**3. Focus on learning disability in every RASopathy**

### **3.1. Neurofibromatosis type 1**

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 seven-

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,

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

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

So far, many studies have neglected the psychological and psychiatric profile of RASopathies,

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

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

but new contributions of literature are proving that it is just the tip of the iceberg [46].

less (SOS) which increase the level of active GTP-bound Ras [36–38].

cellular differentiation, and cellular growth.

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

in cell growth, proliferation, migration, and survival [40–45].

an EVH-1 domain *SPRED1* [16, 35–39].

32 Learning Disabilities - An International Perspective

completely understood.

Neurofibromatosis type I was the first RASopathies identified, it is a genetic disorder caused by mutations in the neurofibromin 1 gene (*NF1*) at locus 17q11.2, resulting in loss-of-function of its protein product. Neurofibromatosis type I has an autosomal dominant inheritance, as homozygous mutations appear to be lethal and has an incidence of approximately 1 in 2600– 3000 individuals [39, 57, 58].

This syndrome is characterized by the presence of café-au-lait maculae (spots), (axillary and inguinal) intertriginous freckling, neurofibromas and plexiform neurofibromas, iris Lisch nodules, osseous dysplasia, optic pathway glioma, and/or a first-degree relative with NF1. Up to 65% of NF1 patients show cognitive impairments which frequently involve executive and higher order cognitive domain [14, 16, 39, 48, 49, 57–59].

Neurofibromin acts as a RAS-GAP (GTPase activating protein) and negatively regulates Ras signaling, also working as an activator of adenylate cyclase. Its mutation brings to a hyperactivation of the RAS-MAPK signaling cascade and to an increased GABA-mediated inhibition of interneurons, significantly reducing long-term potentiation in hippocampus and amygdala. The role of NF1 is crucial in maintaining the balance between RAS- and cAMP-dependent signaling [38, 47, 48, 56–58].

Reading/vocabulary, visuospatial functions, motor coordination, planning, and organizational skills are often impaired. High co-morbidity with attention deficit hyperactivity disorder (ADHD) is explained by frequent impairment of working memory, cognitive flexibility, and inhibitory control. Patients with NF1 show both *nonverbal* (poor performance in tests of visuospatial functioning and spatial learning, impairments in the ability to perceive social cues, poor organizational skills, and increased impulsiveness) and *verbal-type learning disability* (expressive and receptive language, vocabulary, visual naming, and phonologic awareness). A single mutation can give rise to a complex spectrum of learning disabilities [39, 60, 61].

Emerging insights from the pathophysiology of this syndrome have provided new potential targets for learning disability therapy. A randomized, double-blind, placebo-controlled study evaluated the influence of lovastatin on impaired synaptic plasticity in patients with NF1. By decreasing the ERK basal activation, lovastatin reduces RAS-pathway hyperactivity with a significant improvement in verbal and nonverbal memory, visual attention, and efficiency [15, 54, 56, 58, 61].

#### **3.2. Noonan syndrome**

Noonan syndrome is an autosomal dominant genetic disorder with a prevalence approximately of 1 every 1000–2500 live births, caused by activating germline mutations in the *PTPN111* gene in 50% of affected individuals, but other cases have shown to be caused by gain-of-function mutations in *KRAS* (fewer than 50%), *SOS1* (approximately in 13%), *RAF1*, and *RIT1* (in 5%). Other genes have been reported in literature which is associated with Noonan syndrome, for example, NRAS, BRAF, and MAP2K1 [14, 55, 62–66].

Mutation in *PTPN11* induces alteration of function in the protein SHP2 (nonreceptor tyrosine phosphatase) which loses its ability to switch from the active to the inactive protein conformation, causing an increased signaling in Ras/MAPK cascade. Its role is crucial in determining neuronal cell fate and regulating the generation of oligodendrocytes. Hyperactivity of Ras/ MAPK cascade increases delivery of AMPA receptors to the synapses, enhancing the basal excitatory synaptic transmission. Mutations in SOS1 induce loss-of-function in auto-inhibition and gain-of-function in the protein product RAS-GEF protein which acts as a stimulator of the conversion of RAS from the inactive to the active form. Mutations of KRAS and BRAF also determine RAS/MAPK up-regulation [35, 62–66].

