Methylation-specific Multiplex Ligation-dependent Probe Amplification Assay Research Articles

Prospective study of epigenetic alterations responsible for isolated hemihyperplasia/hemihypoplasia and their association with leg length discrepancy

BackgroundHemihyperplasia and hemihypoplasia result in leg length discrepancy (LLD) by causing skeletal asymmetry. Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS) are opposite growth-affecting disorders caused by opposite epigenetic alterations at the same chromosomal locus, 11p15, to induce hemihyperplasia and hemihypoplasia, respectively. Because of their somatic mosaicism, BWS and SRS show a wide spectrum of clinical phenotypes. We evaluated the underlying epigenetic alterations and potential epigenotype-phenotype correlations, focusing on LLD, in a group of individuals with isolated hemihyperplasia/hemihypoplasia.ResultsWe prospectively collected paired blood-tissue samples from 30 patients with isolated hemihyperplasia/hemihypoplasia who underwent surgery for LLD. Methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) and bisulfite pyrosequencing for differentially methylated regions 1 and 2 (DMR1 and DMR2) on chromosome 11p15 were performed using the patient samples. Samples from patients showing no abnormalities in MS-MLPA or bisulfite pyrosequencing were analyzed by single nucleotide polymorphism (SNP) microarray and CDKN1C Sanger sequencing. We introduced a metric named as the methylation difference, defined as the difference in DNA methylation levels between DMR1 and DMR2. The correlation between the methylation difference and the predicted LLD at skeletal maturity, calculated using a multiplier method, was evaluated. Predicted LLD was standardized for stature. Ten patients (33%) showed epigenetic alterations in MS-MLPA and bisulfite pyrosequencing. Of these, six and four patients had epigenetic alterations related to BWS and SRS, respectively. The clinical diagnosis of hemihyperplasia/hemihypoplasia was not compatible with the epigenetic alterations in four of these ten patients. No patients showed abnormalities in SNP array or their CDKN1C sequences. The standardized predicted LLD was moderately correlated with the methylation difference using fat tissue (r = 0.53; p = 0.002) and skin tissue (r = 0.50; p = 0.005) in all patients.ConclusionsIsolated hemihyperplasia and hemihypoplasia can occur as a spectrum of BWS and SRS. Although the accurate differentiation between isolated hemihyperplasia and isolated hemihypoplasia is important in tumor surveillance planning, it is often difficult to clinically differentiate these two diseases without epigenetic tests. Epigenetic tests may play a role in the prediction of LLD, which would aid in treatment planning.

Germline promoter hypermethylation in BRCA1 and BRCA2 genes is not present in hereditary breast cancer patients.

Germline promoter hypermethylation of BRCA1 and BRCA2 genes is an alternative event of gene silencing that has not been widely investigated in hereditary breast and ovarian cancer (HBOC) syndrome. We analyzed germline BRCA promoter hypermethylation in HBOC patients with and without BRCA mutations and control subjects, using a recently developed BRCA methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Neither the patients tested nor the control subjects showed germline hypermethylation of the BRCA1 and BRCA2 promoter regions analyzed. Despite the results achieved at somatic levels by other researchers, these were not confirmed in our study at the germline level. Our results show the need to establish more predictive CpG sites in the BRCA promoter regions to optimize the MS-MLPA assay for the detection of germline hypermethylation as an effective pre-screening tool for whole-BRCA genetic analysis in HBOC, because we can not rule out the existence of germline promoter hypermethylation in BRCA.

Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors.

