22q11.2 recurrent copy number variation-related syndrome: a retrospective analysis of our own microarray cohort and a systematic clinical overview of ClinGen curation

Introduction

A cluster of low-copy repeats (LCRs) from A–H in chromosome 22q11.2 (chr22:17,900,001–25,900,000), also known as LCR22A–H, mediate nonallelic homologous recombination and cause 22q11.2 chromosomal rearrangements. Various intervals occur during this process that are respectively described as “proximal,” “central,” and “distal” on the basis of the corresponding length and region of the recurrent copy number variations (CNVs) (1,2). Both the recurrent regions [(i.e., 3 Mb (A–D: chr22:18,912,231–21,465,672) and 1.5 Mb (A–B: chr22:18,912,231–20,287,208)] included in the proximal CNVs are the most common types for 22q11.2 microdeletions (the small deleted CNVs) or microduplications (the small duplicated CNVs). As previously reported, the proximal A–D or A–B deletion may lead to severe DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS) (3), while identical duplication results in highly variable and nonspecific phenotypes (4).

The 22q11.2 central and distal CNV syndromes also express phenotypic variability, spanning from severe abnormality to mild characteristics and even a completely normal phenotype (5,6). This creates great challenges for genetic counseling and the prediction of clinical consequences. Just recently, the prenatal ultrasound phenotypes of CNVs in different regions of 22q11.2, their parental original, as well as pregnancy outcome, were analyzed and reported by Peixuan group. Interestingly, they found that prenatal phenotypes of the 22q11.2 region CNVs are diverse, which may be related to gene function; nuchal translucency (NT) thickening may be used as an early ultrasound finding of proximal 22q11.2 CNV (7). In this study, we retrospectively analyzed 34 microarray cases with 22q11.2 recurrent CNVs at our own laboratory from September 2018 to August 2021, and systematically reviewed each recurrent 22q11.2 region for which the ClinGen expert group had completed curation (derived from the ClinGen Dosage Sensitivity Map that ClinGen consortium curates genes and regions of the genome to assess whether there is evidence to support that these genes/regions are dosage sensitive and should be targeted on a cytogenomic array). We present the following article in accordance with the STROBE reporting checklist (available at https://dx.doi.org/10.21037/tp-21-560).

Methods

Collection of clinical information

We undertook a retrospective analysis of 34 cases of 22q11.2 recurrent CNVs (including 18 22q11.2 microdeletion cases and 16 microduplication cases) from 8465 microarrays performed at our own laboratory from September 2018 to August 2021. Prenatal amniotic fluid and villus, abortion tissue and villus, as well as peripheral blood derived from children/adults were used in our analysis. Data on gestation/age, the chromosome microarray analysis (CMA) results and corresponding variant evaluation types, clinical phenotypes (prenatal samples conforming to B ultrasound data), and tests were collected and are summarized in Table 1. After post-test counseling, a fraction of parents received a CMA analysis to validate their fetuses’ hereditary mode. Additionally, 5 months after CMA testing, the pregnant women and probands were followed up with by telephone. All the available information is displayed in Table 1. “N/A” represents a loss of communication, an unwillingness to inform, or an unknown hereditary mode. Written informed consent was provided by each pregnant woman, proband, or their parents. The study was approved by ethics board of Ningbo Women and Children’s Hospital (No. EC2020-014). This study conformed to the provisions of the Declaration of Helsinki (as revised in 2013).

CMA

After samples were collected, DNA was extracted using a TIANamp Genomic DNA Kit (Cat.#DP304-02; Lot#U8420). Then, 250ng DNA was amplified, labeled, and hybridized to the GCS 3000Dx v.2 platform (Affymetrix, Santa Clara, CA, USA). The SNP array test was processed with a commercial 750K microarray chip (CytoScan 750K Array; Affymetrix). The chip was washed with buffer, scanned with a laser scanner after hybridization with fragmented DNA. The data were analyzed using Chromosome Analysis Suite v3.2 (Affymetrix).

