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From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Kx: Its Relationship to Chronic Granulomatous Linkage By The Peter Densen, relationship neutrophil chronic Kx. was Xg Wilkinson-Kroovand, between antigen. with Xk, Susan neutrophil as well examined granulomatous function as the linkage in a kindred disease. Four and of the with of the L. Mandell, the male siblings had chronic granulomatous disease by clinical history and tests of neutrophil function, and all four had Kx-negative neutrophils. The remaining four were in good C as HRONIC granulocyte an X-linked granulomatous function recessive disorder in some is transmitted kindreds. recurrent infections neutrophils have ingested inability disease is a disorder of that is inherited primarily trait, although a similar as an autosomal Affected individuals because their defective recessive suffer activity This defect to generate neutrophils that that linked a causal relationship surface antigen and other erythrocyte surface antigen an X-linked mode of inheritance. Evans Department Boston, of The New Supported from and Va. and York Address Room 5) 1981 the Blood in part the National Submitted tal, the Division Medicine lottesville, June reprint E529, by Grune of Pathology, by Grants Institutes Diseases, F. Kimball New York, requests to Dr. siblings been reported.4 75 E. Newton of Red & Stratton, Inc. data on the suggest X chromosome In addiand Xg, and HLO9OI Boston, University Mass. 02118. If ospi- other of the ABO, Rh, groups using have granulomatous of recurrent patients of an four previously disease infections to reduce parameters details of the was and nitroblue the tetra- oxidative burst of phagocytosis. Typing from MN, standard all family members Lutheran, Kell, were Duffy, tested Kidd, for antigens and Xga bkod techniques.5 Typing Erythrocyte indirect Kx determinations antiglobulin Serum from a patient been inadvertently Equal cells was null (K0) with Marsh.6 erythrocytes, ability on the neutrophil blood The neutrophil-rich mm, 22#{176}C),and hypotonic (0.2%) packed neutrophils standard anti-Kx test used were the system performed using of Kx antigen, to reduce and removed, in blood twice 10% suspended in an for I hr Vol. cells equal at 58, dextran. (200 in isotonic incubated Blood, anti-Kx 1 5 ml of ACD-treated centrifuged red washing the of Kx antigen sedimentation of contaminating serum, red erythro- polyspecific as a measure from by was After were blood the final using an excess Neutrophils saline. and The scored red at 37#{176}C for centrifuged saline. neutrophils obtained freed subject’s had cells.2 Kx antigen. was plasma red of commercial have subject’s surface. were Kx who incubated controls lack antiserum test for obtained Kx-positive then drops which that of the of a standard two scored neutrophil hemagglutination Appropriate erythrocytes The was in isotonic and of 32 was were times incubated titer of the mixture a standard being normal serum using result and with anti-Kx three globulin by an anti-Kx suspension and incubation washed pellet McLeod saline the erythrocyte of a 3%-5% The cells with absent performed with transfused volumes 30 mm. Kell with in isotonic blood were technique hemagglutination. 1 with studies disease of chronic history these together function granulomatous during Cell family neutrophil diagnosis exhibit Blood described 2, 198/. Densen. to cells of and from activity blood titer AI16476, METHODS three-generation chronic The Red Kx Char- Institute with or metabolic of Virginia, Research March Street, Kx-negative X chromosome. AND by a compatible zolium Center, Departments of this of neutrophils peripheral Peter reduction had These genes on the features male failure N.Y. AI09504, accepted normally. distinct linked pedigree clinical anti-human of Health. 2, 1980; four Patients established Research, Medical University Lindsley closely The might exist the defective Clinical University of Infectious Center, 0006-497l/81/5801-0005$01.00/0 34 Boston but tetrazolium latter functioned L. Marsh cate that the absence of Kx from neutrophils is strongly associated with chronic granulomatous disease, but does not preclude normal oxidative neutrophil function. In addition, the Xk and Xg loci of the that is known to The data mdi- Department Medicine, Mass.; Internal Memorial nitroblue of these W. code for chronic granulomatous disease and Kx. tion, close linkage was demonstrated between Xk a gene coding for an erythrocyte surface antigen. cyte the closely #{216}yen, and MATERIALS the of We investigated the association of these two abnormalities in a large kindred with X-linked chronic granulomatous disease. We also examined the possibility of linkage between Xk and Xg loci. Xk alleles code for the surface antigen, Kx, on both granulocytes and erythrocytes, while the Xg locus codes for Xga, the From normal one against is due to metabolites to superoxide ion, is believed to account for this defect in oxidative activity, but the precise biochemical abnormality has not been established.’ Recently, granulocytes from patients with X-linked chronic granulomatous disease have also been reported to lack an X-linked, Kell-related surface antigen, Kx. This antigen is present on granulocytes from normal individu- only have Ragnhild had However, appear oxygen that are toxic to ingested bacteria. The dysfunction or absence of a membrane-bound oxidase, which catalyzes the one electron reduction of oxygen als, suggesting that between the absent oxidase activity.2’3 Genetic polymorphonuclear bactericidal microorganisms. of these cells trait from Sullivan, and tests. X-Iinked eight Gail health gene. and Xg With Gerald Disease by saline, volume 37#{176}C,and No. g, 1 (July), 10 lysis in the of the then 1981 From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Kx, CHRONIC GRANULOMATOUS DISEASE, AND Xg 35 Xg(a+) Xg(ci+) Fig. 1 . Partial family pedigree demonstrating the relationship between chronic granulomatous disease (CGD) and neutrophil Kx antigen as well as the genetic linkage between Xg and Xk. The results of erythrocyte Xg’ and neutrophil Kx antigen typing are shown for each individual tested. NT. not tested. removed titer by of centrifugation anti-Kx twofold dilutions 37#{176}C.The globulin red inverse was taken washed, of the examined were highest titer ofanti-Kx mediated previously tous disease above. hemagglutination neutrophils were controls. quantitative described4 sibling at anti-human Normal four male and with all family and bactericidal using neutrophils in this Kx-negative tetrazolium tests’ nitroblue shunt siblings Kx-negative Xg(a-) i(x+ isolated kindred neutrophils, with and were from normal chronic the In addition, performed assessed Kx volun- healthy qualitative Typing 1 , all four male siblings granulomatous disease However, neutrophils from male the last born obtained male Neutrophil Since previously from RESULTS Cell healthy globulin tests on all erythrocyte tive. There were no unusual samples reactions were negawhen the erythrocytes were examined for the blood group antigens described in the methods, and none of these results suggested nonparentage. The crythrocytcs from type all family members were Kx-positive K-k+, Kp(a-b+). No weak or unusual with Kell system antisera were observed. who lacked on two male did Kx when occasions. siblings Therefore, the at the Xk locus. not have a all mother had separate The three Kx-positive must also be het- Function the Kx-negative history of Typing None of the family members’ sera contained atypical blood group antibodies and direct anti-human in generation five of the sibling in generalacked Kx obtained history suggestive of chronic granulomatous and whose qualitative nitroblue tetrazolium neutrophils. erozygous nitro- members. Blood CARRIER As shown in Fig. tion II with chronic on their neutrophils. clinical disease Xq(a.) Ks- granuloma- one on neutrophils as Xg(o+) Ks- CGD remaining reduc- myeloperoxidase- activity neutrophils. were tetrazolium activity, Xg(o.) Ks- #{149} ‘c, NORMAL absence only been chronic granulomatous phil function of the Red Xg(o+) Ks- test was normal, also samples were examined monophosphate iodination, the for 30 mm Xq(o-) i(+ Neutrophil serial as described producing antibody. as positive consumption. hexose teers, still residual incubating polyspecific scored dilution Xg(o-) Kx+ Function Oxygen tion, then 22#{176}C).The by K, erythrocytes hemagglutination concurrently Neutrophil blue cells Xg(o+) Xx- LII 10 mm, determined with and as the g, was of the supernate added, The (1000 antibody Xg(os) NT. and Kell reactions Both I had Xg(a+) red blood cells, eight male siblings in generation parents but only II had quantitative nitroblue of single neutrophil recognized disease, male neutrophils, but repeated infection defect, tetrazolium Kx in antigen patients has with we studied the neutroin this family who had who did not have (Table I ). Neither as measured by test, hexose a a the quantitative monophosphate shunt, or iodination, nor a kinetic defect, as measured by continuously monitored oxygen consumption