numhcr after glucocort~cold~njectionInto l c t t ~ lrahblts to enhance r u r k c t a n t
appearance Pedi:~tric~.5 3 . 358 (1974).
36. I.n\ln. N . K:~chelcf\k!. (; . and Kaplan. S. /\
l n h l h ~ t l o n o f c!cl~c-AMP
pho\phod~esteraccIn human I!rnphoc!te\
ti! ph!rlologlcal concentr:ltlon\ o f
hjdrocortlronc, i l o r m . Metab Kes.. 7 253 ( 1975)
37. Lcvcy. G. S and t p s t e ~ n .S. I:.: M!ocardl:ll adenll c!clase. ~ \ c t l \ ; ~ t ~ oh!n
th!rold hormone\ and ev~dencclor t u o odenkl cyclasc z!\tcms. .I C l l n Inve\t..
4 8 . 1663 ( 1969).
38. 1.1ggins. G . C: Prem;~turedellvery o f foctnl lamhs infused u ~ t glucocort~co~ds.
h
.I
Endocrlnol.. 45. 51 5 (1969).
l ;intcparturn glucocortlco~d
39. 1.1ggins. G . C.. and t-lou~c.R . N '\controlled t r ~ a111
treatment for prcventlon o f the respirator! d~stre\ssyndrome In premature
infants. Ped~ntrics.50: 515 (1972).
40. L.ourry. 0 . [I.. Ro\chrough. N. J.. Farr, A . I.., and Randall. K . J.: P r o t c ~ n
me;isurement ~ ~ thet Folm
h
phenol reagent. J Blol. Chem.. I Y j . 265 (1951).
41. Mang;~n~ello.
V., and Vaughan. M . : A n efl'cct o f dexanietharone on ndenoi~nc
?'.5'-~nonophosphatecontent and adcnos~ne3'.5'-monopho\ph:~tc pho\phodie\terahe acti\it! o f culturctl he pat om:^ cells. J . Clin. In\e\t . 51 2763 (1972).
42. Menon. K . M . J.. (iiese. S.. and . l a f k . R . B.: Hormone- ;~ndlluoride-\eni~tli'c
aden!lnte c\claae\ In human letal tl\eue\. B l o c h ~ m .B~oph!s .\eta. 304. 203
(1972).
43. Moskowitr. .I., and Fain. .l N. Stimulation h? growth hormone and dex:~meth;~sone o f laheled c!clic adcnoh~ne3'.i'-monophosph;~te accumul.~tion h! w h ~ t c
fat cells. J Biol. Chem . 4 5 1101 (1970).
. Knih:~rn. M . . Wu. B.. l ~ g a s ('.
.
44. M o t q a r n a . t. K.. O r r a l e r ~ .M . M . . K i k k - ~ u a Y..
J., and Cook. C. D . Eflect o f cortisol on the maturation o f letal r n h h ~ tlung\
Ped~atr~cs.
4 8 - 547 ( 197 1 )
45. Palmer, G. C : Charactcr~st~cro f the hormonal induced c!cl~c adcnor~nc
3'.5'-rnonophosph:~k re\ponse o f the rat and guinea pig lung ttr ~ r r r o .B l o c h ~ m .
Biophla. Acta. 252. 56 l ( 197 1 ).
46. Palmer. G . C. Cyclic 3',5'-adcnos~ne ~nonophosph~ctc
responac In the rabhlt
lung-adult
propcrtle.; and development. Blochem. Phnrm:lcol.. 21. 2907
(1972).
47. Kedding. R . A,. Douglaa. W . H . I . . and Stein. M . : T h ? r o i d hormonc ~nnuence
upon lung \urfact.~nt rnetabol~rm.Sclence. 175- 994 (1972).
t . K., C r a ~ g J. . M . . and Cook.
48. Ke?nolds, E. 0. R.. Or/alcsi. M. M., Motoy;~rni~,
.
49.
50.
51.
52.
53
54.
55.
56.
57.
58.
59.
60.
61.
C. D: Surfacc propcrtles o f saline extract\ from lungs o i n e u h o r n ~ n f i ~ n t Acta
e.
Paedlat. Scand , 54: 51 1 (1965).
Roone). S A , . (irosr, I.. Moto!an~n, E. K . . and Warshau. .I. B.. tlffects o f
cortisol ; ~ n d th!ro\inc on k ~ t t ! acld and phosphollpld hio\!nthcs~\ In l'ctal
r a h b ~ tlung. Phys~olog~st.
17. 323 (1974)
Russell, B. .I., Nugcnt. L and Chern~ck.V.: Effects ol'steroids on the enzymatic
2 4 - 306 (1974)
pathways o f l e c ~ t h ~product~on
n
In retal rabbits. B ~ o l Neonate,
.
Ru\sell. T . K.. Tcrasak~.W . I. . and Applemi~n.M M . : Separate phosphod~c\terase? for the h!drol!a~? o f c!cllc :~denoa~ns
3'.5'-monopho\phate :~nd c j c l i c
guanosine 3'.5'-monophoaph:~te In rat liver. J. Biol Chem.. 248: 1334 (1973).
Schack. J. A., and Warler. S. t i . : A n ultrav~oletspectrophotonietr~cmethod Ibr
the dctcrminatlon o f theophblline and theohromlne In blood and tissue\. J.
Pharrnacol. t s p Thcr . 97: 283 (1949).
Senft. G., Schultl. G.. Munckc, K.. and Hoffman. M . : t f f e c t \ a f glucocorticoids
and insulin on 3 ' 5 ' - A M P phosphodiesternse activit) In adrennlectom~zedrats.
Dlahctologia. 4 . 330 (1968).
Sh;lp~ro. S.: Some ph)slologic. b~ochem~c;ll
and behavioral consequences o f
neonatal hormone adrnlnistratlon: Cortisol and thyroxin. (;en. Comp. Endocrlnol., 10: 214 (1968).
Snedecor, G. W.. and Cochran. W. Ci.: St:~t~stlc:~I
Methods, Ed 6 ( l o u a State
U n ~ i e r s ~ tPress.
)
Arnes. lou:~. 1967).
Stahlman. M. T., Gra!. M . E.. Llcu. S and Chktll, F.. The role afcycllc A M P In
lamellar hod) a)nthc\~\ and Fecretlon. Pcdiat. Kes.. 8: 470 (1974).
r
change\ In the mbhit placenta
Wcllrn:ln, K . F.. and Volk, B. W.: F ~ n structurnl
Induced h) cortisone. Arch. Pathol.. 94. 147 (1972).
Wu.B.. K i k k a u ; ~ .Y.. Orzale5i. M . M.. Motokama. F. K.. Ka~bnra.M . . Z i p \ . C .
.I..and Cook. C . D.: The effect o f thyroxlnc on the maturation o f k t a l rabbit
lungs. Blol. Neonate. 2 : 161 (1973).
T h ~ cresearch was supported In part h) U n ~ t e dStates Puhlic Health S e r r ~ c eGrant
N o ill>05443. Unlvers~t) General Kerearch Support Fundc, and the
C a l ~ f o r n l a Re\earch and M e d ~ c a lEducation Fund o f the C a l ~ l ~ r n iLau n g
Association
Requests for reprints ahould he :iddre\sed to C . T. Barrett. M.D.. Department o f
Ped~atrlcs,U C L A School o f Med~cine.1.0s Angeleb. C a l ~ f 90024
.
(USA).
Accepted far publication January 14. 1976.
.
.
Prrnred rn U . S . A.
C o p y r ~ g hO
t 1976 1ntern;ltlon;ll Pcd~atricKeiearch Foundat~on.Inc.
Pediat. Res. 10: 625 629 (1976)
Aspartylglucosaminuria
4-L-aspartylglycosylamine amido hydrolase
glycoprotein
leukocytes
lymphocytes
plasma
Enzymatic Diagnosis and Carrier Detection of
Aspartylglucosaminuria Using Blood Samples
PERTTI U L A .
"
KKAl RAIVIO. A N D SEPPO A U T l O
Extract
The activity of the glycoprotein degrading lysosomal hydrolase, 4-L-aspartylglycosylamine amido hydrolase (AADGase,
EC.3.5.1.26). was measured i n plasma, buffy coat leukocytes, and
separated lymphocytes (Ficoll separation) from 16 patients with
aspartylglucosaminuria I A G U ), 29 obligate heterozygotes, and 30
control subjects. I n lymphocytes the A G U patients had unmeasurable or minimal AADGase activity with a mean of 3.9 U. The obligate heterozygotes showed AADGase activities ranging from 5 to 69
U with a mean of 34.1 U. Enzyme activities i n the control group
ranged from 91 to 243 11 with a mean of 127.9 U, and were clearly
separated from the talues of the heterozygotes.
