Congenital heart disease is a leading cause of death in the first year of life (Gillum, 1994). Advances in molecular genetics allow for a genetic defect to be defined in an increasing number of cases. Establishing a genetic abnormality for heart disease not only
defines a cause for the patient and family but can implicate on genetic counseling, prognosis and surveillance for potential complications. The following is a directed approach at genetic and metabolic testing in patients who may present to the
Pediatrician or Pediatric Cardiologist with heart disease. Not included are disorders which will inevitably have non-cardiac features as their primary presentation and for which genetic testing is available.
The traditional approach to genetic analysis involves a standard karyotype which can now be obtained in 24 hours, but is mainly useful for aneuploidies and large deletions. Prophase High Resolution Chromosome Analysis (HRCA) can be obtained within 48 hours if requested and is capable of showing smaller deletions. Fluorescence in situ Hybridization (FISH) can test against specific mutations beyond the resolution of HRCA and can be obtained within 24 hours. However, FISH may not be useful if a genetic defect can be caused by different mutations. Linkage analysis and polymerase chain reaction (PCR) is available in a limited number of facilities, is time-consuming and impractical in isolated cases of disease.
Inherited cardiomyopathies are mainly the result of metabolic disturbances, but a number are also due to Friedrich's ataxia and muscular dystrophies. An inborn error of metabolism should be suspect in cardiomyopathy if there are no other obvious
causes, concomitant myopathy of skeletal muscle, hepatomegaly or hepatocellular dysfunction, resistant metabolic acidosis or encephalopathy. Distinguishing between these disorders based only on clinical grounds is next to impossible and metabolic
screening tests or biopsies are required to establish a diagnosis, but a rational approach can facilitate investigations (Clarke, 1996).
A clinical evaluation for a genetic or metabolic disorder should be performed in all children with congenital heart disease. If there is no obvious syndrome, a standard karyotype and FISH 22q11 should be obtained. If the FISH 22q11 is positive, a
diagnosis can be established and the pending karyotype is unnecessary. If the FISH result is negative, the karyotype should be checked. If the karyotype is normal, HRCA should be performed. Further testing can be directed by the type of heart defects
present or suspected. Cardiomyopathy should be evaluated as described in the text. Consultation with a specialist should be obtained if there are any concerns with diagnosis or investigations.
Down's syndrome is overall the number one cause of congenital heart disease (Grech and Gatt, 1999). Atrial-ventricular canal defects, ventricular septal defects, patent ductus arteriosus, atrial septal defect, tetralogy of Fallot and others are present in up to about half of all cases (Freeman et al, 1998). The vast majority (95%) of cases are due to Trisomy 21. The remainder are due to mosaicism and various translocations.
A routine karyotype is sufficient for trisomy 21, mosaics and most translocations whereas changes beyond the resolution of classical karyotyping such as cryptic partial duplications of the Down's Chromosome Region or undetected mosaics will require a FISH (to 21q22.3) (Iqbal et al, 1997, Pierluigi et al, 1996, Rajangam et al, 1997). FISH can also be useful if a rapid diagnosis is required. There have been associations with COL6A1 deletion also, but the clinical utility of doing RFLP (restriction fragment length polymorphism) for this deletion awaits discovery (Kaisenberg et al, 1998).
A microdeletion in the short arm of chromosome 22 (22q11) is the second leading cause of syndromic cardiac defects but has the strongest association with serious defects including: truncus arteriosus, double outlet right ventricle, interrupted aortic arch, right aortic arch, patent ductus arteriosus, coarctation of the aorta, tetralogy of Fallot, ventricular septal defect and a long list of other defects (Chapelle et al, 1981, Fokstuen et al, 1998, Goldberg et al, 1993, Ryan et al, 1997, Wilson et al, 1993). Molecular genetics has helped polarize a group of synonymous syndromes, DiGeorge, partial DiGeorge, velo-cardio-facial syndrome and CATCH22 in which almost all defects are due to the 22q11 deletion. Only a minority of cases are due to deletion 10q (Gottlieb et al, 1998) and other cytogenetic abnormalities (Johnson et al, 1997). A majority of Shprintzens syndrome cases are also due to the 22q11 deletion.
