Analysis Of X-chromosome Inactivation Patterns Research Articles

Development of canine X-chromosome inactivation pattern analysis for the detection of cell clonality by incorporating the examination of the SLIT and NTRK-like family member 4 (SLITRK4) gene

X-chromosome inactivation pattern (XCIP) analysis can be used to assess the clonality of cell populations of various origin by distinguishing the methylated X chromosome from the unmethylated X chromosome. In this study, the utility of XCIP analysis was improved by incorporating the examination of AC dinucleotide repeats in SLIT and NTRK-like family member 4 (SLITRK4) gene into the previously reported CAG repeat examination of androgen receptor (AR) gene in dogs. The rate of heterozygosity when both genes were analysed (125/150, 83.3%) was higher than AR gene examination alone (86/150, 57.3%). Blood samples from heterozygous dogs in either AC-1 or AC-2 of SLITRK4 gene were examined for the corrected inactivation allele ratio (CIAR), resulting in the determination of a reference range of CIAR <3.8 in non-neoplastic cell/tissue samples. Using this analytical method, 49% (21/43) of neoplastic tissue samples from dogs showed a CIAR >3.8, indicating the presence of a clonal population. Through the present study, the availability of canine XCIP analysis was improved by incorporating the examination of the SLITRK4 gene, providing a highly useful laboratory examination system for the detection of the clonality of various cell/tissue samples in dogs.

Development of ultra-deep targeted RNA sequencing for analyzing X-chromosome inactivation in female Dent disease

The pattern of X-chromosome inactivation (XCI) can affect the clinical severity of X-linked disorders in females. XCI pattern analysis has been conducted mainly by HUMARA assay, a polymerase chain reaction-based assay using a methylation-sensitive restriction enzyme. However, this assay examines the XCI ratio of the androgen receptor gene at the genomic DNA level and does not reflect the ratio of either targeted gene directly or at the mRNA level. Here, we report four females with Dent disease, and we clarified the correlation between XCI and female cases of Dent disease using not only HUMARA assay but also a novel analytical method by RNA sequencing. We constructed genetic analysis for 4 female cases showing high level of urinary low-molecular-weight proteinuria and their parents. Their XCI pattern was analyzed by both HUMARA assay and an ultra-deep targeted RNA sequencing of the CLCN5 gene using genomic DNA and mRNA extracted from both leukocytes and urine sediment. All four cases possessed pathogenic variants of the CLCN5 gene. XCI analysis revealed skewed XCI in only two cases, while the other two showed random XCI. All assay results of HUMARA and targeted RNA sequencing in both leukocytes and urinary sediment were clearly identical in all four cases. We developed a novel XCI analytical assay of ultra-deep targeted RNA sequencing and revealed that skewed XCI explains the mechanism of onset of female Dent disease in only half of such cases.

Demonstration of the cell clonality in canine hematopoietic tumors by X-chromosome inactivation pattern analysis.

X-chromosome inactivation pattern (XCIP) analysis has been widely used to assess cell clonality in various types of human neoplasms. In this study, a polymerase chain reaction-based canine XCIP analysis of the androgen receptor (AR) gene was applied for the assessment of cell clonality in canine hematopoietic tumors. This XCIP analysis is based on the polymorphic CAG repeats in the AR gene and the difference of methylation status between active and inactive X chromosomes. We first examined the polymorphisms of 2 CAG tandem repeats in the AR gene in 52 male and 150 female dogs of various breeds. The 2 polymorphic CAG repeats contained 9 to 12 and 10 to 14 CAGs in the first and second CAG repeats, respectively. Of the 150 female dogs, 74 (49.3%) were heterozygous for the first and/or second polymorphic CAG tandem repeats, indicating the utility of XCIP analysis in these dogs. Canine XCIP analysis was then applied to clinical samples from female dogs with canine high-grade lymphoma, chronic myelogenous leukemia, acute myelogenous leukemia, and benign lymph node hyperplasia. Of 10 lymphoma cell samples, 9 (90%) showed skewed XCIPs, indicating their clonal origins, whereas all the nonneoplastic lymph node samples showed balanced XCIPs. Moreover, bone marrow specimen from a dog with acute myelogenous leukemia and peripheral leukocyte specimens from 2 dogs with chronic myelogenous leukemia showed skewed XCIPs. XCIP analysis was successfully employed to demonstrate the cell clonality of canine hematopoietic tumors in this study and will be applicable to evaluate the clonality in various proliferative disorders in dogs.

