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Identification of tay-sachs disease carriers by acrylamide gel electrophoresis
CLINICA
CHIMICA
ACTA
IDENTIFICATION GEL
397
OF TAY-SACHS
JOAX FRIEDLXND, LARRY AND BRUNO W. VOLK Isaac
DISEASE
CARRIERS
BY
ACRYLAMIDE
ELECTROPHORESIS
Albert
Ic’esearch Institute
Brooklyn.
N. Y. 11203
(Received
December
SCHNECK,
of the
Kingsbvook
ABRAHAM Jewish
SAIFER,
Mrdzcal
MOHAMAD
POURFAR
Center,
(U.S.A.) gth,
1969)
SUMMAKY
I.
For carrier detection
in the general population,
a simple, rapid quantitative
acrylamide gel electrophoretic technique has been developed which employs a fluorogenic (umbelliferyl) substrate and automatic fluorimetric scanning of the gels. 2. With the procedure the leukocytes of 6 Tay-Sachs disease carriers had a range of N-acetylhexosaminidase A values of 33-52% (mean = 49”/ A) while the normal range was 62-760,; (mean = 664: A) with no overlap between the two groups. 3. The presence of two isoenzymes, A and B, of N-acetyl-/5-n-hexosaminidasc (P-2-acetamido-z-deoxy-D-acetamidodeoxyglucohydrolase, EC 3.2.1.30) in the leukocytes of normal individuals (66:; A/34:,; B) an d a b-sence of the A form in leukocytes
of 12 Tay-Sachs
disease patients
has been confirmed.
Robinsen and Stirling1 separated saminidase (P-z-acetamido-z-deoxy-n-glucose
two
isoenzymes of N-acetyl-fl-D-glucoacetamidodeoxyglucohydrolase, EC
3.2.1.30) from human spleen by means of starch gel electrophoresis. Component A and Component B both showed activity toward substrates (glycosides) containing A:-acetylgalactosamine as well as to Wacetylglucosamine but with a glucosaminidase/ galactosaminidase ratio of 8.6. Hexosaminidase activity has been found in many other tissueW and biological fluid..~~95.Okada and O’BrienG, using starch gel electrophoresis and quantitative densitometry of the color developed with the Naphthol AS-BI-derivative of the P-r-x-acetylhexosaminide, found that the A component was absent in leukocytes and other tissues from patients with Tay-Sachs disease. Leukocytes from 5 adult control subjects contained both the A and B isoenzymes in the approximate ratio of 73”/0 A/27::; B6. Since Tay-Sachs disease carriers are widely spread among the Jewish population’, it would be desirable to have a simple, rapid, quantitative procedure for screening purposes. The quantitative acrylamide gel electrophoretic technique described in this paper, which employs the sensitive fluorogenic substrate (4-methylumbelliferyl-Wacetyl-@-glucosaminide), has many technical advantages over the starch gel electrophoretic-calorimetric quantitative procedure for the mass screening
FRIEDLASD
398
et d.
