Germline heterozygous SH2B3-mutations and (idiopathic) erythrocytosis: Detection of a previously undescribed mutation.

Erythrocytosis or polycythemia refers to a true or apparent increase in hemoglobin or hematocrit. When no etiology of erythrocytosis is identified, people are diagnosed with "idiopathic erythrocytosis" (IE). The identification of new contributing genes has recently improved the diagnostic workup of IE. As such mutations within the SH2B3 gene, which codes for the LNK protein and negatively regulates the JAK-STAT pathway, have been identified in cases diagnosed as IE. This reports describes the presence of a previously undescribed germline SH2B3 variant p.(Thr335ArgfsTer4) within IE and emphasizes the advantages of gene panel sequencing as second step in the diagnostic work-up.

Loss of phospholipase PLAAT3 causes a mixed lipodystrophic and neurological syndrome due to impaired PPARγ signaling.

Phospholipase A/acyltransferase 3 (PLAAT3) is a phospholipid-modifying enzyme predominantly expressed in neural and white adipose tissue (WAT). It is a potential drug target for metabolic syndrome, as Plaat3 deficiency in mice protects against diet-induced obesity. We identified seven patients from four unrelated consanguineous families, with homozygous loss-of-function variants in PLAAT3, who presented with a lipodystrophy syndrome with loss of fat varying from partial to generalized and associated with metabolic complications, as well as variable neurological features including demyelinating neuropathy and intellectual disability. Multi-omics analysis of mouse Plaat3-/- and patient-derived WAT showed enrichment of arachidonic acid-containing membrane phospholipids and a strong decrease in the signaling of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipocyte differentiation. Accordingly, CRISPR-Cas9-mediated PLAAT3 inactivation in human adipose stem cells induced insulin resistance, altered adipocyte differentiation with decreased lipid droplet formation and reduced the expression of adipogenic and mature adipocyte markers, including PPARγ. These findings establish PLAAT3 deficiency as a hereditary lipodystrophy syndrome with neurological manifestations, caused by a PPARγ-dependent defect in WAT differentiation and function.

Exome Sequencing and Multigene Panel Testing in 1,411 Patients With Adult-Onset Neurologic Disorders.

Background and Objectives: Owing to their extensive clinical and molecular heterogeneity, hereditary neurologic diseases in adults are difficult to diagnose. The current knowledge about the diagnostic yield and clinical utility of exome sequencing (ES) for neurologic diseases in adults is limited. This observational study assesses the diagnostic value of ES and multigene panel analysis in adult-onset neurologic disorders. Methods: From January 2019 through April 2022, ES-based multigene panel testing was conducted in 1,411 patients with molecularly unexplained neurologic phenotypes at the Ghent University Hospital. Gene panels were developed for ataxia and spasticity, leukoencephalopathy, movement disorders, paroxysmal episodic disorders, neurodegeneration with brain iron accumulation, progressive myoclonic epilepsy, and amyotrophic lateral sclerosis. Single nucleotide variants, small indels, and copy number variants were analyzed. Across all panels, our analysis covered a total of 725 genes associated with Mendelian inheritance. Results: A molecular diagnosis was established in 10% of the cases (144 of 1,411) representing 71 different monogenic disorders. The diagnostic yield depended significantly on the presenting phenotype with the highest yield seen in patients with ataxia or spastic paraparesis (19%). Most of the established diagnoses comprised disorders with an autosomal dominant inheritance (62%), and the most frequently mutated genes were NOTCH3 (13 patients), SPG7 (11 patients), and RFC1 (8 patients). 34% of the disease-causing variants were novel, including a unique likely pathogenic variant in APP (Ghent mutation, p.[Asn698Asp]) in a family presenting with stroke and severe cerebral white matter disease. 7% of the pathogenic variants comprised copy number variants detected in the ES data and confirmed by an independent technique. Discussion: ES and multigene panel testing is a powerful and efficient tool to diagnose patients with unexplained, adult-onset neurologic disorders.

Shortcutting the diagnostic odyssey: the multidisciplinary Program for Undiagnosed Rare Diseases in adults (UD-PrOZA).

