We genotyped 87 single-nucleotide polymorphisms (SNPs) in 222 RD siblings and their parents, within a region of paternal-specific linkage to relative hand skill that we had detected previously in this sample (). The SNPs were targeted within four positional and functional candidate genes on 2p12–p11 (LRRTM4, CTNNA2, LRRTM1 and DNAH6) ( and Table S1
). Following our parent-of-origin linkage data (), we tested for quantitative association of paternally inherited SNP alleles with relative hand skill (using QTDT18
). Four SNPs in distinct locations (rs1517771, rs290015, rs2063436 and rs723524; Table S1
), which were not in significant linkage disequilibrium (LD) with one another, showed nominally significant paternal-specific association with relative hand skill (0.05 > P
> 0.01). This was no more than the expected false-positive rate.
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-p11.9 This corresponds roughly to a 1-LOD unit support interval for the (more ...)
We then aimed to interrogate the underlying haplotype structure by testing haplotypes for association, that were constructed in ‘sliding windows’ representing all sets of three consecutive SNPs (disregarding any instances where SNPs were not in significant LD, that is, P > 0.05 based on χ2-test of pairwise LD). We used the total, paternal and multi-allelic options of QTDT together for the association tests. Significance levels for paternal-specific association were all P > 0.05, except for haplotypes derived from the SNPs rs1446109-rs1007371-rs723524 (P = 0.00002), and the overlapping window rs1007371-rs723524-rs1025947 (P = 0.0007). These SNPs are on 2p12 within a region of strong intermarker LD that spans at least the first exon and 137 kb upstream of the gene LRRTM1, which is located within an intron of CTNNA2 (). There was no significant maternal-specific haplotype association at any location (all P > 0.05).
The rs1446109-rs1007371-rs723524 haplotype 2-2-2 (where 2 is the minor allele of each SNP) was responsible primarily for the paternal-specific association observed with haplotypes derived from these three SNPs. The 2-2-2 haplotype had 9% frequency, and was associated with a mean shift of 1.1 s.d. toward left-handedness in the relative hand skill distribution5,7
when inherited paternally, compared to all other haplotypes. Genotyping of 32 more SNPs, including 12 within 137 kb upstream of LRRTM1, confirmed that rs1446109-rs1007371-rs723524 haplotype 2-2-2 represents a distinct haplotype clade for at least 76 kb upstream of, and including, the predicted promoter of LRRTM1 (Figure S1
and Table S1
We screened the first two exons and predicted promoter of LRRTM1 for polymorphisms in 26 left-handers from the RD sample by denaturing high-performance liquid chromatography and sequencing, but we did not detect any polymorphisms that tagged rs1446109-rs1007371-rs723524 haplotype 2-2-2, or that had overt disruptive effects on the predicted LRRTM1 protein (entirely coded within exon 2). The only novel SNP that we identified was upstream of the first LRRTM1 exon (Table S1
We analyzed the SNPs rs1007371 and rs723524, which together can be used to construct a close proxy to the ‘risk’ haplotype described above (see Figure S1
), in a sample of normal twin-based sibships from Brisbane, Australia, that was derived from 215 independent families. We found no significant evidence for paternal association of the risk haplotype with our quantitative measure of human handedness (P
We genotyped the SNPs rs1446109 and either rs723524 or rs718466 (the latter are equivalent tagging SNPs according to international HapMap data) in four family samples of white European descent that included individuals with schizophrenia or poor-outcome schizoaffective disorder. These comprised a subset of the New York/Oxford sample17
(226 families), an Irish ‘High Density’ sample19
(236 families), a sample collected in Montreal20
(124 families) and an Afrikaner sample21
(416 families) (). The 2-2 haplotype defined by the rare alleles of these SNPs is equivalent to the risk haplotype for left-handedness defined above (Figure S1
). The 2-2 haplotype varied in frequency between 7.6 and 12.1% in the four sample sets. We found that haplotype 2-2 was overtransmitted paternally to affected individuals in a combined analysis of the four samples, P
= 0.0014 (one-tailed test, 38 transmissions to 16 nontransmissions; ). This was a specific hypothesis test that requires no statistical adjustment. There was no significant paternal overtransmission of any other haplotype, nor was there maternal overtransmission of any haplotype. The paternal 2-2 result was derived roughly equally from three of the four samples ().