X-Chromosome Inactivation Pattern Analysis for the Assessment of Cell Clonality in Cats

X-chromosome inactivation pattern (XCIP) analysis has been widely used to assess cell clonality in various types of neoplasms in humans. In the present study, a polymerase chain reaction-based feline XCIP analysis using the feline androgen receptor gene was developed. To construct the system of the analysis, polymorphism in CAG tandem repeats within the feline androgen receptor gene was explored using somatic DNAs from 50 male and 103 female cats. CAG tandem repeats in exon 1 of the feline androgen receptor gene were found to be polymorphic, containing 15 to 22 CAG repeats. Of the 103 female cats, 70 (68%) were heterozygous for the number of CAG repeats, indicating the possible usefulness of XCIP analysis in cats. Application of the feline XCIP analysis to 3 feline mammary gland adenocarcinoma cell lines revealed distinctly skewed XCIPs in these cell lines, indicating their clonal origins. Twelve (80%) of the 15 primary tissue/cell samples obtained from cats with various neoplastic diseases showed skewed XCIPs. Moreover, bone marrow samples from 3 cats with myelodysplastic syndrome were also found to have skewed XCIPs. The polymerase chain reaction-based XCIP analysis developed in this study can provide information on cell clonality in female cats, potentially facilitating the differential diagnosis of various disorders in cats.

Detection of Highly Clonal CD34+, Mononuclear Bone Marrow and Peripheral Blood Cells From Patients with Myelodysplastic Syndrome and Age Related Increase of Clonal Hematopoiesis In Healthy Subjects Using a Novel Transcriptional Pyrosequencing Based Clonality Assay

AbstractAbstract 977X-chromosome inactivation occurs in female cells leading to stochastically random silencing of one X-chromosome. Accordingly, investigation of X-chromosome inactivation patterns (XCIP) in healthy female tissues revealed similar ratios of cells carrying either an active maternal or paternal X-chromosome. Genomic XCIP markers like the HUMARA locus have been used to elucidate clonality of myelodysplastic syndromes (MDS). However, analysis of healthy female peripheral blood (PB) cells has shown a strong age-related increase in skewing of XCIP suggesting the frequent manifestation of clonal hematopoiesis in the elderly. Since the occurrence of clonality in blood cells from healthy individuals remains controversially discussed (Swierczek et al, Blood 2008), we developed a novel highly accurate transcription based approach using Pyrosequencing to detect clonal hematopoiesis. In addition, we present the first comprehensive XCIP analysis of CD34 purified (CD34+) and unfractioned bone marrow (BM) cells of MDS patients and healthy females.BM, CD34+ and PB cells were obtained from patients with MDS (IPSS-low/int-1-risk BM: n=25, CD34+: n=13, PB: n=21, IPSS-int-2/high-risk BM: n=16, CD34+: n=9, PB: n=12) and age related healthy donors (BM: n=19, CD34+: n=15, PB: n=105) after informed consent. Genomic DNA SNP genotyping was carried out in order to screen for heterozygous clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS, MPP1 and G6PD. After PCR-amplification of informative marker loci from cDNA transcripts, SNP allele ratios, representative for XCIP, were quantified using the PyroMark ID system (Qiagen, Hilden, Germany). Clonality was assumed for samples exhibiting skewed XCIP ratios >80% (allelic ratio of >4:1).Standard curves from pyrosequencing reactions with predefined allelic ratios revealed strong correlations for assessment of XCIP ratios in all markers (R2>0.99). Furthermore, excellent inter-marker correlations of XCIP ratios from individuals with multiple informative markers were achieved ranging from R2>0.83 to R2<0.98. These findings indicate a balanced inactivation of the whole X-chromosome and corroborate the suitability of our method. Of note, distinctively increased numbers of clonal PB samples from healthy elderly donors (age: 65–93 years) with skewed XCIP ratios in 55% of all cases were detected as compared to young donors with skewed XCIP ratios in 13% and 25% for age groups ranging from 18–40 and 18–65 years, respectively. Analysis of XCIP ratios in MDS samples revealed clonality in 96% of BM, 100% of CD34+ and 90% of PB specimen from MDS low/int-1-risk and 100% of BM, 100% of CD34+ and 92% of PB specimen from MDS int-2/high-risk patients. In contrast, only 44% of BM and 43% of CD34+ samples from healthy elderly (age range 65–93 years) and 20% of BM and 25% of CD34+ samples from healthy young females (age range 18–65 years) were graded as clonal. Furthermore, we observed extremely skewed XCIP ratios of >95% in 56% of BM, 62% of CD34+ and 57% of PB cells from MDS low/int-1-risk and 50% of BM, 67% of CD34+ and 67% of PB cells from MDS int-2/high-risk patients but only in 22% of BM, 29% of CD34+ and 3% of PB cells from healthy elderly subjects.Our detection of highly clonal cells in MDS patients in 3 hematopoietic cell compartments and frequent detection of extreme XCIP skewing provides strong evidence for the manifestation of a malignant MDS clone even in early progenitor cells. Moreover, detection of non-clonal PB cells in patients with clonal CD34+ cells indicates the presence of residual healthy hematopoietic cells in the peripheral blood. Notably, in our hands, age related increases of XCIP skewing have been detected in PB cells and, for the first time, in CD34+ and BM cells of healthy females suggesting the clonal outgrowth of hematopoietic cells in the early progenitor compartment of healthy elderly. In conclusion, pyrosequencing based clonality assessment has proven to be suitable for quantitative monitoring of clonal and potentially malignant cell populations in hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Low burden of a JAK2-V617F mutated clone in monoclonal haematopoiesis in a Japanese woman with Budd-Chiari syndrome

