Familial hcg Syndrome Production of Variable, Degraded or Mutant Forms of hcg Laurence A. Cole, Ph.D., and Stephen Butler, Ph.D. The Journal of Reproductive Medicine ORIGINAL ARTICLES OBJECTIVE: To examine the properties of the unique degraded forms of hcg produced in familial hcg syndrome and to describe 15 cases referred to the USA hcg Reference Service. familial hcg syndrome is an inherited disorder that is seemingly symptom-free. STUDY DESIGN: Total hcg was detected by Immulite total hcg assay. The molecules missing the β- subunit C-terminal peptide were detected by the Centaur total hcg assay; the proportion of molecules missing the β-subunit C-terminal peptide was determined as Immulite assay minus Centaur assay. Free β-subunit was detected in the FBT11 free β-subunit assay with 5008 anticore-hcgβ tracer. RESULTS: In all cases the syndrome was confirmed by either a mother, father, or sibling exhibiting ectopic hcg production. Serum hcg ranges in cases from 1 216 miu/ml and urine hcg from 1.5 527 miu/ml. It was estimated that 48 100% of molecules were missing the β-subunit C-terminal peptide. Serum hcg free β- subunit was measured, accounting for 52 79% of the total hcg immunoreactivity. Molecules missing the C- terminal peptide and free β-subunit mark this syndrome. Serial serum samples were examined in 4 cases; hcg concentrations varied widely with time from < 1 to 182 miu/ml. CONCLUSION: The proportion of molecules missing the β-subunit C-terminal peptide, 48 100%, is extraordinarily high. Epitope studies and gel filtration studies indicate that the C-terminal peptide may not actually be missing, suggesting that the β-subunit may be a mutant blocking C-terminal peptide recognition. (J Reprod Med 2014;59:435 442) Keywords: chorionic gonadotropin, familial hcg syndrome, genetic defect, hcg syndrome, human chorionic gonadotropin, syndrome. hcg is a heterodimeric glycoprotein hormone composed of a 92 amino acid β-subunit and a 145 amino acid β-subunit joined noncovalently. hcg exists as a heterogeneous mixture of multiple variants sharing a common core amino acid sequence; some variants have been shown to have independent functions. 1 Depending upon the hcg variant, 28 42% of the molecular weight comes from oligosaccharides side chains. 2 The 5 major variants of hcg are (1) the cancer cell autocrine, hcg free β- subunit, 3 (2) the cytotrophoblast cell autocrine, hyperglycosylated hcg, 3 (3) the syncytiotrophoblast cell endocrine, regular hcg, 1 (4) the pituitary gonadotrope cell endocrine, sulfated hcg, 4 and (5) the fetal renal/hepatic cell endocrine, regular hcg. 5 The USA hcg Reference Service was started in From the USA hcg Reference Service, Albuquerque, New Mexico, and the Centre for Investigative and Diagnostic Oncology, Middlesex University, London, U.K. Address correspondence to: Laurence A. Cole, Ph.D., USA hcg Reference Service, 2412 Calle De Panza NW, Albuquerque, NM 87104 (larry@hcglab.com). Financial Disclosure: The authors have no connection to any companies or products mentioned in this article. 0024-7758/14/5909-10 0435/$18.00/0 Journal of Reproductive Medicine, Inc. The Journal of Reproductive Medicine 435
