Multiplex Ligation-dependent Probe Amplification Kit Research Articles

First genetic characteristics of Polish patients with idiopathic pulmonary arterial hypertension

Abstract Introduction Pulmonary arterial hypertension (PAH) is a rare, severe disorder of multifactorial origin. Genetic alterations in BMP/SMAD pathways were previously associated with disease development, however the exact role of other genetic factors from BMP/SMAD signalling is still unclear. Purpose We aimed to search for known PAH-associated mutations and potential novel variants responsible for disease onset in Polish PAH patients. Methods We prospectively recruited 93 consecutive idiopathic PAH patients from a single pulmonary hypertension reference centre between years 2009 and 2020. Eligible patients had pre-capillary pulmonary hypertension with pulmonary vascular resistance >3 Wood units in the absence of other causes of pre-capillary pulmonary hypertension. The presence of large gene rearrangements was analyzed by the Multiplex Ligation-dependent Probe Amplification (MLPA) reactions in the ENG, ACVRL1 and BMPR2 genes (SALSA MLPA Kit, MRC-Holland). We have further sequenced a panel of selected 48 genes from BMP/SMAD and related pathways using next-generation sequencing (SureSelect XT library preparation Kit, Agilent; NextSeq 500 sequencer, Illumina) and assessed causative potential of rare variants (minor allele frequency in non-finish Europeans below 1%) with Mutation Taster web-based tool. Results We identified at least one likely-causative variant of investigated genes in 51 (54.8%) subjects. Large genomic rearrangements were found by MLPA in 2 patients in ENG and ACVRL1 (ALK1) genes. We have found mutations in BMPR2 gene in 12 patients (12.5%) from our cohort with most variants present in one sample and 3 variants present in 2 samples. Mutations were located mostly in protein kinase domain (6 variants) and Activin type I and II receptor domain (2 variants). We have found 5 likely pathogenic variants in 6 patients in genes previously associated with PAH other than BMPR2 (SMAD9, KCNA5, KCNK3, EIF2AK4). In total, potentially disease causing mutations in literature-known genes, were present in 18 patients, which accounted for 18.75% of tested cohort. Medium impact variants were also present in KCNA5, TGFBR2, AQP7, BMP6, EIF2AK4, FBP1, NOTCH1, NOTCH3, SMAD7, TBX4, TGFB2, TOPBP1, GDF5, IL6, PPARA, RXRA, KCNK3, KLF4, BMPR1B, ILK, TGFBR1, SMAD9, PPARD and TGFB3 genes. Additionally, in one patient with clinical diagnosis of pulmonary venoocclusive disease we identified an unknown homozygous frameshift variant (p.Phe1523fs/c.4567_4570delTTTG) in exon 35 of EIF2AK4 gene. Conclusions We found potentially disease causing mutations in 18,75% Polish patients diagnosed with IPAH most of them were present in BMPR2 gene which is in line with data from other European cohorts. Additionally we found an unknown mutation in the EIF2AK4 gene. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Narodowe Centrum Nauki - National Science Centre

RAS pathway mutation is an added-value biomarker in pediatric Philadelphia-negative B-cell acute lymphoblastic leukemia with IKZF1 deletions.

IKZF1deletion is an unfavorable factor in Philadelphia negative (Ph -) B-cell acute lymphoblastic leukemia. However, the effects ofIKZF1deletions co-existing genetic alterations in Ph (-) ALL have not been extensively studied. Bone marrow samples from 368 children with Ph (-) ALL were analyzed by using multiplex ligation-dependent probe amplification kit for detection of gene deletions and Sanger sequencing for mutational analysis of RAS pathway genes. The outcome was analyzed on 215 patients treated with Taiwan Pediatric Oncology Group-ALL-2002 protocol. IKZF1 deletions were present in 12.8% and IKZF1plus in 6.3% of patients. Mutations of RAS pathway genes were detected in 25.0% of IKZF1-deleted patients. The 10-year event-free survival (EFS) of IKZF1-undeleted patients was significantly better compared with IKZF1-deleted patients (80.0%vs. 47.8%, p=0.001). Compared with outcome of patients harboring IKZF1 deletion alone, no difference in EFS was observed in patients with IKZF1plus , whereas three patients carried both IKZF1 and ERG deletions had a superior 10-year EFS (100%). The 10-year EFS of patients with any gene mutation of RAS pathway was worse than that of patients with wild-type genes (79.1%vs. 61.6%, p=0.033). In multivariate analysis, RAS pathway mutations and IKZF1 deletion were independent predictors of inferior EFS. Co-existence of IKZF1 deletion with RAS pathway mutations had a worst 10-year EFS (11.1 ± 10.5%) and 10-year OS (53.3 ± 17.6%). Our results showed that RAS pathway mutation is an added-value biomarker in pediatric IKZF1-deleted Ph (-) ALL patients.

