Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases

1. Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases BIOS 6660 Hung-Chun (James) Yu Shaikh Lab, Department of Pediatrics, University of Colorado Denver Genetic Laboratories, Children’s Hospital of Colorado 11/17/2015

2. Human Genetic Diseases Mutation Penetrance Mutation Frequency Kaiser J. Science (2012) 338:1016-1017.

3. Human Genetic Diseases • Complex Disorder • Polygenic, many genes. • Low penetrance/effect size. • Multifactorial, environmental, dietary. • Examples: heart diseases, diabetes, obesity, autism, etc. • Mendelian Disorder • Monogenic (mostly). • Full or high penetrance/effect size. • Examples: sickle cell anemia (HBB) and cystic fibrosis (CFTR).

4. Complex Diseases • Multiple causes, and polygenic. • Multiple genetics factors with low penetrance individually. Coronary artery disease Coriell Institute for Medical Research. https://cpmc1.coriell.org/genetic-education/diagnosis-versus-increased-risk

5. Mendelian Diseases Veltman J.A. et al. Nat. Rev. Genet. (2012) 13:565-575.

6. Mendelian Diseases • Dominant Inheritance U.S. National Library of Medicine. http://ghr.nlm.nih.gov/

7. Mendelian Diseases • Recessive Inheritance U.S. National Library of Medicine. http://ghr.nlm.nih.gov/

8. Exome Sequencing • Focusing on exons or coding regions of genes Exons Complementary Baits Bamshad, MJ., et al. Nat. Rev. Genet. (2011) 12:745-755.

9. Exome Sequencing • 3,000,000,000bp (3Gb) human genome • ~45% repetitive sequence • ~25% genic region • ~2% exonic, coding region • 20,000 – 30,000 human genes • 3,000 – 5,000 disease genes • ~4,000 human genetic diseases (OMIM) • 114 medically actionable (treatable) genes Michael O. Dorschner., et al. Am J Hum Genet. 2013 93: 631–640.

10. Exome Sequencing • ~40Mb (coding) or 60Mb (coding + UTRs) Gene Read Coverage Individual Reads

11. Mendelian Diseases Identified by Exome Sequencing • Timeline Gilissen C. et al., Genome Biol. (2011) 12:228.

12. Mendelian Diseases Identified by Exome Sequencing Kym M. Boycott et al. Nature Reviews Genetics (2013) 14:681–691

13. Types of Variation • What kind of variation/mutation can be detected by Exome Sequencing? • SNV (single nucleotide variation) • Small InDel, (insertion/deletion <25bp) • Large InDel, CNV (copy number variation) • Possible, but not reliable. • Aneuploidy (loss/gain of entire chromosome) • Possible. • Translocation • Difficult and not reliable. • Complex rearrangement • Very difficult.

14. Exome Variants • SNV (single nucleotide variation) • Synonymous: (1) Silent. • Nonsynonymous: (1) Missense. (2) Nonsense. (3) Stop-loss. (4) Start-gain. (5) Start-loss. (6) Splice-site. http://upload.wikimedia.org/wikipedia/commons/6/69/Point_mutations-en.png http://www.web-books.com/MoBio/Free/Ch5A4.htm

15. Exome Variants • Small InDel (insertion/deletion <25bp) • Frameshift • In-frame NHGRI Digital Media Database (DMD), http://www.genome.gov/dmd/

16. Variant and Population Frequency • Novel/Private variant • Never been reported before. • Rare variant • Minor allele freq. (MAF) < 1%. • Databases • dbSNP (NCBI): http://www.ncbi.nlm.nih.gov/SNP/ • 1000 Genomes: http://www.1000genomes.org/ • ESP (NHLBI): http://evs.gs.washington.edu/EVS/ • ExAC: http://evs.gs.washington.edu/EVS/

17. Exome Variants • How to analyze enormous amount of variants in any given exome? Private/Novel ~100 - 300 Rare, MAF<1% ~500 - 2,000 Protein altering ~4,000 - 15,000 Coding/splice-site ~10,000 - 30,000 All ~20,000 - 200,000 GilissenC. et al. Eur. J. Hum. Genet. (2012) 20:490-497.

18. Exome Analysis Strategies Male Female Affected Heterozygous carrier Sex-linked heterozygous carrier Mating Consanguineous mating Sequenced individual GilissenC. et al., Eur. J. Hum. Genet. (2012) 20:490-497.

19. Trio-based Exome sequencing • Family trio • Both unaffected parents and an affected patient. • Why using trio? • Every inheritance model can be tested • Economical, efficient, single case required. • Access to samples.

