Download TB1 - BIOCHEM, Bidichandani, Genetic Diseases

Review for Biochemistry, Genetic diseases, Dr. B., Chapter C
MUSCULAR DYSTROPHY
Duchene, Becker, and other inherited muscular dystrophies are characterized by progressive muscular
weakness, and classified by mode of inheritance, disease severity, and distribution of affected muscles. There
is growing acceptance of molecular classifications.
1. XLR dystrophies – Duchene, Becker (less severe), and Emery-Dryfus (similar to Becker, with muscular
weakness in spine, neck, elbow and scapulohumeral areas. Also displays joint contractures and cardiac
arrhythmias)
2. AR dystrophies – Limb-Girdle, Congenital-Classic, and Congenital-Fukyama.
3. AD dystrophies – Oculopharyngeal and Fascioscapulohumeral.
1.
Duchene and Becker are characterized my mutations in the dystrophin gene. The dystrophin gene is
the largest gene in the human genome. Increased probability is thought to be the cause for these
dystrophies.
2. Incidence is 1/3000 males, where D has 5 times higher incidence than B.
3. Onset of symptoms is around the ages of 3-5. Patients present with slow running and difficulty rising
from the seated position. Most patients are wheel chair bound by 12 and mean age of death is 17.
Psuedohypertrophy of the calves is seen. Muscle weakness is predominantly in the girdles and
proximal limbs giving rise to the Gowers sign. Patients that live past 17 may develop
cardiomyopathy.
4. BMD is a mild variant of DMD. Patients are wheelchair bound at 16 and cardiomyopathy is more
frequent.
Expression of phenotype by females
1. Can be caused by skewed X inactivation (manifesting carriers) This results in mild forms being
present in 8% of carriers.
2. Another cause in X-autosome translocation that disrupts the dystrophin gene.
Lab findings in DMD, BMD patients
1. Serum creatine kinase is elevated 50-100 fold.
2. histopathalogical examination shows segmental necrosis of muscle fibers which are eventually
replaced by adipose or connective tissue.
Identification of the dystrophin gene by positional cloning
1. The dystrophin gene was initially mapped to Xp21 for several reasons
a. Female with DMD had X-autosomal translocations and breakpoints in Xp21.
b. Rare male patients had deletions of Xp21
c. Polymorphisms known to be near XCp21 were absent with phenotype.
d. Deletions of Xp21 were observed in DMD/BMD patients indicating that they were allele
variants and not distinct diseases.
2. 1/3 of DMD patients are caused by de novo mutations.
Mutations in dystrophin
1. The majority of DMD patients have an out of frame mutation while the majority of BMD patients
have an in frame mutation. This is why DMD is more severe. The frameshift mutation disallows
the protein to perform its job of attachment to the sacrolemmal glycoproteins, while the BMD
mutation only shortens the length of the rod.
2. Multiplex PCR (simultaneous PCR amplification of multiple exons) is a method by which
investigators are able to determine which exons have been removed and can thereby determine if the
mutation is a frameshift.
3. 30% of patients have small deletions or point mutations. RTPCR or PCR followed by screening
for point mutation in the form of heteroduplex analysis, single stranded conformation
polymorphism analysis, or chemical cleavage mismatch analysis can detect these mutations. RTPCR has the advantage of detecting abnormal splicing.
4. You can also perform a protein truncation test to determine premature termination of translation.
This is more efficient than searching exons for point mutations.
5. X linked dilated cardiomyopathy is caused by rare mutations that specifically affect the cardiac
expression of the gene.
6. A western blot can be done with a BMD dystrophin protein to see if it is shorter than a normal
protein.
Histological analysis of DMD/BMD tissue
1. A total lack of dystrophin is seen in DMD patients after staining of tissue.
2. A decrease in concentration of dystrophin is seen in BMD patients.
3. Patchy staining is seen in symptomatic female carriers, this is due to varied X inactivation of cells.
Dystrophin protein
1. Dystrophin has a N terminus that binds to the cytoskeletal actin and a C terminus that is associated
with a group of sacrolemmal glycoproteins. There is also a long chain that connects the two. This is
formed by 4 hinge regions that are proline rich and add flexability to the protein.