436 The Journal of Reproductive Medicine 1997 as a consulting service specializing in problems with hcg assays. The USA hcg Reference Service helps to diagnose women who have a positive hcg assay but who are definitely not pregnant (i.e., no fetal sac, no ectopic pregnancy). We have seen multiple reasons for positive hcg tests other than pregnancy. These include false positive hcg tests, pituitary hcg, quiescent gestational trophoblastic disease, active gestational trophoblastic disease, and cancer. 4,6,7 In 2003 serum and urine were shipped to the USA hcg Reference Service from 2 sisters in Utah who thought that they both had false positive hcg tests. The problem was that they were both positive for hcg in serum and in urine (which excludes false positive hcg), they did not have elevated FSH (which excludes the possibility of pituitary hcg), and they had elevated hyperglycosylated hcg (which excludes quiescent gestational trophoblastic disease). 4 It appeared that we had a new hcg problem when it was confirmed that their mother was also similarly hcg positive and that both daughters had appeared to have inherited the unexplained production of hcg. In all 3 cases the hcg proportions were similar: total hcg was 72% accounted for by free β-subunit, and 79% of molecules were missing the β-subunit C-terminal peptide (Table I, Case 1). We called this strange familial production after finding multiple familial cases familial hcg syndrome. 8,9 The USA hcg Reference Service has now identified this syndrome in 15 referred cases. All cases seem to involve a parent and/or a child confirming its familial nature. In all cases the hcg production involved a mixture of highly dissociated and degraded forms of hcg (Table I). As investigated here, the hcg concentration produced can vary greatly and differ widely from all other USA hcg Reference Service cases and pregnancy serum hcg. Here we examine the properties of the unique degraded forms of hcg produced in familial hcg syndrome. We consider possible explanations for some of the observations, including the possibility that this syndrome arises from a mutant form of hcg. Material and Methods All cases described are those accumulated by the USA hcg Reference Service, a clinical laboratory. Since all cases and their physicians chose to supply the serum and urine samples and agreed on our referral forms to permit research like that presented here, human internal review committee approval was not warranted. Human internal review committee approval was obtained for collecting pregnancy serum (University of New Mexico protocol 03-146, Yale University protocol IRB 8340). Serum was collected from 458 pregnancies attending Yale University and University of New Mexico reproductive endocrinology and maternity clinics. Serum total hcg was measured using the Siemens Immulite 1000 assay (Siemens, Los Angeles, Table I USA hcg Reference Service Results Serum Serum total hcg, Missing C-terminal Serum hcg free β, Urine Confirm total hcg C-terminal peptide peptide, estimated ng/ml, miu/ml total hcg (positive Case Sex miu/ml determinant, miu/ml miu/ml hcg calculated miu/ml hcg) 1 F 43 12 31 (72%) 34 (79%) 3.0 Mother 2 M 1.0 0.5 0.5 (50%) 0.6 (60%) 6.8 Father 3 M 2.0 0.4 1.6 (80%) 1.3 (65%) 26 Father 4 M 2.8 < 1.0 2.8 (100%) 1.7 (61%) 54 Father 5 F 201 29 172 (86%) 113 (56%) 83 Father 6 M 1.8 0.7 1.1 (61%) 1.0 (55%) 2.9 Mother 7 F 216 24 192 (89%) 121 (56%) 24 Father 8 F 32 16 16 (50%) 17 (53%) 27 Son 9 F 9.4 2.5 6.9 (73%) 7.6 (81%) 1.5 Daughter 10 M 2.0 0.8 1.2 (60%) 1.2 (60%) 25 Father 11 M 2.8 < 1.0 2.8 (100%) 1.8 (64%) 6.0 Father 12 M 6.0 3.1 2.9 (48%) 3.1 (52%) 6.5 Father 13 F 17 < 1.0 17 (100%) 13 (76%) 15 Father 14 F 168 50 118 (70%) 168 (100%) 527 Mother 15 F 136 39 97 (71%) 146 (100%) 27 Mother Serum total hcg is Siemens Immulite result, and serum total hcg C-terminal peptide is Siemens Centaur result. Serum hcg free β is measured in ng/ml, miu/ml calculated ( 18). Confirmation of positive hcg was performed by home pregnancy test (2 cases), serum automated total hcg test (9 cases) or urine automated total hcg test (4 cases) (Table II).
