Extracorporeal Membrane Oxygenation Support After Heart Transplantation in Children-Outcomes of a Single Center Cohort.

OBJECTIVES: Extracorporeal membrane oxygenation is used for postcardiotomy low cardiac output but is less established following heart transplantation. We characterized outcomes for children supported with extracorporeal membrane oxygenation after heart transplantation. DESIGN: Single-center retrospective study. SETTING: Large pediatric cardiac referral center. PATIENTS: All patients who received heart transplantation and were cannulated to extracorporeal membrane oxygenation between 1995 and 2016. INTERVENTIONS: Primary outcome measure was mortality 12 months postextracorporeal membrane oxygenation. Patient characteristics were analyzed for association with outcome according to early graft failure (extracorporeal membrane oxygenation ≤ 7 d after heart transplantation), or late graft failure. MEASUREMENTS AND MAIN RESULTS: There were 246 heart transplants during the study period and 50 extracorporeal membrane oxygenation runs in 44 patients. Median time from transplant to extracorporeal membrane oxygenation was 1 day (range, 0-11.7 yr), with early graft failure in 28 patients (median 1, range 0-2 d) and 22 extracorporeal membrane oxygenation runs in 20 late graft failure patients (median, 0.8 yr; range, 8 d to 11.7 yr), including four patients with prior extracorporeal membrane oxygenation for early graft failure. Twenty-six patients (59%) survived to hospital discharge, and survival 12 months postextracorporeal membrane oxygenation was 24 patients (55%), lower in those with late graft failure (40% vs 67%; p 0.02). Independent risk factors for 12-month mortality were congenital heart disease, higher pulmonary vascular resistance indexed to body surface area (> 2.2 Woods U/m), and higher creatinine. Higher panel reactive antibody levels were associated with 12-month mortality in the late graft failure group only. CONCLUSIONS: Extracorporeal membrane oxygenation can be effectively used to rescue patients with graft dysfunction after heart transplantation but is associated with high early mortality. Factors associated with mortality within 12 months include presence of congenital heart disease, renal dysfunction, elevated pulmonary vascular resistance indexed to body surface area and in those supported with extracorporeal membrane oxygenation late after heart transplantation, significant human leukocyte antigen sensitization.

Impact of Heart Transplantation on the Functional Status of US Children With End-Stage Heart Failure.

BACKGROUND: There are limited data describing the functional status (FS) of children after heart transplant (HT). We sought to describe the FS of children surviving at least 1 year after HT, to evaluate the impact of HT on FS, and to identify factors associated with abnormal FS post-HT. METHODS: Organ Procurement and Transplantation Network data were used to identify all US children <21 years of age surviving ≥1 year post-HT from 2005 to 2014 with a functional status score (FSS) available at 3 time points (listing, transplant, ≥1 year post-HT). Logistic regression and generalized estimating equations were used to identify factors associated with abnormal FS (FSS≤8) post-HT. RESULTS: A total of 1633 children met study criteria. At the 1-year assessment, 64% were "fully active/no limitations" (FSS=10), 21% had "minor limitations with strenuous activity" (FSS=9); and 15% scored ≤8. In comparison with listing FS, FS at 1 year post-HT increased in 91% and declined/remained unchanged in 9%. A stepwise regression procedure selected the following variables for association with abnormal FS at 1 year post-HT: ≥18 years of age (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.2-2.7), black race (OR, 1.5; 95% CI, 1.1-2.0), support with ≥inotropes at HT (OR, 1.7; 95% CI, 1.2-2.5), hospitalization status at HT (OR, 1.5; 95% CI, 1.0-2.19), chronic steroid use at HT (OR, 1.5; 95% CI, 1.0-2.2), and treatment for early rejection (OR, 2.0; 95% CI, 1.5-2.7). CONCLUSION: Among US children who survive at least 1 year after HT, FS is excellent for the majority of patients. HT is associated with substantial improvement in FS for most children. Early rejection, older age, black race, chronic steroid use, hemodynamic support at HT, and being hospitalized at HT are associated with abnormal FS post-HT.

