ChromAbn_GenomicRearrangmts_slides_only.pdf

Chromosome abnormalities and genomic rearrangements  Milestones in genetics 1842 - chromosomes observed in plant cells 1850/1860 - Mendelian inheritance 1880-1900 – chromosomes established as the vectors of heredity 1923 – human chromosome number established as 48 1953 - Watson,...

Chromosome abnormalities and genomic rearrangements  Milestones in genetics 1842 - chromosomes observed in plant cells 1850/1860 - Mendelian inheritance 1880-1900 – chromosomes established as the vectors of heredity 1923 – human chromosome number established as 48 1953 - Watson, Crick & Franklin 1956 – Tjio and Levan !46 chromosomes! Aneuploidy = wrong number of chromosomes  Milestones in medical genetics 1959 – chromosomal basis of Down, Turner and Klinefelter syndromes established 1960 – chromosomal basis of Patau and Edwards syndromes established 1962 – Guthrie test 1969 – chromosome banding 1977 – dideoxy (Sanger) DNA sequencing 1983 - PCR The study of chromosomes is called cytogenetics: – number – structure – deletions – duplications – instability Types of chromosome abnormality: chromosome rearrangements whole chromosome aneuploidy copy number imbalance Techniques used to investigate chromosomes Traditional cytogenetics: G-banding cell culture required to collect Some FISH metaphase cells Breakage Molecular cytogenetics: QF-PCR tests carried out on DNA MLPA Array CGH G-banded chromosomes  Chromosomal abnormalities Chromosome rearrangements recurrent miscarriage infertility Copy number imbalance dysmorphism developmental delay learning difficulties specific phenotypes eg epilepsy, diabetes, cardiac malformations Chromosome breakage syndromes Fanconi anaemia, ataxia telangiectasia…  Whole chromosome aneuploidy arises following non-disjunction at mitosis or meiosis large genomic imbalance leads to loss of conceptions, except where the chromosome involved is gene-poor (trisomy 13, 18 and 21 are the only autosomal trisomies that are found at live birth) MEIOSIS I NON-DISJUNCTION MEIOSIS I MEIOSIS II DISOMIC NULLISOMIC 16 17 18 19 20  Mosaicism 47,+21 47,+21 47,+21 Anaphase lag 47,+21 47,+21 47,+21 46,N 47,+21/46,N Alternatively, mosaicism can arise in an initially normal conceptus. This can be due to either non-disjunction or anaphase lag.  Mosaicism Somatic – likely to result in abnormal phenotype. Phenotype may be ameliorated by a normal cell line Gonadal – arises during formation of germ cells and usually only identified following two pregnancies with the same ‘de novo’ abnormality. CPM - mosaicism confined to extraembryonic tissue. Thought to occur in 1-2% of placentas and may go undetected. May be associated with normal outcome or can compromise function of placenta. May result in UPD following trisomy rescue. Chromosome rearrangements Robertsonian translocations reciprocal translocations inversions intrachromosomal insertions ROBERTSONIAN TRANSLOCATIONS result from fusion of two acrocentric chromosomes (13, 14, 15, 21, 22) prevalence of 1 in 1000 most common are der(13;14) and der(14;21) balanced carriers phenotypically normal balanced carriers have reproductive risks – present as – recurrent miscarriages – Patau syndrome – Down syndrome – male infertility 45,XX,der(14;21)(q10;q10) Robertsonian translocation alternate segregation Robertsonian translocation 15% risk of trisomy 21 for female carriers adjacent segregation  Reciprocal translocations can be between any segments of any non-homolgous chromosomes prevalence of 1 in 500 almost always unique to the family only one recurrent RT: 46,XX,t(11;22)(q23.3;q11.2) balanced carriers phenotypically normal balanced carriers have reproductive risks – dependent on size of translocated segments – infertility – miscarriage – child with congenital anomalies Reciprocal translocation: 46,XX,t(12;17)(p13;p13)  Reciprocal translocation alternate segregation normal balanced  Reciprocal translocation adjacent 1 segregation unbalanced unbalanced  Reciprocal translocation 3:1 segregation unbalanced unbalanced Chromosome inversions Pericentric inversion Paracentric inversion  A B C D A B C A A C B D D C B D Paracentric inversions: meiosis G-banding – resolution ~5-10Mb Fluorescence In Situ Hybridization (FISH) developed in 1980s, became a vital part of genetic testing. different types of probe with range of applications.  Fluorescence In Situ Hybridization (FISH) fluorescent marker DNA probe target DNA (complementary to target sequence) denaturation (separation of double-stranded DNA):  Investigation of structurally abnormal chromosomes using FISH whole chromoso me paints subtelomere probes mbanding DiGeorge, VCFS, del22q11 22 del(22) TUPLE 1 locus Control probe locus  Prenatal cytogenetics Traditional test: – G-banded chromosomes BUT - ~2 weeks for results – results needed more quickly for reassurance/pregnancy management FISH QF-PCR Chromosome counting by QF-PCR: 3 5 disomy 1:1 3 3 5 trisomy 2:1 3 4 5 trisomy 1:1:1 3 3 uninformative 1:1 or 1:1:1 Trisomy 21  Dad fetus MumMUM Segmental copy number imbalance normal copy number = 2 (except for sex chromosomes) deletions x1 or x0 duplications x3 triplications x4 …….. G-banded chromosome analysis has a resolution of ~5-10Mb Many small imbalances (microdeletions) cause syndromic disease.  Microdeletion syndromes DiGeorge - del(22q) Prader-Willi – del(15q) Williams – del(7q) Wolf-Hirschhorn – del(4p) Angelman – del(15q)  Microdeletion syndromes Submicroscopic chromosomal deletions 100 kb – 3,000 kb Recognizable as syndromes because they recur at low frequency in all populations Due to genomic structure at the disease locus – predisposes to gene deletion-duplication by unequal recombination Mediated by ‘low copy repeats’ Array CGH: Test DNA Reference DNA Array = many DNA probes attached to a glass slide DNA probes bind to complementary DNA sequences  Oligonucleotide arrays 60,000 probes Red spot – more test material compared to control material, indicating duplication in patient’s genome Green spot – more control material relative to test material, indicating deletion in patient’s genome Array CGH can detect: – whole chromosome aneuploidy – microdeletion/duplication syndromes – subtelomere imbalance – other regions of imbalance (copy number variants, CNVs)  ?benign  ?pathogenic  Human variation research studies using array CGH have shown that “normal” individuals carry multiple small CNVs different combinations of these CNVs may contribute to phenotypic variation between individuals. ?normal genotype Redon et al Nature 2006 444(7118): 444–454. Many patients have imbalance for genes of unknown function, or genes whose function is apparently unrelated to the referral indication. Some of those genes may be associated with other clinical phenotypes, or may predispose to malignancies. How to ascertain the clinical consequence of a previously unreported imbalance? inheritance – de novo - more likely to be pathogenic – inherited from affected parent - more likely to be pathogenic – inherited from unaffected parent – more likely to be benign number of genes (burden) specific gene content (?correlation with phenotype) CNV1 X X CNV2  X CNV1 XX CNV2 resolution If we find imbalance involving a cancer gene, what should we do? Report? raise anxiety in the family → extensive and expensive monitoring Don’t report? miss an opportunity to make a real difference to future health/lifespan  G-banding FISH MLPA Array CGH Whole exome/genome sequencing 1956 1969 2008 2011/12