Aberrant methylation has been associated with transcriptional inactivation of tumor‐related genes in a wide spectrum of human neoplasms. The influence of DNA methylation in oligodendroglial tumors is not fully understood. Genomic DNA was isolated from 61 oligodendroglial tumors for analysis of methylation using methylation‐specific multiplex ligation‐dependent probe amplification assay (MS‐MLPA). We correlated methylation status with clinicopathological findings and outcome. The genes found to be most frequently methylated in oligodendroglial tumors were RASSF1A (80.3%), CASP8 (70.5%), and CDKN2A (52.5%). Kaplan–Meier survival curve analysis demonstrated longer duration of progression‐free survival in patients with 19q loss, aged less than 38 years, and with a proliferative index of less than 5%. Methylation of the ESR1 promoter is significantly associated with shorter duration of overall survival and progression‐free survival, and that methylation of IGSF4 and RASSF1A is significantly associated with shorter duration of progression‐free survival. However, none of the methylation status of ESR1, IGSF4, and RASSF1A was of prognostic value for survival in a multivariate Cox model. A number of novel and interesting epigenetic alterations were identified in this study. The findings highlight the importance of methylation profiles in oligodendroglial tumors and their possible involvement in tumorigenesis.

Methylation of tumor suppressor genes in a novel panel predicts clinical outcome in paraffin-embedded bladder tumors

DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n=14) and paraffin-embedded primary bladder tumors (n=61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p=0.026), TWIST1 (p=0.009), SFRP4 (p=0.002), and CCND2 (p=0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.

MS-MLPA analysis for FMR1 gene: evaluation in a routine diagnostic setting

BackgroundFragile X Syndrome (FXS), the most common cause of familiar mental retardation, is associated in over 99% of cases to an expansion over 200 repeats of a CGG sequence in the 5’ UTR of the FMR1 gene (Xq27.3), leading to the hypermethylation of the promoter. Molecular diagnosis of FXS have been so far based on the use of the Southern Blot (SB) analysis, a low throughput and time consuming technique. In order to update the diagnostic approach for FXS, we evaluated the usefulness of the Methylation-Specific Multiplex-Ligation-dependent Probe Amplification assay (MS-MLPA).MethodsThe study was carried out by retrospectively analysing 44 male patients, 10 Chorionic Villus Sampling (CVS) samples and 10 females previously analyzed by SB. In addition, a prospective study on 98 male subjects, 20 females and 1 CVS sample was carried out for assessing the feasibility and the impact of MS-MLPA in a routine lab work.ResultResults provided by both the retrospective and the prospective parts of this study strongly demonstrate the robustness and reproducibility of the MS-MLPA assay, able to correctly detect the methylation status in all normal and full mutation male samples analyzed, including CVS male samples. On the other hand, MS-MLPA analysis on females samples produced unreliable results.ConclusionBased on our results, we suggest the necessity of a separate workflow for male and female patients with suspected FXS in the routine diagnostic setting. MS-MLPA, in combination with CGG repeat sizing using a single-tube primed FMR1 PCR, represents a reliable diagnostic protocol in the molecular diagnosis of FXS male patients.

1875 METHYLATION OF TUMOR SUPPRESSOR GENES PREDICT RECURRENCE AND SUBCLASSIFY NON MUSCLE-INVASIVE DISEASE: PTA LOW GRADE VERSUS PT1 LOW GRADE AND PT1 HIGH GRADE BLADDER TUMORS

