Rare structural variation of synapse and neurotransmission genes in autism.

Autism spectrum disorders (ASDs) comprise a constellation of highly heritable neuropsychiatric disorders. Genome-wide studies of autistic individuals have implicated numerous minor risk alleles but few common variants, suggesting a complex genetic model with many contributing loci. To assess commonality of biological function among rare risk alleles, we compared functional knowledge of genes overlapping inherited structural variants in idiopathic ASD subjects relative to healthy controls. In this study we show that biological processes associated with synapse function and neurotransmission are significantly enriched, with replication, in ASD subjects versus controls. Analysis of phenotypes observed for mouse models of copy-variant genes established significant and replicated enrichment of observable phenotypes consistent with ASD behaviors. Most functional terms retained significance after excluding previously reported ASD loci. These results implicate several new variants that involve synaptic function and glutamatergic signaling processes as important contributors of ASD pathophysiology and suggest a sizable pool of additional potential ASD risk loci.

Serum alkaline phosphatase reflects post-Fontan hemodynamics in children.

Although survivors of Fontan palliation for a single ventricle are known to have lower cardiac index than patients with two-ventricle surgical reconstructions, it is unclear whether two frequently observed sequelae, short stature and protein-losing enteropathy (PLE), have hemodynamic origins. A serum marker that reflects hemodynamic status would be a tremendous asset in the long-term management of children with these sequelae. The authors recently noted severely reduced total alkaline phosphatase (TALP) levels in two children with early-onset PLE after Fontan operations, both of whom had low cardiac output at cardiac catheterization. Catheter-based or surgical interventions that rapidly increased cardiac output in these two patients resulted not only in relief of PLE but also in a prompt TALP rise. To examine whether the apparent correlation of low TALP with impaired cardiac output also is seen in Fontan patients without PLE, this study retrospectively examined the TALP data from two other Fontan patients who underwent cardiac catheterization specifically to assess the potential benefit of vasodilator therapy. The TALP levels were abnormally low in both cases but increased after up-titration of angiotensin-converting enzyme inhibition. Serum TALP activity, an indicator of osteoblastic function particularly in pre-adolescence, may be a marker of low cardiac output after a Fontan operation.

GATA4 sequence variants in patients with congenital heart disease.

BACKGROUND: Recent reports have identified mutations in the transcription factor GATA4 in familial cases of cardiac septal defects. The prevalence of GATA4 mutations in the population of patients with septal defects is unknown. Given that patients with septal and conotruncal defect can share a common genetic basis, it is unclear whether patients with additional types of CHD might also have GATA4 mutations. AIMS: To explore these questions by investigating a large population of 628 patients with either septal or conotruncal defects for GATA4 sequence variants. METHODS: The GATA4 coding region and exon-intron boundaries were investigated for sequence variants using denaturing high-performance liquid chromatography or conformation-sensitive gel electrophoresis. Samples showing peak or band shifts were reamplified from genomic DNA and sequenced. RESULTS: Four missense sequence variants (Gly93Ala, Gln316Glu, Ala411Val, Asp425Asn) were identified in five patients (two with atrial septal defect, two with ventricular septal defect and one with tetralogy of Fallot), which were not seen in a control population. All four affected amino acid residues are conserved across species, and two of the sequence variants lead to changes in polarity. Ten synonymous sequence variants were also identified in 18 patients, which were not seen in the control population. CONCLUSIONS: These data suggest that non-synonymous GATA4 sequence variants are found in a small percentage of patients with septal defects and are very uncommonly found in patients with conotruncal defects.

Loss-of-function mutations in growth differentiation factor-1 (GDF1) are associated with congenital heart defects in humans.

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.

Chromosome 22q11 deletion in patients with truncus arteriosus.

