Demographic characteristics
We analyzed WES results from 61 deceased fetuses, with 44.3% (27/61) submitted as singleton-only, 55.7 (34/61) as proband-parent trios. All cases terminated the pregnancies due to severe ultrasound abnormalities. The maternal age was from 22 to 38 years old (median 30 years); the mean gestational age was 21 weeks, with a range from 12 to 33 weeks and 1 day. A total of 15 women (25%) had previous pregnancy history of fetus with similar congenital malformations. There were 47.5% (N = 29) males and 52.5% (N =32) females based on fetal sex determined by WES data. The 61 cases were categorized into 10 classes according to the anatomical system affected, including skeletal system (14, 23.0%), multisystem (10, 16.4%), genitourinary (8, 13.1%), nervous system (6, 9.9%), facial region (6, 9.9%), cardiovascular (6, 9.9%), hydrops (4, 6.5%), stillbirth (4, 6.5%), growth abnormality (2, 3.3%), and abdominal (1, 1.6%). [Figure.1(A)].
Potential diagnostic variants and detection rate of WES in deceased fetuses
Among the 61 cases included, 28 cases yielded a definitive diagnosis by WES with a detection rate of 45.9% (28/61), involving 39 variants in 18 genes (10 pathogenic variants, 22 likely pathogenic variants, and 7 VUS). Detailed phenotypic and variant information of the diagnostic cases were summarized in Table 1. Skeletal system abnormalities yielded the highest detected rate of 39.2% (11/28), followed by 17.9% (5/28) in multi-system, 17.9% (5/28) in genitourinary abnormalities, 7.1% (2/28) in facial abnormalities, 7.1% (2/28) in nervous system, and 3.6% (1/28) in fetal hydrops, cardiovascular system abnormalities, and stillbirth, respectively [Figure 1(B)].The diagnostic rate was 48.3% (14/29) for male fetuses and 43.8% (14/32) for female fetuses, the difference was not statistically significant (p=0.723) . The diagnostic rate of single system abnormalities was 45.1% (23/51), and that of two or more systems abnormalities was 50% (5/10). The diagnostic rate of two or more systems fetal abnormalities was slightly higher than that of single-system, but the difference was not statistically significant (p=0.776).
Inheritance mode in the diagnosed cases
Among the 28 cases with diagnostic results, 46.45% (13/28) were associated with autosomal dominant (AD) diseases, 46.45% (13/28) with autosomal recessive (AR) conditions, and 7.1% (2/28) with X-linked recessive diseases. A total of 11 variants had arisen de novo (3 cases with missense variant in FGFR3, 2 cases with missense variant in COL1A1, 4 cases with frameshift variant in SALL4, KAT6B, ARIDA1 and COL1A1, 1 case with nonsense variant in NOTCH2, 1 cases with missense variant in GNAI3), all of them were associated with AD conditions. In remaining 15 cases with recessive pathogenic variants, 12 cases (80%) had biparentally inherited compound heterozygous variants, 1 case had homozygous variant (missense variant in ACE) and 2 fetuses inherited the variant in chromosome X from the mother. Specially, in Case No.9, supratententate hydrocephalus with narrow transparent compartments was detected by ultrasound. A heterozygous variant of L1CAM was found in this female fetus by WES, which was inherited from the mother. The phenotype was consistent with X-linked cerebral edema (HSAS) caused by this gene. Although the condition was X-linked recessive inheritance, considering that similar fetal malformations were found in two previous pregnancies, the phenotypes in the current fetus maybe caused by skewed X-inactivation. In addition, 21 novel variants in 11 gene were found. The inheritance mode of 28 cases was listed in Table2.
Disease categories involved in the diagnosed cases
The most prevalent disease was skeletal disorders involving six genes (FGFR3, DYNC2H1, COL1A1, NEB, SALL4, ECEL1) in 11 cases. FGFR3 associated with achondroplasia (100800) were identified in 3 cases, 4 cases of Asphyxiating Thoracic Dystrophy 3 (613091) were caused by DYNC2H1. In the remaining 4 cases, each was found with pathogenic/likely pathogenic variants in COL1A1 associated with Osteogenesis Imperfecta, Type I (166200), NEB gene associated with Arthrogryposis Multiplex Congenital 6 (619334), SALL4 associated with Duane-radial Ray Syndrome (607323), ECEL1 associated with Arthrogryposis, Distal, Type 5d (615065), respectively. The second most common category was multi-system involving 5 genes-COL1A1, FGFR3, CAD, NOTCH2, KAT6B-in 5 cases. For genitourinary system, variants in 3 genes-PKHD1, JAG1, ACE- were identified in 5 cases, among which 3 cases were Polycystic Kidney Disease 4 With Or Without Polycystic Liver Disease (263200) caused by PKHD1. In the remaining 2 cases, each was found with pathogenic/likely pathogenic variants in JAG1 gene associated with Alagille Syndrome 1 (118450), ACE gene associated with Renal Tubular Dysgenesis (267430). Variants in 2 genes-GNAI3, TFAP2A-associated with facial region were identified in two cases. GNAI3 gene associated with Auriculocondylar Syndrome 1 (602483), TFAP2A gene associated with Branchiooculofacial Syndrome (113620). In addition, variants were identified in the other 4 phenotype categories involving 4 genes, including 2 cases of L1CAM associated with Hydrocephalus Due To Congenital Stenosis Of Aqueduct Of Sylvius (307000), PLAA associated with Neurodevelopmental Disorder With Progressive Microcephaly, Spasticity, and Brain Anomalies (617527), PIEZO1 associated with Lymphedema, Hereditary, Iii (616843), and ARID1A associated with Coffin-siris Syndrome 2 (614607).
In case NO.2, we received a definitive diagnosis with the Alagille syndrome type 1 caused by heterozygous variation of JAG1 gene, which was an autosomal dominant inheritance, and the variation came from the mother. Of the remaining 33 cases without definitive diagnosis, there was one case with VUS. Abnormal development of both kidneys was found in prenatal imaging of one case, and heterozygous variant with uncertain significance of JAG1 was identified by singleton WES. Sanger sequencing indicated that the variant was inherited from the mother who did not show any clinical phenotype.