Evaluating the clinical efficacy of a long-read sequencing-based approach for carrier screening of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the second most common fatal genetic disease in infancy. It is caused by deletion or intragenic pathogenic variants of the causative gene SMN1, which degenerates anterior horn motor neurons and leads to progressive myasthenia and muscle atrophy. Early treatment improves motor function and prognosis in patients with SMA, but drugs are expensive and do not cure the disease. Therefore, carrier screening seems to be the most effective way to prevent SMA birth defects. In this study, we genetically analyzed 1400 samples using multiplex ligation-dependent probe amplification (MLPA) and quantitative polymerase chain reaction (qPCR), and compared the consistency of the results. We randomly selected 44 samples with consistent MLPA and qPCR results for comprehensive SMA analysis (CASMA) using a long-read sequencing (LRS)-based approach. CASMA results showed 100% consistency, visually and intuitively explained the inconsistency between exons 7 and 8 copy numbers detected by MLPA in 13 samples. A total of 16 samples showed inconsistent MLPA and qPCR results for SMN1 exon 7. CASMA was performed on all samples and the results were consistent with those of resampling for MLPA and qPCR detection. CASMA also detected an additional intragenic variant c.-39A>G in a sample with two copies of SMN1 (RT02). Finally, we detected 23 SMA carriers, with an estimated carrier rate of 1/61 in this cohort. In addition, CASMA identified the "2 + 0" carrier status of SMN1 and SMN2 in a family by analyzing the genotypes of only three samples (parents and one sibling). CASMA has great advantages over MLPA and qPCR assays, and could become a powerful technical support for large-scale screening of SMA.

Hereditary haemochromatosis: Prevalence and characterization of the disease in a tertiary hospital in Aragon.

BACKGROUND: The main genetic cause of iron overload is haemochromatosis (HC). In recent years, the study of non-HFE genes (HFE2, HJV, HAMP, TRF2, SLC40A1, and BMP6) has become relevant thanks to next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) techniques. Our objectives were to estimate the prevalence of both HFE (C282Y/HY63D variants) and non-HFE variants attending a tertiary hospital in Aragón, to predict the effect of the variants on the protein, and to establish a genotype-phenotype correlation evaluating with the clinical context. METHODS: Retrospective descriptive study from 2006 to 2020 of patients attended at genetic consultation in a reference hospital for HC in Aragon. We calculated prevalence of HFE and non-HFE variants. We analysed non-HFE genes (HFE2, HJV, HAMP, TRF2, SLC40A1, and BMP6), used bioinformatics tools, consulted different databases and measured clinical parameters (laboratory and imaging). RESULTS: The prevalence of C282Y homozygous was 5.95% respect the total of cases and 0.025% respect our population. The prevalence of non-HFE HC variants was 1.94% respect the total of cases and 0.008% respect our population. We found 27 variants in non-HFE genes and 4 in HFE gene, of which 6 were classified as variant of uncertain clinical significance (VUS), or likely pathogenic or pathogenic according to the ACMG classification criteria. CONCLUSION: Our prevalence results are as expected, and similar to those obtained by other studies. Although some of the genetic findings explain the clinical symptoms of some of our patients, we remain have a high number of patients without a clear molecular diagnosis.

High positive predictive value of CNVs detected by clinical exome sequencing in suspected genetic diseases.

BACKGROUND: Genetic disorders often manifest as abnormal fetal or childhood development. Copy number variations (CNVs) represent a significant genetic mechanism underlying such disorders. Despite their importance, the effectiveness of clinical exome sequencing (CES) in detecting CNVs, particularly small ones, remains incompletely understood. We aimed to evaluate the detection of both large and small CNVs using CES in a substantial clinical cohort, including parent-offspring trios and proband only analysis. METHODS: We conducted a retrospective analysis of CES data from 2428 families, collected from 2018 to 2021. Detected CNV were categorized as large or small, and various validation techniques including chromosome microarray (CMA), Multiplex ligation-dependent probe amplification assay (MLPA), and/or PCR-based methods, were employed for cross-validation. RESULTS: Our CNV discovery pipeline identified 171 CNV events in 154 cases, resulting in an overall detection rate of 6.3%. Validation was performed on 113 CNVs from 103 cases to assess CES reliability. The overall concordance rate between CES and other validation methods was 88.49% (100/113). Specifically, CES demonstrated complete consistency in detecting large CNV. However, for small CNVs, consistency rates were 81.08% (30/37) for deletions and 73.91% (17/23) for duplications. CONCLUSION: CES demonstrated high sensitivity and reliability in CNV detection. It emerges as an economical and dependable option for the clinical CNV detection in cases of developmental abnormalities, especially fetal structural abnormalities.

