Genetic analysis of 55 cases with fetal skeletal dysplasia

Clinical features of cases

Among the 55 families in the present study, the median age of pregnant women was 28 years, and the median gestational age was 23 weeks. According to the clinical characteristics detected using US, fetuses were divided into three groups: 10 (18.18%) had multiple malformations, 41 (74.55%) exhibited short limb deformities (short long bones less than -2standard deviation) with or without long bone bending, fracture, “telephone receiver-shaped” changes, or thoracic hypoplasia, and 4 (7.27%) isolated SD (hypomineralization, premature closure of cranial suture, small mandible, and syndactyly). Detailed information about the clinical features is provided in Additional files 1 and 2: Table S3 and Fig. S1–S12.

Genetic analysis

A total of 55 fetuses with suspected SD were studied, and 45 cases were diagnosed with hereditary diseases, with a diagnostic rate of 81.82% (45/55) (Fig. 1, Table 1). CNV-seq was performed for 1–10 fetuses with multiple malformations, and chromosomal abnormalities were identified in seven fetuses (70%, 7/10), including cases 1–5 with trisomy-18, case 6 with pathological microdeletions and microduplications (Xp22.33p22.12(2700000–19680000) × 1; 11p15.5p15.4 (180000–7100000) × 3; 20q13.2 (50380000–50640000) × 1), and case 7 with triploidy (69-XXX).

Fig. 1figure 1

Flowchart showing the methods and results of 55 cases enrolled in our study. QF-PCR: quantitative fluorescent PCR; CNV-seq: copy number variation sequencing; Panel: a special NGS gene panel analysis (including 811 congenital skeletal anomalies genes); WES: whole exome sequencing

Table 1 Summary of main malformation classification and molecular results for 55 cases with skeletal abnormalities

To provide a molecular diagnosis, targeted skeletal gene panel sequencing accompanied by CNV-seq was applied to fetuses 11–25, and WES to fetuses 8–10 and 26–55. Thirty-eight cases were diagnosed with hereditary diseases, yielding a diagnostic rate of 79.17% (38/48). Thirty-six causative variants were detected in 11 bone development-related genes, including FGFR3 (15/38), COL1A1/COL1A2(9/38), DYNC2H1(5/38), COL2A1(3/38), ALPL (1/38), EVC2(1/38), FGFR2(1/38), GNAI3(1/38), NPR2(1/38) and RMRP (1/38) (Fig. 2, Table 1). Of these, 18 were novel variants. De novo mutations (DNMs) were identified in 27 (71.05%, 27/38) cases with autosomal dominant (AD) inheritance, compound heterozygotes were identified in eight (21.05%, 8/38) cases with autosomal recessive inheritance, and in the remaining three (7.9%, 3/38) cases with AD inheritance, the variants were inherited from the mother. Detailed information is provided in Table 1.

Fig. 2figure 2

Genes in the present study. Eleven genes were diagnosed in 38 fetuses, including FGFR3(15/38), COL1A1/COL1A2(9/38), COL2A1(3/38), DYNC2H1(5/38), ALPL (1/38), EVC2(1/38), FGFR2(1/38), GNAI3(1/38), NPR2(1/38) and RMRP (1/38)

Case examplesCase 42

Case 42 was referred to our hospital because the fetus showed short limbs and fetal hydrops evaluated at 14 weeks and 4 days using US examination (Fig. 3A). Pregnancy was terminated at 17 weeks of gestation. The couple’s second pregnancy was evaluated at 22 weeks for suspected abnormal development of fetal limbs (micromelia; US image unavailable) and terminated. WES analysis confirmed that case 42 was heterozygous for the COL2A1 (NM_001844.4) c.2303G > T variant, resulting in a protein change: p.G768V, consistent with severe type 2 collagenopathy. Sanger sequencing confirmed that the fetus carried the mutation, which was also detected in the mother (the peak of the T allele was much lower than that of the G allele) (Fig. 3E, F). Further detailed clinical assessment of the mother at age 25 revealed mild symptoms compared with those of her fetus, including short stature (height 152 cm, father 175 cm, mother 162 cm), slightly shorter length of the left lower limb than that of the right, and a protruding short fourth toe on her left foot (Fig. 3C). In addition, she showed visual abnormalities, including severe myopia. When she was 1 year old, her left lower limb was shorter than her right lower limb by 2 cm. Her condition seemed to worsen, and she had difficulty walking due to genu varus of the left knee at age 7. Physical examination at age 16 revealed left lower extremity varus deformity, shortening (the length of the right lower limb was 66.8 cm while that of the left lower limb was 58.6 cm), internal rotation deformity of the left leg, and pelvic inclination (Fig. 3B). The patient presented limb claudication and had undergone femoral osteotomy to correct the 8-cm shortening of the left lower extremity. Due to the asymmetrical phenotype and two similar experiences of abortion, we supposed that the mother was affected by mosaicism. For an accurate diagnosis, WES was applied to the mother (II:2), and Sanger sequencing was performed on her parents. The WES results exhibited the mutated allele was present in 11 of 52 (21.2%) reads (Fig. 3G), consistent with the results of Sanger sequencing. COL2A1 c.2303G > T was not detected in her parents and has not yet been recorded in any database. The novel de novo pathogenic variant (COL2A1 c.2303G > T) was highly conserved across different species (Homo sapiens, Pan troglodytes, Macaca mulatta, Canis lupus familiaris, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Danio rerio, and Xenopus tropicalis) (Fig. 3H).

