1. Mary-Kayt Jones Genetics 564 Spring 2014
Larsen Syndrome
Mary-Kayt Jones
Genetics 564
Spring 2014
Hosoe, Hideo et al., 2006

2. What is Larsen Syndrome?
Dobbs, Matthew B. et al., 2008
Bicknell, L. S., et al., 2006
Krakow, D. et al., 2004

3. Management after orthopedic surgery:
A result from severe bone defects

4. The FLNB gene is affected in Larsen Syndrome patients
CH CH Filamin X Filamin X X1
2633 AA
Actin Cytoskeleton organization
Bone Development
Actin/ filamin binding
Filamin most missense mutations found– did MEME and found this conserved region found 4 times in this 522 AA sequence.

5. What are the mutations of FLNB that result in Larsen Syndrome?
13 missense mutations and counting…
Bicknell et, al., 2007
Karakow et, al., 2004

6. How well conserved is FLNB?
The last common ancestor of humans and zebrafish existed approximately 420 million years ago, ?

7. How well conserved is FLNB across species?
AA #
2633
2591
2343
2206
1789
Filamin
Human (Filamin B)
100%
Chimp (Filamin B)
99.6%
Mouse (Filamin B)
95.8%
Zebra Fish (Filamin B)
60.0%
Drosophila (filamin-1)
43.0%
CH domain involved in actin in plants
Nematode (FLN-1)
31.0%
454
Plant (Fimbrin-like protein)
26%
Filamin- like

8. The role of FLNB in bone development is unclear
PNAS mouse paper for aim 2: 3% of embryos reached term. Reduction of cartilage seen in the ribs. Reduced bone mineral density.
Zhou, X. et al., 2007

9. What proteins interact with FLNB?
Ubiquitin proteins
Adhesion to cell ECM
Fibronectin receptors
Ubiquitin is involved in the degradation of proteins- may play a role in proper bone development
FLNB is involved in adhesion to the cells membrane because is in highly involved with the cell cytoskeleton and adhesion of actin to such.
Fibronectin receptors are involoved in cell migration and adhesion and the ECM form is made by fibroblasts, chondrocytes, endothelial cells, macrophages. Importance ***the patterns of FN deposition in developing vertebrate limbs determines the patterns of precartilage cell adhesion to the ECM, thereby specifying limb-specific patterns of chondrogenesis.

10. Primary goal
To understand FLNB’s function across species and time to lead to future medical advances

11. Aim 1: To determine the difference in FLNB between species with and without bones
No Bones!

12. Aim 1: How well conserved is the CH domain in species with and without bones?
: actin binding domain
Very conserved in actin domain. Not any differences between bone and no bones seen. No bones boxed in red.

13. Aim 1: How well conserved is the Filamin domain in species with and without bones?
: actin binding cytoskeletal protein ie. latice
Serine polar and hydrophilic
Proline non-polar and hydrophobic
Hypothesis: removal of phosphorylation site would affect protein folding in no bones which might be why the protein localizes in different areas such as in broad size in nematodes.
Further experiment: Learn how FLNB acts within these organisms as not much is understood.
What can this tell me about bones and no bones?
Bones Phosphorylated Serine
No Bones  Proline

14. What’s going on with FLNB across species?
GFP + protein
Hypothesis:
new functions of FLNB

15. Aim 2: To determine the role of FLNB in bone development with age
filamin actin
Ossification: natural formation of the bone- hardening

16. Aim 2: Mouse Flnb protein network conserved
What I found out about interacting proteins and that mouse would be a good organism to do experiments on
Research: mouse and human very similar protein interactions so mouse good model organism to experiments on to determine effects with age

17. tap tag proteins and sort
Aim 2: How does Flnb interacting proteins change with time in mouse model?
Hypothesis:
Ubiquitin protein function increases with age
Fibronectin receptor function decreases with age
Ubiquitin
Fibronectin receptors
Embyonic
Young
Adult
Low
High
Experiment (tap tag and mass spec.) in mice -hypothesis on what I would expect to see chart at embryonic, young, and adult for WT FLNB and LS FLNB
Future experiment could be done in humans with LS and normal people using iTRAQ to look at proteins and any differences that might lead to an understanding of FLNB in bone development.
tap tag proteins and sort
Mass Spectrometry

18. Future directions Illumina Hi Seq 2000
5.7 million people in Wisconsin. Disease is 1 in 100,000. potentially could be 57 people with LS in Wisconsin which would lead to large scale study say tri state area. Minnesota 53 and Iowa 30 = 140 participants
Closely related to other disorders:
1. Spondylyocarpotarsal syndrome
2. Atelosteogenesis I and III
3. Boomerang Displasia
Illumina Hi Seq 2000

19. Questions

20. References:
Hip surgery:
Bone and cartilage:
Filamin/ actin binding:
Actin cell:
Bone:
Cartilage diagram: engineering-spring-2010/labs/module-3-day-4-preparing-cells-for-analysis/
Actin/ microtubule staining:
Bone “?”:
Hand:
Zebra fish:
Fruit fly:
Arabidopsis:
Large group:
Illumina HiSeq 2000:
Tri-State:
GO:
Mouse image:
Mass Spec: spectrometers-lc-ms html
Blood sample:
“Questions?” Image: