Skewed X-inactivation Research Articles

Case Report of an Infant Female with X-Linked Chronic Granulomatous Disease Due to a De Novo Mutation in CYBB and Extremely Skewed X-Chromosome Inactivation (Lyonization)

Case Report of an Infant Female with X-Linked Chronic Granulomatous Disease Due to a De Novo Mutation in CYBB and Extremely Skewed X-Chromosome Inactivation (Lyonization)

Transplantation from a symptomatic carrier sister restores host defenses but does not prevent colitis in NEMO deficiency

NF-κB essential modulator (NEMO) deficiency causes ectodermal dysplasia with immunodeficiency in males, while manifesting as incontinentia pigmenti in heterozygous females. We report a family with NEMO deficiency, in which a female carrier displayed skewed X-inactivation favoring the mutant NEMO allele associated with symptoms of Behçet's disease. Hematopoietic stem cell transplantation of an affected boy from this donor reconstituted an immune system with retained skewed X-inactivation. After transplantation no more severe infections occurred, indicating that an active wild-type NEMO allele in only 10% of immune cells restores host defense. Yet he developed inflammatory bowel disease (IBD). While gut infiltrating immune cells stained strongly for nuclear p65 indicating restored NEMO function, this was not the case in intestinal epithelial cells — in contrast to cells from conventional IBD patients. These results extend murine observations that epithelial NEMO-deficiency suffices to cause IBD. High anti-TNF doses controlled the intestinal inflammation and symptoms of Behçet's disease.

Skewed X-chromosome inactivation in human miscarriages

Sex ratio in first trimester of pregnancy is skewed due to preferential elimination of female embryos. It could be resulted from aberrant X-chromosome inactivation. X-chromosome inactivation was analyzed in extraembryonic tissues of miscarriages and induced abortions with 46, XX karyotype. In chorion cytotrophoblast of both miscarriages and induced abortions observed either random or skewed X-chromosome inactivation. In extraembryonic mesoderm of the control group, random inactivation was observed, whereas 15% of miscarriages had skewed X-chromosome inactivation. The highest frequency of skewed inactivation of one of the parental homologues was observed in the groups of blighted ovum pregnancy and embryos from women with recurrent pregnancy loss. It was suggested that in these cases compartmentalization of cells in the blastocyst probably leads to predominance of cell with mutant active X-chromosome among the cells of inner cell mass carrying the aberrations that are incompatible with normal embryonic development.

A Bayesian approach for estimating allele-specific expression from RNA-Seq data with diploid genomes.

BackgroundRNA-sequencing (RNA-Seq) has become a popular tool for transcriptome profiling in mammals. However, accurate estimation of allele-specific expression (ASE) based on alignments of reads to the reference genome is challenging, because it contains only one allele on a mosaic haploid genome. Even with the information of diploid genome sequences, precise alignment of reads to the correct allele is difficult because of the high-similarity between the corresponding allele sequences.ResultsWe propose a Bayesian approach to estimate ASE from RNA-Seq data with diploid genome sequences. In the statistical framework, the haploid choice is modeled as a hidden variable and estimated simultaneously with isoform expression levels by variational Bayesian inference. Through the simulation data analysis, we demonstrate the effectiveness of the proposed approach in terms of identifying ASE compared to the existing approach. We also show that our approach enables better quantification of isoform expression levels compared to the existing methods, TIGAR2, RSEM and Cufflinks. In the real data analysis of the human reference lymphoblastoid cell line GM12878, some autosomal genes were identified as ASE genes, and skewed paternal X-chromosome inactivation in GM12878 was identified.ConclusionsThe proposed method, called ASE-TIGAR, enables accurate estimation of gene expression from RNA-Seq data in an allele-specific manner. Our results show the effectiveness of utilizing personal genomic information for accurate estimation of ASE. An implementation of our method is available at http://nagasakilab.csml.org/ase-tigar.

