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Nicked free β-subunit of human chorionic gonadotropin: A potential new marker for Down syndrome screening
Nicked free 13-subunit of human chorionic gonadotropin: A potential new marker for Down syndrome screening Siegfried Rotmensch, M D , a'~ Marco Liberati, MD," Andrew Kardana, PhD," Joshua A. Copel, MD," Zion Ben-Rafael, MD, ~ and Laurence A. Cole, PhD b New Haven, Connecticut, and Petah Tiqva, Israel OBJECTIVE: Our purpose was to investigate maternal serum levels of nicked free ~-subunit in normal and Down syndrome pregnancies. STUDY DESIGN: Serum specimens were obtained from 64 karyotypically normal and 6 Down syndrome pregnancies before amniocentesis. Two immunoenzymometric assays for the ~-subunit of human chorionic gonadotropin were used to determine the level of free #-subunits with peptide linkage breaks ("nicks") between residues 43 to 48. RESULTS: The mean level of nicked free I~-subunit in Down syndrome was 4.76 multiples of the median, which was significantly elevated compared with normal controls (1.07 multiples of the median, p < 0.05). In 5 of 6 cases levels were _>2 multiples of the median, with a range 1.96 to 15.43 multiples of the median, which was 2- to 3-fold higher than the regular free 13-subunit assay (mean level 1.53 multiples of the median, range 0.70 to 3.10 multiples of the median). In one case no nicked free-~ subunits were detectable. CONCLUSION: Our preliminary data indicate that nicked free ~-subunit of human chorionic gonadotropin might be a sensitive marker for Down syndrome screening. (AM J OBSTETGYNECOL1996;174:609-11 .)
Key words: H u m a n chorionic gonadotropin, Down syndrome, serum screening
H u m a n chorionic gonadotropin (hCG) and its free ~-subunit are the most discriminatory markers for Down syndrome screening in maternal serum. ~3 The ~-corehCG is a major breakdown product of the nicked free ~-subunit, which is readily excreted in urine. 4 In this study we investigated the nicked free 13-subunit in serum of patients with normal and Down syndrome pregnancies because a recent study reported that the levels of urine ~-core-hCG are multiple-fold greater in Down syndrome pregnancies than the corresponding levels for maternal serum hCG and free [3-hCG) This finding suggests that [}-core-hCG might be a more efficient Down syndrome marker than hCG or free [}-hCG. Accordingly, if the breakdown product of the nicked free ~3-subunit in urine is elevated in Down syndrome pregnancies, the blood precursor of this c o m p o u n d (i.e., the nicked free [3-subunit) should be elevated as well. The purpose of this study was to investigate nicked free
Prom the Divisions of Maternal-Fetal Medicine" and Reproductive Endocrinology/~Department of Obstetrics and Gynecolog3,, Yale University School of Medicine, and the Department of Obstetrics and Co~necology, Rabin Medical Center (Hasharon Hospital), Sackler School of Medidne7 Received for publication March 8, 1995," revised May 1, 1995; accepted July l I, 1995. Reprint requests: Siegfried Rotmensch, MD, Department of Obstetrics, and Gynecology, Rabin Medical Center, Hasharon Hospital, P.O. Box 121, Petah Tiqva, Israel 49372. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00+ 0 6/1/67719
[3-subunit levels in normal and Down syndrome pregnancies. Material and m e t h o d s
After informed consent was given, blood samples were obtained from 64 women with karyotypically normal fetuses and six women with Down syndrome fetuses. All specimens were obtained before amniocentesis. Indications for amniocentesis included advanced maternal age and low maternal serum c~-fetoprotein levels. Only singleton pregnancies between 13 and 18 weeks' gestation were included in the study. Blood samples were immediately centrifuged and sera were stored a t - 2 0 ° C until retrieved for assay. I~m'ntmoassays. Assays were performed without knowledge of the fetal karyotype. Laboratory technique has been described elsewhere. 4 In brief, free [3-subunit levels were determined with two different immunoenzymomettic assays. (1) The FBTll:anti-13-peroxidase assay measures the total amount of free [3-hCG levels. This assay does not distinguish between the intact (i.e,, without peptide linkage breaks) and nicked (peptide linkage breaks present) free ~-subunit of hCG and accordingly measures the total level of the free [3-subunit.4 (2) The 1E5:anti-~-peroxidase assay is specific for intact (without peptide linkage breaks) free ~3-subunit.4This assay cannot detect free [}-subunits that have broken peptide linkages ("nicks"). Accordingly, the level of the nicked free [3-sub-
609
610
R o t m e n s c h et al.
