Multiplex Ligation-dependent Probe Amplification Screening Research Articles

Severity Scoring Cutoff for MLPA and Its Diagnostic Yield in 332 North Indian Children with Developmental Delay.

Chromosomal aberrations/rearrangements are the most common cause of intellectual disability (ID), developmental delay (DD), and congenital malformations. Traditionally, karyotyping has been the investigation of choice in such cases, with the advantage of being cheap and easily accessible, but with the caveat of the inability to detect copy number variations of sizes less than 5 Mb. Chromosomal microarray can solve this problem, but again the problems of expense and poor availability are major challenges in developing countries. The purpose of this study is to find the utility of multiplex ligation-dependent probe amplification (MLPA) as a middle ground, in a resource-limited setting. We also attempted to establish an optimum cutoff for the de Vries score, to enable physicians to decide between these tests on a case-to-case basis, using only clinical data. A total of 332 children with DD/ID with or without facial dysmorphism and congenital malformations were studied by MLPA probe sets P245. Assessment of clinical variables concerning birth history, facial dysmorphism, congenital malformations, and family history was done. We also scored the de Vries scoring for all the patients to find a suitable cutoff for MLPA screening. In our study, the overall detection rate of MLPA was 13.5% (45/332). The majority of patients were DiGeorge's syndrome with probe deletion in 22q11.21 in 3.3% (11/332) followed by 15q11.2 del in 3.6% (12/332, split between Angelman's and Prader-Willi's syndromes). Also, 3.0% (10/332) of patients were positive for Williams-Beuren's syndrome 7q11.23, 1.8% (6/332) for Wolf--Hirschhorn's syndrome 4p16.3, 1.2% (4/332) for 1p36 deletion, and 1% for each trichorhinophalangeal syndrome type I 8q23.3 duplication syndrome and cri du chat syndrome. The optimum cutoff of de Vries score for MLPA testing in children with ID and/or dysmorphism came out to be 2.5 (rounded off to 3) with a sensitivity of 82.2% and specificity of 66.7%. This is the largest study from India for the detection of chromosomal aberrations using MLPA common microdeletion kit P245. Our study suggests that de Vries score with a cutoff of 3 or more can be used to offer MLPA as the first tier test for patients with unexplained ID, with or without facial dysmorphism and congenital malformations.

Characterization of β-Globin Gene Cluster Deletions Using Multiplex-Gap Polymerase Chain Reaction (PCR) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background : Deletions in the β-globin gene cluster are usually rare, problematic to detect, and subsequently possess a challenge in many diagnostic laboratories. They are normally related to the heterozygous of the delta beta (δβ) thalassemia, hereditary persistence of fetalhemoglobin (HPFH) and some of the hemoglobin variants. These disorders are typically presented by elevated levels of hemoglobin F (Hb F), but with low to normal hemoglobin A2 (Hb A2). However, despite their existence, there is still a limited number of studies focusing on this area, hence no definitive diagnosis could be conclusively established. Therefore, this pilot study was carried out to fill this knowledge gap.
 Methods: In this study, screening of the selected deletional mutations in the β-globin gene cluster among patients with Hb F (>1%) and Hb A2 (<4%) were performed using multiplex Gap-PCR and multiplex ligation-dependent probe amplification (MLPA).
 Results: The results showed that out of 54 samples tested using multiplex Gap-PCR against four target deletions; Thai (δβ)°-thalassemia, HPFH-6, Siriraj J and HbLepore, one sample was found positive with Thai (δβ)°-thalassemia. Further findings from the MLPA screening on 12 randomly selected samples revealed that another patient was positive with double deletions within the region of the β-globin gene cluster. These deletions occur at the gamma-globin gene 1 (HBG1) and gamma-globin gene 2 (HBG2) in exon 3.
 Conclusions: In conclusion, this study highlighted the importance of these deletions’ characterization using multiplex Gap-PCR and MLPA which helps in establishing a definitive diagnosis among this selected group of patients.
 Bangladesh Journal of Medical Science Vol.20(3) 2021 p.618-624

Analysis of Exon Dosage Using Multiplex Ligation-Dependent Probe Amplification in Chinese Patients with Early-Onset Parkinson’s Disease