Tissue-Specific Size and Methylation Analysis in Two Fragile X Families: Contribution to the Clinical Phenotype

Methylation at critical CpG sites on the expanded FMR1 gene is crucial for pathological manifestation of fragile X syndrome and fragile X-related disorders. Methylation status from blood, oral mucosa and root hair was analyzed with the FMR1 mPCR kit (Asuragen). Differential allele expression was studied by TP-PCR. Psychological and neurological explorations were performed in the probands. Patient II-1 of family 1 showed an extremely skewed X-chromosome inactivation of the normal allele in blood, oral mucosa cells and root hair. Analysis of differential expression of both alleles in blood showed the preferential expression of the expanded allele. Similarly, patient II-3 of family 2 showed an extremely skewed X-chromosome inactivation of the normal allele in blood, oral mucosa and root hair. Both females presented clinical features compatible with their skewed methylation toward the normal allele. Methylation analysis at critical CpG sites in the first FMR1 exon may predict clinical manifestations in carriers of premutation or full mutation. Analysis of differential expression of both alleles in women using TP-PCR could contribute to clarify the real impact of skewed methylation on the phenotype.

Correlation of X Chromosome Inactivation Skewing and Bleeding Phenotype in Obligate Carriers of Hemophilia A

Background: Hemophilia A (HA) is an X-linked recessive disorder that affects males, whereas female carriers are presumed asymptomatic if Factor VIII activity levels (FVIII:C) fall within normal range. However, FVIII:C does not always correlate with bleeding phenotype, leading to an underappreciation of bleeding sequelae in females. Therefore, it is clinically important to identify HA carriers at higher bleeding risk. FVIII expression in HA carriers is influenced by X chromosome inactivation (XCI), a process that silences one X in XX females such that each cell has a random probability of inactivating either parental X. However, rare female carriers of X-linked disorders can be severely affected if XCI is skewed and the mutant X is preferentially active. How XCI skewing modulates bleeding in mild/moderate HA is less well understood. HA bleeding may be also affected by variants in factors influencing FVIII binding and clearance, including Von Willebrand Factor (VWF) and ABO blood type. To better understand HA carrier bleeding tendency, we analyzed a family that segregates a mild/moderate HA mutation (F8: c.2167G>A). Four carriers in this pedigree have FVIII:C that approach affected males, necessitating prophylaxis prior to surgical procedures. We hypothesized that bleeding in these carriers can be largely explained by XCI skewing, but additional variants may fine tune FVIII:C.Methodology: FVIII levels were assessed by one stage (FVIII:C) and chromogenic (FVIII:CR) assays. At least two plasma samples spanning >3 years from each female were tested in duplicate with each FVIII assay. To address XCI skewing, we performed methylation-based assays at the Androgen Receptor (AR) and Fragile X Mental Retardation 1 (FMR1) loci. At least three independent PBMC DNA samples from each female were evaluated. Additionally, we screened VWF regions known to influence FVIII:C (exons 18-20, 24-27).Results: For each female, results between XCI assays were indistinguishable (r2 = 0.99). Two of four females had pronounced skewing (≥80:20); a third had measurable skewing (67:33). Importantly, all three predominantly expressed the mutant paternal allele. Familial XCI skewing argues for a genetic cause. However, XIST, the major regulator of XCI, lacked promoter alterations. Importantly, there was linear correlation between XCI skewing and FVIII:C measured by FVIII:C or FVIII:CR assays (r2 = 0.77 and 0.83, respectively). One female with random XCI, had FVIII:C considered hemostatic (median 51%, range 43-67), whereas the other females with skewed XCI had levels <40% (16%, range 14-20, 28%, range 26-32, and 30%, range 23-35). Notably, two females had similar FVIII:C (30% and 28%) but a greater XCI skewing discrepancy (80:20 vs. 67:33). While these two females were heterozygotes for VWF p.Thr789Ala, reported to be associated with 7% higher FVIII:C, neither ABO blood type nor any additional VWF variants known to affect FVIII binding differentiated these two individuals. Therefore, it is likely that XCI skewing primarily explains their bleeding tendency.Conclusions: Our results indicate that HA carrier bleeding phenotypes are multifaceted, and the major determinant of FVIII:C is XCI. These results also suggest that even moderate XCI skewing could influence clinical bleeding in HA carriers. However, random XCI in one female explains FVIII:C but does not fully negate bleeding tendency, emphasizing the complexity of carrier phenotype. These findings provide justification for an expanded study of carriers in unrelated pedigrees using a comprehensive approach to include XCI assays. DisclosuresNo relevant conflicts of interest to declare.

