Excerpt from a grant proposal written by Dr. Warren about autism and genetics,
which an excellent introduction to genetics and autoimmune disorders in general.
(two tables and a gene map within this article should line put if you view it on
Courier font, 10 point size, with the longest possible line length):
A pathogen-autoimmune hypothesis for autism
Some children are susceptible to an environmental pathogen (most likely
a superantigen such as a virus or a bacterium) resulting from an inherited
deficiency of their immune system. Unable to clear the pathogen in a timely
and normal fashion, the child is at higher risk for the pathogen to damage the
developing brain or trigger an autoimmune response resulting in the symptoms
of autism. The autoimmune mechanism, if part of this pathogenesis, would
likely be under the control of the Class II portion of the MHC. The pathogen-
autoimmune mechanism would occur during the second trimester or within a year
or two after birth and may be operative in 50% or more of all autistic cases.
In order to be consistent with clinical observations, the pathogen would not
necessarily create gross neurological damage but have more subtle effects on
portions of the brain controlling behavior. After initial pathogenesis, no
requirement likely exists for the pathogen to remain in the body. However, if
it does persist, it would probably replicate very slowly and/or be maintained
in homeostasis by the immune system.
The pathogen-autoimmune theory can be extended to include the
possibility that, in some cases, an immune deficiency in the mother allows a
pathogen to persist in utero, causing damage to the developing brain of the
fetus with symptoms appearing at birth or sometime after birth. With this
possibility, the developing immune system of the fetus (even if normal) would
not be capable of protecting the fetus in face of the maternal deficiency.
Alternatively, an aberrant maternal immune response also associated with the
MHC may induce pathogenesis in the fetal brain.
The above hypothesis has several key factors: 1) exposure to a certain
pathogen at a vulnerable time, i.e., at the time the central nervous system is
undergoing rapid development; 2) the existence of an immune susceptibility or
deficiency that would allow a pathogen to persist; 3) the genetic constitution
of the immune system (under the control of the Class II region of the MHC)
that would allow certain T cells to react to the pathogen in such a way as to
cause reactivity against the central nervous system or products of the central
nervous system such as neurotransmitters, and 4) in some cases, an immune
susceptibility or deficiency in the immune system of the mother that may
permit a pathogen to be present in utero or allow an immune response against
the fetus. Many normal individuals in the population possess one or more of
the above factors, but it would be only in those subjects in which all of
these factors (plus, probably others) occur simultaneously that the symptoms
of autism develop.
B. BACKGROUND
B1. Findings in autism consistent with a pathogen-autoimmune hypothesis for
some cases of autism
Genetic predisposition:
It is well known that autoimmune disorders show greater concordance in
monozygotic than in dizygotic twins and often cluster in families. In autism,
a number of studies also find a greater concordance in monozygotic than
dizygotic twins (reviewed in 1). Family studies demonstrate that about 2-3%
of autistic probands have autistic siblings, about 50 to 100 times the rate
expected by chance. Most autoimmune disorders are caused by multiple genes
which likely is the case for autism (reviewed in 1).
General Immune abnormalities:
Most autoimmune disorders have varying degrees of immune imbalances,
including altered numbers of helper T cells, abnormal responses to mitogens and
reduced natural killer cell activity (2,3). In many cases these
abnormalities are marginal but significant. In autism, most, if not all
studies, have reported modified general immune function of one type or another
depending on the age of the autistic subjects studied. These include altered
numbers of T cells and T cell subsets (4-8) and depressed (9-10) or, in some
patients, enhanced (11,12) responses to T cell mitogens (the latter being
correlated with high serotonin uptake by platelets (12). Decreased natural
killer cell function has also been observed in some subjects with autism (13).
Disease association with histocompatibility antigens:
Many autoimmune disorders such as rheumatoid arthritis, systemic lupus
erythematosus and insulin dependent diabetes are associated with certain
alleles of the major histocompatibility complex (MHC) or the extended MHC
haplotype on chromosome 6 (see later). Warren et al. have found an
association of the C4B null allele (14) and the extended haplotype B44-SC30-
DR4 with autism (15). Two other studies have attempted to associate the MHC
with autism. The first study (16) investigated only Class I antigens in a
limited number of patients and while their findings were not significant when
corrected for the total number of antigens studied, they did find an excess of
HLA-A2 in their subjects. Interestingly, A2 is often carried on the extended
haplotype B44-SC30-DR4 (see Table 1). The other study (17) attempted to
implicate the MHC in autism by investigating sharing of HLA antigens by
autistic sibling pairs. While not providing evidence for an MHC association
with autism, this study does not rule out this possibility since extended
haplotypes and the C4B gene were not included in this study.
Pathogenic triggers:
Triggering by microorganisms is believed to be an important contributor
to most autoimmune diseases. For example, in multiple sclerosis, (18) viruses
may initiate autoimmunization leading to the destruction of myelin. At least
22 papers (reviewed in 19) report a possible association of prenatal and
postnatal viral and/or bacterial infections with autism including:
cytomegalovirus; herpes simplex; HIV; rubella; and syphilis. Other findings
implicating a possible role of a pathogen in autism are four season-of-birth
studies all of which found an excess of births of autistic children in the
month of March (reviewed in 19). If a pathogen is involved in autism,
conceivably it is more operative or epidemic during the early winter, i.e.,
the second trimester for March babies.
Presence of autoantibodies or cell-mediated immunity:
A hallmark of autoimmune disorders are specific antibodies and cell
mediated immunity reactive to the affected organ or tissue. In autism, T
cell-mediated responses and antibodies to human myelin basic protein (20) and
antibodies to neurofilament proteins (21,22) have been reported.
Sex differences:
Autoimmune diseases are often more common in one sex as exists in autism
where 4-5 times more boys than girls are afflicted. The influence of sex
hormones on immune function is well established.
B2. Complement C4 null alleles and their association with disease
The fourth component of human complement is encoded by two separate
genes, C4A and C4B, closely- linked on chromosome 6 in the middle of the major
histocompatibility complex (MHC) between HLA-B (Class I) and HLA-DR-DQ (Class
II) (Figure 1). C4A and C4B should not be confused with C4a and C4b, the
split or activation products of both C4A and C4B. The Class III C4A and C4B
genes are highly polymorphic with each having several normal alleles as well
as a null allele which is functionally silent (no functionally active protein
is produced). Homozygosity for the null allele at the C4A or C4B gene,
occurring in about 1-2% of the population, is associated with a lack of the
respective C4A or C4B plasma protein and reduced total C4 plasma levels.
Heterozygotes have a null allele on one of their chromosomes (at either C4A or
C4B) at a frequency of approximately 36% and 30%, respectively, in the general
population and C4 protein levels which are usually in the lower end of the
normal range (reviewed in 23).
