A pigmentary manifestation associated with PPP2R5D-related neurodevelopmental disorder: a case report and review of literature

Abstract

Background

The protein phosphatase 2 (PP2A) is one of the major serine/threonine phosphatases in humans. The most frequently reported pathogenic PP2A variants have been identified in PPP2R5D, encoding the regulatory subunit B’ delta, and are known to cause intellectual developmental disorder autosomal dominant 35 (MRD35).

Case presentation

Herein, we describe a unique case of a patient with a heterozygous pathogenic variant, c.592G>A/p.(Glu198Lys) in the PPP2R5D gene which was associated with hyperpigmented skin lesions arising from increased melanin production, known as Café-au-lait macules (CALMs). To our knowledge, this is the first reported case of a PPP2R5D-related neurodevelopmental disorder associated with CALMs.

Conclusions

Our findings suggest that the documentation and reporting of CALMs when associated with one or more physical and/or neurodevelopmental findings are of utmost importance as they could be indicative of an underreported phenotype and may extend the phenotypic spectrum of MRD35.

Background

The identification of de novo pathogenic variants in genes encoding subunits of the protein phosphatase 2 (PP2A) has often been linked to neurodevelopmental disorders (Lenaerts et al. 2021; Houge et al. 2015; Loveday et al. 2015; Reynhout et al. 2019a). PP2A is one of the major serine/threonine phosphatases in humans and has been shown to balance most kinase-regulated signaling pathways, thus regulating cell metabolism, cell cycle, cell proliferation, cell mobility, as well as apoptosis (Seshacharyulu et al. 2013; Alberts et al. 1993; Glenn and Eckhart 1993). PP2A is structurally complex as it forms over 100 different holoenzymes with various tissue and substrate specificity (Yu et al. 2014; Lechward et al. 2001; Biswas et al. 2020). Trimeric PP2A holoenzymes are composed of a scaffold (PP2A-A), a catalytic (PP2A-C) and a regulatory subunit (PP2A-B) (Lenaerts et al. 2021; Kamibayashi et al. 1994). The latter is thought to be the master regulator of PP2A holoenzymes; it is encoded by 15 different genes including 26 distinct splice variants which determine substrate specificity (Seshacharyulu et al. 2013; Zolnierowicz et al. 1994).

The most frequently reported de novo pathogenic PP2A variants have been identified in PPP2R5D, encoding the regulatory subunit B’ delta (B’56δ) (Lenaerts et al. 2021; Shang et al. 2016). PPP2R5D-related neurodevelopmental disorder (NDD), also known as intellectual developmental disorder autosomal dominant 35 (MRD35) and Jordan’s syndrome, is characterized by a broad phenotypic spectrum of mild to severe neurodevelopmental delay (Biswas et al. 2020). Despite a growing number of documented cases, PPP2R5D-related NDD has been linked to a rather limited mutational spectrum (Biswas et al. 2020). In fact, only 17 pathogenic variants have been identified until now (ClinVar [cited 2023]), 9 of which have been extensively documented within 30 cases (Table 1). The first four cases were identified in 2014 after sequencing of 1133 children with undiagnosed severe developmental disorders (Study DDD 2015). These four cases were later well-described along with seven additional cases (Houge et al. 2015). Since then, nine other peer-reviewed publications have reported and described an additional 23 cases for a total of 30 cases (Loveday et al. 2015; Shang et al. 2016; Yeung et al. 2017; Kim et al. 2020; Hetzelt et al. 2021; Madaan et al. 2022; Maines et al. 2021; Walker et al. 2021; Yan et al. 2021). Most cases present with global developmental delay, intellectual disability, hypotonia, and facial dysmorphism with overgrowth (Table 1). Surprisingly, although PP2A holoenzymes modulate the Ras-mitogen-activated protein kinase (Ras/MAPK) signaling pathway, which is associated with physiological and pathological skin pigmentation such as Café-au-lait macules (CALM) (Carvalho et al. 2021; Rzepka et al. 2016; Oiso et al. 2013; Picardo and Cardinali 2011; Zhang et al. 2016), no PP2A-PPP2R5D variants have ever been associated with such skin pigmentary abnormalities. However, two cases associated with CALMs have been described for variants affecting the PP2A-C, specifically in the catalytic Cα subunit gene (PPP2CA) (Reynhout et al. 2019b).

Table 1 Clinical presentation of patients with PPP2R5D variants

Herein, we report the first case of PPP2R5D-related NDD associated with CALMs in a young girl. This finding suggests that CALMs could be associated with MDR35 and may be indicative of an underreported clinical feature.

Case presentation

The patient is a 2-year-old female born prematurely at 33 weeks and 6 days. She presented with global developmental delay, delayed gross motor development, dysmorphic facial features, feeding difficulties, and muscular hypotonia. Her physical examination revealed macrocephaly, tall stature, and the presence of well-delineated CALMs were identified on the right ankle (1 cm × 1.5 cm) and the right flank of the abdomen (1 cm × 2 cm) (Fig. 1A, B). Electroencephalogram, comparative genomic hybridization, fragile-x testing, screening for congenital defect of glycosylation, and metabolic workup including plasma amino acids, acylcarnitine profile, very long-chain fatty acids, pipecolic acid, urine mucopolysaccharidosis, oligosaccharides, organic acid, and purines/pyrimidines were all without significance, thus, not in favor of chromosomal anomalies, fragile X syndrome, or an inborn error of metabolism. Brain magnetic resonance imaging (MRI) revealed non-specific findings such as mild prominence of the cerebral sulci and ventricles as well as mild-to-moderate white matter atrophy (Fig. 2).