Clinical Trial of the Protein Farnesylation Inhibitors Lonafarnib, Pravastatin, and Zoledronic Acid in Children With Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome is an extremely rare, fatal, segmental premature aging syndrome caused by a mutation in LMNA yielding the farnesylated aberrant protein progerin. Without progerin-specific treatment, death occurs at an average age of 14.6 years from an accelerated atherosclerosis. A previous single-arm clinical trial demonstrated that the protein farnesyltransferase inhibitor lonafarnib ameliorates some aspects of cardiovascular and bone disease. This present trial sought to further improve disease by additionally inhibiting progerin prenylation. METHODS: Thirty-seven participants with Hutchinson-Gilford progeria syndrome received pravastatin, zoledronic acid, and lonafarnib. This combination therapy was evaluated, in addition to descriptive comparisons with the prior lonafarnib monotherapy trial. RESULTS: No participants withdrew because of side effects. Primary outcome success was predefined by improved per-patient rate of weight gain or carotid artery echodensity; 71.0% of participants succeeded (P<0.0001). Key cardiovascular and skeletal secondary variables were predefined. Secondary improvements included increased areal (P=0.001) and volumetric (P<0.001-0.006) bone mineral density and 1.5- to 1.8-fold increases in radial bone structure (P<0.001). Median carotid artery wall echodensity and carotid-femoral pulse wave velocity demonstrated no significant changes. Percentages of participants with carotid (5% to 50%; P=0.001) and femoral (0% to 12%; P=0.13) artery plaques and extraskeletal calcifications (34.4% to 65.6%; P=0.006) increased. Other than increased bone mineral density, no improvement rates exceeded those of the prior lonafarnib monotherapy treatment trial. CONCLUSIONS: Comparisons with lonafarnib monotherapy treatment reveal additional bone mineral density benefit but likely no added cardiovascular benefit with the addition of pravastatin and zoledronic acid. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00879034 and NCT00916747.

Clinical trial of a farnesyltransferase inhibitor in children with Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare, fatal, segmental premature aging syndrome caused by a mutation in LMNA that produces the farnesylated aberrant lamin A protein, progerin. This multisystem disorder causes failure to thrive and accelerated atherosclerosis leading to early death. Farnesyltransferase inhibitors have ameliorated disease phenotypes in preclinical studies. Twenty-five patients with HGPS received the farnesyltransferase inhibitor lonafarnib for a minimum of 2 y. Primary outcome success was predefined as a 50% increase over pretherapy in estimated annual rate of weight gain, or change from pretherapy weight loss to statistically significant on-study weight gain. Nine patients experienced a ≥50% increase, six experienced a ≥50% decrease, and 10 remained stable with respect to rate of weight gain. Secondary outcomes included decreases in arterial pulse wave velocity and carotid artery echodensity and increases in skeletal rigidity and sensorineural hearing within patient subgroups. All patients improved in one or more of these outcomes. Results from this clinical treatment trial for children with HGPS provide preliminary evidence that lonafarnib may improve vascular stiffness, bone structure, and audiological status.

Genetic and environmental risk factors in congenital heart disease functionally converge in protein networks driving heart development.

Congenital heart disease (CHD) occurs in ∼1% of newborns. CHD arises from many distinct etiologies, ranging from genetic or genomic variation to exposure to teratogens, which elicit diverse cell and molecular responses during cardiac development. To systematically explore the relationships between CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation and environmental exposures that modulate critical biological systems during heart development.

Nprl3 is required for normal development of the cardiovascular system.

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.

Impact of ABO-incompatible listing on wait-list outcomes among infants listed for heart transplantation in the United States: a propensity analysis.