DQ DR 21-0HB RP2 21-OHA RP1 C2
HSP70 BAT5 TNF BAT1 B
----|------|---//---|----|----|----|----|----|----|------|-------------|------|-----|--|--|-----|----|-----|--|-------|------//------|--
XB C4B XA C4A Bf 2 1 H BAT3 a a
Figure 1. The human extended major histocompatibility complex on the short arm
of chromosome 6 consisting of: the HLA- DQ and DR regions; the 2 Class III
modular structures (XB, 21-OHB, C4B, RP2) and (XA, 21-OHA, C4A and RP1); Bf,
C2; the genes for heat shock protein 70 (2,1, and Hom); BAT5; BAT3; TNF a
and a; BAT1; and the HLA-B region. Spacing of genes in the illustration
approximately corresponds to the spacing of the genes on chromosome 6 except
that the distance between DR and C4B and between the TNF genes and HLA-B are
greater than shown.
The products of the C4A and C4B genes are crucial to the activation of
the other vital components of complement involved in protection against
viruses, bacteria and other infectious agents. Moreover, C4 proteins can
bind to pathogens directly without the assistance of the other complement
components or antibodies. The C4A and C4B proteins share several structural
and functional characteristics identifying them as C4. However, they are
distinct in other characteristics including hemolytic activity; with C4B
proteins being about 4 times more active than C4A. C4A proteins bind more
avidly to amino-rich surfaces (through an amide linkage), whereas, C4B
proteins preferentially form ester linkages with hydroxyl- containing
carbohydrate surfaces. Deficiency in the C4 proteins, especially C4B, has
been associated with increased viral and bacterial infections. For example,
C4 null alleles have been associated with accelerated disease progression
following seroconversion to HIV, and with particular manifestations
associated with AIDS (24-26). Low C4B plasma concentrations have been
reported as a predisposing factor to autoimmune chronic active hepatitis (27).
Total or homozygous C4B deficiency has been associated with infections
causing bacterial meningitis, including streptococcus pneumonia, haemophilus
influenzae and neisseria meningitidis (28,29). Considerable evidence also
indicates that inherited abnormalities of the complement C4 proteins are
linked to certain autoimmune diseases. The frequency of null alleles at
the C4B locus has been reported to be increased in patients with scleroderma
(30) and schizophrenia (31). It is interesting that C4 null alleles have been
associated with spontaneous abortion (32) and C4B null alleles are
underrepresented in people 80 years of age or older suggesting that the
possession of a C4B null has a negative effect on survival (33,34).
B3. Class III region genes that may have relevance to autism.
We described above associations of the C4A and C4B genes with certain
diseases and will describe later an association of the C4B null allele with
autism. Due to their proximity to the C4B gene, other genes within the class
III region also deserve consideration as being involved in the development of
this severe developmental disorder. One candidate gene, the Bf gene is
located very close to the C4 genes and is of interest because one study
associated this gene with reading disorders (35) and a very recent second
investigation (36) using interval mapping, localized a gene for reading
disability to a region between the Bf gene and TNFa gene (see Figure 1).
The genes CYP21A and CYP21B (also called steroid 21-hydroxylase genes
21-OH genes or cytochrome P-450 genes) flank the 3' end of the C4A and C4B
genes. CYP21B codes for an enzyme of the steroidogenesis pathway and CYP21A
is a pseudogene. Located 611 base pairs upstream of each C4 gene are RP1 and
RP2 genes, respectively. RP contains 364 amino acids and has a transcript size
of 1.6-1.8 kb. It is ubiquitously expressed and is theorized to code for a
nuclear protein. The protein product of RP is highly hydrophilic and basic
and may interact with DNA or the acidic domain of transcriptional factors
(37). The physical location of RP suggests that it is probably identical to
the previously described G11 gene that was found to be expressed in many
different cell types, including monocytes, hepatocytes, epithelial cells and T
and B lymphocytes.
Located adjacent to and overlapping the CYP21 A and B genes is a
tenascin-like gene named Gene X (XA and XB). Not much is known about Gene X,
but it does produce an extracellular matrix protein. Data have suggested that
the four tandemly arranged genes RP, C4 CYP21 and Gene X form a structure
"RCCX". The number of these RCCX modules varies from one to three in the
population.
Two genes situated very close together within the extended MHC haplotype
(Figure 1) might be also relevant to autism. These genes are for tumor
necrosis factor (TNF) a and a . TNF a is produced by macrophages whereas
TNF a is produced by T cells, but they have common biological activity.
These proteins are very important to the immune system since they regulate
antibody production, hematopoiesis, fever, interferon production, expression
of the interleukin-2 receptor and have many more functions. TNFa has many
inflammatory effects in common with interleukin-1. TNF has also been directly
implicated in rheumatoid arthritis in the destruction of joint tissue and
cartilage breakdown.
One final gene, the gene for prolactin is (reviewed in 38) located close
to the TNF genes but its exact location is not known. Prolactin is a
pituitary lactogenic hormone with a molecular weight of about 23,000. It is
essential in the induction of lactation in mammals at parturition and is
synergistic with estrogen. Increasing evidence indicates that prolactin has
important regulation properties for the immune system and may be involved in
the pathogenesis and disease expression of autoimmune diseases. Lymphocytes
are known to have receptors for prolactin and also produce prolactin-like
substances suggesting that prolactin may play an important role in
communication between the immune and nervous systems.
B4. The MHC and the relationship of the C4B gene to extended MHC haplotypes and
other genes.
HLA antigens (human leukocyte antigens or histocompatibility antigens or
molecules) are protein structures located on the surface of most human cells.
These antigens, controlled by genes located at various loci within the MHC on
chromosome 6 are termed HLA-A, HLA-B, HLA-DR and HLA-DQ, and are important in
acceptance or rejection of tissue and organ allografts, regulation of human
responses to antigens and predisposition to a series of diseases, most of
which are classified as autoimmune disorders. The C4B gene along with the C4A
gene and the two other complement genes, C2 and B (or Bf), are inherited
together as single genetic units termed complotypes (Figure 1). The complement
genes are so tightly linked that crossover between them in a family has never
been demonstrated. Complotypes segregate as functionally single genetic units
and exhibit linkage disequilibrium with HLA-B and HLA-DR alleles. Linkage
disequilibrium is a poorly understood phenomenon in which certain alleles of
the different MHC regions segregate together more frequently than would be
expected from the relative distance between their loci. Because these alleles
appear as if they are fixed on chromosome 6, they have been termed extended or
ancestral MHC haplotypes. One extended haplotype, the most common one, is
designated B8-SC01-DR3. It is made up of the 8 allele of the HLA-B region,
the S allele of the Bf gene, the C allele of the C2 gene, the 0 (or null
allele) of the C4A gene, the one allele of the C4B gene and the three allele
of the HLA-DR region. In Caucasians, there are 12 or so common sets of
extended haplotypes with a frequency of at least 0.0043 which constitute
25-30% of all MHC haplotypes in this race. In Table 1 are a list of
extended haplotypes that we have observed in our studies in Utah and their
reported associations with disease, IgA deficiency and viral infections.