Investigating the correlation between genotype and phenotype in Prader-Willi syndrome: a study of 45 cases from Brazil.

BACKGROUND: Prader-Willi syndrome (PWS) is a genetic disorder characterized by abnormalities in the 15q11-q13 region. Understanding the correlation between genotype and phenotype in PWS is crucial for improved genetic counseling and prognosis. In this study, we aimed to investigate the correlation between genotype and phenotype in 45 PWS patients who previously underwent methylation-sensitive high-resolution melting (MS-HRM) for diagnosis. RESULTS: We employed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and Sanger sequencing, along with collecting phenotypic data from the patients for comparison. Among the 45 patients, 29 (64%) exhibited a deletion of 15q11-q13, while the remaining 16 (36%) had uniparental disomy. No statistically significant differences were found in the main signs and symptoms of PWS. However, three clinical features showed significant differences between the groups. Deletion patients had a higher prevalence of myopia than those with uniparental disomy, as well as obstructive sleep apnea and an unusual skill with puzzles. CONCLUSIONS: The diagnostic tests (MS-HRM, MS-MLPA, and Sanger sequencing) yielded positive results, supporting their applicability in PWS diagnosis. The study's findings indicate a general similarity in the genotype-phenotype correlation across genetic subtypes of PWS.

Investigation of genotype-phenotype and familial features of Turkish dystrophinopathy patients.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive allelic muscle diseases caused by dystrophin gene mutations. Eight hundred thirty-seven patients admitted between 1997 and 2022 were included in the study. Two hundred twenty patients were analyzed by multiplex PCR (mPCR) alone. Five hundred ninety-five patients were investigated by multiplex ligation-dependent probe amplification (MLPA), and 54 patients were examined by sequencing. Deletion was detected in 60% (132/220) of the cases in the mPCR group only and in 58.3% (347/595) of the cases with MLPA analysis. The rates of deletion and duplication were 87.7% and 12.3%, respectively, in the MLPA analysis. Single exon deletions were the most common mutation type. The introns 43-55 (81.8%) and exons 2-21 (13.1%) regions were detected as hot spots in deletions. It was determined that 89% of the mutations were suitable for exon skipping therapy. The reading frame rule did not hold in 7.6% of D/BMD cases (17/224). We detected twenty-five pathogenic/likely pathogenic variants in sequencing, five of which were novel variants. Nonsense mutation was the most common small mutation (44%). 21% of DMD patients were familial. We detected germline mosaicism in four families (4.3%) in the large rearrangement group and one gonosomal mosaicism in a family with a nonsense mutation. This is the largest study examining genotype and phenotype data in Turkish D/BMD families investigated by MLPA analysis. The reading frame hypothesis is not valid in all cases. Sharing the genotype and phenotype characteristics of these cases in the literature will shed light on the molecular structure of DMD and guide gene therapy research. In genetic counseling, carrier screening in the family and possible gonadal mosaicism should be emphasized.

High prevalence of copy number variations in the Japanese participants with suspected MODY.

Maturity-Onset Diabetes of the Young (MODY) is a diabetes mellitus subtype caused by a single gene. The detection rate of the responsible gene is 27% in the United Kingdom, indicating that the causative gene remains unknown in the majority of clinically diagnosed MODY cases. To improve the detection rate, we applied comprehensive genetic testing using whole exome sequencing (WES) followed by Multiplex Ligation-dependent Probe Amplification (MLPA) and functional analyses. Twenty-one unrelated Japanese participants with MODY were enrolled in the study. To detect copy number variations (CNVs), WES was performed first, followed by MLPA analysis for participants who were negative on the basis of WES. Undetermined variants were analyzed according to their functional properties. WES identified 7 pathogenic and 3 novel likely pathogenic variants in the 21 participants. Functional analyses revealed that 1 in 3 variants was pathogenic. MLPA analysis applied to the remaining 13 undetermined samples identified 4 cases with pathogenic CNVs: 3 in HNF4A and 1 in HNF1B. Pathogenic variants were identified in 12 participants (12/21, 57.1%) - relatively high rate reported to date. Notably, one-third of the participants had CNVs in HNF4A or HNF1B, indicating a limitation of WES-only screening.

