Mutations In Modifier Genes Research Articles

In vivo dissection of the mouse tyrosine catabolic pathway with CRISPR-Cas9 identifies modifier genes affecting hereditary tyrosinemia type 1.

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related "modifier' genes (i.e. genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings.

Somatic Mutations Are Frequently Detected in CML Patients before Treatment-Free Remission (TFR) Attempt with Tyrosine Kinase Inhibitor (TKI) Discontinuation but with Uncertain Predictive Value for Tfr Failure

Treatment-free remission (TFR) has been accepted as a practical treatment goal in chronic myeloid leukemia (CML) patients. TFR is defined as successful discontinuation (DISC) of Tyrosine Kinase Inhibitor (TKI) therapy without losing leukemia control. Multiple clinical studies have reproduced a consistent TFR rate of about 50%. No definite biomarker has been identified or validated yet. Adnan et al (BCJ 2022) recently reported that epigenetic modifier gene mutations at initial diagnosis of CML are associated with TFR success after TKI DISC. The issue is the time gap between mutation profiling at initial diagnosis of CML and the clinical event of a TFR attempt, which is about 5-10 years. Mutation profiles in the samples obtained before TKI DISC have never been investigated as a TFR predictive marker. Next-generation sequencing (NGS) has been incorporated into clinical practice. Conventional targeted sequencing techniques have limitations such as the high error rate, its limit of detection is up to 2-5%. Mutation frequency and allele frequency (AF) were assumed to be very low in the patients who achieved deep molecular response. Thus, conventional NGS likely misses mutations with low AF. Single-molecule molecular inversion probe (smMIP) is a bar-coded, error-corrected NGS approach, can overcome the high error rate issue by tracking all sequence reads derived from a single strand. This study aims to detect somatic mutations using a smMIP panel to evaluate if the mutations assessed can be a biomarkers for TFR failure. Patients and Methods We applied our in-house CML-specific smMIP panel with a limit of detection up to 0.2%. The panel encompasses 37 genes with 332 amplicon probes: epigenetic modifiers (n=7), activation signaling (n=12), myeloid transcription factor (TF; n=5), spliceosome (n=3), tumor suppressor (n=3), cohesion (n=4) and miscellaneous (n=3). Our cohort included 135 patients who attempted TKI DISC for TFR from 4 countries: Argentina (n=28, 20.7%), Brazil (n=30, 22.2%), Czechia (n=3, 2.2%), Canada (n=34, 54.8%). Disease characteristics and outcomes were evaluated and compared according to the mutation profiles prior to TKI DISC. Molecular relapse-free survival (RFS) was calculated from the date of TKI DISC to the date of confirmed loss of molecular response. An episode of major molecular response (MMR) loss or two consecutive episodes of molecular response loss of 4 logs or deeper response (MR4) constituted molecular relapse. Kaplan-Meier survival estimate was used to evaluate RFS and Cox's proportional hazard regression model was applied. Results Median age was 55.5 years (25-72), 55% males, and Sokal Score risks rates were as follows: Low 51.8%, intermediate 26.8%, High 8.33%. All patients were in chronic phase at diagnosis. A total of 102 pts (78%) stopped Imatinib, while 24 pts (22%) stopped 2GTKI: Dasatinib (n=17) and Nilotinib (n=7). There was no difference in RFS between IM DISC vs. 2GTKI DISC: the patients who stopped IM showed RFS rate of 64.3% (40.9-80.4%) vs those who stopped 2GTKI showed 58% (48.5-67.7%; p=0.634). A total of 59 mutations were detected in 43 (31.9%) patients. TET2 was the most commonly mutated gene (n=15, 11.1%) followed by ASXL1 (n=10, 7.4%) and, DNMT3A (n=9, 6.7%). According to its biologic pathways of the mutation, mutations were detected in the Epigenetic modifiers (n=33, 56%) most frequently, followed by Cohesin (n=16, 27%), activated signaling (n=7, 12%), tumor suppressor (n=3, 5%) and splicing (n=1, 2%). According to the mutational profile before TKI DISC for the TFR attempt, no difference in RFS was observed according to the biological pathways of mutations or according to the presence of mutation (fig 1). Overall RFS at 1 year was 56% (95% CI [45.1-66.1%]) in patients without any mutations comparing to 67.5% (50.7-79.7%) in patients with mutation(s) (p=0.49; fig 2). The RFS rate at 1 year was 58.9% (48.3-68%) in patients with epigenetic mutations compared to 63.3% (43.6.-78.7%) in those without it (p=0.73). Even after adjustment for MR4 duration, we could not identify any predictive profile of RFS. Conclusion Somatic mutations were not infrequently detected in the patients qualified for TFR attempt using a technique detecting up to 0.2% VAF. Mutations in epigenetic genes were most frequently detected. However, it does not seem to have any predictive power for TFR success. Its biological relevance remains to be further investigated in future research. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The cAMP-signaling cancers: Clinically-divergent disorders with a common central pathway.