Assessment of Multiplex Ligation-Dependent Probe Amplification (MLPA) as a diagnostic test for Egyptian patients with Williams-Beuren syndrome

BackgroundWilliams-Beuren syndrome (WS) is as a rare multisystem genetic syndrome, caused by the hemizygous deletion of approximately 26–28 genes on chromosome 7q11.23. Fluorescence in-situ hybridization (FISH) is considered the gold standard for its diagnosis. However, FISH does not allow the detection of the exact size of the deletion. This study aimed to compare the efficiency and validity of Multiplex Ligation-Dependent Probe Amplification (MLPA) and (FISH) techniques in diagnosing and confirming clinically diagnosed WS. Patients and methodsThe study comprised twenty-three WS patients they were clinically assessed, cardiovascular evaluation, and laboratory tests, including plasma calcium level, thyroid function tests and glucose tolerance test, were performed. FISH and MLPA analysis were performed for all patients. The MLPA kit (SALSA P029) contained probes for eight genes in the WS critical region: FKBP6, FZD9, TBL2, STX1A, ELN, LIMK1, RFC2, and CYLN2. ResultsNineteen patients out of 23 studied cases (82.6%) showed positive deletion of the WS chromosome region (WBSCR) by both FISH and MLPA techniques. One patient (4.3%) showed negative result by FISH while a duplication of the WBSCR was detected by MLPA. Three patients (13.1%) were negative for both FISH and MLPA. In conclusionThis is the largest cohort of Egyptian patients with WS, where both FISH and MLPA analysis were used to confirm the diagnosis. MLPA analysis has an advantage over FISH as it is less time consuming and detects smaller, atypical deletions and duplications in the WS.

A Multicentric Brazilian Investigative Study of Copy Number Variations in Patients with Congenital Anomalies and Intellectual Disability

Genomic imbalances are the most common cause of congenital anomalies (CA) and intellectual disability (ID). The aims of this study were to identify copy number variations (CNVs) in 416 patients with CA and ID from 5 different genetics centers within 4 different states by using the Multiplex Ligation-dependent Probe Amplification (MLPA) technique and to apply the chromosomal microarray (CMA) methodology in selected cases. The samples were analyzed by MLPA kits P064, P036, P070 and P250. Positive results were found in 97/416 (23.3%) patients. CMA was applied in 14 selected cases. In 6/14 (42.85%) patients, CMA detected other copy number variations not detected by the MLPA studies. Although CMA is indispensable for genotype refinement, the technique is still unfeasible in some countries as a routine analysis due to economic and technical limitations. In these cases, clinical evaluation followed by karyotyping and MLPA analysis is a helpful and affordable solution for diagnostic purposes.

Investigation of major genetic alterations in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. This malignancy shows a wide spectrum of clinical outcome and its prognosis is conditioned by manifold biological and genetic factors. We investigated the tumor genetic profile and clinical data of 29 patients with NB by multiplex ligation-dependent probe amplification (MLPA) to assess therapeutic risk. In 18 of these tumors, MYCN status was assessed by fluorescence in situ hybridization (FISH). Copy number variation was also determined for confirming MLPA findings in two 6p loci. We found 2p, 7q and 17q gains, and 1p and 11q losses as the most frequent chromosome alterations in this cohort. FISH confirmed all cases of MYCN amplification detected by MLPA. In view of unexpected 6p imbalance, copy number variation of two 6p loci was assessed for validating MLPA findings. Based on clinical data and genetic profiles, patients were stratified in pretreatment risk groups according to international consensus. MLPA proved to be effective for detecting multiple genetic alterations in all chromosome regions as requested by the International Neuroblastoma Risk Group (INRG) for therapeutic stratification. Moreover, this technique proved to be cost effective, reliable, only requiring standard PCR equipment, and attractive for routine analysis. However, the observed 6p imbalances made PKHD1 and DCDC2 inadequate for control loci. This must be considered when designing commercial MLPA kits for NB. Finally, four patients showed a normal MLPA profile, suggesting that NB might have a more complex genetic pattern than the one assessed by presently available MLPA kits.

Identification of deletions and duplications in the CYP2E1 gene in a population in Goiânia – GO by MLPA.

Human CYPs are important in physiology, drug metabolism, and pathogenesis of certain diseases, and there is a strong need to explore the genetic variations and regulatory mechanisms of these enzymes. Mutations in CYP genes leading to deficiency in the enzymes result in a wide spectrum of human diseases. The aim of the present study was to investigate, the importance of CYP2E1 genetic polymorphisms in the health population in Goiânia. The study was performed acording approved by the ethics committee of Federal University of Goias (approval CEP: 895.552). In the current survey, 100 patients (35M/65F) of both genders and aged 18-90 (M: 30.2) years old were enrolled after receiving their informed consent. The Multiplex ligation-dependent probe amplification (MLPA) was performed using the P128 CYP450 MLPA kit (Version C1; MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. This gene is located on chromossome 10 and has 9 exons, but only 3 (5, 6 and 8) were analyzed. In the exon 5 was found (2 duplications and 3 deletions), exon 6 (2 duplications and 2 deletions) and the exon 8 (3 duplications and 2 deletions). This gene is the second responsible for the metabolism of alcohol in the body, after the enzyme alcohol dehydrogenase and therefore the importance of checking what these deletions and duplications can cause in the body. Is the first time this methodology is used, and we believe it is a great tool for future studies with other genes.

