Imprinted genes are autosomal genes preferentially expressed from one of the two parental chromosomes. This monoallelic and parent-of-origin expression is due to the presence of differential DNA methylation between the two parental alleles of cis-acting elements known as imprinted differentially methylated regions (iDMRs) (Monk et al., 2019). Dysregulation of imprinted genes results in a group of congenital diseases (Imprinting Disorders, ImpDis) characterized by defective pre- and post-natal growth, neurocognitive development, metabolism, and increased cancer predisposition (Carli et al., 2020).

The Beckwith–Wiedemann spectrum (BWSp, OMIM 130650) and Pseudo-hypoparathyroidism type 1B (PHP1B, OMIM 603233) are ImpDis affecting two clusters of imprinted genes located on chromosome 11p15.5 and chromosome 20q13, respectively (Monk et al., 2019).

The clinical diagnosis of BWSp is based on the manifestation of cardinal features (e.g., macroglossia, exomphalos, lateralized overgrowth) and suggestive features (e.g., neonatal macrosomia, facial naevus flammeus, polyhydramnios, ear creases or pits, abdominal wall defects) (Brioude et al., 2018). The molecular diagnosis relies on the detection of one of the following defects affecting the 11p15.5 imprinted locus: loss of methylation (LoM) of the KCNQ1OT1:TSS-DMR or Imprinting Centre 2 (IC2), found in 50% of cases, mosaic paternal UPD of 11p15 (20%), gain of methylation (GoM) of the H19/IGF2:IG-DMR or Imprinting Centre 1 (IC1) (5%–10%), loss of function mutations of the growth inhibitor CDKN1C (5%), and chromosomal abnormalities in 11p15.5 (1%–5%) (Brioude et al., 2018). In about one-third of the BWSp patients with IC2 LoM methylation defects also affect iDMRs on other chromosomes, a molecular condition known as Multilocus imprinting disturbances (MLID) (Bliek et al., 2009; Brioude et al., 2018).

Diagnostic criteria for MLID have been recently proposed by Ochoa et al. (2022), according to which MLID is diagnosed when methylation abnormalities are detected at an ImpDis-associated iDMR or 2 non-ImpDis-associated iDMR, in addition to the primary ImpDis-associated epimutation. Methylation changes at multiple iDMRs due UPD are not considered MLID because of their genetic origin.”

PHP1B is characterized by end-organ resistance to several endocrine hormones, including parathyroid hormone (PTH) leading to hypocalcaemia and hyperphosphatemia, and thyroid stimulating hormone (TSH) leading to clinical or subclinical hypoparathyroidism. Occasionally, features of Albright hereditary osteodystrophy (AHO) are observed (Garin et al., 2015). At the molecular level, PHP1B is associated with methylation changes of one or more DMRs of the GNAS cluster, including at least the GNAS A/B:TSS-DMR. These methylation defects result in absence of expression of the α-subunit of the stimulatory G protein (Gs-alpha) involved in hormonal signalling pathway in renal proximal tubules. In healthy individuals, the GNAS-A/B:TSS-DMR, GNAS-XL:Ex1-DMR and GNAS-AS1:TSS-DMR are methylated on the maternal allele, and the NESP:TSS-DMR is methylated on the paternal allele. Methylation changes at one or more GNAS DMRs can be caused by inherited deletions, usually associated with autosomal dominant pattern of inheritance through maternal lineage or may occur sporadically without evident underlying genetic abnormality (Mantovani et al., 2018). Around 8%–10% of these sporadic cases are caused by paternal UPD of chromosome 20 [upd(20)pat] and show a paternal-specific methylation pattern on both alleles of all four GNAS DMRs (Mantovani et al., 2018). The upd(20)pat may affect either the long arm [upd(20q)pat] or the entire chromosome 20 (Colson et al., 2019). So far, 8 cases have been reported with upd(20)pat extended to the entire chromosome, 6 of which showing isodisomy and 2 heterodisomy (Fernández-Rebollo et al., 2010; Bastepe et al., 2011; Colson et al., 2019; Choufani et al., 2021). In another subset of PHP1B cases, a MLID profile has been identified (Maupetit-Méhouas et al., 2013). However, the exact prevalence of MLID in PHP1B is uncertain because very few cohorts of patients have been screened for MLID so far. Indeed, an incidence of MLID ranging from 0% to 38% of cases can be found in the few studies reported (Izzi et al., 2010).

A clinically relevant characteristic of imprinted disorders is the heterogeneity of the phenotype that in some cases includes atypical features. This may be caused by the extension of the molecular defect to loci other than the one typically associated with the disease. Examples are represented by cases with MLID or UPD, in particular when the UPD is extended to the whole chromosome or the whole genome in mosaicism (Eggermann and Prawitt, 2022; Grosvenor et al., 2022). In rare cases, the full clinical manifestation of two ImpDis in the same patient has been reported. The diseases most frequently co-existing with BWSp are Transient Neonatal Diabetes (TNDM) (Mackay et al., 2006a; Mackay et al., 2006b; Boonen et al., 2008) and PHP1B (Bakker et al., 2015; Sano et al., 2016; Choufani et al., 2021). All these cases, including three cases of co-occurrence of BWSp and PHP1B, displayed MLID.

