Fang, Han

Lyon,  Gholson J.

Mittelman, David

O'Rawe, Jason

Wu, Yiyang

Cold Spring Harbor Laboratory Institutional Repository

	Whole genome sequencing analysis of a family with familial dysautonomia and neuropsychiatric symptoms	Han Fang1,2, Yiyang Wu1,2, Jason A. O’Rawe1,2, David Mittelman4,5, Gholson J. Lyon1,2,3*	Background 	We report here our discovery and whole genome sequencing (WGS)  analysis  of  a  family  with  dominantly  inherited familial  dysautonomia  (Figure  1).  The  mother  is  affected with  dysautonomia,  hereditary  hemochromatosis,  and obsessive  compulsive  traits.  The  oldest  daughter  reports severe  dysautonomic  syncopal  episodes,  gastroparesis, glaucoma,  visual  and  auditory  hallucinations,  urinary retention,  and  one  prior  stroke.  One  son  is  affected  with dysautonomia,  Tourette  syndrome  (TS),  attention  deficit disorder (ADD), and obsessive-compulsive disorder (OCD). Another  son  reports  dysautonomia,  asthma,  seizure  in response to pertussis, dyslexia, migraine, dysgraphia, ADD, sensory integration disorder, and arthritis. 	1Stanley Institute for Cognitive Genomics, One Bungtown Road, Cold Spring Harbor Laboratory, NY, USA; 2Stony Brook University, 100 Nicolls Rd, Stony Brook, NY, USA; 3Utah Foundation for Biomedical Research, E 3300 S, Salt Lake City, Salt Lake City, UT, USA; 4Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061, USA, 5Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.	Methods 	WGS was performed on two affected siblings in the Illumina CLIA-certified lab. Three other DNA samples from the family also underwent WGS in the research setting at CSHL. DNA samples from the same five people were genotyped using the Illumina HumanOmni2.5-8 BeadChip. Genome-wide parametric linkage analysis was performed using Merlin (adjusted for linkage disequilibrium). Our group previously reported the low concordance rates across different variant calling pipelines. Thus, to reduce algorithm-induced biases, we used multiple pipelines for quality control,  alignment,  assembly,  variant  calling,  genotype  refinement, variant  filtering,  and  variant  annotation  (Figure  2).  The  Empowered Genome Cohort data,   implemented in the Ingenuity variant analysis system,  was  also  used  to  filter  out  WGS-specific  sequencing  errors, which might have been identified as rare variants. 	Results  	Linkage  analysis  showed  not  enough  power  for  identifying  any  disease relevant variant loci in a single family. None of the family members carry any previously reported variants in IKBKAP that have been implicated in the autosomal recessive transmission of familial dysautonomia. The WGS data had good sequence coverage for IKBKAP in all five people (mean coverage ~40x) but we did not identify any novel rare variants in this gene. ERDS and PennCNV did not reveal any CNVs that of relevance to the phenotype of familial dysautonomia. We have generated a list of rare variants of unknown significance (Table 1 & 2). Sanger sequencing results confirmed an insertion in  POU4F1  (Figure  4).  We  find  relatively  low  concordance  across  three variant detection pipelines (Figure 5). Pharmacogenomic analyses reveal the recommended dosages of Coumadin and Simvastatin (Table 3). Five variants in three people with prior clinical evidence are also shown (Table 4).	