LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia

Abstract

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Figure 1

Mapping of an imprinted quantitative-trait-locus (QTL) for human handedness. (a) Top: pointwise significance of linkage derived from paternal sharing across 8 cM of chromosome 2p12-p11. This corresponds roughly to a 1-LOD unit support interval for the QTL. Significance of linkage is shown on a logarithmic scale. Bottom: known genes within the region (adapted from UCSC Genome Browser). Note LRRTM1 (transcribed proximal to distal (right to left)) sited within an intron of CTNNA2 (transcribed distal to proximal). (b) Close up of 286 kb around LRRTM1. Two exons of CTNNA2 are also visible. The final SNP map that we used in this region is shown (see also Table S1), and the three SNPs that showed the initial paternal-specific haplotype associations are highlighted by pink circles (rs1446109-rs1007371-rs723524). Pairwise intermarker LD (Cramer’s V) is shown at the bottom.

Figure 2

LRRTM1 expression is downregulated maternally in humans. (a) Data are shown from A9 cells that each contains a single human chromosome 2 of known parental origin. Products are shown from PCR using primers specific for human LRRTM1. Upper panel: the human gene is present in genomic DNA (labeled ‘DNA’) from all cell lines tested, apart from C1 (mouse A9 cell line containing no human chromosome). Lower panel: the human gene is only expressed (detected by RT-PCR) in three cell lines containing paternally derived human chromosome 2s (P1, P2, P3), and not in three cell lines containing maternally derived human chromosome 2s (M1, M2, M3). (C2 is a human fibroblast cell line). (b) Monoallelic paternal expression of LRRTM1 in a human EBV-transformed lymphoblastoid cell line. Sequence traces derived from genomic DNA (labeled ‘DNA’) surrounding a C/G SNP are shown for a father, mother and child. The child is heterozygous, but the cDNA prepared from mRNA showed expression of only the paternally inherited G allele of LRRTM1.

Figure 3

Developmental increase of Lrrtm1 mRNA expression in mouse thalamus, hippocampus and retrosplenial cortex, and localization of LRRTM1 in neurons. In situ hybridization analysis in sagittal sections of E15 mouse embryos (a and b) and coronal sections of adult mouse brain (c–f). Bright-field images counterstained with hematoxylin (a, c and e) and corresponding dark-field autoradiographs are shown (b, d and f). The white signal in the dark-field images indicates Lrrtm1 expression. (e and f) Higher magnification of the retrosplenial granular cortex (boxed area in d) shows a high level of Lrrtm1 expression in layer III–IV neurons. (g–i) Myc-LRRTM1 was transfected into E17 mouse cortical neurons (g) and nucleofected into rat P18 dorsal root ganglion neurons (h and i) and detected 40 h later with anti-myc antibody. Neurons were visualized with anti-β-tubulin antibody. LRRTM1 is localized to the cell soma and in the neurites; in neurites it is also localized to lamellipodia of the growth cones (arrowheads in h and i). BTe, basal telencephalon; Cb, cerebellum; cc, corpus callosum; CPu, caudate-putamen; Cx, cerebral cortex; HC, hippocampus; HT, hypothalamus; Max, maxilla, Me, medulla; Nas, nasal cavity; OB, olfactory bulb; Pal, palatine; Pir, piriform cortex; Po, pons; RSG, retrosplenial granular cortex; Sc, Spinal cord; Tc, tectum; Th, thalamus; Ton, tongue. Scale bar is 500 μm in (a–d), 100 μm in (e–f) and 20 μm in (g–i).

Figure 4

In situ hybridization analysis of LRRTM1 expression in the developing human brain at 15 weeks’ gestation. In coronal sections of anterior brain (a), expression is strong throughout the cortical plate and otherwise restricted to septum, caudate, and putamen. Transcript distribution is similar in more caudal sections (b) with the addition of signal in dorsolateral thalamus. More caudal still (c), thalamic signal shifts ventrally to a structure consistent with the lateral geniculate body. Abbreviations are as follows: Amyg (Amygdala), Caud (Caudate), Cp (Cortical), Ge (Ganglionic Eminence), Hypothal (Hypothalamus), Lgn (Lateral Geniculate Nucleus), Lv (Lateral Ventricle), Thal (Thalamus), Vz (Ventricular Zone).

Figure 5

LRRTM1 is not localized on the plasma membrane. No cell-surface expression of LRRTM1 is seen in primary sensory neurons electroporated with myc-LRRTM1 construct and detected by live-cell staining with alkaline phosphatase-conjugated anti-myc antibody (a); strong cell-surface staining of myc-Lingo1 expressing neuron is seen (b). Live-cell staining resulted in 20–30 strongly stained DRG neurons per well for myc-Lingo1 but none for myc-LRRTM1. Similarly, in neuro-2a neuroblastoma cells myc-LRRTM1 (c) is not, but myc-lingo1 is (d) localized to plasma membrane as detected by live-cell staining. In permeabilized Cos-7 cells LRRTM1 (anti-myc antibody; e) is colocalized (merge picture; g) with ER-marked dsRED2-ER (f), whereas lingo1 (anti-myc antibody; h) is only partially colocalized with dsRED2-ER, and it is partially localized on the plasma membrane (i; merge picture: j). Scale bars are 50 μm.