Presentation on theme: "Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases"— Presentation transcript:
1 Exome Sequencing as Molecular Diagnostic Tool of Mendelian Diseases BIOS 6660Hung-Chun (James) YuShaikh Lab04/28/2014
2 Human Genetic Diseases Penetrance vs FrequencyKaiser J. Science (2012) 338:
3 Human Genetic Diseases Complex DisorderPolygenic, many genes.Low penetrance/effect size.Multifactorial, environmental, dietary.Examples: heart disease, diabetes, obesity, autism, etc.Mendelian DisorderMonogenic or polygenic.Full or high penetrance/effect size.Examples: sickle cell anemia and cystic fibrosis.
4 Complex Diseases Multiple causes, and polygenic. Multiple genetics factors with low penetrance individually.Coronary artery diseaseCoriell Institute for Medical Research.https://cpmc1.coriell.org/genetic-education/diagnosis-versus-increased-risk
5 Mendelian DiseasesVeltman J.A. et al. Nat. Rev. Genet. (2012) 13:
6 Mendelian Diseases Dominant Inheritance U.S. National Library of Medicine.
7 Mendelian Diseases Recessive Inheritance U.S. National Library of Medicine.
9 Exome Sequencing~40Mb (coding) or 60Mb (coding + UTRs)
10 Mendelian Diseases Identified by Exome Sequencing TimelineGilissen C. et al., Genome Biol. (2011) 12:228.
11 Mendelian Diseases Identified by Exome Sequencing By mid-2012, ~100 genes identified.By mid-2013, >150 genes identified.Rabbani, B., et al. (2012) J. Hum. Genet. 57:
12 Types of VariationWhat kind of variation/mutation can be detected by Exome Sequencing?SNV (single nucleotide variation)Small InDel, (insertion/deletion of <25bp)Large InDel, CNV (copy number variation)Possible, but not reliable.AneuploidySame as CNVTranslocationPossible, but not reliable. Limited.Complex rearrangementNot likely.
14 Exome Variants Small InDel (insertion/deletion <25bp) Frameshift In-frameNHGRI Digital Media Database (DMD),
15 Variant and Population Frequency Novel/Private variantNever been reported before.Rare variantMinor allele freq. (MAF) < 1%.Polymorphic variantMAF > 1% (0.01) or 5% (0.05).DatabasesdbSNP (NCBI):1000 Genomes:ESP (NHLBI):
16 Exome VariantsHow to analyze enormous amount of variants in any given exome?Gilissen C. et al. Eur. J. Hum. Genet. (2012) 20:Private/Novel~Protein altering~4, ,000Coding + splice-site~10, ,000All~20, ,000
18 Exome Analysis Strategies Gilissen C. et al., Eur. J. Hum. Genet. (2012) 20:MaleFemaleAffectedHeterozygouscarrierSex-linkedheterozygouscarrierMatingConsanguineousmating
19 Exome Analysis Strategies LinkageLarge family with multipleaffected individualsPathogenic variant co-segregatewith disorder.HomozygosityAffected patients fromconsanguine parents.Homozygous mutation within ahomozygous stretch in the genome.Ideal for recessive disorders
20 Exome Analysis Strategies Candidate genesBiased approachRequire current biological knowledgeGood for screening or clinical diagnosis of known disorders.OverlapRequire multiple unrelated individuals with identical disorders.Monogenic disorders
21 Exome Analysis Strategies De novoSporadic mutationGermline mutation during meiosisDominant inheritance*
22 Exome Analysis Strategies Double-hitUnaffected parents are heterozygous carriesParental sequence info is very helpfulRecessive inheritance.Compound HeterozygousHomozygous#******#
23 Trio-based Exome sequencing Family trioUnaffected parents and an affected patient.Why we use trio? What can be tested using trio? Advantages?Economical, efficient, single case required.
25 Trio-based Exome sequencing Candidate Genes/VariantsProtein altering variantsRare or novel variantsVariants that fit each inheritance modelRare VariantNovel VariantDominantDe novo0 ~ 1RecessiveCompound Heterozygous0 ~ 200 ~ 3HomozygousX-linked0 ~ 100 ~ 5
26 Case 1 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 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–Opitz syndrome. Sanger sequencing and highresolution CNV analysis with Affymetrix SNP 500K arrays did not identify a FOXL2 mutation.
27 Case 1A-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 2 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 (KMT2D and KDM6A) and Angelman (UBE3A and MECP2) syndromes were suspected in this patient.
30 Case 3 Clinical information Case 3 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 B12 level. Complementation analysis of the patient’s cell line placed the patient into the cblC class. Sequencing and deletion/duplication analysis (microarray) the MMACHC gene was negative in both skin fibroblasts and peripheral blood.
31 Case 3 Feature Combined methylmalonic aciduria and homocystinuria. Severe developmental delay, infantile spasms, gyral cortical malformation, microcephaly, chorea, undescended testes, megacolon
32 Case 3Monster MaxPatient's older sister as a summer student in Shaikh Lab
33 Data for Case Study 3 trios VCF files “Mini” Exome A total of 3 families/cases.Each family/case includes unaffected parents and an affected patient.VCF filesFamilial variants calls in VCF format, mapped to human GRCh37/hg19.2x90bp paired-end reads, with ~50X coverage“Mini” Exome100 genes with/without known disorder association.Validated causative genes, plus randomly selected genes.
35 VCF Format VCF (Variant Call Format) FILTER, INFO, FORMAT ## Meta-information linesFILTER, INFO, FORMAT# Header line
36 VCF Format INFO AA : ancestral allele AC : allele count in genotypes, for each ALT allele, in the same order as listedAF : allele frequency for each ALT allele in the same order as listed: use this when estimated from primary data, not called genotypesAN : total number of alleles in called genotypesBQ : RMS base quality at this positionCIGAR : cigar string describing how to align an alternate allele to the reference alleleDB : dbSNP membershipDP : combined depth across samples, e.g. DP=154END : end position of the variant described in this record (for use with symbolic alleles)H2 : membership in hapmap2H3 : membership in hapmap3MQ : RMS mapping quality, e.g. MQ=52MQ0 : Number of MAPQ == 0 reads covering this recordNS : Number of samples with dataSB : strand bias at this positionSOMATIC : indicates that the record is a somatic mutation, for cancer genomicsVALIDATED : validated by follow-up experiment1000G : membership in 1000 Genomes
37 VCF Format FORMAT GT: Genoetype. 0/0: Homozygous normal 0/1: Heterozygous variant1/1: Homozygous variantPL: the Phred-scaled genotype likelihoods (>0).0/ / /1, ,178GQ : Genotype quality (1-99)