PATHOLOGY

GENETICS 1: CYTOGENETICS DISORDERS

Dr. Araceli jacoba
GENETIC DISORDERS
A. Chromosomal/Cytogenic Disorders Abnormality in number and structure of autosomes and sex chromosomes Uncommon Results from genome mutations or chromosomal mutations Normal (2n): 46XX or 46XY 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) Classified as either: 1. Autosomal dominant/recessive 2. X-linked dominant/recessive C. Multigenic Disorders Combination of multiple genes with environment Family clustering without Mendelian transmission Polygenic inheritance: 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 W/ Non-Mendelian 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 mutation 2. Mutations in mitochondrial genes 3. Associated with gene pairing 4. Associated with gonadal mosaicism e.g. Fragile X Syndrome

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2. FISH (Fluorescent In Situ Hybridization) Fluoresce a certain segment of chromosome Interphase (when not dividing) cytogenetics Uses DNA probes that recognizes sequences that are specific to particular chromosomal region The clones are labelled with fluorescent dyes and applied 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) Basically the same as karyotype (old method) 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= 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) Sex chromosome placed at the end of the karyotype Unidentifiable chromosomes are also placed at the end Chromosomes grouped into letters (AG) are chromosomes having the same appearance (length, centromere location etc.) Karyotypes usually described using short hand system of 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 Method: Mitotic spindle inhibitor (colcemid) Arrests 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 at the edge of nuclei Pyknotic material Here (Text box) 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 do not involve exons May interfere 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 c. Deletions and insertions: Frameshift Mutations Small deletions or insertions involving the coding sequence in the reading frame of a DNA strand d. Trinucleotide-repeat mutations Characterized by amplification of a sequence of three nucleotides Note: Genome Mutations and Chromosome Mutations are the causes of abnormalities in chromosome number

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

Note: 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

Lyons Hypotheis
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

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)

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 Chromosome structural changes Affects chromosome segments Produce cytogenic disorders

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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 number of Info Here (Text box) be a double chromosomes is present, there will 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 didnt 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

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

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

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

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

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 translocation 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

Chromosomal Mutations
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 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.

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

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. 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 Simean crease 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

2 types of chromosome genes affecting carcinogenesis a. Oncogene: Stimulate production of cancer b. Cancer suppressor gene: Inhibits production of cancer 1. Philadelphia chromosome CML Translocation of chromosome 9 and 22 st 1 chromosome identified that is malignancies 2. Rb gene 13q14 (point mutation) 3. Burkitts 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

Important Chromosomes

associated

with

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

CYTOGENENIC DISORDERS (AUTOSOMAL)

1. 2. 3. 4. Trisomy 21- Downs Syndrome Trisomy 18- Edwards Syndrome Trisomy 13- Pataus Syndrome Chrom. 22 deletion SyndromE

Trisomy 21 (Downs 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 <20 years old o IR of 1:25 LB For mothers >45 years old

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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 (Edwards Syndrome)

1:5,000 10,000 LB Rare with multiple congenital abnormalities Severe malformation Very few live beyond 1 year (<13%) Severe mental retardation Severe cardiac and renal anomalies (95%)

Physical Characteristics: 1. Prominent occiput 2. Micrognathia 3. Short neck 4. Low set of ears 5. Hands and feet nd rd th th Overlapping fingers (2 over 3 finger, 5 over 4 ) (also seen in Pataus) Rocker bottom feet

Features: 1. DiGeorge Syndrome features Thymic hypoplasia Absence of T-lymphocytes Patient is exposed to chronic inflammation Parathyroid hypoplasia Hypocalcemia Cardiac malformation Affecting outflow tract Mild facial anomalies 2. Velocardiofacial syndrome features Facial dysmorphism (prominent nose, retrognathia) Cv abnormalities Learning disability Cleft palate 3. Psychotic Illness (high risk) Schizophrenia Bipolar Disorders Attention deficit orders Physical characteristics: 1. Enlarged nose particularly the tip with small nares in most cases 2. Small oral cavity Note: Diagnosis of this condition may be suspected on clinical grounds, but can be established ONLY by detection of the deletion of FISH probes

