There were 23 fetuses with cleft lip (CL), and 82 fetuses with cleft palate with or without cleft lip (CP/CLP) (Table 1). The overall molecular diagnosis yield was 12.4% (13/105). P/LP variants were detected in 13 cases, including 4 CNVs and 10 SNVs. One case (#104) had both CNV and SNV (Appendix Table 1, Appendix Table 2). CL had a significantly lower positive rate than CP/CLP (0% vs 15.9%), although there was no statistical difference (P = 0.067). In comparison with the isolated group of CP/CLP, CP/CLP with additional structural anomalies exhibited a significantly higher detection rate (53.3% vs. 7.5%, P = 0.001).
Table 1
The P/LP variants detected by SNP-array and CES, and pregnancy outcomes in 105 fetuses.
Groups of CL/P
|
Cases (n)
|
P/LP variants
|
Positive rate,
n (%)
|
Pregnancy outcomes
|
CNVs, n (%)
|
SNV/indels, n (%)
|
TOP, n (%)
|
Livebirth, n (%)
|
Lost to follow-up, n (%)
|
Cleft lip (CL)
|
23
|
0 (0%)
|
0 (0%)
|
0 (0%)
|
5 (21.7%)
|
17 (73.9%)
|
1 (4.3%)
|
Isolated CL
|
21
|
0 (0%)
|
0 (0%)
|
0 (0%)
|
4 (17.4%)
|
16 (76.2%)
|
1 (4.8%)
|
CL with additional structural anomalies
|
2
|
0 (0%)
|
0 (0%)
|
0 (0%)
|
1 (50.0%)
|
1 (50.0%)
|
0 (0%)
|
Cleft palate with or without cleft lip (CP/CLP)
|
82
|
4 (4.9%)
|
10 (12.2%)
|
13* (15.9%)
|
42 (51.2%)
|
38 (46.3%)
|
2 (2.4%)
|
Isolated CP/CLP
|
67
|
1 (1.5%)
|
4 (6.0%)
|
5 (7.5%)
|
28 (41.8%)
|
37 (55.2%)
|
2 (3.0%)
|
CP/CLP with additional structural anomalies
|
15
|
3 (20.0%)
|
6 (40.0%)
|
8 (53.3%)
|
14 (93.3%)
|
1 (6.7%)
|
0 (0%)
|
Total
|
105
|
4 (3.8%)
|
10 (9.5%)
|
13* (12.4%)
|
47 (44.8%)
|
55 (52.3%)
|
3 (2.9%)
|
*one fetus (case 104) had both CNV and SNV. P/LP, pathogenic/likely pathogenic; CNVs, copy number variants; SNV, single nucleotide variant; indels, insertion and deletions; VOUS, variants of uncertain significance; TOP, termination of pregnancy.
|
Table 2
Genetic association in CP/CLP location and laterality
Groups of CL/P
|
CLP location and laterality
|
Cases (n)
|
P/LP variants
|
Positive rate, n (%)
|
CNVs, n (%)
|
SNV/indels, n (%)
|
Cleft palate with or without cleft lip (CP/CLP)
|
|
82
|
4 (4.9%)
|
10 (12.2%)
|
13* (15.9%)
|
Isolated CP/CLP(67)
|
Midline
|
54
|
1 (1.8%)
|
4 (7.4%)
|
5 (9.2%)
|
|
Bilateral
|
13
|
0 (0%)
|
0 (0%)
|
0 (0%)
|
CP/CLP with additional structural anomalies(15)
|
Midline
|
13
|
2 (15.4%)
|
5 (38.5%)
|
6* (46.2%)
|
|
Bilateral
|
2
|
1 (50.0%)
|
1 (50.0%)
|
2(100%)
|
*one fetus (case 104) had both CNV and SNV. P/LP, pathogenic/likely pathogenic; CNVs, copy number variants; SNV, single nucleotide variant; |
No genetic abnormalities were detected in the CL group. In the CP/CLP group, 13 cases were positive out of 82 cases. CP/CLP detection rates were significant different for midline and bilateral cleft subgroups based on the location (Table 2). Midline CP/CLP yield (n = 54) was significantly higher than bilateral CP/CLP yield (n = 13) in the isolated CP/CLP group (9.2% vs. 0%, P = 0.574). In the CP/CLP group with additional structural anomalies (n = 15), the yield of midline cleft (n = 13) was lower than that of bilateral cleft (n = 2) (46.2% vs. 100%, P = 0.467).
The detection rate in this study was 3.8% by CMA and 12.4% by CES (Table 3). CES detected all four P/LP CNVs by CMA, but with different sizes and boundaries. In addition to CNV, 9 cases (8.6%) of CL/P fetuses with P/LP SNV were detected by CES. A CES etiology analysis showed a higher discovery rate than a CMA etiology analysis in each subgroup, ranging from 2.5% (apparently isolated CL/CLP) to 33.4% (CP with additional structural anomalies).
Table 3
Positive rate detected by SNP-array and CES.