The cAMP-signaling cancers, which are defined by functionally-significant somatic mutations in one or more elements of the cAMP signaling pathway, have an unexpectedly wide range of cell origins, clinical manifestations, and potential therapeutic options. Mutations in at least 9 cAMP signaling pathway genes (TSHR, GPR101, GNAS, PDE8B, PDE11A, PRKARA1, PRKACA, PRKACB, and CREB) have been identified as driver mutations in human cancer. Although all cAMP-signaling pathway cancers are driven by mutation(s) that impinge on a single signaling pathway, the ultimate tumor phenotype reflects interactions between five critical variables: (1) the precise gene(s) that undergo mutation in each specific tumor type; (2) the effects of specific allele(s) in any given gene; (3) mutations in modifier genes (mutational “context”); (4) the tissue-specific expression of various cAMP signaling pathway elements in the tumor stem cell; and (5) and the precise biochemical regulation of the pathway components in tumor cells. These varying oncogenic mechanisms reveal novel and important targets for drug discovery. There is considerable diversity in the “druggability” of cAMP-signaling components, with some elements (GPCRs, cAMP-specific phosphodiesterases and kinases) appearing to be prime drug candidates, while other elements (transcription factors, protein-protein interactions) are currently refractory to robust drug-development efforts. Further refinement of the precise driver mutations in individual tumors will be essential for directing priorities in drug discovery efforts that target these mutations.

DMRscaler: a scale-aware method to identify regions of differential DNA methylation spanning basepair to multi-megabase features

BackgroundPathogenic mutations in genes that control chromatin function have been implicated in rare genetic syndromes. These chromatin modifiers exhibit extraordinary diversity in the scale of the epigenetic changes they affect, from single basepair modifications by DNMT1 to whole genome structural changes by PRM1/2. Patterns of DNA methylation are related to a diverse set of epigenetic features across this full range of epigenetic scale, making DNA methylation valuable for mapping regions of general epigenetic dysregulation. However, existing methods are unable to accurately identify regions of differential methylation across this full range of epigenetic scale directly from DNA methylation data.ResultsTo address this, we developed DMRscaler, a novel method that uses an iterative windowing procedure to capture regions of differential DNA methylation (DMRs) ranging in size from single basepairs to whole chromosomes. We benchmarked DMRscaler against several DMR callers in simulated and natural data comparing XX and XY peripheral blood samples. DMRscaler was the only method that accurately called DMRs ranging in size from 100 bp to 1 Mb (pearson's r = 0.94) and up to 152 Mb on the X-chromosome. We then analyzed methylation data from rare-disease cohorts that harbor chromatin modifier gene mutations in NSD1, EZH2, and KAT6A where DMRscaler identified novel DMRs spanning gene clusters involved in development.ConclusionTaken together, our results show DMRscaler is uniquely able to capture the size of DMR features across the full range of epigenetic scale and identify novel, co-regulated regions that drive epigenetic dysregulation in human disease.

