Last data update: Sep 16, 2024. (Total: 47680 publications since 2009)
Records 1-2 (of 2 Records) |
Query Trace: Yazdanpanah GK [original query] |
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MALDI-TOF-MS Assay to Detect the Hemizygous 22q11.2 Deletion in DNA from Dried Blood Spots.
Kobrynski LJ , Yazdanpanah GK , Koontz D , Lee FK , Vogt RF . Clin Chem 2015 62 (1) 287-92 BACKGROUND: A hemizygous deletion of 1.5-3 Mb in 22q11.2 causes a distinct clinical syndrome with variable congenital defects. Current diagnostic methods use fluorescent in situ hybridization (FISH) or comparative genomic hybridization by microarray to detect the deletion. Neither method is suitable for newborn screening (NBS), since they cannot be performed on dried blood spots (DBS). We developed a MALDI-TOF-MS assay that uses DBS to measure the hemizygous deletion of UFD1L, located within the 22q11.2 region. METHODS: We used DBS from 54 affected patients, previously tested by FISH or microarray, and 100 cord blood samples to evaluate the performance of the MALDI-TOF-MS assay. With a single primer pair, a 97-base oligonucleotide within UFD1L was amplified, as was a sequence on chromosome 18 that differs by 2 nucleotides. A multiplexed, single-base extension reaction created allele-specific products for MALDI-TOF-MS detection. The products were spotted onto a silicon chip, and the height of the spectral peaks identified the relative amounts of target and reference gene. RESULTS: The median ratio of the spectral peak for each UFD1L target:reference base was 0.96 and 0.99 for controls, compared with 0.35 and 0.53 for 22q11 deletion syndrome patients. There was 100% concordance between FISH/microarray and MALDI-TOF-MS in all patients with 22q11.2 deletion syndrome. CONCLUSIONS: This method can be reliably performed with DBS and is suitable for high sample throughput. This assay may be considered for use in population-based NBS for 22q11.2 deletion. |
Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.
Taylor JL , Lee FK , Yazdanpanah GK , Staropoli JF , Liu M , Carulli JP , Sun C , Dobrowolski SF , Hannon WH , Vogt RF . Clin Chem 2014 61 (2) 412-9 BACKGROUND: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of the motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn bloodspot 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. |
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