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Intermediate CAG (polyQ) expansions in the gene ataxin-2 (ATXN2) are now recognized as a risk factor for amyotrophic lateral sclerosis. The threshold for increased risk is not yet firmly established, with reports ranging from 27 to 31 repeats. We investigated the presence of ATXN2 polyQ expansions in 9268 DNA samples collected from people with amyotrophic lateral sclerosis, amyotrophic lateral sclerosis with frontotemporal dementia, frontotemporal dementia alone, Lewy body dementia and age matched controls. This analysis confirmed ATXN2 intermediate polyQ expansions of ≥31 as a risk factor for amyotrophic lateral sclerosis with an odds ratio of 6.31. Expansions were an even greater risk for amyotrophic lateral sclerosis with frontotemporal dementia (odds ratio 27.59) and a somewhat lesser risk for frontotemporal dementia alone (odds ratio 3.14). There was no increased risk for Lewy body dementia. In a subset of 1362 patients with amyotrophic lateral sclerosis with complete clinical data, we could not confirm previous reports of earlier onset of amyotrophic lateral sclerosis or shorter survival in 25 patients with expansions. These new data confirm ≥31 polyQ repeats in ATXN2 increase the risk for amyotrophic lateral sclerosis, and also for the first time show an even greater risk for amyotrophic lateral sclerosis with frontotemporal dementia. The lack of a more aggressive phenotype in amyotrophic lateral sclerosis patients with expansions has implications for ongoing gene-silencing trials for amyotrophic lateral sclerosis.

We characterized the role of structural variants, a largely unexplored type of genetic variation, in two non-Alzheimer’s dementias, namely Lewy body dementia (LBD) and frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS). To do this, we applied an advanced structural variant calling pipeline (GATK-SV) to short-read whole-genome sequence data from 5,213 European-ancestry cases and 4,132 controls. We discovered, replicated, and validated a deletion in TPCN1 as a novel risk locus for LBD and detected the known structural variants at the C9orf72 and MAPT loci as associated with FTD/ALS. We also identified rare pathogenic structural variants in both LBD and FTD/ALS. Finally, we assembled a catalog of structural variants that can be mined for new insights into the pathogenesis of these understudied forms of dementia.

Objective To identify potential biomarkers of preclinical and clinical progression in chromosome 9 open reading frame 72 gene (C9orf72)-associated disease by assessing the expression levels of plasma microRNAs (miRNAs) in C9orf72 patients and presymptomatic carriers. Methods The PREV-DEMALS study is a prospective study including 22 C9orf72 patients, 45 presymptomatic C9orf72 mutation carriers and 43 controls. We assessed the expression levels of 2576 miRNAs, among which 589 were above noise level, in plasma samples of all participants using RNA sequencing. The expression levels of the differentially expressed miRNAs between patients, presymptomatic carriers and controls were further used to build logistic regression classifiers. Results Four miRNAs were differentially expressed between patients and controls: miR-34a-5p and miR-345-5p were overexpressed, while miR-200c-3p and miR-10a-3p were underexpressed in patients. MiR-34a-5p was also overexpressed in presymptomatic carriers compared with healthy controls, suggesting that miR-34a-5p expression is deregulated in cases with C9orf72 mutation. Moreover, miR-345-5p was also overexpressed in patients compared with presymptomatic carriers, which supports the correlation of miR-345-5p expression with the progression of C9orf72-associated disease. Together, miR-200c-3p and miR-10a-3p underexpression might be associated with full-blown disease. Four presymptomatic subjects in transitional/prodromal stage, close to the disease conversion, exhibited a stronger similarity with the expression levels of patients. Conclusions We identified a signature of four miRNAs differentially expressed in plasma between clinical conditions that have potential to represent progression biomarkers for C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. This study suggests that dysregulation of miRNAs is dynamically altered throughout neurodegenerative diseases progression, and can be detectable even long before clinical onset. Trial registration number NCT02590276.

