Triplet Repeat Disorders and Unusual Genetic Phenomena

Triplet Repeat Disorders

Lecture Objectives

• Explain repeat expansion disorders, in keeping and in deviation from Mendel’s laws.
• Describe the dynamic nature of triplet repeats.
• Explain the threshold concept, anticipation, and parent-of-origin effect.
• Distinguish between coding and non-coding repeat expansions and their effects.
• Contrast normal, intermediate, and affected repeat ranges using one condition as an example.

Genetic Variation

• Types of DNA variation include normal variation and disease-causing variations.
• Disease-causing variations can be:
  • One variant in one gene (Mendelian Inheritance).
  • Many variants in many genes (Multifactorial inheritance).
  • Large variations (Chromosome abnormalities).
• Epigenetics: Variation without a change in DNA sequence.
  • Mechanisms include X chromosome inactivation.
• Tools & Techniques:
  • PCR & qPCR
  • Sequencing
  • Karyotyping
  • FISH
  • Microarrays
  • Next Gen sequencing

Mendelian Inheritance

• Classical patterns of Mendelian inheritance imply:
  • Stable inheritance of mutations – parent and child have identical DNA change.
  • Biparental inheritance (equal contributions) – one allele from each parent.

Triplet Repeat Disorders

• Follow Mendelian rules of inheritance.
• However, because certain patterns of inheritance cannot be explained only on the basis on Mendel’s laws, they fall into the category of non-Mendelian inheritance.
• Do NOT disobey Mendelian principles but cannot be explained by these principles alone.

Repeat Expansion Disorders

• Area in responsible gene with a DNA sequence repeated several times over.
• Different numbers of nucleotides in the repeat stretch:
  • Trinucleotide, Tetranucleotide, Pentanucleotide, Hexanucleotide
• These repeat sequences are found naturally occurring in the genome of humans and many other species.

Mocular Basis

• Repeats occasionally undergo dynamic or expansion mutation during DNA replication.
• The expanded repeat may cause disease.

Triplet (Trinucleotide) Repeat Disorders

• A small group, particularly neuromuscular/neurodegenerative disorders.
• Examples:
  • Fragile X syndrome
  • Myotonic dystrophy
  • Huntington disease
• Inherited but alterations in the mutation can occur between generations, making them dynamic.

Triplet Repeat Disorders (TRDs)

• Characterized within and between families by:
  • Variable disease presentation and progression
  • Anticipation, defined as:
    • Earlier age of onset
    • Increased severity in successive generations
    • Parent of origin is important

TRDs: Molecular Basis

• Number of repeats varies:
  • Between individuals
  • On different chromosomes
• Generally few repeats
• Stable Mendelian inheritance in families

TRDs: Molecular Basis

• Threshold concept
  • Repeat number above a threshold, larger number of repeats
  • Different for different disorders
• Instability becomes manifest
  • Individuals within a single family have different repeat numbers
  • In the same individual, different tissues have different repeat numbers

TRDs: Molecular Basis

• The mutation is dynamic
  • Tends to increase in repeat number between generations
  • Initial change predisposes to further change
• Position of repeats
  • Repeats may be intragenic (within the exon or intron) or extragenic (5’ or 3’ of the gene)

TRDs: Molecular Basis

• If present within exons:
  • Encode a series of identical amino acids, translated to protein
  • Gain-of-function
• If present in the UTR:
  • Disrupt transcription, translation or protein function
  • Loss-of-function

TRDs: Molecular Basis

• Coding repeats
  • Commonly CAG
    • Code for amino acid glutamine (Q)
    • Polyglutamine (polyQ) diseases
  • Gain-of-function mutations
    • Mutant protein has a toxic effect
• Non-coding repeats
  • Variable: CGG, GCC, GAA, CTG, or CAG
  • Very large repeat expansions
  • Usually loss-of-function mutations
    • Multiple tissue dysfunction or degeneration

TRDs: Examples

• Diseases with $(CAG)_n$ repeats in coding regions:
  • Huntington disease
  • Spinocerebellar ataxia (SCA) subtypes 1, 2, 3, 6, 7
  • Spinobulbar muscular atrophy (Kennedy’s disease)
• Diseases with non-coding repeats:
  • Fragile X syndrome (CGG repeat)
  • Myotonic dystrophy (CTG repeat)
  • Friedreich ataxia (GAA repeat)

