Occurrence of Acute Myeloid Leukemia in Young Pregnant Women

Although acute leukaemia is rare in pregnancy its importance lies in its life-threatening potential, both to the child and the mother. The possibility of vertical transmission of leukemic cells increases the attention devoted to these patients and their offspring. Three cases of pregnant young women (15-17 years of age) with AML are presented. This series of cases is the first report where gene abnormalities such as ITD mutations of the FLT3 gene and AML1/ETO fusion genes were screened in pregnant AML patients and their babies, so far. Unfortunately, very poor outcomes have been associated to similar cases described in literature, and the same was true to the patients described herein. Although very speculative, we think that the timing and possible similar exposures would be involved in all cases

Identification of differential regulation of European versus African local ancestry haplotypes surrounding ApoEε4

Background The risk for late‐onset Alzheimer disease (AD) in ApoEε4 carriers differs between ancestral groups. ApoEε4 Non‐Hispanic White (NHW) homozygotes have an odds ratio of 14.9 while the risk is much lower in Africans (AF) (OR 2.2‐5.7). Local ancestry (LA) analyses in ApoEε4 carrier populations have shown the protective effect in Africans relative to NHW is due to factors lying in the LA surrounding ApoEε4. No coding differences between genes in the LA have been observed between AF and NHW ancestries. Thus regulatory differences in LA non‐coding regions are most likely involved in the protective factor(s) lowering the risk for AF carriers of ApoEε4. Enhancers are the most common regulatory element, and thus we sought to identify if any of these variants had functional enhancer effects between the two ancestries. Little functional characterization of genetic regulation in AF ancestries has been investigated. Method We identified 56 significant sequence differences among AF and ApoEε4 haplotypes from the 1000 genomes in a topologically associated area (56kb) surrounding ApoE. None of these differences were identified to be protein coding. We applied Massively Parallel Reporter Assay (MPRA) supplemented with single variant reporter assays using Promega Dual Glo‐Luciferase System in AD relevant cell lines to identify the regulatory potential of these variants and their surrounding regions and to assess the differential effect sizes of the variant alleles on enhancer activity. Result For MPRA and complementary single variants reporter assays, we generated ∼900bp PCR fragments surrounding these variants to ensure full representation of potential regulatory elements. MPRA vector library or single reporter vectors were transfected in three cell lines (SHY‐SY5Y neuronal cells, U‐118 astrocytes and HMC3 microglia). We identified evidence for differential regulation between AF and EU variant haplotypes in intron 5 of PVRL2, TOMM40 intronic regions and a large region located 3’ of APOC1. The latter encompasses a putative enhancer identified through ENCODE analyses in brains with NHW ancestry. Conclusion Our results indicate several areas of differential regulation in this LA region on ApoEε4 haplotypes. Follow‐up of the identified regulatory regions is currently ongoing using publicly available data and in‐house iPSC derived cell lines

Intragenic loci within TOMM40 enhances APOE expression in human microglia

Background Previously, we demonstrated that the ancestry‐related risk for Late Onset Alzheimer’s Disease (LOAD) is driven by a local genomic region (termed Local Ancestry; LA) around APOEε4. Furthermore, we showed that in the brain of individuals bearing European LA there is higher expression of APOEε4 compared to those with African LA. In a follow‐up study, utilizing reporter assays and Capture‐C data we located two intronic regions within the European LA, both in the TOMM40 gene (named B10 and B13), that increased APOE expression in microglia and astrocytes. In this study, we sought to validate their regulatory role in APOE expression using CRISPR interference/activation (CRISPRi/a). Method Human Microglial Clone 3 (HMC3) CRISPRi/a lines were produced by transducing inducible dCas9‐VP64 (Activation), dCas9‐KRAB (Interference) or dCas9 (control) using lentiviral vectors. To direct the dCas9 constructs to our regions of interest, we generated multiplex vectors that encode 4 short‐guide RNAs (sgRNAs) targeting either B10 or B13. We used 4 different sgRNAs in each case to ensure full‐length coverage of the tested regions (∼850bp size). An empty multiplex vector was used as a control. We then transduced either of the multiplex vectors into the HMC3 CRISPRi/a lines. We induced expression of the dCas9 constructs for 2 or 6 days with Doxycycline (2ug/ml). RNA was extracted and the expression of APOE and TOMM40 was measured by qRT‐PCR. Result APOE expression significantly increased when targeting B10 or B13 (p=0.001; p=0.003 respectively) with dCas9‐VP64 after 2 days of Doxycycline treatment. Six days after treatment the significance persisted only when targeting B10 (p=0.01). No significant changes in APOE expression were observed in the cells bearing the dCas9‐KRAB presumably due to low endogenous APOE levels. Expression of TOMM40 did not vary under any treatment. Conclusion These preliminary results support our previous findings that regions B10 and B13 may act as regulators for APOE expression as demonstrated by the elevation of ApoE expression when targeting an activator to these regions. The expression of TOMM40 did not vary across cell lines in the evaluated time points supporting that the effect observed is specific for APOE

