Bio 362 Human Genetics Spring 2008
Bloom syndrome study sheet for February 19th
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Background web reading and Hickson review paper:
In the Hickson paper, don't spend a lot of time on the "RecQ helicases: localization and binding partners" section.
What is the difference between mitotic recombination and sister chromatid exchange?
How can you detect each kind of event?
What are the symptoms of Bloom syndrome at the level of the whole organism?
What type of protein is encoded by the gene associated with Bloom syndrome?
What other disorders are caused by mutations in similar genes? Are the symptoms the same?
What are the structural features of members of the RecQ family?
What types of mutations lead to Bloom syndrome as opposed to those leading to Werner syndrome?
Explain the very last sentence on page 170 of the Hickson paper regarding Werner syndrome mutations.
Why are RecQ helicases considered caretakers and not gatekeepers?
Why is it rare for RecQ-associated cancers to appear somatically and not due to a germline mutation?
Is cancer predisposition in the general population associated with mild alleles of BLM?
What are the cellular defects in Bloom syndrome?
What are two models to explain how hyperrecombination leads to cancer predisposition?
What else can the RecQ helicases unwind besides your standard double helix? Look up these unusual structures so you have a mental picture of what they are.
What are the putative roles for RecQ helicases? Refer to figure 5, the figure legend, and the associated text in the paper.

Ellis et al., 1995
This is obviously an old paper, but it's one of my favorites because of the ingenuity of the experiments. As you read, keep in mind that both mitotic recombination and sister chromatid exchange are at work and that both are used in different ways by the researchers. The approach in this paper is a highly unusual way to do linkage mapping, possible only because of the very nature of the disease itself.
The introduction says that the gene had already been regionally mapped-- in general, how was that done?
How do the researchers assess sister chromatid exchange (SCE) in lymphoblastoid cell lines from Bloom patients?
In most patients, all lymphoblastoid cell lines have high SCE. But some patients (the interesting ones) give rise to a subset of lymphoblastoid lines with low SCE. What is the difference in these patients in terms of their specific Bloom genotypes? How is it possible for them to develop some lymphoblastoid cell lines that are back to normal recombination levels?
What does "reduction to homozygosity" have to do with the gene mapping in this context? How did looking for this phenomenon in low SCE cells lead to the identification of the Bloom gene? Look at Fig 1A, keep in mind that the centromere is on the left, and think about mitotic recombination.
Keep looking at Fig 1. What kinds of polymorphisms are used? Why are there so many bands? Why did it help to find that all low SCE lines were still heterozygous for the D15S1108 polymorphism? And why did it help to show that all low SCE lines were now homozygous for D15S127?
How was the correct gene identified from this region? (Remember that this was before the genome was completed.)
They isolated cDNAs; why weren't all of them full length? Why is it important to make sure you have a full length cDNA? How did they confirm the identity of the 5' end of the gene? (see Fig 3)
Fig 5: How does the transcript size compare with the size of the gene in Fig 2?
Fig 5: What were the sources of all the RNA samples?
Fig 5: Compare with the nature of the mutations as listed in Table 1. Does any aspect of the Fig 5 results fail to match your predictions based on the nature of the mutations? e.g. look at cell line HG1584
Fig 5: In HG1926, how can a deletion of 3 base pairs lead to a stop codon?
How did the researchers confirm that they had identified the right gene?
What do the researchers speculate about possible molecular roles for the BLM gene product?

Luo et al., 2000 (Questions from Mark and Bill)
What is the function of the protein encoded by the BLM gene?
Fig 1: How were the three alleles Blm^(m1), Blm^(m2), and Blm^(m3) made?  What two probes are used to identify them and the wild-type alleles in 1b-f?

Fig 2: What is the difference between 2a and 2c?
Fig 3: What kind of cells were used in this analysis?  What is BrdU?
In what ways does the mutant phenotype resemble a human Bloom syndrome patient?
Fig 4: What tissues are represented in a-f?
Fig 5: What is HAT and what is its relationship with 6TG?  Which type of resistance is seen with random integration of the targeting vector?  Which type is seen with targeted integration?

Fig 6: What is LOH?  What six genetic mechanisms are proposed to account for the loss of the functional PGK-Hprt gene?  Which do the authors consider the most likely?

What are SSLP’s?
Fig 7: What does Apc refer to?  What do the arrows point out in 7a and 7b?  How was allelic loss analyzed in 7d and 7e?
Although this study was performed in mice, what applicability do the authors' findings have for humans?

Note that there are additional papers in the supplementary folder on Louise. In particular, Wu and Hickson's paper (2003) is a difficult but clever piece of work that examines further the role of the Bloom protein. Take a look at it if you would like a bit of a challenge.