Discussion

The importance of genetic factors in the aetiology of CHDs is understood to a better degree each day. With array comparative genomic hybridisation, the aetiology of CHDs is enlightened as a result of the increasing knowledge about genomic variations in patients. In our study, in the array comparative genomic hybridisation analysis of 37 patients with conotruncal heart anomalies, chromosomal deletions were detected in four and duplications in one, and 13.5% of the patients had genomic changes. Although it is observed that the patients in our study were included in the definition of isolated cardiac anomalies due to the absence of distinct dysmorphic features and the absence of extra-cardiac anomalies, the fact that 22 of the patients included in the study were aged 1 year or younger made this definition difficult. Considering that the rate of deletion or duplication detection varied between 3 and 10% in genetic studies conducted with patients with isolated CHD, it was observed that the CNV frequency detected in our study was higher than in other studies. ^{Reference Erdogan, Larsen and Zhang6}

In relation to the advances in medical and surgical treatments in recent years, today, around 85% of children with CHD reach adulthood. ^{Reference Richards and Garg9} In the study by Erdogan et al., CNVs were detected in 17% of patients with isolated CHD, and it was seen that with array comparative genomic hybridisation, a syndromic disorder in a child could be detected without the need to identify additional findings. ^{Reference Erdogan, Larsen and Zhang6} The array comparative hybridisation technique was reported to be the testing method that should be preferred for first-line analysis in patients with apparently isolated CHD by Bachmann and colleagues. Additionally, the literature on this topic states that applying array comparative genomic hybridisation analysis only in patients with syndromic CHD may lead to missed diagnoses of possible chromosomal syndromes, and therefore, it has been suggested that array comparative genomic hybridisation analysis should be the first test applied to patients with CHD. ^{Reference Bachman, DeWard, Chrysostomou, Munoz and Madan-Khetarpal10}

In the present study, a de novo duplication of 0.5 MB was detected in the region of 1p36.32 (2694496-3208375) in patient #2 who had large artery transposition. There is no report in the literature on whether duplication in this region causes conotruncal heart disease. Although the duplication of the 1p36.32 region in databases does not explain the ventricular septal defect in the patient, the PRDM16 gene, one of the genes in the duplication region, is known to be expressed in myocytes, as well as many other tissues. In addition, it has been shown in an animal study that ectopic expression of the PRDM16 gene transforms myoblasts into adipose tissue. ^{Reference Seale, Bjork and Yang11} Left ventricular non-compaction and dilated cardiomyopathy have been reported to be present in patients with 1p36.32 region duplication in the UCSC database (https://genome.ucsc.edu/). In addition, non-cardiac anomalies, such as intellectual delay, developmental delay, ataxia, speech delay, autism, and facial anomalies, have been reported in patients with this particular duplication. Since intellectual delay can be seen in patients with this duplication, it should be recommended that the family be careful in this respect and, if necessary, refer the patient to special education at the earliest possible stage for learning disabilities. However, since the DGV database demonstrates that healthy subjects may also have duplication in this region, it is not possible to directly attribute the cardiac anomaly in our patient to this variation. Even so, considering the possibility that the variation in our patient might show reduced penetrance, it was thought that it might have caused the disease.

In patient #9 who had ventricular septal defect, it was observed that there was de novo deletion in the 22q11.2 region. The relationship between conotruncal heart anomaly and haplo deficiency of the TBX1 gene in the region is well known. ^{Reference McDonald-McGinn and Sullivan12,Reference McDonald-McGinn, Sullivan and Marino13} Although the renal, immunologic, gastrointestinal, palatal and behavioural findings associated with 22q11.2 deletion syndrome were not observed in the patient (except for the cardiac anomaly), they were scheduled for follow-up by the relevant departments for these aspects. 22q11.2 region deletion was detected in this patient who was thought to have an isolated conotruncal heart anomaly. The development of various complications in the perioperative and post-operative periods of cardiac surgery have been shown among individuals with this syndrome – also known as DiGeorge syndrome. For instance, due to heart failure, respiratory problems, infections, and hypocalcaemia, these patients may experience significantly prolonged time on the ventilator and could have extended need for positive inotrope support, ultimately leading to a longer length of stay in the ICU. ^{Reference Yeoh, Scavonetto, Hamlin, Burkhart, Sprung and Weingarten14}

