Multiplex Ligation-dependent Probe Amplification Technique Research Articles

Detection of AZFc gene deletion in a cohort of Egyptian patients with idiopathic male infertility

BackgroundThe deletions of azoospermic factor regions (AZF) are considered risk factor of spermatogenic failure. AZF duplications or complex copy number variants (CNVs) were rarely studied because STS-PCR could not always detect these changes. The application of multiplex ligation-dependent probe amplification (MLPA) as a valuable test for detection of the deletion and or duplication was introduced to investigate the AZF sub-region CNVs. The MLPA technique is still not applied on a large scale, and the publications in this area of research are limited. The aim of this work was to evaluate the efficacy of MLPA assay to detect AZF-linked CNVs in idiopathic spermatogenic failure patients and to evaluate its importance as a prognostic marker in the reproduction outcome. ResultsForty infertile men (37 with azoospermia and 3 with severe oligozoospermia) and 20 normal fertile men were subjected to thorough clinical, pathological, and laboratory assessment, chromosomal study, MLPA, STS-PCR assays, histopathology study, and testicular sperm retrieval (TESE). Out of the 40 patients, 7 patients have shown CNV in the AZFc region, 6 patients have partial deletion, and one patient has partial duplication. Only one of the normal control has AZFc duplication. STS-PCR was able to detect the deletion in only 4 out of the 7 positive patients and none of the control. ConclusionWe concluded that MLPA should be applied on a larger scale for the detection of Y chromosome microdeletion as a rapid, efficient, and cheap test.

Copy Number Variations and Gene Mutations Identified by Multiplex Ligation-Dependent Probe Amplification in Romanian Chronic Lymphocytic Leukemia Patients.

Chronic lymphocytic leukemia (CLL) is known for its wide-ranging clinical and genetic diversity. The study aimed to assess the associations between copy number variations (CNVs) and various biological and clinical features, as well as the survival rates of CLL patients and to evaluate the effectiveness of the multiplex ligation-dependent probe amplification (MLPA) technique in CLL patients.DNA was extracted from 110 patients, and MLPA was performed. Mutations in NOTCH1, SF3B1, and MYD88 were also analyzed. A total of 52 patients showed at least one CNV, 26 had at least one somatic mutation, and 10 presented both, CNVs, and somatic mutations. The most commonly identified CNVs were del(114.3), del(11q22.3), and dup(12q23.2). Other CNVs identified included del(17p13.1), del(14q32.33), dup(10q23.31), and del(19p13.2). One patient was identified with concomitant trisomy 12, 13, and 19. NOTCH1 and SF3B1 mutations were found in 13 patients each, either alone or in combination with other mutations or CNVs, while MYD88 mutation was identified in one patient. Forty-two patients had normal results. Associations between the investigated CNVs and gene mutations and patients' overall survival were found. The presence of NOTCH1 and SF3B1 mutations or the combination of NOTCH1 mutation and CNVs significantly influenced the survival of patients with CLL. Both mutations are frequently associated with different CNVs. Del(13q) is associated with the longest survival rate, while the shortest survival is found in patients with del(17p). Even if MLPA has constraints, it may be used as the primary routine analysis in patients with CLL.

GATA4 rs61277615, rs73203482, and rs35813172 in Newborns with Transposition of the Great Arteries.

Congenital heart disease is the most common malformative pathology in newborns, with a worldwide incidence at 0.4-5%. We investigated the possible relationship between variations in nucleotide sequences and specific cardiac malformations in the GATA-binding factor 4 (GATA4) exon 1 region by using Sanger sequencing. Forty-four newborns from a third-level neonatal intensive care unit who were diagnosed with nonsyndromic, ductal-dependent congenital heart disease (i.e., transposition of the great arteries or ductal-dependent coarctation of the aorta) were enrolled. Their DNA was extracted using commercial methods and tested using the multiplex ligation-dependent probe amplification (MLPA) technique. The Sanger sequencing for GATA4 exon 1 in the newborns' DNA identified rs61277615, rs73203482, and rs35813172 variants not reported in the ClinVar archive of human variations in newborns previously diagnosed with transposition of the great arteries (n=5) and coarctation of the aorta (n=1). The identification of these novel variants in newborns with transposition of the great arteries or ductal-dependent coarctation of the aorta may be the first step in determining the variants' contribution to the occurrence of congenital heart disease. However, these results may be inconclusive, since the observed variants within GATA4 gene were not previously reported.

