Trinucleotide Repeat Disorders and petigrees

Microsatellies

Microsatellite - short segment of DNA, usually 1-6 or more base pairs in length, that is repeated multiple times in succession at a particular genomic location.

•Also known as Short Tandem Repeats (STRs), Simple Sequence Repeats (SSRs).

•Useful polymorphic markers.

Trinucleotide Repeat

• Specific type of microsatellite where the repeating unit is three nucleotides (E.g., CAG-CAG-CAG-CAG).

• Highly polymorphic - mutation rates ~10^-2 to 10^-6 per generation. -Longer repeat tracts and those composed of a single repeating codon - more likely to be unstable and undergo mutation at higher frequencies.

• Thought to arise through replication/repair slippage.

• Slippage causes repetitive triplet sequences to loop out and increase in copy number, forming stable hairpin or secondary structures that evade repair mechanisms. Leads to progressive, inherited lengthening of the DNA tract. Generates tracts with a varying number of repeat units.

• Occur in coding and non-coding regions.

Trinucleotide Repeat Formation

• Daughter strand 5’→3” → (Direction of synthesis of new strand synthesis and parent strand 3’ →5

• Strand separation for a second round of replication

• Second round of replication occurs

• Original length = 9 repeats, new length = 12 repeats

• Increase in the repeat length makes this region further prone to replication slippage and more increase in repeat length

Poly-glutamine or Poly-Q repeat

Short trinucleotide repeats are common in the human genome, but expansions of specific TNRs are responsible for numerous genetic disorders.

The most common pathogenic triplet base pair is CAG•CTG, which occurs in at least 15 known pathogenic TNR loci.

Trinucleotide Repeat Disorders

Trinucleotide repeat disorders are a group of human diseases caused by the abnormal expansion of repetitive sequences.

1991 - Trinucleotide repeat expansion disorders discovered.

•Fragile X Syndrome

•Spinal and Bulbar Muscular Atrophy

Since linked with >30 neurological, neuromuscular, or neurodegenerative disorders. 

Trinucleotide Repeat Disorders are distinguishable by:

•Location within a gene

•Motif of repeat unit

•Overall size of repeat

Inheritance

Stability across generations

Whether the repeat-DNA is transcribed into RNA and translated into protein

Characterizing a repeat expansion: Biallelic: Inheritance, Repeat location, Repeat motif size, Repeat motif, Overall repeat size, If coding, type of protein product

Inheritance: Biallelic (homozygous)

Repeat location: intronic

Repeat motif size: trinucleotide

Repeat motif: e.g CAG, GGC

Overall repeat size: Pre-mutation

If coding, type of protein product: Polyglutamine

Characterizing a repeat expansion: Monoallelic: Inheritance, Repeat location, Repeat motif size, Repeat motif, Overall repeat size, If coding, type of protein product

Inheritance: Monoallelic (heterozygous)

Repeat location: exonic

Repeat motif size: tetranucleotide

Repeat motif: CCTG

Overall repeat size: full expansion

If coding, type of protein product: Polyalanine

Characterizing a repeat expansion: X-linked: Inheritance, Repeat location, Repeat motif size, Repeat motif, Overall repeat size, If coding, type of protein product

Inheritance: X-linked

Repeat location: 5’UTR, 3’UTR

Repeat motif size: pentanucleotide, hexanucleotide

Repeat motif: AAGGG, GGGGGCC

Overall repeat size: interruptions within repeat sequence

If coding, type of protein product: Polyglycine

A STR expansion can be characterised by what?

A STR expansion can be characterised by its location within a gene. Pathogenic repeats in non-coding genic regions tend to be larger than exonic repeats.

A STR expansion can be characterised by its location within a gene. Examples:

• 5’UTR - CGG = Fragile X syndrome and SCA12

• Exon: CAG, Huntingtons disease, SCA1, 2, 3, 6,7,17

• Intron: GAA Friedreich Ataxia

• 3’UTR: CTG: Mytonic dystrophy SCA8

Repeat motif

Repeat motif - whether an STR expansion is pathogenic.

Size of the repeat motif - trinucleotide repeat expansions most common, followed by hexanucleotide repeats.

