Compound Heterozygous Splicing Variants In KIAA0586 Cause Fetal Short-Rib Thoracic Dysplasia And Cerebellar Malformation: The Use of Whole Exome Sequencing In Prenatal Diagnosis

Background: Short-rib thoracic dysplasia (SRTD) and Joubert syndrome (JS) are rare genetic ciliopathies, both patients can manifest cerebellar malformation and variable developmental delays. However, neither could be easily diagnosed during pregnancy due to limited fetal phenotype. Here, we investigated a fetus with short limbs, polydactyly initially and uncovered a compound heterozygous pathogenesis through whole exome sequencing (WES). Results: Merely short limbs and polydactyly of the fetus were detected during second trimester of gestation. Two variants (c.3940+1G>A and c.3303G>A), affecting splicing of KIAA0586 gene, were identied from amniocytes through WES. The presence and effect of these mutations were further validated on DNA and RNA level through Sanger sequencing. More intensive fetal monitoring was applied; deformed cerebellar malformation and restricted thoracic cage of the fetus were additionally uncovered. Conclusion: Herein, we discovered a genetic pathogenesis of KIAA0586 gene associated with SRTD and/or JS in a fetus with mild ultrasound anomalies initially. With the information of prenatal WES and distinct phenotypes of the fetus uncovered by imaging examination, we could reach more accurate clinical diagnosis and provide valuable prognosis information for parents.

validated on DNA and RNA level through Sanger sequencing. More intensive fetal monitoring was applied; deformed cerebellar malformation and restricted thoracic cage of the fetus were additionally uncovered.
Conclusion: Herein, we discovered a genetic pathogenesis of KIAA0586 gene associated with SRTD and/or JS in a fetus with mild ultrasound anomalies initially. With the information of prenatal WES and distinct phenotypes of the fetus uncovered by imaging examination, we could reach more accurate clinical diagnosis and provide valuable prognosis information for parents.
Joubert syndrome (JS) is a neurodevelopmental disorder characterized by a distinctive cerebellar and brain stem malformation called the molar tooth sign (MTS), hypotonia and variable developmental delays. Additional ndings occur in JS patients include retinal dystrophy, respiratory control disturbances, renal disease, ocular colobomas, occipital encephalocele, hepatic brosis, polydactyly, oral hamartomas, and endocrine abnormalities [5,6]. Predominantly, JS is inherited in an autosomal recessive manner, but X-linked and digenic inheritance have also been reported. JS demonstrates extreme phenotypic variability and genetic heterogeneity [7]. To date pathogenic variants in more than 30 genes are known to cause JS. Here, we present a prenatal case of fetal with short limbs and polydactyly. A novel splicing and a known synonymous mutation of KIAA0586 gene (OMIM: 610178) were detected in amniocytes by whole exome sequencing (WES). The mutations were inherited from the fetal parents respectively, establishing a compound heterozygous pathogenesis. Interestingly, though one of which is synonymous, both mutations were proved to affect splicing of KIAA0586 gene, which has been associated with SRTD (type 14) or JS.

Clinical Summary
A primigravid woman was referred for genetic counseling during second trimester, because of abnormal fetal ultrasound ndings: polydactyly, short limbs and persistent left superior vena cava (PLSVC). The ultrasound examination at 24 + 1 weeks of gestation revealed polydactyly of the fetal right hand, a fetal femur length (FL) of 3.9cm (~ 22 + 5w). PLSVC with dilated coronary sinus was detected by fetal echocardiography.
The woman and her husband were non-consanguineous, and there was no family history of congenital malformations or genetic diseases. The pedigree of the family is shown in Fig. 1. Amniocentesis was performed at 25 + 5 weeks of gestation, and DNA extracted from the amniocytes was tested by WES and chromosome microarray analysis (CMA).
Further imaging monitoring was recommended according to genetic mutations found by WES, including serial fetal sonography and the axial brain magnetic resonance imaging (MRI). More distinct phenotypes of the fetus were uncovered, consist with genetic results.

Molecular Results
CMA was successively performed for the fetus but with negative results: no numerical abnormalities of chromosomes, copy number variants (CNVs) or regions of homozygosity were discovered. However, we identi ed two variants in KIAA0586 gene (NM_001244189.2) from the amniocytes in a compound heterozygous state: c.3940 + 1G > A and c.3303G > A (p.P1101P). The presence of these mutations was further validated by Sanger sequencing (Fig. 3).
The novel canonical splice site mutation (c.3940 + 1G > A) inherited paternally has not been reported in any database, including the Human Gene Mutation Database (HGMD), ClinVar or GnomAD databases. Our study showed that the novel canonical splice site mutation (c.3940 + 1G > A) resulted in deletion of exon 26 and also introduced a framshift, producing a premature termination codon. Meanwhile, the synonymous mutation (c.3303G > A; p.P1101P) inherited from the pregnant woman was predicted to be deleterious, according to CADD, Splice AI and dbscSNV. To varify the underlying pathogenicity of these mutations, Sanger sequencing was performed on cDNA from the fetal parents. The results indicated that the synonymous mutation (c.3303G > A) led to deletion of 56 basepairs of exon 23, resulting in a framshift with premature truncation of the protein. This result was consistent with the previous study reported in a JS patient [13] (Fig. 4).

Imaging Findings
Fetal MRI at 31 + 5 weeks of gestation showed deformed cerebellar vermis and characteristic MTS of the fetus (Fig. 2a). Strikingly, hypoplastic cerebellar vermis, enlarged third ventricle, relatively small thoracic cage and short humerus length (HL, 5.03cm, -2.9SD) besides polytactyly were later found at 33 weeks of gestation by fetal sonography (Fig. 2b-2f). Ultimately, the couple chose to terminate the pregnancy after multidisciplinary consultation due to likely poor prognosis after birth.