Detection of CCND1 Gene Copy Number Variations Using Multiplex Ligation-Dependent Probe Amplification and Fluorescence In Situ Hybridization Methods.

The CCND1 locus is located in 11q13 and encodes the G1-S regulatory protein, cyclin D1. Cyclin D1 is frequently amplified in various types of cancers, and is an attractive potential therapeutic target. Multiplex ligation-dependent probe amplification (MLPA) is a new, high-resolution method for the detection of amplification of numerous genes including CCND1 in small amounts of DNA fragments derived from formalin-fixed, paraffin-embedded material in a single reaction. This approach is, however, based on PCR and averages many different cells, so validation by morphological methods such as fluorescence in situ hybridization (FISH) is theoretically mandatory. Here we describe detection of CCND1 gene copy number variations by commercially available MLPA kits and FISH using a bacterial artificial chromosome (BAC) probe.

Analysis of Copy Number Variations in Patients with Autism Using Cytogenetic and MLPA Techniques: Report of 16p13.1p13.3 and 10q26.3 Duplications.

Autism is a common neuropsychiatric disorder affecting 1 in 68 children. Copy number variations (CNVs) are known to be major contributors of autism spectrum disorder (ASD). There are different whole genome or targeted techniques to identify CNVs in the patients including karyotyping, multiplex ligation-dependent probe amplification (MLPA) and array CGH. In this study, we used karyotyping and MLPA to detect CNVs in 50 Iranian patients with autism. GTG banding and 4 different MLPA kits (2 subtelomeric and 2 autism kits) were utilized. To elevate our detection rate, we selected the sporadic patients who had additional clinical features including intellectual disability, seizure, attention deficit hyperactivity disorder, and abnormal head circumference. Two out of 50 patients (4%) showed microscopic chromosome abnormalities and 5 out of 50 (10%) demonstrated copy number gains or losses using MLPA kits. Including one overlapping result between karyotype and MLPA techniques, our overall detection rate was 6 out of 50 (12%). Three out of 6 CNVs were de novo and three others were paternally inherited. Two of CNVs detected by karyotyping and MLPA tests were 16p13.1q13.3 and 10q26.3 duplications, respectively. For these two CNVs genotype and phenotype of the patients were compared with other studies. Although the pathogenicity of cytogenetic results was certain, most of MLPA results needed to be better refined using other more accurate techniques such as array CGH. Our findings suggest that it might be possible to obtain some useful information using MLPA technique but it cannot be used as a single diagnostic tool for the autism.

Genetic Abnormalities and Prognosis in Pediatric B-Precursor Acute Lymphoblastic Leukemia without Conventional Genetic Abnormalities; Tokyo Children's Cancer Study Group