I n leukocytes the AGU patients had unmeasurable enzyme
activity and obligate heterozygotes had enzyme levels closely similar
to those i n the lymphocytes from the same individuals. The
AADGase activity i n the leukocytes of the control group displayed a
n ~ u c hwider rariatioii than i n :hc lymphccytes, raaging from 22 to
132 Li with a mean of 70.7 U.
I n plasma the AGU patients had undetectable AADGase activity.
The mean enzyme letel of obligate heterozygotes was 72.2 U and
that of control indibiduals 107.2 U, but the oterlap between the
groups was extensive. The results indicate that homozygous deficiency of AADGase, i.e., aspartylglucosaminuria, can be reliably
diagnosed using plasma, leukocytes, or separated lymphocytes.
For carrier detection only separated lymphocytes allow a satisfactory differentiation between heterozygous and normal individuals.
A group of 31 siblings of verified A G U cases and 11 children o f
identified carriers, whose spouses had normal AADGase activity,
were investigated using the lymphocyte assay. The observed and
expected frequencies (on the basis o f Mendelian probabilities)
were closely similar, suggesting that the lymphocyte assay can be
used reliably for carrier detection.
626
AULA, KAIVIO, A N D A U T I O
Speculation
Demonstration of AADGase deficiency in plasma or in white
blood cells offers an alternatire method for the diagnosis of
aspartylglucosaminuria. Large scale screening programs for AGU
carrier detection with the lymphocyte enzyme assay are not
considered practical and justified at present. Carrier screening
among relatives of known A G U patients is, however, medically,
econcmically, and psychologically advantageous, particularly since
prenatal diagnosis and the possibility of pretentive abortion can be
offered to carrier-carrier matings.
Deficiency of the lysosomal enryme, 4-L-aspartylglycosylamine
a m i d o hydrolase, causes a disturbance of glycoprotein catabolism
and leads to a clinical disease, A G U . T h e clinical features of A G U
include slowly progressive psychomotor retardation starting at the
age of 2 6 years and resulting in severe mental retardation before
puberty. This psychomotor impairment is associated with the
appearance of typical coarse facial features and degenerative
changes in connective tissue (3). T h e disease was first described in
two mentally retarded siblings in England (10). Subsequent
patients have been found almost exclusively in Finland, where a
total of 82 cases has been verified. Very recently, however, the first
cases of A G U have been detected in the U S A (9) and in Norway
(12). T h e diagnosis of A G U has generally been based on the
demonstration of increased urinary excretion of aspartylglycosylamine by chromatographic o r electrophoretic methods (17). T h e
s a m e glycopeptide is the main storage material in brain and liver
tissue, but small a m o u n t s of some higher glycoasparagines also
accumulate ( 18).
T h e enzyme deficier~cyhas been demonstrated in plasma and
seminal fluid (2 I), a s well a s in liver, brain, and spleen tissue (19) of
A G U patients. T h e enzyme defect is also expressed in cultured
fibroblasts. which offers the possibility of prenatal diagnosis (2).
Fibroblasts from parents of A G U patients have an intermediate
activity of A A D G a s e , which proves the autosomal recessive mode
of inheritance of the disease and provides a basis for heterozygote
detection ( I ) .
In order t o avoid the practical difficulties associated with cell
culture techniques, we have assessed the suitability of plasma,
leukocytes. and lymphocytes for the enzymatic diagnosis and
carrier detection of A G U . Earlier data had already suggested that
A A D G a s e activity in the plasma of obligate heterorygotes was not
different from control individuals (20).
MATERIAL A N D METHODS
T h e subjects studied consisted of 16 patients with clinically and
biochemically verified A G U , 29 parents of patients (obligate
heterozygotes), and 30 control individuals. At a later stage of the
study. 31 healthy sihiings of A G U patients and i l offsprings o i
identified carriers whose spouses had normal A A D G a s e values
were investigated with the lymphocytes assay.
P R E P A R A T I O N OF S A M P L t S
Peripheral venous blood (10 ml) was drawn into a heparinized
syringe and used for the preparation of cell extracts. Leukocyterich plasma was separated from the erythrocytes by letting the
syringe stand at room temperature for 60 min. Leukocytes and
plasma were then separated by centrifugation a t 1,500 rpni for 10
min. T h e cells were washed twice with phosphate-buffered saline
before counting and sonication. Lymphocytes were separated from
another 10-ml blood sample by centrifugation using the one-step
Ficoll-Isopaque method described by Boyum ( 5 ) . T h e yield of
lymphocytes from a 10-nil blood sample runeed
- from 12 t o 8 3 x
l o 6 cells. For elimination of possible erythrocyte contamlnation
both lymphocytes and leukocytes were treated with 0.83C' amrnonium chloride for 15 niin s t 37'.