Massive genetic analysis identifies critical pathways and cell types involved in pathogenesis of amyotrophic lateral sclerosis., Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely, neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated six differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, MAPKAPK3, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

Hannu Laaksovirta konsortion jäsenenä. The Genomics England Research Consortium, The International ALS/FTD Genomics Consortium (iAFGC), The International FTD Genetics Consortium (IFGC), The International LBD Genomics Consortium (iLBDGC), The NYGC ALS Consortium, The PROSPECT Consortium,17 James B. Rowe,17 Luisa Benussi,18 Giuliano Binetti,18,19 Roberta Ghidoni,18 Edwin Jabbari,20,21 Coralie Viollet,22 Jonathan D. Glass,23 Andrew B. Singleton,24 Vincenzo Silani,25,26 Owen A. Ross,27 Mina Ryten,8,28,29 Ali Torkamani,30 Toshiko Tanaka,31 Luigi Ferrucci,31 Susan M. Resnick,32 We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered. We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic cortical interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated five differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

Neurofibromatosis type 2 (NF2) is an autosomal dominant Mendelian tumor predisposition disorder caused by germline pathogenic variants in the tumor suppressor NF2. Meningiomas are the second most common neoplasm in NF2, often occurring in multiple intracranial and spinal locations within the same patient. In this prospective longitudinal study, we assessed volumes and growth rates of ten spinal and ten cranial benign meningiomas in seven NF2 patients that concluded with surgical resection and performed whole-exome sequencing and copy-number variant (CNV) analysis of the tumors. Our comparison of the volume and the growth rate of NF2-associated spinal and cranial meningiomas point to the differences in timing of tumor initiation and/or to the differences in tumor progression (e.g., non-linear, saltatory growth) at these two anatomical locations. Genomic investigation of these tumors revealed that somatic inactivation of NF2 is the principal and perhaps the only driver of tumor initiation; and that tumor progression likely occurs via accumulation of CNVs, rather than point mutations. Results of this study contribute to a better understanding of NF2-associated meningiomas clinical behavior and their genetic underpinnings.

A homozygous AAGGG repeat expansion within the RFC1 gene was recently described as a common cause of CANVAS syndrome. We examined 1,069 sporadic ALS patients for the presence of this repeat expansion. We did not discover any carriers of the homozygous AAGGG expansion in our ALS cohort, indicating that this form of RFC1 repeat expansions is not a common cause of sporadic ALS. However, our study did identify a novel repeat conformation and further expanded on the highly polymorphic nature of the RFC1 locus.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30 % for JMML and 40 % for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a “CBL syndrome”. Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant’s penetrance, function and natural history.

Neurofibromatosis type 2 (NF2) is an autosomal dominant tumor predisposition disorder. It is caused by a germline mutation of the NF2 tumor suppressor gene located on chromosome 22q.1,2 The hallmark of NF2 is the development of bilateral vestibular schwannoma (VS), which is present in 90%–95% of patients. Subsequently, it has been the cardinal focus of research study in NF2, but little is definitively understood about why these tumors differ in gross appearance,3,4 natural history,5 and surgical outcomes6 compared with sporadic VS, despite sharing a common pathobiologic etiology7 (NF2 tumor suppressor gene inactivation)8 and histology. NF2-associated VSs are known to have worse surgical outcomes, including poorer hearing and facial nerve preservation rates9 compared with matched (size) sporadic counterparts. Adverse outcomes in NF2-associated VS resections have been attributed to their propensity to infiltrate the adjacent cochlear and facial nerves rather than displace these structures, as observed with sporadic tumors that respect this histologic cleavage plane.10 Furthermore, synchronous facial neuromas may be identified in at least 20% of cases and may contribute to surgical morbidity when not distinguished at operation.11 A significant disparity in recurrence rates also exists. After complete resection of NF2-associated VS 50%.12 Comparable studies performed in patients with sporadic VS show essentially no recurrence.10 Finally, there is a significant difference in long-term control rate with radiosurgery of sporadic VS (over 95%) compared with NF2-associated VS (∼50%).13,14 In addition to these notable differences in response to treatment, the posterior fossa component of NF2-associated VS is frequently multilobulated (botryoid) and appears phenotypically different from sporadic VS, which is typically ovoid in shape and has smooth contours.3,15 Based on these observations, we hypothesized that the multilobulated VS (MVS) in NF2 arise from numerous, independent, somatic Knudson “second hits”' in NF2. We propose that these multiple, different clonal tumors merge over time in the cerebellopontine angle (CPA) to form a larger, polyclonal, and multilobulated mass (Fig. 1). To investigate this hypothesis, we evaluated the MRIs in a large cohort of untreated NF2-associated VS. To investigate the putative polyclonal origin of NF2-associated MVS, we sequenced NF2 and determined copy-number variation (CNV) in multiple regional samples from 4 tumors resected from 4 patients. Fig. 1. Hypothesis: MVSs in NF2 patients are polyclonal derivatives. Illustration depicting the hypothesis that MVSs in NF2 patients are derived from multiple discrete tumors (top panel) that have collided (middle panel). The premise underlying this investigation ...

