Multiplex Ligation-dependent Probe Amplification Results Research Articles

Efficiency of CNV-seq in detecting fetal DMD gene deletion or duplication in prenatal diagnosis

Objective: To evaluate the diagnostic efficiency of copy number variation sequencing (CNV-seq) to detect the deletion or duplication of DMD gene in prenatal diagnosis. Methods: A retrospective analysis was carried out on the CNV-seq results of 34 544 fetuses diagnosed in the First People's Hospital of Yunnan Province from January 2018 to July 2023. A total of 156 cases of fetuses were collected, including Group 1:125 cases with family history of Duchenne muscular dystrophy or Becker muscular dystrophy (DMD/BMD), and Group 2:31 cases with no family history but a DMD gene deletion or duplication was detected unexpectedly by CNV-seq. Multiplex ligation-dependent probe amplification (MLPA) was used as a standard method to detect the deletion or duplication. Consistency test was carried out basing on the results of CNV-seq and MLPA of all 156 cases. Results: Comparing to MLPA, CNV-seq had a coincidence rate of 92.3% (144/156) for DMD gene deletion or duplication, with a sensitivity and positive predictive value of 88.2%, with a specificity and negative predictive value of 94.3%, a missed detection rate of 3.8%, and a Kappa value of 0.839. CNV-seq missed 4 cases with deletions and 2 with duplications due to involved fragments less than 100 Kb, among 20 cases of deletions and 6 cases of duplications detected by MLPA in Group 1. In Group 2, the deletions and duplications detected by CNV-seq were 42% (13/31) and 58% (18/31), respectively, in which the percentage of duplication was higher than that in Group 1. Among those 18 cases with duplications, 3 cases with duplication locating in exon 42~67 were likely pathogenic; while 9 cases with duplication covering the 5' or 3' end of the DMD gene, containing exon 1 or 79 and with only one breakpoint within the gene, along with the last 6 cases with duplications locating at chrX: 32650635_32910000 detected only by CNV-seq, which might be judged as variants of uncertain significance. Conclusions: CNV-seq has a good efficiency to detect fetal DMD gene deletion or duplication in prenatal diagnosis, while a further verification test by MLPA is recommended. The duplications on chrX: 32650635_32910000, 5' or 3' end of DMD gene detected by CNV-seq should be carefully verified and assessed because those variants appear to be nonpathogenic polymorphisms.

Spectrum of variants in a large Chinese Gitelman syndrome cohort.

Gitelman syndrome (GS) is caused by SLC12A3 biallelic variants. A previous study showed that large rearrangements (LRGs) of SLC12A3 accounted for the low sensitivity of genetic testing. However, a systematic screening for LRGs in Chinese GS patients is lacking. Massively parallel sequencing (MPS) and multiplex ligation-dependent probe amplification (MLPA) were performed to sequence the genomic DNA of patients with clinically diagnosed GS. Of 165 index cases, MPS identified 151 cases with two or more affected alleles and 14 cases with one variant allele. LRGs were detected by MLPA in 20 out of 27 cases, including 15 cases with suspected LRGs by MPS. Among these 20 cases with LRGs, the results of MPS and MLPA were identical in only 8 cases. Additional LRGs in 6 cases were detected by MLPA alone. In 6 cases, E4_E6del was identified by MPS, while E4_E5del and Intron6del were identified by MLPA. Among the 102 distinct variants, 30 are novel. LRGs were found in 20 cases (12.1%). LRGs were found in 12.1% of our Chinese GS patients cohort. We show that MPS and MLPA are two complementary techniques with the ability to improve the diagnostic yield of GS.

Genetic analysis and reproductive intervention of 7 families with gonadal mosaicism for Duchenne muscular dystrophy

