Genetic Interactions and Maternal Genes Modulate Congenital Heart Disease Risk

Congenital heart disease (CHD) is the most common congenital anomaly, which makes it a leading cause of infant mortality. Congenital heart defects are a cluster of distinct developmental malformations that affect the vasculature, musculature and organization of the heart, each with varying clinical severity. Although medical and surgical advances have reduced CHD mortality in newborns and children, these patients grow up and many experience serious morbidity and early mortality. The first step toward reducing this burden is to understand the causes of CHD. Surprisingly, environmental insults and de novo mutations are estimated to explain less than one-third of CHD cases. In many cases, even when a vital cardiac gene is mutated, a heart defect does not occur. This highlights the critical role of genetic and environmental modifiers in CHD pathogenesis. Attempts to identify these modifiers have had marginal success in humans. This motivated us to model CHD in mice, in which we can control the effects of environment and genetics. Using nearly 20,000 Nkx2-5 heterozygous mutant mice from multiple inbred strain crosses, my work provides three key findings that describe how genetic and environmental risk factors modulate CHD risk. First, severe heart defects are rare because they require interactions between multiple risk alleles to manifest disease. Contrarily, mild heart defects can be caused by the Nkx2-5 mutation alone, which allows these defects to be common. Second, genetic robustness to deleterious mutations can result from well-integrated or coadapted genetic networks. In our mouse model, we found that epistatic interaction effects tend to suppress heart defect risk when the interacting alleles originate from the ancestral mouse strain. This suggests that the incomplete penetrance of human CHD-associated mutations is a result of varying levels of robustness to disease across individuals. Third, there is significant genetic variation in the maternal age associated risk of CHD, suggesting that the underlying genes can be identified. We recapitulated the human maternal age risk using a 56th generation advanced intercross mouse mother population and identified one genome-wide significant locus that modulates the age effect across different heart defect types. Modulating the associated molecular pathway may become a fruitful therapeutic target to suppress CHD risk. In conclusion, my work has uncovered multiple factors that contribute to congenital heart disease risk in humans. The importance of epistasis in CHD risk emphasizes the need to consider oligogenic disease models in whole-exome/genome and clinical genetics studies of CHD. Furthermore, maternal effects such as the maternal age effect may help identify modifiable molecular pathways that can suppress CHD risk in human populations. Future studies on the maternal age effect will focus on finalizing our statistical models and validating candidate genes in animal models

The genetic architecture of a congenital heart defect Is related to Its fitness cost

In newborns, severe congenital heart defects are rarer than mild ones. This epidemiological relationship between heart defect severity and incidence lacks explanation. Here, an analysis of ~10,00

Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14. © 2013 Qi et al.Link_to_subscribed_fulltex

Model for outward sorting of HIV Env by the FIP1C-Rab14 complex.

<p>ERC = endosomal recycling compartment.</p

Rab14 interaction with FIP1C is required for HIV-1 Env incorporation.

<p>(A) HeLa cells were transfected with proviral plasmid pNL4-3 or NL4-3 CT144 and either a dominant negative form of Rab14 (Rab14S25N) or a constitutively active form of Rab14 (Rab14Q70L). 48 hours after transfection, cellular and particle content of HIV-1 Env was examined by Western blot. Numbers above Env blots represent densitometry values with leftmost lanes set to 100. (B) shRNA-mediated depletion of Rab11 or Rab14 was performed in HeLa cells. Following selection in puromycin, cells were transfected with either pNL4-3 or pNL4-3 CT144 plasmid. Cellular and particle-associated Env was detected by immunoblotting 48 hours following transfection. (C) shRNA-resistant GFP-FIP1C* WT and GFP-FIP1C* (S580N/S582L) were transfected together with pNL4-3 in control HeLa cells or FIP1C-depleted HeLa cells as in the repletion experiment described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003278#ppat-1003278-g002" target="_blank">Figure 2</a>. Input FIP1C* plasmid levels were 2 µg in lanes 3, 5, 7, and 8 and increased to 4 µg in lanes 4 and 6. In a separate experiment the ability of Rab11 binding mutant GFP-FIP1C* I62E and GFP-FIP1C* D622N to rescue Env particle incorporation in FIP1C-depleted HeLa cells (lanes 7 and 8). Note that gp160 and gp120 were detected with polyclonal antiserum; gp41 particle blots were probed with a monoclonal specific for gp41.</p

FIP1C depletion and HIV-1 Env incorporation in HeLa cells.

