Congenital heart disease (CHD) is the leading noninfectious cause of death in the first year of life (Huang et al., 2010) and is responsible for more deaths than any other type of malformation (Mahle et al., 2009). It affects 1% of all live births. Its significant contribution to pediatric morbidity and mortality has led to efforts toward a better understanding of the etiology and nature of CHD. Although there have been tremendous advances in the diagnosis and treatment of CHD, knowledge of the causes of CHD has been limited and significant morbidity and mortality are still associated with some types of CHD (Pierpont et al., 2007). Therefore, epidemiological studies to elaborate the heritable nature of CHD are necessary in order to improve the understanding of the disease and hence improve management protocols. About 3–5% of CHDs are associated with recognizable genetic syndromes, which are caused by teratogens, a chromosome abnormality, or a single gene disorder. A further significant proportion is associated with other congenital malformations of unknown etiology (Mitchell et al., 2007). Hence, study of the genetic background of syndromic CHD and its interpretation has become increasingly important. To reduce the morbidity and mortality of CHD, the diagnosis of syndromic CHD is extremely important as the prognosis for a child with a congenital cardiac malformation depends not only on the severity of the heart defect, but on whether an underlying syndrome is present that would have implications for other medical issues or cognitive development. Patients with additional extracardiac anomalies and/or associated genetic syndromes are at a greater risk for significant perioperative mortality (Marino and Digilio, 2000). Therefore, a careful evaluation to exclude other major malformations or minor anomalies and dysmorphic features that might lead to the diagnosis of a syndrome is important in every patient with CHD. Because of realization of the fact that many patients with CHD may have subtle dysmorphic features that may be missed unless genetic testing is performed, the concept of genetic testing has become increasingly recognized in the previous years (Goldmuntz, 2004). A genetic evaluation provides a more global approach to the patient and allows the diagnosis and detection of a genetic pattern in individuals with CHD associated with multiple anomalies. It therefore provides an advanced insight into the heritable nature of syndromic CHD and, hence, allows the definition of disease risk and critical elements for disease prevention and to facilitate risk assessment for genetic counseling (Pierpont et al., 2007) – a mandatory step in the efforts to reduce the incidence of CHD and thus the morbidity and mortality rate in infancy and childhood. The understanding of the etiology of congenital cardiac lesions is rapidly progressing from the recognition of embryologic origins to insights into the genetic basis for these disorders. Molecular and genetic analysis of heart development has led to gene discoveries, the identification of essential cardiac regulators and mutations that are linked to human CHD (Nemer, 2008), and are being rapidly translated into meaningful genetic testing. But very few CHD-causing genes have been identified so far. Much remains to be defined. The analysis of genetic syndromes, candidate genes, and novel mutations is crucial to a better understanding of the clinical implications of specific mutations. This will allow not only for more sensitive and specific diagnoses to be made but also for improvements in therapeutics options and efficacy (Calderon Colmenero, 2007).
Aim of the work
This study aims to perform clinical evaluation and genotyping of children with congenital structural heart defects associated with other malformations and/or dysmorphic features to assess the presence of a genetic syndrome on the basis of a high index of suspicion. We hypothesize that a genetic abnormality can be detected in children with structural congenital heart defects associated with other malformations and/or dysmorphic features, and a diagnosis of a syndrome can be made. We were particularly interested to diagnose syndromes that are usually not diagnosed and the patient is managed with respect to the cardiac condition alone. We aimed to provide possible explanations for a delayed genetic diagnosis and to describe the challenges of genetic evaluation.
All newborns, infants, children, and teenagers up to the age of 18 who attended the Medical Genetics Clinic over a 12-month time period were recruited into the study, provided they presented with a congenital structural heart defect associated with other/multiple congenital anomalies and/or dysmorphic features and/or neurocognitive deficits. Our cohort included a total of 66 newborns, infants, children, and teenagers up to the age of 18. This is a descriptive, cross sectional study in which every patient was evaluated clinically and genetically and subjected to detailed medical history, clinical examination, laboratory investigations, and diagnostic imaging to establish a working diagnosis. Information and test results obtained served as the basis for counseling and longitudinal care. Consultations with specialists in relevant fields were sought to clarify diagnostic possibilities. Positive findings in the family history were confirmed by a review of medical records and/or family photographs when available. On the basis of the initial impression, patients were categorized into two categories: (a) multiple malformations, recognizable pattern (syndrome identification), and (b) multiple malformations, pattern not recognized. Diagnostic tests were performed to clarify or establish a clinical diagnosis. Genetic tests were selected in a prioritized order according to the index of suspicion and differential diagnosis and, accordingly, the appropriate genetic tests were performed. Initial results were used to guide the selection of subsequent and more specific tests.
