Transcript No Slide Title

Genetics…
…in the news.
Final
cumulative, but overwhelming focus on…
• Sugar Paper,
• Microarrays, lecture and supported readings
in Chapters 8 and 9,
• Chimp Paper,
• Pheromone Paper,
• PCR, Northerns, Southerns.
Tuesday, Dec.11th, 10:30 - 12:30
Genotypes and Phenotypes
(societies, politics and human nature)
Nature Reviews | Genetics, (3), June 2002, pp 7
Hereditary Fructose Intolerance
• Fructose intolerance was first noted in severely ill infants
with recurrent hypoglycemia (low blood sugar) and
vomiting, occurring at the time of weaning when fructose
or sucrose is added to the diet.
– hypoglycemia,
– depletion of ATP resources,
– degradation of purines (G and A in DNA),
– hypermagnesemia.
Failure to Thrive
Identification
• 1962: a 3-year-old brother of a severely affected infant was found to have
hepatomegaly (enlarged liver), and hypoglycemic shock that was
precipitated by an oral test dose of fructose,
– although he was clinically healthy. He had a marked aversion to sweets and
fruit.
• 1963: 2 adults, aged 33 and 39 years were identified with the same
condition. In addition to the aversion to fructose-containing
foods, remarkable absence of dental caries was noted.
• The defect resides in aldolase B, which catalyzes the
cleavage of fructose-1-phosphate to form dihydroxyacetone
phosphate and D-glyceraldehyde.
Aerobic Respiration 101
“Please start
memorizing
here.”
Glycolysis
205
Prepatory Phase
You’ve learned the
conversion of glucose
into triose phosphates.
300-level
Understanding
aldolase B
We Don’t Live by
Glucose Alone
Liver, Kidney, Intestinal Mucosa
• In aldolase 'B'-deficient tissues, cytoplasmic accumulation of
fructose-1-phosphate leads to sequestration of inorganic
phosphate,
– results in the activation of AMP deaminase that catalyzes the
irreversible deamination of AMP to IMP (inosine monophosphate), a
precursor of uric acid.
• Depletion of tissue ATP occurs through massive degradation to
uric acid, and impairment of regeneration by oxidative
phosphorylation in the mitochondria because of inorganic
phosphate depletion (lost to phosphorylated sugars).
– in the cell, ATP exists largely as a 1:1 complex with magnesium.
Depletion of ATP in tissues leads to higher concentrations of
magnesium.
HFI Metabolic Defects
Fig. 1a
300-level
Understanding
aldolase B
Blocked
How is HFI diagnosed?
• The only definitive way to ascertain if one is
suffering from HFI is to have one of two tests:
1) An enzymatic assay to determine aldolase activity. The
aldolase is obtained from patient liver tissue in an
invasive surgical procedure called a liver biopsy.
2) A fructose tolerance test. Fructose is injected
intravenously under controlled conditions where acute
glucose, fructose, and phosphate levels are monitored.
Genetic Screens
• Three mutations in this Aldolase B apparently account for >75% of all
HFI mutations.
• Genetic screens are performed using the polymerase chain reaction
technique (PCR) followed by hybridization with allele-specific
oligonucleotides (ASOs).
• While this test is relatively harmless and effective, the screen does not
monitor >95% of HFI mutations (many are unkown and require further
research ), and is not considered diagnostic.
– in other words, a negative result does not necessarily mean
someone does NOT suffer from HFI,
– while this test is NOT diagnostic, if it is positive it may aid in
making a clear diagnosis.
HFI Incidence Rate
•
The world-wide incidence rate of HFI remains unknown due to the difficulty
of HFI diagnosis. The first report of an incidence rate was from Switzerland,
where over a five year period that included 100,000 births, five cases of HFI
were reported.
– the degree of deviation in this estimate of 1 in 20,000 births is large and
the incidence rate may range from 1 in 10,000 to 1 in 100,000.
•
It is likely that the incidence rate varies quite widely among different ethnic
groups. Until easier and more effective methods of diagnosis are available
from research involving different ethnic groups, the incidence rate will remain
unclear.
•
Recent data suggest that the incidence rate could be closer to 1 in 10,000 If so,
the carrier frequency would be 1 in 50.
James, C.L., Rellos, P., Ali, M., Heeley, A.F., and Cox, T.M. (1996) Neonatal screening
for HFI: frequency of the most common mutant aldolase B allele (A149P) in the British
population. J. Med. Genet. 33 , 837-841.
Tolan, D.R. (1995) Molecular Basis of Hereditary Fructose Intolerance: mutations and
OMIM
http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=229600
Aldolase B Mutations
Alsolase B, 364 amino acids
• > 21 mutations have been reported;
– 15 of these are single-base substitutions,
• resulting in 9 amino acid replacements,
• 4 (stop) codons,
• and 2 putative splicing defects.
– The other 6 were deletions.
• Recurrent mutations were observed in exons 5 and 9.
Analysis suggests that the A149P and A174D mutations
originated from single founders and achieved a relatively
high frequency through genetic drift.
Mutant Alleles
•
.1 FRUCTOSE INTOLERANCE [ALDOB, ALA149PRO]
–
•
2. FRUCTOSE INTOLERANCE [ALDOB, ALA174ASP]
–
•
In addition, in 11 unrelated Italian patients, researchers found a G-to-C transversion in exon 9
which resulted in substitution of lysine for asparagine at position 334.
