It is becoming increasingly clear that although the neurological symptoms of ASD are the most striking among the behavioral and functional manifestations of affected individuals, there are many associated peripheral physiological symptoms that have often gone unnoticed/ignored and clinically unaddressed. These include gastrointestinal disorders experienced by many on the spectrum (estimated at 50%) as well as immune disorders which have long been described in the literature on ASD 
. The large-scale global gene expression profiling that we have undertaken on LCL derived from peripheral blood lymphocytes of ASD probands and their respective siblings may therefore serve as a window to the underlying biochemical and signaling deficits that may be relevant to understanding the broader symptomatology of autism.
Overall, our study of autistic-nonautistic sib pairs in which the autistic sibling has been subtyped according to severity of language impairment on the basis of cluster analysis of scores from the ADIR diagnostic interview (Hu and Steinberg, Autism Research, 2009, in press), reveals altered expression of genes that participate in cholesterol metabolism and androgen biosynthesis. It is noteworthy that deficiency of 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme in cholesterol biosynthesis, is the genetic cause of Smith-Lemli-Opitz syndrome (SLOS) which is an autosomal recessive disorder characterized by pre- and post-natal growth retardation, distinct facial anomalies, microcephaly, and mental retardation 
. Significantly, over 50% of individuals with SLOS also meet the diagnostic criteria for autism 
. Although the mechanism through which DHCR7 deficiency causes any of these characteristics/phenotypes is unknown, a gene expression study of a DHCR7 knockout mouse model reveals altered expression of numerous genes affecting not only cholesterol biosynthesis, but also neurodevelopment and functions such as Wnt signaling, axon guidance, neuronal cytoskeletal assembly, and neurodegeneration 
. Thus, it can be postulated that disturbance of cholesterol mechanism in either direction (decreased synthesis in the case of SLOS, or increased uptake by SCARB1 and conversion to androgen by SRD5A1 in our study) can have wide-ranging effects on neural development and function. The predicted increase in androgen levels due to increased expression of SRD5A1, on the other hand, is supported by our pilot study on the metabolites within the steroid hormone biosynthetic pathway which shows elevated testosterone in all 3 of the randomly selected autistic siblings relative to his respective nearly age-matched normal sibling as well as by other studies in the literature which show elevated androgen levels in the serum of autistic individuals, including females 
. Our observation that at least 2 of the genes (SCARB1 and SRD5A1) that are involved in cholesterol import into the cell and testosterone metabolism exhibit increased expression in the autistic siblings offers a plausible explanation for elevated androgen levels in ASD.
The biological consequences of elevated testosterone on neurodevelopment and function are just beginning to be understood. While it has been known for more than 10 years that estrogens modulate synaptic plasticity in the hippocampus of female rats 
, it has only recently been shown that androgens likewise play a role in hippocampal synaptic plasticity, but in both
males and females 
. Furthermore, there is increasing evidence for the role of “neurosteroids” (which include DHEA and progesterone) in neurological functions, including rapid modulation of neurotransmitter receptors 
. In contrast to testosterone, DHEA which has been shown to be lowered in ASD 
, plays a neuroprotective role countering the effect of stress-inducing steroids 
. Interestingly, we have observed that the plasma levels of DHEA were lower in several of the autistic siblings relative to their respective nonautistic siblings (unpublished data). Clearly, it will be important to further evaluate the levels of steroid hormones and related molecules in a broader sampling of individuals with ASD as well as to establish a correlation between these metabolite levels and aberrant expression of genes in this metabolic pathway.
Pathway analyses using Pathway Studio 5 also implicated involvement of female hormones in that the estrogens were among the small molecule regulators of the differentially expressed genes (). It is further noted that SRD5A1 is involved in sex determination 
. Thus, the altered expression of genes involved in steroid hormone production and sexual dimorphism, coupled with the differential impact of male and female steroid hormones on brain development in male vs. female animals 
may, in part, underlie the approximately 4
1 male to female ratio in ASD.
