Fig. 2

Molecular algorithm for the confirmation of diagnosis of PHP and related disorders. If patients present with Albright hereditary osteodystrophy (AHO), genetic alterations at GNAS should be studied, including point mutations (sequencing) and genomic rearrangements (such as multiplex ligation-dependent probe amplification (MLPA) and comparative genomic hybridization arrays (aCGH)). Once the variant is found, its pathogenicity should be confirmed according to guidelines¹³⁶, and, when possible, the parental origin should be determined. In the absence of AHO, epigenetic alterations should be analysed first. According to the results obtained for the methylation status, further tests are needed to reach the final diagnosis: if the methylation defect is restricted to transcription start site (TSS)–differentially methylated region (DMR) at exon A/B of GNAS (GNAS A/B:TSS-DMR), STX16 deletions should be screened for, and, if present, the diagnosis of autosomal dominant-pseudohypoparathyroidism type 1B (AD-PHP1B) is confirmed; if the methylation is modified at the four DMRs, paternal uniparental disomy of chromosome 20 (UPD(20q)pat) should be screened for; in absence of UPD(20q)pat, deletions at NESP should be screened for; if no genetic cause is identified as the cause of the methylation defect, the sporadic form of the disease (sporPHP1B) is suspected. After exclusion of the GNAS locus as the cause of the phenotype, and in patients with AHO, pseudohypoparathyroidism (PHP)-related genes (that is, at least PDE4D and PRKAR1A) should be sequenced. Squares in light red indicate the technology; blue, the final molecular confirmation; red, no molecular alteration; and grey, future or research steps are suggested. ICRs, imprinting control regions; MLID, multilocus imprinting disturbance; NGS, next-generation sequencing; PHP1A, pseudohypoparathyroidism type 1A; PHP1B, pseudohypoparathyroidism type 1B; POH, progressive osseous heteroplasia; PPHP, pseudopseudohypoparathyroidism; RT-PCR, reverse-transcription PCR; SNP, single-nucleotide polymorphism; STRs, short tandem repeats (microsatellites); UPD, uniparental disomy; VUS, variant of unknown significance; WES, whole-exome sequencing; WGS, whole-genome sequencing.