Volume 59, Number 9-10/September-October 2014 437 California). This assay uses 2 antibodies to the core of hcg β-subunit and detects nicked hcg and free β-subunit molecules and nicked molecules missing the β-subunit C-terminal peptide. The sensitivity of this assay is 1 miu/ml. As demonstrated, the Immulite assay can be used to measure hcg concentration in serum and urine. 10 The Siemens Immulite 1000 assay was interchangeably used to test serum and urine samples. Serum total hcg was also measured using the Siemens Centaur hcg assay. This assay uses an antibody to the β-subunit C-terminal peptide and so cannot detect molecules missing the C-terminal peptide. The sensitivity of this assay is 2.0 miu/ml. The 2 total hcg assays together (Siemens Immulite result minus Siemens Centaur result) were used to estimate the production of hcg missing the β- subunit C-terminal peptide. The free β-subunit assay used antibody FBT11 (anti-free β-subunit) as capture antibody (gift from D. Bellet, Institut Gustave-Roussy, France) and antibody 5008 (anti-core hcgβ) peroxidase (Bios- Pacific, Inc., Emeryville, California) as tracer antibody in a 96-well microtiter plate immunometric assay. Methods are those described previously. 10 Results were obtained in ng/ml. Result were converted to molar miu/ml hcg for comparison. Values were multiplied by 18, accounting for the molar difference between hcg and hcg free β-subunit. hcg β-subunit standard CR129 (gift from S. Birken, Columbia University) was prepared in miu/ml by multiplying by 18 (100 miu/ml). Using the Siemens 1000 total hcg assay, hcg free β-subunit was measured equally with hcg standard (gift from S. Birken) (4th International Standard), showing that the molar miu/ml calculations were correct. All results were stored in a Microsoft Excel spreadsheet. Statistics were calculated using Microsoft Excel 2007. Results Fifteen cases of familial hcg syndrome have now been identified by USA hcg Reference Service (Table I). In 15 of the 15 cases the familial lineage of this inherited syndrome was confirmed and either a parent or offspring/child was identified with similar levels of hcg (Table II). In 9 cases confirmation that a parent or child was positive was achieved by examining serum samples, in 4 cases by examining urine samples, and in 2 cases it was achieved by supplying over-the-counter pregnancy tests only (First Response, Church and Dwight Inc., Princeton, New Jersey). Interestingly, 5 of the 15 cases are major athletic or sports players. Case 2 (Table I) was a National College Athletic Association player accused of doping with hcg, case 3 was an Olympic silver medal winner accused of doping, case 6 is a National Football League (NFL) player accused of doping with hcg, case 11 is also an NFL player, and case 12 was a professional volleyball player in Greece accused of doping. As shown in Table I, familial hcg syndrome cases had serum hcg ranging from 1.0 216 miu/ ml (median, 9.4 miu/ml) as measured with the Immulite total hcg assay. When measured in the Centaur total hcg test (does not detect molecule missing the β-subunit C-terminal peptide), the results were < 1.0 miu/ml to 29 miu/ml (median, 2.5 miu/ml), indicating that a high proportion of molecules were missing the C-terminal peptide. It was estimated that 48 100% of molecules were seemingly missing the β-subunit C-terminal peptide. Serum hcg free β-subunit was measured; as calculated, 52 79% of the total hcg immunoreacivity was hcg free β-subunit. It is thought that 48 100% of molecules overall, whether dimer or free β-subunit, were missing the β-subunit C- terminal peptide. The predominance of free β- subunit and molecules missing the C-terminal peptide were considered as a diagnostic property of this syndrome. Of the referred cases 8 were female and 7 were male. Of the parents and children, of the cases positive in hcg tests 10 were female and 12 were male. All cases had a positive genetic descendant or ascendant. Data indicated that this syndrome does not appear to be sex-linked and is inherited in a dominant manner. Serial serum samples were obtainable in 4 cases (cases 6, 12, 13, and 14) and serial urine samples in 2 cases (cases 6 and 12). Serial cases are presented in Figure 1. As shown, hcg samples vary in concentration with time greatly. Case 13 varied in serum concentration from < 1 miu/ml to 17 miu/ml. Case 6 varied from < 1 to 1.8 miu/ml in serum, and from < 1 to 3.9 miu/ml in urine. Case 14 varied from 44 to 182 miu/ml in serum. Case 12 varied from < 1 to 2.2 in serum, and from < 1 to 6.0 in urine. The proportion of molecules missing the C- terminal peptide was unusually high in familial hcg syndrome. This extremity has never been observed with USA hcg Reference Service cases previously. As shown in Figure 2, the proportion
