Lyon, Gholson J.

Robison, Reid Justin

English

The University of Utah: J. Willard Marriott Digital Library

Table 1. Summary of SNPs for exome capture samplesReid Robison,1,10 Gholson J. Lyon,1,2,8,10 Tao Jiang,3,4,10 Richard Van Wijk,6 Lin Yang,3 Peng Zhang,3 Renhua Wu,3 Ying Liu,3,4 Xu Yang,3 Jinchuan Xing,5 Barry Moore,5 Joseph Glessner,8 Fred Reimherr,1 Wouter van Solinge,6 Josephine Elia,8 Mark Yandell,5 William McMahon,1 Evan Johnson,9 Mark Clement,9 Hakon Hakonarson,8 Jun Wang,3,4,7 Kai Wang,8,111. Department of Psychiatry, 30 North 1900 East, University of Utah, Salt Lake City, UT 841322. NYU Child Study Center, New York, NY 100163. BGI-Shenzhen 518083, China4. Genome Research Institute, Shenzhen University Medical School, Shenzhen 518060, China5. Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 841326. Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Netherlands7. Department of Biology, University of Copenhagen, Copenhagen, Denmark8. Center for Applied Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA 191049. Department of Statistics, Brigham Young University, Provo, UT 8460210. The first three authors should be regarded as joint first authors.11. Present address: Zilkha Neurogenetic Institute, Department of Psychiatry and Preventive Medicine, University of Southern California, Los Angeles, CA 90089AcknowledgementsWe thank Matthew Bainbridge and Richard Gibbs at Baylor for access to unpublished exome data. This work was funded by University of Utah Department of Psychiatry funds to G.L., R.R. and F.R.  B.M. & M.Y. were supported by NHGRI 1RC2HG005619. K.W. and H.H. were funded by a Pilot/Methodological Study Award from NIH/NCRR GrantUL1 RR025774.Whole exome sequencing was conducted on a father and two sons with severe ADHD, combined hyperactive and inattentive subtype, all of whom improved with treatment with stimulant medication in an adult ADHD clinical trial. Exome capture was done using a commercially available Agilent SureSelect in solution method. Paired end sequencing was performed using the Illumina Genome Analyzer IIx platform with read lengths of 76 base pairs, providing at least 20x-coverage. Sequence reads were aligned to human reference genome builds hg18 and hg19 using both Novoalign 2.07 and SOAPaligner 2.20. Single nucleotide polymorphism (SNP) calling was conducted using both Maq 0.5.0 and SOAPsnp 1.03. SNP results were filtered as followed: Base quality > 20, depth from 4-200, copy number estimate < 2, and distance between two adjacent SNPs no less than 5. Nonsynonymous SNPs (nsSNPs) were identified using ANNOVAR and USeq Alleler, and insertions/deletions (indels) were detected using SOAPindel. Illumina 610k SNP microarrays were also run on each of the three samples, with copy number variant (CNV) detection using PennCNV.ADHD is a common disorder affecting more than 1 in 20 children in the U.S., with as many as 50% remaining symptomatic into adulthood. Genetic factors are thought to play a large role in the etiology of the disorder, but studies thus far have been inconsistent, accounting for a small portion of the risk for ADHD. It is hypothesized that rare, family-specific genetic variants may account for the remaining missing heritability of ADHD, and advances in next-generation sequencing have made whole exome sequencing a potentially viable option for gene variant finding in complex disease pedigrees. Exome sequencing has identified the causes of nearly a dozen Mendelian disorders. Whether exome sequencing can be used to find risk variants for complex traits and has clinical utility as a diagnostic tool remain to be explored. Here we examined the utility of exome sequencing to identify candidate genes for attention deficit/hyperactivity disorder (ADHD) in a Utah pedigree in which ADHD clustered in an apparently Mendelian pattern.Whole Exome Sequencing Reveals the Genetic Basis of a Case of Idiopathic Hemolytic Anemia and Suggests Candidate Rare Variants for ADHD in a Utah PedigreePreliminary results show four interesting candidate rarenonsynonymous variants in brain-expressed genes that are sharedamong 3 individuals (father and two sons) and thus may beassociated with ADHD in this family. Validation of these findings isunderway in each individual, along with expansion of the pedigree toinclude DNA sequence and phenotype data from the unaffectedmother and other siblings, along with functional characterization ofthe nsSNPs and PKLR mutations. These preliminary results showthat exome sequencing can identify genetic causes of mendeliandisorders, as in the case of pyruvate kinase deficiency in onemember of this family, and also has potential to discover rarevariants associated with complex neuropsychiatric conditions suchas ADHD.Collectively, our results identify new candidate genes for ADHD, and suggest that exome sequencing may be an efficient strategy for solving diagnostic unknowns.Analysis and filtering of shared novel variants among three family members with ADHD identified 29 candidate variants. Comparison to variant data from ~6,300 exomes revealed that 12 of these variants have minor allele frequency >0.2%.  