HUMARA Assay Research Articles

Role of Skewed X-Chromosome Inactivation in Common Variable Immunodeficiency.

The term common variable immunodeficiency (CVID) encompasses a clinically diverse group of disorders, mainly characterized by hypogammaglobulinemia, insufficient specific antibody production, and recurrent infections. The genetics of CVID is complex, and monogenic defects account for only a portion of cases, typically <30%. Other proposed mechanisms include digenic, oligogenic, or polygenic inheritance and epigenetic dysregulation. In this study, we aimed to assess the role of skewed X-chromosome inactivation (XCI) in CVID. Within our cohort of 131 genetically analyzed CVID patients, we selected female patients with rare variants in CVID-associated genes located on the X-chromosome. Four patients harboring heterozygous variants in BTK (n = 2), CD40LG (n = 1), and IKBKG (n = 1) were included in the study. We assessed XCI status using the HUMARA assay and an NGS-based method to quantify the expression of the 2 alleles in mRNA. Three of the 4 patients (75%) exhibited skewed XCI, and the mutated allele was predominantly expressed in all cases. Patient 1 harbored a hypomorphic variant in BTK (p.Tyr418His), patient 3 had a pathogenic variant in CD40LG (c.288+1G>A), and patient 4 had a hypomorphic variant in IKBKG (p.Glu57Lys) and a heterozygous splice variant in TNFRSF13B (TACI) (c.61+2T>A). Overall, the analysis of our cohort suggests that CVID in a small proportion of females (1.6% in our cohort) is caused by skewed XCI and highly penetrant gene variants on the X-chromosome. Additionally, skewed XCI may contribute to polygenic effects (3.3% in our cohort). These results indicate that skewed XCI may represent another piece in the complex puzzle of CVID genetics.

X chromosome inactivation skewing is common in advanced carotid atherosclerotic lesions in females and predicts secondary peripheral artery events

Background and aimSex differences in atherosclerosis have been described with female plaques being mostly perceived as stable and fibrous. Sex-specific mechanisms such as mosaic loss of the Y chromosome in men have been linked to cardiovascular health. In women, X-linked mechanisms such as X chromosome inactivation (XCI) skewing is common in several tissues. Yet, information on the role of XCI in female atherosclerotic plaques is lacking. Here, we investigated the presence of XCI skewing in advanced atherosclerotic lesions and its association with cardiovascular risk factors, histological plaque data, and clinical data.MethodsXCI skewing was quantified in 154 atherosclerotic plaque and 55 blood DNA samples of women included in the Athero-Express study. The skewing status was determined performing the HUMARA assay. Then, we studied the relationship of XCI skewing in female plaque and cardiovascular risk factors using regression models. In addition, we studied if plaque XCI predicted plaque composition, and adverse events during 3-years follow-up using Cox proportional hazard models.ResultsXCI skewing was detected in 76 of 154 (49.4%) plaques and in 27 of 55 (67%) blood samples. None of the clinical risk factors were associated with plaque skewing. Plaque skewing was more often detected in plaques with a plaque hemorrhage (OR [95% CI]: 1.44 [1.06–1.98], P = 0.02). Moreover, skewed plaques were not associated with a higher incidence of composite and major events but were specifically associated with peripheral artery events during a 3-year follow-up period in a multivariate model (HR [95%CI]: 1.46 [1.09–1.97]; P = 0.007).ConclusionsXCI skewing is common in carotid plaques of females and is predictive for the occurrence of peripheral artery events within 3 years after carotid endarterectomy.

X-chromosome inactivation patterns in females with Fabry disease examined by both ultra-deep RNA sequencing and methylation-dependent assay.

Fabry disease is an X-linked inherited lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A. Males are usually severely affected, while females have a wide range of disease severity. This variability has been assumed to be derived from organ-dependent skewed X-chromosome inactivation (XCI) patterns in each female patient. Previous studies examined this correlation using the classical methylation-dependent method; however, conflicting results were obtained. This study was established to ascertain the existence of skewed XCI in nine females with heterozygous pathogenic variants in the GLA gene and its relationship to the phenotypes. We present five female patients from one family and four individual female patients with Fabry disease. In all cases, heterozygous pathogenic variants in the GLA gene were detected. The X-chromosome inactivation patterns in peripheral blood leukocytes and cells of urine sediment were determined by both classical methylation-dependent HUMARA assay and ultra-deep RNA sequencing. Fabry Stabilization Index was used to determine the clinical severity. Skewed XCI resulting in predominant inactivation of the normal allele was observed only in one individual case with low ⍺-galactosidase A activity. In the remaining cases, no skewing was observed, even in the case with the highest total severity score (99.2%). We conclude that skewed XCI could not explain the severity of female Fabry disease and is not the main factor in the onset of various clinical symptoms in females with Fabry disease.

