Simple Repeat Expansion Disorders: Huntington’s Disease and Fragile X Syndrome

Classification and Properties of Simple Repeats

  • General Terminology: Simple repeats are also referred to as short tandem repeats (STRs), microsatellites, or minisatellites.

  • Specific Classifications:

    • Microsatellites: Repeat motifs approximately 1−4 bp1-4\,bp in length.

    • Minisatellites: Repeat motifs approximately 5−64 bp5-64\,bp in length.

  • Genetic Instability: Unlike standard genetic mutations that remain stable across generations, simple repeats are unstable and can expand during intergenerational transmission.

  • Clinical Relevance: There are currently over 40 known diseases caused by repeat expansions, most of which primarily impact the nervous system (e.g., Fragile X Syndrome and Huntington’s Disease).

  • Pathogenicity Factors:

    • Repeats can be pathogenic when located in either coding or non-coding regions of a gene.

    • Expandability: Tandem repeat arrays can increase in copy number when passed from parent to offspring.

    • Parental Bias: Certain repeats tend to expand specifically during inheritance from the mother, while others expand through the father.

    • Anticipation: This refers to the phenomenon where a genetic disease increases in severity and shows an earlier age of onset in successive generations due to the increasing number of repeat copies.

    • Somatic Expansion: Repeat arrays can also continue to expand within the somatic cells (non-germline cells) of a patient after birth.

Mechanisms of Repeat Expansion and Pathogenicity

  • Mechanisms of Expansion:

    • Old/Simple Model: Attributed expansion to replication slippage at 3’ end during DNA synthesis.

    • New/Complicated Model: Simple repeats form secondary structures. These structures interfere with and impact DNA replication, transcription, chromatin state, and DNA repair mechanisms. These factors converge differently depending on the specific repeat expansion involved.

  • General Mechanisms of Pathogenicity:

    1. Loss of Function: The formation of secondary structures leads to transcriptional pausing and epigenetic silencing of the affected gene.

    2. Toxic Repetitive RNA: RNA secondary structures can bind to and sequester RNA binding proteins, removing them from their normal cellular functions.

    3. Toxic Protein Gain-of-Function: If the repeat is in a coding region, it can result in the production of insoluble proteins that form cytotoxic aggregates.

Huntington’s Disease (HD)

  • Overview: HD is the most common monogenic neurological disorder in the developed world.

  • Epidemiology and Inheritance:

    • Inheritance: Autosomal dominant, 1 pathogenic allele

    • Onset: Typically occurs in mid-life within the age range of 30−5030-50 years.

    • Progression: The disease is progressive and uniformly fatal.

  • Clinical Presentation:

    • Motor Symptoms: Involuntary spasmodic movements of the limbs and facial muscles (chorea), difficulty with balance, speech, and swallowing. Later stages involve muscle stiffness and slow movements.

    • Cognitive Symptoms: Declines in memory, attention, planning, and decision-making.

    • Emotional symptoms: Mood swings, irritability, apathy, depression, and significant personality changes.

  • Disease Timeline:

    • Prodromal Phase: Up to 15 years15\,years before hallmark motor symptoms, patients may show subtle motor or cognitive changes.

    • Survival: The median survival time after the onset of motor symptoms is approximately 18 years18\,years.

  • Molecular Basis (The HTT Gene):

    • Caused by an abnormal expansion of a CAG trinucleotide repeat array located in Exon 1 of the HTT gene on Chromosome 4.

    • The HTT gene encodes the protein Huntingtin, which is essential for nervous system development.

    • The CAG array encodes a poly-glutamine (poly-Q) tract. While everyone has some CAG repeats, expansion results in an abnormally long poly-Q tract.

  • Repeat Thresholds and Disease Status:

    • <28 repeats: Stable array, Normal disease status.

    • 28−3528-35 repeats: Shows instability but results in a Normal disease status.

    • 36−4036-40 repeats: Increasing instability, Reduced Penetrance (patient may or may not develop symptoms).

    • 41+41+ repeats: Increasing instability, Full Penetrance (patient will definitely develop the disease).

    • CAG expansion instability increases as the array length exceeds 27 repeats.

  • Anticipation in HD:

    • Intergenerational expansion occurs primarily in the male germ line. Male patients are more likely to pass on newly expanded alleles.

    • Offspring have a 50%50\% chance of inheritance if a parent is heterozygous.

    • Anticipation: generation worsening of genetic disease severity and decreasing age of onset by intergenerational expansion.