BACKGROUND: The purported advantage of ABO-incompatible (ABO-I) listing is to reduce wait times and wait-list mortality among infants awaiting heart transplantation. We sought to describe recent trends in ABO-I listing for US infants and to determine the impact of ABO-I listing on wait times and wait-list mortality. METHODS AND RESULTS: In this multicenter retrospective cohort study using Organ Procurement and Transplant Network data, infants<12 months of age listed for heart transplantation between 1999 and 2008 (n=1331) were analyzed. Infants listed for an ABO-I transplant were compared with a propensity score-matched cohort listed for an ABO-compatible transplant through the use of a Cox shared-frailty model. The primary end point was time to heart transplantation. The percentage of eligible infants listed for an ABO-I heart increased from 0% before 2002 to 53% in 2007 (P<0.001 for trend). Compared with infants listed exclusively for an ABO-compatible heart, infants with a primary ABO-I listing strategy (n=235) were more likely to be listed 1A, to have congenital heart disease and renal failure, and to require extracorporeal membrane oxygenation. For the propensity score-matched groups (n=197 matched pairs), there was no difference in wait-list mortality; however, infants with blood type O assigned an ABO-I listing strategy were more likely to undergo heart transplantation by 30 days (31% versus 16%; P=0.007) with a less pronounced effect for infants with other blood types. CONCLUSIONS: The proportion of US infants listed for an ABO-I heart transplantation has risen dramatically in recent years but still appears to be preferentially used for sicker infant candidates. The ABO-I listing strategy is associated with a higher likelihood of transplantation within 30 days for infants with blood group O and may benefit a broader range of transplantation candidates.

Dissecting spatio-temporal protein networks driving human heart development and related disorders.

Aberrant organ development is associated with a wide spectrum of disorders, from schizophrenia to congenital heart disease, but systems-level insight into the underlying processes is very limited. Using heart morphogenesis as general model for dissecting the functional architecture of organ development, we combined detailed phenotype information from deleterious mutations in 255 genes with high-confidence experimental interactome data, and coupled the results to thorough experimental validation. Hereby, we made the first systematic analysis of spatio-temporal protein networks driving many stages of a developing organ identifying several novel signaling modules. Our results show that organ development relies on surprisingly few, extensively recycled, protein modules that integrate into complex higher-order networks. This design allows the formation of a complicated organ using simple building blocks, and suggests how mutations in the same genes can lead to diverse phenotypes. We observe a striking temporal correlation between organ complexity and the number of discrete functional modules coordinating morphogenesis. Our analysis elucidates the organization and composition of spatio-temporal protein networks that drive the formation of organs, which in the future may lay the foundation of novel approaches in treatments, diagnostics, and regenerative medicine.

Clinical, pathological, and molecular analyses of cardiovascular abnormalities in Costello syndrome: a Ras/MAPK pathway syndrome.

Cardiovascular abnormalities are important features of Costello syndrome and other Ras/MAPK pathway syndromes ("RASopathies"). We conducted clinical, pathological and molecular analyses of 146 patients with an HRAS mutation including 61 enrolled in an ongoing longitudinal study and 85 from the literature. In our study, the most common (84%) HRAS mutation was p.G12S. A congenital heart defect (CHD) was present in 27 of 61 patients (44%), usually non-progressive valvar pulmonary stenosis. Hypertrophic cardiomyopathy (HCM), typically subaortic septal hypertrophy, was noted in 37 (61%), and 5 also had a CHD (14% of those with HCM). HCM was chronic or progressive in 14 (37%), stabilized in 10 (27%), and resolved in 5 (15%) patients with HCM; follow-up data was not available in 8 (22%). Atrial tachycardia occurred in 29 (48%). Valvar pulmonary stenosis rarely progressed and atrial septal defect was uncommon. Among those with HCM, the likelihood of progressing or remaining stable was similar (37%, 41% respectively). The observation of myocardial fiber disarray in 7 of 10 (70%) genotyped specimens with Costello syndrome is consistent with sarcomeric dysfunction. Multifocal atrial tachycardia may be distinctive for Costello syndrome. Potentially serious atrial tachycardia may present in the fetus, and may continue or worsen in about one-fourth of those with arrhythmia, but is generally self-limited in the remaining three-fourths of patients. Physicians should be aware of the potential for rapid development of severe HCM in infants with Costello syndrome, and the need for cardiovascular surveillance into adulthood as the natural history continues to be delineated.