In addition, one family member, who had undergone a splenectomy for idiopathic hemolytic anemia, was found to have two rare non-synonymous mutations in PKLR, consistent with a diagnosis of red blood cell pyruvate kinase deficiency.  This finding was confirmed by functional biochemical testing the majority of the SNPs were validated by Sanger sequencing, and validation of the indels is underway.Table 3. 29 candidate variants for ADHD and their population frequencies in other cohortsExomeCapture 84060 84615 92157Number of genomic positions for calling SNPs 52,105,343 52,105,343 52,105,343Number of high-confidence genotypes* 46,651,026 45,555,039 46,393,954Number of high-confidence genotypes in target regions 35,571,754 34,856,090 35,742,301Number of known SNPs in target region 192,415 192,415 192,415Coverage of known SNPs 148,795(77.33%)142,933(74.28%)148,396(77.12%)Total number of novel SNPs 25,523 24,470 25,321Total number of novel indels 335 325 335# chrom Position in HG18 Reference alleleMutant allele Gene Type of MutationAmino acid change# variants in BGI exomes2% in BGI exomes# variants in ~600 Baylor exomes% in Baylor exomeschr17 64384287 A G ABCA8 Nonsynonymous C1387R 0 0.00% 0 0.00%chr11 68323378 G A CPT1A Nonsynonymous L193F 0 0.00% 0 0.00%chr8 101063450 A G RGS22 Nonsynonymous I1084T 0 0.00% 0 0.00%chr18 59805227 G T SERPINB8 Nonsynonymous G287V 0 0.00% 0 0.00%chr1 205267500 - T C1orf116 frameshift insertion 34 1.40% 0 0.00%chr17 42571111 - TGGC CDC27 frameshift insertion 5 0.20% 0 0.00%chr17 42589406 A - CDC27 frameshift deletion 31 1.30% 0 0.00%chr1 109266693 TTC - GPSM2 amino acid deletion 9 0.40% 0 0.00%chr9 8577604 AT - HNRNPK frameshift deletion 37 1.60% 0 0.00%chr11 5432010 - CA OR51I2 frameshift insertion 15 0.60% 0 0.00%chr17 36038691 C - SMARCE1 frameshift deletion 19 0.80% 0 0.00%chr7 98839005 C - PDAP1 frameshift deletion 37 1.60% 1 0.20%chr18 27355154 T G DSG2 Nonsynonymous V158G 1 ~0.0% 1 0.20%chr3 127359980 G A ALDH1L1 Nonsynonymous P107L 2 ~0.0% 0 0.00%chr13 51440681 A G ATP7B Nonsynonymous V536A 1 ~0.0% 1 0.20%chr10 53128652 A C CSTF2T Nonsynonymous C222G 4 0.10% 1 0.20%chr14 21041859 G A METTL3 Nonsynonymous R36W 9 0.20% 1 0.20%chr11 76632438 - A GDPD4 frameshift insertion 36 1.50% 6 1.00%chr7 86998554 A T ABCB1 Nonsynonymous S893T 815 14.3%1 9 1.50%chr11 133634133 C G ACAD8 Nonsynonymous S171C 112 2.00% 20 3.30%chr20 17904347 C T C20orf72 Nonsynonymous R178W 23 0.40% 8 1.30%chr8 33438433 T C FUT10 Nonsynonymous Q27R 15 0.30% 3 0.50%chr13 19695025 A T GJB6 Nonsynonymous S199T 68 1.20% 4 0.70%chr16 69572830 G T HYDIN Nonsynonymous P1491H 77 1.40% dozens >5.0%chr10 22059861 G A MLLT10 Nonsynonymous R713H 15 0.30% 6 1.00%chr17 10355994 A G MYH1 Nonsynonymous Y435H 99 1.70% 14 2.30%chr1 143727234 G T PDE4DIP Nonsynonymous L142I 1256 22.10% hundreds >30.0%chr2 98175864 T C VWA3B Nonsynonymous I513T 15 0.30% 16 2.70%chr5 115230317 AAGA - AP3S1 frameshift deletion 185 7.80% 19 3.20%IntroductionPatient 84060 ControlReference valuesPK (U/gHb) 3.3  L 8.6 6.1 – 12.3HK (U/gHb) 3.2  H 1.1 0.8 – 1.5G6PD (U/gHb) 15.8  H 9.2 6.4 – 10.5Table 2. Biochemical assays of enzyme activities in the patient affected with idiopathic hemolytic anemia confirmed PKLR deficiency. PK, pyruvate kinase; HK, hexokinase; G6PD, glucose-6-phosphate dehydrogenase.MethodsResultsDiscussionConclusionAuthor AffiliationsFigure 1. Identification of family-specific ADHD candidate genes by ANNOVAR stepwise reduction procedure, based on shared variants among 3 patients and assuming a dominant disease model. Figure 2: Pedigree of a Utah family affected with idiopathic hemolytic anemia and ADHD. 

Whole Exome Sequencing Reveals the Genetic Basis of a Case of Idiopathic Hemolytic Anemia and Suggests Candidate Rare Variants for ADHD in a Utah Pedigree

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Whole Exome Sequencing Reveals the Genetic Basis of a Case of Idiopathic Hemolytic Anemia and Suggests Candidate Rare Variants for ADHD in a Utah Pedigree

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