Detection of an Underlying 22q11.2 Duplication in a Female Neonate With Trisomy 18

Abstract

Current guidelines indicate that in patients with developmental disabilities or congenital anomalies, chromosomal microarray (CMA) is a first-tier diagnostic test. However, for patients with obvious chromosomal syndromes such as trisomy 13, 18, and 21, G-banded karyotyping is still recommended over CMA for establishing a diagnosis. In the case presented herein, a female neonate was suspected of having trisomy 18 based on pre- and postnatal evaluations. Karyotyping was requested but not performed due to insufficient cell growth; Interphase fluorescence in situ hybridization (i-FISH) found an extra copy of chromosome 18. CMA analysis uncovered gain of chromosome 18 and an additional duplication in chromosome 22q11.2, which went undetected with FISH. Our patient died within 40 hours after birth, but it is expected that patients with recognizable chromosomal syndromes could benefit from the discovery of coexisting copy number variations (CNVs) using CMA. This case shows that CMA can be a useful test for patients with recognizable chromosomal syndromes because of the potential benefits for patients and their families when co-existing CNVs are found.

Case Report

The patient was an infant female, born preterm (36 weeks of gestation) by spontaneous vaginal delivery. Level II ultrasound examination revealed intrauterine growth restriction (estimated fetal weight <10%) with an elevated systolic/diastolic ratio, strawberry skull, and ventricular septal defect (VSD) consistent with trisomy 18.^1,2 She had a birth weight of 1400 g (< third percentile), length of 39 cm (< third percentile), and head circumference of 28 cm (< third percentile).³ Apgar scores were 6 at the first minute and 8 at the fifth minute. At birth the patient had micrognathia, clenched fists with overlapping fingers, mild calcaneal prominence of heel left > right, mild hypotonia, and rocker bottom feet.

Structural heart defects such as a large VSD and small ostium secundum atrial septal defect (ASD), persistent left superior vena cava, and patent ductus arteriosus (PDA) were present. Head ultrasound revealed a possible mega cisterna magna variation in the central nervous system, and an upper GI series revealed possible nonrotation of the bowel. Echocardiography additionally uncovered thickened, redundant, and dysplastic atrioventricular and semilunar valves; a tricommisural bicuspid, thickened aortic valve; right atrial and ventricular dilatation; and hypoplasia of the transverse aortic arch with an anterior and posterior ductal shelf.

At birth the patient had a weak cry, poor respiratory effort, and decreased air entry. Positive pressure ventilation was started for a heart rate < 100 bpm and low O₂ saturation. By 1 minute, HR was > 100 bpm but still with poor respiratory effort. The patient was placed on nasal intermittent positive pressure ventilation (NCPAP) +5 with improvement in air entry and respiratory effort. The infant was shown to parents and transported to the Infant Special Care Unit (ISCU) for further care. On admission to the ISCU, the infant was maintained on NCPAP+5 at 50% with saturations at 100%. Intravenous fluids were started at 80 ml/kg/day. She was draped, and an umbilical venous catheter was placed. Labs were obtained and antibiotics started. Babygrams revealed mildly hazy lungs bilaterally, pneumomediastinum, subcutaneous emphysema in the soft tissues of the shoulders and neck, an increasingly enlarged heart, a stomach distended with air, and possible malrotation with the majority of the bowel appearing in the right lower quadrant. The patient’s respiratory distress worsened and she was pronounced dead 40 hours after birth.

The mother was 37 years old, G-5, P-4, LC-4, and Ab-1. She had had a ruptured ectopic pregnancy 14 years prior and underwent an open right salpingectomy. She denied drugs, alcohol, or tobacco use. The mother declined amniocentesis or aneuploidy screening for the patient while she was pregnant.

Combination of the phenotypic findings above made trisomy 18 a possible diagnosis. Karyotype and metaphase FISH studies of peripheral blood were ordered but could not be completed due to insufficient metaphase cell quantity and/or quality. Interphase fluorescence in situ hybridization (I-FISH) analysis was performed instead on the patient’s peripheral blood with specific probes for chromosomes 13, 18, 21, X, and Y using the AneuVysion probe kit (Abbott Molecular, Inc). Two hundred interphase cells were scored for each probe. This analysis showed 3 hybridization signals for chromosome 18, consistent with trisomy 18. It confirmed the presence of 2 X chromosomes consistent with a female phenotype. CMA was performed using the patient’s peripheral blood and was examined using the Cytoscan HD microarray (Affymetrix). This microarray consists of 2,696,500 oligonucleotide probes across the genome, including 1,953,246 unique nonpolymorphic oligonucleotide probes, and 743,304 SNP (single nucleotide polymorphism) probes. Microarray results were analyzed using the Chromosome Analysis Suite (ChAS) manufactured by Affymetrix. Results indicated that there were 3 copies of chromosome 18 (Figure 1) as well as a 2.40 Mb gain in chromosome 22q11.21 (18,640,729-21,041,014, arr[hg19]; Figure 2). The gene content of the 22q11.21 duplication was determined using the UCSC Genomic Browser based on the Genome Reference Consortium Human Genome (GRCH; build 37/hg19, accessed November 27, 2016). The duplicated region was found to have 33 genes described in the Online Mendelian Inheritance in Man (OMIM), with 9 of the OMIM genes capable of causing disease. These 9 genes were: PRODH (606810), SLC25A1 (190315), CDC45L (603465), GP1BB (138720), TBX1 (602054), COMT (116790), TANGO2 (616830), RTN4R (605566), and SCARF2 (613619).

Figure 1.