Here, we describe a further case of co-occurrence of BWS and PHP1B, in which the molecular mechanisms underlying the two ImpDis appear to be independent and different from the previously reported cases.

The co-existence of two ImpDis is a rare but not negligible phenomenon that is usually associated with MLID. Three cases of BWS and PHP1B co-occurrence have been described so far, all of them related to MLID. We report and discuss here the fourth case of BWS/PHP1B co-occurrence showing pathogenetic mechanisms of disease different from the previously reported cases.

LoM of the 11p15.5 IC2 and GNAS loci with no evidence of UPD were found in two of the three previously reported cases (case 1 and case 2 in Table 1), suggesting that the two ImpDis have arisen as consequence of MLID (Bakker et al., 2015; Sano et al., 2016). In the third case (case 3 in Table 1), the methylation defects at 11p15.5 and 20q13.32 appear to arise independently, as consequences of MLID (together with additional loci such as the ImpDis-associated iDMR PLAGL1) and paternal isodisomy of the whole chromosome 20 (patUPiD20), respectively (Choufani et al., 2021).

Differently from all the above-mentioned cases, LoM occurs only at the 11p15.5 IC2, and differently from case 3 the paternal UPD of the whole chromosome 20 is a heterodisomy rather than an isodisomy in our patient. Whole-chromosome UPiD and UPhD result from different mechanisms (Eggermann et al., 2015). While patUPiD is mainly the product of zygotic monosomy rescue by endoduplication of the paternal chromosome, patUPhD results from trisomy rescue by loss of the maternal chromosome. Therefore, patUPiD rescues an aneuploidy originated during maternal gametogenesis (nullisomic oocyte) and patUPhD rescues an aneuploidy originated during paternal gametogenesis (disomic sperm). Instead, the mosaic form of IC2 LoM indicates its occurrence as post-zygotic event in all cases. It is intriguing that such rare events may occur independently in the same individual. MLID has been associated with either maternal or zygotic gene variants (Eggermann et al., 2022; Pignata et al., 2022). Because most of the chromosome 20 is completely isodisomic in case 3 and mostly isodisomic in our patient (Figure 3), it is possible that a recessive mutation unmasked by the isodisomy may interfere with maintenance of imprinted methylation in both cases 3 and 4. Thus, although the most probable hypothesis is the independent etiology of UPD20 and IC2 LoM, it is not possible to exclude that these two rare events are interconnected. Although, a direct link between GNAS and IC2 is lacking, a few studies have provided evidence for common regulatory mechanisms. For instance, the two zinc-finger proteins ZFP57 and ZFP445 are able to bind both DMRs and maintain their methylation on the maternal allele, as Zfp57/Zfp445 zygotic inactivation result in their loss of methylation in mouse and in human embryonic stem cells (Quenneville et al., 2011; Takahashi et al., 2019). Also, a meta-analysis of micro-array data reveals that genes of both the IC2 and GNAS loci are members of an imprinted gene network controlling embryonic growth in mice (Varrault et al., 2006).

Despite the different molecular mechanisms underlying the four cases of co-occurrence of BWS and PHP1B described so far, their clinical features appear similar and resulting from the sum of BWS-specific and PHP1B-specific characteristics (Table 1). In particular, macrosomia and umbilical hernia were present in all the 4 cases, macrosomia and hypoglycemia in three of them, lateralized overgrowth in two. Mild intellectual disability affected only the cases with pat(20)upd suggesting that additional loci of chr 20 may be involved in the etiology of this clinical feature. As the previously reported cases, our patient did not experience hypocalcemic tetany or seizures, but differently from them, PHP1B was diagnosed at the age of 5 years and not during adolescence.

In conclusion, this study describes the first case of co-existence of BWSp and PHP1B not associated with MLID. We found 11p15.5 IC2 LoM as molecular cause of BWSp, and paternal UPhD20 as cause of PHP1B. As the former is a postzygotic epigenetic defect and the latter occurs as zygotic trisomy rescue by loss of maternal chromosome 20, the most obvious interpretation is that the two ImpDis have distinct and independent etiologies in our patient. However, further studies will tell if the mechanisms causing the epigenetic defect and the UPD may possibly be interconnected, which may explain why they occurred in the same individual. Moreover, our study adds further evidence to the importance of multilocus molecular testing in ImpDis to reveal not only MLID profiles but also complex combinations of imprinted defects, particularly in the cases with atypical clinical presentations.