MCB I Block 4


  • Sickle cell= caused by point mutation
    • Therefore you can use ASO to detect it,
    • but since this point mutation also alter restriction enzyme site (RFLP), you can also use Southern Blot, or PCR the fragment, then digest/run through gel.
  • Multiple Congenital Anomalies = mutation here is unknown, so use CGH, or SSCP to detect unknown mutations.


  • B-Thalassemia– point mutation that extends the exon, or cause a null mutation
    • Use ASO (since pt. mutation)
    • or Northern Blot (because detecting change in mRNA length)
    • or use Sequencing (all-singing, all-dancing)
  • Hemophilia = intrachromosomal rearrangement → restriction fragment sites change → fragment sizes change (RFLP). Because the mutation is an RFLP, you can use Southern Blot (to detect restriction fragments sizes).
  • HFE mutation= an extra restriction site is made (RFLP)
    • Therefore, use PCR/digest/run through gel to detect the mutation.
  • DiGeorge Syndrome = 22q11 deletion, which is a pretty big chunk of DNA, so you can use FISH or sequencing to detect it.
  • Nail Patella Syndrome = SNP or Null or loss of restriction site. Therefore, use Sequencing.
  • DMD – deletions in entire exons of the dystrophin gene – amplify exons using PCR and run through gel
  • Turner Syndrome= no Y chromosome
    • so detect with either chromosome painting, or PCR with Y-specific probes.
    • Patients, however, may have XY in the gonads, so you can detect gonad cells with Y-specific probe in PCR.
  • SSCP Analysis
    • unlike regular gel electrophoresis (in which double-stranded DNA travel through the gel), the DNA strands in a SSCP test are denatured, separated and run through a gel where they cannot reanneal. Only in this way can they form hairpin loops, and hairpin loops can affect their speed through a gel.
    • Different mutations form different hairpin loops (“conformations”) hence they are called single-strand conformational polymorphism
    • To do the SSCP test, compare how a Test DNA (with hairpin loops) vs. Control DNA (with hairpin loops) will run through the gel!
    • Advantages vs. Disadvantages
      • Advantages: You can test many exons at once.
      • Disadvantages: Not all point mutations will affect secondary structure.
  • DHPLC Analysis
    • like SSCP except you hybridize a single-stranded mutant strand with a normal strand, and they cause “kinks” that causes the whole thing to run differently in the gel.
  • CGH Analysis
    • It’s called Comparative Genomic Hybridization, because you are comparing the mutant with the control ‘s genome, how they compete with each other to hybridize to the array.
    • If much more Control DNA than the Test DNA hybridizes to the BACs on the array then it means that there is no Test DNA at this point of the chromosome, and therefore there is a deletion.



  • During DNA Replication,
    • Backwards slippage → causes insertion
    • Forwards slippage → causes deletion
    • If slip not a multiple of 3, then frameshift mutation!
  • Imbalance in Protein Production causes problems! For every deletion there should also be a corresponding duplication! Hence Smith-Magenis vs. Potocki, or HNPP vs. Charcot-Marie-Tooth.


  • Genomic Diseases
    • DiGeorge Syndrome = 22q11 deletion
    • a-thalassaemia = deletion of entire A-globin gene
    • Williams syndrome = deletion on chrom 7
    • Smith Magenis Syndrome = deletion of 17p11.2
    • Potocki-Lupski Syndrome = duplication of 17p11.2 (PLS)
    • HNPP = null mutation or deletion in PMP22 (also in 17p11.2)(major component of myelin), AD
    • Charcot-Marie-Tooth = duplication in PMP22(also in 17p11.2), AD
    • Williams Syndrome patients have heart problems, and large lips. Remember they are very loquatious and their language ability beats their cognitive abilities.
  • Down Syndrome vs. Williams Syndrome cognitive abilities
    • Down syndrome patients can understand the big picture, but not the details.
    • Williams syndrome patients can understand the details, but not the big picture.



  • FRDA= GAA expansion in Frataxin → unstable transcription. Needed for FeS → Iron in Mito, Free Radicals, loss of energy
    • only one in this list that’s autosomal recessive, so no genetic anticipation
  • FRAXAS= CGG expansion in promoter/5’UTR of FMR1 (needed to develop synaptic connections)→ methylation → MeCP2-HDAC bind to methylated CGG → deactivate gene.
    • maternal grandfather carrier!
    • Despite the name, fragile does not refer to chromosome being broken. Rather, there is a part on the q arm that does not stain with folate.
    • 50% penetrance in females. Not as bad in females!
    • 2nd most common cause of MR
    • Although it is loss-of-function mutation, the disease still displays anticipation because it is x-linked. At least there is anticipation in males.
  • FXTAS = Fragile X that develops in older male premutation carriers.
  • DM1= CTG expansion in 3’UTR of DMPK1 → prevents RBP to bind to other mRNAs (hence “gain-of-function”) → abberant splicing of other mRNAs, hence a lot of symptoms.
    • Maternal Bias
  • DM2 = CCTG expansion in intron 1 of ZNF9 on Chrom3q. Maternal Bias
  • Huntington’s= CAG (glutamine) expansion at Htt → destabilize MT → accumulate in brain. Rhes in striatum sumoylates Htt → soluble (toxic). Disease = 40+ repeats.
    • Paternal Bias.
  • SCA = CAG (glutamine) expansion in ataxin gene.

Why are too many repeats bad (unstable)?

  1. Okazaki fragments can’t be too long → instable.
  2. More repeats → more likely to form hairpin loops → instable.


  • AMINO ACID METABOLISM DISORDERS – mainly autosomal recessive, because missingenzyme
    • Hypophenylalanemia: via BH4 or PKU. Can’t turn phenylalanine into tyrosine.
    • Alkaptonuria: homogentistic acid oxidase deficiency.
    • OCA1 – can’t turn tyrosine into melanin.
    • Homocystinuria – can’t turn Methionine into Cysteine (via CBS) → MR, Osteoporosis
    • Maple Syrup Urine Disease –Defect in ketoacid decarboxylase. Excrete VIL (valine, isoleucine, and leucine) in urine. Common among Mennonites. Mnemonic: Think of Mennonites making maple syrup in their Village (VIL).


    • Hyperammonemia = disrupted urea cycle → too much ammonia in your body… toxic to CNS. OTC deficiency most common, x linked.
    • Galactosemia = lack galactose-1-phosphate uridyltransferase
    • Fructose intolerance = lack fructose-1-phosphate aldolase
    • Hepatic type = deficient in hepatic enzymes. can’t do glycogenolysis (can’t break down glycogen)
    • Myopathic type = deficient in glycogen metabolic enzymes. can’t do glycolysis (can’t break down glucose).
    • GSD I (von Gierke) = lack glucose-6-phosphatase in liver → hepatomegaly. Treat with frequent feeding (since can’t store glycogen). Vulnerable to oxidants. X-linked.
    • G6PD Deficiency= lack glucose-6-phosphate dehydrogenase
      • world’s most common enzyme deficiency!
      • Occurs in 10% African American population.
      • X-linked
    • Congenital Adrenal Hyperplasia (CAH) = deficient in Steroid 21 hydroxylase (CYP21 pseudogene copied into CYP21 gene)→ no negative feedback of ACTH →androgens keep getting produced by adrenal cortex (DHEA) → virilized genitals, and since no aldolase, you get salt wasting.
    • Smith-Lemli-Opitz Syndrome= deficient DHCR7 → low plasma cholesterol (but lots of precursors) → MR, growth retardation, etc.
      • Tx: Zocor Therapy
    • Mucopolysaccharidoses = accumulate polysaccharides
    • Niemann Pick disease= No sphingomyelinase → accumulate sphingomyelin (in myelin sheath), deposited in liver.
      • Type A affects neurons.
      • Type B doesn’t.
    • Tay-Sachs= TATC is inserted in HexA gene → framshift mutation → → can’t break down ganglioside → ganglioside accumulate in brain, Cherry red spot in eye.
      • Symptoms same as Sandhoff.
      • Common among ASHKENAZI JEWS!!
    • Sandhoff disease = missing Hex B → can’t break down ganglioside → gangliosideaccumulate in brain.
      • Symptoms same as Tay-Sachs.
    • Gaucher’s Disease= Deficient glucocerebrosidase → macrophage devour accumulated glucocerebroside → Gaucher cells.
      • not completely recessive. severity depends on how much residual enzymeactivity remains.
      • Treat with enzyme replacement therapy (ERT).
    • Zellweger = Peroxins stop recognizing SKL → no peroxisome enzymes imported → prominent forehead.
    • XALD (X-Linked Adrenoleukodystrophy) = most common peroxisomal disorder. Can’t transport VLCFA into peroxisome → VLCFA accumulate in brain (myelin breakdown) and adrenal cortex (adrenal atrophy)→ apathy, ataxia
    • AIP = hepatic type. Autosomal Dominant. Haploinsufficient in porphobilinogen deaminase, not photosensitive.
    • CEP = Erythropoietic type. Autosomal Recessive. Missing RBC pigments → anemia and photosensitivity
    • VP = hepatic type. Autosomal Dominant. Haploinsufficient in protoporphyrinogenoxidase, photosensitive. Common among AFRIKAANERS!!
    • Lesch-Nyham Syndrome = lose purine synthesis negative feedback → increased purine and uric acid → gout + self-mutilation.
    • CYP2D6 metabolizes Codeine
    • CYP2C9 metabolizes Warfarin
    • CYP2C19 metabolizes Mephenytoin
    • Malignant Hyperthermia = Defective ryanodine receptors → too much calcium released with anaesthetics → too much ATP spent to try to reabsorb the calcium→ excessive heat released (hence hyperthermia) → damage to tissue and depletion of ATP → death from anaesthetics
      • SCID = bubble boy syndrome. Treat with Bone Marrow Transplant.
      • X-SCID = X-linked bubble boy syndrome. Treat with Bone Marrow Transplant.