438 The Journal of Reproductive Medicine Table II Confirmation of Familial hcg Syndrome Serum total hcg, with C-terminal Molecules Affected Serum peptide missing C-terminal Total urine family total hcg determinant, peptide estimated, Serum hcg free β, ng/ml, hcg Case member Sex miu/ml miu/ml miu/ml miu/ml hcg calculated miu/ml 1 Sister F 34 12 22 17 2.0 Mother F 54 15 39 16 2.0 Father M < 1.0 < 1.0 < 1.0 < 1.0 2 Father M < 1.0 < 1.0 < 1.0 < 0.3 10 Mother F < 1.0 < 1.0 < 1.0 < 1.0 3 Brother M 1.1 < 1.0 1.1 < 0.3 4.0 Father M 1.3 < 1.0 1.3 < 0.3 1.6 Mother F < 1.0 < 1.0 < 1.0 < 1.0 4 Father M 3.3 1.0 2.3 1.4 27 Mother F < 1.0 < 1.0 < 1.0 < 1.0 5 Sister F 153 41 112 113 100 Father M 142 30 112 129 670 6 Mother F 2.5 1.6 0.9 1.2 2.8 Father M < 1.0 < 1.0 < 1.0 < 1.0 7 Father M 201 29 181 119 178 Mother F < 1.0 < 1.0 < 1.0 < 1.0 8 Son M 24 11 13 12 12 Father M < 1.0 < 1.0 < 1.0 < 1.0 9 Daughter F Urine only, over-the-counter test > 3.0 10 Father M Urine only, Immulite test 1.6 Brother M Urine only, Immulite test 4.0 Mother F Negative Urine only, Immulite test < 1.0 11 Father F 3.2 1.1 2.1 (66%) 1.8 (56%) 4.2 Mother M < 1.0 < 1.0 Negative < 1.0 < 1.0 12 Father M Urine only, Immulite test 6.0 Mother F Negative Urine only, Immulite test < 1.0 13 Father M Urine only, over-the-counter test > 3.0 Mother F Negative Urine only, over-the-counter test < 3.0 14 Mother F Urine only, Beckman Dxi test 11 Sister F Urine only, Beckman Dxi test 12 Father M Negative Urine only, Beckman Dxi test < 2.0 15 Mother F Urine only, Beckman Dxi test 11 Sister F Urine only, Beckman Dxi test 12 Father M Negative Urine only, Beckman Dxi test < 2.0 In all cases (Table I), confirmation of familial hcg syndrome was sought from family members of referred cases. Where possible, serum was collected and total hcg, hcg free β-subunit, and C-terminal peptide presence determined (9 cases) or urine tested in automated hcg test (4 cases). At a minimum, positive urine over-the-counter test positive hcg (First Response pregnancy test) (2 cases) were accepted. of molecules missing the C-terminal peptide was compared in the 15 cases of familial hcg syndrome (48 100% missing the C-terminal peptide), to serum from 670 other USA hcg Reference Service cases (0 21% missing the C-terminal peptide), including 64 cancer cases. An extremely significant separation was observed distinguishing familial hcg syndrome in Student s t test, p = 1.6 10-256. As shown in Figure 3, we also compared these 15 cases to 458 pregnancy serum samples (0 11% missing the C-terminal peptide). As found by Student s t test, an extreme separation was observed, p = 1.5 10-151. Discussion A total of 15 cases have been referred to the USA hcg Reference Service that have been shown to have familial hcg syndrome. Interestingly, 5 of those cases are major athletic or sports players (Table I). The World Anti-Doping Agency, the NFL Anti-Doping Agency, and the United States Anti- Doping Agency need to be able to avoid detecting familial hcg syndrome cases. The demonstration of familial hcg syndrome explained the hcg presence or the erroneous accusation for doping. In all cases a familial link was demonstrated by showing
Volume 59, Number 9-10/September-October 2014 439 Figure 1 Serial serum total hcg results, case 13 (A), case 6 (B), case 14 (C) and case 12 (D). Results for serial serum total hcg are solid line, open circles, and serial urine total hcg are dashed line, closed circles. that either father or mother, or child, also produced comparable levels of total hcg (Table II). The variability of hcg in familial hcg syndrome patient hcg (Figure 1) suggests that either a secondary factor that controls hcg production, like insulin or hydrocortisone, influences hcg production. This may explain the variability of hcgrelated molecule production. Another option may be that hcg is expressed by swollen cells or inflamed cells, leading to variability in hcg production, possibly concordant with degradation of hcg production. Facing the facts, we do not know what cells are producing placental molecules, hcgrelated molecules, in familial hcg syndrome, let alone how or why values vary each month in familial hcg syndrome. It is likely that there is a genetic origin explaining the production of hcg in these cases and that the protein produced may contain amino acid substitutions or deletions that affect antibody specificity in hcg assays by affecting key epitopes. Regulation and expression of the chorionic gonadotropin β- subunit (CGB) gene cluster is highly complex and studied almost exclusively in normal term placenta and some cancers. Recently, advances in our understanding of the transcription and translation of CGB genes have been made, and the conclusions appear to be that they are highly variable. 11 Aside from the CGB7 paralog, which produces a mature protein with an Ala 117 Asp amino acid substitution, 12 translated CGB gene mutations are rare or largely unreported. This is unsurprising, as pregnancy appears to require biallelic expression of a homozygous allele to be uncomplicated. However, perhaps
440 The Journal of Reproductive Medicine Figure 2 Comparison of proportion of molecules missing the β-subunit C-terminal peptide among 15 cases of familial hcg syndrome (open circles) and 670 other cases managed by the USA hcg Reference Service (closed circles). with modern obstetric interventions, heterozygotes with rare senseless mutations have now been described and may affect structure and/or function: CGB5 p.val 79 Met mutation results in inefficient subunit assembly, 13 CGB5p.Val 56 Leu mutation affects assembly and functionality, 14 CGB8p Pro 73 Arg mutation alters confirmation of the hcg β- subunit, 14 and CGB8p Arg 8 Trp mutation has been described but with no known effect. 14 Further studies are required to characterize the proteins and Figure 3 Comparison of proportion of molecules missing the β-subunit C-terminal peptide among 15 cases of familial hcg syndrome (open circles) and 458 pregnancy serum (closed circles).
