Principles of clinical cytogenetics

1、Principles of clinical cytogeneticsDepartment of Medical GeneticsWanghuiDefinition Clinical cytogenetics: is the study of chromosomes,their structure and their inheritance,as applied to the practice of medical genetics.Introduction Chromosome disorders form a major category of genetic disease. They

2、account for a large proportion of all reproductive wastage, congenital malformations,and mental retardation. They play an important role in the pathogenesis of maligancy. Specific chromosome abnormalities are responsible for more than 100 identifiable syndromes. Cytogenetic disorders are present in

3、nearly 1 percent of live births. Cytogenetic disorders are present in about 2 percent of pregnancies in woman older than 35 years. Cytogenetic disorders are present in fully half of all spontaneous first-trimester abortions.Indications for cytogenetic analysis Problems of early growth and developmen

4、t Stillbirth and neonatal death Fertility problems Family history Pregnancy in a woman of advanced ageNeoplasiaChromosomesDefinitionnGenetic structures of cells containing DNAIdentificationnEach chromosome has a characteristic length and banding patternThe breakdown of a ChromosomenEach autosome is

5、numbered from 1-22, sex chromosomes either X or Yp arm (short arm)q arm (long arm)CentromereMetacentricSub-metacentricAcrocentricMorphology of chromosomesA KaryotypeDefinitionnA photographic arrangement of a complete set of chromosomes of a cell or organism 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

6、18 19 20 21 22 X YImportance of KaryotypesnKaryotypes show the chromosomal makeup of an individual. Knowing the number and structure of chromosomes is essential for identifying chromosomal variations that cause genetic disordersObtaining a samplenRequirementsnCells must be capable of growthnCells mu

7、st be rapid divisionnMost common used cellsnWhite blood cellsChromosome preparationnCollect white blood cells and stimulate to divide;nCulture white blood cells for three days;nArrest the dividing cells in metaphase ;nRelease chromosomes with a hypotonic solution;nFixed,spread and stain chomosomes O

8、btaining a KaryotypenChromosomes are stained for easy visualizationnLight microscope used to view chromosomes in metaphase of mitosisnChromosomes arranged into homologous pairs based on size and banding patternsCytogenetic techniqueObtaining a fetal SamplenFetal samples for cytogenetic analysis are

9、commonly obtained in two ways1. Amniocentesis sample taken from the fluid of the amniotic sac2. Chorionic Villus Sampling sample taken fetal tissue that forms part of the placentaAmniocentesisChorionic villus samplingChromosome identification The 24 types of human chromosomes can be readily identifi

10、ed by a number of specific staining procedure,besides Giemsa banding ,other procedures used in some laboratories or for specific purposes including the following:Q banding,R banding,C banding and high-resolution banding. StainingBanding patterns can be visually identified on chromosomes after staini

11、ng.Traditional Typesn G-Banding Giemsa stainn Q-Banding Fluorescent stainn R-Banding Reverse Giemsa stainNew Typen Fluorescence In Situ Hybridization techniquesG Banding The technique most widely used in clinical cytogenetics laboratories.The chromosomes are treated first with trypsin to digest the

12、chromosomal proteins and then with Giemsa stain.Each chromosome pair stains in a characteristic pattern of light and dark bands.Q Banding This method requires staining with quinacrine mustard or related compounds and examination by fluorescence microscopy,is particularly useful for detecting occasio

13、nal variants in chromosome morphology or staining,called heteromorphisms.Q bandingG bandingR Banding If the chromosomes receive special treatment before staining such as heating,the resulting dark and light bands (R bands) are the reverse of those produced by G banding.It is especially used when exa

14、mining regions that stain poorly by G banding. R BandingG bandingC Banding This method specifically involves staining the centromeric region of each chromosome and other regions containing constitutive heterochromatin namely,sections of chromosomes 1q,9q,and 16q adjacent to the centromere and the di

15、stal part of Yq.R Banding C BandingHigh-Resolution Banding This type of banding is achieved through G-banding or R-banding techniques to stain chromosomes that have been obtained at an early stage of mitosis(prophase or prometaphase),when they are still in a relatively uncondensed state.this method

16、is especially useful when a subtle structural abnormality of a chromosome is suspected.High-Resolution BandingChromosome LabelingChromosome is identified with a number ranging 1-22, or X and YEach arm divided into sub-regions and identified by a numberEach sub-region divided into bands identified wi

17、th a numberIdeogram of chromosome99p129p21Example 1p31,nThe first chromosome, nShort arm, third region nthe first band1q42Fluorescence In Situ HybridizationnProbes can be hybridized to the DNA contained within chromosomes immobilized on microscope slides.This technique is called in situ hybridizatio

18、n.The most common method of labeling probes for in situ hybridization to chromosomes is with a fluorescent dye while the chromosomes are viewed with a fluorescence microscope.Mechanism of situ hybridizationThree-color FISHKaryotype:46,XY,t(11;16)(q24;q23)Del 15q11-13Chromosome AbnormalitynNumerical