On some extended haplotypes it has been shown that conversion of the C4
genes has taken place where a gene originally coding for one of the C4
proteins undergoes a mutation and it begins to produce C4 protein of the other
type (39). Two of the extended haplotypes B44-SC30-DR4 and B35-FC(3,2)-DR1
have been found to have this process take place. The net effect is that a
person inheriting a chromosome of this type will produce much more C4A than
C4B. The clinical effects of such conversions are unknown but may be very
important.
Table 1. List of extended haplotypes observed in this study and their reported
disease associations (see review 40, 41).
Class I Class III Class II_________ _________________ ____________________________________ Disease
A Cw B Bf C2 C4A C4B DR DRB1 DRwa DQA1 DQw DQB1 Abbreviation Associationsb
_________________________________________________________________________________________________________
3 7 7 S C 3 1 15 0501 51 0102 6 0602 B7-SC31-DR15 MS, CD, IgA Def
1 7 8 S C 0 1 3 0301 52 0501 2 0201 B8-SC01-DR3 GMG, IgA Def, SLE
30 6 13 S C 3 1 7 0701 53 0201 2 0201 B13-SC31-DR7
30 5 18 F1 C 3 0 3 0301 52 0501 2 0201 B18-F1C30-DR3 IDDM
3 4 35 F C 3,2c 0 1 0101 51 0101 5 0501 B35-FC(3,2)-DR1 HIV rapid progression
2 5 44 S C 3 0 4 0401 53 0301 7 0301 B44-SC30-DR4 RA (child onset), FS
29 4 44 F C 3 1 7 0701 53 0201 2 0201 B44-FC31-DR7 CD, IgA Def
1 6 7 S C 6 1 7 0701 53 0201 9 0303 B57-SC61-DR7 IgA Def Psoriasis
2 3 62 S C 3 3 4 0401 53 0301 8 0302 B62-SC33-DR4 IDDM, RA
? 8 65 S C 2 1+2 1 0101 51 0101 5 0502 B65-SC2(1,2)-DR1 IgA Def
_________________________________________________________________________________________________________
a. DRw51 = DRB4; DRw52 = DRB2; DRw53 = DRB3.
b. MS, Multiple sclerosis; CD, Celiac disease; SLE, systemic lupus
erythematosus; Def, deficiency; GMG, generalized myasthenia gravis; IDDM,
insulin-dependent diabetes mellitus; RA, rheumatoid arthritis, and childhood
onset, FS, Feltys syndrome.
c. This designation denotes a fairly rare duplication of the C4A gene in which
one allele is expressed as C4A3 and the other allele as C4A2. With the
extended haplotype B35-FC(3,2)-DR1 it has been shown that the gene duplication
results from gene conversion of B to A (380). The extended haplotype
B44-SC30-DR4 has undergone a similar conversion but it is conventionally
designated as B44-SC30-DR4 instead of B44-SC(3,3)0-DR4.
B5. DR molecules and their relationship to the development of T cells
While the HLA-A and -B (Class I) genes are known to be most important in
graft rejection, it is widely accepted that the HLA Class II (DR and DQ)
molecules are intimately involved in the ability of T lymphocytes to
discriminate foreign antigens from self. By selectively binding and presenting
peptide antigens, these HLA molecules determine whether, when and where T
lymphocytes are stimulated (42). A number of structural and functional
studies have demonstrated that these molecules possess a unique binding site
for 6-25 amino acid fragments of degraded antigens (43-46). The polymorphisms
of the HLA genes have been clearly demonstrated to result in a clustering of
polymorphic amino acid residues in the HLA binding site providing selective int
eraction of HLA molecules with antigenic peptides (47).
The functional outcome of the interaction of the HLA molecules with the
T cell receptor (TCR) molecule is highly dependent on the maturation state of
the T cell. When differentiating in the thymus, the pre-T cell undergoes at
least two distinct types of selection, negative and positive (48-50). During
the negative selection process, recognition of self peptides in the context of
self-HLA molecules result in death of cells with anti-self specificities. In
positive selection, engagement of TCR with appropriately high affinity supports
the maturation of the thymocytes into immunocompetent T cells which migrate
into the peripheral tissue.
Both the negative and positive selection processes as well the
interaction of the Class II molecules with the TCR appear basic to many of the
HLA associations to disease, but associations exclusively with the Class I and
Class III have also been demonstrated. The mechanism of Class II association
with disease is speculative but several possibilities exist: 1) The HLA
molecule may merely function as a receptor for an etiologic agent (such as a
virus) with the specificity of the molecule-agent interaction resulting in the
HLA association. 2) The etiologic antigenic fragment may be selectively
embedded into the antigen binding groove of only certain HLA molecules; thus,
only carriers of certain HLA types would be able to react to a particular
etiologic antigen. 3) A third possibility is that the etiologic agent has
molecular mimicry with the HLA molecule. In this model similarities between
the ecological agent and HLA molecule may result in negative selection and the
inability of the person to recognize the pathogen and allow unchallenged disease
progression. Alternatively, similarities between the etiologic agent and the
HLA molecule in a positive- selection manner, induce a vigorous and eventually
specific tissue destruction. Finally, 4) it is possible that HLA are not
directly involved in pathogenesis but act in their role in assembling the TCR
repertoire of specificities. The potential diversity of the TCR is generated
by 'random' somatic recombination events that involve the 5 germ-line variable
segments, Va , Ja , Va, Da and Ja. Pathogenic processes would then result in
the over-representation of certain TCR specificities in individuals with
specific disorders. Associations with polymorphisms in the TCR repertoire
in autoimmune disease have been demonstrated in multiple sclerosis (51),
rheumatoid arthritis (52), juvenile onset arthritis (53) and systemic lupus
erythematosus (54).
MHC class II molecules are composed of an a chain (with an a1 and an a2
domain) and a a chain (with a a1 and a a2 domain) resulting in four external
domains of approximately 90 amino acids each. Structural and functional
studies and sequence analysis indicate that these chains have unique genetic
organization with polymorphic amino acid substitutions clustered in three
hypervariable regions (HVR) 1,2 and 3 (55,56). The a chain of the HLA DR
molecules are highly polymorphic resulting in the more than 50 known allelic
variations while the a chain is conserved. Further, sequence studies indicate
that entire stretches of polymorphic residues are shared among allelic types,
presumably resulting in shared epitopes.
