High-Throughput Multiplexed T-Cell–Receptor Excision Circle Quantitative PCR Assay with Internal Controls for Detection of Severe Combined Immunodeficiency in Population-Based Newborn Screening

Gerstel-Thompson, Jacalyn L.; Wilkey, Jonathan F.; Baptiste, Jennifer C.; Navas, Jennifer S.; Pai, Sung-Yun; Pass, Kenneth A.; Eaton, Roger B.; Comeau, Anne Marie

doi:10.1373/clinchem.2010.144915

Cited by 74 publications

(88 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…All samples showed the typical amplification curves expected for TREC, SMN1, and RPP30. TREC Cq values obtained by the in situ DBS triplex assay showed a slight decrease (mean 0.4 Cq) compared to those from the duplex assay but were consistent with the overall ranges for term newborns reported by public health newborn screening programs (11)(12)(13)(14). The Cq values for SMN1 showed a near-gaussian distribution (Fig.…”

Section: Smn1 Assay Developmentsupporting

confidence: 84%

“…SCID is the first NBS condition for which DNA analysis is the primary (first-tier) screening method. Since the initial pilot experiences in 2 US state public health laboratories (11,12 ), SCID-NBS has expanded to many other state programs (13,14 ) and now covers the majority of newborns in the US as well as many newborns globally (15 ). SCID-NBS prevents infant death through early medical intervention and is highly cost-effective (16,17 ).…”

confidence: 99%

See 1 more Smart Citation

Newborn Blood Spot Screening Test Using Multiplexed Real-Time PCR to Simultaneously Screen for Spinal Muscular Atrophy and Severe Combined Immunodeficiency

et al. 2015

View full text Add to dashboard Cite

BACKGROUND Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. METHOD An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. RESULTS DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. CONCLUSIONS SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.

show abstract

Section: Smn1 Assay Developmentsupporting

confidence: 84%

confidence: 99%

Newborn Blood Spot Screening Test Using Multiplexed Real-Time PCR to Simultaneously Screen for Spinal Muscular Atrophy and Severe Combined Immunodeficiency

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The Wisconsin protocol, a single-plex assay, amplified TRECs from the DBS and the internal control, β-actin, in separate reactions, and only if the TREC value fell below the cutoff on initial testing [6,27]. Prior to implementation in Massachusetts, the second state to undertake SCID screening, a multiplex assay was developed in which the internal control, the RNAse P RNA component H1 gene RPPH1, was amplified and measured from the same punch as the initial TREC sample [28]. Efficiencies of amplification approached 100%, and similar to the single-plex assay, the multiplex assay detected TRECs at low copy number, even in cases of maternal engraftment.…”

Section: Modifications Of the Trec Assaymentioning

confidence: 99%

Newborn Screening for Severe Combined Immunodeficiency-A History of the TREC Assay

Bausch-Jurken

Verbsky

Routes

2017

IJNS

View full text Add to dashboard Cite

Abstract:Infants born with T cell lymphopenias, especially severe combined immunodeficiency (SCID) are at risk for serious, often fatal infections without intervention within the first year or two of life. The majority of these disorders can be detected through the use of the T cell recombination excision circle assay (TREC assay.) The TREC assay detects the presence of non-replicating, episomal DNA that is formed during T cell development. This assay initially developed to measure thymic output during aging and HIV infection, has undergone modifications for the purpose of newborn screening (NBS) for SCID. To meet the requirements for inclusion on NBS panels, the assay needed to utilize blood from dried blood spots on NBS cards, and be both sensitive and specific, avoiding the costs of false positives. Currently, the assay relies upon real time, quantitative PCR (RT-qPCR) to detect TRECs in punches taken from dried blood spots. This review seeks to highlight some of the early work leading up to the initial implementation of the TREC assay for SCID detection, and the subsequent revisions made to optimize the assay.

show abstract

“…В зарубежных исследованиях используются различные подходы к оценке уровня содержа-ния TREC и KREC: количество копий в расчете на микролитр крови, копий на условное количе-ство ядросодержащих клеток, количество копий на реакцию [5,7]. В нашей работе мы определяли количество копий TREC/KREC в объеме пери-ферической крови, взятой для экстракции ДНК и в пересчете на количество ядросодержащих эле-ментов в этом объеме -копий на 10 4 лейкоцитов.…”

Section: результаты и обсуждениеunclassified

Retrospective Diagnosis of Primary Immunodeficiencies for Children in Sverdlovsk Region

Степановна¹,

Tuzankina²,

Vlasova³

et al. 2016

Med. immunol.