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.

Comparison of exon-level copy number variants in CytoScan XON assay and next-generation sequencing in clinical samples.

BACKGROUND AND AIMS: Next-generation sequencing (NGS)-based copy number variants (CNVs) have high false-positive rates. The fewer the exons involved, the higher the false-positive rate. A CytoScan XON assay was developed to assess exon-level CNVs. MATERIALS AND METHODS: Twenty-three clinically relevant exon-level CNVs in 20 patient blood samples found in previous NGS studies were compared with the results from the CytoScan XON and multiplex ligation-dependent probe amplification (MLPA). RESULTS: Fifteen of the 23 exon-level CNVs were consistent with the NGS results. Among these, eight were confirmed using MLPA. In six out of eight discrepancies between the CytoScan Xon and NGS, MLPA was performed, and three were negative, indicating that the CNVs in NGS were false positives. The CytoScan XON exhibits a sensitivity of 72.7% for small exon-level CNVs, along with a specificity of 100%. The assay could not detect the three exon-level CNVs in PKD1 and TSC2 that were detected using both NGS and MLPA. This could be due to the distribution of the probes in some areas, and the CNV-calling regions containing multiple exons. CONCLUSION: The CytoScan XON assay is a promising complementary tool for the detection of exon-level CNVs, provided that the users carefully examine the distribution of probes and calling regions.

The Phenotypic Variability Associated with Hepatocyte Nuclear Factor 1B Genetic Defects Poses Challenges in Both Diagnosis and Therapy.

The evolving landscape of clinical genetics is becoming increasingly relevant in the field of nephrology. HNF1B-associated renal disease presents with a diverse array of renal and extrarenal manifestations, prominently featuring cystic kidney disease and diabetes mellitus. For the genetic analyses, whole exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) were performed. Bioinformatics analysis was performed with Ingenuity Clinical Insights software (Qiagen). The patient's electronic record was utilized after receiving informed consent. In this report, we present seven cases of HNF1B-associated kidney disease, each featuring distinct genetic abnormalities and displaying diverse extrarenal manifestations. Over 12 years, the mean decline in eGFR averaged -2.22 ± 0.7 mL/min/1.73 m2. Diabetes mellitus was present in five patients, kidney dysplastic lesions in six patients, pancreatic dysplasia, hypomagnesemia and abnormal liver function tests in three patients each. This case series emphasizes the phenotypic variability and the fast decline in kidney function associated with HNF-1B-related disease. Additionally, it underscores that complex clinical presentations may have a retrospectively straightforward explanation through the use of diverse genetic analytical tools.

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay.

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis. The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Mutation analysis and clinical profile of South African patients with Neurofibromatosis type 1 (NF1) phenotype.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition with complete age-dependent penetrance, variable expressivity and a global prevalence of ∼1/3,000. It is characteriszed by numerous café-au-lait macules, skin freckling in the inguinal or axillary regions, Lisch nodules of the iris, optic gliomas, neurofibromas, and tumour predisposition. The diagnostic testing strategy for NF1 includes testing for DNA single nucleotide variants (SNVs), copy number variants (CNVs) as well as RNA analysis for deep intronic and splice variants, which can cumulatively identify the causative variant in 95% of patients. In the present study, NF1 patients were screened using a next-generation sequencing (NGS) assay targeting NF1 exons and intron/exon boundaries for SNV and NF1 multiple ligation-dependent probe amplification (MLPA) analysis for CNV detection. Twenty-six unrelated Southern African patients clinically suspected of having NF1, based on the clinical diagnostic criteria developed by the National Institute of Health (NIH), were included in the current study. A detection rate of 58% (15/26) was obtained, with SNVs identified in 80% (12/15) using a targeted gene panel and NF1 gene deletion in 20% (3/15) identified using MLPA. Ten patients (38%) had no variants identified, although they met NF1 diagnostic criteria. One VUS was identified in this study in a patient that met NF1 diagnostic criteria, however there was no sufficient information to classify variant as pathogenic. The clinical features of Southern African patients with NF1 are similar to that of the known NF1 phenotype, with the exception of a lower frequency of plexiform neurofibromas and a higher frequency of developmental/intellectual disability compared to other cohorts. This is the first clinical and molecular characterisation of a Southern African ancestry NF1 cohort using both next-generation sequencing and MLPA analysis. A significant number of patients remained without a diagnosis following DNA-level testing. The current study offers a potential molecular testing strategy for our low resource environment that could benefit a significant proportion of patients who previously only received a clinical diagnosis without molecular confirmation.

