Second Week of Notes Flashcards
Characteristics of Autosomal Recessive Disorders
o Phenotype is only expressed in homozygotes rr
Need to have both mutant alleles
o Males and females are usually equally affected
Does not target based on gender
o Horizontal Inheritance Pattern
-Normally see it across in siblings (they are rare unless there are carrier parents
Parents of an affected child are obligate carriers
o Recurrent risk for each unborn child is ¼ (both parents are carriers)
RR, Rr, Rr, rr (rr is 25%)
o The chance of unaffected siblings being a carrier is 2/3
Carrier =Rr
Only 3 unaffected made RR, Rr, Rr….. 2 of the three are carriers
Calculating Frequencies of AR Disorders (PRACTICE)
o Amount of an autosomal recessive disease 1/10,000
♣ This is the q^2
o Find the square root to get q
♣ 1/100 is the amount of q alleles there are
o Find the amount of carriers (the Rr)
♣ Find 2pq
♣ Rare diseases, just do 2q
• 2* 1/100= 2/100
o REMEMBER, when finding carriers for rare diseases
♣ 2pq~ 2q
• Carrier Frequency vs. Disease Frequency (Practice!!)
o Scotland 1/5,300 1/36 (2pq)
o Finland 1/200,000 1/250
Parental Consanguinity
• Parental Consanguinity The more affected children of an autosomal recessive disease, the more likely there is Parental Consanguinity (common ancestor or inbred
o If the two parents inherited the same mutant allele from a common ancestor, then the disease risk for their child
Much higher risk to have the carrier traits passed down in consanguinity
Compound Heterozygosit
Compound Heterozygosity: presence of multiple common mutant alleles of the same gene in a population. There are different mutant alleles
o Example: is an individual who carries two different mutant alleles of the same gene
♣ Bc// Bs (both of these are mutant alleles in hemoglobin)
• One is sickle cell, other is the hemoglobin C
Simple Heterozygotes
Simple Heterozygotes Have two different alleles, only one of them could be mutant
♣ Ba//Bs (one is normal, the other is mutant sickle-cell)
o Homozygots= Ba//Ba Bs//Bs (one healthy homozygous, other is mutant homozygous)
Phenylketonuria and it’s genetics
Phenylketonuria High phenylalanine in blood due to lacking a critical enzyme, phenylalanine hydroxylase. Will lead to mental retardation, hyperactivity and epilepsy. Caused by mutations for phenylalanine hydroxylase at chromosome 12. Treated by a low phenylalanine diet and possible neutral amino acid supplementation
Cause of Phenylketonuria
♣ High amount of Phenylalanine levels in the blood, due to the defective nature or lack of enzyme phenylalanine hydroxylase
• Phenylalanine Hydroxylase (PAH): Normally found in the liver, is used to turn phenylalanine Tyrosine
• Defects cause 98% of the diseases
Defect in Cofactor BH4 ( a cofactor for PAH) Need the cofactor B4 to bind to the phenylalanine hydroxylase enzyme (and for dopamine/serotonin)
• This causes 1-2% of the cases of Phenylketonuria
• Lacking BH4 Also leads to a loss of neurotransmitters such as dopamine and serotonin
Possible phenotypes from Phenylketonuria
High phenylalanine metabolites in urine, hyperactivity, mental retardation, epilepsy, microcephaly
Molecular defects in PAH The PAH gene is at chromosome 12q22-24. Most mutations in PAH are partial or complete loss-of-function alleles. PAH gene exhibits high allelic heterogeneity; over 400 alleles have been identified. Most PKU patients are compound heterozygotes (i.e. having two different mutant alleles of the PAH gene). Severity of phenotype varies and probably reflects compound heterozygosity
Phenylketonuria (PKU) Defects in BH4 synthesis and Recycle
♣ Defects in different enzymes that give rise to cofactor BH4 (BH4 is for the phenylalanine hydroxylase)
• Enzymes defected to build BH4: GTP-CH, 6-PTS
o Phenylketonuria (PKU) Screening
Detecting Phenylketonuria (PKU) and treating it
Phenylketonuria (PKU) Screening
-Mass Spectrometry(MS/MS) is the current method of choice for many newborn screenings including PKU. A mass spectrometer simultaneously sorts many molecules in a blood specimen by weight (mass) and size, and also measures the quantity of each molecule.
• Sorts molecules in a blood specimen by size, weight, and quantity.
• Fast detection and high sensitivity.
• Measures multiple molecules simultaneously.
Timing of Test: The sensitivity of PKU screening is influenced by the age of the newborn when the blood sample is obtained. Phenylalanine level is typically normal in PKU babies at birth because of normal PAH in maternal supply and increases progressively with the initiation of protein feedings during the first days of life. Early detection and treatment is crucial to prevent irreversible damage to the developing brain. However, if tested too early (within 1-2 days of birth), some affected children can be missed.
• Newborns are tested first after birth and then again at their first pediatrician’s visit days later.
Guthrie test (aka Guthrie bacterial inhibition assay) for PKU was developed in the mid- 1960s. This test is based on the findings that phenylalanine inhibits the growth of the bacterium Bacillus subtilis and that such inhibition can be overcome by a high level of phenylalanine in the blood sample of a PKU baby.
o Phenylketonuria Treatment When treated early with low-phenylalanine diet, the mental retardation can be prevented. Phenylalanine is an essential amino acid and thus cannot be eliminated from the diet. The low-phenylalanine diet should be maintained throughout childhood and school years, and preferably the patient’s whole life. BH4-deficient PKU patients are treated with oral BH4, low-phenylalanine diet, and supplements (L-dopa and 5-hydroxytryptophan etc) to balance neurotransmitter levels.
Low-Phenylalanine Diet:
• From birth through childhood, and beyond
• For women, throughout child-bearing years
BH4 supplementation • Kuvan® : long-term safety and effectiveness?
Other treatments:
• Neutral amino acid supplementation
o Dietary supplementation with large neutral amino acids(LNAAs), with or without the traditional PKU diet is another treatment strategy. The LNAAs (e.g.leu,tyr,trp,met,his,ile,val,thr) compete with phe for specific carrier proteins that transport LNAAs across the intestinal mucosa into the blood and across theblood brain barrierinto the brain .
• Enzyme replacement therapy
• Gene therapy
o PKU: Maternal Effect
♣ Problem: Phenylketonuria women who are off low-Phe diet in pregnancy
• markedly increased risk of miscarriage.
• congenital malformations and mental retardation in babies.
Cause: elevated phenylalanine level in maternal circulation (due to being off diet)
• Prevention: maintain low-Phe diet for females with PKU throughout their child-bearing years.
1-antitrypsin deficiency (ATD)
1-antitrypsin deficiency (ATD) A disease where a protease inhibitor (Serpina1 gene) is inhibited, allowing the protease elastase run rampant, destroying connective tissue elastin in lungs, leading to lung emphysema and liver disease
Phenotype
ATD patients have a 20-fold increased risk of developing emphysema, with more severe symptoms among smokers. This disorder is late-onset, especially in non-smokers, but 80- 90% of deficient individuals will eventually develop disease symptoms. Many patients also develop liver cirrhosis and have increased risk of liver carcinoma due to the accumulation of a misfolded α1-AT mutant protein in the liver
Different conditions it leads to :
• Emphysema of lungs and lung disease
• Liver Cancer and Liver Cirhosis
Earlier and more severe symptoms in smoker (ecogenetics).
Mechanism of 1-antitrypsin deficiency (ATD)
α1-antitrypsin (ATT or SERPINA1) is made in the liver and secreted into plasma. SERPINA1 is a member of serpins (serine protease inhibitor), which are suicide substrates that bind and inhibit specific serine proteases. The main target of SERPINA1 is elastase, which is released by neutrophils in the lung. When left unchecked, elastase can destroy the connective tissue proteins (particularly elastin) of the lung, causing alveolar wall damage and emphysema.
♣ Deficiency in a1-antitrypsin protein (SERPINA1, AAT), a protease inhibitor
♣ SERPINA1 is a suicide substrate of the serine protease elastase.
• Elastase is released by activated neutrophils at the airway, destroying elastin in the connective tissues.
• It’s macrophages that release signals to summon neutrophils the neutrophils will release the Elastase
♣ ATD patients have an imbalance of elastase and SERPINA1 levels
ATD: Molecular Basis The SERPINA1 gene is on chromosome 14 (14q32.13). There are ~20 different mutant alleles, although the Z & S alleles account for most of the disease cases. The Z allele (Glu342Lys) encodes a misfolded protein that aggregates in the endoplasmic reticulum (ER) of liver cells, causing damage to the liver in addition to the lung. The S allele (Glu264Val) expresses an unstable protein that is less effective.
♣ ATD is caused by mutations in a1-AT (SERPINA1) gene.
♣ a1-antitrypsin (elastin inhibitor) is produced in the liver and transported to the lungs via the blood.
Three common M alleles encodes functional proteins.
• Z allele (Glu342Lys) is the most common mutant allele. (Glu at 342 is replaced by Lysine)
o Individuals with Z/Z genotype have ~15% of normal SERPINA1 level.
o The Z allele makes a protein that is not folded properly and tends to accumulate in the endoplasmic reticulum of liver cells, leading to liver damage.
S allele (Glu264Val) makes unstable SERPINA1 protein. o Individuals with S/S genotype has 50-60% of normal SERPINA1 level.
1-antitrypsin deficiency (ATD) Treatment and
effects of smoking on ATD
antitrypsin deficiency (ATD) treatment:
Two approaches of delivering human SERPINA1 to the pulmonary epithelium are being studied: intravenous infusion and aerosol inhalation.
♣ Inhaled bronchodilators and inhaled steroids
♣ O2 therapy and possibly lung replacement
♣ In the future: Enzyme Replacement Therapy and Gene therapy are possibilities
1-antitrypsin deficiency (ATD) and Smoking:
Ecogenetics (Ecogenetics) Smoking accelerates the onset of emphysema in ATD patients. Tobacco smoke damages the lung, prompting the body to send more neutrophils to the lung for protection. More neutrophils release more elastase, causing more severe lung damage. (Dont have α1-antitrypsin to inhibit elastase)
Tay-Sachs Disease (GM2 gangliosidosis type I)
Tay-Sachs is an inherited disorder that progressively destroys neurons in the brain and spinal cord. The most common form of T-S is an early-onset, fatal disorder apparent in infancy. T-S infants appear normal until the age of 3-6 months, when early symptoms such as muscle weakness, decreased attentiveness, and increased startle response appear. As the disease progresses, the T-S children experience symptoms of neurodegeneration including seizures, vision and hearing loss, diminishing mental function, and paralysis. An eye abnormality called “cherry-red spot” is a characteristic of T-S. Children of T-S usually live only till 3-4 years of age.
T-S is caused by a defective α subunit; only HexA activity is affected. Sandhoff disease is caused by a defective Hex A and defective Hex B, leading to defective Hexosaminidase A and B
Phenotype A fatal genetic disorder in children that causes progressive destruction of the central nervous system.
-T-S babies start to develop neurological deterioration around 3 - 6 months of age, and die by 2 - 4 years.
First signs - muscle weakness and a startle response at sudden sound.
Advanced Symptoms: Loss of voluntary movement, muscle seizure, mental retardation, vegetative state
Biochemical defects of Tay-Sachs disease T-S is a lysosomal storage disease. Inability to degrade GM2 ganglioside results in up to 300-fold accumulation of this sphingolipid inside swollen lysosomes in neurons of the central nervous system. A defective hexosaminidase A (HexA) needed for in metabolizing GM2 is responsible for T-S. HexA is a heterodimer of αβ, which are encoded by the HEXA and HEXB genes, respectively. Although HexA is a ubiquitous enzyme, the impact of T-S is primarily in the brain where most of GM2 ganglioside is synthesized.
T-S is a lysosomal storage disorder with (>300x) accumulation of GM2 ganglioside in the lysosome.
• GM2 ganglioside is primarily synthesized in neurons of the brain and a componentof neuron cell membrane.
♣ T-S patients are unable to degrade GM2 ganglioside because of a defective hexosaminidase A.
• Hexosaminidase A consists of two subunits: a and b, which are encoded by the HEXA and HEXB genes, respectively.
o T-S patients usually have mutations in the HEXA gene.
• Mutations in HEX B causes Sandhoff disease (GM2 gangliosidosis II),a similar lysosomal storage disorder.
