The regulatory element READ1 epistatically influences reading and language, with both deleterious and protective alleles

Background Reading disability (RD) and language impairment (LI) are heritable learning disabilities that obstruct acquisition and use of written and spoken language, respectively. We previously reported that two risk haplotypes, each in strong linkage disequilibrium (LD) with an allele of READ1, a polymorphic compound short tandem repeat within intron 2 of risk gene DCDC2, are associated with RD and LI. Additionally, we showed a non-additive genetic interaction between READ1 and KIAHap, a previously reported risk haplotype in risk gene KIAA0319, and that READ1 binds the transcriptional regulator ETV6. Objective To examine the hypothesis that READ1 is a transcriptional regulator of KIAA0319. Methods We characterised associations between READ1 alleles and RD and LI in a large European cohort, and also assessed interactions between READ1 and KIAHap and their effect on performance on measures of reading, language and IQ. We also used family-based data to characterise the genetic interaction, and chromatin conformation capture (3C) to investigate the possibility of a physical interaction between READ1 and KIAHap. Results and conclusions READ1 and KIAHap show interdependence—READ1 risk alleles synergise with KIAHap, whereas READ1 protective alleles act epistatically to negate the effects of KIAHap. The family data suggest that these variants interact in trans genetically, while the 3C results show that a region of DCDC2 containing READ1 interacts physically with the region upstream of KIAA0319. These data support a model in which READ1 regulates KIAA0319 expression through KIAHap and in which the additive effects of READ1 and KIAHap alleles are responsible for the trans genetic interaction.

again with 25 strokes in a Dounce homogenizer. The nuclei were collected by centrifugation (5min, 2500g, 4ºC), washed with 500ul 1X restriction Buffer 2.1 (NEB), collected by centrifugation (same settings), resuspended in 1X Buffer 2.1, and split into 10 aliquots, ~60μl each. To each aliquot, 312μl Buffer 2.1 and 38μl 1% SDS was added, and the aliquots were incubated with rotation at 65ºC for 15min. 44μl 10% Triton X-100 were added to each tube, and 400 units of HindIII restriction enzyme (NEB) were added to 9 of the tubes. The remaining tube was split into two aliquots, and 200 units of HindIII were added to one of them (digested control); the other tube served as an undigested control. All tubes were incubated overnight at 37ºC with rotation. The following morning, an additional 200 units of HindIII were added to the 9 3C digests, and an additional 100 units to the digested control, and the tubes were incubated at 37ºC for an additional 2 hours with rotation. The enzyme was then inactivated by addition of 43μl of 20% SDS to each of the 9 3C digests, and 20μl each to the digested and undigested controls, and incubating at 65ºC with rotation for 30 minutes. Ligation reactions were then set up. Each of the 9 reactions consisted of 1 3C digest, 745μl 10X ligation buffer (500mM Tris-HCl, pH=7.5, 100mM MgCl 2 , and 100mM dithiothreitol), 745μl 10% Triton X-100, 8μl BSA (100mg/mL), 1μl T4 DNA ligase (30 Weiss units/μl), and 5.5mL H 2 O. The reactions were mixed and allowed to proceed at 16ºC for 3 hours. Meanwhile, the digested and undigested controls were treated with 10μg RNAse A and incubated for 1 hour at 37ºC. When ligation was complete, the ligation reactions were treated with 50μl proteinase K (25mg/mL), and incubated overnight at 65ºC to reverse crosslinks and digest protein. The digested and undigested controls were subjected to the same treatment, with 10μl proteinase K. The following morning, an additional 25μl and an additional 5μl of proteinase K was added to each ligation reaction and to each of the controls, respectively; they were then incubated at 65ºC for an additional 2 hours. The digested and undigested controls were stored at -20ºC until further use. The ligation reactions were distributed among 11 MaxTract tubes (Qiagen) for DNA purification. An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added to each tube. The tubes were inverted several times, and spun 5 minutes at 1,500xg. The aqueous phases were decanted and combined into 4 30mL glass centrifuge tubes. To the solution in each tube, 0.7vol isopropanol and 0.1vol 3M sodium acetate (pH=5.2) was added, the tubes were mixed by inversion, and placed at -80ºC for 1 hour. They were the thawed at room temperature for 30min, and spun for 45 minutes at 14,000xg (4ºC). The supernatant was decanted, the pellets were dried at room temperature, and each was dissolved in 250μl 1X TE (10mM Tris-HCl, pH=8.0, 1mM EDTA). The DNA was combined into 1mL total, transferred to a 1.5mL tube, and treated with 100μg RNAse A. The RNAse reaction was allowed to proceed at 37ºC for 1 hour. The DNA was then split into 2 500μl aliquots, and each was added to a 2mL phase-lock tube. The digested and undigested controls were each also added to a phase-lock tube. An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was added to each tube, the tubes were inverted to mix, and spun 5min at 16,000xg (room temperature). The same volume of chloroform was added to the aqueous phase of each tube, and the tubes were again inverted to mix and spun at 16,000xg for 5min. The aqueous phase was then collected and transferred to a fresh 2mL tube, and 0.1vol of 3M sodium acetate (pH=5.2) and 2.5vol of ice cold 100% ethanol was added to each tube. The tubes were inverted to mix, and stored at -80ºC overnight to precipitate the DNA. The following morning, the tubes were spun for 45min, 16,000xg, at 4ºC. The 3C pellets were washed 5X with cold 70% ethanol; the digested and undigested controls, 2X. The pellets were then dried, and each 3C pellet was dissolved in 500μl 1X TE, while the digested and undigested control pellets were dissolved in 150μl 1X TE. The two dissolved 3C pellets were combined into one, and all three samples were quantified with PicoGreen (Life Technologies), as per manufacturer's instructions. Samples were then used as qPCR template. The digested and undigested controls were used to correct for digestion efficiency between the two cell lines, as previously described. [5] qPCR qPCR was done with the QuantiTect SYBRGreen qPCR kit from Qiagen, in 50μl reactions, as per manufacturer's instructions. Primers are listed in Supplementary Table 5. The qPCR reaction is as follows: 15 min at 95ºC, then 45 cycles of 30 sec at 95ºC followed by 30 sec at 60ºC followed by 1 min at 72ºC, then 6 min at 72ºC, and an indefinite hold at 4ºC. For qPCR reactions, 3C template DNA was diluted to a final concentration of 20ng/ul, and each primer was diluted to a final concentration of 0.25µM. qPCR results were normalized across templates to a control amplicon from the gene encoding β-actin (ACTβ). The ACTβ primers amplify across a region without a HindIII, BamH1, or BglII site.

