LINS, a modulator of the WNT signaling pathway, is involved in human cognition

Abstract

Background: Inherited intellectual disability (ID) conditions are a group of genetically heterogeneous disorders that lead to variable degrees of cognition deficits. It has been shown that inherited ID can be caused by mutations in over 100 different genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying the defective gene underlying an autosomal recessive ID in two sibs of an Emirati family.

Methods: A combined approach involving homozygosity mapping and whole-exome sequencing was used to identify the causative mutation. RNA analysis was performed to gain further insight into the pathogenic effect of the detected mutation.

Results: We have identified a homozygous splicing mutation (c.1219_1222+1delAAAGG) in the LINS gene in the affected children. LINS is the human homologue of the Drosophila segment polarity gene lin that encodes an essential regulator of the wingless/Wnt signaling. The identified mutation alters the first consensus nucleotide of the 5' donor splice junction of intron 5 and the 3' end of exon 5. Transcript analysis revealed that this change leads to an exon skipping event resulting in direct splicing of exon 4 to exon 6. Another mutation in LINS has been described very briefly in an Iranian family with autosomal recessive ID and microcephaly.

Conclusion: Our study confirms that LINS, a modulator of the WNT pathway, is an indispensable gene to human cognition and this finding sheds further light on the importance of WNT signaling in human brain development and/or function.

Figure 1

Clinical Data of the studied family. A. Pedigree showing the mode of inheritance for an autosomal recessive intellectual disability phenotype in a consanguineous family from United Arab Emirates. The studied members are indicated by numbers and asterisks. B. Appearance of the 2 affected sibs, 1) II1 at age 8 years and 2) II2 at age 3 years. Note the flattening of the midface.

Figure 2

Genomic mapping and genetic testing of an autosomal recessive intellectual disability phenotype. A. Genotyping the whole genome of both parents (I1 and I2) and affected children (II1 and II2) detected a homozygosity on chromosome 15q26. B. Integrative Genomics Viewer (IGV) visualization of homozygous mutation c.1219_1222+1delAAAGG in LINS gene from exome data. All reads show 5 bp deletion, sequence of wild type gene and exon annotation at bottom. The adjacent homozygous substitution G>A (C>T on reverse strand) is a common variant rs12719734G>A. C. DNA sequencing chromatograms confirmed the segregation of the AAAGG (inside the brown square) deletion detected by exome data with the assessed phenotype. The deletion was found to be homozgous in the patients (II1 and II2) and heterozygous in parents (I1 and I2). The deletion was not found in 100 normal controls. The rs12719734G>A (designated with a red star) was found in all the screened individuals.

Figure 3

Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. A. Agarose gel of RT-PCR reaction products from LINS cDNA amplification in a control (con), parents (I1, I2) and patients (II1, II2) compared to a DNA 100bp ladder (M). The gel showed a ~1014bp band of the wild type LINS transcript encompassing exon 5 in a normal control (con) accompanied with multiple isoforms of varying length (~1000bp). In patients (II1, II2) lanes, only smaller bands were seen (~400bp) suggesting a homozygous deletion of around 600bp. The parents (-I1, -I2) have both upper and lower bands suggesting that they carry the 600bp deletion in a heterozygous state. B. A schematic diagram of the splicing defect seen in patients based on Sanger sequencing data of the cDNA. The upper most band of the higher and lower bands seen in RT-PCR gel were purified and sequenced. This higher band noticed in control and parents was found to include exons 3,4,5 and 6. On the other hand, Exon 5 (E5) was found to be missing in the lower-size band seen in both patients and parents. These results suggested that the genomic deletion at the end of E5 abolished a canonical splicing site masking the exon from the splicing machinery which considered it to be part of intron 4 and cut it out of the nascent mRNA. C. Analyzing the accompanying upper and lower bands amplified by RT-PCR suggested the presence of at least 3 LINS transcripts alternatively spliced in exon 6 with the putative multiple splice junctions are shown. All the lower bands lack exon 5 while the upper bands include it compared to the RefSeq NM_001040616.2.

Figure 4

Lines/LINS plays a putative dual role in WNT canonical pathway. In WNT canonical pathway, the absence of a signal leads to the hyperphosphorylation of arm/CTNNB1 leading to its ubiqitination and degradation by the proteasome in the cytoplasm. Binding of wg/WNT1 ligand to a Fz and arr/LRP receptor complex leads to stabilization of hypophosphorylated arm/CTNNB1, translocating it to the nucleus. In the nucleus, arm/CTNNB1 competes with and displaces gro/TLE interacting with pan/TCF proteins to activate transcription. In Drosophila, lines/LINS was found to act as a modulator of wg/WNT canonical pathway acting in parallel with or downstream of arm/CTNNB1 in response to wg/WNT1 signaling to enhance or represses the transcription of target genes. Frizzled (Fz), arrow (arr), LDL receptor-related protein (LRP), armallido (Arm), β-catenin (CTNNB1), groucho (gro), transducin-like enhancer of split (TLE), pangolin (pan), T-cell factor (TCF).