MUTANTS genetic variation in human development Lecture 8 Fall 2006 Bennington College

1. MUTANTSgenetic variation in human developmentLecture 8Fall 2006Bennington College

2. Some Disorders linked to bone growth cleidocranial dysplasia thanatophoric dysplasia sclerosteosis fibrodysplasia ossificans progressiva (FOP) pseuodoachondroplasia achondroplasia osteogenesis imperfecta osteoporosis osteopetrosis pycnodysostosis

3. cleidocranial dysplasia A rare condition inherited as an autosomal dominant trait and characterized by partial or complete absence of the clavicles, defective ossification of the skull, and faulty occlusion due to missing, misplaced, or supernumerary teeth.

4. cleidocranial dysplasia Current evidence suggests cause is mutation in the Cbfa1 transcription factor (Core binding factor a1)

5. Bone is always a “work in progress” - not a static entity Bone is derived from the same embryonic tissues that make the flesh surrounding bones (connective tissue, muscle) all derived from the mesoderm Osteogenesis - the formation of bones

6. Two major modes of osteogenesis: intramembranous ossification - direct conversion of tissue into bone; this is how flat bones, such as the cranium, are formed. endochondral ossification - cells first differentiate into a cartilage intermediate which is later replaced by bone; this is how long bones, such as the femur and humerus, are formed

7. Intramembranous ossification: layer of osteoblasts secrete a protein matrix. Calcium phosphate forms on this matrix and then encloses the osteoblasts (at which point they become osteocytes)

8. endochondral ossification:chondrocytes produce cartilage as a template for future bone. osteoblasts ultimately come along and turn the cartilage into bone.

9. zoom-in of endochondral ossification

10. Back to the role of cbfa1 in cleidocranial dysplasia…Cbfa1 is a transcription factor that promotes the differentiation of bone-producing cells (osteoblasts and chondrocytes) from mesoderm (mesenchymal cells)

11. If you have one mutant copy of Cbfa1, the result is cleidocranial dysplasia If you have two mutant copies of Cbfa1, the result is death immediately following birth… Defective Cbfa1 means defective bone formation - no bones and all cartilage means no ribs to support the ability of the lungs to expand and contract - this results in asphyxiation.

12. endochondral ossification is what results from the observed “condensations” during embryogenesis. what tells these condensations that they will become bone is BMP (bone morphogenetic protein) BMP tells the mesenchymal cells (cells from the mesoderm) to become bone instead of muscle or connective tissue

13. BMP4 you may recall, earlier in embryogenesis, BMP was telling the developing embryo to form front, not back (while noggin was telling it to form back, not front) Noggin

14. Later in life if we get a fracture - BMP4 will appear and say “make bone” Basically, BMPs can induce bone wherever they are…

15. Chiba et al., Histological study of effect of bone morphogenetic protein derived from bovine tooth on periosteum in rats. Journal of Bone and Mineral Metabolism. 8(3): 24-29. 1990. control injection into femur BMP injection into femur

16. Too little bone is a problem…too much bone is also a problem Sclerosteosis - a rare, potentially lethal, autosomal recessive, progressive sclerosing (hardening) bone dysplasia with characteristic facial and skeletal features. Usually results in massive, thick skulls May become lethal if excess bone crushes a vital nerve

17. Balemans et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Human Molecular Genetics10(5): 537-543, 2001. Identified a gene, SOST (sclerostin), that is the cause of sclerosteosis. sclerostin is a negative regulator of BMPs. Figure 1. Lateral view of a patient from the Brazilian sclerosteosis family showing the characteristic high forehead and the protruding large chin.

18. BMP4 BMP4 BMP4 BMP4 A B B SOST SOST SOST SOST SOST SOST

19. BMP4 BMP4 BMP4 BMP4 A B B go make bone now!

20. fibrodysplasia ossificans progressiva (FOP) An extremely rare disease (~250 worldwide cases known) in which connective tissue and muscle tends to ossify (turn into bone) over time. Also, any injury is repaired with osteoblasts, increasing bone formation. Ultimately leads to complete immobilization of the patient.

21. Harry Eastlack 1930-1973 Appeared normal until he fractured his leg at 5. Soon got stiff in the hip and knee - doctors found bony deposits on his adductor and quadricep muscles By 1946, left leg and hip were frozen. Back muscles were turning into sheets of bone (Torso permanently bent at 33° angle) Any attempts to remove excess bone only made it come back - and get worse

22. By the age of 23 his jaws had entirely seized up and he could only partake of a liquid diet. Had to be institutionalized for care.

23. At the time of his death (due to pneumonia) he could move only his lips. Donated his body to science.

25. Victor Chandler Twitty 1901-1967

26. Twitty aimed to discover where the information governing limb growth came from Experiment: Use a larger species and a smaller species of salamander. Chop off a limb from each. When the limb bud regenerates, excise it and transplant it to the other salamander. What happens? Does the donor (and therefore the limb bud) control the ultimate size of the limb? Or does information from the host (hormones, other signaling pathways) tell the limb what size it should become for that particular species?

