Exome sequencing in 32 patients with anophthalmia/microphthalmia and developmental eye defects.

Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here.

Prenatal diagnosis of primary pulmonary hypoplasia in fraternal twins.

Primary pulmonary hypoplasia is a rare, usually lethal, condition presenting only after birth without other congenital abnormalities. We describe the first case of fraternal twins diagnosed prenatally with primary pulmonary hypoplasia. Both had diffuse hypoplasia of the pulmonary arteries initially identified by fetal echocardiography and confirmed at autopsy following termination. These cases permit examination of the histopathology of this disease in the fetal stage of development.

Multidetector computed tomography for characterization of calcium deposits in reperfused myocardial infarction.

BACKGROUND: Calcium overload is a major cause of reperfusion myocardial injury. Multidetector computed tomography (MDCT) has been previously used in visualizing coronary artery calcium, but not calcium deposits in reperfused infarction. PURPOSE: To assess the ability of MDCT to 1) noninvasively visualize and characterize calcium deposits in reperfused infarcts, and 2) monitor regional wall swelling, regional systolic wall thickening, and infarct resorption. MATERIAL AND METHODS: Reperfused myocardial infarcts were created in seven pigs by 2-hour occlusion of the left anterior descending coronary artery (LAD) after coronary catheterization. A 64-slice MDCT scanner was used for non-contrast images to depict calcium deposits. Furthermore, cine and delayed contrast-enhanced (DE) MDCT imaging were acquired to assess the chronological changes (2-4 hours, 1 week, and 8 weeks) in regional wall swelling, systolic wall thickening, and infarct size. RESULTS: Non-contrast MDCT images depicted calcium deposits as "hot-spots." Attenuation of calcium deposits was greater (89+/-6 Hounsfield units [HU]) than remote myocardium (36+/-3 HU; P<0.05). Calcium deposits were not evident at 2-4 hours and were substantially smaller at 8 weeks compared to 1 week. Correlations were found between the extent of calcium deposits, ejection fraction (R=0.81), and infarction size (R=0.70). Cine MCDT images demonstrated transient wall swelling (edema formation and resorption) at 2-4 hours and differences in regional systolic wall thickening among infarcted, peri-infarcted, and remote myocardium. Calcium-specific von Kossa stain confirmed the presence of calcium deposits in infarcted myocardium. CONCLUSION: 64-slice MDCT has the potential to demonstrate the progression and regression of calcium deposits, interstitial edema, and infarction. The presence of calcium deposits was transient and associated with reperfused recent infarction. The extent of calcium deposits was positively correlated with infarction size and negatively with global left-ventricular function.

In utero angiopoietin-2 gene delivery remodels placental blood vessel phenotype: a murine model for studying placental angiogenesis.

Angiopoietin (Ang)-2, the natural antagonist of the Ang1/Tie2 receptor is a complex regulator of blood vessel plasticity that plays a pivotal role in both vessel sprouting [in the presence of vascular endothelial growth factor (VEGF)-A] and vessel regression (in the absence of VEGF-A). Based on the spatial and temporal expression of Ang2 throughout human gestation, we recently suggested that the Ang2 may play a pivotal role in placental angiogenesis. Further, to examine this tenet we have developed a novel murine model system in which in utero Ang2 gene delivery via a non-replicating adenoviral expression vector has the potential to manipulate the blood vessel phenotype in vivo during pregnancy. Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular extracellular matrix, subsequently promoting plasticity of the maternal and fetal vessels, which appear honeycombed due to a 2-fold increase in blood vessel luminal area. High levels of Ang2 impair endothelial cell adhesiveness, leading to vascular leakiness with perivascular oedema, which increases placental weight. These observations suggest that the Ang2 overexpression may play a key role in placental vascular remodelling. Furthermore, we suggest a novel new model to study the pathobiology of placental vascularization and the effect of placental blood vessels on fetal phenotype.