Skewness of X-chromosome inactivation increases with age and varies across birth cohorts in elderly Danish women

Mosaicism in blood varies with age, and cross-sectional studies indicate that for women, skewness of X-chromosomal mosaicism increases with age. This pattern could, however, also be due to less X-inactivation in more recent birth cohorts. Skewed X-chromosome inactivation was here measured longitudinally by the HUMARA assay in 67 septuagenarian and octogenarian women assessed at 2 time points, 10 years apart, and in 10 centenarian women assessed at 2 time points, 2–7 years apart. Skewed X-chromosome inactivation was also compared in 293 age-matched septuagenarian twins born in 1917–1923 and 1931–1937, and 212 centenarians born in 1895, 1905 and 1915. The longitudinal study of septuagenarians and octogenarians revealed that 16% (95% CI 7–29%) of the women developed skewed X-inactivation over a 10-year period. In the cross-sectional across-birth cohort study, the earlier-born septuagenarian (1917–1923) and centenarian women (1895) had a higher degree of skewness than the respective recent age-matched birth cohorts, which indicates that the women in the more recent cohorts, after the age of 70, had not only changed degree of skewness with age, they had also undergone less age-related hematopoietic sub-clone expansion. This may be a result of improved living conditions and better medical treatment in the more recent birth cohorts.

Genetic analysis for carrier diagnosis in hemophilia A and B in the Mexican population: 25 years of experience.

Our 25 years of experience in carrier diagnosis of hemophilia A (HA) and B (HB) in Mexican population comprises linkage analysis of intragenic F8/F9 neutral variants along with, in severe HA (SHA), detection of F8 int22h and int1h inversions. In symptomatic carriers (SCs) we explored Lyonization to explain their symtomatology. From a DNA-Bank of 3,000 samples, intragenic restriction fragment length (RFLPs) and short tandem repeats (STRs) of F8/F9 genes were assessed by PCR-PAGE and GeneScan. In SHA patients, F8 inversions were detected by inverse shifting-PCR/diagnostic and complementary tests. In SCs, we evaluated hemorrhagic symptoms, clotting FVIII/FIX and X-chromosome inactivation (XCI) patterns were assessed by HUMARA assay and the search of XIST promoter pathogenic variants. Informativeness of linkage analysis for HA carrier diagnosis with RFLP's/STR's increased to 74% and reached 80% with five RFLPs for HB. Combined Inv22/Inv1 diagnosed 113 possible carriers, three de novo Inv22-1, and confirmed 45 mothers as obligate or sporadic carriers. Among 21 SCs, four showed extreme skewed XCI pattern (~80:20) but had normal karyotype and no C43G pathogenic variant in XIST promoter. Clotting FVIII/FIX correlated with the active X in leukocytes. Our data integrate the largest comprehensive research worldwide on the molecular diagnosis of HA and HB carriers in terms of the number of studied and diagnosed cases, in addition to the genetic analysis in SCs. Intragenic RFLPs and STRs of F8/F9 genes along with F8 int22h/int1h inversions in SHA emerge as optimal variants for molecular diagnosis in Mexican population. In counseling SCs, inheritance of skewed X-inactivation should be considered.

Molecular analysis of low-level mosaicism of the IKBKG mutation using the X Chromosome Inactivation pattern in Incontinentia Pigmenti.

