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Origin of Mutations in Two Families With X-Linked Chronic Granulomatous Disease By Uta Francke, Hans D. Ochs, Basil T. Darras, and Anand Swaroop The most common X-linked recessive form of chronic granulomatous disease (X-CGD) is characterized by the absence of cytochrome b, in neutrophils. In a rare variant form of X-CGD, cytochrome b, is present but not functional. The gene (locus symbol CYBB) was localizedto band Xp21 by studies of patients with small chromosome deletions. The gene was cloned based on its location and found t o encode the 91-Kd subunit of the cytochrome b, complex. Most female carriers for X-CGD can be identified by their X-inactivation mosaicism; on average 50% of their neutrophils express the mutant phenotype and fail to reduce nitroblue tetrazolium (NBT). In 2 of 4 familes studied, the maternal grandmothershad normal NBT tests, suggesting either nonrandom X-inactivation or new muta- tions. Restriction fragment length polymorphism analysis using closely linked flanking markers or the Nsil polymorphism detected by the CYBB probe itself. allowed us to identify the X chromosome carrying the mutation as derived from a healthy NBT-positive maternal grandfather. The mothers of the affected boys must have received a paternal X chromosome carrying a new mutation, consistent with the maternal grandmothers‘ normal NBT tests. In all of eight potential carriers studied, the results of the NBT and DNA marker testing were in complete agreement. Prenatal diagnosis by DNA testing can be performed in early gestation obviating the need for fetal blood sampling. 0 1990 by The American Society of Hematology. C factor.I3 The characteristic functional defect in the autosomal forms of the disease is the inability of the cytosol of phagocytes to activate the membrane components of the oxidase system. CGD patients and carrier females for X-CGD can be identified with a simple endotoxin stimulated nitroblue tetrazolium (NBT) slide test.14 Phagocytic cells from affected individuals are unable to generate superoxide and thus fail to reduce NBT from a colorless dye to blue formazan crystals. Because of random X chromosome inactivation, on average only 50% of activated neutrophils from a female carrier are able to reduce NBT as compared with greater than 95% from normal controls. This test and others that demonstrate mosaicism in phagocyte function have been used routinely to identify X-CGD carriers for many years. The precise mapping of the X-CGD gene to sub-band Xp21.1 and the cloning of its product, the cDNA for the 9 1-Kd CYBB subunit, have recently made available molecular tools for the study of the origin and segregation of mutations in this gene.’, W e have studied X-CGD families by using (1) functional assays (NBT reduction); (2) restriction fragment length polymorphisms (RFLP) detectable with closely linked flanking markers; and (3) the CYBB probe that identifies potential gene rearrangements as well as a common RFLP with the restriction enzyme NsiI. In the two families reported here, we were able to identify and trace the X chromosome region carrying the mutant CYBB gene, verify NBT test results, and document the grandpaternal origin of both mutations. HRONIC GRANULOMATOUS disease (CGD) includes a group of inherited disorders, with either X-linked or autosomal recessive inheritance. Affected individuals develop recurrent severe bacterial or fungal infections due to the inability of phagocytic cells to produce superoxide via the membrane-bound NADPH-oxidase system.’.’ The most common X-linked recessive form (X-CGD) is associated with absence of cytochrome b,,,, heterodimeric glycoprotein with tightly associated subunits of 91 and 22 Kd.’ In a rare variant form of X-CGD, cytochrome b,,, spectral activity is present4 but not functional, probably due to a point mutation affecting the 91-Kd subunit gene., The X-CGD gene (CYBB) has been localized to band Xp2I6,’ by studies of male and female patients with partial deletions of this chromosomal band. The gene was subsequently cloned, based on its location and tissue-specific expression.’ It was later shown to encode the 91-Kd subunit of the membrane cytochrome b,,, c~mplex.’.’