Unrelated Pts Research Articles

PB1945: CLINICAL AND MOLECULAR CHARACTERISTICS OF CHILDREN WITH DIAMOND-BLACKFAN ANEMIA (DBA): A SINGLE CENTER EXPERIENCE IN ARGENTINA

Background: DBA is an inherited bone marrow failure characterized by erythroblastopenia due to an intrinsic defect in erythropoietic progenitors. Half the patients (pts) present with physical malformations. To date 20 ribosomal proteins (RP) genes associated with DBA and 4 non-RP genes related to non-classical DBA (GATA1, TSR2, EPO, ADA2) have been described. Aims: To describe clinical and molecular characteristics and outcomes of a pediatric cohort with DBA admitted at a single tertiary pediatric center in Argentina Methods: Patients diagnosed with DBA between 2004 and 2020 were retrospectively analyzed. DBA diagnosis was based on constitutional normocytic/macrocytic anemia, reticulocytopenia, paucity of bone marrow erythroid precursors and congenital anomalies. Demographic, clinical, and laboratory data, genetic evaluation and outcomes were recorded. Pts were screened for DNA changes by using a custom designed NGS panel with 19 genes related to DBA (RPS19, RPL11, RPL15, RPL18, RPL27, RPL31, RPL35, RPL35A, RPL5, RPS10, RPS15A, RPS24, RPS26, RPS27, RPS28, RPS29, RPS7, TSR2 and GATA1). All identified variants were classified according to the American College of Medical Genetics guidelines. Results: Fifteen unrelated pts with DBA were analyzed, 10 (66%) males, median age at diagnosis 3,7months (25days-6yrs). Twelve pts (80%) had physical malformations. All pts showed paucity of bone marrow erythroid precursors, and only two presented with compromise of other linages. Thirteen pts (87%) received treatment with prednisone, 8 (62%) achieved transfusion independence and half of them achieved complete treatment independency, while five pts (33%) did not respond to steroids and are under transfusion regimen. These 5pts developed liver iron overload and are under chelation therapy. No therapy was required in two pts with variants in GATA1 and RPL5. Median follow-up was 7.1years (1.7-16.7 years), no pts received HSCT and no pts died. Molecular variants were detected in 10/15 pts (66%), 8/10 were novel. Eight variants were classified as ¨pathogenic¨ or ¨likely pathogenic” and two as “of uncertain significance”. Three pts (30%) presented variants in RPS19, two (20%) in RPS26, four (40%) in other RP genes and one pt with non-classical DBA, presented a variant in GATA1. Phenotype and molecular results of the pts are summarized in Table1. Image:Summary/Conclusion: In this cohort of pts with DBA, molecular diagnosis was achieved in the majority. RPS19 variants were the most common. Interestingly, all pts with RPS19 variants presented with malformations, one of them had also pancytopenia at onset. As the penetrance of DBA is incomplete, the molecular diagnosis is important for detecting silent phenotype carriers in the family that will allow for an accurate selection of stem cell donors, genetic counseling and tailored treatment.

Prognostic Significance of DDX41 Germline Mutations in Intensively Treated AML Patients: An ALFA-Filo Study

Prognostic Significance of DDX41 Germline Mutations in Intensively Treated AML Patients: An ALFA-Filo Study

Diagnostic utility of genetic testing in restrictive cardiomyopathy a single refferal centre experience

Abstract Introduction Restrictive cardiomyopathy (RCM) is considered to have a genetic background in approximately 50–60% of patients (pts) [1,2,3]. Purpose The aim of the study was to assess the frequency of pathogenic gene variants in Polish patients with RCM. Methods Restrictive cardiomyopathy was diagnosed by transthoracic echocardiography. The analysis included all consecutive unrelated pts with RCM admitted to the cardiology department from January 2015 to December 2016. Light-chain (AL) amyloidosis was diagnosed using tissue biopsy, concentration of serum free light chains and bone marrow biopsy. A genetic test was offered to every patient without AL amyloidosis. In 4 pts coding regions of TTR or GLA genes were screened by Sanger sequencing. Next Generation Sequencing (NGS) was performed: a panel covering coding regions of >4800 disease-associated genes in 18 pts and Whole Exome Sequencing (WES) in 2 pts. Classification of rare variants was made according to ACMG criteria [4]. Results Forty-four pts were enrolled. Eighteen pts were diagnosed with AL amyloidosis. One patient was diagnosed with histologically-proven myocarditis and one patient with transthyretin (ATTR) amyloidosis declined genetic test. The other 24 pts (median age 48 yrs, 6 males) underwent genetic testing, their median values (interquartile ranges) of the following parameters were: NT-proBNP, 2508 (995–5895) pg/mL; hs-TnT, 25 (18–46) ng/L; E/A ratio, 2.5 (1.8–4.5); E wave deceleration time, 135 (98–167) ms; E/e' ratio, 16 (12–19); left ventricular (LV) posterior wall, 13 (12–14.5) mm; interventricular septum, 14 (13–17) mm and LV end-diastolic dimension, 48 (43–49) mm. Pathogenic or likely pathogenic variants were detected in 16 probands: 5 in MYH7 gene, 3 in TNNI3 gene, 2 in MYBPC3 gene, 1 in BAG3 gene, 1 in PRAKG2 gene, 2 in GLA gene and 2 in TTR gene. Conclusions Genetic testing is justified in every patient with RCM, in whom AL amyloidosis is excluded. The molecular diagnosis in RCM may result in causal treatment of Anderson-Fabry disease and ATTR amyloidosis or improve disease management in other types of RCM. MYH7 gene is the most common causative gene in Polish patients with RCM. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): National Institute of Cardiology