Groups of CL/P
|
Cases (n)
|
Positive rate n (%) of P/LP variants
|
Positive rate n (%) of VOUS
|
SNP-array, n (%)
|
CES, n (%)
|
SNP-array, n (%)
|
CES, n (%)
|
Cleft lip (CL)
|
23
|
0 (0%)
|
0 (0%)
|
2 (8.7%)
|
4 (17.4%)
|
Isolated CL
|
21
|
0 (0%)
|
0 (0%)
|
1 (4.8%)
|
3 (14.3%)
|
CL with additional structural anomalies
|
2
|
0 (0%)
|
0 (0%)
|
1 (50.0%)
|
1 (50.0%)
|
Cleft palate with or without cleft lip (CP/CLP)
|
82
|
4 (4.9%)
|
13 (15.9%)
|
0 (0%)
|
4 (4.9%)
|
Isolated CP/CLP
|
67
|
1 (1.5%)
|
5 (7.5%)
|
0 (0%)
|
4 (6.0%)
|
CP/CLP with additional structural anomalies
|
15
|
3 (20.0%)
|
8 (53.3%)
|
0 (0%)
|
0 (0%)
|
Total
|
105
|
4 (3.8%)
|
13 (12.4%)
|
2 (1.9%)
|
8 (7.6%)
|
P/LP, pathogenic/likely pathogenic; VOUS, variants of uncertain significance.
Four P/LP CNVs were detected in 4 fetuses with CP/CLP, including 21q22.13q22.3-del (case #68), 22q12.3q13.33-dup (case #88), 7q11.23-dup (case #104) and 6q22.1q27-dup (case #105) (Appendix Table 2). Nine cases with P/LP SNVs were found, including 6 autosomal dominants, 2 autosomal recessives, and one X-linked. The relevant genes were IRF6, RPS28, CHD7, PIGO, SHOC2, PHF8, UBE3B, and FLNB (Appendix Table 1, Appendix Table 2).
In case # 104, ultrasound at 23 gestation weeks revealed cleft palate, short limbs and low cone of the spinal cord. CMA detected a pathogenic 1.4 Mb duplication at 7q11.23. Due to the short limbs, CES was performed and an NM_001844.5:c.3472G > A variant in COL2A1 was identified (Appendix Table 2). This LP variant could result in achondrogenesis type II or hypochondrogenesis, which would explain the short limbs of a fetus. Consequently, the fetus may have two Mendelian diseases.
In case #97, ultrasound examination at 25 weeks of gestation revealed cleft palate, strephenopodia and hydronephrosis (Fig. 2, Appendix Table 2). According to CES analysis, there is a heterozygous LP SNV in FLNB gene, NM_001164317.2:c.4664G > A, inherited from the mother who has 20% mosaic in peripheral blood. The structural abnormalities of the fetus coincided with Larsen syndrome caused by FLNB variation. However, the mother showed no obvious structural deformity.
VOUS were also detected by CMA and CES. Among the 8 VOUS cases detected, CMA found 2 cases and CES found all 8 cases (Table 3, Appendix Table 3). Two cases had CNVs (8p22-dup, 11q21q22.1-del) and 6 cases had SNVs (CHD7, FLNB, TP63, CTNND1) (Appendix Table 1, Appendix Table 3). All these eight fetuses were live born and without other abnormalities followed up to 1 year.
The pregnancy outcomes of all the fetuses were followed (Table 1, Appendix). A total of 84.6% of P/LP-variant fetuses were terminated (11/13), and only two live births occurred (15.4%, 2/13). Comparatively, the TOP rate for those without a variant was 36.9% (31/84) and 62.5% (5/8) in cases with a VOUS was 62.5% (5/8). Three cases with no variant were lost to follow-up. The live birth rate between CL group and CP/CLP group was significantly different (73.9% vs 46.3%, P = 0.019). Further, the live birth rate was higher in the apparently isolated CP/CLP group than in the CP/CLP group with additional structural anomalies (55.2% vs 6.7%, P = 0.001).
All the livebirth fetuses were clinically reviewed after birth. In 21 cases of CL/P fetuses with P/LP variation or VOUS, five were born alive. Case #3 with LP variation in IRF6(NM_006147.4:c.1234C > T Het)was followed-up at 3 years old. He had post-palatoplasty, without lower lip pits, syngnathia, syndactyly, skin abnormalities, or genital abnormalities in popliteal pterygium syndrome, neither pits and/or sinuses of the lower lip in van der Woude syndrome. Case #55 with pathogenic variation in SHOC2 (NM_007373.4:c.4A > G Het) was followed-up at one year old. Her parents said her hair was fair and they did not want to provide other information. Case #69 with VOUS in CTNND1 (NM_001085458.2:c.655G > T Het) was followed-up at one year old. The affected boy had post-palatoplasty. He also had crooked teeth and ocular hypertelorism without syndactyly or frontal bossing. Case #9 with VOUS in FLNB (NM_001164317.2:c.1448G > A Het) was followed up at 2 years and 10 months old. He was 88 cm in height and 13 kg in weight. He has no deformity of the foot, no abnormality of joint movement, no dislocation of the hip joint, no abnormality of other complications, no phenotype of FLNB-related bone development. Case #71 with VOUS in CHD7 (NM_017780.4:c.5102A > G Het) was followed-up at one year and 4 months old. The boy was 82 cm in height and, 12.5 kg in weight. He had post-palatoplasty without congenital malformations of the external auditory canal or cryptorchidism.