~ The 22-Kd subunit has also been cloned and found to be expressed in most cell types.” Molecular defects for two distinct forms of autosomal recessive CGD have been identified. One group (approximately 90% of cases with an autosomal recessive mode of inheritance) lacks a 47-Kd protein of the cytosol oxidase component.I2 The other group lacks a 67-Kd cytosolic From the Departments of Genetics and Pediatrics and Howard Hughes Medical Institute. Stanford University Medical Center, Stanford, CA; the Department of Pediatrics, University of Washington School of Medicine, Seattle; and the Department of Human Genetics, Yale University School of Medicine, New Haven, CT. Submitted February 16,1990;accepted March 30,1990. Supported by National Institutes of Health Research Grants GM26105 (to U.F.) and AI 07073 (to H.D.O.).U.F. is an investigator of the Howard Hughes Medical Institute. Address reprint requests to Uta Francke, MD. Howard Hughes Medical Institute, Beckman Center, Stanford University Medical Center, Stanford, CA 94305-5428. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1990 by The American Society of Hematology. 0006-4971/90/7603-0012$3.00/0 602 MATERIALS AND METHODS Family 1 contains two affected brothers, born in 1954 and 1956. Both have a history of recurrent pulmonary infections, frequent fevers, lymphadenopathy, and liver absce~ses.’~*’~ In 1969, the younger patient presented with symptoms resembling Crohn’s disease.” The diagnosis of CGD was confirmed by defective bactericidal and metabolic activity of neutrophils, inability to reduce NBT, abnormal superoxide anion formation, and the presence of lipidladen histiocytes in rectal and small bowel biopsies. Neutrophils of both patients had normal levels of cytochrome b55,.4Recent DNA A sequence analysis identified a single nucleotide change, a C transversion that results in a Pro His substitution at residue 415 - -+ Blood, Vol 7 6 , NO 3 (August 1). 1990: pp 602-606 ORIGIN OF MUTATIONS IN X-CGD of the 91-Kd pr~tein.~ Two sisters and one brother are healthy. The mother has discoid lupus and is otherwise healthy. A maternal aunt and the maternal grandfather are alive and well. The maternal grandmother, now deceased, was part of a large sibship; none of her five brothers had unusual infectionsor died in infancy. Family 2 has one affected boy, born in 1984 and diagnosed with CGD at the age of 20 months when he had persistent skin infections, lymphadenopathy, and osteomyelitis. The diagnosis was confirmed by demonstrating decreased bacteriocidal activity of leukocytes, inability to reduce NBT, and absence of cytochrome b,,,. His three sisters and both parents, four maternal uncles, two maternal aunts, and both maternal grandparents are healthy. None of the maternal grandmother's sons died in early infancy or childhood. Methods. NBT reduction was determined histochemically for individual neutrophils using an NBT slide test as previously described.14Coverslips were coated with either endotoxin or phorbol myristate acetate before allowing a drop of blood to clot. After removing the clot and washing off the red blood cells, the coverslips were inverted and incubated on a glass slide with a suspension of NBT, fresh serum, and phosphate-bufferedsaline, pH 7.4. The cells were fixed with methyl alcohol and counterstained with safranin. The proportion of neutrophils capable of reducing NBT was determined by counting at least 500 cells. DNA was extracted from peripheral blood leukocytes and Southern blots were prepared as described previously.18 Samples were tested for the following RFLPs previously mapped to band Xp21: MspI and BumHIIOTC; PstI/754; HindIII/754-6; EcoRII754-11; TuqIIXJ1.1; BstNI and XmnIlpERT87-I; BstXI and TuqI/ pERT87-8; TuqI and XmnIlpERT87-15; BglIIlpERT87-30, BumHIIJ-Bir; EcoRVIC7; and BstNIIB24. Locus symbols, origin of probes, and allelic fragment sizes have been summarized In addition, we have used a human cDNA clone of the X-CGD gene that encodes the large subunit of cytochrome b,,,,8 kindly made available by Dr S.H. Orkin (Boston Children's Hospital), to search for deletions or structural rearrangements and for the recently described Nsil RFLP.