Next Generation Sequencing: technical issues. Francesca Ariani, PhD Medical Genetics, University of Siena

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1 Next Generation Sequencing: technical issues Francesca Ariani, PhD Medical Genetics, University of Siena

2 Advances in knowledge have been driven by the advent of new technologies 1977

3 Advances in knowledge have been driven by the advent of new technologies 2005 A paradigm shift respect to Sanger sequencing that has allowed scalingup by orders of magnitudes

4 NGS: method of the year 2007 It has fundamentaly alterated genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable

5 James Watson s s genome sequenced at high speed 2008

6 NGS technology

7 Template preparation methods Emulsion PCR Bridge amplification

8 Pyrosequencing

9 Sequencing by ligation

10 Sequencing by synthesis Four-colour cyclic reversible termination (CRT) method

Whole Exome Sequencing (WES) More cost-efficient sequencing strategies have been developed to study ~1% of our genome that is protein-coding (the exome),

11 Whole Exome Sequencing (WES) More cost-efficient sequencing strategies have been developed to study ~1% of our genome that is protein-coding (the exome), by using various capturing approaches to enrich before NGS Protein coding genes harbor 85% of the mutations with large effects on disease-related traits.

12 Capture methods Solid-phase hybridization Liquid-phase hybridization

Challenges: data analysis Typically, 20.000-50.

13 Challenges: data analysis Typically, variants identified per sequenced exome need for filtering!!!

14 WES filtering variants Quality criteria (Coverage, % of reads showing the variant ) Exonic and splice site variants Affecting protein sequence Excluding known variants (dbsnp, published studies, in-house databases)

15 WES filtering Additional strategies to find the causative mutation

16 These startegies don t t always work Current challenges in exome or genome-based analysis of Mendelian disorders. Jay Shendure, pltform presentation, ASHG 2012

17 Data analysis: pathogenecity assessment of nonsynonimous variants Current challenges in exome or genome-based analysis of Mendelian disorders. Jay Shendure, pltform presentation, ASHG 2012

18 Data analysis: pathogenecity assessment of nonsynonimous variants Current challenges in exome or genome-based analysis of Mendelian disorders. Jay Shendure, pltform presentation, ASHG 2012

PolyPhen-2 (Polymorphism Phenotyping program version 2) http://genetics.bwh.harvard.

19 PolyPhen-2 (Polymorphism Phenotyping program version 2) FN: 31% FP: 9% TRINARY PREDICTOR Benign Possible damaging Probably damaging

SIFT (Sorting( Intolerant From Tolerant) http://sift.bii.a-star.edu.

20 SIFT (Sorting( Intolerant From Tolerant) star.edu.sg/ FN: 31% FP: 20% BINARY PREDICTOR Tolerated Not tolerated

PhyloP (Phylogenetic P-values) http://genome.ucsc.

21 PhyloP (Phylogenetic P-values) Phylogenetic conservation of a nucleotide at a specific position Numerical value: /+6.94 (negative scores: not C; positive scores: C)

site http://www.fruitfly.org/seq_tools/splice.

22 Data analysis: splice site variants The Berkeley Drosophila Genome Project (BDGP) web site NetGene2

23 NGS technology applied to Alport syndrome (ATS): to improve diagnosis to identify new disease genes

24 Alport syndrome (ATS) A nephropathy characterized by: hematuria with varying degrees of proteinuria progressive renal failure high tone sensorineural hearing loss ocular abnormalities, most typically anterior lenticonus specific ultrastructural lesions of the GBM

25 ATS: a genetically heterogeneous nephropathy AD (autosomal dominant) COL4A4/COL4A3 genes 15% AR (autosomal recessive) COL4A4/COL4A3 genes 85% XL (X-linked) COL4A5 gene COL4 genes (COL4A3/COL4A4/COL4A5) exons

26 ATS is also a clinically heterogeneous condition X-linked form: males severely affected with persistent hematuria, proteinuria, constant progression to ESRD and high incidence of hearing loss and ocular anomalies. Females usually show only urinary abnormalities. AR form: phenotype identical to the X-linked form, but females are severely affected as males. Parents may be completely asymptomatic or may have isolated microhematuria. AD form: also associated to a high risk to develop renal failure but at an older age. Extra-renal manifestations are rarely observed. High inter- and intra-familial phenotypic variability!