In the present study, children with structural CHD were subjected to genetic evaluation and were assessed for the presence of a cardiac syndrome with particular attention to those syndromes that are more likely to escape a diagnosis. Children were initially classified into a group with a recognizable pattern for a syndrome and a nonrecognizable pattern.
Sixty-six children presented with structural congenital heart and extracardiac anomalies over a 12-month time period; a genetic defect was detectable in 65 of 66 children. The genetic defect was consistent with the diagnosis of syndromic CHD in 63 out of 66 children. Two probands showed a nonrecognizable pattern of malformations associated with a genetic defect not inconsistent with a genetic syndrome. In one child with a clinical diagnosis of a cardiac syndrome, a genetic defect was not detectable.
Syndromes identified in our cohort include Marfan syndrome (MS; n=17), Down syndrome (DS; n=12), DiGeorge syndrome (DGS; n=9), Shprintzen syndrome (SS; n=2), Noonan syndrome (NS; n=4), Turner syndrome (TS; n=4), Cornelia de Lange syndrome (n=3), Wolf Hirschorn syndrome (n=3),), Holt–Oram syndrome (HOS; n=3), Alagille syndrome (AS; n=2), Williams syndrome (WS; n=2), Cri du Chat syndrome (n=1), Rubinstein–Taybi syndrome (RTS; n=1), cardiofacialcutaneous syndrome (n=1), deletion 8p syndrome (n=1), and duplication 15q syndrome (n=1). In one child, although clinically diagnosed with a cardiac syndrome, that is, MS, a genetic defect was not detectable.
The most common cardiac defect presented in our cohort was ventricular septal defect (VSD) (27%), followed by aortic dilatation (24%), followed by tetralogy of fallot (TOF) (9%), coarctation of aorta (COA) (9%), complete atrioventricular canal defect (AVCD) (9%), and pulmonary stenosis (PS) (7.5%). The least common cardiac defects were aortic stenosis (AS) (3%), peripheral pulmonary stenosis (PPS) (1%), and truncus arteriosus (1%).
Associated abnormalities and features were prominent and a syndrome was identified on the basis of a recognizable pattern in almost all children (97%).
Among a cohort of 66 children with CHDs, four probands presented with subtle extracardiac features and hence milder forms of DGS (with TOF), AS (TOF), MS (MVR), and TS (MVR).
Genetic screening of our study cohort showed that a genetic diagnosis of cardiac syndrome can be challenging and syndromic features are not uncommonly overlooked. The observation of unique pattern, age-dependent features, nonclassic phenotypes, genetic/phenotypic heterogeneity, and the subtlety of features may be confusing and is greatly responsible for the difficulty in making an accurate and timely diagnosis of a cardiac syndrome.
Subtlety of features was found to be a major challenge and has proven to be the main reason why cardiac syndromes are not recognized and elude diagnosis, and patients are managed with respect to the cardiac condition alone. Among our probands and their parents, affected individuals presented for a genetic evaluation at older ages despite a recognizable pattern and prominent features. This observation shows that a cardiac syndrome may remain undiagnosed beyond school age independent of the severity of the heart disease. This may be due to age-dependent phenotypic or behavioral manifestations, where the features would not appear before a certain age. Another cause for the delayed recognition of a syndrome might be the uniqueness of the pattern of manifestations or genotype/phenotype heterogeneity. Features may be so subtle that they are missed or not even searched for. Families may not recognize those features and describe them as ‘running in the family’ as described below in a child with AGS. It is the subtlety of features and their severity that seemed to delay or draw attention to a syndrome’s existence rather than the pattern of features. This may also explain why parents of affected children sometimes are not diagnosed until their children are (Pierpont et al., 2007). In these situations, the child presents with a more severe form of the disease and more prominent features than their parents, whose condition was overlooked into adulthood. Syndromes that showed this phenomenon in our study population included MS (two fathers and a mother of three patients), DGS (one patient, two fathers, and one mother of three patients), SS (one father), AS (one patient and a father of two affected siblings), NS (one father), and TS (one patient). In agreement with our conclusions, Goldmuntz (2004) had reported that a syndrome may display a nonrecognizable pattern if the features are so mild that they may be overlooked or do not meet the criteria of diagnosis. Therefore, the physician’s awareness and knowledge regarding the variable presentation and severity of cardiac syndromes’ features is a critical factor. But even with suspicion and a clinical diagnosis, a final diagnosis cannot be established unless a genetic test confirms a genetic alteration (Goldmuntz, 2004).
