1. Kym Spencer Liverpool Women’s Hospital
High resolution mapping of the X chromosome pseudoautosomal region in two siblings with Hodgkin Lymphoma and Leri-Weill Dyschondrosteosis
Kym Spencer
Liverpool Women’s Hospital
I am going to talk to you today about my trainee project, in which I undertook a study into genetic susceptibility to Hodgkin Lymphoma.
The aetiology of Hodgkin Lymphoma, or HL, has been the subject of debate since it was first described by Thomas Hodgkin in 1832, and both infectious and genetic influences have been implicated. While the impact of infectious agents such as Epstein Barr virus has been studied in depth, the role of genetic influences remains unclear. In this project I investigated an unusual familial case of HL, and I will talk about this in more detail now.

2. Reed-Sternberg cells & Hodgkin cells
Hodgkin Lymphoma is a form of cancer that arises in the cells of the blood, generally B-cells. It is characterised by the presence of large polyploid multinucleated cells known as Reed-Sternberg cells, and their mononuclear variant Hodgkin cells. These are collectively referred to as HRS cells. HL is unusual among cancers, in that the malignant cells are in the minority within the tumour – often less than 1% of the cell population – while the cellular background appears reactive rather than neoplastic.

3. Clinical Features
The malignancy arises in the lymph nodes, most often in cervical nodes, and from here can spread to the rest of the lymphatic system and eventually other non-lymphatic tissues. The most common symptom of HL is lymphadenopathy, but other symptoms can be seen, including haematological abnormalities like anaemia and lymphocytopenia, fever, night sweats and weight loss and are indicative of more severe disease.

4. Incidence Cancer Research UK figures for 2004:
1,519 new cases - 0.5% of all cancers diagnosed.
Incidence of 2.4/100,000 individuals
2.7/100,000 males
2.1/100,000 females
Bimodal age of incidence
15-40 years
>55 years
According to figures from Cancer Research UK, the incidence of HL for 2004 was 2.4/100,000. If this figure is separated into male and female incidence a male sex bias can be seen. This is generally true in all populations, but the degree of bias varies with subtype. The incidence of HL shows a bimodal age curve – one peak at years and one after 55 years. HL accounts for around 0.5% of all cancers, and has a cure rate of around 75%.

5. A genetic cause of HL Several familial cases of HL reported.
Risk of HL higher in individuals with a family history of the condition.
Higher in siblings.
Highest in gender concordant siblings.
Combination of inherited susceptibility with shared environment?
Male predominance of HL
Horwitz & Weirnik (1999 & 2007) suggest pseudoautosomal link due to recombination mechanism of pseudoautosomal regions.
There is currently a great deal of controversy surrounding the aetiology of HL due to several unusual epidemiological and clinical characteristics of the disease and various infectious and environmental factors have been implicated which unfortunately I do not have time to talk about now. Many investigations into a possible genetic predisposition to HL have been carried out, and there are numerous reported cases of familial HL. However familial studies are difficult as there is a relatively small number of affected families, with few affected cases within each family. The risk of HL is elevated in individuals with a family history of the condition, particularly in siblings and most strikingly in gender concordant siblings. It is well established that there is a male predominance of HL cases, and this fact has lead to the suggestion in two papers by by Horwitz & Weirnik that there may be a complex sex chromosome linked aetiology involving the pseudoautosomal region of the sex chromosomes due to the way in which these regions undergo recombination.

6. Family L
Leri-Weill Dyschondrosteosis (LWD)
Hodgkin Lymphoma (HL)
This study focussed on an unusual family who were first described by Gokhale et al. in 1995, known as Family L. Several members of this family have developed HL, including these two sisters who both developed HL in adolescence. In addition another condition – Leri-Weill Dyschondrosteosis or LWD – is present in this family.

7. LWD - Madelung Deformity
LWD is a type of mesomelic dwarfism characterised by short stature and limb abnormalities such as Madelung deformity as shown here, and occurs in about 1/4000 individuals. As both of these diseases are rare it seemed possible that the two conditions may be linked, and the genetic mutation responsible for their LWD may also confer a susceptibility to HL. The gene responsible for LWD was identified in 1998 as the Short Stature Homeobox or SHOX gene, which is located in the pseudoautosomal region of the short arm of the sex chromosomes.

8. Pseudoautosomal RegionsX Y
PAR1
PAR2 p q

9. Map of PAR1
A study by Shears et al in 2003 determined that the individuals affected by LWD in Family L had a deletion within PAR1 encompassing the SHOX gene. Their results are shown in this picture, with red indicating a deletion and green indicating no deletion. However, as you can see from this picture, it was not entirely clear whether any other genes or regulatory elements were deleted in addition to SHOX, as these genes at either end of the deletion may or may not be fully deleted. Also, advances in the human genome project since this work was completed mean that the probes and markers used are not in the positions they were originally thought to be. The aim of this study was to refine the mapping of the deletion breakpoints in these individuals.

