The genetics of male infertility; What s new? Joris Veltman

Transcription

1 The genetics of male infertility; What s new? Joris Veltman Joris.veltman@radboudumc.nl Joris.veltman@newcastle.ac.uk

2 Male infertility and genetics Opening thoughts o Genetic information is inherited from generation to generation o Infertility cannot be inherited from generation to generation How can genetics play a role? o Genetics only role in inbred populations, recessive inheritance? o Genetics a risk factor, adding little to chances of becoming infertile? Other possibilities???

3 3

4 1989 Genetics is an art

5 2017 Genetics is an industry

6 Genome sequencing: All variation in one experiment! DNA from blood/saliva Genome with all variation Important: - Accuracy - Speed - Price

7 Genome sequencing: All information in one test! deletion point mutation interchromosomal duplication Intellectual disability Cancer What if we apply this to male infertility??

8 Our genome: Full of variation! Single nucleotide variation Additional chromosome Insertion-deletions Deletions of DNA sequence Chromosome rearrangements Microsatellite repeat variations

9 5,996 billion similarities 4 million differences

10

11

12 a Patient-parent trio genetic analysis Patient reads Mother reads Father reads 4 million inherited variants, 100 new mutations per person

13

14 New mutations major cause intellectual disability Unknown cause New mutations Inherited mutations New deletions Gilissen et al. Nature 2014

15 New mutations >75% from dad

16 Old father: more mutations in child 60 Goldmann et al. Nature Genetics 2016

17

18 A C A C T G T

19

20 Back to the genetics of male infertility: Why is this important? Genetic testing Diagnosis Treatment Genetic counselling

21 The current situation: Genetic testing in infertility Cohort of 1,231 infertile men In most cases of severe oligospermia or azoospermia no abnormality is found, but testing strategy outdated! Olesen et al. 2017

22 De novo mutations in male infertility? Many genes known to play a role in reproduction De novo mutations are a known cause of male infertility: - De novo chromosome abnormality: Klinefelter syndrome XXY - De novo deletions of AZF region on chromosome Y Very few autosomal male infertility genes known. Most genes identified cause disease in a recessive manner Recessive inheritance major factor in outbred populations? Study design not yet focused on autosomal dominant genes! Challenge: Parental DNAs normally not available for de novo mutation analysis

23 Genetics of male infertility study in Nijmegen/Newcastle Idiopathic infertility (n=1,000) (reduced sperm count) Characteristic morphological anomalies (n=100) Pre-screening for known genetic causes Exome and genome sequencingn in patient-parent trios to find new causes Replication, data sharing Functional studies

24 Targeted sequencing of known genetic causes of male infertility Assay design Validation of assay in cohort of 26 patients with known genetic causes Next generation sequencing (NGS) Screening 1,119 patients with unexplained male infertility Relevant for male infertility Targeted sequencing

25 Screening for known genetic causes of male infertility Screening for Gene Expected phenotype Inheritance AZF deletions Chromosomal anomalies X/Y All genes on chry All genes on chrx and chry Severe oligozoospermiaazoospermia Oligozoospermia-azoospermia De novo AZF deletion De novo chr. anamaly SNVs/CNVs AURKC Macrozoospermia Autosomal recessive SNVs/CNVs CFTR Obstructive azoospermia (CBAVD) Autosomal recessive SNVs/CNVs DDX3Y Severe oligozoospermiaazoospermia De novo mutation on chr. Y SNVs/CNVs DPY19L2 Globozoospermia Autosomal recessive SNVs/CNVs SYCP3 Azoospermia (meiotic arrest) Autosomal dominant? SNVs/CNVs TEX11 Azoospermia (meiotic arrest) X-linked recessive

26 Gene panel for targeted sequencing ACE ADCY10 AGFG1 AKAP3 AKAP4 APOB ART3 AURKC BPY2 C17orf51 CATSPER1 CATSPER2 CATSPER3 CATSPER4 CCDC39 CCDC40 CDY1 CDY2 CFTR CHD2 COBL CSMD1 CSPG4P1Y CXorf48 CYP21A2 DAZ DDX25 DDX3Y DMRT1 DNAAF1 DNAH1 DNAH11 DNAH5 DNAH7 DNAI1 DNMT3L DPY19L2 EIF1AY ESR1 ESR2 FAM82A1 FHL5 FKBPL GALNT5 H2BFWT HENMT1 HSD17B4 HSF2 HSFY HSPA2 IGSF1 KDM5D KLHL10 LHCGR LIMK2 LIPE LRGUK LRRC32 MAGEA9 MBOAT1 MEI1 MSH5 NLGN4Y NR0B1 NR5A1 OAZ3 PICK1 PIWIL1 PIWIL2 PIWIL3 PLSCR2 PRM1 PRM2 PRY RBMX RBMXL9 RHOFX1 RPSY1 RPSY2 RXFP2 SEPT12 SHBG SIRT4 SLC25A24 SLC26A8 SOHLH1 SPATA16 SRY SYCP3 SYT6 TAC3 TACR3 TAF7L TBL1Y TBPL1 TEKT2 TEX11 TGIF2LY TMSB4Y TSSK2 TSSK4 USP9Y UTP14C UTY VCY XKRY ZFY ZPBP Causal In y-microdeletion region Single case Candidate gene with suggestive evidence but insufficient patients