2. The glycoproteins form two distinct sub complexes, the sacroglycans and the dystroglycans. The
dystroglycans bind to the merosin in the basal lamina (extracellular matrix) of muscle. Dystrophin,
therefore, acts as a mechanical bridge between the actin cytoskeleton and the extracellular matrix.
3. Lack of dystrophin is thought to result in a weakened sacrolemma which is subject to damage during
muscle contracture. Sacrolemmal damage allows an influx of Ca++ that activates intracellular
proteases producing muscle necrosis. The BMD dystrophin is shorter and partially functional
resulting in a milder phenotype.
4. Dystrophin directly interacts with the dystroglycans. Although it does not directly interact with the
sacroglycans, it is important for their assembly, therefore in DMD there is a secondary deficiency in
the sacroglycans.
Diagnostic testing for DMD BMD
1. Confirmation of diagnosis – A positive phonotype showing X linked inheritance, increased CK,
requires one of the following
a. Immunohistochemistry showing deficiency of dystrophin.
b. Western Blot showing shorter of deficient dystrophin.
c. Deletion or duplication shown by multiplex PCR or southern blotting.
d. Proof of other deleterious mutations by screening of gene.
2. Detection of carrier status – This is often difficult to establish. Can be detected by
a. Pedigree analysis may indicate obligate carrier status.
b. Elevated CK may be useful but often overlaps with normal population.
c. Patchy immunohistochemical staining of muscle biopsy.
d. Dosage analysis may show half the signal from deleted exons.
3. Prenatal diagnosis – depends on whether mutation is known or not.
a. If known then DNA testing can test for presence of mutation following chorion villus
sampling, amniocentesis, or preimplantation diagnosis.
b. If mutation is unknown fetal muscle biopsy can test for mutation,
Gonadal mosaicism – means that a mother may have more than one affected son and still not be a carrier with
her somatic cells. A portion of her germline cells may carry the mutation and would put her at a high risk for
having affected sons. For this reason prenatal testing to all mothers is offered at centers regardless of carrier
status.
CYSTIC FIBROSIS
Cystic Fibrosis is a genetic (autosomal recessive) disease characterized by chronic respiratory insufficiency
(usually the cause of death), pancreatic insufficiency, and elevated concentration of sweat electrolytes. The
pathogenesis of CF airway disease is complex, but may include defective antimicrobial activity in airway
surface liquid, altered mucociliary clearance, or abnormal submucosal gland function, all of which leading to
bacterial endobronchitis (especially by Pseudomonas aeruginosa) Caucasians have a 1/2-4000 chance of
getting cystic fibrosis. Most males are infertile due to congenital bilateral absence of vas deferens (CBAVD).
Absence of vas deferens results in azoospermia, therefore CF must be considered a differential diagnosis of
obstructive azoospermia. Meconium ileus (Intestinal obstruction in the fetus and newborn followed by
inspisitation of meconium) is present in 10-20% of patients.
CFTR (cystic fibrosis transmembrane conductance regulator) is the gene that is mutated to lead to the
disease. It was the first to be identified purely by positional cloning. The gene is located on 7q31.2, contains
27 exons, and encodes a 1480 a.a. protein.
CFTR function in a cell
1. CFTR is an ABT (ATP binding cassette transporter). It is broken into halves. Each halve has two
membrane spanning domains that are composed of 6 transmembrane sequences each. Each half
also has an NBD (nucleotide binding domain) that binds ATP, the energy source for the
functionality of the protein. There is one regulatory (R) domain that switches the protein “on and
off” based on the presence of cAMP. Normally this pump acts to channel CL- (and Na+) ions out of
the cell, in CF the secretory coils are unable to perform this function resulting in excess salt
concentration in sweat. This is the basis behind the sweat test.
Mutations of CFTR
1. 508 is the most common mutation in CF. 508 is a 3bp in-frame deletion that leads to the
deletion of phenylalanine at position 508. The carrier frequency for this mutation is 1/22. The
proportion of mutant chromosomes with this mutation increases from southwest to northwest
Europe. This mutation is readily detected by PCR assay that shows the mutant as a shorter product
than the normal allele. Heterozygous carriers can be determined by the presence of a heteroduplex.
