Fetal congenital talipes equinovarus: genomic abnormalities and obstetric follow-up results

Abstract

Objective

The etiology of congenital talipes equinovarus (CTEV) is unknown, and the relationship between chromosome microdeletion/microduplication and fetal CTEV is rarely reported. In this study, we retrospectively analyzed fetal CTEV to explore the relationship among the CTEV phenotype, chromosome microdeletion/microduplication, and obstetric outcomes.

Methods

Chromosome karyotype analysis and single nucleotide polymorphism (SNP) array were performed for the 68 fetuses with CTEV.

Results

An SNP array was performed for 68 fetuses with CTEV; pathogenic copy number variations (CNVs) were detected in eight cases (11.8%, 8/68). In addition to one case consistent with karyotype analysis, the SNP array revealed seven additional pathogenic CNVs, including three with 22q11.21 microdeletions, two with 17p12p11.2 microduplications, one with 15q11.2 microdeletions, and one with 7q11.23 microduplications. Of the seven cases carrying pathogenic CNVs, three were tested for family genetics; of these, one was de novo, and two were inherited from either the father or mother. In total, 68 fetuses with CTEV were initially identified, of which 66 cases successfully followed-up. Of these, 9 were terminated, 2 died in utero, and 55 were live births. In 9 cases, no clinical manifestations of CTEV were found at birth; the false-positive rate of prenatal ultrasound CTEVdiagnosis was thus 13.6% (9/66).

Conclusion

CTEV was associated with chromosome microdeletion/microduplication, the most common of which was 22q11.21 microdeletion, followed by 17p12p11.2 microduplication. Thus, further genomic detection is recommended for fetuses with CTEV showing no abnormalities on conventional karyotype analysis.

Keywords:

• Chromosome microdeletion/microduplication
• fetus
• obstetric follow-up
• congenital talipes equinovarus
• SNP

Introduction

Congenital talipes equinovarus (CTEV) is a common congenital disability, with an incidence of 0.1% in live births [1–4]. The etiology of CTEV remains unknown, and its incidence varies according to sex, with a male-to-female ratio of 2:1 [5]. Prenatal ultrasonography is an important tool for CTEV screening. Diagnostic criteria for CTEV on ultrasound examination of the fetus include the display of plantar and long axial sections of the calf tibiofibula in the same plane, with posture remaining unchanged despite fetal movement of the lower limbs [6]. CTEV is usually diagnosed at or after 20 weeks of gestation [7,8,]. Advances in prenatal ultrasonography have significantly improved the detection rate of CTEV [9,10].

The causes of CTEV are complex and include neuromuscular diseases, chromosomal abnormalities, genetic syndromes, and environmental factors [11–13]. Environmental factors are generally related to limited fetal movement in utero, such as in oligohydramnios, twin pregnancy, and amniotic band syndrome. Approximately 20% of CTEV cases are associated with chromosomal abnormalities (aneuploidy) and known genetic syndromes [1,8,]. The most common aneuploid abnormalities are trisomy 21, trisomy 13, and triploidy [14–16]. Although several epidemiological and genetic studies have examined the causes of CTEV, its specific mechanisms remain unclear [17]. The combination of heredity and environment is a clear pathogenic factor for this disease, and its pathogenesis involves genomic variations as well as vascular and muscle abnormalities [18]. Single nucleotide polymorphism (SNP) arrays, which are mainly used to detect chromosomal microdeletions and microduplications, can increase the detection rate of copy number variations to more than 10% [19]. Few reports have examined the relationship between chromosomal microdeletions/microduplications and fetuses with CTEV [20–22]. Therefore, in this study, SNP arrays and karyotype analyses were performed for 68 fetuses with CTEV, along with follow-up after birth, to explore the relationship among genetic etiology, pregnancy outcome, and neonatal prognosis of fetuses with CTEV and to provide evidence for the clinical management of fetuses with CTEV.

Materials and methods

Patient data

Intotal, 68 fetuses at 18–35 weeks of gestation, with suspected CTEV based on prenatal ultrasonography and invasive prenatal diagnosis at Fujian Maternal and Child Health Hospital, were selected from December 2016 to March 2023. The inclusion criterion for the study was that prenatal ultrasonography showed CTEV as the only fetal abnormality. The exclusion criteria were as follows: prenatal ultrasonography indicating CTEV absence in the fetus or the presence of CTEV along with other structural abnormalities. Under ultrasound guidance, amniotic fluid or umbilical cord blood was extracted at different gestational weeks from the pregnant women, and chromosome karyotype analysis and SNP array detection were performed. Amniocentesis was performed at 18–24 weeks, and umbilical cord blood puncture was performed at 25–35 weeks. The parents of all fetuses underwent prenatal genetic counseling and signed an informed consent form.

