Cytogenetics

Question 1

What are metacentric chromosomes?

Answer 1

centromere is in the middle

Question 2

What are submetacentric chromosomes?

Answer 2

centromere is closer to one end than the other

Question 3

What are acrocentric chromosomes?

Answer 3

centromere at the end of the p arm

Question 4

How do you designate a gain of chromosome (trisomy/ hyperdiploid) in nomenclature?

Answer 4

designated with a “+” (e.g. 47,XX,+21)

Question 5

How do you designate sex chromosome abnormalities?

Answer 5

45,X

47,XXY

Question 6

How do you designate loss of a chromosome (monosomy/hypodiploid)?

Answer 6

designated with “-“ (e.g. 45,XY,-7)

Question 7

How do you designate a translocation?

Answer 7

designated with a “t”

e.g. 46,XX,t(11;22)(q23;q11.2)

Question 8

How do you designate a deletion?

Answer 8

designated by “del”

e.g. 46,XY,del(5)(p15.1)

Question 9

How do you designate an inversion?

Answer 9

designated by “inv”

e.g. 46,XX,inv(20)(p13q13.2)

Question 10

How do you designate mosaicism?

Answer 10

2 different karyotypes are separated by a “/” and [ ] brackets are used to indicate the number of cells in each clone
e.g. Turner syndrome mosaicism- 45,X[15]/46,X,i(X)(q10)[5]

Question 11

Hypotonia
Congenital heart defects (AVSD, endocardial cushion defects, VSD, ASD, TOF)
Skeletal system defects (atlantoaxial instability)
Structural defects of GI system (Hirschsrpung disease 25x more likely)
Myopia, strabismus, nystagmus
Hearing loss
Congenital hypothyroidism
Hypogonadism (men usually sterile)
Developmental Delays
Clinodactyly
Single palmar crease
Leukemia (AML/pre B-ALL)
Premature aging and degenerative brain disorders

Answer 11

Down Syndrome (Trisomy 21)
85-90% due to maternal errors in meiosis (mostly in meiosis I)
3-5% due to paternal meiosis errors (mostly in meiosis II)
3-5% due to mitotic errors
4-5% due to Robertsonian translocations
1% other (including parent with somatic-gonadal or gonadal mosaicism or genetic predisposition to trisomy)

Question 12

IUGR
Congenital heart defects (VSD most common)
GI anomalies (Meckel’s diverticulum or malrotation)
Omphalocele
Microcephaly (with prominent occiput)
Underdeveloped muscles and weak cry
Clenched hand with overlapping fingers (2 over 3, 5 over 4; predominately arches on dermatoglyphics)
Rocker-bottom foot, club foot, syndactyly of 2nd and 3rd toes
Abnormal myelination, hydrocephalus, defects of corpus callosum, cerebellar hypoplasia myelomeningocele
Poor suck
Central apnea
Early lethality (first few days-months)

Answer 12

Edwards Syndrome (Trisomy 18)
Mostly caused by non-disjunction in maternal meiosis (mostly meiosis II)

Question 13

Cleft lip and/or palate
Microcephaly, micropthalmia
Low set ears, external ear malformations
Genital abnormalities
Holoprosencephaly
Sloped forehead
Postaxial polydactyly
Occipital scalp defect
Profound ID
Early lethality (median survival 3 days; 5% survive 6 months)

Answer 13

Patau Syndrome (Trisomy 13)

Question 14

Typically associated with SAB (but can be seen as mosaic)

Deep plantar furrows

Answer 14

Trisomy 8

Question 15

When should mosaic trisomies be considered?

Answer 15

If an abnormality is commonly seen as a mosaic (sex chromosome, trisomy 8, cat eye, Pallister-Killiam syndrome)
Clinical diagnosis and chromosome results are discordant (e.g. if clinical features of Down syndrome but chromosome/array analysis doesn’t detect trisomy 21)
If single cell abnormalities seen in blood correspond with clinical diagnosis, study another tissue

Question 16

Describe the Lyon Hypothesis.