February 1996 Am J Obstet Gynecol
26 24
~
,"a'--....~
°
°
22
g ~o .~_ c e~ ~
16 14
13
14
15
16
17
18
Gestational age (weeks)
Fig. l. Regression curve of total tree ~-subunit levels on gestational age with 95% confidence interval. Individual values for Down syndrorne cases are plotted.
26l 24 22 2O
~
16,
14' 12'
I
10"~
13
• •
14
15
16
17
18
Gestational age (weeks)
Fig. 2. Regression curve of nicked free [~-subunitlevels on gestational age with 95% confidence interval. Individual values for Down syndrome cases are plotted.
unit of hCG is determined by a subtraction of total free [3-subunit levels (FBTll assay) minus intact free ~-subunit levels (1E5 assay). 4 Cross-reactivity of the two free [~-subunit assays with free ~-subunit standard (CR123, further purified by gel filtration to remove hCG) was <0.05%. To allow for gestational age differences in nicked free [~-hCG, levels were also expressed as multiples of the normal median derived from the control series for each gestational age week. Because of the small n u m b e r of controls at any week of gestation, the observed medians were regressed after weighting for the n u m b e r of controls by the standard procedure of the SAS statistical package (SAS Institute, Cary, N.C.). Gestational age was determined by a composite of uhrasonographic biometric measurements. Regression analyses were based on serum samples from 64 karyotypically normal pregnancies. Differences between
patient groups were assessed with the Wilcoxon rank-sum test, with p < 0.05 indicating significance. Results
Fig. 1 shows the regression curve and 95% confidence interval of total free ~-subunit levels on gestational age. The regression equation is Total free [3-subunit = 62.0726 + Gestational age - (-3.0006). A simple linear regression was found adequately to describe the data with no significant advantage offered by higher-order polynomial equations (r=0.39, SE 8.82, p < 0.002). Down syndrome pregnancies are plotted individually. Three of six cases were above the 95% confidence limits. Fig. 2 plots nicked free J3-subunit levels of Down syndrome pregnancies on the respective regression curve and confidence interval. The regression equation is: Nicked free ~-subunit= 23.7023+Gestational
Volume 174, Number 2 Am J Obstet Gynecol
a g e . (-1.2556), ( r = 0.37, SE 3.93, p < 0.003). Five of six cases were above the 95% Confidence interval. In o n e case nicked free [3-subunit levels c o u l d n o t be detected. Table I lists the individual values in each case and selected percentiles in the controls expressed in multiples of the median. T h e m e a n level of total free [3-subunit in the Down syndrome g r o u p was 1.07 multiples of the median. T h e m e a n level of nicked free [3-subunit was 4.76 multiples o f the median, which was significantly elevated in c o m p a r i s o n to n o r m a l controls (p < 0.05, Wilc o x o n rank-sum test). In all cases in which nicked free [~-subunits could be detected, levels were at least 2 multiples of the median, with a range f r o m 1.96 to 15.43 multiples of the median. Comment
This study provides p r e l i m i n a r y data indicating that m a t e r n a l s e r u m nicked free [3-subunit of h C G is remarkably increased in Down s y n d r o m e pregnancies c o m p a r e d with n o r m a l controls. We have o b s e r v e d a greater elevation o f nicked free [3-subunit levels c o m p a r e d with total free [3-subunits a l t h o u g h the small n u m b e r of cases does n o t allow for statistical c o m p a r i s o n between of these two markers. It is striking, however, that in all cases in which nicked free ~-subunit could be d e t e c t e d levels (in multiples o f the m e d i a n ) were 2- to 3-fold h i g h e r than free [3-subunit levels, c o m p a r e d with their respective n o r m a l controls. This raises the possibility that nicked free [3-subunit of h C G could be a m o r e sensitive m a r k e r for Down syndrome screening, similar to the recently described [3-core hCG. 5 In o u r o p i n i o n , these findings are sufficiently e n c o u r a g i n g to warrant a