X-chromosome Inactivation Studies Research Articles

A system to analyze the initiation of random X-chromosome inactivation using time-lapse imaging of single cells

In female eutherian mammal development, X-chromosome inactivation (XCI) of one of the two X chromosomes is initiated early. Understanding the relationship between the initiation of XCI and cell fate is critical for understanding early female development and requires a system that can monitor XCI in single living cells. Traditional embryonic stem cells (ESCs) used for XCI studies often lose X chromosomes spontaneously during culture and differentiation, making accurate monitoring difficult. Additionally, most XCI assessment methods necessitate cell disruption, hindering cell fate tracking. We developed the Momiji (version 2) ESC line to address these difficulties, enabling real-time monitoring of X-chromosome activity via fluorescence. We inserted green and red fluorescent reporter genes and neomycin and puromycin resistance genes into the two X chromosomes of PGK12.1 ESCs, creating a female ESC line that retains two X chromosomes more faithfully during differentiation. Momiji (version 2) ESCs exhibit a more stable XX karyotype than other ESC lines, including the parental PGK12.1 line. This new tool offers valuable insights into the relationship between XCI and cell fate, improving our understanding of early female development.

Clonal dynamics of hematopoietic stem cell compartment in aplastic anemia

Advances in molecular technologies accelerated investigations of the hematopoietic stem cell (HSC) compartment in aplastic anemia (AA). Initially, stem cell biology approaches indicated a profound depletion of HSC pool, while studies of paroxysmal nocturnal hemoglobinuria (PNH), X-chromosome inactivation and cytogenetics provided the first evidence for the presence of clonality in the context of a contracted HSC compartment. More recently, the introduction of deep NGS allowed a more precise assessment of clonal expansions in AA. NGS studies demonstrated that the acquisition of somatic defects, including mutations and copy number alterations, can occur prior to and without a strict consequence of a later evolution of post-AA myelodysplastic syndrome (MDS). Such mutant clones may simply correspond to clonal hematopoiesis of indeterminate potential (CHIP) prematurely uncovered by the depletion of polyclonal “normal” HSCs. However, clonal expansions may also correspond to adaptive responses extrinsically or intrinsically enhancing clonal fitness. These processes could lead to adaptive (clonal non-malignant) or maladaptive (post-AA MDS) attempts at reconstitution of hematopoiesis. The spectrum of molecular events provides many clues as to the evolutionary forces driving the pathogenesis of AA and also valuable lessons as to the function of normal and malignant hematopoiesis. This article summarizes current thinking, controversies and dilemmas in interpreting molecular data obtained from the studies of AA.

A Phase 2 Study to Evaluate the Efficacy and Safety of Selinexor in Patients with Myelofibrosis Refractory or Intolerant to JAK Inhibitors