8. FRUCTOSE INTOLERANCE [ALDOB, ARG3TER]
–
•
A 1-bp deletion in codon 288 causing a frameshift. The mutation seems restricted to Sicilian
subjects.
6. FRUCTOSE INTOLERANCE [ALDOB, ASN334LYS]
–
•
Point mutation found in Italy, Switzerland, and Yugoslavia but not in the UK, France, or the
United States.
3. FRUCTOSE INTOLERANCE [ALDOB, LEU288DEL]
–
•
A G-to-C transversion in exon 5 resulted in a substitution of proline for alanine at position 149
of the protein within a region critical for substrate binding.
A consanguineous family from eastern Turkey, has a C-to-T transition in codon 3 changing arg
to stop codon.
11. FRUCTOSE INTOLERANCE [ALDOB, LEU182DEL, VAL183DEL ]
–
In a 6-year-old patient with a 6-bp deletion in exon 6 of the aldolase B gene that led to
elimination of 2 amino acid residues, leu182 and val183, but left the message in-frame. On the
other allele, the patient carried the asn334-to-lys mutation (#6).
Structure/Mutation Sites
• Aldolase B associates in
quartenary structure as a
homotetramer,
– A149P and A174D mutations
result in a reduced affinity
between sub-units.
• Other mutation may retain
quaternary structure, but
lack enzymatic activity.
Modern Disease
2003 = ~65 kilos (USA)
CLINICAL MANAGEMENT
• Limit fructose and related sugars…sucrose
and sorbitol,
– difficult, if not impossible in modern, Western
society.
G6PD…
…in the news.
Sickle Cell
Malaria
Worldwide distribution of
G6PD deficiency: 1995
Sickling and G6PD Deficiencies
heterozygote benefit
• Heterozygotes carrying alleles for red blood cell sickling
disorders, and for glucose-6-phosphate dehydrogenase
deficiency are resistant to malaria,
– sickle cell allele (Hb S), when expressed, prevents the plasmodium
from breaking down host haemoglobin, slows growth,
– G6PD mutations result in a ~50% reduction of risk for malarial
infection,
• However, these deficiencies have severe consequences in
certain cultural and environmental circumstances.
Slave Trade
conservative estimates
Estimates Range to 20,000,000.
Two-thirds destined for the sugar industry.
Sickle Cell Anemia
• Walter Clement Noel, a first-year dental student at the Chicago College
of Dental Surgery, was admitted to the Presbyterian Hospital in late
1904 where Ernest E. Irons, a 27-year-old intern, obtained a history
and performed routine physical, blood, and urine examinations.
• He noticed that Noel's blood smear contained 'many pear-shaped and
elongated forms' and alerted his attending physician, James B. Herrick,
to the unusual blood findings.
Sickling Disease in Modern Culture
• Homozygotes for sickling disorders have mild to severe
disorders,
–
–
–
–
–
septicemia in infants is the highest cause of death,
Sickle Cell Anemia,
renal failure (adults),
Acute Chest Syndrome (adults),
others,
• Heterozygotes (Sickling Trait), generally do not have
Sickle Cell phenotypes or health problems,
– however, temperate climates (colder temperatures), can induce
Sickle Cell phenotypes in heterozygotes.
Frequency of Heterozygotes
Sickling Trait
• For example, it is estimated that approximately:
•
•
•
•
1 in every 10 Afro-Carib,
1 in every 4 West Africans,
1 in every 50 Asians,
1 in every 100 Northern Greeks...
• Have sickling trait (heterozygote for HbS).
Treatments
• Lifestyle,
– diet, exercise, education,
– and proximity to sickle cell/thalassaemia centers,
• Hydroxyurea,
– drug, relatively effective, however, short term side
effects occur, and long term effects are unknown,
• Gene Therapy. On the horizon.
http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=603903
G6PD Disease in Modern Culture
• First seen in WWII, AfricanAmerican soldiers receiving
an anti-malaria drug
experienced haemolysis.
• Presently,
– Sulphonamides,
– Anti-pyretic drugs,
– Broad Beans.
NADPH producing reactions
Case Study
• 21 yo male medical student with malaria
• Treated with primaquine
• Four days later:
–
–
–
–
–
–
Black colored urine
Low RBC count
Elevated reticulocyte count
RBC with Heinz bodies
Low hemoglobin
Elevated serum bilirubin
• Pt recovered in a few days
G6P is a Research Juggernaut
•
Mary Lyons (Lyons hypothesis, think Barr bodies) used G6P
to demonstrate X-linked inactivation,
•
Evolution... analysis of Mediterranean allele (A-) at this locus
indicates that it evolved independently from other alleles and
has increased in frequency at a rate that is too rapid to be
explained by random genetic drift.
•
–
Tishkoff et al. (2001) used statistical modeling to demonstrate
that the A- allele arose within a past 3,840 to 11,760 years and the
A- allele arose within the past 1,600 to 6,640 years.
–
Tishkoff et al. (2001) concluded that their results support the
hypothesis that malaria has had a major impact on humans only
since the introduction of agriculture within the past 10,000 years
and provide a striking example of the signature of selection on the
human genome.
Others, including DNA “fingerprinting” applications.
http://www.ncbi.nlm.nih.gov:80/entrez/dispomim.cgi?id=305900
Sugar?
Society?
Biotechnology?
Questions?
?
1, and 2 indicate steps that are blocked in HFI patients. What
causes this (genetically, physiologically)?
Monday
• DNA Arrays
• Reading assignment...
• ON LINE TUTORIAL