Fig. 3figure 3

COL2A1 variant caused severe type 2 collagenopathy in case 42 and mother (II2). A The image of US examination of case 42 revealed short limbs. B, C Clinical photograph of the mother with her pelvis inclined on the left side, shortened left lower limb (before extension osteotomy), and shortened fourth toe in the left foot (arrow). D The pedigree map of case 42. E, F Sanger sequencing of case 42 and the mother (II2). G The variant allele fraction and depth of coverage of the COL2A1 pathogenic variant detected by WES in case 42 and the mother. H The variant (COL2A1 c.2303G > T) was highly conserved across different species

Case 49

Due to positive pregnancies of fetal SD, case 49 came to our attention. The pregnancy was terminated because of the abnormal US at 12 weeks of gestation, presenting shortened and curved long bones (Fig. 4A). Two years ago, a scan at 23 weeks of gestation of the first affected fetus exhibited short long bones (< 5standard deviation), polydactylism, and a “common” atrioventricular  valve ring with the absence of the cardiac crux. Compound heterozygous variants (c.871-2A > G and whole EVC2 gene deletion) in EVC2 were found in case 49 using WES, inherited from the mother and father, respectively, which were validated using Sanger sequencing and MLPA analysis (Fig. 4C–F). Both variants were PVS1 + PM2-supporting + PM3 and classified as “pathogenic” according to the ACMG guidelines. Collectively, the affected fetuses were diagnosed with Ellis-van Creveld syndrome (EVC, MIM#225500). WES-based CNV analysis revealed heterozygosity at telomeric SNP rs2301856 (chr4:5461801) and centromeric SNP rs899691 (chr4:5754544), suggesting that the deleted region was smaller than 290 kb. To determine the precise size and breakpoints of the gross deletion, we estimated the number of copies (one or two) using qPCR analysis. We performed long-range PCR at 1.5-kb consecutive intervals to amplify the upstream and downstream regions of the EVC2 gene of the proband. The strategy and results are presented in Fig. 4G, H. Long-range PCR was performed for the affected fetus using the forward primer of D131 and reverse primer of U4, generating a PCR product of 1.5 kb. The aligned sequences revealed a deletion of 205 kb (Chr4:5,548199–5753996) involving the entire EVC2 gene and a part of the EVC gene. To investigate the potential underlying mechanism, the sequences between the two missing boundaries were aligned, and no microhomologous sequence was found. After searching for repetitive elements in the vicinity of the breakpoint, the telomeric breakpoint was found to be in a long interspersed nuclear element (LINE, L1PA4) belonging to the L1 family (Fig. 4I).

Fig. 4figure 4

EVC2 variants caused EVC in case 49. A The image of US examination of case 49 revealed short and curved long bones. B The pedigree map of case 49. C MLPA results of EVC2 in case 49: a heterozygous deletion of EVC2-22 ~ EVC-8. DF Sanger sequencing showed that the proband had a novel variant inherited from the mother. G Schematic presentation of the gross deletion. Arrows indicate genes; Solid horizontal lines indicate retained regions; Broken horizontal lines indicate deleted regions; Multiple primers were designed to be regularly spaced every ~ 25 kb (step 1), ~ 5 kb (step 2), and ~ 1.5 kb (step 3) within breakpoint regions. Long-range PCR with the forward primer of the D131 and the reverse primer of the U4 generates a PCR product of ~ 1.5 kb. H The breakpoint sequences of case 49 and Sanger-sequence of the gross deletion. I UCSC Genome Browser information around the telomeric breakpoint regions (chr4: 5,547,999–5,548,399)

Case 50

Case 50 was admitted to our hospital at 13 weeks of gestation and was diagnosed as micrognathia by US (mandibular length: 8 mm, biparietal diameter: 27 mm). Besides, oligohydramnios was found in case 50 (the single deepest pocket of amniotic fluid was 40 mm). WES demonstrated a heterozygous variant, c.119G > T (p.G40V), in the GNAI3 gene. The verification results of the parents suggested that the variant was a de novo mutation (Fig. 5B–D). The novel variant was classified as “likely pathogenic” based on the interpretation of PM1 + PM2-supporting + PM5 + PM6 + PP3 according to the ACMG guidelines. Structural modeling showed that glycine 40 was located in the random coil structure of the protein secondary structure and formed a hydrogen bond with lysine 46, which possessed a bond length of 2.9 Å (Fig. 5E). After changing to valine, the hydrogen bond was slightly lengthened to 3.3 Å (Fig. 5F). Glycine 40 of Gnai3 is highly conserved among species (Homo sapiens, Pan troglodytes, Macaca mulatta, Canis lupus familiaris, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Danio rerio, and Xenopus tropicalis) (Fig. 5H). In line with the aforementioned results, the etiology of case 50 was attributed to the mutation in GNAI3.

Fig. 5figure 5

GNAI3 variant caused auriculocondylar syndrome (ACS) in case 50. A The pedigree map of case 50. BD Sanger sequencing showed that the proband had a de novo variant. E, F The 3D molecular structure of Gnαi3. The magnified views of the wild-type Gly40 (E) and mutant Val40 (F) are shown respectively. The H-bonds are shown as green dashed lines, and H-bond distances (Å) are shown in red numbers G Gnαi3 domains are depicted in blue: boxes G1–G5. The variant reported here is in red, and previously described variants are in black. H Protein alignment showing conservation of residues GNAI3 p.Gly40Val across multiple species. This mutation occurred at evolutionarily conserved amino acid positions

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