Multiplex Ligation-dependent Probe Amplification Method Research Articles

Mutation spectrum of PAH gene in phenylketonuria patients in Northwest China: identification of twenty novel variants.

This study was performed to analyze the mutational spectrum of the phenylalanine hydroxylase (PAH) gene in phenylketonuria (PKU) patients in Northwest China, to identify mutational hot spots, and to determine the correlation between variants and clinical phenotypes of PKU. A large cohort of 475 PKU families in Northwest China was enrolled to analyze PAH gene variants using Sanger sequencing, Multiplex ligation-dependent probe amplification (MLPA), and gap-PCR. Bioinformatics software was used to predict the pathogenicity of novel variants and analyze the correlations between PAH gene variants and phenotypes of PKU patients. A total of 895 variants were detected in the 950 alleles of 475 patients with PKU (detection rate: 94.21%), 20 of which were novel variants. Other 108, previously known variants, were also identified, with the three most frequent variants being p.Arg243Gln (14.00%), c.611A > G (5.58%), and p.Tyr356* (4.95%). Seven different large deletion/duplication variants were identified by the MLPA method, including the large deletion c.-4163_-406del3758 with high frequency. A correlation analysis between patient phenotype and gene variant frequency showed that p.Arg53His and p.Gln419Arg were correlated with mild hyperphenylalaninemia (MHP). In conclusion, the mutational spectrum underlying PKU in Northwest China was established for the first time. Functional analysis of 20 novel PAH gene variants enriched the PAH gene mutational spectrum. Correlation analysis between variants frequencies in compound heterozygous patients and phenotype severity is helpful for phenotypic prediction.

Molecular Evaluation of Children with Clinical Russell-Silver Phenotypes: The First Report From Iran

Background: Russell-Silver syndrome is a rare heterogeneous genetic disorder that is mostly known because of its prenatal and postnatal growth retardation. Patients with Russell-Silver syndrome have syndromic facial appearance, as well as some other common clinical features. Disrupted methylation of 11p15 is the most common genetic abnormality that is seen in these patients, called as one of the tire molecular studies in diagnostic guidelines. Objectives: In the present study, the methylation status of 11p15 was evaluated in a group of children with clinical diagnosis of Russell-Silver syndrome in Iran. Methods: A total number of 15 children with a clinical diagnosis of Russell-Silver syndrome were enrolled in this descriptive study. Children’s DNA was extracted by the salting-out method and the 11p15 region was assessed by the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method. The correlation of molecular results was then evaluated with clinical features. Results: The mean age of children was 4.5 years and most of them were male. Among 15 children, four children had a confirmed molecular diagnosis of Russell-Silver syndrome according to the MS-MLPA results. All of these four patients had low set ears, high peach voice, micrognathia, failure to gain weight, growth failure, and triangular face. Conclusions: Less than one-third (26.6 ) of our patients had confirmed Russell-Silver syndrome by the MLPA analysis. This experiment showed that Russell-Silver syndrome with abnormal 11p15 methylation was less frequent in our population and showed similar clinical findings in comparison with other studies. © 2019, Author(s).

STUDY ON APPLICATION OF MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA) ASSAY IN MOLECULAR DIAGNOSIS OF RETINOBLASTOMA

Retinoblastoma (Rb) is a malignant retinal tumor on young children which is often founded before the age of 5. This cancer disease appears when both of RB1 alleles on 13q14.2 chromosome are mutated. The aim of this research is to evaluate the ability of Multiplex Ligation-dependent Probe Amplification (MLPA) in screening of RB1 gene insertion/deletions in Vietnamese patients with Rb. Genomic DNA was isolated from peripheral blood of the research subjects and subsequently analyzed by MLPA technique. To prove the results of MLPA, quantitative real-time PCR was used for determining the RB1 gene copy number for all samples. Two significant deletion mutations were identified on two different Rb patients, one is the deletion from exon 4 to exon 27 recorded on KVM38 sample, and the other is the complete removal of an allele on KVM21 sample. The MLPA showed a complete correlation with real-time PCR results. These are the disease causing mutations, can be inherited and they are important evidences for genetic counseling and clinical management. Those results have proven the high speed and reliability of MLPA method in identifying deletion/duplication mutations on Rb patients.