Effects of genetic background on cardiovascular anomalies in the Ts16 mouse.

To investigate the genetic contribution to phenotypic variability in aneuploidy, we generated mice with trisomy 16 (Ts16) by mating [Rb(6.16)24Lub x Rb(16.17)7Bnr]F1 males with females from four inbred strains, BALB/cJ, C3H/HeJ, C57BL/6J, and DBA/2J. Among the four Ts16 strains that were generated, there were no significant differences in survival, weight, or length relative to euploid control littermates at either embryonic day (E) 14.5 or E17.5. All Ts16 fetuses at E14.5 had edema that ranged from mild to severe, increased amniotic fluid volume, and a thickened neck. At E17.5, Ts16 fetuses exhibited two distinct phenotypes, one with an edematous morphology and the other runt-like. None of these gross morphological abnormalities was strain-specific either in occurrence or frequency. At E10.5, there were pharyngeal arch artery (PAA) anomalies in all Ts16 embryos on the C3H/HeJ background, but none in trisomics on the other three backgrounds. However, at E17.5, there was in addition to ventricular and atrioventricular septal defects, a high frequency of aortic arch defects in Ts16 fetuses, irrespective of genetic background. Taken together, these findings indicate that there are at least two mechanistic responses to the presence of three copies of mouse chromosome 16 in the modeling of the cardiovascular system: one, development of PAA defects, is strongly influenced by genetic background; but the second, development of aortic arch anomalies in the absence of preexisting PAA anomalies, is not.

Structure of plaque at carotid bifurcation: high-resolution MRI with histological correlation.

BACKGROUND AND PURPOSE: The composition of carotid atherosclerosis was visualized by using 3D MRI at high resolution with 200-micrometer (3) voxels. Magnetic resonance signal characteristics were correlated with plaque components, including collagenous cap, necrotic core, and calcification, to define resolution and other requirements for future clinical carotid MRI. METHODS: Twenty-one en bloc carotid endarterectomy specimens were imaged ex vivo by 3D gradient-echo MRI by using a 1.5-T clinical scanner with repetition time, echo time, and flip angle of 40 ms, 18 ms, and 20 degrees, respectively. Plaques were placed in Gd-saline and imaged in a solenoid radiofrequency coil. For quantitative tissue-specific signal analysis, techniques were developed to match tissue sections analyzed by MRI and histology. RESULTS: Three-dimensional imaging resolved complex morphological features not visualized by density- or T(2)-weighted 2D spin-echo imaging. The collagenous cap, necrotic core, and areas of focal calcification showed differing signal characteristics: mean contrast-to-noise ratio for cap versus underlying core was 20. The signal distributions for media and necrotic core overlapped but were resolvable in most specimens. The signal from thrombus was variable. CONCLUSIONS: En bloc specimens provide a useful model for studying plaque MRI. By use of isotropic submillimeter resolution, the collagenous cap and underlying necrotic core typically can be distinguished, and calcification can be identified. Thrombus displays a wide variation in signal intensity. The techniques presented could facilitate future clinicohistological correlation studies for atherosclerotic plaque MRI.

Genetic modification of prenatal lethality and dilated cardiomyopathy in Mn superoxide dismutase mutant mice.

Mn superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, has been shown to be essential for animal survival. MnSOD mutant mice (Sod2-/- mice) on the CD1 background develop severe dilated cardiomyopathy and usually die within 10 d after birth. To characterize better the phenotype and understand the mechanism of superoxide-mediated tissue damage in Sod2-/- mice, congenic Sod2-/- mice on inbred backgrounds were generated to ensure genetic homogeneity. When generated on a C57BL/6J background (B6), more than half of the fetuses develop severe dilated cardiomyopathy by embryonic day 15 and die in the uterus. Those that survive to term usually die within 24 h. In contrast, Sod2-/- mice on DBA/2J (D2) and B6D2F1 (B6D2F1) backgrounds develop normally throughout gestation and do not develop dilated cardiomyopathy. However, the D2 mice do develop a severe metabolic acidosis and survive for only up to 12 d after birth. B6D2F1) mice have a milder form of metabolic acidosis and can survive for up to 3 weeks. The marked difference in lifespans and the development of dilated cardiomyopathy in the B6 but not the D2 or B6D2F1 backgrounds indicate the possible existence of genetic modifiers that provide protection to the developing hearts in the absence of MnSOD.

