Case presentation
A 22-year-old P3G4 woman (M1) was referred for genetic testing due to fetal anomalies detected by ultrasound. The fetus (the proband in this report, D4) was found to have dextrocardia, conotruncal anomaly, and pulmonary hypoplasia resulting in total anomalous pulmonary venous return (TAPVR). Previous M1 pregnancies include a phenotypically normal child (D1), one miscarriage (D2), and a child with heterotaxy born with a single ventricle, who passed away the day after delivery (D3) (Figure 1). During the writing of this report, M1 had a fifth pregnancy complicated with fetal pericardial effusion and premature atrial contractions; the child (D5) was born at 37 weeks gestation and is alive and well at one month. M1 presents with sickle cell trait, but is otherwise reportedly healthy, without major surgeries or hospitalizations between pregnancies, and no evidence of heterotaxy syndrome as revealed by a recent echocardiogram. Maternal and paternal families have no known consanguinity.
To characterize the etiology of the fetal dextrocardia found in proband (D4), genetic testing was performed on an amniocentesis sample from M1. Aneuploidy FISH analysis revealed a normal signal pattern for chromosomes X, Y, 13, 18, and 21 (Supplementary Figure 1), however CMA analyses revealed the presence of a duplication at 18q21.1 at g.47858016_48002991 (human genome version GRch38). The duplication, approximately 145 Kbp in size, partially overlapped SMAD2(NM_001003652) from exons one through six (Figure 2A). Subsequent CMA studies on blood of the phenotypically unaffected mother (M1) revealed the duplication to be inherited.
SMAD proteins regulate cell growth and differentiation by mediating TGFβ signaling [16]. In humans, heterozygous loss-of-function mutations of SMAD2 have been linked to congenital heart disease, including dextrocardia [14] and arterial aneurysms and dissections [15]. While SMAD2 has a highly significant genotype-phenotype association in proband, the interpretation of the duplication by CMA alone was uncertain given that CMA does not give positional information for the rearrangement (i.e. the duplication could be located in direct or inverted orientation to the normal SMAD2 allele, or it could be located elsewhere in the genome). To clarify the contribution of theSMAD2 duplication in proband’s phenotype, MPseq analysis was performed on fetal amniocytes. The duplication was mapped to chr18:47,850,720-48,005,918 (hg38). PCR reactions amplified the predicted MPseq junctions and breakpoints are estimated to be located between chr18:47850012-48005625 and chr18:47851237-47852159 (hg38) (Supplementary Figure 2). Structurally, the duplication was found to be in inverted tandem orientation to the normal SMAD2 allele, a pattern predicted to disrupt its coding sequence (Figure 2B, 2C, and Supplementary Figure 3). Gene expression analysis of the partialSMAD2 duplication revealed SMAD2 mRNA levels to be reduced by half compared to a normal male fetal amniocyte control, in agreement with the predicted SMAD2 allele disruption (Figure 3).