Answer 16

Dosage compensation- inactivation of one X in women assures that women and men have equivalent dosages of genes
Women have functional mosaicism- two different X chromosomes with different genes are active in different groups of cells
Random but clonal inactivation of X chromosomes results in variation of the effects of genes on their X chromosomes
About 15% of X-linked genes escape inactivation and 10% show variable expression
Both X chromosomes are initially active before implantation and XCI randomly occurs sequentially, coupled with cell differentiation (first in extraembryonic trophectoderm and primitive endoderm of blastocytes and finally in the fetal precursor cells around the time of implantation)

Question 17

List the commonly mutated genes resulting in X-linked disease.

Answer 17

POG-A (paroxysmal nocturnal hemoglobinuria)
DMD (Duchenne muscular dystrophy)
ATP7A (Menkes syndrome)
COL4A5 (Alport syndrome)
IL2RG (X-linked severe combined immunodeficiency)
TNFSF5 (Immunodeficiency with hyper-IgM)
HPRT1 (Lesch-Nyhan syndrome)
FMR1 (Fragile X syndrome)
ALD (adrenoleukodystrophy)
MECP2 (Rett syndrome)
HEMA (Hemophilia A)

Question 18

Describe the PAR regions.

Answer 18

distal regions of the X and Y chromosomes that contain homologous DNA sequences
During male meiosis crossing over between the X and Y chromosomes occurs only between these regions
Escape X inactivation in females
PAR1 has 24 genes (on the p arm)
PAR2 has 4 genes (on the q arm)

Question 19

What is the function of FiSH?

Answer 19

a cytogenetic technique used to detect and localize the presence or absence of specific DNA regions on chromosomes (identify numerical/structural abnormalities and detect deletions/duplications/rearrangements)
Advantages- highly specific, fast, and does not require dividing cells
Resolution: 50-250 kb

Question 20

List the most common classes of FiSH probes.

Answer 20

locus specific (detects gain or loss of specific genes or small genomic areas)
enumeration (centromere specific and used for aneuploidy analysis)
chromosome paint (used to determine if all material is derived from one chromosome)
rearrangement probes (to detect a rearrangement of a specific locus- can be break-apart or dual color dual fusion)*
*typically used in oncology only

Question 21

Describe the two kinds of rearrangement probes.

Answer 21

Break-apart probes are used when you don’t have a common rearrangement and do not know where it would have gone (as with MYC rearrangements) so you have probes of two different colors that flank the gene of interested and if the probes are broken apart (rather than adjacent/almost fused) you know a rearrangement has occurred (but do not know where it went)
Dual color dual fusion probes are used when you have a common rearrangement partner and can tag both genes to see where they are (such as with BCR on chr 22 and ABL1 on chr 9) so you have probes of two colors linked with two different genes and when they are found overlapping (rather than separated) you know they have rearranged to be together

Question 22

Describe the molecular process of X inactivation.

Answer 22

XIST gene (Xq13) produces Xist RNA (a 17 kb non-coding RNA produced by the X chromosome being inactivated - in cis-) accumulates on or "coats" the future inactive X chromosome
This happens randomly

Question 23

Describe the process of sexual differentiation.

Answer 23

presence of an SRY gene programs male gonadal development, which produces Mullerian inhibiting factor and early androgen production with assures regression of the Mullerian ducts and masculinization of genital structures in males

Question 24

cystic hygromas, generalized hydrops fetalis, coarctation of aorta, edema dorsum of feet, renal anomalies (on prenatal US)
short stature
nuchal skin folds
low posterior hairline
pigmented nevi
decreased birth weight
congenital heat disease (coarctation of aorta)
thyroiditis
delayed or absent menarche, ovarian failure, infertility (only 10% experience puberty and fewer become pregnant without assistance- those that do have high maternal mortality)
delayed or absent secondary sex characteristics
gonadal malignancy (in some mosaic pts)
wide range of severity with high levels of mosaicism

Answer 24

Turner Syndrome
Caused by loss of critical area is p11.2-p22.1 (this includes the SHOX gene)- 45,X (commonly)
99% of conceptions with 45,X result in spontaneous loss
ALWAYS follow-up test individuals with Turner with FISH to check for Y chromosomes (having some accessory Y material increases risk for gonadal malignancy)

Question 25

List the chromosome abnormalities that may result in Turner Syndrome.

Answer 25

Monosomy X- 45,X Mosaic Monosomy X- 45,X/46,XX or 45,X/46XY Isochromosome Xq (most frequent structural alteration- 15-18%)- 46,XX,i(X)(q10) Ring Chromosome X- 46,XX,r(X)(p22q28) 46,X,del(X)(pter-->p11.2)

Question 26

Describe how sex chromosome aneuploidy relates to age.

