Exome Sequencing as Molecular Diagnostic Tool of Mendelian

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

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

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).

Complex Diseases �Multiple causes, and polygenic. �Multiple genetics factors with low penetrance individually. Coronary artery disease Coriell Institute for Medical Research. https: //cpmc 1. coriell. org/genetic-education/diagnosis-versus-increased-risk

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

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

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

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

Exome Sequencing � 3, 000, 000 bp (3 Gb) 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.

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

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

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

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

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/wikipedi a/commons/6/69/Point_mutationsen. png http: //www. webbooks. com/Mo. Bio/Free/Ch 5 A 4. htm

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

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

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

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

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.

Trio-based Exome sequencing � Autosomal dominant � Autosomal recessive ü de novo ü Homozygous * * ** * � Autosomal Gene Male Female ü recessive Compound heterozygous * Sex-linked heterozygous carrier ü recessive Hemizygous in male * * Affected Heterozygous carrier � X-linked ** XY XX

Trio-based Exome sequencing �Candidate Genes/Variants Rare (~500 -2, 000) or novel (~100 -300) protein altering variants • Plus, variants that fit inheritance model • Dominant Recessive Rare Variant Novel Variant de novo 0~2 Compound Heterozygous 0 ~ 20 0~3 Homozygous 0 ~ 20 0~3 X-linked 0 ~ 10 0~5

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 homocysteine and methylmalonic acid and a normal vitamin B 12 level. Complementation analysis of the patient’s cell line placed the patient into the cbl. C 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.

Case 1

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

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

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 syndactyly of toes. Clinical testing demonstrated a normal karyotype (46, XY), and normal FISH studies for 22 q 11. 2 deletion, Cri-du-Chat (5 p deletion) syndrome. Thyroid function was normal. Further, normal 7 -dehydrocholesterol level was used to rule out Smith–Lemli–Opitz syndrome. Sanger sequencing and highresolution CNV analysis with Affymetrix SNP 500 K arrays did not identify a FOXL 2 mutation.

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.

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, gastroesophageal reflux, 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 (KMT 2 D and KDM 6 A) and Angelman (UBE 3 A and MECP 2) syndromes were suspected in this patient.

Case 3

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

Exome analysis Workflow (this class) Variant determination FASTQ sequence 2 x 90 bp (paired-end) Mapping to genome SAM BCF Filter based on Phred score, mapping quality, read depth, etc. Conversion VCF BAM QC: Filter duplicates, artifacts, and unpaired or 100 genes “Mini” Exome unmapped reads, ? BWA (Burrows-Wheeler Aligner) SAMtools

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 2 x 90 bp paired-end Exome sequence reads, and at ~50 X coverage Reads mapped to human GRCh 37/hg 19 and then familial variants calls made in VCF format �“Mini” • • Exome 100 genes with/without known disease association. Validated causative genes and randomly selected disease genes or non-disease genes.

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

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 0/1 174 , 0 , 178 � GQ : Genotype quality (1 -99)

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 more. �Seattle. Seq: http: //snp. gs. washington. edu/Seattle. Seq. Annotation 138/ �VEP (Variant effect Predictor): http: //uswest. ensembl. org/info/docs/tools/vep/ �ANNOVAR: http: //wannovar. usc. edu/

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