Genetics of PC

1. Genetics of PC Frances Smith University of Dundee, UK

2. Pachyonychia Congenita • PC is a genetic disorder caused by mutations in keratin genes • Four keratin genes are linked to PC • PC-1 is caused by mutations in K6a or K16 • PC-2 is due to mutations in K6b or K17

3. What are genes? • Each gene is a DNA recipe for making a particular type of protein • There are ~ 25,000 human genes probably making ~100,000 different proteins • Human Genome has 3,300,000,000 bits of information - in each cell • Big paperback book = 100,000 words. Read one a day for about 100 years

4. What causes a genetic disorder? • Genome has 3,300,000,000 bits of information • Genetic disorders happen when a gene has a single spelling mistake • Defective genes either make a faulty protein or no protein at all

5. DNA is stored in the nucleus of a cell Nucleus

6. How are genes stored? • Genes are stored as a chemical code • This chemical is called DNA • DNA is like a long ladder • The ladder has 4 different types of steps,these are given the letters A, C, G and T • These letters spell out instructions in three letter words

7. DNA chemistry A T G C

8. The genetic code

9. When a cell divides, all the DNA is copied Enzymes in the cell unwind the DNA and copy it into two new stands Very rarely, these enzymes make a mistake and this is a genetic mutation If a mutation occurs in a gene, this usually causes a genetic disorder

10. A gene is a certain region of DNA Exons (coding) DNA Introns (non-coding) RNA Protein

11. A mutation in a gene affects protein production Mutation = Spelling mistake DNA RNA Protein

12. A mutation in a gene affects protein production Mutation = Spelling mistake DNA RNA Protein

13. Dominant mutations: All keratin disorders, including PC Mutation = Spelling mistake Protein

14. Dominant inheritance • A person with a dominant disorder has • 1 normal copy of the gene • 1 mutant copy of the gene • When they have a child it is a 50-50 chance of passing on the mutant gene • 50% of their children are affected • Risk is the same for each pregnancy • can have 3 affected and the next pregnancy has still 50-50 chance • PC is autosomal dominant • no sex bias, males and females equally affected

15. Dominant inheritance Typical Dominant Pedigree e.g. PC New mutation - spontaneous, e.g. PC

16. Genetic diagnosis of PC

17. Pachyonychia Congenita • PC is a genetic disorder caused by mutations in keratin genes • Four keratin genes are linked to PC • PC-1 is caused by mutations in K6a or K16 • PC-2 is due to mutations in K6b or K17

18. Keratins • Keratins are cytoskeletal (structural) proteins • Keratins are found in cells in skin, hair & nails • Two types of keratins - type I (e.g. K16, K17) type II (e.g. K6a, K6b) • Found in specific pairs (a type I and type II)

19. Diagnosis of PC • Blood samples are sent to Dundee • DNA is extracted • Each PC keratin gene is amplified - to make lots of K6a, K16, K6b or K17 • The DNA of each PC keratin gene is sequenced - to look at the DNA code for a genetic spelling mistake

20. Gene analysis

21. PCR schematic After 30 cycles of PCR, you have more than 1 000 000 000 copies of the target sequence

22. Exponential amplification

23. DNA sequencing - decoding DNA

24. Applied Biosystems (ABI) Analyzer ABI 3100 capillary DNA sequencer

25. DNA sequencing

26. DNA sequence trace DNA sequence

27. DNA text of corresponding gene DNA sequence Amino acid code (protein) AAC = N (Asparagine) AAA = K (Lysine) Decode any changes found Some changes can occur that do not cause PC

28. Example of a missense mutation in PC Looking for those genetic spelling mistakes Normal K6a K6a L468P CTG = L (Leucine) CCG = P (Proline) 468 = amino acid number

29. Example of a deletion mutation in PC Looking for those genetic spelling mistakes K16 mutation and normal K16 K16 mutation Normal K16 sequence NNNNNNNN CCT is deleted

30. Type I intermediate filament protein, keratin 16 MTTCSRQFTS SSSMKGSCGI GGGIGGGSSR ISSVLAGGSC RAPSTYGGGL 50 SVSSRFSSGG ACGLGGGYGG GFSSSSSFGS GFGGGYGGGL GAGFGGGLGA 100 GFGGGFAGGD GLLVGSEKVT MQNLNDRLAS YLDKVRALEE ANADLEVKIR 150 DWYQRQRPSE IKDYSPYFKT IEDLRNKIIA ATIENAQPIL QIDNARLAAD 200 DFRTKYEHEL ALRQTVEADV NGLRRVLDEL TLARTDLEMQ IEGLKEELAY 250 LRKNHEEEML ALRGQTGGDV NVEMDAAPGV DLSRILNEMR DQYEQMAEKN300 RRDAETWFLS KTEELNKEVA SNSELVQSSR SEVTELRRVL QGLEIELQSQ 350 LSMKASLENS LEETKGRYCM QLSQIQGLIG SVEEQLAQLR CEMEQQSQEY 400 QILLDVKTRL EQEIATYRRL LEGEDAHLSS QQASGQSYSS REVFTSSSSS 450 SSRQTRPILK EQSSSSFSQG QSS 473 L132

31. Mutations in PC Most mutations in PC occur in one of the hotspot (red) regions L1 L12 L2 S V2 H2 V1 1A 1B 2A 2B ISIS H1 PC-1 mutations in K6a or K16 PC-2 mutations in K6b or K17

32. If no mutation found in hot spot regions • Screen hot spot regions from the two other PC genes • Screen all other regions of the four PC keratin genes • Consider other genes that can lead to PC likesymptoms

33. Why are there different mutationsthat cause PC • The hot spot regions (red domains) are very important for proper keratin filament formation • Mutations in these regions are likely to cause a disorder • Some amino acids are more unstable and more likely to have mutations e.g. K6a N171 • These mutations are found in many families • New mutations will always occur

34. PC Project Thanks to: