>Magister Scientiae - MSc / Cervical cancer is the leading cause of cancer mortality among black women in South Africa. It is estimated that this disease kills approximately 8 women in South Africa every day. Cervical cancer is caused by the human papillomavirus (HPV) with the most common screening method for cervical cancer being Papanicolaou (Pap) smear, test amongst others. However, less than 20% of South African women go for these tests. There are several reasons why women do not go for these tests but the invasiveness of the test is one of the major causes for the low rate of screening. Lateral flow devices offer medical diagnosis at the point- of-care, allowing for the quick initiation of the appropriate therapeutic response. These tests are more cost-effective for the healthcare delivery industry, and can potentially be used by patients to self-test in the privacy of their homes and allow them to make informed decisions about their health. Therefore, the aim of this study was to use computational methods to identify serum biomarkers for cervical cancer that can be used to develop a point-of-care diagnostic device for cervical cancer. An in silico approach was used to identify genes implicated in the initiation and development of cervical cancer. Several bioinformatics tools were employed to extract a list of genes from publicly available cancer repositories. Multiple gene enrichment analysis tools were employed to analyze the selected candidate genes. Through this pipeline, ~28190 genes were identified from the various databases and were further refined to only 10 genes. The 10 genes were identified as potential cervical cancer biomarkers. A subcellular compartmentalization analysis clustered the proteins encoded by these genes as cell surface, secretory granules and extracellular space/matrix proteins. The selected candidate genes were predicted to be specific for cervical cancer tissue in a cancer tissue specificity meta-analysis study. The expression levels of the candidate genes were compared relative to each other and a graph constructed using gene expression data generated by GeneHub-GEPIS and TiGER databases. Further gene enrichment analysis was performed such as protein-protein interactions, transcription factor analysis, pathway analysis and co-expression analysis, with 9 out of the10 of the candidate genes showing co-expression. A gene expression analysis done on cervical cancer cell lines, other cancer cell lines and normal fibroblast cell line revealed differential expression of the candidate genes. Three candidate genes were significantly expressed in cervical cancer, while the seven remaining genes showed over expression in other cancer types. The study serves as basis for future investigations to diagnosis of cervical cancer, as well as for cancers. Thus, they could also serve as potential drug targets for cancer therapeutics and diagnostics.

>Magister Scientiae - MSc / Autism or autism spectrum disorder (ASD) is a very diverse neurological disorder that manifests specifically in children and infants between the ages of two to three years of age. An individual suffering is deemed as autistic and individuals suffering would be classed under the banner of ASD. It is observed that sufferers have impairment in their social and interactive skills. It has both genetic and environmental factors that contribute to its diversity and although the primary cause of autism is still unclear, scientist are investigating both factors. In this study we aimed to investigate the molecular genetics of autism in the South African (SA) population. This was done in two parts, a genetic association study and afunctional genomics (proteomic study). An association study of the 2 single nucleotide polymorphisms (SNPs) of the Cadherin 10 type II gene (CDH10) (rs4307059 and rs4327572) was investigated in the SA healthy and autistic population. The proteomic approach was used to determine the differential expression of genes of the healthy population and compared to the autistic population of African descent. In both parts of the project, objectives were achieved. The SNPs were successfully genotyped however no association was determined for autism in the SA population. The urine protein profiles with 1 dimensional (1D) and 2dimensional (2D) Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDSPAGE)generated in this study has revealed the following proteins, Uromodulin, Vitelline membrane outer layer protein homologue, kinninogen-1, Alpha-1-Antitrypsin, Ig Kappa chain region C, and CD59 glycoprotein that require further investigation. The results indicated that six of the identified proteins were expressed in both groups but were found to be either quantitatively or statistically significant. However, a statistically significant difference was observed in the expression of one protein (Uromodulin) which was observed to be expressed in the healthy group but absent in the experimental group. However further investigation is required validation of these findings.