Variants in the Sequence of the Probe Hybridization Site May Affect MLPA Performance in Patients with Duchenne/Becker Muscular Dystrophy.

The MLPA (multiplex ligation dependent probe amplification) technique is currently the test most widely used to detect mutations in the Duchenne/Becker muscular dystrophy (DMD) gene in the initial assessment. However, several studies have suggested that MLPA results require implementing other detection methods due to false duplication. Our aim was to evaluate variables that could alter the peak ratio in MLPA in individuals with Duchenne/Becker muscular dystrophy (DMD/BMD) who present sequence variants at the probe hybridization site, such as the location of sequence variants (SVs), melting temperature of the probe, and the type of variant. We analyzed patients with clinical suspicion of DMD/BMD through the MLPA technique. The DMD gene was sequenced in patients with normal results in MLPA. Of 111 patients, 72 had an abnormal MLPA result, of which 10 had a single exon abnormal peak, and 39 had a normal peak ratio. Out of 10 patients, 4 (40%) with a single exon abnormal peak ratio had SV at the hybridization site of the probe. In the other 6, the deletion was confirmed. Out of 39 patients with a normal peak ratio, 11 presented SVs at the hybridization site of the probe, and DMD/BMD was confirmed. In cases of abnormal peak ratio results of MLPA in a single exon, it would be valuable to sequence the DMD gene to assess whether variants in the probe hybridization site might result in a false positive that could be interpreted as an exon deletion.

Chromosome 1p status in neuroblastoma correlates with higher expression levels of miRNAs targeting neuronal differentiation pathway.

Neuroblastoma (NB) is characterized by acquired segmental and numerical chromosome aberrations. Although deletions of distal 1p and 11q are frequent alterations, no candidate tumor suppressor gene residing in these chromosomal sites could be identified so far. In the present study, we detected the genomic imbalances of six neuroblastoma cell lines using the multiplex ligation-dependent probe amplification (MLPA) technique and the microRNA (miRNA) expression profiles of the cell lines by a microarray study. According to MLPA results, we aimed to assess the miRNA expression profiles of the cell lines harboring 11q and 1p deletions. The cell lines with 1p deletions revealed statistically significant higher levels of expression for 29 miRNAs in contrast to the cell lines without 1p deletion in microarray study. We also performed GO enrichment analysis for predicted targets of the differentially expressed miRNAs. According to GO enrichment analysis, miRNAs that showed the high change in expression was associated with neuronal differentiation. We showed that hsa-miR-494, hsa-miR-495, and hsa-miR-543 target most of mRNAs in neuronal differentiation pathway. Although limited to the cell lines, our results highly suggest that NBs with different segmental chromosome abnormalities may have different dysregulated miRNA expression signatures that target the genes involved in neuronal differentiation.

Clinical and Molecular Genetic Analysis of Cases with Ectodermal Dysplasia.

Ectodermal dysplasias are a group of >200 clinically and congenitally heterogeneous disorders characterized by abnormal development in the ectodermal structures, such as hair, nails, teeth, and sweat glands. We report here the clinical and molecular genetic analysis of five Greek families with different types of ectodermal dysplasia (ED). The study involved 15 individuals from 5 Greek families that included 8 ED patients, 5 carriers of recessive X-linked or autosomal ED, and 2 healthy relatives. After genetic counseling, the parents signed an informed consent form before subsequent genetic testing. Genomic DNA was isolated from white blood cells of all studied individuals. The search for mutations was realized in patients' DNA samples using next-generation sequencing (NGS) gene panel, whole exome sequencing (WES), chromosomal microarray analysis (CMA), and multiplex ligation-dependent probe amplification (MLPA) technique. The clinical diagnosis of common X-linked recessive hypohidrotic ectodermal dysplasia (HED) was suspected in five male patients with partial anodontia of baby and permanent teeth, hypohidrosis, and thin hair from three families. All HED patients were hemizygous for deletions in the EDA1 gene (Xq13.1): three related patients had a 20bp deletion, one had a 19bp deletion, and one had a 180bp deletion. A female patient had the rare autosomal dominant syndrome of ankyloblepharon-ectodermal dysplasia-cleft lip/palate (AEC) caused by heterozygous missense mutation in the TP63 gene (3q28) that appeared de novo. Two siblings with hypotrichosis and hypodontia, a female and a male, had two pathogenic mutations in compound heterozygosity in the TSPEAR gene (21q22.3); therefore they presented with ectodermal dysplasia type 14 (ECTD14). Clinical and molecular genetic analysis may set an accurate diagnosis of different types of ED. In the reported families, genetic diagnosis and genetic counselling assisted the parents to view their children's condition realistically and to cooperate with the specialists who will contribute to the best possible treatment for their children.