To explore the genetic basis for 7 families with gonadal mosaicism for Duchenne muscular dystrophy (DMD). For the 7 families presented at the CITIC Xiangya Reproductive and Genetic Hospital from September 2014 to March 2022, clinical data were collected. Preimplantation genetic testing for monogenic disorders (PGT-M) was carried out for the mother of the proband from family 6. Peripheral venous blood samples of the probands, their mothers and other patients from the families, amniotic fluid samples from families 1 ~ 4 and biopsied cells of embryos cultured in vitro from family 6 were collected for the extraction of genomic DNA. Multiplex ligation-dependent probe amplification (MLPA) was carried out for the DMD gene, and short tandem repeat (STR)/single nucleotide polymorphism (SNP)-based haplotypes were constructed for the probands, other patients, fetuses and embryos. The results of MLPA showed that the probands and the fetuses/probands' brothers in families 1 ~ 4, 5, 7 had carried the same DMD gene variants, whilst the probands' mothers were all normal. The proband in family 6 carried the same DMD gene variant with only 1 embryo (9 in total) cultured in vitro, and the DMD gene of the proband's mother and the fetus obtained through the PGT-M were normal. STR-based haplotype analysis showed that the probands and the fetuses/probands' brothers in families 1 ~ 3 and 5 have inherited the same maternal X chromosome. SNP-based haplotype analysis showed that the proband from family 6 has inherited the same maternal X chromosome with only 1 embryo (9 in total) cultured in vitro. The fetuses in families 1 and 6 (via PGT-M) were both confirmed to be healthy by follow up, whilst the mothers from families 2 and 3 had chosen induced labor. Haplotype analysis based on STR/SNP is an effective method for judging gonad mosaicism. Gonad mosaicisms should be suspected for women who have given births to children with DMD gene variants but with a normal peripheral blood genotype. Prenatal diagnosis and reproductive intervention may be adapted to reduce the births of further affected children in such families.

Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency

Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in SERPINC1 in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.

Next-generation sequencing approach to molecular diagnosis of Iranian patients with Duchenne/Becker muscular dystrophy: Several novel variants identified.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) constitute the second most prevalent muscular dystrophy, with large deletions or duplications accounting for 66% of cases. No effective treatment exists for DMD/BMD. At present, genetic diagnosis serves as the foundation for gene therapy treatments. In this study, a comprehensive molecular investigation was conducted. The subjects diagnosed with DMD/BMD were initially examined using multiplex ligation-dependent probe amplification (MLPA) technology. The negative MLPA results were analyzed further using next-generation sequencing (NGS) technology. The MLPA detected 201 deletions (65.9%) and 20 duplications (6.6%) along the dystrophin gene among the 305 Iranian patients examined. The deletion of exon 52 in the amenable skipping subgroup was associated with an earlier onset age and a more severe phenotype. Twenty-one of the small mutations found in 58 MLPA-negative patients were novel. The most prevalent variants were nonsense variants (46.5%), frameshift variants (31%), splicing variants (6.9%), missense variants (10.4%), and synonymous mutations (5.1%). Our results demonstrate that MLPA and NGS can be effective diagnostic tools for very young patients with a single exon deletion.

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.

A novel mutation of SPAST gene in a hereditary spastic paraplegia type 4 family

Objective: To clarify the pathogenicity and further explore the association between genotype and clinical phenotype of this variant, analyzing a novel variation of SPAST gene in hereditary spastic paraplegia (HSP) family from Changzhi city, Shanxi Province. Methods: A family with HSP was tracked and collected in Neurology Department of Heping Hospital Affiliated to Changzhi Medical College in October 2019. Peripheral venous blood of 2 ml was extracted from the proband and 8 other members of the family, genomic DNA was extracted from the blood samples, and the genes of spastic paraplegia were screened by next-generation sequencing (NGS). HGMD, 1000G, OMIM databases and PolyPhen2, SIFT and other software were used for bioinformatics analysis of suspected mutations. Multiplex ligation-dependent probe amplification (MLPA) was used to further screen for total deletions/duplications in patients who remained negative after targeting NGS, and Sanger sequencing was performed to verify the suspected pathogenic mutation sites in the family to determine co-isolation of the mutation sites in the family members. Finally, it is necessary to refer to the latest version of The American College of Medical Genetics and Genomics (ACMG) sequence variation interpretation guidelines to interpret the mutation sites to determine pathogenicity. Results: The HSP family consist 47 members of 4 generations and 10 patients, with onset ages ranging from 2 to 44 years. The proband's daughter only showed positive bilateral Babbitt signs on physical examination, and the rest of the patients showed spasticity and weakness of lower limbs with varying severity on this basis. Preliminary screening by next-generation sequencing technology showed that the proband had frame-shift variation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) and missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg). Then, Sanger sequencing was used for in-family verification, which showed SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was detected in the affected members include father, brother, son and daughter, and not detected in the unaffected normal members, the proband's wife, mother, sister and sister-in-law. However, the unaffected of mother detected missense variation of DCTN1 gene c.2213A>G (p.Gln738Arg), while the remaining members did not detect this variation. The results of MLPA showed that no large fragment variation was found. Conclusions: The genetic pattern of the HSP family was autosomal dominant, and the clinical characteristics were consistent with hereditary spastic paraplegia type 4 (SPG4). Co-segregation of SPAST gene c.1057_1058insCC (p.Leu354HisfsTer11) was found in the HSP family and was the pathogenicity cause of this SPG4 family, and it was a newly discovered mutation locus.