▪Background:Selinexor is an oral, small molecule, selective inhibitor of nuclear export (SINE) compound that specifically blocks the karyopherin protein exportin 1 (XPO1, CRM1). In an shRNA library screen, we discovered that the survival of JAK2V167F mutant HEL cells is dependent on XPO1-mediated nuclear-cytoplasmic transport. Selinexor selectively suppressed primary myelofibrosis (MF) cells as compared with normal progenitor cells and induced hematologic responses in an MPN mouse model.Methods:An open label, prospective, investigator-initiated single center study is ongoing in adults with primary or secondary MF with resistance or intolerance to JAK inhibitor (JAKi) therapy with platelets > 30 K/μL and neutrophils > 500/μL. Selinexor was given orally once a week. Spleen volume was assessed by MRI at week 12 and week 24. The study was amended to include additional MRIs every 12 weeks in the year 1 and 24 weeks in the year 2. Primary end point is spleen response, defined as ≥ 35% spleen volume reduction (SVR) by MRI or CT, where applicable) at week 24. Bone marrow was evaluated at baseline and at week 24. The projected sample size of 24 will provide 83% power to reject a response rate of 15% and allow for up to a 25% dropout rate. We provide an interim report after completing 50% enrollment.Results:Between May 2019 and February 2021, 12 patients (pts) were enrolled. JAK2, CALR and MPL mutations were present in 7 (58.3%), 4 (33.3%) and 1 (8.3%) pts respectively. Eight pts (66.6%) had at least one high molecular risk mutation at baseline (Table 1). Median duration of prior JAKi therapy was 22 months (0.5 to 96 months) and 11 out of 12 were refractory to ruxolitinib at study enrollment. Median baseline spleen volume was 1454 cm 3(range 835 to 5792). Selinexor starting dose was 80 mg weekly in the first 6 pts and 60 mg for subsequent pts. At data cutoff, median duration of selinexor therapy was 36 weeks (range 11-114 weeks). One pt was not response evaluable and died due to liver abscess at week 12 (unrelated). One pt discontinued selinexor at week 18 due to grade 3 fatigue and was not evaluable for the primary end point. Of the 11 pts who had week 12 MRI or CT, 6 showed ≥ 10% SVR, 3 showed ≥ 25% SVR and 1 pt had early progression (Figure 1). At week 24, 5/9 (56%) pts had ≥ 25% SVR and 2/9 (22%) had ≥ 35% SVR (Figure 1). In 9 pts who had ≥24 weeks of selinexor, SVR ≥ 25% and ≥ 35% occurred at any point during study treatment in 4 (44%) and 3 (33%) pts, respectively. Two pts were red cell transfusion dependent at baseline; 1 became transfusion independent after 36 weeks of treatment, has not required transfusion for 49 weeks and remains on study treatment to date (114 weeks). Six pts (50%) discontinued selinexor. Reasons for treatment discontinuation are death in 1 pt, progressive disease in 1 pt, alternative treatment in 2 pts, and toxicity in 2 pts. Ten pts required dose reduction due to fatigue (1pt), anemia (1 pt), thrombocytopenia (2 pts), abdominal pain (1pt) and weight loss (5 pts). The most common treatment related adverse event was weight loss (grade 2 in 4 pts and grade 3 in 1 pt). This was manageable with treatment interruption and dose reduction, except in one pt who discontinued selinexor. As yet no changes in reticulin fibrosis MF grade were observed among 9 patients who received at least 24 weeks of treatment.Conclusions:Once weekly, oral selinexor showed single agent activity with sustained spleen responses in pts with JAKi refractory MF. Long-term administration of selinexor was well tolerated over time in MF pts. Correlatives studies including circulating inflammatory cytokine levels and mutant allele burden, as well as clonality studies by X-chromosome inactivation studies in woman, are underway and will be presented. [Display omitted] DisclosuresTantravahi: BMS: Research Funding; Novartis: Research Funding; CTI BioPharma: Research Funding; Abbvie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Consultancy, Honoraria, Research Funding. Patel: Stemline: Research Funding; Genentech: Research Funding; Roche: Research Funding. Chamoun: Karyopharm Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Shah: Karyopharm: Current Employment. George: Celgene: Consultancy; Bristol Meyers Squibb: Consultancy; Incyte Corporation: Consultancy; Blueprint Medicines: Consultancy. Deininger: Fusion Pharma, Medscape, DisperSol: Consultancy; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Novartis: Consultancy, Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Research Funding.