*' RESULTS The results of N B T reduction tests and D N A marker analyses are illustrated in Figs 1 and 2. The numbers above the symbols designating individuals indicate the percent of Fig 1. Results of NBT tests (given in percentage of positive neutrophils) and Xp21 RFLP haplotypes in family 1. The haplotype (black bar) containing the mutant CYBB gene was identified in the affected boys and their carrier mother (circle with dot). For interpretation see text. 603 neutrophils that were positive in the N B T slide test. Numbers greater than 95% represent the normal control range. Affected males are unable to reduce NBT (0% positive). Females with 50% (Fig l ) , or 32%, 53%, and 35% (Fig 2) positive cells were diagnosed as probable heterozygotes. It is noteworthy that in both families the maternal grandmothers had N B T test results in the normal range. While this may indicate that they are not carriers of the X-linked CGD mutation, other possibilities cannot be ruled out, such as nonrandom X-inactivation or loss of heterozygosity due to clonal selection of bone marrow cells. Therefore, molecular genetic studies were performed with the goals to evaluate the NBT slide test for carrier detection, determine the grandparental origin of the mutations, and look for deletions or structural rearrangements of the CYBB gene in affected males. N o deletions or abnormal size fragments were detected on Southern blots in DNA from affected males and carrier females of both families. Both mothers of affected males were informative for four RFLPs, including the CYBB polymorphism in family 2. Maternal grandfathers and fathers were available to unequivocally establish haplotypes in females. The maternal grandmother in family 1 died before the D N A studies were initiated and her alleles are inferred. In family 1 (Fig l), the mother of the two affected males was heterozygous for four RFLPs flanking the CYBB locus. At DXS28 in band Xp21.3, probe C7 distinguishes the EcoRV alleles A1/8.0 kilobases (kb) and A2/7.5 kb. At D X S l 6 4 within the dystrophin ( D M D ) gene, probe pERT87-1 sees XmnI alleles A1/8.7 kb and A2/7.5 kb. At DXS84, located at Xp21.1 close to CYBB, probe 754-11 defines EcoRI alleles D1/2.4 kb and D2/4.2 kb. The ornithine transcarbamylase locus (OTC) is definitely centromeric to CYBB, as patient B.B. who suffered from DMD and CGD, was missing DXS84 but not OTC.' The informative MspI alleles at OTC were A1/6.6 kb and A2/6.2 kb. Haplotypes have been constructed for the Xp21 region as indicated by different bar symbols on Fig 1. The haplotype FRANCICE ET (Kw AL - -1 h prmF b 2. R . w h . o( NOT -0 ago a( m v ionourrophib).nd Xp21 RFLP h.pk typos in f.myY 2. CY88 *rdluros tho MI RFLP d.1.Ct.d with ch. X-COD gwW pob.. Tho mutotm h 0nocin.d rrhh tho A1 C Y 8 8 Jkk tho1 h pori a( tho ohcwn n MKL bor in tho 0ff.et.d mob and tho t h r n trrub c w r w a (ckcl..wtth dol#). Tho 8mte h.p(otyp. h p.um in tho q.nd(0th.r wd (yo of hh 4.ught.r. who Y O not wrrkrr. T h o u (hd(nglplK0 tho or* ol tho X.C<)D mutation )n tho grondp.tunoI m m . that contains the X-CGB mutation. identified as present in both afTccted males. is shown in black. I t is evident that the mutation must have originated in the sperm of the clinically unafTcctcd grandfather who has the black haplotype. Recause the mother’s sister had a normal NRT slide test. her paternal haplotype does not carry the mutation. I n the third generation. the u n a l f ~ ~ t eson d has inherited theothcr maternal haplotype. However. both sisters have received a recombinant chromosome resulting from a crossover between DXSR4 and OTC. Their normal NRT tests indicate that they have not received the mutant CY R R allele. DNIA studies alone would k noninformative regarding thccarricr status of t h m Fb3. rvlrt-h)wns2.th. [email protected].~fmAumg)n k.d.d.fh. #&cood ch.Wl roatrktkrr fr.gmwn8 (in 1 k Y O *dk.c.d on tho kh. A1 11.7 kb .rd A 2113 kb Y O tho O I J l t tr.g”o.” Tho pok r*.. 