The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibility
The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibilityBernadette Olivès1, Marilyn Merriman2, Pascal Bailly1, Stephen Bain3, Anthony Barnett3, John Todd2, Jean-Pierre Cartron1,* and Tony Merriman2,*
1INSERM U76, GIP-INTS, 6 rue Alexandre Cabanel 75015 Paris, France, 2The Wellcome Trust Centre for Human Genetics, Nuffield Department of Surgery, University of Oxford, Windmill Road, Headington, Oxford OX3 7BN, UK and 3Department of Medicine, University of Birmingham, Birmingham Heartlands Hospital, Birmingham B9 5SS, UK
Received February 4, 1997;Revised and Accepted April 23, 1997
The Kidd blood group locus encodes a urea transporter which is expressed on human red cells and in the kidney. This gene is located on chromosome 18q12, and evidence for linkage and association with type 1 diabetes mellitus has been reported. To investigate this further, the genetic basis for the blood group Jka/Jkb polymorphism was first determined by sequencing reverse-transcribed reticulocyte RNAs from Jk(a+b-) and Jk(a-b+) donors. The Jka/Jkb polymorphism was caused by a transition (G838A), resulting in a Asp280Asn amino acid substitution and an MnlI restriction fragment length polymorphism (RFLP). Using the MnlI RFLP, we found that the Jka/Jkb polymorphism was not in linkage disequilibrium with type 1 diabetes in 228 multiplex UK and US families tested.
The Kidd blood group system (Jk) is defined by two alleles, Jka and Jkb (frequency 0.51 and 0.48 in Europeans), whose products were first identified with alloantibodies responsible for haemolytic disease of the newborn or transfusion reactions. There are three common phenotypes Jk(a+b-), Jk(a-b+) and Jk(a+b+) and a rare null phenotype, Jk(a-b-) (1 ).
Since red cells from Jk(a-b-) individuals which lack Jk antigens exhibited an increased resistance to lysis in aqueous 2 M urea (2 ) and showed a defect in urea transport(3 ), it was suggested that both phenotypes could be carried by a single polypeptide. This hypothesis was fully confirmed by the molecular cloning of the urea transporter expressed in human erythrocytes (clone HUT11) (4 ,5 ). The gene encoding the Kidd/HUT11 urea transporter polypeptide has been assigned to chromosome 18q12-q21 by in situ hybridization (5 ), where the Kidd blood group gene locus has been mapped (6 ). The Kidd/urea transport protein is present on human red cells as well as in the kidney, particularly on the endothelial cells of the vasa recta in the inner and outer medulla, but is not present in renal tubules (7 ).
Type 1, or insulin-dependent, diabetes mellitus is a childhood disease affecting 0.4% of children of Caucasian descent. Disease, caused by autoimmune destruction of insulin-producing pancreatic [beta]-islet cells, results from a combination of genetic and environmental factors. The major histocompatibility complex (MHC) locus on chromosome 6p21 is a primary genetic determinant (IDDM1)(8 ). Disease susceptibility is also conferred by the variable number of tandem repeats (VNTR) polymorphism immediately upstream of the insulin gene on chromosome 11p15 (IDDM2)(9 ). Recent genome scanning and other candidate gene studies have provided evidence for the existence of at least five further loci (IDDM4, IDDM5, IDDM6, IDDM8 and IDDM12) that contribute to type 1 diabetes susceptibility(10 -13 ).
Historically, the Kidd blood group locus has been implicated in susceptibility to type 1 diabetes. In 1982, a genome screen by Hodge et al. (14 ) with 27 polymorphic markers revealed linkage of the Kidd blood group locus, in addition to the MHC region, with type 1 diabetes in a US data set of 71 families (parametric lod score = 2.8 under a recessive model of inheritance). Despite a report of linkage disequilibrium of the Kidd blood group Jkballele with type 1 diabetes(15 ), evidence supporting linkage of Jk with disease was not extended when the US data set of Hodge and co-workers was extended to 100 families (16 ), nor was linkage replicated in a separate data set from the US(17 ). However, in 1994, evidence of linkage of the chromosome 18q21 region with type 1 diabetes was obtained in 93 UK affected sib pair families (18 ).
Therefore, in this report, we determine the molecular basis of the historical Jk polymorphism and directly test its association with type 1 diabetes by designing a PCR assay for the polymorphism and by analysing a large number of diabetic families.