B6. The influence of the TCR repertoire on susceptibility to autoimmune disease
How a particular TCR repertoire influences susceptibility to an
autoimmune disease is unknown. However, it is possible that a specific TCR
repertoire either lacks a particular TCR specificity necessary to initiate a
protective immune response to a pathogen, or has a higher potential for
autoimmune responses. Historically, is was assumed that a particular TCR
repertoire could respond to a spectrum of epitopes on a given antigen. Recent
findings, however, indicate that specific antigen recognition is restricted by
Va expression of the TCR (57,58). A number of studies have been carried out
analyzing the influence of the DR antigens on the TCR and Va expression and
have been successful (59). In most autoimmune diseases it has been necessary
to obtain the T cells from the target site of the autoimmune pathogenesis in
order to demonstrate over-representation of certain Va elements. However,
several studies in rheumatoid arthritis have found restricted use of certain
Va types in the peripheral blood as well as in the synovial fluid. One
recent study in rheumatoid arthritis may have relevance to prospective studies
in autism (60). A high frequency of clonotypic (Va) expansion of T cells was
demonstrated in synovial fluid, synovial tissue sections and peripheral blood.
The clonotype expansion was not restricted to one particular Va element, and
in the majority of patients, multiple Va elements were involved. Although the
clonotypic expansion was more evident in synovial fluid and tissue samples
the clonotypes were also found in the peripheral blood.
B7. The role of superantigens in the development of autoimmune disorders
Superantigens are a group of molecules which when bound to Class II
molecules stimulate T cells bearing particular Va bearing TCRs.
Superantigens differ from peptide antigens in several aspects of their
interaction with the TCR: 1) Superantigens-Class II molecules interact
exclusively with the Va domain, whereas peptide antigens bind with all the
variable elements of both the TCR a, a chains; 2) The frequency of T cells
responding to a superantigen is much greater than that to a peptide antigen
and 3) The influence of the DR molecules appears to be less involved with
superantigen than a peptide antigen to the TCR.
Both retroviral and bacterial superantigens have been described and
appear able to activate large numbers of T and B cells leading to the
hypothesis that superantigens may activate autoreactive T and B cells clones
and cause autoimmune symptoms. Thus, a useful approach in establishing a role
of a superantigen in an autoimmune disease is to search for evidence of
restricted Va expression. Another manner in which superantigens may trigger
autoimmune diseases involves cross-reaction between amino acid sequences of
the superantigen and antigens present on self tissue. In this case the
superantigen-activated T cell would react with self-antigen resulting in
tissue damage.
SIGNIFICANCE AND RELATIONSHIP OF SPECIFIC AIMS TO LONG-TERM GOALS.
The major thrust of these studies is to explore more thoroughly the
immunogenetic, pathological and immunological mechanisms that may contribute
to development of autistic symptoms in some patients. It is hoped that a
more thorough understanding of these possible autoimmune mechanisms may lead to
the ability to prevent this severe developmental disorder or identify couples
who are likely to give birth to an autistic child. In addition in some cases
it is possible that a purported immune mechanism may have not caused
irreversible damage to the central nervous system but is only interfering with
brain function such as by binding to various neurotransmitters or their
receptors. The several anecdotal reports of autistic subjects spontaneously
remitting or having their symptoms normalize following treatment with
chemotherapeutic or immunosuppressive regimens give rise to this hope (of
course, chemotherapeutic regimens are known to damage lymphocytes and
interfere with immune processes). Perhaps, a greater specificity in the
ability to diagnose autism and immunotherapeutic intervention can be
developed for these cases of autism. Another exciting aspect of this
research is the potential to develop a new biological marker for autism
and possibly also for related disorders. Finally, these studies might also
help establish biological relationships between autism and other disorders.
C. PROGRESS REPORT For the current funding period (September 1, 1992 *
October 31, 1994).
Personnel who have worked on the project:
Reed P. Warren, P.I., May 6, 1942, 528-54-2899, Sep 1, 1992 - Oct 31, 1994, 20%
time.
Phyllis Cole, Coinvest., 533-28-2288, Sep 1, 1992-Aug 31, 1994, 32% time
Roger Burger, Tech., April 25, 1952, 570-84-6213, Sep 1, 1992-Oct 31, 1994, 50%
time
W. Louise Warren, Reg. Nurse, Jan. 20, 1944, 427-90-9211, Sep 1, 1992 - Oct
31, 1994, 25% time
Alma Maciulis, M.S., Research Associate, Sep 7, 1951, 356-46-5056, Jun 1,
1993- Oct 31, 1994, 50% time
Don Sisson, Statistician, April 18, 1934, 475-32-7662, Sep 1, 1992- Oct 31,
1994, 5% time
The specific aims of the current funding period, modified in response to
budgetary cuts, were: 1. confirm and expand studies on associations of
extended MHC haplotypes and the C4B null allele with autism; 2. investigate a
possible association between the MHC and abnormal immune function and disease
characteristics of autistic subjects and 3. continue an exploration of
autoantibodies against myelin basic protein in the sera of autistic children.
During the first 26 months of the current funding project, 28 additional
autistic subjects (in 23 families), their parents and siblings and 23 healthy
unrelated age-matched subjects were entered into this project. The number of
subjects studied exceeds the projected goals. A number of publications have
already resulted from these studies and other manuscripts are in preparation.
C1. Specific Aim one: Confirmation of associations of extended haplotypes and
the C4B null allele with autism
As presented in Table 1, 10 different extended haplotypes have been
observed in our study of the general Northern Utah population. The
currently-studied autistic subjects had a strikingly increased (chi square
16.2; P LT 0.0001) frequency of their chromosomes carrying one or more of these
10 extended haplotypes as compared to chromosomes of the unrelated normal
population. This result confirms our findings obtained during the initial
funding of this grant that were published in the journal Immunogenetics (15)
Of the 46 chromosomes of the 23 newly studied patients, 27 (58.7%) had an
extended haplotype as compared to the unrelated control group in which 33 of
128 (25.8%) chromosomes carried an extended haplotypes. Also, the frequency
of extended haplotypes on chromosomes of the autistic children was much greater
than that on the family normal chromosomes, i.e., the chromosomes of the
parents which did not segregate to the autistic subjects. Only 30.7% of the
family normal chromosomes expressed an extended haplotype. Amazingly, in
the initial and current studies, only 8 of the 45 autistic subjects have not
had an extended haplotype and 15 autistic subjects carried an extended
haplotype on each of their chromosomes. Also, as seen in the initial
study, the mothers but not the fathers of the autistic children had an
increased representation of extended haplotypes. An additional control
group of mentally retarded subjects had a haplotype frequency (26%) not
unlike that of the unrelated controls.
The phenotypic frequency of the various extended haplotypes are shown
in Table 2. Representation of one extended haplotype B44-SC30-DR4 was highly
increased (P LT 0.001) on autistic and maternal chromosomes also replicating
our initial finding. In the initial investigation, in addition to an
unrelated healthy group, a group of mentally-retarded subjects was
investigated as an additional control group. In order to perform meaningful
statistical analyses at the time, we found it necessary to combine the results
of these two control groups before comparing them to the data of the autistic
subjects. With the inclusion of additional control subjects, we are now able
to analyze, separately, these two control groups, and, remarkably, find B44-SC3
0-DR4 represented only in the mentally-retarded subjects and not in any of the
64 normal subjects. The cause of mental retardation in the four subjects with
B44-SC30-DR4 is unknown. We certainly realize that it was serendipitous to
find no representation of B44-SC30-DR4 among our normal subjects. Other
studies (23,39) have found phenotypic frequencies of this extended haplotype of
about 5- 6%. However, these other studies may have included family members
of autistic or mentally retarded subjects in their control groups. Also, as
far as we know, none of our control subjects had family members of patients
with Felty's syndrome (38) or juvenile-onset arthritis (39), two other
disorders associated with B44-SC30-DR4.