A loss-of-function mutation p.T52S in RIPPLY3 is a potential predisposing genetic risk factor for Chinese Han conotruncal heart defect patients without the 22q11.2 deletion/duplication

BackgroundConotruncal heart defect (CTD) is a complex congenital heart disease with a complex and poorly understood etiology. The transcriptional corepressor RIPPLY3 plays a pivotal role in heart development as a negative regulator of the key cardiac transcription factor TBX1. A previous study showed that RIPPLY3 contribute to cardiac outflow tract development in mice, however, the relationship between RIPPLY3 and human cardiac malformation has not been reported.Methods615 unrelated CTD Chinese Han patients were enrolled, we excluded the 22q11.2 deletion/duplication using a modified multiplex ligation-dependent probe amplification method—CNVplex®, and investigated the variants of RIPPLY3 in 577 patients without the 22q11.2 deletion/duplication by target sequencing. Functional assays were performed to testify the potential pathogenicity of nonsynonymous variants found in these CTD patients.ResultsFour rare heterozygous nonsynonymous variants (p.P30L, p.T52S, p.D113N and p.V179D) were identified in four CTD patients, the variant NM_018962.2:c.155C>G (p.T52S) is referred as rs745539198, and the variant NM_018962.2:c.337G>A (p.D113N) is referred as rs747419773. However, variants p.P30L and p.V179D were not found in multiple online human gene variation databases. Western blot analysis and immunofluorescence showed that there were no significant difference between wild type RIPPLY3 and these four variants. Luciferase assays revealed that the p.T52S variant altered the inhibition of TBX1 transcriptional activity in vitro, and co-immunoprecipitation assays showed that the p.T52S variant reduced the physical interaction of RIPPLY3 with TBX1. In addition to the results from pathogenicity prediction tools and evolutionary protein conservation, the p.T52S variant was thought to be a potentially deleterious variant.ConclusionOur results provide evidence that deleterious variants in RIPPLY3 are potential molecular mechanisms involved in the pathogenesis of human CTD.

Clinical presentation and mutational spectrum in a series of 166 patients with classical 21-hydroxylase deficiency from South China

Classical 21-hydroxylase deficiency (21-OHD) due to mutations in the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene is the most common type of congenital adrenal hyperplasia (CAH). In this study, we analyzed clinical and molecular data of 166 patients with classical CAH in South China. Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) method were used to detect mutations in these 99 salt wasting (SW) patients and 67 simple virilizing (SV) patients. Micro-conversion mutation IVS2-13A/C > G (I2G) was the most frequent mutation in both SW form (42.9%) and SV form (41.8%) in our large cohort, and large gene deletion or large gene conversion also commonly resulted in classical CAH. Rare mutations only account for 8.4% of all alleles, among them four novel variants p.S126X, p.C429X, c.1209_1210insT and c.840delG were responsible for the clinical presentations. CYP21A2 gene duplications linked to the mutation Q319X were found in our cohort, though these cases were rather rare. In this study, we provided detailed clinical data and mutation spectrum to confirm the common mutations in Chinese populations, especially in South China,which will contribute to further genetic consultation and prenatal diagnosis. Sanger sequencing combined with MLPA method could detect most mutation types in the CYP21A2 gene effectively.

