyze the genotypes of two miscarriages. Exomes sequencing was performed on DNA from the two partners and identified variants were validated by Sanger sequencing. RESULTS: STR marker genotyping demonstrated that the two available miscarriages are triploid digynic and resulted from the failure of Meiosis II. SNP microarray analysis revealed an additional Meiosis I abnormality that is the segregation of the two maternal homologous chromosomes in one triploid miscarriage. Whole-exome sequencing on DNA from the two partners identified candidate variants only in the female partner in two genes with roles in female reproduction, a missense in EIF4ENIF1 (OMIM 607445) and a stop gain in HORMAD2 (OMIM 618842). EIF4ENIF1 is a eukaryotic translation initiation factor 4E nuclear import factor required for the oocyte germinal vesicle breakdown, and HORMAD2 is part of the synaptonemal complex that was hypothesized to act as a checkpoint mechanism to eliminate oocytes with asynapsis during meiotic prophase I in mice. CONCLUSION: While both genes may contribute to the phenotype, the Meiosis I abnormalities in the conceptions favor the causal role of HORMAD2 in the etiology of RM in this couple. This report illustrates the importance of comprehensively analyzing the products of conception to guide the search for the genetic causation of RM.

A new blood DNA methylation signature for Koolen-de Vries syndrome: Classification of missense KANSL1 variants and comparison to fibroblast cells.

Pathogenic variants in KANSL1 and 17q21.31 microdeletions are causative of Koolen-de Vries syndrome (KdVS), a neurodevelopmental syndrome with characteristic facial dysmorphia. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes have identifiable patterns of DNA methylation (DNAm) change: DNAm signatures or episignatures. Given the role of KANSL1 in histone acetylation, we tested whether variants underlying KdVS are associated with a DNAm signature. We profiled whole-blood DNAm for 13 individuals with KANSL1 variants, four individuals with 17q21.31 microdeletions, and 21 typically developing individuals, using Illumina's Infinium EPIC array. In this study, we identified a robust DNAm signature of 456 significant CpG sites in 8 individuals with KdVS, a pattern independently validated in an additional 7 individuals with KdVS. We also demonstrate the diagnostic utility of the signature and classify two KANSL1 VUS as well as four variants in individuals with atypical clinical presentation. Lastly, we investigated tissue-specific DNAm changes in fibroblast cells from individuals with KdVS. Collectively, our findings contribute to the understanding of the epigenetic landscape related to KdVS and aid in the diagnosis and classification of variants in this structurally complex genomic region.

Molecular characterization of 13 patients with PIK3CA-related overgrowth spectrum using a targeted deep sequencing approach.

Activating variants in the PIK3CA gene cause a heterogeneous spectrum of disorders that involve congenital or early-onset segmental/focal overgrowth, now referred to as PIK3CA-related overgrowth spectrum (PROS). Historically, the clinical diagnoses of patients with PROS included a range of distinct syndromes, including CLOVES syndrome, dysplastic megalencephaly, hemimegalencephaly, focal cortical dysplasia, Klippel-Trenaunay syndrome, CLAPO syndrome, fibroadipose hyperplasia or overgrowth, hemihyperplasia multiple lipomatosis, and megalencephaly capillary malformation-polymicrogyria (MCAP) syndrome. MCAP is a sporadic overgrowth disorder that exhibits core features of progressive megalencephaly, vascular malformations, distal limb malformations, cortical brain malformations, and connective tissue dysplasia. In 2012, our research group contributed to the identification of predominantly mosaic, gain-of-function variants in PIK3CA as an underlying genetic cause of the syndrome. Mosaic variants are technically more difficult to detect and require implementation of more sensitive sequencing technologies and less stringent variant calling algorithms. In this study, we demonstrated the utility of deep sequencing using the Illumina TruSight Oncology 500 (TSO500) sequencing panel in identifying variants with low allele fractions in a series of patients with PROS and suspected mosaicism: pathogenic, mosaic PIK3CA variants were identified in all 13 individuals, including 6 positive controls. This study highlights the importance of screening for low-level mosaic variants in PROS patients. The use of targeted panels with deep sequencing in clinical genetic testing laboratories would improve diagnostic yield and accuracy within this patient population.

Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies.

Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. We have identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. Our results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

Peripheral blood DNA methylation and neuroanatomical responses to HDACi treatment that rescues neurological deficits in a Kabuki syndrome mouse model.