BACKGROUND: In order to facilitate the diagnostic process for adult patients suffering from a rare disease, the Undiagnosed Disease Program (UD-PrOZA) was founded in 2015 at the Ghent University Hospital in Belgium. In this study we report the five-year results of our multidisciplinary approach in rare disease diagnostics. METHODS: Patients referred by a healthcare provider, in which an underlying rare disease is likely, qualify for a UD-PrOZA evaluation. UD-PrOZA uses a multidisciplinary clinical approach combined with state-of-the-art genomic technologies in close collaboration with research facilities to diagnose patients. RESULTS: Between 2015 and 2020, 692 patients (94% adults) were referred of which 329 (48%) were accepted for evaluation. In 18% (60 of 329) of the cases a definite diagnosis was made. 88% (53 of 60) of the established diagnoses had a genetic origin. 65% (39 of 60) of the genetic diagnoses were made through whole exome sequencing (WES). The mean time interval between symptom-onset and diagnosis was 19 years. Key observations included novel genotype-phenotype correlations, new variants in known disease genes and the identification of three new disease genes. In 13% (7 of 53), identifying the molecular cause was associated with therapeutic recommendations and in 88% (53 of 60), gene specific genetic counseling was made possible. Actionable secondary findings were reported in 7% (12 of 177) of the patients in which WES was performed. CONCLUSION: UD-PrOZA offers an innovative interdisciplinary platform to diagnose rare diseases in adults with previously unexplained medical problems and to facilitate translational research.

Prevalence of Germline Pathogenic Variants in Cancer Predisposing Genes in Czech and Belgian Pancreatic Cancer Patients.

(1) Background: The proportion and spectrum of germline pathogenic variants (PV) associated with an increased risk for pancreatic ductal adenocarcinoma (PDAC) varies among populations. (2) Methods: We analyzed 72 Belgian and 226 Czech PDAC patients by multigene panel testing. The prevalence of pathogenic variants (PV) in relation to personal/family cancer history were evaluated. PDAC risks were calculated using both gnomAD-NFE and population-matched controls. (3) Results: In 35/298 (11.7%) patients a PV in an established PDAC-predisposition gene was found. BRCA1/2 PV conferred a high risk in both populations, ATM and Lynch genes only in the Belgian subgroup. PV in other known PDAC-predisposition genes were rarer. Interestingly, a high frequency of CHEK2 PV was observed in both patient populations. PV in PDAC-predisposition genes were more frequent in patients with (i) multiple primary cancers (12/38; 32%), (ii) relatives with PDAC (15/56; 27%), (iii) relatives with breast/ovarian/colorectal cancer or melanoma (15/86; 17%) but more rare in sporadic PDAC (5/149; 3.4%). PV in homologous recombination genes were associated with improved overall survival (HR = 0.51; 95% CI 0.34-0.77). (4) Conclusions: Our analysis emphasizes the value of multigene panel testing in PDAC patients, especially in individuals with a positive family cancer history, and underlines the importance of population-matched controls for risk assessment.

Prolonged recombinant pregnancy hormone use in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: An early first full-time pregnancy substantially reduces the risk of developing breast cancer later in life. Extensive studies indicate that this protective effect is mediated by the pregnancy hormone human chorionic gonadotrophin (hCG). METHODS: In this proof-of-concept study 33 women with a BRCA mutation received recombinant-hCG (r-hCG). A 4-mm breast biopsy was obtained before (T1) and after 12 weeks of r-hCG injections (T2), as well as 6 months later (T3). The tissue was examined using RNA-sequencing methodology to determine if the 'high-risk' transcriptomic signature was converted to a 'low-risk' signature as in an early first full-time pregnancy. A stringent clinical safety monitoring was performed. RESULTS: The r-hCG administration was well tolerated in all participants. No clinically relevant changes were observed. In 25 women, the RNA quality was good for RNA sequencing in all three breast tissue biopsies. In response to the r-hCG, we observed 1907 differentially expressed genes (DEGs) (1032 up, 875 down) at T2 vs. T1 and 1065 DEGs (897 up, 168 down) at T3 vs. T1 in the group of women (n = 11) not using any hormonal contraceptives during the study. There was no response at T2 vs. T1 and a small number of DEGs, 260 (214 up, 46 down) at T3 vs. T1 in the group of 14 women using contraceptives. CONCLUSIONS: In summary, r-hCG has a remarkable effect on the gene expression profile of breast tissues from BRCA1/2 carriers who did not use any contraception. This opens an opportunity for a novel preventive strategy to reduce the incidence of breast cancer.

Tissue is the issue: when a second biopsy reveals the true diagnosis.

We describe the case of a woman with minimal glomerular changes on initial kidney biopsy. On long-term follow-up, the patient developed nephrotic proteinuria and a second kidney biopsy was performed, which revealed focal segmental glomerulosclerosis (FSGS). Findings from electron microscopy (EM) examination suggested a genetic form of FSGS. Next-generation sequencing showed heterozygosity for a mutation in COL4A3. Collagen IV nephropathies can be linked to late-onset FSGS. By establishing a genetic cause of FSGS, immunosuppressive treatment can be avoided. This case emphasizes the importance of re-biopsy in cases of a non-explained rise in proteinuria. EM can be helpful in differentiating between primary and secondary FSGS and informing treatment strategies. In cases of adult-onset FSGS that cannot be categorized by clinical-pathological assessment, genetic testing should be considered.

