Lack of GNAS Remethylation During Oogenesis May Be a Cause of Sporadic Pseudohypoparathyroidism Type Ib

Abstract

Context

Pseudohypoparathyroidism type Ib (PHP1B) is characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone resistance in the proximal renal tubules. Maternal pathogenic STX16/GNAS variants leading to maternal epigenetic GNAS changes impair expression of the stimulatory G protein alpha-subunit (Gsα) thereby causing autosomal dominant PHP1B. In contrast, genetic defects responsible for sporadic PHP1B (sporPHP1B) remain mostly unknown.

Objective

Determine whether PHP1B encountered after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) causes GNAS remethylation defects similar to those in sporPHP1B.

Design

Retrospective analysis.

Results

Nine among 36 sporPHP1B patients investigated since 2000, all with loss of methylation (LOM) at the 3 maternal GNAS differentially methylated regions (DMRs) and gain of methylation at the paternal NESP DMR, had been conceived through IVF or ICSI. Besides abnormal GNAS methylation, IVF/ICSI PHP1B cases revealed no additional imprinting defects. Three of these PHP1B patients have dizygotic twins, and 4 have IVF/ICSI-conceived siblings, all with normal GNAS methylation; 2 unaffected younger siblings were conceived naturally.

Conclusion

Sporadic and IVF/ICSI-conceived PHP1B patients revealed indistinguishable epigenetic changes at all 4 GNAS DMRs, thus suggesting a similar underlying disease mechanism. Given that remethylation at the 3 maternal DMRs occurs during oogenesis, male factors are unlikely to cause LOM postfertilization. Instead, at least some of the sporPHP1B variants could be caused by a defect or defects in an oocyte-expressed gene that is required for fertility and for re-establishing maternal GNAS methylation imprints. It remains uncertain, however, whether the lack of GNAS remethylation alone and the resulting reduction in Gsα expression is sufficient to impair oocyte maturation.

Pseudohypoparathyroidism type Ia (PHP1A) (OMIM #103580) is caused by inactivating pathogenic variants involving those maternal GNAS exons that encode the alpha-subunit of the stimulatory G protein (Gsα), thereby leading to PTH-resistant hypocalcemia and hyperphosphatemia, as well as resistance to other hormones and to different developmental abnormalities referred to as Albright’s hereditary osteodystrophy (AHO). In contrast, mutations involving the paternal GNAS exons encoding Gsα are the cause of pseudopseudohypoparathyroidism (OMIM #612463), in which certain AHO features occur in the absence of hormonal resistance (1-4).

Individuals affected by pseudohypoparathyroidism type Ib (PHP1B) (OMIM #603233) show PTH-resistant hypocalcemia and hyperphosphatemia similar to the laboratory findings in PHP1A, but these patients present less commonly with resistance to other hormones and rarely with severe AHO features (1-4). Autosomal dominant PHP1B (AD-PHP1B) is caused in cis by maternal mutations, most frequently by STX16 deletions that lead to loss of methylation (LOM) restricted to GNAS exon A/B (3-5). Indistinguishable methylation changes limited to this differentially methylated region (DMR) are also observed in other familial PHP1B cases that are caused either by deletions of GNAS exon NESP and the region centromeric thereof (5,6), an inversion comprising a 1.8-mbp region telomeric of exon XL (7), or duplications involving GNAS (8-10). Other causes of AD-PHP1B, all with complete loss of the 3 maternal GNAS methylation imprints, include different deletions or duplications involving this locus on the long arm of chromosome 20 (3-5).

However, PHP1B occurs most frequently as a sporadic disorder (sporPHP1B) that is characterized by LOM of the 3 maternal GNAS methylation imprints. Furthermore, these patients show a gain-of-methylation (GOM) at GNAS exon NESP, an epigenetic modification that is unique to this PHP1B variant (3-5,11). With the exception of sporPHP1B being caused by paternal uniparental isodisomy or heterodisomy involving chromosome 20q (patUPD20q) (5,12,13), all sporadic cases remain unresolved at the genetic level (3,4).