Sandhoff disease
Sandhoff disease (GM2 gangliosidosis type II) presents the same neurological symptoms as T-S. Sandhoff disease patients have defects in both genes Hexosaminidase A and Hexosaminidase B (HexB) • T-S is caused by a defective α subunit; only HexA activity is affected. Sandhoff disease is caused by a defective Hex A and defective Hex B, leading to defective Alpha and Beta units in Hexosaminidase A • The α subunit gene HEXA and the β subunit gene HEXB reside on chromosomes #15 and #5, respectively.
GM2 ganglioside degradation requires three proteins
The first two proteins are the hexosaminidase A and the hexosaminidase B (the two proteins that enzymatic degrade GM2 ganglioside
GM2A Need this protein to recognize the ganglioside and bring it the hexosaminidase A and B complex for degradation
• If GM2A is defective, can lead to lysosome storage disease
GM2A
GM2A Need this protein to recognize the ganglioside and bring it the hexosaminidase A and B complex for degradation
• If GM2A is defective, can lead to lysosome storage disease
Recognize The effects of Tay-Sachs and Sandhoffs Disease
Tay-Sachs is caused by a defective α subunit; only HexA gene activity is affected.
Sandhoff disease is caused by a defective Hex A and defective Hex B, leading to defective Alpha and Beta units in Hexosaminidase A
If the GM2AP activator is defective, then you cant bring Ganglioside to the hexosaminidase
Screening for Tay-Sachs disease:
o Enzymatic activity assay: both HexA and HexB enzymes are present in the serum. Their activities can be distinguished in such assays because only HexA is inactivated by heat.
o Carrier Screening: primarily among Ashkenazi Jewish population, the enzyme test has 97% accuracy because carriers have lower HexA enzyme levels in the blood.
o Prenatal screening: the enzyme test can also be performed on cultured amniotic fluid cells to detect T-S fetus when both parents are known to be carriers. Notably, this screening has reduced the number of T-S cases by about 95% over the past 30 years.
o DNA testing: The tests currently available can detect about 95% of carriers in the Ashkenazi Jewish population and about 60% of carriers among non-Jewish individuals. Therefore, some carriers will be missed by DNA test alone.
Complex Traits
Complex Traits—> Multifactorial Inheritance
o For a large portion of diseases there are multiple genetic components and environmental components
o Complex traits aggregate in families
Need to distinguish between clustering in families due to genetic factors and those due to shared environmental factors
o Epidemiologic twin, adoption, and immigration studies used
-Each measure of genetic contribution needs to be interpreted carefully, but as a group can provide compelling evidence for genetic contribution to trait.
Concordance
Usually means the presence of the same trait in both members of a pair of twins. However, the strict definition is the probability that a pair of individuals will both have a certain characteristic, given that one of the pair has the characteristic.
o For example, twins are concordant when both have or both lack a given trait
o A twin study compares the concordance rate of identical twins to that of fraternal twins. This can help suggest whether a disease or a certain trait has a genetic cause
Twin Studies
o Compare monozygotic to dizygotic twins
If twins raised together and assume same degree of similar environment, then differences in concordance rate between mono- and dizygotic twins likely due to genetic factors
- Monozygotic Twins Share 100% of the same genetic code
- Dizygotic Twins Shares roughly 50% of the same genetic code
Twin studies provide a potential means of overcoming this problem. Monozygous (MZ) twins are identically matched for DNA sequence, age, and gender, and perhaps closely matched for environmental exposures. Dizygous (DZ) twins on average share 1/2 of their DNA sequences, but may be about as closely matched for other factors as are MZ twins. If it can be assumed that MZ and DZ twins are equally similar with respect to noninherited factors, then twins can be used to get an estimate of the relative contribution of genetic vs. environmental variation to the trait.
Compare Monozygotic twins raised apart
If twins raised apart and assume had different environments, then similarities in trait (high concordance rates) likely due to genetic factors.
• Share 100% same genetics, different environments with Environment 1 and Environment 2
o Adoption Studies
Compare similarity between biological siblings raised apart and adoptive siblings
• If biological sib 2 more concordant with biological sibling than adopted sibling, then have evidence for genetic component as opposed to environmental component.
o This means that despite being in a different environment, the biological twin 2 is more concordant with biological sibling 1 traits are genetic component
Risk of Disease in Relatives
o Can compare the frequency of disease in relatives of patients to see if higher than in general population
o Risk of disease in sibling/ risk of disease in general population= Risk of disease in relative
♣ Risk in general population ~ 0.4%
♣ Risk to sibs of type 1 diabetes patient ~ 6%
o S ~ 15 for type 1 diabetes.
Heritability
Heritability—> Proportion of variance in trait that is due to genetic variation (compared to non-genetic variation)
o The Heritability of a trait is the proportion of total variance in a trait that is due to variation in genes. A high heritability implies that differences among individuals with respect to a trait
♣ such as blood pressure in a population can be attributed to differences in the genetic makeup.
The key to interpreting heritability estimates is to remember that we’re talking about and describing variation in BOTH genetic factors AND non-genetic factors.
♣ If one (alleles or environment) doesn’t demonstrate much variability, then it doesn’t have much potential to explain variability in a trait.
o ***Implication: A high heritability does not imply that non-genetic factors are not important. A low heritability does not imply that environment is not important.
Allele and Locus Difference
Locus is that point on chromosome where the trait or gene is situated. Gene is found at a certain locus. Homologous chromosomes have same genes at the same locus but have different alleles. Gene mapping is used very commonly to determine locus in a certain biological trait. Hence, locus is a maker of DNA. Locus is usually referred as a chromosome marker. It could be called as a gene but it is used specifically to locate the position of chromosome on the gene. Single allele can be found on one single loci and that is what makes it distinct.
Characteristics of Complex Traits
Contains one or more of the following:
♣ Incomplete Penetrance= phenotype does not always show
♣ Variable Expressivity= different ranges/types of expression with the phenotype
♣ Heterogeneity – allele and locus
• The trait exists on different locuses or allels throughout the genome (on different chromosomes)
♣ Presence of phenocopies
Incomplete Penetrance
Penetrance: describes the relationship between trait and genotype
♣ The penetrance of a genotype is the probability that an individual will develop the trait if they have the genotype
Complete penetrance: everyone with pre-disposing genotype will get trait
Incomplete penetrance: some with genotype will not get trait
Susceptibility variants for complex diseases are generally thought to have low-penetrance
♣ Example: Type 1 diabetes: up to 20% of general population has one of two highest-risk haplotypes, but incidence of disease is only .4%
Incomplete penetrance
some with genotype will not get trait
♣ Susceptibility variants for complex diseases are generally thought to have low-penetrance
♣ Example: Type 1 diabetes: up to 20% of general population has one of two highest-risk haplotypes, but incidence of disease is only .4%
Variable expressivity
Variable Expressivity–>individuals with the same variant do not show precisely the same disease or quantitative phenotype characteristics.
o Light dimming: People will show variety of phenotype expressions with the mutated genotype
Allelic heterogeneity
Allelic Heterogeneity
o Different alleles in the same gene result in same trait
o Different alleles in the same gene result in different traits
Example: Cystic Fibrosis
• Many alleles appear to have very similar clinical progression of disease.
o Alleles can be grouped into classes; severity of lung and pancreatic involvement depends on allele class
CTRF Genotype: Example of Heterogeneity Based on the genes active, may or may not have pancreatic disease/ insufficiency. There is also a trait component for lungs
♣ Severe: two F508/F508 genes
• 85% more likely to have severe pancreatic disease
♣ Mild: R117H/F508
• 15% pancreatic insufficiency
Locus Heterogeneity
o Variants in different genes result in very similar clinical presentation
o Classic example: Early onset Alzheimer disease (AD)
♣ Mutations in 3 different genes all result in identical clinical presentation of early-onset AD
♣ Different Loci across chromosomes can bring on different types of Alzheimer’s disease
Phenocopy
Phenocopy—> environmentally caused phenotype that mimics the genetic version of the trait.
o Note that it can be argued that almost everything has some genetic component; intent here is that primary reason for the phenotype is not genetic.
Example: Thalidomide-induced limb malformation vs. genetically-induced
o Type 2 Diabetes as an example
Risk factors:
• increasing age
• obesity, physical inactivity
• family history
• prior gestational diabetes
• impaired glucose tolerance (IGT)
Associated with:
• LDL cholesterol, triglycerides, blood pressure
Control often attained with diet, exercise
o Type 2 Diabetes: Evidence for Genetic Component
♣ Studies in early 1900’s type 2 aggregates in families
• More recent studies
♣ Estimates of concordance rates for MZ twins:
• About 2 times those for dizygotic twins - 35%-100%
• Risk to 1st degree relatives of affected:
o ~ 3 to 4 times that of general population
♣ Intermediate traits of blood glucose and insulin levels have heritability estimates as high as 50%
Summary of Turner’s Syndrom
45,X, is a condition in which afemaleis partly or completely missing anX chromosome.[1]Signs and symptoms vary among those affected. Often, a short andwebbed neck,low-set ears, low hairline at the back of the neck,short stature, andswollenhands and feet are seen at birth. Typically they arewithout menstrual periods, do not developbreasts, and areunable to have children.Heart defects,diabetes, andlow thyroid hormoneoccur more frequently. Most people with TS have normal intelligence. Many, however, have troubles withspatial visualizationsuch as that needed formathematics.[2]Vision and hearing problems occur more often
No cure for Turner syndrome is known. Treatment, however, may help with symptoms.Human growth hormoneinjections during childhood may increase adult height.Estrogen replacement therapycan promote development of thebreastsand hips. Medical care is often required to manage other health problems with which TS is associated.
Inheritance
In the majority of cases where monosomy occurs, the X chromosome comes from the mother.[37]This may be due to anondisjunctionin the father.Meioticerrors that lead to the production of X with p arm deletions or abnormal Y chromosomes are also mostly found in the father.[38]IsochromosomeX orring chromosomeX on the other hand are formed equally often by both parents.
Autosomal Dominant
Manifests in homozygotes and heterozygotes
♣ Will see it in either RR or an Rr, the dominant allele will reign supreme
o Equal in males and females
-Is not gender linked
o Can be passed by either parent
Achondroplasia- Dwarfism
o Autosomal dominant o Most common skeletal dysplasia o 1 in 15,000-40,000 newborns o 80% new mutation rate o 100% penetrance ♣ They will show the disease for sure
o Clinical Meifestation
♣Small stature Males 4’ 3” Females 4’
Rhizomelic limb shortening
Short fingers
Genu varum
Trident hands
Large head/frontal bossing
Midfacial retrusion Their nose and cheeks look bound
Small Foramen Magnum/Craniocervical instability
• Small hole in the head
o Gene of Achondroplasia Mutation of the FGFR3 gene leads to an amino acid substitution (missense mutation) ultimately affecting bone growth by limiting bone formation from cartilage
FGFR3
Fibroblast Growth Factor Receptor 3
Regulates bone growth by limiting the formation of bone from cartilage
Chromosome 4p16.3 nucleotide 1138
♣ Amino acid substitution – missense mutation c.1138G>A;p.Gly380Arg
♣ Mutation increases the activity of the protein interfering with skeletal development
o Nucleotide 1138 of the FGFR3 gene has the highest new mutation rate known in man
Like Down’s Syndrome with women—>As men grow older, the paternal passing down of FGFR3 will mutate
• Aging= greater risk of mutated FGFR3
Retinoblastoma
Malignant tumor of the retina
o 1 in 15,000 live births
o RB1 gene on chromosome 13
RB protein is part of the cell cycle
o Retinoblastoma Associated protein regulates the cell cycle
o 90% penetrance
♣ Will have a 90% chance of showing the disease
Sometimes will not see it in the parents, still are carrying the gene and they will likely pass it down
oThis happen when you see the white reflection (bad) in youths
-Want a healthy red reflection for normal retina health
Neurofibromatosis Type 1
Neurofibromatosis Type 1: Is a tumor disorder that is caused by the mutation of a gene on chromosome 17 that is responsible for control of cell division. NF-1 causes tumors along the nervous system and can grow anywhere on the body. NF-1 is one of the most common genetic disorders and is not limited to any persons race or sex there are at least 100,000 people currently in the U.S. who have been diagnosed with NF. Common symptoms of NF-1 include brownish-red spots in the colored part of the eye called Lisch nodules, benign skin tumors called neurofibromas, and larger benign tumors of nerves called plexiform neurofibromas. o Autosomal dominant o 1 in 3000 births o 50% new mutation rate o Exhibits Variable Expressivity
NIH Diagnostic Criteria 2 or more of the following
6 or more café-au-lait spots
•Little blotches on the skin, will develop over time
2 or more neurofibromas
• Light brown raised skin (larger than moles)
• Will cluster
1 plexiform neurofibroma
♣ Freckling in the axillary or inguinal area
Optic glioma
♣ 2 or more Lisch Nodules
• These are freckling in the eyes
♣ Distinctive osseous lesions
♣ Affected first degree relative
o Mutation for Neurofibromatosis Type 1 NF-1 is a microdeletion syndrome caused by a mutation of a gene located on chromosomal segment 17q11.2 on the long arm of chromosome 17 which encodes a protein known as neurofibromin
♣ NF1
• Neurofibromin- tumor suppressor gene
♣ Chromosome 17q11.2
• Loss of function mutation
• Over 1000 mutations have been described on this c
♣ Although considered dominant must have a mutation in both genes to show the phenotype of NH1
• If you carry this mutation you are still at major risk
• If you have inherited one mutation, you are more likely to develop the second mutation De novo
o Possibly will develop the second mutation and have Neurofibromatosis Type 1
Locus Heterogeneity
A mutation in more than one locus causing the same clinical condition
Summary and Genetic Component of Tuberous Sclerosis
TS is a rare multi-system genetic disease that causes benign tumors to grow in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. A combination of symptoms may include seizures, intellectual disability, developmental delay, behavioral problems, skin abnormalities, lung and kidney disease. TSC is caused by a mutation of either of two genes, TSC1 and TSC2, which code for the proteins hamartin and tuberin respectively. These proteins act as tumor growth suppressors, agents that regulate cell proliferation and differentiation.[1]
Mutation in Tuberous Sclerosis TSC1 encodes for the protein hamartin, is located on chromosome 9 q34. TSC2 encodes for the protein Tuberin, is located on chromosome 16 p13.3 The Hamartin and the Tuberin protein both regulate cell growth and proliferation TSC1, TSC2 -Encode hamartin and tuberin proteins • Regulate cell growth and proliferation Chromosome 9 and 16 -Loss of function mutations
Osteogenesis Imperfecta Type 1
•Osteogenesis Imperfecta Type 1 also known as brittle bone disease or Lobstein syndrome,[1] is a congenital bone disorder characterized by brittle bones that are prone to fracture. People with OI are born with defective connective tissue, or without the ability to make it, usually because of a deficiency of type I collagen.[2] Eight types of OI can be distinguished. Most cases are caused by mutations in the COL1A1 and COL1A2 genes.
♣ Autosomal dominant
♣ 1 in 30,000-50,000
♣ Exhibits Variable Expressivity
o Clinical Manifestations of Osteogenisis Imperfecta Type 1 ♣ Multiple fractures ♣ Mild short stature ♣ Adult onset hearing loss ♣ Blue sclera not always the case o Mutation of Osteogenisis Imperfecta 1 ♣ COL1A1
• Collagen type 1 alpha 1
♣ Chromosome 7q21.3
♣ Reduced production of pro-alpha 1 chains that reduces the type 1 collagen production by half
♣ Molecular and biochemical testing available
Marfan Syndrome
Marfan Syndrome Is a genetic disorder of connective tissue. It has a variable clinical presentation, ranging from mild to severe systemic disease. The most serious manifestations involve defects of the heart valves and aorta, which may lead to early death if not properly managed. The syndrome also may affect the lungs, eyes, dural sac surrounding the spinal cord, the skeleton, and the hard palate. People with Marfan syndrome tend to be unusually tall, with long limbs and long, thin fingers and toes.
♣ Autosomal dominant
♣ 1 in 5000 births
♣ 25% new mutation rate
♣ Variable Expressivity
o Clinical Manifestation of Marfan Syndrome
♣ Systemic disorder of connective tissue
Connective Tissues are the tissues in our body including muscle, ligaments and tendons. Connective tissues are also part of our vascular system, most importantly in the large arteries in our bodies
♣ Ocular
♣ Skeletal
♣ Cardiovascular
o Diagnosis:
o Scoliosis, Thumb and wrist sign, Pectus Excavatum
♣ Signs for the disease
o Ectopia Lentis
♣ Displaced lens
o Cardiovasuclar System
♣ Looking at the Aorta, seeing if it has a larger Diameter
♣ No family History • Aortic root enlargement • Plus one of the following: o Ectopia Lentis ♣ Displaced lens o FBN1 mutation o Systemic score >7 ♣ Positive Family History • Ectopia Lentis • Systemic score of >7 • Aortic root enlargement
Mutation in the Mafran syndrome rfan syndrome is caused by mutations in the FBN1 gene on chromosome 15,[14] which encodes fibrillin-1, a glycoprotein component of the extracellular matrix. Fibrillin-1 is essential for the proper formation of the extracellular matrix, including the biogenesis and maintenance of elastic fibers. The extracellular matrix is critical for both the structural integrity of connective tissue
FBN1
• Fibrillin- extracellular matrix protein
Chromosome 15q21.1
• Dominant negative activity mutation
• A mutation whose gene product adversely affects the normal, wild-type gene product within the same cell
♣ Severe reduction in the number of microfibrils
• Slipped Mispairing
o Mispairing of bases in regions of repetitive DNA replication coupled with inadequate DNA repair systems
o As the repeat grows longer the probability of subsequent mispairing increases
Trinucleotide Repeat Disorders
Trinucleotide Repeat Disorders are a set of genetic disorders caused by trinucleotide repeat expansion, a kind of mutation where trinucleotide repeats in certain genes exceed the normal, stable threshold, which differs per gene. The mutation is a subset of unstable microsatellite repeats that occur throughout all genomic sequences.
o Expansion of a segment of DNA consisting of three or more nucleotides
Just adding a bunch of repeating trio’s of nucleotides
♣ CAGCAGCAGCAGCAGCAG
o Slipped Mispairing
♣ Mispairing of bases in regions of repetitive DNA replication coupled with inadequate DNA repair systems
♣ As the repeat grows longer the probability of subsequent mispairing increases
o Anticipation In genetics, anticipation is a phenomenon whereby as a genetic disorder is passed on to the next generation, the symptoms of the genetic disorder become apparent at an earlier age with each generation. In most cases, an increase of severity of symptoms is also noted. Anticipation is common in trinucleotide repeat disorders
Severity and/or onset of disease increases in next generation
o
Parental Transmission Bias
♣ Trinucleotide expansion more prone to occur in gametogenesis of the male or the female
o AD, AR and X-linked transmission
Anticipation
Anticipation In genetics, anticipation is a phenomenon whereby as a genetic disorder is passed on to the next generation, the symptoms of the genetic disorder become apparent at an earlier age with each generation. In most cases, an increase of severity of symptoms is also noted.
Anticipation is common in trinucleotide repeat disorders
-Severity and/or onset of disease increases in next generation
EX: Huntington disease: More trinucleotides added with each generation, more likely to develop disease sooner with greater severity
o Parental Transmission Bias
♣ Trinucleotide expansion more prone to occur in gametogenesis of the male or the female
o AD, AR and X-linked transmission
Huntington Disease
Huntington Disease is a neurodegenerative genetic disorder that affects muscle coordination and leads to mental decline and behavioral symptoms ♣ Autosomal Dominant ♣ Trinucleotide repeat disorder (CAG) ♣ 1 in 10,000 ♣ Anticipation ♣ Parent of origin • Early onset – paternal • Later onset - maternal ♣ Clinical Manifestations:
Progressive neuronal degeneration causing motor, cognitive and psychiatric disturbances
• Age of onset 35-44
• Death approximately 15 years after onset
♣ Mutation: HD is one of several trinucleotide repeat disorders which are caused by the length of a repeated section of a gene exceeding a normal range. The HTT gene is located on the short arm of chromosome 4at 4p16.3. HTT contains a sequence of three DNA bases—cytosine-adenine-guanine (CAG)—repeated multiple times (i.e. … CAGCAGCAG …), known as a trinucleotide repeat. CAG is the 3-letter genetic code (codon) for the amino acid glutamine
•HTT—> Huntingtin
o Chromosome 4p16.3
• The expansion of glutamine may cause an altered structure or biochemical property of the protein
Myotonic Dystrophy Type 1
Myotonic Dystrophy Type 1 ♣ Autosomal Dominant ♣ Trinucleotide repeat disorder (CTG) ♣ 1 in 20,000 ♣ Anticipation ♣ Maternal transmission
o Clinical Manifestations ♣ Adult onset muscular dystrophy ♣ Progressive muscle wasting and weakness ♣ Myotonia ♣ Cataracts ♣ Cardiac conduction defects
o Mutation In DM1, the affected gene is called DMPK, which codes for myotonic dystrophy protein kinase,[2] a protein expressed predominantly in skeletal muscle.[3] The gene is located on the long arm of chromosome 19.
DMPK—>Myotonic dystrophy protein kinase
♣ Chromosome 19q13.3
♣ Plays important role in muscle, heart and brain cells
o CTG Repeat for Mystonic Dystrophy In DM1, there is an expansion of the cytosine-thymine-guanine (CTG) triplet repeat in the DMPK gene. Between 5 and 37 repeats is considered normal, while individuals with between 38 and 49 repeats are considered to have a pre-mutation and are at risk of having children with further expanded repeats and, therefore, symptomatic disease.[5] Individuals with greater than 50 repeats are almost invariably symptomatic, with some noted exceptions.[ref] Longer repeats are usually associated with earlier onset and more severe disease.
♣ 5-34 normal
♣ 34-49 premutation range
• Risk at having children with more repeats and the disease
♣ >50 full mutation with 100% penetrance
Hemoglobin Structure
Hemoglobin is the main oxygen carrier in the body
o Consists of four subunits called globin: 2 alpha and 2 beta (or beta like) chains
Each subunit has a polypeptide chain (alpha or beta) that is called a globin, a prosthetic group(heme) which carries an Iron
• The iron gives the oxygen transporting capabilities
o Hemoglobin states
♣ Tense state: Deoxygeneated, difficult to bind O2 with high binding affinity, need to have a high pO2 such as in the lungs (will allow the hemoglobin to get past tense state)
♣ Relaxed State: The hemoglobin changes shape, and will have it’s four heme’s carry oxygen, has a much lower binding affinity
• Drop off oxygens in the tissues (pH activasion)
o The Alpha and alpha like genes are located on chromosome 16 while the Beta and Beta like genes are located on chromosome 11
Chromosome 11 Will start with gamma chains in fetal, switch to beta after fertilization
Summary of hemoglobin chromosomes
The Alpha and alpha like genes are located on chromosome 16 while the Beta and Beta like genes are located on chromosome 11
Chromosome 11 Will start with gamma chains in fetal, switch to beta after fertilization
Globin switching
Globin switching—>Expression of various globin through development
o On the Beta chain of chromosome 11, the Genes are in the order that will be produced Start with Hemoglobin F (has 2 alpha and 2 gamma chains) during fetal development.
After birth Hemoglobin A (2 alpha and 2 beta) become predominant
Hb F and Hb A
During Fetal life, the Hemoglobin F is the predominant expressed hemoglobin
• Alpha chains are produced constantly through life on chromosome 16
• The genes of Globin gamma are closest to the locust control (start of the gene) on chromosome 11
o Hence gamma and alpha globin/chains will be made first
Come time of Birth, Beta-chain synthesis will begin to take over, and by 3 months of age, the Hemoglobin A will be dominant (2 alpha 2 beta chains)
•
The Beta globin genes are further down on the DNA expression area
♣ From Yolk Sac to Adult, Different hemoglobin take over
Alpha Hemoglobin is roughly always close to 50% dominant
o During Fetal period, gamma will take up around 40%
o After birth, and over the next several months, The Beta Globin/chains will take over and transition to the 40-50% amount
o Alpha Chains Always Exist around 45-50% throughout life
Need some way to control the Beta globin gene and when its going to be expressed Especially since it doesn’t start till around birth
Locus Control Region A regulatory element of DNA that is upstream of the Beta-Globin complex
o Critical proteins and activators will turn on Locus Control Region during birth
o If the Locus Control Region for Beta globin is deleted Then the patient CANNOT make Beta chains and has the more severe form of Beta thalassemia
♣ Will need to shoot up gamma-globin production if the Locus control region for Beta
o Locus Control Region (LCR), which is located at the most upstream region of each cluster. It is currently thought that the distance between the LCR and a particular globin gene affects its expression. The LCR presumably makes physical contact with the promoter and/or negative regulatory regions via specific transcriptional factors to influence gene expression.
♣ Deletions of the entire LCR of the beta cluster cause beta-thalassemias, a condition in which zero β-globin synthesis leads to precipitation of the α-globin chains.
