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. 2011 Nov;32(11):1278-89.
doi: 10.1002/humu.21568. Epub 2011 Sep 16.

Genotype and Cardiovascular Phenotype Correlations With TBX1 in 1,022 velo-cardio-facial/DiGeorge/22q11.2 Deletion Syndrome Patients

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Genotype and Cardiovascular Phenotype Correlations With TBX1 in 1,022 velo-cardio-facial/DiGeorge/22q11.2 Deletion Syndrome Patients

Tingwei Guo et al. Hum Mutat. .
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Abstract

Haploinsufficiency of TBX1, encoding a T-box transcription factor, is largely responsible for the physical malformations in velo-cardio-facial /DiGeorge/22q11.2 deletion syndrome (22q11DS) patients. Cardiovascular malformations in these patients are highly variable, raising the question as to whether DNA variations in the TBX1 locus on the remaining allele of 22q11.2 could be responsible. To test this, a large sample size is needed. The TBX1 gene was sequenced in 360 consecutive 22q11DS patients. Rare and common variations were identified. We did not detect enrichment in rare SNP (single nucleotide polymorphism) number in those with or without a congenital heart defect. One exception was that there was increased number of very rare SNPs between those with normal heart anatomy compared to those with right-sided aortic arch or persistent truncus arteriosus, suggesting potentially protective roles in the SNPs for these phenotype-enrichment groups. Nine common SNPs (minor allele frequency, MAF > 0.05) were chosen and used to genotype the entire cohort of 1,022 22q11DS subjects. We did not find a correlation between common SNPs or haplotypes and cardiovascular phenotype. This work demonstrates that common DNA variations in TBX1 do not explain variable cardiovascular expression in 22q11DS patients, implicating existence of modifiers in other genes on 22q11.2 or elsewhere in the genome.