Abstract 557: Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia

Abstract Introduction: Not only somatic mutations, but also epigenetic modifications in acute myeloid leukemia (AML) are estimated to have a pivotal role in leukemogenesis and it could be a therapeutic target. Especially, aberrant methylation of the promotor region of tumor suppressor genes (TSGs) were the causative events in leukemogenesis, however, there was only limited information in pediatric myelodysplastic syndrome (MDS) and AML. Materials and Methods: We analyzed the aberrant methylation of promotor region of 25 TSGs in pediatric 7 low grade MDS, 4 advanced MDS and 22 AML patients by FAB classification by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) method (10 Male, 12 female, 0-14 years old, median 6 years ols). Simultaneously, detection of SNVs, In/del, and copy number variants were also analyzed by target sequence of more than 150 leukemia related genes. Especially, specific chimeras were analyzed in each patient. Results: Total 12/22(54.5%) patients had more than one methylated TSGs (number of methylated TSGs was 0-5 in each patient, median 1). More than 20% of patients had methylated TSGs including CDKN2B, CADM1, CDH13 and ESR1. Especially, there was a specific methylation pattern of TSGs, for example, all (4/4) t(8;21) patients had CDH13 methylation, 3/4 (75%) patients with inv(16) had ESR1 mutation and normal karyotype did not. Normal control samples, complete remission samples, and low grade MDS samples were not completely methylated. All advanced MDS patients had 1 or 2 methylated TSGs. Simultaneously, several leukemogenic genes were mutated such as GATA1, IKZF1, KRAS, KIT, FLT3, SH2B3, PIK3CD, PU.1, SMARC1, ALK, etc. Several genes were deleted such as CTCF, RB1, SRP72, ATM, NF1, U2AF1 etc. Discussion: In adult MDS, the increased number of methylated TSGs correlated to the development of AML and the poor prognosis. In this study, a few TSGs were methylated as the adult study (Hess CJ., Leuk Lymphoma 2008), furthermore, many genes related to epigenetic modification such as ASXL1, TET2 and EZH2 were not mutated in this study. Recently, anti-methylation drug were used in adult MDS/AML, larger study will be needed for adjusted use of epigenetic modifier in pediatric MDS/AML. Citation Format: Akira Shimada, Hirotaka Kanzaki, Hisashi Ishida, Takehiro Matsubara, Michinori Aoe. Aberrant methylation of tumor-suppressor genes in pediatric myelodysplastic syndrome/acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 557.

Prevalence of BRCA1/2 large genomic rearrangements in Chinese women with sporadic triple-negative or familial breast cancer.

The prevalence of BRCA1/2 large genomic rearrangements (LGRs) and their underlying mechanisms have not been fully evaluated in Chinese women with breast cancer. In this study, we determined the prevalence of BRCA1/2 LGRs in 834 patients with familial breast cancer (FBC) and 660 patients with sporadic triple-negative breast cancer (TNBC) who were negative for BRCA1/2 small-range mutations using the multiplex ligation-dependent probe amplification method. We found that 20 index patients (2.4%) in the FBC group carried a BRCA1 or BRCA2 LGR, and the frequencies of BRCA1 and BRCA2 LGRs were 1.6% and 0.8%, respectively. Seven index patients (1.1%) carried a BRCA1 LGR in 660 sporadic TNBC patients, whereas no BRCA2 LGRs were found in these patients. Among the BRCA1/2 LGRs, 48.1% (13/27) were novel, and the breakpoints of the majority of the LGRs were identified. ΨBRCA1-mediated homologous recombination (HR) and Alu-mediated HR/non-homologous end-joining (NHEJ) accounted for 40% and 30% of the BRCA1 LGRs, respectively. Alu-mediated HR accounted for 71.4% of the BRCA2 LGRs, and the remaining one-third was generated through Long interspersed nuclear elements (LINE)-mediated NHEJ. Our findings suggest that both FBC patients and sporadic TNBC patients should be tested for BRCA1/2 LGRs.