larger prospective study to d e t e r m i n e the efficacy o f this Down syndrome marker. Peptide linkage breaks in the [3-subunit of h C G occur primarily between a m i n o acids [347 to 48 a n d [344 to 45. ~ T h e cause for free ~-subunit nicking in Down syndrome pregnancies is unknown. Kardana and Cole v have recently identified a g o n a d o t r o p i n ~-subunit nicking enzyme that appears to be specific for p r e g n a n c y and cancer patient serum. No data are available at p r e s e n t on the activity o f this enzyme in Down syndrome pregnancies. T h e n o t i o n of m o l e c u l a r instability o f h C G and its [3-subunit in Down syndrome is s u p p o r t e d by a r e c e n t r e p o r t by Cuckle et al. ~ T h e s e authors f o u n d substantially increased levels of urinary [3-core-hCG, the a p p a r e n t breakdown p r o d u c t o f the nicked [3-subunit. T h e pathway of h C G b r e a k d o w n probably involves dissociation of the ~- and [3-subunit f r o m each other, nicking o f released ~-subunit at multiple sites between residues 43 and 48 and removal of the C-terminal s e g m e n t to p r o d u c e [3-core hCG. 4' 7 T h e latter is readily secreted in urine. For the purpose o f p o p u l a t i o n screening, m a t e r n a l s e r u m nicked [3-subunit m i g h t be preferable to urinary ~-core-hCG because s e r u m screening p r o g r a m s for Down syndrome and neural tube defects have b e e n extensively
Rotmensoh et ai.
611
Table I. Total free [3-subunit levels and nicked free [3-subunit levels for individual Down syndrome cases and selected percentiles for 64 controls
Sample Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Controls (percentile) 10th 25th Median 75th 90th
Total free ~-subunit (multiples of median)
Nicked free ~-subunit multiples of median)
1.15 0.70 0.99 1.71 1.54 3.10
1.96 2.05 3.32 5.81 0 15.43
0.28 0.54 0.97 1.28 1.76
0.00 0.31 0.83 1.68 2.82
i m p l e m e n t e d over the last few years. 8 A t t e n t i o n to p r o p e r collection technique, however, is essential for reliable results. Nicking of [3-subunits tends to increase over time, unless protease inhibitors are used in the collection tube. In this study we have paid careful attention to i m m e d i a t e freezing of the s e r u m until assay. M o r e work is necessary to d e t e r m i n e the screening efficacy of nicked free [3-subunit as a Down syndrome screening marker. Obviously c o m p a r i s o n with o t h e r markers will be necessary. If increased [3-subunit nicking is an intrinsic characteristic of Down syndrome pregnancies, it m i g h t also serve as a first-trimester screening marker.
REFERENCES
1. HaddowJE, Palomaki GE, Knight GJ, et al. Prenatal screening for Down's syndrome with use of maternal serum markers. N EnglJ Med 1992;327:588-93. 2. Wald NJ, Kennard A, DensemJW, Cuckle HS, Chard T, Butler L. Antenatal maternal serum screening for Down's syndrome: results of a demonstration project. BMJ 1992;305:391-4. 3. Phillips OP, Elias S, Shulman LE Andersen RN, Morgan CD, Simpson JL. Maternal serum screening for fetal Down syndrome in women less than 35 years of age using c~-fetoprotein, hCG and unconjugated estriol: a prospective 2-year study. Obstet Gynecol 1992;80:353-8. 4. Cole LA, Kardana A, Park SY, Braunstein GD. The deactivation of hCG by nicking and dissociation. J Clin Endocrinol Metab 1993;76:704-10. 5. Cuckle HS, Iles RK, Chard T. Urinary ~-core human chorionic gonadotrophin: a new approach to Down's syndrome screening. Prenat Diagn 1994;14:953-8. 6. Cole LA, Kardana A, Andrade-Gordon P, et al. The heterogeneity of human chorionic gonadotropin (hCG), lII: the occurrence and biological and immunological activities of n i c k e d hCG. Endocrinology 1991;129:1559-67. 7. Kardana A, Cole LA. Human chorionic gonadotropin ~-subunit nicking enzymes in pregnancy and cancer patient serum. J Clin Endocrinol Metab 1994;79:761-7. 8. Palomaki GE, Knight GJ, McCarthy J, Haddow JE, Eckfeldt JH. Maternal serum screening for fetal Down syndrome in the United States: a 1992 survey. A_MJ OBSXETG'cwEcot.1993; 169: 1558-62.