27 Targeted sequencing Validation Point mutations CNVs Chromosomalanomalies CFTR AZF deletion 47,XXY/47,XYY Up to 80% of obstructive azoospermia Up to 10% of severe oligozoospermia Up to 10% of severe oligozoospermia

28 Validation detection point mutations Validation c.1521_1523del p.phe508del Homozygous c _1210-7del T5-TG12 (c tg(12)t(5)) Heterozygous

29 Validation Point mutations CNVs Chromosomalanomalies CFTR AZF 47,XXY/47,XYY Up to 80% of obstructive azoospermia Up to 10% of severe oligozoospermia Up to 10% of severe oligozoospermia

30 Targeted sequencing design Y chromosome Validation

31 Targeted sequencing design Y chromosome Validation

32 Detection of AZF deletions Validation Classic AZFc deletion

33 Validation Point mutations CNVs Chromosomalanomalies CFTR AZF 47,XXY/47,XYY Up to 80% of obstructive azoospermia Up to 10% of severe oligozoospermia Up to 10% of severe oligozoospermia

34 Validation chromosomal anomalies Validation Klinefelter syndrome (47,XXY)

35 Validation Point mutations CNVs Chromosomalanomalies CFTR AZF 47,XXY/47,XYY Up to 80% of obstructive azoospermia Up to 10% of severe oligozoospermia Up to 10% of severe oligozoospermia

36 Screening of unexplained cohort of infertile men Screening for known causes 1,119 idiopathic male inferility cases AZF deletions Sex chromosome anomalies Point mutations CNVs -Full AZF deletions -47,XXY -CFTR -Partial AZF deletions 5 patients -47,XYY -46,XX male 6 patients -AURKC -DDX3Y -DPY19L2 -SYCP3 -TEX11

37 Screening known genetic causes AZF deletions Sex chromosome anomalies Patient Genetic diagnosis Phenotype Sperm concentration (10 6 per ml) DNA AZFb+c deletion Azoospermia AZFc deletion Severe oligozoospermia AZFc deletion Severe oligozoospermia AZFc deletion Oligozoospermia 5 DNA AZFc deletion Azoospermia 0 DNA , XX male (Y p11.31 and p11.2 positive) Azoospermia 0 DNA , XXY (Klinefelter) Azoospermia 0 DNA , XXY (Klinefelter) Azoospermia 0 DNA ,XXY (Klinefelter) Azoospermia , XYY Oligozoospermia , XYY Oligozoospermia >5

38 Main challenge: Interpretating genetic variation! Point mutations Reliable variant calling from data Variant present <5% of 1,112 patients tested Varient present <1% of general population Variant has an effect on the protein Variant is not a sequence artefact (manual check) 33,415 3,609 2, Classification according to VKGL guidelines Certainly not pathogenic Unlikely pathogenic Pathogenicity uncertain Likely pathogenic Certainly pathogenic

39 Pathogenic variants in known male infertility genes Point mutations

40 SYCP3 is part of the synaptonemal complex Point mutations In prophase I of meiosis crossing over between homologous chromosomes take place SYCP3 p.ile175asnfs*8 Exon 7/9 Burgoyne et al. 2009

41 Targeted gene sequencing: Results Screening for known causes Risk factor Diagnosis Carrier of a mutation 4.0% 1.6% No variant found Likely benign AZF duplication Carrier (likely) benign Carrier VUS Carrier (likely) pathogenic Risk factor partial AZF deletion 21.4% Risk factor 47,XYY Diagnosis AZF deletion Diagnosis 47,XXY (Klinefelter) Diagnosis 46,XX male Diagnosis CFTR Diagnosis SYCP3 73% No variant found Oud et al. Human Mutation 2017

42 Next steps 1. Find genetic causes of idiopathic severe infertility 1. Create cohort >1,000 patients 2. Develop a method for pre-screening (targeted) 3. Pre-screening known causes 4. Pre-screening candidate genes 5. Exome sequencing in patient-parent trios 2. Find genetic causes of rare characteristic forms of infertility 1. Create a cohort of 100 patients 2. Exome sequencing 3. Set-up global collaboration to share data and replicate findings 4. Functional studies of newly discovered genes

43 Overall conclusions Genetics of male infertility poorly established Revolution in genetics allows for - discovery of new genetic causes - development of novel diagnostic tools - insight in risk factors such as advanced paternal age and use assisted reproductive technologies Interpretation of genetic variation is challenging Patient-parent analysis of great benefit International collaboration essential

44 Radboudumc Genetics Manon Oud Lisenka Vissers Alex Hoischen Petra de Vries Dominique Smeets Dorien Lugtenberg Tuula Rinne Jayne Hehir-Kwa Christian Gilissen Stefan Lelieveld Maartje van de Vorst Marc Pieterse Obstetrics and Gynaecology Liliana Ramos Aukje Meijerink Roos Smits Kathrin Fleischer Maastricht UMC+ Michiel Noordam Monash University Moira O Bryan Robert McLachlan Université de Strasbourg Stéphane Viville Özlem Okutman Newcastle University Kevin McEleny Harsh sheth Ludmila Volozonoka Kumara Mastrorosa Bilal Alobaidi