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. 2011 Sep 9;89(3):451-8.
doi: 10.1016/j.ajhg.2011.08.002. Epub 2011 Sep 1.

Mutations Causing Familial Biparental Hydatidiform Mole Implicate c6orf221 as a Possible Regulator of Genomic Imprinting in the Human Oocyte

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Mutations Causing Familial Biparental Hydatidiform Mole Implicate c6orf221 as a Possible Regulator of Genomic Imprinting in the Human Oocyte

David A Parry et al. Am J Hum Genet. .
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Abstract

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome.

Figures

Figure 1
Figure 1
Pedigree of Family L The proband is designated by the arrow; individuals V:3 and IV:5 are the affected individuals. CHM are displayed as clear diamonds. Miscarriages are displayed as small triangles.
Figure 2
Figure 2
Mutations Identified in C6orf221 in Three Cases of FBHM The following abbreviations are used: WT, wild-type; family L, c.3G>T homozygote; family T, c.322_325delGACT homozygote; family W, compound heterozygote c.1A>G + c.322_325delGACT.
Figure 3
Figure 3
Effect of c.3G>T Mutation on C6orf221 HEK293 cells (ECACC, Salisbury, UK) were transfected with Myc-tagged wild-type or c.3G>T C6orf221 containing vectors. Whole-cell protein extracts were resolved with SDS-PAGE and immunoblotted. Blots were probed with a commercial mouse monoclonal c-Myc antibody (Sigma-Aldrich, Irvine, UK) and with a custom-made rabbit polyclonal antibody raised against a peptide antigen (MDAPRRFPTLVQLMQC) containing residues 1–15 of human C6orf221. The following abbreviations are used: C, negative control; Wt, wild-type protein; M1V, site directed mutagenesis produced a protein with the initiation codon mutation. In lanes 1, 2, and 3, the probe used is the anti-c-Myc antibody. The expected 24 kDa wild-type C6orf221 is detected in lane 2; in lane 4 the shorter protein is seen consequent upon the c.3G>T mutation. In lanes 6, 7, and 8, the probe used is the antibody raised against N-terminal peptide. In lane 7 the expected 24 kDa wild-type C6orf221 is detected. The peptide antibody fails to detect the protein produced from the vector containing the c.3G>T mutation run in lane 8. This is consistent with the predicted initiation at codon 14 as a result of the mutation.
Figure 4
Figure 4
C6orf221 Expression in Human Ovarian Follicles, Oocytes, Preimplantation Embryos Expression of C6orf221 and GAPDH was investigated by RT-PCR of cDNA amplified from human ovarian follicles (including granulosa/pregranulosa cells), mature oocytes (granulosa/pregranulosa cell free) and preimplantation embryos. DNA size standard: lane1, primordial follicles (n = 25–40); lane 2, early primary follicles (n = 25–40); lane 3, primary follicles (n = 25–40); lane 4, GV oocyte pool (n = 2); lane 5, MII oocyte pool (n = 5); lane 6, Morula pool (n = 5); lane 7, blastocyst pool 1 (n = 5); lane 8, blastocyst pool 2 (n = 5); negative control (-ve). Diameters used for follicle staging: primordial follicles 34–38 mm, early primary follicles 34–53 mm, primary follicles 52–62 mm, secondary follicles 62–86 mm. The upper panel shows expression of expected 716 bp long C6orf221 mRNA; the lower panel shows expression of the housekeeping gene GAPDH mRNA, used as a control.
Figure 5
Figure 5
Immunohistochemical Staining of Bovine Ovary for C6orf221 Ortholog The primary antibody was raised against the 15 N-terminal amino acids (MASPKRFPTLVQLEQ) of predicted bovine protein XP_002690064.1. (computationally predicted gene, C9H6orf221, Bos taurus ES-cell-associated transcript). The following abbreviations are used: OSE, ovarian surface epithelium; Pr, primordial (nongrowing) follicle; Tr, transitional follicle; 1°, primary follicle; 2°, secondary follicle; A, antral follicle; GC, granulosa cells; TC, thecal cells. Protein is diffusely distributed throughout the cytoplasm of oocytes in the primary and secondary follicles. In the antral follicles localization is again restricted to oocyte cytoplasm, but the staining pattern now takes on a more punctate appearance. There is no staining in surrounding granulosa or stromal cells.

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