BackgroundIncontinentia pigmenti (IP) is a rare X‐linked disorder affecting the skin and other ectodermal tissues that is caused by mutation of the IKBKG/NEMO gene. Previous studies have reported that the overall mutation detection rate in IP is ~75%. We hypothesized that a low‐level mosaicism existed in the remaining cases.MethodsGenomic variations in the IKBKG gene were examined in 30 IP probands and their family members. Standard mutational analyses were performed to detect common deletions, nucleotide alterations, and copy number variations. To assess skewing of the X chromosome inactivation (XCI) pattern, a HUMARA assay was performed. We compared the results of this analysis with phenotype severity.ResultsPathogenic variants were identified in 20 probands (66.7%), the rate of detection was suboptimal. The remaining 10 probands tended to manifest a mild phenotype with no skewed X chromosome inactivation that is generally observed in IP patients. Quantitative nested PCR and digital droplet PCR were performed for the 10 patients and mosaicism of the common IKBKG deletion were identified in five patients.ConclusionOverall, we detected 25 IKBKG mutations (83.3%). Determination of the XCI value in advance of mutational analyses for IP could improve the mutation detection rate. Our improved detection rate for these mutations, particularly those with a low‐level mosaicism, may present opportunities for appropriate genetic counseling.

X Chromosome inactivation: a modifier of factor VIII and IX plasma levels and bleeding phenotype in Haemophilia carriers.

Haemophilia A and B are X-linked hemorrhagic disorders caused by gene variants in the F8 and F9 genes. Due to recessive inheritance, males are affected, while female carriers are usually asymptomatic with a wide range of factor VIII (FVIII) or IX (FIX) levels. Bleeding tendency in female carriers is extremely variable and may be associated with low clotting factor levels. This could be explained by F8 or F9 genetic variations, numerical or structural X chromosomal anomalies, or epigenetic variations such as irregular X chromosome inactivation (XCI). The aim of the study was to determine whether low FVIII or FIX coagulant activity in haemophilia carriers could be related to XCI and bleeding symptoms. HUMARA assay was performed on 73 symptomatic carriers with low clotting activity ≤50 IU/dL. Bleeding Assessment Tool (BAT) from the International Society on Thrombosis and Haemostasis (ISTH) was used to describe symptoms in the cohort of carriers. In 97% of haemophilia carriers, a specific gene variant in heterozygous state was found, which alone could not justify their low FVIII or FIX levels (≤50 IU/dL). A statistical association between XCI pattern and FVIII and FIX levels was observed. Moreover, female carriers with low coagulant activity (≤20 IU/dL) and high degree of XCI ( ≥ 80:20) had a higher ISTH-BAT score than the carriers with the opposite conditions (>20 IU/dL and <80:20). In our cohort of haemophilia carriers, XCI was significantly skewed, which may contribute to the low expression of clotting factor levels and bleeding symptoms.

SUN-137 Clonal Status of Multigland Disease Primary Hyperparathyroidism

Primary hyperparathyroidism (PHPT) is a common endocrine disorder that arises due to single or multiple parathyroid gland disease (MGD). The molecular mechanism(s) of parathyroid neoplasia are incompletely understood and both monoclonal (mono-X) and polyclonal (poly-X) parathyroid tumors have been described using methylation-sensitive PCR of X-linked Human Androgen Receptor (HUMARA) alleles. Our previous investigations of parathyroid tumor clonal status has shown that poly-X tumors are common and are associated with MGD in patients with non-familial PHPT (Shi et al. 2014 & 2018). This work examined the clonal status of the dominant gland and the clonal relationship of multiple tumors from the same patient has not been examined. The goal of the current study was to determine the clonal relationship of parathyroid tumors from PHPT patients with MGD. Banked parathyroid tissues from twenty-nine PHPT patients with MGD were examined in this study. Clonal status (mono-X vs poly-X) of multiple abnormal parathyroid glands from each patient was determined using a modification of the HUMARA assay used in our prior work. Briefly, methylation-sensitive PCR of HUMARA alleles was performed followed by fragment analysis using Capillary-Electrophoresis performed. Raw fragment sizing data analyzed using Peak Scanner software. Classification of samples as either mon-X or poly-X was made as described in (Shattuck et al.) Of 29 PHPT patients with MGD, 13 (45%) had pure mono-X, 5 (17%) had pure poly-X, and 11 (38%) had a mixture of mono-X and poly-X tumors. Five of 29 patients had three or more abnormal glands evaluated: 3 had mixture of poly-X and mono-X, 2 had pure mono-X tumors, and none were pure polyclonal-X. Eighteen (62%) out of 29 patients had paired upper or lower double adenomas. Of these, 9 (50%) were pure mono-X, 4 were pure poly-X, and 5 were mixed mono-X/poly-X. In 2 patients with multiple mono-X tumors, allele distribution was not the same in different abnormal glands. Our previous work has demonstrated that among patients with non-familial PHPT, poly-X parathyroid tumors are common and are associated with MGD. Our new data extend these findings to show that the clonal relationship between multiple parathyroid tumors from the same patient is complex and may reflect the emergence of single or multiple tumors from a background of parathyroid hyperplasia, or other mechanism(s). Future studies to explore the mechanisms behind these apparent clonal relationships are warranted and ongoing. Reference: (1) Shi et al., PNAS 2014, 201319742. (2) Shi et al., Surgery 2018, 9-14. (3) Shattuck. N Engl J Med 2005, 2406-12.

