title: Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit.
abstract: Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls from Estonia, Finland and Denmark] using PCR-restriction fragment length polymorphism. The mutation CGB5 p.Val56Leu (rs72556325) was identified in a single heterozygous RM patient and caused a structural hindrance in the formation of the hCGα/β dimer. Although the amount of the mutant hCGβ assembled into secreted intact hCG was only 10% compared with the wild-type, a stronger signaling response was triggered upon binding to its receptor, thus compensating the effect of poor dimerization. The mutation CGB8 p.Pro73Arg (rs72556345) was found in five heterozygotes (three RM cases and two control individuals) and was inherited by two of seven studied live born children. The mutation caused ~50% of secreted β-subunits to acquire an alternative conformation, but did not affect its biological activity. For the CGB8 p.Arg8Trp (rs72556341) substitution, the applied in vitro methods revealed no alterations in the assembly of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGβ genes CGB5 and CGB8.
authors.affiliation: Human Molecular Genetics Research Group, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.
authors.forename: Liina
authors.initials: L
authors.lastname: Nagirnaja
authors.forename: Česlovas
authors.initials: Č
authors.lastname: Venclovas
authors.forename: Kristiina
authors.initials: K
authors.lastname: Rull
authors.forename: Kim C
authors.initials: KC
authors.lastname: Jonas
authors.forename: Hellevi
authors.initials: H
authors.lastname: Peltoketo
authors.forename: Ole B
authors.initials: OB
authors.lastname: Christiansen
authors.forename: Visvaldas
authors.initials: V
authors.lastname: Kairys
authors.forename: Gaily
authors.initials: G
authors.lastname: Kivi
authors.forename: Rudi
authors.initials: R
authors.lastname: Steffensen
authors.forename: Ilpo T
authors.initials: IT
authors.lastname: Huhtaniemi
authors.forename: Maris
authors.initials: M
authors.lastname: Laan
mesh_terms: D000026:Abortion, Habitual / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D005260:Female; D006801:Humans; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D011247:Pregnancy; D011248:Pregnancy Complications / Q000235:genetics; D011487:Protein Conformation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017422:Sequence Analysis, DNA
chemical_list: D018997:Chorionic Gonadotropin, beta Subunit, Human