From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
The Major Histocompatibility Complex Region Marked by HSP70-1 and
HSP70-2 Variants Is Associated With Clozapine-Induced Agranulocytosis in
Two Different Ethnic Groups
By Deyanira Corzo, Juan J. Yunis, Marcela Salazar, Jeffrey A. Lieberman, Alfreda Howard, Zuheir Awdeh,
Chester A. Alper, and Edmond J. Yunis
Genes of the major histocompatibilitycomplex (MHC) have
been associated with susceptibility to drug-induced adverse
reactions. We previouslyfound
that clozapinainduced
agranulocytosis (CA) is associatedwith the HLA-DRB7*0402,
DRB4*0707, DQB7*0302, DQA7*0307 haplotype in Ashkenazi Jewish patients and
with the HLA-DRB7*7607, DRB5*02,
DOB7+0502, DQA7*0702 haplotype in non-Jewish patients.
In the present study,we tested the hypothesisthat the variants of the heat-shock protein70 (HSP-70) encoded by the
HSP-70 loci located within the MHC region and known to
be involved in apoptosis and regulationof cell proliferation
could play an important role
in molecular mechanisms of
CA.
First, we analyzed HSP70-Zpolymorphism in risk-associated
haplotypes fromHLA homozygous cells and normal individuals and confirmed
that the HSP70-29-kb variant was associated invariablywith DR4 (HLA-DRB7*0402, DQB7*0302)and
DR2 (HLA-DRB7*07607, DOB7*0502, DQA7*0702 and HLA-
DRB7*7507, DOB7*0602) haplotypes, which werethe haplotypes found increased in Jewish and non-Jewish patients
with CA, respectively. The 9.0-kb variant was also found
to be associated with HLA-844, DRB7*0407 and HLA-844,
DRBl*O7 haplotypes. Second, in patients with CA (12 Ashkenazi Jewish and 20 non-Jewish patients),HSP7O-l A and
HSP70-2 9.0-kb variants were associated
with the MHC haplotypes found by us to be markers of susceptibility to CA.
The clozapine-treated control group had an excess number
of HSP70-1 C and HSP70-2 8.5-kb variants, consistent with
genetic resistanceto CA associatedwith those variants. This
finding supports our hypothesis
that a dominant gene
within
the MHC region (marked by HSP70-7 and HSP70-2).but not
necessarily HLA, is associated with CA in two different ethnic groups.
0 7995 by The American Society of Hematology.
T
chotic drugs were studied, as described previously.s Seventy-five
patients previously HLA typed by high resolution DNA techniques
were included in this study. HLA-A, B, C, DR, and DQ generic types
were determined using standard microcytotoxicity. HLA-DRBI, B3,
B4, B5,DQA1, DQBl, and DPBl alleles were determined by PCRSSP, E R - S S O , and RFLP, as described before." There were 32
schizophrenic patients with CA (12 Ashkenazi Jewish and 20 nonJewish) and 43 schizophrenic patients (27 Ashkenazi Jewish and 16
non-Jewish) who also received clozapine and who did not have CA.
HSP70-l and HSP70-2 typing was performed on this population.
HSP70-2 typing was also performed on a panel of 40 HLA homozygous cell lines (68 haplotypes total), most of which were distributed by the Tenth International Histocompatibility Workshop (New
York, NY), and on a panel of 29 Caucasoid families (52 haplotypes
total) from the New England area.
DNAwas extracted by the salting-out method" for FCR-SS0
HREE HSP-70 GENES MAP in the class I11 region of
the major histocompatibility complex (MHC) between
the B and D R loci (Fig 1). They have been defined as HSP70I, HSP70-2, and H S P ~ O - H O ~ HSP70-I
."~
and HSP70-2
genes encode an identical protein of 641 amino acids, the
major heat-inducible heat-shock protein 70 (HSP-70)."6 The
HSP70-Horn gene also encodes a protein of 641 amino acids,
with 90% identity with HSP70-1: HSP70-Horn is expressed
at low levels both constitutively and after heat
HSP70 polymorphism is of interest because of the involvement
of the MHC in disease susceptibility. We previously reported
association of alleles of the HL4-B and D R loci with increased risk of clozapine-induced agranulocytosis
In Ashkenazi Jewish patients, there was an increased frequency of the HLA-B38,DR4, DQ3 (HLA-DRBI*0402,
DQBl*O302,DQAl*0301) haplotype and, in non-Jewish
cases, of the HLA-DR2, DQI (HLA-DRBI *I601,D Q B l *
0502, DQAI *0102 and DRBI *I50I, DRBl*0602, DQAl*
0102) haplotype. In the present study, we tested the hypothesis that these haplotypes share the 9.0-kb and A(9.0-A)
variants encoded by HSP70-2 and HSP70-I, respectively, in
a haplotype and that the 8.5-kb and C variants (8.5-C) are
markers for resistance to CA in Ashkenazi Jewish and nonJewish patients.
We performed restriction fragment length polymorphism
(RFLP) analysis of HSP70-2 genes in haplotypes from HLA
homozygous cells and normal individuals who carry the riskassociated MHC markers. In addition, RFLP analysis of
HSP70-2 and polymerase chain reaction sequence-specific
oligonucleotide (PCR-SSO) analysis of HSP70-I were performed in a population of schizophrenic Ashkenazi Jewish
and non-Jewish patients who developed CA. Results were
compared with those of ethnically matched schizophrenic
patients who did not develop CA. Results indicate that HSP70 alleles are markers for susceptibility or resistance to CA
in both ethnic groups.