Genotype-phenotype association in patients with arrhythmic variant of non-compaction cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: None. Purpose. To evaluate the genotype-phenotype association in Belarusian patients with non-compaction cardiomyopathy (NCCM) and clinically significant ventricular arrhythmias. Methods. The study included 170 unrelated pts with NCCM prospectively observed in the RSPC "Cardiology", who underwent 24-hour Holter ECG monitoring for 12 months after entering the study. The median follow-up was 36 months [6; 152,0]. The median age of pts was 42 [18; 69] years, men – 63,2%. The arrhythmic phenotype of NCСM was diagnosed by the presence of unexplained syncope; nonsustained ventricular tachycardia, defined as ≥3 consecutive ventricular contractions lasting <30 seconds with a frequency of ≥120 bpm; the presence of ≥ 500 ventricular premature beats (VPB) per day. The diagnosis of NCCM was established on the basis of the following criteria: 1) Echocardiography of the R. Jenni criteria; 2) CMR of the S. Petersen and A. Jaquier criteria. The mutations search in the coding sequences of 174 genes was performed in 30 unrelated pts with NCCM using next generation sequencing (NGS). Results. In 76 out of 170 (44,7%) pts, clinically significant arrhythmias were the leading manifestation of the disease. Nonsustained VT was recorded in 54 (71,1%) pts, sustained VT – in 15 (19,7%) pts, VPB more than 500 per day – in 50 (65,8%), chronic AF with episodes of nonsustained VT was noted in 34 (44,7%) pts. During the follow-up period (median follow-up of 36 months), devices (ICD/CRT-D) were implanted in 15 (19,7%) pts, appropriate shocks were observed in two of them; three pts died, among which SCD occurred in one patient with CRT-D therapy, which was ineffective in stopping sustained VT. NGS sequencing revealed 40 changes in the nucleotide sequence (5 pathogenic mutations, 30 variants with uncertain significance (VUS), 5 new substitutions) in 27 genes in 26 (86,7%) probands. The proportion of mutations in sarcomeric proteins genes was 26,9%, and in ion channel proteins genes was 23,1%. Nucleotide changes in genes encoding structural proteins accounted for 11,5%. In 38,5% of cases, not one, but two or more rare mutations were detected, and in 30,8% – amino acid replacements were found in proteins of different functional classes. Conclusions. The frequency of multiple mutations was higher (38,5%) in the group of pts with the arrhythmic NCCM phenotype. In the group of pts with implantable devices (ICD/CRT-D), mutations in the genes of sarcomeric proteins were observed more often. The genetic characteristics of pts, along with their clinical characteristics, are markers of a high risk of developing life-threatening arrhythmias and can be additionally used for predicting adverse events in pts with NCCM, as well as for early diagnosis of the disease in their relatives.

Whole exome sequencing for diagnosis of hereditary thrombocytopenia.

Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management.To evaluate the role of whole exome sequencing (WES) in these Pts.Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM).Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9.This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

Chronic Thrombocytopenia in Children: What Could It Hide?