Chromosome karyotype analysis

The amniotic fluid or umbilical vein blood of the 68 fetuses was cultured, harvested, stained, and prepared according to the standard operation procedure for G-banding karyotype analysis, and G-banding (C-banding and N-banding were added if necessary). Karyotypes were collected and analyzed using the GSL-120 automated chromosome scanning platform. Forty karyotypes were counted in each case, five were analyzed, and the counts and analyses were increased in cases of abnormalities.

Single nucleotide polymorphism array

The experiment was conducted in strict accordance with the standard procedures provided by the manufacturer (Affymetrix Co., USA) for digestion, amplification, purification, fragmentation, labeling, hybridization with chips, washing, scanning, and data analysis of the genomic DNA samples. The CytoScan 750 K chip (Affymetrix, USA) was used for testing. This chip has a single nucleotide polymorphism probe and an oligonucleotide probe, in which the oligonucleotide probe covers the entire genome on average with a 99% detection rate for genomic structural variation. SNP probes can detect genome deletions and duplications, as well as uniparental disomy and loss of heterozygosity. The SNP array results were analyzed using CHAS software and bioinformatics methods; copy number variations (CNVs), deletions, and duplications were then determined according to the scatterplot distribution of the DNA copy number. Fragments with CNV ≥ 100 kb were selected for further analysis. The reference databases used for CNV comparison and analysis included internal and online public databases, including the DGV (http://projects.Tcag.ca/variation), DECIPHER (http://www.sanger.ac.uk/PostGenomics/decipher), and OMIM (http://www. Omim.org) and UCSC (http://www.genome.UCSC.edu/) databases. CNVs can be divided into three categories and five grades based on their nature [23]: (1) pathogenic CNV, (2) possibly pathogenic CNV, (3) variant of uncertain significance (VUS) CNV, (4) possibly benign CNV, and (5) benign CNV.

Pregnancy outcome and post-natal follow-up

All patients were followed up via telephone. Follow-up information included (1) the status of the fetus, in particular whether the fetus was aborted or terminated, along with the reasons; (2) Newborn matrices including body mass and length, Apgar score, abnormal appearance, and presence of CTEV; (3) feeding patterns after birth; (4) physical growth, development, and neurobehavioral progress after birth; (5) whether the final diagnosis of CTEV was confirmed; (6) classification of the clubfoot later as positional clubfoot, syndromic clubfoot, or idiopathic clubfoot, and (7) the treatment administered for CTEV.

Results

Chromosome karyotype analysis of fetuses with CTEV

Chromosomal karyotype analysis was performed successfully for all the 68 fetuses with CTEV, including 45 male fetuses and 23 female fetuses, with a male-to-female ratio of 1.95. Only one abnormal case was detected by karyotype analysis as trisomy 21.

Results of the SNP array

SNP array for the 68 fetuses with CTEV resulted in the detection of eight cases of pathogenic CNV (11.8%, 8/68). In addition to one pathogenic CNV consistent with the karyotype analysis, the SNP array identified seven pathogenic CNVs with fragment sizes ranging from 0.3 to 5.2 Mb. These seven cases of pathogenic CNV included three cases of 22q11.21 microdeletion, two cases of 17p12p11.2 microduplication, one case of 15q11.2 microdeletion, and one case of 7q11.23 microduplication. Of the seven cases with pathogenic CNV, the parents of four fetuses refused family genetic verification. Family genetic testing was performed for the other three cases; the results indicated that one was a denovo case, one was inherited from the father, and one was inherited from the mother (Table 1).

Table 1. Results of pathogenic microdeletion/microduplication in fetuses with CTEV.

Case	SNP-array results	Size (Mb)	Candidate gene(s)	Clinical significance	Inheritance	Obstetric follow-up
1	arr[hg19]22q11.21(18631365_21464764)x1	2.8	DGCR2, TBX1, DGCR8, DGCR6L	P	–	TOP
2	arr[hg19]22q11.21(18,919,477-21,800,471)x1	2.9	DGCR2, TBX1, DGCR8, DGCR6L	P	–	TOP
3	arr[hg19]22q11.21(20,730,143-21,800,471)x1	1.0	ZNF74, SCARF2, MED15, PI4KA, SERPIND1, SNAP29, CRKL	P	–	TOP
4	arr[hg19]17p12p11.2(15,759,453-20,547,625)x3	4.7	RAI1	P	–	5 years old, TE and low intelligence, autism, ADHD, Plaster treatment
5	arr[hg19]17p12p11.2(15491109_20698334)x3	5.2	RAI1	P	Denovo	TOP
6	arr[hg19]15q11.2(22770422_23082237)x1	0.3	TUBGCP5, CYFIP1, NIPA2, NIPA1	P	Paternal	Plaster treatment, healthy
7	arr[hg19]7q11.23(72608900_74242167)x3	1.6	ELN, LIMK1, GTF2IRD1, GTF2I	P	Maternal	Plaster treatment, healthy

CTEV, congenital talipes equinovarus; TOP: termination of pregnancy; ADHD: attention deficit hyperactivity disorder.

