In this study, we conducted CMA on fetuses diagnosed with CL/CP by ultrasound and carried out a subsequent evaluation to elucidate the genetic and clinical significance of CNVs in fetal CL/CP cases. Our findings indicate that the presence of associated structural malformations in fetal CL/CP is associated with a higher likelihood of clinically significant variants. The overall detection rate of clinically significant variants in our study (29/358, 8.1%) is lower compared to previous literature (40/270, 14.8%) 15. That may be attributed to the higher proportion of isolated CL/CP cases included in our study cohort (305/338, 90.2% vs. 125/270, 46.3%). Isolated CL/CP is a genetically complex disease with heterogeneous backgrounds. In cases where CMA produces negative results, further investigation of the underlying etiology may require the use of higher-resolution genetic tools, such as whole-exome sequencing (WES) or whole-genome sequencing (WGS). In our study, we identified nine cases of trisomy 13 and three cases of trisomy 18, findings that align with previous systematic reviews focused on prenatal studies that trisomy 13 and trisomy 18 are the most frequently observed chromosomal abnormalities in fetuses with oral clefts. Furthermore, our study incorporates twin pregnancies and provides a comprehensive comparison between isolated and non-isolated CL/CP cases, as well as between CP-only and CLP-only presentations.
CL/CP can be observed in the context of other syndromic characteristics but frequently occurs as an isolated. In our cohort, the majority of cases (90.2%, 305/338) presented with isolated CL/CP. Isolated CL/CP cannot be attributed to a singular easily identifiable mutation; rather, they are likely influenced by a multitude of risk factors and genetic variants 16,17. Non-isolated CL/CP cases are associated with additional abnormal morphological characteristics, often occurring as part of a syndrome. These syndromic defects are attributed to mutations and deletions that lead to the loss of function of one or more genes 18. In our cohort, there was a significant difference in the detection rate of isolated CL/CP and non-isolated CL/CP (11/33, 33.3% vs. 15/305, 4.9%, p < 0.001), a finding which is similar to previous studies 11.
We conducted inter-group and intra-group comparisons of isolated and non-isolated CP and CLP in singleton births and found no statistically significant differences (3/12, 25.0% vs. 23/288, 8.0%, p = 0.251). Additionally, we compared isolated and non-isolated CL/CP between twin and singleton births and similarly found no statistically significant differences (3/20, 15.0% vs. 26/338, 7.7%, p = 0.433). This observation underscores the decision to pursue prenatal diagnosis should not be based on the type of cleft or whether the pregnancy is singleton or twin, but rather on the presence/absence of associated anomalies during the prenatal period. In situations involving isolated CL/CP unaccompanied by concurrent anomalies and producing negative results on CMA, the use of sequencing methods with higher resolution may be considered as an option.
Further analysis of the cleft subgroups, we found that of the isolated CL/CP cases with ultrasound soft markers, 38.9% (7/18) were identified with chromosomal defects, including aneuploidy and pathogenic CNVs. Among the 7 cases displaying concurrent ultrasound soft markers, the most commonly observed abnormalities were increased nuchal translucency (NT) and nasal bone anomalies. This suggests that soft markers may be indicative of those cases with a higher likelihood of harboring a chromosomal abnormality, thereby highlighting the importance of conducting prenatal diagnostics in such instances. Another interesting finding in our cohort was the presence of 33 cases (9.8%, 33/338) exhibiting a familial history of CL/CP, including relatives in the 1st-degree or 2nd-degree in their families, and the majority of cases were classified as isolated CL/CP (93.9%, 31/33). In these cases, only one instance of trisomy 18 was detected (case 8), and we hypothesize that this may be associated with advanced maternal age. A previous study highlighted a robust correlation between orofacial clefts and familial history 19, which is associated with an elevated risk of cleft occurrence within the family unit 20,21. Moreover, in some situations, familial history can serve as an indicator of an individual's genetic susceptibilities 22, rendering individuals more susceptible to certain external factors, such as environmental/teratogenic factors that may trigger the occurrence of orofacial clefts.
In our study, we identified three cases with simultaneous microdeletions and microduplications. Karyotype testing was performed, with the following specific results: (Case 1: 46,XY,add(18)(q23); Case 9: 46,XY,add(5)(q31.1),add(21)(q22.1); Case 20: 46,XX,del(7)(q32)). Considering the case of Case 9's mother experiencing a spontaneous abortion in the early stages of a previous pregnancy, we recommended karyotype testing for both parents to rule out the possibility of balanced translocations. The results indicated the presence of a balanced translocation variant in Case 9's mother, with the specific karyotype result being 46XX,t(5;21)(q31.1;q22.3). Ultimately, the family decided to undergo third-generation assisted reproductive technology (ART) during their next pregnancy. This situation emphasizes the importance of understanding the limitations of CMA, as it cannot detect chromosomal balanced translocations. When CMA suggests the simultaneous presence of microdeletions and microduplications, alongside a family history of spontaneous abortion, attention should be given to the presence of chromosomal balanced translocations and further parental chromosomal karyotyping should be conducted.