  • XIST and BF
    • XIST inactivates X chromosome by turning it into a heterochromatin.
    • BF blocks XIST from inactivating Xa. This is why X chromosome in guys are not inactivated.
  • Gene inactivation doesn’t inactivate every gene. It doesn’t inactivate the PAR but it also doesn’t inactivate 15% of the rest of the X chromosome, and another 10% only sometimes inactivates.
  • Inactivation is always random, but sometimes it can look as if it is not, because sometimes cells with a mutation on the Active X may be selected out, leaving the good ones behind to populate the body.
  • X-linked Dominant disorders:
    • Craniofrontonasal Syndrome = loss of function in EFNB1, wide eyes. Females more affected than males, even though it’s X-linked!!
    • Rett Syndrome = hand-wringing, mutation in MECP2
    • Vitamin-D-resistant rickets


  • MECP2 = stands for Methylation of CpG Protein – methylates the Cytosine!
  • Deamination of methylated-C (from imprinting of CpG) is a great source of spontaneous mutation. Me-C → T
  • Methyl-transferase recognizes hemimethylated DNA and then methylates the other strand.
  • Sperm and egg are methylated. When sperm and egg combine, they start demethylating until implantation. As somatic cells, methylation begins again and is maintained every time DNA replicates.
  • Imprinting (from parent of origin) done through methylation → turns allele off.
  • glomus tumor is autosomal dominant, but only inherited from father.
  • Beckwith-Weiderman Syndrome only inherited from mother.
  • Imprinting is natural, so if it goes wrong, you’ll have problems.
  • During gametogenesis, old imprints are erased, and re-established in gametes.
  • Here, the trisomic conceptus duplicates, but there is a nondisjunction that causes it to separate into 4n and 2n. The 2n keep multiplying to develop into a baby. Some will turn out normal. Others will be uniparental disomy.
  1. Trisomy Rescue – trisomic cell duplicates into 4n+2n because of nondisjunction (nondisjunction happen in mitosis of cell). 2n→baby
  2. Uniparental Isodisomy – nondisj
    unction happen in meiosis II of gamete, followed by loss of chromosome after fertilization – both alleles from one parent, but identical.
  3. Uniparental Heterodisomy – nondisjunction happen in meiosis I of gamete, followed by loss of chromosome after fertilization – both alleles from one parent, but different.
  • Angelman’s Syndrome vs. Prader-Willi Syndrome
    • Normally, UBE3A is maternally expressed, while father’s is imprinted. You get Angelman’s when mother’s allele is deleted or when there is paternal uniparental disomy.
    • Normally, SNRPN is paternally expressed, while mother’s is imprinted. You get Prader-Willi when father’s allele is deleted or when there is maternal uniparental disomy.
  • Detecting Angelman’s Syndrome vs. Prader-Willi on a gel
    • Prader-Willi patient has maternal band on a gel (because it has deletion in paternal chrom 15)
    • Angelman patient has paternal band on a gel (because it has deletion in maternal chrom 15)
  • Angelman’s patients sometimes have OCA (albinism) because on top of not expression UBE3A, father also has a mutated P gene.
  • Uniparental disomy of entire genome (not just a few genes) can lead to hydatidiform moles (ovarian teratoma)
  • Exons 1-13 of GNAS Gene encodes for production of alpha subunit of Gs protein, needed for PTH Hormone Response.
    • In Renal Proximal Tubule, you need just mother’s expression of 1-13 (because father’s is imprinted). If mutation is in mother, then you get PTH resistance
    • In all other tissues, you need both mother’s and father’s expression of 1-13. If mutation in either, then you get AHO.
    • Note in the diagram that PHP1A has both AHO and PTH resistance.
    • Note that PHPIB is due to imprinting defect, not mutation.
  • PHP vs PPHP
    • PHP1a = AHO w/ PTH resistance
    • PHP1b = PTH resistance w/o AHO
    • PPHP = AHO w/o PTH resistance


  • How to Identify a Mutation through Linkage Analysis
  1. Collect sample DNA from all affected individuals and their families
  2. Find markers over entire genome
  3. Assess each marker to see how close it is to reflecting the behavior of the mutated gene (which we don’t know where it is). Do LOD score to figure this out. Find the markers with the least recombination frequency. Find the region of the marker and look for candidate mutated genes there.
  4. Do SSCP analysis of each exon in each gene. Find which gene/exon has a band shift.
  5. After narrowing in, do sequence analysis to find the exact mutation.



