Ph D Director of Clinical Genetic Laboratory Perkin

Outline • 新生儿筛查的二级筛查及基因诊断 Newborn Screen 2 nd tier Genetic testing • 全外显子以及亚全外显子二代测序的临床应用 Clinical Utility of Whole Exome Sequencing and Focused Exome Sequencing 2

新生儿筛查的二级筛查及基因诊断 Newborn Screen 2 nd tier Genetic testing

新生儿筛查---- 第二阶梯筛查 Primary Screen Dry blood spot sample from newborn • Biochemical • 生化�� 新生儿血斑 Diagnosis Second Tier Testing • Biochemical analysis* • Gene sequencing **基因� 序 • Clinical findings � 床表型 • Gene Sequencing 基因� 序 • Deletion/ duplication analysis 重复与缺失 • Other testing Treatment • Diet • Therapy Monitoring • Regular biochemical tests • Management * *Biochemical Second Tier Testing生化二级筛查 Disorder Biochemical First Tier Biocheical Second Tier Congenital Adrenal Hyperplasia 17 -OHP Extracted 17 -OHP Congenital Hypothyroidism T 4 or TSH with a primary T 4 Galactosemia Total Galactose + quantitative uridyltransferase Fractionated galactose 4

新生儿筛查---- 第二阶梯筛查 Primary Screen Dry blood spot sample from newborn Second Tier Testing • Biochemical analysis* • Biochemical • 生化�� • Gene sequencing **基因� 序新生儿血斑 * Diagnosis • Clinical findings � 床表型 • Gene Sequencing 基因� 序 • Deletion/ duplication analysis 重复与缺失 • Other testing Treatment • Diet • Therapy Monitoring • Regular biochemical tests • Management **Molecular Second Tier Testing • 分子基因测序- Single gene or panel • Sanger sequencing or NGS • 可能需要更长时间：panel> single gene> targeted mutation panel • Introduces the possibility of variants of unknown significance (VOUS) 临床意义不明确的突变 5

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 6

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 7

Example-- Cystic Fibrosis囊肿性纤维化（缺乏二级生化检测） • 常染色体隐性遗传 • NBS新生儿筛查: ◦ Elevated IRT (immunoreactive trypsinogen免疫活性的胰蛋白酶原) ◦ Can not differentiate carrier vs affected • 2 nd tier screen: targeted mutation testing靶向突变panel ◦ ACMG 25 recommended mutation panel ◦ Covers 89% 白人, but only 49% 亚裔 ◦ Some times only one mutation is identified: IRT升高不能区别携带者与患者 • Sanger sequencing/NGS of the CFTR gene ◦ Most exonic mutation 大部分是外显子突变 ◦ Deep intronic mutations c. 1680 -877 G>T ◦ Exon level deletion/duplication ex 20 del (c. 3718 -24_3873+601 del) 8

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 9

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 12

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 14

Example-- Hurler Syndrome Mucopolysaccharidosis Type I (MPS I) 一型粘多糖��症 • • L-α-iduronidase 艾杜糖醛苷酸酶 IDUA gene 常染色体�性�� 病率1: 100, 000 (severe form) to 1: 500, 000 (attenuated form) 临床表型： hepatomegaly, splenomegaly, mental retardation, corneal clouding • 新生儿筛查: L-α-iduronidase 酶活降低 • 二级筛查/诊断：IDUA基因测序(Sanger/NGS) • Common pseudodeficiency alleles: ◦ ◦ c. 235 G>A (p. A 79 T) c. 246 C>G (p. H 82 Q) c. 667 G>A (p. D 223 N) c. 965 T>A (p. V 322 E) • Artificial enzyme abnormality in in vitro assays; normal in vivo； probably due to unstable enzyme in vitro 只有体外才会产生酶活缺陷 ◦ 体外酶蛋白不稳定 ◦ 结构与实验用底物结合问题是主要原因 15

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 16

Example--Methylmalonic Acidemia (MMA) & homocysteinuria 甲基丙二酸血症及高半胱氨酸尿症 Methylmalonyl-Co. A Cbl. B Cbl. A Cbl. F Cbl. C Cbl. D Cbl. E Cbl. G 17

