Genetic Architecture of CHD in NICU patients – UMC Ljubljana Experience

1. Introduction

Congenital heart defects (CHD) are structural anomalies of the heart and great vessels resulting from errors in early embryogenesis. They are the most commonly detected congenital anomaly affecting 0.8–1% of all live-born infants [1] and present a major cause of morbidity and mortality in infancy [2].

The etiology of CHD is deemed to be multifactorial, an interplay of genetic and environmental factors. CHDs can be caused by environmental exposure to teratogens such as drugs, viral infections, and maternal conditions such as obesity and diabetes [3]. Genetic influences are supported by observed relatively high recurrence risk in families and well-established association of CHD with chromosomal abnormalities [4]; however, the cause of the disease remains unexplained in up to 60% of CHD patients [5]. Genetic cause elucidation is also challenging because of CHD's genetic and phenotypic heterogeneity [6]. With current diagnostic methods mainly comprising cytogenetic techniques (karyotyping and copy number variant platforms) and next-generation sequencing (NGS), we can reach a genetic diagnosis in 18–36% of CHD patients [7]. The overall yield of genetic testing depends on the type of congenital heart malformation, the presence of extracardiac congenital anomalies, and the genetic test modality [7,8].

The value of genetic testing in the setting of CHD lies in the defining etiology and consequently ending diagnostic odyssey for patients and families, possible detection of additional health problems associated with genetic diagnosis, prognostic information for clinical outcomes, genetic reproductive risks for the family, and genetic testing of additional family members when appropriate [9,10].

The aim of this study was to assess the diagnostic yield of genetic testing in the clinical evaluation of neonates with diagnosed CHD who were hospitalized at the Department of Neonatology, Division of Paediatrics, University Medical Centre (UMC) Ljubljana.

2. Materials and Methods

2.1. Patient Selection

In the present study, we enrolled neonates under 28 days with abnormal findings on echocardiography who were hospitalized in the Department of Neonatology, Division of Paediatrics, UMC Ljubljana between January 2014 and December 2023. Phenotypic data was obtained through retrospective chart review for each neonate. Neonates with isolated hemodynamically insignificant patent ductus arteriosus (PDA), isolated hemodynamically insignificant patent foramen ovale (PFO), cardiomyopathies, vasculopathies, and neonates exposed to known environmental teratogenic factors were excluded. We also excluded neonates with prenatally or perinatally detected common trisomies, namely trisomy 13, 18, and 21. Neonates were assigned into one of two groups based on the presence of isolated CHD or additional extracardiac anomalies. Neonates with isolated CHD were subdivided into three groups based on the complexity of congenital heart malformation according to Botto classification (simple, association, and complex). Chromosomal microarray analysis (CMA) was performed as a first-tier genetic test in all neonates with CHD, except in 11 neonatal patients in whom high possibility of monogenic disorder based on the phenotype was detected, and next-generation sequencing (NGS) was performed as a first-tier genetic test instead. In most neonatal CHD patients, however, NGS was performed in neonates with normal CMA results and clinical suspicion of monogenic disease. Informed consent was obtained from the parents of the neonates in accordance with guidelines established by the institutional review boards at their primary site of care.

2.2. Genetic and Bioinformatic Analysis

2.2.1. CMA and Classification of Results

According to the manufacturer's protocol, the DNA was isolated from peripheral blood samples using the Qiagen Mini kit (Qiagen, Valencia, CA). The quality and concentration parameters of the DNA were measured on a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific Inc.) and a Qubit 2.0 fluorometer (Life Technologies Inc.). Following the sample extraction, the DNA was processed according to the Agilent protocol (Version 8.0 December 2019) using commercially available male and female genomic DNA (Agilent Technologies, Human Reference DNA, Male and Female) as a reference DNA. The Agilent SurePrint G3 Unrestricted CGH 4x180K microarrays were used, which provide a practical average resolution of 50 kb. The array images were acquired using the Agilent laser scanner G2565CA. The image files were quantified using the Agilent Feature extraction software for Cytogenomics 5.3 and analyzed with the Agilent Cytogenomics 5.3 software (Agilent Technologies). Called copy number variants (CNV) were aligned with known aberrations in publicly available databases—ClinGen (http://dbsearch.clinicalgenome.org/search/), DECIPHER (Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources https://decipher.sanger ac.uk/), Database of Genomic Variants (DGV) (http://dgv.tcag.ca/dgv/app/home), as well as with the in-house database of detected variants and their clinical significance, ascertained by the trained analysts. All called CNVs were classified according to ACMG Standards and Guidelines [11].

2.2.2. Next-Generation Sequencing and Variant Interpretation

NGS was performed on the isolated DNA samples at the Clinical Institute for Special Laboratory Diagnostics, University Children's Hospital, UMC Ljubljana, and/or the Clinical Institute of Genomic Medicine, UMC Ljubljana.

