PATHOLOGY

GENETICS 1: CYTOGENETICS DISORDERS

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Dr. Araceli jacoba July 11, 2011

GENETIC DISORDERS 2. FISH (Fluorescent In Situ Hybridization)

A. Chromosomal/Cytogenic Disorders – Fluoresce a certain segment of chromosome
– Abnormality in number and structure of autosomes and – Interphase (when not dividing) cytogenetics
sex chromosomes – Uses DNA probes that recognizes sequences that are
– Uncommon specific to particular chromosomal region
– Results from genome mutations or chromosomal – The clones are labelled with fluorescent dyes and applied
mutations to metaphase spreads or interphase nuclei
– Normal (2n): 46XX or 46XY – The probe hyberdizes to its homologous genomic
sequence and labels a specific chromosomal region that
B. Mendelian Disorder can be seen under fluorescent microscope
– Disorders related to mutation in single genes – Sample: blood or urine
– Highly penetrant (means presence of mutation is – Reagent: Quinacrine and DNA probe
associated with the disease in a large proportion of – Used for: detection of numeric abnormalities of
individuals) chromosomes, demonstration of subtle microdeletions,
– Classified as either: complex translocations, analysis of gene amplifications
1. Autosomal dominant/recessive and mapping newly isolated genes of interest
2. X-linked dominant/recessive
3. Banding Technique
C. Multigenic Disorders – Identification of chromosome by its banding pattern
– Combination of multiple genes with environment (according to its alleles)
– Family clustering without Mendelian transmission – Basically the same as karyotype (old method)
– Polygenic inheritance: Manifestation of disorder is  In banded karyotypes, each arm of the chromosome
controlled by environmental factors such as diet, exercise, is divided into 2 or more regions bordered by
salt intake, exposure to the disease prominent bands
– Dosage Effect: More number of deletious gene, more  Short arm of chromosome is designated p and long
severe expression of the disease term is referred as q
– e.g. Diabetes mellitus, Carcinoma, HPN, Heart disease,  Each arm of chromosome is divided into two or more
Intelligence regions
 The regions are numbered from centromere outward
D. Single Gene Disorder W/ Non-Mendelian Inheritance  Each region is divided into bands and sub-bands
– Involve only a single gene but transmission is neither which are also numbered.
autosomal dominant/recessive nor X-linked  e.g. Xp21.2= Chromosomal segment located on the
dominant/recessive short arm of X chromosome, in region 2, band 1 and
– Classified into 4 groups: sub-band 2. (refer to the figure below)
1. Caused by trinucleotide repeat mutation – Stain: Giemsa (G banding)
2. Mutations in mitochondrial genes – Best done at prophase
3. Associated with gene pairing
4. Associated with gonadal mosaicism
– e.g. Fragile X Syndrome