Key words: H u m a n chorionic gonadotropin, Down syndrome, serum screening
H u m a n chorionic gonadotropin (hCG) and its free ~-subunit are the most discriminatory markers for Down syndrome screening in maternal serum. ~3 The ~-corehCG is a major breakdown product of the nicked free ~-subunit, which is readily excreted in urine. 4 In this study we investigated the nicked free 13-subunit in serum of patients with normal and Down syndrome pregnancies because a recent study reported that the levels of urine ~-core-hCG are multiple-fold greater in Down syndrome pregnancies than the corresponding levels for maternal serum hCG and free [3-hCG) This finding suggests that [}-core-hCG might be a more efficient Down syndrome marker than hCG or free [}-hCG. Accordingly, if the breakdown product of the nicked free ~3-subunit in urine is elevated in Down syndrome pregnancies, the blood precursor of this c o m p o u n d (i.e., the nicked free [3-subunit) should be elevated as well. The purpose of this study was to investigate nicked free
Prom the Divisions of Maternal-Fetal Medicine" and Reproductive Endocrinology/~Department of Obstetrics and Gynecolog3,, Yale University School of Medicine, and the Department of Obstetrics and Co~necology, Rabin Medical Center (Hasharon Hospital), Sackler School of Medidne7 Received for publication March 8, 1995," revised May 1, 1995; accepted July l I, 1995. Reprint requests: Siegfried Rotmensch, MD, Department of Obstetrics, and Gynecology, Rabin Medical Center, Hasharon Hospital, P.O. Box 121, Petah Tiqva, Israel 49372. Copyright © 1996 by Mosby-Year Book, Inc. 0002-9378/96 $5.00+ 0 6/1/67719
[3-subunit levels in normal and Down syndrome pregnancies. Material and m e t h o d s
After informed consent was given, blood samples were obtained from 64 women with karyotypically normal fetuses and six women with Down syndrome fetuses. All specimens were obtained before amniocentesis. Indications for amniocentesis included advanced maternal age and low maternal serum c~-fetoprotein levels. Only singleton pregnancies between 13 and 18 weeks' gestation were included in the study. Blood samples were immediately centrifuged and sera were stored a t - 2 0 ° C until retrieved for assay. I~m'ntmoassays. Assays were performed without knowledge of the fetal karyotype. Laboratory technique has been described elsewhere. 4 In brief, free [3-subunit levels were determined with two different immunoenzymomettic assays. (1) The FBTll:anti-13-peroxidase assay measures the total amount of free [3-hCG levels. This assay does not distinguish between the intact (i.e,, without peptide linkage breaks) and nicked (peptide linkage breaks present) free ~-subunit of hCG and accordingly measures the total level of the free [3-subunit.4 (2) The 1E5:anti-~-peroxidase assay is specific for intact (without peptide linkage breaks) free ~3-subunit.4This assay cannot detect free [}-subunits that have broken peptide linkages ("nicks"). Accordingly, the level of the nicked free [3-sub-
609
610
R o t m e n s c h et al.
February 1996 Am J Obstet Gynecol
26 24
~
,"a'--....~
°
°
22
g ~o .~_ c e~ ~
16 14
13
14
15
16
17
18
Gestational age (weeks)
Fig. l. Regression curve of total tree ~-subunit levels on gestational age with 95% confidence interval. Individual values for Down syndrorne cases are plotted.