Conclusions	Here  we  show one  example  of  individualized  medicine  in  a  family  with neuropsychiatric symptoms and at least two rare diseases. WGS data provides much more clinically relevant and potentially actionable information when compared to genotyping arrays. Despite limited overall agreement between CLIA certified and non-CLIA certified analysis pipelines, utilizing variants identified by two or more pipelines enables WGS to act as a useful technique for clinical diagnostics. For rare diseases, we argue that family history and other  family  members’  sequencing  data  should  be  incorporated  into downstream analyses so as  to  eliminate false positive findings of  disease-associated variants. We highlight the importance of detailed phenotyping and sharing  of  both  genomic  and  phenotyping  data  because  of  extreme heterogeneity  across  families  and  insufficient  knowledge  of  rare  diseases. Ongoing effort will focus on identifying and proving familial dysautonomia relevant  variants,  extending  the  pharmacogenomic  analyses  to  the  other people in the pedigree, and including more family members in our study.	Questions? Email: GholsonJLyon@gmail.com	Figure 2. The left-hand side is the major analysis work flow while the right-hand side are the details of each procedure.* CADD (Combined Annotation Dependent Depletion, in press) : http://cadd.gs.washington.edu/ 	* Scalpel (in preparation) : Haplotype Micro-assembly for accurate INDEL detection: http://schatzlab.cshl.edu/	Familial dysautonomia		Hemochromatosis		OCD		OCD Subclinical traits		TS = Yes		Migraine headaches = Significant		ADD = Yes	Figure 1. The pedigree in this study. The numbers below the children are their corresponding ages. Five people in the family have thus far been included in our WGS study: the parents, the 24-year-old daughter, the 17-year-old son, and the 15-year-old daughter.	 24            21              19              17              15	2206932 (50.6%)  2618(0.06%)297382 (6.8%)1094243 (25.1%)53922(1.2%)124166  (2.8%)578728 (13.3%)NovoAlign - FreeBayesELAND - CASAVABWA.mem - GATK356571(40.6%)20195 (2.3%)35872 (4.1%)227397 (25.9%)43661(5.0%)81128(9.2%)112980 (12.9%)NovoAlign - FreeBayesELAND - CASAVABWA.mem - GATKFigure 5. Venn diagrams of the variant calling results across three independent alignment and assembly combinations. 2x100 paired end reads for the WGS data were generated on Illumina HiSeq2000 platform. Reads were mapped to the hg19 reference genome. BWA-0.7.5a, GATK v.2.6-5, ELANDv2e, and CASAVA 1.8 were used here. (A) The Venn diagram is for SNP detection comparison. We noticed an even lower concordance rate compared to what we previously reported (57.4% across 5 pipelines). This is because we here use three different alignment steps. (B) The Venn diagram is for INDEL detection, the concordance rate is even lower: 40.6%. We focus on variants called by at least two pipelines, i.e. the central region surrounded by the red dash lines. This allows us to analyze 82.6% of the SNPs and 72.9% of the INDELs in our downstream analyses. 	(A)	 (B)	Table 3. Recommended dosages for Coumadin and Simvastatin dosages based on the oldest daughter's WGS results, in comparison to what she was actually prescribed in the absence of any genetic testing. Pharmacogenomics analyses were performed based on guidelines and algorithms from the International Warfarin Pharmarcogenomics Consortium (IWPC) and the Clinical Pharmacogenomics Implementation Consortium (CPIC) in the PharmGKB database. People who are homozygous for major alleles at both sites in CYP2C9 are designated as *1/*1. 	Table 1. A subset list of rare variants (MAF < 1%) found in at least two affected people. These variants are called by at least two pipelines, within coding regions, with a high VAAST score, and a CADD c-score greater than 20. 	