Novel molecular mechanism in Malan syndrome uncovered through genome sequencing reanalysis, exon-level Array, and RNA sequencing

Abstract

The NFIX gene encodes a DNA-binding protein belonging to the nuclear factor one (NFI) family of transcription factors. Pathogenic variants of NFIX are associated with two autosomal dominant Mendelian disorders, Malan syndrome (MIM 614753) and Marshall-Smith syndrome (MIM 602535), which are clinically distinct due to different disease-causing mechanisms. NFIX variants associated with Malan syndrome are missense variants mostly located in exon 2 encoding the N-terminal DNA binding and dimerization domain or are protein-truncating variants that trigger nonsense-mediated mRNA decay (NMD) resulting in NFIX haploinsufficiency. NFIX variants associated with Marshall-Smith syndrome are protein-truncating and are clustered between exons 6 and 10, including a recurrent Alu-mediated deletion of exons 6 and 7, which can escape NMD. The more severe phenotype of Marshall-Smith syndrome is likely due to a dominant-negative effect of these protein-truncating variants that escape NMD. Here, we report a child with clinical features of Malan syndrome who has a de novo NFIX intragenic duplication. Using genome sequencing, exon-level microarray analysis, and RNA sequencing, we show that this duplication encompasses exons 6 and 7 and leads to NFIX haploinsufficiency. To our knowledge, this is the first reported case of Malan Syndrome caused by an intragenic NFIX duplication.

Keywords: Malan syndrome; NFIX; duplication; overgrowth.

Figure 1.

Patient photos notable for macrocephaly, ataxic gait, bilateral epicanthal folds, downslanting palpebral fissures, prominent philtrum, broad mouth, broad fingers with wrinkled skin on palms, and scarce hair

Figure 2.

Identification of a direct tandem duplication involving exons 6 and 7 of the NFIX gene by genome sequencing (GS) and exon-level XON array. (a) Visualization of the duplication and breakpoints. The duplicated segment is highlighted in a box outlined by a red dash line. The two AluY repeats located in introns 5 and 7 of NFIX are indicated on the top (Figure adapted from the RepeatMasker track in UCSC Genome Browser). GS data visualized in IGV is shown in the middle. The relative location and orientation of GS reads within the duplicated segment are shown at the bottom. The observed mate pair orientation (reverse/forward) of GS reads is consistent with direct tandem duplication. The reference sequence of the NFIX transcript used in this study is NM_001271043.2. (b) XON array data visualized in ChAS. The duplicated segment is highlighted as a blue bar on the top.

Figure 3.

Haploinsufficiency of NFIX confirmed by RNA-Seq. The transcript level of NFIX in this patient is nearly 50% of that in controls, supporting haploinsufficiency due to NMD. (a) Heatmap showing the Log2 fold change of significantly (p<0.05) differentially expressed genes in the patient (PT) compared to controls. (b) Comparison of normalized RNA fragment counts between the patient and controls for NFIX and EGFR (as a fibroblast control) using a student’s t-test. (c) Volcano plot of significantly (p<0.05) differentially expressed genes that are downregulated (blue; Log2FC<−0.5) or upregulated (red; Log2FC>0.5).

Figure 4.

NFIX gene structure and disease associated variants (Figure adapted from Zenker M et al., Am J Med Genet C Semin Med Genet. 2019). Variants associated with Malan syndrome and Marshall-Smith syndrome are shown above and below the schematic for the NFIX gene, respectively. Duplication reported in this study is shown above in purple color. Color scheme of NFIX gene structure, blue: putative DNA binding and dimerization domain; green: MH1 (MAD homology 1) domain and the N-terminal DNA binding domain; orange: CAAT-box transcription factor–nuclear factor I (CTF-NFI) domain.