Role of Von Willebrand Factor in Female Carriers from an Extended Family with Mild Hemophilia A

Background: Hemophilia A (HA) is an X-linked disorder that primarily affects males, although female carriers can exhibit bleeding phenotypes. Factor VIII activity levels (FVIII:C) in XX females are influenced by X chromosome inactivation (XCI), a process that silences each parental X in a proportion of cells. XCI skewing can decrease FVIII expression by preferentially inactivating the normal X chromosome. FVIII:C is further modulated by factors such as ABO blood groups and von Willebrand factor (VWF). The D'/D3 domain of VWF binds circulating FVIII protein, preventing proteolytic degradation. In type 2 Normandy von Willebrand disease (2N VWD), D'/D3 mutations decrease affinity to FVIII and result in bleeding events similar to mild/moderate HA. Variants in VWF also affect the pharmacokinetics of recombinant FVIII. Current clinical screening tests detect the 3 VWF mutations responsible for >90% of 2N, but report variants not directly responsible for 2N as clinically benign. However, common polymorphisms are known to affect FVIII:C in normal individuals. Therefore, a better understanding of how specific alterations in VWF modulate HA phenotype is necessary to interpret clinical presentation and refine management with factor concentrates. This is particularly important in HA individuals carrying mild/moderate mutations. To evaluate VWF variants in HA carriers, we focused on an extended pedigree that includes four obligate carriers from a family with mild/moderate HA (FVIII: p.Ala723Thr). FVIII:C varied and largely correlated with XCI skewing. Nevertheless, the FVIII:VWF interaction prompted us to identify VWF gene variants that could further modulate FVIII:C and contribute to bleeding in this family.Methods: To identify D'/D3 VWF variants that impact FVIII binding, primers were designed to amplify exons 17-20 and 24-27 on chromosome 12. A chromosome 22 pseudogene, with 97% identity to VWF exons 24-34, complicated primer design. Primers specific to VWF were selected by targeting regions that differed from the pseudogene and were verified by digestion with a restriction enzyme unique to each chromosome 12 exon. The PCR products were amplified and sequenced from the four female carriers and a control male relative.Results: After excluding the three most common mutations responsible for 2N, seven other variants were identified. Four of these were intronic polymorphisms and a synonymous variant at p.Asn1138 not associated with VWD and presumed to be clinically benign. All but one of these have been described in normal individuals. Two females were heterozygous for missense variant rs1063856 (p.Thr789Ala) and synonymous polymorphism rs1063857 (p.Tyr795=) that are in linkage disequilibrium and are likely to impact FVIII:C and VWF antigen (VWF:Ag) levels. These common variants, found in up to 36% of Caucasians and 58% of African Americans, are reported to increase VWF:Ag and FVIII:C jn heterozygotes (9 IU/dL and 7 IU/dL respectively). Neither ABO blood groups nor XCI skewing could fully explain the differences in FVIII:C activity observed with this variant.Conclusions: We propose that VWF variants rs1063856/rs1063857 may contribute to FVIII:C differences between two females in the pedigree with similar XCI skewing. We conclude that consideration of VWF variants is important for fully understanding bleeding phenotype and treatment responses in female carriers and males in families with mild/moderate HA. These findings support the need for expanded studies into the role of FVIII and VWF variant interactions in additional unrelated HA individuals. DisclosuresNo relevant conflicts of interest to declare.

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.