10. Family L
Leri-Weill Dyschondrosteosis (LWD)
Hodgkin Lymphoma (HL)
2 (GW)
Cell line DNA was available from individuals RL, GL, PL, ML and GW.

11. Methods
MLPA for SHOX copy number using P018B MLPA kit from MRC Holland
Fluorescent microsatellite analysis
Addition of custom probes into existing MLPA kit
SNP analysis
Microarray analysis
A number of techniques were used to map the breakpoints in this family as I have listed here. As some of you may be aware, MRC Holland now offers an MLPA kit covering SHOX and the surrounding area, so I first tested the family members with the P018B version of the SHOX MLPA kit to get an idea of where the ends of the deletion lay.

12. SHOX MLPA
This is a histogram result showing dosage quotients of the probes from the SHOX MLPA kit from the analysis spreadsheet designed by Andrew Wallace. We have a deletion control, individual RL who is one of the affected siblings and a normal family member. The blue bars represent control probes, the white bars represent the SHOX gene itself, and the green bars represent other parts of the pseudoautosomal region. The red and yellow bars are probes on the X chromosome outside PAR1, and the purple is within PAR2 on the q arm. The spaces here are Y chromosome specific probes, but as I used all female normal controls there are no results for this. As you can see there is clearly a deletion in individual RL.

13. SHOX MLPA
After testing all available family members I determined that the two siblings and their mother all had the deletion - this diagram shows the pseudoautosomal region and the positions of the MLPA probes. Those highlighted in pink were within the deleted region while the green ones were not. You can see that there are no other genes that could be deleted at the 3’ end of the deletion, but this gene, PPP2R3B at the 5’ end, could still be partially deleted.

14. PPP2R3B
PPP2R3B encodes PR48.
B subunit of the PP2A holoenzyme.
Involved in cell cycle regulation - binds to Cdc6 in mammalian cells and restricts DNA replication.
PP2A is a cell cycle regulator and tumour suppressor.
Consists of A, B and C subunits.
B subunits confer substrate specificity. A B C B’
B’’
B’’’
PR65
α or β
PP2A
PR55
α, β, γ or δ
PR61
α, β, γ, δ or ε
PR72/PR130 or
PR48
PR93/SG2NA or
PR110/striatin
The closest gene to the CTCF binding site is PPP2R3B. This gene encodes PR48, a B subunit of the PP2A holoenzyme. PP2A has a wide range of functions, not least of which is its role as a cell cycle regulator. The holoenzyme consists of three sububits, A, B and C - the B subunits confer substrate specificity. PR48 is involved in cell cycle regulation - it binds to Cdc6 in mammalian cells and restricts DNA replication. PP2A itself is believed to act as a tumour suppressor, and several subunits of PP2A have been implicated in various cancers. In addition, a study of a cohort of HL patients by by Goldin et al. in 2006 found linkage of the disease to 4p16.1, which is close to another B subunit of PP2A.

15. Microsatellite Analysis
Some detailed studies on the breakpoints of different SHOX deletions have meant a great deal of information on microsatellites in the area and primer sequences was available. Five microsatellite markers were chosen, three at the 5’ end and 2 and the 3’ end, and primers were ordered according to the sequences published in two papers by Benito-Sanz and Thomas et al. This pedigree shows the results of the microsatellite analysis, showing the inheritance of chromosomes and that all 5 markers were deleted in the three individuals with LWD.

16. Microsatellite Analysis
This map shows the position of these microsatellites, but this side still needed more clarification.
I decided to design custom MLPA probes for exons of the PPP2R3B gene. Probes for exons 1 and 4 were designed, ordered and added to the P018B kit according to MRC-Holland protocols.

17. Custom MLPA
Andrew Wallace very kindly modified the analysis spreadsheet to include the new probes, and here are the results for the same set of individuals as before. The hashed green and white bars represent my custom probes, and they were not deleted in any of the individuals tested.

18. Custom MLPA
So now I had established the majority of PPP2R3B was still present, but as the exon 1 probe was halfway through the exon, I still needed to confirm the very start of it was not deleted. I obtained the entire sequence of the non coding region between PPP2R3B and SHOX from Ensembl, and designed primers to amplify blocks of sequence containing several SNPs. The repetitive nature of this sequence made it a difficult task, but I managed to design three sets of primers to amplify sequences containing a total of 21 SNPs, positioned at 282, 355 and 445kb from the telomere. These 21 SNPs were amplified and sequenced and analysed using mutation surveyor.