Experiment on human subjects was approved by the Ethical Review Board of West China Second
University Hospital, Sichuan University. Genomic DNA of the family members was extracted from amniocytes and parents' peripheral blood leukocytes using QIAamp DNA Blood Mini Kit (QIAGEN). To detect the genetic factors for the fetus, CMA and trio-WES were performed using genomic DNA from the family members (II-1, II-2, III-1).
Genomic DNA (gDNA) from the fetus and parents was subjected to single nucleotide polymorphism (SNP) array with the CytoScan 750K Array (Thermo Fisher Scienti c), following the manufacturer's instructions. Molecular karyotype analysis was performed using Chromosome Analysis Suite v4.1 (Thermo Fisher Scienti c). The GRCh38 (hg38) genome was used for annotation. CNVs larger than 100 kb or those that affected more than 50 contiguous probes were considered. Genetic pathogenicity and clinical signi cance of CNVs were systematically evaluated according to American Society for Medical Genetics and Genomics (ACMG) guidelines [9] and previous publication [10], respectively. Exome capture sequencing was performed using the NanoWES Human Exome V1 (Berry genomics) following the manufacturer's protocol. The enriched library was sequenced through Illunima NovaSeq6000 platform with 150-bp paired-end reads. Next, Burrows-Wheeler Aligner software tool was used for aligning the sequencing reads with GRCh38 (hg38). After that, local alignment and recalibration of base quality of the Burrows-Wheeler aligned reads was performed by the GATK Indel Realigner and the GATK Base Recalibrator, respectively (broadinstitute.org/). Then, Single-nucleotide variants (SNVs) and small insertions or deletions (InDels) were identi ed by GATK Uni ed Genotyper (broadinstitute.org/). Finally, functional annotation was performed using ANNOVAR and the Enliven Variants Annotation Interpretation System (Berry genomics). Public databases using for ltering include gnomAD Candidate variants on each independent gDNA sample (II-1, II-2, III-1) were validated using Sanger sequencing: polymerase chain reaction (PCR) ampli cation was performed using primer pairs (Table 1) designed to cover variants identi ed by WES. Total RNA was extracted from the parents' fresh peripheral blood samples, using RNApure Blood Kit (CWBIO, CW0582S), and then reversed to cDNA through synthesis Kit (Thermo Scienti c, K1622). Target sequences of cDNA were ampli ed using PCR as well, using primers designed using Primer 3 software Version 0.4.0 (http:// bioinfo.ut.ee/primer3-0.4.0/) ( Table 1). Sequencing of PCR products were conducted by ABI 3500 Genetic Analyzer (Thermo Fisher Scienti c) to verify the changes on both DNA and RNA level. Data were evaluated using the Chromas software (2.6.5).

Discussion
It is recommended that laboratories offering prenatal WES report pathogenic and likely pathogenic variants, as determined using ACMG variant interpretation guidelines in known disease genes consistent with the reported fetal phenotype [11,12]. Through DNA and RNA validation, we proved that both variants affected splicing and might lead to frameshift of KIAA0586 gene. Therefore, the compound heterozygous mutations were rated as likely pathogenic combined with low frequency in general population. It is worth noting that the synonymous variant, at the last basepair of exon 23, can be easily neglected while analysis. However, the substitution leads to use of a cryptic splice site in exon 23 and deletion of 56 basepairs, resulting frameshift and in premature truncation of the KIAA0586 protein [13]. Therefore, disease databases such as ClinVar or HGMD and functional researches should be queried for putative pathogenic variants [13][14][15]. Moreover, impact of splice-site variants was con rmed through functional analysis by RNA in our study. RNA veri cation using the parents' blood samples successfully prevented another invasive operation for the fetus and acquired rapid validation results for the family.
Recently, WES may be considered for a fetus with ultrasound anomalies after standard CMA and karyotype analysis have failed to yield a de nitive diagnosis [16]. However, abnormal karyotype and CNVs can merely explain partially the genetic etiology, more than half of fetuses with sonographically identi ed structural anomalies remain without a diagnosis. In addition, WES is increasingly used in both pediatric and adult population, for multiple congenital anomalies, skeletal, neurodevelopmental and neuromuscular disorders [17][18][19][20]. Given the success in these patients, WES, limited to the analysis of coding regions of known genes, is currently applied prenatally. Studies reveal that WES increases the diagnostic yield in structurally abnormal fetuses by about 8-10% after normal karyotype and CMA results, and the detection rate is strongly proportional to the severity of phenotype [21,22]. For our case, multiple fetal anomalies demonstrated a strong indication to implement prenatal WES. Instead of sequential testing, CMA and WES were simultaneously suggested and performed due to late gestation age, leading to a timely and comprehensive genetic diagnosis for the fetus.
Given the analysis of WES is phenotype driven; prenatal reporting of variants is of great challenge due to limited and ambiguous fetal imaging ndings. Accurate identi cation of fetal structural anomalies is sophisticated by gestation age, fetal position, the ultrasound equipment, and the experience of clinicians

Conclusions
In our study, we discovered a compound heterozygous pathogenesis of KIAA0586 gene for a fetus with short limb and polydactyly initially. We reported a novel KIAA0586 variant which expands the spectrum of KIAA0586 in JS and/or SRTDs. Depending on validated WES results, more distinct fetal phenotypes including thoracic dysplasia and MTS were timely uncovered, leading to consistency of genetic and clinical diagnosis during pregnancy. Consequently, valuable prognosis information and future reproductive alternatives were provided for the parents.