RHD genotype and zygosity analysis in the Chinese Southern Han D+, D- and D variant donors using the multiplex ligation-dependent probe amplification assay.

BACKGROUND AND OBJECTIVES: Several comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation-dependent probe amplification (MLPA) assay was validated for RHD genotyping in the Chinese. MATERIALS AND METHODS: The blood samples of 200 D+, 200 D- and 62 D variant Chinese donors were collected. RhD antigen was routinely typed by serological method. D variant phenotype was determined by an anti-D panel (D-Screen), when RBCs were available. The RHD genotype and its zygosity were analysed with the RH-MLPA technique. When the MLPA was unable to identify a RHD variant, direct sequencing of all exons of the RHD gene was performed. RESULTS: In 200 D+ donors, DD (168/200, 84%), D (12/200, 6%), DDD genotype (1/200) and D variant allele carriers (19/200, 9·5%) were found. In 200 D- donors, six reported RHD alleles, RHD*01EL.01, RHD*01N.03, RHD*01N.05, RHD*01N.16, RHD*DFR2 and RHD*weak partial 15 and one novel RHD*1154T allele were identified in 36·5% (73/200) of them. In 62 D variant donors, three novel RHD alleles, RHD*79_81delCTC, RHD*710T and RHD*689A, and twelve reported alleles, RHD*DVI.3, RHD*weak partial 15, RHD*DVI.4, RHD*01EL.01, RHD*01N.03, RHD*DLO, RHD*DV.5, RHD*D-CE(2-10), RHD*730C, RHD*weak D type 25, 33 and 72, were identified, either alone or in combination. CONCLUSION: The RH-MLPA assay correctly identified the common RHD variant alleles in the Chinese population. However, DNA sequencing was required to identify certain alleles; probes to detect these alleles should be added into the assay.

Assessment of p53 and ATM functionality in chronic lymphocytic leukemia by multiplex ligation-dependent probe amplification.

The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.