Table 2. Extended MHC Haplotypes in Autistic Subjects
Number of subjects with an extended haplotype and percentage
______________________________________________________________
Autistic
Mentally Unrelated
Total Current Maternal
Paternal Retardeda Healthy
_________________________________________________________________________________
Extended Haplotype n = 45 n = 23 n = 39 n = 39 n = 25 n = 64
_________________________________________________________________________________
B44-SC30-DR4 9 (20.0)b 3 (13.0)c 8 (20.5)b 3 (7.7) 4 (16.0)d 0 (0)
BX-SC30-DR4e 4 (8.8) 3 (13.0) 4 (7.7) 2 (5.1) 0 (0) 1 (1.6)
B35-FC(3,2)0-DR1 2 (4.4) 1 (4,3) 0 (0) 3 (7.7) 0 (0) 1 (1.6)
B18-F1C30-DR3 2 (4.4) 2 (8.7) 1 (2.6) 1 (2.6) 0 (0) 0 (0)
B62-SC33-DR4 2 (4.4) 2 (8.7) 2 (5.1) 2 (5.1) 0 (0) 2 (3.1)
B44-FC31-DR7 7 (15.5) 3 (13.0) 4 (10.3) 4 (10.3) 2 (8.0) 4 (6.2)
B57-SC61-DR7 3 (6.7) 1 (4.3) 4 (10.3) 1 (2.6) 0 (0) 2 (3.1)
B13-SC31-DR7 3 (6.7) 1 (4.3) 2 (5.1) 1 (2.6) 0 (0) 1 (1.6)
B65-SC2(1,2)-DR1 1 (.2.2) 0 (0) 1 (2.6) 0 (0) 0 (0) 0 (0)
B7-SC31-DR15 7 (17.6) 4 (17.4) 6 (15.4) 6 (15.4) 2 (4.0) 5 (7.8)
B8-SC01-DR3 13 (28.8) 7 (30.4) 9 (23.1) 8 (20.5) 7
(28.0) 17 (26.6)
_________________________________________________________________________________
a. Subjects with idiopathic mental retardation.
b. P < 0.001 as compared to values from unrelated healthy controls.
c. P < 0.05.
d. P < 0.01.
e. Fragment of the B44-SC30-DR4 extended haplotype including SC30 and DR4 but
with a HLA B marker other than B44.
We also discovered in the current funding period that in addition to the
autistic subjects having B44-SC30- DR4, four additional autistic subjects had a
fragment of this extended haplotype including SC30-DR4- DRw53 and DQ7, i.e.,
the Class III and Class II portions of this extended haplotype (Table 1), but
with a different B or Class I region allele. This finding argues that if a
gene (s) within B44-SC30-DR4 is indeed associated with autism, it is likely
located within the Class III or II regions.
Another interesting finding was that three mothers of autistic children
had B44-SC30-DR4 on one of their chromosomes which did not pass to their
children. On the surface this finding argues against a B44- SC30-DR4-autism
association. However, if this extended haplotype is associated with an immune
abnormality/ deficiency, perhaps, in some cases it is sufficient for only the
mother of the autistic child to have this extended haplotype and accords with
the theory that a maternal immune deficiency may have allowed damage to the
developing brain of the fetus. Two of the family normal chromosome also
carried the SC30-DR4 fragment and they too were from mothers.
Interestingly, of the 12 mothers with B44- SC30-DR4 or its fragment, the onset
of autism in their child was either at birth or at the latest 12 months after
birth and 10 of the 12 mothers had histories of troubled pregnancies including
multiple miscarriages, bleeding, and/or toxemia.
The individual components of B44-SC30-DR4 including the B44 allele, the
S allele of Bf, the C allele of C2, the three allele of C4A, the null allele
of C4B, the 4 allele of DR, the 53 allele of DRw and the 7 allele of DQ were
studied independently for association with autism. With the exception of the
C4B null allele and DR4 (to be discussed below) none of these components were
associated with autism.
Most of the extended haplotypes in addition to B44-SC30-DR4 (Table 2)
were also increased on autistic chromosomes but not significantly. However,
it is unlikely that the B44-SC30-DR4 extended haplotype and its fragment
account for the overall increased frequency of extended haplotypes on autistic
chromosomes since significance (P = 0.02) exists even when chromosomes
expressing B44-SC30-DR4 or its fragments are not considered.
Literature Cited
1. Gillberg, C. and Coleman, M. In: The Biology of the Autistic
Syndromes, 2nd Edition, pp. 96-114, Mac Keith Press, Oxford, Blackwell
Scientific Publications Ltd., London, England, 1992.
2. Waksman, B.H., In: Perspectives on Autoimmunity, ed. I.R. Cohen, CRC
Press, Inc., Boca Raton, FL, pp. 59-109, 1988.
3. Klinman, D.M. and Steinberg, A.D., In Immunological Diseases, 4th
Edition, ed. M. Samter, D.W. Talmedge, M.M. Frank, K.F. Austen and
H.N. Claman, Little Brown and Co., Boston, MA, pp. 1335-1363, 1988.
4. Warren, R.P., Yonk, L.J., Burger, R.A., Cole, P., Odell, J.D., Warren,
W.L., White, E. and Singh, V.K.: Deficiency of suppressor-inducer
(CD4+CD45R+) T cells in autism. Immunological Investigations 19: 245-252,
1990.
5. Yonk, L.J., Warren, R.P., Burger, R.A., Cole, P., Odell, J.D. Warren,
W.L., White, E. and Singh, V.K.: CD4+ helper T cell depression in
autism. Immunological Letters 25: 341-346, 1990.
6. Abramason, R.K., Self, S., Genco, P., Smith, N., Pendleton, A.,
Valentine, J., Wright, H.H., Cuccaro, M. and Powell, D. American
Journal of Human Genetics 47(3):A45 (Abstract) (1990).
7. Wood Frei, B., Denny, D., Gaffney, G.R. and O'Donnell, T. Scientific
Proceedings of the Annual Meeting of the American Academy of Child
and Adolescent Psychiatry 7:53, Abstract # 26, 1991.
8. Plioplys, A.V., Greaves, A, Kazemi, K and Silverman E. Lymphocyte
function in autism and Rett Syndrome. Neuropsychobiology 7:12-16,
1994.
9. Stubbs, E.G., Crawford, M.L., Burger, D.R. and Vanderbark, A.A.
Depressed lymphocyte responsiveness in autistic children. Journal
of Autism and Childhood Schizophrenia 7: 49-55, 1977.
