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Introduction
Congenital heart defects (CHD) are one of the most frequent manifestations of 22q11.2 deletion syndrome (22q11.2DS), particularly in those patients evaluated before 2 years of age in whom CHD can be found in 63%-79% of the cases1. In some infants, 22q11.2DS may present as an apparently isolated CHD or combined with other key features, such as typical facial features or developmental and neuropsychiatric disorders that manifest later in life, thus favoring late diagnosis2.
We found relatively few studies specifically aimed at detecting the 22q11.2 deletion in CHD patients, mainly by fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays3-5. However, none of these studies had been performed in Mexico.
The studies in older children and adults have shown variable detection of 22q11.2 deletions attributable to factors such as the type of hospital the patients came from, the type of CHD involved, or its severity. Therefore, such frequencies may not necessarily be representative of the neonatal population1,2. For this reason, this study aimed to determine the frequency of the 22q11.2 deletion in a consecutive sample of newborns with non-syndromic CHD of any type in a public hospital in western Mexico.
Methods
Subjects
We conducted a prospective hospital-based study from January 2017 to March 2021 in the Genetics and Pediatric Cardiology Services of the Hospital Civil de Guadalajara Dr. Juan I. Menchaca (Guadalajara, Jalisco, Mexico) through the Congenital Anomalies Registry and Research Center (CRIAC, for its Spanish acronym). The hospital-based active surveillance program for congenital defects covers all live births of ≥ 20 weeks of gestation and > 500 g of weight. All consecutive newborns identified with any major CHD confirmed by echocardiography performed by a pediatric cardiologist were eligible to participate. A team of certified clinical geneticists reviewed each patient’s information regarding prenatally diagnosed cases, medical records, surgical notes, clinical photographs, imaging studies, and complete dysmorphological examination6.
Each CHD case was classified into three categories, following the guidelines of the US National Birth Defects Prevention Study3,7: (a) isolated CHD, cases with no other major defects; (b) CHD with one extracardiac defect, cases with an additional, unrelated, specific major defect (e.g., gastroschisis, omphalocele, cleft lip, among others); and (c) syndromic, CHD cases with chromosomal abnormalities, monogenic disorders, or three or more major birth defects (e.g., trisomy 13, facio-auricular-vertebral spectrum, VACTERL association).
To determine the frequency of the 22q11 deletion in the population studied, we included only CHD patients with isolated or up to one extracardiac defect, as they presumably have a different etiology than those with specific syndromes. This definition was used because patients with 22q11.2 deletion could have more than 180 clinical features8. After informed consent, blood samples were collected for chromosomal and FISH studies.
Cardiac phenotypes were classified according to the International Paediatric and Congenital Cardiac Code9. Only ostium secundum and sinus venosus types were included as atrial septal defects (ASD), excluding patent foramen ovale and patent ductus arteriosus. Each case of CHD was counted separately. For example, in infants with ASD and ventricular septal defect (VSD), both defects were recorded; however, if a VSD was part of a tetralogy of Fallot (TOF) or double outlet right ventricle (DORV), they were not counted separately. Furthermore, each CHD was classified according to its complexity10. Patients in whom the 22q11.2 deletion was identified were subsequently re-evaluated for the presence of other 22q11.2DS manifestations. The Research Council and the Ethics Committee of our hospital approved this study.
FISH assays
The FISH assay was used to screen for chromosome 22q11.2 deletion in interphase nuclei of standard lymphocyte cultures using the Cytocell® probe (Cytocell Ltd., Cambridge, UK), which maps the TUPLE1 critical region at 22q11.2 combined with the N85A3 control probe at 22q13.3. In each patient, ~ 200 interphase cells were routinely evaluated.
Results
A total of 98 infants with at least one non-syndromic CHD (51 males and 47 females aged between 1 and 30 days) were included. Of these, 75 (76.5%) had no associated defect, and 23 (23.5%) had an additional extracardiac defect (three with cleft lip or palate, three with grade III microtia, two with hypospadias, two with hydrocephaly, and others with microcephaly, encephalocele, cleft palate, scoliosis, hemivertebrae, sacral lipoma, diaphragmatic hernia, omphalocele, intestinal atresia, gastroschisis, anal atresia, multicystic kidney, or cryptorchidism). We found eight patients (8.2%) with CHD carriers of the 22q11.2 deletion detected by the FISH assay. Overall, the number of major CHD observed in the study sample was 164, of which 151 were found in infants with normal FISH and 13 in infants with 22q11.2 deletion.