and bactericidal activity, could be demonstrated. DISCUSSION The KelI determined blood allelic white, blood cells. precursor substance group is a system antigens present of autosomally on red, but not It has been proposed that Kx is a that is converted to the appropri- she is genotypically XgaXg. The only female sibling in the second generation had Xg(a + ) erythrocytes, as would be expected since her father’s erythrocytes were ate Kell antigens by an enzyme coded for on the autosome determining Kell type.2 In contrast to the Kell antigens, Kx is normally present on both red cells and granulocytic white cells, although lesser amounts are detected on red cells, presumably due to the conversion of Kx to Kell system antigens. It has also further Xg(a+) that the Xg(a on their red blood cells mother was heterozygous +). (Fig. I), indicating at the Xg locus, i.e., been suggested that Kx results from the From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 36 DENSEN Table 1 . Neutrophil Function Qualitative NBT NBT CGDt 92±2.7 91 ±4.1 2.8± 1.0 reduction monophosphate (% increase Pat,entt reduction (%increasenormalcontrol) Hexose AL. Studies Normal (%NBT-positivePMN) Quantitative Fun ction El 100 82.5 ± 18.5 29.8 ± 13.1 100 80.0 ± 3.0 26.3 ± 2.0 ± 1,658 ± 226 ± 1.20 ± 0.15 shunt normal control) Iodination (cpm/1O6PMN) Oxygen (MIO2/hr/1O6PMN) Bactericidal 956 9.04 ± 1.92 96.6 ± 2.8 killed) §Patient 7.27 0.43 89.6 for bactericidal activity, each test was performed on the patient chronic granulomatous disease; NBT. nitroblue tetrazolium; PMN. CGD, 8,546 12 SEM. ± tExcept 26,035 activity (% bacteria Mean ± 24,063 consumption but not CGD is within expression Erythrocytes of an and Kx independent 4 siblings with polymorphonuclear CGD 2-3 structural white cells another, gene, Xk.8 may possess depending on which of lytic anemia.9” Individuals chronic Kx from both cell types (chronic granulomatous disease, McLeod phenotype). The X3k allele codes for Kx on red cells but not granulocytes (chronic granulomatous disease phenotype), while the reverse is true for X4k (McLeod phenotype).3 In the family reported here, all members had Kx-positive red cells. However, five of the males in the second generation had Kxnegative granulocytes and three had Kx-positive gran- abnormal ulocytes. These data heterozygous at the lomatous disease. The granulomatous disease genotype. expression The on demonstrated affected males indicate Xk locus consistent both that and the mother must have pattern erythrocytes must be the X’k of Kx antigenic and in these eight male siblings, and in other families having either in other the X3k or X4k allele, indicates that the independent sion of Kx on these two cell types is not due expresto the effect that modi- of an independently segregating fies the expression of Kx. The results of Kx and Xga gene typing were analyzed granulomatous in disease,3 result from the dysfunction The rare patients with the red and are unaware granulomatous Kx positive. phils have appear to X2k or X3k white they have a disorder At the neutrophils evidence expression of chronic kindred appears to be mild in that the four recurrent infections affected males did not until after the age of 5, have all overcome survived into their their susceptibility tion.4 Given the affected brothers relatively with 30s, and appear to to recurrent infec- mild clinical course Kx-negative neutrophils, neutrophils evidence Xg(a-). linkage absence of Kx from neutrophils is closely with chronic granulomatous disease, the for the family is this Xk and between 2. 1 04 score of + at + Xg. the The lod score calculated Xk and Xg loci in this a recombination fraction 2.0 or greater is customarily of zero. accepted level of significance for linkage of X-borne Individuals of the X2k or X4k genotype their ically surface red blood abnormal. cells, is associated A lod as the characters. lack Kx on and their red cells are morphologThis abnormality of the red cell with a mild compensated hemo- in the we investigated the unaffected male sibling with Kxnegative neutrophils for the possibility of a partial defect in the function of his neutrophils. We were three These between linkage we clinical in this unable function. were for genetic time, but does not have of chronic granu- All five males with KxXg(a+) red cells, while all Kx-positive provide strong to oxidase. have both present the eight male siblings. negative neutrophils had with data believed of a membrane X2k genotype cells.3 geno- of Kx has X-linked of any individual with X-linked chronic disease whose neutrophils have been That all persons with Kx-negative neutrochronic granulomatous disease does not be true, since one male sibling in this kindred has Kx-negative either clinical or laboratory relatively experience granulocytes of the type lack Kx on their granulocytes. This lack shown a high degree of association with four recognized Xk alleles occupies the Xk locus. Thus, the X’k allele codes for Kx on both cell types (normal phenotype), but X2k results in the absence of x3k times. neutrophils. 