Introduction: Several studies have identified a number of genes associated with Parkinson’s disease (PD). Genomic rearrangements (exon dosage variations) in these genes have emerged as significant, causing mutations. However, exon dosage variations in several PD genes were rarely investigated in Chinese patients. Objective: This study was aimed at determining the prevalence of PD-causing genes’ exon rearrangements in Chinese sporadic early-onset PD (EOPD) patients. Methods: A total of 150 Chinese sporadic EOPD patients and 100 healthy controls were enrolled. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exon dosage in PD genes, including SNCA, PARKIN, UCHL1, PINK1, DJ1, LRRK2, and ATP13A2. Positive results were verified by real-time quantitative polymerase chain reaction. And exon sequencing was employed to screen for subtle mutations. Novel exon dosage variations were screened in families and controls. Results: PARKIN exon rearrangements were detected in 10 (6.7%) patients, including a novel heterozygous duplication of PARKIN exons 1–4. Clinical investigation showed that the percentage of individuals with PARKIN exon rearrangements was higher in the younger patients. Notably, the MLPA screening detected a heterozygous deletion of UCHL1 exon 1 in a patient. MLPA analysis in the family detected the deletion in an asymptomatic sister, indicating incomplete penetrance. Conclusion: Exon copy number variations (CNVs) in the PARKIN gene are relatively common among Chinese sporadic EOPD patients, whereas exon CNVs in other known PD genes can also be detected. Our findings demonstrate that it is important to perform exon dosage analysis for several known PD genes to obtain a better mechanistic insight into PD pathogenesis.

Mutation Analysis Using Multiplex Ligation-Dependent Probe Amplification in Consanguineous Families in South India with a Child with Profound Hearing Impairment.

Consanguineous marriage, a common practice in South India, increase the incidence of autosomal recessive diseases such as nonsyndromic hearing loss (NSHL) in offspring. This trend was noted in the children with hearing impairment (HI) who received cochlear implants (CI) at our University hospital in Porur, Chennai, India. To ascertain the genetic etiology of HI in these patients, we performed multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 25 families who had a child with NSHL were included in the study. MLPA screening of GJB2, GJB6, and GJB3 was performed for all the recruited individuals. The pathogenic p.W24X* mutation of GJB2 was detected in 2 patients; both of their parents were heterozygous carriers. Both families had a second-degree consanguineous marriage. This study has important implications for molecular-diagnosis strategy and genetic counseling for families with HI in South India.

Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil.

BackgroundApproximately 8–15% epithelial ovarian cancer patients are BRCA1 or BRCA2 germline mutation carriers. Brazilian inhabitants may have peculiar genetic characteristics associated with ethnic diversity, and studies focusing on the entire BRCA1/BRCA2 gene sequencing in Brazilian ovarian cancer patients are still lacking. The aim of this study was to evaluate BRCA1/2 mutations, through entire gene sequencing, in a Brazilian population of women with epithelial ovarian cancer.MethodsIn a cross sectional study performed in one reference centre for cancer treatment in São Paulo, Brazil, 100 patients diagnosed with epithelial ovarian cancer unselected for family history of breast and/or ovarian cancer were included. The complete coding sequence of BRCA1/2 genes was evaluated through Next-Generation or capillary sequencing. Large deletions were investigated through Multiplex Ligation-dependent Probe Amplification (MLPA).ResultsNineteen pathogenic mutations (BRCA1: n = 17 and BRCA2: n = 2) featuring 14 different mutations, including two large deletions in BRCA1 (exon 1–2 deleted and exon 5–7 deleted) were identified. Three mutations were detected more than once (c.3331_3334delCAAG, c.5266dupC and c.4484G > T). Two novel frameshift mutations were identified, one in BRCA1 (c.961_962delTG) and one in BRCA2 (c.1963_1963delC). BRCA1/2 mutations were seen in 35.5% of the patients with first and/or second-degree relatives with breast and/or ovarian cancer. Nineteen variants of uncertain significance (VUS) were detected (BRCA1: n = 2 and BRCA2: n = 17), including five distinct missense variants (BRCA1: c.5348 T > C; BRCA2: c.2350A > G, c.3515C > T, c.7534C > T, and c.8351G > A).ConclusionsAmong epithelial ovarian cancer patients unselected for family history of cancer, 19% were BRCA1/2 germline mutation carriers. Almost ¾ of the BRCA mutations, including two large deletions, were detected only once. Our work emphasizes the need of entire gene sequencing and MLPA screening in Brazil.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2966-x) contains supplementary material, which is available to authorized users.

