Single nucleotide polymorphism (SNP) array testing has been widely used in prenatal molecular diagnosis due to its high resolution and advantage in detecting loss of heterozygosity and uniparental disomy. Recently, there have also been several successive research publications on prenatal diagnosis of fetal cleft lip/palate and cranial anomalies using CMA. However, the progress of research into the clinical relevance of CNVs lags far behind development of the CMA technology itself. Despite the existence of various databases, the clinical pathogenicity of many CNVs is still unclear. Therefore, in this study we attempt to identify chromosomal abnormalities and CNVs in fetuses with CFMs using CMA and karyotype analysis, in order to determine the potential correlation of CNVs with CFMs.
Craniofacial anomalies are common in postnatal cases, especially in patients with nervous system disorders. However, their prenatal detection rate is low due to limitations of prenatal imaging technology and fetal development status. Nicolaides et al(1993) reported a 7% incidence of facial defects in fetal malformations[9], of which orofacial clefts were most common with prevalence of approximately 1 case per 700 deliveries[10]. The detection rate of prenatal cranial abnormalities is unclear, but indicates a high incidence in neonates[11]. Consistent with the above reports, in the 118 CFMs cases in this study it was observed that cranial abnormalities and orofacial clefts were most common, accounting for 40.6% and 43.6%, respectively, in fetuses with single CFMs. In our study, cytogenetic karyotyping revealed abnormal karyotypes in 15.4% of fetuses and the detection rate increased by 12.6% with CMA. In the current study, the incidence of chromosomal aberrations and CNVs was significantly higher than the 4.6% and 6.3%, respectively, reported in a recent study of fetal structural abnormalities[12]. This is because, in contrast to previous reports[13], our study did not merely focus on isolated CFMs or on cases with simple CFMs. When we focused only on isolated CFMs, more similar results of 6.4% and 8.5% were obtained, respectively.
At present, chromosomal karyotype analysis is unable to achieve the same results as CMA for regions less than 10 Mb. However, it failed to detect any abnormalities in case 31 by karyotype, while a 12.49 Mb deletion in 11q24.1q25 and a 12.88 Mb duplication in 16p13.3p13.12 were found by CMA. A similar situation occurred in Case 26 as well; the failure of chromosomal karyotype analysis could have been due to the deletion and duplication regions being similar in size and in band. In addition, chromosomal karyotype analyses were not performed in 14 aborted fetal samples here, including 7 cases of chromosome abnormalities subsequently revealed by CMA. It is well known that cytogenetic karyotype analysis is often made unavailable due to the frequent culture failures of non-sterile tissues or the selective overgrowth of maternal cells during tissue culturing[14]. Although CMA offers obvious advantages in improving the detection rate and identifying the pathogenicity of CNVs, it is limited in detecting balanced translocations and low-level mosaicism. Specifically, we found the enrolled cases of fetal abnormality caused by parental translocation accounted for a certain proportion in this study. Morever, mosaic 45,X[32]/46,XY[3] identified in case 13 by traditional karyotyping was determined to be monosomy X by CMA; this may have a slight impact on genetic counseling. Taken together, these results suggest that the combination of CMA with cytogenetic analysis can achieve more accurate and comprehensive results.