2 SD of the mean for normal. X-linked granulocytic of one or on the to identify These antigen any results is not defect in neutrophil demonstrate that required function. Our data are tion that separate genes chronic granulomatous case, then neutrophils suggesting that associated presence normal of neutrophil consistent with the interpretaon the X chromosome code for disease and Kx. If this is the the normal reflects a chronic granulomatous the recombination did for oxidative while the brother recombination with Kx-negative between the disease and Xk loci. However, not separate the Xk and Xg loci, the order of these three genes on the X From www.bloodjournal.org by guest on June 11, 2017. For personal use only. Kx, CHRONIC chromosome Xk-Xg. GRANULOMATOUS DISEASE. AND Xg 37 may be chronic granulomatous It should be noted that this interpretation that the neutrophils amount of Kx antigen diseasedoes not explain the high degree of association (linkage disequilibrium) of chronic granulomatous disease with Kx-negative granulocytes. Other possible explanations for the separation of Kx-negative neutrophils and chronic granulomatous disease ing must Kx be considered. on The granulocytes measures the standard anti-Kx procedure is an residual for detect- indirect method hemagglutination antiserum after by the test granulocytes. results, the method has that titer it has been Although giving low sensitivity. in absorbed level on the neutrophil membrane have a reduced for functional results full protection from the disease. Kx antigen on neutrophils from as from disease are ities. reproducible It is possible this sibling that is sufficient activity but which is below the level of detection by the present technique. Another possibility is that some compensatory process operating at the phenotypic well a of individuals needed Kx-negative rewarding. with of the neutrophils granulomatous among these descendants may of prove or studies of the individuals as chronic to distinguish Evaluation in partial Further normal possibil- males with to be particularly REFERENCES I . Babior cytes. BM: N EngI 2. Marsh lomatous Oxygen I Med WL, dependent 298:659, 721, microbial #{248}yenR, Nichols disease and the Kell killing by phago- 1978 ME, blood Allen groups. FH: Chronic Br I Haematol method for carriers. I Pediatr granu- 8. 29:247, syndrome, 3. Marsh WL, phenotype, and Sang3l:356, 4. tous ME: Kx disease: antigen, Further the McLeod studies. Vox Mandell GL: of “childhood.” Am Methods Association 6. Marsh WL: 12:352, 1972 HD, WL: and Wimer Dail the BM, WG: chronic 83:77, granulomatous Chronic Kell granulomatous blood Marsh groups. WI, Haematological phenotype of the Kell disease and female 1973 disease, Birth Taswell changes blood HF, Galey associated group system. the Defects McLeod 14:9, 1978 WR, Evan with the AP, McLeod Br I Haematol 36:2 19, I 977 IA, American 7. Ochs Nichols granulomatous 1976 Dilworth disease #{216}yenR, chronic 5. Technical sion Marsh 9. I975 detecting and of Blood Scoring Adults with I Med 63:233, Procedures (ed Banks, chronic granuloma- 1977 7). 10. Galey Cooper RA: cyte. Washington DC, reactions. Iransfu- SB, Iinehan McLeod Igo RP: The NBT slide test: A simple screening 42:575, Van and Vox WA, BI: phenotype 1979 AP, electron properties. 1 1 . Symmans of hemagglutination Evan Morphology I. Scanning transport 1977 WR, PS, Shepherd 34: 1 52, CS. blood WG, and Wimer Mcleod BM, erythro- electrolyte and water WI, R, Shohet 1978 Marsh acanthocytosis Kell Dail of the microscopy Sang Hereditary of the Nice physiology group Oyen associated system. with Br I Haematol the From www.bloodjournal.org by guest on June 11, 2017. For personal use only. 1981 58: 34-37 Kx: its relationship to chronic granulomatous disease and genetic linkage with Xg P Densen, S Wilkinson-Kroovand, GL Mandell, G Sullivan, R Oyen and WL Marsh Updated information and services can be found at: http://www.bloodjournal.org/content/58/1/34.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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