Haploinsufficiency Retrotransposition Dominant-negative mutations Position effect Inversion

1. Briefly explain the meaning of the following terms, giving examples drawn from human molecular pathology: Haploinsufficiency Retrotransposition Dominant-negative mutations Position effect Inversion

2. Key words • Haploinsufficiency • Quantitative relationship • High demand • Retrotransposition • Dominant-negative mutations • Non-sense mediated decay • Position effect • Inversion • Peri / Paracentric • Osteogenesis imperfecta • Aniridia • Campomelic dysplasia

3. Haploinsufficiency (HI) • Loss-of-function mutation in the heterozygous state result in abnormal phenotype • A single functional copy of a gene is insufficient to produce a normal phenotype • Why are 2 copies of a gene barely sufficient? • High quantity of a protein required in a cell • Necessary for a gene product to have a quantitative relationship with other gene products e.g. • Signalling pathways particularly in involved in a developmental or metabolic switch • Interactions with fixed stoichiometry (macromolecular complexes) • Why has this not been selected against in evolution? • It has, there are very few haploinsufficient genes

4. Supravalvular aortic stenosis • Abnormal narrowing of the aortic valve • Caused by HI of ELN gene which encodes for elastin • Affected tissues with high demand for elasticity • e.g. aorta, skin, bladder • Frequent feature of Williams syndrome (Williams-Beuren syndrome) • Microdeletion of 7q11.2 • Includes ELN

5. Osteogenesis Imperfecta (OI) • Fractures caused by minimal or no trauma • Ranging in severity from perinatal lethal to nearly asymptomatic • Caused by mutations in COL1A1 and COL1A2 • Encodes the chains of type I procollagen • A trimer • OI type 1 (mild) caused mainly by COL1A1 premature truncating mutations

6. Alagille syndrome • Cholestasis, cardiac defects, skeletal defects (butterfly verterbae), opthalmologic abnormalities and characteristic facial features. • 99% caused by HI mutations in JAG1 (Alagille syn. type 1) • Jagged-1 functions in the Notch-Delta pathway • Neurogenesis, somitogenesis and imaginal disc development

7. Other haploinsufficiency examples • SHOX gene in Turners syndrome • SHOX (short stature homeobox) gene • Pseudoautosomal region of X and Y (Xp and Yp PAR) • Escapes X-inactivation • i.e. Males and females have 2 active copies • Single copy = skeletal abnormalities • Likely to be a transcription regulator for genes involved in growth • Hereditary liability to pressure palsies (HNPP) • Peripheral neuropathy including carpal tunnel syndrome • PMP22 gene encodes the peripheral myelin protein 22 • Structural role in myelin sheath of peripheral nerves

8. Other e.g. continued • Waardenburg syndrome • Auditory-pigmentary disorder • Nonprogressive sensory neural hearing loss, white forelock • PAX3 gene • TFs expressed in early embryogenesis • Trichorhinophalangeal syndrome • Craniofacial (sparse scalp hair) and skeletal abnormalities (hip malformations, short stature) • TRPS1 gene (8q24) • TF • Acute intermittent porphyria • Abdominal pain, gastrointestinal dysfunction, and neurologic disturbances • PBGD gene • Hydroxymethylbilane synthase • Enzyme deficiency • Campomelic dysplasia and aniridia (see position effect section)

9. Retrotransposition • Transposon – a mobile DNA sequence able to replicate and insert a copy of itself in a new location • Only a tiny proportion are still active • 2 major types – retro and DNA • Retrotransposon • Copied by reverse transcriptase • cDNA copies of RNA transcripts of the retrotransposon

10. Babushok & Kazazian, 2007 Types of retrotransposons • Most common LINE is LINE-1 (L1) • Located in AT rich regions • >500 000 copies in human genome but only 6000 full length • Up to 6 kb long but non-functional ones tend to be shorter • Some L1s still active and responsible for most of the retrotranscription in the genome • Most common SINE is Alu repeats • ~300 bp long • Most abundant sequence in the human genome, >1 million copies • Only found in primates (recent in evolution) • Relatively high GC content and located in GC rich (i.e. gene) regions

11. Retrotransposons and disease • De novo transposition • Inserting into a gene or regulatory region • ~0.3% of all human mutations are attributed to de novo L1, Alu and SVA insertions (Cordaux and Batzer, 2009) • DMD, Apert syndrome, CF, NF, β-thal, hypercholesterolaemia • Potential substrates for unequal crossing over

12. Familial hypercholesterolaemia • High LDL-cholesterol, xanthomas • AD disease mainly caused by variants in the low density lipoprotein receptor (LDLR) gene • 11% LDLR mutations large DNA rearrangements (>100 kb) • Frequently associated with Alu elements • 98 Alu repeats within LDLR gene • Alu repeats used as recombination substrate for nonallelic homologous recombination (NAHR)

13. Dominant negative mutations • Also known as an antimorph • Mutant protein antagonises normal protein function • Normal protein is sequestered into inactive complexes • Resulting in <50% residual function • More severe effect that null mutants • Non-sense mediated decay may have arisen to protect against this. • Particularly common in proteins which function in dimers or multimers

