**2. RASopathies**

RASopathies are a group of genetic developmental syndromes with phenotypic overlapping features caused by germline mutation in genes that encode components or regulators of RAS/ mitogen-activated protein kinase (MAPK) pathway. Approximately, these syndromes affect 1 in 1000 live births, being one of the most common group syndromes. This group includes neurofibromatosis type I (OMIM #162200), Legius syndrome (OMIM #611431), Noonan syndrome (OMIM #163950), Noonan syndrome with multiple lentigines (formerly called LEOPARD syndrome, OMIM #151100), Costello syndrome (CS) (OMIM #218040), cardiofaciocutaneous (CFC) syndrome (OMIM #115150), Noonan-like syndrome, hereditary gingival fibromatosis, and capillary malformation-arteriovenous malformation [9–14]. Several functionally related genes, such as *PTPN11* (OMIM \*176876, mapped in 12q24.13 region), *SOS1* (OMIM \*182530, 2q22.1), *KRAS* (OMIM \*190070, 12p12.1), *BRAF* (OMIM \*164757, 7q34), *RAF1* (OMIM \*164760, 3p25.2), *MAP2K1* (OMIM \*176872, 15q22.31), *MAP2K2* (OMIM \*601263, 19p13.3), *RIT1* (OMIM \*609591, 1q22), *NRAS* (OMIM \*164790, 1p13.2), *RRAS* (OMIM \*165090, 19q13.33), *SOS2* (OMIM \*601247, 14q21.3), *SHOC2* (OMIM \*602775, 10q25.2), *CBL* (OMIM \*165360, 11q23.3), *NF1* (OMIM \*613113, 17q11.2), *HRAS* (OMIM \*190020, 11p15.5), and *SPRED1* (OMIM \*609291, 15q14), have been associated to the pathogenesis of these disorders [12–32].

mathematics skills that are substantially lower than expected for the individual's age, measured intelligence, and age-appropriate education level or when achievement falls below a set standard definition [3]. The International Classification of Disease (ICD) identifies learning disability as a condition of arrested or incomplete development in cognitive functioning or in adaptive behavior in the developmental period [4]. It can be evaluated with the general

Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) is available from 2013. The DSM-V now indicates a unique new category or diagnosis of "specific learning disorder" for issues previously differentiated as: dyslexia, dyscalculia, dysgraphia, and dysorthography. The change was made because there had been no support for a continued distinction among the terms. The single definition joined the "specifiers," and for each of them, the deficit capacities are mentioned with reference to the reading, calculation, and the written language. The DSM-V state classifies the disorder in mild, moderate, and severe. In addition, the risk factors are confirmed as the disturbance of language, familiarity, co-morbidity [5].

During last decades, many studies have been conducted to understand the basis of these neurodevelopmental disorders, leading to the identification of some altered specific neural

In this context, the American Association on Intellectual and Developmental Disabilities (AAIDD) identifies prenatal, perinatal, and postnatal causes. Among the prenatal causes, chromosomal disorders, Syndrome disorders (RASopathies), and inborn errors of metabolism can be taken into account; perinatal and postnatal causes often encompass infectious and traumatic etiologies. Several cognitive deficits may be caused by a single-gene mutation and can be classified into discrete clinical conditions with specific diagnoses [10, 11]. Notwithstanding distinct clinical entities could rise from the interaction between genes and environment.

A better understanding of pathophysiological mechanisms that lead to learning disability could provide new insights in knowledge and therapy of intellectual and learning disabilities.

RASopathies are a group of genetic developmental syndromes with phenotypic overlapping features caused by germline mutation in genes that encode components or regulators of RAS/ mitogen-activated protein kinase (MAPK) pathway. Approximately, these syndromes affect 1 in 1000 live births, being one of the most common group syndromes. This group includes neurofibromatosis type I (OMIM #162200), Legius syndrome (OMIM #611431), Noonan syndrome (OMIM #163950), Noonan syndrome with multiple lentigines (formerly called LEOPARD syndrome, OMIM #151100), Costello syndrome (CS) (OMIM #218040), cardiofaciocutaneous (CFC) syndrome (OMIM #115150), Noonan-like syndrome, hereditary gingival fibromatosis, and capillary malformation-arteriovenous malformation [9–14]. Several functionally related genes, such as *PTPN11* (OMIM \*176876, mapped in 12q24.13 region), *SOS1* (OMIM \*182530, 2q22.1), *KRAS* (OMIM \*190070, 12p12.1), *BRAF* (OMIM \*164757, 7q34), *RAF1* (OMIM \*164760, 3p25.2), *MAP2K1* (OMIM \*176872, 15q22.31), *MAP2K2* (OMIM \*601263, 19p13.3), *RIT1* (OMIM

intelligence functioning and supplemented by scales.