Structural Variants Hemoglobin Disease
Altered components of the globin (the chain protein)
o Protein still made, just defective
o Example: Sickle Cell disease
o HbS or HbC trait Both sickle cell anemia and hemoglobin CC disease are of autosomal recessive inheritance. Sickle cell trait (HbS trait) or hemoglobin C trait (HbC trait) describes the conditions expressed in individuals who are heterozygous of HbS/HbA and HbC/HbA, respectively. They are clinically normal except when under severe low pO2 stress.
Sickle Cell Anemia
Sickle Cell Anemia
Single base mutation at codon #6 in the β-globin gene changes glutamate to valine. HbS is 80% less soluble than HbA when not bound to O2, and polymerizes into long fibers that distort the RBC into a characteristic sickle shape.
o These sickled cells become lodged in the micro-capillaries and further exacerbate the sickling crisis.
-Due to HbS being 80% less sobule than HbA
o The loss of red blood cell elasticity is central to the pathophysiology of sickle-cell disease. Normal red blood cells are quite elastic, which allows the cells to deform to pass through capillaries. In sickle-cell disease, low-oxygen tension promotes red blood cell sickling and repeated episodes of sickling damage the cell membrane and decrease the cell’s elasticity. These cells fail to return to normal shape when normal oxygen tension is restored
o Is typically considered an autosomal recessive disease
Hemoglobin C Disease: HbCC
HbCC
Caused by a single base mutation at codon#6 of the β-globin gene, changing glutamate to lysine.
o A milder form of hemolytic anemia than sickle cell anemia. Hemoglobin C disease (HbCC) A milder form of hemolytic anemia than sickle cell anemia. Caused by a single base mutation at codon#6 of the β-globin gene, changing glutamate to lysine.
♣ HbC is less soluble than HbA and tends to form crystals, reducing the deformability of RBC. (glutamate to lysine at codon six)
♣ Is also an autosomal recessive disease
DNA Diagnosis: Sickle Cell Anemia
DNA Diagnosis: Sickle Cell Anemia Use Southern Blot to find the different sizes based
o Both diseases will sepperate at different sizes on a southern blot test (after they are given southern probes)
o Hemoglobin electrophoresis
♣ is ablood testthat can detect different types ofhemoglobin. It uses the principles ofgel electrophoresisto separate out the various types of hemoglobin and is a type ofnative gel electrophoresis. The test can detect abnormal levels of HbS, the form associated withsickle-cell disease, as well as other abnormal hemoglobin-related blood disorders, such ashemoglobin C. Different hemoglobins have different charges, and according to those charges and the amount, hemoglobins move at different speeds in the gel whether in alkaline gel or acid gel. The hemoglobin electrophoresis is also known to be thalessemia screening, this also can be helpful for the patient who is frequently need of fresh blood transfusion
Symptoms of altered Hb-O2 binding
HbKempsey The O2 affinity of the hemoglobin is too high, not letting O2 go in the tissues
♣ Polycythemia Over production of Red blood cells
• Happens due to less O2 in the tissues, body is signaled to produce more Red blood cells
HbKansas too low of an O2 affinity to the hemoglobin, won’t carry enough O2 on RBC
♣ Have lower O2 in the red blood cells
♣ Cyanosis Bluish skin
Thalassemias
Thalassemias are caused by an imbalance in the relative levels of the α and β globin chains, which leads to the precipitation of the globin in excess and decreases the life span of the RBC.
Alpha Thalassemia
• low or zero alpha-globin
o beta- & gamma-globin in excess & precipitates.
• both fetal and postnatal defects.
• usually caused by deletion of the a-globin gene(s).
Beta Thalassemia
• low or zero beta-globin, alpha-globin in excess & precipitates.
• postnatal defects only.
• usually caused by point mutations in the b-globin gene.
• occasionally caused by deletions in the LCR or the b-gene cluster.
o Low or zero synthesis of one globin chain
♣ Due to the gene being altered
α-Thalassemias
o Mostly caused by deletions of one or both copies of the α-globin gene in the α-cluster. Thus, γ- and β-globin are in excess. Affects the formation of both fetal and adult hemoglobins.
α-thal-1 allele (- -) Common in Southeast Asia. Caused by deletion of both copies of α-globin genes in the α- cluster. Homozygous state (- -/- -) results in “hydrops fetalis” (stillborn). Most fetal hemoglobin is γ4 (Hb Bart’s) although there is enough ζ2γ2 (Hb Portland) to sustain fetal development. Heterozygotes (αα/–) have mild anemia, a.k.a. α-thalassemia-1 trait.
α-thal-2 allele (α -) Common in Africa, Mediterranean, and Asia. Deletion of one of the two α-globin genes in the α-cluster. 50% decrease in α-globin synthesis. No disease phenotype in heterozygote (αα/α-, silent carrier). Mild anemia in homozygotes (α-/α-), a.k.a. α-thalassemia-2 trait.
α-thal-1/α-thal-2 (α -/- -) Compound heterozygous individuals with only 25% of normal α-globin level. Severe anemia. a.k.a. HbH disease. About 5-30% of their hemoglobin is β4 (HbH), which precipitates.
HPFH
HPFH (hereditary persistent fetal hemoglobin) is a benign condition in which significant fetal hemoglobin (hemoglobin F) production continues well into adulthood, disregarding the normal shutoff point after which only adult-type hemoglobin should be produced.
o No δ or β synthesis because of deletions of both genes. Increased γ-globin expression caused by either of the two following mechanisms:
(1) extended deletion of additional downstream sequences, which likely brings a cis-acting enhancer element closer to the γ-globin gene, or
(2) mutations in the promoter region of one of the two γ-globin genes that destroy the binding site of a repressor, thereby relieving postnatal repression of γ.
HPFH individuals are disease free, since adequate levels of γ chains are still made due to the disruption of the perinatal globin switch from γ to β. 100% of hemoglobin is HbF (α2γ2), which is about 17-35% of normal level of total hemoglobin production. HPFH individuals have higher HbF (17-35%) level than δβ0 -thalassemia individuals (5-18%).
Significance of HPFH Understanding the mechanism of HPFH may make it possible to express HbF at high levels postnatally to treat patients of β-thalassemia and sickle cell anemia.
Four major Mutations
1) Loss of Function/Loss of protein function (greatest majority)
-Protein is abnormal (enzyme doesn’t work, it gets degraded
• Decreases necessary function
Typically flaw in coding region (whether it’s DNA or the downstream product RNA)
2) Gain of Function
3) Novel Property mutations
4) Ectopic or heterchronic (Altered Expression Mutation?)
♣ uncommon, except for cancer
Loss of function mutation
Mechanism: Caused by genetic mutations (deletions, insertions, or rearrangements) that eliminate (or reduce) the function of the protein. Of the four major mechanisms, this is the most common genetic mechanism leading to human genetic disease.
Somewhere a Component can be deleted and ruin downstream product
♣ Can have the Gene deleted (at the DNA level)
♣ Can have the RNA defective
♣ Can have no protein or dysfunctional
♣ Example:
Duchenne muscular dystrophy
Duchenne muscular dystrophy (example of loss of function mutation
o DMD Xp21.2 the largest gene in the genome (DMD) can have a variety of mutations, such as deletions, nonsense or frameshift mutations. Can make a non-functional protein. Is a an x-linked gene
• Large deletions (multiple exons)
• Nonsense (stop) mutations / frameshift mutations premature termination
• (in-frame deletions milder Becker muscular dystrophy)
Becker Muscular Dystrophy
o There can be in-frame deletions, a shorted protein that is a little more functional
o Small amount of functional protein better than none
• X-linked inheritance
• Clinically
• Boys with abnormal gait at 3-5 years
• Calf pseudohypertrophy
• Gower maneuver (YouTube)
• Progressive involvement of respiratory muscles
• Median age of death 18 years
• Women may cardiomyopathy
o Duchenne muscular Dystrophy (more severe, earlier onset)
♣ Frameshift deletions of DMD gene = major loss-of-function mechanism in Duchenne Muscular Dystrophy
♣ Make a truncated protein that does not work at all!
Duchenne and Becker Muscular Dystrophy
o Duchenne muscular Dystrophy (more severe, earlier onset)
♣ Frameshift deletions of DMD gene = major loss-of-function mechanism in Duchenne Muscular Dystrophy
♣ Make a truncated protein that does not work at all!
o Becker Muscular Dystrophy (less severe, later onset)
♣ In-frame deletions (and also missense mutations) of DMD gene = major loss-of (i.e. reduction)-function in Becker Muscular dystrophy
♣ Protein that is reduced in function (still exists to some extent)
Hereditary neuropathy with liability to pressure palsies (HNPP) Disease
HNPP Disease
Deletion of PMP22 gene = Loss-of-function Hereditary Neuropathy with Liability to Pressure Palsies (HNPP)
♣ PMP22 protein is an integral membrane glycoprotein in nerves
♣ Notice that deletions often leave to the most dysfunctional or non-existent proteins Lacking the PMP22 protein for glycoprotein membrane
o Clinically:
♣ repeated focal pressure neuropathies (e.g. carpal tunnel syndrome and peroneal palsy with foot drop)
♣ First attack usually in 2nd-3rd decade
• Recovery from acute neuropathy is often complete
• Incomplete recovery mild disability
♣ Autosomal Dominant
PMP22 Gene
Unequal crossing over between two highly homologous repeats on chromosome 17p12 can result in:
• 3 copies of the PMP22 gene with the CMT1A phenotype or
• the reciprocal with 1 copy of the PMP22 gene with the HNPP phenotype.
o (HNPP) Disease will develop
PMP22 Gene
PMP22 Gene
Unequal crossing over between two highly homologous repeats on chromosome 17p12 can result in:
• 3 copies of the PMP22 gene with the CMT1A phenotype or
• the reciprocal with 1 copy of the PMP22 gene with the HNPP phenotype.
o (HNPP) Disease will develop
Osteogeneis Imperfecta Type I
Osteogeneis Imperfecta Type I
Clinically:
♣ Brittle bones, increased fractures (non-deforming)
♣ blue sclerae
♣ normal stature.