Answer 26

As we age, we are at increased risk for sex chromosome aneuploidy (loss of Y in lymphocyte chromosome testing is observed in males 40-50 is 8-10% and loss of X in lymphocyte chromosome testing is age related in females by the age of 30-50 is 3-5%) this DOES NOT indicate low level, clinically significant mosaicism (use clinical features to make best judgement including query for repeat testing)

Question 27

hypogonadism, hypogenitalism most with infertility (asospermia/oligospermia) tall stature variable degrees of gynecomastia differences in body hair, muscle mass, adipose tissue distribution (central adipose distribution) speech delay intellectual disability

Answer 27

Klinefelter Syndrome 47,XXY 15-20% have mosaicism for 46,XY cell line associated with increased maternal age

Question 28

taller than expected (higher frequency of over 6 ft) learning disabilities delayed speech and language skills some delayed motor skills and/or hypotonia hand tremors involuntary motor tics seizures increased behavioral, social, and emotional difficulties (ADHD, depression, anxiety, and autism spectrum disorder) normal puberty, hormone levels, and fertility

Answer 28

47,XYY due to paternal meiotic error When detected prenatally with early intervention, children can be behaviorally normal

Question 29

``` taller stature with longer legs hypotonia joint laxity clinodactyly auditory processing disorders delays in language development problems in forming interpersonal relationships increased liklihood of psychiatric disorders normal puberty and fertility ```

Answer 29

47,XXX | somewhat related to maternal age (but less than Down Syndrome)

Question 30

What is the differential diagnosis for discordant fetal sex (DNA female/US male)?

Answer 30

Sample mix-up or lab error (recommend repeat of both US and cfDNA) Androgen exposure- CAH, aromatase deficiency, androgen producing maternal ovarian tumor, exogenous androgens (from medication/drugs) Translocation of SRY to an X chromosome or autosome (SRY FISH to diagnose) Mosaicism - 45,X/46,XY or 46,XX/46,XY SOX 9 duplication

Question 31

What is the differential diagnosis for discordant fetal sex (DNA male/ US female)?

Answer 31

Androgen receptor defect 5-alpha reductase deficiency- mutation SRD5A2 Swyer syndrome- mutations SRY or other genes that play a role in sexual development (e.g. MAP3K1, NR5A1, DHH) Campomelic dysplasia- SOX9 haploinsufficiency (males develop as female) Mosaic- 45,X/46,XY or 46,XX/46,XY

Question 32

How do you establish clonality?

Answer 32

Requires... 2 cells with the same structural rearrangements 2 cells with the same trisomy 3 cells with the same monosomy

Question 33

How do acquired balanced translocations relate to oncology?

Answer 33

breakage events in two chromosomes with the terminal regions switch places this can have functional consequences (fusion of two genes such as BCR-ABL1 fusion which results in fusion gene that is implicated in constitutive ABL1 kinase activity OR altered expression where a highly regulated gene is brought into close proximity to a strong promoter or enhancer such as IGH-MYC)

Question 34

What is the Philadelphia chromosome?

Answer 34

t(9;22) which results in increased risk of Chronic Myeloid Leukemia can be treated with targeted therapy (tyrosine kinase inhibitor- Imatinib- to inhibit BCR-ABL1 function)

Question 35

congenital abnormalities (including radial ray defects) progressive bone marrow failure increased risk for malignancies (usually AML)

Answer 35

Fanconi Anemia FANC** genes (FANCD1=BRCA2 gene) AR (one AD gene RAD51; X-linked gene FANCB)

Question 36

cerebellar degeneration, oculocutaneous telangiectasias, immunodeficiency, chromosome instability increased sensitivity to x-ray and radiomimetic substances 38% risk for malignancy het carriers have a 6.1 fold relative risk of developing cancer (mostly breast)

Answer 36

Ataxia Telangiectasia ATM AR Characteristic finding is rearrangements identified between chromosome 7 and 14

Question 37

``` immunodeficiency centromere instability (involving chromosomes 1, 9, and 16); multiradial configurations on chromosomal analysis facial anomalies ```