Human Blood Cell Level of 5-Hydroxymethylcytosine (5hmC) Declines Steadily during Aging and Is Multifactorial

Abstract BACKGROUND. The most frequently mutated genes documented in blood cells of aging individuals are known epigenetic regulator genes such as TET2 and DNMT3A, which suggests that alteration of epigenetic homeostasis could be a predisposing factor in the pathogenesis of several age-associated hematological malignancies. This study is aimed at determining if changes in hematopoietic 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) levels occur in normal individuals, and if they are independent of acquired mutations in epigenetic regulators. METHOD. The study population comprised 198 unrelated women randomly selected to form four age categories (neonates, 25-30 years, 70-75 years and &gt;90 years) from the general community. Genomic DNA from total blood cells or cord blood (12.5 ug) was hydrolysed using DNA Degradase Plus and analysed by mass spectrometry (LC-ESI-MS/MS-MRM) to quantify global 5-methyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine levels. Statistical analysis (normality test, outlier detection, descriptive statistic, non-parametric Kruskal-Wallis and Mann Whitney tests) were performed using NCSS 07.1.21 to correlate methylation (5mC and 5hmC) levels with i) X-chromosome inactivation (XCI) patterns (evaluated by HUMARA) in polymorphonuclear (PMN) and ii) telomeres length (measured by the method of Cawthon). All individuals over 70 were sequenced in search for mutations in epigenetic regulator genes including TET2, DNMT3A, ASXL1, IDH1, IDH2 and WT1. RESULTS. 5hmC and 5mC levels: Global 5hmC levels decline steadily with age in human blood cells (30% from birth to old age, P &lt;0.000001). This reduction is progressive between 0 and 75 years of age and plateaued at a low level thereafter (Figure 1). A less severe reduction in 5mC level was also observed between newborn and elderly individuals (3%, P&lt;0.000024). Correlation with biological characteristics: Low level of 5hmC was associated with more important XCI skewing in PMN (age-adjusted, P =0.0304) as well as a reduction of telomere length (age-adjusted, P =0.0354), both surrogate markers of clonal dominance. Correlation with mutational status: Of the 100 individuals over 70 years, 16 had a somatic mutation in TET2, 10 in DNMT3A and none in IDH1, IDH2 or WT1. Individuals with TET2 gene mutation (variant allele frequency (VAF) &gt;10%) had a significant reduction in 5hmC but not the DNMT3A subjects. To evaluate if the TET2 mutated individuals were the sole drivers of the documented reduction in 5hmC, we removed these subjects from the statistical analysis and correlation was preserved. However, correlation with XCI skewing and telomere reduction was no longer significant. CONCLUSION. These results document a significant age-associated reduction in 5hmC during aging in the hematopoietic compartment. DNA hypohydroxymethylation should be considered as a new hallmark of aging hematopoiesis. We also demonstrate that acquired mutations in key epigenetic regulators such as TET2 are not the sole cause of this reduction. Epigenetic drift, which is defined as the cumulative selection of permissive stochastic epigenetic modifications, is responsible for most of the age-associated loss. These results also indicate that the impact on clonal dominance may be different according to the cause of 5hmC (mutation vs drift). Further studies are needed to evaluate the impact of 5hmC reduction on the risk of hematological cancer initiation. Figure 1. Figure 1. Disclosures Busque: Novartis: Consultancy; BMS: Consultancy; PFIZER: Consultancy.

Age-Associated Acquired Mutations in Hematopoietic Cells Predominantly Affect Epigenetic Regulators TET2 and DNMT3A and Are Associated with Distinct Biological and Hematological Profiles