Cardiomyopathy in mice with paternal uniparental disomy for chromosome 12.

Mice inheriting both copies of MMU12 either maternally or paternally demonstrate imprinting effects. Whereas maternal uniparental disomy 12 (matUPD12) fetuses are growth retarded and die perinatally, paternal UPD12 (patUPD12) fetuses die during late gestation and exhibit placentomegaly and skeletal muscle maturation defects. To examine further the developmental consequences of UPD12, we intercrossed mouse stocks heterozygous for Robertsonian translocation chromosomes (8.12) and (10.12). We report that at 13.5-14.5 dg patUPD12 hearts exhibit increased ventricular diameter, thinner, less compact myocardium, and deep intertrabecular recesses when compared to controls. These data provide evidence for cardiac failure, a lethal condition, and suggest a role for an imprinted gene(s) in normal heart development.

Neovascular glaucoma as a complication of retinal vasculitis in Crohn disease.

PURPOSE: To report a case of neovascular glaucoma as a complication of retinal vasculitis in Crohn disease. METHODS: Case report with fluorescein angiogram. RESULTS: A 62-year-old man with biopsy-proven Crohn disease presented with bilateral uveitis, bilateral iris new vessels, and neovascular glaucoma in the left eye. Fluorescein angiography revealed signs of retinal vasculitis and capillary nonperfusion in both eyes. CONCLUSION: Crohn disease may be associated with retinal vasculitis and, thus, neovascular glaucoma. A satisfactory result can be achieved by using corticosteroids to control the retinal vascular inflammation, by applying panretinal photocoagulation and by controlling the increased intraocular pressure surgically.

Evaluation of the morphology of the oval fossa for placement of devices.

OBJECTIVES: First, to examine the morphology of heart specimens with defects of the oval fossa so as to define the factors that facilitate appropriate selection of the size of devices used for inteventional closure. Second, to examine the relationship between morphology and transthoracic and transesophageal echocardiography. BACKGROUND: The success of transcatheter closure is influenced by the variable morphology of deficiencies with the oval fossa, and of the relationship of the fossa itself to adjacent structures. More appropriate selection could reduce the incidence of failures. METHODS: From over 100 specimens in the cardiac registry at the University of California, San Francisco, we judged 16 hearts with atrial septal defects within the oval fossa, either in isolation or associated with other cardiac malformation, to be suitable for this study. We measured the dimensions of the defect and the surrounding rims of the fossa. All values were normalized to the diameter of the aortic root. RESULTS: A fenestrated defect was present in 9 specimens (56%). The shape defect itself was oval in all specimens, with a ratio of major to minor axes of 1.70 + 0.63. The major axis took one of three main directions with respect to the vertical plane: in 11 specimens (69%o) it was at horizontal; in 3 (19%) it was at oblique at an angle of 45 degrees; and in 2 (12%) it was vertical. Discordance was noted in some hearts between the major axis of the defect and that of the oval fossa. Structures closest to the rim of the fossa were the aortic mound, the coronary sinus, and the hinge point of the aortic leaflet of the mitral valve. CONCLUSIONS: Extrapolating from these specimens permitted identification of the major and minor axes of the atrial septal defect by transthoracic and transesophageal echocardiography. Our study has identified landmarks and dimensions that may be employed to improve effectiveness of selection of patients for transcatheter closure of defects within the oval fossa.