Mutation analysis and prenatal diagnosis for 50 pedigrees affected with Duchenne/Becker muscular dystrophy

To establish individualized prenatal diagnosis program for families affected with Duchenne/Becker muscular dystrophy (DMD/BMD) and different clinical background using a variety of methods. Multiplex ligation-dependent probe amplification (MLPA) was performed on 50 patients suspected for DMD/BMD. For single exon deletions of the DMD gene, PCR was used for validating the results. For those without any deletion or duplication, Sanger sequencing was used to screen for DMD gene mutations in the children and their mothers. Prenatal genetic testing was provided to female carriers using chorionic villus, amniocentesis or cord blood samples. To ensure the accuracy of diagnosis, all prenatal specimens were also subjected to linkage analysis. Among the 50 patients with DMD/BMD, 23 harbored large deletions, 11 only had single exon deletions, 10 harbored duplications, and 5 had small scare mutations. No mutation was detected in one family. For 37 women undergoing prenatal diagnosis, 10 fetuses were identified as affected males, 6 were female carriers, while 21 were not found to carry any mutation. Testing of creatine kinase was consistent with the results of prenatal diagnosis. For a patient harboring exon 51 deletion, the same mutation was found in a fetus but not in their mother. The proband and fetus had inherited the same haplotype, which suggested that the mother probably has germline mosaicism for the mutation. Application of individualized methods for analyzing pregnant women with different clinical background can minimize the risk for giving birth to further children affected with DMD/BMD.

Simultaneous detection of ABL1 mutation and IKZF1 deletion in Philadelphia chromosome-positive acute lymphoblastic leukemia using a customized target enrichment system panel.

Recent clinical outcomes of pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) vastly improved owing to tyrosine kinase inhibitor (TKI). However, the genetic status would be different in each case with ABL1 gene mutation or copy number variants (CNVs) such as IKZF1 deletion. In particular, the TKI resistant clone with ABL1 kinase mutation remains problematic. The comprehensive assessment of genetic status including mutation, insertion and deletion (indel) and CNVs is necessary. We evaluated a next-generation sequencing (NGS)-based customized HaloPlex target enrichment system panel to simultaneously detect coding mutations, indel and CNVs. We analysed approximately 160 known genes associated with hematological disorders in 5 pediatric Ph+ALL patients. Mono-allelic IKZF1 deletions were found in 4 patients at diagnosis. Furthermore, the mono-allelic deletions were found in exons of RB1, EBF1, PAX5 and ETV6 genes. Bi-allelic deletions were detected in CDKN2A and CDKN2B genes in 1 patient. ABL1 mutation was also detected in 1 patient at relapse. These results were almost comparable with the results of the multiplex ligation-dependent probe amplification (MLPA) method or Sanger sequence. Next-generation sequencing-based custom HaloPlex target enrichment system panel allows us to detect the coding mutations, indel, and CNVs in pediatric Ph+ALL simultaneously, and its results seem comparable with those of other methods.

TIMP3 Promoter Methylation Represents an Epigenetic Marker of BRCA1ness Breast Cancer Tumours.

Tumours presenting BRCAness profile behave more aggressively and are more invasive as a consequence of their complex genetic and epigenetic alterations, caused by impaired fidelity of the DNA repair processes. Methylation of promoter CpG islands represents an alternative mechanism to inactivate DNA repair and tumour suppressor genes. In our study, we analyzed the frequency of methylation changes of 24 tumour suppressor genes and explored their association with BRCAness profile. BRCA1ness profile and aberrant methylation were studied in 233 fresh frozen breast tumour tissues by Multiplex Ligation-dependent Probe Amplification (MLPA) and Methylation Specific (MS)-MLPA methods, respectively. Our analyses revealed that 12.4% of the breast cancer (BC)patients had tumours with a BRCA1ness profile. TIMP3 showed significantly higher (p = 5.8х10-5) methylation frequency in tumours with BRCA1ness, while methylation of APC, GSTP1 and RASSF1 promoters was negatively associated with BRCA1ness (р = 0.0017, р = 0.007 and р = 0.046, respectively). TIMP3 methylation was also associated with triple negative (TN) BC. Furthermore, TN tumours showing BRCA1ness showed stronger association with TIMP3 methylation (p = 0.0008) in comparison to TN tumours without BRCA1ness (p = 0.009). In conclusion, we confirmed that TIMP3 methylation is a marker for TN tumours and furthermore we showed for the first time that TIMP3 promoter methylation is an epigenetic marker of BRCA1ness tumours.