INTRODUCTION AND OBJECTIVES: Genetic and epigenetic factors are involved in tumorigenesis and bladder cancer progression. The role of epigenetic factors at distinguishing pathological and clinical subgroups is not well defined. We aimed at evaluating the role of methylation of 25 tumor suppressor genes (TSG) to subclassify nonmuscle invasive (NMI) bladder tumor subtypes and as clinical outcome prognosticators. METHODS: A retrospective design included paraffin-embedded tumors (n 251) of patients with primary NMI disease: pTa Low Grade (LG)(n 79), PT1LG(n 81), and PT1 High Grade (HG) (n 91). Methylation of 25 TSG was measured using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression into muscle invasive tumors and diseasespecific survival (DSS) rates were analyzed using competing risks regression analysis. RESULTS: The TSGs most frequently methylated in the overall series were STK11 (96.8%), MGMT2 (64.5%), RARB (63.0%) and GATA5 (63.0%). TSGs methylation significantly correlated to clinicopathological variables within each specific subgroup and the overall series. Methylation of RARB (p 0.027), CD44 (p 0.002), PAX5A (p 0.018), GSTP1 (p 0.001), IGSF4 (p 0.001), PYCARD (p 0.001), CDH13 (p 0.028), TP53 (p 0.001) and GATA5 (p 0.001) allowed classification of PTa versus PT1 tumors while RARB (p 0.001), CD44 (p 0.001), GSTP1 (p 0.001), IGSF4 (p 0.005), CHFR (p 0.032), PYCARD (p 0.001), TP53 (p 0.001), STK11 (p 0.028) and GATA5 (p 0.001) distinguished LG vs HG tumors. Multivariate analyses indicated that PAX5A (p 0.019), WT1 (p 0.042) and BRCA1 (p 0.023) methylation independently predicted recurrence in pTaLG lesions, PAX6 (p 0.044), ATM (p 0.004), CHFR (p 0.035) and RB1 (p 0.007) in pT1LG tumors, and PYCARD (p 0.035) in pT1HG disease, and PAX5A (p 0.01) and RB1 (p 0.008) in the overall NMI series. CONCLUSIONS: Methylation of TSG distinguished NMI bladder tumors depending on their clinicopathological phenotypes. Methylation of TSG correlated to patient outcome and behaved as independent prognosticators of recurrence. Methylation of TSG may select NMI patients who may require closer surveillance and more aggressive therapeutic interventions.

Genetic and epigenetic states of the GNAS complex in pseudohypoparathyroidism type Ib using methylation-specific multiplex ligation-dependent probe amplification assay

Pseudohypoparathyroidism type Ib (PHP-Ib) is a rare disorder resulting from genetic and epigenetic aberrations in the GNAS complex. PHP-Ib, usually defined by renal resistance to parathyroid hormone, is due to a maternal loss of GNAS exon A/B methylation and leads to decreased expression of the stimulatory G protein α (Gsα) in specific tissues. To clarify the usefulness of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), we evaluated genetic and epigenetic changes of the GNAS locus in Japanese PHP-Ib patients. Retrospective case series. We studied 13 subjects with PHP-Ib (three families with eight affected members and one unaffected member and four sporadic cases). The methylation status of GNAS differentially methylated regions (DMRs) was evaluated using MS-MLPA. The main outcome measure was the presence of deletion mutations in the GNAS locus and STX16, which were assessed using MLPA. In all familial PHP-Ib cases, a ~3 kb deletion of STX16 and demethylation of the A/B domain were identified. In contrast, no deletion was detected throughout the entire GNAS locus region in the sporadic cases. Broad methylation abnormalities were observed in the GNAS DMRs. MS-MLPA allows for precise and rapid analysis of the methylation status in GNAS DMRs as well as the detection of microdeletion mutations in PHP-Ib. Results confirm the previous findings in this disorder and demonstrate that this method is valuable for the genetic evaluation and visualizing the methylation status. The MS-MLPA assay is a useful tool that may facilitate making the molecular diagnosis of PHP-Ib.

A methylation assay for the detection of non‐muscle‐invasive bladder cancer (NMIBC) recurrences in voided urine

To develop a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay for the detection of non-muscle invasive bladder cancer (NMIBC) recurrences in voided urine. Genes frequently methylated in NMIBC tumours (n= 37) were selected to develop a BC-specific MS-MLPA assay. Genes methylated in blood from patients with BC (n= 29) and genes methylated in urine from patients with no history of BC (n= 46) were excluded. A four-gene panel with the highest predictive value was selected from the initial assay. This four-gene panel was tested and validated on urine from patients with a histologically confirmed recurrence (n= 68 test set; n= 49 validation set) and urine samples from patients without BC (n= 91, test set) and urine from recurrence-free BC (rec-free BC) patients (n= 60, validation set). A model was developed to predict the probability of having a recurrence based on methylation of the four-gene panel and a threshold probability with the highest sensitivity and specificity was determined. The outcome of the model was validated on BC urine samples (n= 65) and on urine samples from rec-free BC patients (n= 29). The BC MS-MLPA assay consisted of 23 methylation probes. The selected four-gene panel included: APC_a, TERT_a, TERT_b, and EDNRB. This panel reached an area under the receiver operating characteristic curve (AUC) of 0.82 (test set) and AUC 0.69 (validation set). Sensitivity and specificity for the detection of a concomitant tumour were 63.3% and 58.3% respectively (test set) and 72.3% and 55.2%, respectively (validation set). We have developed a methylation detection assay specifically for the detection of recurrences in patients with NMIBC in voided urine. The findings are promising and improvement of this test could eventually contribute to a more individualized patient friendly surveillance.

Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in lung cancer

BackgroundChanges in DNA methylation of crucial cancer genes including tumor suppressors can occur early in carcinogenesis, being potentially important early indicators of cancer. The objective of this study was to examine a multiplexed approach to assess the methylation of tumor suppressor genes as tumor stratification and clinical outcome prognostic biomarkers for lung cancer.MethodsA multicandidate probe panel interrogated DNA for aberrant methylation status in 18 tumor suppressor genes in lung cancer using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Lung cancer cell lines (n = 7), and primary lung tumors (n = 54) were examined using MS-MLPA.ResultsGenes frequently methylated in lung cancer cell lines including SCGB3A1, ID4, CCND2 were found among the most commonly methylated in the lung tumors analyzed. HLTF, BNIP3, H2AFX, CACNA1G, TGIF, ID4 and CACNA1A were identified as novel tumor suppressor candidates methylated in lung tumors. The most frequently methylated genes in lung tumors were SCGB3A1 and DLC1 (both 50.0%). Methylation rates for ID4, DCL1, BNIP3, H2AFX, CACNA1G and TIMP3 were significantly different between squamous and adenocarcinomas. Methylation of RUNX3, SCGB3A1, SFRP4, and DLC1 was significantly associated with the extent of the disease when comparing localized versus metastatic tumors. Moreover, methylation of HTLF, SFRP5 and TIMP3 were significantly associated with overall survival.ConclusionsMS-MLPA can be used for classification of certain types of lung tumors and clinical outcome prediction. This latter is clinically relevant by offering an adjunct strategy for the clinical management of lung cancer patients.

Abstract 4685: Multiplexed methylation profiles of urinary DNA for non-invasive bladder cancer diagnostics

Abstract Changes in DNA methylation of crucial cancer genes including tumor suppressors can occur early in carcinogenesis, being potentially important early indicators of cancer. The objective of this study was to examine a multiplexed approach to assess the methylation of tumor suppressor genes as urinary diagnostic markers for bladder cancer. A multicandidate probe panel interrogated DNA for aberrant methylation status in 25 tumor suppressor genes using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Initial analyses in bladder cancer cell lines (n=14), and fresh-frozen primary bladder tumor specimens (n=31) supported the panel of genes selected to be altered in bladder cancer. MS-MLPA was then optimized for its application in body fluids using two sets of urinary specimens (n=146) including bladder cancer patients (n=96) and controls (n=50). Genes previously reported to be methylated such as BRCA1, CDH13 and RARB, were confirmed to be methylated in the three types of samples analyzed. WT1 and PAX5A were identified as novel tumor suppressor candidates methylated in bladder cancer specimens. The most frequently methylated genes in the bladder tumors analyzed were BRCA1 (71.0%), WT1 (38.7%), and RARB (38.7%), being WT1 methylation significantly associated with tumor stage (p=0.011). BRCA1, WT1 and RARB were also as the most frequently methylated genes in two independent training and validation sets of urinary specimens (n=146). ROC curves analyses revealed significant diagnostic accuracies over 70% in both sets of urinary specimens for BRCA1, RARB, WT1, PAX5A and CDH13. In summary, the novelty of this report deals with applying a multiplexed methylation technique for bladder cancer, and also in body fluids. MS-MLPA represents a promising methylation profiling tool for histopathologic stratification of bladder tumors, and diagnosis using urinary samples. This latter is clinically relevant by offering a non-invasive strategy for the clinical management of patients affected with uroepithelial neoplasias Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4685.