Approximately one-half of the cases of Budd-Chiari syndrome (BCS) are caused by bcr/abl negative chronic myeloproliferative disorders (CMPDs). Furthermore, a mutation in the Janus kinase protein (JAK2-V617F) is detected in half of the patients with BCS. However, whether the JAK2 mutation is the primary event leading to CMPDs and BCS is controversial. We present a report concerning a young woman who suffered from BCS prior to the onset of CMPDs. Analysis of X-chromosome inactivation patterns in this patient, using the human androgen receptor gene demonstrated monoclonal haematopoiesis in her granulocytes. In contrast, she had a low burden of a JAK2-V617F mutation positive clone among granulocyte populations. These results suggest that the JAK2-V617F mutation occurs after the onset of monoclonal haematopoiesis; thus the V617F mutation of JAK2 may not be the primary event in the induction of BCS.

Granulocyte JAK2 (V617F) Mutation Status in Myeloid Neoplasms with Ringed Sideroblasts.

The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts (RARS). We have previously demonstrated that mitochondrial iron accumulation in this condition is in the form of mitochondrial ferritin (MtF). A gain-of-function mutation of JAK2 is found in most patients with chronic myeloproliferative disorders. A high frequency of this mutation has been also reported in RARS associated with marked thrombocytosis (RARS-T), a provisional entity characterized by marked increase in platelet count, hypercellular marrow with increased megakaryocytes, and ringed sideroblasts. In this study, we investigated the granulocyte JAK2 (V617F) mutation status in 73 patients receiving a diagnosis of myeloid malignancy with ringed sideroblasts at the Division of Hematology, University of Pavia Medical School & IRCCS Policlinico San Matteo Pavia, Italy between 2001 and 2006. According to the WHO classification of the myeloid neoplasms, 23 patients had RARS, 17 had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), 16 had refractory anemia with blasts excess, four had MDS with isolated del(5q), and 13 fulfilled the criteria for RARS-T. JAK2 (V617F) mutation status was analyzed on peripheral blood granulocytes through a quantitative real time PCR-based allelic discrimination assay. We compared clinical and biological features of patients with RARS-T with those of patients with refractory anemia or cytopenia with ringed sideroblasts, and found that RARS-T patients had higher neutrophil and platelet counts, lower frequency of cytogenetic abnormalities and higher incidence of the JAK2 (V617F) mutation (P values ranging from <.001 to .02). JAK2 (V617F) was detected in six out of 63 evaluable cases (9.5%), one being diagnosed as MDS with isolated del(5q) and five as RARS-T, resulting in an incidence of mutation in the latter group of 45%. The proportion of mutant alleles ranged between 2.8% and 18.4%, values commonly observed by us in essential thrombocythemia [Blood. 2006 May 1;107(9):3676–82]. Focusing the analysis on RARS-T, a significantly higher hemoglobin level at diagnosis was found in mutated (median value 11.2 g/dL, range 10.1–15.4) compared with non mutated patients (median value 9 g/dL, range 6–9.9) (P=.009). JAK2-positive patients also showed a significantly lower percentage of ringed sideroblasts in the bone marrow (P=.01), and an increased marrow reticulin fibrosis (P=.03). We then evaluated the clonality of hematopoiesis in female patients through analysis of X-chromosome inactivation patterns (XCIPs) in circulating granulocytes and bone marrow CD34-positive cells. Twenty-one out of 23 informative female patients with ringed sideroblasts (91%), as well as 5/6 RARS-T (83%) displayed a clonal pattern of X-chromosome inactivation. These observations suggest that refractory anemia with ringed sideroblasts with marked thrombocytosis is a clonal stem cell disorder significantly associated with the JAK2 (V617F) mutation. This disorder shows both dysplastic and proliferative features, the presence of the mutation being associated with a predominant myeloproliferative phenotype. The recognition of a heterogeneous genetic background in myeloid neoplasms with ringed sideroblasts suggests that different mechanisms might be involved in the induction of mitochondrial ferritin expression in these disorders.