required for carrier identification. With this technique the leukocytes of Tay-Sachs disease carriers gave an average N-acetylhexosaminidase ratio of 4g’& A/51% B, the average value for the control series was 667; A/340/b B while no activity of I\‘-acetylhexosaminidase A was detected in the Tay-Sachs disease children. MATERIALS
AND
METHODS
(I) Lower buffey fey electroplaoresis. The stock buffer was made by dissolving 60.5 g tris-hydroxymethylaminomethane (Tris; Sigma Chemical Co., St. Louis, MO.) in 250 ml of I M HCl and diluted to 500 ml with distilled water. The stock is diluted I : IO with cold distilled water (4’) just before use. The pH is 8.1 f 0.2 at 25”. (2) Upper buffeer fey electrophoresis. The stock buffer was prepared by dissolving 20 g Tris and 36.1 g Tricine (Sigma) in distilled water and diluted to 500 ml. The pH of the solution is 8.1 at 25’. The stock is diluted I :IO with cold distilled water (4’) just before use. (3) Preparatiox of gels. The one-step acrylamide gels, without spacers, were prepared by a modification of the method of Ornsteins and Davis9 using 6 ml each of the following solutions: (a) A solution of 1.8 31 Tris was prepared by dissolving 2.065 g Tris in a 16.8 ml I M HCl and diluted to IOO ml with distilled water. (b) 30 g acrylamide and 0.8 g methylene bis-acrylamide (Distillation Products Industries, Rochester, N.Y.) were dissolved in distilled water and diluted to IOO ml. (c) A 5 rnM stock solution of flavin mononucleotide (FMN ; Sigma) was diluted I :IOO with distilled water. Absorbance at 450 nm = 0.60. (d) Distilled water. To the total volume (24 ml) of the above solutions, pH 6.3, 0.15 ml of a I: IO dilution
of S,
A’, A”“, X’-tetramethylenediamine
(Distillation
Products
Industries)
was added and the mixture de-aerated in a suction flask to prevent bubble formation during polymerization. About 0.25 ml of 40”‘;~ sucrose was added to each of the rubber stopper wells mounted on a wooden block. The gel tubes (7.5 cm x 0.7 cm outer diameter) were then inserted firmly into each rubber stopper. The gel mixture, as prepared above, was layered carefully over the sucrose and the tubes filled to the top. A drop of water was placed on the top of each gel and the gels photopolymerized by illuminating the tubes with a 15-If'fluorescent light for about I 11.The longer time led to further breakdown of the FMN by photoreduction and thus less fluorescent conta mination in the gels. Leukocytes were prepared according to the method of Kampine rt &.I”. The leukocyte volume required for hexosaminidase isoenzyme was based upon the total hexosaminidase activity of the preparation as determined with the 4-methylurnl~elliferyl-2-acetamido-2-deoxy-P_u-glucopyranoside (Koch-Light Co., Colnbrook, England) substrate at pH 4.5 at 37’ (ref. II). An activity of S.O.IO-' pmole of +metll!~lumbelliferone released per 11was used for each gel. For convenience 0.3 ml of l~ukocpte solution diluted with O.OI RI phosphate-saline at pH 7.0 to contain a total activity of 2.4. IO--~/imole of 4-methylumbelliferone released per 11was mixed with 0.15 ml of 40’:; sucrose and 0.15 ml of this solution is applied to the gel. The gel tubes Were first washed free of sucrose with distilled water and inserted, gel side down, into the holes of the upper reservoir of the Canalco electrophoresi~ apparatus. The tubes were completely immersed in the lower buffer, cooled to 4’ After the leukocyte solution was applied to each tube the upper buffer was layered
IDENTIFICATION OF TAY-SACHS DISEASE CARRIERS
399
over the sample and then poured into the upper reservoir. of bromphenol
2 drops of a o.I~/, solution
blue was added to the upper buffer as a tracking
dye. The gels were
run at 4” at 1.0 mA per tube for 5 min and then for I h and 25 min at 2.5 mA per tube migrating toward the anode. The gels were removed from the tubes by loosening them with a syringe, equipped with a No. 22, x-inch needle, filled with cold 0.01 bIvlphosphate buffer, pH 6.0, while submerged in the buffer contained in a glass dish kept in a tray of ice. For the assay of hexosaminidase isoenzymes, the gels were placed on filter paper soaked with 0.1 M sodium citrate buffer, pH 4.5. The substrate consisted of a solution of 7.5 mg of 4-methylumbelliferyl-z-acetamido-z-deoxy-~-~-glucopyranoside dissolved in 12.5 ml of 0.1 RI sodium citrate
(b)
(a)
(cl
(d)
buffer, pH 4.5. This solution was spread
(e)
Fig. I. Fluorimetric tracings of acrylamide gel electrophoretic umbelliferylglycoside patterns of known mixtures of fi-D-h:-acetylhexosaminidase A and B isoenzymes. (a) 100% -4; (b) 509’0 A/507{, B; (c) 333; A/67:; B; ((1) zoo/, A/800,b B; (c) 100”; B. The curves were obtained with a Turner fluorimeter scanning unit using 7-60 (primary) and 3-73 (secondary) filters, at 30 x sensitivity with a 1.5-mm slit width coupled to a Bristol (Technicon) recorder. The experimental values obtaincd are listed above each curve.