Abstract BACKGROUND. Recent studies using whole genome approaches have shown that acquired mutations in genes such as DNMT3A, TET2, ASXL1, JAK2 and TP53 are observed in a subset of elderly subjects without hematologic malignancies, and are associated with an increased relative risk of developing myeloid cancers, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The goal of this study is to precisely determine, using a more sensitive approach, the prevalence of somatic mutations and their allelic frequencies in elderly subjects, and assess the impact of somatic mutations on biological characteristics. METHODS. Cohort: 1101 hematologically healthy women aged 70 to 101 years old were recruited from the general population. All patients in the cohort were characterized with a i) medical questionnaire; ii) complete blood counts (CBC), and iii) X-chromosome inactivation (XCI) patterns in polymorphonuclear (PMN), using HUMARA to assess the clonality of myeloid-derived cells. Sequencing: All individuals were sequenced using a targeted approach at high coverage (95% &gt;500x) on an Ion Proton NGS instrument. Libraries were generated using an amplicon-based panel covering 19 recurrently mutated genes in myeloid malignancies (ASXL1, BRAF, CBL, CEBPA, DNMT3A, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53 and WT1) with 237 amplicons over 22kb. Statistical analyses: Mutational status was correlated with the biological characteristics of subjects using linear regression analysis (GLM). All age-dependent variables were corrected for age (XCI skewing PMN↑, monocytes↑, hemoglobin↓, lymphocytes↓ and platelets↓). RESULTS. Mutations. We identified 249 somatic mutations in 210 of the 1101 individuals (19%) with variant allele frequencies (VAF) ranging from 3.6% to 74.8%. Mutations were found in DNMT3A (n=131), TET2 (n=100), ASXL1 (n=5), JAK2 (N=4), TP53 (n=4), CBL (n=2), IDH1 (n=1), RUNX1 (n=1) and CEBPA (n=1). TET2 and DNMT3A mutations accounted for 93% of all somatic mutations. More than one somatic mutation was observed in 32 individuals (3%), with one subject harboring 5 mutations. Correlation between mutational status and biological characteristics. The presence of mutations significantly increased with age (all genes: P =0.00024, TET2: P =0.00016, TET2 and DNMT3A (double mutations): P =0.03268), although no age effect was documented for DNMT3A mutation alone. The presence of a mutation in TET2, but not in other disease alleles correlated with increased myeloid skewing (P= 0.0008). The presence of a mutation (all genes) or a mutation in DNMT3A correlated with a decreased mean corpuscular volume (MCV) (P= 0.046). TET2 mutations were associated with decreased total white blood cell counts (P =0.0262) and PMN counts (P =0.0137). DNMT3A correlated with increased total white blood cell counts (P =0.035) and increased lymphocyte counts (P =0.0256). No mutational phenotype correlated with alterations in monocyte counts. Despite these global mutation-associated hematological phenotypes, we observed several individuals with a high VAF (&gt;30%) in DNMT3A or TET2 with completely normal CBC. CONCLUSION: Acquired mutations in the normal aging population occur at very high frequency, most commonly TET2 and DNMT3A. This suggests that mutation-driven epigenetic dysregulation is key in the development of age-associated hematological cancers. TET2 and DNMT3A mutations are associated with distinctive characteristics such as increased clonal dominance, aging and lower PMN counts for TET2; increased lymphocyte counts and decreased MCV for DNMT3A. This suggests that these two genes may have a different impact on transformation to malignant phenotype. Prospective evaluation of this cohort and sequential analysis of blood specimens will provide informative insight on the sequence of events leading to age-associated hematological cancers. Disclosures Mollica: Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy. Busque:Novartis: Consultancy; BMS: Consultancy; PFIZER: Consultancy.

Skewed X Inactivation in Women Carrying the FMR1 Premutation and Its Relation with Fragile-X-Associated Tremor/Ataxia Syndrome

Background: Fragile-X-associated tremor/ataxia syndrome (FXTAS) is a late-onset multisystem neurological disorder characterized by intention tremor and cerebellar ataxia. Objective: We hypothesized that in FMR1 premutation females with FXTAS, a normal X chromosome might more frequently be inactivated; therefore, the aim of this study was to determine the relationship between skewed X chromosome inactivation (XCI) and FXTAS. Methods: We studied the XCI patterns of cases of FMR1 premutation in 10 women with FXTAS and 21 without FXTAS. Results: The distribution of XCI patterns in the FXTAS and no-FXTAS groups showed differences regarding the allele presenting severe skewed XCI. In the FXTAS group, all cases preferentially inactivated the non-expanded X chromosome, whereas in the no-FXTAS group, all inactivated the expanded X chromosome. Nevertheless, no significant differences were found on comparing XCI frequencies among FMR1 premutation carriers with and without FXTAS. As expected, we found statistically significant differences in the skewed XCI on comparing FMR1 premutation women and controls. Conclusion: Although the reduced sample size and blood XCI patterns are two limitations of this study, our results suggest that the skewed XCI of the normal FMR1 allele may be a risk factor for the development of FXTAS. Furthermore, our findings also support the protective effect of the expression of a normal FMR1 allele.

