Phenotypic and genotypic characterization of families with complex intellectual disability identified pathogenic genetic variations in known and novel disease genes

Abstract

Intellectual disability (ID), which presents itself during childhood, belongs to a group of neurodevelopmental disorders (NDDs) that are clinically widely heterogeneous and highly heritable, often being caused by single gene defects. Indeed, NDDs can be attributed to mutations at over 1000 loci, and all type of mutations, ranging from single nucleotide variations (SNVs) to large, complex copy number variations (CNVs), have been reported in patients with ID and other related NDDs. In this study, we recruited seven different recessive NDD families with comorbidities to perform a detailed clinical characterization and a complete genomic analysis that consisted of a combination of high throughput SNP-based genotyping and whole-genome sequencing (WGS). Different disease-associated loci and pathogenic gene mutations were identified in each family, including known (n = 4) and novel (n = 2) mutations in known genes (NAGLU, SLC5A2, POLR3B, VPS13A, SYN1, SPG11), and the identification of a novel disease gene (n = 1; NSL1). Functional analyses were additionally performed in a gene associated with autism-like symptoms and epileptic seizures for further proof of pathogenicity. Lastly, detailed genotype-phenotype correlations were carried out to assist with the diagnosis of prospective families and to determine genomic variation with clinical relevance. We concluded that the combination of linkage analyses and WGS to search for disease genes still remains a fruitful strategy for complex diseases with a variety of mutated genes and heterogeneous phenotypic manifestations, allowing for the identification of novel mutations, genes, and phenotypes, and leading to improvements in both diagnostic strategies and functional characterization of disease mechanisms.

Figure 1

Pedigree structures of families presenting with ID syndromes and their corresponding pathogenic mutations. The pedigrees of seven families featuring NDD syndromes are shown. Affected members are represented by either dark squares (males) or circles (females). The only individual who is non-manifesting for the phenotype but is homozygous for the mutation is represented with a white square with a black dot in the middle. Individuals with homozygous mutations are represented as m/m; heterozygous carriers as wt/m; and non-carriers individuals homozygous for the healthy allele as wt/wt. *Indicates those individuals that were subject to WGS analyses. The Sanger chromatogram sequences of the identified pathogenic mutations highlighting the homozygous mutant alleles (red arrow) are shown below each pedigree.

Figure 2

Flow chart for WGS analysis. It shows the steps carried out in the recruited DNA samples for accurate and reliable disease-gene identification.

Figure 3

(A) Conservation of the amino-acid arginine at position 74 of the NSL1 protein across other orthologous. (B) Known and predicted protein-interactions of NSL1 protein according to STRING database. Kinetochore proteins that act as components of the essential kinetochore- associated NDC80 complex, which is required for chromosome segregation and spindle checkpoint activity: NUF2, SPC24, and SPC25. Kinetochore proteins that are part of the MIS12 complex, which is required for normal chromosome alignment and segregation and for kinetochore formation during mitosis: MIS12, PMF1-BGLAP (Polyamine-modulated factor 1), DSN1 (Kinetochore-associated protein DSN1 homolog), NSL1 (Kinetochore-associated protein NSL1 homolog). Kinetochore proteins that are essential for spindle-assembly checkpoint signaling and for correct chromosome alignment: CASC5 (KNL1; Kinetochore scaffold 1) and BUB1 (Mitotic checkpoint serine/threonine-protein kinase BUB1).

Figure 4

Effects of SYN1 R420G mutation on hippocampal neurons. (A) Western blot in HEK293T cells untransfected or transfected with either wild-type of R420G SYN1-V5 mutant confirm expression of both constructs. Original western blot images are provided as Supplementary Material (B) Transfection efficiency on hippocampal neurons was calculated in three independent experiments by counting GFP+ cells. Black solid column shows wild-type SYN1-V5 transfected neurons and solid grey column shows R420G SYN1-V5 mutant transfected neurons C: Microscopy images of hippocampal neurons transfected with either wild-type (upper panel) or mutant (lower panel) SYN1-V5 (Scale bar: 25μM). (D) Neurons transfected with wild-type SYN1-V5 express higher levels of SYN1 (p = 0.0004) and (E) have longer axons (p = 0.0008) than their mutant counterparts. The length of axons and normalized fluorescence were measured using the ImageJ software. The graphs shows the mean normalize fluoresce (upper graph) and the axon length in μM (lower graph) of three independent transfections. Black columns show wild-type SYN1-V5 transfected neurons and solid grey columns show R420G SYN1-V5 mutant transfected neurons. Values represent the means ± SEM. ***p < 0.001; ns = non-significant.