Gene-Specific Lineage Involvement of Age-Related Clonal Hematopoiesis

INTRODUCTIONAge-related clonal hematopoiesis (ARCH), which was initially suggested by X-chromosome inactivation (XCI) studies, is associated with acquired mutations in driver and non-driver genes. ARCH is highly prevalent after the age of 65, and infers an increased risk of hematological cancers and cardiovascular complications. Quantification of these risks in individual subjects is not possible at this time, leading to the creation of a clinical entity named Clonal Hematopoiesis of Indeterminate Potential, or CHIP (Steensma et al., Blood 2015). There is a need to determine factors that modulate the oncogenic penetrance of these mutations. Identifying the origin within the hematopoietic stem cell (HSC) hierarchy of the mutations responsible for ARCH is a crucial initial step. We recently documented that 93% of driver mutations in ARCH occurred in DNMT3A or TET2 (Buscarlet et al., Blood. 2017). We now report the comparative hematopoietic lineage involvement of these two genes in a cohort of 86 individuals with ARCH.METHODSWe have previously identified driver mutations in polymorphonuclear cells (PMN) of 347 individuals from a cohort of 2350 individuals. We have selected 86 individuals on the basis of having a single DNMT3A or TET2 mutation and the availability of DNA from PMN (granulocytes), CD14+ (monocytes), CD19+ (B-cells) and CD3+ (T-cells) allowing lineage involvement analysis. The lineage specific cells were obtained by Fluorescence-activated cell sorting (FACS) from the mononuclear cell fraction of peripheral blood samples. All samples were sequenced at high coverage (95% > 500x) on an Ion Proton sequencing using the AML Ampliseq panel. All mutations were visually inspected on Integrative Genomics Viewer (IGV). Samples were considered mutated if the mutation was detected above 2% VAF (variant allele frequency).RESULTSThe study cohort comprised 86 individuals from 55 to 96 years-old (mean age: 69.0). Sixty two (62) had a single DNMT3A mutation (mean VAF in PMN = 11.2%) and 24 had a single TET2 mutation (mean VAF in PMN = 10.5%). TET2 mutated individuals where older than those with DNMT3A mutations (72.3 vs 67.7, P < .05). From the 62 DNMT3A mutations initially detected in PMN, 32.3% were present in all cell types analysed (PMN, CD14+, CD19+ and CD3+), 37.1% were detected in PMN, CD14+ and CD19+ cells, and 21.0% were found only in PMN and CD14+ cells. The remaining mutations were detected in PMN, CD14+ and CD3+ cells (4.8%), PMN and CD19+ cells (4.8%), or only in PMN (3.2%). For TET2, 45.8% of the mutations were detected in PMN and CD14+ cells, 45.8% were found in PMN, CD14+ and CD19+ cells, and 8.3% only detected in PMN. No mutations were detected in CD3+ cells. The different lineage involvements were statistically different between DNMT3A and TET2 for myeloid restriction (PMN or PMN and CD14+ cells): 21% (13/62) vs 54.2%, (13/24), P < .05; and for mutation in CD3+ cells: 37% (23/62) vs 0% (0/24), P < .001. Although the proportion of subjects with myeloid and B-cell involvement was similar between the two genes, the CD14+/CD19+ VAF ratio was significantly higher for TET2 (3.13, n = 11) than for DNMT3A (1.94, n = 43) (P < .005). Selecting subjects who had a VAF > 10% in PMN (suggesting a longer evolution) had no impact on the myeloid lineage restriction for TET2, but almost all (21/22) DNMT3A samples showed mutations in both myeloid and lymphoid compartments.CONCLUSIONThese results demonstrate a significantly different lineage involvement between DNMT3A and TET2 mutations in ARCH. DNMT3A mutations predominantly affect both the myeloid and lymphoid compartment, including T-cells in a third of subjects. This strongly suggests that DNMT3A mutations occur in a HSC with multipotent proliferative potential. In contrast, we never documented TET2 mutations in T-cells, suggesting the involvement of more committed HSC. This is paradoxical since both DNMT3A and TET2 mutations have been documented in T-cell lymphoma. This could be explained by a different HSC origin for TET2 mutation in myeloid malignancies vs T-cell lymphomas. Alternatively, it could be that in normal aging individuals, the multipotent mutated HSC has a stronger myeloid proliferation bias than DNMT3A . This bias may be in response to homeostatic stimuli as we have documented a slight neutropenia in subjects with ARCH and TET2 mutations (Buscarlet et al., Blood 2017). [Display omitted] DisclosuresBusque:Bristol Myer Squibb: Honoraria; Novartis Canada Inc.: Honoraria; Paladin: Honoraria; Pfizer: Honoraria.

De novo loss-of-function variants in X-linked MED12 are associated with Hardikar syndrome in females

Hardikar syndrome (MIM 612726) is a rare multiple congenital anomaly syndrome characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, but with preserved cognition. Only four patients have been reported previously, and none had a molecular diagnosis. Our objective was to identify the genetic basis of Hardikar syndrome (HS) and expand the phenotypic spectrum of this disorder. We performed exome sequencing on two previously reported and five unpublished female patients with a clinical diagnosis of HS. X-chromosome inactivation (XCI) studies were also performed. We report clinical features of HS with previously undescribed phenotypes, including a fatal unprovoked intracranial hemorrhage at age 21. We additionally report the discovery of de novo pathogenic nonsense and frameshift variants in MED12 in these seven individuals and evidence of extremely skewed XCI in all patients with informative testing. Pathogenic missense variants in the X-chromosome gene MED12 have previously been associated with Opitz-Kaveggia syndrome, Lujan syndrome, Ohdo syndrome, and nonsyndromic intellectual disability, primarily in males. We propose a fifth, female-specific phenotype for MED12, and suggest that nonsense and frameshift loss-of-function MED12 variants in females cause HS. This expands the MED12-associated phenotype in females beyond intellectual disability.