Heterogeneity of neuroendocrine transcriptional states in metastatic small cell lung cancers and patient-derived models

Molecular subtypes of small cell lung cancer (SCLC) defined by the expression of key transcription regulators have recently been proposed in cell lines and limited number of primary tumors. The clinical and biological implications of neuroendocrine (NE) subtypes in metastatic SCLC, and the extent to which they vary within and between patient tumors and in patient-derived models is not known. We integrate histology, transcriptome, exome, and treatment outcomes of SCLC from a range of metastatic sites, revealing complex intra- and intertumoral heterogeneity of NE differentiation. Transcriptomic analysis confirms previously described subtypes based on ASCL1, NEUROD1, POU2F3, YAP1, and ATOH1 expression, and reveal a clinical subtype with hybrid NE and non-NE phenotypes, marked by chemotherapy-resistance and exceedingly poor outcomes. NE tumors are more likely to have RB1, NOTCH, and chromatin modifier gene mutations, upregulation of DNA damage response genes, and are more likely to respond to replication stress targeted therapies. In contrast, patients preferentially benefited from immunotherapy if their tumors were non-NE. Transcriptional phenotypes strongly skew towards the NE state in patient-derived model systems, an observation that was confirmed in paired patient-matched tumors and xenografts. We provide a framework that unifies transcriptomic and genomic dimensions of metastatic SCLC. The marked differences in transcriptional diversity between patient tumors and model systems are likely to have implications in development of novel therapeutic agents.

Identification of potential modifier genes in Chinese patients with Wilson disease.

The mutations in modifier genes may contribute to some inherited diseases including Wilson disease (WD). This study was designed to identify potential modifier genes that contribute to WD. A total of 10 WD patients with single or no heterozygous ATP7B mutations were recruited for whole-exome sequencing (WES). Five hundred and thirteen candidate genes, of which the genetic variants present in at least two patients, were identified. In order to clarify which proteins might be involved in copper transfer or metabolism processes, the isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify the differentially expressed proteins between normal and CuSO4-treated cell lines. Thirteen genes/proteins were identified by both WES and iTRAQ, indicating that disease-causing variants of these genes may actually contribute to the aberrant copper ion accumulation. Additionally, the c.86C > T (p.S29L) mutation in the SLC31A2 gene (coding CTR2) has a relative higher frequency in our cohort of WD patients (6/191) than reported (0.0024 in gnomAD database) in our healthy donors (0/109), and CTR2S29L leads to increased intracellular Cu concentration and Cu-induced apoptosis in cultured cell lines. In conclusion, the WES and iTRAQ approaches successfully identified several disease-causing variants in potential modifier genes that may be involved in the WD phenotype.

Haematologic malignancies with unfavourable gene mutations benefit from donor lymphocyte infusion with/without decitabine for prophylaxis of relapse after allogeneic HSCT: A pilot study.

Relapse is the main cause of treatment failure for leukaemia patients with unfavourable gene mutations who receive allogeneic haematopoietic stem cell transplantation (allo‐HSCT). There is no consensus on the indication of donor lymphocyte infusion (DLI) for prophylaxis of relapse after allo‐HSCT. To evaluate the tolerance and efficacy of prophylactic DLI in patients with unfavourable gene mutations such as FLT3‐ITD, TP53, ASXL1, DNMT3A or TET2, we performed a prospective, single‐arm study. Prophylactic use of decitabine followed by DLI was planned in patients with TP53 or epigenetic modifier gene mutations. The prophylaxis was planned in 46 recipients: it was administered in 28 patients and it was not administered in 18 patients due to contraindications. No DLI‐associated pancytopenia was observed. The cumulative incidences of grade II–IV and III–IV acute graft‐versus‐host disease (GVHD) at 100 days post‐DLI were 25.8% and 11.0%, respectively. The rates of chronic GVHD, non‐relapse mortality and relapse at 3 years post‐DLI were 21.6%, 25.0% and 26.1%, respectively. The 3‐year relapse‐free survival and overall survival (OS) rates were 48.9% and 48.2%, respectively. Acute GVHD (HR: 2.30, p = 0.016) and relapse (HR: 2.46, p = 0.003) after DLI were independently associated with inferior OS. Data in the current study showed the feasibility of prophylactic DLI with/without decitabine in the early stage after allo‐HSCT in patients with unfavourable gene mutations.