MS was the most common cardiac syndrome diagnosed in our study population. One patient had a neonatal Marfan syndrome (MS) presentation. Of the remaining 16 patients, only five (29.4%) were <10 years of age at their last follow-up evaluation and 12 (70.6%) were between 10 and 17 years of age. The presentation at later ages is based on the fact that the majority of clinical manifestations of MFS increase with age and our findings confirm the limited usefulness of international criteria for diagnosis in early infancy as discussed by Faivre et al. (2009) and emphasize the value of FBN1 mutation screening, which confirms the diagnosis. Forty-one percent presented with probable MFS (defined on the basis of unfulfilled clinical international criteria in childhood, i.e., before 18 years of age) and had been followed up by cardiologists without referral for a genetic diagnosis.
In the MFS group, 11 cases (64.7%) were inherited in an autosomal dominant manner and six cases were de-novo mutations. Among the 11 familial cases, only eight patients (47%) had a family history of MFS at the time of presentation, which means that four families were not aware of the existence of their genetic condition. Out of these 11 patients’ parents, three parents, the fathers and mother, respectively, were found to harbor the disease-causing mutation only after their sons with CHD had been diagnosed with the disorder. On further analysis, the parents affected by MS were found to have subtle syndromic features, which were not diagnosed previously and were considered insufficient to meet the diagnostic criteria unless a complete and thorough genetic evaluation was performed. Parents transmitted the genetic defect to their offspring, in whom it was expressed as a more prominent and recognizable phenotype. Our findings demonstrate the phenomenon of marked interfamilial and intrafamilial variability that MS is known for and that suggests the influence of epigenetic factors (Rand-Hendriksen et al., 2007).
The test method applied to establish a definite genetic diagnosis was FBN1 sequence analysis, which has a detection rate of 70–93% (Dietz, 2009), and could detect mutations in all our candidates with a clinical diagnosis. All patients shared a common genetic origin, that is, an FBN1 mutation/deletion with variable locus and mutation type within the gene at locus 15q21.1. Fifteen mutations (88%) were in-frame mutations, whereas two (12%) generated a premature termination codon. Among the missense mutations, seven (46%) of 15 involved cysteine (six replacing cysteine and one creating cysteine). Three mutations (20%) were located in exons 24–32. A genotype–phenotype correlation was not determined for this group. Similarly, intensive international efforts had also failed to establish genotype–phenotype correlations, with the exception of an apparent clustering of neonatal mutations between exons 24 and 32 of FBN1 (De Backer, 2009). No significant phenotypic differences for children with mutations in exons 24–32, compared with children with mutations located elsewhere, were found in our study. We therefore agree with Rand-Hendriksen et al. (2007) that some individuals with this severe presentation may not have identifiable mutations in this region and that many other individuals with mutations in this region have classic or even mild variants of MS. The observation that mutations, which create a premature termination codon and result in rapid degradation of mutant transcripts, can be associated with mild conditions and may fail to meet the diagnostic criteria for MS (Dietz, 2009). We made this same observation in a patient who had a deletion resulting in a stop codon and presented with subtle features and a mild presentation. However, one patient harbored a mutation causing a stop codon, but presented with the classical form of the disease. Substitution of amino acids with intuitive functional significance, such as cysteines that participate in intramolecular linkages and residues that govern the calcium-binding affinity of epidermal growth factor-like domains, tends to cause MS of variable severity. Seven patients in our study population presented with classical moderate forms of the syndrome and variable severity along with genetic alterations in the form of cysteine substitutions. Our findings show that the complete loss of one FBN1 allele does not predict a mild phenotype, and supports the hypothesis that true haploinsufficiency can lead to the classical phenotype of MS as recently confirmed by Hilhorst-Hofstee et al. (2011).
