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.

Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.

The Sod2 gene for Mn-superoxide dismutase (MnSOD), an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation, was inactivated by homologous recombination. Homozygous mutant mice die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis. Cytochemical analysis revealed a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide.

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.

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.

Kawasaki syndrome in an adult: endomyocardial histology and ventricular function during acute and recovery phases of illness.

Kawasaki syndrome, an acute systemic inflammatory illness of unknown origin usually affecting children, may develop into a serious illness complicated by coronary artery aneurysms or myocarditis. This report describes an adult with Kawasaki syndrome studied by right ventricular endomyocardial biopsy and cardiac catheterization during the acute and recovery phases of illness. The initial biopsy specimen showed acute myocarditis and was associated with hemodynamic evidence of biventricular dysfunction, a severely depressed left ventricular ejection fraction and global hypokinesia. With time, there was spontaneous and rapid resolution of the inflammatory cell infiltrate with concurrent return to normal myocardial function. Right ventricular endomyocardial biopsy studies early in the course of the cardiac disease associated with Kawasaki syndrome may correlate with ventricular function and may be useful for monitoring immunosuppressive therapy in patients with this syndrome.

The majority of nitric oxide synthase in pig heart is vascular and not neural.

OBJECTIVE: As a physiological mediator of smooth muscle relaxation and possibly a non-adrenergic non-cholinergic (NANC) neurotransmitter, nitric oxide plays a role in regulating coronary artery blood flow and modulating myocardial contractility. Although recent attention has been directed toward neural tissue, we investigated the possibility that vessels, as well as nerves, may be a source of nitric oxide in the heart. METHODS: The NADPH-diaphorase method was used to localise the synthesis enzyme, nitric oxide synthase (NOS), in the pig heart. RESULTS: All regions showed a similar staining pattern. Muscle fibres had virtually no NOS activity. The endocardium showed reaction product in a lattice configuration without much cytoplasmic staining, suggesting that endothelial cell membranes are the primary site of NOS activity. Every vessel contained reaction product in intima but none in the media or adventitia. Muscular arteries had more NOS activity than veins. Lighter staining capillaries coursed along each muscle fibre. Only a few scattered nerve processes and rare neuronal cell bodies with NOS activity were present in the heart; there was no particular spatial relationship between neural tissue and vessels. CONCLUSIONS: (1) the majority of NOS activity in the pig heart is in vascular endothelium, consistent with the concept of nitric oxide as a regulator of coronary blood flow; less is present in the endocardium; (2) paucity of nerves with NOS activity probably indicates that this particular type of NANC neural tissue does not affect coronary blood flow directly; and (3) although muscle fibres have no discernible NOS activity, the rich vascular supply in close proximity may subserve some myocardial function of nitric oxide.

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.

Nitric oxide synthase is deficient in the aganglionic colon of patients with Hirschsprung's disease.

OBJECTIVES: The cause of Hirschsprung's disease is unknown but defects in nonadrenergic, non-cholinergic innervation could prevent relaxation of aganglionic colon in patients with this disease. Nonadrenergic, noncholinergic nerves induce relaxation by using nitric oxide synthase to produce the smooth muscle relaxant nitric oxide (NO). In this study we asked whether aganglionic colon in patients with Hirschsprung's disease is deficient in NO synthase-containing nerves. METHODOLOGY: Using the tetrazolium blue dye method of demonstrating nicotinamide adenine dinucleotide phosphate-diaphorase enzymes, we examined eight colon specimens (four aganglionic and four ganglionic) from patients with Hirschsprung's disease for the presence of NO synthase. We further quantified NO synthase enzyme activity in these eight specimens by using the [3H]arginine-to-[3H]citrulline conversion assay. RESULTS: The nicotinamide adenine dinucleotide phosphate-diaphorase staining showed that aganglionic colon contained less NO synthase than ganglionic colon. This NO synthase deficiency was located primarily in the nerves of the circular muscle layer of the colon. In addition, there was a striking difference in the NO synthase enzyme activity between aganglionic and ganglionic colon as measured by the [3H]arginine-to-[3H]citrulline conversion assay. Total NO synthase activity, as measured by this assay, was found to be less in aganglionic than in ganglionic colon. When the total activity was divided into its four known isoforms, aganglionic colon was noted to be striking deficient in the isoform derived primarily from nerves. CONCLUSION: We conclude that aganglionic colon is deficient in NO synthase-containing nerves. This deficiency could prevent smooth muscle relaxation in the aganglionic colon of patients with Hirschsprung's disease.

Left atrial isomerism detected in fetal life.