Trisomy 13 (Pataus Syndrome)

1 in 15,000 births Severe malformation (more severe than Trisomy 18) Most die after birth and rarely live to a year Severe cardiac and renal anomalies

CYTOGENIC DISORDERS (SEX CHROMOSOME)

1. 2. 3. 4. Klinefelters syndrome 47XXY, 48XXXY Turners syndrome 45X, 46XXp- or qDouble Y males- 47XYY Multiple X Females 47XXX, 48 XXXX More common More benign Subtle, complaints related to sexual development and fertility Rarely diagnosed at birth

Physical Characteristics: 1. Microcephaly 2. Micropthalmia (small or closed eyes) 3. Cleft lip and palate (hard to feed) 4. Absence of chin 5. Hands and feet Polydactyl Rocker bottom feet

Turners Syndrome
Gonadal Dysgenesis Results from complete or partial monosomy of the X chromosome Characterized by hypogonadism in phenotypic females Most common sex chromosome abnormality in females Types: 1. 45,XO Karyotype (57%) High post natal mortality (classical Turners) Usually live until adulthood nd 2. Defective 2 X chromosome (14%) 46 XXp 46,Xi(Xq) 46 Xq 46,Xr(X) Deletion of the small arm formation of an isochromosome of the long arm: 46Xi(X)(q10) Deletion of portions of both long and short arms ring chromosome formation: 46Xr(X) Deletion of portions of the short or long arm: 46Xdel(Xq),46Xdel(Xp)

Chromosome 22q11.2 Deletion (Di George Syndrome)

Common but unrecognized before since loss of portion of chromosome 22 cannot be easily identified From a small deletion of band q11.2 on the long arm of chromosome 22 1:4000 LB

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3. Mosaicism (29%) Two or more cell lines one of which is 45 XO Dont completely express classical phenotype Wide range of clinical severities Info 45X/46XX Here (Text box) 45X/46XY 45X/47XXX 45X/46Xi(X)(q10) Clinical Features Present During Infancy-Childhood: Edema of the dorsum of the hand and foot due to lymph stasis Swelling of the nape of neck due to distended lymphatic channels Bilateral neck webbing and looseness of skin on back of neck (as infant develops) Congenital heart disease Left sided cardiovascular abnormalities (preductal coarctation of aorta and bicuspid aortic valve) Clinical Features in Adolescent and Adult: Failure to develop normal secondary sex characteristics (in puberty) Infantile genitalia, inadequate breast development, little pubic hair Short stature (barely reach 5ft) o 1 amenorrhea (Turners is the most important cause of this) Others: Infertility, Webbing of neck, Broad chest and wide spaced nipples, Low post hairline, Pigmented nevi, Coarctation of aorta In turner syndrome, Menopause occurs before menarche= ovaries are reduced to atrophic fibrous strands devoid of ova and follicles due to accelerated loss of oocytes (due to absence of second X chromosome)

One of the most common causes of hypogonadism in the male Can rarely be diagnosed before puberty (testicular abnormality does not develop before early puberty) Principal cause of reduced spermatogenesis and male infertility Classic case: 47XXY (90% of cases) o Non-disjunction during meiosis o Maternal non-disjunction at the first meiotic division accounts for more than half of the cases o There is no phenotypic difference between those who receive the extra X chromosome from their father or those who receive it from their mother Others (mosaic patterns): 46,XY/47,XXY; 47XXY/48,XXXY (15%) o Further physiological abnormalities o e.g. Cryptorchidism, hypospadias, more severe hypoplasia of the testes, skeletal changes Clinical Symptoms: 1. Testicular atrophy and azoospermia (*important genetic cause of reduced spermatogenesis and male infertility) Atrophied testicular tubules In some, tubules are primitive and appear embryonic 2. Gynecomastia 3. Female distribution of hair 4. Mental retardation (mean IQ is lower) 5. Eunuchoid bodily habitus With abnormally long legs 6. Most patients have a distinctive body habitus Increased length between soles and pubic bone Appearance of an elongated body 7. Lack of secondary male characteristics (deep voice, beard and male distribution of pubic hair) 8. Plasma gonadotropin levels (FSH) consistently elevates Testosterone is reduced 9. Mean plasma estradiol levels are elevated 10. Increased incidence of type 2 diabetes and metabolic syndrome, higher risk of breast cancer, extragonadal germ cell tumors and auto immune diseases (systemic lupus erythematosus) Note: Hypogonadism Only consistent finding among clinical features Confirmatory Lab Findings: 1. Positive X chromosome 2. Oligospermia or azoospermia 3. Increased urinary excretion of FSH 4. Decrease Serum Testosterone