MATERIALS AND METHODS
Patients with chronic schizophrenia or schizoaffective disorder
(DMS-DXR) who were resistant to standard therapy with antipsyBlood, Vol86, No 10 (November 15), 1995 pp 3835-3840
From theDivision of Immunogenetics,Dana-Farber Cancer lnstitute, Boston, MA:the Departments of Pathology and Pediatrics,
Harvard Medical School, Boston, MA; Hillside Hospital, Long lsland Jewish Medical Center, Glen Oaks, NY;the Department of
Psychiatry, Albert Einstein College of Medicine, Bronx,NY;The
Center for Blood Research, Boston, MA: and the Department of
Pediatrics, Maimonides Medical Center, Brooklyn, NY.
Submitted May IO, 1995; accepted July 11, 1995.
Supported by Grant No. MH-47029 (E.J.Y.) from the National
InstituteofMental
Health, by Research Scientist Development
Award No. MH-00537 (J.A.L.) fromthe National Institute of Mental
Health, by Grant No. MH-41960 to the Mental Health Clinical Research Center of Hillside Hospital, by Grants No. AI-14157 (C.A.A.),
HL-29583 (C.A.A.,Z.A., and E.J.Y.), and HL 48675 (Z.A.) from the
National Institutes of Health, and by theSandoz Pharmaceutical
Corp.
Address reprint requests to Edmond J. Yunis, MD, Dana-Farber
Cancer lnsiitute, 44 Binney St, Boston, MA 02115.
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 1995 by The American Society of Hematology.
ooo6-4971/95/8610-~27$3.00/0
3035
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3836
CORZO ET AL
(dot-blot) analysis of the HSP70-1 gene. One microgram of genomic
Table 1. Significant HLA Associations
DNA wasamplified by the Taq DNA polymerase (Cetus, Emeryville,
in DNA-Typed Patients With CA
CA) in a Model 9600 Thermal Cycler (Perkin Elmer, Norwalk, CT)
Genotype
with primers under conditions described elsewhere." Two microliFrequency
ters of amplified product was spotted on nylon membranes (HybondOdds
Allele or Haplotype
Cases
Controls
PValue
Ratio
N+; Amersham, Arlington Heights, IL), alkali-denatured by 5 minutes of incubation in 0.4 N NaOH, and washed with a 0.75 mom
Ashkenazi Jewish patients
NaCI, 75 mmol/L sodium citrate buffer. DNA was fixed to memHLA-DRBl
6\54
11\24
*0402
.02
6.8
branes using a UV Crosslinker (Amersham). Oligonucleotide probes
4.9 ,008 8\54
HLA-DQBl'O302
11\24
were used to detect sequence variants between position - 1I O and
HLA-DQAl3.1
*0301
12\24
13\54
,003
position + 120, as described elsewhere." Membranes were incubated
HLA-DRBl * l l
13\54
0\24
,007
0.06
at 54°C overnight with a prehybridization solution containing 50
18\54
HLA-DQB1'0301
0\24
,0008 0.04
mmol/L Tris-HCI, 2 mmoVL EDTA, 5 X Denhardt's solution, 0.1%
6\54
HLA-DPB1'0401
11\24
.02
6.8
sodium dodecyl sulfate (SDS), 100 mg/mL salmon sperm DNA, and
IHLA-DRBl*0402, DRB4*0101,
3 m o m TMAC. Probes were 32PdCTP-labeled by the random primer
DQBl'O302, DOA 1'030 1,
method, using the T7 QuickPrime Kit (Pharmacia, Piscataway, NJ). 6\54 11\24
DPBlxO4O11
.02
6.8
A total of 30 to 60 ng of labeled probe was added and membranes
Non-Jewish patients
were incubated at54°C for 1 to 3 hours. Membranes were then
HLA-DRBl *On
3.8.03
4\32
14\40
washed twice for I O minutes at room temperature in a 300 mmol/
HLA-DOE10.3
l,018
*0502
10\40
1\32
L NaCI, 20 mmoVL NaH,PO,, 2 mmol/L EDTA buffer and twice
,007
5.8
HLA-DQA1*0102
3\32 15\40
for 15 minutes at 37°C and 5 8 T , respectively, in a 50 mmol/L Tris[HLA-DRBl1160l,
DRB5'02,
HCI, 3 m o m tetramethylammonium chloride (TMAC) 2 mmol/L
DQB1'0502,
DQA1*01021
10\40
0\32
.002
22.4
EDTA, 0.1% SDS buffer. Autoradiography was performed for 1 to
t Patients carried alleles DRB1'1601 or '1501
2 hours at -70°C using a Kodak X-Omatic film and an intensifying
screen (Eastman Kodak, Rochester, NY).