A female patient with retinoblastoma and severe intellectual disability carrying an X;13 balanced translocation without rearrangement in the RB1 gene: a case report

BackgroundFemale carriers of a balanced X; autosome translocation generally undergo selective inactivation of the normal X chromosome. This is because inactivation of critical genes within the autosomal region of the derivative translocation chromosome would compromise cellular function. We here report a female patient with bilateral retinoblastoma and a severe intellectual disability who carries a reciprocal X-autosomal translocation.Case presentationCytogenetic and molecular analyses, a HUMARA (Human androgen receptor) assay, and methylation specific PCR (MSP) and bisulfite sequencing were performed using peripheral blood samples from the patient. The patient’s karyotype was 46,X,t(X;13)(q28;q14.1) by G-banding analysis. Further cytogenetic analysis located the entire RB1 gene and its regulatory region on der(X) with no translocation disruption. The X-inactivation pattern in the peripheral blood was highly skewed but not completely selected. MSP and deep sequencing of bisulfite-treated DNA revealed that an extensive 13q region, including the RB1 promoter, was unusually methylated in a subset of cells.ConclusionsThe der(X) region harboring the RB1 gene was inactivated in a subset of somatic cells, including the retinal cells, in the patient subject which acted as the first hit in the development of her retinoblastoma. In addition, the patient’s intellectual disability may be attributable to the inactivation of the der(X), leading to a 13q deletion syndrome-like phenotype, or to an active X-linked gene on der (13) leading to Xq28 functional disomy.

EP.62A series of unfortunate events: familial case of DMD, two different mutational events and skewed X chromosome inactivation in a pregnant woman

Duchenne muscular dystrophy (DMD) is a neuromuscular X-linked recessive disease caused by mutations in DMD gene. Here, we present a family with a DMD symptomatic pregnant woman and two affected boys. One of them had a previous multiplex PCR study showing a 45-54 exon deletion. Interestingly, during the prenatal diagnosis another mutation was discovered in the pregnant woman and her fetus, a 38-43 exon duplication. On the basis of this finding, we reanalyzed the initially studied boy, discovering both mutations in his gDNA. In order to unravel this riddle, we performed a complete molecular analysis in family members, applying the following techniques: MLPA, STRs segregation, Humara Assay, CGH Array, Sanger sequencing and WGS. As expected, the Humara assay revealed that the symptomatic female has skewed X-chromosome Inactivation (XCI), while an asymptomatic carrier showed a random XCI. Given the inheritance pattern of the rearrangements, only the affected child carried the del/dup, we could deduce that the deletion was the second mutational event. Furthermore, STRs segregation allowed the detection of a recombinant event in the affected boy, which could be related to the generation of the deletion. We were able to characterize the deletion breakpoints NC_000023.10:g.31664475_32111223del, suggesting the involvement of the non-homologous end joining mechanism. On the other hand, we limited the borders of the duplication up to NC_000023.10: g.(32245444_32247193)_(32380996_32385390)dup by CGH array. However, a thorough characterization of the duplication is currently being done. The profound analysis of complex structural variants, such as the presented one, would allow to detect predisposing mutagenic sequences and widen the understanding on the molecular events that takes place in DMD gene. Finally, this study highlights the importance of retesting patients with identified deletions by PCR, in order to reduce the probability of missing out other rearrangements which could affect the effectiveness of mutation-dependent therapies.

Tenosynovitis With Psammomatous Calcifications: A Distinctive Trauma-Associated Subtype of Idiopathic Calcifying Tenosynovitis With a Predilection for the Distal extremities of Middle-Aged Women-A Report of 23 Cases.