Unlike patients affected by autosomal dominant PHP1B variants, some sporPHP1B cases show, besides epigenetic changes at GNAS, limited methylation changes at other genetic loci that undergo parent-specific methylation, an observation that is referred to as multilocus imprinting disturbance (14-17). The finding that multilocus imprinting disturbance (MLID) can occur in sporPHP1B raises the possibility that an unknown factor impairs GNAS methylation in trans, unlike the different AD-PHP1B variants that are caused by mutations acting in cis.

Recently, 3 unrelated sporPHP1B patients with GNAS methylation changes were reported, who had been conceived through in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) (18-20). IVF/ICSI is associated with an increased incidence of abnormally methylated imprinted loci, but the GNAS locus was not investigated in most of these studies (21-23).

Female infertility, and hence the need for IVF/ICSI, can be caused by recessive mutations in genes encoding members of the subcortical maternal complex (SCMC), which frequently lead to MLID in the conceptus. This raises the possibility that female infertility and impaired DNA methylation at imprinted loci involve similar abnormally regulated pathways. Consistent with this hypothesis, mutations in NLRP7 and other components of the SCMC that cause recurrent molar pregnancies (partial or biparental complete hydatidiform moles or invasive moles), spontaneous abortions, or stillbirths are frequently associated with epigenetic changes at different loci undergoing parent-specific methylation, including GNAS (17,24-30). It is therefore plausible that some pathogenic variants involving members of the SCMC or associated gene products are not only responsible for infertility but can also lead to PHP1B in the offspring, if these mutations prevent proper remethylation at GNAS during oogenesis, yet allow oocytes to mature sufficiently to allow fertilization.

To explore the frequency of IVF/ICSI-conceived PHP1B (IVF/ICSI-PHP1B) patients, we reviewed our cohort of sporPHP1B cases and revealed that assisted reproductive technology (ART) had been used for conception in an unexpectedly large number of cases.

Materials and Methods

Patients had been referred for genetic and/or epigenetic evaluation because PHP1B was considered due to hypocalcemia, hyperphosphatemia, and an elevated PTH level, in the absence of major AHO features. Reproductive history was reviewed for their mothers to exclude or confirm involvement of IVF/ICSI for conception. The parents of each patient have no known calcium and phosphate abnormalities, and there is no family history of such disorders.

Genomic DNA was extracted from peripheral blood, which was evaluated for each patient by multiplex ligation-dependent probe amplification (MLPA) and methylation-specific MLPA (MS-MLPA) to search for copy number variations in STX16 and GNAS, and for methylation changes at the 4 DMRs within GNAS, as previously reported for numerous cases from our cohort (31-33). For the IVF/ICSI-conceived PHP1B patients, other imprinted loci were evaluated by multilocus MLPA and MS-MLPA; these included MEG3, SNRPN, GRB10, PLAGL1, PEG3, MEST, H19, KCNQ10T1, RTL1, and DLK1. MLPA and MS-MLPA was performed using kits ME031 GNAS and ME034 multilocus imprinting, respectively (MRC-Holland, Amsterdam, The Netherlands) following manufacturer’s instructions (https://www.mlpa.com/). In addition, several microsatellite markers in the chr20q13 region were evaluated for each patient and for available parents and siblings at the Center for Human Genetic Research, Massachusetts General Hospital (Boston, MA, USA), as previously described (10,34).

Results

The identification of the genetic locus for AD-PHP1B (35) and our subsequent reports describing the first disease-causing STX16 and GNAS deletions (36,37) led to regular, unsolicited requests to help with the genetic and epigenetic analyses of genomic DNA from patients showing clinical and laboratory abnormalities consistent with PHP1B. These referrals resulted in the identification of different mutations in familial cases (7,10,33,36-40) and furthermore allowed the collection of a large sporPHP1B cohort (n = 108); ie, of patients with PTH-resistance hypocalcemia and hyperphosphatemia for whom no pathogenic variant could be identified (Table 1). Genomic DNA from these unselected patients showed LOM at the 3 maternal GNAS DMRs (i.e., A/B, XL, and AS), and GOM at the paternal NESP DMR; for some patients these epigenetic changes were incomplete ((31-33) and unpublished data). Large duplications involving the long arm of paternal chromosome 20 (patUPD20q) were excluded through the analysis of 6 or more microsatellite markers.

