Putative linkage regions (lod >2.3)
17q22: Our largest linkage signal (lod 2.80, REC model) occurred at 17q22 (Table 1, Fig. 4) with an estimated 15% of families showing linkage to this region. The peak SNP rs2332933 occurred in an intron of the MSI2 (musashi 2) gene encoding an RNA-binding protein that influences generation and/or maintenance of specific CNS stem cell lineages (Sakakibara et al. 2002). Sakakibara et al. demonstrated that MSI2 and the highly homologous MSI1 gene are strongly co-expressed and may be cooperatively involved in proliferation and maintenance of CNS stem cell populations. It is therefore very interesting that our data also produced a linkage peak only 1.1 Mb from MSI1 on chromosome 12q24.23 (lod 1.67, REC model, Table 1). Significant association has been reported between autism and a marker near MSI1 (Lauritsen et al. 2006). Kawase et al. (2011) recently identified an intronic element within MSI1 that regulates MSI1 transcription in neural stem/progenitor cells; since MSI2 may also have an intronic regulator, it is noteworthy that the MSI2 linkage peak localized within the gene. The 17q22 region has also been reported linked to reading ability in ADHD-affected siblings using 404 genome-wide microsatellite markers (Loo et al. 2004). Using a ‘reading recognition’ phenotype, they obtained a lod score of 2.6 near D17S787 (50.6 Mb), which is remarkably close to our linkage maximum at 52.9 Mb. The current study provides the first independent support for a reading disability locus at 17q22, obtained despite using different phenotypic definitions from those of Loo et al. (2004). The 17q22 region has also shown association with autism: rare exonic dup/del variants at the BZRAP1 gene (53.7 Mb, just distal to MSI2; Fig. 4) were increased in autistic cases and unaffected family members, compared to healthy controls (Bucan et al. 2009).
7q36.3: Our second largest lod score (lod 2.57, REC model) occurred on chromosome 7q36.3 at 155.6 Mb, maximizing close to both SHH (sonic hedgehog) and EN2 (engrailed homeobox-2) loci (Table 1, Fig. 5).
SHH is a candidate dyslexia gene, because some individuals with SHH mutations causing holoprosencephaly (incomplete cleavage of the cerebral hemispheres) show delayed speech and learning disabilities as their primary clinical presentation (Hehr et al. 2004). Regarding SHH candidacy, our data also generated a lod score of 1.95 very close to the SIX3 gene on 2p21 (Table 1), and a recent study showed that SIX3 directly regulates SHH transcription, so that SIX3 mutations can also produce holoprosencephaly (Jeong et al. 2008). The 2p21-p22 region has previously shown evidence of linkage (Loo et al. 2004) and association (Francks et al. 2002) with dyslexia. This region appears to be distinct from the more proximal DYX3 dyslexia region at 2p11-p16 (Fagerheim et al. 1999; Kaminen et al. 2003; Petryshen et al. 2002).
The EN2 gene is also of interest because it has been reported associated with autism in several studies (reviewed in Sen et al. 2010). It may be relevant that our data also produced a lod score of 1.4 at 2q14.2 (see above ‘regions with lod scores 1.0–1.5’) only 1.5 Mb from the EN1 gene, since EN2 and EN1 function collaboratively in development of cerebellar organization late in embryogenesis (Cheng et al. 2010). Thus, like MSI2 and MSI1 above, both EN2 and EN1 generated linkage signals, suggesting developmentally interacting pairs of dyslexia-predisposing genes. Future analyses using two-locus disease modelling should address this possibility.
Finally, PTPRN2 is also a regional dyslexia candidate gene. Murine double knock-outs of PTPRN2 and PTPRN show impaired neuroendocrine secretion and changes in behaviour and learning (Nishimura et al. 2009). Furthermore, Lionel et al. (2011) reported a rare inherited duplication of PTPRN2 in two brothers with ADHD. Thus, there are several good candidates in the 7q36.3 region, including SHH, EN2 and PTPRN2. The current study is the first report of suggestive evidence for dyslexia linkage to this region.
7q36.1-q36.2: The next largest linkage signal in our data maximized at the junction of 7q36.1 and 7q36.2, at 152.2 Mb (lod 2.42, REC model, Table 1 and Fig. 5). Conservatively, this linkage peak could reflect the same dyslexia-predisposing locus as the 7q36.3 peak. On the other hand, this peak appears distinct from the 7q36.3 peak using a 1-lod-drop confidence interval (trough lod 1.20 at rs4960690, 154.57 Mb). Furthermore, 7q36.1-q36.2 produces a stronger signal under a DOM model than 7q36.3 (Fig. 5), with one large kindred making a major contribution (family #1913, see Table 2), supporting the possibility of two separate dyslexia loci. The lod score maximized between ACTR3B and DPP6, both good candidate genes. ACTR3B is highly expressed in fetal brain and may influence neuronal morphogenesis and migration during brain development (Jay et al. 2000). Marshall et al. (2008) found dup/dels involving the DPP6 gene in four unrelated children with autism. In addition, studies have reported linkage of the 7q36.1-q36.2 region with autism (Liu et al. 2001, lod 2.1 at D7S483, 151.8 Mb) and autism with developmental dysphasia (Auranen et al. 2002; lod 3.7 at D7S2462, 153.19 Mb). The current study is the first report of suggestive evidence for dyslexia linkage to this region.
16p12.1: The 16p12.1 region showed a linkage peak at 22.6 Mb (lod 2.42, NPL model, Table 1 and Fig. 6) with a secondary peak at 16p12.3, 19.1 Mb (rs916767, lod 2.08, Fig. 6). The major peak is immediately upstream of the HS3ST2 gene (heparan sulphate glucosamine 3-O-sulfotransferase 2), which modifies the structure of heparin sulphate (HS) and shows complex spatial-temporal expression patterns in the developing cerebral cortex and cerebellum (Yabe et al. 2005). HS is critical to midline axon guidance in development of the corpus callosum, hippocampal commissure, and anterior commissure (Inatani et al. 2003). Another regional candidate gene is PRKCB (protein kinase C beta, 23.7 Mb), which has been reported associated with autism (Philippi et al. 2005). Protein kinase C is known to regulate cognitive functioning in the prefrontal cortex (Chen et al. 2009) (note also PRKCE at 2p21 linkage region; Table 1). The secondary 16p12.3 peak lies over another candidate gene, SYT17 (synaptotagmin17), which is abundantly expressed in frontal and temporal lobes (Chin et al. 2006). The 16p11-p13 region has been previously linked to dyslexia in two microsatellite genome-wide scans (16p12.2, D16S3046, lod 2.2–2.4, Loo et al. 2004; 16p11.2, D16S753, lod 2.9, Raskind et al. 2005), to ADHD in two scans (16p13, lod 3.7, Ogdie et al. 2004; 16p13, lod 2.4, Gayan et al. 2005) and to autism (16p13, D16S3102, lod 2.9, IMGSAC 2001). The current study represents additional strong support for dyslexia linkage to the 16p12 region, using a high density SNP map which may assist in finer localization of the susceptibility locus, and highlights again the possible overlapping genetic predisposition of dyslexia with ADHD and autism.