Huntington Disease (HD)

• Autosomal dominant inheritance
• Increased severity in successive generations
• Clinical features:
  • Typically adult onset, 35 to 44 years
  • Median survival, 15 to 18 years after onset
  • Movement disorder (chorea)
  • Psychiatric disturbances
  • Individuals lose ability to walk, talk and reason

Huntington Disease Genetics

• HTT gene on chromosome 4p
• Juvenile form (> 60 repeats), usually inherited from the father

Huntington disease-like 2 (HDL2)

• Also a triplet repeat disorder
• 41 or more CTG trinucleotide repeats in JPH3 are considered diagnostic of HDL2
• HDL2 is rare; so far fewer than 25 pedigrees and 40 affected individuals described worldwide
• Most individuals with HDL2 have been of definite or likely African ancestry
• In South Africa, individuals of African ethnicity with an HD-like phenotype are almost as likely to have HDL2 as HD

Myotonic Dystrophy (MD)

• Most common form of muscular dystrophy in adults (Type I)
  • At least 1/8000
• Autosomal dominant inheritance
• Marked anticipation
• Clinical features:
  • Onset 20s to 30s
  • Myotonia (slow relaxation of muscles)
  • Muscle weakness
  • Cataracts
  • Diabetes
  • Intellectual disability

Myotonic Dystrophy Genetics

• DMPK gene on chromosome 19q
  • CTG repeat expansion in the 3’ UTR
  • Gain-of-function mutation
• Trinucleotide repeat disorder
• Congenital form inherited from the mother
• Paternal transmission uncommon, possibly due to selective pressures against expansions in male gametes

Myotonic Dystrophy Genetics

• Number of CTG repeats in DMPK
  • Normal: 5-35 repeats
  • Intermediate: 35-50 (some instability, no disease)
  • Mild: 50-200 (Cataracts, Asymptomatic)
  • Classic disease: 200-1500 (Classic MD, Developmental Delay)
  • Congenital: 1000+ (Congenital MD) in 40s

Fragile X Syndrome (FRAX)

• Most common inherited form of intellectual disability in males – 1/2000-1/5000 males
• X-linked dominant, FMR1 gene
  • CGG repeat expansion in the 5’ UTR
  • Loss-of-function mutation: silenced transcription
• Large increases with female transmission (anticipation) – increased severity
• Alleles classified as normal, intermediate, premutation or full mutation

FMR1-related Disorders: Molecular Basis

• Number of CGG repeats in FMR1
  • Normal: 5-44 repeats
  • Intermediate: 45-54 repeats
  • Premutation: 55-200 repeats (FXTAS, FXPOI)
  • Full mutation: >200 repeats (FRAX)

Fragile X Syndrome: Affected Males

• Mutation present (>200 repeats)
• Intellectual disability
• Developmental delay
• Poor speech
• Hyperactivity/autistic behaviour
• Long face, large ears, prominent jaw (post-puberty)
• Large testes (post-puberty)

Premutation Carriers

• Normal intellect and appearance
• Some have subtle intellectual or behavioral symptoms including learning difficulties or social anxiety
• May develop Parkinsonian-like disorder in later life: Fragile X-associated tremor ataxia syndrome (FXTAS)
• Females may develop Fragile X-associated premature ovarian insufficiency (FXPOI)
• A male who is a premutation carrier is called a “normal transmitting male”

Full Mutation Carriers: Females

• Fragile X syndrome
• Mutation present on one X chromosome (>200 repeats)
• 50% are symptomatic
• Poorly understood:
  • Either normal or show developmental delay
  • Do not develop FXPOI
  • Greater risk of developmental delay in younger generations

Fragile X Syndrome Genetics

• Normal Transmitting Male
  • Carrier females (Mild ID)
  • Severe ID 2000
• Normal
  • Mild ID 400
  • Normal 9
• Mild ID 6/600
  • Mentally normal 6/650
  • Normal 6/9
• 80
  • 7/9
  • 9
• 80/7
• 80/9
• 6
• 7

Principles for TRDs

• Generally follow Mendelian laws
  • Autosomal dominant (MD, HD)
  • Autosomal recessive (Friedreich ataxia)
  • X-linked (FRAX)
• But:
  • Variable severity correlated to repeat length
  • Exhibit anticipation
  • Parent-of-origin effects
• Often missed because of variability
• Family history is very important