Familial dilated cardiomyopathy secondary to dystrophin splice site mutation.

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM) encompasses a heterogeneous group of disorders, posing significant diagnostic challenges. Genetic etiologies underlie an important subset of DCM, including 20 genes and 5 X-linked disorders to date. We report a family with a rare dystrophin gene alteration, identified after evaluation of asymptomatic children whose extended family history included cardiomyopathy, premature cardiac death, or cardiac transplantation. METHODS AND RESULTS: Record review, clinical evaluations, and DNA samples were obtained from members of a 5-generation pedigree with early onset DCM. Five of 6 affected males experienced death or cardiac transplant in their second or third decades. No affected individuals had skeletal muscle weakness before acute cardiac decompensation. Dystrophin gene analysis of an affected family member revealed sequence alteration at the conserved 5' splice site of exon 1 of the muscle-specific isoform of dystrophin (IVS1 +1 G>T) and co-segregated with cardiac disease in this family. CONCLUSIONS: Young males presenting with apparent isolated cardiomyopathy or acute myocarditis may harbor dystrophin mutations without overt skeletal muscle pathology. The etiology of familial risk was not evident in this pedigree before retrospective cardiovascular genetics assessment, highlighting ongoing diagnostic challenges and limitations of standardized screening panels (which do not include dystrophin) in patients with "idiopathic" DCM.

Whole Exome Sequencing Reveals a Monogenic Cause of Disease in ≈43% of 35 Families With Midaortic Syndrome.

Midaortic syndrome (MAS) is a rare cause of severe childhood hypertension characterized by narrowing of the abdominal aorta in children and is associated with extensive vascular disease. It may occur as part of a genetic syndrome, such as neurofibromatosis, or as consequence of a pathological inflammatory disease. However, most cases are considered idiopathic. We hypothesized that in a high percentage of these patients, a monogenic cause of disease may be detected by evaluating whole exome sequencing data for mutations in 1 of 38 candidate genes previously described to cause vasculopathy. We studied a cohort of 36 individuals from 35 different families with MAS by exome sequencing. In 15 of 35 families (42.9%), we detected likely causal dominant mutations. In 15 of 35 (42.9%) families with MAS, whole exome sequencing revealed a mutation in one of the genes previously associated with vascular disease (NF1, JAG1, ELN, GATA6, and RNF213). Ten of the 15 mutations have not previously been reported. This is the first report of ELN, RNF213, or GATA6 mutations in individuals with MAS. Mutations were detected in NF1 (6/15 families), JAG1 (4/15 families), ELN (3/15 families), and one family each for GATA6 and RNF213 Eight individuals had syndromic disease and 7 individuals had isolated MAS. Whole exome sequencing can provide conclusive molecular genetic diagnosis in a high fraction of individuals with syndromic or isolated MAS. Establishing an etiologic diagnosis may reveal genotype/phenotype correlations for MAS in the future and should, therefore, be performed routinely in MAS.

Mechanisms of premature vascular aging in children with Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome is a rare, segmental premature aging syndrome of accelerated atherosclerosis and early death from myocardial infarction or stroke. This study sought to establish comprehensive characterization of the fatal vasculopathy in Hutchinson-Gilford progeria syndrome and its relevance to normal aging. We performed cardiovascular assessments at a single clinical site on the largest prospectively studied cohort to date. Carotid-femoral pulse wave velocity was dramatically elevated (mean: 13.00±3.83 m/s). Carotid duplex ultrasound echobrightness, assessed in predefined tissue sites as a measure of arterial wall density, was significantly greater than age- and sex-matched controls in the intima-media (P<0.02), near adventitia (P<0.003), and deep adventitia (P<0.01), as was internal carotid artery mean flow velocity (P<0.0001). Ankle-brachial indices were abnormal in 78% of patients. Effective disease treatments may be heralded by normalizing trends of these noninvasive cardiovascular measures. The data demonstrate that, along with peripheral vascular occlusive disease, accelerated vascular stiffening is an early and pervasive mechanism of vascular disease in Hutchinson-Gilford progeria syndrome. There is considerable overlap with cardiovascular changes of normal aging, which reinforces the view that defining mechanisms of cardiovascular disease in Hutchinson-Gilford progeria syndrome provides a unique opportunity to isolate a subset of factors influencing cardiovascular disease in the general aging population.