Length of the repetitive DNA sequence - primary determinant of disease.

What is the relationship between repeat expansion size, age of onset and disease severity?

Age of TNR disease onset, disease severity and rate of disease progression are primarily determined by the length of the corresponding repeat tract at birth, with longer repeats being associated with a less favorable prognosis.

General features of repeat expansion diseases

•Arise from normally existing polymorphic repeats.

•Expansions are unstable, often changing size when transmitted to next generation.

•Longer repeats tend to cause more severe, earlier onset disease.

•Clinical anticipation is common: earlier onset, more severe disease in successive generations.

•Highly variable phenotype, primarily reflecting differences in repeat size.

Genetic Anticipation:

•Phenomenon where a genetic disorder becomes more severe and appears at an earlier age as it's passed down through generations, primarily due to unstable trinucleotide repeat expansions. 

When a parent carrying the expanded repeat sequence (but may not show severe symptoms) has children, there is a risk the repeat sequence will expand further during the transmission of genetic material. 

With every generation, the number of repeats increases drastically, and the age at which the patient presents is inversely related to the number of expansions. The severity worsens with every generation due to a larger repeat sequence. This inheritance pattern of repeat-expansion diseases = anticipation. 

Pathogenic Threshold:

•Threshold number of repeats for each repeat disorder.

•If threshold not exceeded, individual remains unaffected.

•Once threshold surpassed, pathological phenotype manifests.

•Unaffected persons may have a premutation allele, which has a high probability of further expanding in successive generations into the mutant allele that causes the disease.

How Trinucleotide Repeat Disorders cause disease (Pathogenic mechanism):

Loss-of-Function (Transcriptional Silencing): 

•Large expansions can lead to transcriptional silencing of the gene (e.g., in Fragile X syndrome), causing a deficiency in necessary protein levels.

Gain-of-Function (Toxic Protein): 

•In coding regions, expanded repeats cause toxic gain-of-function (e.g., polyglutamine  diseases like Huntington’s), where proteins aggregate and damage cells.

Gain-of-Function (Toxic RNA): 

•Expanded repeats in non-coding regions produce toxic RNA (e.g., in Myotonic dystrophy 1) that binds to and sequesters RNA-binding proteins (RBPs), hindering their function.

Coding Region Polyglutamine Repeat Disorders

expansions of exonic (CAG)n repeats which code for long polyglutamine (polyQ) tracts in proteins. These expansions are relatively short compared to those in noncoding repeat disorders, with lengths only two or three times that of non-mutated alleles.

Huntington’s Disease (HD)

Spinocerebellar Ataxia (E.g., SCA1)

Dentatorubral-Pallidoluysian Atrophy (DRPLA) 

Huntington’s Disease (HD)

•Fatal neurodegenerative disorder.

•Phenotype: Progressive motor, cognitive, psychiatric disturbance.

•Autosomal dominant disease.

•Exhibits genetic anticipation.

•Caused by expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene. 

•Unaffected person: 6-34 repeats

•Affected person: 36-180 repeats

•The longer the length of CAG repeat, the earlier the onset of symptoms.

Spinocerebellar Ataxia (E.g., SCA1)

•Fatal progressive neurological disorder.

•Phenotype: Ataxia, neuropathy, pyramidal sign

•Autosomal dominant disease.

•Exhibits genetic anticipation.

•Results from the expansion of a CAG trinucleotide repeat in the coding region of ataxin-1 gene (ATXN1) 

•Unaffected person: 6-39 repeats

•Affected person: >39

Dentatorubral-Pallidoluysian Atrophy (DRPLA) 

•Progressive neurodegenerative disease, leads to death.

•Phenotype: Ataxia, chorea, dementia, myoclonus, seizure, anticipation, common in Japan (rare elsewhere).

•Autosomal dominant disease.

•Exhibits genetic anticipation.

•Caused by expansion of a CAG trinucleotide repeat in the atrophin 1 gene (ATN1).

•Unaffected persons 7-25 repeats.

•Affected person >49.

Noncoding Region, Non-polyglutamine Disorders 

Nucleotide composition of repeat units in these noncoding, multisystem diseases vary (e.g. GAA, CGG, CTG). Also, can undergo extremely long expansions, with repeats reaching into the thousands.