Principles for TRDs

• Normal range: stable, not expected to expand on transmission
• Intermediate range:
  • No disease
  • May expand upon transmission to next generation
  • Possible grey zone, individual may show mild features of the disorder (reduced penetrance)
• Affected range: disease-associated

Glossary

• Anticipation: Phenomenon associated with triplet repeat disorders where an earlier age of onset and increased severity of symptoms is observed in successive generations with larger repeat expansions.
• Stable mutation: Mutation that does not change when passed down to the next generation
• Dynamic mutation: Mutation that changes when passed down to the next generation
• Threshold concept: When a triplet repeat expands beyond a certain point it causes increased instability and increased likelihood of expanding further. The threshold is disease dependent. Beyond a certain point results in disease.
• Intermediate allele: Triplet repeat expansion that does not cause disease, but is able to expand in the next generation.
• Reduced penetrance allele: Triplet repeat expansion that may or may not result in disease. Penetrance is less than 100%.

Unusual Genetic Phenomena

Exception to the Rule!

• The rules being Mendel's laws of autosomal dominant inheritance

Lecture Objectives

• Explain how the 3 unusual molecular processes:
  • variable/incomplete penetrance (Part 1)
  • variable expressivity (Part 2)
  • new mutation event (Part 3)
    could complicate analysis of an autosomal dominant pedigree
• Explain the difference between variable (incomplete) penetrance and variable expressivity
• Give examples of conditions which are known to exhibit these phenomena

Introduction

• Although in theory autosomal dominant inheritance appears to be the simplest mode of inheritance, in clinical practice AD inheritance can be confusing and unclear
• We know a particular disorder is autosomal dominant
  • single gene
  • on an autosome
  • mutation in one allele is sufficient to cause the phenotype
• But sometimes inconsistency in the pedigree is observed

1. Penetrance

• It is useful to consider the possibility of reduced penetrance when considering apparent inconsistencies in a family history, such as "skipped generations“
• If it is possible for some people to carry a mutation but not develop the disorder, the condition is said to have reduced or incomplete penetrance

Penetrance

• Definition: – measurement of the proportion of individuals in a population who carry a disease-causing allele and express the disease phenotype

Incomplete penetrance

• “Complete” penetrance: means a mutation, when present, is expressed in all members of the population who have that mutation.
• “Incomplete” or ‘reduced’ penetrance: means that a mutation, when present, is expressed in only part of the population.
• Pathogenic mutations are completely penetrant most of the time. However, for certain traits it is now described that a pathogenic mutation might only lead to a phenotype in a subset of individuals from a population.

Measuring Penetrance

• Complete penetrance = 1.0 (or 100%)
  • All individuals with a given genotype have the same phenotype
• Incomplete penetrance
  • E.g. If 10 individuals have a certain genotype, but only 8 of them express the phenotype, the disease is said to have a penetrance of 0.8 (or 80%)

Example: Dominant Retinoblastoma

• A cancer of the retina that primarily affects children
• Retinoblastoma exhibits incomplete penetrance
• RB is 90% penetrant
  • -90% of mutation carriers WILL develop the disease
  • 10% of gene carriers WILL NOT develop the tumour
• but they may pass on the gene (Mendelian laws apply)

Example: Familial Cancers

• Many people with a mutation in the BRCA1 or BRCA2 breast cancer genes will develop cancer during their lifetime
  • But some people will not
  • Penetrance of BRCA1 mutations in females of European descent: approx. 85% by age 70 years
• Clinicians cannot predict which people with these mutations will develop cancer

Genetics of hereditary breast and ovarian cancer syndrome

• Autosomal dominant
• Successive generations affected
• Not all individuals who inherit a mutation in BRCA1 or BRCA2 will develop cancer (reduced penetrance) and the signs, symptoms, cancer type, and age of onset of cancer will vary within families (variable expressivity).