Applying Multiplex ligation-dependent probe amplification technique to identify α-thalassemia carriers

α-thalassemia disease is mostly caused by mutations in the HBA1 and HBA2 genes that lead to the deficiency in the α-globin chain, which builds up the haemoglobin molecule. Depending on the number of missing α chains, the clinical manifestations of the disease are at different levels. This disease is inherited in an autosomal recessive manner, hence identifying healthy individuals carrying mutations in the α-thalassemia gene is essential for prenatal and premarital genetic counselling to reduce the incidence in the community. Multiplex ligation-dependent probe amplification (MLPA) technique was used to determine 15 people who were suspected of carrying the α-thalassemia gene based on their complete blood count. This study identified that therewere 12/15 people carrying --SEA mutation, 1/15 carrier having -α3.7 mutation, 1/15 carrier having -α4.2 mutation and 1/15 person carrying point mutation Hb constant spring -αHbCs.

Detection of Genetic Alterations in Oral Squamous Cell Carcinoma Using Multiplex Ligation-Dependent Probe Amplification (MLPA)

Deletions and amplifications of genes often occur during multistep progression from oral precancer, seen as oral epithelial dysplasia (OED) to cancerous stage. These genetic alterations could be used as markers to aid in detection of oral squamous cell carcinomas (OSCC). This study explored the use of multiplex ligation-dependent probe amplification (MLPA) technique in detecting OSCC and OED specific genetic alterations. MLPA was used to detect gains and losses of 106 genes in DNA extracted from frozen tissue samples of 10 OSCC and 10 non-cancer patients. Two biopsies of OED were analyzed to explore the alterations in oral potentially malignant disorders. There were significant differences (p<0.001) in the number of alterations in OSCC and dysplasia compared to non-cancer samples respectively. The most frequently altered genes in OSCC were PTP4A3, RECQL4, ATM, and KLK3 (60%). Five genes (MYC, SLA, TNFRSF1A, MESDC1, MIF) were altered in 50% of OSCC samples. These nine genes were specific to OSCC samples (p<0.05). Some genes, including MYB, MET, CASP2, SLA and PTEN occurred in 50% of OED samples. MLPA was able to detect genetic alterations, that are present only in OSCC samples and showed potential to be used as an adjunctive tool in early diagnosis of OSCC.

An Easy-to-Use Approach to Detect CNV From Targeted NGS Data: Identification of a Novel Pathogenic Variant in MO Disease.

BackgroundDespite the new next-generation sequencing (NGS) molecular approaches implemented the genetic testing in clinical diagnosis, copy number variation (CNV) detection from NGS data remains difficult mainly in the absence of bioinformatics personnel (not always available among laboratory resources) and when using very small gene panels that do not meet commercial software criteria. Furthermore, not all large deletions/duplications can be detected with the Multiplex Ligation-dependent Probe Amplification (MLPA) technique due to both the limitations of the methodology and no kits available for the most of genes.AimWe propose our experience regarding the identification of a novel large deletion in the context of a rare skeletal disease, multiple osteochondromas (MO), using and validating a user-friendly approach based on NGS coverage data, which does not require any dedicated software or specialized personnel.MethodsThe pipeline uses a simple algorithm comparing the normalized coverage of each amplicon with the mean normalized coverage of the same amplicon in a group of “wild-type” samples representing the baseline. It has been validated on 11 samples, previously analyzed by MLPA, and then applied on 20 patients with MO but negative for the presence of pathogenic variants in EXT1 or EXT2 genes. Sensitivity, specificity, and accuracy were evaluated.ResultsAll the 11 known CNVs (exon and multi-exon deletions) have been detected with a sensitivity of 97.5%. A novel EXT2 partial exonic deletion c. (744-122)-?_804+?del —out of the MLPA target regions— has been identified. The variant was confirmed by real-time quantitative Polymerase Chain Reaction (qPCR).ConclusionIn addition to enhancing the variant detection rate in MO molecular diagnosis, this easy-to-use approach for CNV detection can be easily extended to many other diagnostic fields—especially in resource-limited settings or very small gene panels. Notably, it also allows partial-exon deletion detection.