Glioma 2021 WHO Classification: The Superiority of NGS Over IHC in Routine Diagnostics.

The accurate detection of genetic variants such as single substitutions (IDH1/2, TERT), chromosomal abnormalities (CDKN2A, 1p/19q deletions, and EGFR amplifications), or promoter methylations (MGMT) is critical for glioma patient management, as emphasized in the World Health Organization's (WHO's) most recent classification in 2021 (WHO CNS5). The purpose of this study was to evaluate novel innovative methods for determining IDH1/2 status in the context of WHO CNS5. Multiple biomarkers were simultaneously screened using next-generation sequencing (NGS) on 34 glioma samples. In cases where the IDH1/2 status determined by immunohistochemistry (IHC) or multiplex ligation-dependent probe amplification (MLPA) was inconsistent with the NGS results, quantitative polymerase chain reaction (qPCR) and Sanger sequencing were performed to resolve the adjudicated discrepancy. IDH1/2 NGS results differ from IHC (7/13 samples) as well as MLPA reports (1/4 samples). All NGS findings were confirmed by qPCR and Sanger sequencing. WHO CNS5 requires assessment of multiple mutations for glioma classification. We demonstrated that qPCR or NGS performed in reference genetic laboratories, rather than IHC, is the most reliable method for IDH1/2 analysis. Clinicians should be aware of discrepancies in MLPA or IHC results and seek reconsultation in facilities with extensive access to advanced molecular technologies. Moreover, we proposed a new algorithm for the molecular diagnostic procedures in glioma patients based on the WHO CNS5.

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.

Diagnostic Value of Dystrophin Immunostaining in the Diagnosis of Duchenne and Becker Muscular Dystrophy Patients

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive muscular disorders caused by the absence or reduction of the muscle cytoskeletal protein dystrophin. Standard procedures to detect deletion and duplication of the DMD gene use Multiplex Ligation-Dependent Probe Amplification (MLPA). However, genetic testing, such as MLPA, is not covered by the national insurance scheme in Indonesia. Immunohistochemical (IHC) staining of dystrophin from muscle biopsy in the form of Formalin-Fixed Paraffin-Embedded (FFPE) specimens can be an alternative method to detect dystrophin expression in protein levels to establish the diagnosis of DMD or BMD.
 Objectives: To determinate sensitivity, specificity and accuracy of IHC analysis of dystrophin in DMD/BMD patient in comparison with the standard genetic testing, MLPA.
 Methods: Twenty-six patients enrolled in this study were clinically diagnosed as DMD/BMD in Dr. Sardjito Hospital and Universitas Gadjah Mada Academic Hospital. Genomic DNA was isolated from 3 mL of EDTA-peripheral whole blood samples. The deletion and duplication of DMD genes were detected by MLPA. IHC examination was performed using a specific antibody dystrophin (DYS2). Complete loss of dystrophin staining indicated DMD, while partial loss of dystrophin staining indicated BMD. MLPA result was used as the gold standard to determine sensitivity, specificity, and accuracy of IHC technique using a 2x2 table.
 Results: MLPA results revealed 18 (18/26; 69.3%) patients with deletion and 3 (3/26; 11.5%) patients with duplication. Five (5/26; 19.2%) patients who showed no deletion nor duplication were excluded from the analysis. Among 21 patients with deletion or duplication, 18 (18/21; 85.7%) patients were out-of-frame (DMD) and 3 (3/21; 14.3%) patients were in-frame (BMD). Six patients showed a discrepancy between the IHC and MLPA results with 9.5% (2/21) false positive and 19% (4/21) false negative. The sensitivity of dystrophin IHC was 77.78%, specificity 33.33%, positive predictive value 87.5%, negative predictive value 20%, and accuracy 71.43%.
 Conclusion: Muscle biopsy followed by IHC can be one of the diagnostic tools to diagnose BMD or DMD, with high sensitivity. The protein-based strategy is probably the most efficient way to approach the diagnosis of Duchenne and Becker muscular dystrophy in limited health care settings.