The term "idiopathic calcifying tenosynovitis" (ICT) refers to a clinically and radiologically defined syndrome of pain and tendinous calcifications, most often involving the shoulder joint. A distinctive subset of ICT cases, termed "tenosynovitis with psammomatous calcifications" (TPC), occurs in the distal extremities and shows characteristic morphology, in particular psammomatous calcifications. As only 14 cases have been reported to date, TPC remains poorly recognized by both pathologists and clinicians. Twenty-three well-characterized cases of TPC along with all available radiologic and clinical information, including follow-up, were collected. Cases occurred in 21 females and 1 male (1 patient of unknown sex), aged 16 to 75 years (mean: 41), and almost exclusively involved the fingers and toes, except for one case in the elbow and one in the knee joint. The lesions ranged from 2 to 30 mm in size (mean: 10 mm). Pain was the most common presenting symptom (12/16 patients). A history of trauma or repetitive activity was present in 6 of 15 patients. None of the individuals was known to have disorders in calcium or phosphate metabolism. Radiographic studies showed a nonspecific, calcified mass. Typical morphologic features of TPC were invariably present, with degenerating tendinous tissue containing psammomatous calcifications, surrounded by a variably cellular, CD68/CD163/CD4-positive histiocyte-rich granulomatous host reaction. HUMARA assay in one case showed a polyclonal pattern. Clinical follow-up (19 patients; mean: 5.2 y; range: 1 to 14 y) showed no local recurrences. In this, the largest study of TPC to date, we confirm striking predilection of this distinctive pseudoneoplasm for the fingers and toes of young to middle-aged women. TPC should be rigorously distinguished from other forms of ICT, which typically involve large, proximal joints, and show simply dystrophic calcification involving tendinous tissues, and from tumoral calcinosis, which also involves large joints and often is associated with calcium and/or phosphate abnormalities. TPC appears to be related to trauma and/or repetitive activity and is cured with simple excision.

Clonal Hematopoiesis in Older Women with Extremely Skewed, Non-Random X-Inactivation Is Associated with Previous Cardiovascular and Cerebrovascular Events

Clonal Hematopoiesis in Older Women with Extremely Skewed, Non-Random X-Inactivation Is Associated with Previous Cardiovascular and Cerebrovascular Events

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.

Newly Described Entities in Salivary Gland Pathology.

Salivary glands may give rise to a wide spectrum of different tumors. This review concentrates on 4 salivary gland tumors that have been accepted in the recent literature as new neoplastic entities: mammary analog secretory carcinoma, cribriform adenocarcinoma of minor salivary glands (CASG), sclerosing polycystic adenosis/adenoma (SPA), and the mucinous/secretory variant of myoepithelioma. Mammary analog secretory carcinoma is a distinctive low-grade malignant salivary cancer that harbors a characteristic chromosomal translocation, t(12;15) (p13;q25), resulting in an ETV6-NTRK3 fusion. Cribriform adenocarcinoma (CASG) is a distinct tumor entity that differs from polymorphous low-grade adenocarcinoma by location (ie, most often arising on the tongue), by prominent nuclear clearing, differing alterations of the PRKD gene family, and clinical behavior with frequent metastases at the time of presentation of the primary tumor. Early nodal metastatic disease is seen in most cases of CASG; yet, they are still associated with indolent clinical behavior, making it a unique neoplasm among all low-grade salivary gland tumors. SPA is a rare sclerosing tumor of the salivary glands characterized by the combination of cystic ductal structures with variable cell lining including vacuolated, apocrine, mucinous, squamous, and foamy cells, by prominent large acinar cells with coarse eosinophilic cytoplasmic zymogen-like granules, and by closely packed ductal structures, surrounded by a peripheral myoepithelial layer and stromal fibrosis with focal inflammatory infiltrates. SPA frequently harbors intraductal epithelial dysplastic proliferations ranging from mild dysplasia to severe dysplasia/carcinoma in situ. Moreover, SPA has been proven to be a clonal process by HUMARA assay and is associated with considerable risk of recurrence. Therefore, on the basis of all these newly recognized findings, we believe that SPA is likely a neoplasm, and we suggest the name "sclerosing polycystic adenoma." The mucinous variant of myoepithelioma is a myoepithelial tumor with foci of prominent cytoplasmic clearing frequently containing intracellular mucin material and having signet-ring morphology.

