E3: L21 Cytogenetics

Question 1

Define Cytogenetics

Why is it important?

Answer 1

Cytogenetics: the study of chromosomes and their abnormalities

Why is it important?

Chromosome abnormalities:

• Responsible for significant fraction of genetic diseases: ~ 1 in every 150 live births
• A leading cause of pregnancy loss & intellectual disability
• Seen in 50 % of 1st trimester & 20 % of 2nd-trimester spontaneous abortions

Triploidy:

• (3 x 23 = 69 chromosomes)
• One of the most common abnormalities at conception
• A leading cause of 1st and 2nd trimester abortions
• Most common cause: fertilization of an egg by 2 sperm (called dispermy)

Question 2

What is a Karyotype?

How are they arranged?

Answer 2

Karyotype:

• The ordered display of banded metaphase chromosomes
• Arranged from largest to smallest

Question 3

Describe (3) “variations” of a chromosome that are used to define chromosomes in a karyotype

Answer 3

Chromosomes are sorted by size & position of the centromere

• The centromere is the part of a chromosome that links sister chromatids

Centromere Position:

Metacentric: Centromere is located in the middle of the chromosome

Acrocentric: Centromere is located near the tip of the chromosome

Submetacentric: Centromere is located somewhere between the middle and tip of the Chr.

Telomere: Tip of each Chr.

p arm: Short arm of the Chr.

q arm: Long arm of the Chr.

Question 4

Expand: 13p42.5

Answer 4

Chr. 13

short arm

Region 4

Band 2

Sub-band 5

Question 5

Condense: Chromome 8, long arm, region 8, band 5

Answer 5

8q85

Question 6

What is Comparative Genomic Hybridization?

Why is it used?

Answer 6

CGH:

A widely used technique to detect large scale losses or duplications of chr. regions

More sensitive than fluorescence microscopic techniques, being able to detect deletions/duplications shorter than 100 kb & requires small amounts of DNA (<1 mg)

Highly automated process, requiring little technician time

Question 7

What are different terms associated with changes in the chr. number

Answer 7

Euploidy, Polyploidy, & Aneuploidy

Euploid:

• Having a Chr. number that is an exact multiple of the haploid number for the species
• For humans, the euploid number is a cell that contains a multiple of 23 chrs

Polyploid:

• The presence of a complete set of extra chromosomes in a cell (rarely occurs in humans)
• Can result from a meiotic failure in which a diploid sperm or egg cell is produced, and results in fertilization
• Most polyploid conceptions are spontaneously aborted
• Ex.
• Triploidy: 69 chrs. in the nucleus of each cell
• Tetraploidy: 92 chrs. in a cell nucleus

Aneuploid:

• Otherwise normal diploid cells that w/ an additional or missing individual chrs.
• Usually only one chromosome is affected

Examples:

• Monosomy: Absence of 1 copy of a chr.
  
  • Autosomal monosomies are usually incompatible with survival to term
• Trisomy: One additional copy of a chr.
  
  • Some autosomal trisomies are viable (i.e. trisomy 13, 18, and 21)

Question 8

What are the characteristics associated with Trisomy 21?

Answer 8

Trisomy 21 characteristics:

• Interruption of normal physical & mental development
• Physical growth delays, characteristic facial features, intestinal obstructions, increased risk of leukemia
  
  • Mental retardation: Avg IQ: 50 (similar to an 8 or 9 yo)
  • Congenital heart defects: 80% Atrial-Ventricular or ventricular septal defects
    
    • Greatest cause of mortality in young patients is cardiovascular abnormalities
• Reproduction is uncommon, thus most cases of trisomy 21 are regarded as new mutations
• Males are sterile; Females are fertile
• 75% of trisomy 21 conceptions are spontaneously aborted

Question 9

What are Reciprocal & Robertsonian Translocations?

Answer 9

Reciprocal Translocations:

• Breaks occur in two different chrs. & the material is mutually exchanged
  
  • The resulting chrs. are called derivative chrs.
• Carriers of balanced reciprocal translocations usually have normal phenotypes
  
  • However their offspring might have a partial trisomy or a partial monosomy and an abnormal phenotype

Robertsonian Translocations:

• The short arms (p) of two nonhomologous chrs. are lost & the long arms fuse at the centromere to form a single chr.
• Confined to the acrocentric chrs (13, 14, 15, 21, & 22) because the short arms of these chrs. are very small and contain no essential genetic material
• Usually the carrier of the translation is phenotypically normal with only 45 chr.
  
  • Their offspring, however, may i_nherit an extra or missing long arm of an acrocentric Chr._

Question 10

How does the sex-determing region on the Y chromosome function?