26l 24 22 2O
~
16,
14' 12'
I
10"~
13
• •
14
15
16
17
18
Gestational age (weeks)
Fig. 2. Regression curve of nicked free [~-subunitlevels on gestational age with 95% confidence interval. Individual values for Down syndrome cases are plotted.
unit of hCG is determined by a subtraction of total free [3-subunit levels (FBTll assay) minus intact free ~-subunit levels (1E5 assay). 4 Cross-reactivity of the two free [~-subunit assays with free ~-subunit standard (CR123, further purified by gel filtration to remove hCG) was <0.05%. To allow for gestational age differences in nicked free [~-hCG, levels were also expressed as multiples of the normal median derived from the control series for each gestational age week. Because of the small n u m b e r of controls at any week of gestation, the observed medians were regressed after weighting for the n u m b e r of controls by the standard procedure of the SAS statistical package (SAS Institute, Cary, N.C.). Gestational age was determined by a composite of uhrasonographic biometric measurements. Regression analyses were based on serum samples from 64 karyotypically normal pregnancies. Differences between
patient groups were assessed with the Wilcoxon rank-sum test, with p < 0.05 indicating significance. Results
Fig. 1 shows the regression curve and 95% confidence interval of total free ~-subunit levels on gestational age. The regression equation is Total free [3-subunit = 62.0726 + Gestational age - (-3.0006). A simple linear regression was found adequately to describe the data with no significant advantage offered by higher-order polynomial equations (r=0.39, SE 8.82, p < 0.002). Down syndrome pregnancies are plotted individually. Three of six cases were above the 95% confidence limits. Fig. 2 plots nicked free J3-subunit levels of Down syndrome pregnancies on the respective regression curve and confidence interval. The regression equation is: Nicked free ~-subunit= 23.7023+Gestational
Volume 174, Number 2 Am J Obstet Gynecol
a g e . (-1.2556), ( r = 0.37, SE 3.93, p < 0.003). Five of six cases were above the 95% Confidence interval. In o n e case nicked free [3-subunit levels c o u l d n o t be detected. Table I lists the individual values in each case and selected percentiles in the controls expressed in multiples of the median. T h e m e a n level of total free [3-subunit in the Down syndrome g r o u p was 1.07 multiples of the median. T h e m e a n level of nicked free [3-subunit was 4.76 multiples o f the median, which was significantly elevated in c o m p a r i s o n to n o r m a l controls (p < 0.05, Wilc o x o n rank-sum test). In all cases in which nicked free [~-subunits could be detected, levels were at least 2 multiples of the median, with a range f r o m 1.96 to 15.43 multiples of the median. Comment
This study provides p r e l i m i n a r y data indicating that m a t e r n a l s e r u m nicked free [3-subunit of h C G is remarkably increased in Down s y n d r o m e pregnancies c o m p a r e d with n o r m a l controls. We have o b s e r v e d a greater elevation o f nicked free [3-subunit levels c o m p a r e d with total free [3-subunits a l t h o u g h the small n u m b e r of cases does n o t allow for statistical c o m p a r i s o n between of these two markers. It is striking, however, that in all cases in which nicked free ~-subunit could be d e t e c t e d levels (in multiples o f the m e d i a n ) were 2- to 3-fold h i g h e r than free [3-subunit levels, c o m p a r e d with their respective n o r m a l controls. This raises the possibility that nicked free [3-subunit of h C G could be a m o r e sensitive m a r k e r for Down syndrome screening, similar to the recently described [3-core hCG. 5 In o u r o p i n i o n , these findings are sufficiently e n c o u r a g i n g to warrant a larger prospective study to d e t e r m i n e the efficacy o f this Down syndrome marker. Peptide linkage breaks in the [3-subunit of h C G occur primarily between a m i n o acids [347 to 48 a n d [344 to 45. ~ T h e cause for free ~-subunit nicking in Down syndrome pregnancies is unknown. Kardana and Cole v have recently identified a g o n a d o t r o p i n ~-subunit nicking enzyme that appears to be specific for p r e g n a n c y and cancer patient serum. No data are available at p r e s e n t on the activity o f this enzyme in Down syndrome pregnancies. T h e n o t i o n of m o l e c u l a r instability o f h C G and its [3-subunit in Down syndrome is s u p p o r t e d by a r e c e n t r e p o r t by Cuckle et al. ~ T h e s e authors f o u n d substantially increased levels of urinary [3-core-hCG, the a p p a r e n t breakdown p r o d u c t o f the nicked [3-subunit. T h e pathway of h C G b r e a k d o w n probably involves dissociation of the ~- and [3-subunit f r o m each other, nicking o f released ~-subunit at multiple sites between residues 43 and 48 and removal of the C-terminal s e g m e n t to p r o d u c e [3-core hCG. 4' 7 T h e latter is readily secreted in urine. For the purpose o f p o p u l a t i o n screening, m a t e r n a l s e r u m nicked [3-subunit m i g h t be preferable to urinary ~-core-hCG because s e r u m screening p r o g r a m s for Down syndrome and neural tube defects have b e e n extensively
Rotmensoh et ai.