Reticulocyte RNAs from Jk(a+b-) or Jk(a-b+) blood samples were used as templates to amplify by hemi-nested PCR the entire Jka and Jkb coding sequences, as shown in Figure 1 A. Sequence analysis of the 1234 bp product from each allele revealed that the Jka and Jkb cDNAs differed at only one position by a transition G838A, resulting in the amino acid substitution Asp280Asn (Fig. 1 A), located in the fourth predicted extracellular loop of the human urea transporter(4 ). Sequence comparison of the HUT11 clone encoding the human urea transporter isolated previously(4 ) and Jk alleles revealed two further differences which were not related to the Jk polymorphism since they are shared by both the Jka and Jkb alleles. One change occurred in codon 44 (transition A -> G resulting in a Lys44Glu substitution) and a second corresponded to a hexanucleotide deletion that did not modify the reading frames and resulted in the absence of the dipeptide Val-Gly at amino acid positions 225 and 226. Allele-specific PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed that these differences were not allelic to the Jk locus (data not shown). As the G838A substitution was correlated with the presence or absence of an MnlI restriction site, a hemi-nested PCR-RFLP assay for DNA genotyping of Jk was developed (Fig. 1 B).
The transmission disequilibrium test (TDT)(19 ) was used to test whether the MnlI RFLP, which determines the serological specificity of the Kidd Jka/Jkb polymorphism, also encodes susceptibility to type 1 diabetes mellitus. This test for linkage disequilibrium is family-based so it distinguishes between association due to linkage and association that may arise in the absence of linkage, such as that owing to population stratification. The 228 UK and US families were genotyped with the MnlI RFLP. The frequency of the Jkb allele, which previously had been reported to be associated with type 1 diabetes, was 0.45 in parents. Transmission of this allele from heterozygous parents to affected children was assessed (Table 1 ). There was no significant bias in transmission of the Jkb allele to diabetic offspring when the data sets were analysed separately or together. Previously, association of the Kidd blood group locus with type 1 diabetes was observed only when the data set was partitioned according to genotype at IDDM1-only those type 1 diabetics with zero or one high risk HLA class II alleles (these genotypes are DR 3/X, 4/X and X/X, where DRX is not DR3 or DR4) showed association of the Jkb allele with disease [relative risk = 2.5, P <0.025 (15 )]. In this study, there was no significant bias in transmission of the Jkb allele to diabetics with zero or one high risk IDDM1 alleles (Table 1 ).
Linkage of the MnlI RFLP to diabetes was also tested by the affected sib pair method. In the UK data set, the maximum lod score (MLS) was 0.0, the MLS was 0.1 in the US and was 0.0 in the total 225 families. Thus there was no evidence supporting linkage of the Jka/Jkbpolymorphismto type 1 diabetes in this data set.
Testing for linkage disequilibrium (LD) of the Jka/Jkb polymorphism with type 1 diabetes in UK and US family data sets
Data set
Unstratified
IDDM1-0,1 high risk alleles
T
NT
P value
T
NT
P value
TR>
UK
106
96
ns
39
41
ns
US
120
133
ns
49
51
ns
Total
226
229
ns
88
92
ns
Testing for linkage disequilibrium was done (see Materials and Methods) by the transmission disequilibrium test (TDT)(19). Transmissions (T) and non-transmissions (NT) of the Jkb allele from heterozygous parents are shown to all affected offspring (unstratified), and affected offspring with zero or one of the alleles that confer a high relative risk for type 1 diabetes development at IDDM1 (24) (type 1 diabetics with HLA-DR3/X, -4/X or X/X where X is not 3 or 4). ns = not significant.
This study provides two results. Firstly, the historical Jka/Jkb polymorphism is due to an Asp280Asn amino acid substitution in the fourth extracellular loop of the red blood cell urea transporter. The MnlI PCR-RFLP assay could be applied in the diagnosis of the severe haemolytic disease of newborns that is caused by antibodies to the Jk antigen.