0 3 S k b P n l - K p l tr-t d tho huruo c v t m m u ch.t *wtud..on ol thoooquoneo and moot of tho 3’ untronrlatod ~0 m i u i q w mbormt tr.gnwmr oro ..w,h t h o O H u t d m J . w d ch.thr..h.tworyWU. Thorrwk1.6.Lbtr.g”inth.q0ndmot~ w’r DWA h on m H m . -a two females at risk. The older of the two afTccted males has twodaughters.and both showed mosaicism on the SRTslide test. These results further confirm X-linked inheritance of CGD in this family. The informative RFI-Ps in family 2 (Fig 2) included two within the DMD gene: DXS270. p k J-Rir. with BomWl allelaAI/2I kband A2/5 k h a n d DXS164.prok pERTH730. with &Ill alleles N I / K O kb and N2/30kb. The ,l.’Jil RPLP at the CY R R locus with alleles A I / I .7 kb and A2/ I .3 kb is illustrated in tig 3. Rased on estimates of the s i x of the R.B. deletion. thcse four markers should span no more than 5 megaba.m. Within this region. no recombination event was 605 ORIGIN OF MUTATIONS IN X-CGD identified in the 11 meioses represented in this pedigree. The haplotype associated with the mutant CYBB allele and identified by the A1 NsiI allele (shown in black) was concordant with the NBT test results in the carrier mother, her affected son, and her two carrier daughters. Her noncarrier daughter has received the other haplotype. As in family 1, the haplotype carrying the mutation was traced to the unaffected grandfather. The two noncarrier maternal aunts share the same haplotype without the CYBB mutation as suggested by their normal NBT slide tests. DISCUSSION In this study, the phagocyte functional assays and gene marker studies complement and confirm each other in a remarkable fashion. Because Southern analysis with the CYBB probe did not disclose a specific change in the DNA from the affected males, the X-linked pattern of inheritance of CGD could not have been established in these pedigrees without the NBT slide test that identified heterozygous females. While in family 2 the absence of cytochrome b,,, is consistent with X-CGD, in family 1 the cytochrome b levels were normal. X-linked inheritance was proven by demonstration of X inactivation mosaicism in the mother and in both daughters of the older affected male. However, it remained uncertain whether the mutation involves the CYBB gene or another locus on the X chromosome. The results of the haplotype analysis are consistent with the mutation residing in the Xp21 region. While this report was in preparation, Dinauer et al' reported normal size and abundance of the 91-Kd subunit messenger RNA in the affected males and identification of a nonconservative amino acid substitution in His change at residue 415 does the 91-Kd gene. The Pro not appear to affect a known functional site and no formal - proof exists that it represents the mutational basis of the disorder in this family, other than its absence in normal controls and in cytochrome b negative males. Our data locating the mutation in this family to the Xp21 region lend further support to the notion that there is only one X-linked CGD gene (CYBB). The origin of both mutations has been traced to a grandpaternal sperm. Previous studies of dystrophin gene mutations where the origin could be unequivocally determined have shown grandpaternal origin in 5 of 13 cases.*' Thus, it appears possible that, similar to the dystrophin gene, the CYBB gene undergoes frequent mutations in male gametes. Haplotype analyses in autosomal disorders have also shown predominantly paternal origin of new mutations for retinoblastoma22and ne~rofibromatosis?~ Deletions involving the entire CYBB locus together with the adjacent Xk locus (McLeod syndrome) have been reported."^^' In contrast to DMD, partial deletions or structural abnormalities resulting in a truncated gene product seem to be rare in X-CGD.~ Our determinations of carrier status by the NBT slide test and by DNA haplotype analysis are in complete agreement. Prenatal diagnosis of X-CGD, which previously required fetal blood sampling and NBT test in mid-gestation, can now be done early in pregnancy by chorionic villus biopsy and DNA haplotype analysis, in particular, using the NsiI RFLP at the X-CGD locus. ACKNOWLEDGMENT We are grateful to L.M. Kunkel, S.H. Orkin,J.-L. Mandel, G.