This study showed 9 cases (cases 1523) with CNVs relating to known chromosomal MMSs, of which 22q11 deletion/duplication syndrome was the most common with an overall prevalence of 3.4% (4/118; 2 proximal deletion, 1 proximal duplication and 1 distal deletion). 22q11 proximal deletion, also known as DiGeorge syndrome or velocardiofacial syndrome, involves more than 30 Mendelian genes; potential genes such as TBX1, COMT, UFD1L, GNB1L, TRXR2, MED15, and RANBP1 were researched to explore the phenotype/CNV correlation. Cleft palate is among the most common problems in patients with this microdeletion, while cleft lip is only occasionally found[15]. According to previous reports[16], TBX1 is considered to be responsible for cleft lip/palate phenotypes in either 22q11 deletion or 22q11 duplication. Fetus 17 with heart abnormalities and cleft lip was found to carry 22q11 distal deletion syndrome. Heart problems are a usual finding but cleft lip only, without cleft palate, has never been reported within the clinical spectrum of this syndrome. This suggested that simple cleft lip may need to be included in the phenotype spectrum of 22q11 distal deletion syndrome. Although researchers such as Spineli-Silva supported that the cause of CHDs and craniofacial anomalies in patients with distal 22q11 deletion may be associated with haploinsufficient MAPK1 expression[17], the underlying mechanisms are still largely unknown. We identified five distinct CNVs associated with rare MMSs including two microdeletion syndromes (3q29 and 17q12) and three microduplication syndromes (7q11.23, 8p23.1 and 16p11.2). These syndromes are associates with a range of mental and physical disabilities as well as craniofacial abnormalities. We screened several candidate genes located in these regions involved in craniofacial development, such as PCYT1A (locus 3q29)[18], DLG1 (locus 3q29)[19], and LHX1 (locus 17q12)[20,21]. Although TBX6 is considered to be a key gene that results in several major phenotypes in 16p11.2 duplication, potential genes associated with orofacial cleft of this region still need further exploration. Additionally, there is no reported correlation between 7q11.23 duplication and skull defects resulting from anencephaly, but this fragment has been confirmed as a pathogenic CNV of central nervous system development.
Other rare CNVs detected in the present study are also believed to contribute to the pathogenesis of CFMs. ZIC2 in 13q23.3 (cases 2325) has been identified as a key gene associated with several major CFMs resulting from holoprosencephaly[22]. Deletion in chromosome 7q34q36.3 encompassing gene SHH was identified in cases 26 and 27; SHH is involved in the organization and morphology of the developing embryo and is known to be a key gene in craniofacial abnormalities such as microcephaly, hypotelorism, midface hypoplasia, and cleft lip/palate[23]. In case 33 with isolated micrognathia, a 0.44 Mb deletion in region 1q21.1 was identified; haploinsufficiency of gene SF3B4in 1q21.1 has been confirmed to be associated with micrognathia[24]. Additionally, there has been evidence of the pathogenicity of haploinsufficient FOXC1 expression as well. Heterozygous deletion of FOXC1 in 6p25.3 (cases 28 and 32) can lead to Axenfeld-Rieger syndrome (6p25 deletion syndrome); ocular hypertelorism and flat midface were prevalent in the affecting postnatal cases[25]. Case 31, which was characterized by a maxillary protrusion, midface hypoplasia, and flat nose, had a 12.49 Mb deletion in chromosome 11q24.1q25 and a 12.88 Mb duplication in chromosome 16p13.3p13.12 involving the gene CREBBP. There are several literature reports suggesting that duplication of the 16p13.3 region containing the CREBBP gene results in a distinct similar facial dysmorphism[26], but to date still no case with duplication only encompassing the CREBBP gene has been reported. Case 29 had microcephaly < 2 SD and had a 21.21 Mb mosaic deletion in chromosome 20p13p11.21. Among 141 protein coding genes within this deletion region, mutations only in SNRPB and CSNK2A1 had been reported as associated with autosomal dominant microcephaly[27,28]. However, to date there is no evidence supporting their pathogenicity in haploinsufficiency. In case 30, we could not identify a gene specifically associated with the observed cleft palate; we only identified an autosomal recessive gene, HPGD, associated with a high-arched palate and without dose-sensitive reports[29]. We suspected a single mutation on the other chromosome may explain the observed phenotype.
There are several limitations of our study that should be discussed. First, we detected chromosomal abnormalities in fetuses with ultrasonographic features of cranial or facial abnormality; however, the majority of enrolled cases were cranial malformations and orofacial clefts due to the rarity of other CFMs features. Second, the function of candidate genes within the identified CNVs was not further investigated. Finally, not all parental samples were available, resulting in insufficient information for the interpretation of CNVs identified in these fetuses.