Information about:
Pseudohypoparathyroidism Ib
Pseudohypoparathyroidism Typ Ib (PHP1B, OMIM 603233)
by Susanne Thiele1 and Agnes Linglart2
Clinical description:
Pseudohypoparathyroidism type Ib (PHP1B) is a group member of disorders united by PTH resistance in the renal proximal tubule, i.e. pseudohypoparathyroidism (PHP). PTH resistance is manifested by elevated serum PTH levels, hyperphosphatemia, and hypocalcemia. In most cases of PHP1B the clinical phenotype is limited to renal PTH resistance and in some cases TSH resistance has been reported (Levine et al., 1983, Mantovani et al., 2007). In addition, patients with PHP1B may rarely show some features of AHO (de Nanclares 2007, Mariot et al., 2008), including short stature, brachymetacarpia, subcutaneous ossifications, and mental retardation, typically seen in patients with the PHP1a subtype.
Molecular genetic and epigenetic background: ↓ Such as most PHP subtypes, PHP1B is caused by Gsalpha (Gsa) deficiency. Gsa is the alpha subunit of trimeric stimulatory G proteins mediating the extra to intracellular signal transduction of more than 800 different G protein coupled receptors (Fredriksson et al., 2003). The Gsa encoding gene GNAS is located on the long arm of chromosom 20 and comprises not only the coding regions of the Gsa protein, but also of alternative transcript variants termed as XLalpha-s (XLas), A/B transcript (A/B), antisense transcript (AS), and neurosecretory protein 55 (NESP55) (Hayward et al., 1998; Hayward and Bonthron, 2000). The expression of these alternative variants underlies an imprinting, leading to a predominantly expression of XLas, A/B, and AS from the paternal and NESP55 from the maternal allele and to silencing of the expression of the opposite allele, respectively. In agreement with their parental specific expression, the promoters and first exons of the alternative variants lie into differentially methylated regions (DMRs), characterized by cytosine methylation. In contrast to XLas, A/B, AS, and NESP55 Gsa is biallelically expressed in most tissues. Exceptions are the proximal renal tubules, brown adipose tissue, the pituitary, the gonads, and the thyroid, in which Gsa is predominantly expressed from the maternal allele, but silenced on the paternal allele. However, silencing of Gsa paternal expression is not determined through methylation of its promotor, but through a mechanism still to be concealed, involving the GNAS-DMRs of the alternative variants. The methylation pattern of these DMRs is controlled by different imprinting control regions (ICRs) lying either in the GNAS locus itself, or in the about 220 kb upstream localized STX16 gene. STX16 is coding for a member of the SNARE-(soluble N-etylmaleimide-sensitive factor attachment protein receptor) protein family, important for transport and vesicle function in the cell. Since STX16 is not an imprinted gene it is thought that it intronically contains an ICRs regulating the establishing or maintanance of the methylation pattern of exon A/B. For a scheme of the STX16 gene and the GNAS gene locus see Figure 1.
More than 20 alternative spliced isoforms are encoded by the GNAS gene locus, partly created due to the use of alternative first exons and promotors. The scheme shows the most important alternative first exons and promotor regions, the corresponding transcripts and their direction of transcription (arrows), as well as their methylation pattern (+++) on the maternal (Mat) and on the paternal (Pat) allele. 220 kb upstream of GNAS the STX16 gene is located, containing an imprinting control region important for the methylation of the DMR of Exon A/B (adapted from Thiele et al., 2010).