<p>(A) The efficiency of depletion of Rab11a-FIPs was measured by real time PCR (top). Infectivity of released particles following depletion of each FIP family member is shown below. (B) Depletion of FIP1C was confirmed at the protein level by immunoblotting with a FIP1C-specific antiserum. Env levels in both cell lysates and virions harvested from the FIP-depleted HeLa cells were assayed by immunoblotting. (C) Restoration of Env incorporation following shRNA-mediated depletion. Two constructs were used in this experiment: FIP1C cDNA is an shRNA sensitive GFP-tagged plasmid; while FIP1C* includes silent mutations in the shRNA target sequence rendering it shRNA-resistant. 2 ug and 4 ug of each FIP1C construct were used in the repletion assay as indicated at the top of the blot. (D) Infectivity of viral particles from the experiment shown in panel D as evaluated using TZM-bl indicator cells. Lanes are numbered and correspond to the blot above. Statistical comparisons utilized the unpaired t-test, * = p<.05; ** = p<.01.</p

The incorporation of Env onto HIV-1 particles is saturable in a cytoplasmic tail-dependent fashion.

<p>(A) Increasing amount of wildtype and the tailless mutant (CT144) of NL4-3 Env protein expressing plasmids were co-transfected with fixed amount of pNL4.3Env- proviral plasmid into HeLa cells. Progeny viral particles were harvested two days after transfection through a 20% sucrose cushion and the particles were lysed with SDS loading buffer. Proteins were analyzed by immunoblot using Env and CA specific antibodies. (B) The intensity of each Env band and Gag band in the particle immunoblot shown in A was quantified by Licor software. Background-subtracted pixel intensity values were plotted on the graph as Env band intensity versus CA band intensity. (C) The absolute quantity of Env and CA/P24 on particles was measured by gp120 ELISA and p24 ELISA. The number of Env molecules per particle was calculated as an approximation, assuming that 5000 CA molecules are present in one HIV-1 virion. (D) The infectivity of the progeny viral particles was measured using TZM-bl reporter cells. Infectivity is plotted as number of blue cells per nanogram of p24 antigen used in the infection assay.</p

FIP1C is required for Env incorporation in MDMs and enhances Env content of HIV-1 particles when overexpressed with Rab14.

<p>(A) MDMs were prepared from peripheral blood of healthy donors and depleted of FIP1C using two administrations of siRNA. MDMs were then infected with VSV-G-pseudotyped NL4-3 or NL4-3delCT144. Cell lysates and purified particles were examined 8 days following infection. Shown are Western blot results from two donors. (B) Particle infectivity from the experiments described above was measured in TZM-bl reporter cells as before. (C) Parental HeLa T-REx cells (Invitrogen) and HeLa-T-REx cells stably transfected with plasmids for inducible expression of FIP1C and Rab14Q70L were transfected with a constant amount of Gag expression plasmid and increasing amounts of NL4-3 Env plasmid (numbers above gp160 blot represent µg of input plasmid). Cell lysates and purified particles were examined by Western blotting. In these experiments, virus lanes were loaded with equal amounts of p24 as measured by ELISA. The bottom Env immunoblot represents cell surface Env as determined by biotinylation and precipitation with streptavidin beads, using the same input Gag and Env DNA as above. (D) Env and Gag content of released viral particles was quantified by densitometry using the LiCor instrument and plotted as background-subtracted Env pixel intensity to Gag pixel intensity (left) and by p24 and gp120 antigen-capture ELISA (right). The number of Env molecules per particle was calculated as an approximation, assuming that 5000 CA molecules are present in one HIV-1 virion.</p

HIV-1 Env redistributes FIP1C to plasma membrane in a gp41 cytoplasmic tail-dependent manner.

<p>HeLa cells were transfected with GFP-FIP1C (A–C) and either proviruses NL4-3 WT Env (B,C) or NL4-3delCT144 Env mutant (D). 24 hours after transfection, cells were fixed with 4% PFA and stained for HIV-1 Env using human monoclonal antibody b12. The distribution of GFP-tagged FIP1C, FIP2 and Env was examined using a wide-field deconvolution immunofluorescent microscope. Quantitation of tight perinuclear distribution versus peripherally-distributed GFP-FIP1C was evaluated by blinded observers on a per-cell basis, presented graphically as the % of evaluable cells in each category (E). We also tested if HIV-1 Env protein alone redistributes FIP1C using same method described above and plotted the result in (F).</p