Identification of the genes responsible for multiple sulphatase deficiency (MSD) Charlotte Alston

1. Identification of the genes responsible for multiple sulphatase deficiency (MSD) Charlotte Alston NCG Mitochondrial Diagnostic Laboratory Newcastle upon Tyne

2. Keywords: Multiple sulfatase deficiency SUMF1 Formylglycine Generating Enzyme Post-translational modification Microcell mediated chromosome transfer

3. Multiple sulfatase deficiency (MSD) ► Introduction to MSD ► Clinical symptoms ► MSD and isolated sulfatase deficiencies ► Elucidation of the pathogenic mechanism in MSD ► Discovery of the gene involved in MSD pathology

4. What is multiple sulfatase deficiency (MSD)? ► Rare metabolic disorder - defective lysosomal storage Fewer than 30 cases reported to date (Zafeiriou et al., 2008) Estimated 1 MSD case every 4-5 years in the UK ► Sulfatases are lysosomal enzymes that catalyse the hydrolysis of sulfate esters - break down and recycle complex sulphate-containing sugars from lipids and mucopolysaccharides ► In MSD patients, activities of all known sulfatases are greatly reduced or abolished completely ►MSD leads to accumulation of macromolecules (proteins, polysaccharides, lipids) in lysosomes

5. What is multiple sulfatase deficiency (MSD)? MSD is characterised by: ► Metachromatic leukodystrophy “Leukodystrophy” from the Greek leukos = white dys = lack of Troph = growth Leukodystrophy refers to diseases that affect CNS myelin ► Mucopolysaccharidosis / dysostosis multiplex ► Ichthyosis

6. Clinical symptoms associated with MSD ► Metachromatic leukodystrophy Diagnosed on MRI High signal intensity in deep white matter of cerebral hemispheres & subcortical white matter

7. Clinical symptoms associated with MSD ► Metachromatic leukodystrophy Age 22 months Zafeiriou et al. 2008 Eur.J.Ped Neur.

8. Clinical symptoms associated with MSD ► Metachromatic leukodystrophy Age 22 months Age 29 months Zafeiriou et al. 2008 Eur.J.Ped Neur.

9. Clinical symptoms associated with MSD ► Metachromatic leukodystrophy Age 22 months Age 29 months Age 36 months Zafeiriou et al. 2008 Eur.J.Ped Neur.

10. Clinical symptoms associated with MSD ► Mucopolysaccharidosis Build up of glycosaminoglycans in the cells, blood and connective tissues. Caused by lack of sulfatase activity Results in permanent, progressive cell damage which affects - physical appearance - physical abilities - organ function - mental development. Zafeiriou et al. 2008 Eur.J.Ped Neur.

11. Clinical symptoms associated with MSD ► Ichthyosis excessive growth of the outer layer of skin resulting in dryness and scaly appearance

12. Multiple vs isolated sulfatase deficiency MSD patients have phenotypes often including many or all of these symptoms, and more besides. 12 sulfatases have currently been described in the literature. Each of the clinical symptoms in MSD can be ascribed to a particular single sulfatase deficiency; occur as monogenic diseases.

13. Multiple vs isolated sulfatase deficiency Symptom Monogenic deficiency & gene Metachromatic leukodystrophy → arylsulfatase A deficiency ARSA1 gene at 22q13.31-qter Mucopolysaccharidosis → mucopolysaccharide sulfatase deficiency e.g. IDS gene at Xq28 Ichthyosis → steroid sulfatase deficiency STS gene at Xp22.32

14. Isolating the defect in MSD Horwitz (1979) assayed sulfatase activity in MSD fibroblasts. Complementation studies showed sulfatase activities were impaired in fibroblasts derived from two unrelated MSD patients.

15. Isolating the defect in MSD – Horwitz 1979 Adapted from Horwitz AL. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6496-9. All sulfatase levels are decreased in MSD fibroblasts vs wildtype fibroblasts. b-galactosidase used as non-sulfatase control enzyme.