Overview of ClinGen curation

A ClinGen Dosage Sensitivity Map was retrieved for “22q11.2”. The information of each 22q11.2 recurrent region was collected. The score of each interval’s haploinsufficiency/triplosensitivity in current ClinGen curation, and respective clinical features, hereditary mode, and penetrance were all systematically classified and are summarized in Table 2.

Statistical analysis

The data were analyzed using Microsoft Excel 2010, and not involved in complex statistical analysis.

Results

Case presentation of 22q11.2 recurrent CNVs

We performed a retrospective analysis of 8,465 microarray cases at our laboratory from September 2018 to August 2021. We identified 34 patients carrying 22q11.2 recurrent CNVs (including 18 microdeletion cases and 16 microduplication cases), and examined their genotype-phenotype correlations. Their detailed clinical information is summarized in Table 1. Similar to previous reports, the 22q11.2 CNVs containing the proximal A–D interval comprised the most common recurrent region in our CMA data (17/34, 50%). In addition, the aberrant 22q11.2 CNVs of the other 6 cases (including 4 deleted fragments and 2 duplicated fragments) overlapped with the A–D region (6/34, 17.6%). As expected, all of these 22q11.2 CNVs were assessed as pathogenic variants. The central B–D interval (chr22:20,731,986–21,465,672) was found to be involved in the deletion/duplication regions of 5 cases (5/34, 14.7%). Additionally, 6 distal 22q11.2 CNVs were found to contain type II (E–F) (3/34, 8.8%) and type III (F–G) (2/34, 5.9%) regions and overlap with the type I (D–E/F) (1/34, 2.9%) interval. Notably, partially due to their variable phenotypes, incomplete penetrance, low dosage, or incomplete sensitivity curation, the majority of these CNVs were evaluated as variants of uncertain significance according to the American College of Medical Genetics and Genomics (ACMG) criteria.

Our cases were also accompanied by clinical phenotypes (ultrasound data), fractional follow-up information, and hereditary mode data. Across these 22q11.2 microdeletion samples, the congenital cardiovascular defect was identified as the most common feature (13/18, 72%), and mainly included tetralogy of fallot, ventricular septal defect, and patent foramen ovale. In particular, the pathogenic microdeletion variants concerning these congenital heart diseases generally spanned the proximal A–D interval. Most of the samples involved comprised prenatal amniotic fluid, so the clinical characteristics of the fetus were mainly distinguished by a B ultrasound combined with postnatal follow-up data. First, by detection with amniotic fluid, we exemplified cases 1 and 5. In the prenatal B ultrasound of the case 1 fetus with central B–D deletion, we observed strong light spots in the left ventricle and a patent foramen ovale. The postnatal follow-up information revealed more severe features, including a premature infant, anemia of prematurity, neonatal respiratory distress syndrome, neonatal pneumonia, and a low birth weight. In case 5, the pregnant woman had ever before given birth to an abnormal child with tetralogy of fallot. Unfortunately, the B ultrasound of the case 5 fetus indicated a more complicated congenital heart disease (tetralogy of fallot and pulmonary atresia) combined with an absent thymus, but the validated parental result suggested that the proximal A–D CNV of case 5 was inherited from the mother, despite the fact that >90% A–D deleted cases are de novo (3).

Among the 18 microdeletion cases, case 12 was a 10-year-old child who was confirmed to carry a pathogenic A–D deletion that was responsible for his multiple malformations of abnormal sexual development (i.e., a micropenis and microrchidia), intellectual/physical retardation, and congenital heart disease. Except for amniotic fluid and peripheral blood detections, the microdeletion CMA data of the abortion tissues and abortion villi are also set out in Table 1. As we observed, cases 13–15 contained the proximal (A–D) region and cases 16–17 overlapped with the proximal (A–D) region, and all of these cases presented with complicated congenital heart disease, including a severe tetralogy of fallot, ventricular septal defect, persistent truncus arteriosus, or perpetuate left superior vena cava (PLSVC). Parental validation of cases 14 and 15 indicated de novo variants.