For RFLP analysis of the HSP70-2 gene, 10 pg of DNA for each
sample was digested with the restriction enzyme Pst I (10 U/mg) at
HLA homozygous cells. HSP-70 allele assignments to patients' hap37°C overnight. A 10% v01of 3 molL Na acetate and 2 v01of
lotypes were deduced from known nonrandom association of alleles
absolute ethanol (-20°C) were added to digested DNAand incuwith DR alleles or extended haplotypes. In most patients with CA,
bated for 2 hours at -20°C. DNA was collected by centrifugation
assignment of HSP-70 alleles to haplotypes was simple. However,
at 15,000 rpm for 20 minutes at 4°C and washed once with 70%
the majority of haplotypes of the control group were those known
ethanol. DNAwas resuspended in 50 pL of 100 mmol/L Tris, 1
to be without significant delta (A) values.
mmol/L Na2EDTA buffer, pH 7.4. Samples were subjected to elecTo test the fit ofthe distribution of homozygotes and heterozygotes
trophoresis in 1% agarose gels at 30 V overnight with a 40 mmol/
for individual markers to that predicted by a simple dominant or
L Tris-acetate, 1 mmol/L EDTA buffer. HindIII-digested bacteriorecessive model of inheritance, calculations for expected frequencies
phage-A DNAwas run in parallel for molecular weight markers.
were performed as described el~ewhere.'~Differences between
Gels were washed once for 10 minutes in 0.15 N HCI, rinsed with
groups and deviation of observed homozygote/heterozygotedistribuwater for 3 minutes, and washed again twice for 30 minutes with a
tions from those expected by recessive or dominant inheritance were
0.4 N NaOH, 0.6 N NaCl solution. DNA was blotted onto nylon
determined using x' analysis. Significant differences in frequency
membranes (Hybond-N+; Amersham) by the Southern method for
of alleles were estimated using Fisher's exact test.
8 to 12 hours using a 0.4 N NaOH, 0.6 mol/L NaCl solution. UV
DNA binding and prehybridization and labeling of probes and moRESULTS
lecular weight markers were performed as described above. Fifty
nanograms of labeled probe and 5 ngof labeled HindIII-digested
The data showing significant associations of HLA class
bacteriophage-A DNA were added and membranes were incubated
I1 alleles with CA are summarized in Table 1; the data are
overnight at 42°C. Membranes were then washed as described above
from a previous report." It shows that in Ashkenazi Jewish
and exposed for 5 to 7 days at -70°C using Kodak X-Omatic film.
patients, HLA-DRBlW402,HLA-DQBl"O302,
and HLAThe BarnHVHindIII 2.3-kb insert subcloned in pAT153 used as a
DQAl*O301
and
HLA-DPB1*0401,
independently
or toprobe was kindly donated by Richard Morimoto (Northwestern Unigether
as
a
haplotype,
were
increased
significantly
in
patients
versity, Evanston, IL).I3
with CA as compared with those without. In addition, alleles
HSP-70 allele frequencies were determined by direct counting of
HLA-DQB1*0301 and D R B l * l 1 were significantly underhaplotypes. Just one haplotype was counted in related individuals
represented in the Ashkenazi Jewish patients with CA ( P <
of normalNew England family members and in consanguineous
H U C U S S II
CLASS
HLA
111
HLACLASSI
"
U
c1
m
A
Io(
A B
U
I*W
Fig 1. Human MHC region, including complement, HSP7O (HSP70l,HSP70-2 and HSP7O-Horn), and tumor necrosis factor (TNFA and
TNFB) loci in the non-HLA or class 111 region. CS, loci (A and B) for
complement protein C 4 ; C2, locus for complement protein C2; 210H.
loci (A and B) for the enzyme 21 hydroxylase.
.01 in both cases). In non-Jewish patients, the HLA-DR2,
DQBl*O502, and DQAl'O102 alleles, independently or together as a haplotype, were increased significantly in patients
with CA as compared with those without.
Amplification of the HSP-70 gene plus hybridization with
oligonucleotide probes detected three allelic forms, named
HSP70-l A, B, and C." Using Pst I RFLP analysis, two
polymorphic bands of 8.5 and 9 kb were detected within the
HSP70-2 gene.' Comparing HSP70-l and HSP70-2 polymorphism assignment in our population of 75 schizophrenic
patients (data not shown) and HSP70-l and HSP70-2 (performed by us, Table 2 ) polymorphism assignments in 16 of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3831
MAJOR HISTOCOMPATIBILITY COMPLEX IN AGRANULOCYTOSIS
Table 2. HSP70-2 Variant Distribution in Haplotypes From HLA
Homozygous Cell Lines and in Normal Individuals
No. of Positive
Haplotypes
HSP70-2
Variant
9.0-kb
group
8.5-kb
group
Mixed
group
Total
Cell
Linest
Norm.
Indiv.
26
10
HLA-DR2
HLA-838, DRB1'0402
HLA-844, DRB1'0401
HLA-844, DRB1'07
16
8
8
6
1
2
0
9
10
6
HL4-198, DR3 (*0301)*
HLA-818, DR3 ('0301)
HLA-813, DR7 ('0701)
12
11
0
3
0
2
15
11
2
HLA Alleles or Haplotype
HLA-DRI, 9.0 kb
HLA-DRI, 8.5 kb
HLA-DR3, 9.0 kb
HLA-DR3, 8.5 kb
HLA-DR4, 9.0 kb
B381
HLA-DR4, 8.5 kb
HLA-DR5, 9.0 kb
HLA-DR5, 8.5 kb
HLA-DR6, 9.0 kb
HLA-DR6, 8.5 kb
HLA-DR7, 9.0 kb
844)
HLA-DR7, 8.5 kb
HLA-DR8, 9.0 kb
HLA-DR8, 8.5 kb
lnot 88)
(not €38)
lnot 844,
Total
Haplotypes
0
2
0
0
0
4
1
1
2
4
1
1
0
0
2
3
3
4
4
2
3
3
4
6
2
7
4
1
3
1
2
52
3
1
2
120
0
4
1
(not 813,
0
0
0
0
68
1
t Among the cells typed, the following are from the 10th Workshop:
9013,9083,9081,9065,9026,9035,9062,9057,9027,9050,9051,9063,
9014,9008, and 9038 had the HSP70-2 9.0-kb variant;9053,9022,9023,
9086, 9018, 9020, 9085, and 9019 had the HSP70-2 8.5-kb variant.