Possible mechanisms underlying the phenomenon of reduced penetrance

• Mutation type – severe vs mild mutations
• Environment – breast ca – age at first pregnancy, breast feeding, BMI
• Age – you might still get the disease later in life
• Gender – breast ca if female
• Modifier genes – genes at other loci influence trait

End of part 1

• Unusual Genetic Phenomena
  • Part 1: Incomplete Penetrance
  • Part 2: Variable Expressivity
  • Part 3: New Mutations

2. Variable Expressivity

• Penetrance is different to expressivity
• Think of penetrance as a light switch that can only be on or off (an individual either has the disease or not)
• and expressivity as a dimmer on that light switch (individuals who have the disease may have it with varying degrees of severity)

Variable Expressivity

• Variable expressivity refers to the range of symptoms that can occur in different people with the same genetic condition

Example of variable expressivity: Neurofibromatosis

• Neurofibromatosis:
  • Café-au-lait spots; Neurofibromas (benign tumors which grow along nerves)
• Family members who carry the same mutation may have varying degrees of the phenotype!

Example of variable expressivity: Marfan Syndrome

• Connective tissue disorder
• Some people have only mild symptoms
  • such as being tall and thin with long, slender fingers
• Others also experience life-threatening complications involving the heart / blood vessels
• All have mutations in the same gene → FBN1

Summary

• For disorders which exhibit variable expressivity: the same genotype can lead to a range of phenotypes
• This can make diagnosis difficult, which in turn, can complicate a pedigree/family history (as mildly affected individuals may be missed)

Visual Representation of Penetrance and Expressivity

• Variable penetrance
• Variable expressivity
• Variable penetrance and expressivity
• All these individuals have the same genotype

End of part 2

• Unusual Genetic Phenomena
  • Part 1: Incomplete Penetrance
  • Part 2: Variable Expressivity
  • Part 3: New Mutations

3. New Mutations

• In addition to the genetic information passed on from generation to generation (egg and sperm), each of us is born with a small number of novel genetic changes: de novo (or ‘new’) mutations
  • 44 to 82 de novo single-nucleotide mutations when compared to the two parental genomes
  • 1 - 2 usually affect the coding sequence
• These occurred either during the formation of the gametes or post-zygotically in the early embryo
• This is a natural occurrence, leading to normal variation and allows for evolution

Scenario

• A couple presents at Genetic Counselling Clinic. They have just had a child affected with achondroplasia (autosomal dominant inheritance). Both parents are normal and there is no family history.

New mutations

• No family history → sporadic
• Laboratory testing reveals:
  • De novo (new) mutation present in the affected individual
  • Neither parent carries the mutant allele

Achondroplasia – often a new mutation

• About 80% of cases are due to a de novo (new) dominant mutation

Recurrence risk

• For parents who have a child with a disorder resulting from a new mutation event
  • This was a random/chance event
  • Recurrence risk is exceptionally low
• For the individual who now has an autosomal dominant disorder resulting from a new mutation event
  • Recurrence risk is as per the Mendelian mode of inheritance: so for AD disorders, they have a 50% chance of having an affected child, with each pregnancy

Apert Syndrome

• almost always a new mutation
• craniosynostosis
• mitten hands (syndactyly)

End of part 3

• End of lecture: Unusual Genetic Phenomena
• One of these can usually explain inconsistencies seen in an autosomal dominant pedigree:
  • Incomplete Penetrance
  • Variable Expressivity
  • New Mutations

POPULATION SPECIFIC CONDITIONS

LEARNING OBJECTIVES

1. Explain the importance of studying diversity of genetic diseases in different population groups.
2. Discuss the reasons why certain mutations are more frequent in specific population groups, including founder effect, heterozygous advantage and consanguinity.
3. Demonstrate heterozygotic advantage as it pertains to sickle cell anaemia.
4. Give examples of conditions specific to the population groups of South Africa
5. Recognize which genetic testing would be appropriate in different scenarios with regards to management and prevention.

INTRODUCTION

• Research opportunities
• Major relevance when offering diagnostic testing.
• Frequencies of disease may differ between groups, and the mutational basis may be different.
• Inappropriate testing may be performed and important diagnoses may be missed.
• Different mutation-specific therapies may also be required in the future.
• Why is it important to study diversity of genetic diseases in different population groups?
• Many genetic conditions have population-specific distributions

WHY ARE SOME CONDITIONS MORE PREVALENT IN CERTAIN POPULATION GROUPS?