>Magister Scientiae - MSc / Prostate cancer (PCa) is the second most common form of cancer in men around the world. In many parts of Africa, data on prostate cancer is sparse. This is attributed to poor access to testing and diagnostics. The International Agency for Research on Cancer (GLOBOCAN) estimated that 28,000 deaths occurred as a result of PCa in Africa in 2008, 4500 of which were in South Africa. This figure (28,000) is predicated a rise to 57,000 over the next two decades. Currently, the most commonly used diagnostic tests for PCa are the DRE and PSA tests. The former is highly invasive and both have low specificity and poor sensitivity. Therefore, the need for a less invasive early detection method with the ability to overcome the lack of specificity and sensitivity is required. Biomarkers have recently been identified as a viable option for early detection of disease. Examples of biological indicators for disease are miRNAs. miRNAs are small non-coding RNA molecules which play a key role in controlling gene expression and certain biological processes. Studies have shown that aberrantly expressed miRNAs are a hallmark of several diseases like cancer. miRNA expression has been shown to be associated with tumour development, progression and response to therapy, suggesting their possible use as diagnostic, prognostic and predictive biomarkers. The study aimed to investigate the potential of miRNAs implicated in prostate cancer as putative biomarkers for the disease and evaluating these miRNAs in a panel of prostate as well as several other cancer cell lines using qRT-PCR. An in silico approach was used to identify 13 putative miRNAs implicated in prostate cancer of which 8 were further analysed in a parallel study and 5 in this study. Two publicly available target prediction software were used for target gene prediction of the 5 identified miRNAs. The target genes were subjected to functional analysis using web-based software, DAVID. Functions which were clustered with an enrichment score of 1.3 and greater were considered significant. Targets with gene ontologies linked to “transcription regulation”, “regulation of “apopotosis”, “extracellular region” and “metal ion binding” were considered for further analyses. Protein gene interaction analysis was performed to determine the pathways the target genes are involved in using STRING. Expression profile analysis of the genes in various tissues was also carried out using in silico methods through the TiGER and GeneHub-GEPIS databases. Analysis using DAVID resulted in 9 gene targets for the 5 miRNAs. It was found that miR3 seemed the most promising miRNA for biomarker validation based on the in silico analyses. Its target gene MNT was found to be abundantly expressed in prostate tissue from the TiGER results. The GeneHub-GEPIS results also indicated that the gene’s expression is up-regulated during prostate cancer. The expression levels of the miRNAs analysed using qRT-PCR indicated that miR3 is significantly over-expressed in prostate cancer cells when compared to the other cancer cell lines used in this study, corroborating the results observed from the in silico analyses. Another miRNA with interesting results was miR5. It was predicted to target two genes, YWHAZ and TNFSF13B. In TiGER, both were found to be expressed in prostate tissue. The genes were also found to be up regulated during prostate cancer in GeneHub-GEPIS. The expression level of miR5 in LNCaP was 15.32; it was significantly up-regulated in the cell line using qRT-PCR. However, miR5 was also present in HEPG2-7.06, MCF7-0.79, HT29-1.61 and H157-3.59. Thus, it was concluded it can be used as a biomarker in combination with other miRNAs. The miRNA miR2 was found to target the actin filament protein encoding gene AFAP1. The gene was predicted to be upregulated with a DEU of 33.25 in GeneHuB-GEPIS. The qRT-PCR analysis showed that the expression ratio in LNcaP was 8.79. However, miR2 expression was up-regulated in MCF7-0.85 and HT29-1.09 as well. The expression level of miR1 in BHP1 was found to be 4.85. It can be considered as an indicator for benign prostate hyperplasia. Future work would include investigating the expression of miR3 in a larger panel of cancer cells as well as in patient samples. In addition, analysis of the UTR sequences of the miRNAs targets experimentally to prove that the target genes identified using in silico methods, are indeed regulated by these miRNAs. Furthermore, performing gene “knock-out” studies on the genes that code for the miRNAs to study their roles in prostate cancer development.