Mutation of JAK2 in the myeloproliferative disorders: timing, clonality studies, cytogenetic associations, and role in leukemic transformation

AbstractThe identification of an acquired mutation of JAK2 in patients with myeloproliferative disorders has raised questions about the relationship between mutation-positive and mutation-negative subtypes, timing of the JAK2 mutation, and molecular mechanisms of disease progression. Here we demonstrate that patients with V617F- essential thrombocythemia do not commonly progress to become V617F+. Consistent with the concept of distinct pathogenetic mechanisms, we show that patients with and without the JAK2 mutation have different patterns of cytogenetic abnormality, with virtually all patients carrying the 20q deletion or trisomy 9 being V617F+. We also investigated the existence of a “pre-JAK2” phase by comparing the proportion of clonally derived granulocytes, estimated from X-chromosome inactivation patterns (XCIPs), with the proportion of V617F+ granulocytes. Our results demonstrate that inherent XCIP variability between granulocytes and T cells produces a systematically biased pattern of results that may be misinterpreted as evidence for an excess of clonally derived granulocytes, an observation that limits the utility of XCIP analysis in this context. Lastly, we studied 4 patients with V617F+ myeloproliferative disorders who subsequently developed acute myeloid leukemia. In 3 patients the leukemic cells were V617F-, suggesting that in these patients the leukemia arose in a V617F- cell.

JAK2 (V617F) as an acquired somatic mutation and a secondary genetic event associated with disease progression in familial myeloproliferative disorders

A somatic gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been identified in chronic myeloproliferative disorders, which appear to have a sporadic occurrence in most individuals. The authors studied the biologic significance of the JAK2 (V617F) mutation in familial myeloproliferative disorders. Twenty pedigrees with familial chronic myeloproliferative disorders were identified through an investigation of family history in 264 patients with sporadic myeloproliferative disorders. A quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay was employed for the detection of the V617F mutation in circulating granulocytes and T lymphocytes. An analysis of X-chromosome inactivation pattern was performed in female patients. Fourteen families had homogeneous phenotypes, and 6 families had mixed phenotypes. By using a qRT-PCR-based allelic discrimination assay, the JAK2 (V617F) mutation was detected in circulating granulocytes from 20 of 31 patients, but the mutation was not detected in T lymphocytes. Granulocyte mutant alleles ranged from 2.1% to 91.5% and, on average, increased with time. Discordant distribution of the JAK2 (V617F) mutation was observed in siblings with polycythemia vera. The proportion of granulocytes that carried the JAK2 (V617F) mutation was lower than the proportion of clonal granulocytes, as determined in an analysis of X-chromosome inactivation patterns in female patients. The current findings indicated that the JAK2 (V617F) mutation represents an acquired somatic mutation in patients with familial chronic myeloproliferative disorders and probably occurs as a secondary genetic event in the background of preexisting clonal hematopoiesis. Thus, a genetic predisposition to acquisition of JAK2 (V617F) is inherited in families with myeloproliferative disorders.

Telomerase and Telomeres in Polycythemia Vera and Essential Thrombocythemia.