19. SNP Analysis
Having very little information on allele frequency for all these SNPs, I wasn’t sure what to expect, but most of the results turned out to be informative. 20 of the 21 SNPs were of sufficient quality to be identified, and the results show that the SNPs at 282kb from the telomere were not deleted, but the SNPS at 355 and 445kb were deleted, as shown on the map here.

20. SNP Analysis
So now I had established that no other genes were included in this deletion. But then my mind turned to regulatory elements. An Ensembl search revealed a CTCF binding site here.

21. CTCF

22. CTCF CTCF (CCCTC - binding factor) Transcriptional regulator through
chromatin remodelling
epigenetic modification
control of transcriptional machinery
insulating promoter interaction with enhancers /silencers
Can bind to a diverse array of DNA sequences using different combinations of its 11 zinc finger domains
CTCF (CCCTC - binding factor) is a multifunctional protein that can bind to a diverse array of DNA sequences using different combinations of its 11 zinc finger domains, one of which is shown here, and can regulate transcription by a number of mechanisms including chromatin remodelling, epigenetic modification, control of transcriptional machinery and by insulating promoter interaction with enhancers and silencers. When bound by CTCF, CTCF binding sites act as regulatory elements. Deletion of this CTCF binding site could result in deregulation of a nearby gene.
Further SNP analysis was carried out around the CTCF binding site, and another 22 SNPs in three amplicons at 300, 305 and 309kb from the telomere were sequenced.
CTCF zinc finger domain

23. More SNP analysis
These results confirmed that the CTCF binding site has in fact been deleted in these individuals.

24. Microarray analysis
In addition, microarray analysis was carried out on individual RL, and the probes are indicated on the map here – red being deleted.
Overall the deletion is kb, and while no genes other than SHOX are deleted, a nearby CTCF binding site is deleted.

25. Conclusions
Deletion of this CTCF binding site could affect regulation of its target gene.
Could the target be PPP2R3B?
Deregulation of PPP2R3B could result in deregulation of the cell cycle
Another B subunit of PP2A is present at 4p16.1, a region linked to HL in a study by Goldin et al. (2006).
The CTCF binding site could regulate another, more distant gene.
Further studies required.
If the CTCF binding site controls the expression of its nearest gene, PPP2R3B, its deletion could affect the function of PP2A and cell cycle regulation. Interestingly, a recent linkage study by Goldin et al. on a cohort of HL patients found evidence of linkage to a marker on 4p16.1, which is quite close to a gene encoding another of the B subunit of the PP2A holoenzyme. However, CTCF has many functions, including long range interactions involving chromatin looping facilitated by CTCF homodimerisation, so this binding site could have a different function altogether. In fact, Shears et al had suggested following their work that a more distant gene, like CSF2RA, IL3RA or even CD99, could be responsible for HL in this family due to some transacting factor. Further studies are needed to determine the function and target of this CTCF binding site and its relevance to HL.

26. Map of PAR1

27. Conclusions
Deletion of this CTCF binding site could affect regulation of its target gene.
Could the target be PPP2R3B?
Deregulation of PPP2R3B could result in deregulation of the cell cycle
Another B subunit of PP2A is present at 4p16.1, a region linked to HL in a study by Goldin et al. (2006).
The CTCF binding site could regulate another, more distant gene.
Further studies required.
If the CTCF binding site controls the expression of its nearest gene, PPP2R3B, its deletion could affect the function of PP2A and cell cycle regulation. Interestingly, a recent linkage study by Goldin et al. on a cohort of HL patients found evidence of linkage to a marker on 4p16.1, which is quite close to a gene encoding another of the B subunit of the PP2A holoenzyme. However, CTCF has many functions, including long range interactions involving chromatin looping facilitated by CTCF homodimerisation, so this binding site could have a different function altogether. In fact, Shears et al had suggested following their work that a more distant gene, like CSF2RA, IL3RA or even CD99, could be responsible for HL in this family due to some transacting factor. Further studies are needed to determine the function and target of this CTCF binding site and its relevance to HL.

28. Acknowledgements Thanks to the following people for their help:
David Gokhale
Vicky Stinton
G Malcolm Taylor
Ciaron McAnulty
Frances White
Una Maye
Julie Sibbring
Emma McCarthy
Roger Mountford
Andrew Wallace
Simon Thomas
Kevin Baker
Gareth Evans
John Radford
All at Liverpool Molecular Genetics Laboratory for their help, advice, interest and patience. 

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