Non-Invasive Prenatal Detecting of Achondroplasia In Earlier Stage of Pregnancy By Droplet-Digital PCR

Yipeng Wang Beijing Obstetrics and Gynecology Hospital, Capital Medical University Wei Wang Beijing Obstetrics and Gynecology Hospital, Capital Medical University Chengrong Wang Beijing Obstetrics and Gynecology Hospital, Capital Medical University Shanshan Li Beijing Obstetrics and Gynecology Hospital, Capital Medical University Meng Zhang Beijing Obstetrics and Gynecology Hospital, Capital Medical University Chenghong Yin (  yinchh@ccmu.edu.cn ) Beijing Obstetrics and Gynecology Hospital, Capital Medical University


Introduction
Achondroplasia (ACH, MIM 100800), characterized by disproportionate short stature, is a common type of skeletal dysplasia with the prevalence of 5-15/100,000 [1] . The clinical features of achondroplasia include rhizomelic shortening of the limbs, macrocephaly, and characteristic facial features with frontal bossing and midface retrusion [2] . Though intelligence and life span are usually near normal, craniocervical junction compression increases the risk of death in infancy [2] .
ACH is an autosomal dominant disorder, resulting from mutation in the broblast growth factor receptor 3 gene (FGFR3) with 100% penetrance. More than 99% of ACH is caused by the heterozygous glycine-toarginine substitution at codon 380 in the transmembrane domain of FGFR3, of which about 98 % of cases are the G to-A transition at nucleotide 1138 of coding sequence (c. 1138G>A) and about 1 % of cases are G-to-C transversion at this position (c. 1138G>C) [3]. Most cases appear to be de novo mutations with no prior familial history of skeletal dysplasia [4] .
Usually, ACH is detected by abnormal prenatal ultrasound ndings in the third trimester of pregnancy, and then con rmed by molecular genetic testing of fetal genomic DNA obtained by percutaneous umbilical blood or amniotic uid sampling [2] . The postponed diagnose of fetal ACH increases risk of clinical decision and anxiety of the family. To screen fetal ACH noninvasively in earlier stage of pregnancy should be a better solution for the clinic and the family.
Since its discovery in maternal blood, cell-free fetal DNA (cfDNA) originated from placenta trophoblasts was soon used for fetal disease testing, and rapidly adopted in clinical practice known as the noninvasive prenatal screening (NIPS) [5] . With higher sensitivity and speci city than conventional biochemical approaches, NIPS is broadly used to screen for fetal trisomy 21 (T21), trisomy 18 (T18), and trisomy 13 (T13) as early as 12 weeks of pregnancy [6][7][8] . In recent years, cfDNA was used to screen for sex chromosome aneuploidy and chromosome copy number variants (CNVs) [9,10] . Studies have demonstrated the utility of NIPS in fetal sex determination, fetal Rhesus D genotyping in D-negative mothers, and in some monogenic disorders [11][12][13] . Achondroplasia is of the rst monogenic disorders that can be noninvasively prenatal diagnosed thought cfDNA, because genetic absence of de novo mutation from the maternal and the high prevalence of FGFR3 single point hot spot mutations makes the detection easier to get high speci city and high coverage. Since fetal FGFR3 c.1138G>A was rst detected through mass spectrometry (MS) in 2000, several approaches have been demonstrated capable of detecting FGFR3 mutations from cfDNA, including MS, qPCR, droplet digital PCR (ddPCR), and Next generation sequencing [14][15][16][17][18] . All the reported tests aimed at non-invasive prenatal diagnosis of ACH after abnormal ultrasound ndings in the third trimester. In this work, we developed a ddPCR method of detecting FGFR3 p.G380R (c.1138G>A and c.1138G>C), and screened pregnant women in earlier stage of pregnant women in parallel of NIPS for T21/T18/T13. After tested 5927 pregnant women, we detected a 22-week pregnant woman positive for c.1138G>A, and further Sanger sequencing con rmed de novo mutation.

Subjects
Pregnant women who visited Beijing Obstetrics and Gynecology Hospital for NIPS could choose to have ACH detection simultaneously to NIPS for chromosome aneuploidy screening after informed consent. A total of 5927 pregnant women who voluntarily had ACH detection from November 2019 to October 2021 were included in the study. The study was carried out under the approvement by the Hospital Ethics Committee of the Beijing Obstetrics and Gynecology Hospital.

Exclusion criteria
Pregnant women who were ACH patients were excluded. Besides, because ACH detection was conducted synchronously with NIPS, exclusion criteria of NIPS were also followed [9] . And the recommended sampling time was at gestational age 12 +0 to 22 +6 weeks. In order to prevent false negative results due to low fetal fraction, in case of the fetal fraction of cfDNA <4%, a new analysis is recommended on a second sample 2 weeks later [8] .

Collection and treatment of blood samples
Maternal peripheral blood samples (5 ml) were collected in EDTA tubes, fully mixed, stored temporarily in 4 °C refrigerator. Samples were excluded if hemolysis or storage beyond 8 hours before plasma separation. The blood samples were treated as follows: centrifuged at 4 °C, 1600 g for 10min and the plasma was collected carefully and dispensed into 2.0 ml Eppendorf tubes (0030108078, Eppendorf, Germany). The plasma was centrifuged again at 4 °C, 16,000 g for another 10 min. The upper plasma was carefully divided into new 2.0 ml Eppendorf tubes, and each contained approximately 600 ml plasma, −80 °C refrigerator to save. The plasma should avoid repeated freezing and thawing before experiment.