BACKGROUND: Recent findings from studies of mouse models of Mendelian disorders of epigenetic machinery strongly support the potential for postnatal therapies to improve neurobehavioral and cognitive deficits. As several of these therapies move into human clinical trials, the search for biomarkers of treatment efficacy is a priority. A potential postnatal treatment of Kabuki syndrome type 1 (KS1), caused by pathogenic variants in KMT2D encoding a histone-lysine methyltransferase, has emerged using a mouse model of KS1 (Kmt2d+/ßGeo). In this mouse model, hippocampal memory deficits are ameliorated following treatment with the histone deacetylase inhibitor (HDACi), AR-42. Here, we investigate the effect of both Kmt2d+/ßGeo genotype and AR-42 treatment on neuroanatomy and on DNA methylation (DNAm) in peripheral blood. While peripheral blood may not be considered a "primary tissue" with respect to understanding the pathophysiology of neurodevelopmental disorders, it has the potential to serve as an accessible biomarker of disease- and treatment-related changes in the brain. METHODS: Half of the KS1 and wildtype mice were treated with 14 days of AR-42. Following treatment, fixed brain samples were imaged using MRI to calculate regional volumes. Blood was assayed for genome-wide DNAm at over 285,000 CpG sites using the Illumina Infinium Mouse Methylation array. DNAm patterns and brain volumes were analyzed in the four groups of animals: wildtype untreated, wildtype AR-42 treated, KS1 untreated and KS1 AR-42 treated. RESULTS: We defined a DNAm signature in the blood of KS1 mice, that overlapped with the human KS1 DNAm signature. We also found a striking 10% decrease in total brain volume in untreated KS1 mice compared to untreated wildtype, which correlated with DNAm levels in a subset KS1 signature sites, suggesting that disease severity may be reflected in blood DNAm. Treatment with AR-42 ameliorated DNAm aberrations in KS1 mice at a small number of signature sites. CONCLUSIONS: As this treatment impacts both neurological deficits and blood DNAm in mice, future KS clinical trials in humans could be used to assess blood DNAm as an early biomarker of therapeutic efficacy.

Multiple Germline Events Contribute to Cancer Development in Patients with Li-Fraumeni Syndrome.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer-predisposition disorder. Approximately 70% of individuals who fit the clinical definition of LFS harbor a pathogenic germline variant in the TP53 tumor suppressor gene. However, the remaining 30% of patients lack a TP53 variant and even among variant TP53 carriers, approximately 20% remain cancer-free. Understanding the variable cancer penetrance and phenotypic variability in LFS is critical to developing rational approaches to accurate, early tumor detection and risk-reduction strategies. We leveraged family-based whole-genome sequencing and DNA methylation to evaluate the germline genomes of a large, multi-institutional cohort of patients with LFS (n = 396) with variant (n = 374) or wildtype TP53 (n = 22). We identified alternative cancer-associated genetic aberrations in 8/14 wildtype TP53 carriers who developed cancer. Among variant TP53 carriers, 19/49 who developed cancer harbored a pathogenic variant in another cancer gene. Modifier variants in the WNT signaling pathway were associated with decreased cancer incidence. Furthermore, we leveraged the noncoding genome and methylome to identify inherited epimutations in genes including ASXL1, ETV6, and LEF1 that confer increased cancer risk. Using these epimutations, we built a machine learning model that can predict cancer risk in patients with LFS with an area under the receiver operator characteristic curve (AUROC) of 0.725 (0.633-0.810). Significance: Our study clarifies the genomic basis for the phenotypic variability in LFS and highlights the immense benefits of expanding genetic and epigenetic testing of patients with LFS beyond TP53. More broadly, it necessitates the dissociation of hereditary cancer syndromes as single gene disorders and emphasizes the importance of understanding these diseases in a holistic manner as opposed to through the lens of a single gene.

Imprinting disorders.

