Browsing by Author "Singh, Susmita"

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    Detection of large expansions in myotonic dystrophy type 1 using triplet primed PCR
    (Frontiers Media, 2014-04-24) Singh, Susmita; Zhang, Amy; Dlouhy, Stephen; Bai, Shaochun; Medical and Molecular Genetics, School of Medicine
    Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disease caused by expansion of a CTG trinucleotide repeat in the DMPK gene. Methodology for genetic testing of DM1 is currently not optimal, in particular for the early-onset patients in pediatric populations where large expanded (CTG)n alleles are usually common. Individuals who are homozygous for a normal allele and individuals who are heterozygous for one normal and one large expanded allele are indistinguishable by conventional PCR, as both generate a single product of the normal allele. Thus, reflex Southern blot has often been needed to distinguish these cases. With the aim to decrease the need for reflex Southern blot tests, a novel, single-tube CTG repeat primed PCR technology was designed to distinguish the true homozygous patients from the individuals whose large alleles are missed by conventional PCR. The method utilizes two gene-specific primers that flank the triplet repeat region and a third primer set complementary to the repeated region to detect the large alleles. Compared to traditional PCR, this novel Triplet-repeat Primed PCR can detect the presence of large expanded alleles with demonstrating a ladder pattern. Using this single-step protocol, 45 specimens were tested. The alleles with sizes~í~85 repeats were determined by the gene specific primers. 13 abnormal alleles, which were missed by conventional PCR, were successfully detected by the Triplet-repeat Primed PCR. All the abnormal alleles were confirmed and measured by Southern Blot analysis. In summary, optimized Triplet-Primed PCR (TP-PCR) can accurately detect the presence of the large expanded alleles. With the ability to distinguish the true homozygous patients from the false negative homozygous individuals, the application of the optimized TP-PCR can significantly reduce the need of Southern Blot tests.
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    Pretransplant HLA typing revealed loss of heterozygosity in the major histocompatibility complex in a patient with acute myeloid leukemia
    (Elsevier, 2019) Lobashevsky, Andrew L.; Krueger-Sersen, Mary; Britton, Rebecca M.; Littrell, Courtney A.; Singh, Susmita; Cui, Connie P.; Kashi, Zahra; Martin, Russ K.; Breman, Amy M.; Vance, Gail H.; Farag, Sherif S.; Medicine, School of Medicine
    Introduction Chromosomal abnormalities are frequent events in hematological malignancies. The degree of HLA compatibility between donor and recipient in hematopoietic stem cell transplantation is critical. Purpose of the study In this report, we describe an acute myeloid leukemia case with loss of heterozygosity (LOH) encompassing the entire HLA. Materials and methods HLA molecular typing was performed on peripheral blood (PB) and buccal swabs (BS). Chromosomal microarray analysis (CMA) was performed using a whole genome platform. Results Typing results on PB sample collected during blast crisis demonstrated homozygosity at the -A, -B, -C, -DR, and -DQ loci. A BS sample demonstrated heterozygosity at all loci. A subsequent PB sample drawn after count recovery confirmed heterozygosity. The CMA performed on PB samples collected during and after blast crisis revealed a large terminal region of copy-neutral LOH involving chromosome region 6p25.3p21.31, spanning approximately 35.9 Mb. The results of the CMA assay on sample collected after count recovery did not demonstrate LOH. Conclusions LOH at the HLA gene locus may significantly influence the donor search resulting in mistakenly choosing homozygous donors. We recommend confirming the HLA typing of recipients with hematological malignancies when homozygosity is detected at any locus by using BS samples, or alternatively from PB when remission is achieved.