28 Learning Disabilities - An International Perspective

networks although the mechanisms are not fully understood [6–9].

**2. RASopathies**

Although clinical presentation can be similar, every disorder has its peculiar features (as shown in **Table 1**). They share common central nervous system dysfunction leading to learning disability-intellectual disability, cardiovascular abnormalities, dismorphic features, short stature, skeletal malformation, coetaneous lesions (tumors, spots, vascular malformation), and increasing risk of benign or malignant tumors (e.g. **Figure 1**).


**Table 1.** Classification of RASopathies with gene correlation and phenotypic features.

**Figure 1.** Typical signs of RASopathies: a) Pterigium colli (the clinical signs are wanted); b) Pectus escavatum; c) Cubitius valgus; d) Axillary freckling: a sign which initially show; e) From strains to nodules and fibroids.

Such complex phenotypes derive from mutation in the Ras/mitogen-activated protein kinase (MAPK) pathway which plays an essential role in regulation of cell cycle, differentiation, growth and cell senescence [12, 15, 16]. Focusing on these signaling alterations, there was a hyperactivation of extracellular-regulated kinase 1/2 (ERK1/2; member of the MAPK superfamily) in all of these disorders. This kind of signal could be induced by mutations in positive regulators, producing gain-of-function alleles or in negative regulators (neurofibromin 1), lossof-function alleles, of the RAS/ERK signaling pathway. Oyshi et al. ruled out this mechanism only in Noonan syndrome with multiple lentigines (LEOPARD syndrome), where mutation in the gene *PTPN11* (Y729C and T468M) encoding for protein tyrosine phosphatase SHP-2 results in a loss-of-function and a decrease in the level activity of ERK1/2 [33].

Variation across the Ras/MAPK pathway syndromes suggests that different mutant alleles of gene can have markedly various developmental effects, flowing in several syndromes. At the same time, the same allele mutant can produce different phenotypes because of the interaction with the environment, the epigenetic variation, and the action of others gene.

Recent advances in genetic analysis technologies, including whole-exome sequencing, have identified potential new genes for RASopathies [12].

#### **2.1. The Ras/mitogen-activated protein kinase (MAPK) pathway**

Such complex phenotypes derive from mutation in the Ras/mitogen-activated protein kinase (MAPK) pathway which plays an essential role in regulation of cell cycle, differentiation, growth and cell senescence [12, 15, 16]. Focusing on these signaling alterations, there was a hyperactivation of extracellular-regulated kinase 1/2 (ERK1/2; member of the MAPK superfamily) in all of these disorders. This kind of signal could be induced by mutations in positive regulators, producing gain-of-function alleles or in negative regulators (neurofibromin 1), lossof-function alleles, of the RAS/ERK signaling pathway. Oyshi et al. ruled out this mechanism only in Noonan syndrome with multiple lentigines (LEOPARD syndrome), where mutation in the gene *PTPN11* (Y729C and T468M) encoding for protein tyrosine phosphatase SHP-2 results

**Figure 1.** Typical signs of RASopathies: a) Pterigium colli (the clinical signs are wanted); b) Pectus escavatum; c) Cubitius

valgus; d) Axillary freckling: a sign which initially show; e) From strains to nodules and fibroids.

30 Learning Disabilities - An International Perspective

in a loss-of-function and a decrease in the level activity of ERK1/2 [33].

The Ras/mitogen-activated protein kinase (MAPK) pathway has a crucial role in regulating cell cycle and development, transducting signals from membrane receptors activated by growth factors to the cytoplasm and nucleus. This cascade is tightly regulated [16, 23, 34, 35]. For a better understanding, see **Figure 2**.

**Figure 2.** The Ras/MAPK signal transduction pathway of protein kinases is critically involved in cellular proliferation, differentiation, motility, apoptosis, and senescence. Mutation of genes encoding components or regulators of the Ras/ MAPK pathway (indicated by *dashed lines*) cause medical genetics syndromes named RASopathies. These disorders include neurofibromatosis type 1 (NF1). Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NSML), capillary malformation-arteriovenous malformation syndrome (CM-AVM), Costello syndrome (CS), cardiofacio-cutaneous syndrome (CFC), Leopard syndrome (LeoS), Legius syndrome (Legs), and ALPS (Autoimmune lymphoproliferative syndrome). RTK is the Receptor Tyrosine Kinase.

RAS genes, including *HRAS*, *NRAS* and *KRAS*, encode for small guanosine nucleotide-bound GTPases which are positively matched with different kind of receptors, inducing a transformation in an active GTP-bound form and an inactive Guanosine Diphosphate (GDP) bound form. Activation of RAS through receptor tyrosine kinases (RTKs) occurs thanks to recruitment of the adaptor protein growth factor receptor bound protein 2 (GRB2) and son of sevenless (SOS) which increase the level of active GTP-bound Ras [36–38].

