Multiplex Ligation-dependent Probe Amplification Analysis Research Articles

A Novel Microduplication Spanning Exons 8-16 of ATP2C1 That Was Undetectable by Standard Sanger Sequencing in a Japanese Patient With Hailey-Hailey Disease.

Hailey–Hailey disease (HHD) is genetic skin disorder characterized by repeated and exacerbated skin lesions in friction regions. ATP2C1, encoding SPCA1, was demonstrated to be the responsible gene for HHD pathogenesis. However, for some cases, no ATP2C1 mutation could be determined by standard Sanger sequencing, thereby obscuring the cause and diagnosis of HHD. In this study, we investigated the possibility that HHD is caused by complex ATP2C1 defects using multiplex ligation-dependent probe amplification (MLPA) analysis for 10 of 50 cases in our institute without ATP2C1 mutations. In one female Japanese patient and her father, who also show HHD, MLPA followed by polymerase chain reaction (PCR) analyses revealed a novel duplication of exons 8–16 of ATP2C1. The duplication was predicted to add 20,615 base pairs, 882 nt, and 294-amino-acid residues to the genome, mRNA and SPCA1 protein, respectively. By reverse transcriptase–PCR using patient skin RNA, we could confirm that a larger transcript was produced and we found that the abundance of the normal transcript was clearly reduced in the patient. Putative structures of wild-type and duplicated proteins revealed differences in arrangement of SPCA1 domains that may have functional consequences. Strikingly, the phosphorylation and the nucleotide-binding domains were interrupted by insertion of a partial actuator, transmembrane, and phosphorylation domains. The additional 294 amino acids appear to disrupt SPCA1 structure and function, causing HHD. Our study expands the spectrum of genetic defects in HHD and showed that disruption of SPCA1 structure and function by the microduplication caused HHD in the patient and her father.

Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic and pituitary tumors, and is due to mutations in the coding region of the MEN1 gene, which encodes menin. We investigated a family with identical twins that had MEN1, with different MEN1 tumors. DNA sequence analysis of the MEN1 coding region had not identified any abnormalities and we hypothesized that deletions and mutations involving the untranslated regions may be involved. Informed consent and venous blood samples were obtained from five family members. Sanger DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses were performed using leukocyte DNA. This revealed a heterozygous 596bp deletion (Δ596bp) between nucleotides -1087 and -492 upstream of the translation start site, located within the MEN1 5' untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5'UTR deletion on MEN1 promoter activity, we generated luciferase reporter constructs, containing either wild-type 842bp or mutant 246bp MEN1 promoter, and transfected them into human embryonic kidney HEK293 and pancreatic neuroendocrine tumor BON-1 cells. This revealed the Δ596bp mutation to result in significant reductions by 37-fold (p < 0.0001) and 16-fold (p < 0.0001) in luciferase expression in HEK293 and BON-1 cells, respectively, compared to wild-type. The effects of this 5'UTR deletion on MEN1 transcription and translation were assessed using qRT-PCR and Western blot analyses, respectively, of mRNA and protein lysates obtained from Epstein-Barr-virus transformed lymphoblastoid cells derived from affected and unaffected individuals. This demonstrated the Δ596bp mutation to result in significant reductions of 84% (p < 0.05) and 88% (p < 0.05) in MEN1 mRNA and menin protein, respectively, compared to unaffected individuals. Thus, our results report the first germline MEN1 5'UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Editors' note: Discrepancy in redetermination of SMN2 copy numbers in children with SMA

