Characteristics of patients
During the study period we identified 126 cases of oligohydramnios. Of all cases in the study, 62 (49.2%, 62/126) cases were isolated oligohydramnios, while 64 (50.8%, 64/126) were non-isolated (fetuses had additional ultrasonography anomalies such as soft markers or structural abnormality). The mean age at diagnosis was 26.8 ±4.1weeks (range 17-35 weeks), and the mean maternal age was 29.5 ± 4.8 years (range 20–43 years). Overall, 19.8% (25/126) of prenatal samples were performed following amniocentesis and 80.2% (101/126) following cordocentesis sampling.
Results of CMA
Of the 124 cases with CMAs performed, one pathogenic CNVs and one likely pathogenic CNVs were identified. Overall, the detection rate of P/LP CNVs was 1.6% (2/124). In addition, four (3.2%, 4/124) VOUS findings were noted (Table 1).
Fetus 1 was presenting with oligohydramnios at 17 weeks. CMA revealed a duplication on chromosome 10q23.31q26.3 and a deletion at 4p16.3p16.2 in the fetus, which indicated that the karyotype of one of the parents may be balanced chromosomal translocation. CMA revealed a 3.0-Mb duplication on chromosome 9q34.11q34.13 in fetus 2 with oligohydramnios and IUGR.
Results of WES
WES was successfully performed in 32 fetuses. Pathogenic/likely pathogenic CNVs were identified in 7 fetuses, yielding a detection rate of 21.8% (7/32). De novo variants were identified in 1 cases (14.3%, 1/7), and 6 (85.7%, 6/7) variants were functional homozygous or compound heterozygous mutations in accordance with their recessive patterns of inheritance. Three (42.9%, 3/7) variants were involved in the renin-angiotensin-aldosterone system (RAAS), which are the known genetic causes of ARRTD (Table 2).
Case 1 was a fetus presenting with severe oligohydramnios, hyperechogenic kidneys and enlarged kidneys. AGT exon 3-4 (E3_E4 del) homozygous deletion was detected by WES, indicating parental heterozygosity. In case 2, ultrasound showed oligohydramnios and hyperechogenic kidneys. WES showed a heterozygous point variation c.598C>T (p.Q200*) (novel variant) from the mother and the heterozygosity E3_E4 del from the father, forming a compound heterozygosity. In case 3, ultrasound showed severe oligohydramnios. WES revealed a homozygous point variation c.1028G>A in the ACE gene. All the three fetuses were finally diagnosed as ARRTD.
We also found a homozygous point variation c.199-10T>G in the SLC25A20 gene in case 4. Mutation of SLC25A20 is causative for Carnitine-acylcarnitine translocase deficiency (CACTD). Case 5 presented with oligohydramnios, hyperechogenic kidneys and enlarged kidneys. WES revealed a compound heterozygous mutation in PKHD1, a gene associated with PKD4. Case 6 had bilateral renal dysplasia and hypoplastic nasal bone in addition to oligohydramnios. WES revealed a compound heterozygous mutation in FRAS1. Mutations in the FRAS1 gene may cause Fraser syndrome. Case 7 carried a heterozygous mutation of (c.1406_1413dup8) in the HNF1B gene. Mutations in this gene cause renal cysts and diabetes syndrome. Ultrasound showed severe oligohydramnios, bilateral renal dysplasia and enlarged heart.
In addition, one fetus showed genetic variants that were not P/LP, but merited further clinical and molecular investigations, and it was classified as VOUS variant. The total proportion of fetuses with VOUS variants is 3.1% (1/32).
Breakpoint PCR detection:
WES found fetus 1 and fetus 2 had the same E3_E4 del in AGT gene. Then both point mutation and deletion were confirmed by Sanger sequencing.
Located in the upstream of intron 2 primer IN2F (5 '- CAGGTCTGCCACTGCC
TTCTTAC -3') and downstream primer in intron 5 IN5R (5'- AGACTCTGTGGGCT
CTCTCATCC - 3') amplified a shorter product than the wild fragment in the samples with E3_4del. The wild fragment was 4280bp, and 2870bp region deleted (chr1:230,839,696-230,842,565, GRCh37/hg19). The PCR product containing the breakpoint was 1419bp band, which included a 9bp insertion (Fig 2A,2B), the same as the previously reported deletion type .
Prenatal diagnosis of the fetus was performed by amniocentesis at the third pregnancy of Family 1. Breakpoint PCR detection was performed in amniotic fluid DNA, and no 1419bp fragment was found, replaced by the wild fragment length (Fig 2B). It suggested that the fetus did not inherit the AGT deletion from the parents. Follow-up amniotic fluid monitoring of the fetus was normal.