When do we speak about galactosaemia? Galactose Glucose (D) Lactose at the root of the intestinal villi ( lactase (β galactosidase ) enzyme Glucose ( disacchar ide ) monosacchari de monosacchari de monosaccharide + H 2 O hydrolys is inhibiti d
When do we speak about galactosaemia?
Galactose Glucose (D)
Lactose
at the root of the intestinal villi ( lactase (β galactosidase ) enzyme
Glucose
( disaccharide )
monosaccharide monosaccharide
monosaccharide
+ H2O
hydrolysis
inhibitid
galactose aldosereductase galactitol
galactokinase
galactose-1-P
gal.-1-P uridyl transferase.
UDP galactose
UDP gal.-4-epimerase
UDP glucose
UDPG pyrophosporilase
glucose-1-P
Total galactose
The process of conversion
cytosol
GLUT
GALT
glucose
galactose
cytosol
The glucose-6-phosphate and the galactose-1-phosphate fall into an ambush in the cell.
THE PHOSPHORYLATION
impermeable
Determination of galactosaemia : based on
Guthrie microbiological metabolic inhibition
method
Principle: the blood-soaked filter paper spots
are placed on agarose medium witch
contains galactose sensitive
mutant E.coli (W5) strain
„Microbiological newborn screening”
Weidemann test: galactose over 20 mg %
Principle: The galactose – dehydrogenase
( Gal-DH ) enzyme transfers H+
from galactose to NAD, to form
fluorescent NADH
Control (mg % ) Sample
20 25 30 35 40
Interfering factors:
False-positive result:
1. if antibiotic was given to the newborn which
inhibits the growth of E-coli
2. any contamination which gives a clear zone
around the disk
False-negative result:
1. if the blood sample was taken before breast
feeding was started
2. if the filter paper was not blood soaked properly
3. if the baby was transfused
Total Galactose Screening Assay/ galactose and galactose-1- phosphate /
R &D Diagnostics Ltd Greece
alkaline phosphatase galactose-1- phosphate Galactose
galactose dehydrogenase
Galactose + NAD+H2O Galaktono (1,4) lakton + NADH++ + H+
diaphorase
NADH + NBT ( yellow ) NBTH ( violet ) + NAD / nitroblu-terazolium / / formazan /
Beutler probe: determination the
deficit of galactose-1-phosphate –galactose-1-phosphate –
uridil- transferase uridil- transferase ( G-1-PUT )
enzyme to confirm the diagnosis of
classical galactosaemia
Samples: heparinised total blood collected into
gas capillary and a healthy control blood
which is collected, transported, stored,
and measured under the same conditions
Principle: In the presence of G-1-PUT the NADP is reduced forming fluorescent NADPH
blood + galactose-1-phosphate + NADP
30‘ 60‘ 90‘ 120‘
Control:
Sample:
Time ( minute ) 0 30 60 90 120
Control
Galaktosaemia .
heterozygote Galaktosaemia classic
Duarte variant
Duarte variant
heterozygote
Detection of Galactose-1-phosphate uridil-transferase /GALT/ enzyme
GALT
GAL-1-P + UDPG Galactose-1-phosphate uridil-transferase G-1-P + UDP-GAL
PGM
G-1-P phosphoglucomutase G-6-P
G-6-PD
G-6-P + NADP glucose-6-phosphate dehydrogenase 6-PGA + NADPH+
6-PGD
6-PGA + NADP 6-fphosphogluconate dehydrogenase R-5-P + NADPH+
ResultsGALT enzyme aktivity :
Normal : > 3,8 U/gHb Transitional: 2,9 -3,8 U/gHb Abnormal: < 2, 9U/gHb
Beteg B.I. T.K. A.G. M.I. M.A F.G. S.A. S.I. T.A.GALT 3.40 3.11 1.05 2.93 3.51 1.77 3.34 1.75 1.25
Galactose tolerance test ( 1,5 g/kg laktóz; max 50g )
30 perc
3,67 3,10 3,12 3,47 3,83
60 perc
4,01 3,33 3,22 3,91 4,11
120 perc
3,81 3,52 3,28 3,88 3,97
Screening year
Screenedsaces
Screening year
Screenedsaces
Screening year
Screenedsaces
1986 1 G1PUT 1996 2 G1PUT5 G-kinase
2006 4 G-kinase
1987 1 G1PUT 1997 2 G1PUT3 G-kinase
2007 4 G-kinase
1988 1 G1PUT 1998 3 G1PUT2 G-kinase2 Duarte?
2008 4 G-kinase
1989 1 G1PUT 1999 2 Duarte?3 G-kinase
2009 4 G-kinase
1990 0 2000 3 G1PUT1 G-kinase
2010 4 G-kinase
1991 1 G1PUT2 G-kinase
2001 02 G-kinase
2011 1 G1PUT
1992 1 G-kinase 2002 2 G1PUT2 G-kinase
1993 1 G1PUT1 G-kinase
2003 1 G1PUT2 G-kinase
1 Duarte?
1994 1 G1PUT1 G-kinase
2004 2 G1PUT0
1995 1 G1PUT1 G-kinase
2005 1 G1PUT 10 G-kinase
Screened galactosaemic cases
Most common point mutations
G-1-PUTransferase Galactokinase
GALT GalK1 GalK2 9p13 17q24 15q21.1
Classical Duarte
Q188R G1391A S142I G171VK285N N314D G148C
P28T
country Screening starting
Austria 1966
Ireland 1972
United Kingdom 1972 Only in Scotland
Switzerland 1975
Hungary 1975
Belgium 1978 Only the Flemish provinces
Germany 1978 Not compulsory in some provinces
Denmark 2002
France no
Netherlands no
Norway no
Poland no
Spain no
Newborn screening of galactosaemia in Europe