Prognostic significance of copy number alterations detected by multi-link probe amplification of multiple genes in adult acute lymphoblastic leukemia.

The multiplex ligation-dependent probe amplification (MLPA) method was used to detect the copy number alterations (CNAs) of IKAROS family zinc finger 1 (IKZF1), paired box 5 (PAX5), ETS variant 6 (ETV6), RB transcriptional corepressor 1 (RB1), BTG anti-proliferation factor 1 (BTG1), early B-cell factor 1 (EBF1), cyclin dependent kinase inhibitor 2A/2B (CDKN2A/2B) and cytokine receptor like factor 2 (CRLF2) genes in 87 adults with acute lymphoblastic leukemia (ALL) in China. The effects of CNAs on prognosis were analyzed. Gene deletions were detected in 58/87 (66.7%) ALL patients. The most common deletions were observed in the following genes: IKZF1 (40.6%), CDKN2A (31.9%), CDKN2B (29%), PAX5 (21.7%), RB1 (14.5%) and BTG1 (10.1%). B cell-ALL (B-ALL) patients with CDKN2A/2B deletions exhibited poor 2-year overall survival (OS; P=0.055) and relapse-free survival (RFS; P=0.054) rates. CDKN2A/2B deletions were associated with poor 2-year OS (P=0.045) and RFS (P=0.071) rates in Philadelphia chromosome positive (Ph+) B-ALL patients, as well as in the high risk (HR) B-ALL group (P=0.037 and P=0.047, respectively). Patients with PAX5 deletions displayed poor 2-year OS (P=0.004) and RFS (P=0.016) rates in Philadelphia chromosome negative (Ph-) B-ALL patients. Patients with ≥3 gene deletions exhibited a poorer prognosis than other patients (OS, P=0.001; RFS, 0.002).

Clinical and Molecular Risk Factors for Glioblastoma Multiforme in the Era of New WHO CNS Tumors Classification

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and its prognosis, despite the best efforts, remains poor. Taking into consideration the paucity of reliable clinical predictors in patients with GBM and because of the large molecular heterogeneity of cases, increasing importance is given to molecular subclassifications of GBM. Limited findings in gliomas biology (e.g. MGMT methylation or 1p/19q codeletion) now allow the identification of a group of patients with a better prognosis. The aim of our study was to analyze the influence of selected clinical factors and molecular prognosis in patients with GBM. The study group consisted of 244 patients treated for GBM in the years 2007-2015. This cohort was retrospectively analysed in order to evaluate the influence of selected clinical and molecular factors on patients overall survival (OS). The assessment of molecular abnormalities was done on DNA was extracted from formalin-fixed and paraffin-embedded tissue with utilization of multiplex ligation-dependent probe amplification method and Sanger sequencing. The study group consisted of 103 women (42.21%) and 141 men (57.79%). The average age of the patients was 55.3 ± 10.7 years. Higher preoperative Karnofsky Performance Scale scores (HR=0.89, 95% CI: 0.87-0.91; p <0.001) and concurrent radiochemotherapy with temozolomide (HR=0.38, 95%CI: 0.25-0.61; p<0.001) were associated with a better prognosis for patients. Higher age at diagnosis was found to be significantly associated with worse prognosis - HR=1.06 (95% CI: 1.04-1.07); p <0.001. In terms of molecular markers, we have shown that the incidence of chromosome 7 polysomy significantly worsened prognosis (HR=2.37, 95% CI: 1.20-5.84; p = 0.022) while EGFRvIII expression was associated with a better prognosis (HR = 0.71, 95%CI: 0.54-0.97; p = 0.041). Despite many years of research, GBM remains the most lethal tumor of the central nervous system with very poor natural history. The results of our study indicate that the modern classification of gliomas should take into account both clinical and molecular factors thereby allowing more precise diagnosis and tailored therapy in this group of patients.