Introduction; Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease that can be subdivided according to primary recurrent genetic abnormalities that are strongly associated with characteristic biological and clinical features. In approximately two-thirds of pediatric patients with BCP-ALL, well-characterized genetic abnormalities can be detected by standard genetic analyses. In the remaining BCP-ALL patients, major pathogenic or driver cytogenetic abnormalities have yet to be clarified and they are called B-others. Recent studies using advanced analytical approaches have stratified a variety of subgroups harboring novel genetic abnormalities. For example, Ph-like ALL, iAMP21, deletion of ERG and rearrangements of ZNF384 and MEF2D have been identified in B-others. However, a certain portion of B-others still remains as genetically unclassified.Patients and Methods; 291 children with de novo BCP-ALL were enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials (between 2004-2007). Conventional genetic abnormalities, including BCR-ABL1, MLL-AF4, MLL-AF9, TCF3-PBX1, ETV6-RUNX1, hyperdiploidy and hypodiploidy were detected in 16 (5.5%), 3 (1.0%), 2 (0.7%), 18 (6.2%), 54 (18.5%), 65 (22.3%) and 3 (1.0%) cases by G-banding, fluorescent in situ hybridization and molecular analyses. 131 cases (44.9%) were classified B-others. We studied 121 (93.1%) out of 131 B-others ALL of the L0416/L0616 cohort. We performed whole transcriptome sequencing (WTS) of 35 pediatric B-others cases. For the confirmation and screening of the fusion transcripts we performed RT-PCR in a total of 121 B-others cases. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327. We also analyzed the known genetic mutations or copy number alterations associated with these patients using the data from WTS and MLPA kit P335 and P200.Results; A total of 47 candidate in-frame gene rearrangements were identified in 29/35 (82.8%) B-others samples. The fusion genes including ZNF384 (n=12, 4.1%) or MEF2D (n=6, 2.1%) were frequently found in this cohort. All of patients with ZNF384-related fusion genes were found to have a weak or negative expression of CD10. In addition, 7 and 17 patients out of 22 patients with ZNF384-related fusion genes exhibited more than 20% expression of CD13 and CD33. We identified 6 recurrently mutated genes, including NRAS, KRAS, PTPN11, EZH2, MLL2 and ASH1L. Especially, activating mutations in RAS signaling pathway genes, including NRAS, KRAS and PTPN11 were detected in 4 out of 10 ZNF384-translocated patients. Mutations in B-cell developmental genes such as PAX5, BTG1 and IKZF1 were not detected at all in DNA samples with ZNF384-related fusion genes. We identified a total of 6 (1.8%) patients with the fusion genes related with Ph-like ALL, including IGHM-CRLF2, P2RY8-CRLF2, IGH@-EPOR and PAX5-JAK2. Three of 6 patients with Ph-like ALL related fusion gene relapsed and two patients died. We identified iAMP21 in 6 (2.1%) patients and deletion of ERG in 7 (2.4%). One of 6 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 6 patients with iAMP21, three patients relapsed, and two patients died. No patients with ERG deletion relapsed or died.Conclusions; WTS analysis unmasked a complexity of gene rearrangements in pediatric BCP-ALL without conventional genetic abnormalities. In this study, 10.6% of genetic alterations were revealed in Japanese pediatric B-others cohort. Apart from increasing our understanding of the pathogenesis of pediatric BCP-ALL, this may help improve risk stratification and eventually increase the therapeutic option. DisclosuresNo relevant conflicts of interest to declare.

Improved Diagnosis of Intrachromosomal Amplification of Chromosome 21 (iAMP21) By Copy Number Profiling

Abstract Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of 2% childhood acute lymphoblastic leukaemia (ALL). iAMP21-ALL patients have precursor B-cell ALL, are older (median age 9 years), generally present with low white cell counts and have an inferior outcome when treated with standard therapy. Stratification to high risk treatment arms has significantly reduced their relapse risk, thus accurate diagnosis is essential. We have identified iAMP21 as a complex structure of one copy of chromosome 21, comprising multiple regions of gain, amplification, inversion and deletion, initiated through breakage-fusion-bridge cycles and chromothripsis. Currently, fluorescence in situ hybridisation (FISH), using probes directed to RUNX1, is the most reliable and convenient detection method, in which four or more copies of RUNX1 on a single abnormal chromosome 21 defines iAMP21. From our examination of several hundred cases of iAMP21-ALL, we have noted that, in the absence of metaphase FISH and/or in cases with an unusual cytogenetic presentation, reliance on FISH alone for accurate detection may be problematic. Among a collection of 210 patients with iAMP21-ALL, we performed SNP6.0 and Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA kit P327 iAMP21-ERG (MRC Holland) on 57 and 45 patients, respectively. Although chromosome 21 structure is highly variable between patients, a characteristic copy number profile emerged from the SNP6.0 data (Figure 1). In common, all cases were amplified across a 5.1Mb region, between 32,813,553 and 37,941,425bp that includes RUNX1 and 46 other known protein-coding genes. Asub-telomeric deletion occurred in 88% patients. We propose that this distinctive chromosome 21 copy number profile is used in addition to FISH for the definitive diagnosis of iAMP21-ALL in problematic cases. Support for this approach is provided by results from 5 patients from the Children's Oncology Group (North America, Australia and New Zealand) and ALL2003 (UK) treatment trials who met the iAMP21 FISH criteria but had atypical karyotypes or an unusual distribution of the additional RUNX1 signals that made confident diagnosis challenging. Clinical and cytogenetic data were collected and SNP6.0 and MLPA were performed to clarify the genomic alterations present in these cases. In patient #1, although five RUNX1 signals were observed per cell, two normal copies of chromosome 21 (each with one RUNX1 signal) were present with only three signals located to the abnormal chromosome 21. In patient #2, the additional signals were distributed between 2 different abnormal copies of chromosome 21. Metaphase FISH of patient #3 indicated that the RUNX1 signals were distributed between one normal copy of chromosome 21 and four small chromosomes, which were identified to originate from chromosome 21 by chromosome painting with a chromosome 21 specific probe (wcp21). In a further 2 cases (patients #4 and #5) the signals were too tightly clustered for the number to be discerned. In these 5 cases the characteristic SNP6.0 profile definitively confirmed the suspected diagnosis of iAMP21-ALL (Figure 2). Our previous SNP6.0 data have shown that the copy number profile of the iAMP21 chromosome remains stable between diagnosis and relapse, as well as in serial xenografts successfully transplanted with iAMP21-ALL cells over several generations of mice. However, while FISH analysis of patient #6 at diagnosis and relapse showed the same signal pattern at both time points, there was a change in the karyotype, involving translocation of part of the iAMP21 chromosome onto chromosome 11 at relapse. These observations indicate that the iAMP21 chromosome may become fragmented and distributed throughout the genome without changing its genomic profile. Collectively these observations indicate that the amplified segments resulting from the formation of iAMP21 chromosomes, with typical copy number profiles, can be distributed throughout the genome, either by translocation or fragmentation. Thus FISH together with chromosome 21 copy number profiling provide more accurate diagnosis of iAMP21-ALL than cytogenetics, which may be misleading. For laboratories with no access to SNP6.0 or other copy number arrays, we have shown that MLPA, with a kit specifically designed to detect chromosome 21 copy number changes, provides a reliable alternative (Figure 3). Disclosures No relevant conflicts of interest to declare.