Molecular Genetic Analysis of Patients with Duchenne/Becker Muscular Dystrophy by Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing Techniques

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular genetic disorders, which are characterized by mutations in the dystrophin gene. Large deletions or duplications in the dystrophin gene are found in approximately 60 to 70% of cases, and the remaining have point mutations, small deletions, or insertions. Objectives: The aim of this study was to assess mutations in the dystrophin gene in clinically suspicious subjects of DMD/ BMD by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS) techniques. Methods: This study consisted of 13 clinically suspected DMD/BMD patients. MLPA, NGS, and Sanger sequencing were used to determine mutations in the dystrophin gene. In order to minimize the time and cost, MLPA (SALSA P034/P035 DMD test kit) was performed as the first step to detect large deletions or duplications, followed by NGS and Sanger sequencing for MLPA-negative cases. Results: This study included nine males and four females with clinical suspicion of DMD or BMD. The MLPA was performed for all cases, among which ten cases were definitely diagnosed with DMD/BMD. NGS and Sanger sequencing were performed for the three remaining subjects to find out possible point mutations and small deletions, which could not be detected by MLPA. Nine intragenic deletions were identified, mostly in exons 46 to 47 and 4 to 7. Furthermore, an intragenic triplication was detected in exon 71 by MLPA. Conclusions: In agreement with other reports, our findings indicated that exonic deletions of the dystrophin gene are more frequent than duplications and point mutations in Duchenne/ Becker muscular dystrophy. Most deletions were demonstrated to occur in exons 46 to 47 and 4 to 7.

CNV Analysis Using Multiplex Ligation-Dependent Probe Amplification in Iranian Families with Non-Syndromic Congenital Heart Defects: Early Diagnosis of Non-Syndromic Patients

Background and Aims: Congenital heart defects (CHD) are the most common type of congenital disability. Copy number variations (CNVs) have been found as one of the genetic etiology of non-syndromic CHD, and researchers have detected several pathogenic CNVs in patients with cardiac defects.
 Materials and Methods: In the present study, 70 patients with familial (20 patients) and sporadic (50 patients) non-syndromic CHD were evaluated to find whether CNVs in the GATA4, NKX2-5, TBX-5, CREL, BMP4 genes, and 22q11.2 region contribute to the pathogenesis of non-syndromic CHD. We have used the Multiplex Ligation-dependent Probe Amplification (MLPA) technique as a molecular method to identify CNVs in predefined loci.
 Results: Normal MLPA results were demonstrated for GATA4, NKX2-5, TBX-5, CRELD, and BMP4 genes for all sporadic and familial cases. However, we found three patients with imbalances for the 22q11.2 region. One patient with 22q11.2 deletion showed tetralogy of fallot, and the other had ventricular septal defects/ pulmonary atresia/ multiple aortopulmonary collateral arteries. A duplication of the 22q11.2 region was detected in one patient with patent ductus arteriosus.
 Conclusion: Identifying genomic imbalances in 6% of the non-syndromic sporadic patients indicates that recurrent CNVs could be associated with non-syndromic CHD. It seems that it is the first CNV analysis using MLPA carried out in Iranian patients with cardiac defects. We suggest that 22q11.2 imbalances should be considered in patients with cardiac lesions to provide an accurate diagnosis and appropriate genetic counseling in affected families.

The Genetic Analysis of Cystic Fibrosis Patients With Seven Novel Mutations in the CFTR Gene in the Central Anatolian Region of Turkey.