Chromosome Analysis Suite (ChAS) karyoview depicting gain of chromosome 18. The blue bar indicates the gain of chromosome 18.

Figure 2.

Chromosome Analysis Suite (ChAS) depicting a 2.40 Mb gain in chromosome 22q11.21. ISCN: arr[hg19] 22q11.21(18,640,729-21,041,014) x3.

Discussion

Although the chromosomal microarray is increasingly utilized because of its advantages over traditional cytogenetic methods, G-banded karyotyping is still considered first-tier testing in current guidelines when a patient has a recognizable chromosomal syndrome such as a common aneuploidy (e.g. trisomy 21, trisomy 18) or Turner syndrome.⁴ Reasons for this preference include the relatively rapid turnaround time, lower cost, sensitive detection of low-level mosaicism, and ability to distinguish free trisomy from translocation-associated trisomy associated with traditional cytogenetic methods.⁴ In our patient, karyotype and metaphase FISH analysis were not possible because of an unviable metaphase cell quantity and/or quality. Although interphase FISH confirmed a diagnosis of trisomy 18, only CMA detected the coexisting 22q11.2 duplication. Had karyotype analysis been possible, it would have likely missed this imbalance because its lower limit of resolution is typically only 3 Mb.⁴ Below 3 Mb, imbalances become extremely difficult to detect by karyotype.⁵ This underscores the value of CMA testing, even in patients suspected of having a common chromosomal syndrome, because the CMA is capable of detecting underlying, smaller CNVs that may go undetected with traditional cytogenetic methods, as in our patient.

Despite the presence of a 22q11.2 duplication, our patient’s phenotype was most consistent with that of trisomy 18 postnatally. This is not surprising, given the generally mild nature of the 22q11.2 duplication phenotype compared with that of trisomy 18. Phenotypic features of the patient consistent with trisomy 18 included virtually all findings; rocker bottom feet, clenched fists with overriding fingers, congenital heart malformations (including VSD, ASD, PDA), microcephaly, low birth weight, and micrognathia.^1,2 However, because 22q11.2 duplication syndrome has such a variable phenotype,⁶ it is possible that some phenotypic features were a product of the 22q11.2 duplication instead of the trisomy. Similar to cases of trisomy 18, infants with 22q11.2 duplication syndrome may have micrognathia, muscular hypotonia, congenital heart defects, and microcephaly, all of which this patient exhibited.^6-8 Additionally, reports of 22q11.2 duplication cases with specific heart defects such as VSD and ostium secundum ASD have been made.^9,10 These overlapping features make it difficult to determine the exact genotype-phenotype correlation that attributed to 22q11.2 duplication or the gain of chromosome 18 in this patient. Delineation of an increasing number of microdeletion and microduplication syndromes with potentially highly variable and unpredictable phenotypes makes it difficult to determine which combination of phenotypic features are syndromic for which possible genetic imbalances. Thus, a diagnosis of trisomy 18 does not rule out the presence of smaller CNVs.

Although the clinical significance of detecting the 22q11.2 gain in this reported patient would have been negligible because of the relative severity of the trisomy 18 phenotype and because of her early mortality, it may have been more significant if the patient had a condition such as trisomy 21 or Klinefelter syndrome, which implicate the use of karyotype and interphase FISH as first-tier diagnostic tests instead of CMA.⁴ Traditional cytogenetic analysis would very likely detect the predominant genetic abnormality but not the smaller 22q11 duplication (or other similar CNV). In cases such as trisomy 21, in which patients have a relatively high life expectancy, the influence of microdeletions and microduplications such as the 22q11.2 duplication could be clinically significant, perhaps through having a complementary or synergistic effect on phenotype leading to a different prevalence of certain phenotypic features. Certain phenotypic manifestations, such as congenital heart defects, muscular hypotonia, microcephaly, and dysmorphic features, are found in multiple different genetic syndromes, and it is likely that many patients clinically suspected of having a certain aneuploidy, for example, may have underlying, “hidden” CNVs with phenotypes that overlap with the phenotype of the suspected syndrome. These “hidden” CNVs are likely missed in many patients with genetic imbalances for which CMA testing is not performed. This is particularly likely for CNVs such as the 22q11.2 deletion or duplication, which are associated with remarkably variable and unpredictable phenotypes.^6,11

Although initial karyotype or FISH analysis remains invaluable for infants with obvious chromosomal syndromes (eg, Edwards, Down, and Patau syndromes), prior studies have recommended concurrent use of CMA in most instances of suspected trisomy 13 or trisomy 18.¹² Concurrent CMA could identify coexisting, smaller, clinically significant CNVs in patients who have classical chromosomal syndromes, as in our patient. Detection of the gain in chromosome 22q11.2 in the reported patient had important familial implications. Because roughly 70% of 22q11.2 duplications are inherited from parents with a normal or mild phenotype, it is possible that one of the patient’s parents carried this CNV.⁶ Thus, it was recommended that the parents undergo genetic testing themselves in order to determine if one of them carried the duplication, especially if there were intentions to have children in the future. Detecting underlying, clinically significant CNVs with the CMA in patients with a recognizable chromosomal syndrome for which karyotype and/or FISH testing remains first-tier could thus remain valuable for the families of patients, when these variations can be inherited. LM

Funding

This work was supported in part by the National Institutes of Health [5TL1TR001440-02].

Abbreviations

CMA

chromosomal microarray

iFISH

Interphase fluorescence in situ hybridization

CNVs

copy number variations

VSD

ventricular septal defect

ASD

atrial septal defect

NCPAP

nasal intermittent positive pressure ventilation

ISCU

Infant Special Care Unit

ChAS

Chromosome Analysis Suite