MG-121 Complexity of phenotypes of females with unbalanced x-autosomal translocations exemplified by a case with 46, x,der (x)t (x;16)(p11.2;p13.2) karyotype

Background Females with unbalanced X-autosomal translocations frequently exhibit skewed X-chromosome inactivation, as cells with the least functional imbalance have selective advantage. However, predicting the inactivation status of the translocated autosomal segment and its impact on phenotype is challenging. Active vs inactive state can depend on translocation breakpoint and sequence features of the translocated segment. Objective Report a case with 46,X,der (X)t (X;16)(p11.2;p13.2) karyotype and discuss karyotype/phenotype correlations. Results We report a 18 month old girl with intrauterine growth restriction, failure to thrive, cardiac anomalies and dysmorphic features. Oligonucleotide microarray has revealed a terminal ~9 Mb gain of chromosome 16p and terminal loss of most of Xp. A de novo unbalanced translocation t (X;16)(p11.2;p13.2) was confirmed by karyotype analysis and family studies. Androgen receptor assay further showed complete skewing of X-inactivation, suggesting that derivative X-chromosome was inactive. Phenotype of our patient was not fully consistent with either Xp deletion or 16p13.3 duplication, leaving a question about inactivation status of the translocated 16p material. A case with 46, X,der (X) t (X;16)(q28;p12) karyotype and phenotype consistent with 16p13.3 duplication syndrome was previously reported. Replication studies have shown skewed inactivation of the der (X), not spreading into 16p translocated segment. 1 Conclusions Our patient phenotype suggests that while Androgen Receptor assay provides valuable information in regard of X-inactivation skewing, it has limitations for karyotype/phenotype correlations for patients with unbalanced X-autosomal translocation. Additional analysis of inactivation status of the genes of the translocated segment is underway to understand the phenotype of our patient. Reference Preis W, Barbi G, Liptay S, Kennerknecht I, Schwemmle S, Pohlandt F. X/autosome translocation in three generations ascertained through an infant with trisomy 16p due to failure of spreading of X-inactivation. Am J Med Genet . 1996; 61 (2):117–21

The Role of X-Chromosome Inactivation in Retinal Development and Disease.

The expression of X-linked genes is equalized between males and females in mammalian species through X-Chromosome inactivation (XCI). Every cell in a female mammalian embryo randomly chooses one X Chromosome for epigenetic silencing at the 8-16 cell stage, resulting in a Gaussian distribution of XCI ratios with a peak at 50:50. At the tail extremes of this distribution, X-linked recessive mutations can manifest in disease in female carriers if the mutant allele is disproportionately active. The role of XCI skewing, if any, in X-linked retinal disease is still unknown, although many have speculated that such skewing accounts for phenotypic variation in female carriers of X-linked retinitis pigmentosa (XlRP). Some investigators have used clinical findings such as tapetal-like reflex, pigmentary changes, and multifocal ERG parameters to approximate XCI patches in the retina. These studies are limited by small cohorts and the relative inaccessibility of retinal tissue for genetic and epigenetic analysis. Although blood has been used as a proxy for other tissues in determining XCI ratios, blood XCI skews with age out of proportion to other tissues and may not accurately reflect retinal XCI ratios. Future investigations in determining retinal XCI ratios and the contribution of XCI to phenotype could potentially impact prognosis for female carriers of X-linked retinal disease.