TABLE
I
PERCENTAGEDISTRIBUTIONOF ~-D-~-AcETYLI~ExoS.~~II~ID.~SE
ISOENZYMES 2% AXD B IK LEUKOOFTAY-SACHST~I~~.~~EPATIENT~A~DTHEIRPARENT~(CARRIERS)
CYTE~OFNORMALSUBJECT~AND
B-n-A’-Acetylhexosamivz~dase
Subject
A (‘%
Normal,
Carrier,
Tay-Sachs
of total)
activity .____--
B (‘0 of total)
R.P.
76.0
1T.C. N.F.
70.4 64.8
n1.s. S.K. M.P. A.1’. B.H.
65.2 66.6 6X.6 62.0 63.0
34.8 33.-l 31.2 38.0 37.0
F.S. 11,s. F.T.
46.9
53.1
52.0
&O jj.0
1I.T.
46.6
P.H. M.H.
33.0
53.1 67.0
46.0
54.0
0.0
100.0
(I 2 cases)
45.0
24.0 29.6 35.’
C/in. Chum. Acta,
28 (1970) 397-402
FRIEDLASD Ct cd.
400
Fig. L. ITppcr : Fluorimetric tracings of /i-u-S-acetylhexosaminitlase 1’1and I%isoenzymc thstribution from leukocytes of (a) ;L normal atlult; (b) a Tay-Sachs disease child (homozygotr) ; (c) a
father of TX)--Sachs diseaw child (heterozygote) ; (cl) a mother of Tay-Sachs discasc child (hcterozygote), Curves were obtained exactly as described in the legend of Fig. T. Lower: Corresponding acrylamitle ~~‘1swith fluortscent lx~ntls photographed under ultraviolet light.
evenly r.5 h moist Each
over the gels on the filter paper placed in a glass dish and incubated at 3:” for so as to develop fluorescent bands at the site of activity. The gels were kept at 4’ until the readings were made. Readings should be made the same day. gel was placed in a small rectangular quartz cell containing sufficient buffer
to prevent air bubbles from forming. The cell was then mounted on the door of the Turner automatic scanning unit and the scan of the fluorescent bands recorded on a Bristol recorder. Quantitation of the area under each curve was performed by triangulation and the results expressed as a percentage of the total area. Relatively pure hexosaminidase ,4 was isolated from normal brain and hexosaminidase B from Tay-Sachs disease brain and the enzymatic activity concentrated more than so-fold by a combination of salt fractionation, isoelectric pH precipitation, Sephadex chromatography and ultrafiltration, the details of which will be published later. Known mixtures of these purified isoenzymes were prepared and run with the fluorescent gel procedure as described above. The results obtained are illustrated in Fig.
I.
Leukocytes obtained from the blood of 8 normal adults, 6 carriers (3 sets of Tay-Sachs disease parents) and 12 Tay-Sachs disease children were run with the quantitative procedure and the results obtained are given in Table I. Typical enzyme patterns for normal, carrier and Tay-Sachs disease leukocytes are shown in Fig. 2. RESULTS
AND
DISCUSSIOK
A number of investigators’2313 have postulated that the probable metabolic defect in Tay-Sachs disease resides in a defective or missing enzyme which normally Clzn. Chinz. Acta,
28 (1970) 397-402
IDENTIFICATION OF TAY-SACHS DISEASE CARRIERS converts
one type of ganglioside
minal N-acetylgalactosamine
(Gbr,) into another
residue.
