Fang, Han

Lyon,  Gholson J.

Mittelman, David

O'Rawe, Jason

Wu, Yiyang

Cold Spring Harbor Laboratory Institutional Repository

Uncovering	  gene+c	  components	  	  of	  a	  previously	  un-­‐described	  syndrome	  	  Jason	  O’Rawe1,	  Yiyang	  Wu1,	  David	  Mi8elman2,	  Han	  Fang1,	  Gholson	  J.	  Lyon1,3	  	  	  1Cold	  Spring	  Harbor	  Laboratory,	  Human	  Gene8cs,	  Cold	  Spring	  Harbor,	  NY,	  11724,	  2Virginia	  Tech,	  Department	  of	  Biological	  Sciences,	  Blacksburg,	  VA,	  24061,	  3Utah	  Founda8on	  for	  Biomedical	  Research,	  UFBR,	  Salt	  Lake	  City,	  UT,	  84106	  Abstract		Background:  We describe a whole genome sequencing study of  one family containing two affected male children,  aged 10 and 12,  with severe intellectual disability, autism-like behavior, and very distinctive facial  features.  High  accuracy  is  of  paramount  importance  in  the context of detecting or discovering the genetic influencers of human disease, yet each sequencing and analysis pipeline is still imperfect.  In this study, we leverage data from two sequencing platforms and many data processing and downstream analysis pipelines to more confidently identify variants that may play an influential role in the disease state of these two children.	Presentation of the phenotype		The  propositi  are  two  affected  male  brothers  (Fig.  1),  aged  10  and  12 respectively,  with  severe  intellectual  disability,  autism-like  behavior, attention  deficit  issues,  and  very  distinctive  facial  features  (Fig.  1), including  broad,  upturned  nose,  sagging  cheeks,  downward  sloping palpebral fissures, relative hypertelorism, high-arched palate, and prominent ears.  Their  parents are nonconsanguineous and are both healthy,  and the family history does not demonstrate any members with anything resembling this current syndrome.	Fig. 1  Facial phenotype of the younger brother at age 19 months (A), 3.5 years (a) and 7 years (a) ; and the elder brother at age 3 years (B), 5 years  (b)  and  9  years  (b).   A  pedigree  displaying  intra  familial relationships.		Complete Genomics WGS		Whole  genomes  of  the  mother,  father  and both  affected  boys  were  sequenced  and analyzed  with  the  Complete  Genomics WGS  sequencing  and  bioinformatics pipeline.  Reads  were  mapped  to  the Genome  Reference  Consortium  assembly GRCh37.  Due  to  the  proprietary  data formats,  all  the  sequencing  data  QC, alignment  and  variant  calling  were performed by CG as part of their sequencing service,  using  their  version  2.0  pipeline. Complete Genomics WGS was optimized to cover 90% of the exome with 20 or more reads  and 85% of  the  genome with  20 or more reads. 	Illumina WGS		Whole  genomes  of  the  entire  pedigree were  sequenced  using  the  Illumina HiSeq2000.  WGS  covered  >90%  of  the genome with 30 reads or more and with >80% of the bases having a quality score of >30. Illumina reads were mapped to the hg19  reference  genome  using  BWA  v. 0.6.2-r126,  and  variant  detection  was performed using the GATK v.  2.4-9.   A second  analytical  pipeline  was  used  to map reads to the hg19 reference genome using  Novoalign,  and  variants  were detected using the FreeBayes caller. 	Methods: Whole genome sequencing (WGS) was performed on ten members of this family using the Illumina HiSeq2000 platform, with four (the two affected boys  and  their  parents)  being  additionally  sequenced  using  the  Complete Genomics (CG) WGS platform. CG data analysis was performed by CG, using their version 2.0 pipeline. Illumina reads were mapped to the hg19 reference genome using BWA v.0.6.2-r126, and variant detection was performed using the GATK v. 2.4-9. A second analytical pipeline was used to map the Illumina reads and detect variants using Novoalign and the FreeBayes caller. Disease variant  discovery  procedures  included  traditional  filtering  techniques  (using ANNOVAR  and  Golden  Helix  SVS),  and  a  statistical  framework  for identifying the likely disease causing variants (using VAAST). 	Results: CG WGS covered >85% of the genome and >90% of the exome, both with 20 or more reads. Illumina WGS covered >90% of the genome with 30 reads or more and with >80% of the bases having a quality score of >30. We found a ~2 to 5-fold difference in the number of variants detected as being relevant for various disease models when using different sets of sequencing data and analysis pipelines. In one instance, employing a ‘quad’ study design reliably identified three putative variants that followed an X-linked disease model, in TAF1, ZNF41 and ASB12 respectively.  However, ZNF41 and ASB12 variants were subsequently found to be false positive findings  when  the  study  expanded  to  include  more  family  members  and more data from each. 	Bioinformatics analysis		Bioinformatics  analyses  of  multiple  disease  model  pathways  were  performed in order  to  prioritize  and  identify  any  putative  mutations  that  might  aid  in  better understanding the pathogenesis of the described syndrome. We performed analyses to interrogate variants conforming to a de-novo, autosomal recessive and x-linked model of disease transmission. 		We used several methods to prioritize and identify potentially disease contributing germ-line  mutations,  including  VAAST,  Golden  Helix  SVS,  ANNOVAR  and CADD,  a  integrative  tool  for  scoring  single  nucleotide  variants  and  insertion/deletions.  VAAST employs a likelihood-based statistical framework for identifying the most likely disease contributing variants given genomic makeup and population specific  genomic  information.    SVS  and  ANNOVAR  employ  more  traditional filtering techniques that leverage data stored in public genomic databases.	Conclusions: Using multiple sequencing and bioinformatics pipelines provides greater power in reducing false positive findings in the context of WGS studies - biological conclusions can shift between sequencing smaller to larger portions of a family.	Using information from a greater portion of the family structure: 17726 Variants were found to be de-novo in the two affected boys  40 were coding : 32 non-synonymous missense 3 splicing  2 frame-shift deletions 1 stop-loss 1 frame-shift insertion 1 frame-shift substitution 2824 Variants were found to conform to an X-linked disease model 4 were coding: 3 non-synonymous missense 1 splicing Using only nuclear family: 55195 Variants were found to be de-novo in the two affected boys  122 were coding : 107 non-synonymous missense 4 splicing  3 frame-shift deletions 3 frame-shift insertions 2 frame-shift substitutions 2 stop-gain  1 stop-loss 26514 Variants were found to conform to an X-linked disease model 28 were coding: 27 non-synonymous missense 1 splicing 

Uncovering genetic components of a previously un-described syndrome

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