Clonal hematopoiesis as determined by the HUMARA assay is a marker for acquired mutations in epigenetic regulators in older women.

Recent large cohort studies revealed that healthy older individuals harbor somatic mutations that increase their risk for hematologic malignancy and all-cause cardiovascular deaths. The majority of these mutations are in chromatin and epigenetic regulatory genes (CERGs). CERGs play a key role in regulation of DNA methylation (DNMT3A and TET2) and histone function (ASXL1) and in clonal proliferation of hematopoietic stem cells. We hypothesize that older women manifesting clonal hematopoiesis, defined here as a functional phenomenon in which a hematopoietic stem cell has acquired a survival and proliferative advantage, harbor a higher frequency of somatic mutations in CERGs. The human androgen receptor gene (HUMARA) assay was used in our study to detect the presence of nonrandom X inactivation in women, a marker for clonal hematopoiesis. In our pilot study, we tested 127 blood samples from women ≥65years old without a history of invasive cancer or hematologic malignancies. Applying stringent qualitative criteria, we found that 26% displayed clonal hematopoiesis; 52.8% displayed polyclonal hematopoiesis; and 21.3% had indeterminate patterns (too close to call by qualitative assessment). Using Illumina MiSeq next-generation sequencing, we identified somatic mutations in CERGs in 15.2% of subjects displaying clonal hematopoiesis (three ASXL1 and two DNMT3A mutations with an average variant allele frequency of 15.7%, range: 6.3%-23.3%). In a more limited sequencing analysis, we evaluated the frequency of ASXL1 mutations by Sanger sequencing and found mutations in 9.7% of the clonal samples and 0% of the polyclonal samples. By comparing several recent studies (with some caveats as described), we determined the fold enrichment of detecting CERG mutations by using the HUMARA assay as a functional screen for clonal hematopoiesis. We conclude that a functional assay of clonal hematopoiesis is enriching for older women with somatic mutations in CERGs, particularly for ASXL1 and TET2 mutations and less so for DNMT3A mutations.

Clonal Hematopoiesis in AML Patients in Hematological CR Is Present in Many Patients with Intermediate Risk AML and Is Associated with a High Prevalence of DNMT3A gene Mutations

Background De novo acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells usually characterized by a rapid clinical onset. Despite this clinical manifestation, recent evidence suggests that premalignant stem cells might be present in patients with AML, which can persist after chemotherapy in some patients and induce clonal hematopoiesis. Little is known about the prevalence of clonal persistence and about the molecular basis. In order to study the prevalence of this phenomenon and to better understand the underlying molecular mechanisms, we investigated AML patients in remission for clonal persistence of cells after chemotherapy.Patients and methods: All patients included in this analysis were treated within a prospective treatment protocol of the Study Alliance Leukemia (SAL). The primary study cohort consisted of 61 female patients with intermediate risk cytogenetics achieving hematologic complete remission (CR), whose DNA material was available at CR. Clonality analysis was based on X-chromosome inactivation testing using the HUMARA assay. DNA from diagnostic samples of patients presenting with evidence for X-chromosome skewing in CR was analyzed using amplicon based resequencing on a MiSeq next generation sequencing (NGS)-system for DNMT3A, ASXL1, ASXL2, TET1, TET2, EZH1, EZH2, IDH1 and IDH2.Results: Of the 61 patients included, 52 were heterozygous for the STR in the human androgen receptor gene. In CR, 22 of these 52 patients (42%) showed evidence for a skewed X-chromosome representation, indicating persistence of clonal hematopoiesis in remission. The NGS-based analysis of genes involved in epigenetic regulation revealed mutations in 13/22 (59%) of the patients. DNMT3A was most frequently mutated (11/13 patients), either alone or in combination with other alterations (TET2, EZH2). Interestingly, two patients showed somatic alterations in the TET1 gene. In remission, clonal persistence of these alterations was detected in all 13 patients with mutations at diagnosis at levels between 0.8 and 50% as documented using ultradeep-NGS. To get an idea on the prevalence of clonal persistence in other cytogenetic groups, we analyzed 22 low risk (i.e. CBF-leukemias) as well as 18 poor risk (-7, complex karyotype) patients using the HUMARA assay. Here we observed similar results, with 13/19 informative patients showing clonal persistence in low-risk group (68%) compared to 7/14 patients (50%) in the poor risk population. Since all these analyses were confined to female patients and potentially limited by the sensitivity of the HUMARA method, we went on to look for persistence of clonal molecular markers using more sensitive ultra-deep NGS. Because DNMT3A exon 23 was the common alteration in this initial analysis, we screened a cohort of 48 patients with mutations in NPM1 and comutations in DNMT3A. In this separate cohort, persistence of the DNMT3A mutations at CR or during follow-up (FU) was detected in 42 patients (87.5%) at levels between 0.5 and 50% (median 11.1%). No difference was seen between male and female patients, the median age was 51 years, persistence was seen even in young patients at 26 years of age. During FU, the DNMT3A VAF level rose further in all patients analyzed, arguing for a clonal advantage of the mutant cells. All patients with relapse and available material showed high levels of DNMT3A at time of relapse. However, correlation of DNMT3A mutant allele levels at CR1 with the incidence of relapse showed no significant impact of the VAF for the development of relapse.Conclusions: Our data indicate that clonal persistence of premalignant cells carrying clonal alterations in epigenetic regulator genes is a common phenomenon in patients in continuous CR. DNMT3A is the most common lesion persisting, the majority of patients with this mutations retain it at CR and during FU. These data indicate that de novo AML develops from preleukemic stem and progenitor cells in many patients. Preliminary data indicate that this persistence per se is not associated with inferior outcome. DisclosuresSchuster:AgenDix GmbH: Employment. Thiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