In the article, “Discrepancy in redetermination of SMN2 copy numbers in children with SMA,” Drs. Schorling et al. evaluated the accuracy of initial SMN2 copy number determination by repeating genetic testing in 20 patients with spinal muscular atrophy (SMA) using Multiplex Ligation-dependent Probe Amplification (MPLA) analysis. They found that the results of the second testing were discrepant to initial testing in 45% of patients, concluding that reliability testing and quality control procedures are essential before SMN2 copy numbers can be used for clinical decisions and interpretation of outcome data. In response, Drs. Dangouloff et al. reported having observed high rates of discrepancy between the results for SMN2 copy number obtained from their laboratory and other laboratories where their patients were initially tested. They report finding an overall agreement rate of just 31% for SMN2 copy number for 35 patients when testing their samples using 2 different MPLA methods in their own laboratory. They conclude that better standardization of SMN2 copy number testing is needed before these results guide decision-making in the setting of newborn screening. Responding to these comments, the authors note that they switched from the P021-A2 kit the P021-B1 kit (both methods used by Drs. Dangouloff et al.) in March 2019 and found identical results when running 3 probes with variable SMN1 / SMN2 copies with both kits and found an extra copy of exon 1-6 in 12% of the 77 individuals whose samples they subsequently analyzed. They suggest that it is critical to use reliable controls with DNA isolated from the same tissue in every MPLA run. This exchange highlights the difficulties of standardizing quantitative genetic testing techniques across different laboratories—particularly for relatively rare diseases for which it may be challenging to build domain-specific laboratory expertise, but the results nevertheless guide potentially game-changing decisions for patients. In the article, “Discrepancy in redetermination of SMN2 copy numbers in children with SMA,” Drs. Schorling et al. evaluated the accuracy of initial SMN2 copy number determination by repeating genetic testing in 20 patients with spinal muscular atrophy (SMA) using Multiplex Ligation-dependent Probe Amplification (MPLA) analysis. They found that the results of the second testing were discrepant to initial testing in 45% of patients, concluding that reliability testing and quality control procedures are essential before SMN2 copy numbers can be used for clinical decisions and interpretation of outcome data. In response, Drs. Dangouloff et al. reported having observed high rates of discrepancy between the results for SMN2 copy number obtained from their laboratory and other laboratories where their patients were initially tested. They report finding an overall agreement rate of just 31% for SMN2 copy number for 35 patients when testing their samples using 2 different MPLA methods in their own laboratory. They conclude that better standardization of SMN2 copy number testing is needed before these results guide decision-making in the setting of newborn screening. Responding to these comments, the authors note that they switched from the P021-A2 kit the P021-B1 kit (both methods used by Drs. Dangouloff et al.) in March 2019 and found identical results when running 3 probes with variable SMN1 / SMN2 copies with both kits and found an extra copy of exon 1-6 in 12% of the 77 individuals whose samples they subsequently analyzed. They suggest that it is critical to use reliable controls with DNA isolated from the same tissue in every MPLA run. This exchange highlights the difficulties of standardizing quantitative genetic testing techniques across different laboratories—particularly for relatively rare diseases for which it may be challenging to build domain-specific laboratory expertise, but the results nevertheless guide potentially game-changing decisions for patients.

Phenotype and genotype analysis of 55 children patients with Wilson's disease

Objective: To understand the clinical phenotype and spectrum of ATP7B gene mutation in children with Wilson's disease (WD). Methods: A total of 55 cases diagnosed with WD at the Children's Hospital Affiliated to Nanjing Medical University from June 2012 to June 2018 were taken as the research subject. ATP7B gene point mutation was detected by direct sequencing after PCR amplification. Heterozygous mutation in children was discovered by sequencing. Furthermore, the long segment mutation of exon was analyzed by multiplex ligation-dependent probe amplification (MLPA). Results: All 55 WD children had varying degree of liver damage symptoms. Among them, 2 cases had combined neurological symptoms. The positive rates of K-F ring (21%), 24-hour urine copper (97.7%), and ceruloplasmin were all abnormal. The results of ATP7B gene had identified 8 homozygous, 41 compound heterozygous and 6 heterozygous in 55 cases. Direct sequencing method had detected ten cases of ATP7B heterozygotes. In addition, MLPA analysis showed that other allele in four cases had a deletion of the ATP7B gene exon. In all cases, 35 different ATP7B gene mutations were detected, including 23 missense mutations, 3 frameshift mutations, 4 nonsense mutations, 3 exon deletions and 2 splicing changes. The most common allele mutation was c.2333G > T/p.R778L in exon 8, with an allele frequency of 36.54%, followed by c.2975C > T/p.P992L in exon 13, with an allele frequency of 14.42%. Conclusion: ATP7B gene c.2333G > T/p.R778L and c.2975C > T/p.P992L mutations are the most common mutations in children with WD in China. WD patients report shows that there are three long deletion mutations in the exon of the ATP7B gene. For WD children whose DNA sequencing is heterozygous ATP7B gene, it is suggested to further use MLPA method to detect deletion mutations of exons.

Integrated Genomic Analysis of Chromosomal Alterations and Mutations in B-Cell Acute Lymphoblastic Leukemia Reveals Distinct Genetic Profiles at Relapse.