All homozygous cells were '0301.
*
the HLA homozygous cell lines studied, we confirmed that
the 9.0-kb variant is in linkage disequilibrium with the A
variant and that the 8.5-kb variant is in linkage disequilibrium with C.I6 In 1 non-Jewish patient without CA, HSP70I AB corresponded to HSP70-2 918.5 kb.
The haplotypes of the homozygous cell lines and normal
individuals determined by family studies were assigned to
three groups (Table 2).In the group with 9.0-A variants,
there were many independent examples of HLA-DR2; HLAB44, DRBI *0401; HLA-B38, DRBl*0402; and HLA-B44,
DRBI *07 haplotypes. In the group of 8.5-C variants, the
main haplotypes were HLA-B8, DRBl*O301; HLA-Bl8,
DRBl *OIO2; and HLA-B13, DRBI *07. There was an increased frequency of the 9.0-A alleles in the CA group because of the excessive number of high-A value and DR2
haplotypes. The HSP-70 9.0-A haplotype frequency was significantly increased in Ashkenazi Jewish patients with CA
(83.3% [20 of 241 compared with 59.2% [32 of 541 Ashkenazi Jewish controls; P = .04, Fisher's exact test). In nonJewish patients with CA, the 9.0-A frequency was also increased, but the difference was not significant (75% [30 of
401 of cases compared with 59.3% [ l 9 of 321 of controls).
None of the 32 total CA cases studied was homozygous for
HSP-70 8.5-C, compared with 7 of 43 total controls (P =
.01, Fisher's exact test). The frequency of high-A value
haplotypes was compared in CA groups and controls, as
shown in Tables 3 and 4. Results of these comparisons are
given in Table 5. It is seen that the number of high-A value
haplotypes with 9.0-A was increased in the CA group and
that the number of random haplotypes associated with the
8.5-C variants is higher in the group without CA.
The results of analysis for mode of inheritance of HLADR4 and DR2 when Ashkenazi Jewish and non-Jewish populations are studied separately or together showed a tendency
to fit a dominant model (lower Ex2) and reject a recessive
mode of inheritance (higher Ex2).However, the Ex2 values
for the recessive model were not high enough to reach a
significant P value and categorically reject a recessive mode
of inheritance. Analysis of mode of inheritance for HSP70 9-A variants when Jewish and non-Jewish patients were
analyzed together also tended to fit a dominant model, but
a recessive model could not be rejected (data not shown).
DISCUSSION
In preliminary studies including a total of 11 patients, we
reported that CA is associated with the HLA-B38, DR4, DQ3
haplotype in Ashkenazi Jewish cases and with the HLADR2, DQl haplotype in non-Jewish
In 75 additional
patients typed by high resolution DNA techniques (32 cases
and 43 controls), we confirmed and defined the association
of CA with the DRBI *0402, DRB4*0101, DQBl*O302,
DQAl*O301,DPBl*O401 haplotype in Ashkenazi Jewish
patients and with the HLA-DRBI *1601, DRB5*02, DQBI *
0502, DQAl*0102haplotype in non-Jewish patients." There
was no significant increase of any HLA-DPB1 allele in the
non-Jewish CA patients. The finding of increased frequency
of HLA-DPBI *0401 in Jewish patients is consistent with the
known nonrandom association of HLA-DPBI *0401 with the
haplotype HLA-B38, DRBI*0402.15 At present, there is no
clear-cut explanation for the finding of different HLA haplotypes or allele associations with the same disease in populations of different ethnic backgrounds.16-" A possibility is
that non-HLA alleles in linkage disequilibrium with HLA
class I or I1 alleles are involved." The ratio of DRBl*1501,
DQBl*O602 to DRBl*1601, DQBI *0502 on DR2 haplotypes is high (5:l to 10:l) in Northern European populat i o n ~ . The
* ~ ratio is lower in Southern European populations
(l:l).23 In our patients with CA, the ratio is 1:2. This may
reflect a Southern European origin of the MHC susceptibility
gene for CA. HSP-70 and TNF are candidate genes to explain
the association of CA with HLA-DRBI *0402 in Ashkenazi
Jewish patients and HLA-DR2 in non-Jewish patients. First,
we tested this hypothesis by studying HSP70-2 polymorphism in normal individuals and HLA homozygous cell lines
carrying the haplotypes associated with CA. The 9.0-kb allele was invariantly associated with HLA-DR4 (HLA-B38,
DRBl*0402, DQBl*O302,
DPBl*O401;
and HLA-B44,
DRBl*O401), with HLA-DR2 (HLA-DBI *1601, DQBl*
0502, and HLA-DRBI *1501, DQBl*O602), and with HLADR7 (HLA-B44, HLA-DRBI *07). DR2 and DR4 haplotypes
were significantly overrepresented in the patients with CA
and underrepresented in the control group. Results of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CORZO ET AL
3838
Table 3. Assignment of Haplotypes in Ashkenazi Jewish Patients With CA
Patient
No.