Genomic profile at diagnosis by targeted high throughput sequencing, and real-world clinical outcomes in patients diagnosed with acute myeloid leukemia at a tertiary care cancer center.

e19515 Background: Acute Myeloid Leukemia (AML) is a highly heterogeneous disease. High throughput sequencing (HTS) has led to the discovery of a number of recurrently mutated genes in AML & has provided a detailed molecular landscape of the disease. This information is now extensively utilised & interpreted in AML. Methods: Retrospective analysis of 47 patients with newly diagnosed AML for those who had both cytogenetic and targeted HTS information at diagnosis, [median age: 44y (12-80y); 24 females] & treated at Tata Medical Center, India between 2013-2019. HTS was performed using Illumina Trusight myeloid panel and/or Thermo Fisher Scientific Oncomine Myeloid panel, and mutation data from the common 28 genes were used. Patients were stratified by European Leukemia Net (ELN) 2010 and 2017 guidelines. Standard of care clinical outcome data was available for all patients. Results: 45 of 47 patients had mutations. The most frequently mutated gene was FLT3 (n = 13, 28.9%), followed by NPM1 (n = 8, 17.8%), and others [ IDH1, IDH2, TET2, CEBPA, and NRAS; n = 7, 15.6%). ELN-2010 classified 6 patients as Favourable (12.8%), 10 as Intermediate-I (21.3%), 25 as Intermediate-II (53.2%) and 6 as Adverse (12.8%) risk groups. One patient migrated to the adverse group from Intermediate-II when ELN-2017 was applied. ELN 2017 re-classification: Favorable (n = 6, 12.8%), intermediate (n = 34, 72.3%), and adverse risk groups (n = 7, 14.9%). Co-occurrence of driver gene mutations was found in NPM1 and FLT3 (8.2%) frequently. Patients with mutations in epigenetic modifiers ( ASXL1, IDH1, IDH2, DNMT3A) had high Variant Allele Frequency (VAF, 40-50%) while those with signaling pathway gene mutations ( NRAS, KRAS) had low VAF (9-25%). At a median follow up of 3.25 years, median overall survival in the three risk groups (favorable, intermediate, adverse) were 11.25 mons, 7 mons and 8 mons respectively, and relapse rates were 16%, 29% and 42% respectively. Conclusions: Early experience from a middle-income real-world setting suggests feasibility of integration of HTS information in practise, as additional prognostic/ predictive information. The pattern of a high VAF in epigenetic modifier gene mutations, potentially as a result of early acquisition during clonal development, needs further evaluation. This data set has helped us initiate a prospective multimodal omics study at our institution.

Epigenetic modifier gene mutations-positive AML patients with intermediate-risk karyotypes benefit from decitabine with CAG regimen.

It remains unclear whether there is a relationship between therapeutic effects of hypomethylating agents (HMAs) and epigenetic modifier gene mutations (EMMs) in patients with cytogenetically intermediate-risk acute myeloid leukemia (IR-AML). Based on targeted-capture sequencing, we retrospectively analyzed the correlation between EMMs and prognosis in 83 IR-AML patients treated with decitabine in combination with cytarabine, aclarubicin hydrochloride and granulocyte colony-stimulating factor (DCAG, n = 35) or "7 + 3" induction regimens (n = 48). In the multivariate analyses, EMM (+) patients did not show any statistically significant difference in remission rates from EMM (-) patients in the DCAG group (p > 0.05), but achieved inferior complete remission (CR; p = 0.03) and overall remission rates (ORR; p = 0.04) after the first course of standard induction regimens (p < 0.05). In the EMM (-) cohort, the DCAG group showed the tendency of adverse total CR (p = 0.06). Besides, DCAG group with EMMs achieved the best survival outcome independent of baseline characteristics, whereas it was opposite in EMM (+) patients receiving standard induction regimens (p < 0.05). Additionally, in the EMM (+) cohort, the survival rate of isolated DCAG group was statistically similar to that of the combination of standard chemotherapies and allogeneic hematopoietic stem cell transplantation (allo-HSCT) (p > 0.40), whereas patients who received only standard regimens had the worst survival rate (0.0%, p < 0.01). It can be concluded that the EMMs might be regarded as the potentially predictive biomarkers of better response to DCAG in IR-AML patients.