Molecular and epigenetic changes in PHP1B: ↓ The knowledge about the ICRs comes mainly from patients with PHP1B, in whom deletions removing these regions lead to distinct changes of DMR methylation patterns hence diminished Gsa expression in the imprinted tissues. Since the loss of methylation (LOM) at the A/B DMR of the maternal GNAS allele is a consistent finding in patients with PHP1B, this epigenetic defect seems to be mandatory for the pathogenesis of PHP1B and accounts for decreased Gsa expression from the affected allele (Liu et al., 2000). Most PHP1B cases (about 80%) occur sporadically (spor-PHP1B) (Liu et al., 2000; Maupetit-Mehouas et al., 2011), while, in some kindred, maternally autosomal dominant inheritance has been reported (AD-PHP1B). Independent of their inheritance pattern, both forms lead to the same clinical manifestations of PTH resistance (Linglart et al., 2007). In spor-PHP1B methylation changes affect several GNAS–DMRs including A/B. The underlying molecular cause for spor-PHP1B has not been detected yet, but it is assumed, that it is caused by disruptive mutations of regulatory elements other than STX16 or GNAS. Rarely, UPD can be found in spor-PHP1B cases (Bastepe et al., 2001; Fernández-Rebollo E et al., 2010; Bastepe et al, 2010; Dixit et al., 2013). In contrast to spor-PHP1B, most cases of AD-PHP1B present with an exclusive loss of methylation (LOM) of Exon A/B, frequently associated with a 3-kb microdeletion located about 220 kb upstream of Exon A/B and removing the exons 4-6 of STX16 (Bastepe et al., 2003). A second microdeletion (4.4 kb length) was detected in 2005 in a family with AD-PHP1B, removing the exons 2-4 of STX16 (Linglart et al., 2005). Very recently, a third deletion in the STX16 region has been described starting in intron 1 and removing exon 2 to 8, overlapping with the above mentioned deletions (Elli et al., 2014). In two families with AD-PHP1B a deletion within GNAS ablates the NESP55 DMR and the exon 3 and 4 of AS (delNESP55/delAS3-4) and leads after maternal inheritance to LOM of the XLas, A/B, and AS DMRs and biallelic expression of these transcripts (Bastepe et al, 2005). An additional deletion removes selectively exon 3 and 4 (delAS3-4) of the AS transcripts and overlapps by 1.5 kb with the deletion delNESP55/delAS3-4. In case of maternal inheritance, not only the methylation level at the maternal methylated DMRs (XLas, AS, and A/B) is diminished, but also the paternally methylated NESP is hypermethylated. While all other described deletions influence the methylation pattern only through maternal inheritance, delNESP55/delAD3-4 leads after paternal inheritance to partial LOM at the NESP55-DMR and to partial gain of methylation at exon A/B-DMR (Chillambi et al., 2010). Another deletion of about 19 kb starting at NESP55 and removing a large part of AS intron 4 has been associated to a LOM restricted to the A/B DMR without affecting the other GNAS DMRs. This deletion overlaps with the previously reported delNESP55/delAS3-4 and delAS3-4 by only 342 bp (Richard et al., 2012). For an overview of the described deletions in AD-PHP1B see Figure 2 and Table 1.
Figure 2 demonstrates the localization of the currently reported deletions in STX16 and GNAS associated with an abnormal methylation pattern at the GNAS-DMRs. While deletions described by Bastepe in 2003, Linglart, Elli, and Richardson affect only the A/B-DMR, deletions identified by Bastepe in 2005 and by Chillambhi affect all GNAS-DMRs, a pattern similar to that of spor-PHP1B. Because deletions are associated with methylation changes, it is assumed, that ICRs are located in the deleted regions.