The correlation between Pax5 deletion and patients survival in Iranian children with precursor B-cell acute lymphocytic leukemia.

Despite advances in treatment, children with acute lymphoblastic leukemia (ALL) still experience drug resistance and relapse. Several gene mutations are involved in the onset of this disease and resistance to therapy. The present study examines the incidence of IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, JAK2, and Xp22.33 gene deletions/duplications associated with pediatric ALL in Iran and investigates the possible effect of these mutations on drug resistance. Three-year disease-free survival (3DFS) was evaluated for children diagnosed with Philadelphia negative precursor-B-cell ALL hospitalized at Sayed-al-Shohada Hospital, Isfahan-Iran, from January 2009 until December 2012. DNA was extracted from bone marrow slides, and ALL correlated gene deletions and duplications were measured using Multiplex Ligation-dependent Probe Amplification (MLPA) method. The correlation between gene mutations and 3DFS was then assessed. Among the nine aforementioned investigated genes, 63% of samples showed at least one gene mutation. At least two concomitant genomic mutations were observed in 42% of samples. Pax5 deletion was the most prevalent gene mutation observed in 45% of cases, and showed significant negative impact on response to treatment. CDKN2A/B (9p21.3) gene deletion, and ETV6 (12p13.2) gene duplication also demonstrated negative effect on patient survival and contributed to a worse prognosis if concomitant with Pax5 gene deletion. ALL patients with one of the gene deletions including Pax5 and CDKN2A/B (9p21.3) or ETV6 (12p13.2) gene duplication are classified as high-risk patients and need more intensified protocols of treatment to improve their chance of survival.

The prevalence of SHOX gene deletion in children with idiopathic short stature. A multicentric study

The isolated haploinsufficiency of the SHOX gene is one of the most common cause of short stature determined by monogenic mutations. The heterozygous deviation of the gene can be detected in 2-15% of patients with idiopathic short stature (ISS), in 50-90% of patients with Leri-Weill dyschondrosteosis syndrome (LWS), and in almost 100% of patients with Turner syndrome. The aim of our study was to evaluate the frequency of SHOX gene haploinsufficiency in children with ISS, LWS and in patients having Turner syndrome phenotype (TF), but normal karyotype, and to identify the dysmorphic signs characteristic for SHOX gene deficiency. A total of 144 patients were included in the study. Multiplex Ligation-dependent Probe Amplification (MLPA) method was used to identify the SHOX gene haploinsufficiency. The relationships between clinical data (axiological parameters, skeletal disorders, dysmorphic signs) and genotype were analyzed by statistical methods. 11 (7.6%) of the 144 patients showed SHOX gene deficiency with female dominance (8/11, 81% female). The SHOX positive patients had a significantly higher BMI (in 5/11 vs. 20/133 cases, p<0.02) and presented more frequent dysmorphic signs (9/11vs 62/133, p = 0.02). Madelung deformity of the upper limbs was also significantly more frequent among the SHOX positive patients (4/11, i.e. 36%, vs. 14/133, i.e. 10%, p = 0.0066). There were no statistically significant differences between the mean age, mean height and auxological measurements (sitting height/height, arm span/height) between the two groups of patients. The occurrence of SHOX gene haploinsufficiency observed in our population corresponds to the literature data. In SHOX positive patients, in addition to short stature, the dysmorphic signs have a positive predictive value for SHOX gene alterations. However, the SHOX deletion detected in a patient with idiopathic short stature without dysmorphic signs suggest that SHOX deletion analysis can be recommended in patients with ISS. Orv Hetil. 2017; 158(34): 1351-1356.

A Clinical and Molecular Genetic Study in 11 Chinese Children With Peutz-Jeghers Syndrome.