Multiplex Ligation-Dependent Probe Amplification Accurately Detects Turner Syndrome in Girls with Short Stature

Aims: We aimed at evaluating a standard multiplex ligation-dependent probe amplification (MLPA) probe set for the detection of aneuploidy to diagnose Turner syndrome (TS). We first fixed an MLPA ratio cutoff able to detect all cases of TS in a pilot TS group. We then tested this value on a second group of TS patients and a short-stature population to measure specificity and sensitivity. Methods: 15 TS patients with X mosaicism or X structural abnormalities (Pilot TS Group), 45 TS karyotype-assessed patients (TS Group), and 74 prepubertal female patients with apparent idiopathic short stature (Short-Stature Group) were enrolled. All subjects underwent MLPA and karyotype analysis. In the TS and Short-Stature Groups, MLPA testing was performed in blind. Results: The choice of an MLPA threshold ratio of 0.76 for at least 1 probe allowed us to detect all TS cases, including mosaicisms. Sensitivity and specificity were 100% (CI 95%, 0.92-1) and 88.89% (CI 95%, 0.79-0.94), respectively. The positive predictive value was 88.5%, and the negative predictive value was 100%. MLPA detected the presence of Y chromosome material in 2 patients. Conclusion: MLPA is an accurate and inexpensive tool to screen for TS in girls with short stature. A customized MLPA kit may be useful for the screening of an even larger population.

The use of two different MLPA kits in 22q11.2 deletion syndrome

22q11.2 deletion syndrome (22q11DS) is one of the most common recurrent copy-number variant disorder, caused by a microdeletion in chromosome band 22q11.2 and occurring with a population prevalence of 1 in 2000. Until today there has been no evidence that the size of the deletion has an influence on the clinical phenotype. Most studies report that 22q11DS is associated with mild or borderline intellectual disability. There are a limited number of reports on 22q11DS subjects with moderate or severe intellectual disability.In this study we describe 63 adult patients with 22q11DS, including 22q11DS patients functioning at a moderate to severe intellectual disabled level. Deletion size was established with an experimental Multiplex ligation-dependent probe amplification (MLPA) mixture (P324) in addition to the commonly used MLPA kit (P250). We compared deletion size with intellectual functioning and presence of psychotic symptoms during life. The use of the experimental MLPA kit gives extra information on deletion size, only when combined with the common MLPA kit. We were able to detect eleven atypical deletions and in two cases the deletion size was shorter than all other “typical ones”. We conclude that the use of the experimental kit P324 gives extra information about the deletion size, but only when used together with the standard P250 kit. We did not found any relation of deletion size with intelligence or presence of psychosis.

Identification of an NPHP1 deletion causing adult form of nephronophthisis.

Nephronophthisis (NPHP) is an autosomal recessive cystic disease of the kidney with main characteristic features of polyuria/polydipsia, mild or absent proteinuria, interstitial fibrosis, and tubular cysts. NPHP is responsible for 5-10% of inheritable end-stage renal disease (ESRD) cases. We investigated the clinical features and genetic cause of NPHP in a Persian family with three siblings affected by tubulointerstitial nephropathy reaching ESRD in adulthood. Uromodulin (UMOD), known to be involved in adult medullary cystic kidney disease, and nephronophthisis 1 (NPHP1) were investigated in the genomic DNA of the probands using DNA sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis and molecular karyotyping. No mutation was detected in UMOD. Copy number variation analysis of the NPHP1 gene using the commercially available MLPA kit identified a recurrent large homozygous deletion encompassing all NPHP1 exons. The parents were heterozygous for this deletion. Whole genome array-CGH analysis confirmed a homozygous deletion on chromosome 2q13, NPHP1 site, and revealed that the size of the copy number loss was approximately 102 Kbp. This is the first report of determination of an NPHP1 deletion size using routine diagnostic methods. The results of this study expand the knowledge about the genotype-phenotype correlations in NPHP1, and have implications for genetic counseling and family planning advice for other affected families. This is the first molecular analysis of NPHP1 in an Iranian kindred.

