Senogles 5

Senogles 5
Fragile X syndrome

Review questions are posted on Blackboard under Assignments with bullets for important points. We will review after lecture tomorrow-short. You can also email her.
Tomorrow –Huntingdon’s disease.
Today-fragile X. These are trinucleotide expansion diseases (the only new development in genetics in a long time- dseases in which a repeat of three nucleotides is generated too many times, separating a gene from its promoter, and a disease results). This mechanism explains some diseases with weird inheritance.
Decreased penetrance can occur with an X-linked dominant disease. The phenotype is not manifested the same in each individual. More severe in males. Oddball is the normal transmitting male. Flag: carrier is not affected, but subsequent generations are severely affected.
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Sherman’s paradox will be on the exam. Understand basic evidence for #2.
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Sherman was a clinical psychologist who discovered aberrations in pedigrees. For X-linked dominant disorder, risk should not change in pedigree for succeeding generations. Something was wrong here. This is unusual genetics.
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X-linked dominant disorders- all female children of affected males are carriers.
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Review slide of normal X-linked transmission.
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Sherman’s paradox pedigree
Risk gets higher with succeeding generations. Grandchildren at higher risk than siblings of the normal transmitting male (T).
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Look at karyotype of patient- deprive cells of folate in vitro- X chromosome fragments at fragile sites. Name of the disease comes from karyotype.
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Used perturbation to clone by somatic cell hybrids. Breakage is always at same place on chromosome. Take panel of rodent cells and fuse human cells to them. Used fragile X patient cells. X breaks under thymidine stress and reforms with rodent cells.
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Heterokaryon is fused cell.
Mouse tumor cell is immortal and clonal. Form heterokaryons, select, then grow fused cells on permissive medium that selects only for hybrid cells. Wind up with hybrid cells that are clonal- each group grows from single cell. Can use RT-PCR to see what human chromosomes are carried in the heterokaryons.
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Get a rodent chromosome with a bit of a human chromosome at the end.
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When mapped out, get this diagram. The fragile X site is near telomeric end. B is a higher resolution map. CpG islands are normally unmethylated regions 5’ to a mammalian gene. Probes used are shown in D. Hypothesis was that something odd was going on near CpG island.
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Southern analysis of 2 fragile X pedigrees. Filled in squares are affected individuals. Females indicate various degrees of clinical manifestation. Took blood, looked at DNA. Normal individual- Eag1 should cut, indicating a non-methylated CpG island.
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Different pedigree. Affected males and carrier females have higher fragment size from CpG islands being methylated. Region is larger in some affected males and carrier females. Two things are happening (methylation of the CpG islands and increased numbers of repeats).
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Track through pedigree analysis and look at size of insertions- size on Southern blot gets larger (higher in gel). Gets substantially larger in terms of base pairs. The insertion is at the fragile X locus.
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Cloned region. Circled area is CpG island. Repeat of CCG is shown by arrows.
Repeat could be basis of increase of fragment size, or a nearby gene could be changing.
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Flanking regions did not change size. Repeat region changes, not flanking regions.
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Examine repeat sequence in more detail. Is it polymorphic in normal individuals, or just in fragile X families?
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Is polymorphic in normal population. Run sequencing gel to determine size of repeats. PCR through- get normal repeat size (29 repeats on average).
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Look at fragile X pedigrees- centers on several hundred repeats.
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Studied number of pedigrees. Found risk of expansion to full mutation was correlated through premutation of mother . Expansion occurs in carrier female. Graph shows repeat size in mother. Does not correlate with paternal allele size. Imprinting- differential expression depending on parent allele comes from.
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This is consistent with Sherman’s observations. Expansions go through females, but only when allele is transmitted through female does risk increase. NTM (normal transmitting male) has expanded allele, but not fragile X. NTM is key. He has premutation repeat size. Size of allele correlates well with retardation in males, but X inactivation in females complicates things, producing diverse phenotypes.
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3 is at 53/29 copies (females have 2 X chromosomes, so for a female, there can be two sets of repeat values). Daughter 4 is at 29/83 copies. 29 size does not change. Other one changes. Sons relevant DNA will not enter gel- off scale in terms of number of repeats.
Pedigree 2:
1 has 83 and 29 repeats. 3 has a reduction in size, which is possible. Repeat sizes are dynamic. Nobody knows how the expansion and reduction occur. Curent thinking is that repeats form odd structures so they can’t copy accurately.
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premutation size is also dynamic. Normal family shown. Premutation size shown. There is flux even in premutation alleles.
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Affected gene was cloned. Original start site in the literature was wrong. Originally reported repeat in coding region. It is actually in the 5’UTR. Called Fragile X Mental Retardation Gene (FMR).
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Methylated CpG island and repeat size change in fragile X patients. How are they linked? Look at methylation status. By Southern blot analysis we see complete cleavage (unmethylated DNA) in normal individuals. Carrier females- methylated and unmethylated. Affected males- not digested by BssHII. They have methylated CpG island close to gene. Carrier females- half methylated. They have 2 copies. Fragile X males- CpG is completely methylated.
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Expression or transcription of gene: there is a correlation between methylation and gene expression- inactivated by methylation.Silences DNA. Transcription of FMR1 was measured as follows: Isolated RNA, reverse transcriptased it,Used RT-PCR. No message- no band on PCR. All had HPRT housekeeping gene. N are normal individuals. Asterisks are fragile X patients- no transcript.Carriers also lack the transcription of the gene.
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Scan gel with densitometer-2 blips for normal individual. In patient 411- no FMR1 product. Patient 412- reduced over normal population. 410 carrier female also has reduced quantities.
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Correlates expression and methylation status.
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Normal repeat size puts AUG close to CpG island unmethylated, allowing transcription. As repeat size gets larger, still get transcription.
Full mutation size, cell methylates CpG island and shuts down gene.
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Resolved Sherman’s paradox with a mechanistic explanation to some degree. Repeat size gets large through mitotic expansion in the females. There is a threshold size of repeat.