ACMG(NBS) MMA 诊断策略 18

Importance of Molecular Testing分子��的重要性 • Some disorders do not have biochemical 2 nd tier test 缺乏生化检测方法 • Molecular testing is cheaper and easier than biochemical 2 nd tier test in some disorders 基因��更��便捷 • Biochemical test may not be reliable in some patients 生化��在某些患者中可靠 • Pseudodeficiency alleles 假缺陷突� • Treatment specific to the gene (genetic heterogeneous disease) 同一疾病的治�方法基于致病的不同基因有差异 • Response to treatment or adverse event depend on mutation type �物�效或副作用因不同突�而有差异 • Clinical surveillance of individuals at risk of genetic disease �度或者晚�基因型患者的�期�床�督 • Provide information to family members about the genetic disease for screening/prevention/reproductive choices �患者及家属提供相关信息：��疾病、��、�防、治�以及�助生殖 19

Example– Elaprase for Hunter syndrome • Elaprase is an enzyme replacement therapy produced by Shire • Patients with Hunter syndrome (Mucopolysaccharidosis II) can be treated with Elaprase • Treatment cost ~$375 k per year (Shire revenue of $353 M in US 2010) • Biochemical analysis and sequencing of LSD genes (IDS gene) • Patients with complete gene deletion, large gene rearrangement, nonsense, frameshift or splice site mutations experienced a higher incidence of hypersensitivity reactions, serious adverse reactions and anti-idursulfase antibody development than Hunter syndrome patients with missense mutations 20

PE NBS panel: 新生儿筛查遗传病及代谢病panel 设计思路及特点 • 276 genes & 182 diseases • 所有ACMG 新生儿筛查core panel and expanded panel • 常见新生儿发病的代谢病和遗传病 • NGS二代测序+ long range PCR for real genes (e. g. CYP 21 A 2 先天性肾上腺皮质增生症, GBA 戈谢病) • 捕获探针覆盖范围： ◦ Exon± 50 ◦ Reported intronic, splicing and regulatory variants: - e. g. CFTR c. 1680 -877 G>T ◦ Common deletions and duplications using breakpoint probes - e. g. PKU PAH c. -4173_-407 del • Any low coverage regions failed NGS will be considered Sanger fill- in • Customize single-/multi-gene panel个性化定制单基因/多基因检测 21

PE NBS panel: 新生儿筛查遗传病及代谢病panel 276 genes & 182 diseases • Endocrine diseases 内分泌疾病 (e. g. Primary congenital hypothyroidism, Congenital adrenal hyperplasia, Permanentneonatal diabetes mellitus etc. ) • Hemoglobin disorders 血红蛋白病 (地中海贫血, 镰刀状红细胞贫血) • Fatty acid oxidation disorders 脂肪酸氧化(e. g. CPT I/II, MCAD, LCHAD, VLCAD etc. ) • Organic acidemia 有机酸血症 (e. g. Isovaleric acidemia, PA, MMA, Biotinidase deficiency) • Amino acidemia 氨基酸酸血症 (e. g. Tyrosinemia I/II/III, Homocystinuria, MSUD, PKU, Citrullinemia I/II etc. ) • 代谢病：Galactosemia 半乳糖血症 • SCID 连锁重症联合免疫缺陷 (XL-SCID 23 genes, Bare lymphocyte syndrome) • Lysosomal storage disease 溶酶体贮积病 (e. g. Fabry disease, Gaucher disease, Hunter disease, Hurler disease, Niemann-Pick Disease) • Glycogen storage disease 糖原累积病 • Genetic disease 遗传病 (Cystic fibrosis, chronic leukemia & focal dermal hypoplasia, Menkes syndrome, Wilson disease) • Hearing impairment 耳聋 (e. g. Usher syndrome, Pendred syndrome, branchiootorenal (BOR) syndrome 22

Evolution of Gene Sequencing & Testing Options Development of NGS Whole exome Disease • 22, 000 focused genes panel of • Coding Several genes (10 s regions only individual • No introns – 100 s) genes • Only 1% of Single genes 25 Gene sequencing 1990 s to present the genome • But ~85% of diseasecausing mutations Whole genome • Entire genome, coding and noncoding • Greater resolution with no replication bias • Able to detect large deletions and duplications

全外显子学的临床意义 • End the expensive and stressful diagnostic odyssey • Gene-specific prognostication • Anticipatory management for other comorbidities • Redirect therapy to treat the underlying genetic etiology • More accurately estimates recurrence risk in the family & guide future pregnancy • Identify other at-risk family members and provide available treatment at presymptomatic phase 26

Pediatric Setting 小儿科 Mitochondrial; 0. 2% • One WES program of 2000 patients X-linked; 12. 3% ◦ Diagnosis achieved in 25% pts ◦ 58% findings not previously reported • Recent studies using trio WES (家系) reached at least 35% diagnostic rate 27 autosomal recessive; 34. 3% autosomal dominant; 53. 1%

