Jknull alleles in two patients with anti-Jk3

Abstract

Background: As of publication, a total of 41 null alleles have been acknowledged by the International Society of Blood Transfusion (ISBT) to cause the rare Jk_null phenotype, but none have been discovered in Austria thus far.

Materials and methods: Two patients with anti-Jk3 were serologically identified by a positive antibody screening and typed as Jk(a-b-). The initial genotyping using an SSP-PCR method for the common 838A/G polymorphism indicated a JK*02/02, or JK*01/02 genotype, respectively. To find the disruptive mutations, Sanger sequencing was performed and results were compared to the reference sequence. The patient's antibodies were characterized with a monocyte monolayer assay (MMA) for their potential clinical significance.

Results: Three novel null-mutations of the SLC14A1 gene were found in two patients. Patient 1 was homozygous for a 10bp deletion in exon 4 (c.157_166del on JK*02). Testing of her family members revealed Mendelian inheritance of the deletional allele. The other patient was compound heterozygous for two mutations: one allele carrying a single base deletion in exon 4 (c.267delC on JK*01) and the other a splice site mutation in intron 3 (c.152-1g>a on JK*02). The MMA results suggest high clinical significance of the anti-Jk3 in both patients.

Discussion: The detected mutations led to Jk_null phenotypes and are the first description of JK_null alleles in the Austrian population.

Figure 1

Mendelian inheritance of a 10bp deletion in patient 1 and family members (A) Pedigree of patient 1’s family indicating the zygosity of the JK c.157_166del mutation. ABO, RhD blood group and serological Jk phenotype are given and below the result of urea lysis test. The father of patient 1’s children was not tested. (B) Partial DNA Sequencing chromatogram of SLC14A1 exon 4, Patient 1. Top panel shows the wildtype sequence with the 10 bases deleted in Patient 1 shown in grey. (C) SSP-PCR for detection of c.157_166del and corresponding wildtype using primer pairs JK-x5-ndel-F/JK-i5R and JK-x5-del-F/JK-i5-R respectively. Numbers indicate the persons as given in pedigree. wt: Jk(a+b+) blood donor; A.D: water control; M: 100bp marker.

Figure 2

Detection of two mutations on separate alleles in patient 2 (A) Partial sequencing chromatogram of SLC14A1 exon 4. Top panels shows wildtype sequence and overlapping deletional allele c.267delC. (B) Partial DNA sequencing chromatogram of SLC14A1 intron 3 and exon 4. Top panels shows wildtype sequence and overlapping allele with SNP −1g>a in intron 3. Lower case letters are used for intron sequences. (C 1–4) SSP-PCR detection of the mutations shown in A and B. Schematic representation of exon 4 genomic region and SNPs are shown above the gel. Arrows indicate primers used in each assay (black: wildtype, outlined: mutation). Agarose gel is shown below for SSP-PCRs using the primer pairs indicated above. P2: patient 2; wt: Jk(a+b+) blood donor; M: 100bp marker; AD: water control.

Figure 3

Proposed effect of mutated alleles on the urea transporter protein Protein sequences were derived from reference sequence NM_015865.7 using the translate tool from ExPASy bioinformatics resource portal. Amino acid positions 41 to 120 are shown. For in silico translation of c.152 −1g>a, exon 4 was omitted from the reference sequence. Altered amino acids are written bold. Stop is indicated by an X.