1. Founder effect

• The loss of genetic variation that occurs when a new population is established by a very small number of individuals from a larger population.
• New population may be distinctively different
• Genotypically
• Phenotypically

Elaboration on Founder Effect

• New population often very small population
• ↑ inbreeding
• ↓ genetic variation
• Rare alleles will move to one of two extremes
  1. Allele is soon lost altogether
  2. Allele survives and become more dispersed throughout population
• ↑ frequency of recessive alleles
• ↑ number in individuals homozygous for recessive traits

EXAMPLE OF A FOUNDER POPULATION IN SA

• The Afrikaner population of SA has many features of a founder population.
• They originated from a small number of individuals,
• Then underwent a period of rapid growth.
• Genetic mutation frequencies in the new population may differ significantly from those in the original population.

WHY ARE SOME CONDITIONS MORE PREVALENT IN CERTAIN POPULATION GROUPS?

2. Heterozygotic advantage

• The heterozygous genotype has a higher relative fitness than either the homozygous dominant or homozygous recessive genotype
• Sickle cell anaemia
  • Affects haemoglobin
  • Causes RBC to become sickle shaped
• Signs and symptoms
  • Anaemia
  • Shortness of breath
  • Fatigue
  • Delayed growth and development
  • Jaundice
  • Painful episodes
  • Organ damage
  • Autosomal recessive inheritance

HETEROZYGOUS ADVANTAGE

• 10-20% of people in certain parts of Africa are carriers of SCA
• Strong heterozygotic advantage
• Carriers (sickle cell trait) are resistant to malaria
• Expected that the allele frequency will decrease overtime if it only has a negative result.
• However, areas with malaria outbreaks carriers have a distinct advantage.

Visual Representation: Malaria Prevalence and HS Allele Frequency

• Correlation between areas where malaria is common and the frequency of the HS allele.

WHY ARE SOME CONDITIONS MORE PREVALENT IN CERTAIN POPULATION GROUPS?

3. Consanguinity

• Prevalent in many Middle Eastern and Arab cultures and societies
• Offspring may be at ↑ risk for genetic disorders because of the expression of AR gene mutations
• CLOSER the relationship the ↑ the probability that offspring will inherit identical copies of faulty recessive genes
• Marriage or reproductive relationship between two closely related individuals

Elaboration on Recessive Mutations

• Most genetic disorders are caused by recessive mutations,
• Recessive disease mutations are much more common than dominant diseases mutations
• “Dominant" mutations are more easily eliminated by natural selection.
• Humans carry an average of one to two disease causing mutations

DIFFERENT POPULATIONS IN SOUTH AFRICA

• Comprehensive data on South Africa's population in 2017, broken down by race and gender.

AFRICAN POPULATION: ALBINISM

• Common among black African populations in Southern Africa
• The average carrier rate for albinism in South Africa is 1 in 30, resulting in 1 in 4000 black South Africans being born with albinism.
• Clinical manifestation
  • Fair skin and light colored hair
  • Sun exposure increases the risk of skin damage and skin cancer
  • Reduced pigmentation of the iris and the retina
  • Visual problems
  • Nystagmus
  • Photophobia
• Genetics
  • Genes associated is involved in producing pigment (melanin)

AFRICAN POPULATION: OTHER MENTIONS

• Trinucleotide repeat disorders
• Neither myotonic dystrophy nor Friedreich’s ataxia has ever been reported in an black patient in South Africa
• Absence of a predisposing chromosomal background (haplotype)
• Huntington disease shows some unique features in black individuals.
  • Previously thought to be rare in this ethnic group.
  • HD in people of African ancestry has been shown to be genetically heterogeneous.
  • HD due to mutations in the HTT gene occurs, but on African-specific haplotypes that differ from those in white patients.
  • In addition, a second HD gene (JPH3) has been implicated in African patients with an HD-like phenotype (HDL2)
  • Individuals with HDL2 share many clinical features with individuals with HD - clinically indistinguishable

CAUCASIANS: CYSTIC FIBROSIS

• CF the most common life-limiting disease among people of Northern European heritage.
• One of the most common autosomal recessive disorders.
• Occurs in all of SA’s diverse population groups
• CF is a multifaceted condition with
• wide clinical variability,
• hundreds of causative CFTR variants

CAUCASIANS: CYSTIC FIBROSIS (cont.)