4q13.1: This linkage signal maximized at 59.7 Mb (lod 2.34, DOM model, 20% of families linked, Table 1 and Fig. 7) in a ‘gene desert’ immediately upstream of the large LPHN3 gene (latrophilin 3, 61.7–62.6 Mb), encoding a brain-specific G-protein coupled receptor that is expressed in both fetal and adult brain. Arcos-Burgos et al. (2004) reported linkage of ADHD to this same region. Subsequently, in a large case–control study, Arcos-Burgos et al. (2010) showed significant association of ADHD to three SNPs within the LPHN3 locus (OR 1.21–1.23) and demonstrated that these variants were associated with differences in metabolic brain activity and in response to stimulant medication used to treat ADHD. The association of LPHN3 with ADHD has been confirmed in adult cases (Ribasés et al. 2011). This same chromosomal region was also reported linked to a latent class cluster including ADHD, co-morbid behavioural disorders, and alcohol/nicotine dependence (lod 4.0, D4S3248, 59.7 Mb, Jain et al. 2007) and linked to a dyslexia ‘nonword repetition’ phenotype – maximizing at the same microsatellite as the ADHD latent class (lod 2.43, D4S3248, 59.7 Mb, Brkanac et al. 2008). Our linkage signal maximized upstream of LPHN3; this is consistent with a recent LPHN3 mutation screen showing that coding variants did not account for the observed ADHD association (Domené et al. 2011). We tested for association at 393 SNPs located in the 5 Mb region around the linkage peak, across the LPHN3 gene, and 500 kb downstream of the gene, and found only one SNP with nominal P < 0.01 (rs13141378, P = 0.0036, OR 1.67), located 707 kb upstream of LPHN3. In summary, the current study provides the first independent data supporting linkage of dyslexia to LPHN3, a gene strongly implicated in ADHD and ADHD-related behavioural traits. Given the convincing evidence of LPHN3 involvement in dyslexia, it is interesting that our families also produced a linkage signal at 1p31, only 42 kb from the LPHN2 locus (latrophilin 2, lod 1.5, Table 1) which is also expressed in fetal brain.
Other regions (lod 1.5–2.3)
Six of the ten regions with lod scores between 1.5 and 2.3 are interesting since they have been previously reported linked to dyslexia or ADHD. However, these results must be interpreted with caution because they are not statistically significant.
Four of these weaker linkage peaks are in regions implicated in dyslexia by other research groups. Peaks at 2p21 (lod 1.95) and at 4q35.1 (lod 1.78) have already been discussed above (Table 1). Our peak at 4p15.32 (lod 1.95, 16.7 Mb, Table 1) is very close to a linkage peak for reading ability found by Bates et al. (2007) at 4p15.33 (lod 2.1, D4S403, 13.4 Mb). Our signal maximized adjacent to LDB2 (LIM-domain binding 2), which is expressed in fetal and adult brain and implicated in early neuronal differentiation pathways (Azim et al. 2009). Our families also generated a linkage signal at 9q31.2 (lod 1.52, 109.9 Mb, Table 1), which is close to a previous report of linkage to spelling ability at 9q31.3 (lod 1.2, D9S1677, 111.0 Mb, Loo et al. 2004). Our localization is also supported by results of the first high density QTL association genome screen for early reading ability (Meaburn et al. 2008), which found and replicated an association with rs1323381 at 110.10 Mb (although this association was not replicated by Luciano et al. 2011). A possible regional candidate is AL390170 at 110.93 Mb, an uncharacterized clone highly expressed in fetal and adult brain.
Two of our weaker peaks are in regions previously linked to ADHD. The 9q33.3 peak localized right at LHX2 (lod 2.16, 125.9 Mb, 22% of families linked, Table 1), a gene critical to development of cerebral cortex, particularly hippocampus (Mangale et al. 2008). The 9q33.3 region has been previously reported linked to ADHD in two microsatellite genome screens (Bakker et al. 2003, lod 2.1, D9S1825, 126.9 Mb; Arcos-Burgos et al. 2004, 9q33.3, marker not stated). Similarly, our peak at 5p13.3-p13.1 (lod 1.69, 32.1–40.2 Mb, Table 1) has also shown linkage in two independent genome screens of ADHD (Ogdie et al. 2004, lod 2.6, D5S418, 40.1 Mb; Arcos-Burgos et al. 2004, 5p13.3, marker not stated) as well as autism (D5S2494, lod 2.55, 40.3 Mb, Liu et al. 2001).