Philosophiae Doctor - PhD / The current diagnostic, prognostic, predictive and therapeutic monitoring methods used for breast cancer are limited. Thus, research into more specific, sensitive and effective strategies is required. Breast cancer is the most prevalent form of cancer in women worldwide and accounts for the most common cause of death in women every year. Cancer development is characterized by a wide spread of genetic abnormalities of gene sequences that can be used in detecting and monitoring treatment of the disease as a result of altered gene expression patterns which leave a trail of biomarkers. Seven candidate genes (Gene 1-7) were identified from a previous in silico study and their gene products (BRG 1-7) were annotated to be good candidate breast cancer biomarkers. Differential gene expression analysis using quantitative real-time PCR (qRT-PCR) validated the over-expression of Gene 3, Gene 4 and Gene 7 in a breast cancer cell line (MCF7), of which Gene 7, annotated as a Natural killer group 2, member D (NKG2D) ligand, was observed to be the most over-expressed gene. The innate immune system is the first line of the body's physiological defense against diseases and the natural killer (NK) cells, are central to mediating this type of immunity. NK cells are activated when a specific surface receptor such as the NKG2D receptor binds its ligands expressed by tumor cells. To evade being detected by the immune system, cancer cells are reported to shed off the NKG2D ligands and are expected to be present in the bodily fluids of cancer patients. Also, chemotherapeutics have been reported to suppress the natural anti-tumour immune response, thus should be taken into account when designing optimal therapy for cancer patients. The aim of this research was to validate these candidate genes as effective breast cancer biomarkers using several in silico methods as well as molecular techniques and study the effect of Gene 7 on modulating the effect of several pro-apoptotic compounds. The in silico part of the study investigated the functional, protein interaction, pathways, and tissue expression specificity of the candidate biomarkers using computational software such as DAVID, STRING, KEGG, Genecards and GEA. Also an in silico validation of the prognostic/predictive values of the genes was analysed using SurvExpress, KMplot, and GOBO. Protein expression of selected genes was analysed by Western blot, and immunofluorescence analysis. BRG 7 gene was cloned into pcDNA3.1 vector using recombinant DNA technology while commercial shRNA construct was used to 'knock-down' Gene 7 expression. The two constructs were used to transfect MCF-7 and MCF-12A cells. Over-expression and 'knock down' Gene 7 in transfected cells was confirmed using western blot analysis. Stably transfected cells were then treated with three pro-apoptotic compounds (Camptothecin, Doxorubicin and DMSO) for 24 hours. The apoptotic cells were stained with 3, 4, 5, 6-tetrachloro-2', 4', 5', 7' tetraiodofluorescein (TCTF) and then analysed using flow cytometry. Functional analysis linked Gene 1, Gene 2, Gene 4, Gene 6 and Gene 7 to different cancer related processes. The pathway analysis showed Gene 1, Gene 2, Gene 4 and Gene 7 were involved in pathways that can be linked to cancer modulation. The protein-protein interaction analysis showed only BRG 2 was directly linked to two major hallmarks of cancer (Apoptosis and Autophagy). Breast cancer associated Transcription factors were shown to regulate these genes. Gene 1 and Gene 5 as well as the three genes observed to be highly expressed in the qRT-PCR study were validated to differentially express in breast cancer. An additional protein (BRG 8) was identified and postulated to be a good biomarker candidate for breast cancer based on its direct interaction with BRG 7 and estrogen receptor protein (ESR). The prognostic value of the candidate genes were monitored in two datasets (DATA1 and DATA2) in SurvExpress. DATA1 showed that Gene 6 and Gene 8 while DATA2 showed that Gene 3, Gene 6 and Gene 7 were valuable candidate genes in breast cancer prognosis. The survival curves from the two datasets showed the combined genes could predict the outcome of breast cancer patients undergoing treatments. A plot box output from SurvExpress showed most of the genes were differentially expressed comparing two risk groups. The Kaplan Meier plotter confirmed, Gene 1, Gene 3, Gene 4 and Gene 7 have a significant P-value in predicting the survival outcome based on gene differential expression value. GOBO analysis showed the genes may accurately predict the survival outcome of estrogen positive subtype, ERBB2 subtype of estrogen receptor negative and lymph node negative subtype of ER- tumours, but not all subtype of ER- tumours. Western blot analysis showed BRG 7 may be highly expressed in MCF-7 as compared to MCF-12A, BRG 8 was found to be expressed in all cancer cell types analyzed except for MCF-7 and HT29. BRG 2 was found to be expressed in all cancer types analyzed. immunofluorescence analysis showed BRG 3, BRG 4 and BRG 7 are differentially expressed in breast cancer cell line and are more localized on the cell membrane when compared to the breast non-cancer cell line. Over-expression and gene knock down in cells were successfully confirmed with Western blot analysis. Stably transfected MCF-12A cell for over-expression of BRG7 protein, resulted in cell senescent and the cell stopped growing while stably transfected MCF-7 over-expressing BRG7 did not show any morphological changes. Apoptosis was enhanced in cells treated with camptothecin, doxorubicin and DMSO overexpressing BRG7. Apoptosis was reduced in camptothecin and DMSO treated gene 'knock-down' cells but not doxorucin treated. BRG7 gene 'knock down' in transfected cells showed varying response to all three pro-apoptotic compounds. From this study Gene 3, 5, 7 and 8 and their protein levels were confirmed to be differentially expressed in breast cancer cells and could serve as putative biomarkers for breast cancer. However the variance in the effectiveness of individual genes suggests that the set of genes would perform better than individual gene. The modulating role of BRG7 in drug induced apoptosis, suggest it could probably play an important role in personalised medicine and could serve as a biomarker to monitor the prognosis and/or therapeutic outcome of pro-apoptotic drugs in breast cancer patients. These findings will be further investigated in human breast tissues to validate these data.