The clonal basis of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is complex and not fully understood. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis–relapse samples from 13 BCP-ALL patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of the BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (p = 0.019). MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. TP53 was the most frequently mutated gene at relapse. Two TP53 mutations were detected only at relapse, whereas the three others showed an increase in their mutational burden at relapse. Clonal evolution patterns were heterogeneous, involving the acquisition, loss and maintenance of lesions at relapse. Therefore, this study provides additional evidence that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. Integrative NGS, aCGH and MLPA analysis enables better molecular characterization of the genetic profile in BCP-ALL patients during the evolution from diagnosis to relapse.

Assessment of Multiplex Ligation-Dependent Probe Amplification (MLPA) as a diagnostic test for Egyptian patients with Williams-Beuren syndrome

BackgroundWilliams-Beuren syndrome (WS) is as a rare multisystem genetic syndrome, caused by the hemizygous deletion of approximately 26–28 genes on chromosome 7q11.23. Fluorescence in-situ hybridization (FISH) is considered the gold standard for its diagnosis. However, FISH does not allow the detection of the exact size of the deletion. This study aimed to compare the efficiency and validity of Multiplex Ligation-Dependent Probe Amplification (MLPA) and (FISH) techniques in diagnosing and confirming clinically diagnosed WS. Patients and methodsThe study comprised twenty-three WS patients they were clinically assessed, cardiovascular evaluation, and laboratory tests, including plasma calcium level, thyroid function tests and glucose tolerance test, were performed. FISH and MLPA analysis were performed for all patients. The MLPA kit (SALSA P029) contained probes for eight genes in the WS critical region: FKBP6, FZD9, TBL2, STX1A, ELN, LIMK1, RFC2, and CYLN2. ResultsNineteen patients out of 23 studied cases (82.6%) showed positive deletion of the WS chromosome region (WBSCR) by both FISH and MLPA techniques. One patient (4.3%) showed negative result by FISH while a duplication of the WBSCR was detected by MLPA. Three patients (13.1%) were negative for both FISH and MLPA. In conclusionThis is the largest cohort of Egyptian patients with WS, where both FISH and MLPA analysis were used to confirm the diagnosis. MLPA analysis has an advantage over FISH as it is less time consuming and detects smaller, atypical deletions and duplications in the WS.

Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Mutation analysis of 419 family and prenatal diagnosis of 339 cases of spinal muscular atrophy in China

BackgroundSpinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disease caused by mutations in the survival motor neuron 1 (SMN1) gene. At present, gene therapy medicine for SMA, i.e., Spinraza (Nusinersen), has been approved by the FDA, bringing hope to SMA patients and families. Accurate diagnosis is essential for treatment. Our goal was to detect genetic mutations in SMA patients in China and to show the results of the prenatal diagnosis of SMA.MethodsIn this study, we examined 419 patients in our hospital from January 2010 to September 2019. Multiplex ligation-dependent probe amplification analysis was used to determine the copy numbers of SMN1 and SMN2. Long-range PCR combined with nested PCR was used to detect point mutations in SMN1. In addition to the above detection methods, we also used QF-PCR in prenatal diagnosis to reduce the impact of maternal contamination. We conducted a total of 339 prenatal diagnoses from January 2010 to September 2019.ResultsHomozygous deletion of SMN1 exon 7 was detected in 96.40% (404/419) of patients. Homozygous deletion of SMN1 exon 7 alone was detected in 15 patients (3.60%). In total, 10 point mutations were detected in the 15 pedigrees. Most patients with SMA Type I have 1 ~ 2 copies of the SMN2 gene. Patients with SMA Type II have 2 or 3 copies of the SMN2 gene. The results of prenatal diagnoses showed that 118 fetuses were normal, 149 fetuses were carriers of heterozygous variants, and the remaining 72 fetuses harbored compound heterozygous variants or homozygous variants.ConclusionsOur study found that the most common mutation in SMA was homozygous deletion of SMN1 exon 7 in our study. We suggest that detecting only the deletion of exon 7 of SMN1 can meet most of the screening needs. We also believe that SMN2 copy numbers can help infer the disease classification and provide some reference for future treatment options.