10. Warren, R.P., Margaretten, N.C., Pace, N.C. and Foster, A. Immune
abnormalities in patients with autism. Journal of Autism and
Developmental Disorders 16: 189-97, 1986.
11. Singh, V.K., Fudenberg, H.H., Emerson, D. and Coleman, M.
Immunodiagnosis and immunotherapy in autistic children. Annals of the
New York Academy of Sciences 540:602-604, 1988.
12. Ferrari, P., Marescot, M.R., Moulias, R., Bursztejn, C., Deville
Chabrolle, A, Thiollet, M., Lesourd, B., Braconnier, A., Dreux, C. and
Zarifian, E. Immune status in infantile autism: Correlation between the
immune status, autistic symptoms and levels of serotonin. Encephale
14:339-344, 1988.
13. Warren, R.P., Foster, A. and Margaretten, N.C. Reduced Natural Killer
Cell Activity in Autism. Journal of the American Academy of Child
Psychiatry 26:333-335, 1987.
14. Warren, R.P., Singh, V.K., Cole, P., Odell, J.D., Pingree, C.B.,
Warren, W.L. and White, E.: Increased frequency of the null allele
at the complement C4B locus in autism. Clinical and Experimental
Immunology 83:438-440, 1991.
15. Warren, R.P., Singh, V.K., Cole, P., Odell, J.D., Pingree, C.B.,
Warren, W.L., DeWitt, C.W. and McCullough, M. Possible Association of
the Extended MHC Haplotype B44-SC30-DR4 with autism. Immunogenetics
36:203-207, 1992.
16. Stubbs, E.G. and Magenis, R.E. HLA and Autism. Journal of Autism and
Developmental Disorders 10:15-19, 1980.
17. Spence, M.A., Ritvo, E.R., Marazita, M.L., Funderburk, S.J.,
Sparkes, R.S. and Freeman , B.J. Gene mapping studies with the syndrome of
autism. Behavioral Genetics 15:1-13,1985.
18. Waksman, B.H., In: Perspectives on Autoimmunity, ed. I.R. Cohen, CRC
Press, Inc., Boca Raton, FL.
19. Gillberg, C. and Coleman, M. In: The Biology of the Autistic
Syndromes, 2nd Edition, pp. 218- 225, Mac Keith Press, Oxford,
Blackwell Scientific Publications Ltd., London, England, 1992.
20. Weizman, A., Weizman, R., Szekely, G.A., Wijsenbeek, H. and Livini, E.
Abnormal immune response to brain tissue antigen in the syndrome of autism.
Am J Psychiatry 139: 1462-1465, 1982.
21. Singh, V.K., Warren, R.P., Odell, J.D., Warren, W.L. and Cole, P.
Antibodies to Myelin Basic Protein in Children with Autistic
Behavior. Brain, Behavior and Immunity 7:97-103, 1993.
22. Plioplys, A.V., Greaves, A., Kazemi, K. and Silverman, E.
Immunoglobulin reactivity in autism and Rett's syndrome. Developmental
Brain Dysfunction 7:12-16, 1994.
23. Alper, C.A., Awdeh, Z.L. and Yunis, E.J. Complotypes and extended
haplotypes in laboratory medicine: Complement in laboratory medicine.
Complement Inflammation 6: 8-18, 1989.
24. Cameron, P.U., Mallal, S.A., French, M.A.H. and Dawkins, R.L. Major
histocompatibility complex genes influence the outcome of HIV infection:
Ancestral haplotypes with C4 null alleles
explains diverse HLA associations. Human Immunology, 29, 282-295, 1990.
25. Pollack, M.S. Immunogenetics of AIDS. In: Farid NR, ed. The
Immunogenetics of Autoimmune Diseases. Volume 2. pp. 311-320, Boca Raton,
FL, CRC Press, 1991.
26. Hentges, F., Hoffman, A., Dearaujo, F.O. and Hemmer, R. Prolonged
clinically asymptomatic evolution after HIV-1 infection is marked by the
absence of complement-C4 null alleles at the MHC. Clinical and
Experimental Immunology, 88, 237-242, 1992.
27. Vergani, D., Larcher, V.F. and Davies, E.T. Genetically determined low
C4: A predisposing factor to autoimmune chronic active hepatitis. Lancet,
August 10, 294-298, 1985.
28. Rowe, P.C., McLean, R.H., Wood, R.A., Leggiadro, R.J. and Winkelstein
JA. Association of homozygous C4B deficiency with bacterial meningitis.
Journal of Infectious Disease, 160, 448-451,1989.
29. Bishof, N.A., Welch, T.R. and Beischel, L.S. C4B deficiency: A risk
factor for Bacteremia with encapsulated organisms. Journal of
Infectious Disease, 162, 245-250, 1990.
30. Mollenhauer, E,. Schmidt, R., Heinrichs, M. and Rittner, C.
Scleroderma: possible significance of silent alleles at the C4B locus.
Arthritis Rheumatology, 27, 711-712, 1984.
31. Rudduck, C., Beckman, L., Franzen, G., Jacobsson, L. and Lindstrom, L.
Complement factor C4 in schizophrenia. Human Heredity, 35, 223-226,
1985.
32. Laitinen, T., Lokki, M.L., Tulppala, M., Ylikorkala, O.,Koskimies, S.
Increased frequency of complement C4 'null' alleles in recurrent
spontaneous abortions. Human Reproduction 1991 Nov; 6 (10):
1384-1387, 1991
33. Kramer, J., Rajczy, K., Fust, G. Low incidence of null alleles of the
fourth component of complement (C4) in elderly people. Immunology
Letters 20: 83-5, 1989.
34. Kramer, J., Fulop, T., Rajczy, K., Nguyen, A.T., Fust, G.A. Marked
drop in the incidence of the null allele of the B gene of the fourth
component of complement (C4B*Q0) in elderly subjects: C4B*Q0 as a
probable negative selection factor for survival. Human Genetics 86:
595-598, 1991.
35. DeFries, J.C., Olson, R.K., Pennington, B.F. and Smith, S.D. Colorado
Reading Project: an update (1991). In: The Reading Brain: The Biological
Basis of Dyslexia, edited by D.D. Duane and D.B., pp. 53-87, Gray, York
Press, Parkton, Maryland.
36. Cardon, L.R., Smith, S.D., Fulker, D.W., Kimberling, W.J., Pennington,
B.F., DeFries, J.C. Quantitative trait locus for Reading Disability on
Chromosome 6. Science, 266:276-279, 1994.
37. Shen, L., Wu, L., Sanlioglu, S., Chen, R., Mendoza, A.R., Dangel, A.W.,
Carroll, M.C., Zipf, W.B. and Yu, C.Y. Structure and genetics of the
partially duplicated gene RP located immediately upstream of the
complement C4A and C4B genes in the HLA Class III region. The Journal of
Biological Chemistry 269:8466-8476, 1994.