HSP-70
Haplotypes
First Haplotypet
938, DR4 *0402, DPBl*O401
838, *0402,
DR4
1'040
DPB
l
838, DR4 *0402, DPB1*0401
B
DPB 1'040 1
838, DR4 '0402, DPB l '040 1
938, DR4 '0402, 1'040
DPB
1
638, DR4 *0402, 1'040
DPB
1
838, DR4 '0402, DPBl'O401
938, DR4 *0402, DPB l '040 l
838, DR4 '0402, DPB 1'040 1
838, DR4 '0404, DPB1*0401
845, DR13 '1301, DPBl*0402
1
2
3
4
5
6
7
8
9
10
11
12
Second Haplotype
844, DR14 *l401 DPB1'0401
98, DR2 * 150l, DPB l *030 l
98, DR3 '0301, DPBl'0407
95, DR2
DPB
* 1502,
l * 170l
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
-
Bx, DRl0 '1001, DPBI*0401
844, DR7 '0701, DPBI'0402
837, DR7 '0701, DPB1'0401
844, DR4 '0402, DPB1*0401
855, DR2 '1501, DPB1'0201
98, DR3 '0301, DPBl'O401
841, DR14 '1401, DPB1'0401
98, DR3
I,
'030
1'0202
DPB
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
-
HSP-70
Haplotypes
9.0-A
9.0-A
8.5-C
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
8.5-C
8.5-C
8.5-C
-
t Haplotypes with high-A values and all DR2 haplotypes are underlined.
HSP70-I and HSP70-2 polymorphism analysis in patients
confirmed the hypothesis that the 9.0-A haplotypes are in
linkage disequilibrium with different h i g h 4 value haplotypes found increased in schizophrenic patients with CA.
9.0-A variants were also associated with D R l l and D Q B l *
0301, which were identified as protective markers of CA in
Ashkenazi Jewish patients." Therefore, 9.0-Ahaplotypes are
part of the DR4 and DR2 haplotypes and a marker for CA
in both ethnic groups, but can be found among control patients and haplotypes. Our findings also suggested that the
9.0-A variants associated with specific HLA-DR4 and DR2
haplotypes are dominantly inherited markers of CA. Conversely, there may have been underrepresentation of common high-A value haplotypes and an excess of 8.5-C homozygotes in the control group, indicating protection from CA.
It is unknown if HSP-70 variants are associated with differences in the cell response to stressful stimuli. Consistent
with this possibility is the finding that peripheral blood mononuclear cells from individuals homozygous for the 8.5-kb
allele have lower HSP70-2 mRNA expression after heat
shock than 9.0-kb homozygous individuals.m Certain findings must bementioned before the discussion of the potential
role of HSP-70 in CA. First, using the enzyme myeloperoxidase, human activated polymorphonuclear cells (PMNs) are
capable of metabolizing clozapineZ5and c h l ~ r p r o m a z i n e . ~ ~ ~ ~ ~
During the process, some free radical intermediates are produced.26-2a
Second, there is evidence that HSP-70 gene expression can be induced in cells after exposure to antipsychotic drugs. In Escherichia coli, chlorpromazine induces
DnaK and GroEL proteins, the bacterial homologues of HSP70 and HSP-60 in mammal^.^^^^^ In rat neurons, haloperidol
induces HSP-70 tran~cription.~'Finally, changes in HSP
gene expression could be associated with modificationof the
proliferative activity of granulocyte precursors. It has been
Table 4. Assignment of Haplotypes in Non-Jewish Patients With CA
HLA-B
Locust
Patient
No.
~~
87,
97,
97,
97,
97,
1
2
3
4
5
~
'1601
* 160l
*l501
'1501
'1501
827, DR2 ' l 6 0 1
DR2 *l601
DR2 '1601
DR2 '1601
DR2 '1601
DR2 *l601
835, DR2 * 160l
644, DR7 '0701
DR7 '0701
844, DR4 '0401
844, DR4 '0404
844, DR4 '0401
627, DR11 '1107
DR17 '1101
840, B60
851, Bx
B44,Bx
835, B60
814, B39
8
9
10
11
12
13
14
15
16
l?
18
19
20
~
DR2
DR2
DR2
DR2
DR2
~~
8
-
6
7
~
HSP-70
Haplotypes
First Haplotype
6x, Bx
87,644
~
~~~
~
~
~
9.0-A
9.0-A
9.0-A
-
9.0-A
-
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
-
9.0-A
9.0-A
9.0-A
9.0-A
_(__
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
9.0-A
~~~
Haplotypes with high-A values and all DR2 haplotypes are underlined.
t HLA-B, DR haplotypes could not be assigned.