Cell extracts were prepared by sonication after adding Triton
X- 100 to a final concentration of 0. I % t o the cell suspension. T h e
sonicates were used for enLyme assays and protein determination
(13). F o r the plasma assays, aliquots of 200
were used.
E N L Y M E ASSAYS
A A D G a s e assay was originally described by M a k i n o and
coworkers (14). and a detailed account of o u r assay system has
been reported ( I ) . T h e synthetic substrate (2-acetamido-N-(Laspart-4'-oyl)-2-deoxy-P-_plucopyranosqlamine)
was kindly provided by Professor I . Yamashina (Kyoto University. Kyoto,
Japan).
O n e unit of enryme activity is defined a s I nmol 1%'-acetjlglucosamine libernted/min/g sonicate protein; in plasma 1 U
activity is expressed per P I ! plasma. h'-Acetyl-@-glucosaminidase
was assayed in all samples 3s a cell viability control using the
commerciall) available p-nitrophenll derivative a s substrate (19).
I n most cases lymphocytes and leukocytes were separatcd and used
for enzyme determinations immediatelj after the blood was drawn,
otherwise the extracts were stored at 7 0 ' for 1 7 days without
loss of activity. In a few cases, the blood sample was kept overnight
at room temperature. Storage of blood for periods longer than 24
hr was shown t o decrease the enzyme activities.
R ES CJ L T S
AADCinse A C T I V I T Y I N L Y M P H O C Y T E S
All the A G U patients studied had unmeasurable o r minimal
A A D G a s e activity in lymphocytes, with a mean of 3.9 U (Fig. I ) .
This represents 3 % of the mean activity in control samples. T h e
highest activity of 16 U was found in a 22-)ear-old female patient.
Unfortunatel!, the leukocytes and plasma ol' this case were not
available for study.
T h e parents of A G U patients (obligate heterozygotes) showed
A A D G a s e activities ranging from 5 to 69 U , with a nieun of 34.1
U . An exceptionally low value of 5 U was found in one mother.
T h e blood in this case was stored overnight before lymphocyte
separation which possibly accounts !-r the low enzyme ~!c!ivit!
AADCiase activities in the Iqmphocytes of the control group
ranged from 91 to 243 U. with a mean ol' 127.9 U, and were clearly
separated from the values of the heterozygotes.
AADCiasr A C T I V I T Y I N L E U K O C Y T E S
Ail four A G U patlents studied had unmeasurable AADCrase
activity in leukocytes. Obligate heterorygotes had enzyme levels
closely similar to those in the lymphocytes from the s a m e
individuals, ranging from I to 65 U with a mean of 38.8 U .
However, the A A D G a s e activity in the leukocytes of the control
group displayed a much wider variation than that in the lymphocytes, ranging from 22 to 132 U with a mean of 70.7 U . As
depicted in Figure 1 , there is considerable overlap between the
leukocyte A A D G a s e values of obligate heterozygotes and control
individuals.
AADCiart: A C T I V I T Y I N PI.ASMA
A G U patients had undetectable o r minimal activity of
A A D G a s e in their plasnia samples also. T h e mean e n r y m e level
of obligate heterozygotes was 72.2 U and that of control indi-
ASPAKTYLGI I I C OSA
and 0.1 '8 and restudy using a neu Iymphocqte sample o r CLIItured skin fihroblasts is required. A D D G a s e activ~tqless than 1.5
S D above the mean of obllgate heterorygotes ( <60.7 U ) was considered t o Indicate a carrier genotqpe. Using this value a s the cutoff point, the risk of a normal indiv~dualb c ~ n glabeled a hetero,
on the distribut~on
zqgote M;IS calculated to be 2.5'; or l e s ~ based
of value\ In the control group. A simrlar c;irrier assessment s!stern has been used by Kaback and coworkers in heterorqgotc
screening projects for Tay-Suchs gene ( I I ) .