♣ First fracture may occur with diapering, but more typically once infant begins to walk (and fall)
• Easy to assume baby abuse, usually because of weak bones
Autosomal Dominant
Due to dffect of the type 1 procollagen protein
♣ Affected individuals may have anywhere from a few fractures to more than 100M
♣ Progressive hearing loss in adults
Genetic Cause
♣ The structure of type I procollagen. Note that type I procollagen is composed of two proα1(I) chains and one proα2(I) chain = 3 chains total (2 pro alpha1 and 1 pro alpha2)
• Normally the alleles COLA1 and COL1A1 (2 of them) pump out 2 pro-alpha1 chains
• COL1A2 and COL1A2 (2 of them) make only 1 pro-alpha 2 chain
• These four genes will make the three necessary genes
Very important structure in bones
♣ OI Type I: Premature termination codons (nonsense and frameshift) in COL1A1 mRNA unstable mRNA degraded reduction of normal COL1A1 protein
• One of the COL1A1 genes is knocked out (frameshift or nonsense), making only 1 pro-alpha1 chains when you need 2 chains
o Makes very brittle bones from missing the pro-alpha1 chain (you need two of them)
Hemoglobin Kempsey
Hemoglobin Kempsey example of a “gain of function” mutation, will have a higher oxygen affinity. Problem is that the hemoglobin will bind to oxygen too well, won’t release it in cells. Body may produce too much RBC and cause polycythemia (too much red blood cells)
o Beta hemoglobin gene
Asp99Asn missense mutation
Higher oxygen affinity
• In normal hemoglobin binding of oxygen allowing for shift from tense (deoxygenated) to relaxed (oxygenated) form
♣ 99Asn mutation prevents this shift
♣ Hemoglobin remains ‘locked’ in the relaxed state (which has higher oxygen affinity) = Gain Of Function (kinda)
o Consequences:
♣ Hb Kempsey unloads less oxygen in tissues
♣ Body ‘thinks’ it needs more oxygen makes more red blood cells polycythemia
Charcot Marie Tooth Syndrome Type IA
Charcot Marie Tooth Syndrome Type IA A large amount of muscle waste due to nerve atrophy, the CMT1A protein “gains a new function” mutation by destroying nerve
o Duplication of PMP22 gene = Gain-of-function Charcot Marie Tooth Syndrome type IA (CMT1A)
o PMP22 protein is an integral membrane glycoprotein in nerves
o Clinically:
♣ Demyelinating motor and sensory neuropathy
♣ Often presents in lower extremities with weakness and muscle atrophy and mild sensory loss
♣ Progressive; typical patterns on nerve conduction studies
♣ Autosomal Dominant
Sickle Cell anemia
“Novel Mutation”
• Sickle Cell anemia (example of Novel Property Mutation)
Mutation:
♣ No effect on oxygen carrying ability of hemoglobin
♣ Novel property of polymerizing under low oxygen conditions–> makes it clump up in specific areas
• “New Property” now that it can polymerize under low oxygen (change it’s 3D shape)
Osteogenesis Imperfecta Types II,III, IV (type 2-4 will have novel mutations)
With these different types, will have point-mutations
These point mutations in Osteogenisis Imperfect Type II, III, and IV will lead to new proteins/ Chains made for the procollagen
o In Osteogenesis type I, loss of function mutations ½ the amount of total collagen trimers, but it is all normal mild phenotype
♣ Osteogensis type 1 is less severe, still have some functional collagen (not causing problems)
o In Osteogenesis types II, III, IV novel property mutations relatively ‘normal’ amount of total collagen trimers, but ½ is abnormal severe phenotype
♣ The abnormal collagen causes more abnormal/bad effects
o Lesson: better to have ½ the amount of normal collagen, than produce abnormal collagen trimers
Hereditary Persistence of fetal hemoglobin
HPFH
Remember that as you go through life (fetal adulthood)
♣ Produce different hemoglobin (go from Hemo F to Hemo A)
Gamma hemoglobin to beta hemoglobin
HPFH Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition in which significant fetal hemoglobin (hemoglobin F) production continues well into adulthood, disregarding the normal shutoff point after which only adult-type hemoglobin should be produced
Tri/tetra nucleotide repeat disorders (unstable repeat disorders)
They are typically repeat nucleotides that insert in the gene, the longer they are greater chance of producing the disease
o Correlation between CAG repeat number and age-of-onset for Huntington disease
The gene contains a trinucleotide repeat (CAG) that is expanded within HTT on at least one chromosome of individuals with Huntington disease (HD). The CAG repeat length is highly polymorphic in the population and unaffected alleles have CAG repeat size ranges from ten to 35. The median size allele is p.Gln18(18). The most common alleles in all populations contain repeats of 15-20 CAG in length
• Will be normal with 25 or lower CAG repeats (can pass on to offspring)
• 27-40 CAG repeats, greater risk of expansion
• greater than 40 CAG, or close to 50 Risk of disease
Pedigree of Huntington Disease
♣ CAG repeats are unstable >27
♣ Risk of expansion
♣ Longer repeats ~earlier onset
• anticipationis a phenomenon whereby as a genetic disorder is passed on to the next generation, the symptoms of thegenetic disorderbecome apparent at an earlier age with eachgeneration.
♣ Expansion paternally in Huntington disease
Myotonic Dystrophy 1 (tri nucleotide repeat disease)
In DM1, there is an expansion of thecytosine-thymine-guanine(CTG) triplet repeat in theDMPKgene. Between 5 and 37 repeats is considered normal, while individuals with between 38 and 49 repeats are considered to have a pre-mutation and are at risk of having children with further expanded repeats and, therefore, symptomatic disease.[5]Individuals with greater than 50 repeats are almost invariably symptomatic, with some noted exceptions.[ref] Longer repeats are usually associated with earlier onset and more severe disease.
DMPKalleleswith greater than 37 repeats are unstable and additional trinucleotide repeats may be inserted during cell division inmitosisandmeiosis.
Summary of Thalassemia Alpha and Beta
o The α-thalassemias involve the genesHBA1[11]andHBA2,[12]inherited in aMendelian recessivefashion. Twogene lociand so four alleles exist. It is also connected to the deletion of the 16p chromosome. α Thalassemias result in decreased alpha-globin production, therefore fewer alpha-globin chains are produced, resulting in an excess of β chains in adults and excess γ chains in newborns.
o Beta thalassemias are due to mutations in theHBB geneon chromosome 11, also inherited in an autosomal, recessive fashion. The severity of the disease depends on the nature of the mutation.
Mutations are characterized as either βoor β thalassemia major if they prevent any formation of β chains, the most severe form of β-thalassemia; as either β+or β thalassemia intermedia if they allow some β chain formation to occur; or as β thalassemia minor if only one of the two β globin alleles contains a mutation, so that β chain production is not terribly compromised and patients may be relatively asymptomatic.
Fetal: Hemoglobin F
o Fetal: Hemoglobin F Start with 2 alpha and 2 gamma
Will sometimes see it adults, mostly fetal
60-90% at birth, less than 2 % adults
Adult: Hemoglobin A
Adult: Hemoglobin A then have 2 alpha and 2 beta
♣ Most common after 2 years of age (only have some of it at birth
♣ Over 95% of adult
Newborn Screens
Newborn Screens Use to detect hemoglobin disease and issues
♣ Most babies will have a lot of Hemoglobin F and some hemoglobin A
♣ Use it to find early impact from hemoglobin disease
♣ Babies with sickle cell disease What to give to present infection?
• Give the babies to penicillin= Save many lives!
Qualitative Hemoglobinopathies
Qualitative Hemoglobinopathies—> Usually changes in structure (a change in amino acid)
Hb S (sickle cell) a valine replacing a glutamine in the 6th position of the beta chain of globin
Hb C which substitution of a glutamic acid residue with a lysine residue at the 6th position of the β-globin chain has occurred
Hb E At position 26 there is a change in the amino acid, from glutamic acid to lysine.
Hemoglobin S
Hemoglobin S
The most common type of abnormal hemoglobin and the basis of sickle cell trait and sickle cell anemia.Hemoglobin S differs from normal adult hemoglobin (called hemoglobin A) only by a single amino acid substitution (a valine replacing a glutamine in the 6th position of the beta chain of globin).
o Homozygous SS disease “Sickle Cell Anemia”
o S heterozygous: AS, sickle trait
Sickle cell trait (or sicklemia) describes a condition in which a person has one abnormal allele of thehemoglobinbeta gene (is heterozygous), but does not display the severe symptoms ofsickle cell diseasethat occur in a person who has two copies of that allele (is homozygous).
Hemoglobin C
Hemoglobin C is an abnormal hemoglobin in which substitution of a glutamic acid residue with a lysine residue at the 6th position of the β-globin chain has occurred o Homozygous CC hemoglobinopathy o Heterozygous C, e.g. AC, or C trait ♣ Traits depend on what you inherit o C-Beta thalassemia
Hemoglobin E
Hemoglobin E is an abnormal hemoglobin with a single point mutation in the β chain. At position 26 there is a change in the amino acid, from glutamic acid to lysine. Hemoglobin E has been one of the less well known variants of normal hemoglobin. It is very common in Southeast Asia but has a low frequency amongst other ethnicities. HbE can be detected on electrophoresis.
o Homozygous EE
o Heterozygous AE
o Combination: E-Beta thalassemia
Mutation by region (Why did the mutation of hemoglobin help survival?)
• The mutation is because it gives a selective advantage, these different hemoglobin mutations in these regions are used to combat Malaria. These genetic disorders are related to surviving Malaria in different regions.
o #1 – SE Asia: α, β thalassemia and E
o #2 Africa – S, C, α and β thalassemia
o #3 West Pacific – α and β thalassemia and E
o #4 East Mediterranean – β thalassemia and S
αThalassemia types:
α thalassemia major unable to make alpha chains (worst one)
♣ you don’t make any alpha globin chains
α thalassemia 3 gene deletion (Hgb H disease)
♣ missing 3 genes, only have 1 gene making alpha chains
♣ Only making small amount of alpha chains
α thalassemia 2 gene deletion (α thalassemia trait)
♣ Missing two of the alpha making genes
♣ Still have a ~50% alpha production
♣ Called alpha thalassemia trait not clinically significant
α thalassemia 1 gene deletion – clinically insignificant
♣ Usually only important for genetic counciling
α thalassemia + Hgb Constant Spring
♣ Can have disease with Hgb Constant Spring, can lead to disease
β Thalassemias and it’s types
β thalassemia major “Cooley’s anemia”
♣ Major Disease, lacking all the capabilities making Beta chains
β thalassemia intermedia
♣ You have clinically disease that could be significant
β thalassemia trait
♣ only 1 Beta chain allele missing/mutated
Hemolysis
meaning a “loosing”, “setting free” or “releasing”, is the rupturing of erythrocytes (red blood cells) and the release of their contents (cytoplasm) into surrounding fluid (e.g. blood plasma). Hemolysis may occur in vivo or in vitro (inside or outside the body).
Possible Test’s for Blood Disorders
Possible Tests: Based on symptoms, tests are ordered for a differential diagnosis. These tests include:
o complete blood count
o hemoglobin electrophoresis
o serum transferrin, ferritin, Fe Binding Capacity; urine urobilin and urobilogen;
o peripheral blood smear;
o hematocrit; and serum bilirubin.
Beta Thalassemia Treatment
Beta Thalassemia Treatment o Red cell transfusions o Iron chelators o Vitamin C o Splenectomy/cholecystectomy o Bone marrow transplant
• Beta Thalassemia Major treatment:
o Affected children require regular lifelongblood transfusionand can have complications, which may involve the spleen.Bone marrow transplantscan be curative for some children.[27]Patients receive frequentblood transfusionsthat lead to or potentiateiron overload.[28]Iron chelation treatment is necessary to prevent damage to internal organs. Advances in iron chelation treatments allow patients with thalassemia major to live long lives with access to proper treatment. Popular chelators includedeferoxamineanddeferiprone.
Beta Thalassemia Intermedia[edit]
o Patients may require episodic blood transfusions. Transfusion-dependent patients developiron overloadand requirechelationtherapy[35]to remove the excess iron. Transmission isautosomal recessive; however, dominantmutationsandcompound heterozygoteshave been reported.Genetic counselingis recommended andprenatal diagnosismay be offered.[36]
Beta Thalassemia Minor
o Patients are often monitored without treatment. While many of those with minor status do not require transfusion therapy, they still risk iron overload, particularly in theliver. Aserumferritin test checks iron levels and can point to further treatment.[38]Although not life-threatening on its own, it can affect quality of life due to the anemia.
X vs Y Chromosome
A normal chromosomal complement
♣ 22 pairs of autosomes
♣ 1 pair of sex chromosomes (XX or XY)
X vs Y Chromosome
o Y chromosome
♣ ~200 genes
Males still use the X chromosome, necessary genes on it
-That is why males are always affected by x-recessive diseases
o X chromosome
♣ >1000 genes
♣ X chromosome is drastically larger
o 5-10 fold difference!
X Chromosome Inactivation
The X chromosome
Only ONE copy of the majority of genes on the X chromosome is necessary for normal function
♣ Two copies of some of the genes may actually be detrimental
♣ In somatic cells of females (not males), one copy of the X chromosome randomly turned off
“Random X inactivation”
• Do NOT want both X-chromosomes working, need on deactivated
o Occurs during the 1st week of embryogenesis
♣ A week after fertilization, one of the X-chromosomes is deactivated (becomes a Barr body)
Barr Body is the inactive X chromosome in a female somatic cell, rendered inactive
Either the paternal X or the maternal X will become activated, the other is a Barr Body
o Females are functionally mosaic for their X chromosomes meaning that different cells in the body will express either the maternal or paternal x chromosome (roughly 50% split), and deactivate the other
Half of their cells express the maternally-inherited X, half express the paternally-inherited X
♣ This can also be demonstrated in some breeds of female cats
• Will see that the Barr body (deactivated X) will be either paternal or maternal on different cells
Nonrandom X chromosome inactivation
Occurs when there is a structurally abnormal X chromosome
• more of the normal Xs are turned on
This occurs to inactivate a defunct X chromosome
Skewed X inactivation
Skewed X inactivation occurs when the inactivation of one X chromosome is favored over the other, leading to an uneven number of cells with each chromosome inactivated. It is usually defined as one allele being found on the active X chromosome in over 75% of cells, and extreme skewing is when over 90% of cells have inactivated the same X chromosome
♣ This is of medical significance due to the potential for the expression of disease genes present on the X chromosome that are normally not expressed due to random X inactivation.