Pregnancy outcome and postnatal follow-up

Out of the initially identified 68 cases of fetuses with CTEV, 66 were successfully followed up, while 2 were lost to follow-up, yielding a follow-up rate of 97.1% (66/68) (Table 2). Among the 66 successfully followed-up cases, 8 were diagnosed with syndromic varus (1 with trisomy 21 and 7 with pathogenic CNV), 9 were born without foot varus (resulting in an overall false-positive rate of 13.6%), 7 were diagnosed with positional clubfoot, and 42 were diagnosed with idiopathic clubfoot. The treatment administered for clubfoot include manual reduction treatment for positional clubfoot fetus and surgical treatment for idiopathic clubfoot (specific surgery is unknown). Further, of the 66 follow-up cases, 9 pregnancies were terminated (5 carried pathogenic CNVs and 4 had normal CNVs), 2 resulted in intrauterine deaths, and 55 were live births. Among the 55 live births, four were found to have other abnormalities besides CTEV during post-natal follow-up, including one complicated with polydactyly of the thumb and biliary atresia; one complicated with intestinal obstruction, multi-joint contracture of the hands and feet, and cryptorchidism; one with language retardation; and one with autism and hyperactivity (with 17p12p11.2 microduplication).

Table 2. Pregnancy outcomes and follow-up of 68 fetuseswith CTEV.

	Pregnancy outcomes andfollow-up
SNP-array	Loss to follow-up	Stillbirth	TOP	CTEVafter birth	Normal after birth	CTEV and abnormal growth and developmentafter birth	Total
Normal CNV	2	2	4	40	9	3	60
pCNV	0	0	5	2	0	1	8

CTEV, congenital talipes equinovarus; TOP: termination of pregnancy; pCNV: pathogeniccopy number variation.

Discussion

In this study, among the 68 fetuses suspected of having CTEV, 45 were male and 23 were female, showing a ratio of 1.95, consistent with that reported in the literature, indicating that the incidence of CTEV differs according to sex, with a male-to-female ratio of 2:1 [5]. G-banding karyotype analysis indicated that only one fetus had trisomy 21. However, SNP array analysis of 68 fetuses revealed 7 additional cases of pathogenic CNV in addition to 1 case of pathogenic CNV consistent with karyotype analysis; the detection rate was 11.8% (8/68) (8 cases were diagnosed with syndromic varus), and the CNV fragment size was between 0.3 and 5.2 Mb, all of which could not be recognized by conventional karyotype banding. Therefore, the SNP array can compensate for the shortcomings of traditional chromosome karyotype analysis and improve the detection rate of pathogenic CNVs in fetuses with CTEV.

The question of whether a fetus with CTEV should receive a prenatal diagnosis remains controversial. Lauson et al. [24] consider that prenatal diagnosis is unnecessary for CTEV because the detection rate of chromosomal aneuploidy is very low, ranging from 1.7% to 3.6%. In this study, the detection rate of pathogenic CNV in CTEV was 11.8% using the SNP array. Further, studies have reported that the false-positive rate in CTEV fetuses ranges from 11.8% to 40.0% [25,26,]. In this study, the overall false-positive rate was 13.6%. Considering the detection rate of pathogenic CNVs and the false-positive rate of CTEV in this study, we suggest that a continuous ultrasound review should be performed for fetuses with CTEV. Invasive prenatal diagnosis and SNP array should be performed when ultrasound results still indicate CTEV. Continuous ultrasound observation of CTEV fetuses has the potential to reduce the false-positive rate and the invasive prenatal diagnosis rate.