Our analysis revealed the presence of two cases exhibiting a 4p16.3 deletion (associated with Wolf-Hirschhorn syndrome (WHS) (OMIM 194190)) among isolated CLP cases, accounting for 6.9% (2/29) of all cases. WHS is a well-documented genetic disorder resulting from a partial deletion of the short arm of chromosome 4, spanning a range of sizes from 2.62 Mb to 17.25 Mb, and involving genes such as LETM1, WHSC1, and FGFR3 23. Phenotypic manifestations in WHS patients typically encompass characteristic facial features, intellectual disability, growth retardation, and the presence of seizures (or EEG anomalies). The clinical presentation of WHS is typically associated with the size of the 4p deletion, with the more common category ranging between 5 and 18 Mb, leading to the manifestation of the widely recognizable WHS phenotype. The majority of prenatally diagnosed cases of WHS reported in the medical literature are delineated by facial abnormalities, symmetric intrauterine growth retardation, microcephaly and oligohydramnios 24,25. However, both cases (Case 2 and Case 4) in our study with WHS were not combined with other structural abnormalities. The widespread clinical use of CMA has increased the detection rate of submicroscopic chromosomal abnormalities linked to the WHS phenotype, particularly in cases presenting solely with facial abnormalities during mid-pregnancy. No pertinent literature has addressed the potential association between isolated CLP and the deletion of this chromosomal fragment. Therefore, it is imperative to conduct additional investigations to elucidate their correlation. Regrettably, these two cases chose to terminate of pregnancy and declined autopsy. Consequently, we are unable to ascertain whether other characteristic phenotypes associated with this syndrome were present.
In our data, case 6 exhibited a microdeletion of 610Kb in the chromosomal region 16p11.2, while case 7 exhibited a microduplication of 597Kb in the same region. Both aberrations have been suggested to possess pathogenic implications based on information derived from the OMIM and DECIPHER databases. The 16p11.2 (BP4-BP5, OMIM: 611913 and 614671) deletions and duplications encompassing 30 genes have been linked to varying levels of cognitive impairment, epilepsy, and psychiatric disorders, such as autism spectrum disorder and schizophrenia, each displaying distinctive clinical features and facial gestalt 26,27. Prior studies have provided evidence for the quantitative developmental effects of the 16p11.2 dosage, specifically impacting morphometric characteristics, such as head circumference and body mass index (BMI) 28,29. Deletions of this region have been associated with increased head size and BMI, whereas duplications are associated with reduced head size and BMI. Remarkably, both cases involved fetuses with isolated CLP, It is reasonable to hypothesize that there may be a potential association with the genetic underpinnings of craniofacial anomalies, yet further investigation is required to elucidate this connection.
The predominant coexisting major anomaly identified in conjunction with oral clefts was cardiac defects, present in 30.3% (10/33) of cases. The close association between CL/CP and congenital cardiovascular defects is not surprising considering the mechanisms of early embryonic development, the aortic arches of the primitive heart encircle the pharyngeal arches, which subsequently undergo morphogenesis to form the facial structures 30. This also suggests that careful screening of the cardiac development in fetuses with CL/CP should be conducted during the prenatal and postnatal periods to promptly identify any abnormal cardiovascular development. Indeed, Rittler's study demonstrated that 7.2% of infants with oral clefts were subsequently reclassified as having major coexisting anomalies, with cardiovascular defects being particularly prevalent, upon evaluation during a 1-year follow-up period 31.
In the majority of cases, the outcome of pregnancy is largely influenced by the presence of combined malformations detected by prenatal ultrasound and the results of chromosomal analysis. Parents often opt for pregnancy termination in cases where a prenatally diagnosed pCNV carries a poor prognosis, such as the 4p16.3 deletion syndrome. Similarly, when subsequent ultrasound assessments reveal worsening conditions for fetuses with VUS, parents frequently choose TOP. Conversely, a negative result obtained through CMA can enhance parents' confidence in continuing the pregnancy. A notable distinction was observed between the rates of pregnancy termination and live birth in cases where clinically significant variants were identified through gene testing, in comparison to cases with negative test results (28/29, 96.6% vs. 110/302, 36.4%, p < 0.001). The findings from genetic testing in fetuses diagnosed with CL/CP appear to have an impact on parental decision-making. Among cases where clinically significant variants were detected via genetic diagnosis, a small proportion of patients opted for pregnancy continuation following genetic counseling. Conversely, the majority elected for pregnancy termination due to potential postnatal phenotypic manifestations and unfavorable prognosis. Consequently, it is essential to conduct genetic testing in patients who receive a prenatal ultrasound diagnosis of fetal CL/CP. This approach equips physicians and parents with additional insights regarding potential postnatal phenotypes, which in turn, aid in informed decision-making regarding pregnancy outcomes and post-birth management.
This study is subject to several limitations. Firstly, it should be noted that this study is retrospective, and as a consequence, certain critical parameters, such as parental examination, are absent from the results. Second, although our study included both singleton and twin pregnancies in the prenatal investigation of CL/CP, as well as cases with a family history of CL/CP in the fetus, the number of cases in these two groups was relatively limited. This limitation hampers our ability to draw conclusive inferences. We aim to gather additional cases of twin pregnancies to gain further insight into potential differences between them. Furthermore, it is worth noting that CL/CP may be associated with a single gene, but we did not conduct further examinations such as WES.