Novel SMC1A frameshift mutations in children with developmental delay and epilepsy

Cornelia de Lange syndrome (CdLS) is a rare dominantly inherited genetic multisystem developmental condition with considerable phenotypic and allelic heterogeneity. Missense and in-frame deletions within the SMC1A gene can be associated with epilepsy and milder craniofacial features. We report two females who presented with developmental delay and developed isolated medically refractory seizures with unrevealing initial laboratory, imaging and genetic evaluations. Whole exome sequencing (WES) analyses were performed and were instrumental in uncovering the genetic etiology for their conditions. WES identified two novel de novo heterozygous frameshift mutations in the SMC1A gene [c.2853_2856delTCAG (p.Ser951Argfs*12) and c.3549_3552dupGGCC (p.Ile1185Glyfs*23)]. We also observed marked skewing of X-inactivation in one patient. The individual with the p.Ser951Argfs*12 mutation represents an extreme on the CdLS phenotypic spectrum, with prominent neurological involvement of severe developmental delay and refractory epilepsy, with mild craniofacial features. Both individuals eventually had incomplete clinical responses to therapy with valproic acid. We review previous reports of SMC1A mutations with epilepsy. SMC1A should be included in clinical gene panels for early infantile and early childhood epileptic encephalopathy.

Stochastic epigenetic mutations (DNA methylation) increase exponentially in human aging and correlate with X chromosome inactivation skewing in females.

In this study we applied a new analytical strategy to investigate the relations between stochastic epigenetic mutations (SEMs) and aging. We analysed methylation levels through the Infinium HumanMethylation27 and HumanMethylation450 BeadChips in a population of 178 subjects ranging from 3 to 106 years. For each CpG probe, epimutated subjects were identified as the extreme outliers with methylation level exceeding three times interquartile ranges the first quartile (Q1-(3 x IQR)) or the third quartile (Q3+(3 x IQR)). We demonstrated that the number of SEMs was low in childhood and increased exponentially during aging. Using the HUMARA method, skewing of X chromosome inactivation (XCI) was evaluated in heterozygotes women. Multivariate analysis indicated a significant correlation between log(SEMs) and degree of XCI skewing after adjustment for age (β = 0.41; confidence interval: 0.14, 0.68; p-value = 0.0053). The PATH analysis tested the complete model containing the variables: skewing of XCI, age, log(SEMs) and overall CpG methylation. After adjusting for the number of epimutations we failed to confirm the well reported correlation between skewing of XCI and aging. This evidence might suggest that the known correlation between XCI skewing and aging could not be a direct association but mediated by the number of SEMs.

Concomitant partial exon skipping by a unique missense mutation of RPS6KA3 causes Coffin–Lowry syndrome

Coffin–Lowry syndrome (CLS) is an X-linked semi-dominant disorder characterized by diverse phenotypes including intellectual disability, facial and digital anomalies. Loss-of-function mutations in the Ribosomal Protein S6 Kinase Polypeptide 3 (RPS6KA3) gene have been shown to be responsible for CLS. Among the large number of mutations, however, no exonic mutation causing exon skipping has been described. Here, we report a male patient with CLS having a novel mutation at the 3′ end of an exon at a splice donor junction. Interestingly, this nucleotide change causes both a novel missense mutation and partial exon skipping leading to a truncated transcript. These two transcripts were identified by cDNA sequencing of RT-PCR products. In the carrier mother, we found only wildtype transcripts suggesting skewed X-inactivation. Methylation studies confirmed X-inactivation was skewed moderately, but not completely, which is consistent with her mild phenotype. Western blot showed that the mutant RSK2 protein in the patient is expressed at similar levels relative to his mother. Protein modeling demonstrated that the missense mutation is damaging and may alter binding to ATP molecules. This is the first report of exon skipping from an exonic mutation of RPS6KA3, demonstrating that a missense mutation and concomitant disruption of normal splicing contribute to the manifestation of CLS.

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

X-chromosome inactivation in female patients with Fabry disease.