Subtlety of features was also seen in the patient group diagnosed with 22qdeletion syndrome. These probands presented with a recognizable pattern consistent with DGS/SS and cardiac phenotypes including TOF, VSD, truncus arteriosus, and coarctation of aorta. All of these cardiovascular defects are among the most common cardiovascular defects associated with a 22q11 deletion (del 22q11) (Fomin et al., 2010). Features were prominent in all our probands, except for patient 26, a 6-year-old girl, who presented with learning difficulties at school and had been managed for TOF since infancy. The challenge that subtle features impose in diagnosis is especially evident in the parents of this group. Parents of four patients harbored the genetic defect and were diagnosed with del 22q11 only after their child with CHD had been diagnosed with the syndrome. On further evaluation and analysis, these parents were found to have subtle syndromic features that had not been recognized previously, which is a not rare phenomenon for 22qdel syndrome (Yang et al., 2009). A genetic diagnosis of probands and parents was confirmed by fluorescent in situ hybridization (FISH) and all nine patients in our cohort with clinical features of a DiGeorge phenotype, except patient 21, showed a 22q11.2 deletion of slightly variable sizes. The genetic heterogeneity that characterizes the 22qdeletion syndrome explains the inability to find a genetic origin. Possibilities are that the patient does not have a mutation in the tested gene and sequence alteration exists in another gene at another locus or the patient has a sequence alteration that could not be detected using the tests applied.
A 13-year-old girl with short stature and mitral valve regurge was considered to have a rather subtle presentation. She sought medical advice only because of delayed thenarche and was not aware of the cardiac defect until further examination detected it. As anomalies were not prominently obvious, her features can be considered as subtle, which would explain the delayed diagnosis until other age-dependent signs and symptoms appear and allow a diagnosis to be made. On karyotyping, she showed an XO genotype, the complete absence of a second sex chromosome. TS is another cardiac syndrome in which the age dependence of features causes a delay in diagnosis.
Genetic testing of first-degree family members revealed affected individuals with subtle features, parents as well as siblings. A 6-month-old male infant presented with peripheral pulmonary stenosis and typical facial features for AS. His 6-year-old brother, who had a history of TOF, was recruited for assessment and showed subtle facial features. He had never been genetically evaluated as he was considered to ‘look like his father’. The brothers were clinically diagnosed with AS. Interestingly, the brothers had the same genotype (JAG1 gene: c.686G>A) detected by DNA sequencing but showed different phenotypes. Phenotypic variability among the brothers is an example of the highly variable expressivity demonstrated by AGS, with clinical features ranging from subclinical to severe. Modifiers leading to organ-specific features are suggestive of being responsible for the heterogeneous behavior with complete haploinsufficiency of missense mutants (Bauer et al., 2010). Mutations of JAG1 have been identified in patients with a broad spectrum of clinical phenotypes of AS, including patients with a predominant cardiac phenotype (Spinner et al., 2010). More than 90% of individuals with the classic phenotype of AS have a JAG1 mutation (Hofmann et al., 2010). No genotype–phenotype correlation exists between clinical manifestations of AGS and specific JAG1 mutation types or location within the gene (McElhinney et al., 2003).
Intrafamilial and interfamilial variability of expression is another reason why family members of affected individuals remain undiagnosed. NS is a cardiac syndrome with frequently subtle features that vary in expressivity. Many affected adults are diagnosed only after the birth of a more obviously affected infant (Pierpont et al., 2007). We observed the father of one proband with NS who was tested after his son’s diagnosis as part of the son’s genetic evaluation and found to have the disease-causing mutation. On examination, the father was found to have a pointed chin and a triangular but full face, difficult to detect unless suspected and looked for. The cardiac involvement observed in all four probands with clinical features of NS was valvar pulmonary stenosis. The same genetic basis, a PTPN11 mutation, was detected by sequencing analysis in all four probands with NS. PTPN11 encodes protein tyrosine phosphatase SHP-2, which plays an important role in signal transduction for a wide variety of biological processes, including the formation of the semilunar valves. Therefore, PTPN11 mutations are more likely to be found when pulmonary valve stenosis is present, and are observed in 40–50% of NS patients (Wolf and Basson, 2010). Very recently, Allanson et al. (2010) determined in their study that some individuals with mutations in the most commonly affected gene, PTPN11, which is correlated with the cardinal physical features, may have a quite atypical face. Conversely, some individuals with KRAS mutations, which may be associated with a less characteristic intellectual phenotype and a resemblance to Costello and cardiofaciocutaneous syndromes, can have a very typical face. Thus, the facial phenotype alone is insufficient to predict the genotype, but certain facial features may facilitate an educated guess in some cases. The cardiac phenotype, namely pulmonary stenosis with dysplasia, is more predictive of a genotype (Romano et al., 2010).
