Genetics

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Genetics in the news.
2
Finalcumulative, 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, 1030 - 1230
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Genotypes and Phenotypes (societies, politics and
human nature)
Nature Reviews Genetics, (3), June 2002, pp 7
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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
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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.

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Aerobic Respiration 101
Please start memorizing here.
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Glycolysis 205

• Prepatory Phase
• Youve learned the conversion of glucose
  into triose phosphates.

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We Dont Live by Glucose Alone
300-level Understanding
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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 11 complex
  with magnesium. Depletion of ATP in tissues leads
  to higher concentrations of magnesium.

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HFI Metabolic Defects
Fig. 1a
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300-level Understanding
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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.

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Genetic Screens

• Three mutations in this Aldolase B apparently
  account for gt75 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 gt95 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.

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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 polymorphisms in the
human aldolase B gene. Hum. Mutat. 6, 210-218.
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OMIM
http//www.ncbi.nlm.nih.gov80/entrez/dispomim.cgi
?id229600
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Aldolase B MutationsAlsolase B, 364 amino acids

• gt 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.

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Mutant Alleles

• .1 FRUCTOSE INTOLERANCE ALDOB, ALA149PRO
• 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.
• 2. FRUCTOSE INTOLERANCE ALDOB, ALA174ASP
• Point mutation found in Italy, Switzerland, and
  Yugoslavia but not in the UK, France, or the
  United States.
• 3. FRUCTOSE INTOLERANCE ALDOB, LEU288DEL
• A 1-bp deletion in codon 288 causing a
  frameshift. The mutation seems restricted to
  Sicilian subjects.
• 6. FRUCTOSE INTOLERANCE ALDOB, ASN334LYS
• 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 consanguineous family from eastern Turkey, has
  a C-to-T transition in codon 3 changing arg to
  stop codon.

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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.

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Modern Disease
2003 65 kilos (USA)
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CLINICAL MANAGEMENT

• Limit fructose and related sugarssucrose and
  sorbitol,
• difficult, if not impossible in modern, Western
  society.

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G6PD in the news.
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Worldwide distribution of G6PD deficiency 1995
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Sickling and G6PD Deficienciesheterozygote
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.

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Slave Tradeconservative estimates
Estimates Range to 20,000,000.
Two-thirds destined for the sugar industry.
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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.

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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.

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Frequency of HeterozygotesSickling 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).

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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.

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(No Transcript)
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http//www.ncbi.nlm.nih.gov80/entrez/dispomim.cgi
?id603903
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G6PD Disease in Modern Culture

• First seen in WWII, African-American soldiers
  receiving an anti-malaria drug experienced
  haemolysis.
• Presently,
• Sulphonamides,
• Anti-pyretic drugs,
• Broad Beans.

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NADPH producing reactions
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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

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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.

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http//www.ncbi.nlm.nih.gov80/entrez/dispomim.cgi
?id305900
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Sugar?Society?Biotechnology?Questions?
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?
1, and 2 indicate steps that are blocked in HFI
patients. What causes this (genetically,
physiologically)?
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Monday

• DNA Arrays
• Reading assignment...
• ON LINE TUTORIAL