SAT-LB17 Triplication of SHOX Downstream Region in Mild Short Stature

Deletions of the downstream flanking regions of the SHOX gene that contain conserved non-coding cis-regulatory DNA elements are a known cause of Leri-Weill dyschondrosteosis or idiopathic short stature (ISS). Functional and comparative genomic studies have demonstrated the existence of four CNE enhancers downstream of SHOX which have transcriptional activity, and a fifth has recently been suggested at approximately X:970,000. Duplications of these downstream regions have also been described in patients with ISS, although with a variable phenotype. We present a family with ISS and the first ever reported SHOX downstream triplication. The index case was a 12-year-old Caucasian female of unrelated parents, born with normal weight and length, and normal hearing/vision and development. At the age of 12, her height was -1.87 SD (-1.35 SD adjusted for parental height). Her growth chart showed a height on the 90th centile at birth, slowly crossing centiles for the first 5 years to the 2nd centile, which she then followed. She had normal proportions and head circumference, and no dysmorphic features apart from inverted nipples. Baseline investigations for short stature were normal, including IGF1, karyotype and celiac screen. Bone age was 1.5 year delayed. Family history: arthritis in the father, early menopause in the mother. Analysis of SHOX and its flanking regions was carried out using Multiplex Ligation-dependent Probe Amplification (MLPA) and direct sequencing of all coding exons. MLPA analysis showed a SHOX downstream triplication with a minimum size of 66kb (X:963670-1029779) and a maximum size of 428kb (X:899389-1327688). The size of the duplication was further defined using array comparative genome hybridization (ACGH) analysis which showed the minimum size to be ~325kb (X:907457-1232802). This triplication includes the proposed regulatory element at ~ X:970,000. The triplication was subsequently shown to be present in the proband’s mother and brother who also have mild ISS. Further analysis of these and other family members is ongoing. The triplication is of a single-sized fragment, and the fragment size is identical to one that has been seen in duplicated form in several other patients, increasing the likelihood that the triplicated fragments are in tandem. The precise mechanism of the pathogenic effect of the triplication is unknown, but it likely exerts a negative effect on SHOXtranscription, reducing its expression and ultimately resulting in SHOX haploinsufficiency. In conclusion, we describe a family with mild ISS and a downstream triplication of the proposed X:970,000 SHOX downstream regulatory element. This provides further evidence for this previously proposed regulatory element downstream of SHOX and adds additional proof that increased dosage of this regulatory element region is a cause of short stature.

Multiplex Ligation-Dependent Probe Amplification for Simultaneous Identification of Bungarus multicinctus and Its Common Adulterants in a Single Assay.

Bungarus multicinctus, an important traditional Chinese medicine, possesses remarkable medicinal activities, while lots of adulterants from other species were misused as B. multicinctus for its large demand and resource starvation. In order to accurately identify B. multicinctus and its common adulterants such as Sinonatrix annularis, Xenochrophis flavipunctatus, Deinagkistrodon acutus, and Naja atra, a simultaneous identification method was designed with multiplex ligation-dependent probe amplification (MLPA) analysis. Five species-specific MLPA probe-couples for B. multicinctus and its common adulterants were designed based on the universal primer amplified COI sequences, which can specifically detect the five species with no mutual interference, and sensitivity analysis showed as less as 5% B. multicinctus or 8.75% adulterants in the mixed samples can be identified in a MLPA assay, especially, the relative quantity of the adulterants can be also inferred based on the MLPA peak area values. Moreover, the results of the present study confirmed the effectiveness of this technique in terms of simultaneous identification of B. multicinctus and its common adulterants in an assay, which has great potential for ensuring the safety of this commercially valuable snake species.

Genetic analysis of 90 families affected with spinal muscular atrophy

To carry out genetic testing and prenatal diagnosis for 90 families affected with spinal muscular atrophy (SMA), and discuss the necessity for carrier screening. All families were subjected to multiplex ligation-dependent probe amplification (MLPA) analysis. Combined MLPA and allele-specific PCR (AS-PCR) was used for prenatal diagnosis of the pregnant women. Among the 90 couples, 84 (93%) had a negative family history, 85 (94%) had given birth to an affected child before. Eighty-five husbands and 88 wives carried heterozygous deletion of exon 7 of the SMN1 gene. Two wives had homozygous deletion of exon 7 of the SMN1 gene and were affected. Prenatal diagnosis showed that 19 fetuses were SMA patients, 48 fetuses were carriers, and 23 fetuses were normal. Of note, eighteen affected fetuses were conceived by couples without a family history, which accounted for 20% of all pregnancies and 95% of all affected fetuses. To screen SMA carriers using MLPA and carry out prenatal diagnosis using combined MLPA and AS-PCR can ensure accurate diagnosis, which has a significant value for the prevention of SMA affected births.

