Identification of a novel gross deletion of TCOF1 in a Chinese prenatal case with Treacher Collins syndrome

Abstract Background Treacher Collins syndrome (TCS) is the most common mandibulofacial dysostosis with an autosomal dominant or rarely recessive manner of inheritance. It is still challenging to make a definite diagnosis for affected fetuses with TCS only depending on the ultrasound screening. Genetic tests can contribute to the accurate diagnosis for those prenatal cases. Methods Targeted exome sequencing was performed in a fetus of a Chinese family, who presenting an abnormal facial appearance by prenatal 2D and 3D ultrasound screening, including micrognathia, nasal bridge pit, and abnormal auricle. The result was validated with multiplex ligation‐dependent probe amplification (MLPA) and real‐time quantitative PCR (qPCR). Results A novel 2–6 exons deletion of TCOF1 gene was identified and confirmed by the MLPA and qPCR in the fetus, which was inherited from the affected father with similar facial anomalies. Conclusion The heterozygous deletion of 2–6 exons in TCOF1 results in the TCS of this Chinese family. Our findings not only enlarge the spectrum of mutations in TCOF1 gene, but also highlight the values of combination of ultrasound and genetics tests in diagnosis of craniofacial malformation‐related diseases during perinatal period.


| INTRODUCTION
Treacher Collins syndrome (TCS, OMIM 154500) is a rare craniofacial malformation that occurs with an estimated prevalence of 1/50000 live births. The minimal diagnostic criteria were defined as hypoplasia of the zygomatic arches and downslanting palpebral fissures (Teber et al., 2004). Other frequent findings include mandibular hypoplasia, colobomata of the lower eyelids, and ear malformations often associated with bilateral conductive hearing loss (Trainor & Andrews, 2013;Wong, Pfaff, Chang, Travieso, & Steinbacher, 2013). Most cases are inherited as an autosomal dominant trait, although an autosomal recessive form of this syndrome has been reported (Dauwerse et al., 2011;Schaefer et al., 2014).
Treacher Collins syndrome has been proven to be genetically heterogeneous, and three genes (TCOF1, POLR1D, and POLR1C) have been identified in the etiology of TCS. Among the causative genes, TCOF1 (OMIM 606847) mutations are most common, which account for approximate 70%-93% of studied individuals with autosomal dominant cases (Bowman et al., 2012;Splendore et al., 2000). To date, more than 300 distinct mutations have been identified within the TCOF1 gene and most of the mutations are family specific collected in the Human Gene Mutation Database ® (HGMD). However, pathogenic mutations in about 11% of TCS cases remain unknown (Chen et al., 2018).
A remarkable feature of TCS is the large amount of inter-and intra-family variation in phenotype severity, and no genotype-phenotype correlation has been found based on the evaluation of the clinical variability in TCS (Dixon, Ellis, Bottani, Temple, & Dixon, 2004). It has been reported that TCS expressivity varies from non-affected and mildly affected to severely affected individuals or perinatal deaths (Schlump et al., 2012). These factors contribute to the difficulties of definitive clinical diagnosis and genetic counseling for TCS patients, especially for those prenatal fetuses. Currently, prenatal TCS may be detected by means of conventional two-dimensional (2D) ultrasound or by the more refined three-dimensional (3D) scanning, however, it is rarely effective for mild to moderate TCS, particularly in sporadic cases (Hsu, Hsu, Chang, & Chang, 2002;Ruangvutilert, Sutantawibul, Sunsaneevithayakul, & Limwongse, 2003;Tanaka et al., 2002). Thus, identification of causative genes and mutations for the affected fetus is of great importance to precisely prevent TCS.
Here, we describe a 24-week fetus with mandibulofacial dysostosis via ultrasound testing, who was clinically suspected to be TCS after physical examination for his father with similar abnormal facial features. A novel gross deletion of 2-6 exons in TCOF1 was identified in both of the fetus and his father by targeted sequencing, and confirmed via multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative PCR (qPCR), providing important information for genetic counseling on this family. To our knowledge, this is the first report on the genetic diagnosis for a fetus with TCS in Chinese population.