Second Haplotype
HLA-DPB1
Locus
HSP-70
Haplotypes
~~
827, DR2 1501
Bx, DR1 '0101
68, DR4 '0404
837, DRI * O l O l
Bx, DR13 '1302
862, DR4 '0401
835, DR4 '0408
DR4 "0401
DR4 '0402
DRI '0102
DR4 '0404
DR4 '0405
813, DR7 '0701
87, DR7 '0701
DR 13* 1302
813, DR7 *0701
627, OR3 '0301
68, DR3 '0301
Ss, DR3 *0301
DR9 *0901
'0401/*0402
*020 1/*030 l
*0401/*0401
'040 1/*040 1
'040 1/*040 1
'040 1/*0402
'040U' 150l
*0401/*0301
*040U*0402
*0401/*1401
*0401/*0301
*040 l/* I 70l
*0401/*0402
*0401/*040 I
'040 I/x
'040 1/*030 I
*0401/*040 l
*0401/'0401
'040 1/'020 l
'030 1/'020 1
9.0-A
9.0-A
8.5-C
8.5-C
8.5-C
9.0-A
8.5-C
8.5-C
9.0-A
9.0-A
9.0-A
9.0-A
8.5-C
9.0-A
9.0-A
8.5- C
9.0-A
8.5-C
8.5-C
8.5-C
-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IN AGRANULOCYTOSIS
3839
Tabla 5. Association of HSP-70Variants WRh DR2 and Nonrandom
(Hiah-A Value) and Random Haplotypes in CA
DRBl*0402,9.OA with TNF 10,4,1 and HLA-DR2,g.OAwith
TNF 11,4,1),they should have a higher representation in the
agranulocytosis patients, whereas the genotypes TNF 2,3,1,
which are associated with the high A haplotype B8, DR3
(absent in CA patients), should be overrepresented in the
control group, independently of ethnicity. Previous studies
have shown that lymphocytes from individuals carrying certain extended HLA haplotypes have higher TNFa secretion
level^^'^^^ and that TNFa induces apoptosis in human
PMNs:~ Therefore, it is necessary to study whether patients
with risk-associated haplotypes produce abnormal levels of
TNF, thus putting them at greater risk for CA.
MAJORHISTOCOMPATIBILITYCOMPLEX
~
~~
Nonrandom Haplotypes
With hsp-70 9.0-A
Haplotypes\Total
Group
-~
Jewish
Non-Jewish
All patients
~
Cases
Controls
PValue
16\24
19\40
35\64
10\54
6\32
16\86
<.0001
.01
Odds
Ratio
~~
<.0001
8.8
3.9
5.2
PValue
Odds
Ratio
.008
0.09
Random Haplotypes
With hsp-70 8.5-C
Haplotypes\Total
Cases
Group
-
~~~
Controls
~
Jewish
Non-Jewish
All patients
Abbreviation: NS, not significant.
1\24
6\40
7\64
17\54
11\32
0.25
28\86
NS
,002
ACKNOWLEDGMENT
We thank Rosalind Felber, RN, and Judy Dunlop, RN, for their
assistance in the collection of lymphocytes from subjects.
REFERENCES
1. Goate AM, Cooper DN, Hall C, h u n g TKC, Solomon E, Lim
reported that the promyelocytic leukemia cell line HL-60
has a decreased HSP-70 level3’ and increased HSP-70 and
HSP-27 levels33after pharmacologic induction of differentiation along the granulocyte-neutrophil pathway.
We believe that, in individuals carrying risk-associated
MHC markers, clozapine or its metabolites could induce the
expression of HSP-70and that the upregulated proteins could
act as a signal to decrease the proliferative capacity” or
induce a p o p t o ~ i in
s ~granulocyte
~
precursors of the bone marrow. Another mechanism could involve a decrease in the
level of cytokines, such as granulocyte-macrophage colony
stimulating factor (GM-CSF) and interleukin-2 (IL-2),36-38
which are important for granulocyte production and granulocyte protection from induced apoptosis. Because heat reduces the secretion of GM-CSF and IL-2,39it is possible that
HSP-70 could mediate this effect. Alternatively, failure to
increase the level of HSP and not its upregulation could
be the mechanism involved in clozapine-induced toxicity in
granulocyte precursors in the bone marrow. Clozapine or a
free radical metabolite can act as a noxious stimulus and
failure to develop an appropriate stress response in individuals carrying risk-associated MHC markers could result in
cellular injury@ or induction of apoptosis. Low levels of
expression of other genes, such as bcl-2, with products with
the capacity to defend against oxidative injury, could also
be involved?’ Finally, an HSP-70 variant could act as a
receptor for clozapine in PMN or their precursors in the
bone marrow. Some HSP can be found in the membrane of
cells,42and it has been reported that HSP-70 acts as a receptor
HSP-70 could
for 15-deoxyspergualin in T ~ e l l s . 4Thus,
~
become a target of autoantibodies or autoreactive T cells
against stress proteins restricted through MHC class I1 and
I antigens.
A second candidate to explain MHC susceptibility genes
for CA are TNFa and TNFP genes. Three polymorphic microsatellite allelic systems (TNFa, TNFb, and TNFc) have
been identified in or near the TNFP gene,” and their linkage
disequilibria with some MHC alleles and extended haplotypes have been d e ~ c r i b e d ? Because
~,~
TNF genotypes are
associated with certain high A HLA haplotypes (B38,
L: Localisation of a human heat-shock HSP70 gene sequence to
chromosome 6 and detection of two other loci by somatic-cell hybrid
and restriction fragment length polymorphism analysis. Hum Genet
75:123, 1987
2. Harrison GS, Drabkin HA, Kao FT, Hartz J, Hart IM, Chu
EHY, Wu BJ, Morimoto RI: Chromosomal location of the human
genes encoding major heat-shock protein HSI’IO. Somat Cell Mol
Genet 13:119, 1987
3. Sargent CA, Dunham I, Trowsdale J, Campbell RD: Human
major histocompatibility complex contains genes for the major heat
shock protein HSP70. Proc Natl Acad Sci USA 86:1968, 1989
4. Hunt C, Morimoto RI: Conserved features of eukaryotic hp70
genes revealed by comparison with the nucleotide sequence of human hsp70. Roc Natl Acad Sci USA 825455, 1985
5. Milner CM, Campbell RD: Structure and expression of the
three MHC-linked HSP70 genes. Immunogenetics 32:242, 1990
6. Milner CM, Campbell W.Polymorphic analysis of the three
MHC-linked HSP70 genes. Immunogenetics 36:357, 1992
7. Dwomiczak B, Mirault M: Structure and expression of a human
gene coding for a 71 kD heat shock ’cognate’ protein. Nucleic Acids
Res 15:5181, 1987
8. Lieberman JA, Yunis JJ, Egea E, Canoso RT, Kane JM, Yunis
ET: HLA-B38, DR4, DQw3 and clozapine-induced agranulocytosis
in Jewish patients with schizophrenia. Arch Gen Psychiatry 47:945,
1990
9. Yunis JJ, Lieberman JA, Yunis El: Major histocompatibility
complex associations with clozapine-induced agranulocytosis. Drug
Safety 7:7, 1992 (suppl 1)
10. Yunis JJ, Corzo D, Salazar M, Yunis ET, Howard F, Lieberman JA: HLA associations in clozapine-induced agranulocytosis.
