TY - JOUR
T1 - Improved HUMARA for the Detection of X-Linked Agammaglobulinemia Carriers
AU - Carrillo-Tapia, Eduardo
AU - Espinosa-Padilla, Sara E.
AU - Perez-Perez, Daniela
AU - Gonzalez-Serrano, Maria E.
AU - Berron-Ruiz, Laura
AU - Espinosa-Rosales, Francisco J.
AU - Rodriguez-Alba, Juan C.
AU - Mújica-Guzman, Fabiola
AU - Yokoyama-Rebollar, Emiy
AU - García-Flores, Jose R.
AU - Herrera-González, Norma E.
AU - Scheffler-Mendoza, Selma
AU - Yamazaki-Nakashimada, Marco A.
AU - Staines-Boone, A. Tamara
AU - Lopez-Herrera, Gabriela
N1 - Publisher Copyright:
© Copyright 2022, Mary Ann Liebert, Inc., publishers 2022.
PY - 2022/4
Y1 - 2022/4
N2 - Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.
AB - Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.
KW - HUMARA
KW - X-chromosome inactivation
KW - XLA
KW - carriers
UR - http://www.scopus.com/inward/record.url?scp=85129581968&partnerID=8YFLogxK
U2 - 10.1089/gtmb.2021.0139
DO - 10.1089/gtmb.2021.0139
M3 - Artículo
C2 - 35394812
AN - SCOPUS:85129581968
SN - 1945-0265
VL - 26
SP - 220
EP - 227
JO - Genetic Testing and Molecular Biomarkers
JF - Genetic Testing and Molecular Biomarkers
IS - 4
ER -