Objectives The aims of this thesis were to (1) investigate the association of gestational exposure to environmental phthalates with maternal and perinatal outcomes, and (2) explore phthalate-induced changes to maternal inflammatory responses as potential mediators of possible health effects. Methods A systematic review was performed to summarize existing evidence on the association of gestational exposure to phthalates with obstetrical outcomes, including pre-eclampsia (PE), pregnancy-induced hypertension (PIH), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR), birth weight (BW), head circumference (HC), gestational age (GA), preterm birth (PB), and Apgar scores (AS). Additionally, a secondary analysis of data from the MIREC Study was conducted to evaluate the association of phthalate metabolites with clinical outcomes in the mother and infant using multiple linear and logistic regression, and with inflammatory biomarkers using multinomial logistic regression. Results The systematic review identified a total of 24 articles, and observed inconsistent evidence on BW, HC, GA, and PB, a paucity of research on IUGR, PE, GDM, and AS, and a lack of studies on PIH. However, among studies with statistically significant (p<0.05) results, most suggest an association of phthalates with decreased BW and GA, and increased HC and PB. Findings from the MIREC Study indicate a significant (p<0.01) positive association between MBP and HC among female infants; however, null results were identified for BW, GA, PB, AS, and PIH. In relation to the exposure to phthalates, general trends among suggestive associations (p<0.05) for head circumference showed consistent increases in females and decreases in males, and for gestational age displayed decreases in both stratums. Additionally, a significant positive association of MBzP and ∑DEHP was observed with high MMP-2 and low VCAM levels, respectively. Results approaching statistical significance demonstrated a positive association of ∑DEHP with low MCP1 and ICAM levels, MCPP with low GMCSF levels, MBzP with low CRP and high ICAM levels, and MEP with high MMP-7 and IL-2 levels. Conclusion From the systematic review, the effects of phthalates on maternal and perinatal health remain unclear, possibly due to sources of heterogeneity and challenges in exposure assessment. In the MIREC Study cohort, phthalate levels were associated with GA and HC in infants in a sex-specific manner. Phthalates also appear to influence the circulating inflammatory marker levels, possibly explaining the observed adverse effects. Future research is needed to validate these findings.

A report from the National Institutes of Health defines a disease biomarker as a "characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention." Early diagnosis is a crucial factor for incurable disease such as cancer and Alzheimer’s disease (AD). During the last decade researchers have discovered that biochemical changes caused by a disease can be detected considerably earlier as compared to physical manifestations/symptoms. In this dissertation electrochemical detection was utilized as the detection strategy as it offers high sensitivity/specificity, ease of operation, and capability of miniaturization and multiplexed detection. Electrochemical detection of biological analytes is an established field, and has matured at a rapid pace during the last 50 years and adapted itself to advances in micro/nanofabrication procedures. Carbon fiber microelectrodes were utilized as the platform sensor due to their high signal to noise ratio, ease and low-cost of fabrication, biocompatibility, and active carbon surface which allows conjugation with bio-recognition moieties. This dissertation specifically focuses on the detection of 3 extensively validated biomarkers for cancer and AD. Firstly, vascular endothelial growth factor (VEGF) a cancer biomarker was detected using a one-step, reagentless immunosensing strategy. The immunosensing strategy allowed a rapid and sensitive means of VEGF detection with a detection limit of about 38 pg/mL with a linear dynamic range of 0–100 pg/mL. Direct detection of AD-related biomarker amyloid beta (Aβ) was achieved by exploiting its inherent electroactivity. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Triple barrel carbon fiber microelectrodes were used to simultaneously detect Aβ1-40 and Aβ1-42 in cerebrospinal fluid from rats within a detection range of 100nM to 1.2μM and 400nM to 1μM respectively. In addition, the release of DNA damage/repair biomarker 8-hydroxydeoxyguanine (8-OHdG) under the influence of reactive oxidative stress from single lung endothelial cell was monitored using an activated carbon fiber microelectrode. The sensor was used to test the influence of nicotine, which is one of the most biologically active chemicals present in cigarette smoke and smokeless tobacco.