It is observed that the parathyroid gland is underdeveloped or absent altogether in patients with DiGeorge syndrome, and as a result, hypocalcaemia may develop in patients. ^{Reference Cuneo, Langman, Ilbawi, Ramakrishnan, Cutilletta and Driscoll15} More importantly, hypocalcaemia may develop in the perioperative period and could lead to hemodynamic instability. In addition, hypocalcaemia has been reported to be a cause of epileptic seizures in the post-operative period. ^{Reference Flashburg, Dunbar, August and Watson16–Reference V.P.Singh, Sanyal, Waghray, Luthra and Borcar18} Epileptic seizures unrelated to hypocalcaemia have also been reported in some patients with DiGeorge syndrome. ^{Reference Kao, Mariani and McDonald-McGinn19} Nonetheless, in these patients, parathyroid gland function usually develops within 1 year after birth, and hypertrophy of the gland is often observed. Accordingly, during this period, patients may have a period of normocalcaemia. ^{Reference Perez and Sullivan20} However, in patients with parathyroid gland hypertrophy, there is a decreased parathyroid hormone response during physiological stress and hypocalcaemic hypoparathyroidism; in other words, latent hypoparathyroidism may occur. ^{Reference Cuneo, Langman, Ilbawi, Ramakrishnan, Cutilletta and Driscoll15,Reference Cuneo21}

Apart from hypocalcaemia, another problem encountered in the post-operative period in patients with DiGeorge syndrome is infection. In DiGeorge syndrome, infections are more common than expected due to susceptibility to bacterial and viral agents. IgG, IgA, IgM levels may be low in these patients. ^{Reference Goldmuntz22} Graft versus host disease can be prevented by using irradiated blood products in patients with DiGeorge syndrome, in whom immune deficiency is detected. ^{Reference Castro23} In addition, in these patients, it is recommended to prevent possible cytomegalovirus infections by using blood products that do not contain cytomegalovirus seronegative or leukocytes in the post-operative period. ^{Reference Ljungman24}

In patient #17, with outlet-type ventricular septal defect and pulmonary stenosis, a deletion of 1.8 MB in the 4q21.22q21.23 region was detected. 4q21 deletion syndrome (MIM: 613509) is characterised by apparent growth retardation, mental retardation, speech delay, cerebral hypoplasia, cerebellar anomalies, and ventricular dilatation. ^{Reference Bonnet, Andrieux and Beri-Dexheimer25} CHD has not been reported in 4q21 deletions, except for our case. There is a need for more reports of similar cases/findings in order to determine whether the cardiac anomaly in our patient was a rare component of the 4q21 deletion syndrome.

In patient #19, a 4.1 Mb deletion in the 6p22.3 region was detected. In the literature, developmental delay, craniofacial malformations, brain, kidney, heart and, eye anomalies have been reported in patients with 6p22 deletion. In addition, dysmorphic findings such as clinodactyly, syndactyly, and short neck structure were observed in these patients. ^{Reference Bremer, Schoumans, Nordenskjöld, Anderlid and Giacobini26} It has been established that the HIST1H1E gene mutation in the deletion region causes Rahman syndrome. The HIST1H1E gene encodes the Histone E1.4 protein, which is one of the epigenetic regulatory proteins. The main dysmorphological findings of the syndrome are high frontal hairline, full cheeks, bi-temporal narrowing, frontal bossing, and deep-set eyes. Among the clinical findings of the syndrome, intellectual disability, hypotonia, corpus callosum anomalies in the brain, and skeletal anomalies have been reported. In a study of individuals with this syndrome, cardiac anomalies were reported in 43% of patients. ^{Reference Burkardt, Zachariou and Loveday27} No finding was observed in our patient, except for ventricular septal defect and malalignment.

