CYTOGNETICS

Tony Wynshaw-Boris, M.D., Ph.D.SOM 204 Genetics Lecture 7

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Clinical Cytogenetics

CBB Human GeneticsDr. Anthony Wynshaw-Boris

Clinical CytogeneticsBegan ~35 years ago with discovery that somatic cells in humans contain 46 chromosomes (1956)Simple procedure—hypotonic treatment for spreading the chromosomes of individual cellsAbility to microscopically examine and study chromosomes in single cells rather than tissue sections Between 1956 and 1959: visible changes in the number or structure of chromosomes could result in a number of birth defects

Down, Turner, Klinefelter, and Cri du Chat syndromes Chromosome disorders: a large proportion of fetal loss, congenital defects and mental retardationNumber of clinical indications for chromosome analysis

Clinical Indications for Chromosome Analysis

Problems of early growth and developmentStillbirth and neonatal deathFertility problemsFamily history of chromosome abnormalityNeoplasiaPregnancy in woman of advanced age (>35 years)


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Chromosome IdentificationG bands (from Giemsa staining): most widely used banding technique.Q bands (quinacrine mustard and other fluorochromes): identical to G bands. Particularly useful for identifying the Y chromosomes.R bands ("reverse" banding): opposite to that in G and Q banding.

Light bands produced on G and Q banding are dark on R banding, and dark bands on G and Q banding are light on R banding.

T bands: specific for telomeric regions of the chromosome.C bands: constitutive heterochromatin located primarily on the pericentric regions of the chromosome.

Ideogram of G-Banded Metaphase Chromosomes (400 Bands)

G-Banded Metaphase Chromosomes (550 Bands)


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High Resolution BandingX Chromosome

Molecular Cytogenetics: FISH (Fluorescence In Situ Hybridization

T FISH: Telomeres


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Spectral Karyotyping (SKY)

SKY Analysis- Medulloblastoma

Prader-Willi Syndrome15q11-13 Deletion


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Chromosome AbnormalitiesAbnormalities of chromosome number (diploid is normal, or euploid)

Triploidy and tetraploidy: multiple of haploid (3n or 4n) Aneuploidy: extra or missing chromosome

Abnormalities of chromosome structureUnbalanced: more or less than normal complement

Deletions, duplications, marker chromosomes, ring chromosomes, isochromosomes

Balanced: no net loss of gain or genetic material, but packaged differently

Inversions, translocations (reciprocal, Robertsonian, insertions)Mosaicism: two or more different chromosome complements present in an individual (commonly caused by mitotic nondisjunction in zygote)

Less severely affectedIf germline mosaicism, can have abnormal offspring

Abnormalities of Chromosome Number: Aneuploidy

Most common and clinically significant type of chromosome disorder

3-4% of all clinically recognized pregnanciesAutosomes: Trisomy 21, 18 and 13 seen in live borns with trisomy 21 most common (Down syndrome)Sex chromosomes: both X and Y can be missing or in multiple copies (Turner and Klinefelter syndromes)Most common chromosomal mechanism: meiotic non-disjunction

Failure of a pair of chromosomes to disjoin properly during one of the two meiotic divisions (usually meiosis I)

Male Patient with Down Syndrome (47 XY +21)


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Three Color FISH: Human SpermYellow: Ch. 18; Red: X; Green: Y

23,X and 23,Y 24,XX 24,XY

Three Color FISH: Amniocytes

Aqua: Ch. 18; Green: X; Red: YGreen: Ch. 18

Red: Ch. 21

Meiotic Nondisjunction and Aneuploidy


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Abnormalities of Chromosome Structure

Result from chromosome breakage and reconstitution in abnormal combinationLess common than aneuploidy (1 in 375 newborns)Some rearrangements stable, others unstableBalanced if normal complement of chromosomal material

Usually normal phenotype, but pose a risk to future generationsInversions, translocations (reciprocal, Robertsonian, insertions)

Unbalanced if there is additional or missing materialCan be associated with abnormal phenotype, but may be normal phenotype Deletions, duplications, marker chromosomes, ring chromosomes, isochromosomes

Types of Structural Abnormalities

Paracentric and PericentricInversions

Paracentric inversionslow risk of livebornchild with abnormal karyotype

Pericentric inversion: high risk of livebornchild with abnormal karyotype (5-10%)


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Offspring of Carrier of PericentricInversion with Abnormal Karyotype

Quadrivalent Formation in Meiosis: Balanced 3, 11 Translocation

Cryptic TranslocationRed: 3p telomere; Green: 11q


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Chromosome Paint ProbesBalanced Translocation

Incidence of Chromosome Abnormalities: Newborn Survey

Chromosome Abnormalities: Spontaneous Abortions


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Incidence of Chromosome Abnormalities: 10,000 Conceptions

Down Syndrome Dysmorphic featuresHypotoniaShort statureHands: short and broad, single palmar crease, incurved 5th digitsFeet: wide space between 1st and 2nd toes, proximal plantar furrowMental retardation: IQ 30-60, but verbalCongenital heart disease: present in 1/3Duodenal atresia and tracheoesophageal fistula: more commonLeukemia risk (15-fold elevation)Significant risk of Alzheimer disease at younger ageChromosomes: 95% trisomy 21; 4% Robertsonian translocation (between 21 and either 14 or 22); 1% 21q21q translocation, Mosaic Down syndrome, partial trisomy 21

Down Syndrome

BrachycephalyFlat occiputShort neck (loose skin) Flat nasal bridgeLow-set earsBrushfield spots (iris margins)Open mothProtruding tongue Tongue furrowsEpicanthal folds


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Dermatoglyphics- Down Syndrome

Down Syndrome- Maternal Age

Other Liveborn TrisomiesTrisomy 18: 1/7500 births Trisomy 13:

1/20-25,000 births


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Microdeletion/Duplication Syndromes

Mechanism of Duplication and Deletion via Repeat Sequences

Y Chromosome in Sex Determination and Differentiation


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Sex Determination and Differentiation

XX Male and XY Females Due to Unequal X-Y Exchange

X Inactivation- Barr Body

XX Female XX Female XY Male


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X Inactivation Distribution

Xist RNA is Expressed from and Binds to the Inactive X

X Inactivation with Abnormal X Chromosomes


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Disorders of Sex Chromosomes Klinefelter syndrome (47,XXY; 1:1000 male births)

Tall, thin, with long legs, hypogonadism with infertility, mild learning disabilities, psychosocial adjustment

Klinefelter variants (48,XXXY; 49,XXXXY) Generally more severe phenotypes

47,XYY (1:1000 male birthsNormal phenotype and fertility, except for lower IQ

Trisomy X (47,XXX; 1:1000 female births)Tall stature, usually fertile, lower IQ

Turner syndrome (45,X and variants; 1:4000 female live births, 99% fetal demise, so in 1-2% of all conceptuses): Distinct phenotypeHigh frequency of mutations or microdeletions on X that cause X-linked mental retardation

Klinefelter Syndrome- 47,XXY

• Tall• Thin• Long legs• Hypogonadism with

infertility• Mild learning disabilities • Mild problems with

psychosocial adjustment

Turner Syndrome- 45,X• Short stature• Gonadal dysgenesis (streak

gonads)• Characteristic facies• Webbed neck• Low posterior hairline• Broad chest with widely

spaced nipples• Frequent renal and

cardiovascular problems• Average to above average

intelligence• Nonverbal IQ<<Verbal IQ • Growth hormone therapy

adds 6-10 cm to adult height

CYTOGNETICS

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