VEGF gene delivery to myocardium: deleterious effects of unregulated expression.

BACKGROUND: Vascular endothelial growth factor (VEGF) is being investigated for therapeutic angiogenesis in ischemic myocardium. Primarily, transient delivery systems have been tested. The goal of this study was to investigate the effects of continuous expression of VEGF in myocardium by use of myoblast-mediated delivery. METHODS AND RESULTS: Primary murine myoblasts (5 x 10(5) cells in 10 microL of PBS with 0.5% BSA) expressing both the murine VEGF gene and the beta-galactosidase (beta-gal) gene from a retroviral promoter were implanted in the ventricular wall of immunodeficient mice (n=11) via a subdiaphragmatic approach. Control immunodeficient mice (n=12) were injected with the same number of myoblasts expressing only the beta-gal gene. Between days 14 and 16, surviving mice were euthanized and the hearts processed for histology. In the experimental group, 11 of 11 mice demonstrated failure to thrive by day 13; 5 deaths occurred between days 8 and 15. There were no complications in the control mice. Histochemistry documented successful implantation of myoblasts (positive beta-gal reaction product) in 6 of 6 surviving experimental mice and 12 of 12 controls. Histology disclosed intramural vascular tumors resembling hemangiomas in the VEGF-myoblast-injected myocardium in 6 of 6 surviving mice. beta-Gal-expressing cells were present at the site of the vascular tumors. Immunohistochemistry localized abundant endothelial nitric oxide synthase and CD31 (platelet and endothelial cell adhesion molecule) within the lesion, consistent with the presence of endothelial cells. CONCLUSIONS: In this model, unregulated continuous expression of VEGF is associated with (1) a high rate of failure to thrive/death and (2) formation of endothelial cell-derived intramural vascular tumors in the implantation site. These results underscore the importance of regulating VEGF expression for therapeutic angiogenesis.

Matrix metalloproteinases and collagen ultrastructure in moderate myocardial ischemia and reperfusion in vivo.

Severe ischemic injury or infarction of myocardium may cause activation of matrix metalloproteinases (MMPs) and damage the interstitial matrix. However, it is unknown whether MMP activation and matrix damage occur after moderate ischemia and reperfusion that result in myocardial stunning without infarction, and if so whether such changes contribute to postischemic myocardial expansion and contractile dysfunction. To address these questions, open-chest anesthetized pigs underwent 90 min of regional ischemia (subendocardial blood flow 0.4 +/- 0.1 ml. g(-1). min(-1)) and 90 min of reperfusion. After ischemia plus reperfusion, histological and ultrastructural examination revealed no myocardial infarction or inflammatory cell infiltration. Myocardial MMP-9 content increased threefold with a fourfold increase in the active form (P < 0.001). Myocardial collagenase content doubled (P < 0.01) but remained in latent form. MMP-2 and tissue inhibitors of metalloproteinases were unaffected. Despite increases in MMPs, collagen ultrastructure (assessed by cell maceration scanning electron microscopy) was unaltered. Intracoronary administration of the MMP inhibitor GM-2487 did not prevent or attenuate myocardial expansion (assessed by regional diastolic dimensions at near-zero left ventricular pressure) or contractile dysfunction. We conclude that although moderate ischemia and reperfusion alter myocardial MMP content and activity, these effects do not result in damage to interstitial collagen, nor do they contribute to myocardial expansion or contractile dysfunction.

Posterior capsular opacification with hydrogel, polymethylmethacrylate, and silicone intraocular lenses: two-year results of a randomized prospective trial.

