Patho1-5_Cytogenetics.pdf

PATHOLOGY Dr. Araceli jacoba

July 11, 2011

GENETIC DISORDERS A. Chromosomal/Cytogenic Chromosomal/Cytogenic Disorders Disorders   of autosomes and  – A b n o r m a l i t y i n n u m b e r a n d s t r u c t u r e  of sex chromosomes

 –  Uncommon  – Results from

genome mutations mutations  – Normal (2n): 46XX or 46XY

or

chromosomal

B. Mendelian Disorder  – Disorders related to mutation in single genes  – Highly penetrant (means presence of mutation is associated with the disease in a large proportion of individuals) as either:  – Classified as 1. Autosomal dominant/recessive dominant/recessive 2. X-linked dominant/recessive C. Multigenic Multigenic Disorders multiple genes with environment environment  – Combination of multiple without Mendelian transmission transmission  – Family clustering without  – P o l y g e n i c i n h e r i t a n c e :  Manifestation of disorder is controlled by environmental factors such as diet, exercise, salt intake, exposure to the disease  – Dosage Effect : More number of deletious gene, more severe expression of the disease  –  e.g. Diabetes mellitus, Carcinoma, HPN, Heart disease, Intelligence D. Single Gene Disorder Disorder W/ Non-Mendelian Inheritance Inheritance  –  Involve only a single gene but transmission is neither autosomal dominant/recessive nor X-linked dominant/recessive  – Classified into 4 groups: 1. Caused by trinucleotide repeat repeat mutation 2. Mutations in mitochondrial genes 3. Associated with gene gene pairing 4. Associated with gonadal mosaicism  – e.g. Fragile X Syndrome

2. FISH (Fluorescent In Situ  Hybridization)  Hybridization) chromosome  – Fluoresce a certain segment of chromosome  – Interphase (when not dividing) cytogenetics  –  Uses DNA probes that recognizes sequences that are specific to particular chromosomal region fluorescent dyes and applied  – The clones are labelled with fluorescent to metaphase spreads or interphase nuclei  – The probe hyberdizes to its homologous genomic sequence and labels a specific chromosomal region that can be seen under fluorescent microscope  – Sample: blood or urine  –  Reagent: Quinacrine and DNA probe  – Used for: detection of numeric abnormalities of chromosomes, demonstration of subtle microdeletions, complex translocations, analysis of gene amplifications and mapping newly isolated genes of interest 3. Banding Technique  – Identification of chromosome by its banding pattern (according to its alleles) alleles ) same as karyotype karyotype (old method)  – Basically the same  In banded karyotypes, each arm of the chromosome is divided into 2 or more regions bordered by prominent bands  Short arm of chromosome is designated p  and long term is referred as q  Each arm of chromosome is divided into two or more regions  The regions are numbered from centromere outward  Each region is divided into bands and sub-bands which are also numbered.  e.g. Xp21.2= Xp21.2= Chromosomal segment located on the short arm of X chromosome, in region 2, band 1 and sub-band 2. (refer to the figure below)  – Stain: Giemsa (G banding)  – Best done at prophase

Diagnosis of Genetic Disorders 1. Karyotype  –  Standard chromosome spread after arresting mitosis at metaphase arranged in chromosomal pairs in order of decreasing length (numbered or labelled with letters) at the end of the karyotype  – Sex chromosome placed at Unidentifiable chromoso chromosomes mes are also placed at the end  –  – Chromosomes grouped into letters (AG) are chromosomes having the same appearance (length, centromere location etc.) described using short hand system of  – Karyotypes usually described notations (total number of chromosomes are stated first, followed by sex chromosome, and finally the description of abnormality)  e.g. Male with Trisomy 21 disorder = 47, XY, +21  – Sample: Blood  –  Arrests  –  Method: Mitotic spindle inhibitor (colcemid)  –  dividing cells

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4. Barr Body or X chromatin  – Inactive X chromosome  – Number of sex chromosome minus active X  e.g. 4 Barr bodies  5 X chromosomes  49 Info Here (Text box)  – Pyknotic material at the edge of nuclei  – Seen in interphase nucleus as darkly staining small mass in contact with nuclear membrane  – Basis of inactivation involves gene called XIST, “coats” X chromosome that it is transcribed from and initiates a gene-silencing process by chromatin  – Sample: Buccal smear   – Only seen in females  – One X active and the other X is inactive (males only have inactive X)  – Modification and DNA methylation  – Sample: Buccal smear 