Another challenge to genetic screening and establishing a straightforward diagnosis is the existence of unknown patterns. A 9-year-old girl presented with learning difficulties and prominent dysmorphic features including a small upturned nose and hypotelorism. Her history revealed TOF, for which she had been seen by pediatric cardiologists since her infancy. The pattern was nonrecognizable for a specific syndrome and her karyotype was negative. A later comparative genomic hybridization (CGH) diagnosed a de-novo 1.1 Mb deletion of Chromosome 2p. CGH was also the test of choice for the second patient with a nonrecognizable pattern: a 16-year-old boy, who presented with dysmorphic features such as large ears, small philtrum, and prominent and large upper central incisors. Further examination and imaging revealed a VSD. Karyotyping was negative and a subsequent CGH diagnosed a de-novo 4p22 deletion suggested by clinical findings and confirmed by detection of a deletion of the most distal band of the short arm of chromosome 4.
Atypical, nonclassic phenotypes and/or unique patterns of features are a challenge to making a diagnosis. This phenomenon has been studied in RTS and was observable in our study population, emphasizing the importance and complexity of genotype–phenotype heterogeneity. A 6-year-old girl was diagnosed with RTS on the basis of characteristic facial dysmorphia, broad thumb and hallux, and VSD with an unusual normal IQ. Classical and nonclassical forms of RTS have been described, with the nonclassical form being a milder form, lacking skeletal manifestation and with improved mental functions (Bartholdi et al., 2007; Zimmermann et al., 2007). Recently, an ‘incomplete’ form has been described as another nonclassic presentation of RTS, characterized by MR, skeletal features, and characteristic but incomplete facial features (Li and Szybowska, 2010). Over 56 mutations within the CREBBP gene have been linked to RTS (Van Belzen et al., 2011) compared with only eight mutations in EP300 (Roelfsema et al., 2005; Bartholdi et al., 2007; Zimmermann et al., 2007; Foley et al., 2009; Bartsch, 2010a; Tsai et al., 2011 and Van Belzen et al., 2011). The fact that only very few mutations have been found to date in EP300 might be because EP300 has been studied in a smaller subset of RTS patients and EP300 mutations present with unique phenotypes that have not been associated and recognized as forms of RTS. There is an increasing observation that carriers of EP300 mutations show a milder phenotype, showing normal hands and feet and normal mental development. It remains unknown whether EP300 mutations are responsible for milder or nonclassical forms of RTS or whether EP300 haploinsufficiency results in a unique phenotype.
Sequence analysis/mutation scanning detects CREBBP mutations in another 30–50% of affected individuals, whereas mutations in EP300 are identified in approximately 3% of individuals with RTS. The mutation detection rate in RTS is generally 50%. Possibilities are that the mutations reside in parts of the genes that are not screened, such as noncoding regulatory sequences, or mutations occur in other genes. The recent finding of mosaic patients leads to the idea, that mosaicism might explain the failure to detect mutations. (Bartsch et al., 2010b). Nevertheless, several missing mutations would need to be explained by a phenomenon that is generally considered rare. CREBBP and EP300 encode paralogous nuclear histone/factor acetyltransferases (HATs) that activate signal-dependent transcription through acetylation of histones and DNA-binding transcription factors and are important for chromatin remodeling, cell differentiation, and proliferation, and the HAT domain of the genes is crucial o their function (Schorry et al., 2008). Somatic mosaicism is increasingly being considered to be responsible for the low mutation detection rate as well as expression variability (Bartsch et al., 2010b). On the basis of the fact that our proband presented with a nonclassic form of RTS, EP300 sequencing analysis was carried out and a novel point mutation was identified in our proband and her mother with RTS in the HAT domain of EP300, the first reported missense mutation in this gene (at position 3386 A>G in exon 15, changing isoleucine to valine). Our finding confirms the genetic heterogeneity of RTS and the contributing role of EP300 in the phenotypic heterogeneity of this syndrome. The mutation is expected to affect HAT activity and/or p300 protein stability.
Another challenge to the diagnosis of a cardiac syndrome is the fact that there is not always a straight link from the genetic cause to the phenotype, and it can be seen in our study population that the same etiologic cause may produce dissimilar anatomic defects. Del 22q11 may cause VSD, TOF, and CoA, but also TA and interrupted aortic arch. The same findings have been confirmed by Marino and Digilio (2000). HOS presented with ASD as well as VSD. AS presented with peripheral pulmonary stenosis but also TOF.