Polycythemia Vera (PV) and Essential Thrombocythemia (ET) are chronic myeloproliferative disorders (CMPD) with a relatively favorable prognosis and a long natural history. They originate from a multipotent progenitor cell with dominance of the transformed clone over normal hematopoiesis. However, the heterogeneity of these diseases with respect to clonal development from a common progenitor has been established, and no specific molecular or cytogenetic markers are so far considered useful prognostic indicators. In the current study we correlate telomerase activity (TA) and telomeres [terminal restriction fragment (TRF)] lengths in granulocytes (PMN) and mononuclear cells (MNC), with the results of the clonality status, as determined by investigation of X chromosome inactivation patterns (XCIPs), from 22 informative female patients with ET and PV. All patients were in chronic phase without immature myeloid cells in the peripheral blood. After testing for heterozygosity at the HUMARA locus PMN expressed a monoclonal XCIP in 9 out of 13 subjects with ET (69%), and in 6 out of 9 with PV (67%). MNC were always polyclonal. The amount of TA for each reaction was expressed in Total Product Generated (TPG) units. ΔTRF length was calculated by subtracting the telomer length of PMNs from that of MNCs and expressed as kilobases. TA and ΔTRF results were blindly compared with those from XCIP analysis. Monoclonal PMN displayed a TA ranging between 47.6 and 212.5 TPG units (mean 118.2 ± 45.5 SD), whereas polyclonal PMN had a TA between 1.0 and 33.3 TPG units (mean 14.4 ± 11.9 SD) (p<0.001). MNC had a TA similar to that of polyclonal PMN. Mean ΔTRF of clonal PMNs was 4.6 kilobases, while that of polyclonal PMNs was 0.44 kilobases (p<0.001). PMNs from normal control subjects had a mean ΔTRF of 0.1 kilobases, similar to that of polyclonal PMNs (p= n.s.). The results of our study clearly show the existence of two distinct subgroups of CMPD, with polyclonal or monoclonal granulopoiesis, each of them correlating with a peculiar pattern of TA and telomere lengths. Although none of the patients analyzed showed any difference in clinical characteristics with respect to the parameters considered, we believe that TA and ΔTRF analysis and their relationship with clonality status may represent an useful tool for further investigation, to predict survival in larger cohorts of patients with PV and ET, over an extended period of follow up.

Analysis of JAK2 Mutation in Essential Thrombocythemia (ET) Patients with Monoclonal and Polyclonal X-Chromosome Inactivation Patterns (XCIPs).

Background: ET is considered as a hematopoietic stem cell disorder, closely related to polycythemia vera (PV). Several studies in female ET patients, using XCIPs analysis, have shown the persistence of polyclonal patterns in a subset of patients, the significance of this finding being unclear (Blood : 1997; 89:128and 1999;93:417). Patients with polyclonal patterns were suggested to be at lower risk of thrombosis. The mechanism of sustained megakaryocytic proliferation, in the absence of a reactive cause, is still to be found in this disease. The recent description of an activating V617F JAK2 mutation in PV prompted us to study its relevance in ET, especially with respect to XCIPs analysis results.Methods: We looked for V617F JAK2 mutation in whole blood DNA in a series of 43 ET female patients for whom XCIPs had previously been reported by our group (Blood : 1997; 89:128). Monoclonal pattern was defined by greater than 75% expression of one allele of an X-chromosome polymorphic gene. ET diagnosis was made according to PVSG criteria, after careful exclusion of PV by red cell mass measurement, CML by karyotype and/or BCR-ABL RT-PCR, and idiopathic myelofibrosis by bone marrow biopsy. V617F JAK2 mutation was screened by direct DNA sequencing (sensitivity of the assay: about 5%).Results: V617F JAK2 mutation was found in 12 of the 43 samples (28%), in agreement with recent reports. No significant differences were found between JAK2 mutated and non-mutated patients for age (43 vs 57 years), clinical symptoms at presentation (33% vs 35%), platelet count (866 vs 907 G/l), or cytoreductive therapy (41% vs 61%), respectively. Of the 35 patients with a monoclonal pattern in granulocytes (and polyclonal T lymphocytes), 8 (23%) had JAK2 mutation, as compared to 4 of the 8 patients (50%) with polyclonal pattern in granulocytes (p=0.185, Fisher's exact test).Conclusion: The 50% incidence of JAK2 mutation we found in ET patients with a polyclonal pattern of hematopoiesis may be due to a better sensitivity of the JAK2 mutation assay by comparison to XCIPs clonality assays. It shows that many ET with a XCIPs polyclonal pattern are in fact clonal disorders. On the other hand, the absence of JAK2 mutation in about 75% of patients with a monoclonal pattern suggests that other molecular events can lead to clonal megakaryocytic proliferation in a large proportion of ET patients.