2. 508 mutation occurred once in history and several theories for this have been proposed
a. Heterozygote advantage – Mice with a single CFTR mutation are more resistant to
secretory diarrhea by Cholera, humans may be similar.
b. Hitch hiker effect – The mutation may be a byproduct of a nearby, advantageous mutation.
This is supported by the fact that females with this mutation have shown increased survival
rates in Europe.
3. More than 1000 mutations have been discovered in the CFTR gene. (800 of which cause CF)
a. Most of these are small mutation (1% is large deletions)
b. Only 4 mutations, other than 508, account for >1% of CF alleles worldwide.
c. Certain mutations are more prevalent in certain areas.
d. Most labs test for more than 20 mutations. These labs claim to ID 90% of mutant
chromosomes in Caucasians in the US. Testing for Caucasian and Native African mutations
detects 75% of mutation in African Americans.
e. At least 2 individuals have been identified as obtaining CF by uniparental disomy (where
one heterozygous parent passed on the deficient gene and the child inherited two copies of
that gene.)
f. The core point mutations can be detected by PCR followed by restriction digest or direct
sequencing.
g. Rare mutations can be detected by single stranded conformation polymorphism analysis and
heteroduplex analysis.
Diagnostic criteria for CF – The CF foundation established the following criteria for diagnosis, both of these
must be met to diagnose.
1. One or more of the following features
a. Typical pulmonary disease
b. Typical GI manifestations due to pancreatic insufficiency.
c. Salt loss syndrome
d. CBAVD
2. And, Either lab evidence of CF, or definitive family history of CF.
a. Lab evidence includes the following
i. Positive sweat test
ii. Homozygosity for deleterious mutations in CF gene
iii. Abnormal ion transport in nasal epithelium
3.
CFTR gene testing
a. Carrier testing is done to assess the risk of transmitting CF when there is a family history of
CF.
b. Prenatal genetic testing is performed when both parents are known carriers.
c. CFTR testing is also done in the case of atypical patients, i.e. those that show signs of CF,
with a negative sweat test.
d. CFTR genetic testing is done for those patients with azoospermia.
Four general classes of mutations
1. Class I, Deficiency of CFTR – i.e. nonsense or frameshift mutations that result in complete
deficiency of CFTR
2. Class II, Defective processing of CFTR – Mutations that prevent the transfer of CFTR to the golgi
complex, leading to a deficiency of appropriately glycosylated mature CFTR at the cells surface.
508 is a Class II mutant.
3. Class III, defective regulation – Mutations of the NBD or R domains that result in mutant CFTR
arriving at the membrane with poorly regulated function.
4. Class IV, defective conduction – Missense mutations of the MSD that may interfere with the
amount of CL- flow, reducing channel function.
Genotype-phenotype correlation in CF
1. CFTR genotypes (mutations) show poor predictability of pulmonary phenotype.
2. CFTR genotypes show positive predictability with pancreatic function and CBAVD.
a. 5-15% of CF patients are pancreatic sufficient.
b. Concordance is seen among siblings indicating effects of genotype.
c.  508 patients are rarely pancreatic sufficient, however patients with partial CFTR deficiency
are often pancreatic sufficient.
d. 2% of patients with obstructive azoospermia have CBAVD and 75% of them are
heterozygous for CFTR mutations. (intron-8 variant)
3. The intron-8 variant
a. A method by which splicing of the 8th intron is facilitated is the presence of a poly T region
(numerous thymidine DNA bases) on the CFTR chromosome. The longer the Poly T tract
the more efficient the splicing of exon 9. Most chromosomes have 7 or 9 T’s, which is fine,
but 5% have 5 T’s. The 5 T variant results in a significant amount of defective exon 9
transcript. In 5T homozygotes, 90% of exon 9 is missing.
b. The Inron-8 variant influences the type and severity of CF.
c. The 5T variant is often associated with CBAVD. Furthermore the R117H mutation, that
normally results in CBAVD, if associated with the 5T variant results in CBAVD and CF.
