Exome sequencing reveals neurodevelopmental genes in simplex consanguineous Iranian families with syndromic autism

Abstract

Background and objective: Autosomal recessive genetic disorders pose significant health challenges in regions where consanguineous marriages are prevalent. The utilization of exome sequencing as a frequently employed methodology has enabled a clear delineation of diagnostic efficacy and mode of inheritance within multiplex consanguineous families. However, these aspects remain less elucidated within simplex families.

Methods: In this study involving 12 unrelated simplex Iranian families presenting syndromic autism, we conducted singleton exome sequencing. The identified genetic variants were validated using Sanger sequencing, and for the missense variants in FOXG1 and DMD, 3D protein structure modeling was carried out to substantiate their pathogenicity. To examine the expression patterns of the candidate genes in the fetal brain, adult brain, and muscle, RT-qPCR was employed.

Results: In four families, we detected an autosomal dominant gene (FOXG1), an autosomal recessive gene (CHKB), and two X-linked autism genes (IQSEC2 and DMD), indicating diverse inheritance patterns. In the remaining eight families, we were unable to identify any disease-associated genes. As a result, our variant detection rate stood at 33.3% (4/12), surpassing rates reported in similar studies of smaller cohorts. Among the four newly identified coding variants, three are de novo (heterozygous variant p.Trp546Ter in IQSEC2, heterozygous variant p.Ala188Glu in FOXG1, and hemizygous variant p.Leu211Met in DMD), while the homozygous variant p.Glu128Ter in CHKB was inherited from both healthy heterozygous parents. 3D protein structure modeling was carried out for the missense variants in FOXG1 and DMD, which predicted steric hindrance and spatial inhibition, respectively, supporting the pathogenicity of these human mutants. Additionally, the nonsense variant in CHKB is anticipated to influence its dimerization - crucial for choline kinase function - and the nonsense variant in IQSEC2 is predicted to eliminate three functional domains. Consequently, these distinct variants found in four unrelated individuals with autism are likely indicative of loss-of-function mutations.

Conclusions: In our two syndromic autism families, we discovered variants in two muscular dystrophy genes, DMD and CHKB. Given that DMD and CHKB are recognized for their participation in the non-cognitive manifestations of muscular dystrophy, it indicates that some genes transcend the boundary of apparently unrelated clinical categories, thereby establishing a novel connection between ASD and muscular dystrophy. Our findings also shed light on the complex inheritance patterns observed in Iranian consanguineous simplex families and emphasize the connection between autism spectrum disorder and muscular dystrophy. This underscores a likely genetic convergence between neurodevelopmental and neuromuscular disorders.

Keywords: De novo variants; Consanguineous simplex family; Exome sequencing; Iran; Muscular dystrophy; Neurodevelopmental disorder; Syndromic autism.

Fig. 1

Pedigrees of four simplex families. Pedigrees of four families showing affected females (solid circles) and males (solid squares). Carrier parents are indicated by open circles/squares with black dots

Fig. 2

Genetic variants in four genes in Iranian families. The chromatograms display variants within the four genes across four simplex Iranian families, with variant positions indicated by asterisks

Fig. 3

IQSEC2 protein structure and location of the Trp546Ter variant. Schematic representation of the human IQSEC2 protein (NP_001104595), highlighting its IQ-like domain, Sec7 domain, pleckstrin homology domain (PH), and PDZ binding motif. The location of the Trp546Ter variant is indicated in the linker between IQ-like domain and Sec7 domain

Fig. 4

FOXG1 structure and location of de novo variant Ala188Glu. A three-dimensional model of FOXG1 amino acids 168–280 (based on PDB 1vtn.1.C) is depicted, showing the overall protein structure. A The Alanine residue at position 188 is highlighted in green. B The mutated Glutamic acid at position 188 is illustrated in red. The presence of this bulky Glutamic acid likely causes misfolding due to spatial hindrance. C Ala188 resides within a highly intolerant region, as identified by MetaDome web server analysis

Fig. 5

Schematic representation of the Leu211Met substitution in DMD’s N-terminal. A Depicts the cartoon structure of both wild-type (in green) and mutated (in cyan) DMD. The presence of a Methionine residue at position 211 induces misfolding within the core of the DMD protein B Offers a close-up view of the Leucine (in blue) and Methionine (in red) residues at position 211, highlighting the substitution. C Compares the surface electrostaticity of the wild-type DMD and D the mutated DMD

Fig. 6

Schematic representation of CHKB structure and glutamic acid at position 128. A Illustrates the dimerized CHKB structure (in cyan) with Glutamic acid at position 128 (in green), serving as a negatively charged residue. B Emphasizes the significance of the Glu128 residue (in green) for dimerization of CHKB monomers (in red). C Provides a magnified view of Figure A, highlighting the crucial role of Glu128 (in green) in CHKB dimerization (in cyan)

Fig. 7

Disease gene transcript levels in human tissues using RT-qPCR. A The expression of FOXG1 and IQSEC2 is elevated in fetal brain tissue, while CHKB and DMD expression exhibit their highest expression levels in skeletal muscles. B The expression of FOXG1, IQSEC2, DMD, and CHKB varies across different parts of the brain, demonstrating distinct levels of expression