Volume 59, Number 9-10/September-October 2014 441 genes produced in familial hcg cases to directly establish an inheritable trait. We may not be looking for mutations within only the mature peptide; changes within the promoter regions may also be implicated. One of the strangest findings associated with familial hcg syndrome is the extent of hcg dissociation and degradation in the molecules produced. All molecules are either primarily a free β-subunit or primarily a molecule missing the β-subunit C- terminal peptide. As we have learned, leukocyte elastase initially nicks the β-subunit at β47-48 and then cleaves the entire β-subunit C-terminal peptide at β92-93 (Figure 4). 15,16 As such, it can be assumed that all of these molecules missing the C- terminal peptide are nicked. Nicking deactivates hcg biological activity. 15 As such, free β-subunit is not biologically active at the hcg receptor, nor is nicked hcg missing the C-terminal peptide. Thus, all the molecules in serum in familial hcg syndrome are biologically inactive. As such, with fertility this syndrome is seamlessly passed on from generation to generation. The hcg made in familial hcg syndrome appears to be a mixture of nicked hcg dimer and nicked free β-subunit missing the C-terminal peptide (Table I). The free β-subunit has been demonstrated by our free β-subunit assay. That the remaining hcg immunoreactivity is hcg dimer has been proven by a Roche Elecsys intact hcg assay run on the serum in just 2 cases, cases 6 and 13, and by our 2119 α-subunit/β-subunit intact hcg assay run on the serum in 5 cases, cases 1, 2, 3, 4, and 5 (data not shown). What cells produce hcg in familial hcg syndrome? How are they able to combine hcg subunits? Placental trophoblast cells and pituitary gonadotrope cells make hcg dimer. Efficient dimer formation in these cells is facilitated using enzymes that rapidly complete the last 2 critical disulfide bridges β93-100 and β26-110 on hcg β-subunit critical to dimer formation. 17,18 Cancer cells secrete mostly a free α-subunit and a hyperglycosylated hcg free β-subunit. Combination of subunits in cancer cells fails because of absence of these critical enzymes. 17,18 All 15 cases of familial hcg syndrome are producing a primary molecule seemingly missing the β-subunit C-terminal peptide (48 100% of immunoreactive hcg is missing the C-terminal peptide), in that they are not bound by the C-terminal antibody used in the Siemens Centaur assay (which recognizes the terminal β130-145 residues). Molecules missing the C-terminal peptide in such high proportion have never been described in any other dis- Figure 4 hcg 3-D structure (20), showing epitopes on the β-subunit C-terminal peptide. Scissors show β-subunit residues 92-93, the normal site of cleavage by leukocyte elastase (16,17).