Congenital heart disease is the leading cause of mortality in these groups (Chapelle et al, 1981, Goldberg et al, 1993, Ryan et al, 1997). Most carrier parents are asymptomatic and most cases are so mild that they can escape diagnosis in infancy (Du Montcel et al, 1993). FISH is the most rapid and reliable method for screening for the 22q11 deletion. A positive result will verify the diagnosis whereas a negative result should be followed by high resolution chromosome analysis (prophase) to check for the minority of other causes (Johnson et al, 1997). Because of its protean manifestations, all cases of congenital heart disease with no other obvious cause should be screened for the 22q11 deletion. The recent discovery of a deletion for a transcription factor, UFD1L, in the 22q11 deletion may have future implications on screening (Yamagashi et al, 1999).
Turner's Syndrome is characterized by mental retardation, ambiguous genitalia, risk of gonadal malignancy and coarctation of the aorta. The diagnosis is evident clinically in the majority of cases. Forty to sixty per cent of cases are due to 45,X karyotype, the remainder are due to mosaics, deletions, translocations, isochoromosomes and marker chromosomes resulting in haploinsufficiency or imbalance in activation of X and/or Y chromosome regions. Cytogenetics confirms the diagnosis in the vast majority of cases and a Turner's karyotype will distinguish from Noonan's syndrome in equivocal cases. However, milder phenotypes (associated with Y linkage), mosaics and non-aneuploid changes may escape karyotype detection (Schwartz et al, 1997). X and Y chromosome FISH probes have been designed which have a higher sensitivity and take less time than
karyotyping (Schwartz et al, 1997). HRCA should always be ordered; however, if cytogenetics are negative, the clinical presentation is atypical, there is concern about the risk of gonadoblastoma or a faster result is desired, FISH or PCR analysis may be performed (Kuznetzova et al, 1995).
Marfan's syndrome is a connective tissue disorder resulting in skeletal, ocular, and cardiovascular defects. The diagnosis is based on clinical grounds (Gilchrist, 1994, Paepe et al, 1996). Mutations in the fibrillin-1 gene on chromosome 15 (15q15-21.3) are responsible for 50-60% of cases with autosomal dominant inheritance, high penetrance and clinical variability (Dietz et al, 1991). Up to 40% of cases may be due to a 3 deletion (3p24.2p25; Milewicz et al, 1998). Fibrillin-1 monomers make microfibrils which are critical to the integrity of elastin and non-elastin tissues. The most fatal cardiovascular consequence is aortic dissection resulting from progressive aortic root dilatation in classic Marfan's syndrome (Paepe et al, 1996), but different clinical variances such as the MASS phenotype also result in mitral valve prolapse (Montgomery et al, 1998). A Cystine to Arginine amino acid substitution in the fibrillin-1 gene has been associated with a more severe phenotype and may have cardiovascular implications: aortic root dissection, mitral valve prolapse (MVP), and others (Paepe et al, 1996). Because of a large number of mutations and the large size of the gene (Kainulainen et al, 1994), a FISH test would not be practical. Instead, a method called haplotype segregation analysis, determines whether alleles are inherited from affected individuals in families. This type of linkage analysis depends on identifying a defective gene in an affected family member when the gene in unaffected members can be used for comparison. Therefore, testing is most useful in familial cases and can be used to identify
family members who may have the defective gene and be at risk for the cardiovascular complications. Studies have shown that prophylactic beta-adrenergics started when there is aortic root dilatation reduce development of aortic complications in some patients (Shores et al, 1994). If haplotype segregation analysis fails to detect the affected individual, PCR can be performed to determine the gene sequence (Pereira et al, 1994) and a list of centersis provided by Milewicz (1998). A preimplantation
testing method using fluorescent PCR and automated laser fluorescence DNA has also been successful in identifying a mutated fibrillin-1 gene (Sermon et al, 1999).