Clonal granulocytes in polycythaemia vera and essential thrombocythaemia have shortened telomeres

Summary The purpose of this study was to evaluate telomere length in peripheral blood granulocytes and mononuclear cells collected from 22 women with polycythaemia vera (PV) and essential thrombocythaemia (ET). PV and ET are chronic myeloproliferative diseases whose heterogeneity of stem cell origin and clonal development has been established through analysis of X-chromosome inactivation patterns. The results from clonality assay and determination of telomere length show that only clonal granulocytes have shortened telomeres.

Association Between Androgen Receptor Gene CAG Repeat Polymorphism and Plasma Testosterone Levels in Postmenopausal Women

The biologic action of androgens in target cells depends on plasma androgen levels and receptor transcriptional activity. We investigated the relationship between androgen receptor (AR) CAG repeat polymorphism, serum androgen levels, and anthropometric, metabolic, and hormonal variables in 39 postmenopausal women, taking into consideration the patterns of X-chromosome inactivation. Genomic DNA was extracted from peripheral leukocytes. Polymerase chain reactions (PCRs) were performed to amplify the polymorphic (CAG)n repeat of the human AR gene, which were analyzed with GeneScan software (Applied Biosystems, Foster City, CA). The X-chromosome inactivation analysis was based on the AR gene methylation patterns. The mean age of participants was 54.7 years; mean age at menopause was 48 years. The number of CAG repeats ranged from 15 to 30, with a median length of 23. Analysis of X-chromosome inactivation patterns showed 19 cases with a degree of skewing (DS) > or =70% and seven with a DS > or =90%. The X-weighted CAG repeat biallelic mean was significantly lower in individuals with total testosterone (TT) greater than 0.56 ng/mL (group mean) than in the group with TT < or =0.56 (P=.018). No difference was observed between the groups regarding dehydroepiandrosterone sulfate (DHEA-S). Plasma TT was significantly higher in the group with the smaller X-weighted CAG repeat biallelic mean (P=.01). Free androgen index (FAI) was also significantly higher in this group (P=.033). Testosterone levels and FAI were inversely correlated to X-weighted CAG repeat biallelic mean. Our data indicate an association between testosterone plasma levels and AR CAG repeats in postmenopausal women, and suggest that plasma levels of androgens in postmenopausal women may be related to variants of the AR gene.

Clonal hemopoiesis and risk of thrombosis in young female patients with essential thrombocythemia.

Several studies demonstrated a high prevalence of nonrandom X-chromosome inactivation pattern (X-CIP) in essential thrombocythemia (ET). This study explored the incidence of clonal hemopoiesis in myeloid precursors and endogenous erythroid colonies (EECs) in ET patients and its correlation with thrombotic manifestations. Clonal analysis of hemopoiesis using X-CIP was performed in 40 female patients with ET. Median age was 40.5 years (range 20-64), and median platelet count at testing time was 700 x 10(9)/L (range 220-1300 x 10(9)/L). Patients older than 65 years were excluded to reduce age-related skewing. Clonality was assessed on neutrophils, platelets, EECs, and bone marrow CD34(+) cells. Eight (20%) of 40 patients developed thrombosis mainly at diagnosis. Clonal hemopoiesis was found in 17 (42.5%) patients, 15 (37.5%) had polyclonal hemopoiesis, and 8 (20%) were considered uninterpretable due to constitutive skewing. Clonality was confirmed on purified CD34(+) subpopulations from bone marrow, documenting that clonality does not appear lineage-restricted. There were no statistical differences in age at diagnosis, median platelet count at testing time, and length of follow-up. Thrombotic episodes were significantly more frequent in the monoclonal group (p = 0.04, Fisher exact test). Young female patients with ET exhibiting a clonal pattern of hemopoiesis by X-CIP analysis are at higher risk for thrombosis. X-CIP analysis may contribute to defining the individual risk leading to appropriate treatment. X-CIP will allow a correct diagnosis in patients with latent myeloproliferative disorders and thrombosis in unusual sites. Clonal hemopoiesis is easily recognized by X-CIP, but its applicability is limited to the female sex and is hampered by the presence of age-related or constitutive skewing.