Background Cystic fibrosis, a pulmonary disease which is an autosomal recessive, inherited, multisystemic genetic disease commonly seen in the Caucasian race, is the most frequent cause of mortality and morbidity. So far, more than 2000 disease-causing gene variants have been found and this number has been increasing with the studies conducted. Although there is not yet enough data that include the Turkish population, the recent increase of studies is noteworthy.Aims To discover the genetic variation in patients diagnosed with cystic fibrosis in the Central Anatolian region.Study Design Cross-sectional study.Methods The study was carried out in the Central Anatolian region in 3 pediatric pulmonology departments (Kayseri, Konya, and Ankara) in Turkey between July 2014 and December 2017. The Sanger and Next Generation Sequence analyses were used for exon and exon–intron boundaries in the cystic fibrosis transmembrane conductance regulatory (CFTR) gene, and in selected patients, mutation analysis was performed using the Multiplex Ligation-dependent Probe Amplification technique for large deletions and duplications.Results CFTR gene analysis was performed for 316 patients and 215 of them were genetically diagnosed with cystic fibrosis. Sixty-three different variants were defined in these patients and 7 of these were large deletions/duplications detected with the MLPA method. The most frequent variants were F508del (29.6%), G85E (8.2%), N1303K (8.2%), Y515* (7.5%), and G542* (3.4%).Conclusion Using sequencing and Multiplex Ligation-dependent Probe Amplification methods, the identification of seven new mutations that were not previously reported in the literature contributes to a better understanding of the heterogeneous nature of CFTR mutations in the Turkish population. When no mutations are detected (pathogenic/probably pathogenic) in clinically compatible cases, Multiplex Ligation-dependent Probe Amplification analysis contributes significantly to the diagnosis.

Cytogenomics Investigation of Infants with Congenital Heart Disease: Experience of a Brazilian Center.

ResumoFundamentoAlgumas síndromes têm características específicas e facilmente reconhecíveis, enquanto outras podem ser mais complexas de se identificar e podem apresentar diferentes manifestações fenotípicas, por exemplo. Um diagnóstico etiológico é importante para entender a natureza da doença, para estabelecer o prognóstico e para começar o tratamento, permitindo a inclusão de pacientes na sociedade e reduzindo o custo financeiro dessas doenças.ObjetivoA proposta inicial deste estudo foi a triagem citogenética para detectar a síndrome de deleção 22q11.2 (SD22q11.2) em recém-nascidos e crianças com doença cardíaca congênita utilizando a técnica da amplificação multiplex de sondas dependente de ligação (MLPA). Assim, por meio da pesquisa, outras mudanças genômicas foram identificadas nesses pacientes cardíacos. Nosso objetivo se estendeu a investigar essas outras mudanças citogenéticas.MétodosInvestigamos 118 recém-nascidos com doenças cardíacas congênitas nascidos consecutivamente durante um ano, utilizando a técnica da MLPA.ResultadosA técnica da MLPA permitiu a detecção da SD22q11.2 em 10/118 pacientes (8,5%). Outras alterações genômicas foram identificadas em 6/118 pacientes (5%): 1p36 del, 8p23 del (2 casos), 7q dup, 12 dup e 8q24 dup.ConclusãoEste estudo ressalta a relevância da detecção de alterações genômicas que estão presentes em recém-nascidos e crianças com doenças cardíacas congênitas por meio de ferramentas citogenômicas.

Comprehensive Analysis of the Tumoral and Microenvironmental Transcriptome of Lymph Nodes and Bone Marrow in Mantle Cell Lymphoma