DNA extraction
The cfDNA was extracted by QIAamp Circulating Nucleic Acid(55114,Qiagen) according to the manufacturer's introduction. cfDNA was concentrated by Agencourt AMPure XP paramagnetic bead (A63881 Beckman, USA) and stored in Nuclease-Free water(P1195,Promega,USA).
Droplet-digital PCR ddPCR method based on the ampli cation of the two possible mutant alleles A allele or C allele and the wildtype G allele was developed. Primer sequences are described in Table 1. The presence or absence of ampli cation for the mutant allele signs the presence or absence of mutant FGFR3 sequences of fetal origin in the maternal plasma. ddPCR was performed using the BioRad QX200TM droplet digital PCR System (BioRad, USA) according to the manufacturer's instructions. In each well, 10μL of cfDNA solution were mixed with 11 μL of ddPCR master mix (BioRad, dd PCR supermix for probe, 1863028, Biorad, USA) and 1μL of each duplex PCR primers/probes. The reaction mix was dispensed into the 96-well PCR plate (0030128575, Eppendorf, USA). The PCR plate was then placed in QX200 Droplet Generator (BioRad, USA) to generated droplets.
The PCR plate was then heat-sealed with a foil seal by PX1 heat sealer (BioRad, USA), and the sealed plate was placed in the PCR thermocycler. Thermal conditions were: 1 cycle of 95 °C for 5 min, 40 cycles of 94 °C for 30s, 57 °C for 60s, and 1 cycle of 98 °C for 10 min. After the reaction, the droplets were read using the Droplet Reader, and QUANTASOFT software (BioRad, USA) converted the data into concentrations using Poisson distribution statistical analysis. Brie y, the wildtype allele (G) and the mutant alleles (C and A) were indicated with different uorescences, and detecting a uorescence indicates the DNA target was initially present in the droplet and negative of uorescence indicates no DNA target was initially present in the droplet. The concentrations of DNA targets could be calculated when >10,000 droplets were generated in each well. Result was considered positive when two replicates showed more than ve positive droplets for the mutant allele each. The absence of ampli cation of A-allele and C-allele, together with the positive ampli cation of the G-allele indicated a fetus free of the hotspot mutation of nucleotide 1138 of FGFR3 gene.

Con rmation of FGFR3 gene mutation of the fetus
Prenatal ultrasound and amniocentesis were then performed to con rm the positive screening result of ACH cases. Fetal DNA was extracted by using amniotic uid cells. The mutation sites of the fetus were then identi ed via Sanger sequencing.

Clinical outcome follow-up
For the women screened negative of FGFR3 mutation, length of the limb and nger length, shape of the hand and face, muscular tension of the newborns were recorded to exclude fetal achondroplasia. Ultrasound examination in the third trimester and pregnancy outcome follow-up of 2950 pregnant women negative in the screen con rmed true negative of ACH. The sensitivity and speci city of the test was 100%(5.5%-100%) and 100%(99.8%-100%), respectively. Discussion cfDNA is becoming a crucial tool for diagnosis and management in various clinical disorders. In the eld the noninvasive prenatal testing, cfDNA has been applied fetal gender determination, RHD genotyping, and detecting of chromosomal aneuploidies, chromosomal aneuploidies, and increasing number of monogenic disorders [5][6][7][8][9][10][11][12][13] . NIPS is becoming a routine clinical management screening more and more genetic disorders [19] .
In this work, we developed a high-e cient and low-cost method for noninvasive prenatal ACH detecting, and rst applied noninvasive ACH detection in screening of earlier stage pregnant women. 5927 pregnant women voluntarily had this additional test at the time they were having NIPS for chromosomal aneuploidies screening. One woman was diagnosed carrying a FGFR3 c.1138G>A mutant. Following up of 1 positive and 2950 negative results showed the sensitivity and speci city of the test was high, 100% (5.5%-100%) and 100%(99.8%-100%) respectively.
Interestingly, in consistence with the conclusion of previous studies, that advanced father age increases the risk of disease caused by de novo mutations, the husband the ACH positive pregnant woman was 49 years old, suggesting that pregnant women with advanced-age husband are recommended to have this screen [4,20,21] .
Diagnosis of ACH in earlier stage of pregnancy before the abnormal ultrasound ndings will give the patients more time to make decision and to get prepared for an ACH baby. Besides, there are less ethical concerns and less physical and phycological harm to the family if they choose termination of pregnancy in earlier stage of pregnancy compared to doing that in the third trimester of pregnancy.
In addition to ACH, there are other severe monogenic disorders caused by de novo mutations, for example four types of osteogenesis imperfecta caused by mutations in COL1A1/2, Pfeiffer syndrome caused by mutations in FGFR1/2, and Noonan Syndrome caused by mutations in one of several Ras/MAPK pathway genes. In the future, the screen can be expanded to cover more disorders caused de novo mutations.
In conclusion, we developed a rapid, cheap, and convenient method for noninvasive ACH screening in earlier stage (12-22+6 weeks) of pregnancy, and earlier diagnosis of ACH bene ts the affected families a lot. We prospect a broad clinical application of this method in the future. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication
All patients in this report provided their consent for publication.

Availability of supporting data
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.