Optimizing gDNA extraction from fresh frozen meningioma tissue for downstream genetic analysis

ObjectiveMeningioma is the most common brain tumor. Genetic mutations in meningioma that include deletion of the neurofibromatosis type 2 gene, (NF2), offer diagnostic information on tumor behavior, recurrence and potential response to treatment. Obtaining high-grade genetic material is critical for accurate, sensitive and robust molecular testing. Currently, no standardized procedure exists for extracting gDNA from meningioma, and this problem was addressed in this report. MethodThis study compared the yield and quality of extracted gDNA from patient meningioma specimens using an optimized phenol chloroform method and two commercial silica column-based extractions kits and tested respective performances as template in qPCR tests and multiplex ligation-dependent probe amplification (MLPA) NF2 screening. ResultsMean gDNA yields were comparable for each method tested; however, phenol chloroform extraction outperformed column-based kits in all other quality assurance metrics examined. Phenol chloroform extracted gDNA was highly pure, and of a higher fragment size species when compared to column prepared gDNA. qPCR of GAPDH, B2MG, and RPL37A housekeeping genes demonstrated variance in cycle thresholds between patient samples was much lower in the phenol chloroform group. Similarly, primer efficiencies were significantly improved in this sample group which translated to a broader qPCR linear dynamic range and much improved qPCR performance at low concentrations of template. MLPA screening identified NF2 gene deletions in 6 of 12 meningioma samples. Inconsistencies in copy number data for NF2 and reference regions of the genome were observed between gDNA sample extraction groups that included both false negative and positive errors in silica column derived gDNA samples. ConclusionsThis study outlines a highly robust phenol chloroform extraction method for obtaining high-quality gDNA from frozen meningioma tissue and highlights the significance of performing adequate quality assurance when using gDNA for downstream genetic analysis. Most importantly, we demonstrate using gDNA extracted with silica column based kits can lead to diagnostic errors when screening NF2 deletions in meningiomas with MLPA.

Recurrent fetal syndromic spina bifida associated with 3q26.1-qter duplication and 5p13.33-pter deletion due to familial balanced rearrangement

ObjectiveNeural tube defects belong to the second most common group of congenital anomalies, after heart defects, which can be diagnosed by prenatal ultrasonography. Rarely, neural tube defects can be associated with chromosomal abnormalities, including full and partial aneuploidies. We report a familial fetal case with syndromic spina bifida and discuss its association with partial 3q duplication and partial 5p deletion. Materials and methodsClinical findings of three affected family members in two generations and two carriers of the balanced translocation are described. Conventional cytogenetic and fluorescence in situ hybridization (FISH) analysis of the carrier, as well as subtelomeric multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (CGH) analysis on the DNA extracted from affected family members was performed. ResultsSubtelomeric FISH analysis of the proposita revealed balanced reciprocal translocation between the long arm of chromosome 3 and short arm of chromosome 5. Subtelomeric MLPA screening of the first child revealed the deletion in 5p15.33 and duplication in 3q29 chromosomal loci, the finding consisting of the unbalanced rearrangement involving the short arm of chromosome 5 and long arm of chromosome 3. Array CGH analysis of the DNA of the second affected child revealed a 31.1Mb duplication of 3q26.1-qter and a 33.6Mb deletion of 5p13.33-pter. ConclusionOur report serves to emphasize the consistency in the prenatal sonographic feature of spina bifida in consecutive pregnancies with fetuses associated with partial trisomy 3q (3q26.1-qter) and partial monosomy 5p (5p13.33-pter). The use of molecular cytogenetic technologies such as array CGH and FISH is important for clarifying any type of unbalanced chromosome rearrangement.

Analysis of chromosome 22q11 copy number variations by multiplex ligation-dependent probe amplification for prenatal diagnosis of congenital heart defect.