Fig. 1

Clinical photographs of the patient showing: A frontal bossing, bi-temporal narrowing, large depressed nasal bridge, low-set ears, hypertelorism, and mild prognathism. B Images of well-defined Café-au-lait macules on the right thorax (1by 2 cm) and ankle (1 by 1.5 cm)

Fig. 2

(i) Axial T2 prop. (ii) Sagittal T1 Flair brain MRI: macrocephaly with slight ventricular extra-axial CSF prominence with prominent cerebral sulci and mild-to-moderate white matter atrophy at 12 months of age

Weaver, Sotos, Banayan-Ruvalcaba syndromes, and in particular, Neurofibromatosis (NF1/NF2) were the primary clinical suspicions due to the presence of CALMs, even though clinical criteria were not fully met. A targeted exome sequencing panel including copy number variants did not reveal any variants in genes associated with the initial suspected genetic disorders. However, the exome sequencing revealed a pathogenic PPP2R5D c.592G>A/p.(Glu198Lys) variant, confirming the genetic diagnosis of MRD35. Both parents were also tested, but results came back negative supporting the de novo occurrence of this variant. Due to the presence of CALMs, a re-analysis of genes covering the initial clinical suspicions was performed; however, the results were all negative (Table 2). Also, no family history of neurocutaneous disorders was documented or reported. Surveillance and management of patient, more specifically, monitoring for seizures, vision issues, developmental progress, and education needs were recommended. Furthermore, a follow-up with a speech-language therapist was also recommended.

Table 2 Genes re-analyzed by sequencing

Discussion

Including this patient, a total of 31 cases of PPP2R5D-related NDD have been reported in case reports and 48% (15/31) carry the same c.592G>A/p.(Glu198Lys) variant (Table 1). Interestingly, a new study by Oyama et al. (2022) has recently analyzed clinical data collected from the Simons Searchlight Single Gene Dataset V.7 and have revealed 73 PPP2R5D-related NDDs. Unfortunately, detailed growth measurements, clinical photographs, or details on physical examinations were not included, and thus, the presence of CALMs was not assessed. CALMs are hyperpigmented skin lesions arising from increased melanin production by melanocytes in the basal layer of the epidermis (Anderson 2020). While CALMs are usually benign skin hyperpigmentation, careful attention should always be given when they are associated with one or more physical and/or neurodevelopmental findings as they have been found to be associated with rare genetic conditions (Carvalho et al. 2021; Zhang et al. 2016; Anderson 2020). In fact, a recent study that aimed at identifying documented diseases that may be present simultaneously with CALMs (Carvalho et al. 2021) was able to identify 60 rare genetic diseases where CALMs were reported in the clinical presentation. For most cases in this list, CALMs were an occasional or unexpected finding, similarly to our case, with an insufficient number of affected patients to establish a clear association with the disease in question (Carvalho et al. 2021). Furthermore, apart from Neurofibromatosis type 1, a disease where CALMs are very well characterized, no clear guidelines seem to exist regarding their number or size except that their presence could highlight a potential genetic disease (Ponti et al. 2012). Thus, it is of great importance to document CALMs or other skin pigmentary abnormalities when they are associated with NDD or other rare diseases as they might be underreported or missed in physical examinations. Systematically reporting CALMs when they are present with other phenotypic characteristics or when a genetic disease is suspected/diagnosed could help in establishing if they underlie a specific disease and request appropriate genetic testing since current neurofibromatosis multigene panels do not include PPP2R5D gene.