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund., The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1-/- and the M1/M3-/- knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing.

Losses, gains and regions with allelic imbalance in the grade I meningioma and all four fragments of the grade II meningioma. (PDF 173 kb)

Genes affected by gains and losses in the grade II meningioma. (PDF 884 kb)

Phred scores for Sanger sequencing of the heterozygous mutant (red font) and surrounding homozygous wt nucleotides. Normal control sample is shown on the top (green fill). Phred scores for both forward and reverse sequencing reactions are shown. Note that a heterozygous nucleotide would usually affect (decrease) phred scores of a few adjacent wt homozygous nucleotides. (PDF 44 kb)

Cancer genes affected by losses or gains in the grade II meningioma. (PDF 62 kb)

Mutations selected for Sanger verification. (PDF 115 kb)

Describe the relationship between cochleovestibular schwannoma (CVS) volume, audiovestibular characteristics, and magnetic resonance imaging (MRI) findings in patients with neurofibromatosis type 2 (NF2).Subgroup analysis of NF2 prospective natural history study from 2008 to 2011.Quaternary medical research institute.NF2 patients with small treatment-naive CVSs (volume1000 mm(3)) by ear; N = 49 ears (32 patients). Cross-sectional analysis of the following parameters was performed: tumor size, auditory brainstem response (ABR), 4-frequency pure-tone average (4f-PTA; 0.5, 1, 2, and 4KHz), cervical vestibular evoked myogenic potential (cVEMP), caloric testing, 240° velocity step test (VST), and MRI findings.For all physiologic measures but the 4f-PTA, larger tumors correlated with abnormal responses (P.05). For abnormal ABR, mean tumor volume was 405 vs 151 mm(3) (P = .0007) for normal ABR. Similarly, larger tumors correlated with weak caloric responses (mean 521 vs 165 mm(3); P = .0007) and weak cVEMP (mean 357 vs 192 mm(3); P = .0262). Tumor volume was not significantly correlated with 4f-PTA. Elevated intralabyrinthine protein on MRI fluid-attenuated inversion recovery sequences was correlated with larger tumor volume (mean 333 vs 55 mm(3); P = .001) and abnormal ABR and 4f-PTA (P.05) but did not correlate with cVEMP, VST, or caloric responses.In our cohort, ABR, caloric response, cVEMP, and elevated intralabyrinthine protein correlated with tumor volume, but 4f-PTA did not. Abnormal ABR and 4f-PTA correlated with elevated intralabyrinthine protein. These findings may provide insight on the effect of small CVS on the inner ear and cochleovestibular nerves, which may aid in their optimal management.