Consistent with ClinGen triplosensitivity phenotype comments, the 22q11.2 recurrent microduplications exhibited a broad range of highly variable phenotypes. Of these affected amniotic fluid cases, 46% of the pregnant women primarily sought CMA help due to the high risk of 21 trisomy in fetal serological screening. The B ultrasound results revealed multiple variable phenotypes, including a ventricular septal defect, undetected right kidney, fetal cerebral ventriculomegaly, fissure in the gall bladder, and small and obscured transparent diaphragmatic cavity. Another abortion tissue with a duplicated variant of uncertain significance (VUS) inherited from the mother contained a distal type II (E–F) region, and presented with shorter long bones of the limbs, a slightly weakened echo of the spine and other body bones.

Clinical overview of the ClinGen curation

The proximal A–D/A–B recurrent CNVs are responsible for the most common microdeletions or microduplications in chromosome 22q11.2. In particular, A–D/A–B regional deletions can lead to severe DGS/VCFS syndrome for which clinical phenotypes typically include congenital heart disease (particularly conotruncal malformations), palatal abnormalities (particularly velopharyngeal incompetence, cleft palate, and bifid uvula), characteristic facial features, developmental delay and intellectual disability (DD/ID), behavior problems, immune deficiency, and hypocalcemia (3,8). The score of haploinsufficiency sensitivity in the ClinGen gene curation reached “3”. Among the 30 involved protein coding genes, TBX1 was identified as the most crucial morbid gene for DGS/VCFS syndrome. Yagi et al. found that a heterozygous 1 base pair deletion (1223delC) in the TBX1 gene caused a frameshift leading to a stop codon, and thus induced conotruncal anomaly face syndrome/VCFS (9). Paylor et al. reported that a heterozygous 23 base pair deletion (1320–1342del23) in the TBX1 gene could result in a frameshift and the extension of the protein from 504 to 616 amino acids, for which a mutation was found in a mother and her 2 sons who presented with VCFS (10). Nevertheless, patients with proximal duplication in 22q11.2 share a more variable clinical phenotype, ranging from apparently normal to nonspecific phenotypes (e.g., intellectual disability, learning disability, developmental delays, autism, psychiatric disorder growth delays, and hypotonia) (2,4). According to the literature, >90% 22q11.2 proximal (DGS/VCFS) deletions are de novo (3), while A–B/A–D duplications are frequently inherited (4). Notably, cat eye syndrome (CES) results from the tetrasomy/triplication of the CES critical region (CESCR) that overlaps with the chromosome 22q11.2’s proximal region. The CESCR includes the CECR1 and CECR2 genes, which are responsible for heart/facial and neurologic/eye features, respectively, but does not involve the DGS/VCFS syndrome region of 22q11.2 (11,12).

22q11.2 central recurrent CNVs contain B–D and C–D regions. The current haploinsufficiency score of B–D reaches “2” for the emerging evidence of phenotypic variability, including dysmorphic facial features, growth restriction/short stature, central nervous system (CNS) anomalies/seizures, developmental delay (including language delay), intellectual disability, psychiatric/behavioral problems, skeletal anomalies, cardiovascular defects, genitourinary anomalies, and immune deficiency/recurrent infections. Among the collected cases, 60% of the deletions were confirmed to be de novo events by parental testing (1,5,13). Similarly, the current triplosensitivity score for the B–D region is only “1” due to the limited number of patients reported in the literature, phenotypic variability, incomplete penetrance, and the lack of case-controlled data.

The distal CNVs of 22q11.2 mainly include 3 types, and currently, only the haploinsufficiency and triplosensitivity of type I (D–E/F) have been curated. ClinGen experts summarized the variable clinical phenotypes of D–E/F deletion: preterm birth, pre- and/or postnatal growth restriction, DD/ID, behavioral problems, cardiovascular defects, skeletal anomalies, and mild dysmorphic facial features. The deletions are de novo events in the majority of cases, but occasionally a few carrier parents are reported to have a mild or normal phenotype (14,15). Conversely, the variable clinical phenotypes of D–E/F microduplication mainly include developmental delays and facial dysmorphisms that exhibit incomplete penetrance in the clinical population (16,17). The above contents are included in the ClinGen dosage sensitivity curation page, and have been further summarized in Table 2. The dosage sensitivity curation of type II (E–F), type III (D–H or F–G) CNVs have not been completed, but sporadic cases have been reported.