• Caused by variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene
• Function: regulation of movement of chloride across the epithelial membranes in the body
• Movement of water across cell membranes is dependent on the correct function of the chloride and sodium channels.
• The defects in CFTR result in poor movement of water across the epithelial cell surfaces; which causes the secretions produced by many organs of the body to be dehydrated and sticky.

CAUCASIANS: CYSTIC FIBROSIS (cont.)

• In South Africa, approximately
  • 1 in 27 individuals in the Caucasian population,
  • 1 in 55 in the population of mixed ancestry
  • and up to 1 in 90 black Africans carry a CF mutation.
• It is estimated that 1 in 2 000 Caucasian babies, 1 in 12 000 babies of mixed ancestry and up to 1 in 32 000 black African are born with CF in South Africa.

ASHKENAZI JEWISH POPULATION

• Several autosomal recessive genetic disorders that are more common in Jewish populations
• Due to population bottlenecks as well as practice of consanguineous marriages
• Lead to a decrease in genetic diversity
• Higher likelihood that two parents carrying the same mutation will have a child who will then have both mutations

COMMON CONDITIONS IN THE ASHKENAZI JEWISH POPULATION

Several organizations, such as DorYeshorim, offer screening for Ashkenazi genetic diseases, and these screening programs have had a significant impact, in particular by reducing the number of cases of Tay–Sachs disease.

Common conditions in the Ashkenazi Jewish population and the carrier rates are listed in a table:

ASHKENAZI JEWISH: TAY-SACHS DISEASE

• Rare autosomal recessive condition
• Progressively destroys nerve cells in the brain and spinal cord
• Typically appear normal until the age of 3-6 months
• Development slows and muscles weaken
• With disease progression:
  • Seizures
  • Vision and hearing loss
  • Intellectual disability
  • Paralysis
• Usually only survive into early childhood

AFRIKANERS

• Several diseases with an unusually high frequency in Afrikaners have been suggested to be the result of founder effects:
  • Sclerosteosis
  • Cystic fibrosis
  • Gaucher's disease
  • Autosomal recessive polycystic kidney disease
  • Familial colonic polyposis
  • Variegate porphyria
  • Familial hypercholesterolemia
  • Huntington’s disease
  • Fanconi anemia
  • Pseudoxanthoma elasticum

AFRIKANER: SCLEROSTEOSIS

• Autosomal recessive disorder characterized by bone overgrowth.
• Clinical features
  • Syndactyly
  • Facial distortion
  • Tall stature
  • Recurrent facial palsies
  • Increased intracranial pressure
  • Hearing loss
• Majority of individuals affected are Afrikaners (Dutch ancestry)
• Carrier rate 1:100
• Prevalence 1:60 000
• Extremely rare outside SA

INDIAN: THALASSAEMIA

• Thalassemia is most prevalent in populations having Mediterranean ancestry
• Inherited blood disorder characterised by fewer red blood cells (RBC) in the body than normal and less haemoglobin in RBC
• Symptoms include:
  • fatigue, weakness, paleness and slow growth.
• Mild forms may not need treatment.
• Severe forms may require blood transfusions or a donor stem-cell transplant

INDIAN: THALASSAEMIA (cont.)

• Thalassaemia in Natal
• The Indian people who came to Natal originated in regions of the Indian subcontinent where malaria was endemic, and many were heterozygous for a thalassaemia trait that protected them from malaria.
• It is not surprising that thalassaemia is the most common single gene disorder found in their descendants in Natal, where malaria was also endemic during the 19th and early 20th centuries

MANAGEMENT AND GENETIC TESTING

• Carrier screening
  • Before a couple gets married or have children
    • Important that BOTH partners are tested
• Mainly to prevent recessive conditions
  • Discourage marriages between carriers

MANAGEMENT AND GENETIC TESTING (cont.)

• Prenatal Genetic Diagnosis (PGD)
  • Individuals affected with a condition to have unaffected children
• Both parents are carriers of a condition and want to ensure an unaffected baby
  • IVF and genetic testing before implantation of embryo

MANAGEMENT AND GENETIC TESTING (cont.)

• Prenatal genetic testing
  • Women who are already pregnant and want to test the fetus to determine if the fetus is affected or not.
  • Patient would need to decide if they want to continue with the pregnancy or want to terminate the pregnancy
• Predictive Testing
  • Adult onset conditions