Table 1 compares the distribution of specific subtypes of CHD according to the presence of 22q11.2 deletion. The most frequent types of CHD in the studied sample were VSD (32.6%), ASD (27.5%), DORV (15.3%), pulmonary valve stenosis (9.2), and TOF (9.2%). All eight infants with 22q11.2 deletion had a CHD classified as a conotruncal defect compared to 37/90 (41.1%) of infants without the deletion (p = 0.001). The particular subtypes of conotruncal defects that showed a significant difference were truncus arteriosus (p = 0.013), TOF (p = 0.024), and pulmonary atresia with VSD (p = 0.031). No other statistically significant differences were found, even considering the number of CHD presented or the severity of the cardiac phenotype.
Table 1 Congenital heart defects and 22q11.2 deletion in the studied sample
| Subtypes of cardiac defects | Total (n = 98) n (%) | Normal (n = 90) n (%) | 22q11.2 deletion (n = 8) n (%) | p-value* |
|---|---|---|---|---|
| Dextrocardia | 2 (2) | 2 (2.2) | 0 (0) | 0.843 |
| Conotruncal defects | ||||
| Tetralogy of Fallot | 9 (9.2) | 6 (6.7) | 3 (37.5) | 0.024 |
| Truncus arteriosus | 4 (4.1) | 2 (2.2) | 2 (25) | 0.013 |
| Interrupted aortic arch | 6 (6.1) | 5 (5.5) | 1 (12.5) | 0.445 |
| Double outlet right ventricle | 15 (15.3) | 14 (15.5) | 1 (12.5) | 0.646 |
| Dextro-transposition of the great arteries | 7 (7.1) | 7 (7.8) | 0 (0) | 0.540 |
| Pulmonary stenosis with VSD | 4 (4.1) | 2 (2.2) | 2 (25) | 0.031 |
| Atrioventricular canal defect | 3 (3.1) | 3 (3.3) | 0 (0) | 0.772 |
| Anomalous pulmonary venous return | 7 (5.9) | 7 (6.7) | 0 (0) | 0.778 |
| Left ventricular outflow tract obstruction | ||||
| Bicuspid aortic valve | 1 (1) | 1 (1.1) | 0 (0) | 0.918 |
| Aortic valve stenosis | 1 (1) | 1 (1.1) | 0 (0) | 0.918 |
| Coarctation of the aorta | 5 (5.1) | 5 (5.5) | 0 (0) | 0.647 |
| Supravalvular aortic stenosis | 1 (1) | 1 (1.1) | 0 (0) | 0.918 |
| Hypoplastic left heart syndrome | 3 (3.1) | 3 (3.3) | 0 (0) | 0.772 |
| Mitral valve stenosis | 1 (1) | 1 (1.1) | 0 (0) | 0.918 |
| Right ventricular outflow tract obstruction | ||||
| Tricuspid valve atresia/stenosis | 5 (5.1) | 5 (5.5) | 0 (0) | 0.647 |
| Pulmonary valve atresia | 5 (4.1) | 4 (4.4) | 1 (12.5) | 0.353 |
| Pulmonary valve stenosis | 10 (9.2) | 9 (10.0) | 0 (0) | 0.647 |
| Hypoplastic right heart syndrome | 3 (3.1) | 3 (3.3) | 0 (0) | 0.772 |
| Ebstein malformation of the tricuspid valve | 1 (1) | 1 (1.1) | 0 (0) | 0.772 |
| Septal defect (isolated) | ||||
| Atrial septal defect | 27 (27.5) | 25 (27.8) | 2 (25) | 0.615 |
| Ventricular septal defect | 31 (31.6) | 30 (33.3) | 1 (12.5) | 0.285 |
| Single atrium | 3 (3.1) | 3 (3.3) | 0 (0) | 0.772 |
| Other specified heart defects | ||||
| Single ventricle | 6 (5.9) | 6 (6.7) | 0 (0) | 0.591 |
| Pulmonary artery stenosis | 3 (3.1) | 2 (2.2) | 0 (0) | 0.644 |
| Summary | ||||
| Number of major cardiac defects† | 164 | 151 | 13 | 0.231 |
| Cardiac defects per infant | ||||
| 1 | 58 (59.2) | 54 (60) | 4 (50) | 0.423 |
| 2 | 29 (29.6) | 26 (28.9) | 3 (37.5) | 0.610 |
| 3 | 7 (7.1) | 6 (6.7) | 1 (12.5) | 0.546 |
| ≥ 4 | 4 (4.1) | 4 (4.4) | 0 (0) | 0.707 |
| Severity of congenital heart defects‡ | ||||
| Great complexity | 53 (54.1) | 47 (52.2) | 6 (75) | 0.194 |
| Moderate complexity | 15 (15.3) | 14 (15.5) | 2 (25) | 0.493 |
| Simple | 30 (30.6) | 29 (32.2) | 0 (0) | 0.154 |
| Cases with one extracardiac defect | 23 (23.4) | 22 (24.4) | 1 (12.5) | 0.231 |
*X2 with Yates’s correction
†Since each CHD was counted separately, the total in each group may be > 98, > 90, or > 8, respectively.