2086260 The Prenatal Diagnosis of Microdeletion/Microduplication Syndromes in Increased Nuchal Translucency with Normal Karyotype: Unselected Korean Populations

Though the presence of aneuploidy is ruled out, nuchal thickening is clinically associated with many problems. Some reports have examined the value of microdeletion testing in increased NT fetuses, normal karyotype and no structural anomalies, but the results have showed limited value. The purpose of this study was to assess the value of testing for microdeletion/micoduplication syndromes with NT greater or equal to 95th percentile according to crown-rump length and normal karyotype. During April 2010 to December 2013, a total of 3051 samples had a routine karyotyping and MLPA analyses performed for several indications. Among them a total of 974 pregnancies were collected the inclusion criteria of increased NT. All samples were processes for G-banding karyotyping in additional to rapid aneuploidy testing and Multiplex Ligation dependent-Probe Amplification (MLPA). The MLPA analysis for detection of microdeletion or microduplication syndrome were performed using commercial MLPA kits (SALSA P245,MRC- Holland, Netherland). Application of the screening detected 26 cases of microdeletion/microduplication syndromes in 3051 samples (0.85% incidence). Among the 974 in increased NT samples with a normal karyotype, we identified 3 cases of microdeletion syndromes; 1 cases of DiGeorge syndrome (22q11 deletion), 1 cases of Wolf- Hirschhorn syndrome (4p 16.3 deletion), 1 Sotos syndrome and 9 cases of microduplication syndromes(3q,7q,10p,15q,17q,22q). The diagnostic yield was 1.23%(12/974) in the thick NT, 0.73%(4/545) in advanced maternal age and 0.35%(3/843) in the high risk result following maternal serum biomarker screening. This is the largest single reported study of microdeletion/microduplication syndromes in increased NT fetuses with normal karyotype. The prevalence of microdeletion/microduplication syndromes with increased NT is obviously higher than that expected in the general population. For this reason, the additional MLPA analyses for selected microdeletion/microduplication syndrome are valuable and useful in counselling parents of pregnancies with increased NT.

Analysis of SMN1 gene partial deletion of spinal muscular atrophy based on MLPA

To explore the diversity of mutations in survival motor neuron gene 1 (SMN1) by analyzing seven cases of partial deletion of SMN1 gene. Seven patients suspected spinal muscular atrophy (SMA) were recruited from 2011 to 2013. Multiplex ligation-dependent probe amplification (MLPA) for genetic testing of SMA was based on the commercially available SALSA MLPA kit P021-A2. Then the data were analyzed by the software Coffalyser.Negative control samples were chosen with two copies of SMN1 and SMN2. Positive control samples were chosen with zero copies of SMN1 and two copies of SMN2. According to the product description (www.mlpa.com): for exon 7 and 8 of SMN1 and SMN2: a ratio of <0.7 indicates 1 copy, a ratio of 0.7-1.3 2 copies, a ratio of 1.3-1.7 3 copies and a ratio of 1.7-2.3 4 copies. For exon 1, 4, 6, 8 of SMN gene (SMN1+SMN2): a ratio <0.4 indicates 1 copy, a ratio of 4.0-0.6 2 copies, a ratio of 0.7-0.9 3 copies and a ratio of 0.9-1.1 4 copies. All samples were analyzed in duplicate. Using MLPA for clinical diagnostics, two types of partial deletions of SMN1were identified in 7 patients.Since exon 8 is not translated and has no effect on the function of SMN protein, exons 1, 4, 6, 7 were targeted.One had an isolated deletion of exon 7 while the other ones were caused by the deletions of exon 1, 4 and 7. These mutations were not detected by conventional diagnostic methods. Both types of partial deletions of SMN1 gene contained a deletion of exon 7. Two types of partial deletions of SMN1 gene indicate that the structure of SMN gene is unstable leading to a variety of mutation forms. But the major cause of SMA lies in a deletion of exon 7 of SMN1 gene.

Genetic Abnormalities and Prognosis in Pediatric B–precursor Acute Lymphoblastic Leukemia Treated on Tokyo Children′s Cancer Study Group Protocol