Reproducibility of echocardiographic diagnosis of left ventricular noncompaction.

BACKGROUND: Left ventricular noncompaction (LVNC) cardiomyopathy is variably defined by numerous trabeculations, deep intertrabecular recesses, and noncompacted-to-compacted (NC/C) ratio >2. Limited studies exist on the reproducibility of diagnosing LVNC. METHODS: Clinical records of patients diagnosed with LVNC by echocardiography were reviewed. Blinded review of the index echocardiogram for all patients and a 1:1 match without LVNC was performed independently by two observers, measuring the number of trabeculations and the NC/C ratio. RESULTS: A total of 104 patients with LVNC were included in the study, 52 with no congenital heart disease (NCongHD) and 52 with congenital heart disease (CongHD). The duration of follow-up was 7.2 years (range, 0.5-23.1 years) for NCongHD and 8.2 years (range, 0-33.3 years) for CongHD. Agreement between observers in determining zero to three versus more than three trabeculations was 59% (NCongHD) and 73% (CongHD). Agreement in measuring an NC/C ratio ≤ 2 versus > 2 was 79% (NCongHD) and 74% (CongHD). Agreement with the original reader in diagnosing LVNC was 67%. There was no association between the number of trabeculations or the NC/C ratio and the likelihood of a major event. Patients with moderate or severe left ventricular dysfunction at the time of diagnosis were more likely to undergo cardiac transplantation or die compared with those with normal or mild dysfunction (NCongHD, 22% vs 0%, P = .01; CongHD, 39% vs 3%, P = .001). CONCLUSIONS: The reproducibility of making measurements to diagnose LVNC by accepted criteria is poor. Heart transplantation and death are associated with significant ventricular dysfunction and not with increased trabeculations or NC/C ratios.

Association of graft ischemic time with survival after heart transplant among children in the United States.

BACKGROUND: Previous studies have found no association between graft ischemic time (IT) and survival in pediatric heart transplant (HTx) recipients. However, previous studies were small or analyzed risk only at the extremes of IT, where observations are few. We sought to determine whether graft IT is independently associated with graft survival in a large cohort of children with no a priori assumptions about where the risk threshold may lie. METHODS: All children aged <18 years in the U.S. undergoing primary HTx (1987 to 2008) were included. The primary end point was graft loss (death or retransplant) within 6 months. Multivariate analysis was performed to analyze the association between graft IT and graft loss within 6 months after transplant. A secondary end point of longer-term graft loss was assessed among recipients who survived the first 6 months after transplant. RESULTS: Of 4,716 pediatric HTxs performed, the median IT was 3.5 hours (interquartile range, 2.7-4.3 hours). Adjusted analysis showed that children with an IT > 3.5 hours were at increased risk of graft loss within 6 months after transplant (hazard ratio, 1.3; 95% confidence interval, 1.1-1.5; p = 0.002). Among 6-month survivors, IT was not associated with longer-term graft loss. CONCLUSIONS: IT beyond 3.5 hours is associated with a 30% increase in risk of graft loss within 6 months in pediatric HT recipients. Although the magnitude of risk associated with IT is small compared with the risk associated with recipient factors, these findings may be important during donor assessment for high-risk transplant candidates.

Safety and early outcomes using a corticosteroid-avoidance immunosuppression protocol in pediatric heart transplant recipients.