Answer 37

ICF Syndrome DMNT3B (20q11.2) AR

Question 38

severe pre and postnatal growth restriction, proportionately short stature sun sensitivity, erythematous facial skin lesions immunodeficiency increased predisposition for cancer increased levels of sister chromatid exchange

Answer 38

Bloom Syndrome BLM (15q26.1) AR

Question 39

Monosomy 7

Answer 39

common cytogenetic abnormality in myeloid neoplasms (e.g. MDS, AML) germline mutations have been associated with familial monosomy 7 (germline GAT2 mutations on 3q21.3 are seen in 10% of children with primary myelodysplastic syndrome; SAMD9 and SAMD9L localized to 7q21 - GOF toxic to cell function- are also associated because of monosomy 7 rescue mechanism)

Question 40

What level of deletion/duplications can be detected on karyotype analysis?

Answer 40

5-10 Mb

Question 41

``` US findings that show... Disproportionately large head size compared to body Hydrocephalus/brain malformations 3-4 syndactyly of hands Open neural tube defects Placental thickening with cystic changes Ovaries with theca lutein cysts ```

Answer 41

Triploidy | Live born individuals are rare and only survive a short period of time

Question 42

Describe Trisomy 16.

Answer 42

Most common trisomy in pregnancy loss Fetuses not viable by end of first trimester May result in placental thickening and theca lutein cysts of ovary Rare live births are usually mosaic and those with trisomy rescue who have maternal UPD; typically have fetal growth restriction

Question 43

Describe the difference between paracentric and pericentric inversions.

Answer 43

Paracentric inversions include information all on one side of the centromere; difficult to detect (array does not detect balanced rearrangements) Pericentric inversions include information on both sides of the centromere; very large pericentric inversions may result in very small terminal del/dups that are consistent with viable offspring BUT small inversions are more likely to result in perinatal loss (e.g. Pericentric inversion of chromosome 9 which happens 1-3% of the time, can also have pericentric inversion of Y- these are not associated with reproductive failure BUT all others are)

Question 44

List the recurrent identifiable Marker Chromosomes.

Answer 44

der(22) t(11;22)- most common recurrent translocation Cat Eye Syndrome dicentric (22;22) (q11.2;q11.2) isodicentric 15 tetrasomy 15 Pallister Killian isochromosome 12p

Question 45

Describe small supernumerary marker chromosomes.

Answer 45

high rate of mosaicism (because they are frequently lost in cell division) 1/3 are familial and can be seen in unaffected relatives abnormal outcomes noted in non-familial cases in spite of normal US about 5-18%- it is important then to identify the origin of the marker (usually use centromeric probes but does not always give a result) Chromosome 15, 17, and 22 appear to be over represented among high risk group

Question 46

How are marker chromosomes managed?

Answer 46

parental testing evaluate any family members with marker and concern for associated problems request lab advise on identifying marker by FiSH using alpha satellite probe, array or both low % mosaicism and small size often precludes identification of the source perform serial US for growth and other concerning findings (if pregnancy is continued)

Question 47

Describe Robertsonian translocation

Answer 47

centric fusion between two acrocentric chromosomes (13, 14, 15, 21, 22) p arms are most often lost and sometimes you get two centromeres 13/14 translocation is most common about 4% of Down syndrome is due to unbalanced Robertsonian translocations (most commonly 14/21)- maternal risk is higher (m-10-15%; p-2%) due to risk for UPD due to trisomy rescue you should test for this in children if suspected (this is especially the case with 14 and 15)

Question 48

When should you consider evaluating someone for UPD?

Answer 48

When there is a low percentage trisomy mosaicism on amnio or CVS (though note that CVS always confers a higher risk for mosaicism) because trisomy rescue can result in UPD When a chromosome involved in parental translocation has an imprinted region

Question 49

Which chromosomes have imprinting regions that are clinically relevant?

Answer 49

6 (paternal UPD)- Transient Neonatal DM 7 (maternal UPD)- Russell Silver Syndrome 11 (loss of paternal methylation)- Russell Silver Syndrome 11 (paternal UPD)- Beckwith Wiedemann 14 (paternal UPD)- Temple syndrome 14 (maternal UPD)- Milder than paternal UPD syndrome 15 (paternal UPD)- Angelmann syndrome 15 (maternal UPD)- Prader Willi Syndrome 20 (paternal UPD)- Albrights Hereditary Osteodystrophy