Ancestry-specific intronic variants on the APOEɛ4 haplotype influence enhancer activity and interaction with APOE promoter

The risk for late-onset Alzheimer disease (AD) in APOEε4 carriers differs between ancestral groups, where APOEε4's odds ratio for AD risk is lower in African (AFR) homozygous carriers than in non-Hispanic White (NHW) or Japanese (JPT) carriers (odds ratio ∼2-5 vs >15). Local ancestry (LA) analyses in APOEε4 carrier populations have shown the protective effect in AFR relative to EUR/JPT is due to noncoding factors lying in the LA surrounding APOEε4. Thus, regulatory differences between risk and protective LA haplotypes are most likely involved in the differential risk effect seen for APOEε4 on different backgrounds. We identified 56 significant sequence differences between AFR and EUR/JPT APOEε4 haplotypes from the 1000 genomes in the immediate topologically associated domain surrounding APOE. We performed two different Massively Parallel Reporter Assay (MPRA) designs; one assessing small haplotype (∼900bp) effects and one based upon single variant effects. We supplemented these results with single fragment luciferase reporter assays. All assays were performed in at least duplicate in HMC3 (microglia), U118 (astrocytes) and SH-SY5Y (neurons) cell lines. Additionally, we integrated chromatin interaction information from promoter capture C chromatin conformation assays in the same cell types. We identified a region in the first introns of TOMM40 with increased EUR/JPT enhancer activity, supported by both MPRA analyses and APOE promoter interaction in astrocytes and microglia. Two additional regions with differential enhancer activity in neurons, but no promoter interaction, were identified; downstream of APOE and in PVRL2 introns upstream of APOE presenting with higher EUR or higher JPT haplotype variant enhancer activity compared to AFR, respectively. Our results indicate several areas of differential regulation in this LA region on APOEε4 haplotypes dependent on cell type. As APOE is mostly expressed in glial cells, the data in TOMM40 introns points to this region as having the biggest impact on APOE expression in our study and thus highly supports the involvement of this region in the differential risk effects seen for APOEε4. Follow-up of the identified regulatory regions is currently ongoing using in-house iPSC derived cell lines

Clinical significance of filamin A in patients with acromegaly and its association with somatostatin and dopamine receptor profiles

Abstract Filamin-A (FLNA) plays a crucial role in somatostatin receptor (sst) subtype-2 signaling in somatotropinomas. Our objective was to investigate the in vivo association between FLNA and sst2 expression, sst5 expression, dopamine receptor subtype-2 (D2) expression, somatostatin receptor ligand (SRL) responsiveness and tumor invasiveness in somatotropinomas. Quantitative real-time PCR was used to evaluate the absolute mRNA copy numbers of FLNA/sst2/sst5/D2 in 96 somatotropinomas. FLNA, sst2 and sst5 protein expression levels were also evaluated using immunohistochemistry. The Knosp-Steiner criteria were used to evaluate tumor invasiveness. Median FLNA, sst2, sst5 and D2 copy numbers were 4,244, 731, 156 and 3,989, respectively. Thirty-one of the 35 available tumors (89%) were immune positive for FLNA in the cytoplasm and membrane but not in the nucleus. FLNA and sst5 expression were positively correlated at the mRNA and protein levels (p < 0.001 and p = 0.033, respectively). FLNA was positively correlated with sst2 mRNA in patients who were responsive to SRL (p = 0.014, R = 0.659). No association was found between FLNA and tumor invasiveness. Our findings show that in somatotropinomas FLNA expression positively correlated with in vivo sst5 and D2 expression. Notably, FLNA was only correlated with sst2 in patients who were controlled with SRL. FLNA was not associated with tumor invasiveness