4or (Gnln) Z&Zhydrolysis
of the ter-
However,
analysis of total @-u-A’-acetylhexosdisease 14y15showed elevated values of this
aminidase brain tissue levels in Tay-Sachs enzyme as compared to normal controls. Robinson and Stirling’ using starch gel electrophoresis and DEAE-cellulose chromatography showed that N-acetyl-b-glucosaminidase existed in biological fluids and tissues as two isoenzymes, A and B. Okada and O’BrienG, by means of starch gel electrophoresis and densimetric analysis, found that the A component was absent in various Tay-Sachs disease tissues. These authors indicated that the heterozygotes (carriers) should have leukocyte S-acetylhexosaminidase A values which were intermediate between those of Tag;-Sachs disease patients and normal controls. The detection of carriers in the general population requires a simple, rapid and accurate quantitative procedure suitable for mass screening purposes. Using the quantitative acrylamide gel electrophoretic-fluorimetric paper we have confirmed the basic findings of Okada
technique described in this and O’Brien6 with respect to
the absence of the A form of N-acetylhexosaminidase from the tissues of patients with Tay-Sachs disease (Fig. 2). We have also found that the difference between N-acetylhexosaminidase A levels of the heterozygotes (parents) and normal controls can be in the order of only IO 9/o(Table I). The data obtained for 6 carriers (3 sets of Tay-Sachs
disease parents) gave an average leukocyte enzyme value of 4q”,, A, with as compared to a normal control group (8 adults) whose average a range of 33-52:;, value was 6674 A with a range of 62-76:: (Fig. z and Table I). The use of known mixtures of pure A and B isoenzymes proves the acrylamide gel procedure to have an accuracy between I and 20/, of the predicted values (Fig. I). Further studies are presently in progress to statistically validate these preliminary findings. The carrier test for Tay-Sachs disease described here is more sensitive and specific than the one based upon a fructose-r-phosphate aldolase deficiency16. Studies are now in progress to validate the hypothesis that a Tay-Sachs disease fetus can he detected amniotic
by the complete fluidIT.
absence of the A form of the enzyme in exfoliated
cells from
ACKKOWLEDGEMENTS The authors wish to acknowledge the technical assistance of Mrs. Guta Perle with the gel electrophoretic-fluorimetric analyses. We are also grateful to Mrs. Lillian Salowitz for the typing and editing of the manuscript. This study was supported National Tay-Sachs Association.
by Grants
from the N.I.H.
(XB285-C17)
and the
RBFERENCES I D. RORI~XOEANDJ. L. STIRLING, Biochrwz. J., 107 (1968) 321. z N. DANCE, R. G. PRICE, D. ROBIKSON AND J. L. STIRLING, Clin. Chim. Acta, 21 (1969) 3 J. COXCHIE,J. FIXDLAY ANDG. A. LEVVY, Biochem. J., 61 (1959) 318. 4 J. W. WOOLENANDP. TURNER. Clin. Chim. Acta, 12 (1965) 671, 5 J. C. CAYGILL AND F. R. JEVONS, Clin. Chins. Acto, 13 (1966) 61. 6 S. OKADA AND J, S. O’RRIEK, Science, 165 (1969) 698. 7 N. C. hlYRIAKTHOPOULOS AND S. Nl. ARONSON, in S. M. AROXOK AND B. W.VOLK. Inborn orders of Sphingolipid Metabolism, Pergamon Press, New York, 1967, p. 431. Clin. Chim.
Acta,
ZQ (1970)
189.
Dis-
397-402
FRIEDLAND
402 8 9 IO II 12 13 14 15 16 17
Pt d.