Blood 86: 1177, 1995
11. Miller SA, Dykes DD, Polesky HF: A simple salting out
procedure for extracting DNA from human nucleated cells. Nucleic
Acids Res 85:1215, 1988
12. Cascino I, Sorrentino R, Tosi R Strong genetic association
between HLA-DR3 and a polymorphic variation in the regulatory
region of the HSWO-1 gene. Immunogenetics 37:177, 1993
13. Wu BJ, Morimoto RI: Structure and expression of the human
gene encoding major heat shock protein HSP70. Mol Cell Biol5:330,
1985
e
d AR, Yunis ET, Khatri K, Wagner R, Notani G, Awdeh
14. b
Z, Alper CA: Major histocompatibility complex haplotype studies
in Ashkenazi Jewish patients with pemphigus vulgaris. Proc Natl
Acad Sci USA 87:7658, 1990
15. Salazar M, Yunis I, Alosco SM, Chopek M, Yunis ET: HLA-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3840
DPB 1 allele mismatches between unrelated HLA-A,B,C,DR (generic) DQAl-identical unrelated individuals with unreactive MLC.
Tissue Antigens 39:203, 1992
16. Pugliese A, Awdeh ZL, Galluzzo A, Yunis EJ, Alper CA,
Eisenbarth GS: No independent association between HSP70 gene
polymorphism and IDDM. Diabetes 41:788, 1992
17. Nepom GT,Hansen JA, Nepom BS: The molecular basis for
HLA class I1 associations with rheumatoid arthritis. J Clin Immunol
7:1, 1987
18. Schiff B, Mizrachi Y, Orgad S, Yaron M, Gazit E: Association
of HLA-Aw31 and HLA-DR1 with adult rheumatoid arthritis. Ann
Rheum Dis 41:403, 1982
19. Thorsby E, Helgesen A, Solheim BG, Vandvik B: HLA antigens in multiple sclerosis. J Neurol Sci 32:187, 1977
20. Naito S, Kurowa Y, Hoyama T, Tsubaki T, Horikawa A,
Sasazuki T, Naguchi S, Ohtsuki S, Tokuomi H, Miyatake T, Takahyata N, Kawanami S: HLA and Japanese MS. Tissue Antigens 12:19,
1978
21. Ahmed AR, Wagner R, Khatri K, Notani G, Awdeh Z, Alper
CA, Yunis EJ: Major histocompatibility complex haplotypes and
class I1 genes in non-Jewish patients with pemphigus vulgaris. Proc
Natl Acad Sci USA 885056, 1991
22. Johnson AH, Hurley CK, Hartzman RJ, Alper CA, Yunis EJ:
HLA: The major histocompatibility complex of man, in Tomar RH,
Henry JB (eds): Clinical Diagnosis and Management by Laboratory
Methods (ed 18). Philadelphia, PA, Saunders, 1991, p 761
23. Imanishi T, Azaka T, Kimura A, Tokunaga K, Gojobori T:
Allele and haplotype frequencies for HLA and complement loci in
various ethnic groups, in Tsuji K, Aizawa M, Sasazuki T (eds): HLA
1991, Proceedings of the Eleventh International Histocompatibility
Workshop and Conference. Yokohama, Japan, Oxford, 1992
24. Pociot F, Ronningen KS, Nerup J: Polymorphic analysis of
the human MHC-linked heat shock protein 70 (HSP70) and HSP70Horn genes in insulin-dependent diabetes mellitus (IDDM). Scand
J Immunol 38:491, 1993
25. Uetrecht JP: Metabolism of clozapine by neutrophils. Drug
Safety 751, 1993 (suppl 1)
26. Kelder PP, De Mol NJ, Hart BA, Janssen LH: Metabolic
activation of chlorpromazine by stimulated human polymorphonuclear leukocytes. Induction of covalent binding of chlorpromazine
to nucleic acids and proteins. Chem Biol Interact 79:15, 1991
27. van Zyl JM, Basson K, Kriegler A, van der Walt BJ: Activation of chlorpromazine by the myeloperoxidase system of the human
neutrophil. Biochim Pharmacol 40:947, 1990
28. Mason RP, Fisher V: Possible role of free radical formation
in drug-induced agranulocytosis. Drug Safety 7:45, 1992 (suppl 1)
29. Tanji K, Mizushima T, Natori S, Sekimizu K: Induction by
psychotropic drugs and local anesthetics of DnaK and GroEL proteins in Escherichia coli. Biochem Biophys Acta 1129:172, 1992
30. Mizushima T, Natori S, Sekimizu K: Relaxation of supercoiled DNA associated with induction of heat-shock proteins in
Escherichia coli. Mol Genet 238:1, 1993
31. Sharp FR, Butman M, Wang S, Koistinaho J, Graham SH,
Sagar SM, Berger P, Longo FM: Heat shock proteins used to show
that haloperidol prevents neuronal injury produced by ketamine,
MK801, and phencyclidine. Ann NY Acad Sci 679:288, 1993
32. Beere HM, Morimoto RI, Hickman JA: Investigations of