Amyotrophic lateral sclerosis is a relentlessly progressive and fatal neuromuscular disease with no effective biomarkers, treatments or cure. In the early stages of ALS, it can be difficult to provide a diagnosis as patients do not meet diagnostic criteria until they become symptomatic, a sign of neuron degeneration. Early detection is therefore crucial to provide access to therapeutics prior to significant neuron loss. Extracellular vesicles are an ideal source of biomarkers as they contain a mix of proteins and nucleic acids reflective of the physiological state and are released from all cell types. We identified valosin-containing protein, integrin-beta 1 and gelsolin as potential biomarkers for ALS14 through proteomic analysis of EVs isolated from cell lines carrying the ALS-associated VCP-R155H mutation. My results indicate that EVs may serve as a valuable source of protein biomarkers in diagnostic, prognostic and predictive applications.

Introduction: Biomarkers of tuberculosis (TB) diagnosis and treatment response in patients coinfected with human immunodeficiency virus (HIV) are a necessity to ensure early diagnosis and adequate monitoring of TB treatment response. We conducted 3 sub-studies: study 1 was a bioavailability study; study 2 was a PK study in HIV-TB co-infected persons, and study 3 evaluated a WHO-recommended treatment algorithm in TB-HIV co-infected persons. Study 1 and 2 contributed to the study of 2 (NAT2) polymorphisms. Study 1 was leveraged to evaluate Quantiferon Gold in tube (QFT-GIT) and a quality of life instrument as a longitudinal biomarker in smear and culture positive TB-HIV co-infected patients. Study 3 was leveraged to study urine lipoarabinomannan (LAM) as a diagnostic adjunct in smear-negative HIV-infected patients treated for TB. Methods: Blood was collected from participants with HIV-infection only and TB-HIV coinfection for NAT2 polymorphisms at baseline, and for QFT-GIT at baseline, month 3, 6 and 12; a health-related quality-of-life (HRQOL) instrument was applied at the same timepoints to monitor treatment response in Study 1. An additional 40 TB-HIV co-infected participants (Study 2) were included in the analysis for the assessment of NAT2 polymorphisms and its effect on isoniazid plasma levels and hepatotoxicity. Urine was collected from seriously ill HIV-infected patients with confirmed smear-negative presumptive-TB (Study 3) prior to anti-TB treatment and tested using a commercially available LAM-ELISA. Blood and sputum were collected and processed for TB culture. Results: One hundred and twenty participants (100 TB-HIV co-infected and 20 non-TB but HIVinfected) from Study 1 and Study 2 with genotype results and were evaluated. Percentage of metabolisers in each category were: slow 52.5% (63/120), (NAT2*5/*5); intermediate 35.8% (43/120), (NAT2*4/*5 and NAT2*5/12); and rapid 11.7% (14/120), (NAT2*4/*11, NAT2*11/12 and NAT2*12/12). In general, isoniazid area under the concentration curve (AUC)0-∞ and maximum concentration (Cmax) were lower amongst the study 1 compared to study 2 participants. INH and AcINH PK parameters across genotypes were not statistically significantly different within each study. The log AcINH: log INH ratio, calculated as a measure of acetylation at two and four hours post-dose, showed no statistically significant difference between genotypes.