Imprinting disorders (ImpDis) are congenital conditions that are characterized by disturbances of genomic imprinting. The most common individual ImpDis are Prader-Willi syndrome, Angelman syndrome and Beckwith-Wiedemann syndrome. Individual ImpDis have similar clinical features, such as growth disturbances and developmental delay, but the disorders are heterogeneous and the key clinical manifestations are often non-specific, rendering diagnosis difficult. Four types of genomic and imprinting defect (ImpDef) affecting differentially methylated regions (DMRs) can cause ImpDis. These defects affect the monoallelic and parent-of-origin-specific expression of imprinted genes. The regulation within DMRs as well as their functional consequences are mainly unknown, but functional cross-talk between imprinted genes and functional pathways has been identified, giving insight into the pathophysiology of ImpDefs. Treatment of ImpDis is symptomatic. Targeted therapies are lacking owing to the rarity of these disorders; however, personalized treatments are in development. Understanding the underlying mechanisms of ImpDis, and improving diagnosis and treatment of these disorders, requires a multidisciplinary approach with input from patient representatives.

Missense variant in SRCAP with distinct DNA methylation signature associated with non-FLHS SRCAP-related neurodevelopmental disorder.

Floating-Harbor syndrome (FLHS) is a neurodevelopmental disorder (NDD) caused by truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein gene (SRCAP). Truncating variants proximal to this location in SRCAP result in a non-FLHS SRCAP-associated NDD; an overlapping but distinct NDD characterized by developmental delay with or without intellectual disability (ID), hypotonia, normal stature, and behavioral and psychiatric issues. Here, we report a young woman who initially presented in childhood with significant delays in speech and mild ID. In young adulthood, she developed schizophrenia. On physical examination, she had facial features suggestive of 22q11 deletion syndrome. After non-diagnostic chromosomal microarray and trio exome sequencing (ES), a re-analysis of trio ES data identified a de novo missense variant in SRCAP that was proximal to the FLHS critical region. Subsequent DNA methylation studies showed the unique methylation signature associated with pathogenic sequence variants in non-FLHS SRCAP-related NDD. This clinical report describes an individual with non-FLHS SRCAP-related NDD caused by an SRCAP missense variant, and it also demonstrates the clinical utility of ES re-analysis and DNA methylation analysis for undiagnosed patients, in particular, those with variants of uncertain significance.

Severe obesity and global developmental delay in preschool children: Findings from a Canadian Paediatric Surveillance Program study.

Background: The co-presentation of severe obesity (SO) and global developmental delay (GDD) in Canadian preschool children has not been examined. However, SO and GDD may require syndromic diagnoses and unique management considerations. Objectives: To determine (1) minimum incidence; (2) age of onset and risk factors; and (3) health care utilization for co-presenting SO and GDD. Methods: Through the Canadian Paediatric Surveillance Program (CPSP), a monthly form was distributed to participants from February 2018 to January 2020 asking for reports of new cases of SO and GDD among children ≤5 years of age. We performed descriptive statistics for quantitative questions and qualitative content analysis for open-ended questions. Results: Forty-seven cases (64% male; 51% white; mean age: 3.5 ± 1.2 years) were included. Age of first weight concern was 2.5 ± 1.3 years and age of GDD diagnosis was 2.7 ± 1.4 years. Minimum incidence of SO and GDD was 3.3 cases per 100,000 for ≤5 years of age per year. Identified problems included school and/or behavioural problems (n = 17; 36%), snoring (n = 14; 30%), and asthma/recurrent wheeze (n = 10; 21%). Mothers of 32% of cases (n = 15) had obesity and 21% of cases (n = 10) received neonatal intensive care. Microarray was ordered for 57% (n = 27) of children. A variety of clinicians and services were accessed. As reported by CPSP participants, challenges faced by families and health service access were barriers to care. Conclusion: Children with SO and GDD have multiple comorbidities, and require early identification and referral to appropriate services. These cases may also benefit from additional testing to rule out known genetic obesity syndromes.

DNA methylation signatures for chromatinopathies: current challenges and future applications.

Pathogenic variants in genes that encode epigenetic regulators are the cause for more than 100 rare neurodevelopmental syndromes also termed "chromatinopathies". DNA methylation signatures, syndrome-specific patterns of DNA methylation alterations, serve as both a research avenue for elucidating disease pathophysiology and a clinical diagnostic tool. The latter is well established, especially for the classification of variants of uncertain significance (VUS). In this perspective, we describe the seminal DNA methylation signature research in chromatinopathies; the complex relationships between genotype, phenotype and DNA methylation, and the future applications of DNA methylation signatures.

Assessment of a multisite standardized biospecimen collection protocol for immune phenotyping in neurodevelopmental disorders.