♣ Observed when a female shows signs or symptoms of an X-linked recessive condition, such as Duchene Muscular Dystrophy
♣ Instead of a random inactivation pattern more of the X chromosome with normal gene is turned off
X-linked Disorders
o Mutation on the X-chromosome
o Mostly males affected
o No male to male transmission
o Males considered to be “hemizygous” for mutations in X-linked genes
X-linked Recessive Inheritance
o Phenotype expressed in all males who carry the affected genotype
o Phenotype expressed in homozygous females only
♣ Have to get from affected father and mother carrier of disease
o Heterozygote females are carriers
X-linked Dominant Inheritance
o Phenotype expressed in females who carry the disorder
o Phenotype expressed in all males
Father can only pass the X-linked dominant to the duaghters
o Phenotype may be so severe in males, it may not be compatible with life
♣ X-Linked dominant diseases can be more severe in males than females
Hypophosphatemic Rickets
Hypophosphatemic Rickets
An X-linked dominant form of rickets that differs from most cases of rickets in that ingestion of vitamin D is relatively ineffective. It can cause bone deformity including short stature and genu varum (bow leggedness). It is associated with a mutation in the PHEX gene sequence (Xp.22) and subsequent inactivity of the PHEX protein
♣ X-linked Dominant
♣ 1 in 20,000
o Clinical Manifestation of Hypophosphatemia Rickets
♣ Hypophosphatemia low levels of phosphate
♣ Short stature
♣ Bone deformity
Mutation in Hypophasphatemic rickets a mutation in the PHEX gene sequence, located on the human X chromosome at location Xp22.The PHEX protein regulates another protein called fibroblast growth factor 23 (produced from the FGF23 gene). Fibroblast growth factor 23 normally inhibits the kidneys’ ability to reabsorb phosphate into the bloodstream.
♣ Gene: PHEX
♣ Regulates fibroblast growth factor
♣ Inhibits the kidneys ability to reabsorb phosphate into the blood stream
Fragile X Syndrome
Fragile X Syndrome a genetic syndrome. Nearly half of all children with fragile X syndrome meet the criteria for a diagnosis of autism.[1] It is an inherited cause of intellectual disability especially among boys. It results in a spectrum of intellectual disabilities ranging from mild to severe as well as physical characteristics such as an elongated face, large or protruding ears, and large testes
X-linked dominant
Gene: FMR1
Trinucleotide repeat disorder- CGG (will have anticipation)
♣ 1 in 2500-4000 males
more predominant in males
♣ 1 in 7000-8000 females
Most common cause of inherited developmental delay
♣ Anticipation
♣ Maternal transmission bias
Clinical Manifestation ♣ Intellectual disabilities ♣ Dysmorphic features: large ears, long face, macroorchidism ♣ Autistic behavior ♣ Social anxiety ♣ Hand flapping/biting ♣ Aggression
Genetic composition Fragile X Fragile X syndrome is a genetic disorder which occurs as a result of a mutation of the fragile X mental retardation 1 (FMR1) gene on the X chromosome, most commonly an increase in the number of CGG trinucleotide repeats in the 5’ untranslated region of FMR1.
♣ Fragile X Associated Tremor Ataxia Syndrome(FXTAS)
• White matter lesions on MRI
• Intention tremor, Gait ataxia
♣ FMR1- related Primary Ovarian Insufficiency
• Cessation of menses before age 40
o CPG repeats for Fragile X
♣ 6-45 Normal range
♣ 46-55 Grey Zone
• Grey Zone – can expand when passed onto the mother – do not expand to full mutation but can to premutation
♣ 56-200 Premutation
• Premutation – Causes FXTAS and POI
♣ >200 Full Mutation
Rett Syndrome
Rett Syndrome is a rare genetic postnatal neurological disorder of the grey matter of the brain[2] that almost exclusively affects females but has also been found in male patients. The clinical features include small hands and feet and a deceleration of the rate of head growth (including microcephaly in some). Repetitive stereotyped hand movements, such as wringing and/or repeatedly putting hands into the mouth, are also noted.[3] People with Rett syndrome are prone to gastrointestinal disorders and up to 80% have seizures.[4] They typically have no verbal skills, and about 50% of affected individuals do not walk. Scoliosis, growth failure, and constipation are very common and can be problematic. ♣ X-linked Dominant ♣ 1 in 10,000 females ♣ 95% new mutation rate o Clinical manifestations ♣ Loss of normal movement and coordination ♣ Acquired microcephaly ♣ Loss of communication skills ♣ Failure to thrive ♣ Seizures ♣ Abnormal hand movements
Mutation of Rett is caused by mutations in the gene MECP2 located on the X chromosome (which is involved in transcriptional silencing and epigenetic regulation of methylated DNA), need these proteins for normal nerve cell function
Gene: MECP2
Methyl CpG binding protein
Essential for the normal function of nerve cells
Lesch-Nyhan Syndrome
Lesch-Nyhan Syndrome is a rare inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), produced by mutations in the HPRT gene located on the X chromosome. ♣ X-linked recessive ♣ 1 in 380,000 o Clinical Manifestations of Lesch-Nyhan Syndrome ♣ Cerebral palsy ♣ Cognitive and behavioral disturbances ♣ Overproduction of uric acid ♣ Self injury o Mutation on Lesch-Nyhan ♣ Gene: HPRT1 ♣ Hypoxanthine phosphoribosyltransferase 1 • Recycling of purines
Dystrophinopathies
Dystrophinopathies -X-linked recessive Spectrum of muscle disease from mild to severe • Duchenne Muscular Dystrophy • Becker Muscular Dystrophy • DMD-associated dilated cardiomyopathy
o Mutation
♣ Gene: DMD
♣ Chromosome Xp21-21.1
♣ Dystrophin
Dystrophin is a protein located between thesarcolemmaand the outermost layer ofmyofilamentsin the muscle fiber (myofiber). It is a cohesive protein, linkingactinfilaments to another support protein that resides on the inside surface of each muscle fiber’s plasma membrane
Largest human gene, located on the x-chromosome
o Early onset and late onset of different muscular dystrophy
♣ Early= Duchenne Muscular Dystrophy
♣ Later =Becker Muscular Dystrophy
Becker Muscular Dystrophy
Becker Muscular Dystrophy
is an X-linked recessive inherited disorder characterized by slowly progressive muscle weakness of the legs and pelvis. It is a type of dystrophinopathy, which includes a spectrum of muscle diseases in which there is insufficient dystrophin produced in the muscle cells, resulting in instability in the structure of muscle cell membrane. This is caused by mutations in the dystrophin gene, which encodes the protein dystrophin.
♣ Progressive muscular weakness proximal > distal
♣ Dilated cardiomyopathy
♣ CK levels 5x normal
♣ Later onset
• Wheelchair bound after 16
• Death in their 40’s
Abnormal quantity or quality of Dystrophin
Duchenne Muscular Dystrophy
Duchenne Muscular Dystrophy a recessive X-linked form of muscular dystrophy, affecting around 1 in 3,600 boys, which results in muscle degeneration and premature death.[1] The disorder is caused by a mutation in the gene dystrophin, located on the human X chromosome, which codes for the protein dystrophin. Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. ♣ Progressive muscular weakness proximal > distal ♣ Calf hypertrophy ♣ Dilated cardiomyopathy • CK levels 10x normal ♣ Onset before the age of 5 • Wheelchair bound before 13 • Death in their 30’s ♣ Absence of Dystrophin
Hemophilia A
Hemophilia A is a genetic deficiency in clotting factor VIII,[1] which causes increased bleeding and usually affects males. About 70% of the time it is inherited as an X-linked recessive trait, but around 30% of cases arise from spontaneous mutations. ♣ X-linked recessive ♣ I in 4000 male births ♣ 10% carrier females affected
Clinical Manifestations
Blood disorder where blood fails to clot appropriately due to a deficiency of Factor VIII
♣ Spontaneous bleeds into joints, muscles or intracranial
♣ Excessive bruising
♣ Prolonged bleeding after injury or incision
♣ Delayed wound healing
♣ Royal family
Mutation in Hemophilia A ♣ Gene: F8 ♣ Chromosome: Xq28 ♣ Deficiency of Factor VIII ♣ 22A inversion causes 50%
Mitochondria
Intracellular organelles found in most eukaryotic cells
♣ Oxidative phosphorylation
o Mitochondrial DNA (mtDNA)
♣ Encodes 37 genes
♣ The majority of these produce components of the respiratory chain
Mitochondiral DNA ♣ Maternal inheritance ♣ Replicative segregation ♣ Homoplasmy/Heteroplasmy ♣ Threshold effect
Replicative Segregation
At cell division the multiple copies of mtDNA replicate and sort randomly among newly synthesized mitochondria
o This could be normal DNA or mutated
Mitochondrial Disease o Group of disorders caused by dysfunction of the respiratory chain o These disorders tend to affect tissues that heavily rely on oxidative phosphorylation, brain, retina, skeletal muscle and heart Examples: • Kearns-Sayre Syndrome • MELAS • MERRF • Leber Hereditary Optic Neuropathy
Kearns-Sayre Syndrome
Kearns-Sayre Syndrome
is a mitochondrial myopathy with a typical onset before 20 years of age. KSS is a more severe syndromic variant of chronic progressive external ophthalmoplegia (abbreviated CPEO), a syndrome that is characterized by isolated involvement of the muscles controlling movement of the eyelid
Mitochondrial inheritance
♣ 1-3 in 100,000
♣ Most commonly caused by a somatic mutation
Clinical Manifestation Kears-Sare Syndrome ♣ Triad • Pigmentary Retinopathy • Progressive External Ophthalmoplegia • And onset before age 20y ♣ Cardiac conduction defects ♣ Ataxia ♣ Deafness ♣ Kidney problems
Mutation of Kearns-Sayre Syndrome
♣ Single large deletion of mtDNA (mitochondrial)
♣ Most common deletion removes twelve genes
MELAS
MELAS
Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes
♣ Mitochondrial inheritance
♣ 1 in 300,000
♣ Low new mutation rate
o Melas clinical manifestation ♣ Starts between age 2 & 10 y ♣ Muscle weakness ♣ Seizures ♣ Repetitive stroke-like episodes ♣ Elevated lactic acidosis
o Mutation of Melas caused my mutations in the mtDNA, ome of the genes (MT-ND1, MT-ND5) affected in MELAS encode proteins that are part of NADH dehydrogenase (also called complex I) in mitochondria, that helps convert oxygen and simple sugars to energy. ♣ Mitochondrial genes • MT-ND1 • MT-ND5 • MT-TH • MT-TL1 (80%) • MT-TV
MERRF
MERRF (or Myoclonic Epilepsy with Ragged Red Fibers) is a mitochondrial disease. It is extremely rare
♣ Myoclonic epilepsy with ragged-red fibers
♣ Mitochondrial inheritance
♣ 1 in 400,000
♣ Low new mutation rate
o Clinical Manifestations of MERRF ♣ Muscle symptoms ♣ Seizures ♣ Ataxia ♣ Dementia ♣ Ragged-red fibers
o Mutation for MERRF
♣ Mitochondrial genes
♣ MT-TK gene is mutated
Leber Hereditary Optic Neuropathy
Leber Hereditary Optic Neuropathy
is a mitochondrially inherited (transmitted from mother to offspring) degeneration of retinal ganglion cells (RGCs) and their axons that leads to an acute or subacute loss of central vision; this affects predominantly young adult males.
♣ Mitochondrial inheritance
♣ 1 in 30,000-50,000 Europeans
o Clinical Manifestation
♣ Bilateral subacute vision failure
♣ Occurs during young adulthood
Mutation of Leber Hereditary Optic Neuropathy These genes code for the NADH dehydrogenase protein involved in the normal mitochondrial function of oxidative phosphorylation. ♣ mtDNA • m.3460G>A • m.1178G>A • m.14484T>C
Summary of Genetic Counseling
o Genetic counseling is a communication process to promote understanding of and adaptation to genetic contributions to disease
o There are many different patients appropriate for genetic counseling and many different providers who can provide this service
o There is more to consider than just a diagnosis when it comes to genetics: ethical, legal, psychological, and utility discussions may need to be addressed
Chromosomal Analysis
General Uses and Indications: Suspected abnormality of chromosome number or structure (deletion, insertion, rearrangements). Frequently obtained from pregnant women > 35 years (amniocentesis or chorionic villus sampling), from patients with congenital abnormalities (dysmorphisms, structural organ defects, mental and/or growth retardation), from families with multiple miscarriages and/or fertility problems, and directly from certain cancer biopsies.
Can Diagnose: aneuploidies (abnormal chromosome number), chromosome deletions, duplications, and insertions of moderate to large size (>3,000-5,000 kb / 3-5 Mb), and rearrangements.
Cannot Diagnose: single gene deletions, point mutations, small deletions, duplications, and insertions, methylation defects, trinucleotide repeat abnormalities.
