Proximal tubular resistance to parathyroid hormone (PTH) resulting in hypocalcemia and hyperphosphatemia are preeminent abnormalities in pseudohypoparathyroidism type Ib (PHP1B), but resistance toward other hormones as well as variable features of Albright’s Hereditary Osteodystrophy (AHO) can occur also. Genomic DNA from PHP1B patients shows epigenetic changes at one or multiple differentially methylated regions (DMRs) within GNAS, the gene encoding Gαs and splice variants thereof. In the autosomal dominant disease variant, these methylation abnormalities are caused by deletions in STX16 or GNAS on the maternal allele. The molecular defect(s) leading to sporadic PHP1B (sporPHP1B) remains in most cases unknown and we therefore analyzed 60 sporPHP1B patients and available family members by microsatellite markers, single nucleotide polymorphisms (SNPs), multiplex ligation-dependent probe amplification (MLPA), and methylation-specific MLPA (MS-MLPA). All investigated cases revealed broad GNAS methylation changes, but no evidence for inheritance of two paternal chromosome 20q alleles. Some patients with partial epigenetic modifications in DNA from peripheral blood cells showed more complete GNAS methylation changes when testing their immortalized lymphoblastoid cells. Analysis of siblings and children of sporPHP1B patients provided no evidence for an abnormal mineral ion regulation and no changes in GNAS methylation. Only one patient revealed, based on MLPA and microsatellite analyses, evidence for an allelic loss, which resulted in the discovery of two adjacent, maternally inherited deletions (37,597 and 1427 bp, respectively) that remove the area between GNAS antisense exons 3 and 5, including exon NESP. Our findings thus emphasize that the region comprising antisense exons 3 and 4 is required for establishing all maternal GNAS methylation imprints. The genetic defect(s) leading in sporPHP1B to epigenetic GNAS changes and thus PTH-resistance remains unknown, but it seems unlikely that this disease variant is caused by heterozygous inherited or de novo mutations involving GNAS.

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Pseudohypoparathyroidism type Ib (PHP1B) is characterized primarily by resistance to parathyroid hormone (PTH) and thus hypocalcemia and hyperphosphatemia, in most case without evidence for Albright Hereditary Osteodystrophy (AHO). PHP1B is associated with epigenetic changes at one or several differentially methylated regions (DMR) within GNAS, which encodes the α-subunit of the stimulatory G protein (Gsα) and splice variants thereof. Heterozygous, maternally inherited STX16 or GNAS deletions leading to isolated loss-of-methylation (LOM) at exon A/B alone or at all maternal DMRs are the cause of autosomal dominant PHP1B (AD-PHP1B). In this study, we analyzed three affected individuals, the female proband and her two sons. All three revealed isolated LOM at GNAS exon A/B, while the proband’s healthy maternal grandmother and uncle showed normal methylation at this locus. Haplotype analysis was consistent with linkage to the STX16/GNAS region, yet no deletion could be identified. Whole genome sequencing of one of the patients revealed a large heterozygous inversion (1,882,433 bp). The centromeric breakpoint of the inversion is located 7,225 bp down-stream of GNAS exon XL, but its DMR showed no methylation abnormality raising the possibility that the inversion disrupts a regulatory element required only for establishing or maintaining exon A/B methylation. Because our three patients presented phenotypes consistent with PHP1B, not PHP1A, the Gsα promoter is probably unaffected by the inversion. Our findings expand the spectrum of genetic mutations that lead to loss-of-methylation at exon A/B alone and thus biallelic expression of the transcript derived from this alternative first GNAS exon.

Hypocalcemia and hyperphosphatemia because of resistance towards parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder that is caused by GNAS methylation changes, resistance can occur towards other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3-20 years later. Although anti-TPO antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL and A/B, and gain of methylation at exon NESP55, i.e. findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications.

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Inadequate maternal iodine intake affects thyroid function and may impair fetal brain development. This study investigated the association between maternal iodine intake during pregnancy and neurodevelopmental delay in offspring at 1 and 3 years of age using a nationwide birth cohort: the Japan Environment and Children’s Study. We assessed dietary iodine intake during pregnancy using a food frequency questionnaire and child neurodevelopment using the Japanese translation of the Ages and Stages Questionnaire, Third Edition. The risk of delay (score below the cut-off value) for fine motor domain at 1 year of age was increased in the lowest quintile iodine intake group compared with the fourth quintile iodine intake group. The risk of delay for problem-solving at 1 year of age was increased in the lowest and second quintile iodine intake group and decreased in the highest quintile iodine intake group. The risk of delay for communication, fine motor, problem-solving, and personal–social domains at 3 years of age was increased in the lowest and second quintile iodine intake group compared with the fourth quintile iodine intake group, while the risk of delay for fine motor and problem-solving domains was decreased in the highest quintile iodine intake group. Low iodine intake levels in pregnancy may affect child neurodevelopment.

Pseudohypoparathyroidism 1B (PHP1B) is caused by maternal epigenetic defects in the imprinted GNAS cluster. PHP1B can follow an autosomal dominant mode of inheritance or occur sporadically (spor-PHP1B). These latter patients present broad methylation changes of two or more differentially methylated regions (DMR) that, when mimicking the paternal allele, raises the suspicious of the occurrence of paternal uniparental disomy of chromosome 20 (upd(20)pat). A cohort of 33 spor-PHP1B patients was screened for upd(20)pat using comparative genomic hybridization with SNP-chip. Methylation analyses were assessed by methylation specific-multiplex ligation-dependent probe amplification. Upd(20)pat was identified in 6 patients, all exhibiting typical paternal methylation pattern compared to normal controls, namely a complete loss of methylation of GNAS A/B:TSS-DMR, negligible methylation at GNAS-AS1 :TSS-DMR and GNAS-XL:Ex1-DMR and complete gain of methylation at GNAS-NESP:TSS-DMR. The overall frequency of upd(20) is 18% in our cohort when searched considering both severe and partial loss of imprinting. However, twenty five patients displayed severe methylation pattern and the upd(20)pat frequency reaches 24% when searching in this group. Consequently, up to day, upd(20)pat is the most common anomaly than other genetic alterations in spor-PHP1B patients. Upd(20)pat occurrence is not linked to the parental age in contrast to upd(20)mat, strongly associated with an advanced maternal childbearing age. This study provides criteria to guide further investigations for upd(20)pat needed for an adequate genetic counseling.