‡Classified according to Stout et al.10
VSD: ventricular septal defect.
Except for one infant with hypocalcemia and another with hypocalcemia and thymic aplasia, the diagnosis of 22q11.2DS was not clinically suspected on reevaluation of any of the patients in our cohort. Additional clinical features found retrospectively after the confirmation of 22q11.2 deletion are described in Table 2. In particular, it is noteworthy that upon reevaluation of the facial phenotype, we found a bulbous nasal tip (Figure 1) as the most common morphological feature in our cohort of patients with 22q11.2 deletion.
Table 2 Clinical findings in infants with congenital heart defects and 22q11.2 deletion
| Findings | Patient | Total (n = 8) | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||
| Age at first examination (days) | 1d | 1d | 1d | 1d | 1d | 1d | 1d | 1d | |
| Gender | |||||||||
| Male | + | - | - | - | - | - | - | - | 1/8 |
| Female | - | + | + | + | + | + | + | + | 7/8 |
| Congenital heart defects | |||||||||
| Conotruncal cardiac defects | + | + | + | + | + | + | + | + | 8/8 |
| Tetralogy of Fallot | - | + | + | - | - | - | + | - | 3/8 |
| Pulmonary stenosis with VSD | - | - | - | + | - | - | - | + | 2/8 |
| Truncus arteriosus | - | - | - | - | + | + | - | - | 2/8 |
| Double outlet right ventricle TOF type | + | - | - | - | - | - | - | - | 1/8 |
| Atrial septal defects | - | - | + | - | + | - | - | - | 2/8 |
| Ventricular septal defects | - | - | - | - | - | + | - | - | 1/8 |
| Interrupted aortic arch | - | - | - | - | - | - | - | + | 1/8 |
| Pulmonary valve atresia | - | - | - | - | + | - | - | - | 1/8 |
| Typical facial features | |||||||||
| Hooded eyelids | - | - | - | - | - | - | - | - | 0/8 |
| Upslanting palpebral fissures | + | - | - | - | - | - | + | + | 3/8 |
| Malar flatness | - | - | + | + | - | - | - | - | 2/8 |
| Tubular nose | - | - | - | - | - | - | - | - | 0/8 |
| Bulbous nasal tip | + | + | + | + | + | - | + | + | 7/8 |
| Microtia (grade I) | - | + | - | + | - | - | + | - | 3/8 |
| Cleft lip and palate | - | - | - | - | - | + | - | - | 1/8 |
| Hypocalcemia* | + | - | - | - | + | - | - | - | 2/11 |
| Thymic aplasia* | - | - | - | + | + | - | - | - | 2/11 |
| Others | |||||||||
| Seizures* | - | - | - | - | + | - | - | - | 1/8 |
| Hypothyroidism* | - | - | - | + | - | - | - | - | 1/8 |
*Identified during the first week of life.
d: days; TOF: tetralogy of Fallot; VSD: ventricular septal defects.
Figure 1 Facial features of newborns with congenital heart defects and 22q11.2 deletion. Except for the patient in panel (D), a regular appearance is observed in the others but with an evident bulbous nasal tip. The lobe is forward-facing and uplifted on the patient in panel (C). Notably, the cleft lip and palate in the patient in panel (D) are not suggestive of the facial gestalt of 22q11.2 deletion syndrome.