BackgroundCongenital heart defects (CHD) represent one of the most common birth defects. This study aimed to evaluate the value of multiplex ligation-dependent probe amplification (MLPA) as a tool to detect the copy number variations (CNVs) of 22q11 in fetuses with CHD.ResultsA large cohort of 225 fetuses with CHD was screened by fetal echocardiography. Once common chromosome abnormalities in 30 fetuses were screened out by conventional G-banding analysis, the CNVs of chromosome 22q11 in the remaining 195 fetuses were determined by MLPA for prenatal genetic counseling. In 195 CHD fetuses with normal karyotype, 11 cases had pathological CNVs, including 22q11.2 deletion (seven cases), the deletion of 22q11 cat eye syndrome (CES) region (one case), 22q11.2 duplication (one case), 22q13.3 deletion (one case) and 17p13.3 deletion (one case). In total, our findings from MLPA screening represented 4.9 % in our cohort. Among these, three cases were inherited CNVs, and eight cases were de novo. These CNVs were further verified by single nucleotide polymorphism (SNP)-array analysis, and their chromosomal location was refined.ConclusionThis study indicated that MLPA could serve as an effective test for routine prenatal diagnosis of 22q11 in fetuses with CHD.

Multiplex ligation-dependent probe amplification and array comparative genomic hybridization analyses for prenatal diagnosis of cytogenomic abnormalities.

BackgroundThe aims of this study were to evaluate the clinical utility of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH) analyses on prenatal cases and to review prenatal ultrasound findings of cytogenomic syndromes.ResultsOf the 54 prenatal cases analyzed, cytogenomic abnormalities were characterized in 14 cases. In four fetuses with abnormal ultrasound findings, a 40.701 Mb duplication of 8q22.3-q24.3 and a 23.839 Mb deletion of 7q33-q36.3 derived from a paternal balanced translocation, a de novo 13.062 Mb deletion of 11q24.1-q25 for Jacobsen syndrome, a de novo 19.971 Mb deletion of 7q11.23-q21.3 for type 1 split-hand/foot malformation (SHFM1), and a de novo 28.909 Mb duplication of 3q21.1-q25.1 were detected. A 699.8 Kb deletion at 5p15.33 for Cri du Chat syndrome was confirmed in a fetus with abnormal MLPA result. A fetus with abnormal maternal screening was detected with a de novo distal 1.747 Mb duplication at 2q37.1-q37.2 and a 6.664 Mb deletion at 2q37.2-q37.3. Of the eight cases referred by history of spontaneous abortions, derivative chromosomes 11 from paternal carriers of a balanced 8q/11q and a 10q/11q translocation were noted in two cases, simple aneuploids of trisomy 2 and trisomy 21 were seen in three cases, and compound aneuploids of two or three chromosomes were found in three cases. Post-test genetic counseling was performed with detailed genomic information and well characterized postnatal syndromic features.ConclusionsThese results demonstrated that coupling MLPA screening and aCGH analysis are a cost-effective approach to detect cytogenomic abnormalities in a prenatal setting. The aCGH analysis provided not only genomic maps of breakpoints and gene content of imbalanced regions but also better inference of related phenotypes for genetic counseling. Prenatal ultrasound findings reported in the literature for Jacobsen syndrome, SHFM and Cri du Chat syndrome were summarized for use as diagnostic references.

SPAST mutations in Australian patients with hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is often caused by mutations in the SPAST gene. The frequency of SPAST mutations causing HSP in Australian patients is currently unknown. We aimed to determine the frequency of SPAST gene mutations in our cohort of HSP patients. We recruited 30 unrelated patients with HSP for clinical and genetic assessment. DNA or RNA was extracted from patients' samples to perform direct DNA sequencing of the SPAST gene, multiplex ligation-dependent probe amplification (MLPA) and/or cDNA analysis. We identified 13 heterozygous SPAST mutations in 16 unrelated patients. Most mutations (75%) were detected by DNA sequence analysis. We identified nine-point mutations (n = 9), insertion (n = 1), one type of splice site mutation (n = 2), one type of exonic deletion (n = 2) and one type of exonic amplification (n = 2). Missense mutations (n = 7) were the most frequent mutation type (44%). Heterozygous exonic deletion (n = 2) and heterozygous exonic amplification (n = 2) were identified by MLPA and cDNA screening (25%). We also identified the single heterozygous p.Ser44Leu polymorphism in two other patients without pathogenic mutations in SPAST. We conclude that SPAST mutations are responsible for the majority of HSP in Australia. Most of the patients with SPAST mutations had pure forms of HSP and a positive family history to suggest autosomal dominant (AD) HSP. Not all mutations were identified by direct sequencing of the SPAST gene, necessitating further molecular analysis. Given that SPAST mutations cause AD-HSP, these findings are important when providing genetic counselling for affected patients.