| CLINICAL REPORT
A gravid 1, para 1 woman came to our clinical genetics center for genetic counseling at 24 weeks of gestation. This is her first pregnancy (Figure 1a). A first round prenatal twodimensional and three-dimensional ultrasound revealed that the male fetus (II:1) showed multiple defects, including micrognathia (Figure 1b), asymmetric nasal bone with 0.28 cm in left side and 0.44 cm in right side (Figure 1c), as well as abnormal auricle in both sides with only 0.76 cm and 0.67 cm high echo sound (Figure 1d,e). Other indicators of the fetus seemed normal, with biparietal diameter of 6.1 cm (50-90th centile), head circumference of 22.7 cm (50-90th centile), abdominal circumference of 19.7 cm (50-90th centile), femora length of 4.3 cm (50-90th centile),and humerus length of 4.0 cm (50-90th centile).
The mother (I:2) was 35 years old and her husband (I:1) was 39 years old. Both of them were non-consanguineous Chinese. When her husband appeared in our center, we found that he also had an abnormal facial appearance, with slanting palpebral fissures, coloboma of the eyelid, hypoplastic zygomatic arches, but without atresia of the external ear canal and cleft palate. A hearing loss of left side was also mentioned. They denied a family history with same phenotypes.
Based on the clinical features of the fetus and his father, a clinical diagnosis of TCS was highly suspected. Therefore, the family was referred for molecular diagnosis. Fetal cord blood and peripheral blood from the parents were obtained. After sufficient genetic counseling and totally informed the prognosis of TCS, the parents still decided to terminate the pregnancy at 25 weeks. The craniofacial features of the aborted fetus were consistent with the ultrasonic findings and additional down-slanting palpebral fissures were discerned Figure 1f), but further autopsy was refused.

SEQUENCING FOR SKELETAL SYSTEM DISEASE GENES
The patients provided an informed consent to publish all clinical information. The research was approved by the Ethics Committee of Hunan Provincial Maternal and Child Health Care Hospital. DNA was extracted from blood samples of the family. A minimum of 3 μg DNA was used to create the indexed Illumina DNA libraries, according to the manufacturer's protocol. All the exons of the 248 genes known to be | 3 of 7 LIU et aL. involved in skeletal system diseases were enriched using the GenCap custom enrichment kit (MyGenostics Inc.). The enriched exome libraries were sequenced on an Illumina HiSeq 2000 sequencer (Illumina), using the paired-end method, for the 100-bp reads.
Data analysis: The Trim Galore and Solexa cutadapt programs were used to filter out the low-quality reads and adapter sequences. Only the reads with a sequencing quality >20, a depth >90, and a read length >80 bp were retained. The clean reads were aligned to each human reference genome (hg19) using the SOAPaligner program (BGI). The duplicated reads were removed using the Sequence Alignment/Map tools (SAMtools) V3 and only the uniquely mapping reads were used for detection of variation. The single nucleotide variants (SNVs) were detected and genotyped with the SOAPsnp program (BGI). Subsequently, a Burrows-Wheeler Aligner (BWA) was used to realign the reads to the reference genome. The insertions or deletions (InDels) were identified using the GATK software package. The identified SNPs and InDels were annotated using multiple databases, including the Consensus CDS database, the human genome builder NCBI 37, the dbSNP database V147, and the In-house sequencing data of 300 local normal Asians.

PCR DETECTION
The SALSA MLPA P310 TCOF1 probe mix kit (MRC-Holland) was performed to determine the DNA copy number of a single DNA sequence in TCOF1 regions and 10 different autosomal chromosomal locations. MLPA analysis was performed following the manufacturer's instructions. The products were examined by ABI 3500dx genetic analyzer (Thermo Fisher). Quantitative data were analyzed using the software of Coffalyser V8.0. Validation of the result was carried out by qPCR (Primers are listed in Table S1).