Haploinsufficiency of A20 with a novel mutation of deletion of exons 2–3 of TNFAIP3

Objectives Haploinsufficiency of A20 (HA20) due to loss-of-function mutations of TNFAIP3 leads to an autoinflammatory disease. These mutations produce a premature termination codon in most cases of HA20. However, exon deletion has not been reported. Methods Genomic DNA was extracted from the peripheral blood of the patient clinically suspected of HA20. We examined autoinflammatory disease-causing genes and performed a multiplex ligation-dependent probe amplification (MLPA) assay for copy number analysis. Next, to determine the disconnection point, genomic DNA was amplified with long-range PCR and sequenced. Finally, western blotting was carried out to measure A20 protein expression in mitogen phytohaemagglutinin (PHA)-induced T-cell blasts from the patient and a healthy volunteer. Results Targeted next-generation sequencing found no pathogenic mutation, but copy number variation (CNV) analysis suggested a heterozygous deletion of exons 2–3. The MLPA assay and long-range PCR confirmed the mutation. Western blotting analysis indicated a marked decrease in expression of A20 protein from the patient compared to a normal control. The results showed that this deletion was a pathogenic mutation. Conclusion This study demonstrates a novel mutation resulting in a deletion of exons 2–3 of TNFAIP3. MLPA analysis is a useful initial screening method for HA20 patients.

54. Towards better characterisation of molecular alterations in glioneuronal tumours

Background: Glioneuronal tumours constitute a subtype of brain tumour characterised by neoplastic cells with both glial and neuronal differentiation. The lack of specific biomarker panels has resulted in large inter-observer variability in their diagnosis using morphological and existing immunohistochemical criteria. Preliminary findings indicate that paediatric glioneuronal tumours may be subclassified based on detection of certain molecular abnormalities. In combination with histopathological analysis, better molecular characterisation of glioneuronal tumours has the potential to improve diagnostic accuracy and provide data relevant for treatment and prognostication. Aim: To characterise genomic and genetic alterations in paediatric and adult glioneuronal tumours. Methods: DNA extracted from 30 FFPE low grade glioneuronal tumours was analysed using combinations of low pass whole genome sequencing (LP-WGS) and multiplex ligation-dependent probe amplification (MLPA) for brain tumour associated genetic and genomic alterations. Results: LP-WGS of 22 glioneuronal tumours detected no whole or partial chromosomal losses or gains in 9 of the tumours. Chromosome 7 gain was identified in 6 of the tumours and was the most common genomic alteration, with other chromosomal abnormalities (1p–, 5+, 8q+, 9–, 10p+) evident in single tumours. Two tumours, an unclassified high grade glioneuronal tumour and a low grade ganglioglioma exhibited multiple chromosomal abnormalities, 1p–/3p–/14–/15–/16q–/17q+ and 5+/7+/8+/9+/10+/12+/13+/16+/20+/21+, respectively. MLPA analysis confirmed regions of 7p gain (EGFR) and 8p gain (FGFR1/TACC) and identified BRAFV600E mutations in 3 tumours. CDKN2A loss, a marker of poor prognosis, was detected in one tumour. Conclusions: Findings of this study indicate glioneuronal tumours to be a molecularly heterogeneous group of neoplasms that may be subtyped on the basis of chromosomal and genetic alterations, and using laboratory procedures available in diagnostic pathology practice. Refinement of molecular profiling, and the detection of markers of poor prognosis or targetable mutations may improve tumour classification algorithms and enable better management of this rare tumour entity.