Klinefelters Syndrome
Testicular Dysgenesis Most frequent genetic disease of sex chromosome IR 1:660 LB Male hypogonadism that occurs when there are two or more X chromosomes and one or more Y chromosomes One of the most frequent forms of genetic disease involving the sex chromosomes
Fig. Klinefelters (LEFT) : Turners (RIGHT)

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OTHER SEX-LINKED CHROMOSOMAL ABNORMALITIES

Terms: 1. Genetic sex Determined(Text box) absence of Y Info Here by presence or chromosome 2. Gonadal sex Based on histologic characteristics of gonads 3. Ductal sex Depends on the presence of derivatives of the mullerian or wolffian ducts 4. Phenotypic sex Based on the appearance of external genitalia Double Y Males 47 XXY Phenotypically normal Excessively tall and prone to acne Multiple X Females Phenotypically normal Tendency to mental retardation with increase of each X Amenorrhea and menstrual irregularities True Hermaphrodite Presence of both ovarian and testicular tissue Some cases testis on side, ovary on other; in some, combination called ovotestes Extremely rare Karyotype is 46,XX IN 50% of patients; remaining are Mosaics with a 46,XX/46XY Implies that those with 46,XX karyotype might possess SRY gene which dictates testicular differentiation Pseudohermaphrodite Disagreement bet. gonadal and phenotypic sex Female PH Genetic sex: XX, normal internal genitalia but ambiguous external genitalia Ovaries with excessive androgenic stimulation No vagina or uterus During conception, mother was exposed to high androgenic stimulation Fetal adrenal affected by congenital adrenal hyperplasia (autosomal recessive trait) Male PH Most complex of all disorders of sexual differentiation Possess Y chromosome, gonads are exclusively testes but external genital are either ambiguous or completely female Common cause is defective virilization of male embryo from genetically determined defects in androgen synthesis or action Xq12 Location of gene encoding the androgen receptor and its mutation results to Complete androgen insensitivity syndrome (testicular feminization) Not enough testosterone produced or testosterone receptors are inactive

REFERENCES
1. Dr. Jacobas lecture: Cytogenetic Disorder 2. Robbins Pathologic Basis of Disease 3. 2013 B Trans: Cytogenetics

REVIEW QUESTIONS
1. What stage of mitosis does arresting is done during karyotyping? 2. Type of mutation that produces Mendelian disorder 3. What chromosome that if problem occurs, will show manifestation of CML 4. Type of chromosomal mutation that a segment of 1 chromosome is transferred to another 5. When a chromosome lags behind before the cytoplasmic membrane is formed, it will cause this type of genome mutation. 6. Simean crease is a clinical feature of this cytogenetic disorder 7. Another name for Gonadal Dysgenersis 8. When there is another extra copy of chromosome 13, this disorder is formed 9. Disagreement between phenotypic and gonadal sex 10. The classic case of Klinefelters Syndrome.

SUMMARY

REMARKS
The greatest disease is not TB or leprosy; it is being unwanted, inloved, and uncared for. We can cure physical diseases with medicine, but the only cure for loneliness, despair and hopelessness is love. There are many in the world who are dying for a pieces of bread, but there are many more dying for a little love Mother Teresa

SUMMARY
1. Cytogenetic disorders a. Abnormal chromosome number and structures b. Brough about by genome mutations gametogenesis or embryogenesis 2. Cytogenic abnormalities of autosome 3. Cytogenic abnormalities of sex chromosome during

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