Table 1.

SporPHP1B patients referred to our laboratory from the United States or non-US countries

	Year of birth
	Before 1980	1980-2000		After 2000a		Total
sporPHP1B	17	55		36		108
		US	Non-US	US	Non-US
		27 (7)	28 (3)	20 (2)	16 (2)
IVF/ICSI PHP1B	0	1	1	6	3	11

The number of IVF/ICSI-conceived PHP1B cases, among the total number of sporPHP1B patients, is indicated for each time period. In parentheses are the sporPHP1B cases for whom physicians and/or parents could not be reached to verify natural conception.

Abbreviations: IVF/ICSI, in vitro fertilization/intracytoplasmic sperm injection; PHP1B, pseudohypoparathyroidism type Ib; sporPHP1B, sporadic pseudohypoparathyroidism type Ib.

^aAfter 2000, 0.9% to 4.1% of all children every year had been born after pursuing assisted reproductive technology (ART) in the United States, Europe, and Australia/New Zealand. Before 2000, ART was used less frequently and publically available records are less complete (43).

Review of medical records, information provided by the referring physician, and/or direct contact with the parents revealed that 11 sporPHP1B patients had been born after their parents had decided to pursue IVF/ICSI for conception because of several factors, which included reduced oocyte quality, recurrent ectopic pregnancies, or a retroverted uterus, as well as reduced sperm count and/or motility, or biobanking of sperm before surgery for prostate malignancy (Table 2); IVF/ICSI was excluded for 83 patients, but for 14 cases, parents or physicians could no longer be reached to verify that IVF had not been involved. Among the 11 PHP1B patients who had been conceived through IVF/ICSI, none was born before 1980; note that IVF and ICSI were first reported in 1978 and 1992, respectively (41,42). Only 2 of our sporPHP1B cases among the 55 patients born between 1980 and 2000 had been conceived through ART [i.e., during the first 2 decades after regulatory approval for IVF was granted in the United States (1981), Canada (1980), and subsequently in other countries]. Between 1999 and 2002, ICSI was pursued more frequently than IVF in Australia/New Zealand, the United States, and Europe (43). After 2000, 6 of the investigated PHP1B patients had been born in the United States to mothers after IVF/ICSI, while 14 sporPHP1B cases were conceived naturally. Among the 16 PHP1B patients who were born since 2000 in other countries, 3 had been conceived through IVF/ICSI. Among the IVF/ICSI-PHP1B cases in our cohort of patients (6 boys, 5 girls), 9 are Caucasian, 1 is Asian, and 1 is African American (Table 2).

Table 2.

Information related to the investigated IVF/ICSI-conceived PHP1B patients, their siblings, and mothers

Patient code	Country	Ethnicity	Sex	Birth year	Unaffected twin	Maternal age (years)	IVF vs. ICSI	Reason for IVF/ICSI	IVF/ICSI-conceived, unaffected siblings	Naturally conceived, unaffected siblings	Maternal GNAS allele shared with healthy twin or sibling	Ref.
135/II-1	USA	Asian	F	1988		36	IVF	Reduced sperm count/motility	—	—	NA	(32,33)
150/II-1	Chile	Caucasian	M	2007	Yesa	39	ICSI	Reduced sperm count/motility	—	—	NA	(18,33)
157/II-1	USA	Caucasian	F	2011	Yes	30	ICSI	Reduced oocyte quality	1b	—	Yes	(33)
174/II-1	Sweden	Caucasian	M	1998		38	IVF	Reduced oocyte quality	—	1	Yes	This report
204/II-2	USA	Caucasian	F	2005	Yes	36	IVF	Sperm collected before prostate surgery for malignancy	—	—	No	This report
234/II-1	USA	Caucasian	M	2008		32	ICSI	Unknown	2c	—	Yes	This report
241/II-1	USA	African American	F	2013		41	ICSI	Reduced sperm count/motility	—	—	NA	This report
247/II-1	Albania/Greece	Caucasian	M	2009		34	ICSI	Reduced oocyte quality	1	—	Yes	This report
252/II-1	Turkey	Caucasian	M	2006		35	ICSI	Retroverted uterus	—	—	NA	This report
269/II-1	Turkey	Caucasian	F	2012		35	ICSI	Reduced sperm count/motility	—	—	NA	This report
273/II-1	USA	Caucasian	M	2002	Yes	25	IVF	Prior ectopic pregnancies; normal hysterosalpingogram	—	1	To be done	(19)

^aDied in utero.

