Download Origin of Mutations in Two Families With X-Linked

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