NICU setting 新生儿重症室 • WES as first-tier molecular tests ◦ ◦ 80 infants Prospective study In parallel to standard investigations (including single or multigene panel) 57% received a molecular diagnosis by WES, compared to 14% by standard investigation ◦ 33% changed clinical management ◦ 12 relatives received a genetic diagnosis following cascade testing ◦ 28 couples were identified at high risk of recurrence in future pregnancy 28 Lancet Respir Med. 2015 May; 3(5): 377 -87. doi: 10. 1016/S 2213 -2600(15)00139 -3 Genet Med. 2016 Mar 3. doi: 10. 1038/gim. 2016. 1

From Single Gene to Genome Single gene (<0. 0001% of genome, <3000 bp) � 全外全外� 子 Known disease genes – 5, 000 genes (<0. 5% of genome, <15, 000 bp) Exome – 22, 000 genes (<2% of genome, <60, 000 bp) Genome – 22, 000 genes, regulatory regions, everything else – ~3 billion base pairs 29

Sequencing Options Targeted panels Exome Genome Smaller target region Targets the entire coding region of the genome (the exome) Targets the entire genome Highest depth of coverage Lower compared to targeted Lowest compared to targeted and exome Difficult for detection of CNVs, indels, trinucleotide repeats, pseudogenes Can detect CNVs and indels; Difficult to detect trinucleotide repeats, pseudogenes No incidental findings Need to address incidental findings 30

How many variants? • Single gene ◦ Average 5 variants • Gene panel ◦ XL-Intellectual Disability panel 91 genes – Average 200 variants • Exome ◦ Average 17, 000 variants • Genome ◦ 3 million average SNPs, 350, 000 average indels 31

How many variants? Pre-screen • • gnomad db. SNP Clin. Var • HGMD Annotated variants WES ~20 k changes WGS Millions of changes 32 • Filter out common variants Correct gene symbols Re-annotate/ update genedisease associations Inheritance patterns ~1, 800 changes Tens of thousand of changes Phenotype review Variant interpretation Clinical report Few dozen changes <Dozen changes Hundreds of changes Tens of changes 32

ACMG secondary findings • 60 medically actionable genes • Recommend clinical diagnostic labs to report pathogenic mutations in these genes • Labs usually provide “opt-out” option for patients who do not want to receive these results 33

Who are We? Perkin. Elmer Genetics Global team • Madhuri Hegde, Ph. D, FACMG ◦ Vice President and Chief Scientific Officer, Global Laboratories • Alice Tanner, Ph. D, MS, CGC, FACMG ◦ Director, Laboratory Testing and Clinical Education • Yang Wang, Ph. D ◦ Director，Clinical Genetic Laboratory • Zeq Ma, Ph. D ◦ Director of Clinical Informatics, Diagnostics Perkin. Elmer Genetics Site Leaders • Suzhou, China ◦ Nicky Xu, General Manager, Diagnostics ◦ Fang, Laboratory Director • Branford, CT ◦ Alexander Valencia, Scientific Laboratory Director ◦ Gerard Irzyk, Senior Director of Operations • Pittsburg, PA ◦ Joseph Quashnock, Laboratory Director ◦ PJ Borandi, Site Leader, Information Technology • Chennai, India 34 ◦ Raghu Basiboina, Director, Diagnostic Laboratory Services ◦ Rajiv Rose, Manager, Molecular Diagnostics

Useful website • • Genereviews OMIM HGMD (requires subscription) Clin. Var Gnomad (replacing Ex. AC by broad institute) www. orpha. net 罕见病查询(gene or disease) Carrier Frequency Calculator 计算携带率 ◦ http: //perinatology. com/calculators/Hardy-Weinberg. htm 35

Variant analysis, annotation & interpretation • • Bioinformatics pipeline: variants calling and annotation Variant filtering ◦ ◦ Variant analysis-- quality of reads: automatic + manual review ◦ ◦ ◦ Coverage: decision for Sanger fill-in Variant read depth wt/mut ratio Mutation type (del/dup BAM file) Strand bias Phase (BAM file) Variant interpretation ◦ ◦ ◦ • Population frequency (public databases and internal database frequency) Phenotypic filter set Population databases: 1000 genome, db. SNP, Ex. AC, gnomad Public knowledge databases: genereviews, HGMD, Clin. Var, LOVD, gene-specific databases (DMD, BRCA 1/2 …. ) Internal databases: PKIG global knowledge database - Reporting ◦ ◦ ◦ Phenotype review Variants review Select for reporting - 36 Curated GAD and GOUS (genes associated with disease/unknown significance) Curated variants and notes Variants related to phenotype ACMG secondary findings (57 genes- medically actionable diseases)