442 The Journal of Reproductive Medicine order, including cancer cases and pregnancies (Figures 2 and 3). Why do these familial hcg syndrome cases stand out as being so extreme and so unusual? One possibility is that the β-subunit is an alternative structure molecule produced by an alternative hcg β-subunit gene to that expressed in pregnancy. There are 8 hcg β-subunit genes on chromosome 19, and only 4 CGβ, CGβ5, CGβ7 and CGβ8 are expressed in pregnancy. The alternative β-subunit could lead to greater degradation or to lack of recognition by C-terminal peptide antibody. Another option that has to be considered is expression of a mutant hcg β-subunit in familial hcg syndrome. This may also not be recognized by different total hcg assays. It was noteworthy that the FBT11 hcg free β- subunit assay binds residues β92-110 in the β- subunit C-terminal peptide (Figure 4). As demonstrated, the majority of molecules, 52 79% hcg free β-subunit in familial hcg syndrome cases, were detected by this assay. If the C-terminal peptide were cleaved at β92-93 as occurs normally, then β92-110 would be absent. This was not the case since molecules were detected in FBT11 assay. This observation suggests that either C-terminal peptide was cleaved at an alternate site, like β122-123 the tryptic site, or that the β-subunit was a mutant arising in amino acid substitutions leading to nonrecognition in the Siemens Centaur total hcg C-terminal peptide assay. Ulf Stenman, Ph.D., has studied many of our cases of familial hcg syndrome in Finland. He claims that gel filtration shows the size of the β- subunit to be a normal β-subunit size (Ulf Stenman, Ph.D., personal communication). It is inferred that familial hcg syndrome may not be exceptionally degraded as indicated but rather has mutations blocking recognition by a C-terminal peptide antibody. Mutant hcg may be the explanation for everything: nonrecognition by the Siemens Centaur C-terminal peptide assay and variable hcg production as indicated in Figure 1. Variable production could be due to variable recognition of mutant hcg. Genetic studies are urgently needed with familial hcg syndrome cases. In conclusion, familial hcg syndrome is an inherited disorder that is seemingly symptom-free. According to antibody profiling, the hcg forms produced in this disorder are dissociated hcg free β-subunit and nicked forms of hcg missing the β-subunit C-terminal peptide. Possible explanations include expression of a mutant hcg β-subunit gene in familial hcg syndrome. The syndrome is also characterized by low, highly variable levels of hcg varying week by week. These variations may be an artefact of variable recognition of a mutant hcg or low level basal expression of one or more of the CGB genes. References 1. Cole LA: hcg, five independent molecules. Clin Chim Acta 2012;413:48-65 2. Elliott MM, Kardana A, Lustbader JW, et al: Carbohydrate and peptide structure of the α- and β-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997;7:15-32 3. Cole LA, Butler SA: Hyperglycosylated hcg, hcgß and hyperglycosylated hcgβ: Interchangeable cancer promoters. Molec Cellul Endcrinol 2012;349:232-238 4. Cole LA, Laidler L, Muller C: USA hcg reference service, 10 year report. Clin Biochem 2010;43:1013-1022 5. Goldsmith PC, McGregor WG, Raymoure WJ, et al: Cellular localization of chorionic gonadotropin in human fetal kidney and liver. J Clin Endocrinol Metab 1983;57:54-61 6. Rotmensch S, Cole LA: False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. Lancet 2000; 355:712-715 7. Butler SA, Cole LA: Falsely elevated hcg leading to unnecessary therapy. Obstet Gynecol 2002;99:515-516 8. Cole LA: Familial hcg. J Reprod Immunol 2012;93:52-57 9. Cole LA, Laidler LL: Inherited hcg. J Reprod Med 2010;55: 99-102 10. Cole LA, Khanlian SA: The need for a quantitative urine hcg assay. Clin Biochem 2009;42:676-683 11. Butler SA: The molecular genetics of hcg. In Human Chorionic Gonadotropin. Edited by LA Cole, SA Butler. Burlington, MA, Elsevier, 2010, pp 37-48 12. Policastro P, Ovitt CE, Hoshina M, et al: A map of the hcgβ- LHβ gene cluster. J Biol Chem 1986;261:5607-5616 13. Miller-Lindholm AK, Bedows E, Bartels CF, et al: A naturally occurring genetic variant in the human chorionic gonadotropin-beta gene 5 is assembly inefficient. Endocrinol 1999;140:3496-3506 14. Nagirnaja LL, Ceslovas CV, Laan MM: Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit. Mol Hum Reprod 2012;18: 379-390 15. Cole LA, Kardana A, Andrade-Gordon P, et al: The Heterogeneity of hcg: III. The occurrence, biological and immunological activities of nicked hcg. Endocrinol 1991;129:1559-1567 16. Cole LA, Kardana A: The biological and clinical significance of nicks in human chorionic gonadotropin and its free β- subunit. Yale J Bio Med 1991;64:627-627 17. Beebe JS, Huth JR, Ruddon RW: Combination of the chorionic gonadotropin free beta-subunit with alpha. Endocrinol 1990;126:384-391 18. Ruddon RW, Krzesicki RF, Norton SE, et al: Detection of a glycosylated, incompletely folded form of chorionic gonadotropin beta subunit that is a precursor of hormone assembly in trophoblastic cells. J Biol Chem 1987;262:12533-12540