20. Trio-based Exome sequencing • Autosomal recessive • Homozygous • Autosomal dominant • de novo * * * * * • Autosomal recessive • Compound heterozygous • X-linked recessive • Hemizygous in male Male Female Gene Affected * * Heterozygous carrier * Sex-linked heterozygous carrier X X Y X * * * Y X

21. Trio-based Exomesequencing • Candidate Genes/Variants • Rare (~500-2,000) or novel (~100-300) protein altering variants • Plus, variants that fit inheritance model

22. Case 1 • Clinical information Case 1 was the result of a non-consanguineous union and he presented to care at four months of age with a seizure disorder, hypotonia and developmental delay. The patient underwent a left parietal craniotomy and partial resection of the frontal cortex without complete resolution of the seizure disorder. Initial laboratory studies included an elevated homocysteineand methylmalonic acid and a normal vitamin B12 level. Complementation analysis of the patient’s cell line placed the patient into the cblC class. Severe developmental delay, infantile spasms, gyral cortical malformation, microcephaly, chorea, undescended testes, megacolon. Sequencing and deletion/duplication analysis (microarray) the MMACHC gene was negative in both skin fibroblasts and peripheral blood.

23. Case 1

24. Case 1 9News Colorado: Student joins first-grade class via web (May 15, 2011) http://archive.9news.com/news/local/article/198634/346/Student-joins-first-grade-class-via-web

25. Case 1 • Monster Max http://www.maxwatson.org/ • Patient's older sister as a summer student in Shaikh Lab

26. Case 2 • Clinical information The patient was a 7-month-old boy when first evaluated. He was diagnosed with BPES by a pediatric ophthalmologist. In addition to blepharophimosis, ptosis, and epicanthus inversus normally associated with BPES, he had cryptorchidism, right hydrocele, wide-spaced nipples, and slight 2–3 syndactylyof toes. Clinical testing demonstrated a normal karyotype (46,XY), and normal FISH studies for 22q11.2 deletion, Cri-du-Chat (5p deletion) syndrome. Thyroid function was normal. Further, normal 7-dehydrocholesterol level was used to rule out Smith–Lemli–Opitzsyndrome. Sanger sequencing and highresolutionCNV analysis with Affymetrix SNP 500K arrays did not identify a FOXL2 mutation.

27. Case 2 • A-D: 2-month old. note blepharophimosis, ptosis, epicanthus inversus (A), posteriorly angulated ears with thickened superior helix and prominent antihelix (B), and slight 2–3 syndactyly of toes in addition to overlapping toes (C, D) • E-F: 3.5-year old. Following oculoplastic surgery to correct ptosis; note right-sided preauricular ear pit (F, indicated by arrow). • G-I: 12-year old. Note the recurrence of ptosis (L>R), arched eyebrows, abnormal ears, thin upper lip vermilion, small pointed chin, downsloping shoulders, and wide-spaced and low-set nipples.

28. Case 3 • Clinical information The proband is a nine year old girl who presented with microcephaly, unilateral retinal coloboma, bilateral optic nerve hypoplasia, nystagmus, seizures, gastroesophagealreflux, and developmental delay including not yet saying specific words (at 29 months old). On exam, she has microcephaly with a normal height, a down-turned upper lip, and fingertip pads. A karyotype and CGH analysis have been normal. Kabuki (KMT2D and KDM6A) and Angelman (UBE3A and MECP2) syndromes were suspected in this patient.

29. Case 3

30. Exome NGS Workflow Exome Sequencing Mapping and variant detection Variant prioritization Genomic DNA Sequence read processing QC Annotation (General) Exome capture QC Library construction Mapping Annotation (In-house) Exome Enrichment SAM Illumina Sequencer Sequencing Filtering BAM Galaxy/FASTX Toolkit • Galaxy/BWA QC Inheritance test, candidate genes, ect. QC • Galaxy/Samtool Variant calling Galaxy

31. ExomeanalysisWorkflow (this class) FASTQ sequence 2x90bp (paired-end) Variant determination BCF Filter based on Phredscore, mapping quality, read depth, etc. Mapping to genome SAM Conversion Conversion VCF BAM QC: Filter duplicates, artifacts, and unpaired or unmapped reads, 100 genes “Mini” Exome ? BWA (Burrows-Wheeler Aligner) SAMtools

32. Data for Case Study • 3 trios • A total of 3 families/cases. • Each family/case includes both unaffected parents and an affected patient. • VCF files • Generated from 2x90bp paired-end Exome sequence reads, and at ~50X coverage • Readsmapped to human GRCh37/hg19and then familial variants calls made in VCF format • “Mini” Exome • 100 genes with/without known disease association. • Validated causative genes andrandomly selected disease genes or non-disease genes.

33. VCF Format • Variant Call Format http://www.1000genomes.org/wiki/Analysis/Variant%20Call%20Format/vcf-variant-call-format-version-41 ## Meta-information lines FILTER, INFO, FORMAT # Header line

34. VCF Format • FORMAT • GT: Genoetype. 0/0: Homozygous normal 0/1: Heterozygous variant 1/1: Homozygous variant • PL: the Phred-scaled genotype likelihoods (>0). 0/0 0/1 1/1 174 ,0 ,178 • GQ : Genotype quality (1-99)

35. Annotation Tools • Annotate variants with useful information • Mutation effect • Population frequency • Clinical association • Genomic sequence and protein domain • Pathogenicity prediction • Gene expression, protein interaction. • …..and many many more. • SeattleSeq:http://snp.gs.washington.edu/SeattleSeqAnnotation138/ • VEP (Variant effect Predictor): http://uswest.ensembl.org/info/docs/tools/vep/ • ANNOVAR: http://wannovar.usc.edu/

36. Question ?