Applicability of Real-Time PCR Methodology in the Neonatal Detection of Turner Syndrome

Rocha, Mylene Neves; Longui, Carlos Alberto; Kochi, Cristiane; Corrêa, Clovis; Faria, Claudio; Richeti, F.; Melo, Mariana

doi:10.1055/s-0030-1253351

Cited by 7 publications

(5 citation statements)

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“…We previously studied the real-time PCR technique in the identification of TS (Rocha et al, 2010) by quantifying the ARSE/GAPDH genes ratio. In our previous study, we obtained 100% sensitivity and specificity for TS patients and the 45,X karyotype, but only 56% sensitivity in patients with mosaicism.…”

Section: Discussionmentioning

confidence: 99%

“…PCRs were carried out in a 7500 Real-Time PCR System (Applied Biosystems), and the parameter setting was conducted with the aid of the SDS-Sequence Detection System version 1.2, 7500 Systems SDS Software (Applied Biosystems). The thermocycling conditions used were 50°C for 2 min; 95°C for 10 min; 40 cycles at 95°C for 15 s; and 60°C for 1 min; fluorescence readings were recorded during the final step of each cycle (Rocha et al, 2010).…”

Section: Molecular Analysismentioning

confidence: 99%

“…Karyotyping for neonatal screening has important limitations, such as running time, cost, and need for specialized personnel, among others (Fröhling et al, 2002;Siegel and Sybert, 2005;Klein, 2011). Therefore, various molecular methods have been proposed for diagnosis or neonatal screening of TS, including Southern blotting, polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), fluorescent PCR genotyping, GeneScan-based genotyping, pyrosequencing, and real-time PCR (Gicquel et al, 1998;Longui et al, 2002;Cirigliano et al, 2004;Meng et al, 2005;Figueiredo et al, 2008;Rocha et al, 2010;Rivkees et al, 2011;Aksglaede et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Neonatal detection of Turner syndrome by real-time PCR gene quantification of the ARSE and MAGEH1 genes

Corrêa¹,

Rocha²,

Richeti³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Turner syndrome (TS) is characterized by the presence of one full X chromosome and total or partial deletion of the second sex chromosome. Diagnosis of TS is often delayed, resulting in inappropriate treatment. Early diagnosis of TS using a neonatal screening test may improve preventive measures and treatment, thus improving patient quality of life. The goal of this study was to standardize a neonatal TS screening algorithm. Two study genes (ARSE and MAGEH1) and 1 normalizing gene (HBB) were used to detect the second X chromosome. We screened 996 newborns whose peripheral blood was collected and stored in filter paper. In addition, samples from 20 patients with confirmed diagnosis of TS were included in the study. Relative amounts of ARSE/HBB were determined using real-time polymerase chain reaction. The cutoff at the 5th percentile was arbitrarily set to indicate repetition of the test. The test was repeated in 51/1016 patients with ARSE/HBB < 0.81. For 10 samples with values persistently <0.81, we quantified the MAGEH1/HBB ratio. Values below the 95th percentile in TS patients (MAGEH1/HBB < 1.24) were considered to be inadequate. Only 6/996 NB showed inadequate values for the 2 studied genes, which were recalled for clinical evaluation and karyotype testing. Analysis of 20 patients diagnosed with TS allowed for identification of falsenegatives and true-positives, establishing 95% sensitivity when the indicated cutoff values were used. In conclusion, our algorithm reached 95% detection sensitivity with an acceptable recall rate (0.6%), allowing for the detection of suspected TS cases in the neonatal period.

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Section: Discussionmentioning

confidence: 99%

Section: Molecular Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Neonatal detection of Turner syndrome by real-time PCR gene quantification of the ARSE and MAGEH1 genes

Corrêa¹,

Rocha²,

Richeti³

et al. 2014

Genet. Mol. Res.