BACKGROUND: Long-term oral corticosteroids have been a mainstay of maintenance immunosuppression in pediatric heart transplantation. In this study, we report early clinical outcomes in a cohort of pediatric heart transplant recipients managed using a steroid-avoidance protocol. METHODS: Of the 70 patients who underwent heart transplantation during the study period, 55 eligible recipients, including 49 non-sensitized and 6 sensitized (all 55 with negative crossmatch) patients, entered a steroid-avoidance immunosuppression protocol consisting of thymoglobin induction followed by a 2-drug, tacrolimus-based, corticosteroid-free regimen. The primary outcome variable was freedom from moderate rejection (International Society for Heart and Lung Transplantation [ISHLT] Grade 2R/3A or antibody-mediated rejection). RESULTS: The median age at transplant was 7.1 years (range 2 weeks to 22 years) and median follow-up was 19 months (range 2 to 46 months). Fifty patients survived to discharge after transplantation. Of these patients, 2 (4%) were discharged on steroids and 8 (16%) started on maintenance steroids at follow-up. Rejection was diagnosed in 8 patients (Grade 2R cellular rejection in 3 and antibody-mediated rejection in 5). Freedom from rejection was 92% at 6 months (95% confidence interval [CI] 80% to 97%) and 87% at 1 year (CI 73% to 94%). Post-transplant survival was 91% at 6 months (CI 79% to 96%) and 88% at 12 and 24 months (CI 75% to 95%). There was 1 death due to rejection (antibody-mediated) 8 months after transplantation. CONCLUSIONS: An immunosuppression protocol consisting of induction followed by corticosteroid avoidance appears to achieve acceptable rejection rates during the first year post-transplant in pediatric heart transplant recipients.

Outcomes of heart transplantation using donor hearts from infants with sudden infant death syndrome.

BACKGROUND: Uncertainty exists whether hearts from infants who have died of sudden infant death syndrome (SIDS) are acceptable for transplantation because the mechanism of death in SIDS remains unclear. We analyzed post-transplant outcomes in infants who received a heart from a donor where SIDS was the primary cause of brain death. METHODS: This retrospective multicenter cohort study used data from the Organ Procurement and Transplant Network (OPTN). All infants aged < 12 months undergoing heart transplant between 1994 and 2008 were included. A Cox proportional hazards model was used to determine whether donor SIDS was independently associated with post-transplant graft loss (death or retransplant). RESULTS: During the study period, 66 of 1033 infants (6.4%) who underwent heart transplant received an allograft from a SIDS donor. These infants were similar to the remaining infants with respect to age, diagnosis, blood type, and invasive support. In multivariable analysis, graft loss was associated with congenital heart disease (hazard ratio [HR], 1.6; 95% confidence interval [CI], 1.2-2.1), ventilator (HR, 1.4; 95% CI, 1.1-1.9), and extracorporeal membrane oxygenation support (HR, 3.0; 95% CI, 2.2-4.3), but not donor SIDS (HR, 1.0; 95% CI, 0.6-1.5), suggesting graft survival in SIDS-donor heart recipients was similar to the remaining infants. Primary causes of post-transplant death in infants receiving SIDS-donor hearts and the remaining infants were similar. CONCLUSIONS: Graft survival was similar in infants who received SIDS-donor hearts compared with those who received hearts from donors who died of other causes. There was no increase in incidence of non-rejection-related cardiac deaths after transplant in these children.

Spectrum of heart disease associated with murine and human GATA4 mutation.

The transcription factor GATA4 is essential for heart morphogenesis. Heterozygous mutation of GATA4 causes familial septal defects. However, the phenotypic spectrum of heterozygous GATA4 mutation is not known. In this study, we defined the cardiac phenotypes that result from heterozygous mutation of murine Gata4. We then asked if GATA4 mutation occurs in humans with these forms of congenital heart disease (CHD). In mice, heterozygous Gata4 mutation was associated with atrial and ventricular septal defect (ASD, VSD), endocardial cushion defect (ECD), RV hypoplasia, and cardiomyopathy. Genetic background strongly influenced the expression of ECD and cardiomyopathy, indicating the presence of important genetic modifiers. In humans, non-synonymous GATA4 sequence variants were associated with ECD (2/43), ASD (1/8), and RV hypoplasia in the context of double inlet left ventricle (1/9), forms of CHD that overlapped with abnormalities seen in the mouse model. These variants were not found in at least 500 control chromosomes, and encode proteins with non-conservative amino acid substitutions at phylogenetically conserved positions, suggesting that they are disease-causing mutations. Cardiomyopathy was not associated with GATA4 mutation in humans. These data establish the phenotypic spectrum of heterozygous Gata4 mutation in mice, and suggest that heterozygous GATA4 mutation leads to partially overlapping phenotypes in humans. Additional studies will be required to determine the degree to which GATA4 mutation contributes to human CHD characterized by ECD or RV hypoplasia.