Left and right atrial isomerism, comprising congenital heart defects with disturbances in normal left-right asymmetry, are phenotypically distinct after birth, although animal models suggest a common embryologic origin. We postulated that the prenatal phenotype may indeed be similar in both syndromes but that differential fetal loss is responsible for the distinct postnatal phenotypes. Distinctive fetal echocardiographic features of these syndromes have not been described in detail. We therefore sought markers of left atrial isomerism that could be recognized prenatally by echocardiography and compared our results with postnatal data to identify unique intrauterine features. We reviewed 10 cases at our center and 28 published cases of cardiac malformations with atrial isomerism detected by fetal echocardiography. Postnatal imaging and autopsies provided definitive diagnoses. Ninety-five percent of cases exhibited left atrial isomerism and formed the primary study population. Echocardiographic markers included a large azygos continuation of an interrupted inferior vena cava, atrioventricular block with structural heart disease, and viscerocardiac heterotaxy. At least 1 of these markers was seen in all of our center's cases. The incidences of most cardiac lesions detected prenatally were similar to those detected postnatally. However, although the incidences of atrioventricular septal defect and pulmonary outflow obstruction in live births were 50% and 45%, respectively, they were found much more frequently among stillbirths (80% each). In summary, we identified key fetal echocardiographic features highly sensitive for left atrial isomerism. Fetal loss selects against certain lesions such as atrioventricular septal defect. The spectrum of cardiac disease suggests a greater primitivity of the fetal heart than previously shown; the typical cardiac phenotypes are closer to right atrial isomerism than are their extrauterine presentations.

Malignant neurogenic tumor of the heart.

This is the first report of the fine structure of a malignant neurogenic tumor in the heart. The tumor, arising in the infundibulum of the right ventricle of the heart, was resected and examined by both light and electron microscopy. The histologic appearance of the originally resected tumor was consistent with that of a neurofibroma, while that of the recurrent tumor suggested a malignant schwannoma. Ultrastructurally, the presence of basement membrane investing some cells confirmed the neurogenic nature of the tumor. The discontinuous nature of the basement membrane in these cells may provide a clue as to the malignant nature of this tumor.

Diagnosis of acute rheumatic carditis by endomyocardial biopsy.

This report is the first to document the definitive diagnosis of acute rheumatic carditis by endomyocardial biopsy. An adolescent girl with mitral regurgitation and biventricular congestive heart failure thought to be secondary to rheumatic heart disease was admitted to the hospital for mitral valve replacement. An endomyocardial biopsy performed at cardiac catheterization demonstrated and Aschoff's body in the endocardium and thus confirmed a clinical suspicion of active carditis. Following a course to steroids, the mitral valve was replaced. The surgically excised valve showed gross and microscopic features of acute and chronic valvulitis consistent with rheumatic endocarditis.

Disseminated intravascular meconium in a newborn with meconium peritonitis.

A 3-day-old premature infant with meconium peritonitis, periventricular leukomalacia, and pulmonary hypertension died with respiratory insufficiency. An autopsy disclosed intravascular squamous cells in the lungs, brain, liver, pancreas, and kidneys. Numerous pulmonary capillaries and arterioles were occluded by squamous cells, accounting for pulmonary hypertension. Brain parenchyma surrounding occluded cerebral vessels showed infarct and gliosis. A mediastinal lymph node filled with squamous cells alluded to the mechanism by which these cells from the peritoneal cavity likely entered the bloodstream--namely, via diaphragmatic pores connecting with lymphatics. Thus, disseminated intravascular meconium rarely may complicate meconium peritonitis and have devastating consequences.

Hepatitis B virus and myocarditis.

Myocarditis may be a serious extrahepatic complication of hepatitis. In this fatal case of serologically documented hepatitis B viral hepatitis, acute myocarditis was present, with histologic features consistent with a viral pathogenesis. Hepatitis B surface antigen was demonstrated by immunoperoxidase methods in small intramyocardial vessels, suggesting that hepatitis B virus infected the heart. The resulting inflammatory heart disease may have been caused either directly, by virus infecting the myocardium, or indirectly, by an immune-mediated mechanism.

Utility of endomyocardial biopsy in the diagnosis of cardiac sarcoidosis.

Cardiac involvement in sarcoidosis can be demonstrated in about 25 percent of autopsied cases, but antemortem diagnosis is uncommon. To evaluate the usefulness of the endomyocardial biopsy in detecting cardiac sarcoid disease, the medical records of ten patients with sarcoidosis who underwent endomyocardial biopsy for routine clinical indications were reviewed. The patients fell into two groups: patients with known sarcoidosis and presumed cardiac involvement (n = 8), and patients in whom the biopsy finding of sarcoid disease was unexpected (n = 2). Four patients in the first group had positive endomyocardial biopsy results (granulomas and/or marked mononuclear cell infiltrate) and were treated with glucocorticoid therapy with improvement in three; the fourth was disabled with lung disease. The diagnoses of three other patients were revised on the basis of the biopsy results; their therapy was tailored accordingly. The remaining patient may represent a false-negative biopsy result, based on clinical criteria. The two patients in the second group presented with symptomatic ventricular tachycardia and restrictive cardiomyopathy respectively, and in neither case was sarcoidosis considered prior to biopsy results. Overall, a change in treatment strategy based on biopsy results occurred in eight of ten cases. Thus, endomyocardial biopsy is useful for the diagnosis of cardiac sarcoidosis; treatment strategies may be affected by biopsy findings; and rarely, endomyocardial biopsy can provide the first clinical evidence of sarcoid disease that is otherwise occult.