Hypomethylation of Tumor Suppressor Genes in Acute Myeloid Leukemia: Characteristic of Cell Lines with MLL Abnormalities?.

Abstract 365▪▪This icon denotes an abstract that is clinically relevant. Background:Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines. Results:Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion:These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins. Disclosures:No relevant conflicts of interest to declare.

Hypomethylation and expression of BEX2, IGSF4 and TIMP3 indicative of MLL translocations in Acute Myeloid Leukemia

BackgroundTranslocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines.ResultsUsing MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLLmu AML cells, while 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu AML cell lines. MLLwt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene.ConclusionThese results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

Reply to Camprubí et al

Reply to Camprubí et al

MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a single-tube experiment

Human chromosome 11p15.5 harbours a large cluster of imprinted genes. Different epigenetic defects at this locus have been associated with both Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). Multiple techniques (Southern blotting, COBRA and microsatellite analysis) have been used so far to detect various DNA methylation abnormalities, uniparental disomies and copy number variations, which are characteristics of these two diseases. We have now evaluated a methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) for the molecular diagnosis of BWS and SRS. Seventy-three samples derived from BWS- and SRS-affected individuals and 20 controls were analysed by conventional tests and MS-MLPA in blind. All cases that were found positive with conventional methods were also identified by MS-MLPA. These included cases with paternal UPD11, hyper- or hypo-methylation at the Imprinting Centre 1 or Imprinting Centre 2 and rare 11p15.5 duplications. In summary, this MS-MLPA assay can detect both copy number variations and methylation defects of the 11p15.5 critical region within one single experiment and represents an easy, low cost and reliable system for the molecular diagnostics of BWS and SRS.

Use of multiplex ligation‐dependent probe amplification increases the detection rate for 11p15 epigenetic alterations in Silver–Russell syndrome

Silver-Russell syndrome (SRS) describes a malformation syndrome with severe intrauterine and postnatal growth retardation. Currently, two major (epi)mutations have been described: while approximately 10% of patients carry a maternal uniparental disomy of chromosome 7 (UPD7), 35-60% show a hypomethylation at the H19 differentially methylated regions (DMRs) in 11p15. Until recently, a Southern-blot based test was routinely used to identify epimutation carriers. Nevertheless, this test was time consuming and hampered by the huge amount of genomic DNA needed. With the methylation-specific multiplex ligation-dependent probe amplification assay (MLPA) for SRS, a PCR-based test is now available, allowing the analysis also of small amounts of DNA. Probes in this assay hybridize to the H19 DMRs but do not cover the genomic target of the Southern-blot probe. We now screened 72 patients with SRS by MLPA. Hypomethylation of the H19 DMRs was confirmed in all patients analyzed by Southern blot. In addition, we identified six individuals with hypomethylation of the H19 DMR who had previously normal blot results. This discrepancy can be explained by the observed generally lower degree of demethylation in this group, possibly not detectable by the less sensitive Southern-blot method but also with a varying degree of methylation at different DMRs in the same individual. Apart from hypomethylation in the H19 DMR, we observed a slight demethylation for one of the IGF2 probes. The total detection rate of 11p15 hypomethylation is now increased to >38%. Considering maternal UPD7 and chromosomal aberrations, (epi)genetic alterations now account for more than 50% of SRS patients. In summary, MLPA represents an easy, low cost and reliable system in the molecular diagnostics of SRS.

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) robustly detects and distinguishes 11p15 abnormalities associated with overgrowth and growth retardation

Background:A variety of abnormalities have been demonstrated at chromosome 11p15 in individuals with overgrowth and growth retardation. The identification of these abnormalities is clinically important but often technically difficult. Methylation-specific...