-J. van Ommen, R. Worton, and A. Honvich for DNA probes; and to L. Battat and E. Chang for technical assistance. REFERENCES 1 . Klebanoff SJ, Clark RA: The metabolic burst, in: The Neutrophil; Function and Clinical Disorders. Amsterdam, The Netherlands, Elsevier, 1978 2. Forrest CB, Forehand JR, Axtell RA, Roberts RL, Johnston RB Jr: Clinical features and current management of chronic granulomatous disease. Hematol Oncol Clin North Am 2:253, 1988 3. Parkos CA, Allen RA, Cochrane CG, Jesaitis AJ: Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest 80732, 1987 4. Okamura N, Malawista SE, Roberts RL, Rosen H, Ochs H, Babior B, Curnutte J: Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual cytochrome negative and X-linked cytochrome-positive types of chronic granulomatous disease. Blood 72:8 1 1, 1988 5. Dinauer MC, Curnutte JT, Rosen H, Orkin SH: A missense mutation in the neutrophil cytochrome b heavy chain in cytochromepositive X-linked chronic granulomatous disease. J Clin Invest 84:2012, 1989 6. Francke U: Random X inactivation resulting in mosaic nullisomy of region Xp21.1-p21.3 associated with heterozygosity for ornithine transcarbamylase deficiency and for chronic granulomatous disease. Cytogenet Cell Genet 38:298, 1984 7. Francke U, Ochs HD, de Martinville B, Giacalone G, Lindgren V, Disteche C, Pagon RA, Hofker MH, van Ommen G-J, Pearson PL, Wedgwood RJ: Minor Xp21 chromosome deletion in a male associated with expression of Duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome. Am J Hum Genet 37:250,1985 8. Royer-Pokora B, Kunkel LM, Monaco AO, Goff SC, Newburger PE, Baehner RL, Cole FS,Curnutte JT, Orkin SH: Cloning the gene for an inherited human disorder-chronic granulomatous disease-on the basis of its chromosomal location. Nature 322:32, 1986 9. Dinauer MC, Orkin SH, Brown R, Jesaitis AJ, Parkos CA: The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature 327:717, 1987 10. Teahan C, Rowe P, Parker P, Totty N, Segal A W The X-linked chronic granulomatous disease gene codes for the b-chain of cytochrome b-245.Nature 327:720, 1987 11. Parkos CA, Dinauer MC, Walker LE, Allen RA, Jesaitis AJ, Orkin SH: Primary structure and unique expression of the 22kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci USA 85:3319,1988 12. Lomax KJ, Let0 TL, Nunoi H, Gallin JI, Malech H L Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science 245:408,1989 13. Clark RA, Malech HL, Gallin JI, Nunoi H, Volpp BD, Pearson DW, Nauseef WM, Curnutte J T Genetic variants of chronic granulomatous disease: Prevalence of deficiencies of two cytosoliccomponents of the NADPH oxidase systems. N Engl J Med 321:657,1989 14. Ochs HD, Igo R P The NBT slide test: A simple screening 606 method for detecting chronic granulomatous disease and female carriers. J Pediatr 83:77,1973 15. Orkin SH: Molecular genetics of chronic granulomatous disease. Ann Rev Immunol7:277,1989 16. Pincus SH, Klebanoff SJ: Quantitative leukocyte iodination. N Engl J Med 284:744,1971 17. Ament ME, Ochs HD: Gastrointestinal manifestations of chronic granulomatous disease. N Engl J Med 288:382, 1973 18. Darras BT, Harper JF, Francke U: Prenatal diagnosis and detection of carriers with DNA probes in Duchenne’s muscular dystrophy. N Engl J Med 316:985, 1987 19. Kidd KK, Bowcock AM, Schmidtke J, Track RK, Ricciuti G, Hutchings G, Bale A, Pearson P, Willard H F Report of the DNA committee and catalogs of cloned and mapped genes of DNA polymorphisms. Cytogenet Cell Genet 51:622, 1989 20. Battat L, Francke U: NsiI RFLP at the X-linked chronic granulomatous disease locus (CYBB). Nucl Acids Res 17:3619, 1989 21. Darras BT, Blattner P, Harper JF, Spiro AJ, Alter S, Francke U: Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/ FRANCKE ET AL Becker muscular dystrophy (BMD) families studied with the dystrophin cDNA: Location of breakpoints on Hind111 and BgIII exoncontaining fragment maps, meiotic and mitotic origin of the mutations. Am J Hum Genet 43:620, 1988 22. Dryja TP, Mukai S , Petersen R, Rapaport JM, Walton D, Yandell DW: Parental origin of mutations of the retinoblastoma gene. Nature 339556, 1989 23. Jadayel D, Fain P, Upadhyaya M, Ponder MA, Huson SM, Carey J, Fryer A, Mathew CGP, Barker DF, Ponder BAJ: Paternal origin of new mutations in Von Recklinghausen neurofibromatosis. Nature 343:558, 1990 24. Frey D, Machler M, Seger R, Schmid W, Orkin SH: Gene deletion in a patient with chronic granulomatous disease and McLeod syndrome: Fine mapping of the Xk gene locus. Blood 71:252, 1988 25. de Saint-Bade G, Bohler MC, Fischer A, Cartron J, Duffer JL, Griscelli C, Orkin SH: Xp21 DNA microdeletion in a patient with chronic granulomatous disease, retinitis pigmentosa, and McLeod phenotype. Hum Genet 80:85,1988