^bOne older sister conceived after retrieval of separate batch of eggs for ICSI.

^cSame batch of embryos used for patient and two older brothers

NA, not applicable because single child.

When the diagnosis of PHP1B was first considered (age range at diagnosis: 3-23 years), the IVF/ICSI-conceived patients had calcium, phosphate, and PTH levels that were indistinguishable from the laboratory findings in other sporPHP1B patients, who had been conceived naturally (31-33, 44) (Fig. 1). Furthermore, all IVF/ICSI-conceived patients showed the typical, broad methylation abnormalities at the 4 GNAS DMRs, including patient 273/II-1, whose epigenetic investigations had initially been restricted to the exon A/B DMR (19). Importantly, no methylation abnormalities were detected at the other investigated loci that undergo parent-specific methylation, which is different from previous reports describing IVF/ICSI-conceived offspring (21-23). The mothers had no evidence for an abnormal regulation of calcium and phosphate homeostasis.

Figure 1.

Parathyroid hormone (PTH), calcium (Ca), and phosphate (P) levels when pseudohypoparathyroidism type Ib (PHP1B) patients who had been conceived naturally or through in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) (striped left and open right bars, respectively) were first diagnosed (grey boxes, reference ranges). % methylation at the 4 GNAS differentially methylated regions (stippled line, normal methylation). Individual laboratory data and GNAS methylation are provided (44). All data are shown as mean ± SE; both patient groups revealed no statistically significant difference for PTH, Ca, and P levels when assessed by 2-tailed t-test.

Seven of the IVF/ICSI-facilitated pregnancies resulted in single offspring (see Table 2). Three IVF/ICSI-PHP1B patients have a healthy dizygotic twin; note that the twin of 150/II-1 had shown no progression and died by gestational week 8. Two cases have younger or older siblings, who were also conceived through IVF/ICSI using embryos that had been cryopreserved after the mother had initially undergone superovulation for egg retrieval and fertilization. Importantly, 2 younger siblings of 2 IVF/ICSI-PHP1B patients had been conceived naturally, suggesting maternal subfertility. All siblings (n = 10) lack clinical or laboratory evidence for an abnormal regulation of mineral ion homeostasis, and those available for testing (n = 8) showed no evidence for GNAS methylation changes. Analysis of several microsatellite markers centromeric and telomeric of GNAS using DNA from the IVF/ICSI-PHP1B cases revealed no evidence for a paternal duplication involving a large portion of chromosome 20q. Furthermore, four IVF/ICSI-conceived PHP1B patients have siblings with whom they share the same maternal allele, thus making a maternally inherited disease-causing mutation within the STX16/GNAS region unlikely.

Discussion

Different genetic mutations involving the maternal STX16-GNAS region and the associated methylation changes affecting one or several GNAS DMRs are the cause of several AD-PHP1B variants (3,4). In contrast, the genetic defect(s) responsible for sporPHP1B remains unknown, except for patients with patUPD20q. The recently reported IVF/ICSI-PHP1B cases showed epigenetic GNAS changes that are characteristic for sporPHP1B (18-20) suggesting that abnormal re-methylation at GNAS was already present when oocytes had been retrieved for fertilization.

Several imprinting disorders, including Beckwith-Wiedemann, Angelman, Prader-Willi, and Silver-Russell syndromes occur more frequently after IVF/ICSI (22,45). We therefore sought to determine how many of our sporPHP1B cases had been conceived by ART and whether IVF/ICSI-conceived patients revealed epigenetic changes at imprinted loci other than GNAS. In our large sporPHP1B cohort, 11 patients were born to mothers who had undergone IVF or ICSI. Several of these patients have unaffected, dizygotic twins, as well as 4 unaffected older or younger IVF/ICSI-conceived siblings.