L. ORNSTEIN, Ann. N.Y. Acad. Sci., 121 (1964) 321. B. J, DAVIS, Ann. N.Y. .4cad. Sci., IZI (‘964) 404. P. J. KAMPIXE, R. 0. BRADY, J. N. KANFER, M. FELD ANI) D. SHAPIRO, Science, 155 (1966) 86. D. H. LEABACK AND P. G. WALKER, Bi~chem. J., 78 (1961) IsI. L. SVEKKERHOLM, in S. M. AROXSO~- +.ND B. W. VOLK, Inbovlz Disorders of Sphingollpzd .11&nholism, f-‘ergamon Press, New York, 1967, p. 169. L. SCHNECK, B. W. VOLK AND A. SAIFER, Am. j. Med., 46 (1969) ‘45. K. SAXDHOFF, 77. ANDREAE AND H. JATZKEWITZ, Pathol. Euvopaea, 3 (1968) 278. L. SCHXECK, J. FRIEDLASD, M. POUFAR, .-\. SAIFER AXL) B. \V. \‘OLK, Pvoc. Sm. I:‘x@. Hiol. ,Wed., in the press. S. M. ARONSON, G. PERLE, A. SAIFER ASI) B. W. YOLK, Pvoc. Sm. Exptl. Biol. Ued., I II (1962) 664. L. SCIINXK, J. FRIEDLAXD, (‘. V:~LEKTI, RI. ADACHI, D. AI\ISTERDARIASU B. \v. POLIO, La?zcet. in the press.
Clin. Chinz.
z4Cta,
28
(1970) 397-40’
CHIMICA
ACTA
IDENTIFICATION GEL
397
OF TAY-SACHS
JOAX FRIEDLXND, LARRY AND BRUNO W. VOLK Isaac
DISEASE
CARRIERS
BY
ACRYLAMIDE
ELECTROPHORESIS
Albert
Ic’esearch Institute
Brooklyn.
N. Y. 11203
(Received
December
SCHNECK,
of the
Kingsbvook
ABRAHAM Jewish
SAIFER,
Mrdzcal
MOHAMAD
POURFAR
Center,
(U.S.A.) gth,
1969)
SUMMAKY
I.
For carrier detection
in the general population,
a simple, rapid quantitative
acrylamide gel electrophoretic technique has been developed which employs a fluorogenic (umbelliferyl) substrate and automatic fluorimetric scanning of the gels. 2. With the procedure the leukocytes of 6 Tay-Sachs disease carriers had a range of N-acetylhexosaminidase A values of 33-52% (mean = 49”/ A) while the normal range was 62-760,; (mean = 664: A) with no overlap between the two groups. 3. The presence of two isoenzymes, A and B, of N-acetyl-/5-n-hexosaminidasc (P-2-acetamido-z-deoxy-D-acetamidodeoxyglucohydrolase, EC 3.2.1.30) in the leukocytes of normal individuals (66:; A/34:,; B) an d a b-sence of the A form in leukocytes
of 12 Tay-Sachs
disease patients
has been confirmed.
Robinsen and Stirling1 separated saminidase (P-z-acetamido-z-deoxy-n-glucose
two
isoenzymes of N-acetyl-fl-D-glucoacetamidodeoxyglucohydrolase, EC
3.2.1.30) from human spleen by means of starch gel electrophoresis. Component A and Component B both showed activity toward substrates (glycosides) containing A:-acetylgalactosamine as well as to Wacetylglucosamine but with a glucosaminidase/ galactosaminidase ratio of 8.6. Hexosaminidase activity has been found in many other tissueW and biological fluid..~~95.Okada and O’BrienG, using starch gel electrophoresis and quantitative densitometry of the color developed with the Naphthol AS-BI-derivative of the P-r-x-acetylhexosaminide, found that the A component was absent in leukocytes and other tissues from patients with Tay-Sachs disease. Leukocytes from 5 adult control subjects contained both the A and B isoenzymes in the approximate ratio of 73”/0 A/27::; B6. Since Tay-Sachs disease carriers are widely spread among the Jewish population’, it would be desirable to have a simple, rapid, quantitative procedure for screening purposes. The quantitative acrylamide gel electrophoretic technique described in this paper, which employs the sensitive fluorogenic substrate (4-methylumbelliferyl-Wacetyl-@-glucosaminide), has many technical advantages over the starch gel electrophoretic-calorimetric quantitative procedure for the mass screening
FRIEDLASD
398
et d.