38. Myal, Y., DiMattia, G.E., Gregory, C.A., Friesen, H.G., Hamerton, J.L
and Shiu, R.P. A BgIII RFLP at the human prolactin gene locus on
chromosome 6. Nucleic Acids Research, 19:1167-1171, 1991.
39. Campbell, R.D., Dunham, I., Kendall, E. and Sargent, C.A. Polymorphism
of the human complement component C4. Experimental and Clinical
Immunogenetics 7:69-84, 1990.
40. Degli-Esposti, M., Leaver, A.L., Christiansen, F.T., Witt, C.S.,
Abraham, L.J. and Dawkins, R.L. Ancestral haplotypes: Conserved
population MHC haplotypes. Human Immunology 34:242-252,1992.
41. Fraser, P.A., Stern, S., Larson, M.G., Marcus-Bagley, D., Awdeh, Z.,
Glass, D.N. and Alper, C.A. HLA extended haplotypes in childhood and
adult onset HLA-DR4-associated arthropathies. Tissue Antigens
35:56-59,1990.
42. Schwartz, R.H. T-lymphocyte recognition of antigen in association with
gene products of the major histocompatibility complex. Annual Review of
Immunology 3:237-261, 1985.
43. Guillet, J.-G., Lai, M.-Z., Briner, T.J., Smith, J.A. and Gefter, M.L.
Interaction of peptide antigens and class II major histocompatibility
complex antigens. Nature 324:260-262, 1986.
44. Babbit, B.P., Matsueda, G., Haber, E., Unanue, E.R. and Allen, P.M.
Antigenic competition at the level of pedtide-Ia binding. Proceedings
of the National Academy of Sciences, U.S.A. 83:4509-4513, 1986.
45. Guillet, J.-G., Lai, M.-Z., Briner, T.J., Buus, S., Sette, A., Grey,
H.M., Smith, J.A. and Gefter, M.L. Immunological self, non-self
discrimination. Science 235, 865-870, 1987.
46. Chicz, R.M., Urban, R.G., Lane, W.S., Gorga, J.C., Stern, L.J.,
Vignali, D.A.A. and Strominger, J.L. Predominant naturally processed
peptides bound to HLA-DR1 are derived from MHC-related molecules and
heterogeneous in size. Nature 358:764-768, 1992.
47. Rothbard, J.B., and Gefter, M.L. Interaction between immunogenetic
peptides and MHC peptides. Annual Review of Immunology 10:527-565, 1991.
48. von Boehmer, H., Teh, H.S., and Kisielow, P. The thymuus selects the
useful and destroys the harmful. Immunology Today 10:57-61, 1989.
49. Schwartz, R.H. Acquisition of immunological self tolerance. Cell
57:1073-1081, 1989.
50. Fowlkes, B.J. and Pardoll, D.M. Molecular and cellular events of T cell
development. Advances in Immunology 44:207-264,1989.
51. Beall, S.S., Diddison, W.E., McFarland, H.F., and Hood, L.E.
Susceptibility for multiple sclerosis is determined, in part, by
inheritance of a 175 kb region of the TCR-beta chain locus and HLA class II
genes. Journal of Neuroimmunology. 45:53-60, 1993.
52. Weyand, C.M., Oppitz U., Hicok, K., and Goronzy, J. Selection of T cell
receptor V beta elements by HLA-DR determinants predisposing to
rheumatoid arthritis. Arthritis Rheum 35(9):990-998.1992.
53. Berstein, B.H., Miltenburg, A.M., van Laar, J.M., Hertzberger, R. and
Breedveld, F. T cell receptor rearrangements in juvenile rheumatoid
arthritis: a search for oligoclonality. Clinical and Experimental
Rheumatology 11(2):209-1314, 1993.
54. Ikaheimo I.L.,Tiilikainen, A.S., Hameenkorpi R., and
Silvennoinen-Kassinen, S. Different distribution of T cell receptor
beta-chain haplotypes in mixed connective tissue disease and systemic lupus
erythematosus. Diabetes 43(4):599-6063, 1994.
55. Bell, J.I., Denney, D., Jr., Foster, L., Belt, T. and McDevitt, H.O.
Allelic variation in the DR subregions of the human major
histocompatibility complex. Proceedings of the National Academy of
Sciences, U.S.A. 84:6234-6238, 1987.
56. Gorski, J. and Mach B. Polymorphism of human Ia antigens: Gene
conversion between two DRB loci results in a new HLA D/DR specificity.
Nature 322:67-70, 1986.
57. Nanda, N.K., Apple, R. and Sercarz, E.E. Limitations in plasticity of
the T-cell receptor repertoire. Proceedings of the National Academy
of Sciences U.S.A. 88:9503-9507, 1991.
58. Nanda, N.K., Arzoo, K.K., Sercarz, E.E. In a small multideterminant
peptide, each determinant is recognized by a different VB gene
segment. Journal of Experimental Medicine 176:297-302, 1992.
59. Loveridge, J.A., Rosenberg, W.M.C., Kirkwood, T.B.L. and Bell, J.I. The
genetic contribution to human T-cell receptor repertoire. Immunology
74:246-250,1991.
60. Yamamoto, K., Sakoda, H., Nakajima, T., Kato, T., Okubo, M., Dohi, M.,
Mizushima, Y., Ito, K. and Nishioka, K. Accumulation of multiple T cell
clonotypes in the synovial lesions of patients with rheumatoid arthritis
revealed by a novel clonality analysis. International Immunology 4:1219-1226.
61. Warren, R.P., Burger, R.A., Odell, D, Torres, A.R. and Warren, W.L.
Decreased Plasma Concentrations of the C4B Complement Protein in
Autism. Archives of Pediatric and Adolescent Medicine 148:180-183,
1994.
62. Gaither, T.A., Alling, D.W. and Frank, M.M. A new one-step method for
the functional assay of the fourth component (C4) of human and guinea pig
complement. Journal of Immunology 113:574-583,1974.
63. Gillberg, C. and Coleman, M. In: The Biology of the Autistic
Syndromes, 2nd Edition, pp. 115-130, Mac Keith Press, Oxford,
Blackwell Scientific Publications Ltd., London, England, 1992.
64. Schaffer, F.M., Palermos, J., Zhu, Z.B., Barger, B.O., Cooper, M.D. and
Volankis, J.E. Individuals with IgA and common variable immunodeficiency
share polymorphisms of major histocompatibility complex class III genes.
Proceedings of the National Academy of Sciences U.S.A. 86:8015-
8019,1989.
65. McClelland, R.J., Eyre, D.G., Watson, D., Clavert, G.J. and Sherrard,
E. Central conduction time in childhood autism. British Journal of
Psychiatry 160:659-663,1992.
66. Burmester, G.R., Jahn, B., Gramatzki,M., Zacher, J. and Kalden, J.R.
Activated T cells in vivo and in vitro: Divergence in expression of
Tac and Ia antigens in the non-blastoid small T cells of inflammation
and normal T cells activated in vitro. Journal of Immunoliogy
133:1230-1231,1984.
