Date of Award

8-2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Committee Member

Charles Rice, Committee Chair

Committee Member

Lisa Bain

Committee Member

Yanzhang Wei

Abstract

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition affecting approximately 1% of the population. It occurs roughly four times more in males than females, and includes individuals with classic autism, as well as those with "incomplete" forms. Behavioral characteristics include, but are not limited to: restricted, stereotyped, and repetitive actions, as well as impairments in communication and social interaction. At the Greenwood Genetics Center, Greenwood, SC, ASD patients and control donors provided peripheral blood as a source of lymphocytes, and a skin-scrape sample as a source of fibroblasts for genetic analysis. A severely autistic patient, coded as B26, his two parents (mother Y17, father VT8), and two donor controls (JOR, RT9) were compared. Patient and donor lymphocytes were transformed into immortal lymphoblast lines using EBV-transformation procedures. In addition to routine genetic analysis, both fibroblasts and lymphoblasts were evaluated by GGC researchers as a possible platform to generate neuronal cells through iPS steps. Attempts were successful with fibroblasts, but not lymphoblasts. In this thesis, these five lymphoblast lines were evaluated for possible association with ASD and both parents, along with two donor controls. These cells were also examined for their potential in other areas of research, including the autocrine production of inflammatory and anti-inflammatory cytokines, reactivation of RAG-1 as a consequence of EBV-transformation, pharmacological and toxicological applications mediated through the AHR, and their metabolic responses to tryptophan exposure. All five cell lines produce significant quantities of the autocrine B-cell growth factor IL-6, and, to a lesser degree, IL-10, before and after further activation with phorbol ester in combination with calcium ionophore. Patient B26 and his two parents produced the most cytokine in response to activation, while the control donors had a higher baseline production. All five cells lines expressed RAG-1 protein, indicating reactivation of B-cell receptor diversity process that may lead to a different BCR specificity. In pharmacology and toxicology, many xenobiotics and endogenous metabolites of tryptophan act through the AHR leading to induction of phase I, II, and III enzymes. The classical marker of AHR activity is the induction of CYP1B1. In response to FICZ, a potent tryptophan intermediate, patient B26 had the highest level of CYP1B1 induction, followed by Y17, the mother. Because tryptophan is metabolized by IDO2 in activated normal lymphocytes, the induction of IDO2 was quantified after activation with phorbol ester and calcium ionophore. Expression of IDO2 was highest in patent B26, followed by Y17, the mother. Recent research at GGC suggests that tryptophan is metabolized differently in ASD patients when compared to donor controls. A commercially available PCR array of genes associated with tryptophan metabolism was then employed and revealed that key genes involved in tryptophan metabolism are not altered in the severely autistic patient B26, either of B26's two parents (mother Y17, father VT8), nor the two donor controls (JOR, RT9). The array also showed that several genes involved in mitochondrial function were highly responsive to tryptophan treatment in B26, and similarly in the mother, Y17. Genes not only involved in energy metabolism, but also inflammation and different pathways stemming from tryptophan metabolism (serotonin and melatonin pathways) were increased. This study shows that lymphoblasts generated from ASD patients and donor controls may have use outside of the field of autism research, and may lead to the development of novel biomarkers for screening individuals for ASD.

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