Secondly, we have shown that the Jkb allele, previously associated with type 1 diabetes, does not show either linkage or linkage disequilibrium with disease in 228 families. Recently we have provided evidence for linkage disequilibrium of the chromosome 18q12 region to type 1 diabetes, but to a locus 13 cM telomeric of the Jk locus (12 ). Using linkage disequilibrium mapping, the type 1 diabetes susceptibility locus IDDM6 has been located near the D18S487 marker. The 13 cM interval between IDDM6 and the Jk locus is compatible with linkage of the Jk locus to type 1 diabetes observed previously (14 ), since regions of linkage to polygenes can be broad, encompassing 20 cM (10 ,11 ). The results presented here indicate that the Jk locus is not IDDM6.
Nevertheless, more polymorphic markers in and around Jk would have to be analysed in multiple different ethnic groups before it could be firmly concluded that the Jk locus is not involved in type 1 diabetes. It is likely that many common variants in multiple genes will contribute to autoimmune diabetes, and the frequencies of these variants will in some cases vary considerably in different populations.
Blood samples were collected on EDTA, and the Jka/Jkb typing was determined by Antigen-Profil DiaMed-ID Micro Typing System (Diamed SA. Morat, Switzerland).
All families used in this study were Caucasian with two affected siblings per family and both parents included. The 95 families recruited from the UK were restricted to those with grandparents born in the UK, and were affected sib pair families in which at least one sibling was diagnosed with IDDM under age 17 years and the other under age 29 years(20 ). The 133 US families were selected according to the same age-of-onset criterion and were from the Human Biological Data Interchange (HBDI) repository of type 1 diabetic families(21 ).
Total reticulocyte RNA (5 [mu]g) extracted by the acid-phenol-ammonium method(22 ), was used to produce first cDNA strands using the commercial kit from Pharmacia (Uppsala, Sweden). The coding region of the Kidd/urea transporter (clone HUT11) was enzymatically amplified using one-sixth of the cDNA products, by a hemi-nested PCR carried out first between primers SP-1 (sense, nt -9 to + 15) and AS-1 (antisense, nt 1266-1243), as follows: 30 cycles of 30 s at 94oC, 30 s at 60oC and 1.5 min at 72oC. The second PCR was performed with 1/25 of the first PCR products in the same conditions, using primers SP-1 and AS-2 (antisense, nt 1231-1205). Final PCR products were subcloned into the EcoRV-digested pT7TS vector (provided by P. Krieg, Austin, TX). Nucleotide sequences of three independent clones from each phenotype were determined on both strands by the dideoxy chain termination method (Sanger) with the Sequenase kit version 2.0 (US Biochemical Corp., Cleveland, OH). For primer designation, nt +1 was taken as the first nucleotide of the initiation codon of HUT11 cDNA clone(4 ).
Determination of the Jka/Jkb polymorphism by PCR-RFLP was carried out by a hemi-nested PCR reaction [30 cycles of 30 s at 94oC, 30 s at 66oC and 10 s at 72oC in the optimized buffer Tris-HCl 300 mM, (NH4)2S04 75 mM, MgCl2 1.5 mM, pH 8.5], using 40 ng of leukocyte DNA. The first amplification was performed between primers SP-A (sense, 5'-tcctgtcttaacagGACTCAGTC-3', exonic nt 818-826) and AS-B (antisense, nt 952-933) and the second PCR between the primers SP-A and AS-C (antisense, nt 893-870), except that annealing was done at 55oC. For routine genotyping, the 90 bp PCR products were digested with 5 U of MnlI restriction enzyme, and electrophoresed on a 4% (w/v) agarose gel stained with ethidium bromide.
Maximum lod scores were calculated as described by Risch(23 ). Transmission disequilibrium testing was performed as described previously(19 )-the test statistic has a [chi]2 (1df) distribution.
We thank Véronique van Huffel (INTS, Paris) for the supply of DNA from Jk donors. We also thank A. Wilson, S. Jenkins, Z. Atta, B. Rowe, J. Carr-Smith and C. Smyth for their help and contribution to this work. This work was funded by the Wellcome Trust, British Diabetic Association, UK Medical Research Council and Juvenile Diabetes Foundation International (JDFI). The BDA and HBDI are thanked for the collection of families. T.R.M. was a Wellcome Trust-New Zealand Health Research Council Overseas Postdoctoral Fellow and is currently a JDFI Postdoctoral Fellow. J.A.T. is a Wellcome Trust Principal Research Fellow.
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*To whom correspondence should be addressed. Tel: +44 1865 740024; Fax: +44 1865 742193; Email: tonym{at}well.ox.ac.uk
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