16. Isolating the defect in MSD – Horwitz 1979 Mixing of MSD fibroblasts with wildtype fibroblasts did not affect residual ASA enzyme activity. Conclude that mutation does not act as a diffuse sulfatase inhibitor. Horwitz AL. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6496-9.

17. Isolating the defect in MSD – Horwitz 1979 • Conclusions • Activities of all sulfatase enzymes are grossly impaired in MSD • Some residual sulfatase activity may be present • MSD phenotype is not caused by a inhibition of sulfatase action • Possible defect at the level of sulfatase activation

18. Isolating the defect in MSD – Schmidt et al. 1995 ► MSD patients produce apparently wild-type, full length sulfatase polypeptides that interesting lack enzymatic activity. ► Schmidt et al. searched for a protein modification present in catalytically active sulfatases but deficient in inactive sulfatases using Arylsulfatase A (ARSA) and Arylsulfatase B (ARSB). ► SDS PAGE and isoelectric focusing showed no difference ► Reverse phase HPLC on trypsinised ASA proteins; separated fragments of the protein on basis of polarity (polar -> quicker elution)

19. Isolating the defect in MSD – Schmidt et al. 1995 ► Fragments characterised by mass spectrophometry - Size (Da) of all fragments were in accordance with wildtype sizes, apart from one. ► Aberrantly sized fragment involved amino acids 59-73 of Arylsulfatase A protein

20. Isolating the defect in MSD – Schmidt et al. 1995 ► Fragments characterised by mass spectrophometry - Size (Da) of all fragments were in accordance with wildtype sizes, apart from one. ► Aberrantly sized fragment involved amino acids 59-73 of Arylsulfatase A protein Residues 59-73 Residues 59-73 Unmodified, expected MW ? Associated with MSD Modified, lower MW, Wildtype

21. The discovery of the modification involved in MSD pathogenesis – Schmidt et al. 1995 ► Showed that oxidation of conserved p.Cys69 in ARSA to 2- amino-3-oxopropionic acid, termed a C-Formylglycine residue. ► Enzyme involved in catalysing the p.Cys69 -> C-Formylglycine reaction was named Formylglycine Generating Enzyme (FGE)

22. The discovery of the modification involved in MSD pathogenesis – Schmidt et al. 1995 ► Showed that oxidation of conserved p.Cys69 in ARSA to 2- amino-3-oxopropionic acid, termed a C-Formylglycine residue. ► Enzyme involved in activating all sulfatases was named Formylglycine Generating Enzyme (FGE)

23. Clinical phenotype associated with MSD ► MSD patients produce apparently wild-type, full length sulfatase polypeptides that interesting lack enzymatic activity. ► MSD is caused by a deficiency to generate FGly residues ► Caused by defects in Formylglycine Generating Enzyme (FGE) =>search for the gene encoding FGE.

24. SUMF1 gene identified as the cause of MSD by two separate groups in 2003. Cosma et al. 2003 Andrea Ballabio Naples, Italy Dierks et al. 2003 Kurt von Figura Göttingen, Germany

25. Multiple sulfatase deficiency caused by mutations in SUMF1 ► Caused by mutations in the SUMF1gene ►sulfatase modifying factor 1 ►encodes formylglycine-generating enzyme (FGE) ► SUMF1 defect affects activity of all human sulphatases ► Autosomal recessive inheritance

26. The path to elucidating the causative gene in MSD ► Rarity of MSD patients and lack of familial cases ► linkage was not feasible ►Different approaches to identify the SUMF1 gene employed by the two groups ► Cosma et al. identified the chromosome that harboured the elusive MSD-associated gene using functional complementation using microcell-mediated chromosome transfer ► Narrowed the candidate region by repeating complementation studies, this time using irradiated chromosome 3 fragments.