Figures

Figure 1
Figure 1
Cardiovascular phenotype enrichment groups. The stacked bar chart represents the total number of samples used for TBX1 resequencing (Cohort). The number in each group is also indicated above the bar chart (Cohort). Those with a significant intracardiac or aortic arch malformation are referred to as having a congenital heart defect (CHD). Total samples of isolated individual malformations were shown on the top of the figure (Isolate). For example, the total number of patients with atrial septal defects (ASDs) is 80. Among the 80, 16 had an ASD but no other defects (not shown in the table). The rest had ASD as well as other cardiovascular defects. TBX1 sequencing was performed on 48 ASD patients, while genotyping for common SNP variants was performed in all of them. The number 32 derives from subtracting 48 from 80. There were 385, 22q11DS subjects with VSDs (ventricular septal defects; 52 samples with VSD only). VSD occurs as part of tetralogy of Fallot (TOF) and persistent truncus arteriosus (PTA). Among those in the VSD enrichment group, 150 had a VSD that was not associated with TOF or PTA (52 samples had VSD but no other intracardiac defect). A separate designation was made for those with a VSD but not in association with TOF or PTA (VSD2). There were 150 with VSD as the only intracardiac defect. A total of 181 patients had TOF; 75 were subjected to TBX1 resequencing and all were genotyped. Sixty 22q11DS patients had TOF but no aortic arch defect or ASD (not shown in the table). Pulmonic stenosis (PS) occurs in association with TOF and it can co-occur with a VSD. In some of the echocardiogram summaries, PS and VSD were indicated but TOF wasn’t. In those situations, we did not assume the patient had a TOF. A total of 124 comprised the PS enrichment group (pulmonic atresia or stenosis). Sixty were subjected to TBX1 resequencing and all were genotyped. Among those with PS, only 4 had this malformation and no other intracardiac or aortic arch anomaly. Only 28 of 55 had a PTA but no other anomaly. Fifty-four patients had an interrupted aortic arch type B (IAAB); 34 were sequenced and all were genotyped. Only 4 total had an IAAB but no other intracardiac or aortic arch defect. A total of 137 had a right-sided aortic arch (RAA) and 77 were subjected to TBX1 resequencing, while all were genotyped. Thirty-three patients had RAA as the only cardiovascular defect. In total, 664 patients had an intracardiac and/or aortic arch malformation (CHD; cases) and were genotyped while 231 were subjected to TBX1 resequencing. A total of 358 patients had no detectable CHD (controls), and 112 were sequenced, while all were genotyped. Defects such as small patent foramen ovale or abnormal branching of the subclavian arteries were not always detected or noted and were not included in the analysis.
Figure 2
Figure 2
DNA variations in TBX1 exons. Three TBX1 isoforms (TBX1 isoform B, top, 18,124,226-18,151,112; TBX1 isoform A, bottom, 18,124,226-18,147,068; TBX1 isoform C, middle, 18,124,226-18,134,855 on human genome hg18) were shown on the “RefSeq Genes” track in the UCSC Genome Browser. Custom track “Variations in TBX1 exons” was added to visualize the DNA variations found in the exons from DNA sequence analysis of 360 22q11DS patients. Each bar represents a variation, color coded as red, green and blue to represent non-synonymous, synonymous and UTR variations, respectively.
Figure 3
Figure 3
Comparison of frequency SNPs to public databases. We performed a comparison of data on the MAF (Minor allele frequency) of SNPs between the 22q11DS study and 1,000 Genome Project (http://www.1000genomes.org/;) (Durbin, et al., 2010). Three custom tracks are shown in addition to the RefSeq track in a snapshot of the UCSC Genome Browser (hg18). The “1,000Genome” track shows the MAFs of all DNA variations identified in the TBX1 locus. The data were extracted from pilot data (release 2010_07) from the 1,000 Genome Project. The next two tracks, entitled “1,000 Genome-2″ and “ 22q11DS” track show the MAFs of DNA variants indentified in the same regions of TBX1 that were sequenced in our cohort. The height of the lines representing each variant depicts the MAF, which turn out to be similar in both datasets. Detailed information on DNA variations identified by TBX1 resequencing is in Supp. Table S2.
Figure 4
Figure 4
(A) Enrichment of very rare DNA variations. “Control”, “ CHDs” (Congenital heart defects=CTDs+ASD), “RAA” and “PTA” custom tracks were added to the UCSC Genome Browser as shown. The custom tracks indicate the type of rare variations (MAF<0.01) identified by TBX1 resequencing analysis in each phenotype enrichment group (total number of subjects and definition of each phenotype group is in Figure 1). Each line or bar represents a different rare SNP. The orange vertical lines represent variations from the putative promoter region, black represents intronic variants and blue are variants from the 3′-UTR. The red bar represents a nonsynonymous variant and a green bar denotes a synonymous variation. Detailed information of rare variations used for DNA enrichment analysis is shown in Supp. Table S2. (B) Multiple sequence alignment of 45 very rare TBX1 variants (MAF<0.01). Multiple alignment data from 44 vertebrate species generated using multiz and other tools in the UCSC/Penn State Bioinformatics comparative genomics alignment pipeline were extracted using Galaxy (http://main.g2.bx.psu.edu/), a web-based genome analysis tool (Blankenberg, et al., 2010; Goecks, et al., 2010). Both Galaxy and then Jalview (Waterhouse, et al., 2009) were used to edit alignments. The species in the database with significant missing sequence were discarded and only results from 24 mammals are shown. The first two rows indicate the major or minor allele identified in the 22q11DS patient cohort. Each row below represents one of the 24 mammalian species. In general, each column represents a rare DNA variation except for the 8th variation that has a three base and 24th that has a four base indel. The most intense blue color corresponds to the most conserved SNPs. A total of 30 major alleles from the 22q11DS cohort show evolutionary conservation. Five minor alleles show conservation as indicated. The minor allele (TCGC), the only novel variation (novel 23) found in the sample, was conserved among most mammalian species. Detailed information on the 45 rare variations is shown in Supp. Table S2.
Figure 5
Figure 5
Linkage disequilibrium (LD) plot of 17 common SNPs from the TBX1 resequencing data. An LD matrix was generated from DNA sequence data, using the X-chromosome model in Haploview 4.2. Seventeen common SNPs with a MAF >0.05 were used to generate the plot. The SNPs highlighted in green are those selected to genotype in the whole cohort of 1,022 patients. Standard (D′/LOD) models were used to create the LD color scheme. The value in each pairwise box is the r2 value. We found that there were three LD blocks in the TBX1 locus.
Figure 6
Figure 6
Haplotypes of common SNPs (MAF>0.05) and 54 rare variants (MAF<0.05). A total of 54 rare variants (MAF<0.05) identified by TBX1 resequencing were shown in each column (the order of variants is according to Supp. Table S2). Each row represents a patient that carries one or more of the 54 rare variants (the major allele is a black dot; the minor allele is indicted in bold red font). Each haplotype group is indicated as Hap1–21 (Hap22–49 which didn’t occur in any patient with rare variations, is not shown.). They were determined by the allele pattern of the 17 common SNPs. Some patients shared common haplotypes. Twenty-one of 49 haplotypes captured all rare variations (MAF<0.05) in a total of 77 subjects. Hap1 (CAGGGTCTCAAAACGCA), Hap2 (CTCGGTCTCAAAACGCA) and Hap3 (TTCGACCCCGGACCTCG) were common haplotypes (frequency>0.05) generated by 17 common SNPs from TBX1 resequencing data. Hap4 to Hap7 were less common haplotypes (0.01<frequency<0.05) and hap8 to hap21 were very rare haplotypes (frequency<0.01). For each variant, the boxes highlighted in different colors represent the minor allele of rare variants observed in the subjects with the given haplotypes indicated (Hap1–21).

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