Screening of Spinal Muscular Atrophy Carriers and Prenatal Diagnosis in Pregnant Women in Yancheng, China.

Spinal muscular atrophy (SMA) is a neuromuscular disorder with an autosomal recessive inheritance pattern. Patients with severe symptoms may suffer respiratory failure, leading to death. The homozygous deletion of exon 7 in the SMN1 gene accounts for nearly 95% of all cases. Population carrier screening for SMA and prenatal diagnosis by amniocentesis for high-risk couples can assist in identifying the risk of fetal disease. We provided the SMA carrier screening process to 55,447 pregnant women in Yancheng from October 2020 to December 2022. Among them, 8185 participated in this process, with a participation rate of around 14.76% (95% CI 14.47-15.06%). Quantitative real-time polymerase chain reaction (qPCR) was used to detect deletions of SMN1 exons 7 and 8 (E7, E8) in screened pregnant women. 127 were identified as carriers (111 cases of E7 and E8 heterozygous deletions, 15 cases of E7 heterozygous deletions, and 1 case of E7 heterozygous deletions and E8 homozygous deletions), resulting in a carrying rate of around 1.55% (95% CI 1.30-1.84%). After genetic counseling, 114 spouses of pregnant women who tested positive underwent SMA carrier screening; three of them were screened as SMA carriers. Multiplexed ligation-dependent probe amplification (MLPA) was used for the prenatal diagnosis of the fetuses of high-risk couples. Two of them exhibited two copies of SMN1 exon 7 (normal), and the pregnancy was continued; one exhibited no copies of SMN1 exon 7 and exon 8 (SMA patient), and the pregnancy was terminated. Analyzing SMN1 mutations in Yancheng and provide clinical evidence for SMA genetic counseling and birth defect prevention. Interventional prenatal diagnosis for high-risk families can promote informed reproductive selection and prepare for the fetus's early treatment.

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

Case report: A family of atypical hemolytic uremic syndrome involving a CFH::CFHR1 fusion gene and CFHR3-1-4-2 gene duplication.

Mutations in the complement factor H (CFH) gene are associated with complement dysregulation and the development of atypical hemolytic uremic syndrome (aHUS). Several fusion genes that result from genomic structural variation in the CFH and complement factor H-related (CFHR) gene regions have been identified in aHUS. However, one allele has both CFHR gene duplication and CFH::CFHR1 fusion gene have not been reported. An 8-month-old girl (proband) presented with aHUS and was treated with ravulizumab. Her paternal grandfather developed aHUS previously and her paternal great grandmother presented with anti-neutrophil cytoplasmic antibody-associated vasculitis and thrombotic microangiopathy (TMA). However, the proband's parents have no history of TMA. A genetic analysis revealed the presence of CFH::CFHR1 fusion gene and a CFHR3-1-4-2 gene duplication in the patient, her father, and her paternal grandfather. Although several fusion genes resulting from structural variations of the CFH-CFHR genes region have been identified, this is the first report of the combination of a CFH::CFHR1 fusion gene with CFHR gene duplication. Because the CFH-CFHR region is highly homologous, we hypothesized that CFHR gene duplication occurred. These findings indicate a novel pathogenic genomic structural variation associated with the development of aHUS.

A Pilot Study of Multiplex Ligation-Dependent Probe Amplification Evaluation of Copy Number Variations in Romanian Children with Congenital Heart Defects.