Discussion

Multiple studies have conducted systematic reviews of 22q11.2 microdeletion/microduplication syndrome, performed statistical analyses of their own CMA data and elucidated possible genotype-phenotype contributions (18-20). For example, Pinchefsky et al. reported on the clinical phenotype and cytogenetic studies of a 3-year-old girl with a de novo distal 22q11.2 duplication, reviewed the literature associated with distal 22q11.2 duplication, and compared the clinical features of 28 previously published cases (6). Burnside’s review article on Gytogenetic and Genome Research systematically classified each the CNV of 22q11.2 region and provided a detailed overview of patients’ clinical features (as available in the literature). Additionally, their own cohort of postnatal and prenatal microarray cases with the 22q11.2 CNVs abnormality were also included in their review (1). Emerging reports of patients with 22q11.2 variants in the literature provide favorable evidence for the ClinGen dosage sensitivity curation experts, who are curating genes and regions of the genome to assess whether there is evidence that these genes/regions are dosage sensitive. The score of haploinsufficiency or triplosensitivity in ClinGen gene curation could further support the pathogenicity evaluation of chromosome CNVs.

According to the ClinGen dosage sensitivity curation and clinical overview of 22q11.2 recurrent CNVs, we learned that patients with 22q11.2 microdeletion/microduplication syndrome presented with a wide range of phenotypes concerning the abnormality of multiple systems, of which congenital heart diseases and facial dysmorphisms are the most common congenital malformations. Data on the penetrance of these recurrent CNVs is highly incomplete; some patients appear phenotypically normal, while others with the same genotype have mild to severe abnormalities. Thus, more evidence on the genotype-phenotype contributions of different 22q11.2 deleted/duplicated regions needs to be gathered.

In our summary of the abnormal 22q11.2 CNV cohort, the variants associated with proximal (A–D) region showed more severe clinical phenotypes, while those associated with the central (B–D) and distal type I (D–E/F)/type II (E–F)/type III (F–G) regions exhibited more mild or even normal features. In our laboratory, the majority of microarray cases used prenatal amniotic fluid. Due to the pathogenicity, potential terrible ending for the child, huge financial and psychological burden, most pregnant women choose odinopoeia, and only a fraction eventually give birth to their children. However, the postnatal newborns displayed various abnormalities, including neonatal dyspnea syndrome, and neonatal pneumonia. Among the 14 cases that received parental validation, 6 cases had de novo variants (6/14, 42.9%) and the other 8 were inherited from their parents (8/14, 57.1%) (7 of the 8 from the mother). In conclusion, this study provides clinical overview of the ClinGen curation and data support for ACMG evaluation of the pathogenicity of each interval involved in 22q11.2 recurrent deleted and duplicated CNVs. Certainly, more evidences on the genotype-phenotype contributions of different 22q11.2 recurrent regions need to be gathered.

Acknowledgments

Funding: This work was supported by the Social Development Public Welfare Foundation of Ningbo (Grant No. 202002N3150, 2019C50070), the First Municipal Medical and Health Brand Foundation of Ningbo (Grant No. PPXK2018-06), the Medical and Health Project of Zhejiang Province (Grant No. 2022503086).

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://dx.doi.org/10.21037/tp-21-560

Data Sharing Statement: Available at https://dx.doi.org/10.21037/tp-21-560

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/tp-21-560). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work, including ensuring that questions related to the accuracy or integrity of any part of the work have been appropriately investigated and resolved. Written informed consent was provided by each pregnant woman, proband, or their parents. The study was approved by ethics board of Ningbo Women and Children’s Hospital (No. EC2020-014). This study conformed to the provisions of the Declaration of Helsinki (as revised in 2013).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)