  • Carrier frequency can “artificially” increase via founder effect.
  • Autosomal dominant disorders are selected. Recessive alleles stay under the radar because of the time they don’t affect the phenotype to be selected. X-linked recessive alleles only get selected when they are in males.


  • SOD1 mutation causes ALS, but CNTF +/- can affect the age of onset (but only if there is already a SOD1 mutation to begin with)
  • Digenic Inheritance = retinitis pigmentosa caused by being heterozygous in two different genes. If you are only heterozygous in one of those genes, you won’t get the disease.
  • MTHFR = Methylene Tetra Hydro Folate Reductase → this gene encodes for an enzyme needed by the body to process folate. If a pregnant woman has a missense mutation in her MTHFR gene, it can cause her to be deficient in folate. If she ever gets pregnant, she can be at risk of having low folate, and therefore her fetus has a risk of neural tube defect.  She needs to eat more than the recommended 400 micrograms per day of folate to decrease her baby’s risk of neural tube defect.
  • It’s the susceptibility to the disease that is the continuous character, not the disease itself (that is continuous, like height). You either have diabetes or you don’t, but your susceptibility is continuous.
  • Heritability
    • H=0 means disease is completely environmental.
    • H=1 means disease is completely genetic.
    • Most multifactorial diseases have Heritability of 0.4-0.7


  • You can use these as association markers when doing Linkage Analysis:
  1. SNPs – most stable, and best kind of marker to use – doesn’t keep changing from generation to generation like CA-repeats.
  2. Structural Variants
  3. Copy Number Variants.
  • Celiac Disease
    • HLA-DQ2/8 = a gene responsible for celiac disease. (Mnemonic: Think about people with celiac disease can’t go to Dairy Queen (DQ) to (2) eat (8) ice cream cones.)
    • However, Celiac disease is multifactorial!
    • Using sibpair analysis, they found that a mutation on chrom 6 was not the only defect responsible but also a mutation on chrom 19!
    • Found disease in gene Myo9B of chrom 19!
  • Hirschsprung Disease
    • RET normally causes neural crest cells (which develop into autonomic nervous system) to migrate to intestines → but a mutation in RET causes Hirschsprung Disease.
    • Whereas RET is dominant gene, EDNRB is recessive. EDNRB is responsible for a lot of cases of Hirschsprung disease among Mennonites!! Mnemonic: Think of two mennonite fellas named “Ed-n-Rob”
    • Lots of ways these responsible genes can interact. So mutation to some of them or all of them can cause problems large and small.
    • In summary, RET, SOX, and EDNRB is to blame for Hirschsprung Disease. (Mnemonic: Think about Ed and Rob watching a Red Sox Game and they see a giant doodoo in the stadium bathroom)
  • A lot more guys get Hirschsprung Disease than girls. If a girl has it, then chances are her genes are “bad enough” for her siblings to have a higher chance to get it as well (vs. if she were a guy).
  • There’s also some evidence HLA-DQ2/8 (same as in celiac disease) also involved in Diabetes Type I.
  • MODY = basically it is MONOGENIC DIABETES TYPE II. Diabetes Type II that doesn’t depend on environment and solely on genes.
  • PParGamma = a candidate gene for Diabetes Mellitus Type II
  • FTO is the only gene linked between obesity and Type II Diabetes so far.


  • First Trimester Screening:
    • FIRST = First Trimester Integrated Screening For Trisomies
    • Maternal serum screening of hCG and Pregnancy Associated Protein-A (PAP-A)
  • Chorionic villus sampling (10-14 wks) or amniocentesis (16-20 wks) for DNA analysis, probably because you don’t have enough amnionic fluid yet at 10-14 wks.
  • Second Trimester Maternal Serum Screening:
    • Screen for AFP, uE3, and hCG (vs. hCG and PAP-A in First Trimester Serum Scr
    • Downs Syndrome is likely if only detect 70% AFP, 75% uE3, and slightly increased hCG.
  • High AFP levels can mean neural tube defect.

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