Immune thrombocytopenic purpura (ITP) is one of the most common hemorrhagic disorders in childhood, often caused by an acute self-limiting event. However, 30% of these children develop chronic ITP. Identification of the underlying causes in ITP is an important challenge. Inherited thrombocytopenia (IT) is a rare, underdiagnosed disease, included among the chronic platelet disorders. Next-Generation-Sequencing (NGS) could be an efficient way of discovering potential IT-associated mutations in children with chronic ITP. The purpose of this retrospective study was to investigate children with chronic ITP using a targeted NGS, in order to identify IT-associated mutations. Between June 2017 and April 2020, mutational screening by a targeted NGS was performed on 19 children, either with a familial history of IT [4 unrelated patients (pts)], or with chronic ITP (15 pts), after all other causes of thrombocytopenia were excluded. Nineteen relatives were also investigated. This study was carried out in collaboration with the Laboratory of Genetics, IRCCS Burlo Garofolo in Trieste, that developed a targeted NGS method for the simultaneous analysis of 28 IT genes. The cost of the NGS tests were supported by the public healthcare service. We retrospectively divided our cohort of 19 pts, into three subgroups: Group I included 4 unrelated pts with familial IT; Group II consisted of 6 pts with chronic ITP and a clinical history and/or laboratory features associated with familial IT; and, Group III included 9 pts with chronic ITP refractory to several treatments (Table 1). The median age at the initial diagnosis of thrombocytopenia was lower in Group I than in Groups II and III (19/12 years vs 1310/12 years and 9 years, respectively, p=0.33). The median time between the diagnosis of thrombocytopenia, and the time of the study, was shorter in Group I compared to Groups II and III (11.7 months vs 45.3 and 51.7 months, respectively, p=0.16). Median platelet count at the disease onset was lower in Group III than in Groups I and II (21 x 109/L vs 99 x 109/L and 38 x 109/L, respectively, p=0.28). The median MPV values were 12.5 fL, 9.85 fL and 8.8 fL in Groups II, III, and I respectively. Bleeding symptoms requiring treatment were present at diagnosis in 1/6 (16%) and in 5/9 (55%) children of Groups II and III, respectively. Genetic variants, usually detected in IT, were found in heterozygosity in all children in Groups I and II, and in 7/9 (78%) in Group III. Two out of 4, 2/6 and 2/9 children in Groups I, II, and III, respectively, presented ≥2 variants. Among the 4 children of Group I, ANKDR26 variant was found in 2 pts, together with GP1BA and NBEAL2 (pt#1) and TUBB1 (pt#2). ANKDR26 variant was also recorded as a single mutation in their relatives. Two different variants involving GP1BA (c.98T>A and c.515C<T) were detected in the remaining 2 children of Group I and in their relatives. Pt #4 with GP1BA c.515C>T mutation with mild macrothrombocytopenia had relatives with a previous diagnosis of monoallelic Bernard-Souliers syndrome. As shown in table 1, ABCG8, ACTN1, ETV6, GP1BA, MYH9, SLFN14, or WAS variants, found in combination in 2 pts (pt#5, pt#8), were also detected in the children in Group II, as well as, at least one of the relatives (for a total of 7 cases). ABCG5, ABCG8, ETV6, FLNA, GP1BA, NBEAL2, or SLFN14 were found as variants in patients of Group III. The peripheral blood smear evaluation confirmed the diagnosis of grey platelet syndrome with two NBEAL2 mutations in pt #16. SLFN14, as a single variant, was associated with macrothrombocytopenia in one pt (#10). Three pts (#5, #6, #17), with variants of ABCG8 had hypercholesterolemia. In the cohort of pts with chronic ITP, 4 (#12, #13, #14, #15) had relatives with thrombocytopenia, and 2 (#11, #13) had a familial history of hematological malignancies. Segregation analysis in families, and functional studies to evaluate the pathogenic role of the variants reported, are still in progress. The clinical significance of IT-associated mutations in chronic ITP is uncertain, and yet to be clarified. However, our experience has shown that an in-depth clinical history, and accurate peripheral blood smear examinations, are important to better characterize chronic ITP in children. A targeted NGS method for the simultaneous analysis of different IT genes, has demonstrated to be an effective approach to explore in-depth the IT-associated mutations in children with chronic ITP, refractory to treatment. Table 1. Disclosures Giona: Novartis: Research Funding; Takeda: Speakers Bureau; Sanofi Genzyme: Research Funding, Speakers Bureau.

Non-compaction cardiomyopathy: genetic and clinical features, 5-years outcomes

Abstract Presence of morphological sign as a left ventricular non-compaction (LVNC) only, without supporting clinical criteria, does not determine diagnosis of non-compaction cardiomyopathy (NCCM). Objective To study of the spectrum of NCCM-associated genes, analysis of phenotype-genotype correlations and predictors of life-threatening ventricular tachyarrhythmia (ltVTA), myocardial fibrosis, and adverse outcome. Methods Of 93 pts with identified (Echo/MRI) morphological criteria for LVNC (follow-up median 5,1 years), 60 unrelated pts were included in the study (aged 38.5±13.8 years; 33/55% male; LVEF 42.1±12.9%) with clinical confirmed NCCM (presence any one obligate criteria): family history, neuromuscular disorder, abnormal 12-lead ECG, arrhythmia, HF or thromboembolism (Figure). Genetic testing by NGS (174 genes) was performed; all variants considered as pathogenic (PV) and likely pathogenic (LPV) were confirmed by a Sanger sequencing. Baseline and follow-up data (ECG, HM, Echo, MRI, device interrogation) were collected. Combined adverse outcomes (HF death; SCD; LVAD; HTx; and ltVTA: VT/VF, successful resuscitation, ICD shock) were accepted as composite endpoint. Results PV and LPV were detected in 33 (55%) pts. The most common variants were identified in sarcomere genes – TTNtv, MYBPC3, and MYH7 (47.4%); ion channel genes – 18.2%; digenic mutations were found in 21.6% pts. Gene positivity was associated with systolic dysfunction (LVEF≤49%); the highest risk of low LVEF revealed for digenic carriers (OR 38; 95% CI 4.74–305; p=0.0001). According to CATREG analysis, predictive model was built (R=0,80; R2=0,65; F=10,1; p=0,0001); the presence of disease-causing PV/LPV (β=0.46; F=15.2; p=0,0001) along with low LVEF (β=−0.28; F= 5.96; p=0,018), fibrosis (β=0.21; F= 3.05; p=0,037), wide QRS (β=0.22; F= 4.11; p=0,011) were identified as independent predictors of adverse outcomes. As a result of ROC analysis, independent predictors of ltVTA were determined: fibrosis (nLGE≥2: AUC 0.824; 95% CI: 0.716–0.931; p=0.0001; sen 69%, spe 79%), systolic dysfunction (LVEF≤39%: AUC 0.832; 95% CI: 0.720–0.943; p=0.0001; sen 85%, spe 70%) and nsVT (HR≥150 bmp: AUC 0.829; 95% CI: 0.719–0.940; p=0.0001; sen 76%, spe 83%). According to ROC curves analysis, independent markers of myocardial fibrosis (LGE) were found: nsVT (HR≥150 bpm: AUC 0.766; 95% CI: 0.635–0.897; sen 80%, spe 77%); QRS fragmentation (nQRSfr≥4 leads ECG: AUC 0.822; 95% CI: 0.706–0.938; sen 76%, spe 92%); QTc duration (QTc≥450 ms: AUC 0.828; 95% CI: 0.722–0.935; sen 80%, spe 72%) and native MRI T1-relaxation (T1≥1086 ms: AUC 0.752; 95% CI: 0.626–0.879; sen 70%, spe 70%). Conclusion This results show a basically genetic causing NCCM with predominant mutations in sarcomere genes. As per predictive model, the strongest predictor of poor outcome was gene positivity. Identifying the genetic cause allows risk stratification and management optimization with counseling NCCM pts and their relatives. Study design Funding Acknowledgement Type of funding source: None

