2What are ChromosomesChromosomes are structures found in the nucleus of cellsChromosomes carry all of our genes, and therefore all of our genetic informationHumans have 46 chromosomes, or 23 pairs, to carry our approximately 25,000 genesThe first 22 pairs are called autosomesThe 23rd pair are the sex chromosomes or gonosomes this pair will either be XX or XY
3KaryotypingDuring mitosis, the 23 pairs of human chromosomes begin condensing during prophase, and reach their most condensed state during metaphaseA karyotype analysis usually involves blocking cells in mitosis and staining the condensed chromosomes with Giemsa dye.The dye stains regions of chromosomes that are rich in the base pairs Adenine (A) and Thymine (T) producing a dark band
4KaryotypesA karyotype is a pattern or picture of chromosomes. The chromosomes are paired and arranged according to sizeEach chromosome is paired with its homologous chromosomeits exact match in size and structure, though the homologous chromosomes may carry different alleles of the same geneThen the chromosome pairs are labeledThe autosomes are numbered 1 through 22 according to sizeThe sex chromosomes are a different storyThe X and X or the X and Y are paired, then placed at the end, even though they are not necessarily the smallest chromosomesThese chromosomes do not receive a number - just XX or XY
6Human KaryotypingA karyotype is an actual photograph of the chromosomes from one cell.Karyotypes are usually done using blood cells, fetal skin cells (from amniotic fluid or the placenta) and occasionally bone marrow cellsIt takes at least one week to complete a karyotype
7Clinical Steps to Karyotyping 1. Sample Collection2. Separating cells3. Growing Cells4. Synchronizing Cells5. Releasing Chromosomes from their cells6. Staining Chromosomes7. Analysisa. Counting & Sorting of chromosomesb. Looking at Chromosomes Structure
8Sample CollectionIn newborns, a blood sample which contains red bloods cells, white blood cells, serum and other fluids is collected.A karyotype will be done on the white blood cells which are actively dividing (a state known as mitosis).
9How do we get blood samples from a fetus During pregnancy, the sample can either be amniotic fluid collected during an amniocentesisThe amniotic fluid contains fetal skin cells which are used to generate a karyotypeor a piece of the placenta collected during a chorionic villi sampling test (CVS).The important thing is to choose a cell type that is actively dividing, some cells, like nerve cells, divide infrequently or never!
10Separating the CellsIn order to analyze chromosomes, the sample must contain cells that are actively dividing (or in mitosis).In blood, the white blood cells are actively dividing cells.Sometimes adult cells are treated with chemicals such as phytohemaglutenin (PHA) to induce mitotisMost fetal cells are actively dividing.Once the sample reaches the cytogenetics lab, the non-divided cells are separated from the dividing cells using special chemicals.
11Growing CellsIn order to have enough cells to analyze, the dividing cells are grown in special media or a cell culture.This media contains chemicals and hormones that enable the cells to divide and multiply.This process of “culturing” the cells can take 3 to 4 days for blood cells, and up to a week for fetal cells
12Synchronizing CellsThe best mitotic phase for chromosome analysis is prometaphase or metaphaseIn order to get all the cells to this specific stage of cell division, the cells are treated with a chemical which stops cell division at the point where the chromosomes are the most compact.cholicine
13Releasing Chromosomes From Their Cells In order to see these compact chromosomes under a microscope, the chromosomes have to be out of the white blood cells.This is done by treating the white blood cells with a hypotonic solution that causesChromosomes to spread apart for easier viewingAnd causes cells to burstThis is done while the cells are on a microscopic slide. The leftover debris from the white blood cells is washed away, and the chromosomes are fixed to the slide.
14Staining the Chromosomes Chromosomes are naturally colorless.In order to be able to tell one chromosome from another, a special dye called Giemsa dye is applied to the chromosomes on the slide.Giemsa dye stains regions of chromosomes that are rich in the bases adenine (A) and thymine (T).When stained, the chromosomes look like strings with light and dark bands.Each chromosome has a specific pattern of light and dark bands which enables cytogeneticist to tell one chromosome from another.
15G Banding 1st use a protease to digest any protins Giemsa reagent consists of a mixture od dyes includingAminophenothiazine dyes(basic)Eosin (acidic)Produces light & dark bandsPatterns of banding change with the stage of mitosisThere may be as many as 1,000 bands in early prophase, and as few as 300 on a given chromosome in metaphase
16G Banding GC rich Most genes are in light bands Dark BandssGC richMost genes are in light bandsLight bands represent relatively open (uncoiled) regions of DNA making transcription easierAT richHighly coiled regions of DNAContains few genesMost genes in these regions are tissue specific, and so not transcribed in all cells
17AnalysisOnce chromosomes are stained, the slide is put under the microscope and the analysis of the chromosomes begins.A picture is taken of the chromosomes and at the end of the analysisThis is known as a “metaphase spread”Take a picture of the spread using a special microscope with attached camera
19Critical Thinking!When we draw chromosomes, we typically draw them in the shape of the letter XWhat does this represent (ie, label the important parts of the X structure)Why, in the above metaphase spread are the chromosomes NOT in the X shape, but rather linear?