Krüppel-Like Factor 1 Gene Mutations in Thalassemia Patients from North Iran: Report of a New Mutation Associated with β-Thalassemia Intermedia

Thalassemia is a hereditary disease with an autosomal recessive inheritance pattern resulting in reduced production of globin chains. Mutations in modifier genes can cause or affect thalassemia. Krüppel-like factor 1 (KLF1) is a modifier gene that was investigated in this study. Thirty-five Iranian β-thalassemia (β-thal) minor patients with hematological symptoms including Hb A2 3.0%, mean corpuscular volume (MCV) <75.0 fL, mean corpuscular hemoglobin (Hb) (MCH) <25.0 pg, and two β-thal intermedia (β-TI) patients in 50 subjects who carried no mutations on the HBB and HBA2 or HBA1 genes were investigated for all exons of the KLF1 gene by polymerase chain reaction (PCR) and sequencing methods. Of the 35 patients with a β-thal minor phenotype, one patient was heterozygous for the c.544T>C mutation in exon 2 of KLF1 and HBB: c.380T>G variant, Hb Dhonburi [also known as Hb Neapolis or codon 126 (T>G)]. The c.340T>C mutation was also found in exon 2 of the KLF1 gene with an allele frequency of 16.6% in the studied β-thal carriers. The two β-TI patients were homozygous for a new mutation c.942delA in exon 3 of KLF1. Mutations in modifier genes can cause or affect thalassemia. Therefore, exact investigation of globin genes and modifiers such as KLF1 is necessary in areas where globin gene disorders are most prevalent to understand the reason of clinical and hematological symptoms of thalassemia and facilitate newborn screening or prenatal diagnosis (PND) programs.

Chidamide, Decitabine, Cytarabine, Aclarubicin, and Granulocyte Colony-Stimulating Factor (CDCAG) in Adults with Relapsed/Refractory Acute Myeloid Leukemia: Final Results from a Phase 1/2 Study