The schematic in suggests that bile acid synthesis might also be affected by some of the differentially expressed genes in ASD, particularly SCARB1 and SRD5A1, which respectively internalize cholesterol and participate enzymatically in bile acid synthesis. This suggests that altered expression of genes in this pathway may also be responsible for the digestive and hepatic disorders associated with ASD. Indeed, in a separate case-control study of a large number of unrelated individuals (total of 116), hepatic cholestasis and fibrosis are strongly indicated on the basis of the gene expression profiles of the autistic probands vs. unrelated controls (Hu et al., Autism Research, 2009, in press). Changes in metabolite profiles thus may be predicted and tested on the basis of a functional analysis of altered gene interactions that arise from increases or decreases in gene expression within a specific metabolic pathway. Indeed, this would be a complementary approach to that used by James et al. who used targeted metabolite profiling of the methionine transmethylation and transsulfuration pathways to identify potential gene defects in ASD 
. Such metabolomic analyses, guided by gene expression studies, may in turn lead to a diagnostic screen for ASD based on metabolite profiling of serum or other easily accessible tissues (e.g., steroid hormone, bile acid, or redox molecule assays).
Aside from genes involved in cholesterol metabolism and steroid hormone biosynthesis, we also confirmed the altered expression of several other novel genes that may play a role in the pathophysiology of autistic disorder (). The use of LCL cells as a surrogate tissue to study potential changes in brain gene expression that may mechanistically underlie autism or other neurological disorders is not unprecedented. Gene expression profiles of different brain regions have been shown to exhibit the highest similarity to whole blood 
. Moreover, a meta-analysis of studies performed in blood and post-mortem brain demonstrated convergent gene expression changes 
, although further studies are warranted 
. Because of their role in neuronal development, migration, and morphology (), we were particularly interested in confirming the differential expression of CXCR4, CD9, and GATA3. Although the chemokine receptor CXCR4 is most frequently associated with inflammatory processes and leukocyte trafficking in the immune system, recent studies show that it, along with the chemokine stromal cell-derived factor 1 (SDF-1), are important regulators of neuronal migration and axonal pathfinding, particularly in the cortex and cerebellum 
where it is involved in the development and organization of Purkinje cells, which are notably deficient in ASD 
. CD9 is yet another molecule involved in cell migration, both in the immune system as well as nervous system 
, with its expression in Schwann cells regulated by axonal contact 
. Interestingly, androgens have been shown to induce CD9 in human prostate 
, suggesting yet another mechanism for increased expression in autism. GATA3 is a transcription factor that is involved in both allergic inflammation (like CXCR4) and cytokine production 
. In addition, GATA3 has also been shown to be involved in the development of the central nervous system in mice 
and the induction of dopamine beta-hydroxylase (DBH) in primary neural crest stem cells 
. With respect to the latter activity, it is of interest to note that DBH genotype has been associated with autism in some families 
and that DBH activity has been noted to be elevated in a subgroup of autistic patients 
. Like the 3 genes discussed above, NFKBIZ, a nuclear regulator of NFKB activity, is also involved in inflammation and immunity. In particular, it is induced upon stimulation of the innate immune system 
and, in turn, stimulates IL-6 production 
. These characteristics of NFKBIZ are noteworthy in light of studies by Pardo and colleagues demonstrating activation of the innate immune system in brain tissues from autistic patients, with a notable increase in IL-6 
. Thus, the elevation of NFKBIZ in peripheral cells derived from autistic probands may be a reflection of a systems-wide activation of the innate immune system in autism, providing strong support for the use of LCL as a surrogate model to examine gene dysregulation in ASD.
In summary, gene expression profiling of LCL from autistic and nonautistic siblings reveals alteration of genes involved in both metabolic and signaling pathways in ASD that is consistent with the known pathophysiology of autism which includes inflammation as well as disturbances in axon guidance, neuronal survival, and differentiation, biological themes also implicated in our earlier study on monozygotic twins discordant in diagnosis and severity of autism 
. The involvement of genes affecting both the immune and nervous systems, coupled with the pleiotropic effects of dysregulated steroid hormone biosynthesis, may further explain some of the systemic disorders associated with autism. The genes, metabolites, and pathways identified in this study moreover suggest novel targets for therapeutics. Thus, gene expression profiling, which provides a global view of functional gene networks in the context of living cells from individuals with ASD, not only allows for the elucidation of compromised pathways but also provides a meaningful and complementary (with respect to genetics) approach towards understanding the complex biology of ASD.