required for carrier identification. With this technique the leukocytes of Tay-Sachs disease carriers gave an average N-acetylhexosaminidase ratio of 4g’& A/51% B, the average value for the control series was 667; A/340/b B while no activity of I\‘-acetylhexosaminidase A was detected in the Tay-Sachs disease children. MATERIALS
AND
METHODS
(I) Lower buffey fey electroplaoresis. The stock buffer was made by dissolving 60.5 g tris-hydroxymethylaminomethane (Tris; Sigma Chemical Co., St. Louis, MO.) in 250 ml of I M HCl and diluted to 500 ml with distilled water. The stock is diluted I : IO with cold distilled water (4’) just before use. The pH is 8.1 f 0.2 at 25”. (2) Upper buffeer fey electrophoresis. The stock buffer was prepared by dissolving 20 g Tris and 36.1 g Tricine (Sigma) in distilled water and diluted to 500 ml. The pH of the solution is 8.1 at 25’. The stock is diluted I :IO with cold distilled water (4’) just before use. (3) Preparatiox of gels. The one-step acrylamide gels, without spacers, were prepared by a modification of the method of Ornsteins and Davis9 using 6 ml each of the following solutions: (a) A solution of 1.8 31 Tris was prepared by dissolving 2.065 g Tris in a 16.8 ml I M HCl and diluted to IOO ml with distilled water. (b) 30 g acrylamide and 0.8 g methylene bis-acrylamide (Distillation Products Industries, Rochester, N.Y.) were dissolved in distilled water and diluted to IOO ml. (c) A 5 rnM stock solution of flavin mononucleotide (FMN ; Sigma) was diluted I :IOO with distilled water. Absorbance at 450 nm = 0.60. (d) Distilled water. To the total volume (24 ml) of the above solutions, pH 6.3, 0.15 ml of a I: IO dilution
of S,
A’, A”“, X’-tetramethylenediamine
(Distillation
Products
Industries)
was added and the mixture de-aerated in a suction flask to prevent bubble formation during polymerization. About 0.25 ml of 40”‘;~ sucrose was added to each of the rubber stopper wells mounted on a wooden block. The gel tubes (7.5 cm x 0.7 cm outer diameter) were then inserted firmly into each rubber stopper. The gel mixture, as prepared above, was layered carefully over the sucrose and the tubes filled to the top. A drop of water was placed on the top of each gel and the gels photopolymerized by illuminating the tubes with a 15-If'fluorescent light for about I 11.The longer time led to further breakdown of the FMN by photoreduction and thus less fluorescent conta mination in the gels. Leukocytes were prepared according to the method of Kampine rt &.I”. The leukocyte volume required for hexosaminidase isoenzyme was based upon the total hexosaminidase activity of the preparation as determined with the 4-methylurnl~elliferyl-2-acetamido-2-deoxy-P_u-glucopyranoside (Koch-Light Co., Colnbrook, England) substrate at pH 4.5 at 37’ (ref. II). An activity of S.O.IO-' pmole of +metll!