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 (AG) 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 3. Gene Mutation
– Inactive X chromosome – Partial or complete deletion of a gene
– Number of sex chromosome minus active X – Produce Mendelian disorders
 e.g. 4 Barr bodies  5 X chromosomes  49 a. Point Mutations within coding sequences
Info Here
– Pyknotic material (Text
at the edge box)
of nuclei  Alter the code in a triplet of bases and lead to the
– Seen in interphase nucleus as darkly staining small mass replacement of one amino acid by another in the
in contact with nuclear membrane gene product
– Basis of inactivation involves gene called XIST, “coats” X  Often termed missense mutations
chromosome that it is transcribed from and initiates a  2 types:
gene-silencing process by chromatin a. Conservative (substituted AA causes little
– Sample: Buccal smear change in function)
– Only seen in females b. Nonconservative (normal AA replaced by a
– One X active and the other X is inactive (males only have very different one)
inactive X)  Example: Sickle mutation affecting B-globin
– Modification and DNA methylation chain of haemoglobin. CTC (glutamic acid)
– Sample: Buccal smear 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
Fig. Barr bodies shown in nuclei of cells from buccal smear
c. Deletions and insertions: Frameshift Mutations
Note:
 Small deletions or insertions involving the coding
 Barr bodies: Condensed substance that is adherent to
sequence in the reading frame of a DNA strand
nuclear border, based upon LYONS HYPOTHESIS
 In peripheral smears  Dumbell-likebodies d. Trinucleotide-repeat mutations
 Best to assess with buccal smear  Characterized by amplification of a sequence of
three nucleotides
Lyons Hypotheis
1. Only 1 of the X chromosome is genetically active Note:
2. The other X chromosome (from either paternal or maternal Genome Mutations and Chromosome Mutations are the
origin) undergoes heteropyknosis to become the Barr body causes of abnormalities in chromosome number
(inactive)
3. Inactivation of either maternal or paternal X occurs at random Genome Mutations
th
among all cells of blastocyst on or about the 16 day of Terms:
embryonic life 1. Euploid – Any exact multiple of the haploid number
4. Inactivation of the same X chromosome persists in all cells 2. Aneuploidy – Cell acquires a chromosome complement that
derived from each precursor cell is not an exact multiple of 23
3. Mosaicism – Condition in which mitotic errors in early
Types of Mutations development give rise to two or more populations of cells
 Mutation – Permanent change in the DNA with different chromosomal complement in the same
 Those that affect germ cells usually give rise to inherited individual
diseases while those that affect somatic cells usually are A. Non-disjunction
important in genesis of cancers or congenital malformations – Failure of chromosome to separate in anaphase
1. Genome Mutations – Can occur in either spermatogenesis or oogenesis
– Subtraction or addition of whole chromosomes (meiosis)
– Affects number – Gametes formed extra chromosome (n + 1) or one less
– Produce cytogenic disorders chromosome (n – 1).
– Results in 2 types of zygotes:
2. Chromosome Mutations a. Trisonomic (2n + 1)
– Rearrangement of genetic material  Chromosome b. Monosomic (2n – 1)
structural changes
– Affects chromosome segments
– Produce cytogenic disorders

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Note: 1. Inversion
 Ex. Chromosome 21 failed to join the other daughter cell – 2 breaks in a single chromosome with reincorporation of
(both chromosomes join one cell) the inverted, intervening segment
 If this is an ova, where it is expected that only half of the – Paracentric – Inversion involving only one arm of
number of Info Hereis (Text
chromosomes present, box)
there will be a double chromosome
dose of chromosomes – Pericentric – Breaks on opposite sides of centromere
 If combined to a sperm with a normal number, the individual – Often compatible with normal development
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 2. Isochromosomes
– When one arm is lost and the remaining arm is duplicated
i. Meiotic (in parents) – 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 3. Deletion

 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
– 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
Note: 46,XX,t(2;5)(q31;p14)
 Chromosome that lag behind + normal sperm = Monosomic – No loss of genetic material (if ever, only a small portion)
 Autosomal monosomies generally causes loss of too much
genetic information to permit birth/embryogenesis but a. Balanced reciprocal translocation
several trisomies do permit survival  Size of both chromosomes appeared the same after
translocation/transfer
Chromosomal Mutations  There are single breaks in each of the two
 Severity of manifestation depends on volume of genetic chromosomes with exchange of material
material lost  A balanced translocation is of great risk of
 If only a small portion is lost, patient may be asymptomatic producing abnormal gametes
and may live  Example: 46,XX,t(2;5)(q31;p14) – Between the
 If a significant portion is lost, patient will die after birth or in long arm chromosome 2 and one of the
utero chromosome 5
 No loss of genetic material, individual likely to be
phenotypically normal