The association between truncus arteriosus and chromosome 22q11 deletion is well recognized, but the frequency of a chromosome 22q11 deletion has not been characterized in a large series of patients with truncus arteriosus, and little is known about cardiovascular morphologic features associated with a chromosome 22q11 deletion in this group of patients. We prospectively enrolled 50 consecutive patients with truncus arteriosus who were admitted to The Children's Hospital of Philadelphia between November 1991 and December 2001. Patients were studied for chromosome 22q11 deletion using fluorescence in situ hybridization. Correlations between anatomic features and chromosome 22q11 deletion were assessed. A chromosome 22q11 deletion was detected in 20 of the 50 patients (40%). Anatomic features that were significantly associated with a chromosome 22q11 deletion included a right-sided aortic arch, an abnormal aortic arch branching pattern, both abnormal sidedness and branching of the aortic arch, and the combined category of either abnormal sidedness or branching of the aortic arch. There was a trend toward the association of discontinuous pulmonary arteries with a chromosome 22q11 deletion. Interruption of the aortic arch and truncal valve morphology and function did not correlate significantly with the presence of a chromosome 22q11 deletion. In conclusion, a chromosome 22q11 deletion is common in patients with truncus arteriosus, and those with abnormal sidedness and/or branching of the aortic arch are significantly more likely to have a deletion. Clinically important anatomic variables, such as abnormalities of the truncal valve and interrupted aortic arch, were not associated with a chromosome 22q11 deletion in this series.

Cardiovascular anomalies in patients diagnosed with a chromosome 22q11 deletion beyond 6 months of age.

OBJECTIVE: Cardiovascular anomalies are present in 75% to 80% of patients with a chromosome 22q11 deletion. In the majority of cases, the cardiovascular defect becomes evident in the neonatal period and is often the initial manifestation of the chromosome 22q11 deletion syndrome. However, a 22q11 deletion may also be associated with cardiovascular defects that are less obvious, such as a vascular ring, which may not be diagnosed until the patient is older. The objective of this study was to determine the frequency and types of cardiovascular anomalies in patients diagnosed with a chromosome 22q11 deletion beyond 6 months of age. METHODS: We studied 29 patients diagnosed with a chromosome 22q11 deletion at a median age of 6.2 years (9 months to 45 years) who were subsequently referred for cardiovascular evaluation. Comprehensive cardiologic evaluation was performed, with transthoracic echocardiography (N = 28) and/or magnetic resonance imaging (N = 6), including imaging of the aortic arch. The frequency of cardiovascular anomalies diagnosed in these patients and the need for intervention were assessed. RESULTS: Cardiovascular anomalies were detected in 11 (38%) patients: 3 with a vascular ring formed by a right aortic arch with an aberrant left subclavian artery and left-sided ligamentum arteriosum, 3 with a right aortic arch with mirror-image branching of the brachiocephalic arteries (no vascular ring; 1 with a patent ductus arteriosus), 4 with a left aortic arch with an aberrant right subclavian artery (no vascular ring; 1 with a patent ductus), and 1 with a left superior vena cava draining to the coronary sinus. The median age at diagnosis in these 11 patients was 3 years (9 months to 28 years). The remaining 18 patients had normal cardiovascular anatomy. All 3 patients with vascular rings subsequently underwent surgical repair, and 1 patient with a ductus arteriosus underwent transcatheter coil occlusion. CONCLUSIONS: The frequency of cardiovascular anomalies necessitating intervention in patients referred for cardiovascular evaluation after diagnosis of a chromosome 22q11 deletion beyond 6 months of age is 14% in our experience. Routine screening for cardiovascular anomalies, including echocardiography and other imaging studies to identify the laterality and branching pattern of the aortic arch, is indicated in patients diagnosed with 22q11 deletion beyond 6 months of age and is particularly critical for patients with respiratory or feeding disorders.

NKX2.5 mutations in patients with tetralogy of fallot.