~lumbelliferone released per 11was used for each gel. For convenience 0.3 ml of l~ukocpte solution diluted with O.OI RI phosphate-saline at pH 7.0 to contain a total activity of 2.4. IO--~/imole of 4-methylumbelliferone released per 11was mixed with 0.15 ml of 40’:; sucrose and 0.15 ml of this solution is applied to the gel. The gel tubes Were first washed free of sucrose with distilled water and inserted, gel side down, into the holes of the upper reservoir of the Canalco electrophoresi~ apparatus. The tubes were completely immersed in the lower buffer, cooled to 4’ After the leukocyte solution was applied to each tube the upper buffer was layered
IDENTIFICATION OF TAY-SACHS DISEASE CARRIERS
399
over the sample and then poured into the upper reservoir. of bromphenol
2 drops of a o.I~/, solution
blue was added to the upper buffer as a tracking
dye. The gels were
run at 4” at 1.0 mA per tube for 5 min and then for I h and 25 min at 2.5 mA per tube migrating toward the anode. The gels were removed from the tubes by loosening them with a syringe, equipped with a No. 22, x-inch needle, filled with cold 0.01 bIvlphosphate buffer, pH 6.0, while submerged in the buffer contained in a glass dish kept in a tray of ice. For the assay of hexosaminidase isoenzymes, the gels were placed on filter paper soaked with 0.1 M sodium citrate buffer, pH 4.5. The substrate consisted of a solution of 7.5 mg of 4-methylumbelliferyl-z-acetamido-z-deoxy-~-~-glucopyranoside dissolved in 12.5 ml of 0.1 RI sodium citrate
(b)
(a)
(cl
(d)
buffer, pH 4.5. This solution was spread
(e)
Fig. I. Fluorimetric tracings of acrylamide gel electrophoretic umbelliferylglycoside patterns of known mixtures of fi-D-h:-acetylhexosaminidase A and B isoenzymes. (a) 100% -4; (b) 509’0 A/507{, B; (c) 333; A/67:; B; ((1) zoo/, A/800,b B; (c) 100”; B. The curves were obtained with a Turner fluorimeter scanning unit using 7-60 (primary) and 3-73 (secondary) filters, at 30 x sensitivity with a 1.5-mm slit width coupled to a Bristol (Technicon) recorder. The experimental values obtaincd are listed above each curve.
TABLE
I
PERCENTAGEDISTRIBUTIONOF ~-D-~-AcETYLI~ExoS.~~II~ID.~SE
ISOENZYMES 2% AXD B IK LEUKOOFTAY-SACHST~I~~.~~EPATIENT~A~DTHEIRPARENT~(CARRIERS)
CYTE~OFNORMALSUBJECT~AND
B-n-A’-Acetylhexosamivz~dase
Subject
A (‘%
Normal,
Carrier,
Tay-Sachs
of total)
activity .____--
B (‘0 of total)
R.P.
76.0
1T.C. N.F.
70.4 64.8
n1.s. S.K. M.P. A.1’. B.H.
65.2 66.6 6X.6 62.0 63.0
34.8 33.-l 31.2 38.0 37.0
F.S. 11,s. F.T.
46.9
53.1
52.0
&O jj.0
1I.T.
46.6
P.H. M.H.
33.0
53.1 67.0
46.0
54.0
0.0
100.0
(I 2 cases)
45.0
24.0 29.6 35.’
C/in. Chum. Acta,
28 (1970) 397-402
FRIEDLASD Ct cd.
400
Fig. L. ITppcr : Fluorimetric tracings of /i-u-S-acetylhexosaminitlase 1’1and I%isoenzymc thstribution from leukocytes of (a) ;L normal atlult; (b) a Tay-Sachs disease child (homozygotr) ; (c) a
father of TX)--Sachs diseaw child (heterozygote) ; (cl) a mother of Tay-Sachs discasc child (hcterozygote), Curves were obtained exactly as described in the legend of Fig. T. Lower: Corresponding acrylamitle ~~‘1swith fluortscent lx~ntls photographed under ultraviolet light.
