Pathogenic copy number variations are associated with foetal short femur length in a tertiary referral centre study

Abstract Shortened foetal femur length (FL) is a common abnormal phenotype that often causes anxiety in pregnant women, and standard clinical treatments remain unavailable. We investigated the clinical characteristics, genetic aetiology and obstetric pregnancy outcomes of foetuses with short FL and provided a reference for perinatal management of such cases. Chromosomal microarray analysis was used to analyse the copy number variations (CNV) in short FL foetuses. Of the 218 foetuses with short FL, 33 foetuses exhibited abnormal CNVs, including 19 with pathogenic CNVs and 14 with variations of uncertain clinical significance. Of the 19 foetuses with pathogenic CNVs, four had aneuploidy, 14 had deletions/duplications, and one had pathogenic uniparental diploidy. The 7q11.23 microdeletion was detected in three foetuses. The severity of short FL was not associated with the rate of pathogenic CNVs. The duration of short FL for the intrauterine ultrasound phenotype in foetuses carrying a pathogenic CNV was independent of the gestational age. Further, maternal age was not associated with the incidence of foetal pathogenic CNVs. Adverse pregnancy outcomes occurred in 77 cases, including termination of pregnancy in 63 cases, postnatal dwarfed foetuses with intellectual disability in 11 cases, and three deaths within 3 months of birth. Pathogenic CNVs closely related to foetal short FL were identified, among which the 7q11.23 microdeletion was highly associated with short FL development. This study provides a reference for the perinatal management of foetuses with short FL.


| INTRODUC TI ON
Foetal femur length (FL) is a primary ultrasound measure used to assess foetal growth and skeletal development during pregnancy.
Short FL is a common abnormal phenotype in foetuses, which often causes anxiety in pregnant women, and ultrasonographic diagnostic criteria for short FL have not yet been standardized. Short FL has been defined based on several criteria, including foetal FL below the 5th percentile 1,2 or below two standard deviations (SDs), 3 a difference between foetal biparietal diameter and FL >4 cm (measured via ultrasound), 4 or a FL/foot length <0.88. 5 Foetal FL varies considerably depending on race, ethnicity, nutritional status and congenital diseases. Common causes of short FL include physiological short FL, foetal growth restriction (FGR), congenital skeletal hypoplasia and genomic abnormalities, 6,7 and obstetricians often face challenges in providing clinical treatment. Thus, it is necessary to establish diagnostic indicators, conduct timely prenatal diagnoses, identify various possible pathological factors that shorten FL and conduct foetus risk assessments to provide appropriate treatment.
Conventional karyotyping has low sensitivity and is a timeconsuming method for detecting inherited disorders. Chromosomal microarray analysis (CMA) is an effective method for detecting chromosomal microdeletions and microduplications at the genome level.
Owing to its rapid, high-throughput and high-resolution capability, it is considered a powerful tool for the diagnosis of developmental and intellectual disabilities, autism and multiple congenital anomalies [8][9][10] ; however, its efficiency in the prenatal diagnosis of short FL remains to be investigated. Therefore, we performed CMA to analyze the copy number variations (CNVs) in 218 foetuses with short FL and investigated their clinical characteristics, genetic aetiology and obstetric pregnancy outcomes to provide a reference for perinatal management of such cases.

| Chromosomal microarray analysis
For amniotic fluid collection, approximately 20 mL of amniotic fluid was collected using ultrasound-guided amniocentesis, whereas for umbilical cord blood collection, 1-2 mL of umbilical cord blood were collected using ultrasound-guided cordocentesis. Genomic DNA was extracted from the amniotic fluid or foetal umbilical cord

| Follow-up and pregnancy outcomes
All patients were followed up by telephone regarding the pregnancy outcomes and postnatal conditions. The follow-ups obtained data on postnatal physical growth and neurobehavioural development.
All adverse pregnancy outcomes, such as miscarriage, stillbirths, intrauterine growth retardation, developmental abnormalities and perinatal or infant death, were recorded.

| Subgroup analysis according to the presence of other ultrasonographic abnormalities
Foetuses with short FL were classified into three groups according to the presence of other ultrasonographic abnormalities: isolated short FL, short FL with ultrasonographic soft markers and short FL with ultrasonographic structural anomalies. The rates of pathogenic CNVs in foetuses with isolated short FL, short FL with ultrasonographic structural malformations and short FL with ultrasonographic soft markers were 9.6% (5/52), 11.1% (5/45) and 7.4% (9/121), respectively. No significant differences were observed in the rate of pathogenic CNVs among the three groups (χ 2 = 0.626, p = 0.731; Table 3).

| Subgroup analysis with respect to the degree of femoral shortening
Foetuses with short FL were classified into two groups according to the degree of femoral shortening: those with relatively mild shortening (-2SD < FL ≤ −4SD) and those with severe shortening (FL < −4SD). The rates of pathogenic CNVs in the mild and severe FL shortening groups were 8.4% (16/191) and 11.1% (3/27), respectively. No significant difference was observed in the rate of pathogenic CNVs between the two groups (χ 2 = 0.222, p = 0.895; Table 3).

| Subgroup analysis of different gestational ages at the initial diagnosis of short FL
Foetuses with short FL were classified into two groups according to their gestational age at the time of initial diagnosis: those presenting with short FL in the second trimester and those presenting with short FL in the third trimester. In total, 106 foetuses were initially diagnosed with short femurs in the second trimester (gestational age range, 16-28 weeks), and the remaining 112 foetuses were diagnosed in the third trimester (gestational age, ≥28 weeks). The rate of pathogenic CNVs was 7.5% (8/106) for foetuses diagnosed in the second trimester and 9.8% (11/112) for those diagnosed in the third trimester. No significant difference was observed in the rate of pathogenic CNVs between the two groups (χ 2 = 0.354, p = 0.552; Table 3).

| Subgroup analysis with respect to maternal age
Foetuses with short FL were classified into two groups according to maternal age: those <35 and those ≥35 years of age. The rate of pathogenic CNVs in the foetuses of younger mothers (<35 years old) was 7.7% (15/196), whereas that of the older mothers was 18.2% (4/22). No significant difference was observed in the rate of pathogenic CNVs between the two groups (p = 0.109; Table 3). This study was limited by a small sample size, with only 218 foetuses with short FL retrospectively analysed, as well as our reliance on CMA because of the lack of suitable methods for data analysis.

| Follow-ups and obstetrical outcomes
Furthermore, while the chromosomal pathogenic causes of short FL were excluded, genetic mutations were not and may have influenced the results. Indeed, it has been reported that short FL may be associated with congenital skeletal malformations, among which mutations in fibroblast growth factor receptor 3 are the most common. 17,32 Therefore, future studies should evaluate larger cohorts and perform single-gene detection to improve the perinatal management of foetuses with short FL.
In conclusion, this study analysed the aetiology of CNVs in foetuses with short FL and identified pathogenic CNVs that were closely related to short FL, including a microdeletion in 7q11.23.
Further, this study analysed the clinical characteristics and follow-up of foetuses with short FL to provide a reference for perinatal management of such foetuses. Our findings provide valuable information for the evaluation and management of foetuses with short femurs by obstetricians, allowing them to provide good consultation to prospective parents.