Differences in Expression of IQSEC2 Transcript Isoforms in Male and Female Cases with Loss of Function Variants and Neurodevelopmental Disorder

Abstract

Pathogenic hemizygous or heterozygous mutations in the IQSEC2 gene cause X-linked intellectual developmental disorder-1 (XLID1), characterized by a variable phenotype including developmental delay, intellectual disability, epilepsy, hypotonia, autism, microcephaly and stereotypies. It affects both males and females typically through loss of function in males and haploinsufficiency in heterozygous females. Females are generally less affected than males. Two novel unrelated cases, one male and one female, with de novo IQSEC2 variants were detected by trio-based whole exome sequencing. The female case had a previously undescribed frameshift mutation (NM_001111125:c.3300dup; p.Met1101Tyrfs*5), and the male showed an intronic variant in intron 6, with a previously unknown effect (NM_001111125:c.2459+21C>T). IQSEC2 gene expression study revealed that this intronic variant created an alternative donor splicing site and an aberrant product, with the inclusion of 19bp, confirming the pathogenic effect of the intron variant. Moreover, a strong reduction in the expression of the long, but also the short IQSEC2 isoforms, was detected in the male correlating with a more severe phenotype, while the female case showed no decreased expression of the short isoform, and milder effects of the disease. This suggests that the abnormal expression levels of the different IQSEC2 transcripts could be implicated in the severity of disease manifestations.

Keywords: IQSEC2 gene; SpainUDP; exome; gene expression; intron variant; neurodevelopment syndrome; transcript isoforms.

Figure 1

(A) Representation of IQSEC2 isoforms. Schematic representation of large isoform (NM_001111125) which include 15 exons; intermediate isoform (NM_015075) which include 14 exons; and short isoform (NM_001243197) which include 3 alternative exons. Primers used for the quantitative expression of the long and short isoforms are represented by arrows. (B) Isoforms expression of IQSEC2 in different tissues obtained from GTEX database (https://www.gtexportal.org, accessed on 30 May 2022). TPM values corresponding to the larger IQSEC2 isoform (NM_001111125) and the short isoform (NM_001243197) are represented as the expression values in brain cerebellum and cortex, skeletal muscle and whole blood.

Figure 2

Variant effect on splicing in ND0673 patient. Conventional RT-PCR expression of IQSEC2 mRNA in peripheral blood samples from the patient and his parents, and patient muscle sample. M: Molecular weight marker (100 bp DNA Ladder, Promega). Schematic representation of the mutation (c.2459+21C>T) in NM_001111125 transcript and the newly created donor slicing site. Sanger sequencing of aberrantly spliced products reveals the insertion of 19 bp of intron 6 in the patient.

Figure 3

Relative quantification of IQSEC2 transcripts isoforms in the two families. Quantitative QT-PCR expression analysis for IQSEC2 isoforms in ND067 and ND144 families, showing expression levels of patients (ND0673 and ND1443) compared to their fathers (ND0671 and ND1441) and mothers (ND0672 and ND1442). Left panels represent expression levels based on primers amplifying between exons 2 and 3, specific for larger isoform (NM_001111125). Middle panel shows the expression using primers for exons 12–13, which do not discriminate between large and intermediate isoforms. Right panels show the expression of short IQSEC2 isoform (NM_001243197) with a specific assay designed between alternative exons 2′ and 3′, only present in this isoform.

Figure 4

In silico prediction of transcription factor binding. Transcription factors with differential binding to variant c.2459+21C>T (Alt. allele) and the wild-type sequence (Ref. allele) are depicted. Binding motifs and p-values are also shown.