Comparative genetic, clinical features, and 5-years outcomes for dilated vs noncompaction cardiomyopathy

Abstract Non-compaction cardiomyopathy (NCCM) and dilated cardiomyopathy (DCM) are associated with high risk of adverse cardiovascular events. Objective To investigate the genetic architecture of NCCM and DCM with comparing the incidence rate of NCCM/DCM related cardiovascular events. Method The study enrolled 120 unrelated pts with NCCM (n=60; age 38.5±13.8 years; 33/55% male; LVEF 42.1±12.9%) and DCM (n=60; age 40.5±13.5 years; 37/61.7% male; LVEF 30.5±11.1%). Diagnosis of NCCM was confirmed by any three accepted criteria as Echo (Jenni/Stöllberger) and MRI (Petersen/Jacquier) with supporting by clinical evidence (family history, neuromuscular disorders, abnormal ECG, arrhythmia, heart failure or thromboembolism). All pts were examined by ECG, HM, Echo, and MRI. Genetic testing was performed by NGS (174 genes) with Sanger verification. The composite end point was accepted as a major adverse cardiac event (MACE) during follow-up: SCD, HF death, LVAD, HTx, successful resuscitation, sustained VT/VF or appropriate ICD/CRT-D shock. Clinical, genetic data, and MACE during a median follow-up of 5.1 years were compared. Results Sarcomere gene (SG) variants (TTNtv, MYBPC3, and MYH7) were the most common pathogenic mutations (47.4%) found in gene-positive NCCM (n=33/55% pts). In general, a similar gene structure (SG – 44.4% vs 47.4%; ion channel protein genes – 25.9% vs 18.2%) was detected for genetic DCM (n=27/45% pts) except for LMNA gene (29.6% vs 6.06%, χ2=4,6; p<0.05). Risk of systolic dysfunction (LVEF<49%) was high for NCCM carriers and higher for multiple mutations (Cochran test: OR=38; 95% CI 4.74–305; p=0.0001) and association of gene positivity with a composite end point was determined at the level of prognostic significance (McNemar's test χ2=22.9; p=0.0001). NCCM patients had a similar MACE risk compared with DCM pts (OR 0.93; 95% CI 0.45–1.92). The incidence rates of all-cause mortality, stroke, HTx, VT/VF, and cardiac device implantation was comparable in DCM vs NCCM groups (Fig.A). As a result of multiple regression analysis, MACE risk models were constructed with step-by-step inclusion: similar common predictors for NCCM et DCM such as low LVEF (β=−0.37 vs β=−0.32), myocardial fibrosis (nLGE: β=0.19 vs β=0.23), width QRS (β=0.17 vs β=0.21), and a strong predictor for NCCM only - the gene positivity (β=0.85; p=0.0001). Kaplan-Meier analysis confirmed this (Fig. B,C). So, amazing fact revealed for NCCM such as systolic dysfunction (but not extent/number trabeculation or NC/C) and disease-causing gene mutations (β=0.85) that were associated with high MACE risk (R=0.90; R2=0.81; F(5.41)=34.8; p<0.00001). Conclusions NCCM pts had a similar cardiovascular risk and mainly genetic spectrum when compared with DCM pts. The unifying MACE predictor was LV dysfunction for both groups. However, gene positivity was associated with adverse outcomes for NCCM pts only (without detailed analysis the specific/localization causing genetic variants). Funding Acknowledgement Type of funding source: None

P341Life-threatening events in patients with non-compaction cardiomyopathy and mutations in ion channel genes

