A fine inheritance, but it s not all in the bag
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1 A fine inheritance, but it s not all in the bag Dr Guy Besley, formerly Willink Biochemical Genetics Unit, Manchester Children s Hospital, Manchester M27 4HA, UK.
2 The Willink Unit, Manchester - a fine inheritance
3 June 1979
4 Brian Fowler Joined George Komrower s group in 1965 Mental Retardation Unit, Royal Manchester Children s Hospital. Later renamed Willink Biochemical Genetics Unit. Working with Michael Griffiths and Ann Lambert to set up screening tests for amino acidaemias. Cath Bridge joined group in 1967 Gained Higher National Certificate in 1967 and MIBiol in 1970 and subsequently PhD
5 Brian Fowler On MRC travel scholarship Research at Yale with Rosenberg, Packman and Kraus on CBS Returned to Manchester in 1977 Continued interest in CBS and MMA Willink Unit moved to a purpose-built establishment in 1984 Left the new Willink Unit in 1990
6 Phenylketonuria Patient Images removed
7 Newborn Screening Mental Retardation Unit (RMCH) Guthrie test felt to be too limited Scriver chromatography would identify other than PKU A community pilot survey for amino acidopathies (1967) Would this be workable using liquid blood?
8
9 Newborn Screening Tandem MS purchased in 1998 Initially used butylated samples for acylcarnitines PKU screening in 2001 and MCAD roll out on non-butylated in 2004 However, continued to look at methionine and branched-chain amino acids UK still trying to expand newborn screening
10 Amino acid Screening 2D TLC Paper chromatography (iodoplatinate, Pauly and ninhydrin/ehrlich stains) Spot tests Labour-intensive but excellent method
11 Leucine/ Isoleucine Interpretation of amino acid TLC patterns Methionine Alanine Phenylalanine Valine Tyrosine Threonine Serine Histidine Basics Homocystine Glycine Glutamic acid Aspartic acid Glutamine Asparagine Cystine
12 +ve CN/NP
13
14 2D-urine AA Cystinuria Hyperprolinaemia Homocystinuria Tyrosinaemia type I
15 2D-urine AA Citrullinaemia PKU Ornithinaema Argininosuccinic aciduria
16 Edinburgh SSIEM 1976
17 SSIEM Council hard at work June 1989
18 At ease with Royalty
19 But a European at heart
20 Expanding European interests
21 ERNDIM and lysosomal EQA But it s not all in the bag Developing an EQA for lysosomal enzyme assays has proved something of a challenge Activities are both cell and substratespecific
22 Lysosomal function
23 Evidence of lysosomal storage Vacuolated cells in blood and bone marrow Cherry-red spot in eye
24 Lysosomal storage EM showing MCBs PAS staining showing ballooned neurones
25 TLC of brain lipids GM3 GM2 GM1 C Sandhoff GM1 GM1 GM1 C
26 GM1-ganglioside and -galactosidase N Glc Gal GalNAc Gal beta-galactosidase assay GM1-gangliosidosis NANA 4MU Gal 4MU beta-galactoside
27 Enzyme activities in tissues (nmol/min per mg protein) Tissue GM1 brain Control brain GM1 liver Control liver 4MU- galactosidase GM1- galactosidase
28 Enzyme activities in fibroblasts (nmol/min per mg protein) Cells 4MU- -galactosidase GM1-gangliosidosis 0.12 and 0.03 Carriers 1.78 and 2.28 Controls
29 4MU -galactosidase assay Diagnostic assays normally on leukocytes Very simple assay B-gal usually used as reference enzyme when other lysosomal assays performed But the enzyme is a complex containing a protective protein and is stabilised by chloride ions
30 4MU -glucosidase assay For diagnosis of Gaucher disease Hydrophobic membrane-bound enzyme Requires activator in vivo And in vitro, the detergent taurocholate is required for leukocyte assays The assay conditions are critical for diagnostic testing
31 Leukocyte beta-glucosidase Raghavan et al (1980)
32 4MU acid -glucosidase assay Diagnostic assay for Pompe disease Problems using leukocytes due to interference from neutral/renal enzyme Can be overcome using lymphocytes or specific inhibitor
33 Diagnostic lysosomal enzyme assays Usually performed on mixed leukocytes and plasma A group of enzymes are assayed in parallel But a full understanding of specificities and optimum conditions for each are required Different methods of enzyme extraction and optimisations used
34 EQA requirement Ideally real samples should be used Activities in fibroblasts very different to leukocytes Activities in transformed lymphocytes also very different and not normally used This is a big challenge! Maybe plasma and DBS in the future To Kees Schoonderwoerd: Good luck
35 Munich SSIEM Herzogstand
36 Good-Bye and Good Luck, Brian
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