PURPOSE: To compare the visual outcome, percentage of posterior capsular opacification, and laser capsulotomy rates with polymethylmethacrylate, silicone, and hydrogel intraocular lens implants at 1 and 2 years postoperatively. METHODS: Ninety-three eyes of 93 patients were randomized to receive a polymethylmethacrylate, silicone, or hydrogel intraocular lens implant. A standardized surgical protocol was followed by a single surgeon using phacoemulsification with capsulorhexis; any patients with surgical complications were excluded, and all patients received standardized medication and follow-up. Patients were examined at days 1 and 7, months 1, 3, and 6, and years 1 and 2 after surgery. At each assessment, best-corrected logMAR visual acuity and Pelli-Robson contrast sensitivity were measured. Posterior capsular opacification was objectively assessed by digital retroillumination imaging with the use of a dedicated software program and calculated as the percentage area of opacified capsule. Laser capsulotomy was performed if the eye had lost 2 lines of visual acuity with a clinically opaque capsule. RESULTS: At 2 years postoperatively, the mean percentage area of posterior capsular opacification for hydrogel lenses was 63%; for polymethylmethacrylate, 46%; and for silicone, 17%. Hydrogel intraocular lenses were associated with 17% more posterior capsule opacification than were polymethylmethacrylate lenses (95% confidence interval, 1-33; P =. 037) and 45% more than were silicone lenses (95% confidence interval, 33-58; P <.0001) at 2 years. Polymethylmethacrylate lenses had 28% more posterior capsule opacification than silicone lenses (95% confidence interval, 13-43; P <.0001) at 2 years. Twenty-eight percent of patients with hydrogel intraocular lenses required an Nd:YAG laser posterior capsulotomy at 2 years, compared with 14% with polymethylmethacrylate, whereas no patients with silicone lenses needed a capsulotomy (P =.014). Visual acuity was not significantly different among the three groups, but patients with silicone intraocular lenses had significantly better contrast sensitivity than those with hydrogel lenses (P =.046). CONCLUSIONS: Intraocular lenses made of this specific hydrogel were associated with a significantly higher degree of posterior capsular opacification and more laser capsulotomies than polymethylmethacrylate and silicone intraocular lenses.

Reperfused rat myocardium subjected to various durations of ischemia: estimation of the distribution volume of contrast material with echo-planar MR imaging.

PURPOSE: To estimate and compare the fractional distribution volume (fDV) of gadodiamide injection and technetium 99m-diethylenetriaminepentaacetic acid (DTPA) in the reperfused myocardium of rat hearts subjected to various durations of ischemia. MATERIALS AND METHODS: Magnetic resonance (MR) imaging and autoradiography were performed in rats subjected to 20, 30, 40, or 60 minutes of regional ischemia followed by 1 hour of reperfusion. The fDVs of gadodiamide injection and (99m)Tc-DTPA were measured and compared by using inversion-recovery echo-planar imaging and autoradiographic phosphor imaging, respectively. RESULTS: The mean fDV of both tracers (gadodiamide and (99m)Tc-DTPA) in normal myocardium was 18% +/- 1, whereas that in the entire area at risk increased significantly (P <.05) with 20, 30, 40, and 60 minutes of ischemia to 32% +/- 1, 57% +/- 4, 66% +/- 2, and 68% +/- 2, respectively. The fDV was significantly (P <.05) greater in the core of infarction-78% +/- 4, 89% +/- 5, and 88% +/- 5 with 30, 40, and 60 minutes of ischemia, respectively-than in the normal myocardium or in the area at risk. CONCLUSION: The fDV of MR contrast material in the periinfarcted rim was significantly (P <. 05) greater than that in the normal myocardium, but significantly less than that in the core of infarcted myocardium.

Aortic vasculopathy with aneurysm: a rare cause of fetal hydrops.

An hydropic fetus seen at 28-weeks gestation had a saccular aortic aneurysm in the descending thoracic aorta. Histology disclosed marked fibrointimal hyperplasia, thrombus, and attenuation of the tunica media. The remainder of the descending thoracic aorta showed fibrointimal hyperplasia. We speculate that the narrowed lumen and rigid aortic wall resulting from this vasculopathy provided an increased afterload leading to cardiac failure.