Answer 10

SRY gene (sex-determining region on the Y)

• Located just centromeric to the pseudoautosomal region on the Y chr.
• Expressed during embryonic development
• Encodes an SRY protein transcription factor
  
  • Promotes the development of a male phenotype
  • Represses the expression of DAX1 (which promotes female differentiation)

SRY present: DAX1 expression is suppressed so a male embryo is produced

SRY absent: DAX1 expression represses male differentiation genes so a female embryo is produced

Loss-of-function mutation to SRY gene:

• Individual has an XY karyotype, but a female phenotype
• ~1/20,000 human males show a Klinefelter syndrome phenotype, but have a XX karyotype that might have undergone faulty crossover between an X chr & the SYR gene during meiosis

Question 11

What is PKU?

Answer 11

Phenylketonuria (PKU)

• Autosomal recessive metabolic genetic disorder
• Characterized by a loss-of-Function mutation in the hepatic enzyme [phenylalanine hydroxylase] (PAH) gene
  
  • PAH is only produced in the liver
  • Necessary to metabolize the AA phenylalanine (Phe) to Tyrosine (Tyr)
• When PAH activity is reduced, Phe accumulates and is converted into phenylpyruvate (AKA phenylketone), which can be detected in the urine

Question 12

How is PKU harmful?

Answer 12

PKU is a hepatic phenotype, however the major clinical effect is on brain development & function

• Elevated Phe is probably the chief villain with respect to neurotoxicity; derivatives of phenylalanine are not found to be toxic
  
  • No abnormal metabolites in PKU, only normal metabolites in abnormal amounts
• People with PKU have less white matter in CNS (primary culprit for cognitive effects)
  
  • However, Phe has no direct negative effect on myelin sheath formation, nor on the oligodendrocytes that make myelin

Hypothesis 1: Phe inhibits an important enzyme in cholesterol synthesis in the brain, which is an important component of myelin

Hypothesis 2: A transporter of large neutral AA’s has a high affinity for Phe, and consequently blocks other AA’s transport into the brain (blocking the blood brain barrier)

However, the exact etiology is unknown

Question 13

How do you treat PKU?

Answer 13

Dietary modification is the general treatment of choice.

• Care must be taken that the restriction of phe is not complete or other problems can occur
• Since phe is necessary for the synthesis of many proteins, it is required for appropriate growth but levels must be strictly controlled in PKU patients
• Supplements (i.e. infant formulas, pills, & specially formulated foods) are used to provide the necessary nutrients that would otherwise be lacking in a low-phenylalanine diet

Enzyme replacement therapy:

• PKU is enzyme substitution therapy with a recombinant [phenylalanine ammonia lyase] (PAL)
• PAL is a bacteria-derived enzyme that catalyses the conversion of L-phenylalanine to transcinnamic acid & ammonia
• Subcutaneous delivery of PAL to hyperphenylalaninemic mice models successfully converted Phe to harmless metabolites
• However PALs metabolite effect is not sustained due to immune responses & Phe levels have to be stringently monitored to prevent hypophenylalanemia
• New chemical modifications of the recombinant PAL have been shown to suppress immunogenicity
  
  • Currently being investigated in clinical trials

Gene Therapy:

• Experimental, yet very promising approach for PKU treatment
• By delivering a functional PAH gene to the liver in vivo, its activity should be reconstituted leading to normal clearance of Phe in the blood, therefore eliminating the need for dietary restrictions or frequent enzyme replacement therapies.
• Reported that reconstitution of 10-20% of normal PAH enzymatic activity is sufficient to restore normal serum Phe levels

Question 14

Name 2 classes of Lysosomal Storage Diseases

Answer 14

Lysosomal Storage Diseases: Mucopolysaccharidoses & Sphingolipidoses

• Lysosomal enzymes process (degrade) unwanted materials into substances that can be recycled for future use
• When these enzymes are missing or deficient in their reactions, this causes an abnormally high accumulation of substances both inside the lysosome & the cell, eventually leading to cell degradation and death

Typical Symptoms;

• Bone abnormalities
• Dementia
• Movement disorders
• Hepatomegaly & splenomegaly

Question 15

What are the major biochemical and phenotypic problems associated with mucopolysaccharidoses and sphingolipidoses?

Answer 15

Mucopolysaccharidoses:

• Mutations in lysosomal enzymes involved in glycoaminoglycan (long chains of carbohydrates) degradation.
  
  • Carbohydrates includes dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin sulfate.
• The primary phenotype;
  
  • Bone & skeletal abnormalities
  • CT abnormalities
  • Neurological complications
  • Hepato/spleno-megaly

Sphingolipidoses:

• Lysosomal diseases that affect lipid & sphinogolipid storage & processing
• Many of the enzymes that are deficient in these disease are centered around ceramide metabolism
• The phenotype associated with these diseases include;
  
  • hepato/spleno-megaly
  • Mental impairment & atrophy
  • Optic deterioration
• Other symptoms include;
  
  • Skeletal and bone disease
  • Anemia
  • Reduced Erythrocytes & Lymphocytes