| RESULTS
As the clinical manifestations of the father highly match the typical features of TCS, fetal, and paternal DNA samples were examined by targeted exome sequencing (cover 248 genes known to be involved in skeletal system diseases), which would detect not only the SNVs, but also large indels among the targeted genes according to the optimized bioinformatics analysis. After targeted exon sequencing, three candidate mutations in different genes were screened 2), respectively. According to further genotype-phenotype analysis, we found that heterozygous mutations of EVC2 gene are responsible for Weyers acrofacial dysostosis (OMIM 193530), which were mainly characterized by mild short stature, postaxial polydactyly, and so on. The FLNB-related disorders include a spectrum of phenotypes, including atelosteogenesis types I (OMIM 108720), type III (OMIM 108721, Larsen syndrome (OMIM 150250), and Boomerang dysplasia (OMIM 112310), but these disorders are mainly characterized by either severe short limbs, or dislocations of the hip, knee, and elbow, or underossification of the limb bones and vertebrae, etc. However, the proband and his father in our case did not show any malformation in their trunk and limbs, except the craniofacial abnormalities, thus both EVC2 and FLNB are unlikely to be the genetic cause for the family. Actually, the clinical features of the family highly match the TCS caused by TCOF1 mutations, and based on the mode of inheritance, mutation frequency, and pathogenicity prediction through multiple softwares, the novel heterozygous deletion of 2-6 exons in TCOF1 was thereby classified as a pathogenic variant according to the ACMG (the American College of Medical Genetics and Genomics) guideline ( Figure 2a) (Richards et al., 2015). The deletion was then confirmed by MLPA and qPCR testing. Both results revealed that the copy number of 2-6 exons was only half of other exons in TCOF1, suggesting a heterozygous deletion in 2-6 exons (Figure 2b,c). This gross deletion has not been reported yet, nor recorded in related databases. Cosegregation analysis demonstrated that the fetus inherited the same deletion from his affected father, while the mother showed normal genotype in TCOF1. Therefore, the affected fetus and his father were molecularly diagnosed to be the TCS, due to the pathogenic mutation of 2-6 exons in TCOF1.

| DISCUSSION
Treacher Collins syndrome exhibits great phenotypical variability ranging from perinatal death to clinically undiagnosed cases. When structural anomalies are present in prenatal cases, usually it is a task for the sonographer to first clinically diagnose a genetic syndrome due to incomprehensive information from the imaging detection, especially for those sporadic cases. Although TCS is characterized with craniofacial malformation, several syndromes involved with facial dysmorphism have been described, among which the main differential diagnoses F I G U R E 2 Identification of a gross deletion of TCOF1 in the fetus by genetic testing. (a) Targeted exome sequencing revealed a heterozygous deletion of 2-6 exons of TCOF1 as indicated by the red circle. (b) MLPA also showed a 50% relatively decreased bar height of 2-6 exons in TCOF1 gene, indicating a heterozygous deletion. (C) qPCR confirmed a half-dose for 2-6 exons of TCOF1 in the affected fetus compared with the normal individual. Exon 12 was set up as a normal control. MLPA, ligation-dependent probe amplification; qPCR, real-time quantitative PCR of TCS are with the Goldenhar and Nager syndromes. Main symptoms of Goldenhar syndrome include hemifacial microsomia, incomplete development of the ear, cleft lip and/or palate, ocular, and vertebral abnormalities (Barisic et al., 2014). Nager syndrome is characterized by malar hypoplasia, ear abnormalities, micrognathia, downslanting palpebral fissures, radial hypoplasia of the limbs, and absence of thumbs, and/or other fingers (Lansinger & Rayan, 2015).
In our case, the male fetus presented with facial malformations, including micrognathia, abnormal nose, ear anomalies, and suspected downslanting palpebral fissures by the first round 2D and 3D ultrasound. Although limbs and fingers of the fetus were normal, it was still hard to differentially define a specific syndrome only depending on the ultrasound results. Until the father came to our genetic outpatient at the second visiting, displaying visible slanting palpebral fissures, coloboma of the eyelid, hypoplastic zygomatic arches, the father, and fetus were highly suspected to be suffered from TCS, as there is no additional hemifacial microsomia or limbs abnormalities, which usually presenting in Goldenhar syndrome or Nager syndrome.
Among known causative genes of autosomal dominant TCS, TCOF1 is responsible for approximately 70%-93% of studied individuals (Dauwerse et al., 2011). POLR1D and POLR1C mutations account for 11%-23% of patients without TCOF1 mutations (Fan et al., 2019). TCOF1 encodes for a protein called treacle, which is part of a complex implicated in ribosomal RNA biogenesis and is involved in the proliferation and differentiation of neural crest cells in the first and second branchial arches during early embryogenesis (Dixon et al., 2006). To date, more than 300 different mutations of TCOF1 gene have been reported in patients with TCS according to HGMD data. The mutations include missense, nonsense, small deletions, and duplications. In particular, the most common classes of TCOF1 alleles are small deletions (60%) and duplications (25%) resulting in frameshifts (Conte et al., 2011). Though it has been suggested that five exons (10, 15, 16, 23, and 24), are defined as a hot spot region of the TCOF1 gene mutations (Splendore, Fanganiello, Masotti, Morganti, & Passos-Bueno, 2005), a distribution of pathogenic variations along all the gene was reported by different authors.
Large deletions encompassing the TCOF1 gene or gross deletions in TCOF1 were rare in literature (Table 1) (Beygo et al., 2012;Bowman et al., 2012;Vincent et al., 2014Vincent et al., , 2016. Beygo et al. (2012) reported the first single exon deletion T A B L E 1 Previously reported gross deletion mutations in patients with TCS within TCOF1. Another study conducted by Bowman et al described five pathogenic rearrangements in TCOF1, which suggested that TCOF1 rearrangements are responsible for a significant proportion (5.2%) of TCS-associated mutations, and they therefore recommended that dosage analysis by MLPA or a comparable method should be undertaken as part of a TCOF1 gene screening service (Bowman et al., 2012). Vincent et al reported on two patients presenting with mandibulofacial dysostosis characteristic of TCS, associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene (Vincent et al., , 2016. According to previous studies, most mutations reported in Chinese TCS patients were in TCOF1 gene. Majority of these pathogenic variations were SNVs and small Indels ranging in size from 1 to 38 nucleotides, only one gross deletion (exon 9-13) was reported before (Table S2) (Chen et al., 2018;Fan et al., 2019;Li et al., 2012Li et al., , 2019Wang et al., 2014;Yan et al., 2018;Zhang, Fan, Zhang, Xue, & Chen, 2013).

