Array-based molecular karyotyping in fetuses with isolated brain malformations identifies disease-causing CNVs
Fetuses with prenatal conventional karyotyping
Fetus 2 (GA 25?+?6) was diagnosed by prenatal ultrasound with isolated, symmetric bilateral ventriculomegaly. The fetus carried a 0.39-Mb duplication of chromosomal region 2q32.1-2q32.2 (chr2:189,363,100-189,750,863) comprising the complete coding regions of the two RefSeq genes GULP1 (PTB domain containing engulfment adaptor protein 1, MIM 608165) and DIRC1 (mRNA disrupted in renal carcinoma 1, MIM 606423). This fetus carried an additional 0.1-Mb deletion of chromosomal region 16q12.11 (chr16:47,074,235-47,174,275) comprising exons 2–9 of the NETO2 gene (neuropilin and tolloid (TLL)-like 2, MIM 607974).
Fetus 3 (GA 12?+?6) was diagnosed by prenatal ultrasound with agenesis of the corpus callosum (ACC) (colpocephaly and absent septum pellucidum). The fetus carried a 0.51-Mb duplication of chromosomal region 3q26.1 (chr3:165,553,646-166,065,099) affecting BCHE gene exon 1 (butyrylcholinesterase, MIM 177400).
Fetus 4 (GA 24?+?0) was diagnosed by prenatal ultrasound with symmetric bilateral ventriculomegaly, cerebellar hypoplasia, ACC, and signs of lissencephaly. The fetus carried a 0.07-Mb deletion of chromosomal region 3p26.3 (chr3:1,436,664-1,511,108), comprising the last three exons (21–23) of the CNTN6 gene (contactin 6, MIM 607220). CNVs comprising CNTN6 have recently been associated with neurodevelopmental discorders and brain anomalies [17, 18].
Fetus 5 (GA 21?+?3) was diagnosed by prenatal ultrasound with occlusive hydrocephalus (with dilatation of the lateral and third ventricles, suggesting aquaeductal stenosis) and secondary macrocephaly. The fetus carried a 0.18-Mb deletion of chromosomal region 4q31.23 (chr4:148,727,355-148,903,388), comprising the single RefSeq gene ARHGAP10 (Rho GTPase activating protein 10, MIM 609746).
Fetus 6 (GA 23?+?4) was diagnosed by prenatal ultrasound with alobar holoprosencephaly. The fetus carried two duplications. The first duplication was 0.25Â Mb in size affecting chromosomal region 4q13.3 (chr4:73,812,806-74,058,406) comprising the complete coding region of the COX18 gene (cytochrome c oxidase assembly factor, MIM 610428) and part of the ANKRD17 gene (ankyrin repeat domain 17, MIM 615929). The second duplication was 0.09Â Mb in size affecting chromosomal region 8q24.3 (chr8:142,295,965-142,382,757). The region comprises the complete GPR20 gene (G protein-coupled receptor 20, MIM 601908) and the long non-coding RNA LOC7311779.
Fetus 7 (GA 27?+?5) was diagnosed by prenatal ultrasound with Dandy-Walker malformation (agenesis of the vermis cerebelli) with bilateral symmetric ventriculomegaly and microcephaly. The fetus carried a 1.25-Mb duplication of chromosomal region 9p23 (chr9:10,144,584-11,398,305) affecting the PTPRD gene (protein-tyrosine phosphatase, receptor-type, delta, MIM 601598) and its complete long non-coding RNA, antisense RNA 2 (head to head) (PTPRD-AS2).
Fetus 9 (GA 21?+?6) was diagnosed by prenatal ultrasound with symmetric bilateral ventriculomegaly and secondary macrocephaly. This fetus carried two duplications. The first duplication was 0.1 Mb in size affecting chromosomal region 2q37.3 (chr2:241,623,894-241,722,445) comprising the two RefSeq genes KIF1A (kinesin family member 1A, MIM 601255) and AQP12A (aquaporin 12A, MIM 609789). Here, the KIF1A gene has been associated with hereditary sensory and autonomic neuropathy type II and in autosomal recessive spastic paraparesis as well as spastic paraplegia [19–21]. The second duplication was 0.08 Mb in size affecting chromosomal region 3q13.32 (chr3:118,730,933-118,812,027) comprising intron 3 of the IGSF11 gene (immunoglobulin superfamily, member 11, MIM 608351).
