Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

Racial disparities in CD4 counts at initial HIV-1 diagnosis: Analysis of the Adult Spectrum of HIV disease dataset and public health implications

Introduction. The HIV/AIDS disease burden disproportionately affects minority populations, specifically African Americans. While sexual risk behaviors play a role in the observed HIV burden, other factors including gender, age, socioeconomics, and barriers to healthcare access may also be contributory. The goal of this study was to determine how far down the HIV/AIDS disease process people of different ethnicities first present for healthcare. The study specifically analyzed the differences in CD4 cell counts at the initial HIV-1 diagnosis with respect to ethnicity. The study also analyzed racial differences in HIV/AIDS risk factors. ^ Methods. This is a retrospective study using data from the Adult Spectrum of HIV Disease (ASD), collected by the City of Houston Department of Health. The ASD database contains information on newly reported HIV cases in the Harris County District Hospitals between 1989 and 2000. Each patient had an initial and a follow-up report. The extracted variables of interest from the ASD data set were CD4 counts at the initial HIV diagnosis, race, gender, age at HIV diagnosis and behavioral risk factors. One-way ANOVA was used to examine differences in baseline CD4 counts at HIV diagnosis between racial/ethnic groups. Chi square was used to analyze racial differences in risk factors. ^ Results. The analyzed study sample was 4767. The study population was 47% Black, 37% White and 16% Hispanic [p<0.05]. The mean and median CD4 counts at diagnosis were 254 and 193 cells per ml, respectively. At the initial HIV diagnosis Blacks had the highest average CD4 counts (285), followed by Whites (233) and Hispanics (212) [p<0.001 ]. These statistical differences, however, were only observed with CD4 counts above 350 [p<0.001], even when adjusted for age at diagnosis and gender [p<0.05]. Looking at risk factors, Blacks were mostly affected by intravenous drug use (IVDU) and heterosexuality, whereas Whites and Hispanics were more affected by male homosexuality [ p<0.05]. ^ Conclusion. (1) There were statistical differences in CD4 counts with respect to ethnicity, but these differences only existed for CD4 counts above 350. These differences however do not appear to have clinical significance. Antithetically, Blacks had the highest CD4 counts followed by Whites and Hispanics. (2) 50% of this study group clinically had AIDS at their initial HIV diagnosis (median=193), irrespective of ethnicity. It was not clear from data analysis if these observations were due to failure of early HIV surveillance, HIV testing policies or healthcare access. More studies need to be done to address this question. (3) Homosexuality and bisexuality were the biggest risk factors for Whites and Hispanics, whereas for Blacks were mostly affected by heterosexuality and IVDU, implying a need for different public health intervention strategies for these racial groups. ^

Role of {\it Mycobacterium avium-intracellulare\/} complex infection in AIDS mortality

Disseminated MAC (dMAC) is the third most prevalent opportunistic infection in AIDS patients. In order to understand the role MAC infection plays in affecting survival of AIDS patients, a cohort of 203 suspected dMAC veterans seen at the Houston Veterans Affairs Medical Center between August 14, 1987 and December 31, 1991 were analyzed. The criteria for suspected dMAC infection was HIV+ men having a CD4+ level $\le$200 cells/mm$\sp3,$ on zidovudine treatment $\ge$1 month and who had any of the following: (a) a confirmed respiratory MAC infection, (b) fever $\ge$101$\sp\circ\rm F$ for $\ge$48 hours, (c) unexplained weight loss of 10 lbs or $\ge$10% BW over 3 months or (d) Hgb $\le$7.5 g/dl or decrease in Hgb $\ge$3.0 g/dl, while on 500-600 mg/day AZT. The study was conducted before the commencement of an effective MAC anti-mycobacterial therapy, so the true course of MAC infection was seen without the confounder of a therapeutic regimen. Kaplan-Meier and Cox regression survival analysis was used to compare 45 MAC culture positive and 118 MAC culture negative veterans. The 1 year survival rate of veterans with documented dMAC infection was 0.37 compared to 0.50 for veterans not acquiring dMAC infection. Significant differences between subgroups were also seen with the variables: PCP prophylaxis, the AIDS indicator disease Candida esophagitis, CD4+ lymphocyte level, CD4 percent lymphocyte level, WBC level, Hgb and Hct levels. Using multivariate modeling, it was determined that PCP prophylaxis (RR = 6.12, CI 2.24-16.68) was a predictor of survival and both CD4% lymphocytes $\le$6.0% (RR = 0.33, CI 0.17-0.68) and WBC level $\le$3000 cells/mm$\sp3$ (RR = 0.60, CI 0.39-0.93) were predictors of mortality. CD4+ level $\le$50 cells/mm$\sp3$ was not a significant predictor of mortality. Although MAC culture status was a significant predictor of mortality in the univariate model, a positive dMAC culture was not a significant predictor of AIDS mortality in the multivariate model. A positive dMAC culture, however, did affect mortality in a stratified analysis when baseline laboratory values were: CD8+ lymphocytes $>$600 cells/mm$\sp3,$ Hgb $>$11.0 g/dl, Hct $>$31.0% and WBC level $>$3000 cells/mm$\sp3.$ ^