Analysis of complex structural variants in the DMD gene in one family

This work describes a family with Duchenne muscular dystrophy (DMD) with a rare case of a symptomatic pregnant woman. The main aim was to perform prenatal molecular diagnosis to provide genetic counseling. The secondary aim was to suggest the molecular mechanisms causing the complex structural variant (cxSV) identified. To accomplish this, we used a multi-technique algorithm including segregation analysis, Multiplex Ligation-dependent Probe Amplification, PCR, X-chromosome inactivation studies, microarrays, whole genome sequencing and bioinformatics. We identified a duplication of exons 38–43 in the DMD gene in all affected and obligate carrier members, proving that this was the DMD-causing mutation. We also observed a skewed X-chromosome inactivation in the symptomatic woman that explained her symptomatology. In addition, we identified a cxSV (duplication of exons 38–43 and deletion of exons 45–54) in the affected boy. The molecular characterization and bioinformatic analyses of the breakpoint junctions allowed us to identify Double Strand Breaks stimulator motifs and suggested the replication-dependent Fork Stalling and Template Switching as the most probable mechanisms leading to the duplication. In addition, the de novo deletion might have been the result of a germline inter-chromosome non-allelic recombination involving the Non-Homologous End Joining mechanism. In conclusion, the diagnostic strategy used allowed us to provide accurate molecular diagnosis and genetic counseling. In addition, the familial molecular diagnosis together with the in-depth characterization of the cxSV helped to determine the chronology of the molecular events, and propose and understand the molecular mechanisms involved in the generation of this complex rearrangement.

Many XCI-ting routes to reach the eXACT dose.

X-chromosome inactivation studies have revealed striking species specificities in terms of the players, kinetics and mechanisms. Transcriptomic profiling of human pre‐implantation development and germ cell differentiation suggests a peculiar dosage compensation mechanism with yet undefined contributions from XIST and XACT lncRNAs.

Versatility of RNA-Binding Proteins in Living Cells

This conference paper aims at demonstration of complexity or multiplicity of RNA-binding protein (RBP) and its related RNA. The data in the manuscript are based upon the third Annual Meeting of the RNA study group, held at the Tokyo office of the Kyoto University on May 23 in 2019. The purpose of the meeting is to present recent progress of the study group and have discussion regarding RNA and related molecules, especially focusing on RBP in this year. We had fourteen sessions and documented ten of them in the manuscript. There were exciting and fruitful debates. Actual presentations are shown as follows. Specific RBP Sfpq and Qk are involved in neuronal development in mouse system. Also in mouse, liquid- and solid-like RNA granule formation plays pivotal roles in neuronal functions. Biochemical experiments demonstrate that affinity profiles categorize RNA-Binding proteins into distinctive groups. This presents basis for biological divergence of RBPs. In the context of pathology, RNA aptamers against prion and Alzheimer’s diseases may present therapeutic outcome. Computational analysis is another utility for approaching riddle of RBPs. The study of X-chromosome inactivation is pioneering field of long noncoding RNAs. Redundant triplex interactions between lncRNA and LINE-1s are supposed to be crucial in X-chromosome inactivation. Myogenesis-related lncRNA, <i>Myoparr</i> promotes skeletal muscle atrophy caused by denervation through the regulation of <i>GDF5/BMP14</i> expression. In neurodegenerative disorders functional loss of TLS/FUS induces onset of frontotemporal lobar degeneration. Conformational change of TLS is induced with binding of various nucleic acid molecules. TLS is also involved in phase separation into aggregation to pathogen generation of neurodegenerative diseases. The meeting has been successfully prorogued. We utilize the data to boost the activity of the field of RNA and RBP. The achievements of the meeting have confirmed pivotal roles of RBPs in living cells. It should be in complexity. We will be able to analyze intricate phenomena of RBP biological actions and contribute to uncover the veiled rules in divergent biological programs.