β-globin Control Experiment
To assess the effectiveness of our 3C protocol, and to eliminate any systematic differences between Raji and GM17831 cells, we performed 3C, according to the above protocol, with a set of previously described intrachromosomal interactions in the β-globin locus. Vu et al. (2010) detail two interactions and one non-interaction with an LCR region in the locus (flanked by anchor primer C). [6] One is a strong local interaction with a nearby region (flanked by prey primer B), one is a weaker long-range interaction (flanked by prey primer A), and one is a noninteraction with a distant region (flanked by prey primer D). Globin primer sequences are listed in Supplementary Table 6. Because the globin primers flank BglII or BamH1 sites rather than HindIII sites, fixed cells were subjected to double-digests with these enzymes in NEB restriction  Non-word reading task, age 9 Spelling at 7 Single-word spelling task, age 7 Spelling at 9 Single-word spelling task, age 9

B.
Phenotype Description Severe RD Cases defined as having a score less than or equal to 2 standard deviations below the mean on the phoneme deletion task Severe LI Cases defined as having a score less than or equal to 2 standard deviations below the mean on either the WOLD verbal comprehension task or the non-word repetition task

Supplementary Table 2: (A)
List of phenotypes used in ALSPAC analyses. Reading measures in the ALSPAC include a phoneme deletion task at age 7, single-word reading at ages 7 and 9, spelling at ages 7 and 9, single non-word reading at age 9, and passage comprehension, speed and accuracy at age 9. The phoneme deletion task measures phoneme awareness, [7] which is widely considered to be a core deficit in RD. [8] For the phoneme deletion task the child listens to a word spoken aloud, and is then asked to remove a specific phoneme from that word to make a new word (e.g. what word is created when the /b/ sound is removed the word 'block'? 'Lock'). This task is also known as the Auditory Analysis Test, and was developed by Rosner and Simon.
[9] Single-word reading was assessed at age 7 using the reading subtest of the Wechsler Objective Reading Dimensions (WORD).
[10] At age 7 and 9, spelling was assessed; the child was asked to spell a set of 15 age-adjusted words. At age 9, single-word reading was again assessed by asking the child to read ten real words and ten non-words aloud. The words and nonwords used are a subset of a larger list of words and non-words taken from research conducted by Terezinha Nunes and others at Oxford. [11] The non-word repetition (NWR) task was ascertained at 8 years of age. This is a verbal language measure wherein the child was asked to repeat recorded non-words. This task measures short-term phonological memory and processing; [12] children with LI consistently perform poorly. [13] Verbal, performance, and total IQ were assessed at age 8, using the Wechsler Intelligence Scale for Children (WISC-III). [14] (B) Case/control definitions used in association analysis (Table 1). showing statistical significance of differences between means for the four genotype classes listed in Figure 1, for the indicated phenotype and READ1 single or composite allele. P-values below 0.05 are shown in bold. READ1  5'-AGCCCTCCCTACTGACGGAAACACAT-3'  5'-TTGCAGGGTGAAAATGAGGAGTTGAAAT-3'   NRSN1  5'-TGCCCGGTACTCCCTCCAATCAGC-3' 5'-CCAAGCCAAGGCCGCAGTGTTC-3'

Control Primers
ACTβ 5'-GCCCTAGGCACCAGGGTGTGA-3' 5'-ACAGGGTGCTCCTCAGGGGC-3' Table 5: Primer sequences for 3C primers. Primers in black were used to assess fusion fragments for 3C template (anchor + prey). Primers in red are reverse primers with respect to their cognate 3C primers. 3C + reverse primers amplify across the relevant restriction site, and these short amplicons were used with the digested and undigested control template to assess digestion efficiency. Control primers do not amplify across a restriction site; they generate a short amplicon from the ACTβ gene, which was used to normalize across different qPCR templates.

Anchor Primer
Globin_C 5'-CGGTCATCCTCACGGTGACTAACGCA-3'  Supplementary Figure 1: Results of the β-globin control 3C experiment. The y-axis shows fold-enrichment of the indicated fusion fragment relative to the control ACTβ primers, which were used to normalize across 3C templates. Error bars represent standard error among three replicates. These results agree with previously reported findings for this locus, [6] and indicate an effective 3C protocol.