Background:There is no standard cytoreductive regimen for patients with relapsed/refractory (R/R) acute myeloid leukemia (AML). Increasing evidence indicates the importance of epigenetic modification in the pathogenesis of myeloid neoplasms, and the epigenetic changes can be pharmacologically reversed by epigenetic modulating drugs (Halpern, Othus et al. 2017). Hypomethylating agents and histone deacetylase inhibitors showed chemo-sensitization when combined with conventional chemotherapeutics in patients with R/R AML (Halpern, Othus et al. 2017, Li, Wang et al. 2017). We conducted a phase 1/2 study of chidamide (a histone deacetylase inhibitor), decitabine (a hypomethylating agent), cytarabine, aclarubicin, and granulocyte colony-stimulating factor (CDCAG) for patients with R/R AML.Methods:This multicenter clinical trial evaluated the safety and efficacy of CDCAG regimen in patients with R/R AML (NCT02886559). Adults (age: 18-60) with R/R AML were eligible if they had not received treatment within 4 weeks prior to the enrollment. ECOG performance status ≤ 3 and expected survival time ˃ 3 months were required. All patients were treated with chidamide 30 mg on days 1, 4, 8, and 11; decitabine 20 mg/m2 on days 1-5; cytarabine 50 mg/m2 on days 1-7 if WBC ≥ 20×109/L, and on days 3-7 if WBC < 20×109/L; aclarubicin 10 mg/m2 on days 3-7; and granulocyte colony-stimulating factor, 300 ug/d until WBC > 20×109/L. Treatment cycles were repeated every 28 days. Patients could be treated for up to two cycles. Genomic DNA from patients at enrollment was examined for mutations by the target sequencing panel, which covered the coding sequences of 127 known genes relevant to the pathogenesis of AML (Annoroad Gene Technology Co., Ltd).Results:Ninety-three patients, median age 40 (range: 18-60) years, with primary refractory (n = 37), early relapsed disease (n = 38), or late relapsed disease (n = 18) were enrolled. Among the 56 patients with relapsed AML, 40 were experiencing their first relapse, 15 the second relapse, and 1 patient the third. They received a median of 5 (1-17) therapies cycles prior to the enrollment. Patients were enrolled on median 8.8 (1.3-63.8) months after initial diagnosis.Overall, 24/93 (26%, 95% confidence interval [CI]: 16.9-34.7%) patients achieved a complete remission (CR) and 19/93 (20%, 95% CI: 12.2-28.4%) patients achieved a CR with incomplete blood count recovery (CRi), for an objective overall response rate of 46% (95% CI: 36.1-56.4%). Among the 43 CR/CRi patients, the 6-month disease-free survival (DFS) rate was 56% (95% CI: 41.0-70.7%), and the median DFS was 305 (95% CI: 258-NA) days. Patients achieving an CR/CRi had a significant improvement in median overall survival (OS) (259 vs 563 days; P < 0.001; hazard ratio [HR], 3.6) compared with nonresponders. The 6-month OS estimate was 52% (95% CI: 41.5-61.8%), and the median OS was 266 (95% CI: 235-398) days. Overall, we detected 184 mutations in 76 patients. Most commonly mutated genes were FLT3-ITD, CEBPA, WT1, RUNX1, and DNMT3A. Within patients with mutation, the CR/CRi rate was 55.6% in patients with epigenetic modifier gene mutations, and only 36.7% in patients with other mutations. Serious adverse events (SAE) occurred in 12 (13%) patients, with 4 patients experienced early death (death within 28 days). Only 1 patient died during consolidation.Conclusions:The CDCAG regimen was well tolerated and effective in relapsed/refractory AML. Compared with patients with other mutations, patients with epigenetic modifier gene mutations might benefit from the CDCAG regimen.DisclosuresChidamide tables were provided by Chipscreen Biosciences Ltd and decitabine was provided by Shandong New Time Pharmaceutical Co., Ltd. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chromatin-regulatory genes served as potential therapeutic targets for patients with urothelial bladder carcinoma.

Urothelial bladder carcinoma is the ninth most common cancer in the world, with an estimated 150,000 deaths per year. Two comprehensive analysis based on The Cancer Genome Atlas urothelial bladder carcinoma reported that chromatin modifier gene mutations were common in bladder cancer. We aimed to find how the mutations and transcriptional profiles of the genes involving in chromatin modification affected the prognosis of patients. The data were retrieved from the GenomicDataCommons data portal and the gene list in pathway Chromatin Modifying Enzymes were obtained from Reactome. The expression levels and mutational profiles of the genes involving in the chromatin were utilized altogether to construct a fusion patient similarity network by similarity network fusion. The genes that were differentially expressed in one clustered group or two were identified. Fifty chromatin-regulating genes had nonsilent mutations in at least 10 patients. KMT2D, KDM6A, CREBBP, ARID1A, and ARID2 had enriched inactivating mutations. Among 399 cases where both the single-nucleotide polymorphism information and the messenger RNA expression profiles were available, 326, 23, and 50 patients were clustered into Groups 1, 2, and 3, respectively. The survival analysis suggested that the patients in these three groups had a different prognosis. Thity-one genes were identified as differentially expressed in any group. The Gene Ontology term enrichment showed that the differentially expressed genes were enriched in the immune response especially in the complement activation. Altogether, chromatin-regulatory genes were key in bladder cancer and can serve, with the differentially expressed genes, as potential therapeutic targets.