27. Results of Twitty’s limb bud grafting experiment The limb bud grafted from the donor contained all the information to govern the ultimate size of the limb. The host, and therefore the host’s hormones, did not influence this process.

28. Twitty thus demonstrated local control of bone growth But how does the bone know how long it should grow to be? Perhaps by studying what happens when they fail to do so we can gain some insight… dwarfism

29. This student was Josef Mengele Shortly after the time that Twitty spent in Hans Spemann’s laboratory in Germany, another young scientist was beginning his career at the Kaiser Wilhelm Institute of Anthropology. 1935 dissertation described work measuring hundreds of jawbones looking for distinctive racial differences The world does not remember Mengele for his doctoral work, but for the horrific career path he then followed.

30. Mengele was encouraged by his mentor, von Verschuer, to go and take advantage of the “extraordinary research opportunities” at Auschwitz. The medical practice paradigm during the Hitler period saw the physician as a "selector" acting on behalf of the state in order to improve the “health of the nation” (Volksgesundheit). People defined as “underclass” and therefore a “risk to the genetic or racial health of the population”. These people could be selected for enforced sterilization, incarceration, and eventual extermination. Research on eugenics and racial hygiene was conducted in university research institutes and those of the Kaiser-Wilhelm organization. The helpless human quarry incarcerated by the state was viewed by medical science as a unique opportunity for the kinds of research which under German law were not even permitted on animals. These victims were exploited in the name of “science” both before death for inhuman research, and their bodies were exploited after death.

31. When Mengele arrived at Aushwitz in May 1943, there were >100,000 prisoners. Killing took place daily. • Aushwitz survivors usually remember Mengele - • he was a very complicated entity: • very attractive • well groomed • charming • could speak nicely to a child then • send it off to the gas chamber • (sociopathic?) Mengele often performed the initial “sorting” to life or death of prisoners arriving by train. He kept the most interesting “specimens” for his “research”

32. One such “lucky” (or unlucky?) group of “subjects” was a family afflicted with dwarfism. The father of the Ovitz family had a type of dwarfism known as psuedoachondroplasia Of his 9 children, 7 were dwarfed. After his unexpected death, the mother got the children musical training to engineer a way for them to earn a living. They hid their Jewish heritage for a few years, but were eventually captured and sent to Auschwitz - much to the delight of Mengele

33. They became Mengele’s favorite experimental subjects • those who were married were subjected to gynecological experiments • uterine injections, blood samples, other tissue samples • all subjected to numerous other “medical” tests including • extraction of spinal fluid, flushing of ears with extremely hot or • cold water, hair extraction, painful brain and face examinations. • forced to stand naked on display while Mengele lectured about • them to Nazi higher-ups

34. Auschwitz was liberated in January 1945. The Ovitz family survived with their lives (but at what cost?) Mengele was also fascinated with other subjects, especially twins, who he would kill just to dissect and compare the similarity of their organs. Mengele was never tried for his crimes against humanity. He died on a beach in Brazil in 1979.

35. psuedoachondroplasia Dominantly inherited form of dwarfism. The gene mutated in this condition is cartilage oligomeric protein (COMP). Growth plates are the active sites of bone growth at the ends of developing/growing bones. Growth plates remain uncalcified until around the age of 18 (when we stop growing).

36. normally, there are columns of chondrocytes that secrete a cartilagenous matrix, then die. if COMP is mutated, it fails to be secreted to the matrix and prematurely kills the chondrocytes in the process (halting growth ) therefore, COMP and other proteins that control the activity of the growth plate play a large role in determining the final length of the bone.

37. achondroplasia Inherited in an autosomal dominant fashion, but over 80% of cases arise from spontaneous mutation. Phenotypically characterized by short stature with disproportionately short arms and legs, a large head, and characteristic facial features

38. achondroplasia The mutation responsible is a single amino acid change in FGFR3resulting in a glycine to arginine change at position 380. glycine arginine

39. achondroplasia The mutation responsible is a single amino acid change in FGFR3resulting in a glycine to arginine change at position 380. This mutation makes the Fibroblast Growth Factor Receptor inappropriately active (mimics excess FGF) Too much FGF would inhibit chondrocytes from travelling up the growth plate - thus this mutation results in lack of bone growth from the growth plates. (no chondrocytes, no secreted matrix, no future bone)

40. extremely short limbs folds of extra skin on the arms and legs narrow chest short ribs underdeveloped lungs enlarged head with a large forehead wide-spaced eyes. thanatophoric dysplasia This disorder is lethal. It is caused by different mutations in the same FGFR3 receptor. Condition occurs if both copies of the receptor carry the mutation.

41. HAVE A NICE WEEKEND!!!