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 b. Robertsonian translocation Causes:
 Translocation between two acrocentric chromosomes 1. Meiotic Non-Disjunction
 If unequal in sizes after translocation – 90% in either parent
 A translocation between two acrocentric – Maternal age affects this disorder
Info Here (Text box)
chromosomes – Occurs in gametogenesis – Parents are normal
 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 2. Translocation
Note: – 4%
We may not be able to distinguish a balanced translocation – t(21,22 or 14)
in a karyotype (possible on spectral karyotype). It is relatively – Extra chromosomal material derives the presence of
easy to check what took place in Robertsonian type of Robertsonian translocation of the long arm of
translocation. chromosome 21 to another acrocentric chromosome
 The translocated material provides the same triple
Cause of Mutations gene dosage as in trisomy 21
1. Increasing age  The translocated chromosome is inherited from one
2. Chromosomal instability of the parents
3. Ionizing radiation 3. Mosaic
4. Drugs – 1%
5. Viruses – Non-disjunction in baby
– HPV, HIV, EBV – individual with more than one cell line
– Can change chromosome  unstable  produce cancer – Having a mixture of cells with 46 and 47 chromosomes
2 types of chromosome genes affecting carcinogenesis – Results from mitotic disjunction of chromosome 21
a. Oncogene: Stimulate production of cancer during an early stage embryogenesis.
b. Cancer suppressor gene: Inhibits production of cancer – Variable and milder symptoms
Clinical Features:
Important Chromosomes 1. Mental retardation
1. Philadelphia chromosome – Severe
– CML – 80% have IQ 25 to 50
– Translocation of chromosome 9 and 22 2. Facial profile
st
– 1 chromosome identified that is associated with – Oblique palpebral fissures (slanted upwards)
malignancies – Flat facial profile
2. Rb gene – 13q14 (point mutation) – Epicanthic folds (normally stops before inner canthus
3. Burkitt’s lymphoma – t(8;14)(q24) eyes)
4. WT-1 gene – 11p13 – Dysplastic ears (folded or misplaced downwards)
5. DCC gene – 18q21 – Protruding tongue
6. APC gene – 5q21
– Abundant neck skin
7. p53 - cancer suppressor gene 17p13.1 3. Motor abnormality
8. NF-1 gene – 17q11.2
– Loss of Moro reflex (startle reflex)
Note:  Initiated by a banging sound (clapping of hands)
1-5 are oncogene; 6-8 are cancer-suppresor gene – Muscle hypotonia
4. Hands and feet
CYTOGENENIC DISORDERS (AUTOSOMAL) – Simean crease – Straight crease in palm (normal in 3%
1. Trisomy 21- Down’s Syndrome of population)
2. Trisomy 18- Edward’s Syndrome – Short broad hands
3. Trisomy 13- Patau’s Syndrome – Hypoplasia of middle phalanx
st nd
4. Chrom. 22 deletion SyndromE – Gap between 1 and 2 toe
Note:
Trisomy 21 (Down’s Syndrome) Mosaics – Phenotypically mild
 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: Features:
1. Congenital heart disease – 40% (atrial septal defects, 1. “DiGeorge Syndrome” features
ventricular septal defects, atrioventricular valve – Thymic hypoplasia
malformatios, ostium primum (endocardial cushion defect)  Absence of T-lymphocytes
Info Here
2. Acute lymphoblastic (Text
and acute box)leukemia
myeloid  Patient is exposed to chronic inflammation
3. Premature Alzheimer disease – Parathyroid hypoplasia  Hypocalcemia
4. Decreased immune response – Frequent infection – Cardiac malformation  Affecting outflow tract
5. GIT – Atresia, stenosis, imperforate anus – Mild facial anomalies
2. “Velocardiofacial syndrome” features
Trisomy 18 (Edward’s Syndrome) – Facial dysmorphism (prominent nose, retrognathia)
 1:5,000 – 10,000 LB – Cv abnormalities
 Rare with multiple congenital abnormalities – Learning disability
 Severe malformation – Cleft palate
 Very few live beyond 1 year (<13%) 3. Psychotic Illness (high risk)
 Severe mental retardation – Schizophrenia
 Severe cardiac and renal anomalies (95%) – Bipolar Disorders
– Attention deficit orders
Physical Characteristics:
1. Prominent occiput Physical characteristics:
2. Micrognathia 1. Enlarged nose particularly the tip with small nares in most
3. Short neck cases