Several unaffected twins or siblings of PHP1B cases had also been conceived through ART. It therefore appears unlikely that retrieval of oocyte, their fertilization, and in vitro maintenance before implantation, as well as cryopreservation of embryos or environmental factors had contributed to the epigenetic GNAS changes observed for the IVF/ICSI-PHP1B cases. However, factors with an impact on individual oocytes during development cannot be excluded. In addition, if IVF/ICSI itself were a risk factor for acquiring epigenetic GNAS defects, as has been proposed for other imprinting disorders (46), many more PHP1B cases should be identified among ART-conceived infants, which represent, depending on the country, 1.9% to 4.1% of all newborns (43). Approximately one quarter of the cases in our sporPHP1B cohort was born after IVF or after ICSI, once the latter procedure had become more widely available. It is therefore conceivable that more ART-conceived cases will be diagnosed with PHP1B in the coming years, possibly in conjunction with advanced maternal age.

The complex GNAS locus gives rise to the messenger RNA encoding Gsα as well as several other transcripts (2-4). After global demethylation, the murine DMRs at exons AS, XL, and 1A (A/B in humans) are remethylated before meiosis I (maternal imprints), while methylation at the NESP DMR is re-established after fertilization (paternal imprint) (29,47-52) (Fig. 2). For patients affected by AD-PHP1B, the known pathogenic variants (deletions, duplications, or inversions), if located on the maternal allele, prevent in cis remethylation of the maternal DMRs during oogenesis, which reduces Gsα expression, thus limiting the formation of 3′,5′-cyclic adenosine 5′-monophosphates (cAMP), the second messenger mediating hormonal activity at different GPCRs. In mice, Gnas methylation at the maternal exon 1A, and less so at maternal exons XL and AS, is re-established only if oocytes express Nesp transcripts (51). In contrast, patients with AD-PHP1B due to deletions involving the maternal exon NESP and the region centromeric thereof show LOM that is restricted to the exon A/B DMR (5,6).

Figure 2.

GNAS remethylation during oogenesis and postfertilization. (Left panel) After demethylation at GNAS (and other loci that undergo parent-specific methylation) in the oogonium, methylation is re-established in secondary oocytes in mice and humans at the maternal AS, XL, and A/B differentially methylation regions (DMRs; increasing intensity of red color); after fertilization, the paternal NESP DMR is remethylated (increasing intensity of blue color); 2n, diploid; n, haploid (29,47-52). (Upper right panel) During oogenesis, NESP expression facilitates remethylation at GNAS exons AS, XL, and A/B thereby allowing Gsα expression. We postulate that the mothers of sporadic pseudohypoparathyroidism type Ib (sporPHP1B) patients, including those who underwent in vitro fertilization/intracytoplasmic sperm injection, have impaired NESP expression thus preventing remethylation at the three maternal DMRs. Sperm DNA shows no GNAS methylation and very low transcript levels (52,60). In oocytes, the NESP DMR remains unmethylated thus allowing active NESP transcription, which is required for remethylation at exons AS, XL, and A/B; methylation at the A/B DMR allows Gsα expression. It is conceivable that a genetic mutation(s), which impairs NESP transcription, prevents remethylation of the maternal GNAS DMRs during oogenesis, thereby allowing AS transcription and consequently methylation of the maternal NESP DMR postfertilization. (Lower right panel) After fertilization, the normal zygote undergoes remethylation at the paternal NESP DMR, while the paternal exons AS, XL, and A/B remain unmethylated. This allows active transcription from these paternal promoters, which reduces in some tissues or cell types Gsα expression from this parental allele later in life. The maternal NESP DMR remains unmethylated in the zygote, while the 3 other maternal DMRs had been remethylated during oogenesis; methylation at the A/B DMR allows Gsα expression. Mutation(s) leading to sporPHP1B presumably prevent NESP transcription during oogenesis, thereby preventing methylation of the DMRs at exons AS, XL, and A/B. Active transcription from the AS promoter was previously shown to be required for methylation of the NESP DMR postfertilization.

Methylation at the Nesp DMR occurs only after fertilization and only in the presence of active AS transcription (52-54). In sporPHP1B cases, lack of remethylation at the maternal AS DMR during oocyte maturation is predicted to allow generation of the AS transcript, which is required for NESP methylation postfertilization (53,54). It is therefore conceivable that an oocyte-specific defect or defects that impair NESP transcription would prevent methylation at exon A/B, thereby reducing or eliminating maternal Gsα protein and consequently impairing agonist-induced cAMP formation in oocytes and other tissues postfertilization.