William's Syndrome (WS) is a connective tissue and brain disorder characterized by elfin facies, mental retardation, gregarious personality and congenital heart defects such as supravalvular aortic stenosis, supravalvular pulmonic stenosis, VSD, PDA and systemic hypertension (Morris et al, 1988), as well as diffuse arterial wall thickening including the cornary arteries. The cardiac defects often escape detection until developmental abnormalities bring this disorder to attention at about 6 years of age. Supravalvular aortic stenosis, which can be present in about 2/3 of cases (Morris et al, 1988) and is associated with disruption of the elastin gene either from a deletion at 7q11.23 in the majority of cases (Ewart et al, 1993) or from an autosomal dominant 6;7 translocation on chromosome 7 (Curran et al, 1993). HRCA may only be able to detect deletions about 2 megabases or larger but a number of mutations can be submicroscopic (Jalal et al, 1996). A FISH probe has be designed to the flanking regions of elastin to detect the absence of the gene (Wu et al, 1998). In classic WS, 96% showed elastin deletion, and of those with normal cytogenetics 38% showed elastin deletion by FISH and 60% showed deletion by FISH when no clinical information was available (Lowery et al, 1995). The sensitivity of both tests is higher if there is a stronger clinical suspicion (Brewer et al, 1996, Nickerson et al, 1995), SVAS in non-dysmorphic children can sometimes test positive for the deletion (Curran et al, 1993). Nevertheless, point mutations, minute deletions within the elastin gene and isolated autosomal dominant inheritance can still escape current FISH tests (Jalal et al, 1996) and these findings have been confirmed in a variety of studies (Lowery et al, 1995, Nickerson et al, 1995). Having both a negative cytogenetic test and FISH provides strong evidence but does not exclude the diagnosis of WS since studies using PCR show that polymorphisms in the elastin gene can result in Williams syndrome but escape both chromosome analysis
and FISH. PCR is not feasible yet on a routine basis. However, it is the hemizygosity of the elastin gene that shows clinical correlation with congenital heart disease and no clinical correlation yet has been made with the different elastin gene deletions (Nickerson et al, 1995, Wu et al, 1998). Therefore, in all cases where a clinical diagnosis of Williams syndrome is made and in all cases of SVAS where there is no apparent syndrome, both HRCA and FISH should be performed (Fryssira et al, 1997).
Danlos Syndrome Type IV
Ehler's Danlos Syndrome Type IV is an autosomal dominant connective tissue disorder. There is hyperextensibility of the skin and hypermobility of the joints, but the disorder can present initially as a fatal event from aortic (or other arterial) rupture, bowel rupture or uterine rupture during pregnancy. The defect is due to mutations in the COL3A1 gene at 2q31-q32 which encodes for type III collagen (Kontusaari et al, 1990, Pope et al, 1975, Superti-Furga et al, 1988). Because there are different mutations (polymorphisms), siblings may not be equally affected. Initial screening is best done with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) on cultured skin fibroblasts to check for abnormally low levels of collagen III (Pope et al, 1975, Superti-Furga et al, 1988). If there is known disease in the family, it would be worthwhile to do SDS-PAGE but also do mutation analysis by PCR from cultured skin fibroblasts to identify the type of mutation and any prospective genotype / phenotype correlation since disease severity can cluster in families (Gilchrist et al, 1999, Raghunath et al, 1996, Schwarze et al, 1997). If the genetic defect is not detected, an unaffected sibling can receive greater reassurance that they did not inherit the mutation.
Holt-Oram Syndrome is a developmental disorder with upper limb and cardiac defects such as atrial septal defect, ventricular septal defect, tetralogy of Fallot and conduction abnormalities. A deletion in the HOS1 gene (TBX5) on chromosome 12 (12q24.1) leads to haploinsuffiency and presentation of this syndrome (Basson et al, 1997, Li et al, 1997). In particular, a glycine to arginine mutation within the gene has been linked to significant cardiac malformations (Basson et al, 1999). The gene has an autosomal dominant mode of transmission and cases therefore cluster in families (Newbury-Ecob et al, 1996). Because the cardiac defects themselves are non-specific, genetic testing should be undertaken if there is clinical suspicion for this syndrome. A FISH has been developed for HOS1 deletions and would be the test of choice (Terrett et al, 1996).
Wolf-Hirschhorn Syndrome is manifest by characteristic facial dysmorphia, severe growth and mental retardation, microcephaly, hypotonia and weak suck and sporadic cardiac defects. The diagnosis is clinical and confirmed by cytogenetics.
The defect is caused by a 4p16.3 deletion which can sometimes escape detection by cytogenetics and a commercial FISH probe (Johnson et al, 1994) is available if there are equivocal cases (Fagan et al, 1994).