IRF2BPL Is Associated with Neurological Phenotypes.

Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

Accurate detection and quantification of epigenetic and genetic second hits in BRCA1 and BRCA2-associated hereditary breast and ovarian cancer reveals multiple co-acting second hits.

BACKGROUND: This study characterizes the second hit spectrum in BRCA1 and BRCA2-associated breast and ovarian cancers at both gene loci to investigate if second hit mechanisms are mutually exclusive or able to coincide within the same tumor. METHODS: Loss of heterozygosity, somatic point mutations and copy number alterations along with promoter methylation were studied in 56 breast and 15 ovarian cancers from BRCA1 and BRCA2 germline mutation carriers. A mathematical methodology was introduced to quantify the tumor cell population carrying a second hit. RESULTS: Copy neutral LOH was the most prevalent LOH mechanism in this cohort (BC 69%, OC 67%). However, only 36% of BC and 47% of OC showed LOH in all cancerous cells. Somatic intragenic deletions and methylated subclones were also found in combination with (partial) loss of heterozygosity. Unequivocal deleterious somatic point mutations were not identified in this cohort. CONCLUSION: Different mechanisms inactivating the wild type allele are present within the same tumor sample at various extents. Results indicate that BRCA1/2-linked breast and ovarian cancer cells are predominantly characterized by LOH, but harbor a complex combination of second hits at various frequencies.

Future perspectives of genome-scale sequencing.

INTRODUCTION: In recent decades, we witnessed a revolution in genetic technology. Some 20 years ago, analysing a single gene was quite laborious and time-consuming. In addition, diagnostic testing was only available for selected genes. Nowadays, whole exome analysis - a technique enabling sequencing of all protein coding sequences in the entire genome - is gradually introduced in a clinical setting. Whole genome sequencing forms the ultimate exponent of this evolution and offers an even broader application. METHODS: A review of the application of these technologies in a diagnostic setting is presented. RESULTS: Whole exome sequencing has a prominent place in modern clinical diagnostics. It offers a cost- and time-efficient way to interrogate all protein coding portions of the genome leading to a quick and adequate diagnosis, also in cases of phenotypic heterogeneity. As sequencing costs continue to drop, whole genome sequencing will take over in the near future guaranteeing a further improvement of the quality of genetic testing. CONCLUSION: Due to technological advances in the past decades, the field of clinical diagnostics has changed dramatically. With techniques such as whole exome and whole genome sequencing, the diagnostic yield increases serving both the patient and the health care system.

Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11.

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay.

Breast cancer risk drastically increases in individuals with a heterozygous germline BRCA1 or BRCA2 mutation, while it is estimated to equal the population risk for relatives without the familial mutation (non-carriers). The aim of the present study was to use a G2 phase-specific micronucleus assay to investigate whether lymphocytes of healthy BRCA2 mutation carriers are characterized by increased radiosensitivity compared to controls without a family history of breast/ovarian cancer and how this relates to healthy non-carrier relatives. BRCA2 is active in homologous recombination, a DNA damage repair pathway, specifically active in the late S/G2 phase of the cell cycle. We found a significantly increased radiosensitivity in a cohort of healthy BRCA2 mutation carriers compared to individuals without a familial history of breast cancer (P=0.046; Mann-Whitney U test). At the individual level, 50% of healthy BRCA2 mutation carriers showed a radiosensitive phenotype (radiosensitivity score of 1 or 2), whereas 83% of the controls showed no radiosensitivity (P=0.038; one-tailed Fisher's exact test). An odds ratio of 5 (95% CI, 1.07-23.47) indicated an association between the BRCA2 mutation and radiosensitivity in healthy mutation carriers. These results indicate the need for the gentle use of ionizing radiation for either diagnostic or therapeutic use in BRCA2 mutation carriers. We detected no increased radiosensitivity in the non-carrier relatives.

Increased chromosomal radiosensitivity in asymptomatic carriers of a heterozygous BRCA1 mutation.