Congenital heart defects (CHDs) have had an increasing prevalence over the last decades, being one of the most common congenital defects. Their etiopathogenesis is multifactorial in origin. About 10-15% of all CHD can be attributed to copy number variations (CNVs), a type of submicroscopic structural genetic alterations. The aim of this study was to evaluate the involvement of CNVs in the development of congenital heart defects. We performed a cohort study investigating the presence of CNVs in the 22q11.2 region and GATA4, TBX5, NKX2-5, BMP4, and CRELD1 genes in patients with syndromic and isolated CHDs. A total of 56 patients were included in the study, half of them (28 subjects) being classified as syndromic. The most common heart defect in our study population was ventricular septal defect (VSD) at 39.28%. There were no statistically significant differences between the two groups in terms of CHD-type distribution, demographical, and clinical features, with the exceptions of birth length, weight, and length at the time of blood sampling, that were significantly lower in the syndromic group. Through multiplex ligation-dependent probe amplification (MLPA) analysis, we found two heterozygous deletions in the 22q11.2 region, both in patients from the syndromic group. No CNVs involving GATA4, NKX2-5, TBX5, BMP4, and CRELD1 genes were identified in our study. We conclude that the MLPA assay may be used as a first genetic test in patients with syndromic CHD and that the 22q11.2 region may be included in the panels used for screening these patients.

Clinical and genetic characteristics in a Chinese cohort of complex spastic paraplegia type 4.

Spastic paraplegia type 4 (SPG4), caused by SPAST mutations, is the most predominant subtype of hereditary spastic paraplegia. Most documented SPG4 patients present as pure form, with the complex form rarely reported. We described the clinical and genetic features of 20 patients with complex phenotypes of SPG4 and further explored the genotype-phenotype correlations. We collected detailed clinical data of all SPG4 patients and assessed their phenotypes. SPAST gene mutations were identified by Multiplex ligation-dependent probe amplification in combination with whole exome sequencing. We further performed statistical analysis in genotype and phenotype among patients with various manifestations and different variants. Out of 90 SPG4 patients, 20 patients (male:female = 16:4) with additional neurologic deficits, namely complex form, were included in our study. The bimodal distribution of age of onset at 0-10 and 21-40 years old is concluded. On cranial MRI, obvious white matter lesions can be observed in five patients. We identified 9 novel and 8 reported SPAST mutations, of which 11 mutations were located in AAA (ATPase associated with various cellular activities) domain. The AAA cassette of spastin is the hottest mutated region among complex SPG4. All patients with cognitive impairment (CI) are males (n = 9/9). Additionally, 80% patients with ataxia are due to frameshift mutations (n = 4/5). Overall, our study summarized and analyzed the genetic and phenotypic characteristics of complex SPG4, making up over 1/5 of in-house SPG4 cohort, among which CI and ataxia are the most common features. Further studies are expected to explore the underlying mechanisms.

Application of a Multiplex Ligation-Dependent Probe Amplification-Based Next-Generation Sequencing Approach for the Detection of Pathogenesis of Duchenne Muscular Dystrophy and Spinal Muscular Atrophy Caused by Copy Number Aberrations.

Both Duchenne muscular dystrophy (DMD; OMIM no. 310200) and spinal muscular atrophy (SMA; OMIM no. 253300/253550/253400/271150) are genetic disorders characterized by progressive muscle degeneration and weakness. Genetic copy number aberrations in the pathogenetic genes DMD and SMN1 lead to alterations in functional proteins, resulting in DMD and SMA, respectively. Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for the detection of common copy number aberrations (CNAs), including DMD and SMN1 deletions, both of which are associated with poor clinical outcomes. However, traditional MLPA assays only accommodate a maximum of 60 MLPA probes per test. To increase the number of targeted sequences in one assay, an MLPA-based next-generation sequencing (NGS) assay has been developed that is based on the standard MLPA procedure, allows high-throughput screening for a large number of fragments and samples by integrating additional indices for detection, and can be analyzed on all Illumina NGS platforms.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

OBJECTIVES: Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. METHODS: Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. RESULTS: The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. CONCLUSIONS: Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

Comparison of the accuracy of multiplex digital PCR versus multiplex ligation-dependent probe amplification in quantification of the survival of motor neuron genes copy numbers.