Cardiac Registry screening for DiGeorge Critical Region deletion using loss of heterozygosity analysis.

DiGeorge (DGS), velocardiofacial, and conotruncal anomaly face syndromes comprise a phenotypic spectrum that is associated with a submicroscopic 22q11.2 deletion in the majority of cases. These syndromes variably express complex congenital heart disease, cellular immune deficits, hypocalcemia, craniofacial anomalies, and learning disabilities. This retrospective study correlates the presence of a deletion in this region with autopsy and clinical findings in a cohort of patients selected from the Cardiac Registry at Boston Children's Hospital. DNA was extracted from formalin-fixed paraffin-embedded cardiac tissue sampled from 189 patients with conotruncal anomalies. Polymerase chain reaction (PCR) was performed using 4 fluorescently labeled oligonucleotide primer pairs for unique short tandem repeat polymorphisms in the DGS critical region. The PCR products were analyzed for loss of heterozygosity (LOH), and a deletion was assumed when at least 3 consecutive loci demonstrated homozygosity. Of the 189 cases, 16 (8%) met our criteria for LOH and were assumed to have a deletion. These patients included 6 (35%) of 17 patients diagnosed clinically with DGS prior to death. Of the 10 non-DGS patients with LOH, 4 had aortic atresia and 3 had tetralogy of Fallot, both frequently seen in DGS. Polymerase chain reaction is a useful screening alternative to fluorescence in situ hydridization for detecting 22q11.2 deletions in archived tissue samples. This study identified a probable deletion in a subset of cases from a cardiac registry with cardiac defects associated with the DGS phenotype.

Connection between elastin haploinsufficiency and increased cell proliferation in patients with supravalvular aortic stenosis and Williams-Beuren syndrome.

To elucidate the pathomechanism leading to obstructive vascular disease in patients with elastin deficiency, we compared both elastogenesis and proliferation rate of cultured aortic smooth-muscle cells (SMCs) and skin fibroblasts from five healthy control subjects, four patients with isolated supravalvular aortic stenosis (SVAS), and five patients with Williams-Beuren syndrome (WBS). Mutations were determined in each patient with SVAS and in each patient with WBS. Three mutations found in patients with SVAS were shown to result in null alleles. RNA blot hybridization, immunostaining, and metabolic labeling experiments demonstrated that SVAS cells and WBS cells have reduced elastin mRNA levels and that they consequently deposit low amounts of insoluble elastin. Although SVAS cells laid down approximately 50% of the elastin made by normal cells, WBS cells deposited only 15% of the elastin made by normal cells. The observed difference in elastin-gene expression was not caused by a difference in the stability of elastin mRNA in SVAS cells compared with WBS cells, but it did indicate that gene-interaction effects may contribute to the complex phenotype observed in patients with WBS. Abnormally low levels of elastin deposition in SVAS cells and in WBS cells were found to coincide with an increase in proliferation rate, which could be reversed by addition of exogenous insoluble elastin. We conclude that insoluble elastin is an important regulator of cellular proliferation. Thus, the reduced net deposition of insoluble elastin in arterial walls of patients with either SVAS or WBS leads to the increased proliferation of arterial SMCs. This results in the formation of multilayer thickening of the tunica media of large arteries and, consequently, in the development of hyperplastic intimal lesions leading to segmental arterial occlusion.