Variantes que mantienen el marco de lectura en el dominio Rod 1 proximal del gen FLNA se asocian con un predominio del fenotipo valvular

Introduction and objectivesX-linked cardiac valvular dysplasia is a rare form of male-specific congenital heart defect mainly characterized by myxomatous degeneration of the atrioventricular valves with variable hemodynamic consequences. It is caused by genetic defects in FLNA-encoded filamin A, a widely expressed actin-binding protein that regulates cytoskeleton organization. Filamin A loss of function has also been associated with often concurring neurologic and connective tissue manifestations, with mutations in the first half of the Rod 1 domain apparently expressing the full cardiac phenotype. We contribute to previous genotype-phenotype correlations with a multidisciplinary approach in a newly-described family. MethodsCardiologic, dysmorphologic, and genetic evaluation of available members were complemented with transcriptional and X-chromosome inactivation studies. ResultsA novel FLNA mutation c.1066-3C>G cosegregated with a male-expressed, apparently isolated, cardiac phenotype with no skewed X-inactivation pattern in female carriers. This variant was shown to result in an in-frame deletion of 8 amino acid residues near the N-terminal region of the protein. ConclusionsA nonimprinted, partial loss of function of filamin A proximal Rod 1 domain seems to be the pathogenetic mechanism of cardiac valvular dysplasia, with some cases occasionally expressing associated extracardiac manifestations.Full English text available from: www.revespcardiol.org/en

RNA-FISH and Immunofluorescence of Mouse Preimplantation and Postimplantation Embryos.

There are two modes of X chromosome inactivation (XCI) in the mouse. One mode is imprinted XCI: it is initiated at around the four-cell stage in favor of the paternal X chromosome, and is maintained in the extraembryonic tissues. The other mode is random XCI, which takes place in the epiblast lineage at the periimplantation stage. X-linked noncoding Xist RNA, which becomes upregulated on the X chromosome to be inactivated at the onset of XCI and plays a critical role in both imprinted and random XCI, and its accumulation in the nucleus have been referred to as one of the hallmarks of the presence of the inactivated X chromosome. RNA-FISH has therefore been an invaluable method for the study of XCI. As XCI status changes dynamically during periimplantation development in the mouse, analysis using samples from these developmental stages is absolutely necessary for elucidation of the molecular basis of XCI mechanisms. However, dissection of the embryos at around the periimplantation stages is not easy, and this impedes in vivo analysis of the kinetics of XCI. Here, we describe our methods for dissecting the periimplantation stage embryo and subsequent procedures for RNA-FISH and immunostaining.

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs

During female mouse embryogenesis, two forms of Xchromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI invivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

In-frame Variants in FLNA Proximal Rod 1 Domain Associate With a Predominant Cardiac Valvular Phenotype

Introduction and objectivesX-linked cardiac valvular dysplasia is a rare form of male-specific congenital heart defect mainly characterized by myxomatous degeneration of the atrioventricular valves with variable hemodynamic consequences. It is caused by genetic defects in FLNA-encoded filamin A, a widely expressed actin-binding protein that regulates cytoskeleton organization. Filamin A loss of function has also been associated with often concurring neurologic and connective tissue manifestations, with mutations in the first half of the Rod 1 domain apparently expressing the full cardiac phenotype. We contribute to previous genotype-phenotype correlations with a multidisciplinary approach in a newly-described family. MethodsCardiologic, dysmorphologic, and genetic evaluation of available members were complemented with transcriptional and X-chromosome inactivation studies. ResultsA novel FLNA mutation c.1066-3C>G cosegregated with a male-expressed, apparently isolated, cardiac phenotype with no skewed X-inactivation pattern in female carriers. This variant was shown to result in an in-frame deletion of 8 amino acid residues near the N-terminal region of the protein. ConclusionsA nonimprinted, partial loss of function of filamin A proximal Rod 1 domain seems to be the pathogenetic mechanism of cardiac valvular dysplasia, with some cases occasionally expressing associated extracardiac manifestations.

Rare GABRA3 variants are associated with epileptic seizures, encephalopathy and dysmorphic features.

Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.