For over two decades, multiplex ligation-dependent probe amplification (MLPA) has served as the gold standard for genetic testing of spinal muscular atrophy. However, there is emerging evidence questioning the reliability of MLPA in determining the copy numbers (CNs) of the survival of motor neuron (SMN) gene in certain cases. Recently, digital polymerase chain reaction (dPCR) has shown potential for better performance in copy number variant detection. This study aimed to compare MLPA and dPCR in quantifying SMN1 and SMN2 CNs, identify reasons for observed discrepancies, and explore the clinical implications of false results. A total of 733 DNA samples, previously subjected to MLPA analysis, were tested using multiplex droplet dPCR assays. Samples exhibiting inconsistent results between the two methods underwent repeated dPCR assays. When inconsistencies persisted, a third method was employed for verification. Digital PCR yielded results consistent with those of MLPA in 94.4% (692/733) of samples. Forty-one cases exhibited quantitative disparities in SMN1 and/or SMN2 CNs between the two methods. Confirmatory tests revealed that 37 inaccurate results were produced by the MLPA analysis, whereas four were attributed to the dPCR method. The dPCR technique exhibits better accuracy than MLPA and is qualified for SMA genetic testing across various clinical scenarios.

Amplificación de sondas múltiples dependientes de ligación: actualización y revisión del principio, variantes y aplicaciones / Multiplex ligation-dependent probe amplification: update and review of principles, variants, and applications / Amplificação de múltiplas sondas dependentes de ligação: atualização e revisão do princípio, variantes e aplicações

Resumen La amplificación de sondas múltiples dependientes de ligación (MLPA) es una valiosa herramienta en el estudio de alteraciones en el número de copias para distintas patologías de origen genético. La existencia de una amplia oferta de kits comerciales, el fácil y rápido procesamiento de laboratorio, su alta sensibilidad y, en general, los buenos resultados informados han permitido que su uso se encuentre expandido en muchos laboratorios de biología molecular alrededor del mundo. El principio de esta técnica ha sido adaptado para distintas aplicaciones como la MLPA metilación específica para el estudio de enfermedades relacionadas con la impronta epigenética y la MLPA digital que se acopla a equipos de secuenciación de nueva generación para aumentar la cantidad de regiones analizadas en un solo ensayo. Al contar con 10 años de experiencia en el uso de esta técnica en el Laboratorio Nacional de Tamizaje Neonatal y Alto Riesgo se realiza esta revisión con el fin de analizar los principios, variantes de la técnica, análisis de los datos, algunas aplicaciones, ventajas y desventajas de la MLPA en comparación con otras tecnologías disponibles.

Abstract Multiplex ligation-dependent probe amplification (MLPA) is a valuable tool in the study of copy number alterations for different pathologies of genetic origin. The availability of a wide range of commercial kits, the easy and fast laboratory processing, its high sensitivity and, in general, the good results reported have allowed its use to be expanded in many molecular biology laboratories around the world. The basic principle of this technique has been adapted for different applications such as specific methylation MLPA for the study of diseases related to epigenetic imprinting and digital MLPA that is coupled to next-generation sequencing equipment to increase the number of regions analysed in a single trial. With 10 years of experience in the use of this technique, the National Laboratory for Neonatal and High Risk Screening performs this review in order to analyse the principles, variants of the technique, data analysis, some applications, and advantages and disadvantages of MLPA compared to other available technologies.

Resumo A amplificação de múltiplas sondas dependentes de ligação (MLPA) é uma ferramenta valiosa no estudo das alterações do número de cópias para diferentes patologias de origem genética. A existência de uma ampla gama de kits comerciais, processamento laboratorial fácil e rápido, alta sensibilidade e, em geral, os bons resultados relatados, permitiram que seu uso se encontre expandido em muitos laboratórios de biologia molecular ao redor do mundo. O princípio desta técnica foi adaptado para diferentes aplicações como a MLPA metilação específica para o estudo de doenças relacionadas com o imprinting epigenético e a MLPA digital que é acoplada a equipamentos de sequenciamento de nova geração para aumentar o número de regiões analisadas em um único ensaio. Com 10 anos de experiência no uso da técnica, o Laboratório Nacional de Triagem Neonatal e de Alto Risco realiza esta revisão visando a analisar os princípios, variantes da técnica, análise dos dados, algumas aplicações, vantagens e desvantagens da MLPA comparado com outras tecnologias disponíveis.