The Reverse Transcription Multiplex Ligation-dependent Probe Amplification (RT-MLPA) technique is a method allowing for the semi-quantitative concomitant analysis of a number of transcripts of interest in cells or tissues. It relies on the amplification of complementary DNA (cDNA) derived from messenger ribonucleic acid (mRNA) after binding and ligation of specific probes on adjacent regions. It is thus applicable to the relatively fragmented low-quality RNA that can be derived from formalin-fixed paraffin embedded samples (FFPE), ill adapted to other methods such as RNASeq. Adequate tagging provides specific amplicon sizes for each marker, thus becoming readily identifiable after capillary electrophoresis. Signal normalization further allows to compare relative levels of transcripts in a given run.Here we report on the application of this method to explore the tumoral and microenvironmental, i.e. intrinsic and extrinsic, transcriptome of mantle cell lymphoma (MCL). Data were obtained from i)bone marrow (BM) and lymph nodes (LN) from normal individuals, ii)BM and LN from MCL patients at diagnosis (n=33) and iii)BM and LN from relapsing MCL patients (n=19). Samples were either frozen BM and frozen of FFPE LN. mRNAs were extracted and reverse-transcripted in cDNA before being processed for MLPA investigating for 17 transcripts of interest. The latter explored the intrinsic tumoral expression of CCND1, SOX11 and MKI67. The extrinsic parameters of the microenvironment investigated concerned the monocyte/macrophage compartment (CD14, CD163), T-cells (CD3E/CD3, CD8A/CD8), NK cells (CD94), the costimulation molecule CD40-ligand (CD40LG/CD40L) and immune-checkpoint molecules (CD152/CTLA4, PDC1/PD1, CD274/PDL1, PDCD1LG2/PDL2, INDO1/IDO) as well as cytokines (CSF1/MCSF , IL10, TGFB1).The samples studied were 39 MCL LN, 20 MCL BM, 10 non-tumoral LN and 9 normal BM.RT-MLPA readily differentiated MCL samples from non-tumoral samples by showing high levels of CCND1 and SOX11 transcripts in patients. Extrinsic markers expression, analyzed in principal component analysis (PCA) clearly segregated diseased and normal LN, while the difference was less clear in BM. Macrophage markers transcript levels were lower in patients' LN which also showed a down regulation of global T- and NK-cells yet increased levels of CD8 transcripts. PDL1 expression was higher in MCL BM compared to controls.LN from MCL patients with aggressive characteristics displayed lower CCND1 but higher MKI67 transcripts than other MCL patients' samples. These aggressive forms were found to be significantly associated with high CD14 transcripts in LN and high CD163 in BM. In addition, blastoid morphology was significantly associated with high CD14 (p=.005) and CD163 transcript levels in LN. Regarding T-cells, MCL aggressive forms had significantly lower amounts of CD3E transcripts, yet increased CD8 expression.LN from patients with a high proliferative index displayed higher levels of CD8 but also of CTLA-4, PD-1 and PDL1, suggesting that these T-cells are exhausted.A significant upregulation of the levels of CCND1 transcripts was observed in relapse LN. Conversely, CD163, CD3E, CD40L and CD94 levels were lower in relapsed samples compared to diagnosis, which could reflect immune depletion secondary to immuno-chemotherapy.This study confirms, in an original transcriptomic approach, the great heterogeneity of the MCL microenvironment. Besides being a useful tool at diagnosis, RT-MLPA successfully allowed for a concomitant approach of a large number of transcripts and thus a precise evaluation of intrinsic prognostic and extrinsic features of MCL. Important, yet seldom described, modulations of T-cells, NK cells, macrophages and cytokines were observed, with different profiles segregating MCL with aggressive morphology and those with resistance to treatment. DisclosuresNo relevant conflicts of interest to declare.

Copy number variants within AZF region of Y chromosome and their association with idiopathic male infertility in Serbian population.

Results of numerous studies gave contradictory conclusions when analysing associations between copy number variants (CNVs) within the azoospermia factor (AZF) locus of the Y chromosome and idiopathic male infertility. The aim of this study was to identify the presence and possible association of CNVs in the AZF region of Y chromosome with idiopathic male infertility in the Serbian population. Using the multiplex ligation-dependent probe amplification technique, we were able to detect CNVs in 24 of 105 (22.86%) infertile men and in 11 of 112 (9.82%) fertile controls. The results of Fisher's exact test showed a statistically significant difference between cases and controls after merging g(reen)-r(ed)/g(reen)-r(ed) and b(lue)2/b(lue)3 partial deletions identified in the AZFc region (p=0.024). At the same time, we observed a trend towards statistical significance for a deletion among gr/gr amplicons (p=0.053). In addition to these, we identified a novel complex CNV involving inversion of r2/r3 amplicons, followed by b2/b3 duplication and b3/b4 deletion, respectively. Additional analyses on a larger study group would be necessary to draw meaningful conclusions about associations among CNVs that presented with higher frequency in the infertile men than the fertile controls.