Clonal Events in Normal Aging Hematopoiesis

Chronological aging of the hematopoietic compartment is associated with decreased bone marrow cellularity, reduced lymphopoiesis, increased anemia, a myeloid proliferation bias and an increased incidence of myeloid cancers. Beerman et al. proposed that this age-related myeloid lineage favoritism may be explained by clonal expansion of intrinsically myeloid-biased hematopoietic stem cells with robust self-renewal potential(1). This age-associated clonal expansion was initially suspected by X-chromosome inactivation (XCI) studies performed in the normal aging population, which documented a skewed XCI pattern in a significant proportion of women over 60 year-old(2). More recently, genome wide approaches led several groups to document au augmented prevalence of acquired clonal copy number changes (3,4,5) or clonal somatic mutations with increasing age (6,7,8,9). The most frequently mutated genes are the same as those documented in myeloid cancers, such as TET2, DNMT3A, ASXL1, PPM1D, GNAS, TP53, JAK2 and SF3B1 among others. The prevalence of these age-associated mutations may reach &gt; 10% of older individuals, and is associated with an 11-12 fold increased relative risk of developing hematological malignancies. However, the actual problematic is to define the prognostic significance of these clonal mutations in the aging population. Steensma et al. proposed to consider these mutations as «Clonal Hematopoiesis of Indeterminate Potential (CHIP)»(10). The goal of our research group is to define the oncogenic penetrance of CHIP by applying a precision medicine approach in a large prospective cohort (n=4000) of aging individuals comprised of related and unrelated subjects. The variables under investigation include, clonality by XCI in women, deep sequencing (NGS) of myeloid cancer associated genes, epigenetic markers (5hmC, 5mC), telomere length, blood counts, heritability and outcome. PRELIMINARY RESULTS. XCI analyses Acquired skewing of XCI predominantly affects the myeloid lineage with a prevalence of 41.4% for PMN and is age dependent (r=0.15, P&lt;10-4), in contrast to T cells 22.5%. These results support the idea of an age-associated clonal myeloid expansion. NGS of myeloid gene panel. We documented a prevalence of 17.9% of mutated individuals. Mutations were mainly documented in TET2 and DNMT3A which accounted for 90% of all identified mutations. Other significantly mutated genes included JAK2, ASXL1, CBL, TP53 and KRAS. Double mutations were identified in 2.5% of individuals (14% of the mutated individuals) and half of them had concomitant mutation in TET2 and DNMT3A. Age and XCI skewing was similar between subjects with mutation in TET2 or DNMT3A, but slightly higher in double mutants. Epigenetic markers. Subjects with mutation in TET2 had a significant reduction in 5hmC level that correlated with Variable Allele Frequency (VAF) of the mutation. No specific global epigenetic phenotype was documented in the DNMT3A mutation subgroup. We also documented an age-associated reduction in 5hmC that was independent of acquired mutation in the TET2 gene. Taken together these results indicate that age-associated clonal mutations involves predominantly two genes (TET2 and DNMT3A), suggesting that alteration of epigenetic maintenance is a central to the initiation of clonal dominance. Completion of investigation of the aging cohort and prospective follow-up will help characterize the link between aging hematopoiesis and the development of myeloid cancers. 1. Beerman I, Maloney WJ, Weissmann IL, et al. Stem cells and the aging hematopoietic system. Curr Opin Immunol. 2010;22(4):500-506. 2. Busque L, Mio R, Mattioli J, et al. Non-random X-inactivation patterns in normal females: lyonization ratios vary with age. Blood. 1996;88(1):59-65. 3. Forsberg LA, Rasi C, Razzaghian HR, et al. Age-related somatic structural changes in the nuclear genome of human blood cells. AJHG, 2012;90:217-228. 3. Laurie CC, Laurie CA, Rice K, et al. Detectable clonal mosaicism from birth to old age and its relationship to cancer. Nat Genet. 2012;44(6):642-650. 4. Jacobs KB, Yeager M, Zhou W, et al. Detectable clonal mosaicism and its relationship to aging and cancer. Nat Genet. 2012;44(6):651-658. 5. Busque L, Patel JP, Figueroa ME, et al. Recurrent somatic TET2 mutation in normal elderly individuals with clonal hematopoiesis. Nat Genet. 2012;444(11):1179-1181. 6. Xie M, Lu C, Wang J, et al. Age-related mutations associated with clonal hematopoietic expansion and malignancies. Nat Med. 2014;20(12):1472-1478. 7. Genovese G, Kähler AK, Handsaker RE, et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med. 2014;371(26):2477-2487. 8. Jaiswal S, Fontanillas P, Flannick J, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014;371(26):2488-2498. 9.Steensma DP, Bejar R, Jaiswal S, et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood. 2015;126(1):9-16 Disclosures Busque: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Two‑gene mutation in a single patient: Biochemical and functional analysis for a correct interpretation of exome results.