Evaluation of clonal origin of malignant mesothelioma.

BackgroundThe hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy.Methods and resultsTo investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM.ConclusionsOur results show that MM originate as polyclonal tumors and suggest that the carcinogenic “field effect” of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0301-3) contains supplementary material, which is available to authorized users.

Identification of novel informative loci for DNA-based X-inactivation analysis

The HUMARA assay, the most common method for evaluation of X-inactivation skewing in blood cells, has been reported to be usable in only about 80% of females, emphasizing the need for alternative methods for testing of HUMARA-uninformative individuals. We conducted an in silico search for potentially polymorphic tri-to-hexanucleotide repeats in the proximity of CpG islands located in 5' regions of X-chromosome genes to design five candidate assays (numbered I, II, III, IV, and V) combining methylation-specific restriction digest with PCR amplification in a manner similar to the HUMARA assay. The results obtained by these assays in 100 healthy females were compared to X-inactivation skewing measured by the AR-MSP method which is based on methylation-specific PCR amplification of the first exon of the AR gene. On the basis of statistical evidence, three of the novel assays (II, IV, and V), which were informative in 18%, 61%, and 55% of females in the cohort, respectively, may be used as alternatives or conjointly with the HUMARA assay to improve its reliability. The three new assays were combined with the HUMARA assay into a novel X-inactivation test leading to the increase of informative females in the cohort from 67% to 96%.

Abstract 3188: Evaluation of clonal origin of malignant mesothelioma/polyclonal origin of malignant mesothelioma

Abstract Tumors are generally thought to be monoclonal in origin, although this paradigm stemmed decades ago mostly from the study of hematopoietic malignancies and sarcomas. The clonal origin of malignant mesothelioma, a deadly cancer resistant to the current therapies, has never been investigated before. We used the HUMARA (human androgen receptor) assay to examine the pattern of clonality in 14 sporadic and 2 familial Malignant Mesotheliomas (MM). Of 16 specimens studied, 14 were informative and nearly all (13/14) revealed two electrophoretically distinct methylated HUMARA alleles, indicating a polyclonal origin for these tumors. This discovery has important clinical implications, in that an accurate assessment of tumor clonality is key to the design of novel molecular strategies for the treatment of MM. Citation Format: Sabahattin Comertpay, Rosanna Mezzapelle, Mika Tanji, Oriana Strianese, Harvey I. Pass, Tracey Weigel, Joseph Friedberg, Paul Sugarbaker, Thomas Krausz, Ena Wang, Giovanni Gaudino, Haining Yang, Amy Powers, Barbara Parsons, Sandra Pastorino, Michele Carbone. Evaluation of clonal origin of malignant mesothelioma/polyclonal origin of malignant mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2014-3188

5meCpG epigenetic marks neighboring a primate-conserved core promoter short tandem repeat indicate X-chromosome inactivation.

X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.