Abstract Purpose to analyze life-threatening events during follow-up in patients with non-compaction cardiomyopathy (NCCM) who have mutations in the genes responsible for the functioning of ion channels. Methods the study included 64 unrelated pts (aged 38.63 ± 14.72 years, 34/53.12% male, LVEF 43.02 ± 8.65%) with criteria of NCCM confirmed by imaging techniques (echocardiography (Jenni) and cardiovascular magnetic resonance (Petersen). Mutation search was carried out by the next generation sequencing (NGS). Pathogenic mutations or rare substitutions with possible clinical significance were found in 12 unrelated pts in the ion channel genes: KCNH2, KCNJ2, CACNA1C, RYR2, HCN4, TRPM4, PKP2, TRPM4. Mutations in other genes (PLN, DSP, NOTCH1, RBM20) were detected in 4 pts (1 pathogenic and 3 VUS). Results mutations in genes associated with long QT syndrome (LQTS) were found in 5 pts with NCCM: c.3107G > A (p.Gly1036Asp), с.2653С>T (p.Arg885Cys) in KCNH2 gene (16.7%); c.T28076 (p.F936C) in CaCNA1C gene (8.3%); c.1165G > C (p.Asp389Asn) in KCNJ2 gene (8.3%). There was no prolongation of the QTc interval > 480 msec on ECG; frequent episodes of nonsustained ventricular tachycardia (VT) were recorded on 24-hour ECG-monitoring. During the follow-up period (median follow-up of 2.4 years), two pts with mutations in KCNH2 genes with suddenly developed sustained VT were implanted with ICD. A 32-year-old patient with a mutation in CACNA1C gene and ST-elevation myocardial infarction of thromboembolic genesis underwent PCI. 58-year-old patient with widening of QRS > 130 msec and frequent paroxysms of nonsustained VT was implanted with CRT-D. An ICD was implanted in a 60-year-old patient with a mutation c.12665_12667del(p.4223del) in the RYR2 gene and suddenly developed a sustained VT. In the remaining two pts with RYR2 gene mutations c.12272C > T (p.Ala4091Val), c.12944G > A(p.Gly4315Glu), frequent episodes of nonsustained VT were recorded, which were corrected medically. Orthotopic heart transplantation and Accucinch transcatheter direct mitral valve annuloplasty system implantation were performed in two pts with ventricular tachyarrhythmias and severe systolic and diastolic dysfunction of the left ventricle with mutations c.3224T > C (p.Leu1075Pro), c.1459_1494del36 (p.Lys487_Leu498del) in the TRPM4 gene. In other pts with mutations in HCN4 (8.3%), DPP6 (8.3%), PKP2 (8.3%) genes ventricular tachyarrhythmias were treated with antiarrhythmic drugs. Conclusion the presence of mutations in the genes controlling the functioning of ion channels is an additional risk factor for adverse events in patients with NCCM.

P1774Genetic heterogeneity and clinical outcomes of non-compaction cardiomyopathy

Abstract The clinical outcomes of non-compaction cardiomyopathy (NCM) range from mild-symptomatic to progressive HF, arrhythmias, thromboembolism, and SCD. Objective The genetic spectrum and clinical outcomes of verified NCM were investigated. Methods The study enrolled 72 unrelated pts (aged 39.2±14.1 years; 43/59.7% male; LVEF 44.7±13.2%) with morphological signs of non-compaction myocardium, confirmed by any three accepted criteria: Echo (Jenni and Stöllberger), MRI (Petersen and Jacquier). During the follow-up 59,7±16,2 months, diagnosis of NCM has been revised by supporting diagnostic elements based on clinical evidence: a family history of cardiomyopathy, coexisting neuromuscular disorders, abnormal 12-lead ECG findings, presence of arrhythmias, heart failure or thromboembolic events. After exclusion 19 cases with isolated asymptomatic and reversible (athletic/peripartum) LVNC, a group (53 persons) for NGS (174 genes) was formed. All variants considered as pathogenic and likely pathogenic were confirmed by a Sanger sequencing. Results Forty-nine (likely) pathogenic variants were identified in 38/71,7% carriers (gene positive pts), including 27/50,9% pts with single variant and 11/20,8% pts with complex genotypes. The most prevalent mutations involved genes were TTNtv (11), MYBPC3 (8), LMNA (4), then MYH7 (3), and two each ILK, HCN4, LAMP2, RAF1, SDHA, RYR2. One mutation is found in the genes TAZ, HADHA, ACTC1, ACTN2, MYH6, DSP, SLC25A4, FBN1, BAG3, SCN5A, TBX20. Clinical features and adverse outcomes during follow-up were compared between groups of the gene-positive and gene-negative pts. Combined adverse clinical outcomes were taken as composite endpoints: death due to HF, SCD, HTx, life-threatening VTA (VT/VF, successful resuscitation, ICD discharges). To analyze composite endpoints, a multivariate regression analysis was performed with step-by-step exclusion. As a result, a prognostic model was constructed (R=0.89; R2=0.78; F=32.9; p<0.ehz748.05261) and independent predictors of adverse outcomes were identified: gene-positivity (β=0.45; p=0.ehz748.05261), 6MWT (β=−0.30; p=0.0002), PQ interval (β=0.23; p=0.0008), syncope history (β=0.29; p=0.ehz748.05263), mTWA (β=0.23; p=0.0005), LVDD (β=0.27; p=0.0001) and RVEF (β=−0.40; p=0.ehz748.05261). As a strongest factor of adverse outcome was gene-positivity, Kaplan-Meier survival curves were constructed. According to the Cox F-criterion (F=24.9) a high level of significance of differences to achieving combined endpoints in the groups (p=0.0001) was determined. Conclusions These results show that NCM is basically a genetic disease with significant heterogeneity and predominant TTNtv mutations; the patients with (likely) pathogenic variants have a worse prognosis with an increased risk for the composite end point of death, transplantation, and defibrillator implantation. Genetic evaluation is an important for prediction and subsequent management NCM pts considering genetic status.