Clonality analysis using X-chromosome inactivation patterns by HUMARA-PCR assay in female controls and patients with idiopathic thrombocytosis in Taiwan.

Analysis of X-chromosome inactivation patterns (XCIPs) is a useful tool in the diagnosis of clonal disorders. The human androgen receptor (HUMARA) locus is especially useful for clonality study. The present study was conducted 1) to determine the heterozygosity rate for HUMARA locus in Taiwanese women, 2) to determine the frequency of excessive skewing in different cell types, and 3) to determine the utility of XCIPs in the differential diagnosis of thrombocytosis. XCIPs by HUMARA-PCR assay were performed on purified granulocytes and T cells from 73 female patients presenting with idiopathic persistent thrombocytosis (IT), 10 patients with reactive thrombocytosis (RT), and 46 bone marrow samples from female controls. XCIPs of buccal mucosa cells were also compared with those of T cells in 57 patients with IT. The percentage of clonal granulocytes was calculated after correcting for the degree of Lyonization in T cells. The heterozygosity rate for the HUMARA gene was 89.1% in Taiwanese females. The median age of informative IT patients and controls was 59 (18-92) and 58 (19-89), respectively. Excessive skewing (allele ratio <0.33) was more frequent in granulocytes than in T cells in both controls (12/43 vs 9/43, p = 0.080) and IT patients (56/64 vs 25/64, p < 0.001). XCIPs were the same for both buccal mucosa and T cells in 43 patients but were different in 14 patients. Of the 43 informative controls, 31 had a polyclonal pattern; an ambiguous pattern was found in nine; and the remaining three, aged 71, 73, and 80, respectively, had a clonal pattern. A clonal pattern was found in 42 IT patients, a polyclonal pattern in 12, and an ambiguous pattern in 10 of the 64 IT patients. The frequency of clonal, polyclonal, and ambiguous patterns in the 40 IT patients with age < or = 65 was 55.0%, 30.0%, and 15.0%, respectively. None of the IT patients aged >65 had a polyclonal disease. IT patients aged >65 had a significantly higher frequency of clonal pattern (p = 0.030) and a significantly lower frequency of polyclonal pattern (p = 0.002) than those with age <65. Of the eight heterozygous patients with RT, one aged 80 exhibited a clonal pattern, and the remaining seven had a polyclonal pattern. The present study on Taiwanese females showed a heterozygosity rate of 89.1% for the HUMARA gene. Our results confirmed that IT is a heterogeneous disorder in terms of clonality. Twenty-three percent of IT patients exhibited a greater than 20% difference in allele expression for buccal mucosa and T cells. Presence of a clonal XCIP in young patients with IT can serve as a positive marker for the diagnosis of clonal thrombocytosis, and elderly patients with polyclonal XCIPs are unlikely to have essential thrombocythemia.

X-chromosome inactivation analysis of isolated Reed-Sternberg cells in nodular sclerosing Hodgkin's disease.

In spite of several studies of immunoglobulin (Ig) gene rearrangements in whole Hodgkin's disease (HD) tissues and in isolated single Reed-Sternberg (RS) cells, the issue of clonality of the RS cell in HD remains incompletely resolved. Analysis of X-chromosome inactivation patterns (XCIPs) can be used to determine whether cell populations are clonal in origin. By PCR amplification of the human androgen receptor (HUMARA) loci using nested primers, we have studied XCIPs in six cases of HD of the nodular sclerosing (NS) subtype in which individual RS cells were isolated by micromanipulation from formalin-fixed, paraffin-embedded tissue sections immunostained with CD30 or CD15. In order to assess whether a clonal population of RS cells might be present in NSHD tissues, we compared the XCIPs obtained from whole NSHD tissues with those obtained from single RS cells harvested from the same tissues. Whole tissues from all six cases of NSHD showed balanced HUMARA allelic patterns, whereas an average of 83% (range 77-91%) of single RS cells from each of the six tissues expressed the same high- or low-molecular weight allele, suggesting that a clonal population of RS cells was likely to be present in each case. These data are consistent with the presence of a clonal population of RS cells in NSHD tissues.