The goal of the present work was to identify the candidate genetic markers predictive of alloimmunization in sickle cell disease (SCD). Red blood cell (RBC) transfusion is indicated for acute treatment, prevention, and abrogation of some complications of SCD. A well-known consequence of multiple RBC transfusions is alloimmunization. Given that a subset of SCD patients develop multiple RBC allo-/autoantibodies, while others do not in a similar multiple transfusional setting, we investigated a possible genetic basis for alloimmunization. Biomarker(s) which predicts (predict) susceptibility to alloimmunization could identify patients at risk before the onset of a transfusion program and thus may have important implications for clinical management. In addition, such markers could shed light on the mechanism(s) underlying alloimmunization. We genotyped 27 single nucleotide polymorphisms (SNPs) in theCD81,CHRNA10,andARHGgenes in two groups of SCD patients. One group (35) of patients developed alloantibodies, and another (40) had no alloantibodies despite having received multiple transfusions. Two SNPs in theCD81gene, that encodes molecule involved in the signal modulation of B lymphocytes, show a strong association with alloimmunization. If confirmed in prospective studies with larger cohorts, the two SNPs identified in this retrospective study could serve as predictive biomarkers for alloimmunization.

Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia.

Although schwannoma and neurofibroma tumors are generally reported as distinct pathologic diagnoses, sporadic schwannoma/neurofibroma hybrid nerve sheath tumors have been reported in the general population with components of both entities. We report the clinicopathological features of these hybrid nerve sheath tumors in patients with neurofibromatosis type 2 (NF2). A retrospective review of nerve sheath tumor surgical specimens from patients with NF2 enrolled at the National Institutes of Health was performed. Those specimens reported to have schwannoma-like and neurofibromalike features were selected for further characterization by morphology, immunohistochemical panel (CD34, S100, neurofilament triplet protein (immunostain) (NFTP), epithelial membrane antigen (EMA)), and confirmation as hybrid tumors. Of 43 total NF2 patients undergoing resection of nerve sheath tumors, 11 specimens from 11 (26%) patients were found to be benign nerve sheath tumors exhibiting hybrid features of both neurofibroma and schwannoma. Immunohistochemical studies showed the schwannoma component to be S100+, CD 34- while the neurofibroma component was CD34+, variable S100+. Our experience emphasizes the importance of including this distinct tumor subtype, the schwannoma/neurofibroma hybrid tumor, in the differential diagnosis of nerve sheath tumors in NF2 patients and suggests that the relationship between neurofibroma and schwannoma tumors is closer than previously suspected..

2051 Background: Neurofibromatosis type 2 is a heritable tumor predisposition syndrome characterized by the growth of multiple tumor types in the nervous system, including bilateral vestibular schwannomas, meningiomas and ependymomas. Recent genomic sequencing studies have revealed that NF2 inactivation is the most frequent genetic event in sporadic meningiomas. In line with Knudson’s hypothesis, it is accepted that somatic inactivation of the wildtype NF2 allele initiates tumor growth in NF2 patients, but little is known of what other genes or pathways influence meningioma tumorigenesis. Methods: To investigate this question, we performed whole exome sequencing (WES) (Illumina Hi-Seq 2500 platform, 96 Mb SeqCap EZ Exome + UTR Library, NimbleGen) and SNP-array analysis (HumanOmniExpressExome-8, v1.2 arrays, Illumina) of twenty spinal and cranial meningioma samples from seven NF2 patients. Mutation validation was completed via orthogonal sequencing (IonTorrent, ThermoFisher). Results: We identified NF2 germline mutations in all patients, including five nonsense, one splice site and one likely pathogenic intronic mutation. We found that the predominant mechanism of somatic NF2 inactivation in the tumors was loss of heterozygosity (LOH): we identified large chromosome 22 deletions containing NF2 in nineteen out of twenty meningiomas. The second most frequent chromosomal aberration was a deletion within chromosome 1p, followed by entire chromosome X deletion and rearrangements in chromosome 17q. The remaining samples exhibited normal diploid genomic architecture. Somatic mutations included about twenty point substitutions and small indels. Conclusions: Our study revealed that somatic inactivation of NF2 is the most frequent and only recurrent genetic event in NF2-associated meningiomas. Large LOH events are the most prevalent second hit mechanism and may also represent a common path of meningioma progression. Somatic single nucleotide substitutions and small indels are rare in these tumors. Interestingly, we did not identify mutations in TRAF7, KLF4, AKT1, or SMO, which have been found to be critical for non-NF2 meningioma growth.