A group of 3 1 siblings of verified A G U cases and I I children ol'
identified carriers hose spouses had normal AADCiase activity
were investigated using the Iqniphocyte e n l q m e assa). T h e o h served number and frequencb of heterorygotes was compared with
those expected on the basis of Mendelian probabilities. The re\ults
a r e given in Table I . T h e expected and observed frequencie\ of
A G U carriers are closely similar, suggesting that the lymphocqte
assay can be used reliably for carrier detection.
T w o cases out of a total of 4 2 fell within the "~nconclusive"
range 01' A A D G a s e activit). a s s h o n n in Figure 2. O n e daughter of
an identified carrier had A A D G a s e activ~t! of 71 U in the first
assaq, but in a later repeat stud! the activit! was 123 U , indicating
a norrn:rl A A D G a s e genotype. T h e other "inconclusive" case Mas
a female sibling of a n A G U patient having A A D G a s e activit) of
76 U . It has not yet been possible to perform a repeat test, but she
is verq likel! a carrier because her daughter was shown t o have
Iymphockte A A D G a s e activit! of 40 U and her husband, normal
activtt! of I03 U .
LYMPHOCYTES
LEUKOCYTES
PLASMA
The demonstration of A A D G a s e defic~cncy in plasma o r in
white cells h a a number of practical iniplications. It offers an
alternative method for the diagnosis of aspart! lglucosaminur~a.As
mentioned earlier. this has so far been hased on the demonstration
of increased amounts 01' aspartqlglucosqlam~ne ( A A D G ) in the
urine. Screening tests for this purpose have been developed ( 8 ) .
Even though incrnsed A A D G excretion has not heen encountered
under other conditions. demonstration of the enrqrne defect is a
viduals 107.1 U , but the overlap bet\\c.cn the groups \\:IS extenmore basic k a t u r e of the disease and thus prekrahle for diagno'itic
sive. a s seen in Figure I .
purpo\es. Furthermore, it is not known whether A A D G excretion
T o conclude, the results indicate that hornorygous del'iciencq of
is elev:~ted alreadq during the "s~lent" period between birth and
A A D G i t x . i . r . . ;~sp:trtqIgIucosani~nuria,can be reliably diagnosed
appearance of the first s y n s of the d ~ s e a s e .The age of onset of
using plasma. leukocyte\, o r separated lymphocytes. However.
clinical manifestations is known to be variable. ranging froni some
fur carrier detection. only separated I!mphocqtes allon it satisfacmonths t o 6 years (3). O u r results demonstrate that A G U patients
tory differentiation between hetero/)fotes ~ t n dnormal ~ndividuals. have undetectable o r minimal enzyme activity both in plasma.
buff) coat leukocytes. and separated lymphocytes. There was.
however, some overlap between iymphocyte i\i\liGabe aciivit! of
4C;U hornoz)gote\ and heterozy:otes. It is therefore advisable t o
A jeveral-fold increase in the ~ ~ c t i v i tof
q this e n r y m e \$as earller
aasaq for A A D G a s i both in plasma and in l!mphoc!trs
in the case
found in liver and brain tissue of A<iU patients (19). In the presof an asymptomatic newborn o r infant suspected of being affected
ent stud) a slight11 elevated le\,el of '2'-acetyl-fl-glucosaminidase
by A G U .
was demonstrated in both 1eukoc)tes and I>mphocytes, with a
A nractical and reliable carrier test of ACiU is needed. at least in
mean * S D of 18.0 * 5.3 and 20.3 i 14.6 nriiol/rnin/nip protein.
~ i n l a ' n d .where the disease is the most common known metabolic
respectively. T h e corresponding values In the control group n e r e
disorder leading to severe psychomotor retardation. T h e urinary
9.3
4.8 and 12.3 * 5 . 5 , respectlvel!. The activit! o f t h e enLqnit
excretron of AADCi in obligate heterozqgotes is not different from
in obligate hetero/ygotes was not different froni the control
normal individuals ( 3 ) and thus the only possibilit! to show
group.
heterorygosity is the demonstration of intermediate levels of
enzyme activity. T h e accuracy of our lynlphocyte assay in A G U
carrier identification appears to be adequate, a s the observed
frequency of carriers among the A G U rel:ttives was in agreement
O n the basis of the data presented above, A A D G a s e assa!
with the theoretical expectations. This is the optimal method of
using lymphocyte extracts was considered to fulfill the criteria of
evaluating a hetero/ygote detection method. a s mentioned by
a reliable heterorygote test. The assessment of the genotype was
Kaback and coworkers ( I I).