67. Bergroth, V., Konttinen, Y.T., Pelkonen, P., Haapala, M., Haapasaari,
J., Nordstrom, D., Kunnamo, I. and Friman, C. Synovial fluid
lymphocytes in different subtypes of juvenile rheumatoid arthritis.
Rheumatology 31:780-783, 1989.
68. Hafler, D.A., Hemler, M.E., Christenson, L., Williams, J.M., Shapiro,
H.M., Strom, T.B., Strominger, J.L. and Weiner, H.L. Investigation
of in vivo T cells in multiple sclerosis and inflammatory central nervous
system diseases. Clinical Immunology and Immunopathology 37:163-171, 1985.
69. Gillberg, C. and Coleman, M. In: The Biology of the Autistic
Syndromes, 2nd Edition, pp. 115-130, Mac Keith Press, Oxford,
Blackwell Scientific Publications Ltd., London, England, 1992.
70. Roy, B.F., Benkelfat, C., Hill, J.L., Pierce, P.F., Dauphin, M.M.,
Kelly, T.M., Sunderland,T., Weinberger, D.R. and Breslin, N. Serum
antibody for somatostatin-14 and prodynorphin 209-240 in patients with
obsessive-compulsive disorder, schizophrenia, Alzheimer's disease, multiple
sclerosis, and advanced HIV infection. Biological Psychiatry 35:
335-44,1994.
71. Folstein, S.E. and Rutter, M.L. Autism:Familial Aggregation and
Genetic Implications. Journal of Autism and Developmental Disorders 18,
No. 1:3-30, 1988.
72. Geschwind, N., and Galaburda, A.M. Cerebral Lateralization.
Biological Mechanisms, Associations, and Pathology:I. A hypothesis
and a program for research. Archieves of Neurology 42, May:428-452,
1985.
73. Piven, J., Gayle, J., Chase, G.A., Fink, B., Landa, R., Wzorek, M.W.,
and Folstein, S.E. A family history study of neuropsychiatric disorders
in the adult siblings of autistic individuals. Journal of the
American Academy of Child and Adolescent Psychiatry 29, No. 2:177-183,
1990.
74. Gross-Tsue, V., Shalev, R.S. and Amir, N. Attention deficit disorder:
association with familiar-genetic factors. Pediatric Neurology
7(4):258-261, 1990.
75. Barkley, R.A. Attention deficit hyperactivity disorder:a handbook for
diagnosis and treatment. The Guilford Press, NY, 1990.
76. Pitzalis, C., Choy, E., and Kingsley, G. Monoclonal antibody therapy
in rheumatic disease. Presse Med 23(11):532-93, 1994.
77. Le Couteur, A., Rutter, M., Lord, C., Rios, P., Robertson, S.,
Holdgrafer, M., McLennan, J.J. Autism Diagnostic Interview: a
standardized investigator-based instrument. Journal of Autism and
Developmental Disorders 19 (3):363-387, 1989.
78. Goodman, R., and Stevenson J. A twin study of hyperactivity:II. The
aetiological role of genes, family relationships, and perinatal
adversity. Journal of Child Psychology and Psychiatry 30:691-709, 1989.
79. Biederman, J., Faraone, S.V., Keenan, K., Benjamin, J., Krifcher, B.,
Moore, C., Sprich-Buckminster, S., Ugaglia, K., Jellinek, M.S.,
Steingard, R., et al. Further evidence of family-genetic risk factors in
attention deficit hyperactivity disorder. Patterns of comorbidity in
probands and relatives psychiatrically and pediatrically referred
samples. Archives of General Psychiatry 49(9):728-738.
80. Achenbach, T.M. Manual for the Teacher's Report Form and 1991 Profile.
Burlington, VT:University of Vermont Department of Psychiatry, 1991.
81. Achenbach, T.M. Manual for the Child Behavior Checklist/4-18 and 1991
Profile. Burlington, VT:University of Vermont Department of Psychiatry,
1991.
82. Greenberg, L.M. T.O.V.A. Manual, Minneapolis, MN:University of
Minnesota Department of Psychiatry, 1993.
83. Inoko, H. and Ota, M. PCR-RFLP. In: K.M. Hui and J.L. eds. Handbook
of HLA Typing Techniques, pp. 9-70, CRC Press, Boca Raton, 1993.
84. Nutman, T.B. Measurement of Polyclonal Immunoglobulin Synthesis using
ELISA. In: J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M.
Shevach, and W. Strober, W. eds. Current Protocols in Immunology, p.
7.12, Green Publishing and Wiley-Interscience, New York, 1991.
85. Schneider, P.M., Carroll, M.C., Alper, C.A., Rittner, C., Whitehead,
A.S., Yunis, E.J. and Colten, H.R. Polymorphism of the human complement C4
and steroid 21-hydroxylase genes. J. Clin. Invest. 78:650-657, 1986.
86. Banerji, J., J. Sands, J.L. Strominger and T. Spies. Gene pair from
the human major histocompatibility complex encodes large proline-rich
proteins with multiple repeated motifs and a single ubiquitin-like
domain. Proceeding of the National Academy of Science, USA 87:2374-2378,
1990.
87. Volanakis, J. E., Zhu, A-B., Schaffer, F.M., Macon, K.J., Palermos,
J., Barger, B.O., Go, R., Campbell, R.D., Schroeder, Jr. H.W. and
Cooper, M.D. Major histocompatibility complex class III genes and
susceptibility to Immunoglobulin A deficiency and common variable
immunodeficiency. J. Clinical Investigations 89(June):1914-1922, 1992.
88. Milner, Caroline M. and R. Duncan Campbell. Polymorphic analysis of
the three MHC-linked HSP70 genes. Immunogenetics 36:357-362, 1992.
89. Degli-Esposti, M.A., C. Leelayuwat and R.L. Dawkins. Ancestral
haplotypes carry haplotypic and haplospecific polymorphisms of BAT1:
possible relevance to autoimmune disease. European Journal of
Immunogenetics 19:12 1-127, 1992.
90. Nedospasov, Sergei A., Irina A. Udalova, Dmitry V. Kuprash and Regina
L. Turetskaya. DNA sequence polymorphism at the human tumor necrosis
factor (TNF) locus. The Journal of Immunology 147(3):1053-1059,
1991.
91. Davey, M.P., Meyer, M.M., Munkirs, D.D., Babcock D., Braun, M.P.,
Hayden, J.B. and Bakke, A.C. 1991. T-cell receptor variable =DF genes
show differential expression in CD4 and CD8 T cells. Human Immunology
32:194-198, 1991.
92. Spinella, D.G. and Robertson, A. Analysis of human T-cell repertoires
by PCR. In: The Polymerase Chain Reaction, K. Mullis, F. Ferre
and R. Gibbs, eds. Birkhauser, 1994.
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