Clinical and Molecular Characteristics of DDX41-Mutated Patients in a Large Cohort of Sporadic MDS/AML

Background:MDS and AML are mostly found in elderly patients. However, even in this population there is increasing evidence of predisposing genetic conditions, which have been underdiagnosed so far. Identifying inherited predisposition to myeloid disorders can be crucial especially in the context of hematopoietic stem cell transplantation (HSCT).Germline mutations in the DEAD/H-box helicase gene DDX41 have been identified in families with multiple cases of MDS or AML but also in sporadic cases. We aimed to analyze the prevalence and clinical features of DDX41-related myeloid malignancies within an unselected cohort of pts diagnosed with MDS or AML (MDS/AML).MethodsBetween March 2017 and June 2018, mutation screening was performed in 842 consecutive pts with a diagnosis of MDS/AML in a single center at Hôpital Saint-Louis, Paris. DNA was obtained from bone marrow or peripheral blood. Targeted sequencing of all exons of a panel of 80 genes recurrently mutated in myeloid malignancies was performed using custom capture-based library preparation (Agilent SureSelect) and Illumina sequencing. Sanger sequencing was performed on selected pts' cultured skin fibroblasts to check for the putative germline origin of the variants.ResultsWe identified a DDX41 gene variant in 36 unrelated pts (4% of 842). We focused on the 32 pts having at least one DDX41 variant with a variant allele frequency (VAF) ranging from 40 to 60% highly suggestive of a germline origin, which was subsequently confirmed in all available cases (N=7). Sixteen variants were classified as pathogenic or likely pathogenic based on major predicted changes in protein sequence while the 16 others were missense variants of unknown significance (VUS), which scored deleterious in most algorithms (Figure 1A). An additional, likely somatic DDX41 mutation (VAF < 40%) was present in 18 of 32 pts (56%). Overall, 22 pts could be unambiguously considered as having a DDX41-related malignancy based on the presence of a major disturbing mutation and/or a second DDX41 mutation, while 10 pts had a single VUS. Twenty-six variants were newly described, including a recurrent one, G173R found in 5 pts, all having a second DDX41 mutation.Median age of the 32 patients was 70 years (35-88). Only 4 pts (12%) had a familial history of hematologic disorders. According to revised WHO classification, 4 (12.5%) had MDS-MLD, 8 MDS-EB (25%), 12 AML (37.5%), 6 MDS/MPN (18.7%), one 5q syndrome and one aplastic anemia. Strikingly, 15/32 (47%) pts had a history of cytopenia several years before blastic evolution and the 5 pts with G173R presented with hypoplastic MDS or initially isolated cytopenias, suggesting a specific functional effect of this mutation. Karyotype was normal in 16 pts (44%), complex in one, 12 pts had an isolated abnormality, and three had cytogenetic failure. Additional driver mutations were identified in most (27/32,84%) pts (Figure 1B), but we noticed that they were less frequent and at lower VAF in pts having both germline and somatic DDX41 mutations as compared to pts with a single variant (median 1.5 vs 3 mutations, median VAF 7% vs 29.5%, p<0.001). This suggests distinct oncogenic pathways, with DDX41 double-hit oncogenesis being relatively independent of other oncogenic drivers.Seven low-risk MDS pts were untreated, 7 received ESA and 5 (71%) responded. Ten high-risk MDS/AML pts received a hypomethylating agent and 8 (80%) achieved hematological response. Nine AML pts received intensive chemotherapy, with a complete response rate of 100% (7/7, 2 ongoing) and 5 of them had HSCT, all of them being alive with tolerable toxicity. Five pts died, median OS was 87 months, and 2-y OS was 89%. No difference on OS was observed between single and double-DDX41 mutated pts.Conclusions:DDX41 germline variant carriers represent a significant part of MDS/AML pts, the vast majority presenting without familial history. The predicted change in protein and/or the presence of a second somatic mutation strongly support the causality of the germline variant in most pts. By contrast with previous reports, pts frequently presented a phase of cytopenia before overt malignancy. Finally, outcome regarding response to treatment and OS in this DDX41-mutated cohort appeared relatively favorable. DisclosuresPeffault De Latour:Pfizer Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Fenaux:Otsuka: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

In Patient with Chronic Lymphocytic Leukemia the Overall SURVIVAL Is NOT Different WHEN Analysed by Cause of Death, Related or NOT Related to the Disease

AbstractAbstract 4587Chronic Lymphocytic Leukemia (CLL) is the most common lymphoprolipherative disorder of the elderly population in Western countries. It shows a highly variable clinical course. In fact, some patients may die within few months from the diagnosis because of CLL itself or disease-related complications. Other patients do not require any treatment for many years and have a long-standing disease. Many of them could die because of disease different from CLL. The identification of subgroups of patients with peculiar features predictive of the clinical behaviour of the disease is important. This retrospective analysis has the purpose to study patients affected by CLL, diagnosed and followed at our single centre of Haematology, focusing our attention on their causes of death.We selected 340 patients affected by CLL from our data-base, diagnosed from January 1999 to December 2010 and followed until March 2012. We distinguished the causes of death in two groups: one related to CLL (as progression of the disease, evolution to Richter’s Syndrome, infections due to chemotherapy) and the other not related to CLL (i.e. cardiovascular diseases, solid tumours, old age). Statistical analysis, conducted using SPSS version 16.0 for Windows and “GraphPad Prism” GraphPad Software Inc., compared these two groups and tried to select other subgroups.We recorded 69 deaths: 47 related to CLL (68.1%) and 22 unrelated to CLL (31.9%). The median age of death of our cohort of patients was 76 years (range 40–92); those patients with a CLL-related death (related pts) died at a median age of 76 years (range 40–89) and the patients with a CLL-unrelated death (unrelated pts) died at a median age of 76 years (range 61–92). No differences in terms of median age of death were found analysing the data by gender.Also, considering the overall survival time from diagnosis to death, it was 58 months (range 9–155) in the related group and 43 months (range 14–121) in the unrelated group (p=0.185).When divided our population by the disease behaviour, we obtained 3 subgroups of patients: patients who progressed and died for CLL related causes (group A), patients who progressed and died for CLL unrelated causes (group B) and patients who did not progress and died for CLL unrelated causes (group C) (Table 1). The only statistical significant difference was found among the median overall survival times in un-progressive patients who died for non CLL related causes (p=0.043).The median age of death was not affected by the cause of death in our CLL population. Moreover, patients with un-progressive CLL showed an overall survival time shorter than the progressive CLL subgroups. Patients with un-progressive CLL probably had a shorter survival because unrelated CLL diseases could are more difficult aggressive than CLL itself.Table 1group Agroup Bgroup CpN° patients47139Median age at diagnosis (years) (range)70 (39–85)71 (56–83)77 (67–86)n.s.Median age at death (years) (range)76 (40–89)75 (61–84)79 (68–92)n.s.Overall survival time (months) (range)58 (9–155)48 (18–121)22 (14–78)0.043 Disclosures:No relevant conflicts of interest to declare.