The Cri-du-Chat Syndrome is characterized by striking craniofacial dysmorphia, "cat-like" cry, low birth weight, failure to thrive and developmental delay. Ventricular septal defects, atrial septal defects, bicuspid aortic valve, tetralogy of Fallot and
patent ductus arteriosus have been reported in certain cases. The 5q deletion has been associated with this syndrome (Niebuhr, 1978). There are 2 distinct critical regions: 5p15.2 for facial features, mental and developmental delay and 5p15.3 for cat-like
cry. FISH testing can be done for either deletion, but no association has been made yet between the type of deletion and congenital heart disease (Gersh et al, 1997). The diagnosis should be obvious on clinical grounds and confirmed with cytogenetics (Baccichetti et al, 1988). Currently there is no basis for using only the FISH test to screen for this deletion, but FISH and PCR can be used when there is clinical suspicion but the karyotype is normal (Kuznetzova et al, 1995).
Cardiomyopathy with hepatic involvement
Patients with cardiomyopathy tend to have hepatic involvement secondary to impaired cardiac function making it difficult to distinguish from primary metabolic causes. Lysosomal storage diseases such as glycogen storage disease type II (Pompe's),
mucopolysaccharide storage (MPS) diseases and fucosidosis tend to produce hepatomegaly (Clarke, 1996). MPS storage diseases will inevitably have dysmorphic facies. In Pompe's disease, there can be electrocardiographic abnormalities such as
left axis deviation, shorted PR interval, tall QRS complexes and T wave inversion (Gillette et al, 1974) although we have experience in our institution with a newborn who did not have any of these findings but was diagnosed with Pompe's disease
based on endomyocardial and skeletal muscle biopsy and confirmed with low leukocyte alpha-glucosidase activity. Disorders of fatty acid oxidation, carnitine deficiency and organic acidopathies are associated with hepatocellular dysfunction and can present with a non-lactic acid metabolic acidosis. Cardiomyopathy from amino-acidopathies is rare. These disorders can produce hyperammonemia (which can lead to encephalopathy if severe) and vascular disease (homocysteinemia and alkaptonuria).
Initial screening should involve a urine MPS screen, oligosaccharides (Piraud et al, 1998) and beta-hexosaminidase to screen for MPS disease and endomyocardial and skeletal muscle biopsy. Intralysosomal storage of macromolecules can be seen in
lysosomal storage diseases (i.e.glycogen storage disease type II (Ninomiya et al, 1984) and Fabry's disease). Confirmation is required with peripheral blood leukocyte acitivities for alpha-glucosidase (Pompe's) and beta-galactosidase (Fabry's). Microvesicular lipid accumulation can be seen in fatty acid oxidation defects and Barth syndrome. A case of tetralogy of Fallot has also been reported in Fabry's disease (Lewin et al, 1999). Fatty acid, organic acid and carnitine defects can be screened with urine organic acids, plasma free, esterified and total and acyl- carnitines (Vianey et al, 1997). The carnitine profile is
especially useful for organic acidopathies since organic acid esters of carnitine are often more sensitive and specific tests than plasma organic acids. Evaluation for aminoacidopathies should involve plasma quantitative amino acids and plasma ammonia
(preferably arterial and transported immediately to laboratory on ice).
Cardiomyopathy and Skeletal Myopathy and / or Lactic Acidosis
If there is any suspicion of skeletal myopathy in addition to cardiomyopathy or a persistent anion gap metabolic acidosis then mitochondrial diseases, glycogen storage disease types II and IV and fatty acid oxidation defects or Barth syndrome should be suspect (Clarke, 1996). A high CPK with fractionation may be provide a clue to muscle involvement but will probably be elevated due to cardiomyopathy. Combined endomyocardial biopsy and skeletal muscle biopsy can show increased mitochondria and ragged red fibers in mitochondrial myopathies, increased lysosomal glycogen or fat in lysosomal diseases and increased cytoplasmic glycogen in peripheral blood lymphocytes in glycogen storage disease. If mitochondrial diseases are highly suspect, further investigations can involve testing for MELAS and MERRF from peripheral blood
leukocytes but require at least a week for a result. An arterial plasma lactate level should be performed since a lactic acidosis from mitochondrial diseases may not be result in a metabolic acidosis depending on the clinical status and ongoing therapy.