Fragile-X Syndrome (FRAXA)

Myotonic Dystrophy 1 (DM1)

Spinocerebellar Ataxia - Italian Spinone

Fragile-X Syndrome (FRAXA)

•Most common form of inherited intellectual impairment.

•Phenotype: Developmental delay, learning disability, autism spectrum disorder.

•X-linked dominant disease.

•Exhibits genetic anticipation.

•Caused by expansion of a CGG trinucleotide repat in the fragile-x mental retardation gene (FMR1).

•Unaffected persons: 6-52 repeats.

•Premutation: 55-200 repeats.

•Affected persons: >200 repeats.

Number of repeats determines severity of syndrome.

Myotonic Dystrophy 1 (DM1)

•Most common form of muscular dystrophy in adults. 

•Phenotype: Mild: cataracts, mild myotonia. Classic: weakness, myotonia, cataracts, cardiac abnormalities. Congenital: hypotonia, severe weakness at birth, respiratory difficulties.

•Autosomal dominant disease.

•Exhibits genetic anticipation.

•Caused by expansion of a CTG trinucleotide repeat in the dystrophia myotonica protein kinase gene (DMPK).

•Unaffected persons: 5-37 repeats.

•Affected persons: 50-several thousand repeats.

Spinocerebellar Ataxia - Italian Spinone

•Progressive neurodegenerative disease characterised by incoordination, a “prancing” gait, and loss of balance often resulting in falling.

•Clinical signs start to appear at four months of age and progress to a degree of dysfunction which leads to euthanasia at one year of age on average.

•Autosomal recessive inheritance.

•Likely caused by an intronic GAA repeat expansion in the ITPR1 gene.

Diagnosis - Detecting Repeat Expansion Disorders

•Patient history & Physical exam

•Family history

•Neuroimaging

•Genetic Testing

•Polymerase Chain Reaction (PCR)

•Repeat Primed PCR (RP-PCR)

•Whole Genome Sequencing (WGS)

•Southern Blotting

Potential Treatment for Huntington’s

•Small clinical trial – 29 patients.

•Funded by uniQure.

•Treatment (AMT-130) is a combination of gene therapy and gene silencing technologies.

•One-time treatment.

•12 to 18 hours of brain surgery.

•Data shows the disease was slowed by 75% in patients.

•Cost anticipated at > US$1 million/person.

Problems with the FDA

How does huntingtons gene therapy work?

1. Huntingtions mutation leads to toxic proteins in brain cells

2. Gene therapy infused into the brain

3. Brain cells become their own drug factory

4. Lowers levels of toxic protein in the brain

 Why pedigrees?

Geneticists Often Use Pedigrees to Study the Inheritance of Characteristics in Humans

•Difficulties with studying human genetics:

•People have children for many reasons, but usually not to understand the genetic mechanisms of inheritance.

•Long lifecycle (about the length of a human lifespan).

•Long inter-generational time (~20 years).

•Relatively small family size.

•Pedigrees are a good mechanism of displaying personal (genetic) relationships between human individuals.

Pedigree -

Pictorial representation of a family history, essentially a family tree that outlines the inheritance of one or more characteristics.

Autosomal Recessive Traits

Autosomal recessive is a pattern of inheritance characteristic of some genetic disorders. “Autosomal” means that the gene in question is located on one of the numbered, or non-sex, chromosomes. “Recessive” means that two copies of the mutated gene (one from each parent) are required to cause the disorder.

Recessive traits should occur relatively infrequently, because they are recessive, they are easily masked. You need two of the alleles to show the trait whereas you’d only need one dominant allele to show a dominant trait.

Autosomal recessive traits normally appear with equal frequency in both sexes and often skip generations

more likely to appear aming progeny with related parents

•E.g. Sickle Cell, Tay-Sachs, Cystic Fibrosis

Autosomal Dominant traits

Autosomal dominant traits normally appear with equal frequency in both sexes and do not skip generations.