20Analysis: Step 1The first step of the analysis is counting the chromosomes.Most humans have 46 chromosomes.People with Down syndrome have 47 chromosomes.It is also possible for people to have missing chromosomes or more than one extra chromosome.By looking at just the number of chromosomes, it is possible to diagnose different conditions including Down syndrome, Turners, Kleinfelters
21Analysis: Step 2 Sort the chromosomes by comparing chromosome length,the placement of centromeres (the areas where the two chromatids are joined),the location and sizes of G-bands.The chromosomes pairs are numbered from largest (number 1) to smallest (number 22).There are 22 pairs of chromosomes, called autosomes, which match up exactly.2 sex chromosomestwo X‘s is a femaleX and a Y is a male.
22Analysis: Step 3In addition to looking at the total number of chromosomes and the sex chromosomes, the cytogeneticist will also look at the structure of the specific chromosomes to make sure that there is no missing or additional material, no structural abnormalities likeTranslocationsDeletionsDuplications
23What we will doUnfortunately in high school we aren’t allowed to stick needles in our students and/or work with human bodily fluidsPossible spread of infectious diseasParents complainWe will practice karyotyping from the analysis standpoint
243 Criteria used to Identify a Chromosome The analysis involves comparing chromosomes for theirLengthplacement of centromeres (areas where the two chromatids are joined)location and sizes of G-bands.
25Typical Chromosome Structure Each chromosome has 2 arms separated by a constriction point called the centromereMetacentric: term used to describe chromosomes with centromere at or near the center of the chromosomeSubmetacentric: term used to describe chromosomes with centromere slightly displaced from the center
26Typical Chromosome Structure Very submetacentric: term used to describe chromosomes with centromere halfway between the middle and the tip of the chromosomeAcrocentric: term used to describe chromosomes with centromere very near one end
28Typical Chromosome Structure Telocentric: term used to describe chromosomes with centromere at the very tip of one endThese are absent in human chromosomes
29Typical Chromosome Structure Notice, the centromere divides the chromosome into 2 separate regionsIn generalThe shorter chromosome arm is designated p for petiteThe longer arm is designated the q arm, because the letter q comes after the letter p in the alphabet
32How To Start Open your worksheet titled Key to Human Chromosomes It is a dichotomous keyStart by cutting chromosomes out of your metaphase spreadDivide all chromosomes into one of the 2 class sizesClass 1 = 1st 12 chromosome pairs (24), and X chromosomesSo class 1 will contain 25 chromosomes for a boy and 26 chromosomes for a girlClass 2 = the rest of the smaller chromosomes
33Nomenclature Normal female 46, XX Normal Male 46, XY Telomeres, centromeres, and a number of prominent bands areused as landmarks on a chromosome
34NomenclatureA section of a chromosome between 2 landmarks is called a regionRegions are numbered 1,2,3 in both directions starting with the centromereBands within a region are numbered according to the same ruleExample: the 1st band in the 2nd region of the p arm of chromosome #1 is indicated as follows1p21
35So what does this mean: 14q32 14 refers to chromosome # 14q refers to the q arm (the longer arm) of chromosome 143 refers to region 3 of the q arm of chromosome 14And 2 refers to the 2nd band in the 3rd region of the q arm in chromosome 14
36Nomenclature There may also be sub-bands Decimal points are used to indicate what sub-bandExample: 14q32.3Refers to the 3rd (last) sub-band within the 2nd band of region 3 in the q arm of chromosome 14
37Numerical Chromosome Abberations for Sex Chromosomes 45, X 45 chromosomes, 1 X chromosome47, XXY 45 Chromosomes, two X chromsomes one Y chromosome
38Numerical Chromosome Abberations Use a + or – signbefore the symbol to indicate additional or missing whole chromosomesAfter a symbol to indicate additional or missing segments of a chromosome47, XY, +21 an additional 21st chromosome46,XX, 1q+ female karyotype with 46 chromosomes showing an increase in the length of the long arm of chromosome 1
39Nomenclature Question marks are used to indicate uncertainty 45, XY, -?845 chromosomesXY is maleMissing a chromosome, probably #8There is special nomenclature for inversions, deletions, translocations, insertions, etc.; but its is beyond the scope of this class
40Other Types of Staining C-Banding: stains constitutive heterochromatinRegions of chromosomes that are highly compacted containing highly repetitive DNAResults from selective removal of the chromosomes except from regions of the C-BandsUsed for determining presence or absence of a centromere, as well as for studying abnormalities of the centromeric region
41Other Types of Staining Silver Staining for NORRibosomal DNA, which codes for ribosomal RNA, occurs in multiple copies on the short arms of all acrocentric chromosomesSilver stain results in the deposition of silver grains on the active nucleolar organizer regions (NORs)In other words, NORs which were actively transcribing their rRNA’s in the preceding interphase, contain the proteins to which thre silver grains bind