Urine organic acids are useful in screening for Barth syndrome which has been shown to have increased levels of urinary 3-methyl glutaconate, 3-methyl glutarate, 2-ethyl hydracylate. Barth syndrome is X-lined to region Xq28 and a defective gene, G4.5, has recently been identified (Bione et al, 1996). Patients show dilated cardiomyopathy, skeletal myopathy, short stature and neutropenia and often succumb to congestive heart failure or fatal infection.
Cardiomyopathy Not Associated with Inborn Errors of Metabolism
Friedrich's ataxia, muscular dystrophies and myotonic dystrophies will initially present as a clinical syndrome. Friedrich's ataxia can be identified using PCR for unstable GAA repeats on 9q13 which are present in 97% of affected individuals (Cosee et al, 1999, Pandolfo et al, 1998, Wood 1998). Testing should be performed on clinical grounds or in asymptomatic individuals with a positive family history.
For muscular dystrophy, the initial screen can still be performed by CPK levels and immunohistochemical stain and can help differentiate non-Xp21 muscular dystrophies such as limb-girdle dystrophy, spinal muscular atrophy and myotonic dystrophy and the Xp21 dystrophies (DMD and BMD) (Nicholson et al, 1993). A combination of PCR immunoblotting and immunohistochemistry can help differentiate between autosomal or X-linked forms of the dystrophies (Essen et al, 1997). Cardiac disease in myotonic dystrophy is related to the size of the CTG repreats on chromosome 19 (Annane et al,
1996) which can be determined using PCR. Because of this correlation, molecular testing should be performed when clinically suspect. Autosomal dominant and autosomal recessive causes of familial dilated cardiomyopathy have been found but routine genetic testing is not yet established (Bachinski and Roberts, 1998, Mestroni et al, 1998).
Errors Associated with Cardiovascular Disease
Hyperhomocystienemia can cause premature atherosclerosis and thrombosis. The most severe defect is due to homozygous cystathione-beta-synthetase deficiency (Vilaseca et al, 1998), but other defects in the remethylation pathway can also lead to this disorder (Rees and Rodgers, 1993). Knowledge of this inherited disorder formed the basis for which screening in asymptomatic adults is now established. Criteria for screening children have not been well-established. If found, treatment with vitamin B6 and folic acid may be helpful in certain cases (Jacques eta al, 199). Plasma quantitative amino acids should definitely be checked in families with inherited disorders of cystathione or methione metabolism, megaloblastic anemia, Marfinoid features and those with ischemic events such as stroke, myocardial infarction or thromboses (Vilaseca et al, 1998).
Severe hypercholesterolemia can lead to xanthomas but most cases of hyperlipidemia are subclinical and lead to accelerated atherosclerosis and a higher risk for early myocardial infarction. The typical lipoprotein profile and triglycerides should be used
to screen children with a defined familial hyperlipidemia, at 2 years of age in children with a family history of premature myocardial infarction or other stigmata of athersclerotic disease.
Alkaptonuria is due to deficiency of homogentisic acid oxidase. High levels of plasma homogentesic acid are found in affected patients and accumulation in valvar tissue can lead to valvular incompetence as the presenting problem. (Deutsch and Santhosh-Kumar, 1996, Kragel et al, 1990)
Other Chromosomal Disorders
and Genetic Abnormalities
Other Chromosomal Aneuploidies such as trisomy 13, trisomy 18, Klinefelter's, tetrasomy 22p and tetrasomy 12p are associated with congenital heart disease. The diagnosis is made mainly on clinical grounds for each of the disorders and confirmation by karyotyping is generally sufficient, however FISH is available for trisomy 13 and trisomy 18 if a rapid diagnosis is required (Eiben et al, 1998). A strong association has been found with recombinant chromosome 8, tetralogy of Fallot and other forms of congenital heart disease and can be detected using cytogenetics (Gelb et al, 1991). Dysrrhythmias can be secondary to other disease but are also seen in carnitine deficiency (Pande), muscular dystrophies (Lazarus et al, 1999), fatty acid oxidation defects (Saudubray et al, 1992). Long QT syndrome is the best known primary disorder linked to genetic defects, but a routine test to detect defects is yet to be defined (Vincent, 1995, Wattanasirichaisoon and Beggs, 1998).
Hereditary hemorrhagic telangiectasia, will present with obvious clinical findings (Marchuk, 1997). A large number of genetic disorders are associated with congenital heart disease, but will likely present with non-cardiac findings.
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