Immunohistology of the distribution of the α5(iv) collagen chain in the skin Control Male with X- linked ATS Female with X-linked ATS Case with AR ATS But: 20% of males show normal expression;

27 Immunohistology of the distribution of the α5(iv) collagen chain in the skin Control Male with X- linked ATS Female with X-linked ATS Case with AR ATS But: 20% of males show normal expression; females display a discontinuous α5 staining but only in 60-70% of cases discordance of expression between the SBM and GBM has been reported Identification of the underlying mutation remains the gold standard for diagnosis

28 2010: NGS protocol for ATS diagnosis Method coupling selective amplification to the Roche DNA sequencing platform (454 junior Sequencer)

29 NGS primer design

30 454 GS Junior procedure

31 Flowchart for 454 variants filtering

32 Roche 454 mutation detection

33 NGS impact on ATS diagnosis in 80 patients Identification of 47 mutations, 33 novel COL4A3 COL4A4 COL4A5

34 NGS impact on ATS diagnosis Frequency of AD cases much higher (38%) than expected, indicating that this form is presently underestimated The use of NGS was conclusive for diagnosis in 22 cases where clinical data and family history were not sufficient to select the specific test. In 5 families with a clinical diagnosis of AD forms NGS conversely detected COL4A5 mutations Formal genetic analysis NGS results N of cases sporadic XL 5 AD 2 XL/AD XL 7 AD 8 XL XL 10 AR AR 1 AD AD 7 AD XL 5

early treatment with ACE inhibitors has been proven to delay renal failure and to

35 NGS impact on ATS diagnosis Importantly, this test will allow a rapid and cost effective diagnosis also in oligosymptomatic ATS children and this is extremely important since early treatment with ACE inhibitors has been proven to delay renal failure and to improve life expectancy in a time-dependent manner Submitted to Nephrology Dialysis Transplantation

36 Identification of new genes Overlap strategy WES in 5 mutation-negative ATS patients? 3 2 Family 1 (BER) Family 2 (ZZA) Family 3 (BAU)? 6 3 Family 4 (DEG) Family 5 (CLA)

37 WES in 5 mutation-negative negative ATS patients Illumina platform Prof. V. Nigro

38 WES in 5 mutation-negative negative ATS patients Family 5 (CLA) Family 2 (ZZA) COL4A5: p.g1107r Polyphen2: probably damaging SIFT: not tolerated PhyloP: conserved

39 WES in 5 mutation-negative negative ATS patients Family 5 (CLA) Family 2 (ZZA) COL4A4 : p.l1482fs COL4A5: p.g1107r Missed by previous analysis!!! 41 50? ? Family 5 (CLA) COL4A4: p.l1482fs ESRD Hearing loss Family 2 (ZZA) COL4A5: p.g1107r Microhematuria Proteinuria ATS GMB lesions

40 454 Junior data analysis of COL4A4 : p.l1482fs AVA Version 2.6 was not able to identify del/ins of one base AVA 2.6 AVA Version 2.7 (June 2012) is now able to identify del/ins of one base AVA 2.7 c.4443_4445delc

41 454 Junior data analysis of COL4A5 : p.l1482fs 1 st experiment 16X 2 nd experiment 5000X c.g3319a Increasing coverage we were able to identify the substitution!

42 Search for variants in 3 ATS patients Smith Magenis syndrome (OMIM#182290)

43 Smith Magenis syndrome

44 Variants in 3 ATS patients: RAI1 (c.840delg) Prof. M. Zollino Lab (Medical Genetics, Hospital "A. Gemelli, Roma) did not confirmed RA1 variant by Sanger sequencing FALSE POSITIVE RESULTS of WES!!! Probably due to a region of CAG repeats causing misalignment!

45 Family 4 (DEG) Not reported as SNP (dbsnp, 1000 Genome and in house database) Polyphen2: probably damaging SIFT: not tolerated PhyloP: conserved

46 Fibronectin 1 (FN1( FN1) Glycoprotein present on cell surfaces, in extracellular fluids, connective tissues, and basement membranes. Fibronectins interact with other extracellular matrix proteins and cellular ligands, such as collagen, fibrin, and integrins. Fibronectins are involved in adhesive and migratory processes of cells.