Identification of a rare tri-allelic inheritance at the D8S1179 locus in a case of paternity testing

Short Tandem Repeat (STR) typing is routinely used in forensic casework, paternity disputes and in the detection of aneuploidies. Anomalous peak patterns are occasionally encountered in STR typing due to genetic variations, mutations and, other abnormalities. We report here a rare tri-allelic variant at the D8S1179 locus in a 6-month-old female child, observed during routine paternity testing in the Indian Tamil population.DNA eluted from the blood samples collected on FTA elute microcards were subjected to STR typing for autosomal and sex STR loci using the commercial AmpFlSTR Identifiler Plus kit and the Promega PowerPlex® Fusion kit. Capillary electrophoresis was carried out in ABI 3130xl Genetic Analyzer and genotyped using GeneMapper ID-X Software v1.5. Copy number variations and aneuploidy status of chromosome 8 were analyzed using Multiplex ligation-dependent probe amplification (MLPA) and Prenatal BoBs assays. In this case of paternity exclusion, the child displayed a Type II tri-allelic pattern (10,13,14) at the D8S1179 locus. In addition, the mother displayed an abnormal 10,14 genotype (with heterozygous peak imbalance - 2:1) at the D8S1179 locus suggesting 10,10,14 pattern. MLPA and prenatal BoBs analyses did not detect any abnormalities on chromosome 8. These results suggest an intrachromosomal segmental duplication event at the D8S1179 locus (8q24.13) as the reason behind tri-allelic pattern and its inheritance. Understanding anomalous cases such as these can help in appropriate interpretation of the results in STR typing.

Clinical manifestations and neurofibromatosis type 1 gene mutations of 25 patients with neurofibromatosis type 1 from 10 Chinese pedigrees

This study enrolled 25 patients with neurofibromatosis type 1 (NF1) from 10 Chinese pedigrees. Sanger sequencing analysis and multiplex ligation-dependent probe amplification analysis were used to detect mutations and large fragment losses of the NF1 gene. This study identified 10 NF1 mutations, which comprised six novel and four recurrent mutations. Majority of the mutations can lead to termination codon production, which results in the synthesis of the truncated gene product neurofibromin.

Identification of PCDH19 Gene Mutations/Deletions in Patients with Early Onset Epilepsy.

Background and Aims:PCDH19 gene, which encodes protocadherin 19, is associated with epilepsy and intellectual disability, mainly in affected females. The clinical manifestations are heterogeneous and the main features include early onset seizure, generalized or focal seizures sensitive to fever, and brief seizures occurring in clusters. The disorders exhibit a unique and unusual X-linked pattern of expression. We aimed to investigate PCDH19 mutations/deletions in patients with epilepsy and describe the clinical/molecular features.Methods:PCDH19 gene was analyzed in 35 Turkish female patients from 34 families with early-onset epilepsy via direct sequencing and multiplex ligation-dependent probe amplification analysis. Additionally, array comparative genomic hybridization analysis was performed in patients with whole gene deletion.Results:We identified 2 different heterozygous mutations in 2 unrelated probands (5. 7%) which were located in exon 1. Additionally, whole gene deletions were detected in dizygotic twin girls (5. 7%), who had distinct clinical features and the deletion was inherited from the unaffected father. The second twin suffered more severe clinical manifestations including autistic features, behavioral problems, mild-moderate mental retardation and seizures, which were under control with multidrug regimen when compared with the first twin.Conclusion:PCDH19 is a major causative gene in patients with epilepsy and further data is required to gain a better understanding of phenotype-genotype correlation. In addition to gene sequencing, deletion/duplication analysis will improve the molecular diagnosis in patients with clinical findings.

Newborn Screening for Spinal Muscular Atrophy in China Using DNA Mass Spectrometry.

Background: Spinal muscular atrophy (SMA) is the most common neurodegenerative disorder and the leading genetic cause of infant mortality. Early detection of SMA through newborn screening (NBS) is essential to selecting pre-symptomatic treatment and ensuring optimal outcome, as well as, prompting the urgent need for effective screening methods. This study aimed to determine the feasibility of applying an Agena iPLEX SMA assay in NBS for SMA in China. Methods: We developed an Agena iPLEX SMA assay based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and evaluated the performance of this assay through assessment of 167 previously-genotyped samples. Then we conducted a pilot study to apply this assay for SMA NBS. The SMN1 and SMN2 copy number of screen-positive patients were determined by multiplex ligation-dependent probe amplification analysis. Results: The sensitivity and specificity of the Agena iPLEX SMA assay were both 100%. Three patients with homozygous SMN1 deletion were successfully identified and conformed by multiplex ligation-dependent probe amplification analysis. Two patients had two SMN2 copies, which was correlated with severe SMA type I phenotype; both of them exhibited neurogenic lesion and with decreased muscle power. Another patient with four SMN2 copies, whose genotype correlated with milder SMA type III or IV phenotype, had normal growth and development without clinical symptoms. Conclusions: The Agena iPLEX SMA assay is an effective and reliable approach for population-based SMA NBS. The first large-scale pilot study using this assay in the Mainland of China showed that large-scale implementation of population-based NBS for SMA is feasible.