The second messenger cAMP plays an important role in oocyte maturation (55,56), thus raising the possibility of an association between the need for IVF/ICSI in the investigated 11 females and the development of PHP1B in more than half of their offspring. In fact, cAMP appears to be essential for fertility, since mice with oocyte-specific ablation of Gsα are healthy, but completely infertile due to premature resumption of meiosis and poor oocyte quality (57). It is therefore conceivable that a single genetic defect impairs Gsα expression because of failure to remethylate GNAS exon A/B, thereby potentially reducing oocyte quality and leading to offspring affected by PHP1B, if ART is pursued.

However, females affected by PHP1A and pseudopseudohypoparathyroidism have provided no evidence for reduced fertility (58). This makes it less likely that heterozygous, inactivating GNAS mutations, which reduce Gsα levels by approximately 50%, lead to female infertility or subfertility. Instead, another factor may be required not only for re-establishing maternal GNAS methylation but also for normal oocyte maturation.

Pathogenic variants involving different SCMC genes can cause female infertility and some of these defects lead to methylation abnormalities at multiple loci, including the GNAS locus (14-17,27). However, in our IVF/ICSI-conceived PHP1B cohort, no methylation changes were identified by multilocus MS-MLPA, a technique that provides results comparable to those obtained by pyrosequencing and Southern blot analysis (59). The molecular defect causing female infertility/subfertility and PTH1B in the offspring, if ART is pursued, thus impairs only GNAS remethylation.

Inherited biallelic mutations in the females undergoing ART that involve an as-yet-unknown gene are an unlikely cause of PHP1B given that some IVF/ICSI-conceived children are healthy and show normal GNAS methylation. Furthermore, 4 IVF-conceived children share the same STX16/GNAS haplotype with their dizygotic twin or with another sibling, thus making it doubtful that a genetic defect within this locus is responsible for the epigenetic GNAS abnormalities. In addition, reduced sperm count and/or viability are unlikely to explain PHP1B encountered in the offspring of females undergoing ART because failure to re-establish maternal GNAS methylation imprints occurs most likely during oogenesis. Consistent with this conclusion are the findings in 1 patient (204/II-2) and her unaffected twin sister (204/II-1), who were IVF-conceived using their father’s presumably normal sperm that had been banked before he underwent surgery for prostate cancer. Abnormal GNAS remethylation during oogenesis was thus most likely responsible also for this case of PHP1B.

Thus far, all investigated siblings of sporPHP1B cases and all children of adult females and males with this disease variant are unaffected (see Table 2 in (33) and unpublished cases). It is therefore plausible that the mothers of sporPHP1B patients had inherited a genetic mutation from only 1 unaffected parent. If this variant is combined with a second de novo mutation, it may lead in some eggs, prior to meiosis, to homozygosity or compound heterozygosity of the disease-causing variant. The resulting offspring would therefore be affected by PHP1B, yet are heterozygous for the defect, and IVF/ICSI-conceived PHP1B females will have only unaffected children.

Conclusion

In summary, IVF/ICSI-conceived PHP1B patients revealed epigenetic changes at the 4 GNAS DMRs that are indistinguishable from those encountered in other sporPHP1B cases. Both PHP1B variants could thus be caused by a defect or defects in an oocyte-expressed gene that is required for re-establishing maternal GNAS methylation imprints during oogenesis. Whether some females show reduced fertility because of impaired Gsα expression during oogenesis or whether the underlying genetic defect affects fertility through an independent mechanism remains to be determined.

Financial Support

This work was supported by grants from the National Institutes of Health, NIDDK (DK046718 and DK11794, subproject III), by unrestricted anonymous gifts, by a fellowship grant from the European Society for Pediatric Endocrinology (ESPE) (to P.H.), and by the K20 Association (https://www.associationk20.com).

Additional Information

Disclosures: The authors have no conflict of interest.

Data Availability

Some or all data generated or analyzed during this study are included in this published article or in the data repositories listed in the references.