Moreover, morphologically similar anomalies may result from markedly different genetic causes. Atrioventricular canal defects, ASD, and VSD are frequently due to trisomy 21, del 22q11, HOS, and RTS but may also occur in relation to other chromosome defects or may be present in nonsyndromic patients (Chehab et al., 2010). Moreover, Tetralogy of Fallot can be caused by a 22q11 deletion or other syndromes and can be present in nonsyndromic children (Griffin et al., 2010). But if we consider the anatomic subtypes of these cardiac defects, the associations with the single genetic conditions become more specific and specific congenital heart defects can be associated with specific syndromes as indicated earlier by Marino and Digilio (2000), and we can see that peculiar anatomic subtypes are related to specific genetic conditions. The phenotypical evaluation of patients with atrioventricular canal defects may reveal a huge variety of chromosomal abnormalities, Mendelian syndromes, and malformation complexes (Chehab et al., 2010). The majority of children with atrioventricular canal defect have DS, and the atrioventricular canal defect is the ‘classic’ heart defect in children with DS (Chehab et al., 2010). Muscular ventricular septal defect, seen in our probands with HOS, is a congenital heart malformation specifically associated with HOS and is quite rare in children with DS (McDermott et al., 2004). This can be explained by the fact that TBX5 expression is particularly evident at the atrial level and in the left-sided endocardium of the ventricular septum (Bruneau et al., 1999), whereas a perimembranous ventricular septal defect is thought to be related to anomalies of the intracardiac blood flow (Bruneau et al., 1999). In children with del 22q11, the presence of a subarterial ventricular septal defect is frequently associated with the right or the cervical aortic arch with an aberrant subclavian artery consisting of a peculiar cardiac pattern (Toscano et al., 2000) and probably due to ectomesenchymal tissue migration abnormalities. On the basis of our findings that patients diagnosed with del 22q11 had a cardiac defect in the form of subarterial VSD, we confirm this pathophysiologic association.
In our cohort, we found the subtype of Tetralogy of Fallot with a right aortic arch, absent infundibular septum, or absent pulmonary valve specifically associated with patients with 22q11, which can be considered peculiar to this syndrome (Rauch et al., 2010). 22q11.2 deletion represents the most common known cause of TOF, and the associated cardiac phenotype is distinct for obstruction of the proximal pulmonary artery, hypoplastic central pulmonary arteries, and subclavian artery anomalies. An atrioventricular septal defect associated with TOF is very suggestive of trisomy 21 and almost excludes 22q11.2 deletion (Rauch et al., 2010). Severe dysplasia of the pulmonary valve is seen exclusively in our probands diagnosed with NS. The cardiac defect is a specific anatomic marker of NS as it is very unusual in nonsyndromic patients with pulmonary valve stenosis (Pierpont et al., 2007).
A proband showed supra-aortic stenosis in association with peripheral pulmonary artery stenosis, a peculiar cardiac phenotype characteristic that is almost exclusive to patients with WS (Schubert, 2009). Ahmad and Vettukattil (2010) recently reported that the finding of a pulmonary artery diverticulum is considered a pathognomonic feature of WS, which was found in one of our probands diagnosed with WS. Our probands with TS mainly presented with AC. TS is the most common genetic condition associated with AC and, conversely, AC is one of the prevalent cardiac malformations found in patients with TS, particularly those with 45, X karyotype (Gedikbasi et al., 2010). One proband with truncus arteriosus was diagnosed with 22q11 syndrome. Truncus areteriosus is frequently associated with extracardiac anomalies (40%) and the association with del 22q11 is particularly frequent (30%) (Momma, 2010). In patients with truncus arteriosus and 22q11, there is a prevalence of additional anomalies of the aortic arch (interruption type B, right sided or double aortic arch) or of the pulmonary arteries (discontinuity stenosis or ‘crossed’ pulmonary arteries, which was a documented finding in our proband). Also similar to our findings, Detaint et al. (2010) found ascending aortic dilatation (AOD) in 90% of their cohort of MFS, and concluded that the cardiovascular risk remains significant in patients with an FBN1 gene mutation.
Our findings show that the initial specific and detailed description of the cardiac defects and a thorough examination of extracardiac features are the first crucial steps to identification of a genetic syndrome. Proper investigations of family members and strategic specific genetic testing are keys to a timely and accurate diagnosis. A genetic test is selected on the basis of an index of suspicion, which is often supported by a recommended strategy of testing as provided by Genetests or a genetic algorithm.