Clonality of Isolated Eosinophils in the Hypereosinophilic Syndrome

The idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII andHha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression.

Clonality of Isolated Eosinophils in the Hypereosinophilic Syndrome

AbstractThe idiopathic hypereosinophilic syndrome (IHES) is a rare disorder characterized by unexplained, persistent eosinophilia associated with multiple organ dysfunction due to eosinophilic tissue infiltration. In the absence of karyotypic abnormalities, there is no specific test to detect clonal eosinophilia in IHES. Analysis of X-chromosome inactivation patterns can be used to determine whether proliferative disorders are clonal in origin. Methylation of HpaII andHha I sites near the polymorphic trinucleotide repeat of the human androgen receptor gene (HUMARA) has been shown to correlate with X-inactivation. In this study, we have used the polymerase chain reaction (PCR) with nested primers to analyze X-inactivation patterns of the HUMARA loci in purified eosinophils from female patients with eosinophilia. Peripheral blood eosinophils were isolated by their autofluoresence using flow cytometric sorting. Eosinophils purified from a female patient presenting with IHES were found to show a clonal pattern of X-inactivation. Eosinophil-depleted leukocytes from this patient were polyclonal by HUMARA analysis, thus excluding skewedness of random X-inactivation. After corticosteroid suppression of her blood eosinophilia, a clonal population of eosinophils could no longer be detected in purified eosinophils. In contrast, eosinophils purified from a patient with Churg-Strauss syndrome and from six patients with reactive eosinophilias attributed to allergy, parasitic infection, or drug reaction showed a polyclonal pattern of X-inactivation by HUMARA analysis. The finding of clonal eosinophilia in a patient presenting with IHES indicates that such patients may have, in reality, a low-grade clonal disorder that can be distinguished from reactive eosinophilias by HUMARA analysis. Further, the method described can be used to monitor disease progression.

Quantification of X-chromosome inactivation patterns using RT-PCR of the polymorphic iduronate-2-sulphatase gene and correlation of the results obtained with DNA-based techniques.

Most studies using X-chromosome inactivation patterns (XCIPs) to determine clonality in females have used polymorphic DNA loci, but interpretation may be complicated by the complexity of the differential methylation patterns necessary to distinguish active and inactive X-chromosomes. Recent description of polymorphisms within the transcribed region of three X-chromosome genes has enabled XCIP analysis of allele expression at the RNA level, which should circumvent this problem. We report here a quantitative RT-PCR assay for one of these loci, the iduronate-2-sulphatase (IDS) gene, using a mismatch primer to introduce a Bc/l cutting site in one of the alleles. DNA samples from 150 females were screened and 61 (41%) were found to be informative for the polymorphism. Reproducible results were obtained from clonal analysis of 56 RNA samples covering the complete spectrum of XCIP ratios. The values correlated closely with those obtained from the corresponding DNA samples analyzed using either PGK or HUMARA (r=0.98) provided that the number of amplification cycles was limited to 25 to reduce heteroduplex formation. However, sample purity is of greater importance than in the DNA-based assays as contaminating red cells and platelets will still contain the relevant transcripts and could influence the result.

Acquired skewing of X-chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age.

More frequent skewing of X-chromosome inactivation patterns (XCIPs) occurs in the white blood cells of elderly females; this study was performed to determine whether this occurs in myeloid or lymphoid lineages. XCIPs were analysed in purified neutrophils and T cells from 80 females > 75 years and the results were compared with 23 cord blood and 94 younger adult blood samples. The degree of XCIP skewing in cord blood and younger adult blood cells was similar, with 3-4% having > 90% expression of one allele. Skewing was markedly increased in the neutrophils of elderly females, with 33% having > 90% expression of one allele (P < 0.0001). Extreme skewing was present in only 9% of the elderly T-cell samples and no evidence of T-cell clonality was found by PCR analysis of the TCR gamma gene. The high level of acquired skewing of the XCIPs in myeloid cells of the elderly suggests that with time there is a change in stem cell usage with stochastic loss of some of the original stem cells. This has major implications for the use of XCIP analysis in the diagnosis of myeloid malignancies in the elderly and for gene therapy into haemopoietic stem cells.