Fabry disease (FD) is an X-linked genetic disorder caused by the deficient activity of lysosomal α-galactosidase (α-Gal). While males are usually severely affected, clinical presentation in female patients may be more variable ranging from asymptomatic to, occasionally, as severely affected as male patients. The aim of this study was to evaluate the existence of skewed X-chromosome inactivation (XCI) in females with FD, its concordance between tissues, and its contribution to the phenotype. Fifty-six females with FD were enrolled. Clinical and biological work-up included two global scores [Mainz Severity Score Index (MSSI) and DS3], cardiac magnetic resonance imaging, measured glomerular filtration rate, and measurement of α-Gal activity. XCI was analyzed in four tissues using DNA methylation studies. Skewed XCI was found in 29% of the study population. A correlation was found in XCI patterns between blood and the other analyzed tissues although some punctual variability was detected. Significant differences in residual α-Gal levels, severity scores, progression of cardiomyopathy and deterioration of kidney function, depending on the direction and degree of skewing of XCI were evidenced. XCI significantly impacts the phenotype and natural history of FD in females.

Pathology and Function of Conduction Tissue in Fabry Disease Cardiomyopathy

Cardiac arrhythmias are common in Fabry disease (FD) and may occur in prehypertrophic cardiomyopathy suggesting an early compromise of conduction tissue (CT). Therefore, FD X-linked and CT may be variously involved in male and female patients with FD cardiomyopathy, affecting CT function. Among 74 patients with endomyocardial biopsy diagnosis of FD cardiomyopathy, 13 (6 men; 7 women; mean age, 50.1±13.5 years; maximal wall thickness, 16.7±3.7 mm) had CT included in histological specimens and 6 also at electron microscopy. CT glycolipid infiltration was defined as focal, moderate, extensive, or massive, if involved ≤30%, ≤50%, >50%, or 100% of cells; identified as loosely arranged small myocytes positive to HCN4 immunostaining, supplied by a centrally placed thick-walled arteriole. CT involvement was correlated with age, sex, and α-Gal gene mutation. CT function was evaluated by electrophysiological study and arrhythmias at Holter registration. CT infiltration was focal/moderate in 4 women with no arrhythmias and normal electrophysiological study, extensive in 3 women with atrial or ventricular arrhythmias and short HV interval, and massive in 6 men with atrial fibrillation or ventricular arrhythmias and short HV. Short PR/AH with increased refractoriness was additionally found in 3 patients with extensive/massive CT infiltration. A male patient with the shortest HV presented infra-Hissian block during decremental atrial stimulation. There was no correlation with age, maximal wall thickness, and type of gene mutation. CT infiltration in FD cardiomyopathy is constant in men and variable in women because of skewed X-chromosome inactivation; its extensive/massive involvement causes accelerated conduction with prolonged refractoriness and electric instability.

Genetic Evaluation of Copy Number Variations, Loss of Heterozygosity, and Single-Nucleotide Variant Levels in Human Embryonic Stem Cells With or Without Skewed X Chromosome Inactivation.

Human embryonic stem cells (hESCs) exhibiting skewed X chromosome inactivation (XCI) have been reported. The copy number variations (CNVs), loss of heterozygosity (LOH), or single-nucleotide variant (SNV) events in those epigenetically distinct cells remain unknown, and whether such genetic abnormalities will influence the XCI status of hESCs is unclear. In this study, three hESCs with skewed XCI, three with random XCI, and two male hESC lines at different passages were analyzed for CNVs and LOH levels using a high-resolution genotyping microarray. Whole-exome sequencing was used to investigate the potentially damaging SNVs. On average, 17.6 CNVs and 5.3 cases of LOH were identified in the skewed hESCs, which were similar to the rates observed in random hESCs. Five recurrent CNV regions were uniquely identified in the skewed hESCs, but all of them were considered polymorphisms. With the exception of a nongenic CNV, no additional CNVs were detected on the X chromosome in the skewed hESCs. Although the XCI status in two hESC lines was observed to be changed from random to skewed, no significant CNV difference was identified before and after the XCI change. SNV analysis indicated that normal alleles are maintained for most genes within copy-neutral LOH regions. Three types of expression patterns were observed in heterozygous alleles, and the damaging SNVs in skewed hESCs favored the expression of the wild-type alleles. In conclusion, in the present study, we did not find genetic differences in the CNV and LOH levels between hESCs with and without skewed XCI. Wild-type allele expression in the presence of damaging SNVs on the X chromosome in skewed hESCs might alleviate adverse effects in those hESCs.