3. Gene Mutation  – Partial or complete deletion of a gene  –  Produce Mendelian disorders a. Point Mutations within coding sequences  Alter

the code in a triplet of bases and lead to the replacement of one amino acid by another in the gene product  Often termed missense mutations  2 types: a. Conservative (substituted AA causes little change in function) b. Nonconservative (normal AA replaced by a very different one)  Example: Sickle mutation  affecting B-globin chain of haemoglobin. CTC (glutamic acid) changed to CAC (valine) gives rise to sickle cell anemia  Can also change an amino acid codon into a chain terminator/stop codon (nonsense mutation)  Example: Premature termination of B-globin gene translation gives rise to B-thalassemia b. Mutations within noncoding sequences  Mutations that  May interfere

do not involve exons binding of transcription factors leading to reduction/total lack of transcription  Example: Hereditary anemia  May also lead to defective splicing of intervening sequences resulting to failure to form mature mRNA

Fig. Barr bodies shown in nuclei of cells from buccal smear

c . D e l et i o n s a n d i n s e r ti o n s : F r a m e s h i f t M u t a t i o n s

Note:

 Small

deletions or insertions involving the coding sequence in the reading frame of a DNA strand

: Barr bodies 

Condensed substance that is adherent to nuclear border, based upon LYONS HYPOTHESIS  In peripheral smears   Dumbell-likebodies  Best to assess with buccal smear 

d . T r i n u c l e o t i d e -r e p e a t m u t a t i o n s  Characterized

by amplification of a sequence of three nucleotides

L y o n s H y p o t h ei s 1. Only 1 of the X chromosome is genetically active 2. The other X chromosome (from either paternal or maternal origin) undergoes heteropyknosis to become the Barr body (inactive) 3. Inactivation of either maternal or paternal X occurs at random th among all cells of blastocyst on or about the 16   day of embryonic life 4. Inactivation of the same X chromosome persists in all cells derived from each precursor cell

Types of Mutations 

Mutation – Permanent change in the DNA  Those that affect germ cells usually give rise to inherited diseases while those that affect somatic cells usually are important in genesis of cancers or congenital malformations 1. Genome Mutations  – Subtraction or addition of whole chromosomes  – Affects number  –  Produce cytogenic disorders 2. Chromosome Mutations  – Rearrangement of genetic material structural changes  – Affects chromosome segments  –  Produce cytogenic disorders

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Chromosome

Note: Genome Mutations and Chromosome Mutations are the causes of abnormalities in chromosome number

Genome Mutations Terms: 1. Euploid – Any exact multiple of the haploid number 2.  Aneuploidy – Cell acquires a chromosome complement that is not an exact multiple of 23 3. Mosaicism  –  Condition in which mitotic errors in early development give rise to two or more populations of cells with different chromosomal complement in the same individual A. Non-disjunction  – Failure of chromosome to separate in anaphase  – Can occur in either spermatogenesis or oogenesis (meiosis)  –  Gametes formed extra chromosome (n + 1) or one less chromosome (n – 1).  – Results in 2 types of zygotes: a. Trisonomic (2n + 1) b. Monosomic (2n – 1)

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Note: 

Ex. Chromosome 21 failed to join the other daughter cell (both chromosomes join one cell)  If this is an ova, where it is expected that only half of the Hereis (Text number of Info chromosomes present, box) there will be a double dose of chromosomes  If combined to a sperm with a normal number, the individual after fertilization will be trisomic for chromosome 21  The other cell which didn’t have chromosome 21, when it combine with a sperm of normal number will be monosomic for chromosome 21 i. Meiotic (in parents)

1. Inversion  –  2 breaks in a single chromosome with reincorporation of the inverted, intervening segment  – Paracentric  –  Inversion involving only one arm of chromosome  – Pericentric – Breaks on opposite sides of centromere  – Often compatible with normal development

2. Isochromosomes  – When one arm is lost and the remaining arm is duplicated  –  Leads to a chromosome consisting of two short arms or two long arms  – Have morphologically identical genetic information in both arms  – Most common isochromosome is i(X)(q10)

Fig. Results in Trisomy or Monosomy

ii. Mitotic  Can occur after fertilization (in babies) even when sperm and ova are both normal

Fig. Results in a Mosaic 47 XXX/45XO

B. Anaphase lag  – A chromosome lags behind before cytoplasmic membrane is formed  – One homologous chromosome in meiosis or one chromatid in mitosis lags behind and is left out of the cell nucleus  –  Result: One normal cell and one cell with monosomy

Note:  Chromosome that lag behind + normal sperm = Monosomic  Autosomal monosomies generally causes loss of too much genetic information to permit birth/embryogenesis but several trisomies do permit survival

C h r o m o s o m a l M u t a ti o n s  

3. Deletion  –  Most are interstitial   where there are two breaks within the chromosome arm followed by a loss of chromosomal material between the breaks and fusion of the broken ends  – 46,XX,14p- or 46,XX,del14(p.11.2p13.1)   Describes breakpoints in the short arm of chromosome 14 at 14p11.2 and 14p13.1 with the loss of material between breaks  – Terminal deletions result from single break in chromosome arm producing a fragment with no centromere (lost in next cell division) and a chromosome bearing a deletion

