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).