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries

Conventional karyotyping detects anomalies in 3–15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cut-off for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams–Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome.

Partial loss of heterozygosity events at the mutated gene in tumors from MLH1/MSH2 large genomic rearrangement carriers

BackgroundDepending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts.MethodsThe main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes.ResultsWe found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region.ConclusionLGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.

Low Frequency of MLL-PTD Detected in Pediatric Acute Myeloid Leukemia Using MLPA Screening.

Mixed-lineage leukemia (MLL)-partial tandem duplications (PTD) consist of an inframe repetition of MLL exons, which seems to be the result of mispairing of repetitive regions with high homology. MLL-PTD is found in 3–5% of adult acute myeloid leukemia (AML) and is associated with poor prognosis. MLL-PTD is mutually exclusive with other known aberrations, except for trisomy 11 and FLT3-ITD in adult AML. Shimada et al. found a frequency of 13% for MLL-PTD in pediatric AML patients (n=185) with an adverse outcome. However 5/21 MLL-PTD cases were found in MLL-rearranged AML, and 4/21 cases in patients with a t(8;21). Screening was performed for MLLPTD transcripts only, which have also been detected in healthy adults and cord blood samples in up to 100% of the cases, although these expression levels were much lower. In this study we screened for MLL-PTD in pediatric AML on the genomic level. Retrospectively, a cohort of 248 children with AML was screened for MLL-PTD using multiplex ligation-dependent probe amplification (MLPA), which is a method to detect copy number differences of specific sequences. If possible, screening was also performed for MLL-PTD transcripts with RT-PCR. We designed a reaction mix for MPLA-analysis containing probes for exon 2 to 13 of MLL for MLL-PTD detection and exon 17 of MLL as internal control. A probe in the serpinB2 gene was used as external control. The patient samples were analyzed according to the manufacturer's protocol (MRC Holland). Data were analyzed using GeneMarker v1.5. We detected MLL -PTD in 7/248 patients (2.8%), indicating a low frequency in pediatric AML. In all these patients also MLL-PTD transcripts were present. Three patients had normal cytogenetics; 1 patient had a trisomy 11, while for 3 patients no conventional cytogenetic data were available. Moreover, in 4 patients a FLT3-ITD was detected, in 1 other patient a FLT3 tyrosine kinase domain mutation, and in another patient a mutation in NRAS. MLL-PTD transcripts were also detected in some patients with an MLL-rearrangement. However, using MLPA, only exon deletions and amplifications were detected around the break-point region of MLL, but there was no evidence for MLL-PTD on the genomic level. MLL-PTD did not show preference for sex. The median age of patients with MLL-PTD was 7.5 (4.8–18) years and the median white blood cell count 95 × 109/l (44–170). Five of the 6 patients were treated with uniform DCOG and BFM protocols. Two patients were in continuous complete remission (CR) after 3 years. Two patients achieved CR; one however died after hematopoietic stem cell transplantation (HSCT) from an infection while the other one relapsed and was salvaged successfully. One patient had refractory disease and died from progressive disease following two HSCT's. One patient was enrolled in the French LAME protocol, but died within 2 days from cerebral hemorrhage. Survival analysis was restricted to the subset of patients treated according to uniform DCOG and BFM treatment protocols (n=184). In this cohort, patients with MLL-PTD (n=5) had similar 3-years event-free survival rates (pEFS) compared to patients without MLL-PTD (40 vs. 44%, p=0.98). In conclusion, the frequency of MLL-PTD is lower than in adult AML and than previously described in another pediatric AML cohort. Moreover, MLPA can more accurately detect MLL-PTD compared to RT-PCR. In concordance with adult AML, it seems that MLL-PTD does not co-exist with other known aberrations, except for trisomy 11 and mutations in FLT3 or RAS. Due to the small sample-size of patients with MLL-PTD no clear conclusion on prognosis can be made, indicating that larger prospective studies are needed for the prognostic relevance and stratification of MLL-PTD in pediatric AML

MLPA screening in theBRCA1gene from 1,506 German hereditary breast cancer cases: novel deletions, frequent involvement of exon 17, and occurrence in single early-onset cases