Transgenic APOEε4/4 overexpression induces reactivity in astrocytes with a European APOEε4/4 local ancestry, but not in astrocytes with an African APOEε4/4 local ancestry

Recently we demonstrated that local genomic ancestry (LA) drives the difference in AD risk between European (EU) and African (AF) carriers of APOEε4/4. As a follow-up study using single-nuclei RNAseq, we found that AD APOEε4 homozygotes with EU Local Ancestry (LA) had a significantly increased APOEε4 expression compared to AD APOEε4/4 homozygotes with AF LA. In two of the EU LA patients, an astrocyte cluster with the highest APOEε4 expression was observed and expressed a panel of genes consistent with A1 reactive astrocytes (A1RA). No such cluster was seen in the AF LA. As a previous study in mice suggested that APOEε4 expression was a contributor to the development of A1RA, we sought to explore the relationship of increased APOEε4 expression and A1RA using inducible pluripotent stem cells (iPSC)-derived astrocytes. APOEε4/4 astrocytes from one European and one African LA were derived from iPSC lines. The astrocytes were brought to maturity (Day 54 in-vitro), and then each group was treated with either a cytokine cocktail (IL-1a, hTNFa, C1q) or overexpressed with APOEε4 by lentiviral transduction for 14-days (two replicates per line). mRNA was extracted and qPCR was performed to measure changes in APOEε4 and markers of A1RA (C3, GBP2, IFITM3). iPSCs were validated by immunocytochemistry (ICC) and qPCR for Nanog, SOX2 and OCT4 and subsequently differentiated into astrocytes. Astrocyte validation was performed by GFAP and βS100 ICC. Astrocytes treated with the cytokine cocktail had a 100- and 600-fold increase in C3 in EU and AF LA astrocytes respectively, as well as significant increase in GBP2 and IFITM3 compatible with A1RA. When overexpressing APOEε4, a significant increase in C3 was observed in the EU LA astrocytes, while no increase was observed in the AF LA astrocytes. Our preliminary results support the hypothesis that APOEe4 overexpression can increase the conversion of astrocytes to the toxic A1RA state in EU LA astrocytes. It is intriguing that this effect was not seen in the African LA astrocytes. By increasing the sample size, we will increase the significance of the association between APOEe4 overexpression and A1RA induction in EU LA astrocytes compared to their AF counterparts

Using Oligodendrocytes for studies in Alzheimer disease

Background Genomic regulatory architecture (GRA) has been primarily studied in European ancestry. As part of the functional Consortium of the Alzheimer Disease Sequencing Project, we are determining the GRA in African and Amerindian ancestries. Oligodendrocytes (OLs) are the largest glial population in the adult central nervous system. While their main role is to support neuronal metabolism and connectivity, few studies have examined their importance in Alzheimer’s disease (AD), despite reports of low numbers of oligodendrocytes and reduced myelin in the early stages of AD and a demonstrated role of myelinating oligodendrocytes in learning and memory. Several studies have reported the derivation of OLs from pluripotent cells, such as induced pluripotent stem cells (iPSC) to be challenging. Here, we optimized a protocol to derive oligodendrocytes from iPSC for studies in AD patients with different ancestries and how ancestry‐specific genomic differences drive the onset and pathogenesis of AD. Method iPSC lines derived from AD patients were cultured and differentiated into oligodendrocytes using different induction media, as well as different seeding densities. The cells in each treatment were compared at multiple time points using immunocytochemistry (ICC) and qRT‐PCR for oligodendroglia lineage markers with the goal of identifying the culture conditions that increase the yield of O4+ cells and myelinating oligodendrocytes. Result Our results showed that increasing the initial seeding density positively correlates with the number of Olig2+ cells that subsequently transitioned into mature O4+ cells capable of producing myelin. Additionally, we showed that the addition of N2 supplement to the induction media was necessary to maintain the cell viability during the initial stage of differentiation. Conclusion We have optimized a protocol to derive OLs from human iPSC lines. We determined that both the seeding density and the media supplements used during the initial stage of differentiation directly influence viability and, consequently the amount Olig2+ cells that could be obtained to be terminally differentiated into O4+ and myelinating cells. Our optimized protocol will be used to evaluate the GRA and functionality of potential GWAS driving loci in cultured oligodendrocytes from individuals of African and Amerindian ancestries