  • Pathogenic Mechanisms:

    • Translation produces full-length Huntingtin and fragments containing the expanded poly-Q tract.

    • These proteins self-associate to form aggregates in the nucleus and cytoplasm.

    • Aggregates impair transcription, intracellular signaling, transport, secretory pathways, endocytic recycling, mitochondrial function, synaptic function, and immunity.

  • Somatic Expansion Research:

    • Specifically targets striatal projection neurons (SPNs), which are the cells lost in HD.

    • SPN CAG arrays can somaticallly expand to over 500500 repeats.

    • Beyond the 500500 repeat threshold, SPNs undergo a ‘de-repression crisis’ leading to cell death.

Fragile X Syndrome (FXS)

  • Overview: The leading cause of inherited intellectual disabilities.

  • Epidemiology and Inheritance:

    • Inheritance: X-linked dominant (though females show variable and milder symptoms due to random X-inactivation).

    • Prevalence: 1 in 5,0005,000 males; 1 in 4,000−8,0004,000-8,000 females.

    • Diagnosis: Average age is 35−37 months35-37\,months for boys and 42 months42\,months for girls.

    • Prognosis: Lifelong condition, no cure, but life expectancy is normal.

  • Phenotypic Characteristics:

    • Intellectual disability, language delays, anxiety, and sensory over-reactivity.

    • Physical traits: Prominent ears, long face, flat feet, double-jointed thumbs, soft skin, and macro-orchidism.

  • Molecular Basis (The FMR1 Gene):

    • Caused by a CGG repeat array expansion within the promoter/5’ UTR of the FMR1 gene (Xq27.3).

    • The gene encodes FMRP, an RNA-binding protein that controls the transport and translation of mRNAs in the brain, impacting learning and the endocannabinoid system.

  • Repeat Thresholds and FXS Status:

    • 5−445-44 repeats: Stable, Normal status.

    • 45−5445-54 repeats: Shows instability, Normal status.

    • 55−20055-200 repeats: Premutation Carrier. At risk of passing on full mutation. Individuals are at risk for FXTAS (Fragile X-associated tremor/ataxia syndrome), FXPOI (Fragile X-associated primary ovarian insufficiency), and FXAND (Fragile X-associated neuropsychiatric disorders).

    • >200 repeats: Increasing instability, Full Mutation (FXS affected).

  • Transmission and Expansion:

    • The CGG array expands in the female germ line. Female premutation carriers are more likely to pass on expanded alleles.

    • Unlike HD, FXS does not show progressive generational worsening (the primary issue is the jump from premutation to full mutation).

  • Silencing Mechanism:

    • Expansions over 200200 repeats form secondary structures during transcription (R-loops).

    • These structures recruit repressive chromatin modifiers and lead to DNA methylation of CpGCpG sites.

    • The resulting epigenetic silencing prevents the production of FMRP mRNA and protein.

Sequencing Technologies in Repeat Expansion Research

  • Short-Read Sequencing:

    • Requires 100 ng100\,ng of DNA; takes ~$7\,hours$.

    • DNA is cut into small fragments, ligated, and amplified.

    • Repeat size is estimated by tiling short reads together. It is often inaccurate for large expansions.

  • Oxford Nanopore Sequencing (Long-Read):

    • Requires 1 μg1\,\mu g of DNA; takes ~$2-3\,hours$.

    • Long DNA fragments are fed through nanopores. Ion current signals are recorded as bases pass through.

    • Provides accurate sizing as a single read can cover the entire STR region.

Case Study / Practice Exam Question

  • Scenario: Ethan has clinical symptoms of Fragile X (intellectual disability, elongated face, large ears). His brother Steve has no symptoms. Their mother Sara is a premutation carrier.

  • A. What gene is mutated? The FMR1 gene.

  • B. Nature and location of mutation? It is a CGG trinucleotide repeat expansion found in the promoter/5’ UTR (non-coding region) of the gene.

  • C. What is a ‘premutation carrier’? An individual with between 55−20055-200 CGG repeats. They do not have FXS themselves but have an unstable allele that can expand to a full mutation (>200) in their children.

  • D. Predicted results for siblings?

    • Ethan: Testing will likely reveal a full mutation allele with >200 CGG repeats. Reasoning: His symptoms indicate the gene is silenced by massive expansion and subsequent methylation.

    • Steve: Testing will likely reveal a normal allele (5−545-54 repeats) or a premutation allele (55−20055-200). Reasoning: Since he is asymptomatic, he lacks the full mutation required to cause FXS silencing.