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“…Schmidt et al (2000) reported a method for the prenatal diagnosis of somatic aneuploidies and SCAs based on the gene copy number. Other reports include the analysis by quantitative polymerase chain reaction (qPCR) of different genes for the diagnosis of TS, such as the androgen receptor (AR) gene (Rocha et al, 2010), dose-sensitive sex reversal-adrenal hypoplasia congenital critical region on the X chromosome gene 1 (DAX1), and the arylsulfatase E gene (ARSE) (Rocha et al, 2005), and for the diagnosis of KS, such as the AR and short stature homeobox (SHOX) genes (Ottesen et al, 2007;Aksglaede et al, 2012). We have previously published, in already diagnosed patients, that the identification of TS could be done by means of quantifying copy number of SHOX and VAMP7 (vesicle-associated membrane protein 7) pseudoautosomal genes together with the SRY (Ibarra-Ramirez et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Gene Copy Number Quantification of SHOX, VAMP7, and SRY for the Detection of Sex Chromosome Aneuploidies in Neonates

Ibarra‐Ramírez

Lugo-Trampe

Campos-Acevedo

et al. 2020

Genetic Testing and Molecular Biomarkers

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Aims: To explore the feasibility of detecting sex chromosome aneuploidies (SCAs) by means of gene copy number quantification of short stature homeobox (SHOX), vesicle-associated membrane protein 7 (VAMP7), and SRY in newborns. Materials and Methods: Gene doses of SHOX, VAMP7, and SRY were determined by quantitative polymerase chain reaction (qPCR) using DNA obtained from dried blood samples from newborns. Relative quantification values were obtained. An aneuploidy profile was established according to cutoff values. Samples with ‡2 gene doses (out of range) were reanalyzed, and those with aneuploidy profiles were confirmed by karyotyping. Sensitivity, specificity, and positive and negative predictive values were obtained. Results: A total of 10,033 samples were collected (4945 females and 5088 males). Of 244 (2.43%) samples with ‡2 gene doses that were retested, 20 cases were confirmed. The overall incidence of SCAs was 1 in 500 live newborns. There were six cases of Turner syndrome (1/824), 3 cases of XXX (1/1648), 7 cases of Klinefelter syndrome (1/726), and 4 cases of of XYY (1/1272). The sensitivity was 0.952 (95.24%), specificity of 0.975 (97.56%), positive predictive value of 0.909 (90.91%), and negative predictive value of 0.987 (98.77%). Conclusions: Gene copy number analyses of VAMP7, SHOX, and SRY genes by qPCR from blood samples spotted onto filter paper is a highly reliable method for the early detection of male and female SCAs.

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“…The dosage reduction of genes located in PARs1 and 2, which is inherent to the complete or partial loss of the second sex chromosome, can be sensitively quantified by quantitative polymerase chain reaction (qPCR), as reported for the ARSE gene in Xp22.33 (Rocha et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Detection of Turner Syndrome by Quantitative PCR of SHOX and VAMP7 Genes

Ibarra‐Ramírez

Zamudio-Osuna²,

Campos-Acevedo³

et al. 2015

Genetic Testing and Molecular Biomarkers

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Turner Syndrome (TS) is an unfavorable genetic condition with a prevalence of 1:2500 in newborn girls. Prompt and effective diagnosis is very important to appropriately monitor the comorbidities. The aim of the present study was to propose a feasible and practical molecular diagnostic tool for newborn screening by quantifying the gene dosage of the SHOX, VAMP7, XIST, UBA1, and SRY genes by quantitative polymerase chain reaction (qPCR) in individuals with a diagnosis of complete X monosomy, as well as those with TS variants, and then compare the results to controls without chromosomal abnormalities. According to our results, the most useful markers for these chromosomal variants were the genes found in the pseudoautosomic regions 1 and 2 (PAR1 and PAR2), because differences in gene dosage (relative quantification) between groups were more evident in SHOX and VAMP7 gene expression. Therefore, we conclude that these markers are useful for early detection in aneuploidies involving sex chromosomes.

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Applicability of Real-Time PCR Methodology in the Neonatal Detection of Turner Syndrome

Cited by 7 publications

References 5 publications

Neonatal detection of Turner syndrome by real-time PCR gene quantification of the ARSE and MAGEH1 genes

Neonatal detection of Turner syndrome by real-time PCR gene quantification of the ARSE and MAGEH1 genes

Gene Copy Number Quantification of SHOX, VAMP7, and SRY for the Detection of Sex Chromosome Aneuploidies in Neonates

Detection of Turner Syndrome by Quantitative PCR of SHOX and VAMP7 Genes

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