Characterization of an African ancestry‐specific protective allele of the APOE ε4 allele for Alzheimer’s disease risk

Abstract Background We recently identified a protective interaction between the APOE ε4 allele and an African ancestry‐specific allele (rs10423769_A) significantly reducing the odds ratio for Alzheimer’s disease by 75% in APOE ε4 homozygous carriers. rs10423769 is located approximately 2 mB upstream of APOE in a large area of segmental duplication, within a cluster of pregnancy specific beta‐1 glycoproteins (PSG) genes and a long noncoding RNA, lncRNA (ENSG00000282943). Hi‐C analysis in multiple brain cell types showed no direct chromatin interactions between APOE and the rs10423769 region. rs10423769 has been reported to be a splicing quantitative trait locus for the transmembrane protein 145 gene (TMEM145), which has the highest expression in cerebellum. Methods Since this interaction region is enriched for segmental duplications, long‐read whole genome sequencing was performed on 16 individuals (nine heterozygous and seven homozygous rs10423769_A carriers) using the Oxford Nanopore PromethION24 to more precisely map this variant. Expression of PSG genes (2, 4, 5 and 11), ENSG00000282943, and TMEM145 isoforms were analyzed by qPCR in cerebellum and frontal cortex of carriers and non‐carriers of rs10423769_A. Hi‐C analysis was conducted in these brain samples as well. Results Nanopore long‐read sequencing showed that rs10423769 is present as a single copy on chromosome 19. In the frontal cortex, preliminary Hi‐C results supported the presence of transcriptionally active chromatin over the PSG region in some individuals but not in others. Both PSG genes and the lncRNA had low expression in the frontal cortex and cerebellum. We observed higher TMEM145 expression in the cerebellum compared to frontal cortex and detected alternative splicing forms of TMEM145. There was a trend of increased expression of TMEM145 in rs10423769_A carriers, which was not statistically significant likely due to sample size. Conclusion We established that only one copy of rs10423769 exists in the modifying region, which will facilitate future haplotype analysis. The Hi‐C analysis suggested inter‐individual variations on chromatin structure over PSG genes in frontal cortex, although no expression of these genes was detected. Further analysis of regulatory activity of the region is ongoing. This modifier locus appears to represent a novel protective mechanism for APOE ε4

Identifying differential regulatory control of APOE eP on African versus European haplotypes as potential therapeutic targets

We previously demonstrated that in Alzheimer's disease (AD) patients, European apolipoprotein E (APOE) epsilon 4 carriers express significantly more APOE epsilon 4 in their brains than African AD carriers. We examined single nucleotide polymorphisms near APOE with significant frequency differences between African and European/Japanese APOE epsilon 4 haplotypes that could contribute to this difference in expression through regulation. Two enhancer massively parallel reporter assay (MPRA) approaches were performed, supplemented with single fragment reporter assays. We used Capture C analyses to support interactions with the APOE promoter. Introns within TOMM40 showed increased enhancer activity in the European/Japanese versus African haplotypes in astrocytes and microglia. This region overlaps with APOE promoter interactions as assessed by Capture C analysis. Single variant analyses pinpoints rs2075650/rs157581, and rs59007384 as functionally different on these haplotypes. Identification of the mechanisms for differential regulatory function for APOE expression between African and European/Japanese haplotypes could lead to therapeutic targets for APOE epsilon 4 carriers

African Locus Reduces the Effect of ApoE ɛ4 Allele in Alzheimer’s Disease (S15.003)

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