4. Ring chromosomes  –  Break at both ends of a chromosome with fusion of damaged ends  – Expressed as 46,XX,r(14)

5. Translocation  –  Segment of one chromosome is transferred to another 46,XX,t(2;5)(q31;p14)  – No loss of genetic material (if ever, only a small portion) a. Balanced reciprocal translocation  Size of both chromosomes appeared the same after translocation/transfer   There are single breaks in each of the two chromosomes with exchange of material A balanced t ranslocation is of great risk of producing abnormal gametes   Example: 46,XX,t(2;5)(q31;p14)  –  Between the long arm chromosome 2 and one of the chromosome 5 No loss of genetic material, individual likely to be phenotypically normal 

Severity of manifestation depends on volume of genetic material lost  If only a small portion is lost, patient may be asymptomatic and may live  If a significant portion is lost, patient will die after birth or in utero

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b. Robertsonian translocation  Translocation between two acrocentric chromosomes  If unequal in sizes after translocation A translocation between two acrocentric Info Here (Text box) chromosomes The breaks typically occur close to the centromeres of the chromosomes and transfer of segments lead to one very large chromosome and one extremely short one   Loss of small product is compatible with normal phenotype (because it usually carries highly redundant genes) Occurs 1/1000 individual 

Causes: 1. Meiotic Non-Disjunction  – 90% in either parent  – Maternal age affects this disorder  – Occurs in gametogenesis – Parents are normal

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Note: We may not be able to distinguish a balanced translocation in a karyotype (possible on spectral karyotype). It is relatively easy to check what took place in Robertsonian type of translocation.

Cause of Mutations 1. 2. 3. 4. 5.

Increasing age Chromosomal instability Ionizing radiation Drugs Viruses  – HPV, HIV, EBV  – Can change chromosome  unstable  produce cancer 

2 types of chromosome genes affecting carcinogenesis a. Oncogene: Stimulate production of cancer b. Cancer suppressor gene: Inhibits production of cancer

Im ortant Chromosomes

1. Philadelphia chromosome  –  CML  – Translocation of chromosome 9 and 22 st  – 1   chromosome identified that is malignancies 2. Rb gene – 13q14 (point mutation) 3. Burkitt’s lymphoma – t(8;14)(q24) 4. WT-1 gene – 11p13 5. DCC gene – 18q21 6. APC gene – 5q21 7. p53 - cancer suppressor gene 17p13.1 8. NF-1 gene – 17q11.2

associated

with

Note: 1-5 are oncogene; 6-8 are cancer-suppresor gene

CYTOGENENIC DISORDERS (AUTOSOMAL) 1. 2. 3. 4.

Trisomy 21- Down’s Syndrome Trisomy 18- Edward’s Syndrome Trisomy 13- Patau’s Syndrome Chrom. 22 deletion SyndromE

Trisomy 21 (Down’s Syndrome) 

Most common chromosomal disorder Major cause of mental retardation Chromosome count is 47 in 95% w/ trisomy 21  FISH with chromosome 21 specific probes reveals extra copy of chromosome 21  Maternal age- strong influence on the incidence of trisomy 21 o IR of 1:1500 LB – For mothers 45 years old

2. Translocation  – 4%  – t(21,22 or 14)  –  Extra chromosomal material derives the presence of Robertsonian translocation of the long arm of chromosome 21 to another acrocentric chromosome  The translocated material provides the same triple gene dosage as in trisomy 21  The translocated chromosome is inherited from one of the parents 3. Mosaic  – 1%  – Non-disjunction in baby  – individual with more than one cell line  – Having a mixture of cells with 46 and 47 chromosomes  –  Results from mitotic disjunction of chromosome 21 during an early stage embryogenesis.  – Variable and milder symptoms Clinical Features: 1. Mental retardation  –  Severe  – 80% have IQ 25 to 50 2. Facial profile  – Oblique palpebral fissures (slanted upwards)  – Flat facial profile  –  Epicanthic folds (normally stops before inner canthus eyes)  – Dysplastic ears (folded or misplaced downwards)  – Protruding tongue  – Abundant neck skin 3. Motor abnormality  – Loss of Moro reflex (startle reflex)  Initiated by a banging sound (clapping of hands)  – Muscle hypotonia 4. Hands and feet  – S i m e a n c r e a s e   – Straight crease in palm (normal in 3% of population)  – Short broad hands  – Hypoplasia of middle phalanx st nd  – Gap between 1  and 2  toe Note: Mosaics – Phenotypically mild

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Associated Defects: 1. Congenital heart disease  –  40% (atrial septal defects, ventricular septal defects, atrioventricular valve malformatios, ostium primum (endocardial cushion defect) Info Here (Text box) 2. Acute lymphoblastic and acute myeloid leukemia 3. Premature Alzheimer disease 4. Decreased immune response – Frequent infection 5. GIT – Atresia, stenosis, imperforate anus

Trisomy 18 (Edward’s Syndrome)       

1:5,000 – 10,000 LB Rare with multiple congenital abnormalities Severe malformation Very few live beyond 1 year (