Fetus 11 (GA 28?+?3) was diagnosed by prenatal ultrasound with ACC (colpocephaly and absent septum pellucidum) and interhemispheric arachnoidal cysts. This fetus carried a 0.57-Mb deletion of chromosomal region 16p12.2 (chr16:21,839,340-22,409,463). This region comprises seven RefSeq genes namely UQCRC2 (ubiquinol-cytochrome c reductase core protein II, MIM 191329), PDZD9 (PDZ domain containing 9), C16orf52, VWA3A (von Willebrand factor A domain containing 3A), EEF2K (eukaryotic elongation factor 2 kinase, MIM 606968), POLR3E (polymerase (RNA) III (DNA directed) polypeptide E (80kD)), and CDR2 (cerebellar degeneration-related protein 2, MIM 117340).
Fetus 12 (GA 22?+?3) was diagnosed by prenatal ultrasound with Dandy-Walker malformation (agenesis of the vermis cerebelli with bilateral, symmetric ventriculomegaly and secondary macrocephaly). The fetus carried a 4.65-Mb deletion of chromosomal region 6p25.1-6p25.3 (chr6:212,548-4,864,581) comprising 35 RefSeq genes. CNV validation via MLPA confirmed the deletion and analysis of parental DNA established its de novo occurrence. Among the deleted genes resides FOXC1 (forkhead box C1, MIM 601090). Point mutations and deletions of FOXC1 have been shown to play a major role in the formation of Dandy-Walker malformations [22, 23].
Prompted by our finding of a deletion in fetus 12 comprising FOXC1, we used a region-specific high-density MLPA probe (see above) to screen 19 additional fetuses with syndromic non-isolated Dandy-Walker malformation-related brain anomalies of whom no array-based molecular karyotyping had been previously performed. However, this additional analysis failed to detect any further deletions (or duplications) of the FOXC1 region.
Fetus 13 (GA 23?+?4) was diagnosed by prenatal ultrasound with ACC. The fetus carried a 0.21-Mb duplication of chromosomal region Xp22.11 (chrX:23,799,472-24,012,381). The region comprises four RefSeq genes encoding SAT1 (spermidine/spermine N1-acetyltransferase, MIM 313020), APOO (apolipoprotein O, MIM 300753), CX0rf58, and KLHL15 (kelch-like family member 15). Interestingly, KLHL15 was most recently identified as a potential X-linked intellectual disability (ID) gene [24]. Investigation of parental DNA showed transmittance of this CNV from the healthy mother.
Fetus 14 (GA 28?+?5) was diagnosed by prenatal ultrasound with ACC. The female fetus carried a 7.2-Mb deletion of chromosomal region Xp22.2-Xp22.32 (chrX:4,642,016-11,935,042) comprising 23 RefSeq genes. Among these genes is the midline 1 (MID1) gene. Mutations in MID1 have been associated with the X-linked form of Opitz syndrome [25], which is characterized by midline abnormalities such as cleft lip, laryngeal cleft, heart defects, hypospadias, and ACC [26]. Furthermore, and on the same X-chromosome, the fetus carried a 1.93-Mb duplication, involving chromosomal region Xp22.32-Xp22.33 (chrX:2,700,157-4,628,697), comprising the following 12 RefSeq genes: GYG2 (glycogenin 2, MIM 300198), XG (Xg blood group, MIM 300879), ARSD (arylsulfatase D, MIM 300002), ARSE, ARSH, ARSF (arylsulfatases E, H, and F, MIM 300180, 300586, 300003), MXRA5 (matrix-remodeling associated 5, MIM 300938), PRKX (protein kinase, X-linked, MIM 300083) and its long non-coding RNA (PRKX-AS1), long intergenic non-protein coding RNA 1546 (LINC01546), and the uncharacterized long non-coding RNAs LOC101928201 and LOC389906. qPCR CNV validation and segregation analysis of the fetus and the parents confirmed both the deletion and the duplication and revealed their de novo occurrence in the fetus.