•E.g. Huntington’s Disease, Marfan Syndrome

Autosomal dominant is a pattern of inheritance characteristic of some genetic disorders. “Autosomal”, again, means that the gene in question is located on one of the numbered, or non-sex, chromosomes. “Dominant” means that a single copy of the mutated gene (from one parent) is enough to cause the disorder. 

X-linked recessive traits

X-linked recessive inheritance refers to genetic conditions associated with mutations in genes on the X chromosome. A male carrying such a mutation will be affected, because he carries only one X chromosome. A female carrying a mutation in one gene, with a normal gene on the other X chromosome, is generally unaffected. Females would have to inherit two copies of the allele, one from each parent, to be affected. Because of this males are more often affected than females.

X-linked recessive traits appear more often in males than in females and are not passed from father to son.

•E.g. Colorblindness, haemophilia A

hemophilia

Classic hemophilia in the royal families of Europe

This blood-clotting disorder is inherited as an X-linked recessive trait.

X-linked dominant traits

X-linked dominant inheritance refers to genetic conditions associated with mutations in genes on the X chromosome. A single copy of the mutation is enough to cause the disease in both males (who have one X chromosome) and females (who have two X chromosomes). These traits tend to affect females more often than males, so if we see a female bias, we can start thinking maybe its x-linked dominant.

X-linked dominant traits affect both males and females. An affected male must have an affected mother. -affected males pass on the trait to all their daughters and none of their sons

•E.g. Rett syndrome

doesnt skip generations

If affected female is heterozygous pass the trait onto half of their sons and half of their daughters

Y-linked traits

Y-linked traits appear only in males and are passed from a father to all his sons.

•E.g. ‘Maleness’

Y-linked traits exhibit a specific, easily recognized pattern of inheritance. Only males are affected, and the trait is passed from father to son. If a man is affected, all his male offspring should also

be affected.

Pedigree characteristics of autosomal recessive trait

1. Usually appears in both sexes with equal frequency

2. Tends to skip generations

3. Affected offspring are usually born to unaffected parents

4. When both parents are heterozygous, approximately one fourth of the offspring will be affected

5. Appears more frequently among the children of consanguineous marriages

Pedigree characteristics of autosomal dominant trait

1. Usually appears in both sexes with equal frequency

2. Both sexes transmit the trait to their offspring

3. Does not skip generations

4. Affected offspring must have an affected parent unless they posses a new mutation

5. When one parent is affected (heterozygous) and the other parent is unaffected, approximately half od the offspring will be affected

6. Unaffected parents do not transmit the trait

Pedigree characteristics of X-linked recessive trait

1. Usually more males than females are affected

2. Affected sons are usually born to unaffected mothers; thus the trait skips generations

3. Approximately half of a carrier (heterozygotes) mothers sons are affected

4. Never passed from father to son

5. All daughters of affected fathers are carriers

Pedigree characteristics of X-linked dominant trait

1. Both males and females are usually affected; often, more females than males affected

2. Does not skip generations, Affected sons must have an affected mother, affected daughters must have an affected mother or an affected father

3. Affected fathers pass the trait to all their daughters

4. Affected mothers (if heterozygous) pass the trait to half their sons and half of their daughters

Y-linked traits

1. only males are affected

2. Passed from father to all sons

3. Does not skip generations

Microsatellite -

short segment of DNA, usually 1-6 or more base pairs in length, that is repeated multiple times in succession at a particular genomic location.

Trinucleotide Repeat -

specific type of microsatellite where the repeating unit is three nucleotides

Loss-of-Function (Transcriptional Silencing)

Large expansions can lead to transcriptional silencing of the gene (e.g., in Fragile X syndrome), causing a deficiency in necessary protein levels.

Gain-of-Function (Toxic Protein) -

In coding regions, expanded repeats cause toxic gain-of-function (e.g., polyglutamine  diseases like Huntington’s), where proteins aggregate and damage cells.

Gain-of-Function (Toxic RNA)

Expanded repeats in non-coding regions produce toxic RNA (e.g., in Myotonic dystrophy 1) that binds to and sequesters RNA-binding proteins (RBPs), hindering their function.

Pedigree

- Pictorial representation of a family history, essentially a family tree that outlines the inheritance of one or more characteristics

Proband

The person from whom the pedigree is initiated.