mechanisms of drug-induced changes in gene expression: N-methylformamide-induced changes in synthesis of the Mr 72,000 constitutive heat shock protein during commitment of HL-60 cells to granulocyte differentiation. Cancer Res 53:3034, 1993
COR20 ET AL
33. Shakoori AR, Oberdof AM, Owen TA, Weber LA, Hickey
E, Lian JB, Stein GS: Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia
cells. J Cell Biochem 48:277, 1992
34. Spector NL,Ryan C, Samson W, Levine H, Nadler LM,
Arrigo AP: Heat shock protein is a unique marker of growth arrest
during macrophage differentiation of HL-60 cells. J Cell Physiol
156:619, 1993
35. Filippovich I, Sorokina N, Khanna KK, Lavin MF: Butyrateinduced apoptosis in lymphoid cells preceded by transient overexpression of HSP70 mRNA. Biochem Biophys Res Commun
198:257, 1994
36. Brach MA, deVos S, Gruss HJ, Hermann F: Prolongation of
survival of human polymorphonuclear neutrophils by granulocytemacrophage colony-stimulating factor is caused by inhibition of programmed cell death. Blood 80:2920, 1992
37. Whetton AD, Dexter MT: Influence of growth factors and
substrates on differentiation of haemopoietic stem cells. Curr Op
Cell Biol 5:1044, 1993
38. Pericle F, Liu JH, Diaz JI, Blanchard DK, Wei S, Forni G,
Djeu JY: Interleukin-2 prevention of apoptosis in humanneutrophils.
Eur J Immunol24:440, 1994
39. Dinarello CA, Dempsey RA, Allegretta M, Lo Preste G, Dainiak N. Parkinson DR, Mier W: Inhibitory effects of elevated temperature on human cytokine production. Cancer Res 46:6236, 1986
40. Buttke TM, Sandstrom PA: Oxidative stress as a mediator of
apoptosis. Immunol Today 15:7, 1994
41. Jarvis D, Turner AJ, Povirk LF, Traylor RS, Grant S: Induction of apoptotic DNA fragmentation and cell death in HL-60 human
promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res 54:1707, 1994
42. Koga T, Wand Wurttenberger A, DeBruyn J, MunkME,
Schoel B, Kaufmann S: T cells against a bacterial heat-shock protein
recognize stressed macrophages. Science 245: 1 112, 1989
43. Mazzucco CE, Nadler SG: A member of the Hsp70 family
of heat-shcok proteins is a putative target for the immunosuppressant
15-deoxyspergualin. Ann NY Acad Sci 685:202, 1993
44. Nedospasov SA, Udalova IA, Kuprash DV: DNA sequence
polymorphism at the human tumor necrosis factor (TNF) locus. J
Immunol 147:1053, 1991
45. Jongeneel CV, Briant L, Udalova IA, Sevin A, Nedospasov
SA, Cambon-Thomsen A: Extensive genetic polymorphism in the
human tumor necrosis factor region and relation to extended haplotypes. Proc Natl Acad Sci USA 88:9717, 1991
46. Abraham LJ, Marley JV, Nedospasov SA, Cambon-Thomsen
A, Crouau-Roy B, Dawkins RL, Giphart MJ: Microsatellite, restriction fragment-length polymorphism, and sequence-specific oligonucleotide typing of the tumor necrosis factor region. Hum Immunol
38:17, 1993
47. Santarnaria P, Gehrz RC, Bryan MK,Barbosa JJ: Involvement
of class I1 MHC molecules in the LPS-induction of IL-l/TNF secretions by human monocytes. Quantitative differences at the polymorphic level. J Immunol 143:913, 1989
48. Jacob CO, Fronek 2, Lewis GD, Koom M, Hansen JA, McDevitt HO: Heritable major histocompatibility complex class 11-associated differences in production of tumor necrosis factor a:Relevance
Natl
to genetic predisposition to systemic lupus erythematosus. ROC
Acad Sci USA 87:1233, 1990
49. Takeda Y, Watanabe H, Yonehara S, Yamashita T, s i t 0 S,
Sendo F: Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-alpha. Int Immunol 5:691, 1993
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1995 86: 3835-3840
The major histocompatibility complex region marked by HSP70-1 and
HSP70- 2 variants is associated with clozapine-induced
agranulocytosis in two different ethnic groups
D Corzo, JJ Yunis, M Salazar, JA Lieberman, A Howard, Z Awdeh, CA Alper and EJ Yunis
Updated information and services can be found at:
http://www.bloodjournal.org/content/86/10/3835.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.
Copyright 2011 by The American Society of Hematology; all rights reserved.