Background Thymic involution is a prominent characteristic of an aging immune system. When thymic function is reduced/absent, the peripheral T cell pool is subject to the laws of peripheral T cell homeostasis that favor survival/expansion of T cell receptors with relatively higher functional avidity for self-peptide/MHC complexes. Due to difficulties in assessing the TCR avidity in polyclonal population of T cells, it is currently not known whether high avidity T cells preferentially survive in aging individuals, and what impact this might have on the function of the immune system and development of autoimmune diseases. Results The phenotype of T cells from aged mice (18-24 months) indicating functional TCR avidity (CD3 and CD5 expression) correlates with the level of preserved thymic function. In mice with moderate thymic output (> 30% of peripheral CD62Lhi T cells), T cells displayed CD3lowCD5hi phenotype characteristic for high functional avidity. In old mice with drastically low numbers of CD62Lhi T cells reduced CD5 levels were found. After adult thymectomy, T cells of young mice developed CD3lowCD5hi phenotype, followed by a CD3lowCD5low phenotype. Spleens of old mice with the CD3low/CD5hi T cell phenotype displayed increased levels of IL-10 mRNA, and their T cells could be induced to secrete IL-10 in vitro. In contrast, downmodulation of CD5 was accompanied with reduced IL-10 expression and impaired anti-CD3 induced proliferation. Irrespective of the CD3/CD5 phenotype, reduced severity of experimental allergic myelitis occurred in old mice. In MTB TCRβ transgenic mice that display globally elevated TCR avidity for self peptide/MHC, identical change patterns occurred, only at an accelerated pace. Conclusions These findings suggest that age-associated dysfunctions of the immune system could in part be due to functional erosion of T cells devised to protect the hosts from the prolonged exposure to T cells with high-avidity for self., National Institutes of Health (U.S.), National Heart, Lung, and Blood Institute (1U54 HL090503 (ZT-C)), National Institute of Allergy and Infectious Diseases (U.S.) (Grant 1R01 AI48837), National Institute of Allergy and Infectious Diseases (U.S.) (Grant 1R01 AI41573 )

Familial acute myeloid leukemia (AML) is rare and linked to germline mutations in RUNX1 or CCAAT/enhancer binding protein-α (CEBPA). We conducted whole-exome sequencing on a large family with AML originally evaluated at NIH in 1969 and uncovered a CEBPA Q311P mutation in all tested, affected members. The Q311P variant, located in the C-terminal bZip domain of CEBPA, was predicted to be highly deleterious by in silico algorithms; the Q311 position was highly conserved among CEBPA orthologs and paralogs. This variant has not been observed in the NHLBI's Exome Sequencing Project or the Broad Institute's Exome Aggregation Consortium. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mutant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation of a promoter with a tetramer of CEBP sites, consistent with a loss-of-function mutation. In addition, there was decreased colony survival in fibroblasts with CEBPA Q311P, associated with the clinical radiosensitivity postulated in one carrier. From 45 years of follow-up, we observed incomplete penetrance (46%) of CEBPA Q311P; we did not observe other cancers or leukemias segregating with the mutation. Thus, we present a comprehensive clinical and molecular characterization of a novel AML-associated variant in CEBPA in a NCI family with nearly half a century of follow-up. This study reveals that a germline mutation in the C-terminal bZip domain can alter the ability of CEBPA to bind DNA and reduces transactivation, leading to AML, though with lower penetrance than the canonical N-terminal frameshift germline mutations. Citation Format: Anand Pathak, Katja Seipel, Alexander Pemov, Ramita Dewan, Christina Brown, Sarangan Ravichandran, Brian T. Luke, Meredith Yeager, Richard A. Gatti, Neil Caporaso, John J. Mulvihill, Lynn Goldin, Thomas Muller Pabst, Mary Lou McMaster, Douglas R. Stewart. Whole-exome sequencing reveals a novel germline variant in CEBPA-associated familial acute myeloid leukemia: 45-year follow-up of a large family. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2756. doi:10.1158/1538-7445.AM2015-2756