Discussion
The distribution by subtype of CHD observed in the sample studied was representative and corresponded with the frequency reported in other population-based studies11, in which VSD, ASD, TOF, and pulmonary stenosis are the predominant defects (Table 1), which allows us to generalize our results.
The frequency of 22q11.2 deletion (8.2%) in 98 newborns with unselected (i.e., either conotruncal or non-conotruncal heart defects) and apparently non-syndromic CHD was slightly higher than that reported in other studies in older children. In those studies, also performed with a FISH assay, the frequency ranged from 1.3% to 5.7%, probably due to differences in the population studied and the selection criteria3,4. However, our results agree with the frequency reported in another study (9.6%) in which MLPA identified the 22q11.2 deletion5.
Consistent with previous studies2-5, 22q11.2 deletions caused a substantial proportion of cases with TOF, pulmonary atresia or pulmonary stenosis with ventricular septal defects, truncus arteriosus, DORV, and interrupted aortic arch, but not a transposition of the great arteries (Table 1). Notably, the detection rate of 22q11.2 deletion among patients with CHD did not vary significantly when including patients with or without extracardiac malformations (5.7% vs. 1.3%, respectively)12,13. This indicates that CHD cases with extracardiac anomalies should not be excluded from 22q11.2 microdeletion studies because other features characteristic of 22q11.2DS may not be evident in neonates or very young infants3,4,12.
Moreover, our findings do not differ substantially from those reported in a previous large study of 62 older children from Mexico with 22q11.2DS, in whom CHD was found in 97%, with a predominance of conotruncal defects (77%) followed by other lesions (20%)13. The fact that we did not find cases with defects such as isolated aneurysmal cervical aortic arch may be explained because our sample of newborns was studied for the CHD complaint and does not necessarily represent a cohort of infants with 22q11.2DS.
No studies in Mexico have investigated the appearance of newborns with 22q11.2DS. However, two studies analyzed facial features in Mexican patients13,14. Although they included only a few newborns, both studies concluded that typical facial characteristics such as a long face, short and up-slating palpebral fissures, hooded eyelids, bulbous tubular nose, and downward labial commissures are distinctive in Mexican patients with 22q11.2DS. Both studies recognize that these facial features are difficult to observe in newborns.
Based on these findings, our study confirms the importance of investigating the presence of a 22q11.2 deletion in all infants with CHD, particularly of conotruncal type, even in the absence of other vital manifestations also easily detectable during the neonatal period, such as hypocalcemia or thymic aplasia (Table 2). This could help to reduce the late diagnosis of 22q11.2DS since, for example, in countries such as Canada or the United States is performed until an average age of 4.7 years and after visiting at least seven different medical specialties15. Although the information on the age at diagnosis in Mexico is unknown, only a minority of 22q11.2DS patients are diagnosed during the neonatal period.
We also emphasize that the presence of a bulbous nasal tip (Figure 1) on the initial examination of a newborn with a conotruncal defect may help support the suspicion of 22q11.2DS (Table 2) because the other minor anomalies of typical 22q11.2DS facial dysmorphia are usually apparent or recognizable in older children or adults2,13.
A limitation of this study is the relatively small number of patients studied. Therefore, our estimates of 22q11.2 deletion frequency and the phenotypic frequencies observed in infants with 22q11.2 deletion may have been underestimated. The 22q11.2DS is usually diagnosed by FISH using commercial probes, including TUPLE1 (flanked by LCR A-B of 22q11.2 deletion). Another limitation of the study is that this probe has a detection yield of 85% for classic 2.55 Mb deletion spanning LCR A-D but only 5% and 2% of the atypical deletions spanning LCR A-B and A-C, respectively4,13,14,16. Therefore, it is possible that our interphase FISH studies missed some cases with deletions without the involvement of the A-B region.
In conclusion, in our sample of infants with unselected and apparently non-syndromic CHD, almost one in ten had the 22q11.2 deletion, increasing this proportion to approximately one in five among those with conotruncal defects. This confirms the relevance of the contribution of FISH studies in diagnosing patients with 22q11.2DS at increasingly younger ages.