BACKGROUND: Recent reports have implicated mutations in the transcription factor NKX2.5 as a cause of tetralogy of Fallot (TOF). To estimate the frequency of NKX2.5 mutations in TOF patients and to further investigate the genotype-phenotype correlation of NKX2.5 mutations, we genotyped 114 TOF patients. METHODS AND RESULTS: Patients were recruited prospectively (November 1992 through June 1999) and tested for a 22q11 deletion; those with 22q11 deletion or recognized chromosomal alteration were excluded from the present study. Patients were screened for NKX2.5 alterations by conformation-sensitive gel electrophoresis and sequencing of fragments with aberrant mobility. Four heterozygous mutations were identified in 6 unrelated patients with cases of TOF, including 3 with pulmonary atresia and 5 with right aortic arch; none had ECG evidence of PR interval prolongation. Three of 4 mutations (Glu21Gln, Arg216Cys, and Ala219Val) altered highly conserved amino acids, of which 2 mapped in the conserved NK2 domain. The fourth mutation (Arg25Cys) was identified in 3 unrelated probands in the present study and has been previously reported. No homeodomain mutations were identified. CONCLUSIONS: NKX2.5 mutations are the first gene defects identified in nonsyndromic TOF patients. NKX2.5 mutation is present in >/=4% of TOF patients. Mutations identified in the present study mapped outside of the homeodomain, were not associated with atrioventricular conduction disturbances, and were not fully penetrant, in contrast to mutations previously reported that impair homeodomain function.

Patterns of right aortic arch and mirror-image branching of the brachiocephalic vessels without associated anomalies.

It is unusual to have a right aortic arch with mirror-image branching of the brachiocephalic vessels and no associated congenital cardiac anomalies. Pathogenetic factors of this anomaly, and associated development of the derivatives of the pharyngeal arches, have not been explored extensively. Eleven patients with a mirror-image right aortic arch and no intracardiac anomalies have undergone evaluation at our institution since 1987. Of these, 4 had stenosis (n = 2) or atresia (n = 2) of the proximal left pulmonary artery. These included 2 with bilateral ductus arteriosus. Six patients had a vascular ring formed by a ductus arteriosus or ligamentum arteriosum from the descending aorta to the left pulmonary artery. Six patients were evaluated for deletion within the DiGeorge critical region of chromosome 22q11. All 6 of the patients with a vascular ring underwent division of the ductus arteriosus or ligamentum through a left lateral thoracotomy. None of the patients with stenosis or atresia of the LPA have undergone intervention. Two of the 6 patients who underwent evaluation for chromosome 22q11 deletion were found to have the deletion. At a median follow-up of 7 years (15 months to 12 years), the patients who underwent division of the ring are all alive and asymptomatic. Two of these patients have undergone repeat magnetic resonance imaging, 1 of whom had mild residual tracheal compression. A right aortic arch with mirror-image branching of the brachiocephalic vessels and no associated intracardiac anomalies is extremely uncommon. We have observed two basic patterns of this lesion: (1) with associated abnormalities of the proximal left pulmonary artery, (2) with a vascular ring formed by a ductus arteriosus or ligamentum from the descending aorta to the left pulmonary artery. Pathogenetic considerations are explored, with a focus on flow-related vascular development and the role of deletions within chromosome 22q11.

Association of chromosome 22q11 deletion with isolated anomalies of aortic arch laterality and branching.

OBJECTIVES: The purpose of this study was to determine the frequency of chromosome 22q11 deletions in patients with isolated anomalies of the aortic arch and its branches. BACKGROUND: Chromosome 22q11 deletions are often present in patients with certain forms of congenital cardiovascular disease, including tetralogy of Fallot, truncus arteriosus and interruption of the aortic arch. Among patients with these anomalies, chromosome 22q11 deletion is more common in those with abnormal aortic arch laterality or branching. METHODS: We studied 66 patients with isolated anomalies of the aortic arch and no associated intracardiac defects for deletions within chromosome 22q11, using fluorescence in situ hybridization with the cosmid probe N25 (D22S75). Arch anomalies included: double aortic arch (n = 22); right aortic arch with aberrant left subclavian artery (n = 28); right aortic arch with mirror-image branching and a vascular ring formed by a left-sided ductus from the descending aorta (n = 5); right aortic arch with mirror-image branching and no vascular ring (n = 4); and left aortic arch with aberrant right subclavian artery (n = 7). In addition, four patients had a cervical aortic arch, four had aortic coarctation and six had hypoplasia/atresia of the proximal pulmonary arteries. RESULTS: Chromosome 22q11 deletions were found in 16 patients (24%) across the full spectrum of anomalies studied. Among the morphologic variables analyzed, only hypoplasia/atresia of the proximal pulmonary arteries correlated with the deletion (p = 0.03). Among patients with a double arch, the frequency of chromosome 22q11 deletion was higher in those with an atretic minor arch than it was in those with a patent minor arch (p = 0.02). CONCLUSIONS: Chromosome 22q11 deletion is associated with isolated anomalies of laterality or branching of the aortic arch in 24% of cases in our series. These findings should alert the clinician to consider deletion screening in patients with isolated anomalies of the aortic arch.

The epidemiology and genetics of congenital heart disease.

The studies summarized demonstrate that CHD is a common, major malformation. The genetic cause of each specific lesion is heterogeneous. In addition, different types of CHD can result from the same chromosomal alteration or from mutations in the same gene. Although one might predict that genotype influences clinical outcome, further studies are required. At this time, routine clinical diagnostic tests to identify the specific genetic cause are available in only a few cases, namely, those with abnormal karyotypes or those with a 22q11 deletion. In those cases with single-gene defects, genetic testing is not clinically available at this time and most likely will not become available until we can predict the significance of each mutation and until technologic advances are made that allow for large-scale, accurate screening. In the meantime, continued research on the genetic cause of CHD promises to augment our understanding of the mechanisms underlying the normal and abnormal development of the cardiac structures. These investigations also promise to augment our ability to counsel families on the recurrence risk with greater accuracy and, in the future, will allow the physician to modify his or her clinical management based on genetic cause. Finally, identifying the cause and understanding the disease mechanism allows for early intervention that may modify the degree of cardiac maldevelopment or avoid cardiac malformation altogether.

Recent advances in understanding the genetic etiology of congenital heart disease.

The genetic etiologies of multiple cardiovascular disorders have been identified recently. For the most part, familial cardiomyopathic, vascular, or arrhythmogenic disorders have been studied given the opportunity to identify the disease gene by linkage analyses, positional cloning, and analysis of candidate genes. Given that structural congenital heart disease rarely occurs in the context of large families, alternative approaches to understand the possible genetic etiologies have been taken. In particular, molecular evaluations of genetic syndromes in which cardiac defects are a cardinal feature are providing new insights into disease-related genes and developmental pathways. The identification of rare families with multiple affected members also has provided some insight into the genetic contribution to structural congenital heart defects. This review highlights the newest findings on the genetic etiology or implications in each of the subcategories of congenital cardiovascular disorders, and will provide the reader with both a brief overview and update. Particular note will be made of the genotype/phenotype analyses of hypertrophic cardiomyopathy and the long QT syndromes, as well as the identification of new disease-related genes for dilated cardiomyopathy, idiopathic ventricular fibrillation, and structural heart disease.

Cognitive and behavior profile of preschool children with chromosome 22q11.2 deletion.

A microscopic deletion of chromosome 22q11.2 has been identified in most patients with the DiGeorge, velocardiofacial syndrome, conotruncal anomaly face syndrome, and in some patients with isolated conotruncal cardiac anomalies. This study presents the neurodevelopmental outcome, including cognitive development, language development, speech, neuromuscular development, and behavioral characteristics of 40 preschool children (ages 13 to 63 months) who have been diagnosed with the 22q11.2 deletion. The impact of cardiac disease, cardiac surgery, and the palatal anomalies on this population was also studied. In the preschool years, children with a 22q11.2 deletion are most commonly found to be developmentally delayed, have mild hypotonia, and language and speech delays. The more significantly delayed children are at high risk to be subsequently diagnosed with mild or moderate mental retardation. The global delays and the variations in intelligence found are directly associated with the 22q11.2 deletion and are not explained by physical anomalies such as palatal defects or cardiac defects, or therapeutic interventions such as cardiac surgery. Our findings demonstrate that there is a pattern of significant speech disorders within this population. All of the children had late onset of verbal speech. Behavioral outcomes included both inhibition and attention disorders. Early intervention services are strongly recommended beginning in infancy to address the delays in gross motor skills, speech and language, and global developmental delays.

Jagged1 mutations in patients ascertained with isolated congenital heart defects.

Mutations in Jagged1 cause Alagille syndrome (AGS), a pleiotropic disorder with involvement of the liver, heart, skeleton, eyes, and facial structures. Cardiac defects are seen in more than 95% of AGS patients. Most commonly these are right-sided defects ranging from mild peripheral pulmonic stenosis to severe forms of tetralogy of Fallot. AGS demonstrates highly variable expressivity with respect to all of the involved systems. This leads us to hypothesize that defects in Jagged1 can be found in patients with presumably isolated heart defects, such as tetralogy of Fallot or pulmonic stenosis. Two patients with heart defects of the type seen in AGS and their relatives were investigated for alterations in the Jagged1 gene. Jagged1 was screened by a combination of cytogenetic and molecular techniques. Patient 1 was studied because of a four-generation history of pulmonic stenosis. Molecular analysis showed a point mutation in Jagged1 in the patient and her mother. Patient 2 was investigated owing to the finding of tetralogy of Fallot and a "butterfly" vertebra on chest radiograph first noted at age 5 years. She was found to have a deletion of chromosome region 20p12 that encompassed the entire Jagged1 gene. The identification of these two patients suggests that other patients with right-sided heart defects may have subtle findings of AGS and Jagged1 mutations.

The Philadelphia story: the 22q11.2 deletion: report on 250 patients.

A submicroscopic deletion of chromosome 22q11.2 has been identified in the majority of patients with the DiGeorge, velocardiofacial, and conotruncal anomaly face syndromes, and in some patients with the Opitz G/BBB and Cayler cardiofacial syndromes. We have been involved in the analysis of DiGeorge syndrome and related diagnoses since 1982 and have evaluated a large number of patients with the deletion. We describe our cohort of 250 patients whose clinical findings help to define the extremely variable phenotype associated with the 22q11.2 deletion and may assist clinicians in providing genetic counseling and guidelines for clinical management based on these findings.

Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.

We have studied 92 patients with Alagille syndrome (AGS) to determine the frequency of clinical manifestations and to correlate the clinical findings with outcome. Liver biopsy specimens showed paucity of the interlobular ducts in 85% of patients. Cholestasis was seen in 96%, cardiac murmur in 97%, butterfly vertebrae in 51%, posterior embryotoxon in 78%, and characteristic facies in 96% of patients. Renal disease was present in 40% and intracranial bleeding or stroke occurred in 14% of patients. The presence of intracardiac congenital heart disease was the only clinical feature statistically associated with increased mortality (P <.001). Initial measures of hepatic function in infancy including absence of scintiscan excretion were not predictive of risk for transplantation or increased mortality. The hepatic histology of these AGS patients showed a significant increase in the prevalence of bile duct paucity (P =.002) and fibrosis (P <.001) with increasing age. Liver transplantation for hepatic decompensation was necessary in 21% (19 of 92) of patients with 79% survival 1-year posttransplantation. Current mortality is 17% (16 of 92). The factors that contributed significantly to mortality were complex congenital heart disease (15%), intracranial bleeding (25%), and hepatic disease or hepatic transplantation (25%). The 20-year predicted life expectancy is 75% for all patients, 80% for those not requiring liver transplantation, and 60% for those who required liver transplantation.

Frequency of 22q11 deletions in patients with conotruncal defects.