GATK FreeBayes CASAVABWA-MEM NovoAlign ELANDScalpel*Whole Genome Sequencing – 40x mean Coverage 2.5 M Genotyping ArrayERDS PennCNVGenome StudioSNPs+INDELsINDELsSNPs+INDELsSNPs+INDELsCNVs SNPs CNVsSequencing + GenotypingWGS QC+AlignmentAssembly+Variant CallingRaw VariantsPoolHigh Quality VariantsReady-to-UseVAAST 2.0 CADD* Ingenuity ANNOVAR Omicia OpalMiSeqResequencing (To do)Sanger LobSTRRepeatSeqGenotypeRefinementVariantFilteringVariantAnnotationIngenuityVariant Analysis PipelineOmicia Opal PharmGKBDiagnosisSupportTable 2. The insertion in POU4F1 is found in two children with familial dysautonomia. There is no coverage in the mother’s WGS data. Follow-up Sanger sequencing confirmed the existence of the homozygous insertion in all three people : affected mother, affected daughter, and affected son. POU4F1 encodes a member of the POU-IV class of neural transcription factors. This protein is expressed in a subset of retinal ganglion cells and may be involved in the developing sensory nervous system.	Table 4. At least seven mutations with substantial clinical relevance were found in our study. The “Daughter” means the oldest daughter, and the “Son” means  the 17-year-old son. “hom” = homozygous and “het” = heterozygous. “WT” = wild type. The mother is homozygous for the C282Y variant in HFE, and, consistent with the lack of symptoms for hemochromatosis in the children, we found that all three children are carriers for the C282Y variant, but not carrying any other reported hemochromatosis related variants. 	Figure 4. Sanger sequencing results confirmed the homozygous insertion in exon 2 of POU4F1. Sanger sequencing results of (A) the mother, (B) the oldest daughter, and (C) the 17-year-old son.	 G    G    C    G    G    C   G    G    C    C    C    C    G    G    G                 Insertion	Unassembled Samples: Traces for 1 sampleNovember 11, 2013 10:55:53 AM ESTPage 1 of 2yiwu-E5-F-formG G G G G G C G G T G G C G G C C220C G G G C G G C G G230C G G C G G C C C G240G G C G G C G G C G250G C C C C G G G G G260A G G CG    G    C    G    G    C                        C    C    C    G    G    G                    WT	Unassembled Samples: Traces for 2 samplesNovember 11, 2013 11:23:25 AM ESTPage 1 of 2yiwu-E2-F_1G C G G C C C G G G C G220G C G G C G G C G G230C C C G G G C G G C240G G C G G C C C C G250G G G G A G G C G G260C G G T G G C G Gyiwu-E1-FT G G C G G C C C220G G G C G G C G G C230G G C G G C C C G G240G C G G C G G C G G250C C C C G G G G G A260G G C G G C G G T G270GUnassembled Samples: Traces for 2 samplesNovember 11, 2013 11:23:25 AM ESTPage 1 of 2yiwu-E2-F_1G C G G C C C G G G C G220G C G G C G G C G G230C C C G G G C G G C240G G C G G C C C C G250G G G G A G G C G G260C G G T G G C G Gyiwu-E1-FT G G C G G C C C220G G G C G G C G G C230G G C G G C C C G G240G C G G C G G C G G250C C C C G G G G G A260G G C G G C G G T G270GThe mother	The oldest daughter	The 17 year-old son	(A)	(B)	(C)	Gly              Gly              Pro                                         Amino Acids	Gene Chr Position Region Transcript Variant Protein Variant Inferred ActivityPOU4F1 13 79176323 Exonic c.484_486dipGGC p.162_163_ins LossDrug Recommend dosages based on genotypesPrevious prescriptionsFDA recommendationsGenotypesCoumadin (Warfarin Sodium)5.85 mg/day 5 mg/day2 to 10 mg dailyConsider genetic testing resultsVKORC1: A/G(rs9923231)CYP2C9: *1/*1(rs1799853, rs1057910)Simvastatin20 mg/dayIncreased risk of myopathy with 40 mg Simvastatin20 mg/day 80 mg/day SLCO1B1: T/C(rs4149056)GenenameGenomiccoordinatesAmino AcidchangeZygosityEffectQualityCoveragePopulationfrequencyEPS15 chr1:51829571 Gly>ArgHeterozygousnon-synon19747:24:23 UnknownMYL7 chr7: 44179406 Gly>SerHeterozygousnon-synon7123:16:7 0.69%ERI1 chr8: 8865561 Pro>SerHeterozygousnon-synon15649:30:19 UnknownCCIN chr9: 36170317 Arg>GlnHeterozygousnon-synon22336:14:22 0.14%PCTP chr17: 53844742 Cys>TyrHeterozygousnon-synon22347:22:25 0.98%

Whole genome sequencing analysis of a family with familial dysautonomia and neuropsychiatric symptoms

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