Atrial morphologic features in tricuspid atresia.

We studied the atrial morphology in 110 hearts removed at autopsy from patients with tricuspid atresia. Ten of the patients had had a Fontan operation. We compared the findings in these hearts with those in 30 normal hearts. Prominent eustachian valves were common (40%). The tricuspid "dimple," present in 33 of 100 specimens, denoted the membranous atrioventricular septum and was never related to the right ventricle. Most interatrial communications (85/100) were nonobstructive. A minority of patients with obstructive interatrial communications (13/100 = small, 2/100 = severely obstructive) would be expected to benefit from balloon atrial septostomy early in life. Right atrial hypertrophy was greatest in specimens with restrictive interatrial communications (severely obstructive = 3.2 +/- 0.3 mm, small = 2.1 +/- 0.6 mm) and in those that had been subjected to a Fontan operation (3.2 +/- 0.7 mm), but thickness was also increased in hearts with nonobstructive interatrial communications (1.6 +/- 0.5 mm) compared with normal hearts (1.1 +/- 0.2 mm; p less than 0.001 versus all groups). Of the patients in the series, 11 of 100 had left juxtaposition of the atrial appendages, which may have important implications for the Fontan operation.

Surface cytologic features on intraocular lenses: can increased biocompatibility have disadvantages?

OBJECTIVE: To compare the anterior surface cytologic features and effect on blood-aqueous barrier of polymethyl methacrylate, silicone, and hydrogel intraocular lens (IOL) implants to give an indication of their biocompatibility. METHODS: This prospective study was performed at an English-teaching hospital. Ninety eyes were randomized to receive a polymethyl methacrylate, silicone, or hydrogel implant. A standardized surgical protocol was performed by a single surgeon using phacoemulsification. Patients were seen at intervals for 1 year. Measurements of visual acuity, contrast sensitivity, and anterior chamber laser flare and cells were obtained; and an assessment of lens cytologic features using specular microscopy of the anterior IOL surface was performed. RESULTS: Visual acuity and contrast sensitivity were not significantly different among the 3 groups. Hydrogel IOLs were associated with fewer inflammatory cells on their surface than polymethyl methacrylate and silicone IOLs (P<.001), but with significantly more lens epithelial cells (LECs) (P<.001). Patients with hydrogel implants without LECs had greater blood-aqueous barrier breakdown than those with LECs. CONCLUSIONS: The hydrogel IOLs were associated with a reduced inflammatory cell reaction but had many more LECs on their anterior surface. Those IOLs associated with increased blood-aqueous barrier damage did not develop LECs. If an IOL is too biocompatible, then it may incite the growth of LECs over its surface, which could have disadvantages.

Development of a model of complete heart block in rats.

Atrioventricular (AV) block is a useful substrate for the study of cardiac physiology. The objective of this investigation was to develop a straightforward and reproducible model of permanent AV block in rats. Working through a sternotomy, we used an epicardial fat pad between the aortic root and the right atrial wall of the rat as a landmark for the site for injection of 70% ethanol (5-10 microl) into the myocardium 3 mm below the epicardial surface. Stable, complete heart block was produced in 23 of 28 rats (82%) with a success rate of 100% in the last 16 rats of the series. Saline injection produced no heart block in 15 rats. A separate group of 14 animals was allowed to recover. Chronic heart block was achieved in all ethanol-injected animals for up to 7 days before death. The survival rate in the recovered rats was 90% in the ethanol-injected group and 100% in the saline-injected control group. Acute hemodynamic changes following the production of heart block consisted of an increase in central venous pressure, a decrease in systolic blood pressure, a decrease in left ventricular pressure, and a decrease in change in pressure over time. Chronic hemodynamic changes demonstrated a return to baseline of the central venous pressure, a persistent decrease in systolic blood pressure, and a decrease in left ventricular pressure. After the rats were killed and the hearts were dissected, discrete areas of myocardial damage were identified histologically in the atrial septum near the AV conduction axis tissue in the ethanol-injected hearts. Complete heart block was associated only with lesions extending into the specialized muscle of the AV node or His bundle. Focal mild hemorrhage, inflammation, and damaged myocardial fibers were observed in the acute stage, whereas healing lesions were characterized by granulation tissue and fibrosis replacing conduction tissue. The simple technique described provides a reproducible model for permanent, complete heart block and the study of cardiac function.

Traumatic rupture of extracardiac valved conduit: unusual late complication producing outflow tract obstruction.

A 22-year-old woman died suddenly 15 years after successful repair of truncus arteriosus with a valved Dacron conduit. At autopsy there was complete obstruction of the right ventricular outflow tract by a large organizing thrombus between the outer aspect of the conduit and the adherent pericardial tissue. This rare late complication may have resulted from an unrecognized deceleration injury occurring at the time of a serious automobile accident 5 months before death.