A homozygous SH3TC2 mutation in a Korean patient with Charcot–Marie–Tooth disease type 4C

Charcot–Marie–Tooth disease type 4C (CMT4C) is an autosomal recessive neuropathy associated with SH3TC2 mutations, resulting in slow conduction velocity via hypomyelination. The occurrence of CMT4C in demyelinating Charcot–Marie–Tooth (CMT) varies among ethnicities, and several variants have been reported as the founder mutation. In Korea, the incidence of CMT4C was calculated as approximately 2%, and all patients have compound heterozygous mutations, which is partly due to the prohibition of consanguineous marriage. Herein, we describe a 25-year-old male who presented a slowly progressive limb weakness and impaired vibration sensation. Whole-exome sequencing revealed homozygous variants c.929G>A of SH3TC2 after identifying negative multiplex ligation-dependent probe amplification results of PMP22. Based on our literature review, this is the first CMT4C patient with a homozygous variant with each allele inherited from both the parents.

Evaluation of MLPA as a comprehensive molecular cytogenetic tool to detect cytogenetic markers of chronic lymphocytic leukemia in Egyptian patients

BackgroundChronic lymphocytic leukemia (CLL) is the most common form of adult leukemia. This disease is genetically heterogeneous, and approximately 85% of patients with CLL harbor chromosomal aberrations that are considered effective prognostic biomarkers. The most frequent aberrations include deletions in 13q14, followed by trisomy 12, and deletions in 11q22.3 and 17p13 (TP53). Currently, fluorescence in situ hybridization (FISH) is the most widely used molecular cytogenetic technique to detect these aberrations. However, FISH is laborious, time-consuming, expensive, and has a low throughput. In contrast, multiplex ligation-dependent probe amplification (MLPA) is a reliable, cost-effective, and relatively rapid technique that can be used as a first-line screening tool and complement with FISH analysis. This study aimed to evaluate the contributions of MLPA as a routine standalone screening platform for recurrent chromosomal aberrations in CLL in comparison to other procedures. Thirty patients with CLL were screened for the most common genomic aberrations using MLPA with SALSA MLPA probemix P038-B1 CLL and FISH. ResultsIn 24 of the 30 cases (80%), the MLPA and FISH results were concordant. Discordant results were attributed to a low percentage of mosaicism. Moreover, the MLPA probemix contains probes that target other genomic areas known to be linked to CLL in addition to those targeting common recurrent CLL aberrations. ConclusionsThe usage of MLPA as the first screening platform followed by FISH technique for only the negative cases is the most appropriate approach for CLL diagnosis and prognosis.

Abstract P6-08-13: Personalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing

Abstract Background: Germline copy number variants (CNV) of hereditary breast and ovarian cancer genes are a known class of clinically significant mutations. However, failure to detect CNV is a known limitation of Sanger sequencing and conventional amplicon next-generation sequencing (NGS). Multiplex ligation-dependent probe amplification (MLPA), while robust for CNV detection, is labor-intensive, costly to scale up and has limited breakpoint resolution at exon level only. We evaluated the clinical utility of molecular-barcode NGS and long-read nanopore sequencing in our genetic testing algorithm, specifically on CNV detection and breakpoint characterization of probands and family members. Methods: Both molecular-barcode NGS (BRCA1, BRCA2, TP53, PTEN, PALB2 and CDH1) and MLPA (BRCA1 and BRCA2) were performed in parallel on 200 high-risk breast and/or ovarian cancer probands and 11 CNV positive controls. CNV calling from MLPA and NGS data was performed by Coffalyser.Net and a novel in-house bioinformatics algorithm BAMClipper-SE, respectively. Nanopore amplicon or whole-genome sequencing was attempted to map the exact breakpoint of CNV controls and any newly identified CNV positive probands (up to May 2019). Personalized breakpoint PCR test for specific CNVs was designed and validated on probands and subsequently applied to corresponding family members for carrier testing. Results: Molecular-barcode NGS was 100% sensitive in detecting all BRCA1/2 CNVs from 11 positive controls. A total of 4 BRCA1/2 CNVs were detected by NGS from the 200 probands and matched the corresponding MLPA results. In addition, a PALB2 CNV (exons 4-6 deletion) was also detected by NGS during the parallel comparison. Up to May 2019, a total of 22 probands with detectable CNV in BRCA1, BRCA2, PALB2 or CHEK2 were accrued. Exact CNV breakpoint at nucleotide resolution was identified from 20 probands by nanopore and Sanger sequencing (BRCA1 n=12, BRCA2 n=5, PALB2 n=2 and CHEK2 n=1). High-resolution breakpoint mapping was applicable to inform variant pathogenicity and to enable recurrent CNV identification. Personalized breakpoint PCR designed for the 20 probands were applied to 42 family members, of whom 16 were CNV carriers and 26 were non-carriers. Breakpoint PCR results and any available MLPA results of these family members were 100% concordant. Conclusion: Molecular-barcode NGS allows a streamlined workflow to detect both sequence mutations and CNVs. It proves to be a reliable and cost-effective replacement of BRCA1/2 MLPA in genetic testing algorithm of new probands. Simultaneously the NGS workflow expands CNV detection coverage to additional genes (e.g. PALB2) without costly scale-up of MLPA gene panels. Nanopore sequencing is a practical alternative to MLPA in orthogonal validation of newly detected CNV. The high-resolution breakpoint mapping transforms CNV carrier testing to a personalized, simple and cost-effective breakpoint PCR. Citation Format: Ava Kwong, Chun H Au, Dona N Ho, Elaine YL Wong, Yvonne Chung, Fian BF Law, Cecilia YS Ho, Jiawei Chen, Isabella W Cheuk, Vivian Y Shin, Tsun L Chan, Hextan YS Ngan, Edmond SK Ma. Personalized and cost-effective detection of copy number variants by molecular-barcode next-generation sequencing and long-read nanopore sequencing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-13.

Constraints of carrier screening in spinal muscular atrophy: Co-existence of deletion and duplication in SMN1 gene and false negative MLPA result

Spinal Muscular Atrophy (SMA) is the second most common autosomal recessive genetic disorder. Deletion/duplication changes in SMN1 gene are common features in SMA patients. MLPA (Multiplex Ligation-dependent Probe Amplification) is a robust method for investigation of copy number changes because it not only deals with investigating the SMN1 gene but also adjacent genes such as NAIPE, GTFH2 and SMN2. This study was carried out by MLPA and STR markers to SMA carrier screening on people were referred to Dr. Zeinali's Medical Genetics laboratory. Analysis of 150 unrelated families with at least one affected child suspected of having SMA identified causative mutations in 106 families. Among the nighty-nine patients with a homozygous deletion of SMN1 gene (93.3%), there were ten patients with only one parent showing this deletion as heterozygote, and the other parent had two copies of the gene with normal MLPA result. The results were unexpected findings in an autosomal recessive disorder. Paternity and maternity were tested and confirmed.Further analysis of the grandparents showed that the parents inherited both deletion and duplication from one of their parents. So they have two copies of SMN1 genes in a chromosome and no copy on the other one.Due to high carrier frequency of SMA in highly consanguineous population as Iran, co-existence of deletion and duplication must be highly considered. It seems that performing genetic testing only in patients and parents is not sufficient and investigation of the grandparents or autozygosity mapping is also necessary to have reliable results especially for next pregnancies.

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 novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

Application of Multiplex Ligation-Dependent Probe Amplification Assay for Genotyping Major Blood Group Systems Including DEL Variants in the D-Negative Korean Population.

BackgroundThe DEL blood type, a very weak D variant, is a major concern in the field of transfusion medicine because of its potential to cause anti-D alloimmunization. We investigated the molecular basis of serologically D-negative phenotypes, including the DEL type, and the distribution of other blood group systems in the Korean population using the recently developed multiplex ligation-dependent probe amplification (MLPA) assay.MethodsBlood group genotyping using the MLPA assay and RhCE phenotyping were performed on randomly selected 95 D-negative red blood cell products. The MLPA results were verified by multiplex PCR for the RHD promoter, exons 4, 7, and 10 and by direct sequencing of RHD exon 9.ResultsOut of 95 cases, total deletion of the RHD was observed in 74 cases (77.9%) and four cases (4.2%) had an RHD-CE-D hybrid allele. The other 17 cases (17.9%) had an RHD(1227G>A) allele, which was further confirmed by sequencing analysis. The RhCE phenotypes of RHD(1227G>A) alleles were composed of 14 Cce and 3 CcEe, and all 60 cases of the ce phenotype were revealed to have a total deletion of the RHD. Genotyping results and allele distribution of the other 17 blood group systems were consistent with previous reports on the East Asian population.ConclusionsMLPA assay correctly determined RHD genotype, including RHD-CE-D hybrid alleles or RHD(1227G>A) allele, and other clinically relevant blood group genotypes in D-negative Koreans. The use of MLPA assay on serologically D-negative individuals may help improve transfusion safety by preventing anti-D alloimmunization.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 21