Multisite collection and preservation of peripheral blood mononuclear cells (PBMCs) for centralized analysis is an indispensable strategy for large cohort immune phenotyping studies. However, the absence of cross-site standardized protocols introduces unnecessary sample variance. Here we describe the protocol implemented by the Province of Ontario Neurodevelopmental Disorders (POND) Network's immune platform for the multisite collection, processing, and cryopreservation of PBMCs. We outline quality control standards and evaluate the performance of our PBMC processing and storage protocol. We also describe the Child Immune History Questionnaire results, an assessment tool evaluating pre-existing immune conditions in children with neurodevelopmental disorders (NDDs). Cell viability was assessed in samples from 178 participants based on strict quality control criteria. Overall, 83.1% of samples passed quality control standards. Samples collected and processed at the same site had higher quality control pass rates than samples that were collected and subsequently shipped to another site for processing. We investigated if freezer time impacted sample viability and found no difference in mean freezer time between samples that passed and failed quality control. The Child Immune History Questionnaire had a response rate of 87.1%. The described protocol produces viable samples that may be used in future immune phenotyping experiments.

Multiomics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and noncanonical Wnt signaling dysregulation.

ASXL1 (additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo protein-truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS; OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, distinctive facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies, including heart defects and severe skeletal defects giving rise to a typical BOS posture. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia. We used primary cells from individuals with BOS (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multiomics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data show that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, which encodes a planar cell polarity pathway protein that acts through noncanonical Wnt signaling to direct tissue patterning and cell migration. This multiomics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.

A mutational hotspot in AMOTL1 defines a new syndrome of orofacial clefting, cardiac anomalies, and tall stature.

AMOTL1 encodes angiomotin-like protein 1, an actin-binding protein that regulates cell polarity, adhesion, and migration. The role of AMOTL1 in human disease is equivocal. We report a large cohort of individuals harboring heterozygous AMOTL1 variants and define a core phenotype of orofacial clefting, congenital heart disease, tall stature, auricular anomalies, and gastrointestinal manifestations in individuals with variants in AMOTL1 affecting amino acids 157-161, a functionally undefined but highly conserved region. Three individuals with AMOTL1 variants outside this region are also described who had variable presentations with orofacial clefting and multi-organ disease. Our case cohort suggests that heterozygous missense variants in AMOTL1, most commonly affecting amino acid residues 157-161, define a new orofacial clefting syndrome, and indicates an important functional role for this undefined region.

ANKRD11 pathogenic variants and 16q24.3 microdeletions share an altered DNA methylation signature in patients with KBG syndrome.

Pathogenic variants in ANKRD11 or microdeletions at 16q24.3 are the cause of KBG syndrome (KBGS), a neurodevelopmental syndrome characterized by intellectual disability, dental and skeletal anomalies, and characteristic facies. The ANKRD11 gene encodes the ankyrin repeat-containing protein 11A transcriptional regulator, which is expressed in the brain and implicated in neural development. Syndromic conditions caused by pathogenic variants in epigenetic regulatory genes show unique patterns of DNA methylation (DNAm) in peripheral blood, termed DNAm signatures. Given ANKRD11's role in chromatin modification, we tested whether pathogenic ANKRD11 variants underlying KBGS are associated with a DNAm signature. We profiled whole-blood DNAm in 21 individuals with ANKRD11 variants, 2 individuals with microdeletions at 16q24.3 and 28 typically developing individuals, using Illumina's Infinium EPIC array. We identified 95 differentially methylated CpG sites that distinguished individuals with KBGS and pathogenic variants in ANKRD11 (n = 14) from typically developing controls (n = 28). This DNAm signature was then validated in an independent cohort of seven individuals with KBGS and pathogenic ANKRD11 variants. We generated a machine learning model from the KBGS DNAm signature and classified the DNAm profiles of four individuals with variants of uncertain significance (VUS) in ANKRD11. We identified an intermediate classification score for an inherited missense variant transmitted from a clinically unaffected mother to her affected child. In conclusion, we show that the DNAm profiles of two individuals with 16q24.3 microdeletions were indistinguishable from the DNAm profiles of individuals with pathogenic variants in ANKRD11, and we demonstrate the diagnostic utility of the new KBGS signature by classifying the DNAm profiles of individuals with VUS in ANKRD11.