611
Table I. Total free [3-subunit levels and nicked free [3-subunit levels for individual Down syndrome cases and selected percentiles for 64 controls
Sample Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Controls (percentile) 10th 25th Median 75th 90th
Total free ~-subunit (multiples of median)
Nicked free ~-subunit multiples of median)
1.15 0.70 0.99 1.71 1.54 3.10
1.96 2.05 3.32 5.81 0 15.43
0.28 0.54 0.97 1.28 1.76
0.00 0.31 0.83 1.68 2.82
i m p l e m e n t e d over the last few years. 8 A t t e n t i o n to p r o p e r collection technique, however, is essential for reliable results. Nicking of [3-subunits tends to increase over time, unless protease inhibitors are used in the collection tube. In this study we have paid careful attention to i m m e d i a t e freezing of the s e r u m until assay. M o r e work is necessary to d e t e r m i n e the screening efficacy of nicked free [3-subunit as a Down syndrome screening marker. Obviously c o m p a r i s o n with o t h e r markers will be necessary. If increased [3-subunit nicking is an intrinsic characteristic of Down syndrome pregnancies, it m i g h t also serve as a first-trimester screening marker.
REFERENCES
1. HaddowJE, Palomaki GE, Knight GJ, et al. Prenatal screening for Down's syndrome with use of maternal serum markers. N EnglJ Med 1992;327:588-93. 2. Wald NJ, Kennard A, DensemJW, Cuckle HS, Chard T, Butler L. Antenatal maternal serum screening for Down's syndrome: results of a demonstration project. BMJ 1992;305:391-4. 3. Phillips OP, Elias S, Shulman LE Andersen RN, Morgan CD, Simpson JL. Maternal serum screening for fetal Down syndrome in women less than 35 years of age using c~-fetoprotein, hCG and unconjugated estriol: a prospective 2-year study. Obstet Gynecol 1992;80:353-8. 4. Cole LA, Kardana A, Park SY, Braunstein GD. The deactivation of hCG by nicking and dissociation. J Clin Endocrinol Metab 1993;76:704-10. 5. Cuckle HS, Iles RK, Chard T. Urinary ~-core human chorionic gonadotrophin: a new approach to Down's syndrome screening. Prenat Diagn 1994;14:953-8. 6. Cole LA, Kardana A, Andrade-Gordon P, et al. The heterogeneity of human chorionic gonadotropin (hCG), lII: the occurrence and biological and immunological activities of n i c k e d hCG. Endocrinology 1991;129:1559-67. 7. Kardana A, Cole LA. Human chorionic gonadotropin ~-subunit nicking enzymes in pregnancy and cancer patient serum. J Clin Endocrinol Metab 1994;79:761-7. 8. Palomaki GE, Knight GJ, McCarthy J, Haddow JE, Eckfeldt JH. Maternal serum screening for fetal Down syndrome in the United States: a 1992 survey. A_MJ OBSXETG'cwEcot.1993; 169: 1558-62.