Introduction; Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Most childhood cases of B-precursor ALL (BCP-ALL) may be subclassified by the presence of either gross or submicroscopic genetic alteration. Recent genomewide analyses have identified genetic or cytogenetic subtypes, such as Ph-like ALL, deletion of ERG, intrachromosomal amplification of chromosome 21 (iAMP21) in BCP-ALL. Deletion of ERG and iAMP21 were associated with frequent IKZF1 deletions, which were found in Ph-like ALL associated with poor prognosis. However, the frequency and prognostic implications of these genomic abnormalities have not been defined in Japan.Patients and Methods; We studied 208 children with de novo BCP-ALL enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials. All patients had a diagnosis of BCP-ALL without BCR-ABL1 phenotype confirmed by morphology, immunophenotyping and mRNA Real-time PCR. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327-B1. To identify Ph-like ALL and BCR-ABL1 like ALL, We investigated gene expression of 235 BCP-ALL cases enrolled on TCCSG L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using clustering analysis as described previously. To assess whether these genomic alterations overlap with known gene abnormalities, such as mutations of CRLF2,JAK1, JAK2 and IL7R, rearrangement of P2RY8-CRLF2, PAX5-JAK2 and PDGFRB-EBF1 genes and copy number alteration of IKZF1, CDKN2A/2B, PAX5, EBF1 and SH2B3 genes, mutational analyses of these genes were also performed using direct sequencing, RT-PCR or MLPA kit P335-A1 and P200.Results; We identified Ph-like signature in 9 (3.8%) patients and BCR-ABL1 like ALL signature in 11 (4.7%) patients. Five (55.6%) of 9 Ph-like ALL patients and 3 (27.3%) of 11 BCR-ABL1 like ALL patients have IKZF1 deletions. Five patients were classified into both Ph-like ALL and BCR-ABL1 like ALL. Among these 5 patients, one patient had CRLF2 high expression, JAK2 R683S/R683G mutation, CDKN2A/2B deletion and EBF1 deletion, and another patient have PAX5-JAK2 rearrangement. These two patients relapsed, and the latter patient died. Three of 9 Ph-like ALL patients relapsed and 2 died, while 7 of 11 BCR-ABL1 like ALL patients relapsed and 2 died. We identified iAMP21 in 5 (2.4%) patients and deletion of ERG in 8 (3.8%). One of 5 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. One patient with iAMP21 had BCR-ABL1 like ALL signature, but not Ph-like ALL signature. CRLF2 high expression was identified in two of 5 patients with iAMP21, and P2RY8-CRLF2 rearrangement and SH2B3 deletion was identified in one of them. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 5 iAMP21 patients, patient with BCR-ALB1 signature and IKZF1 deletion relapsed, and one patient died. No patients with ERG deletion relapsed or died.Conclusions; Using GeneChip microarray analysis, low frequency of Ph-like ALL signature and BCR-ABL1 like ALL signature was found. Using MLPA as a novel screenings technique, frequency of iAMP21 and ERG deletion in pediatric BCP-ALL was similar to that of previous studies. BCR-ABL1 like ALL and iAMP21 was associated poor prognosis, while ERG deletion was associated good prognosis in pediatric ALL. These data suggest that screening for these genetic abnormalities in pediatric patients with BCP-ALL is critical not only for outcome prediction but also for risk-adapted therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Diagnosis of MECP2 duplication syndrome with molecular genetic techniques

To investigate whether the four boys with delayed motor development and intellectual disability suffer from MECP 2 duplication syndrome. Blood specimens and clinical data of four patients and mothers of patient 2 and patient 4 were collected. Genomic DNA was extracted from peripheral blood using DNA extraction kit. At first multiplex ligation-dependent probe amplification (MLPA) was employed in 4 patients, two distinct kits SALSA P036 and P070 for sub-telomere screening, and SALSA P245 for the 22 common microdeletion and microduplication syndromes. Then array-CGH analysis was carried out. Two mothers of patients were tested by array- comparative genomic hybridization (CGH) and X chromosome inactivation analysis. All the 4 patients presented with severe hypotonia, delayed motor development, intellectual disability and absent or limited language. Three patients manifested recurrent pneumonia in infancy except patient 2. Four patients had duplication on chromosome Xq28 with MLPA kit SALSA P245. Array-CGH identified the size of each duplication on Xq28. The precise size of each duplication was different in the four patients: patient 1, 14.931 Mb, patient 2, 0.393 Mb, patient 3, 0.482 Mb and patient 4, 0.299 Mb. To compare Xq28 duplications with UCSC database (http://genome.ucsc.edu/) revealed that each duplication harbors the MECP 2 and HCFC 1 gene. Mothers of patient 2 and patient 4 also carried microduplication on Xq28. X chromosome inactivation analysis demonstrated completely skewed inactivation (0: 100) and it is the inactive allele that passed on to the patients. For patients that present with delayed motor development, intellectual disability, hypotonia, absent or limited language and recurrent infection, combination of MLPA and array- CGH is effective and specific diagnostic methods of MECP 2 duplication syndrome.

Investigation of microdeletions in syndromic intellectual disability by MLPA in Iranian population.