Patients Deletion Region Clinical manifestations References
In this study, we performed targeted exome sequencing on the family with a requirement for prenatal diagnosis, due to the high suspicion of TCS for the fetus and his father. A novel gross deletion of 2-6 exons in TCOF1 was identified and confirmed the diagnosis of TCS for the family. This gross deletion was never reported in literatures and might lead to loss of protein function consistent with a mechanism of haploinsufficiency. Regarding to the genotype-phenotype correlation, a previous report with a large cohort has proved that although TCOF1 has a LisH domain from exon 1 to 2 and a Treacle domain from exons 2 to 24, the localization of the mutations in the LisH or Treacle domains had no different effects on clinical features or severity of TCS (Vincent et al., 2016). In our case, we also found no special phenotypes compared with other patients with different mutations.
Molecular diagnosis plays a significant role for patients with TCS, in both prenatal and postnatal stages, and has an undeniable impact on the development of genetic counseling. In this case, we combined 3D ultrasound and targeted exome sequencing together to identify a TCS fetus successfully. A novel gross deletion of 2-6 exons in TCOF1 was ascertained in this Chinese family, which enlarged the spectrum of mutations in TCOF1 gene. Our study also indicates that when facing with a similar case with craniofacial malformation in prenatal stage, molecular genetics testing would help a lot to make definite diagnosis to guide the following genetic counseling.

ACKNOWLEDGMENTS
We thank this family for their contribution. We also thank laboratory staff at Prenatal Diagnosis Center of Hunan Province, Hunan provincial Maternal and Child Health Care Hospital. This study was supported by grants from the Program of Health and Family Planning Commission of Hunan Province (B20180083), the Natural Science Foundation of Hunan Province (No. 2018JJ3275) the Major Scientific and Technological Projects for Collaborative Prevention and Control of Birth Defects in Hunan Province (2019SK1010, 2019SK1014) and the National Natural Science Foundation of China (81970829).