47 Family 4 (DEG) I 56 II III Sp VI settimana Microhematuria Proteinuria ATS GMB lesions

48 FN1 segregation analysis by Sanger sequencing c.c1535t (p.s512l) Proband (III8) Healthy brother(iii10) Healthy father!(ii2) Healthy mother (II6) FN1 variant does not cosegregate with disease status

49 Family 1 (BER) Not reported as SNP (dbsnp, 1000 Genome and in house database) Polyphen2: probably damaging SIFT: not tolerated PhyloP: conserved

50 Uromodulin (UMOD( UMOD) a GPI-anchored glycoprotein and the most abundant protein in normal urine. Uromodulin, uropontin, and nephrocalcin are the 3 known urinary glycoproteins that affect the formation of calcium-containing kidney stones UMOD-associated kidney disease (uromodulin-associated kidney disease): - hyperuricemia and gout -progressive interstitial kidney disease early in life - elevations in serum creatinine (5-40 y) leading to ESRD usually between the 4th and 7th decade

51 Family 1 (BER) I II 85 ESRD 71 ESRD ESRD ESRD ESRD 84 III 3 72 ESRD, hearing loss ESRD IV V months Microhematuria Proteinuria GMB lesions compatible with ATS Hearing loss Renal cysts Elevations in serum creatinine Uric acid levels: 6mg/dl

52 UMOD segregation analysis by Sanger sequencing c.g115a (p.a39t) Proband (III8) Affected paternal aunt (II4) Affected brother (III9) Affected father (II2) Affected paternal aunt (II6) UMOD variant cosegregates with disease status

53 Family BER: UMOD atypical phenotype Prof. Mario De Marchi s lab. (Medical Genetics, University of Torino) :.. the UMOD gene variant that you have identified is known to us for a long time This is not a classical mutation (not involving a Cysteine) of UMOD gene and for this reason we are doing to further investigations, in collaboration with the Consortium of the Medullary Cystic Disease Also from the clinical point of view is not a typical diagnosis of uromodulin-associated disease and the phenotype described in your family is very similar to that observed in our family. It would be interesting to know the geographical origin of your family to see if it is a founder effect, we have available the microsatellite region adjacent to UMOD and we can investigate a possible founder effect of the mutation

54 Family 3 (BAU) Not reported as SNP (dbsnp, 1000 Genome and in house database) Polyphen2: probably damaging SIFT: not tolerated PhyloP: conserved

nephrin Podocytes are a major site of biosynthesis of Gal1 in the glomerulus and the

55 GALECTIN (LGALS1( LGALS1) an autocrine-negative growth factor not previously associated to diseases Gal1 is a component of the glomerular slit diaphragm (SD) directly binding to nephrin Podocytes are a major site of biosynthesis of Gal1 in the glomerulus and the expression patterns and levels of Gal1 are altered in patients with minimal change nephrotic syndrome

56 Potential mechanism underlying chronic renal disease in ATS

57 Family 3 (BAU) I II III Microhematuria IV Microhematuria GMB lesions compatible with ATS Microhematuria Microhematuria

58 LGALS1 segregation analysis by Sanger Sequencing c.g10t (p.g4c) Proband (III2) Affected doughter (IV3) Affected doughter (IV4) LGALS1 variant cosegregates with disease status Good candidate gene!

59 Conclusions NGS technology is improving diagnosis and patients management it also provides an exciting opportunity to solve thousands of Mendelian and non-mendelian disorders This will allow to better understand the pathogenetic mechanisms of diseases and to design therapeutic strategies Going forward there will be enormous opportunity and challenges in the lab and in the clinic

Acknowledgments Medical Genetics, University of Siena Dr.

Laura Bianciardi "Cell lines and DNA bank of Rett

diseases biobank supported by TELETHON grant GTB07001 A

60 Acknowledgments Medical Genetics, University of Siena Dr. Laura Dosa Dr. Chiara Fallerini Dr. Caterina Lo Rizzo Dr. Laura Bianciardi "Cell lines and DNA bank of Rett Syndrome, X-linked mental retardation and other genetic diseases biobank supported by TELETHON grant GTB07001 A donation in favour of Graziano and Marco Laurini Tigem Prof. Vincenzo Nigro Dr. Margherita Mutarelli Medical Genetics, University of Torino Prof. Mario De Marchi, Prof. Daniela Giachino

62 NGS impact on ATS diagnosis Pedigrees compatible with both an X-linked and an AD inheritance Pedigrees with suspected AD inheritance

Unbiased next generation sequencing analysis confirms the existence of autosomal dominant Alport syndrome in a relevant fraction of cases

$Unbiased next generation sequencing analysis confirms the existence of autosomal dominant Alport syndrome in a relevant fraction of cases$ Clin Genet 2014: 86: 252257 Printed in Singapore. All rights reserved Short Report 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12258 Unbiased next