based on the distribution of values in obligate heteroqgotes: any
T h e incidence of A G U in Finland has been estimated roughly to
individual with A A D G a s e activity 3 S D o r more above the mean
be in the order of 1 in 26,000 (3), suggesting the overall carrier
of obligate heterorygotes ( > 8 7 . 2 U ) was designated normal. T h e
frequency of A G U t o be about 1 in 80. W e consider this too low t o
probability of a false negative carrier assessment with this crijustify large scale screening programs for heterozygote detection,
terion is 0.1% o r less. Individuals with enzyme activitk between
at least with the presently available methods. However, carrier
1.5 S D and 3 S D above the mean of obligate hetero/ygotes (from
detection a m o n g the relatives of known A G U patients is medi60.7 to 87.2 U ) were called "inconclusive." In these cases the
probability of a false negative carrier assessment is between 7.9% cally, economically, and psychologically advantageous, particubig. I . Activit) 01' 4-1.-aspart)Igl~cu\~
lani~nciirnldo h)drola\c in wparated I!n~phucyte~,Icuhocqtei. and pl:i\rn;~ 111 three htud) group\. ('o:
control individual\. Iff,: obl~gatehetcro/ygorcs (parent\ of p;~t~cnt\):
Ho:
horno/qgouh aspartylglucosaniinuria patient>.f:n/qme actlvltc I S exprc\bed
a \ nanomoles 01' N-iicct!lglucosarnine lihcrated per min per g honicate
protcln (or per ml pl:~\m;~).
Mean a c t ~ ~i SI)
~ t I~i giben in \cpar;itc bar.
628
AULA, RAIVIO, AND AUTlO
Table I . Carrier screeriing in close relniives qfaspnriylglucosan~iti~trin
( A G U )paiietrt.~hy !,~r~lphocj.tetrsi
Relationsh~p
to patient
Parent
S~bling
Offspring of siblingZ
Husbandlwife of sibling
Frequency
expected
No. tested
Carriers
Inconclusive
Noncarriers
29
31
29
22
5
I'
1
8
I .O
0.67
0.5
Unknown
II
Frequency
observed
I .O
9
5
9
0.7 1
0.45
0
' 4-L-Aspartylglucosamine amido hydrolase (AADGase) 76 U; repeat test not performed; probable carrier (see text).
One parent (sibling of AGU patient) is identified carrier, the other parent has normal AADGase activity.
AADGase 71 U; repeat test 123 U ; noncarrier.
Activity
with phytohemagglutinin stimulation a distinct differentiation was
possible (16). Also in glycogenosis type I 1 unstimulated lymphocytes were not suitable for the demonstration of the heterozygous
level of a-glucosidase activity, but in phytohemagglutininstimulated lymphocytes the heterozygotes could be distinguished
(7).
The lymphocytes constitute a more homogeneous cell population than leukocytes, which probably accounts for the better
distinction of AADGase activity in heterozygotes and normal
individuals. It is possible that enzyme assays in separated lymphocytes could offer possibilities for heterozygote detection in some
other lysosomal storage diseases in which serum or leukocytes are
not suitable for that purpose. For instance, in mannosidosis,
heterozygote and normal levels of n-mannosidase activity in serum
and leukocytes had considerable overlap, and distinction of
heterozygotes and normal individuals could be achieved only by
calculating ratios between a-mannosidase and other lysosomal
enzymes (15).
SUMMARY
0
I
i
8
S i b l i n g s of
AGUpatients
Offspring of
heterozygotes
Fig. 2. Activity of 4-L-aspartylglycosylamine amido hydrolase i n lymphocytes of close relatives of aspartylglucosam~nuria patients. Values
above the upper dotted line showing the mean act~vity t 3 SD of obligatc
heterorygotes Indicate normal genotype, values below mean heterotygote
activity + 1.5 S D indicate carrier genotype. Enryme activities falling
between the two lines comprise the "inconclusive" cases (see text).
larly since prenatal diagnosis and the possibility of preventive
abortion can be offered t o carrier-carrier matings.
The use of separated lymphocytes for heterozygote detection has
previously been reported only in Gaucher's disease (4). Distinction
of heterozygotes from normal individuals by P-glucosidase activity
was clearly better with Ficoll-separated lymphocytes than with
mixed leukocytes or granulocytes. Even with lymphocytes there
was some overlap between the enzyme activities in the two groups.