Molecular diagnosis in PHP1B patients: ↓ The molecular diagnosis of PHP1B is a three-step strategy, starting with investigation of the phenotype, going over to determinate the epigenetic changes and ending by the identification of the underlying structural change on the genetic level: First step: identify the patient’s phenotype compatible with PHP1B. As written, in most cases the phenotype of patients affected with PHP1B is restricted to the renal resistance to the action of PTH, i.e. the association of normal or low calcium levels, elevated serum phosphate, elevated PTH and normal kidney function. Since PTH resistance develops overtime and may be absent in young individuals, it is of importance to investigate also carefully siblings of PHP1B patients, especially when they display slightly elevated phosphate level, low utinary calcium and/or high-normal PTH. Careful attention should be paid to the biochemical characterization of PTH resistance as other conditions may lead to increased PTH levels and normal calcium levels (normocalcemic hyperparathyroidism) or increased PTH levels and hypocalcaemia (vitamin D deficiency). The diagnosis should be considered also in patients with resistance to hormones that signal through GPCRs and features of Albright osteodystrophy in absence of loss-of-function mutation in the coding GNAS gene. Second step: prove the loss of imprinting at the A/B promoter of GNAS Since the LOM of Exon A/B seems to be mandatory for the pathogenesis of PHP1B, the determination of the methylation status of the A/B-DMR should be the first step for investigating the epigenetic changes involved in PHP1B. To assess the methylation pattern, today, most techniques differentiate the methylated and unmethylated alleles through the chemical modification of unmethylated cytosines by sodium bisulfite followed by DNA amplification. The qualitative, semi-quantitative or quantitative assessment of the methylated cytosines (not modified by the sodium bisulfite) or unmethylated cytosines (converted into thymines) may be done through numerous tactics including enzymatic restriction (COBRA or Combined Bisulfite restriction enzymatic analysis), Sanger sequencing, pyrosequencing, MethylQuant or EpiTYPER. Alternatively, other methods such as Methylation-Specific MLPA, or MS-MLPA, profile the methylation semi-quantitatively using methyl-sensitive restriction of the DNA combined to copy number detection (see below). Third step: identify the underlying cause of the loss of maternal imprint at the A/B promoter of GNAS As described above, the known causes to be discovered of loss of maternal imprint at GNAS are: The deletion of an ICR of GNAS Identification of ICRs deletions in GNASor STX16 has been done through various methods including Southern-blot (Mariot et al., 2008), quantitative genomic PCR (Mariot et al., 2008), Comparative Genomic Hybridization, Multiplex long-range PCR (Bastepe et al., 2003) and MS-MLPA (Elli et al., 2014). The latter is now widely used as it assesses both the methylation profile and copy number of GNAS alleles in a single experimental run and it is commercially available. However, the large size of the imprinted GNAS locus and the likelihood of necessary long-acting regulator elements of GNAS prevent the complete search for ICRs deletions with these methods. Cytogenetic errors involving chromosome 20q Because the discovery of patUPD will allow informing the patient of the near-absent risk of transmission, we propose to investigate patients with broad loss of imprinting (LOI) at the GNAS locus, especially if they also display LOI at the nearby imprinted locus L3MBTL1 (Maupetit-Mehouas et al., 2013). Isodisomy is characterized by a perfect homozygosity along segments or the entire chromosome. All methods used to evidence isodisomy feature the loss of heterozygosity (LOH) like microsatellite analysis or Single Nucleotide Polymorphism (SNP) arrays. The search for heterodisomy requires analyzing parents and proband’s samples and perform haplotype analysis. Identification of LOI will therefore trigger genetic and cytogenetic investigations depending on the GNAS methylation profile. Loss of methylation restricted to the A/B promoter of GNAS leads to a thorough analysis of the STX16 and NESP regions. Broad LOI at GNAS implies to look for patUPD, deletion of the locus and deletion of an ICRs of GNAS. However, in most cases of spor-PHP1B, and some families of AD-PHP1B, the molecular genetic hit leading to the GNAS epigenetic changes remains to be found. To reveal the underlying cause in these cases and the mechanism leading to the methylation changes will be the main focus of our scientific work.
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Author affiliations: ↓ 1) Devision of Experimental Pediatric Endocrinology and Diabetes, Department of Pediatrics, University of Lübeck, Germany; thiele@paedia.ukl.mu-luebeck.de 2) Service d´Endocrinologie et Diabétologie pédiatrique, Hopital de Bicétre, France; secretariat.linglart@bct.aphp.fr