Next-generation sequencing (NGS) has generated a large amount of sequence data with the requirement of frequent critical revisions of reported mutations. This innovative tool has proved to be effective in detecting pathogenic mutations; however, it requires a certain degree of experience to identify incidental findings. In the present study, whole exome sequencing analysis was performed for the molecular diagnosis and correct genotype/phenotype correlation between parents and a patient presenting with an atypical phenotype. In addition, mevalonic acid quantification and frequency analysis of detected variants in public databases and X‑chromosome inactivation (XCI) studies on the patient's mother were performed. V377I as well as the S135L mutations were identified on the mevalonate kinase deficiency gene and the levels of mevalonic acid in the patient were 5,496 µg/ml. A D59G variation, reported in ESP6500 in two healthy individuals, was found on the Martin Probst syndrome gene (RAB40AL). Based on XCI studies on the patient's mother, it is likely that RAB40AL escapes XCI, while still remaining balanced. In conclusion, the results of the present study indicated that the Martin Probst syndrome is an X‑linked condition, which is probably not caused by RAB40AL mutations. Although NGS is a powerful tool to identify pathogenic mutations, the analysis of genetic data requires expert critical revision of all detected variants.

Pathogenicity of C-terminal mutations in CDKL5

It was with great interest that we read the report by Martinez et al. [1] entitled “CDKL5 in different atypical Rett syndrome variants: Description of the first eight patients from Spain”, and in particular of a late C-terminal missense mutation in CDKL5. The mutation described, p.Pro976Leu (c.2927C>T) was identified in a female patient with Rett syndrome (RTT) with regression of late onset. The patient had very mild symptoms in comparison with the others in the report and most strikingly, had never had any seizure episodes. This is in contrast to the general description of patients with CDKL5 mutations [2–4], characterized by early-onset seizures, usually difficult to control. The mutation in this patient was de novo and X-chromosome inactivation (XCI) in the patient’s lymphocytes showed a random pattern of inactivation. We propose that the effect of this missense mutation is minimal due to its position along the CDKL5 gene. Recently, we reported an alternate CDKL5 protein isoform, which differs from the recognized isoform at the C-terminal end at exon 18 (GenBank reference sequence NM_003159.1: c.2713) onwards [5]. The C-terminus of the newly-identified isoform is encoded by an alternatively-spliced exon 18, which extends into intron 18 by at least 170 bases. The presence of an in-frame termination codon in this sequence means that translation beyond exon 18 does not occur in this isoform. As the missense mutation p.Pro976Leu is located on exon 20, the mutation would only affect the previouslyrecognized isoform (GenBank reference sequences NM_001037343 and NM_003159). Expression analysis in different human tissues indicates that the initially recognized isoform is the minor isoform [5], and most abundantly expressed in testis. In contrast, the newlyidentified isoform is expressed in all tissues, highest in parts of the brain, and is likely to be of greater physiological importance. To the best of our knowledge, only three other C-terminal CDKL5 variations in exons 19, 20 or 21 have been reported in patients with RTT or a related disorder. One of these is also a missense variation, p.Val999Met (c.2995G>A), reported as a polymorphism by Intusoma et al. [6] and has a heterozygosity of 0.118 (NCBI dbSNP, rs35693326). The other two variations are nonsensemutations, p.Arg952X and p.Arg970X. The latter mutation (p.Arg970X, c.2908C>T)was reported in a female patient with a Rett-like phenotype, and late-onset seizures at 17 mo [7]. Parental screening was limited only to the mother, and XCI studies were not carried out. The other nonsense mutation p.Arg952X (c.2854C>T) was identified in a female with severe mental retardation without RTT features, and seizures starting from 11 mo [6]. Familial screening found the same mutation in the grandmother, mother and half-sister of the patient. A difference in phenotypes could not be attributed to skewed XCI. Furthermore, population screening revealed an allele frequency of 0.8%, suggesting that although rare, the truncating variation is not likely to be pathogenic. In all, the existing data indicate that C-terminal mutations in CDKL5 should be interpreted with care. *Corresponding author: John Christodoulou, Disciplines of Pediatrics and Child Health and Genetic Medicine, Sydney Medical School, University of Sydney, Sydney, Australia. Tel.: +61 2 9845 3452; Fax: +61 2 9845 1864; E-mail: john.christodoulou@health.nsw.gov.au. Journal of Pediatric Epilepsy 1 (2012) 185–186 DOI 10.3233/PEP-2012-029 IOS Press 185

Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells.