BACKGROUND: Breast cancer risk increases drastically in individuals carrying a germline BRCA1 mutation. The exposure to ionizing radiation for diagnostic or therapeutic purposes of BRCA1 mutation carriers is counterintuitive, since BRCA1 is active in the DNA damage response pathway. The aim of this study was to investigate whether healthy BRCA1 mutations carriers demonstrate an increased radiosensitivity compared with healthy individuals. METHODS: We defined a novel radiosensitivity indicator (RIND) based on two endpoints measured by the G2 micronucleus assay, reflecting defects in DNA repair and G2 arrest capacity after exposure to doses of 2 or 4 Gy. We investigated if a correlation between the RIND score and nonsense-mediated decay (NMD) could be established. RESULTS: We found significantly increased radiosensitivity in the cohort of healthy BRCA1 mutation carriers compared with healthy controls. In addition, our analysis showed a significantly different distribution over the RIND scores (p = 0.034, Fisher's exact test) for healthy BRCA1 mutation carriers compared with non-carriers: 72 % of mutation carriers showed a radiosensitive phenotype (RIND score 1-4), whereas 72 % of the healthy volunteers showed no radiosensitivity (RIND score 0). Furthermore, 28 % of BRCA1 mutation carriers had a RIND score of 3 or 4 (not observed in control subjects). The radiosensitive phenotype was similar for relatives within several families, but not for unrelated individuals carrying the same mutation. The median RIND score was higher in patients with a mutation leading to a premature termination codon (PTC) located in the central part of the gene than in patients with a germline mutation in the 5' end of the gene. CONCLUSIONS: We show that BRCA1 mutations are associated with a radiosensitive phenotype related to a compromised DNA repair and G2 arrest capacity after exposure to either 2 or 4 Gy. Our study confirms that haploinsufficiency is the mechanism involved in radiosensitivity in patients with a PTC allele, but it suggests that further research is needed to evaluate alternative mechanisms for mutations not subjected to NMD.

Novel biological insights in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of blood cancer that accounts for about 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. It is considered as a paradigm for the multistep nature of cancer initiation and progression. Genetic and epigenetic reprogramming events, which transform T-cell precursors into malignant T-ALL lymphoblasts, have been extensively characterized over the past decade. Despite our comprehensive understanding of the genomic landscape of human T-ALL, leukemia patients are still treated by high-dose multiagent chemotherapy, potentially followed by hematopoietic stem cell transplantation. Even with such aggressive treatment regimens, which are often associated with considerable acute and long-term side effects, about 15% of pediatric and 40% of adult T-ALL patients still relapse, owing to acquired therapy resistance, and present with very dismal survival perspectives. Unfortunately, the molecular mechanisms by which residual T-ALL tumor cells survive chemotherapy and act as a reservoir for leukemic progression and hematologic relapse remain poorly understood. Nevertheless, it is expected that enhanced molecular understanding of T-ALL disease biology will ultimately facilitate a targeted therapy driven approach that can reduce chemotherapy-associated toxicities and improve survival of refractory T-ALL patients through personalized salvage therapy. In this review, we summarize recent biological insights into the molecular pathogenesis of T-ALL and speculate how the genetic landscape of T-ALL could trigger the development of novel therapeutic strategies for the treatment of human T-ALL.

Molecular basis and clinical significance of genetic aberrations in B-cell precursor acute lymphoblastic leukemia.

B-Cell precursor acute lymphoblastic leukemia (BCP-ALL) arises from recurrent genetic insults that block precursor B-cell differentiation and drive aberrant proliferation and cell survival. Risk-adapted intensive chemotherapy is effective in curing the majority of children with BCP-ALL (>85%), but some children, not considered "high risk" and treated accordingly, experience a hematologic relapse. Moreover, survival rates in adults are significantly lower (∼40%) than those in children. Recent developments in genomewide genetic analysis have provided a wide range of chromosomal and genomic abnormalities characterizing BCP-ALL, several of which are associated with patient outcome. These findings provide an opportunity to adapt risk stratification and treatment schedules and to identify new druggable targets. In this review, we discuss the established and novel genetic alterations in BCP-ALL, their molecular background, and their potential use in risk stratification and treatment of BCP-ALL.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

Epigenetics in T-cell acute lymphoblastic leukemia.

Normal T-cell development is a strictly regulated process in which hematopoietic progenitor cells migrate from the bone marrow to the thymus and differentiate from early T-cell progenitors toward mature and functional T cells. During this maturation process, cooperation between a variety of oncogenes and tumor suppressors can drive immature thymocytes into uncontrolled clonal expansion and cause T-cell acute lymphoblastic leukemia (T-ALL). Despite improved insights in T-ALL disease biology and comprehensive characterization of its genetic landscape, clinical care remained largely similar over the past decades and still consists of high-dose multi-agent chemotherapy potentially followed by hematopoietic stem cell transplantation. Even with such aggressive treatment regimens, which are often associated with considerable side effects, clinical outcome is still extremely poor in a significant subset of T-ALL patients as a result of therapy resistance or hematological relapses. Recent genetic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in T-ALL, suggesting that epigenetic homeostasis is critically required in restraining tumor development in the T-cell lineage. In this review, we provide an overview of the epigenetic regulators that could be implicated in T-ALL disease biology and speculate how the epigenetic landscape of T-ALL could trigger the development of epigenetic-based therapies to further improve the treatment of human T-ALL.

ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.