After RAS activation, the signaling cascade is turned on with the activation of RAF (ARAF, BRAF, and/or CRAF), which activates, phosphorylating the MAPK kinases, MEK1 and/or MEK2 and, in turn, ERK1 and ERK2. ERK1 and ERK2 are the effectors of the cascade and control a large number of nuclear and cytosolic molecules owning as target cell cycle progression, cellular differentiation, and cellular growth.

Among the negative regulators of this cascade, neurofibromin 1 (*NF1*) is a GTPase-activating protein that is a negative regulator of RAS (RAS-GAP) and the Sprouty-related protein with an EVH-1 domain *SPRED1* [16, 35–39].

#### **2.2. Basis of learning disability in RASopathies**

Since 1997 the central role of RAS-ERK signaling has been identified in long-term potentiation (LTP), in long-term depression (LTD), in synaptic plasticity, in memory formation and learning during the development, including spatial learning and fear conditioning, therefore not only in cell growth, proliferation, migration, and survival [40–45].

So far, many studies have neglected the psychological and psychiatric profile of RASopathies, but new contributions of literature are proving that it is just the tip of the iceberg [46].

In central nervous system (CNS), synaptic plasticity is a prerequisite for learning and memory. First studies in animal models of RASopathies have provided interesting findings on the biological basis of these disabilities, examining the RAS/ERK functions that unfortunately are not completely understood.

The long-term potentiation (LTP) and the long-term depression (LTD) are prerequisite for synaptic plasticity in key areas as hippocampus, amygdala, insular cortex, and prefrontal cortex. N-methyl-d-aspartate (NMDA) receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA) are closely related to these mechanisms. After high-frequency release of glutamate and activation of AMPA receptors, NMDA magnesium block is removed resulting in a more sustained excitatory. NMDA receptors lead to intracellular calcium influx and AMPA phosphorylation of AMPA receptors and movement to the cell surface bringing to an increasing answer to glutamate release. Furthermore, calcium influx can promote the transcription of crucial gene for the LTP trough cAMP-dependent signaling cascade involving PKA, mitogenactivated protein kinases (MAPK), and the transcription factor cAMP-responsive element binding protein (CREB). On the other hand, low-frequency stimulation induces LTD through a weak calcium influx inducing dephosphorylation and endocytosis of AMPA receptors [47].

Increased activity of RAS-ERK pathway in keys areas of the brain (as hippocampus, parahippocampus, amygdale, prefrontal) can lead, on the one hand, to an increased activity of GABAergic interneurons, and on the other hand, to an impaired signaling in glutamatergic synapses and consequently to dysruption of synaptic plasticity through LTP or LTD. In GABAergic synapses, RAS-MAPK pathway regulates the phosphorylation of synapsin I in presynaptic neurons, where it is critically involved in maintaining the vesicle reserve pool and regulating the rate of neurotransmitter vesicle release. Neurofibromin 1 negatively regulates Ras/MAPK signaling pre-synaptically in hippocampal-GABAergic neuron; as a matter of fact, mutations in the gene of NF1 induce an enhanced GABA release. As for glutamatergic synapses, RAS-ERK pathway is activated by tyrosine kinase receptors (TRK) or calcium influx activated through N-methyl-d-aspartate (NMDA) receptors or voltage-gated calcium channels, playing a key role in the transcription of many crucial genes for long-term potentiation. Modulation of glutamatergic synapses is necessary to modify AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) receptor expression on cells surface, enhancing the basal excitatory synaptic transmission, blocking further potentiation of synaptic strength. Consistently, an hyper-activation of RAS-ERK signaling, for example, could be also due to a SHP2 mutation, enhancing the basal excitatory synaptic transmission, facilitating the synaptic trafficking of AMPA receptors to synapses with the subsequent events before described (for more information see: [45, 47–53]).

Recently, the importance of RAS-MAPK pathway has been also revealed in differentiation of neuron progenitor cells. Disruption of this cascade can result in an imbalance between neurogenesis and glycogenesis [35].

Several neurophysiological studies have been conducted in patients with RASopathies by using transcranial magnetic stimulation (TMS), a noninvasive and safe way to investigate neuronal plasticity. Several experimental paradigms applying the so-called paired associative stimulation (PAS) have demonstrated that patients with neurofibromatosis type I and Noonan syndrome have reduced LTP-like synaptic plasticity depending on an increased intracortical inhibition. On the contrary, TMS studies in Costello syndrome (CS) patients have shown enhanced LTP-like synaptic plasticity related to reduced inhibition [51, 54–56].

In summary, there are strong evidences that the deregulation of activity of RAS-MAPK signaling can lead to LTP impairment and altered neuronal plasticity resulting in learning and memory impairment.