In a majority of inherited enzyme defects, particularly those of
lysosomal hydrolases, heterozygous levels of the enzyme activity
a r e demonstrable in an easier fashion using either serum (plasma)
a s in Tay-Sachs' disease and Krabbe's disease or buffy coat leukocytes a s in metachromatic leukodystrophy, Fabry's disease, Niemann-Pick's disease, Wolman's disease, and in some other genetic
enzyme defects (see Reference 6).
In acid phdsphatase deficiency, separated lymphocytes (glass
bead column separation) could not be reliably used t o distinguish
heterozygotes from normal individuals, but after 56 hr of culture
Assay of activity of 4-L-aspartylglycosylamine aniido hydrolase
in peripheral blood lymphocytes was found t o be reliable for
heterozygote detection of aspartylglucosaminuria. Enzyme levels
in buffy coat leukocytes, on the other hand, could not distinguish
heterozygotes from normal individuals. A G U patients had minimal or nondetectable activity of AADGase in lymphocyte^,
leukocytes. and plasma. A group of siblings of A G U patients and
of children of identified carriers were investigated with the
lymphocyte assay for AADGase. The observed number of heterozygotes was in good agreement with expectations based on
Mendelian probabilities. AADGase assay in separated lymphocytes offers possibilities for heterozygote screening among selected
high gene frequency populations.
REFERENCES AND NOTES
I. Aula, P.. Autio. S., Raivio. K.. and NlntB. V.: Detection of heteroqgotes for
aspartylglucosaminur~a(ACiU) in cultured fihroblasta. Humangcnctik, 2 5 . 307
(1974).
2. Aula, P., NlntB, V.. Laipio, M.-L., and Autio, S: Aspartylglucosaminur~n.
Deficiency of aspartylglucosamin~dasein cultured fibroblasts of patients and
their heterozygous parents. Clin. Genet.. 4. 297 (1973).
3. Autio, S . : Aspartylglucosam~nuria: Analysis of thirty-four patlent\. J . Ment
Defic Res Monogr. Ser., I: 1 (1972).
4. Beutler, E and Kuhl, W.: T h e diagnosis of the adult type of Gaucher's disease
and its carricr stare by demonstration of deficiency of 8-glucosidase activity in
peripheral blood leukocytes. J. Lah. Clin. Mcd.. 76. 747 (1970).
5. BByum, A,: Separation of lcukocyteb from blood and bone marrow. Scand. J .
Clln. Lab. Invest.. 2l(suppl 97): 1 (1968)
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(Academic Press, New York, 1973).
7. Hlrschhorn, K., Nadler, H . L., Walthe. W. 1 , Brown, B.. and Hirschhorn, R.:
Pompe's disease Detection of hetero~ygotes by lymphocyte stimulat~on.
Sclence, 166. 1632 (1969).
8. Humbel, R., and Marchal. C.: Screening test for aspartylglucosaminuria. J.
Pediat.. 84. 456 (1974).
9. Isenberg, J . N.. and Sharp, 1-1. L.: Aspartylglucosaminuria: Psychomotor
retardation masquerading as a mucopolysaccharidosis. J. Pediat.. 86: 713
(1975)
629
HURLER S Y N D R O M E
10. Jenner, F. A , . and Pollitt. R . J.. Large q u a n t ~ t i e of
s 2-acelnm1do-I-(betn-L-asparlamido)-1,2-d~deoxyglucoseIn the urine of mentally retarded siblings. Bioohem.
J.. 103. 48 (1967)
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t o the control and prevention of Tay-Sachs d ~ s e a s e .In: A. G . Steinberg and A.
G . Bearn: Progress In Medical Genetics, Vol. X , p. 103 (Grune and Stratton.
New York. 1974).
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measurement with Folin phenol reagent. J . Biol. Chem., 190: 265 (1951).
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15. Masson, P.. Lundblad, A , and Autio, S . Mannosldosls: Detection o r the disease
and of heterozygotes uslng serum and leukocytes Blochem. B~ophys.Res.
Commun., 56: 296 (1974).
16. Nadler, H L.: Acid phosphatasc deficiency. In: H . G. Hers and F. Van Hoof:
Lysosomes and Storage Diseases ( A c a d e m ~ cPress, New York. 1973).
17. Palo. J., and Mattson, K.: Chromatographic isolation of 2-acetamido-I-(beta1-Laspartamid0)-1.2-dideoxyglucose from urine. J . Chromatogr., 50. 534 (1970).
18. Palo, J . . Pollitt, R . J.. Pretty. K M.. and Savolainen, H: Glycoa$paragine
metabol~tes in patients with aspartylglucosam~nur~aComparison between
English and Finnlsh patients w ~ t hspecial reference to storage materials. Clin.
Chim. Acta. 47- 69 (1975).
19. Palo, J . . R ~ e k k l n e n ,P.. Arstila. A. U.. Autio. S., and Kivimaki, T.: Aspartylglucosam~nuria.I I. Biochemical s t u d ~ e son brain, liver, kidney and spleen. Acta
Neuropathol. (Berlin), 20. 217 (1972).
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fluid: Failure to detect the heterozygous state for aspartylglucosam~nuria.Clin
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22. This study was supported by grants from Sigrid Juselius Foundation and the
National Research Council for Medlcal Sciences. Finland.
23. Requests for reprints should be addressed to. P. Aula. M D.. Children's Hospital.
00290 Helsinki 29 (Finland).
24. Accepted for publication January 14, 1976
Prir~redin U.S.A.
Copyright O 1976 International Pediatric Research Foundations, Inc.
Pediat. Res. 10: 629-632 (1976)
/3-Galactosidase
heterozygote
Hurler syndrome
a-L-iduronidase
leukocytes
Hurler Syndrome: a-L- Iduronidase Activity in
Leukocytes as a Method for
Heterozygote Detection
REBECCA S. WAPPNER'Z9' A N D IRA K . BRAND7
Departn~entsof Pediatrics and Medical Geneiics, Indiana University School of Medicine, Indianapolis, Indiana, U S A
Extract
cu-L-lduronidase activity and P-galactosidase activity were determined in mixed leukocyte preparations in 10 families in which the
Hurler syndrome had occurred. Affected patients, heterozygotes,
and normal subjects were clearlg distinguished by a-l-iduronidase
activity alone. Patients had &3%, obligate heterozygotes 19-6070,
and normal subjects 83-121% of the mean normal activity. There
was no oversap between heterozygotes and normal subjects. Although the mean a-l-iduronidase to p-galactosidase ratio was
significantly lowered in heterozygotes when compared with that of
normal subjects, appreciable overlap was noted between the two
groups.
Speculation
Carriers and affected individuals for Hurler syndrome can be
reliably detected using mixed leukocyte preparations for the
determination of 0-L-iduronidase activity. As technology improves
enzyme assays will become reliable enough for determination of
heterozygotes for an increasing number of disorders, thus enabling
more thorough and accurate genetic counseling.
The Hurler and Scheie syndromes (rnucopolysaccharidosis IH
and IS, respectively) are autosomal-recessive disorders characterized by deficient activity of the lysosomal enLyme (ec. 3.2.1.76)
a-L-iduronidase (2, 9, 14, 16). This enzymatic defect results in
generalized lysosomal accumulation and excessive urinary excretion of partially degraded mucopolysaccharides containing
iduronic acid residues: heparan sulfate and dermatan sulfate
(6, 8).
Using phenyl-cu-L-iduronide as a substrate, cu-L-iduronidase
activity has been demonstrated to be reduced in leukocytes, serum, and cultured skin fibroblasts from obligate neterozygotes
for the Hurler and Scheie syndronies (9, 14). IIall and Neufcld
(9) demonstrated that the cu-L-iduronidase activity of Hurler
heterozygous cdtured skin fibroblasts varied from 20 t o 95% of
normal, with a mean value of 46%. This range overlapped that of
material obtained from normal control subjects. Liem and Hooghwinkel (14) demonstrated a-L-iduronidase activity of 30 and 53%
of normal control subjects in mixed leukocyte preparations from
two Hurler obligate heterozygotes. Singh et al. (17) have reported cu-L-iduronidase activity in serum o t one Hurler obligate
heterozygote and one Scheie obligate heterozygo~eof I2 and 19%
of normal, respectively.
Although it is known that other lysosomal enzymes differ in
specific activity among different leukocyte cell types (3, 12) and
that factors resulting in a change from the normal differential may
affect the reliability of carrier detection when mixed leukocyte
preparations are used, no data have been published concerning the
relative distribution of cu-L-iduronidase among various leukocyte
cell types.
We report a modified assay for a-L-iduronidase activity which
employs mixed leukocyte preparations. The assay was used for
controlled studies of families in which the Hurler syndrome has
occurred and appears to be sufficiently accurate and precise to
enable the detection of heterozygosity with greater than 95% confidence.