19、abnormalityn Euploidn AneuploidnStructural abnormalityn Balanced n UnbalancedHeteroploidGeneral rules to chromosome abnormalitiesnLoss is more severe than gain.nGain or loss of sex chromosomes are tolerated much better.n In most cases are de novo mutations. Numerical abnormalitynA chromosome complem

20、ent with any chromosome number other than 46 is called heteroploid.nAn exact multiple of the haploid chromosome number is called euploid.nAny other chromosome number is aneuploid.EuploidnTriploid:Three chromosome sets are involved in an nucleus (3n).The karyotype can be 69,XXY,69,XYY,or 69,XXX.nTetr

21、aploid:Four chromosome sets are involved in an nucleus (4n).The karyotype are always 92,XXXX or 92,XXYY.MechanismnTriploid DispermyDiploid egg or sperm三极纺锤体DispermyDiploid eggFormation of TrioploidThe origin of triploidMechanismnTetraploidnEndomitosis- Chromosomal replication without nuclear or cell

22、ular division that results in cells with many copies of the same chromosome. EndoreduplicationEndomitosis Endoredupliation:repeated replication of DNA during the S phase of the cell cycle without forming new nuclei in telophase. (Endoreduplication is also known as endoreplication.)AneuploidynMonosom

23、y-Only one representative of a particular chromosome (2n-1).nTrisomy-Three instead of normal pair of a particular chromosome (2n+1).nMultiple aneuploidy-Has an extra representative of more than one chromosome (2n+2).Monosomy-45,XTrisomy-47,XX,+21Viable aneuploidy in humans Outcome of 10,000 concepti

24、onsMechanismnNondisjunction:refer to the failure of a pair of chromosomes to disjoin properly during one of the two meiotic divisions,usually during meiosis.nPremature separation:premature separation of sister chromatids in meiosis instead of meiosis .The separated chromatids may by chance segregate

25、 to the oocyte or to the polar body,leading to an unbalanced gamete. Gain or loss of sex chromosomes are tolerated much better. why?Dosage compensation inhuman X-inactivationnOne copy of the X chromosome is inactivated in females of placental mammals.nObservation: patches of coat color of calico cat

26、s.n“X-inactivation center”(XIC) on XqBarr bodyNot all genes on X chromosome are inactivatiedAbnormalities of chromosome structurenStructure rearrangements result from chromosome breakage,followed by reconstitution in an abnormal combination.structure abnormalities are present in about 1 in 375 newbo

27、rns.nBalanced structure rearrangement:if the chromosome set has the normal complement of chromosome material.nUnbalanced structure rearrangement:if there is additional or missing material in the chromosome set. Unbalanced rearrangementnDeletions nDuplicationsnRing chromosomesnIsochromosomesnDicentri

28、c chromosomesDeletionnLoss of some segment of a chromosomenMost are lethal or cause serious disorderTerminal deletion46，XX,del (1) (q21)46，XX,del (1) (pterq21）Interstital deletion46,XX,del(3)(q21q31）46,XX,del(3)(pter q21:q31 qter)Unequal crossing over between misaligned homologous chromosomesduplica

29、tiondeletionDeletionDuplicationnGene sequence that is repeated several to hundreds of times noriginate by unequal crossing over or by abnormal segregation from meiosis in a carrier of a transolcation or inversion.nMay have adaptive advantagenPhenotypes appear to be associated with duplications of pa

30、rticular chromosomal region.Duplicationnormal chromosomeone segment repeatedthree repeats DuplicationMarker and ring chromosomesnVery small,unidentified chromosomes,called “marker chromosome”. referred to as supernumerary chromosomes or extra structurally abnormal chromosomes(ESACs). consist of litt

31、le more than centric heterochromatincontain some material from one or both chromosome arms.lack identifiable centromeric DNA sequences,including alpha satellite Ring chromosomesnRing chromosomes are formed when a chromosome undergoes two breaks and the broken ends of the chromosome reunite in a ring

32、 structure.Ring chromosomeIsochromosomesnIs a chromosome in which one arm is missing and the other duplicated in a mirror-image fashion.A：46,X,i(Xp)46,X,i(ptercenpter)B:46,X,i(Xq)46,X,i(qtercenqter)Dicentric chromosomesnIs a rare type of abnormal chromosome in which two chromosome segments ,each wit

33、h a centromere ,fused end to end,with loss of their acentric fragments.Banlanced rearrangementsnInversionsnTranslocationsninsertionsInversionsnAn inversion occurs when a single chromosome undergoes two breaks and is reconstituted with the segment between the breaks inverted. paracentric-not inculding the centromere pericentric-including the centromereParacentricA 1 2 3 4 5 6 7 8 9 10 B 1 2 6 5 4 3 7 8 9 10配 子 类 型a 1 2 3 4 5 6 7 8 9 10b 1 2 3 4 5 6 2 1 c 1 2 6 5 4 3