CTEV is a common phenotype associated with some microdeletion/microduplication syndromes [27]. In this study, seven cases of pathogenic CNV were detected, all of which were microdeletion/microduplication syndromes, with 22q11.2 microdeletion syndrome (22q11.2 DS) (3/7) being the most common. Previous studies have shown that 22q11.2 DS is primarily associated with cardiac malformations, cleft palate, and thymic development. Deletion of the T-box1 transcription factor (TBX1) is generally considered an important cause of 22q11.2 DS. In this study, the SNP array revealed missing fragments of 2.8 Mb, 2.9 Mb, and 1.0 Mb, in three cases of CTEV, all of which were located in the 22q11.2 DS. Homans et al. [28] showed that the incidence of CTEV in patients with 22q11.2 DS (3.3%, 48/1 466) was 30 times that in the general population (approximately 1%), suggesting that CTEV is highly correlated with 22q11.2 DS. Accelerated interchondral mineralization of the occipital bone in newborn mice with TBX1 deletion is reported to cause fusion [27]; however, the mechanism underlying the correlation between CTEV and 22q11.2DS remains unclear and requires further study. In this study, 17p12p1.2 microduplication was detected in two CTEV fetuses, encompassing the RAI1gene, resulting in Potocki–Lupski syndrome. The main clinical features of patients after birth in this syndrome include mild-to-moderate mental retardation, developmental delay, short stature, autism, and other abnormalities [29]. The 15q11.2 microdeletion detected in a CTEV fetus involved a fragment size of approximately 312 Kb containing the TUBGCP5, CYFIP1, NIPA2, and NIPA1 genes. The penetrance of the15q11.2 microdeletion is approximately 8–10%, with a varying clinical phenotype includinga normal phenotype, as well as developmental delays, epilepsy, autism spectrum disorders, congenital heart disease, and other abnormalities [30,31,]. The 15q11.2 microdeletion, in this case, was inherited from a phenotypically normal father. This study also included a CTEV fetus with 7q11.23 microduplication involving a fragment size of approximately 1.6 Mb containing ELN, LIMK1, GTF2IRD1, GTF2I, and other genes. The penetrance of the 7q11.23 microduplication syndrome is approximately 60%, and the clinical phenotype of patients is heterogeneous, manifesting as intellectual impairment, language retardation, autism, brain abnormalities, epilepsy, macrocephaly, and other abnormalities [32,33,]. Owing to genetic heterogeneity, clinical phenotypes are not completely explicit and expressive. This case of 7q11.23 microduplication was inherited from a phenotypically normal mother.

During clinical prenatal consultation, pregnant women are often conflicted due to the detection of CTEV in the fetus, and, there are often cases of pregnancy termination because of CTEV. Among the 66 successful follow-up cases, forty-two cases of CTEV were diagnosed with idiopathic clubfoot, treated with surgery at birth (the specific surgery is unknown), and are currently in good health. Combined with the 13.6% false-positive rate, and the success of postnatal treatment, pregnant women should be counseled regarding the next management after diagnosing CTEV fetuses, such as continuous ultrasound review, SNP array, and multidisciplinary consultation to evaluate fetal prognosis. In this study, abnormal phenotypes were also found in four cases during postnatal follow-up, of which three carried normal CNVs, and one carried 17p12p11.2 microduplication. CTEV may be caused by genetic mutation [34–36]. In this study, eight (12.1%, 8/66) pregnant women chose to terminate their pregnancies, including four with pathogenic CNVs and four with normal CNVs. Meanwhile, the three patients with normal CNV showed an adverse postnatal clinical phenotype, which should be ruled out as the cause of the gene mutation. Simultaneously, two cases of paternal (maternal) CNV were treated after birth and are currently in a healthy condition.Thus, for CTEV fetuses with abnormal CNV, the genetic origin of the CNV should be verified, detailed genetic counseling should be provided to the parents in combination with imaging examinations and parental phenotypes, and long-term follow-up should be conducted for infants born.

This study has some limitations. First, this study was retrospective and included a small number of patients, which may lead to error bias; thus, further prospective, multicenter, and large-case studies are needed. Second, the second-generation sequencing, a new technology for genetic testing, is used to detect gene mutations and CNV, which may provide a more comprehensive prenatal genetic diagnosis for fetuses with CTEV and better evaluate fetal prognosis [37,38,]. Third, the follow-up time was short, and the long-term treatment effect on recurrence could not be evaluated. Subsequently, we can continuously accumulate data through multicenter cooperation and improve the detection methods and post-natal follow-ups to accumulate clinical evidence for the fetal evaluation of CTEV[39].

In conclusion, CTEV was found to be associated with chromosome microdeletion/microduplication, the most common of which was 22q11.21 microdeletion, followed by 17p12p11.2 microduplication.Thus, further genomic detection is recommended for fetuses with CTEV showing no abnormalities on conventional karyotype analysis. After excluding abnormal genomic factors, fetuses diagnosed with CTEV have a good prognosis after treatment, and blind termination of pregnancy is not recommended.

Ethics approval and consent to participate

The studies were approved by the ethics committee at Fujian Provincial Maternal and Child Health Hospital. All patients consented to participate and signed writteninformed consents. All subjects and/or their legal guardian(s) provided informed consent for both study participation and the publication of identifying information/images in an online open-access publication. All methods were performed in accordance with the relevant guidelines and regulations.

Acknowledgments

We thank the patients who participated in this study.

Disclosure statement

The authors report there are no competing interests to declare.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Additional information

Funding

Fujian Provincial Natural Science Foundation; Fujian Provincial Health Technology Project.