Inflammatory Bowel Disease (IBD), divided into Crohn’s disease (CD), ulcerative colitis (UC), and indeterminate colitis (IC), is a chronic, crippling autoimmune condition characterized by gastrointestinal (GI) inflammation. The methods used to diagnose IBD and assess its activity can be invasive and costly and typically include a combination of histologic, endoscopic, radiologic, clinical, and biochemical measures. Currently, there is an increasing need for the development of noninvasive assessment measures to detect an interval response to prescribed therapy. Previous studies have found serial and fecal biomarkers to be reliable, but non-specific indicators of GI tract inflammation. At present, they cannot be used to distinguish between inflammation resulting from infection and that caused by chronic inflammation in patients with IBD. The aim of this study is to measure changes in serum and fecal biomarkers over time in individual children and adolescents with CD, UC, and IC initiating infliximab therapy while investigating any parallels between fluctuations in biomarker levels and endoscopic, clinical, and biochemical outcomes. The inflammatory biomarkers evaluated in this study include fecal and serum anti-Saccharomyces-Cerevisiae Antibody (ASCA), fecal and serum lactoferrin, fecal hemoglobin, fecal calprotectin, fecal IL1-α, fecal IL1-β, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). The data for this study was collected from a multicenter longitudinal prospective cohort study following pediatric patients over the course of six consecutive infliximab infusion appointments. Study sites include Boston Children’s Hospital and Riley Children’s Hospital in Indianapolis. Participants were recruited from a pool of CD, UC, and IC patients who were either new to infliximab, had been receiving infliximab for less than six months, or had been receiving infliximab for more than one year. Patients brought in stool samples at each of their scheduled infliximab infusions, biochemical labs (ESR and CRP) were obtained, and patients completed a health-related quality of life survey (IMPACT-III Questionnaire). Forty-three patients (26 with CD, 16 with UC, and one with IC) completed this study. There was no significant difference in mean serum or fecal ASCA levels between participants with CD and those with UC. However, average serum and fecal ASCA were higher in patients with CD than those with UC at almost every infusion. The baseline mean CRP level in patients with CD was significantly higher than that observed in patients with UC (p<0.05). In patients with CD, the mean IMPACT-III score was significantly higher (improved quality of life) at Infusion 5 than at baseline. The data collected in this study suggest serial biomarker measurements may be useful in monitoring a patient’s response to infliximab therapy. This study is not yet complete and requires further data analysis to more definitively conclude if a single or a composite metric including several fecal and/or serum inflammatory biomarkers would provide a more robust assessment of disease activity in children and young adults with IBD.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s Disease (AD) is one of the most common forms of dementia and is known to have a strong genetic component, but known genetic loci do not fully account for the observed genetic heritability of late onset AD. This genetic complexity is further complicated by disease heterogeneity, with non-uniform presentation and progression of AD neuropathology. Endophenotypes lie upstream of observed AD clinical outcomes and downstream of genetic contributors, allowing for a biological understanding of genetic effects. Understanding the genetic architecture of AD endophenotypes can aid in breaking down AD genetic complexity and heterogeneity. In this study we utilized a variety of models to evaluate the genetic contributors to pathological change and heterogeneity in the top markers of AD pathology: amyloid, tau, neurodegeneration, and cerebrovascular (A/T/N/V framework). Additional composite quantitative measures of cognitive performance were used to relate to downstream AD presentation. These biomarkers allow the investigation of genetic effects contributing to the disease over the stages of disease progression from amyloid deposition to neurofibrillary tangle formation, disruption of metabolism, brain atrophy, and finally to clinical outcomes. First, we performed genome-wide association studies (GWAS) for AD endophenotypes at baseline using a cross-sectional regression model. This method identified sixteen novel or replicated loci, with six (SRSF10, MAPT, XKR3, KIAA1671, ZNF826P, and LOC100507506) associated across multiple A/T/N biomarkers. Cross-sectional data was further utilized to identify three genetic loci (BACH2, EP300, PACRG-AS1) that showed disease stage specific interaction effects. We built upon those results by performing a longitudinal association analysis with linear-mixed effects modeling. Gene enrichment analysis of these results identified 19 significant genetic regions associated with linear longitudinal change in AD endophenotypes. To further break down longitudinal heterogeneity, a latent class mixed model approach was utilized to identify subgroups of longitudinal progression within cognitive and MRI measures, with 16 genetic loci associated with membership in different classes. The genetic patterns of these subgroups show biological relevance in AD. The methods and results from this study provide insight into the complex genetic architecture of AD endophenotypes and a foundation to build upon for future studies into AD genetic architecture. / 2022-11-26