Transmission disequilibrium statistics for two-marker haplotypes (see main text), for families affected with schizophrenia/schizoaffective disorder
We genotyped rs1446109 (almost tagging for the risk haplotype; Figure S1
), in two case–control collections of European descent (461 cases and 459 controls from Munich, Germany, and 429 cases and 428 controls from Scotland (all cases had Diagnostic and Statistical Manual of Mental Disorders
, fourth edition diagnoses of schizophrenia, and both sample sets were recruited according to the same protocol)).22
As the parental origin of the alleles could not be established and the paternal and maternal alleles were confounded, we expected this analysis to be low-powered to detect an imprinted effect. Nonetheless, this SNP showed a trend toward association (P
= 0.09) when tested under a genotypic 2-df logistic regression model using covariates for gender and the collection site (Munich/Aberdeen). The additive component of this model was significant at P
= 0.036, and the direction of allelic effect was the same as in the family samples. When we repeated the analysis using only those cases (151) who reported a positive family history of schizophrenia or bipolar disorder, with the aim of removing sporadic environmental cases, rs1446109 showed significant association with P
= 0.013, and the additive component of the model showed P
= 0.0038, again in the expected direction. We also tested for association in a sample of 270 Han Chinese families23
but we found no significant bias in paternal or maternal transmission of any haplotype to schizophrenic people (there were 65 paternal transmissions to 78 paternal nontransmissions of the 2-2 haplotype).
No imprinted genes were known previously on chromosome 2p. We found that human LRRTM1 is imprinted (paternal-only expression) in hybrid A9 cells24
(mouse cell lines containing single human chromosomes of known parental origin) (). Despite certain limitations with hybrid cells in the context of imprinting studies,25
mouse A9 cells have been used for the reliable identification and verification of human imprinted genes.24
To validate the chromosome 2 hybrid cells, we analyzed the expression of four additional biallelically expressed genes, GGCX (2p11.2), BCL2L11 (2q13), TCF7L1 (2p11.2) and RALB (2q14.2). We detected the expression of all four genes in chromosome 2 hybrid cells, regardless of the parental origin of the human chromosome 2 (Figure S2
). No detection of these genes was obtained in RT- controls. We also used transcribed polymorphisms to show mono-allelic LRRTM1 expression in tissue samples from a minority of unrelated postmortem human brains (3 out of 18) by RT-PCR and sequencing or restriction digestion (not shown). However, biallelic expression was found in 15 out of 18 brains, and LRRTM1 was expressed at similar levels from both alleles in the adult human cerebral cortex of five individuals showing biallelic expression (analyzed by quantitative PCR). All brain tissue was obtained from normal controls that were unaffected by major neurological or psychiatric disease. These results suggest that imprinting of LRRTM1 is either variable between individuals (as for the imprinted genes IMPT1 or IGF226
), and/or variable between different brain regions or cell/tissue types (as for UBE3A27
). We also found mono-allelic paternal expression of LRRTM1 in four out of four unrelated EBV-transformed human lymphoblastoid cell lines that were heterozygous for a transcribed SNP ().
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 (more ...)
By use of in situ
hybridization in the mouse, we found that Lrrtm1 is expressed predominantly in the nervous system by postmitotic neurons, but also in some nonneuronal tissues (; Figure S3
). Expression is upregulated in the mouse brain during embryonic development and early postnatally. In adult brain, Lrrtm1 expression is most prominent in the forebrain, particularly in the thalamus (in most or all nuclei), and in cortical areas including hippocampus, piriform and posterior cingulate (; Figure S3
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 (more ...)
