Prenatal Detection of Novel Compound Heterozygous Splice Site Variants of the KIAA0825 Gene in a Fetus with Postaxial Polydactyly Type A

Abstract

Postaxial polydactyly (PAP) is a common abnormality characterized by extra digits on hands and/or feet. To date, sequence variants in seven genes have been identified in non-syndromic PAP. In the present study, a fetus manifesting non-syndromic postaxial polydactyly type A (PAPA) was found by fetal ultrasonography. To better evaluate fetal prognosis, SNP array analysis and trio whole-exome sequencing (trio-WES) were performed to identify the underlying etiology. Although SNP array analysis revealed no abnormality, trio-WES identified compound heterozygous splice site variants in KIAA0825, c.-1-2A>T and c.2247-2A>G in intron 2 and intron 12, respectively. These two splice site variants were absent in control databases and were predicted to influence splicing by in silico analysis. To confirm the potential pathogenicity of the variants, in vitro splicing assays using minigene and RNA from peripheral leukocytes of the heterozygous parents were conducted. Minigene and RT-PCR assays demonstrated that the c.-1-2A>T variant led to the loss of the initiation codon, and the c.2247-2A>G variant mainly resulted in exon 13 skipping. Prenatal WES and subsequent functional studies are important approaches for defining the genetic etiology of fetuses with PAPA and are also essential for accurate genetic counseling and decision making. Taken together, this study expands the spectrum of KIAA0825 variations in PAPA patients and increases the knowledge of the molecular consequences of KIAA0825 splice site variants.

Keywords: KIAA0825; aberrant splicing; functional study; limb anomaly; postaxial polydactyly type A; whole-exome sequencing.

Figure 1

Family pedigree and Ultrasound imaging data of the fetus showed PAPA. (a) A Pedigree of this family with segregating PAP in an autosomal recessive manner. Circles and squares represent females and males, respectively. Clear symbols represent unaffected members, while filled symbols show affected members. (b,c) Ultrasound imaging for right hand (b) and left hand (c) displaying PAPA. (d,e) Ultrasound imaging for right foot (d) and left foot (e) exhibiting PAPA.

Figure 2

Sanger sequencing chromatogram of KIAA0825 variants in the family. (a) The variant c.-1-2A>T in KIAA0825 is identified in II-2 (proband) and I-1 (father). Arrows represent the variant. (b) The variant c.2247-2A>G in KIAA0825 is identified in II-2 (proband) and I-2 (mother). Arrows represent the variant.

Figure 3

Blood RT-PCR and minigene assay analysis of the KIAA0825 c.-1-2A>T. (a) Electrophoresis results of the RT-PCR products with the father’s peripheral leukocytes; the band amplified from the control (wt) is labeled as a and the bands amplified from the father (mut) are labeled as a, b and c. (b) RT-PCR electrophoresis results of the minigene assay; bands from WT and the c.-1-2A>T constructs were labeled as d and e, respectively, in both HeLa and 293T cells. (c) The corresponding Sanger sequencing results of excised bands of a, b, c from the blood RT-PCR and excised bands of d, e from the minigene assay. (d) The diagram of alternative splicing events observed from the sanger sequence in the blood RT-PCR and minigene assay. The alternative splicing events from the father (mut) are labeled as a, b and c. The alternative splicing events from WT and the c.-1-2A>T constructs in minigene were labeled as d and e. Red arrow indicates the variant location.

Figure 4

Blood RT-PCR and minigene assay analysis of the KIAA0825 c.2247-2A>G variant: (a) Electrophoresis results of the RT-PCR products with the mother’s peripheral leukocytes, the band amplified from the normal control (wt) was labeled as a and the bands amplified from the mother (mut) were named as a and b. (b) RT-PCR electrophoresis results of the minigene assay. Bands from the WT and c.2247-2A>G constructs were labeled as c, d and e, respectively, both in HeLa and 293T cells. (c) The corresponding Sanger sequencing results of excised bands of a, b from the blood RT-PCR and excised bands of c, d, e from the minigene assay. (d) The diagram of alternative splicing events observed in the blood RT-PCR and minigene assay. The alternative splicing events from the mather (mut) are labeled as a and b. The alternative splicing events from WT and the c.2247-2A>G constructs in minigene were labeled as c, d and e. Red arrow indicates the variant location.