The 3PAs: An Update on the Association of Pheochromocytomas, Paragangliomas, and Pituitary Tumors.

Pituitary adenomas (PA) and pheochromocytomas/paragangliomas (PHEO/PGL) are rare tumors. Although they may co-exist by coincidence, there is mounting evidence that genes predisposing in PHEO/PGL development, may play a role in pituitary tumorigenesis. In 2012, we described a GH-secreting PA caused by an SDHD mutation in a patient with familial PGLs and found loss of heterozygosity at the SDHD locus in the pituitary tumor, along with increased hypoxia-inducible factor 1α (HIF-1α) levels. Additional patients with PAs and SDHx defects have since been reported. Overall, prevalence of SDHx mutations in PA is very rare (0.3-1.8% in unselected cases) but we and others have identified several cases of PAs with PHEOs/PGLs, like our original report, a condition which we termed the 3 P association (3PAs). Interestingly, when 3PAs is found in the sporadic setting, no SDHx defects were identified, whereas in familial PGLs, SDHx mutations were identified in 62.5-75% of the reported cases. Hence, pituitary surveillance is recommended among patients with SDHx defects. It is possible that the SDHx germline mutation-negative 3PAs cases may be due to another gene, epigenetic changes, mutations in modifier genes, mosaicism, somatic mutations, pituitary hyperplasia due to ectopic hypothalamic hormone secretion or a coincidence. PA in 3PAs are mainly macroadenomas, more aggressive, more resistant to somatostatin analogues, and often require surgery. Using the Sdhb +/- mouse model, we showed that hyperplasia may be the first abnormality in tumorigenesis as initial response to pseudohypoxia. We also propose surveillance and follow-up approach of patients presenting with this association.

Histone modifier gene mutations in peripheral T-cell lymphoma not otherwise specified.

Due to heterogeneous morphological and immunophenotypic features, approximately 50% of peripheral T-cell lymphomas are unclassifiable and categorized as peripheral T-cell lymphomas, not otherwise specified. These conditions have an aggressive course and poor clinical outcome. Identification of actionable biomarkers is urgently needed to develop better therapeutic strategies. Epigenetic alterations play a crucial role in tumor progression. Histone modifications, particularly methylation and acetylation, are generally involved in chromatin state regulation. Here we screened the core set of genes related to histone methylation (KMT2D, SETD2, KMT2A, KDM6A) and acetylation (EP300, CREBBP) and identified 59 somatic mutations in 45 of 125 (36.0%) patients with peripheral T-cell lymphomas, not otherwise specified. Histone modifier gene mutations were associated with inferior progression-free survival time of the patients, irrespective of chemotherapy regimens, but an increased response to the histone deacetylase inhibitor chidamide. In vitro, chidamide significantly inhibited the growth of EP300-mutated T-lymphoma cells and KMT2D-mutated T-lymphoma cells when combined with the hypomethylating agent decitabine. Mechanistically, decitabine acted synergistically with chidamide to enhance the interaction of KMT2D with transcription factor PU.1, regulated H3K4me-associated signaling pathways, and sensitized T-lymphoma cells to chidamide. In a xenograft KMT2D-mutated T-lymphoma model, dual treatment with chidamide and decitabine significantly retarded tumor growth and induced cell apoptosis through modulation of the KMT2D/H3K4me axis. Our work thus contributes to the understanding of aberrant histone modification in peripheral T-cell lymphomas, not otherwise specified and the stratification of a biological subset that can benefit from epigenetic treatment.