We prepared NGS libraries according to standard Illumina protocols (Illumina DNA Prep with Enrichment). Sequencing was performed using Illumina instruments (NovaSeq 6000, NextSeq 550, or MiSeq). We processed, mapped, and aligned raw-read data and identified variants using standard bioinformatics pipelines used at the Clinical Institute for Special Laboratory Diagnostics, University Children's Hospital, UMC Ljubljana, or at the Center for Mendelian Genomics, CIGM, UMC Ljubljana. The interpretation of sequence variants was based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) Standards and Guidelines [12].

3. Results

The present study included 188 neonates diagnosed with CHD who underwent genetic testing when hospitalized at the Department of Neonatology, Division of Paediatrics, UMC Ljubljana. The cohort comprised 36% of neonates with isolated CHD and 64% of neonates with CHD and additional extracardiac congenital anomalies. Neonates clinically diagnosed with isolated CHD were assigned to one of the three groups according to Botto classification; 15% of neonates were diagnosed with simple isolated CHD, 10% with an association, and 11% with complex CHD (Table 1). CMA was performed as a first-tier test in 94% of neonates; in 6% of neonates, NGS was performed instead due to the high phenotype specificity of a monogenic genetic cause. In two cases of abnormal CMA results, karyotype and FISH were employed to delineate chromosomal aberrations further (Figure 1).

We established the genetic diagnosis of 22 distinct genetic syndromes in 17% of neonates. Genetic cause of CHD was identified in 24.8% of neonates with CHD and additional extra-cardiac anomaly and 3% of neonates with isolated CHD. For neonates with isolated CHD, the diagnosis was made in 5% of complex isolated CHD and 5.6% of associations, and none of the patients with simple isolated CHD. The diagnosis was reached by CMA in 11% of neonates. The most common microdeletion syndromes were 22q11.2 microdeletion syndrome (22.7%) and Williams syndrome (9.1%) (Table 2). By using NGS either sequentially to CMA or as a first-tier genetic test, the diagnosis was reached in 7.4% of neonates with CHD. The most common monogenic conditions identified were CHARGE syndrome (22.7%) and Noonan syndrome (9.1%) (Table 2).

In one patient, we have established a dual genetic diagnosis of 17q12 microduplication syndrome and Weaver syndrome. The phenotype present has characteristics of both conditions. Phenotypes and genetic diagnosis are described in detail in Table 2. Additionally, we detected variants of uncertain significance in 4.8% of neonates with CHD (Table 3).

4. Discussion

In a cohort of 188 neonates with CHD, we identified a genetic cause in 17% of patients. As all phenotypic features of genetic diseases are not always fully present at birth, but may only become apparent during the course of the child's development (e.g. global developmental delay), neonatal CHD patients present a diagnostic challenge that differs from paediatric or adult patients. The general recommendation for clinical genetic testing in CHD includes CMA as a first-tier and exome sequencing as a second-tier genetic test [13,14]. In this paper, we report on a clinical experience of using the recommended protocol in a cohort of neonates in the Slovenian National Tertiary Centre.

The diagnostic yield of CMA in our experience was 11%. The most frequently detected copy number variants associated with syndromic CHDs were those present in 22q11.2 microdeletion syndrome and Williams syndromes. Reported diagnostic yields in other studies varied considerably among different subgroups of CHDs [13,15,16].

The most frequently detected monogenic causes of syndromic CHD were Noonan and CHARGE syndromes. Exome and whole genome sequencing are increasingly used in research, but also in the clinical setting. However, diagnostic rates still differ across the studies and tested phenotypes [17,18,19]. In addition, rapid WGS demonstrated the yield in 27% of individuals with CHD, leading to changes in clinical management in 62% of patients with diagnostic results [20].

The incremental yield of whole genome sequencing over QF-PCR and CMA was estimated to be 26% for the cohort of congenital anomalies patients, with no significant yield increase over exome sequencing [21].

Interestingly, a dual genetic diagnosis was established in one patient in our cohort with a combination of clinical signs of both 17q12 microduplication syndrome and Weaver syndrome, highlighting the complexity of making a genetic diagnosis.

We found a genetic diagnosis in 3% of neonates with isolated CHD. It was estimated that 13.4% of infants with isolated CHD with identifiable genetic causes would have been missed if genetic testing had not been offered [22]. Although the yield of genetic testing in newborns with isolated CHD is relatively low, it is still important to offer them genetic testing because of the clinical benefit of molecular diagnosis. Timely diagnosis provides information about the prognosis and enables us to organise better surveillance for patients. It also provides us with information about the risk of recurrence in future pregnancies of the parents.

5. Conclusions

In a cohort of 188 neonates with CHD, we were able to identify a genetic cause in 17% of patients using CMA and NGS. Timely genetic diagnosis is important for the detection of syndrome-related comorbidities, prognosis, reproductive genetic risks and, when appropriate, genetic testing of other family members.