Silent KEL alleles identified from Japanese individuals with the Ko phenotype.

BACKGROUND AND OBJECTIVE: The rare Ko phenotype lacks all 36 antigens in the Kell blood system. The molecular basis of the Ko phenotype has been investigated, and more than 40 silent KEL alleles are reported by many investigators. The majority of silent alleles are the KEL*02 background. Here, we report molecular genetic analysis of the KEL gene in Japanese individuals with the Ko phenotype. MATERIALS AND METHODS: The Ko phenotype was screened from Japanese blood donors for several years using monoclonal anti-Ku or anti-K14 by an automated blood grouping system PK7300. Kell-related antigens were typed by standard tube tests. Genomic DNA was extracted from the blood samples, and KEL gene was analysed by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: We collected 35 Ko blood samples with K-k-, Kp(a-b-), Js(a-b-) and K14-. PCR and sequence analysis revealed that 11 individuals were homozygous for a mutant KEL allele with a c.299G>C (p.Cys100Ser) mutation (rs. 200268316). Three individuals were homozygous for the KEL*02N.24 allele that is c.715G>T (p.Glu239*), and one individual was homozygous for the KEL*02N.40 allele that is c.1474C>T (p.Arg492*). Five individuals were homozygous for novel KEL alleles with single-nucleotide mutations, four individuals had a c.2175delC (p.Pro725 fs*43), and one individual had a c.328delA (p.Arg110 fs*79). The remaining 15 individuals were compound heterozygous, and eight new alleles were identified from them. CONCLUSIONS: We identified three known and ten new silent KEL alleles from Japanese individuals with the Ko phenotype. The KEL allele with the c.299G>C (p.Cys100Ser) mutation was the most frequent.

Molecular basis for D- Japanese: identification of novel DEL and D- alleles.

BACKGROUND AND OBJECTIVES: The occurrence of D- is approximately 0.5% in Japanese, but DEL in apparently D- individuals is relatively common compared with that in Caucasian populations. On the basis of molecular genetics, we examined D- Japanese blood donors. METHODS: A standard serological technique was used for RhD typing, and we selected 3526 D- blood samples. Genomic DNA obtained from whole blood was used for RHD analysis by polymerase chain reaction (PCR) and sequencing. Multiplex PCR to detect all of the RHD exons and use of PCR-sequence-specific primer (PCR-SSP) to detect RHD deletion (RHD*01N.01) and c.1227G>A mutation (for RHD*01EL.01) were performed. RESULTS: Multiplex PCR and PCR-SSP revealed that 3091 of 3526 D- individuals (87.7%) were homozygous for RHD*01N.01, and 318 individuals (9.0%) had the RHD*01EL.01/RHD*01N.01 or RHD*01EL.01/RHD*01EL.01 genotype. The other 103 in the 3526 individuals (2.9%) had the known D-CE-D hybrid allele, RHD*01N.04, and the association of RHCE*Ce with RHD*01EL.01 as well as RHD*01N.04 was observed. The remaining 14 individuals had RHD*01N.01 hemizygous with one of the following alleles: RHD*01N.06 (3), RHD*01N.07 (1), RHD*04N.01 (1), RHD*DEL8 (1), RHD with c.761C>G (p.Ser254Ter) (2), RHD with c.1252T>A (p.Ter418Lysex26) (2) and apparently common RHD (4). Adsorption and elution tests with anti-D revealed that the individuals with c.761C>G mutation were D- while the individuals with c.1252T>A mutation were DEL. CONCLUSIONS: The RHD genotype of more than 96% of D- Japanese could be determined by conventional PCR-SSP. In addition, we identified a novel DEL allele having c.1252T>A mutation and a novel RHD silencing allele having c.761C>G nonsense mutation.

Elevated receptor for activated C kinase 1 expression is involved in intracellular Ca2+ influx and potentially associated with compromised regulatory T cell function in patients with asthma.

BACKGROUND: Regulatory T cells (T(regs)) are activated during anergy in response to T cell receptor (TCR) activation and functional immune suppression. Anergy of paediatric T(regs) is partially dependent on intracellular calcium mobility; following TCR activation, T(regs) do not exhibit increased intracellular Ca(2+) concentration ([Ca(2+) ](i)). OBJECTIVE: We determined whether [Ca(2+) ](i) in adult T(regs) defined their anergy, if intracellular Ca(2+) movement was linked to regulatory functions, whether [Ca(2+)](i) was indicative of asthma pathology, and the potential molecular mechanism responsible for Ca(2+) movement in T(regs). METHODS: T(regs) were purified by the magnetic bead method, and their regulatory functions were assessed by monitoring carboxyfluorescein succinimidyl ester-labelled responder T cell proliferation. The Ca(2+) response of Fura-2-labelled cells was measured using a video image analysis system. To analyse the functions of T(regs) at the molecular level, we generated Jurkat Tet-On(®) clones with doxycycline (Dox)-induced forkhead box P3 (FOXP3) protein expression. RESULTS: CD4(+) CD25(+) CD127(-/low) T(regs) from participants without asthma did not elicit Ca(2+) influx in response to TCR activation, exhibited little proliferation and suppressed proliferation of CD4(+) CD25(-) T cells. In contrast, under similar conditions, T(regs) from patients with asthma exhibited increased [Ca(2+)](i) and robust proliferation with partial loss of regulatory functions. FOXP3 protein levels in Tet-On(®) clones were high after both 2- and 5-day Dox treatment; however, 5-day cells were comparable with T(regs) from patients with asthma, whereas 2-day cells were similar to T(regs) from participants without asthma. Increasing [Ca(2+)](i) induced a high level of receptor for activated C kinase 1 (RACK1) expression in 5-day cells. CONCLUSIONS AND CLINICAL RELEVANCE: We confirmed that T(regs) in patients with asthma are functionally impaired and that the abnormal regulatory functions of these cells can be analysed by [Ca(2+)](i) following TCR engagement. Furthermore, the impaired functioning of T(regs) evident in patients with asthma may be due to a high level of RACK1.