Small supernumerary marker chromosomes (sSMCs) are small supernumerary aberrant chromosomes that are generally equal in size or smaller than a chromosome 20, and cannot be identified or characterized by conventional cytogenetic banding techniques [1–3]. sSMCs can appear in 0.044% of newborn infants and in 0.075% of prenatal cases [1,3,4]. About 70% of sSMCs are caused by a de novo event [5], about 70% of sSMCs are originated from acrocentric chromosomes [1,6], and about 70% of de novo sSMCs are associated with no phenotypic effects [4]. Prenatal diagnosis of sSMCs gives rise to difficulties in genetic counseling, and identification of the nature of the aberrant chromosome requires molecular cytogenetic technologies [4,7–10]. We present our experience of the prenatal diagnosis and molecular cytogenetic characterization of an sSMC derived from chromosome 21 using fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA). A 36-year-old woman, gravida 2, para 1, underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed an sSMC, which was C-band positive and nucleolar organizing region-stain positive. The parental karyotypes were normal. The karyotype was 47,XX,+mar (Figure 1). The sSMC hybridized with a centromere 13/21-specific α-satellite DNA probe (D13Z1/D21Z1) (cep13/21) (Cytocell, Adderbury, Oxfordshire, UK) (Figure 2). MLPA was used to determine the origin of the sSMC using a SALSA MLPA P181 centromere kit (MRC-Holland, Amsterdam, the Netherlands) (Figure 3). The results of MLPA indicated a duplication of the 21q11.2 segment containing the STCH and SAMSN1 genes. FISH determination of the duplications of the STCH and SAMSN1 genes was performed using the bacterial artificial chromosome clone probes RP11-138O15 and RP11-392H8 at 21q11.2. FISH determination of 21q21.1 involvement utilized the 21q21.1-specific bacterial artificial chromosome clone probes RP11-89M24 and RP11-109H14. FISH revealed STCH and SAMSN1 gene duplications in the fetus with sSMC(21) (Figure 4). No RP11-89M24or PRENATAL DIAGNOSIS AND MOLECULAR CYTOGENETIC CHARACTERIZATION OF A SMALL SUPERNUMERARY MARKER CHROMOSOME DERIVED FROM CHROMOSOME 21

Use of multiplex ligation-dependent probe amplification (MLPA) for screening of wheat, barley, rye and oats in foods

A multiplex ligation-dependent probe amplification (MLPA) approach is described for the simultaneous detection of DNA from four common cereal ingredients in foods: wheat, barley, rye and oats. The method uses species-specific MLPA half-probes targeting DNA fragments from the ribosomal internal space transcriber (ITS) region for the detection of barley and oats, and from the genes encoding the low molecular weight (LMW) glutenin and the ω-secalin for wheat and rye, respectively. After hybridization, the probes are ligated and amplified by polymerase chain reaction (PCR) into specific and size-differential amplicons that are simultaneously detected by capillary electrophoresis. The cereal MLPA technique showed an optimal specificity against a representative number of plant and animal species. A sensitive detection of the targets (LOD of 50 mg kg−1) was achieved in a reference model cake experimentally spiked with different levels of each wheat, barley, rye and oats seeds. MLPA applicability was assessed through the analysis of 40 commercial food products with different labeling declarations regarding the targets (contain, may contain and do not declare/gluten free), indicating the presence of non-declared cereal ingredients in some of the tested samples. MLPA results were further confirmed by simplex Taqman real-time PCR assays. The described MLPA technique is a reliable and sensitive tool for screening the presence of low amounts of wheat, barley, rye and oats in processed foodstuffs, contributing to compliance with authenticity legal requirements and protecting consumers from fraudulent commercial practices or adventitious contamination which may lead to allergic reactions associated to cereal proteins ingestion.