FISH
General Uses and Indications: Used to diagnose deletions, some translocations, and abnormalities of copy number. Often used to detect cytogenetic changes that are at or beyond the limits of resolution obtained by high-resolution chromosomal analysis. FISH for duplications works better on cells in interphase than metaphase (metaphase the chromatin is very compact)
Can Diagnose: recognized microdeletion syndromes, recognized chromosomal rearrangements (in cancers), and gene copy numbers (cancers). Also useful in diagnosing anueploidies (e.g. trisomy 13, 18, 21) in the prenatal setting. Roughly 100-200 kb in size for detection range.
Cannot Diagnose: deletions, rearrangements that are not specifically tested for (i.e. FISH probes are specifically designed for each condition). FISH is not always able to detect duplications of gene region
Examples of Microdeletion Syndromes: Cri-du-chat, Smith-Magenis, DiGeorge (22qdel), Williams syndrome, Wolf-Hirschhorn, Prader-Willi syndrome, Angelman syndrome.
Sanger Sequencing
Widely used method of mutation detection based on Sanger-Gilbert method. Able to identify the genetic mutation causing many disease conditions.
General Uses and Indications: Used to identify sequence changes (mutations) in specific genes. In general you need the following:
Can Diagnose: Mutations in known genes (mutation can be previously reported or can be novel), polymorphic variants, small (1 to ~100 nucleotide) deletion/insertions. Ideal for looking at the sequence of a known disease gene
Cannot Diagnose: The technique is very specific, assaying only the region of the gene(s) for which the test has been designed. Frequently, many clinical genetic tests do NOT routinely sequence all parts of a gene (e.g. promoters, introns). This means that although the approach is often very specific, clinical sensitivity is frequently below 100% (this is an important concept to understand). This technique cannot easily detect larger deletions/insertions, rearrangements, and most chromosomal abnormalities.
Microarrays
Expression Arrays: Used to test the RNA expression of genes (i.e. which genes are turned ‘on’ or ‘off’). These are semi-quantitative and test the activity of genes (see figure) rather than just the presence or absence of a gene or genetic variant (expression arrays). These have a small, but likely growing role, in oncology.
Chromosomal Microarray Analysis (CMA): These have a big role in clincal genetics currently. These look for chromosomal DNA losses and gains (so called ‘deletion/duplication’ studies). Sometimes this is also called array comparative genomic hybridization (aCGH) analysis.
General Uses and Indications of CMA: CMA has become fairly standard for looking for small genomic deletions/insertions. You can think of this as a superior method to looking for chromosomal gains than losses than traditional chromosomal analysis because the resolution of the CMA is vastly superior to chromosomal analysis. The probe size used these days is between 100-200 Kb so they can pick up smaller changes than can be appreciated by chromosome analysis. Currently, some labs use >~200 Kb for deletions and >~400 Kb for duplications.
Can Diagnose: aneuploidies, unbalanced chromosomal rearrangements, chromosome deletions and duplications > 200 Kb and 400 Kb, respectively.
Cannot Diagnose: Deletions/Duplications below the resolution of CMA, nucleotide mutations, balanced chromosomal rearrangements
Trisomy 21 Downs Syndrome
and how it’s detected
Short stature, midface hypoplasia, small ears, hearing loss, upslanted palpebral fissures, epicanthal folds, protruding tongue, heart disease, ‘Brushfield Spots”, duodenal atresia, C1-2 instability, 5th finger hypoplasia, single palmar crease (Simian), mental retardation, Leukemia, Dementia.
While normaly chromosome 21 nondisjunction is the cause…. Trisomies of 13 and 18 also liveborns
o
Detecting Trisomy 21 Downs Syndrome:
♣ Chromosome analysis is useful for identifying aneuploidies, like trisomy 21
♣ Also suitable for identifying large chromosomal structural changes (duplication, deletion, rearrangements)
♣ Resolution of about 3-5 Mb
WAGR syndrome
WAGR Syndome
is a rare genetic syndrome in which affected children are predisposed to develop Wilms tumour (a tumour of the kidneys), Aniridia (absence of the coloured part of the eye, the iris), Genitourinary anomalies, and Retardation.[1] The G is sometimes instead given as “gonadoblastoma,” since the genitourinary anomalies are tumours of the gonads (testes or ovaries). The condition results from a deletion on chromosome 11 resulting in the loss of several genes. As such, it is one of the best studied examples of a condition caused by loss of neighbouring (contiguous) genes.
o Wilms Tumor, Aniridia (black eye), malformation, retardation
o A Deletion of chromosome 11
o Detecting: FISH for PAX6 Locus in Child with WAGR and normal chromosomes
Use a FISH probe to see the deletion of PAX6 for WAGR syndrome
o Some individuals with isolated Aniridia have PAX6 mutations
♣ Black eyes with no oether syndromes
♣ Isolated Aniridia is familial ~70% of the time
80% of Aniridia without PAX6 deletion have detectable mutations in PAX6 by DNA sequence analysis
o Micro Array and Fish cannot detect this, too small
♣ Use Sanger Sequencing to find this small gene
DNA Sequencing: Mutations in known genes (mutation can be previously reported or can be novel), polymorphic variants, small (1 to ~100 nucleotide) deletion/insertions. Can find and sequence small, specific changes
FISH Testing and it’s uses
FISH test of chromosomes is specific, you know what disease you are looking for
o If you know (or strongly suspect) the diagnosis of a micro-dup/del syndrome then FISH may be your best (most specific, most cost-efficient) option
o When you do not know the diagnosis then consider a ‘genomic’ test
Chromosomal Microarray Analysis (CMA)
Test and reference DNA samples (targets) are labeled with different colors, mixed, and passed over an array with (oligonucleotide probes) containing DNA fragments from the whole genome.
♣ Abnormal ratios of the colors are indicative of deletions or duplications
♣ Can tell that colors will show differences of the ratio between the target DNA and the reference DNA (will tell of duplication or deletion)
o aCGH has replaced many chromosomal studies
(Array Comparative Genomic Hybridization, a.k.a. ‘microarray’)
DNA Sequencing:
DNA Sequencing: Mutations in known genes (mutation can be previously reported or can be novel), polymorphic variants, small (1 to ~100 nucleotide) deletion/insertions. Ideal for looking at the sequence of a known disease gene. CAN detect NOVEL mutations.
o DNA sequencing now a workhorse technique for many single gene defects
o Can detect novel mutations
o May miss larger deletions
Diagnostic Testing
Patient with SIGNS or SYMPTOMS of genetic disease
o Positive genetic test result confirms diagnosis
If disease diagnosis is already suspected on clinical grounds then test is ‘confirmational’. If symptoms are present, but clear diagnosis is unknown, test results can diagnose the underlying and current disease
Predictive testing
Patient with No SIGNS or SYMPTOMS of genetic disease
o Positive genetic provides estimate of future disease risk
Patient (or in case of prenatal testing, a developing baby) has some underlying ‘risk’ of disease (based on family history or ethnic background). The genetic test result further classifies the risk of a future disease
Heterogeneity (Allelic and Loci)
o Allelic Heterogeneity: Different mutations (alleles) at single locus
o Genetic Heterogeneity: Different mutations (alleles) at different loci
♣ Can throw off the Test completely!
♣ The test can be negative for one family, positive for one another
Genes such as Alzheimers, Cystic Fibrosis, and Sickle Cell Enemia
♣ These all have Heterogeneity mutations at different loci in the genome
• Can make test’s difficult as there are different possible regions of genomic defect
Why cant we cure genetic diseases yet?
Usually the large number of cells are affected since conception (fertilization) Too much of a massive scale problem
o Chromosomal defect
♣ Cannot fix billions to trillions of cells
♣ We cannot move or fix chromosomes on the massive scale
o Single Gene defect
♣ We cannot yet inset genes on the massive scale
♣ Also cannot silent or regulate genes on massive scale
o Complex (many genes + environment) ♣ Large scale mutations or gene heterogeneity makes it difficult to defeat diseased multiple genes ♣ Hard to understand the gene and environment combined issue
Trisomy 21: Improving health without addressing the underlying genetic defect
Trisomy 21: Frequent identified cause of intellectual disability
o >35 years ago, median age of death for patients with Trisomy 21 was ~1 year
Better cardiotherapy has allowed much longer life
o In 1983 the median age of death for patients with Trisomy 21 was 25 years; by 1997 the median age had risen to 49. (Lancet 2002;359:1019-1025)
Median age has gone up drastically
o Critical: Supportive care and better cardiac surgery responsible for the improvements in survival
-Cardiac therapy and longer life span has allowed us to realize that Alzheimer’s appears later
Multiple Endocrine Neoplasia and Genetic Testing
Multiple Endocrine Neoplasia
We use genetic testing to manage this disease, realize that you may need to remove thyroid to avoid consequences with the disease. Using management to change outcomes.
presymptamatic carriers prophylactic thyroidectomy
-improved survival
MEN —>encompasses several distinct syndromes featuring tumors of endocrine glands, each with its own characteristic pattern. In some cases, the tumors are malignant, in others, benign. Benign or malignant tumors of nonendocrine tissues occur as components of some of these tumor syndromes.
Can dettect MEN1, MEN2, ect. early with genetic testing, and treat before it causes fatality
Surgery: Many times, the affected gland can be surgically removed to treat symptoms caused by MEN. Hyperparathyroidism caused by MEN1 is typically treated with surgical removal of three-and-a-half of the four parathyroid glands,
PKU Disease and indirect therapy
PKU Disease
Young baby is lacking the phenylalanine, mostly survive on mother
Knowing the Diagnosis, able to give necessary low Phenylalanine diet to prevent the harshness of the symptoms
♣ Usually Autosomal Recessive
♣ Usually due to enzyme deficiency
♣ Some amenable to dietary and/or pharmacological manipulation.
♣ Newborn screening (heel stick) done for early diagnosis early treatment less morbidity.
Treatment Strategies for metabolic Disorders:
o Avoidance
♣ Antimalarial drugs
♣ Barbiturates
o Dietary Restrictions
♣ Phenyalanine
♣ Galactose
o Replacement
♣ Thyroxine
♣ Biotin
o Diversion
♣ Sodium Benzoate
♣ Oral resins
o Inhibition
♣ Satin drugs
o Depletion
♣ LDL Apheresis
Protein Replacement Approaches (enzyme therapy)
Alpha-1 AT a disease that leads to lung and liver problems (early emphysemia) ♣ Autosomal recessive ♣ Deficiency of alpha-1 antitrypsin Elastases unchecked • Early empysema • Hepatic fibrosis / cirrhosis Recombinant AT1 therapy • Is a protein infusion therapy that works okay • AT1 inhibits elastase
Fabry Disease
♣ X-linked disorder
♣ Deficiency of alpha-galactosidase A enzyme activity will build up the Gl3 glucolipid
• Microvascular Disease
• Neuropathy
• Nephropathy
• Cardiomyopathy
Mean survival 40 years prior to dialysis and renal transplantation
Recombination Alpha-Gal
•Can have Alpha-Galactosidase A enzyme transfusions
Chaperone-Based Therapy
Chaperone-Based Therapy :
Molecular chaperone therapy is one of the latest pharmacological approaches to lysosomal storage diseases. It fixes defective protein as an alternative to Stop codon suppression treatment. These chaperones are minute molecules that can enter the central nervous system ( via Blood Brain Barrier). Once in the CNS, they attach to the enzyme (inactive form) and fix it so that it takes the correct functional shape. Limitation of the therapy is that it only works with certain mutations :
Gaucher’s disease, Fabry disease, Pompe disease and Late-onset Tay-Sachs disease.
Take chemical cahpperones, helps bind to misfolded proteins and fix their folding
♣ The proteins that are not working in diseases Chaperone is able to fix the native protein/enzyme
o Chapperone protein is able to fix missense or non-sense made proteins that are defective
How Costly for Chapperone Therapy?
♣ Can be roughly 20-25,000$ a dose, can be around 300-600,000 dollars a year
Have a patient in my practice with Pompe disease
♣ Pompe: an enzyme deficiency disease
• AR disease, due to lack of acid alpha-glucosidase
• Progressive muscular failure
♣ Patient is wheelchair bound and ventilator dependent
Protein Replacement Therapy:
o Protein replacement therapies have greatly changed the treatment of a modest number of genetic illnesses (most autosomal recessive)
o Challenges of protein-based therapy include: production, delivery, targeting, immunological reactions, cost
The Progeria Story (Super Rare)
Farnesyl Transferase Inhibitors: possible treatment for Progeria disease
Showing promise in Lamin A/C mutations in Progeria, a premature aging syndrome.