Diseases that are known to be associated with CALMs or other skin pigmentary abnormalities often present with mutations in genes implicated in the modulation of the Ras/MAPK pathway (Carvalho et al. 2021; Tajan et al. 2018; Silverstein et al. 2002). PP2A holoenzymes have clearly been shown to regulate this pathway (Silverstein et al. 2002; Letourneux et al. 2006; Ory et al. 2003; Ciccone et al. 2015; Adams et al. 2005; Sieburth et al. 1999; Walter 2003); however, some have shown PP2A to be a key negative regulator of MAPK signaling (Silverstein et al. 2002; Zhou et al. 2002; Alessi et al. 1995; Sonoda et al. 1997; Sontag et al. 1993), while others seem to indicate that it functions as a positive regulator of this kinase cascade (Ory et al. 2003; Adams et al. 2005; Abraham et al. 2000; Kubicek et al. 2002; Jaumot and Hancock 2001; Strack 2002; Dougherty et al. 2005). These discrepancies seem to be due to the complexity of the PP2A holoenzymes’ structure, their tissue dependence, and the regulatory subunits involved. In fact, specific PP2A holoenzymes seem to be able to target multiple steps in the Ras-MAPK signal transduction cascade and appear to do so in a cell type-specific manner (Walter 2003). It is unclear whether PP2A holoenzymes simultaneously play opposing roles at different sites in the MAPK signaling, both positive and negative regulations, or if these occur in a cell type-dependent manner or in response to different external stimuli (Walter 2003). These knowledge gaps highlight a crucial need for mechanistic studies in the roles of specific PP2A holoenzymes, such as PP2A-PPP2R5D in Ras-MAPK signaling which could lead to a better understanding of PPPP2R5D-related NDD and associated CALMs. Some studies have also demonstrated that additionally to the convoluted roles of specific PP2A-holoenzymes in Ras-MAPK regulation, some PP2A complexes seem to have direct roles in melanin synthesis (Reilein et al. 1998; Kim et al. 2005). Reilein et al. (1998) showed that melanosome aggregation could be mediated by PP2A as overexpression of PP2A inhibited pigment dispersion by α-melanocyte-stimulating hormone. Furthermore, in a murine cell model, Kim et al. (2005) showed that PP2A holoenzymes-mediated melanin synthesis through a mechanism that seemed to be dependent on ERK regulation. Thus, these results also suggest direct roles for PP2A holoenzymes in melanin synthesis and could explain the presence of CALMs in gene variants affecting specific subunits in PP2A holoenzymes.

It is of note that PP2A-PPP2R5D holoenzymes have also been shown to regulate the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway by direct AKT dephosphorylation. It is thought that variants disrupting PP2A holoenzyme lead to loss of PI3K-AKT-mTOR growth regulation and affect intellectual skills (Loveday et al. 2015; Yeung et al. 2017). A functional study showed that a constitutively active AKT-mTOR signaling leads to increased cell size and an uncoordinated cellular growth associated with the PPP2R5D c.1258G>A/p.E420K variant generated in HEK-293 cells using CRISPR-cas9 biotechnology (Papke et al. 2021).

To the best of our knowledge, CALMs have never been described with PPP2R5D variants. We cannot eliminate the possibility that this occurrence might be coincidental because of the higher incidence of CALMs being reported in the general population; however, we strongly believe that cases of CALMs associated with one or more physical and/or neurodevelopmental findings may be underreported. The systematic dermatological review and documentation of patients with mutations in PPP2R5D is clearly difficult in such a rare disorder. The only reported cases of CALMs associated with PPP2R5D are of two patients with variants in the PPP2CA catalytic subunit (Reynhout et al. 2019a). It is of note that this subunit has also been shown to be a regulator of the Ras/MAPK signaling pathway (Wlodarchak and Xing 2016). Interestingly, the identified PPP2R5D c.592G>A/p.(Glu198Lys) variant in our patient is one of the most characterized biochemically and has been shown to affect the catalytic subunits of PP2A (Houge et al. 2015; Loveday et al. 2015; Biswas et al. 2020; Shang et al. 2016). Patients with the PPP2R5D c.592G>A/p.(Glu198Lys) variant present with more severe intellectual disability because of the conferred degree of biochemical defect in PP2A activity (Houge et al. 2015; Biswas et al. 2020). Specifically, this variant localizes in a highly conserved acidic loop where the substitution of a negatively charged glutamine for a positively charged lysine significantly affects the binding of the catalytic subunit with the scaffolding subunit (i.e., holoenzyme formation) through what is thought to be a dominant-negative mode of action (Lenaerts et al. 2021; Houge et al. 2015). Because the PPP2R5D c.592G>A/p.(Glu198Lys) is known to directly affect the catalytic subunit of PP2A, it is possible that the CALMs identified in this case may be associated with the same defective molecular mechanisms underlying the PPP2CA catalytic subunit variant.

Because the regulatory subunit of PP2A is the most structurally diverse, it is challenging to study mechanistically. However, we strongly believe that documenting this PP2A-PPP2R5D variant associated with CALMs is of utmost importance. A clear limitation of this study remains the lack of molecular and functional studies linking the PPP2R5D pathogenic variant to melanogenesis. Future functional studies will thus be needed to uncover the roles of PP2A-PPP2R5D in Ras-MAPK signaling as well as in the various paracrine networks mediating melanogenesis which may lead to a clearer clinical association between CALMs and PPP2R5D-associated NDD.

Conclusions

This is the first report of PPP2R5D-related NDD associated with CALMs. Encouraging the systematic documentation and reporting of CALMs when associated with one or more physical and/or neurodevelopmental findings is of utmost importance to better characterize the phenotype of specific disorders in order to improve differential diagnosis and consequently request appropriate genetic testing. In fact, for most rare genetic cases, CALMs are an occasional or unexpected finding, similarly to our case, with an insufficient number of affected patients to establish a clear association with the disease in question. Further studies will be needed to characterize the roles of PP2A-PPP2R5D holoenzymes in Ras/MAPK and AKT/mTOR pathways and may shed light on the association of CALMs with PPP2R5D-related NDD.

Exome sequencing

Buccal swab was performed and sent to LifeLabs Genetics (Toronto, ON) who performed the DNA extraction, sequencing, and data analysis in accordance with their protocol.