4. Low set of ears 2. Small oral cavity
5. Hands and feet
nd rd th th Note:
– Overlapping fingers (2 over 3 finger, 5 over 4 )
(also seen in Patau’s) Diagnosis of this condition may be suspected on clinical
– Rocker bottom feet grounds, but can be established ONLY by detection of the
deletion of FISH probes
Trisomy 13 (Patau’s Syndrome)
 1 in 15,000 births CYTOGENIC DISORDERS (SEX CHROMOSOME)
 Severe malformation (more severe than Trisomy 18) 1. Klinefelter’s syndrome – 47XXY, 48XXXY
 Most die after birth and rarely live to a year 2. Turner’s syndrome – 45X, 46XXp- or q-
 Severe cardiac and renal anomalies 3. Double Y males- 47XYY
4. Multiple X Females – 47XXX, 48 XXXX
Physical Characteristics:
 More common
1. Microcephaly
 More benign
2. Micropthalmia (small or closed eyes)
 Subtle, complaints related to sexual development and fertility
3. Cleft lip and palate (hard to feed)
 Rarely diagnosed at birth
4. Absence of chin
5. Hands and feet Turner’s Syndrome
– Polydactyl
– Rocker bottom feet  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 Turner’s)
– 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:
Chromosome 22q11.2 Deletion 46Xdel(Xq),46Xdel(Xp)
(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
– Don’t completely express classical phenotype  Wide
range of clinical severities
Info Here (Text box)
 45X/46XX
 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 (Turner’s 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)
Klinefelter’s 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
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 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
Fig. Klinefelter’s (LEFT) : Turner’s (RIGHT)
Pathology 6 | 7
 OTHER SEX-LINKED CHROMOSOMAL REFERENCES
ABNORMALITIES 1. Dr. Jacoba’s lecture: Cytogenetic Disorder
Terms: 2. Robbin’s Pathologic Basis of Disease
1. Genetic sex – Determined
Info Here (Text box)
by presence or absence of Y 3. 2013 B Trans: Cytogenetics
chromosome
2. Gonadal sex – Based on histologic characteristics of REVIEW QUESTIONS
gonads
1. What stage of mitosis does arresting is done during
3. Ductal sex – Depends on the presence of derivatives of
karyotyping?
the mullerian or wolffian ducts
2. Type of mutation that produces Mendelian disorder
4. Phenotypic sex – Based on the appearance of external
3. What chromosome that if problem occurs, will show
genitalia
manifestation of CML
Double Y Males 4. Type of chromosomal mutation that a segment of 1
– 47 XXY chromosome is transferred to another
– Phenotypically normal 5. When a chromosome lags behind before the cytoplasmic
– Excessively tall and prone to acne membrane is formed, it will cause this type of genome
mutation.
Multiple X Females 6. Simean crease is a clinical feature of this cytogenetic
– Phenotypically normal disorder
– Tendency to mental retardation with increase of each X 7. Another name for Gonadal Dysgenersis
– Amenorrhea and menstrual irregularities 8. When there is another extra copy of chromosome 13, this
disorder is formed
True Hermaphrodite
9. Disagreement between phenotypic and gonadal sex
– Presence of both ovarian and testicular tissue 10. The classic case of Klinefelter’s Syndrome.
– Some cases testis on side, ovary on other; in some,
combination called ovotestes SUMMARY
– 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 REMARKS
 During conception, mother was exposed to high The greatest disease is not TB or leprosy; it is being
androgenic stimulation  Fetal adrenal affected by unwanted, inloved, and uncared for.
congenital adrenal hyperplasia (autosomal recessive We can cure physical diseases with medicine, but the
trait) only cure for loneliness, despair and hopelessness is love. There
– Male PH are many in the world who are dying for a pieces of bread, but
 Most complex of all disorders of sexual differentiation there are many more dying for a little love
 Possess Y chromosome, gonads are exclusively testes Mother Teresa
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

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

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