The process of X chromosome inactivation (XCI) during reprogramming to produce human induced pluripotent stem cells (iPSCs), as well as during the extensive programming that occurs in human preimplantation development, is not well‐understood. Indeed, studies of XCI during reprogramming to iPSCs report cells with two active X chromosomes and/or cells with one inactive X chromosome. Here, we examine expression of the long noncoding RNA, XIST, in single cells of human embryos through the oocyte‐to‐embryo transition and in new mRNA reprogrammed iPSCs. We show that XIST is first expressed beginning at the 4‐cell stage, coincident with the onset of embryonic genome activation in an asynchronous manner. Additionally, we report that mRNA reprogramming produces iPSCs that initially express XIST transcript; however, expression is rapidly lost with culture. Loss of XIST and H3K27me3 enrichment at the inactive X chromosome at late passage results in X chromosome expression changes. Our data may contribute to applications in disease modeling and potential translational applications of female stem cells. Stem Cells 2015;33:1771–1781

Heterogeneity of clonal expansion and maturation-linked mutation acquisition in hematopoietic progenitors in human acute myeloid leukemia.

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML), but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34(+)/CD33(-) cells revealed polyclonal growth in highly curable AMLs, suggesting that mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34(+)/CD33(-) cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications.

MECP2duplication: Possible cause of severe phenotype in females

MECP2 duplication syndrome, originally described in 2005, is an X-linked neurodevelopmental disorder comprising infantile hypotonia, severe to profound intellectual disability, autism or autistic-like features, spasticity, along with a variety of additional features that are not always clinically apparent. The syndrome is due to a duplication (or triplication) of the gene methyl CpG binding protein 2 (MECP2). To date, the disorder has been described almost exclusively in males. Female carriers of the duplication are thought to have no or mild phenotypic features. Recently, a phenotype for females began emerging. We describe a family with ∼290 kb duplication of Xq28 region that includes the MECP2 gene where the proposita and affected family members are female. Twin sisters, presumed identical, presented early with developmental delay, and seizures. Evaluation of the proposita at 25 years of age included microarray comparative genomic hybridization (aCGH) which revealed the MECP2 gene duplication. The same duplication was found in the proposita's sister, who is more severely affected, and the proband's mother who has mild intellectual disability and depression. X-chromosome inactivation studies showed significant skewing in the mother, but was uninformative in the twin sisters. We propose that the MECP2 duplication caused for the phenotype of the proband and her sister. These findings support evidence for varied severity in some females with MECP2 duplications.

P.2.14 Isolated cognitive abnormalities associated to DMD mutations

The dystrophinopathies are a group of X-linked muscle diseases caused by mutations in the DMD gene. Clinical phenotypes vary from asymptomatic high CK levels and cramps to severe progressive skeletal and cardiac muscle disorders such as Duchenne (DMD) and Becker (BMD) muscular dystrophies, and X-linked dilated cardiomyopathy (XLCM). About one third of patients present mental retardation. Most disease-responsible mutations are large intragenic rearrangements (exonic deletions and duplications) that account for 65–75% of cases, while the remaining cases are caused by single point mutations or small rearrangements [3,4]. In most patients, the clinical outcome can be predicted according to the reading-frame rule. The majority of DMD patients carry truncating mutations while BMD patients usually carry in-frame mutations allowing the expression of semi-functional dystrophins. We present a male patient 23 years old with mental retardation and no muscle weakness. The Western blot with Dys 1 and Dys 2 antibodies showed a reduced amount of dystrophin. An Inframe deletion of exons 56 and 57 was identified by MLPA and cDNA sequencing, affecting different dystrophin isoforms: Dp424m, Dp260, Dp140 and Dp116. In addition, we diagnosed three symptomatic female carriers of dystrophinopathy presenting isolated cognitive abnormalities without muscle involvement. Each of them carried DMD mutations (Duplication exons 13–27, Subexonic deletion exon 46 and Deletion exons 46–55, respectively). A study of X-chromosome inactivation was performed exhibiting a clear bias in the inactivation of the paternal chromosome: 99:1, 88:12 and 100:0, respectively. These cases suggest that isolated mental retardation could be more frequent than expected.