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Unravelling the genetic component of male infertility Alexandra Lopes May 2015 Researcher @ Pop Genetics, i3S/IPATIMUP Nov. 2011 Postdoc fellow @ Pop Genetics & Dep. Genetics, WUSTL, USA May 2010 PI: “Search for genomic structural variants in azoospermia: a study in the Portuguese population”, IPATIMUP 2007-2011 Postdoc fellow @ Pop Genetics & Mammalian Mol. Genetics lab U. Cambridge, UK 2002 – 2006 PhD student @ Pop Genetics lab, IPATIMUP, PT 2001 Biology degree, FCUP, PT X&Y BIOLOGY Nabeel Affara Cambridge, UK Lounes Chikhi IGC POPULATION GENETICS & EVOLUTION CHR. REARRANG. João Alves GABBA PhD António Amorim IPATIMUP Ana Lima Don Conrad GABBA PhD WUSTL FUNCTIONAL GENOMICS EPIGENETICS X-INACT. Laura Carrel Penn State REPROD. BIOLOGY Catarina Seabra UA MSc Genetics of Male Infertility Initiative (GEMINI) International consortium to study the genetics of male infertility Analysis of large number of patients by high density arrays and exome sequencing Genetics wiki DNA Human Chromosomes Single base variant Single Nucleotide Polymorphism (SNPs) • Change a single DNA letter • Most frequent genetic variant • 1 per 300 base pairs • Common (MAF>5%) • Less common (1-5%) • Rare ‘variants’ (<1%) •“SNV” Genotypes BB or AB or AA Locus 4 Alleles at locus 4 A B Each somatic cell is diploid Locus: chromosomal location that’s polymorphic. Alleles: different variants @ locus (two copies of each autosome) Thus, 3 genotypes at locus 4 PAR 1 X X and Y Chromosomes Y PAR 1 ~2.7 Mb X chromosome: ~ 150 Mb MSY Usp9y ~ 1000 functionally diverse genes Ross et al. (2005) Y chromosome: Male determining locus SRY PAR 2 ~300 Kb ~ 23 Mb (coding region) ~ 156 transcripts; many multi-copy gene families expressed in testis Skaletsky et al. (2003) PAR 2 B Y chr Alleles at A B locus or A Genetics of male infertility Human fertility facts ▪ As many as 8% of children in some Western countries are born as a result of assisted reproductive techniques ▪ 1 out of 7 European couples suffer from reproductive disorders resulting in infertility ▪ In 30-50% of the cases the male is the infertile partner Known genetic causes of non-obstructive azoospermia (NOA) 15% 15% 70% ? Some sporadic mutations have been associated with male infertility ANEUPLOIDY Y CHR DELS UNKNOWN Genetic architecture of spermatogenic failure Genetic architecture of a trait • the number of genes/loci modulating the trait • the number of risk alleles at each gene/locus • their frequencies and effect sizes Our Aim To detect rare variants across the genome with strong association with infertility Approach Genome-wide analysis in infertile patients (azoospermic) to detect rare variants single base variants (exome sequencing – NGS) Previous Results New generation sequencing era New generation sequencing (NGS) wiki Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:110-122 Copyright © American Heart Association, Inc. All rights reserved. Variant calling NGS technologies have inherent error – uncertainty in the data Benjamin Meder et al. Circ Cardiovasc Genet. 2011;4:110-122 Copyright © American Heart Association, Inc. All rights reserved. Genotypes BB or AB or AA Locus 4 Alleles at locus 4 A B Each somatic cell is diploid Locus: chromosomal location that’s polymorphic. Alleles: different variants @ locus (two copies of each autosome) Thus, 3 genotypes at locus 4 Assembly and mapping Assembly and mapping depends on the quality of the reference genome sequence Association tests Opportunities and challenges of the analysis of sex chromosomes’ variants by NGS Opportunities 1. Sex chromosomes are prime candidates for variants involved in male reproduction – support from previous results 2. Biological properties of the Y chr allow specific strategies Before eggs and sperm are formed mom and dad’s chromosomes exchange information DAD’s CHR MOM’s CHR our children will carry new combinations of alleles Not on the Y chromosome!!! Y chr is inherited in a block accumulating mutations linearly with time Zhang M, Wang CC, Yang C, Meng H, Agbagwa IO, et al. (2016) Epigenetic Pattern on the Human Y Chromosome Is Evolutionarily Conserved. PLoS ONE 11(1): e0146402. doi:10.1371/journal.pone.0146402 http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0146402 A group of mutations defines a haplogroup (lineage) Well defined phylogeny (tree of related sequences) Using haplogroup information to call new variants Challenges 1. Variant calling Y chr is hemizygous (one copy in males) – lower signal-to-noise ratio 2. Variant filtering to eliminate false positives 3. Mismapping - Y chr sequence is still incomplete and has many repetitive regions - Homology to X chr in some regions Até 31 de Março 2016!! Contactem-nos!! Alexandra Lopes [email protected] Eduardo Sousa UM/FCUP [email protected]