P59.24 Genomic Abnormalities in Malignant Pleural Effusion of Lung Cancer Patients Detected by Multiplex Ligation-Dependent Probe Amplification

Malignant pleural effusion related to lung cancer (MPE-LC) is a common occurrence in medical practice and its diagnosis is a challenge for clinical laboratories. It is known that most of metastatic tumors to the pleura present chromosomal gains and losses, which can be detected by cytogenomic techniques such as the Multiplex Ligation-dependent Probe Amplification (MLPA). The aim of this study is to evaluate genomic abnormalities in tumor cells from pleural fluid (PF) of patients with lung cancer (LC) by MLPA technique. We evaluated 23 samples of PF: 16 samples from MPE of LC patients (13 with positive cytology, 2 negative and one suspicious) and 7 samples from patients with benign PE (cardiac transudate, parapneumonic and tuberculosis). DNA was extracted using the QIAMP DNA Blood kit (QIAGEN, Valencia, California) and the cytogenomic analysis was performed using the MLPA P175-B1 Tumor Gain kit (MRC-Holland®, Amsterdam, Netherlands), in search of pathogenic CNVs and specific point of BRAF mutations. The results were analyzed with the GeneMarker® software (SoftGenetics, LLC, State Collage, PA). From the 16 cases of MPE-LC, MLPA identified genomic changes in 7 cases with cancer cells in the PF: 1 case with point mutation BRAF (7q34) p.V600E (c.1799T>A) and MDM2 (12q15) duplication; 1 with CDK4 (12q14), MDM2 (12q15) and CCND1 (11q12) duplications (fig 1); 1 with AURKB (17p13) deletion; 1 with AURKB (17p13) deletion and AURKA (20q13), MDM4 (1q32) duplications; 1 with EGFR (7p11), ERBB2 (17q12) and TOP2A (8p12) duplications; 1 with MDM2 (12q15) duplication and 1 case with FGFR1 (8p12) deletion, MDM2 (12q15), CCND2 (12p13) duplications and point mutation BRAF (7q34) p.V600E (c.1799T>A). MLPA analysis of control group showed normal results (fig2). The MLPA technique detected genomic abnormalities in seven MPE-LC samples, when at least 30% of tumor cells were present in the cytological examination. The findings of pathogenic variants in MPE-LC can be valuable to explore new targets for the treatment of lung cancer patients.

Detection of Del/Dup Inside SHOX/PAR1 Region in Children and Young Adults with Idiopathic Short Stature.

Short stature is a common growth disorder defined as a body height two standard deviations (SD) or more below the mean for a given age, gender, and population. A large part of the cases remains unexplained and is referred to as having idiopathic short stature (ISS). One of the leading genetic causes of short stature is variants of short stature homeobox-containing gene (SHOX) and is considered to be responsible for 2–15% of ISS. We aimed to analyse the regulatory and coding region of SHOX in Slovenian children and young adults with ISS and to investigate the pathogenicity of detected variants. Our cohort included 75 children and young adults with ISS. Multiplex ligation-dependent probe amplification (MLPA) was performed in all participants for the detection of larger copy number variations (CNVs). Sanger sequencing was undertaken for the detection of point variants, small deletions, and insertions. A total of one deletion and two duplications were discovered using the MLPA technique. Only one of these four variants was identified as disease-causing and occurred in one individual, which represents 1.3% of the cohort. With Sanger sequencing, two variants were discovered, but none of them appeared to have a pathogenic effect on height. According to the results, in the Slovenian population of children and young adults with ISS, SHOX deficiency is less frequent than expected considering existing data from other populations.

Isolated Macroprolactinomas in Four Young Adult Males Harboring a Variant of the Aryl Hydrocarbon Interacting Protein (AIP) Gene: Isn’t It Too Much of a Coincidence?