27. The path to elucidating the causative gene in MSD ► Dierks et al. used a biochemical approach to isolate the FGly generating enzyme from bovine testis using ARSA peptide fragments to bind the FGE enzyme. ► The amino acid sequence of the purified enzyme was established and homologous human cDNA was identified. ► Screening a panel of MSD patients for mutations in the candidate gene

28. Microcell mediated chromosome transfer Individual tagged normal human chromosomes are fused in serial manner with an immortalised cell line from a patient with MSD. Vector has a selectable marker gene – HyTK – used to confer resistance to hygromycin B (antibiotic). Only hybrids that contain the insertion (including transfected specific chromosome) grow in presence of hygromycin. Hybrid cells are grown up and enzyme activities for ARSA, ARSB, and ARSC can be assayed.

29. Microcell mediated chromosome transfer Cosma et al. Cell. 2003 May 16;113(4):445-56.

30. Microcell mediated chromosome transfer Increased activity of Arylsulfates A, B and C Cosma et al. Cell. 2003 May 16;113(4):445-56.

31. Microcell mediated chromosome transfer The increase in sulfatase activity following chr3 complementation suggests the gene involved in sulfatase activation is located on chromosome 3. Cosma et al. Cell. 2003 May 16;113(4):445-56.

32. Microcell mediated chromosome transfer Some individual clones from chr3 transfer had low sulfatase activity. 23 microsatellite markers along Chr3 were used to determine whether whole chr3 was transfected into clones. (+ or –) Cosma et al. Cell. 2003 May 16;113(4):445-56.

33. Microcell mediated chromosome transfer Clones with low sulfatase activities did not contain an entire wt chr3. Incidental chromosome breakage => critical region for sulfatase activation was lost but HyTK resistant marker gene was retained. The search for the critical gene is narrowed down to chromosome 3… Cosma et al. Cell. 2003 May 16;113(4):445-56.

34. Identifying the critical region on chromosome 3 Neo-tagged chromosome 3 hybrids were used; susceptible to spontaneous breakage. Neo-tag conveys neomycin resistance. Repeated microcell-mediated chromosome transfer of irradiated HyTK-tagged human chromosome 3. Microsatellite analysis of clones that were able to grow in hygromycin-containing media (i.e. confirming insertion of HyTK marker gene)… …All clones with high ARSA, ARSB and ARSC activities retained some 3p26 markers Cosma et al. Cell. 2003 May 16;113(4):445-56.

35. Identifying the critical region on chromosome 3 Repeated process at higher resolution using microsatellites across 3p26 identified the region between markers D3S3630 and D3S2397 as the candidate region. Cosma et al. Cell. 2003 May 16;113(4):445-56.

36. Identification of the SUMF1 gene Sequencing of the exons and exon-intron boundaries of all 7 genes in the candidate region in 12 unrelated MSD patients. Numerous unclassified variants were detected in anonymous cDNA sequence PSEC0152 (gbk AK075459) Cosma et al. Cell. 2003 May 16;113(4):445-56.

37. Identification of the SUMF1 gene – Dierks et al. • Dierks et al. employed a different, biochemical approach to identify the SUMF1 gene. • Developed a fast in vitro assay to measure FGly-activity • Utilised a 23mer peptide substrate containing the sulfatase signature of arylsulfatase A: CTPSRAALLTGR (residues 65-80 of ARSA) • Used MALDI-TOF mass spectroscopy to measure substrate and product after the reaction. Dierks et al. Cell. 2003 May 16;113(4):435-44. .

38. Identification of the SUMF1 gene – Dierks et al. • Four stage chromatography was employed to purify formylglycine-generating enzyme(FGE) from bovine testis • Two critical steps involved purification on affinity matrices using 16mer peptides. - Firstly the 16mer peptide was variant of ARSA residues p.65-80; critical residues for FGly formation (p.Cys69, p.Pro71and p.Arg73) were scrambled. Purification involved removal of peptide binding proteins. Dierks et al. Cell. 2003 May 16;113(4):435-44. .