evenly r.5 h moist Each
over the gels on the filter paper placed in a glass dish and incubated at 3:” for so as to develop fluorescent bands at the site of activity. The gels were kept at 4’ until the readings were made. Readings should be made the same day. gel was placed in a small rectangular quartz cell containing sufficient buffer
to prevent air bubbles from forming. The cell was then mounted on the door of the Turner automatic scanning unit and the scan of the fluorescent bands recorded on a Bristol recorder. Quantitation of the area under each curve was performed by triangulation and the results expressed as a percentage of the total area. Relatively pure hexosaminidase ,4 was isolated from normal brain and hexosaminidase B from Tay-Sachs disease brain and the enzymatic activity concentrated more than so-fold by a combination of salt fractionation, isoelectric pH precipitation, Sephadex chromatography and ultrafiltration, the details of which will be published later. Known mixtures of these purified isoenzymes were prepared and run with the fluorescent gel procedure as described above. The results obtained are illustrated in Fig.
I.
Leukocytes obtained from the blood of 8 normal adults, 6 carriers (3 sets of Tay-Sachs disease parents) and 12 Tay-Sachs disease children were run with the quantitative procedure and the results obtained are given in Table I. Typical enzyme patterns for normal, carrier and Tay-Sachs disease leukocytes are shown in Fig. 2. RESULTS
AND
DISCUSSIOK
A number of investigators’2313 have postulated that the probable metabolic defect in Tay-Sachs disease resides in a defective or missing enzyme which normally Clzn. Chinz. Acta,
28 (1970) 397-402
IDENTIFICATION OF TAY-SACHS DISEASE CARRIERS converts
one type of ganglioside
minal N-acetylgalactosamine
(Gbr,) into another
residue.
4or (Gnln) Z&Zhydrolysis
of the ter-
However,
analysis of total @-u-A’-acetylhexosdisease 14y15showed elevated values of this
aminidase brain tissue levels in Tay-Sachs enzyme as compared to normal controls. Robinson and Stirling’ using starch gel electrophoresis and DEAE-cellulose chromatography showed that N-acetyl-b-glucosaminidase existed in biological fluids and tissues as two isoenzymes, A and B. Okada and O’BrienG, by means of starch gel electrophoresis and densimetric analysis, found that the A component was absent in various Tay-Sachs disease tissues. These authors indicated that the heterozygotes (carriers) should have leukocyte S-acetylhexosaminidase A values which were intermediate between those of Tag;-Sachs disease patients and normal controls. The detection of carriers in the general population requires a simple, rapid and accurate quantitative procedure suitable for mass screening purposes. Using the quantitative acrylamide gel electrophoretic-fluorimetric paper we have confirmed the basic findings of Okada
technique described in this and O’Brien6 with respect to
the absence of the A form of N-acetylhexosaminidase from the tissues of patients with Tay-Sachs disease (Fig. 2). We have also found that the difference between N-acetylhexosaminidase A levels of the heterozygotes (parents) and normal controls can be in the order of only IO 9/o(Table I). The data obtained for 6 carriers (3 sets of Tay-Sachs
disease parents) gave an average leukocyte enzyme value of 4q”,, A, with as compared to a normal control group (8 adults) whose average a range of 33-52:;, value was 6674 A with a range of 62-76:: (Fig. z and Table I). The use of known mixtures of pure A and B isoenzymes proves the acrylamide gel procedure to have an accuracy between I and 20/, of the predicted values (Fig. I). Further studies are presently in progress to statistically validate these preliminary findings. The carrier test for Tay-Sachs disease described here is more sensitive and specific than the one based upon a fructose-r-phosphate aldolase deficiency16. Studies are now in progress to validate the hypothesis that a Tay-Sachs disease fetus can he detected amniotic
by the complete fluidIT.
absence of the A form of the enzyme in exfoliated
cells from
ACKKOWLEDGEMENTS The authors wish to acknowledge the technical assistance of Mrs. Guta Perle with the gel electrophoretic-fluorimetric analyses. We are also grateful to Mrs. Lillian Salowitz for the typing and editing of the manuscript. This study was supported National Tay-Sachs Association.
by Grants
from the N.I.H.
(XB285-C17)
and the
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