Peutz-Jeghers syndrome (PJS) is caused by the germline mutations in serine/threonine kinase 11 (STK11) gene. The aim of the present study was to investigate the spectrum of STK11 gene mutations using multiplex ligation-dependent probe amplification (MLPA) assay in combination with direct sequencing in Chinese children with PJS. Nine children who met the clinical criteria for PJS and 2 presumed patients with PJS were enrolled in the present study. Patients' clinical information on polyp characteristics, polyp-related complications, family histories, and so on were reviewed and analyzed. After obtaining informed consent, we performed a mutation analysis of STK11 gene in 11 Chinese patients using MLPA assay and direct sequencing. By means of MLPA method, we detected exonic deletions in 5 patients. In details, 1 patient had the complete deletion of all 10 exons, 3 patients showed deletions of promoter region and exon 1, and 1 patient had exon deletions from 1 to 9. By direct sequencing of the coding region of STK11 gene, we identified point mutations in 4 patients at c.548T>G/p.Leu183Arg, c.580G>T/p.Asp194Tyr, c.152_153insGG/Asp53GlyfsX12, and c.631delC/Arg211GlyfsX76, respectively, and 3 of them are novel mutations. We failed to find any mutation in left 2 patients who met the clinical criteria of PJS. MLPA plus direct sequencing revealed large genomic deletions of STK11 gene in Chinese children with PJS and increased the detecting rate of STK11 gene mutations in Chinese patients with PJS. MLPA combined with direct sequencing could serve as a better strategy for the genetic diagnosis of PJS in Chinese population.

Evidence of metachronous development of ovarian teratomas: a case report of bilateral mature cystic teratomas of the ovaries and systematic literature review

BackgroundMature cystic teratomas are usually found in the ovaries. They are bilateral in 10 to 15% of cases and multiple cystic teratomas may be present in one ovary. The aim of this study is to clarify if development of mature cystic teratomas of the ovaries in a single host is metachronous or due to autoimplant or recurrence.Case presentationWe report a woman with bilateral mature cystic teratomas of the ovaries. DNA profiles of these teratomas were investigated via short tandem repeat (STR) analysis and methylation statuses were determined via methylation sensitive multiplex ligation-dependent probe amplification methods. The results showed that the cystic teratomas originated from different stages of oogonia or primary oocyte before germinal vesicle stage failure of meiosis I in female gametogenesis.Potentially relevant literature was searched in PubMed database. Cases of bilateral or multiple mature cystic teratomas of the ovaries were analyzed. To date, there has been no reported case of multiple mature cystic teratomas in which clarification of the origin was achieved using molecular genetic methods.ConclusionsThe results of this case study provide evidence of metachronous development of mature cystic teratomas of the ovaries and may serve as a reference in the management of patients following laparoscopic cystectomy.

The Utilization of Karyotyping, iFISH, and MLPA for the Detection of Recurrence Genetic Aberrations in Multiple Myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by abnormal accumulation of clonal plasma cells in the bone marrow. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for detection of common genetic alterations in MM patients. Here, we aimed to confirm MLPA utility for this purpose and furthermore to test the feasibility of a combination of karyotyping, interphase fluorescence in situ hybridization (iFISH) and MLPA methods for diagnosis, prognostic assessment and risk stratification of MM. Thirty-five genomic DNA samples isolated from CD138-enriched plasma cells from bone marrow of MM patients were analyzed using the MLPA method. We found that amp (1q) was the most frequent genetic alteration (48.6%) in the tested samples, followed by del (1p) and del (13q) (34.3%). Moreover, concordant results between sensitivity and specificity of iFISH and MLPA for the detection of del (13q) (p-value >0.05) and del (17p) (p-value >0.05) were obtained. In summary, we could provide evidence of MLPA assay utility for the detection of common genetic alterations in MM. The combination of karyotyping, iFISH, and MLPA proved very helpful for clinical risk stratification.

Ankle-Foot Orthosis in Duchenne Muscular Dystrophy: A 4year Experience in a Multidisciplinary Neuromuscular Disorders Clinic.