Genotype-Phenotype Characterization of Wolf-Hirschhorn Syndrome Confirmed by FISH: Case Reports

The Wolf-Hirschhorn syndrome (WHS) is a multiple malformation and contiguous gene syndrome resulting from the deletion encompassing a 4p16.3 region. A microscopically visible terminal deletion on chromosome 4p (4p16→pter) was detected in Case 1 with full blown features of WHS. The second case which had an interstitial microdeletion encompassing WHSC 1 and WHSC 2 genes at 4p16.3 presented with less striking clinical features of WHS and had an apparently “normal” karyotype. The severity of the clinical presentation was as a result of haploinsufficiency and interaction with surrounding genes as well as mutations in modifier genes located outside the WHSCR regions. The study emphasized that an individual with a strong clinical suspicion of chromosomal abnormality and a normal conventional cytogenetic study should be further investigated using molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) or array-comparative genomic hybridization (a-CGH).

Breast Cancer Risk Assessment and Genetic Testing: Complexities, Conundrums, and Community

This special issue of Breast Disease is a testament to how far the field of genetic counseling for breast cancer susceptibility has advanced since the mid-1990s, following the cloning of two major breast (and ovarian) cancer susceptibility genes, BRCA1 and BRCA2. Although perhaps not fully appreciated at the time, individuals who pursued genetic testing shortly after it became available were truly pioneers. They were seeking important, indeed life-changing information, when in fact few specifics about several key issues could be provided. For example, the only available data regarding cancer risks in mutation carriers were derived from the highest risk families, most of which were not representative of the families seen in clinical practice. Then, largely due to the discovery of three founder mutations in Ashkenazi Jews, population-based data revealed a range of breast and ovarian cancer risks in carriers. Subsequently, additional information about mutation penetrance, genetic epidemiology, and risk modifiers, some of which is now integrated into routine clinical counseling, has also facilitated the development of elaborate models to predict the likelihood that an individual will test positive for a BRCA1/2 mutation. For women who test positive, several recent studies confirm what we suspected early on – namely that prophylactic mastectomy and oophorectomy confer significant protection against the development of breast and ovarian cancer. In premenopausal women, the benefits of oophorectomy are particularly significant, given the associated risk reduction for both breast and ovarian cancer. In addition, for those women who opt against prophylactic mastectomy, screening with a combination of mammography and magnetic resonance imaging appears to frequently detect breast cancer at an early stage. Effective screening strategies for ovarian cancer, however, remain elusive. Ongoing research to develop better screening measures for ovarian cancer is especially critical now given the substantial number of women who are identified by BRCA1/2 positive status as being at high risk for this disease – a risk not necessarily intuitive based on their family history, which often does not include ovarian cancer. Perhaps one of the most unanticipated findings subsequent to the identification of BRCA1 and BRCA2 was the number of high-risk families who do not harbor a detectable mutation in these genes. Recently, the availability of testing for large rearrangements in BRCA1 and BRCA2 has increased the sensitivity of commercial testing; however, the overall yield of deleterious mutations detected with this new method is small. In other high risk families, rare hereditary cancer syndromes may be implicated. Although these syndromes often have a distinct phenotype, they may be underrecognized by clinicians. In addition, we are learning more about other genetic contributions to familial breast cancer risk – including low penetrance gene mutations, SNPs (single nucleotide polymorphisms), and mutations in modifier genes. Given that no other ma-

Protoplasmic incompatibility and self-lysis in Podospora anserina: enzyme activities associated with cell destruction

In the fungus Podospora anserina protoplasmic incompatibility, a phenomenon of cell destruction resulting from the presence in a common cytoplasm of nonallelic incompatible genes, is associated with the appearance of specific proteolytic activities and a phenoloxidase absent from normal cells. We have now shown that during cell lysis, the levels of four additional enzymes increase significantly (3- to 10-fold): a second phenoloxidase, a malate dehydrogenase, a reduced nicotinamide adenine dinucleotide dehydrogenase, and an amino acid oxidase. These increases were inhibited by cycloheximide but unaffected by 5-fluorouracil and actinomycin D or β-phenyl pyruvic acid, an inhibitor of the proteases specific to disintegrating cells. The increase of these four enzymes was shown to be suppressed by mutations in modifier genes known to prevent cell destruction and to inhibit the formation of the proteolytic enzymes.