Mouse models of CF
1. Knockout miceand mice with the 508 mutation have been created.
2. Surprisingly they all have severe intestinal phenotype, and they all lack significant pulmonary,
pancreatic, or male genital phenotypes.
Fragile X syndrome
Fragile X syndrome is an X linked dominant disease with reduced penetrance. It causes mental
retardation, affecting 1/4500 males and 1/9000 females. There is a classical triad of features associated with
Fragile X syndrome; a long face, prominent ears, and enlarged testicles in post pubertal males. Prepubertal males present with autism-like symptoms, hyperactivity, and language delay. Fragile X syndrome
in females is typically less severe, as a result of only a percentage of defective X chromosomes as a result of X
inactivation of a good X chromosome.
1. FXS derives its name from a fragile site at Xq27.3 present in a portion of metaphase chromosomes
from cultured cells of males. It was noticed early on, that there were a significant higher number of
mentally retarded males. This accounts for X linked mental retardation.
2. Dr. Sutherland showed that cell culture that was deficient in folic acid or containing thymidine
expressed a fragile site that appears as a break in the chromosome. At this time, diagnosis was ID of
a patient with at least 2% of chromosomes with the fragile site. There was found to be another
fragile site in Xq that gave false positive readings. The fragile “gap” may be due to uncondensed
DNA or altered chromatin structure.
Differences in Fragile X syndrome and classic X linked inheritance
1. 20% of males with FXS were clinically unaffected.
2. A relatively high % of females with the mutation were affected.
3. Daughters of normal transmitting males were almost never affected.
4. Daughters of non expressing females had a 25% chance of being affected.
5. Clinically affected females were at a high risk of transmitting the disease.
6. The risk of having an affected child increased with generations. (anticipation)
7. ***The subsequent discovery of CGG triple repeat expansion as the cause of the disease would later
explain the above phenomena.
Genetics of Fragile X Syndrome
1. The FMR1 gene was identified by positional cloning in 1991.
2. The mutation responsible for the disease
a. Was a CCG triple repeat expansion in the 5’ UTR of the FMR1 gene.
b. Is a highly methylated version of the TRE.
c. Other patients with the disease (2%) show loss of function mutations, large deletions,
frameshift mutations, and splice mutations. These patients however, do not express the
fragile site, indicating that the triple repeat expansion is the cause for the fragile site.
Four allele variants of the FMR1 gene
1. Normal – polymorphic 6-40 CCG repeats interspersed with 1-3 AGG sequences every 9 to 10
CGG repeats. These are stable when transmitted from parent to offspring.
2. Intermediate – 40-60 unmethylated CCG repeats that shows limited stability and have the
potential to expand to premutation alleles and some even full mutation alleles.
3. Premutation alleles – An increase in the number of pure CGG repeats from a normal allele, this
can expand to a full mutation allele when transmitted from mother to child. The creation of these
alleles is caused by an increase in CGG repeats in the 3’ region and often loss of one of the
intervening AGG sequences. Maternal transmission of PM’s has a definite risk of becoming FM’s.
This makes female carriers of PM’s have a high risk of having children with FXS. Whereas
paternally transmitted PM’s never become FM’s.
4. Full mutation – the disease causing variant of the TRE’s. This version is highly methylated and is
also unstable which leads to the shutting off of the FMR1 gene transcription. No mRNA is
produced, this results in a deficiency of the FMR1 protein. Contain more than 230 CGG repeats.
Because of random X inactivation, females are typically less severely affected.
Detection of triple repeat expansions
1. Southern Blot analysis – has the added benefit of detecting the methylation status of the mutation,
and is therefore the preferred method of diagnosis.
2. PCR – Allows for more accurate size measurement, this accounts for the more accurate risk
predictability.
Types of mosaics among carriers of the full mutation
1. Expansion mosaics – (15% of patients) carry non methylated PM’s resulting in production of
FMR1 gene product, decreasing the negative affects of the disease.