42FISH Fluorescence In Situ Hybridization Involves precise annealing of ssDNA probes to complementary target sequencesCan be visualized with a fluorescent microscope by using a probe directly labeled with a fluorophoreCan be used to findIf a specific gene is present (and or duplicated)And if it is in the correct region of the correct chromosome
45WCP Whole Chromosome Painting Same idea as FISH, but you use a mixture of many fluorophore labeled probesMust also use unlabeled blocking DNA to supress highly repetitive regions common to many different chromosomesAllows forChromosome enumerationIdentification of chromosomal structural rearrangements
47WCPParticularly useful in solid tumor and linical genetic research because it has the ability to aid in the analysis of complex and cryptic translocationsAlso helpful in research that is evaluating the extent of chromosomal breakage and translocations following exposure to mutagenic chemicals or radiation
49Using A KaryotypeA karyotype allows a cyto-geneticist or lab technician to examine the chromosomes and see if there is anything extra or missing, or if the structure of the chromosomes is grossly different than usual
50Trisomy 21 Trisomy 21 is the presence of 3 chromosome 21’s. Trisomy 21 causes the condition commonly known as Down syndromeThe extra chromosome leads to the specific characteristics of Down syndrome, some of which are very familiarnot all individuals with Down syndrome will show the exact same characteristics there is a great deal of variability
51Trisomy 21/Down syndrome Individuals with Down syndrome have a typical facial appearanceAll have some degree of mental retardation, but for most it is mild or moderateCan learn to read, write, do some math, and live day-to to-day with minimal assistance from othersOthers require a lot of attention and careThere are several possible health concerns, includingheart problems, hearing loss, feeding concerns, and othersIndividuals with Down syndrome are now living longer than in the past – into their 50’s and 60’sWe are now discovering that those individuals who survive to this age are at very high risk of developing Alzheimer Alzheimer’s disease
52Trisomy 13Trisomy 13 is the presence of 3 chromosome 13’s, and causes the condition sometimes known as Patau syndromeTrisomy 13 is a very serious condition with only about 5% of babies with the disorder survive past their first yearMost pregnancies involving Trisomy 13 end in miscarriageAll individuals with Trisomy 13 have severe mental retardationOther characteristics commonly seen in people with Trisomy 13 includea small headextra fingers and/or toesand a cleft lip or cleft palateTrouble breathing, especially in their sleepseizures
53Trisomy 18Trisomy 18 is the presence of 3 chromosome 18’s, & causes the condition sometimes known as Edwards syndromeOnly about 10% of babies with the disorder survive past their first year.A majority of babies who survive are female.Most pregnancies involving Trisomy 18 end in miscarriage
54Trisomy 18Children with Trisomy 18 usually have breathing problems, difficulty eating, and many have seizures. Some have serious heart conditions.All individuals with Trisomy 18 have severe mental retardation.Most babies with Trisomy 18 are very small and have certain recognizable facial features. They also tend to overlap their fingers in a very distinct pattern
5547, XXYAn individual with the genotype 47, XXY is male The person has 47 chromosomes just like someone with Trisomy 21, 13, or 18, but does not have a typical “trisomy,” as he has two of one chromosome and one of anotherOne could say, though, that such an individual has a sex chromosome trisomyThe extra X chromosome leads to features of the condition commonly known as Klinefelter syndrome
57Klienfelters Syndrome Affects about 1 in 1000 malesMost males are taller than average and may have a different distribution of body fat (e.g. more than usual in the hips or chest)Also tend to have sparse facial and body hairSome have a degree of mental retardation, but many have normal intelligenceThe most common feature is infertilityIt is estimated about 2% of infertile men have Klinefelter SyndromeSince the characteristics of the syndrome are not always obvious, many males with Klinefelter will never be diagnosed
5845, X An individual with the genotype 45, X is phenotypically female The person has 45 chromosomes instead of the usual 46 Instead of a trisomy, this would be called a monosomy XOne copy of an X chromosome = monosomyMonosomy X is the only monosomyknown to be compatible with lifeHaving only one copy of an X chromosome leads to the features of the condition known as Turner syndrome
60Turner Syndrome Affects about 1 in 5000 newborn females Physical featuresSwelling of the hands and feetwebbing of the neckbroad chestMay also have features which affect their health, includingheart diseasea“horseshoe-shaped”kidneyIndividuals with Turner syndrome will not typically have mental retardation, but may have specific learning disabilities
61Discussion QuestionsWhy would the presence (or absence) of a chromosome lead to certain characteristics in a person?Why wouldn’t the physical, mental, and behavioral features be exactly the same for two people with the same extra (or missing) chromosome?Why are babies with trisomy 21 more likely to survive than babies with trisomy 13 or 18?Why do you think monosomy X (Turner syndrome) is the only monosomy in which the individual survives?