In northern blot analysis of adult human brain, LRRTM1 also showed predominant expression in forebrain regions including thalamus and cerebral cortex (Figure S4
). By in situ
hybridization in coronal sections of the post-mortem developing human brain (14–16 weeks gestation; ), strong expression was observed in anterior sections throughout the cortical plate and in septum, caudate and putamen. The absence of signal in the subventricular zone argues against a direct involvement in neurogenesis (). Transcript distribution was similar in more caudal sections with the addition of signal in dorsolateral thalamus (). More caudal still, thalamic signal shifted ventrally to a structure consistent with the lateral geniculate body (). The striking signal in caudate and putamen in human () was not present in mouse, at least not at E15 and in adult. In addition, expression within the human thalamus was more restricted as compared to the mouse, with staining relatively limited to dorsomedial regions.
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, (more ...)
No consistent asymmetric expression was observed in any of three human developing brains examined by in situ
hybridization (14–16 weeks’ gestation), regardless of whether cerebral hemispheres were analyzed in aggregate or cortical sub-regions were examined in isolation (dorsolateral, temporal, ventrolateral or cingulated). Similarly to embryonic brain, LRRTM1 was expressed at similar levels (that is, symmetrically) in all analyzed regions of left and right adult human cerebral cortex (several different cortical regions from five individuals were analyzed by quantitative PCR). We also quantified left- and right-brain Lrrtm1 mRNA expression levels in rats and embryonic mice (see Materials and methods), but did not detect evidence for asymmetrical expression in rodents. In addition, we tested cerebral cortex, cerebellum, brain stem, olfactory bulb, thymus, heart, lung, liver, intestine, pancreas, spleen, kidney, muscle and testis of two reciprocal crossed F1 mice between C57BL/6J and JF1 strains for allele-specific expression. Each of the F1 mice was 30 weeks old. Expression of Lrrtm1 was detected in cerebral cortex, cerebellum and brain stem, but it was biallelic (Figure S5
In rodent primary sensory (DRG) and cortical neurons (), and in cerebellar granular neurons (data not shown), overexpressed LRRTM1 localized to the cell soma, neurites and lamellipodia of growth cones, suggesting a function in axon guidance and/or synaptogenesis. Unexpectedly, in transfected MRC5, Cos-7 and Neuro-2a cells, LRRTM1 colocalized with endoplasmic reticulum (ER) markers ( and Figure S6
). Live-cell staining for overexpressed LRRTM1 in DRG neurons revealed that the protein is not accessible on the plasma membrane under conditions that allowed surface detection of a related member of the LRR protein superfamily, Lingo1 (). These results suggest that endogenous LRRTM1 may have a role in intracellular trafficking within axons. However, it remains possible that LRRTM1 is localized to plasma membrane in cells expressing an unidentified LRRTM1 chaperone or coreceptor protein that promotes its processing and/or transport.
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 (more ...)
We analyzed methylation within 2 CpG islands that correspond to the predicted promoter and coding exon of LRRTM1, and a third island roughly 18 kb upstream of LRRTM1, in 17 lymphoblastoid cell lines and 17 human post-mortem brain samples, but we did not find evidence that these CpG islands are differentially methylated regions (DMRs) (data not shown).
We tested CTNNA2 for mono-allelic expression in 10 post-mortem brain samples from normal, unrelated individuals, by use of a transcribed SNP in exon 12, but expression was always biallelic, in contrast to LRRTM1 (data not shown). We did not detect CTNNA2 expression in the A9 hybrids, or in human lymphoblastoid cell lines, by RT-PCR. The first CTNNA2 exon is over 750 kb from LRRTM1 (), and several LD blocks away, further suggesting that the regulation of these genes is distinct. There are no other genes within 1.13 Mb of LRRTM1, and the next closest gene expressed in the forebrain (expression data available via the UCSC genome browser) is over 2.75 Mb distally (LRRTM4; ).
In order to analyze the evolution of LRRTM1, we compared it between humans and chimpanzees. There is no fixed amino acid difference between human LRRTM1 and chimpanzee LRRTM1, and it is not among the genes that are differently expressed between adult humans and adult chimpanzees in brain, liver, kidney, heart or testis.28
Furthermore, biallelic expression of LRRTM1 was detected in two out of two chimpanzee brain samples (see Materials and methods).