2. Methylation mosaics – (1% of patients) Carry a partially of totally unmethylated FM, having
similar effects as the expansion mosaics. These types of patients are referred to as high functioning
males.
Intergenerational instability of the CGG triple repeat
1. All full mutations are derived from premutations.
2. The number of repeats increases from one generation to the next.
3. There is a sex bias in determining intergenerational stability
a. Expansion of PM to FM is a seen almost always in maternal transmission.
b. Maternal transmission of FM results in offspring with FM.
c. Paternal transmission of FM results in daughters with PM.
d. This reversion in type of mutation is strictly in male transmission and male sperm show PM
alleles even if their blood DNA shows FM’s
4. Anticipation – occurs due to a continuous increase in length of TRE by generation
5. The risk of mental retardation depends on the clinical status of the carrier mother
a. If mother is affected, the risk of affected male/females is 50/25%
b. If mother is unaffected, the risk is lower, 40/15%.
c. This is because maternal transmission of FM is certain, however maternal transmission
of PM is less than 100%.
d. The risk of mental retardation is higher for daughters of clinically normal females than
that for normal transmitting males
i. This is because normal transmitting males have PM alleles and passing this to
offspring never results in an FM allele.
ii. Also because a clinically normal female with a PM allele will transmit an FM allele
depending on the size of the PM allele.
e. Sherman Paradox - The risk of mental retardation in offspring is different depending on
whether or not a normal transmitting male is present as a sibling
i. A clinically normal female in FXS can carry an FM or PM.
ii. Risk of mental retardation in offspring of normal female is 40% for sons and 15% for
daughters.
iii. However if the female is a sibling of a normal transmitting male, the risk of mental
retardation is substantially lower, 10% for males and 5% for females.
iv. This is b/c???????
Who should be offered genetic testing
1. Persons with X linked mental retardation – 30% of them will have FXS.
2. Persons that have mental retardation or autism of unknown origin.
3. Any Person who is clinically diagnosed with Prader-Willi or Tourette syndrome and psychiatric
conditions.
a. ***In rare cases mental retardation can be caused by a fragile site at the Xq end of the
chromosome due to CCG repeats at other loci.
FMRP protein
1. The KH domains of this protein are used to bind RNA and the RGG box enhances the activity of
the KH domains.
2. FMRP selectively binds brain mRNA and is associated with ribosome’s and protein translation, this
is the reason that the defective protein can cause mental retardation.
3. A rare case can also cause mental retardation with the normal CGG repeat. This happens when
there is a missense mutation in the KH2 domain causing decreased RNA binding activity.
Mouse model of FXS
1. Knockout mice and mice that carry the FXS mutation have been created and subject to the Morris
Water maze test.
a. The Morris water test is used to test short term memory and visual spatial abilities.
i. Mice are placed in a small pool, with a platform to stand on hidden in opaque water.
ii. The test, determines the mice’s ability to locate the platform after training.
b. The mice with the FXS mutation showed that they took a longer amount of time to find the
platform when compared to normal mice.
c. This indicates that the knockout mice might be able to act as animal species to test the
effectiveness of drug therapies.
Huntington Disease
Huntington disease is a slow progressing, autosomal dominant, neurodegenerative disease. Onset is late,
usually around 40. For juvenile HD around 20, and some patients develop the disease at a later age, around
50. Death usually occurs 15-20 years after onset. The prevalence among European Caucasians is around 37/100,000. Phenotypes include involuntary movements, progressive dementia, and psychiatric
disturbances. Chorea (spasmodic movements of the limbs and facial muscles) is also seen in 90% of patients.
The disease is more severe, the earlier it begins. In contrast to Alzheimer’s, language remains intact and the
patient is aware spatially and temporally.
Detection of Huntington disease
1. Positron Emission tomography (PET) scans shows decreased glucose metabolism in the striatum.
This may be helpful in establishing the clinical diagnosis of HD in early stages when the signs and
symptoms are non-specific.