39. Identification of the SUMF1 gene – Dierks et al. • Use of a wildtype 16mer, using wildtype ARSA residues p.65-80 bound FGE efficiently but no activity remained following recovery from the affinity matrix. - Secondly, a different 16mer was used for the affinity matrix – again modelled on ARSA residues p.65-80 but critical residue p.Cys69 was replaced with p.Ser69. Following removal by dialysis, some FGE activity was retained. Following purification, 5% FGE activity and 0.0006% of the starting protein was recovered; quoted as 8333-fold purification Dierks et al. Cell. 2003 May 16;113(4):435-44. .

40. Identification of the SUMF1 gene – Dierks et al. Result of chromatography is the purified FGE enzyme molecules indicated by arrows, sized 41.5 and 39.5kDa. Both shown by MALDI-TOF mass spectroscopy to largely overlap, suggesting they are from products from same gene. Dierks et al. Cell. 2003 May 16;113(4):435-44. .

41. Identification of the SUMF1 gene – Dierks et al. Digestion of FGE enzyme by trypsin yielded peptide fragments of varying size. Amino acid sequence of these peptide fragments was determined by mass spectroscopy and despite derivation from bovine testis, comparison with known human cDNAs revealed significant homology to human cDNA AK075459. cDNA AK075459 was therefore deemed to be a candidate gene for the cDNA encoding the SUMF1 gene… Dierks et al. Cell. 2003 May 16;113(4):435-44. .

42. Mutations in the SUMF1 gene Changes absent in 100 control individuals (200 chromosomes); less likely to correspond to neutral polymorphisms. RTPCR of patient mRNA confirmed exon skipping due to sequence variants occuring at or near a splice site. Cosma et al. Cell. 2003 May 16;113(4):445-56.

43. Mutations in the SUMF1 gene Dierks et al. Cell. 2003 May 16;113(4):435-44. .

44. Confirming the pathogenicity of mutations in the SUMF1 gene Repeated their complementation work using MSD cell line: - transfection MSD cell line with wildtype SUMF1 cDNA produced normal ARSA, ARSB and ARSC activities - transfection of MSD cell line with p.R327X cDNA did not result in functional complementation of ARSA, B or C - transfection of MSD cell line with skipped exon 3 SUMF1 cDNA did not restore activity of ARSA, B or C Cosma et al. Cell. 2003 May 16;113(4):445-56.

45. Evolutionary conservation of the SUMF1 gene Cosma et al. Cell. 2003 May 16;113(4):445-56.

46. Immunofluorescence proves SUMF1 protein is co-localised with ER Transfection of Cos7 cells with Myc tagged SUMF1 cDNAs Anti-Myc antibodies fluoresce green; anti-ER antibodies fluoresce red. Co-expression of ER and tagged-SUMF1 transcripts show as yellow signal. Fits with protein function in post-translational modification. Cosma et al. Cell. 2003 May 16;113(4):445-56.

47. The role of FGE in sulphatase activation Nat Rev Mol Cell Biol. 2004 Jul;5(7):554-65

48. References Horwitz AL. Genetic complementation studies of multiple sulfatase deficiency. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6496-9. Schmidt B, Selmer T, Ingendoh A, von Figura K. A novel amino acid modification in sulfatases that is defective in multiple sulfatase deficiency. Cell. 1995 Jul 28;82(2):271-8. von Figura K, Schmidt B, Selmer T, Dierks T. A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease. Bioessays. 1998 Jun;20(6):505-10. Landgrebe J, Dierks T, Schmidt B, von Figura K. The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes. Gene. 2003 Oct 16;316:47-56. Dierks T, Schmidt B, Borissenko LV, Peng J, Preusser A, Mariappan M, von Figura K. Multiple sulfatase deficiency is caused by mutations in the gene encoding the human C(alpha)-formylglycine generating enzyme. Cell. 2003 May 16;113(4):435-44.