Background: Germline mutations in the Aryl hydrocarbon receptor-Interacting Protein (AIP) gene are associated with pituitary adenomas in young patients usually in the setting of Familial Isolated Pituitary Adenomas (FIPA). The majority of these adenomas are somatotropinomas followed by prolactinomas, and rarely non-secreting adenomas. AIP-mutation-related prolactinomas predominantly affect men, as opposed to sporadic prolactinomas, that typically affect women. Clinical Case: We previously described an AIP gene mutation in two patients affected by prolactinomas. During the past years, we continued our study and have identified two more male patients with macroprolactinomas originally from the same small village and harboring the same AIP gene mutation. These male patients aged 19 to 44 years at the time of diagnosis. Two of them had neurological manifestations as the first clinical manifestation of the disease, one was studied because of hypogonadism and two patients had visual field defects. All of them had prolactin levels above 1000 ng/dl (mean 2946.5±948.7 ng/dl, reference range 10-21). In the imaging exams (CT/MRI) they presented pituitary adenomas larger than 20 mm (macroprolactinomas) and in two of the cases, the adenomas were even larger than 40 mm (giant prolactinomas). In order to exclude mutations most often associated with prolactinomas, DNA samples were obtained and analyzed by Next Generation Sequencing (NGS) using TruSightCancer Gene Set (Illumina) methodology. Investigation of significant deletions and/or duplications was performed using the MLPA (Multiplex ligation-dependent probe amplification) technique. None of the patients were positive for mutations of Multiple Endocrine Neoplasia type 1 (MEN1) gene. A variant of the AIP gene c.47G>A, expecting to lead to a substitution of arginine by histidine at position 16 (p.Arg16His) of the AIP was found in these four patients, including a father and his son. Seven asymptomatic carriers were identified among their first-degree relatives. In silico analysis and the information available in the literature, as well as in databases is not in agreement with the pathogenicity of this variant of the AIP gene. However, our findings point to a founder effect transmitted as a dominant trait with incomplete penetrance (4 out of 11 patients, 36%). Conclusion: The variant of the AIP gene identified in our patients behaved as a pathogenic mutation and was only associated with prolactinomas, including two giant prolactinomas.

Detection of Aneuploidies in Products of Conception and Neonatal Deaths in Iranian Patients Using the Multiplex Ligation-Dependent Probe Amplification (MLPA).

Background:Around 70% of all pregnancies (Including 15% of clinically-recognized ones) are lost due to various fetal or maternal disorders. Chromosomal aneuploidies are among the most common causes of pregnancy loss. Standard chromosome analysis using G-banding technique (Karyotype) is the technique of choice in studying such abnormalities; however, this technique is time-consuming and sensitive, and limited by vulnerabilities such as cell culture failure. The use of molecular cytogenetic techniques, including array-based techniques and Multiplex Ligation-Dependent Probe Amplification (MLPA), has been proposed to overcome the limitations of this method to study the products of conception. This study has been designed to investigate the feasibility of using MLPA technique as a standalone genetic testing, with histopathologic examinations and genetic counseling to detect aneuploidies in products of conception and neonatal deaths.Methods:Forty-two verified fetal and neonatal samples were studies and genetic counseling was scheduled for all parents. Histopathologic examinations were carried out on the products of conception, and appropriate fetal tissues were separated for genetic studies. Following DNA extraction and purification, MLPA was carried out to investigate chromosomal aneuploidies.Results:Nine samples (21.42%) were diagnosed to be affected with aneuploidy. Detected aneuploidies were trisomy 22 (n=3), trisomy 21(n=1), trisomy 18 (n=2), trisomy 16 (n=1), trisomy 13 (n=1), and monosomy of chromosome X (n=1). The MLPA analysis results were conclusive for all of the fetal samples (Success rate: 100%).Conclusion:These results suggest that MLPA, as a standalone genetic testing, is an accurate, rapid, and reliable method in overcoming the limitations of standard cytogenetic techniques in genetic investigation of products of conception.

A Pilot Study of Identification Genetic Background of Craniosynostosis Cases.

The early fusion of the cranial sutures was described as a craniosynostosis. The early diagnosis and management of craniosynostosis is very important. Environmental factors and genetic abnormalities plays a key role during the development of craniosynostosis. Syndromic craniosynostosis cases are related with autosomal dominant disorders but nearly half of the affected cases carry a new mutation. In this study, in order to identify the genetic etiology of craniosynostosis the authors analyzed 20 craniosynostosis patients by using conventional karyotype, aCGH, sanger sequencing, next generation sequencing (NGS) and Multiplex ligation-dependent probe amplification (MLPA) techniques. The authors identified mutations on FGFR2 and FGFR3 genes which were associated with Muenke syndrome, Crouzon syndrome and skeletal dysplasia syndromes. NGS applied all of the cases and 7 clinical variations in 5 different gene were detected in %20 of cases. In addition to these abnormalities; del(11)(q14.1q22.2), del(17)(q21.31), dup(22)(q13.31) and t(2;16)(q37;p13) have been identified in our cohort which are not previously detected in craniosynostosis cases. Our study demonstrates the importance of detailed genetic analysis for the diagnosis, progression and management of the craniosynostosis.