To assess Ankle-Foot-Orthosis (AFO) requirement and ambulation in Duchenne Muscular Dystrophy (DMD) patients seen over a period of 4 y at a multi-disciplinary Neuromuscular disorders clinic (NMD). A study was conducted in university quaternary research hospital with DMD patients confirmed by MLPA (multiplex ligation - dependent probe amplification) method and were evaluated between January 2012 and December 2015. Their ambulatory status, detailed neurological and functional status were recorded. Requirement of AFOs was determined and provided. In total 126 DMD children reported to the NMD clinic. Mean age at presentation was 7.6 y (range 2 to12 y, SD 2.1). Mean duration of illness at first evaluation was 3.4 y (range 0.5 to 10 y, SD 2.0). AFO's were advised at a mean age of 8.5 y (range 7 to 12 y, SD 1.8). Fifty-nine patients were advised AFO as resting or walking splint. At last follow-up 113 patients were still ambulatory whereas 13 had become wheel chair bound. Out of 59 patients, 48 were still wearing AFOs and the remaining discontinued AFOs for various reasons. Children with DMD require wearing of AFOs as resting or walking splint, mostly in first or early second decade of life. As there is some gap between onset of clinical signs and requirement of orthosis, follow-up preferably at a multidisciplinary clinic at regular intervals is desirable for timely intervention in the form of AFOs or other splints to prolong ambulatory status in these patients.

Use of a multiplex ligation-dependent probe amplification method for the detection of deletions/duplications in the GBA1 gene in Gaucher disease patients

Gaucher disease (GD) is caused by the deficient activity of β-glucocerebrosidase due to pathogenic mutations in the GBA1. This gene has a pseudogene (GBAP) with 96% of sequence homology. Recombination (Rec) events in the GBA1 seem to be facilitated by an increased degree of homology and proximity to the GBAP. The objectives of this study were to validate the P338-X1 GBA kit (MRC-Holland) for Multiplex Ligation-dependent Probe Amplification (MLPA) and to detect larger deletions/duplications present in GBA1 in GD patients from Brazil. Thirty-three unrelated Brazilian GD patients, previously genotyped by the Sanger method (both pathogenic alleles identified=29 patients, only one allele identified=3 patients, no pathogenic alleles identified=1 patient), were evaluated by the MLPA assay. MLPA was compatible with the previous results obtained by Sanger sequencing and identified an additional allele (a heterozygous deletion in intron 7 in one patient with only one mutation identified by Sanger). Our data suggest that, although larger deletions/duplications do not appear to be frequent in GD, the P338-X1 GBA kit for MLPA appears to be a good method for GBA1 analysis. Additional investigations should be performed in order to characterize the remaining four uncharacterized alleles of our sample.

Detection of SHOX gene aberrations in routine diagnostic practice and evaluation of phenotype scoring form effectiveness.

Heterozygous aberrations of SHOX gene have been reported to be responsible for Léri-Weill dyschondrosteosis (LWD) and small portion of idiopathic short stature. The study was established to assess effectiveness of using phenotype 'scoring form' in patients indicated for SHOX gene defect analysis. The submitted study is based on a retrospective group of 352 unrelated patients enrolled as a part of the routine diagnostic practice and analyzed for aberrations affecting the SHOX gene. All participants were scanned for deletion/duplication within the main pseudoautosomal region (PAR1) using the multiplex ligation-dependent probe amplification (MLPA) method. The phenotype 'scoring form' is used in our laboratory practice to preselect patients for subsequent mutation analysis of SHOX gene-coding sequences. The overall detection rate was 11.1% but there was a significant increase in frequency of SHOX gene defect positive with increasing achieved score (P<0.0001). The most frequent aberration was a causal deletion within PAR1. In three probands, MLPA analysis indicated a more complex rearrangement. Madelung deformity or co-occurrence of disproportionate short stature, short forearm and muscular hypertrophy had represented the most potent markers to determine the likelihood of SHOX gene defect detection. We conclude that appliance of phenotype 'scoring form' had saved excessive sample analysis and enabled effective routine diagnostic testing.