P2-13-07: Prevalence of Germline BRCA1 and BRCA2 Deleterious Mutations in Brazilian Patients with High-Risk of Breast and Ovarian Cancer.

Abstract Background: Germline mutations in the BRCA1/2 genes account for the majority of hereditary breast ovarian cancer (HBOC). Distribution and prevalence of clinically relevant mutations in BRCA1/2 differ among different populations. All of the Brazilian studies published to date have not performed complete sequencing of both genes, restricting their analyses to selected exons. In addition, a highly heterogeneous mutation spectrum was observed. The lack of data concerning the prevalence and profile of BRCA1/2 deleterious mutation and the appropriated screening criteria for DNA sequencing in the Brazilian population complicates the design of screening strategies. Objective: To accurately determine the contribution of BRCA1/2 germline mutations in a well characterized sample of Brazilian breast and ovarian high-risk pts. Methodology: A total of 73 unrelated pts attending a Brazilian public hospital from September 2005 to July 2006, with HBOC risk ≥10% according to Frank, Evans or BRCAPRO algorithms, underwent complete sequencing and MLPA analysis of BRCA1/2 genes. Demographic data and tumor pathological features were extracted from prospectively registered medical records. Results: Mean age, median of cancer cases in families and median score by Frank, Evans and BRCAPRO algorithms were respectively 48y (24-78), 4 (0-13), 11.2 (0-56,3), 13.4 (0,1-100) and 16 (2-75). A total of 79 unknown clinical significance mutations were identified: 44 in BRCA1 and 35 in BRCA2. Known deleterious mutations were identified in 8 pts. Four in BRCA1: R71G at exon 5, 5382insC at exon 20 (2 patients) and R1751X at exon 20; and four in BRCA2: R2318X at exon 13, R3128X at exon 25, 5844del5 and 6633del5 at exon 11. Several polymorphisms were found in all pts. No mutation was detected by MLPA technique. One novel frameshift deleterious mutation in BRCA2: 6610insTT at exon 11 which results in a stop codon was detected in a breast cancer patient and in four of her relatives from a family with high-risk of HBOC. Taking into account the novel mutation, the prevalence of BRCA1/2 mutation in high-risk pts in our cohort is 12,3%. Pts characteristics presented in [table 1]. Conclusion: To the best of our knowledge, the study represents the first report of the complete sequencing and MLPA analysis of BRCA1/2 genes in large Brazilian series of HBOC high-risk pts. The prevalence of germline mutations found in the present study was similar to the reported by other studies examining high-risk populations. B1=BRCA1; B2=BRCA2; BC=breast cancer; IDC=invasive ductal carcinoma; MC=medullary carcinoma; ILC=invasive lobular carcinoma; HR=hormone receptor; (b)=bilateral, N/A=not available; N/C=never had cancer; pos=positive; neg=negative, ind=indeterminate Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-13-07.

Reduced Intensity Conditioning (RIC) Regimen Followed By Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) In Adult Patients (PTS) With Acute Lymphoblastic Leukemia (ALL)