ENVIRONMENTAL AND GENETIC EVALUATION IN 46,XY DISORDERS OF SEX DEVELOPMENT

The phenotype of Disorders of Sex Development (DSD)patients depends on many factors including the presence of Copy Number Variation (CNVs) of different genes. The unbalanced rearrangement and the presence of deletions or duplications affect dramatically the phenotypic sex. The aim of this study is to correlate genotypic abnormalities with clinical phenotype in 46,XY DSD patients by Multiplex Ligation dependant Probe Amplification (MLPA) technique for accurate diagnosis in these patients and study possible paternal and maternal exposure to environmental risk factors in these cases. This study reported on forty patients with variable presentations of disorders of sex development (DSD) presenting with ambiguous genitalia, hypospadias, micropenis or with female phenotype with primary amenorrhea or short stature, with exclusion of cases with disorders in androgen synthesis. A complete personal, family history and clinical examination were done. Parents were asked to fill a questionnaire about frequency of dealing with some environmental factors including smoking, caffeine and using some materials having estrogenic effect as plastics, insecticides, and others. Conventional cytogenetics studies and fluorescence in situ hybridization (FISH) on peripheral blood as well as DNA extraction and Multiplex Ligation-dependent Probe Amplification (MLPA) were done for all cases for some genes; DMRT1, CYP17A1, SRD5A2, HSD17B3, DAX1, CXor21, SOX9, SRY, ZFY, WNT4 and SF. Results showed 46,XY was shown in all patients. MLPA analysis showed Copy Number Variation (CNVs) in 15% of cases. The study showed duplication of DMRT1 in 5% patients, deletion of SRY 2.5% patients, deletion of SOX9 in 5% patients, duplication of DAX1 in 12.5% patient, duplication of CYP17A41 in 5% patients, deletion of DMRT1 in 12.5% patient, duplication of SRD5A in 2.5% patient and duplication of HSD17B3 in 2.5% patient. Maternal exposure during pregnancy and paternal exposure to Endocrine Disrupting Compounds (EDCs) were insignificantly associated with DSD compared to control cases. This study demonstrates the importance of proper detection of genetic mutation in DSD patients showing a discrepancy between their karyotype and gonadal phenotype. It was concluded that using MLPA is recommended for better understanding of the phenotype with other recent techniques as for better diagnosis and follow up.

Phenotype categorization of neurofibromatosis type I and correlation to NF1 mutation types.

Neurofibromatosis type 1 (NF1) is caused by heterozygous mutation in the NF1 gene. NF1 is one of the most common human genetic diseases. However, the overall genotype-phenotype correlation has not been known, due to a wide spectrum of genotypic and phenotypic heterogeneity. Here we describe the detailed clinical and genetic features of 427 Korean NF1 patients from 389 unrelated families. Long range PCR and sequencing of genomic DNA with multiplex ligation-dependent probe amplification analysis identified 250 different NF1 mutations in 363 families (93%), including 94 novel mutations. With an emphasis on phenotypes requiring medical attention (classified and termed: NF1+), we investigated the correlation of NF1+ and mutation types. NF1+ was more prevalent in patients with truncating/splicing mutations and large deletions than in those with missense mutations (59.6%, 64.3% vs. 36.6%, p = 0.001). This difference was especially significant in the patients younger than age 19 years. The number of items in NF1+ was a higher in the former groups (0.95 ± 0.06, 1.18 ± 0.20 vs. 0.56 ± 0.10, p = 0.002). These results suggest that mutation types are associated not only with higher prevalence of severe phenotypes in NF1 but also with their earlier onset.

Identification of novel BRCA1 large genomic rearrangements by a computational algorithm of amplicon-based Next-Generation Sequencing data.

BackgroundGenetic testing for BRCA1/2 germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients.MethodsWe first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel BRCA1 LGRs was obtained via long-range PCR and direct sequencing of the DNA products.ResultsIn our cohort, the newly defined DQ-based algorithm detected 3/3 BRCA1 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6–99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6–99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel BRCA1 deletions, involving exons 16–17, 21–22 and 20, have been characterized.ConclusionsOur study defined a DQ-based algorithm to identify BRCA1 LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.