Intellectual Disabilities (ID), defined as a state of developmental deficit, result in significant limitation of intellect and poor adaptation behavior. A number of genetic factors can result in ID, such as chromosomal abnormalities, copy number variation, and single gene defect. Karyotyping is the routine method for detecting chromosomal abnormalities in patients with ID. More recently, the Multiplex Ligation-dependent Probe Amplification (MLPA) method has been applied for detecting microdeletion/duplication in patients with dysmorphism and ID. A total of 100 patients with dysmorphism and ID have been referred to us since 2011. All patients were first evaluated clinically and a number of these individuals had normal karyotypes. We investigated duplications and deletions for 21 different microdeletion syndromes using MLPA kit (MRC-Holland). We were able to identify aberrations in 12 (12%) patients clinically ascertained as follows: 5 Williams syndromes, 3 Miller- Dieker syndromes, 1 Sotos syndrome, 1 Angelman Syndrome, 1 Di-George syndrome and one patient with an abnormal 4p chromosomal region. Our MLPA results indicate a high degree of concordance between the clinical data and the genotype. We suggest MLPA as the first screening method for children suffering from MR with normal karyotypes. In those cases where clinical findings were not compatible with the microdeletion syndrome identified by MLPA investigation, further studies such as FISH and aCGH were performed.

Exonic deletions in the NF1 gene in patients with neurofibromatosis type I from the lower Silesian region of Poland.

Neurofibromatosis type I (NF1, Recklinghausen's disease) is an autosomal dominant disorder characterized by the following clinical features: café au lait spots, neurofibromas, Lisch nodules, freckling of the axillary and inguinal regions, optic nerve gliomas, bone dysplasia and increased risk of certain tumors. NF1 is diagnosed on the basis of clinical criteria, while identifying the genetic background of the disease is important mainly for genetic counseling. NF1 genetic analysis is based on searching for NF1 exon deletions/duplications using Multiplex ligation-dependent probe amplification (MLPA), searching for microdeletions of the critical region using fluorescence in situ hybridization (FISH), searching for point mutations by gene sequencing (in most cases) and analyzing mRNA. The aim of this study was to estimate the frequency of single and multi-exon deletions/duplications in the NF1 gene in Polish patients, and to evaluate the usefulness of MLPA as a cheap and easy method for NF1 molecular diagnosis, despite the fact that such changes may be found in only a small group of NF1 patients. The study included 65 patients suspected of NF1 or with recognized NF1 on the basis of clinical criteria. Cytogenetic analysis were carried out for all the patients, and for one patient with a translocation [46,XY,t(17;22)(q11.2;q11.2)], a FISH analysis was performed. All patients were tested for deletions/duplications in the NF1 gene using two MLPA kits for neurofibromatosis I. The MLPA analysis showed deletions in the NF1 gene in 7.7% of the cases (5/65). The results indicate that an MLPA analysis may be performed in patients with a clinical diagnosis of NF1 or patients with suspected NF1 as an easy and inexpensive first molecular test, enabling the exclusion of about 7% of NF1 patients from expensive and time-consuming molecular diagnosis by DNA sequencing.

Genomic Heterogeneity In Pediatric Acute Lymphoblastic Leukemia

Pediatric B cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is characterized by recurrent chromosomal aneuploidies and translocations, which are important for diagnosis and risk stratification. Classical cytogenetic and FISH are routinely used for the diagnosis of most of these alterations, particularly those involving balanced translocations, and numerical changes. Recently, genome wide analysis, such as aCGH and SNP technologies have identified copy number alterations (CNA) in genes involved in lymphoid differentiation: IKZF1, PAX5, EBF1 BTG1; and in cell cycle control: CDNK2A/B, RB1 and ETV6. Multiplex ligation–dependent probe amplification (MLPA) technique has been used for the identification of those focal genetic aberrations in a single assay but it cannot reliably detect aberrations present in less than 50% of cells. Cytoscan HD SNP array (Affymetrix) can overcome this limitation and is the only array which enables independent confirmation of CNA with SNP allelic information. Thus minor genetic subclones missed by other methods, can be detected.The aim of this work was to compare between SNP array and MLPA platforms in a routine diagnostic setting.Samples of 39 pediatric BCP-ALL patients well characterized by classical cytogenetic and FISH at diagnosis, were studied by MLPA P335 (MRCHolland) and SNP array (Affymetrix). In total 273 genomic loci were evaluated, 13 (5%) were not informative, of them 7 were not covered by the MLPA kit and 6 gave not evaluable MLPA results. 231/260 (89%) of the loci showed concordance, 39/260 (15%) with CNA and 192/260 (74%) normal. A total of 68 CNA were detected, 29 were discordant and 39 concordant.IKZF1 deletion was identified by both methods in 5 cases (13%). However, a deletion was identified in 3 additional samples, only by cytoscan. In these cases, the deletion was present in a minor subpopulation (in 45% or less of the cells), while the main cytogenetic aberration was present in the majority of cells (60%-90%).We conclude that although MLPA is a useful technique, it is not sensitive enough to detect small populations. In this small cohort, 3 IKZF1 deletions were missed since they were present only in a minor subclone. Interestingly, one of these patients relapsed, and the relapse clone harbored the IKZF1 deletion. ConclusionGenome-wide sensitive methods identify different subpopulations in each patient, thus detecting previously unrecognized genetic heterogeneities. Disclosures:No relevant conflicts of interest to declare.