14. Severe OI (types II, III & IV) • COL1A1 and COL1A2 missense mutations in the Triple helical domain • Contains 1014 amino acids in the repeating sequence of Gly-X-Y • Replacing glycine for any other AA causes disruption of helical packing • Strong dominant negative

15. Familial adenomatous polyposis (FAP) • Colon polyposis and cancer predisposition • APC gene (5q22) • Important in negative regulation of wingless signalling pathway • Truncating mutations at codon 1309 severely inhibit normal APC function and cause severe polyposis (Dihlmann et al., 1999) • Escape NMD and cause dominant negative effect because homodimerise with wild type APC

16. Marfan syndrome • Connective tissue disorder • FBN1 gene encodes for fibrillin-1 • Contributes to microfibrils • Large multimeric structures • Mutant monomers disturb the function of the multimeric microfibrils.

17. Position effect • A change in chromosome position leads to alteration in gene expression despite the transcriptional unit remaining intact. • Gene expression can be changed by; • Separating the gene from its regulatory elements • Long range regulatory elements can affect a gene up to 1 Mb away • Developmental genes have particularly complex set of distant enhancers • Juxtaposing a gene with another genes regulatory elements • Relocating a gene and it’s regulatory elements next to another gene which competes for the enhancers • Altering the chromatin structure around the gene. • Translocating a gene to a heterochromatic region • Position effect variegation (PEV) • Heterochromatin may spread into the euchromatin silencing nearby genes

18. Aniridia • Congenital malformation of the eye (absence of iris) • PAX6 (11p15) • TF involved in ocular development • Majority of mutations point mutations and gene deletions cause HI • Some patients have translocations 3’ of PAX6 • Up to 125 kb beyond the final exon • A cluster of enhancers have been identified and called the ‘Down stream regulatory region’ • No difference in phenotype compared to loss of function aniridia.

19. Campomelic dysplasia • Skeletal dysplasia, distinctive facies, Pierre Robin sequence with cleft palate, shortening and bowing of long bones, and club feet • SOX9 gene • TF (involved in regulation of COL2A1 and COL11A2) • ~90% mutations within coding region (causing HI) • ~5% are deletions, inversions or translocations • Disrupt the 5’ cis-acting regulatory effect. • Regulatory region 1 Mb upstream of the gene

20. Burkitt’s lymphoma • Lymphoma of the jaw • Seen in children in Africa • 90% of affected children have a translocation of the c-myc gene from 8q24 to chromosome 14 • C-myc is an oncogene • Translocation moves c-myc under the control of the immunoglobulin gene regulation • This allows over-expression of the gene

21. Facioscapulohumeral muscular dystrophy (FSHD) • Weakness of the facial muscles, stabilizers of the scapula or the dorsiflexors of the foot • Caused by deletions of the tandem repeat D4Z4 at 4q • Very complicated disease mechanism • The deletion is thought to affect expression of FSHD-associated genes through position effect mechanism

22. Inversions • A two break chromosome rearrangement involving a single chromosome • A segment is reversed in position • ‘balanced’ rearrangements • Pericentric – Includes the centromere • Paracentric – Involves only one chromosome arm • Rarely cause phenotype unless a gene is disrupted by the breakpoints • However, they cause problems during meiosis if a cross over occurs.

23. Chromosome inversions Pericentric Paracentric

24. Haemophilia A • ~50% severe cases (i.e. those with <1% factor VIII activity) are caused by a 400 kb flip inversion mutation in the factor VIII gene • The most common severe mutation • Arises from a recombination between a small gene called A located in intron 22 and other copies of the A gene located upstream near the telomere • Flip inversions show >10-fold higher mutation rate in male germ cells than female • Perhaps because there is not an X homolog to pair with during meiosis so bigger opportunity for intrachromosome recombination

26. Hunter syndrome (Mucopolysaccharidosis type II) • Hearing loss, recurrent infection, diarrhoea, poor growth, Coarsening facial features • Caused by mutations in IDS gene (Xq28) • IDS2 pseudogene located 20 kb telomeric of the IDS gene is involved in an inversion event with the IDS gene in about 13% of patients with the Hunter syndrome

27. Inversions predisposing to disease • Wolf-Hirschhorn syndrome • Recurring constitutional translocation t(4;8)(p16;p23) • Parents of translocation offspring are heterozygous carriers of inversions at both locations

28. References • Cordaux and Batzer, Nat. Rev. Genet., 10, 691-700, 2009 • de Greef et al., Mutat. Res., 647, 94-102, 2008 • Dihlmann et al., Cancer Res. 59(8), 1857-60, 1999. • Emery, Elements of Medical Genetics, 13th Edition • Feuk, Carson and Scherer, Nat. Rev. Genet., 7, 85-97, 2006 • Goldman et al., BMC Med. Genet. 11 115-122 2010 • Kleinjan and Coutinho, Brief. Funct. Genom. Proteom., 8(4), 317-332, 2009 • Kleinjan and Heyningen, HMG, 7(10), 1611-1618, 1998 • Strachan and Read, HMG, 4th Edition • Veitia, J. Path., 218, 409-418, 2009 • Veitia and Birchler, J. Path., 220, 174-185, 2010