We showed that with comprehensive evaluation and strategic genetic testing including other family members, the exact genetic risks faced by families and the affected individual are revealed. Proper investigation of family members is of utmost importance to define the exact causes and risks and identify at-risk family members as we succeeded in establishing for nine individuals (eight parents and a sibling) and their families as discussed above. Almost 75% of the cardiac syndromes occurred de novo and another 25% were inherited in an autosomal manner. It is expected that the percentage of parent-to-child transmission may increase if family members are comprehensively evaluated, if somatic mosaicism is considered, and the knowledge of nonclassic and subtle presentations expands.
Availability and affordability of genetic tests are important factors to ensure proper screening and evaluation. Some cardiac syndromes such as RTS are caused by mutations in large, multiexon genes. Genetic testing for these disorders is labor intensive and expensive. We sought to develop a more rapid, comprehensive, and cost-effective approach. Therefore, we had used an RNA-based sequencing protocol with newly designed primers and thus showed that RNA-based sequencing can be an efficient, cheaper, and faster alternative to exonic DNA sequencing for mutation screening and genotype–phenotype discovery in RTS. This method had been successfully used by Miller et al. (2007) for long QT syndrome, MS, hypertrophic, and dilated cardiomyopathy.
The diagnosis of cardiac syndromes is often delayed because it can be challenging to make a specific genetic diagnosis. The reasons for this are age-dependent phenotypic or behavioral manifestations as seen in MS and DGS. Uniqueness of the pattern of manifestations or genotype/phenotype heterogeneity as is known for RTS is partially responsible for a low detection rate. Genotype–phenotype correlation rules are insufficient and causative genes and mutations are yet to be defined. Another cause for the delayed recognition of a syndrome is variable expressivity as a result of somatic mosaicism and the phenomenon of interfamilial and intrafamilial variability, which is frequently seen in AS and DGS. Many syndromes are not recognized and evade diagnosis due to the subtlety of the features, and patients are managed with respect to the cardiac condition alone. It is the subtlety of features and their severity that seem to delay or draw attention to a syndrome existence rather than the pattern of features. This may also explain why parents of affected children sometimes are not diagnosed until their children are. MS, DGS, SS, AS, NS, and TS are the syndromes that presented with subtle features in children and also their parents and eluded a diagnosis. The physician’s awareness and knowledge of the variable presentation and severity of cardiac syndromes’ features is a critical factor. But even with suspicion and a clinical diagnosis, a final diagnosis is not established unless a genetic test confirms a genetic alteration. Thus, it is important to screen family members and infants at risk as there is not always a straight link from the genetic cause to phenotype; an initial specific and detailed description of the cardiac defects and a thorough examination of extracardiac features are the first crucial steps toward identification of a genetic syndrome. Proper investigations of family members and strategic specific genetic testing are keys to a timely and accurate diagnosis for peculiar anatomic subtypes related to specific genetic conditions, and therefore a detailed description of the CHD is mandatory in the genetic evaluation of children with CHD and the diagnosis of a cardiac syndrome. RNA-based sequencing is a cheap and easy screening method for RTS and can be used as an alternative to DNA sequencing, saving half the cost and time. We diagnosed a proband with nonclassic RTS and found a novel mutation in EP300: the first missense mutation and ninth reported mutation in EP300 in RTS patients and the fifth case of familial RTS. This finding confirms the heterogeneity of this syndrome and the contribution of EP300 to it.
Every child with CHD should be carefully examined for extracardiac malformations and dysmorphic features. The use of an algorithm on the basis of the initial presentation to assess for the presence of noncardiac abnormalities may be helpful. An interdisciplinary approach of different specialties to the child with CHD should be encouraged. A child with CHD should be evaluated by pediatricians, cardiologists, and geneticists. Medical personnel, including pediatric cardiologists, pediatricians, adult cardiologists, internists, obstetricians, nurses, and thoracic surgeons, need to be updated about the genetic aspects of CHD, nonclassic, and subtle presentations. The approach to a newly diagnosed patient with CHD should include routine examination of all first-degree relatives for a potential genetic contribution. The anatomic, clinical, and surgical description of congenital heart defects must include the phenotypical aspect and the possible presence of genetic anomalies, and a complete understanding of the interactions between abnormal cardiac physiology and derangements in other organs is important for appropriate management and counseling in such patients. A detailed description of the heart defect is mandatory as peculiar anatomic subtypes are related to specific genetic conditions and facilitate the diagnosis of a genetic syndrome. Further analysis of large cohorts of individuals with cardiac syndromes is essential to delineate the genetic alterations in heterogenic syndromes in detail and to clarify genotype–phenotype correlations, thereby permitting more accurate prognosis and minimization of testing costs and efforts. In syndromes where features are age-dependent, diagnostic criteria should be revised and set according to age groups. Efforts should be directed toward the use of RNA-based sequencing as an alternative to DNA-based sequencing to establish cheaper and easier genetic tests to facilitate screening of syndromes that are expensive or labor intensive to screen for.