We present a comprehensive analysis of 1,506 German families for large genomic rearrangements (LGRs) in the BRCA1 gene and of 450 families in the BRCA2 gene by the multiplex ligation-dependent probe amplification (MLPA) technique. A total of 32 pathogenic rearrangements in the BRCA1 gene were found, accounting for 1.6% of all mutations, but for 9.6% of all BRCA1 mutations identified in a total of 1,996 families, including 490 with small pathogenic BRCA1/2 mutations. Considering only high risk groups for hereditary breast/ovarian cancer, the prevalence of rearrangements is 2.1%. Interestingly, deletions involving exon 17 of the BRCA1 gene seem to be most frequent in Germany. Apart from recurrent aberrations like del ex17, dupl ex13, and del ex22, accounting for more than 50% of all BRCA1 LGRs, we could fully characterize 11 novel deletions. Moreover, one novel deletion involving exons 1-7 and one deletion affecting the entire BRCA1 gene were identified. All rearrangements were detected in families with: 1) at least two breast cancer cases prior to the age of 51 years; 2) breast and ovarian cancer cases; 3) ovarian cancer only families with at least two ovarian cancer cases; or 4) a single breast cancer case prior to the age of 36 years, while no mutations were detected in breast cancer only families with no or only one breast cancer case prior to the age of 51 years. Analysis for gross rearrangements in 412 high-risk individuals, revealed no event in the BRCA2 gene and only two known CHEK2 mutations. However, in an additional 38 high-risk families with cooccurrence of female breast/ovarian and male breast cancer, one rearrangement in the BRCA2 gene was found. In summary, we advise restricting BRCA1 MLPA screening to those subgroups that revealed LGRs and recommend BRCA2 MLPA screening only for families presenting with cooccurrence of female and male breast cancer.

Deletions of NF1 gene and exons detected by multiplex ligation-dependent probe amplification

To estimate the contribution of single and multi-exon NF1 gene copy-number changes to the NF1 mutation spectrum, we analysed a series of 201 Italian patients with neurofibromatosis type 1 (NF1)....

Holoprosencephaly and preaxial polydactyly associated with a 1.24 Mb duplication encompassing FBXW11 at 5q35.1

Holoprosencephaly (HPE) is the most common developmental defect affecting the forebrain and midface in humans. The aetiology of HPE is highly heterogeneous and includes both environmental and genetic factors. Here we report on a boy with mild mental retardation, lobar HPE, epilepsy, mild pyramidal syndrome of the legs, ventricular septal defect, vesicoureteral reflux, preaxial polydactyly, and facial dysmorphisms. Genome-wide tiling path resolution array based comparative genomic hybridisation (array CGH) revealed a de novo copy-number gain at 5q35.1 of 1.24 Mb. Additional multiplex ligation-dependent probe amplification screening of a cohort of 31 patients with HPE for copy-number changes at the 5q35.1 locus did not reveal any additional genomic anomalies. This report defines a novel 1.24 Mb critical interval for HPE and preaxial polydactyly at 5q35.1. The duplicated region encompasses seven genes: RANBP17, TLX3, NPM1, FGF18, FBXW11, STK10, and DC-UbP. Since FBXW11 is relatively highly expressed in fetal brain and is directly involved in proteolytic processing of GLI3, we propose FBXW11 as the most likely candidate gene for the HPE and prexial polydactyly phenotype. Additional research is needed to further establish the role of genes from the 5q35.1 region in brain and limb development and to determine the prevalence of copy number gain in the 5q35.1 region among HPE patients.

Deletion and duplication screening in the DMD gene using MLPA

We have designed a multiplex ligation-dependent probe amplification (MLPA) assay to simultaneously screen all 79 DMD gene exons for deletions and duplications in Duchenne and Becker muscular dystrophy (DMD/BMD) patients. We validated the assay by screening 123 unrelated patients from Serbia and Montenegro already screened using multiplex PCR. MLPA screening confirmed the presence of all previously detected deletions. In addition, we detected seven new deletions, nine duplications, one point mutation, and we were able to precisely determine the extent of all rearrangements. To facilitate MLPA-based screening in laboratories lacking specific equipment, we designed the assay such that it can also be performed using agarose gel analysis and ethidium bromide staining. The MLPA assay as described provides a simple and cheap method for deletion and duplication screening in DMD/BMD patients. The assay outperforms the Beggs and Chamberlain multiplex-PCR test, and should be considered as the method of choice for an initial DNA analysis of DMD/BMD patients.