View full text Add to dashboard Cite

Адрес для переписки:Дерябина Светлана Степановна ГБУЗ СО «Клинико-диагностический центр «Охрана здоровья матери и ребенка» 620041, Россия, г. Екатеринбург, ул. Флотская, 52. Тел./факс: 8 (343) 374-31-10. E-mail: ssderyabina@gmail.com Address for correspondence: Deryabina Svetlana S. Medical Centre "Healthcare of Mother and Child" 620041, Russian Federation, Yekaterinburg, Flotskaya str., 52. Phone/Fax: 7 (343) 374-31-10. E-mail: ssderyabina@gmail.com // Медицинская иммунология, 2016. Т. 18, № 6. С. 583-588. doi: 10.15789/1563-0625-2016-6-583-588 © Дерябина С.С. и соавт., 2016 For citation: S.S. Deryabina, I.A. Tuzankina, E.V. Vlasova, S.G. Lavrina, V.N. Shershnev "Retrospective diagnosis of primary immunodeficiencies for children in Sverdlovsk Region", Medical Immunology (Russia) /Meditsinskaya Immunologiya, 2016, Vol. 18, no. 6, pp. 583-588. doi: 10.15789/1563-0625-2016 Резюме. С целью обоснования необходимости внедрения массового обследования новорожденных на первичные иммунодефицитные состояния (ПИД) методом определения количества копий коль-цевых участков ДНК Т-клеточного (TREC) и В-клеточного (KREC) рецепторов лимфоцитов прове-дено ретроспективное исследование архивных образцов крови детей (n = 43), умерших на первом году жизни. Выраженное снижение количества TREC и (или) KREC выявлено в 16 случаях (37,0%). Кли-ническая картина и обнаружение микроделеции 22q11.2 у одного ребенка подтвердили диагноз ПИД. Еще в 5 образцах обнаружены изменения нуклеотидной последовательности ДНК в гене RAG1: два варианта миссенс-замены Lys820Arg и His249Arg (для трех образцов в гетерозиготном состоянии и для одного -в компаундном) и один уникальный, ранее не описанный вариант -c. 1315C>G (Leu439Val) в гетерозиготном состоянии. Совокупность клинических, гематологических и молекулярно-генети-ческих данных позволяет ретроспективно верифицировать первичную иммунную недостаточность в рассматриваемых случаях. В результате проведенного исследования подтверждена необходимость внедрения метода определения TREC/KREC в программы неонатального скрининга на ПИД для их своевременной диагностики и лечения. Abstract. In order to justify a need for mass screening of primary immunodeficiencies (PID) in a regional program for the newborns, we performed a retrospective study of blood spots (archived screening cards) from

show abstract

High-Throughput Multiplexed T-Cell–Receptor Excision Circle Quantitative PCR Assay with Internal Controls for Detection of Severe Combined Immunodeficiency in Population-Based Newborn Screening

Cited by 74 publications

References 15 publications

Newborn Blood Spot Screening Test Using Multiplexed Real-Time PCR to Simultaneously Screen for Spinal Muscular Atrophy and Severe Combined Immunodeficiency

Newborn Blood Spot Screening Test Using Multiplexed Real-Time PCR to Simultaneously Screen for Spinal Muscular Atrophy and Severe Combined Immunodeficiency

Newborn Screening for Severe Combined Immunodeficiency-A History of the TREC Assay

Retrospective Diagnosis of Primary Immunodeficiencies for Children in Sverdlovsk Region

Contact Info

Product

Resources

About