Genetics of Huntington disease
1. HD is an autosomal dominant disease. Recurrence risk is 50%.
2. Risk is complicated by delayed onset of the disease.
3. Presymptomatic diagnosis is accurate, but often refused.
4. Risk decreases for healthy people as they age.
Neuropathological alterations
1. Most characteristic finding in HD is neuronal loss and gliosis in the causdate nucleus and
putamen. Collectively called the neostriatum or striatum.
2. Memory impairment correlates with caudate atrophy.
3. Chorea and involuntary movement correlate with changes in putamen.
4. Macroscopically the brain appears atrophic.
Neurochemical alterations
1. The earliest and most extensively affected neurons are the medium sized spiny neurons.
a. These neurons express gama-aminobutyric acid (GABA) and enkephalin or substance P
as neurotransmitters.
b. There cell bodies are located in the striatum and they project into the globus pallidus.
c. Degeneration of the GABA/ENK projection , which causes chorea and is normally
inhibited by dopamine (hence the alteration of dopaminergic neurotransmission in the
degenerating striatum causing chorea), is more severe than the GABA/SP connection
which causes rigidity and bradykinesia.
d. The primary pathological condition contributing to chorea in HD is the inhibition of
subthalmic neurons due to hyperactivity of the pallidal-subthalmic GABA projection.
i. This is supported in that lesions of the subthalmic nucleus in humans produce chorea
on the opposite side of the body.
Huntington gene
1. Located on chromosome 4p16 and contains 67 exons.
2. It produces 2 transcripts that differ in size of their 3’UTR.
3. The CAG encoding polyglutamine tract is located in exon 1.
a. The mutation in HD is an expansion of the polyglutamine encoding CAG tract in the first
exon.
i. Normal chromosomes contain 18 CAG repeats.
ii. HD chromosomes contain 40 CAG repeats.
iii. The CAG repeat does not affect gene expression.
Genotype-phenotype correlation
1. As CAG repeat length increase, the age of onset decrease (this can be viewed as a longer repeat
leading to a more sever version of HD.)
2. It is still difficult to predict age of onset due to the fact that any given repeat length is associated
with a wide range of onset age.
3. CAG repeat length correlates with amount of straital neuronal pathology.
4. Anticipation is seen in the form of earlier age of onset in successive generations.
Instability if CAG repeat in HD – there are 4 groups of repeat alleles
1. Normal – (up to 28 CAG repeats) – patients never develop nor transmit HD.
2. Non penetrant (with paternal meiotic instability) – (29-35 CAG repeats) – patients do not
develop HD, but some males may transmit HD related repeat.
Reduced penetrance (with meiotic instability) – (36-39 CAG repeats) – some patients will
develop HD and males will transmit CAG repeat.
4. HD – (40 or more CAG repeats) – all patients develop HD and offspring have a 50% chance of
inheriting the genotype.
Sex bias of HD
1. Maternal alleles undergo small expansions.
2. Paternal alleles undergo large expansions (10 or more).
a. Male patients have longer CAG repeats in sperm compared to blood.
3. This is the basis for two observations
a. Anticipation is mainly seen when transmission is through male germ line.
b. Juvenile HD patients often inherit mutation from their father.
Gene testing in HD – commonly done in 3 scenarios
1. presymptomatic testing – When there is a family history and informed decision has been made.
Usually after two counseling sessions.
2. To confirm a clinical diagnosis of HD.
3. Prenatal testing – Can test for mother and child and positive results can have a major effect on
mother, child, and marriage. Given the potential issues of discrimination, prenatal testing is
strongly discouraged if abortion is not an option.
Pathophysiology of HD
1. Western blotting shows enlarged Huntington protein due to enlarged POLG tract.
a. Mutant POLG tracts form intracellular and perinuclear aggregates
i. Aggregates are also seen in cerebellum and muscle of HD patients that are not
pathologically affected.
b. Polyglutamine containing Huntington interferes with neuronal specific transcription by
interfering with transcription factors.
c. Polyglutamine tracts are toxic and are the cause of cell death.
i. When HD gene with expanded CAG repeats is introduced to cells in culture,
apoptosis with nuclear aggregates results.
ii. Transgenic mice with HD show neurodegenerative phenotype. The mice also show
the presence of nuclear aggregates.
3.