Tetralogy of Fallot was detected on the echocardiography of patient #32. In the patient’s array comparative genomic hybridisation study, a 3.9 Mb deletion was observed in the 1q21.1 region. The 1q21.1 deletion (MIM: 612474) may show significant phenotypic variability. Patients may have neuropsychiatric findings such as microcephaly, mental retardation, autism, and schizophrenia, whereas some individuals may demonstrate normal neurologic development. Cardiologically, bicuspid aorta, truncus arteriosus, transposition of great arteries, and aortic coarctation are observed in these patients. Dysmorphologically, frontal bossing, deep-set eyes and bulbous nasal tip, and wide fingers and toes have been reported. ^{Reference Mefford, Sharp and Baker28,Reference Walsh, McClellan and McCarthy29} In our patient, it was detected that there was GJA5 gene in the region where the deletion was observed. The GJA5 gene encodes a gap junction protein called Connexin40. Gap junctions are channels that allow the passage of ions and small molecules between adjacent cells. These channels are necessary for mammalian cardiomyocytes to communicate electrically, enabling rapid and coordinated propagation of the cardiac action potential. ^{Reference Jansen, van Veen, de Bakker and van Rijen30} There are three different isoforms of connexin in the human heart, which we can count as connexin40, connexin43, and connexin45. Connexin40 is known to be expressed in atrial myocytes, the atrioventricular node, His-bundle, and ventricular conduction system (Purkinje fibres). ^{Reference Vozzi, Dupont, Coppen, Yeh and Severs31} It has been reported that polymorphisms in the Connexin40 promoter region may increase the risk for idiopathic atrial fibrillation. ^{Reference Firouzi, Ramanna and Kok32–Reference Juang, Chern and Tsai34} Familial atrial fibrillation type 11 (MIM:614049) is seen in humans in GJA5 heterozygous mutations. Cardiac anomalies such as the bifid atrial appendix, ventricular septal defect, tetralogy of Fallot, and aortic arch anomaly have been reported in GJA5 heterozygous knockout mice. ^{Reference Gu, Smith, Taffet and Delmar35} In studies conducted with individuals with Velocardiofacial syndrome and Thrombocytopenia-Absent Radius syndrome associated with cardiac anomalies, it has been reported that deletion can be detected in the 1q21.1 region and may be associated with the GJA5 gene-phenotype. ^{Reference Brunet, Armengol and Heine36,Reference Klopocki, Schulze and Strauß37} It was observed that the GJA5 gene was located in the deletion region we detected in our patient, and it was thought that the cause of the cardiac anomaly in the patient might be haploinsufficiency of the GJA5 gene.

The frequency of intellectual disability in developing countries is estimated to range between 10 and 15 per 1000 children, with 85% of these children having a mild intellectual disability. In Western societies, intellectual disability is observed at a rate of 1-3%. Precise estimation of the incidence is difficult since mild cases usually cannot be detected until late childhood. ^{Reference Maulik, Mascarenhas, Mathers, Dua and Saxena38} Genetic factors play an important role in the aetiology of intellectual disability. Chromosomal anomalies (aneuploidies), submicroscopic deletions and duplications (copy number variations), and monogenic disorders are responsible for its genetic aetiology. ^{Reference Sherr, Michelson, Shevell, Moeschler, Gropman and Ashwal39} It is thought that early diagnosis of intellectual disability and initiation of treatment may contribute to the prevention of learning and behavioural problems that may present during academic life in the following years. ^{Reference Jacob, Olisaemeka and Edozie40} It has been reported that CHD adversely affects brain development in the embryological period and intellectual disability can be seen in these individuals. ^{Reference Riehle-Colarusso, Autry and Razzaghi41} Besides structural heart disease, the possible presence of chromosomal or monogenic genetic factors that cause intellectual disability in the aetiology of the disease may increase disease severity and could cause additional problems, as demonstrated in our patients. For this reason, it was thought that performing genetic tests on individuals when they were diagnosed with CHD would help alleviate future complications. It has been seen that a significant portion of the patients can be diagnosed with array comparative genomic hybridisation testing after conventional chromosome analysis.