Farnesyl transferase inhibitors appear to reduce progerin sequestration at the nuclear membrane
Gene Therapy
Definition:
♣ Introduction of DNA (or RNA) molecules into human cells to treat an acquired or inherited disease
Principle:
♣ Introduction of a gene (and its product) should cure or slow down the progression of a genetic disease
o Gene therapy can approaches include in vivo and ex vivo strategies
♣ Advantages and disadvantages (prior table) of different methods should be learned
Delivery of Gene therapy
Delivery: Several delivery mechanisms are currently being studied
o Viral: Adenoviral and Retroviral
o Non-Viral: Liposomal, protein-DNA conjugates, injection of naked DNA, artificial chromosomes
Note: Current gene therapy efforts are largely directed at gene ‘replacement / deficiency’ models, where a gene (protein) is missing or non-functional. Approaches to correct diseases due to dominant negative and/or gain of function mutations are expected to be more challenging
Achondroplasia (dwarfism):
FGFR3 Gly380Arg gain of function mutation leads to achondroplasia, Spinal cord compression (apnea/death) is the most feared complication, paternal age effect
PRINCIPLES: Gain-of-function mutations
♣ Advanced paternal age
♣ De novo mutation
o MAJOR PHENOTYPIC FEATURES
♣ Age at onset: prenatal
♣ Rhizomelic short stature
♣ Megalencephaly
♣ Spinal cord compression
♣ (trident hand)
Skull growth abnormal: midface hypoplasia (otits, sleep apnea)…small cranial foramina (hydrocephalus, brainstem compression (10% of patients), increased frequency of hypotonia, quadriparesis, failure to thrive, central apnea, and sudden death).
♣ Between 3% and 7% of patients die unexpectedly during their first year of life because of brainstem compression (central apnea) or obstructive apnea
o Genetic component of Achondroplasia
♣ Autosomal dominant disorder
♣ Caused by specific mutations in Fibroblast Growth Factor Receptor 3 (FGFR3);
♣ Two mutations, 1138G>A (∼98%) and 1138G>C (1% to 2%), account for more than 99% of cases of achondroplasia, and both result in the Gly380Arg substitution.
Pathogensis of Anchondroplasia
♣ FGFR3 is a transmembrane tyrosine kinase receptor binds fibroblast growth factors initiates a signaling cascade ~ inhibits bone growth (over-simplified)
♣ FGFR3 c.1138 G>A Gly380Arg = gain-of-function mutations that cause ligand-independent activation of FGFR3. FGFR3 turned ON inappropriately inhibits bony growth
♣ c.1138G is ~#1 most mutable nucleotide in human gene nearly 100% of achondroplasia (single gene / single nucleotide)
♣ De novo mutations of FGFR3 guanine 1138 occur exclusively in the father’s germline and increase in frequency with advanced paternal age (>35 years)
Summary of Achondroplasia
♣ FGFR3 Gly380Arg gain of function mutation leads to achondroplasia
♣ Spinal cord compression (apnea/death) is the most feared complication
♣ Paternal age effect
Summary of Achondroplasia:
- FGFR3 Gly380Arg gain of function mutation leads to achondroplasia
- Spinal cord compression (apnea/death) is the most feared complication
- Paternal age effect
Genetic component of Achondroplasia:
Autosomal dominant disorder
Caused by specific mutations in Fibroblast Growth Factor Receptor 3 (FGFR3);
Two mutations, 1138G>A (∼98%) and 1138G>C (1% to 2%), account for more than 99% of cases of achondroplasia, and both result in the Gly380Arg substitution.
Non Syndromic Deafness
Non Syndromic Deafness
Genetic, autosomal recessive, nonsyndromic deafness due to GJB2 mutation is most common category of congenital deafness, Be able to recognize the syndromic forms
o Allelic heterogeneity with both dominant and recessive inheritance patterns
Newborn screening
Cultural sensitivity in counseling
o MAJOR PHENOTYPIC FEATURES
♣ Congenital deafness in the recessive form
♣ Progressive childhood deafness in the dominant form
o Genetic Partition
♣ ~ 1/2 congenital deafness = genetic
Of genetic:
♣ 3/4 nonsyndromic
♣ 1/4 syndromic
♣ Non Syndromic
♣ GJB2 mutations most common ½ of all nonsyndromic autosomal recessive deafness
Syndromic Deafness
Systems outside the ears are involved:
Important (clinically and on exams) to recognize some syndromic forms of deafness
General:
• Intellectual disability, seizures, dysmorphic syndromes
Specific:
With retinitis pigmentosa suggests Usher (AR)syndrome
With thyroid goiter suggests Pendred (#2 AR) syndrome
• With arrhythmia or sudden death suggests Jervell and Lange-Nielson (AR) syndrome
• With white forelock suggests Waardenburg (#1 AD)syndrome
• With 8th nerve schwannomas suggests Neurofibromatosis type II
Genetics Continued: GJB2 Mutations:
♣ Recessive inheritance for severe congenital
♣ Parents typically carriers
♣ Loss of function mutations predominate
♣ Autosomal dominant for nonsyndromic progressive deafness with childhood onset (hearing ~normal at birth)
♣ One parent typically affected
Each type is numbered in the order in which it was described. For example, DFNA1 was the first described autosomal dominant type of nonsyndromic deafness. Mitochondrial nonsyndromic deafness involves changes to the small amount of DNA found in mitochondria, the energy-producing centers within cells.
Most forms of nonsyndromic deafness are associated with permanent hearing loss caused by damage to structures in the inner ear.
Fragile X Syndrome
Fragile X Syndrome
Fragile X syndrome is hypermethylated loss of function, Premature Ovarian Failure and FXTAS occur with premutations and a gain of transcript / gain of function, Fragile X triplet repeat is in 5’ UTR region
o X-Linked PRINCIPLES ♣ Full Triplet repeat expansion ♣ Somatic mosaicism ♣ Sex-specific anticipation ♣ DNA methylation ♣ Haplotype effect
o MAJOR PHENOTYPIC FEATURES ♣ Age at onset: childhood ♣ Mental deficiency ♣ Dysmorphic facies ♣ Male postpubertal macroorchidism
o fragile X-associated tremor/ataxia syndrome (FXTAS)
♣ X-Linked
♣ Premutation Triplet repeat expansion
♣ Age at onset: Adulthood
♣ Ataxia, tremor
♣ Memory loss, parkinsonism, peripheral neuropathy
♣ Men»_space; women
o Premature Ovarian Failure
♣ X-linked
♣ Premutation Triplet repeat expansion
♣ Women (not men)
o FMR1 (Gene) and FMRP (protein):
♣ FMRP expressed most abundantly in neurons, may chaperone mRNAs from nucleus to translational machinery. (mRNA and protein have function)
♣ 99% of FMR1 mutations are 5’ CGG expansions
♣ Normal: 6-50 CGG repeats
♣ Normal FMRP protein amount
o Fragile X: Premutations and Full Mutations
The molecular pathogenesis is different in the premutation diseases, compared with the full mutation that leads to FXS. FMR1 mRNA expression levels are increased with the premutation and decreased or absent with the full mutation. FMRP levels are absent or decreased with the full mutation and normal or close to normal with the premutation.
The CGG repeat in the Methylation due to extensive elongation of the CGG repeat in the 5′-ÚTR of the FMR1 gene is depicted as a lock
Summary of Fragile X syndrome
♣ Fragile X syndrome is hypermethylated loss of function
♣ Premature Ovarian Failure and FXTAS occur with premutations and a gain of transcript / gain of function
♣ Fragile X triplet repeat is in 5’ UTR region
Epigenetic phenomenon goes across generations
Overkalix, Sweden= has kept very good documentation and medical documentation of generations
♣ Have kept records during feast and famine
♣ Found that with paternal grandparents (pre-adolescence) went through time of famine
Found that grandchildren were at greater cardiovascular risk and diabetes
• These exposures are male descendants from several generations ago
Same paper looked at children in UK who have high BMI
• They found that many of the high BMI children had fathers who started smoking around 11
• Found that smoking and diet can affect DNA methylation
o Can silent certain genes
Mice testing and epigenetics
Feed mice the precursors for DNA methylation
o Genetically comparable mothers who were fed slightly different diet (low or high “methyl donor” ingredients) during gestation and nursing produced very different offspring
♣ Methyl donor ingredients:
♣ folic acid, vitamin B12, choline, betaine
The AGOUTI gene is turned off in the darker, smaller mouse. (From the high MD mother)
These mice had the large amount of methyl donor content
Agouti protein regulates both coat color and feeding habits
o Normal diet= would have just normal amount of methylation
o Abnormal diet= increased methylation which lead to the smaller and darker mice, turning off the AGOUTI gene
Can have effects on eating habits and metabolism
Epigenetic Characteristics: A Definition:
1)Different gene expression pattern/phenotype, identical genome
2) Inheritance through cell division, even through generations
- seen passed down multiple generations like the swedish family generations in starvation
3) Like a Switch: ON/OFF
♣ Genes are either on/off (like a gradient)
4) Erase-able (inter-convertible) Therapeutic potential
♣ plasticity is possible, able to change and reset epigenetic marks
♣ Important for disease: Cancer and other diseases will replicate without differentiation, ignoring epigenetic markers
Need the epigenetic markers
♣ HUGE opportunity for therapy potential, can reset diseases or diseased cells, turn it into healthy cells
Epigenetic Visualization
There are infinite possibilities/pathways for cells to develop before being specific
Different cell states based on epigenetics
The hill makes it difficult for cells to go back, keeps the cell specific (maintains its identity) after it specializes
• Think of it in terms of energy states in chemistry. Each cell state is a stable “low energy” state
Light-switch Example= A bunch of light switches represent the gene with epigenetics
o Whatever combination of “on” and “off” switches are activated= will decide what the cells will ultimately turn into
o Induced Pluripotent Cells Goal is to eventually turn all the switches “on” so they can be used to turn into any cell possible
One day for therapy?
Why is erasure and resetting of methylation patterns of imprinted genes during gametogenesis essential?
Erasure must occur, removing all the epigenetic markers (such at DNA methylation)
Than there must be the Sex-Specific Gene silencing after the reset
♣ Make sure the right genes are methylated before they are passed down and fertilized
Paternal silencing= For the sperm gametes
Maternal silencing= for the ovum
Need only 1 active copy of certain genes for a specific maternal or paternal chromosome, without reset: possibility of incorrect gene amount
DNA methylation
DNA methylation occurs only on cytosines of CpG.
♣ Does not affect base paring of 5-meC with G.
o Contributes to gene silencing by solidifying the repressed state
Methyltransferases:
Propagate Epigenetic Marks Through Somatic Cell Division (DNA replication)
Most important during DNA replication
♣ The old strand has the methylated part after replication
The new strands are NOT methylated yet after DNA replication
• Specific enzymes will go in and reset the methyl groups on the New Strand (Methyltransferase)
• Otherwise you would lose the methylation/ gene silencing
Packaging eukaryotic DNA into chromatin
Majority of the chromosomes are packaged around histones
o There are repressive and active histone marks
Repressive Histone Marks: These will often have methyls on them, difficult to reach
• Methyl usually deactivates, sometimes activates
Active Histone Marks: There are other markers that will activate, such as acetyl groups on histone tails or phosphorylation
Inheritance of a Chromatin State
Begins at DNA replication, at the replication fork
♣ Happening During the S-phase
♣ Need to interrupt the nucleosome organization
♣ Need to make newly make nucleosomes/histones with DNA replication
Problem for maintaining an epigenetic state (DNA methylation or histone modification)
♣ DNA is half old/half new
♣ Histones are half old/half new
o There nucleosomes are able to communicate via enzymes to tell the other histones/nucleosomes to change their epigenetic nature
Epigenetic phenomenon is strongly being considered in diseases
o Cancers Taking away the epigenetic capabilities (or affecting/mutating them)
o Silencing of a tumor suppressor gene (TSG) by 5meC can lead to cancer
Normally there are tumor suppressor genes that are activated
• These regions will become methylated, it is unable to create the tumor suppressing proteins
These methylations can be caused by environment
Histone Deacetylases as a future therapy tool
Histone Deacetlyases been used to help combat these genes that become methylated and silenced, especially tumor suppressers that are silenced (possible therapy)
Bart’s and other random hemoglobin
Rare cases with lack of any alpha hemoglobin:
Most fetal hemoglobin is γ4 (Hb Bart’s) although there is enough ζ2γ2 (Hb Portland) to sustain fetal development.