Complete evaluation and comprehensive assessment of a child with CHD and multiple congenital anomalies is an essential key to appropriate care and reducing pediatric morbidity and mortality. Genetic testing of cardiac patients has become increasingly important. It is mandatory to evaluate for associated noncardiac features and to identify a cardiac syndrome in a timely manner and to provide accurate genetic counseling. The purpose of this study was to screen children with structural CHDs associated with extracardiac anomalies for the presence of a syndrome with a particular focus on those syndromes that are challenging to screen and diagnose. The children in our cohort, ranging in age from 2 months to 16 years, were initially classified into those with a recognizable pattern for a syndrome and those with a nonrecognizable pattern and on the basis of an index of suspicion; the specific genetic test was applied accordingly. We used karyotyping, FISH, DNA sequencing, and CGH, which detected a genetic alteration in 98.5% of the cases. A clinical diagnosis was made and followed by a genetic test to confirm a laboratory diagnosis. In probands in whom clinical features were suspicious but not fulfilling the diagnostic criteria, genetic testing helped establish a diagnosis. In our cohort of 66 children, the majority (97%) presented with a recognizable pattern of malformations and a diagnosis could be confirmed by genetic testing in 98% of these. We identified 64 children with cardiac syndromes. Two probands showed a nonrecognizable pattern of cardiac and extracardiac malformations and showed new genetic alterations detected by CGH. Cardiac syndromes identified included MS, DS, DGS, SS, NS, TS, Cornelia de Lange syndrome, Wolf Hirschorn syndrome, HOS, AS, WS, Cri du Chat syndrome, RTS, cardiofacialcutaneous syndrome, deletion 8p syndrome, and duplication 15q syndrome. We found that the diagnosis of cardiac syndromes is often delayed. A specific genetic diagnosis can be challenging due to age-dependent phenotypic or behavioral manifestations as seen in MS and DGS. Uniqueness of the pattern of manifestations or genotype/phenotype heterogeneity as known for RTS is partially responsible for a low detection rate. Genotype–phenotype correlation rules are insufficient and causative genes and mutations are yet to be defined. Another cause for the delayed recognition of a syndrome is variable expressivity as a result of somatic mosaicism and the phenomenon of interfamilial and intrafamilial variability that is frequently seen in AS and DGS. Many syndromes are not recognized and evade diagnosis due to the subtlety of the features, and patients are managed with respect to the cardiac condition alone. It is the subtlety of features and their severity that seem to delay or draw attention to the existence of a syndrome rather than the pattern of features. This may also explain why parents of affected children sometimes are not diagnosed until their children are. Marfan syndrome, DiGeorge syndrome, Shprintzen syndrome, Noonan syndrome and Turner syndrome may present with subtle features and therefore may remain undiagnosed. The physician’s awareness and knowledge of the variable presentation and severity of cardiac syndromes’ features is a critical factor. But even with suspicion and a clinical diagnosis, a final diagnosis is not established unless a genetic test confirms a genetic alteration. Thus, it is important to screen family members and infants at risk.
As there is not always a straight link from the genetic cause to phenotype, an initial specific and detailed description of the cardiac defects and a thorough examination of extracardiac features are the first crucial steps toward identification of a genetic syndrome. Proper investigations of family members and strategic specific genetic testing are keys to a timely and accurate diagnosis for peculiar anatomic subtypes related to specific genetic conditions, and therefore a detailed description of the CHD is mandatory in the process of genetic evaluation of children with CHD and the diagnosis of a cardiac syndrome. In our attempts to develop an easy and cheap screening method for RTS, we successfully showed that RNA-based sequencing can be used instead of DNA, saving half the cost and time. Using the RNA-based sequencing protocol, we were able to genotype a proband with nonclassic RTS and found a novel mutation in EP300: the first missense mutation and the ninth reported mutation in EP300 in RTS patients and the fifth case of familial RTS. This finding confirms the heterogeneity of this syndrome and the contribution of EP300 to it.
This work has been sponsored by the Egyptian Government as a scholarship for a doctoral thesis.
Conflicts of interest
There are no conflicts of interest.
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