Background: RIC regimens are less myelosuppressive, but still allow for successful engraftment with acceptable treatment-related mortality (TRM) in frail pts who otherwise would not be suitable candidates for HSCT. This is particularly relevant in ALL, since pts often sustain toxicity from dose-intense regimens, or may be diagnosed in advanced age. The antitumor effect of this approach is not well-established in ALL. Methods: We evaluated 30 ALL pts (19 M/11 F) treated from 1996 to 2008 with fludarabine 120 mg/m2, melphalan 140 mg/m2 and unmanipulated stem cells. GVHD prophylaxis consisted of tacrolimus and mini-methotrexate. Anti-thymocyte-globulin was added to matched unrelated pts. Results: Median age was 44 years (range 23–64). ECOG status at time of HSCT was 0 (n = 16), 1 (n = 10) or 2 (n = 4) with median co-morbidity score of 3 (range 0–7) by Charlson Comorbidity Index. 24 pts had B-lineage and 6 had T-lineage disease. Cytogenetic data were available for 26 pts; 19 had high-risk cytogenetics, including 9 with Ph+ disease. Disease stage at time of study entry was CR1 (n = 5), ≥CR1 (n = 12), or primary or refractory relapse (n = 13), with median 2 prior chemotherapy regimens (range 1–4); five pts had a prior allogeneic HSCT. Donor type was matched related (n = 13) or matched unrelated (n = 17) and stem cell source was bone marrow (n = 14) or peripheral blood (n = 16). The median total nucleated cell dose and CD34+ cell dose were 3.80 × 108 cells (range 0.68 – 17.16) and 4.15 × 106 cells (range 1.78 – 12.03), respectively. Median time to ANC 0.5 × 109/L was 13 days (range 10–24). Median time to platelet count 20 × 109/L was 18 days (range 10–57). Eight pts were alive at a median follow up of 12 months from HSCT (range 3–59). OS and DFS were 32% and 29%, respectively, at 1 year. Of note, only 1 among 5 pts in CR1 had disease progression, compared to 8 among 13 with refractory disease at time of HSCT. The cumulative incidence of acute GVHD, grades II-IV and III-IV were 40% and 13%, respectively, and chronic GVHD was 22% (7% for extensive). The cumulative incidence of TRM at 100 days and 1 year were 17% and 33%, respectively. There were 22 deaths: recurrence 14, infection 4, GVHD 4. Conclusion: RIC HSCT can provide disease control in patients with ALL, and merits further evaluation. Alternative treatment strategies need to be explored in pts with advanced disease. The observed TRM rate is comparable to previously reported rates in in heavily pre-treated leukemia patients.

Abstract 2966: Should a Minimum Corrected QT Interval (QTc) be a Prerequisite for Long QT Syndrome Genetic Testing?

Background: Long QT Syndrome (LQTS) genetic testing has been available clinically since August 2004. In cases of bona fide LQTS, the yield of genetic testing is approximately 75%. Recently (HRS 2007), Priori and colleagues have suggested that referral for LQTS genetic testing should be based on the screening QTc citing a yield of &lt; 2% for cases referred to their research program with a QTc &lt; 440 ms. Here, we analyze the effect of QTc on the yield in two of the largest assembled cohorts of unrelated patients (pts) referred for LQTS genetic testing. Methods: From August 1997 to July 2006, 1125 unrelated pts (717 females, average age at diagnosis 23.5 yrs, average QTc 473 ms) were referred to either the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory (N = 541) for research-based LQTS genetic testing or to PGxHealth (N = 574) for the FAMILION ® LQTS genetic test. Comprehensive open reading frame and splice site analysis (60 exons) for LQTS-causing mutations involving KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) was conducted by either dHPLC/DNA sequencing (Mayo), or by direct DNA sequencing (FAMILION). Results: The probability of a positive genetic test increased dramatically with increasing QTc, ranging from 5% for the 38 pts referred for genetic testing despite a QTc &lt; 400 ms to 60% for the 168 pts with a QTc &gt; 500 ms (p &lt; 0.00001). Akin to the Priori cohort, the yield of the genetic test was similar among the subset of pts with a QTc &lt; 470 ms (64% Priori, 61% Mayo cohort, and 53% FAMILION, p = NS). However, the yield for those pts with a QTc between 440 and 470 ms was much greater in the Mayo (49%) and FAMILION (36%) cohorts compared to the Priori cohort (14%, p &lt; 0.0005). In contrast to the 2% yield observed in Italy, 22% of pts with QTc &lt; 440 ms referred to Mayo Clinic and 14.5% of the FAMILION referrals had a positive genetic test (p &lt; 0.001). Conclusions: Whereas only 2% of patients with a QTc &lt; 440 ms had a positive genetic test in Italy, the yield was ten times greater among those referred to Mayo Clinic despite the same QTc cut-off. The cumulative index of suspicion for LQTS rather than a pre-specified QTc cut-off should guide clinical decision making as to the appropriate utilization of the genetic test.

STUDIES OF A NEW GENETIC DISEASE CHARACTERIZED BY A DEFICIENT PMN LEUKOCYTE SURFACE GLYCOPROTEIN(S)

Four unrelated pts (3 F, 1 M) with granulocytosis, delayed umbilical cord severance, periodontitis, &/or recurrent soft tissue infections were identified. Adhesion-dependent PMN functions (chemotaxis, adherence-spreading, aggregation, phagocytosis, orientation, & antibody-dependent cellular cytotoxicity) were significantly (p <.001 for each assay) diminished in each pt. All fathers, mothers or siblings (2 M, 1 F) were asymptomatic & demonstrated normal PMN functions. PMN homogenates of each patient demonstrated a severe deficiency (<5% normal) of a high M.W. glycoprotein (GP138) (SDS-PAGE). A NaB3H4 labelling technique indicated that GP138 represents a major surface GP complex of normal PMNs (11.4±7 μg/mg total protein), but was undetectable on the surface of pt PMNs. GP138 content of maternal or female sibling PMNs was diminished (mean = 3.3 μg/mg), but was normal (mean = 13.6 μg/mg) in paternal or male sibling suspensions. Monoclonal antibodies directed at the α & common β subunits of 3 leukocyte "adhesive" GPs (OKM1, LFA-1, P150,95) were employed to demonstrate the absence of each of these GPs on the surface of patient PMNs & diminished (10-50% normal) surface OKM1 & β on maternal or female siblings' PMNs (flow microfluorimetry). Thus, GP138 deficiency syndrome represents a new disorder(s) related to the absence of critical adhesive PMN GPs. Since males & females are affected & the carrier state can be detected only in females, its mode of genetic transmission is uncertain.