OBJECTIVES: This study was designed to determine the frequency of 22q11 deletions in a large, prospectively ascertained sample of patients with conotruncal defects and to evaluate the deletion frequency when additional cardiac findings are also considered. BACKGROUND: Chromosome 22q11 deletions are present in the majority of patients with DiGeorge, velocardiofacial and conotruncal anomaly face syndromes in which conotruncal defects are a cardinal feature. Previous studies suggest that a substantial number of patients with congenital heart disease have a 22q11 deletion. METHODS: Two hundred fifty-one patients with conotruncal defects were prospectively enrolled into the study and screened for the presence of a 22q11 deletion. RESULTS: Deletions were found in 50.0% with interrupted aortic arch (IAA), 34.5% of patients with truncus arteriosus (TA), and 15.9% with tetralogy of Fallot (TOF). Two of 6 patients with a posterior malalignment type ventricular septal defect (PMVSD) and only 1 of 20 patients with double outlet right ventricle were found to have a 22q11 deletion. None of the 45 patients with transposition of the great arteries had a deletion. The frequency of 22q11 deletions was higher in patients with anomalies of the pulmonary arteries, aortic arch or its major branches as compared to patients with a normal left aortic arch regardless of intracardiac anatomy. CONCLUSIONS: A substantial proportion of patients with IAA, TA, TOF and PMVSD have a deletion of chromosome 22q11. Deletions are more common in patients with aortic arch or vessel anomalies. These results begin to define guidelines for deletion screening of patients with conotruncal defects.

Molecular characterization of a serine/threonine kinase in the DiGeorge minimal critical region.

The majority of patients with DiGeorge, velocardiofacial or conotruncal anomaly facial syndromes share a common genetic etiology, deletion of chromosomal region 22q11.2. This report describes a computational approach toward the identification and molecular characterization of a newly identified serine/threonine kinase from the minimal critical deleted region (MDGCR). A cosmid contig of the minimal critical region has been assembled and sequenced in its entirety. Database searches and computer analysis of one cosmid (111f11) for coding sequences identified two regions with high similarity to the mouse serine/threonine kinase, Tsk1. Our investigations demonstrate that one of these regions contains a testis-specific gene that undergoes differential splicing, while the other region is most likely a pseudogene. Northern blot analysis and cDNA cloning demonstrate that there is alternate processing of the 3'UTR without altering the conserved kinase domains within the open reading frame. Serine/threonine kinases can play a regulatory role and have been found to be expressed during early embryogenesis. Based on its position in the MDGCR and possible function, the gene reported here is a candidate for the features seen in the 22q11 deletion syndrome.

Comparison of patterns of pulmonary venous blood flow in the functional single ventricle heart after operative aortopulmonary shunt versus superior cavopulmonary shunt.

In this study we investigated the patterns of pulmonary venous flow in children with functional single ventricles to obtain a better understanding of the determinants of transpulmonary blood flow. Sixty-eight patients with functional single ventricles and aortopulmonary shunt (n = 34, group I), or superior cavopulmonary connection (n = 34, group II) underwent transesophageal Doppler echocardiographic assessment of flow in the left upper pulmonary vein before undergoing the next stage of surgery. Twelve patients from group II also underwent simultaneous evaluation of superior vena caval flow. Biphasic forward pulmonary venous flow was noted in 62 patients in sinus rhythm (S wave in systole, D wave in diastole); in 6 patients with junctional rhythm, significant early systolic reversal of flow was present. Both the S- and D-wave velocity-time integrals (VTI) were greater in group I than in group II (S(VTI) 9.9 +/- 4.2 vs 8.0 +/- 2.6, p = 0.02; D(VTI) 8.0 +/- 3.5 vs 4.2 +/- 2.6, p <0.001). In both groups, pulmonary venous flow was predominantly systolic; however, the proportion of flow during ventricular systole was significantly greater in group II than in group I (S(VTI)/D(VTI) group II: 2.4 +/- 1.5; group I 1.4 +/- 0.5, p = 0.001; percent systolic fraction of pulmonary venous flow group II = 67%, group I = 56%, p <0.001). Analysis of superior vena caval flow in group II revealed a single predominant wave with onset at early systole and peak in late systole at a mean of 150 ms after the pulmonary venous S-wave peak. Our data suggest that ventricular systole (i.e., atrial relaxation, atrioventricular valve descent) asserts great influence on transpulmonary blood flow in the functional single ventricle.