This syndrome is typically characterized by facial anomalies, short stature, family history, chest carinatum/excavatum, congenital heart defects (pulmonary valve stenosis, septal defects and hypertrophic cardiomyopathy, atrial and ventricular septal defects, branch pulmonary artery stenosis, and tetralogy of Fallot), lymphatic dysplasia, cryptorchidism, varied coagulation defects, and learning disabilities [64, 65].

A wide variability of cognitive complaints has been recognized ranging from absent or mild learning problems to severe intellectual disabilities and depends on type of mutation. Patients with *SOS1* mutations performed significantly higher on both verbal and nonverbal cognitive tests than individuals with PTPN11 and other kinds of mutations. Several studies have demonstrated that these patients have a greater risk to have impaired performance in verbal free recall task than in visual and spatial recognition memory tasks. Furthermore, Pierpont et al. showed that children with Noonan syndrome have different performance on verbal memory tasks, on visual memory, or working memory. Better performances have been obtained in immediate verbal memory than in delayed free recall tasks; a more pronounced hippocampal and prefrontal cortex dysfunction may probably reflect RAS-MAPK aberration in memory formation and consolidation [62, 63, 65–67].

Likewise, NF1 promising studies on the therapeutic effect of lovastatin in mice with mutation in PTPN11 are currently underway thanks to its action in decreasing basal Erk activation and seem to represent a therapeutic strategy for learning deficits Noonan syndrome [68].

#### **3.3. Legius syndrome (NF1 like)**

nodules, osseous dysplasia, optic pathway glioma, and/or a first-degree relative with NF1. Up to 65% of NF1 patients show cognitive impairments which frequently involve executive

Neurofibromin acts as a RAS-GAP (GTPase activating protein) and negatively regulates Ras signaling, also working as an activator of adenylate cyclase. Its mutation brings to a hyperactivation of the RAS-MAPK signaling cascade and to an increased GABA-mediated inhibition of interneurons, significantly reducing long-term potentiation in hippocampus and amygdala. The role of NF1 is crucial in maintaining the balance between RAS- and cAMP-dependent

Reading/vocabulary, visuospatial functions, motor coordination, planning, and organizational skills are often impaired. High co-morbidity with attention deficit hyperactivity disorder (ADHD) is explained by frequent impairment of working memory, cognitive flexibility, and inhibitory control. Patients with NF1 show both *nonverbal* (poor performance in tests of visuospatial functioning and spatial learning, impairments in the ability to perceive social cues, poor organizational skills, and increased impulsiveness) and *verbal-type learning disability* (expressive and receptive language, vocabulary, visual naming, and phonologic awareness). A single mutation can give rise to a complex spectrum of

Emerging insights from the pathophysiology of this syndrome have provided new potential targets for learning disability therapy. A randomized, double-blind, placebo-controlled study evaluated the influence of lovastatin on impaired synaptic plasticity in patients with NF1. By decreasing the ERK basal activation, lovastatin reduces RAS-pathway hyperactivity with a significant improvement in verbal and nonverbal memory, visual attention, and efficiency

Noonan syndrome is an autosomal dominant genetic disorder with a prevalence approximately of 1 every 1000–2500 live births, caused by activating germline mutations in the *PTPN111* gene in 50% of affected individuals, but other cases have shown to be caused by gain-of-function mutations in *KRAS* (fewer than 50%), *SOS1* (approximately in 13%), *RAF1*, and *RIT1* (in 5%). Other genes have been reported in literature which is associated with

Mutation in *PTPN11* induces alteration of function in the protein SHP2 (nonreceptor tyrosine phosphatase) which loses its ability to switch from the active to the inactive protein conformation, causing an increased signaling in Ras/MAPK cascade. Its role is crucial in determining neuronal cell fate and regulating the generation of oligodendrocytes. Hyperactivity of Ras/ MAPK cascade increases delivery of AMPA receptors to the synapses, enhancing the basal excitatory synaptic transmission. Mutations in SOS1 induce loss-of-function in auto-inhibition and gain-of-function in the protein product RAS-GEF protein which acts as a stimulator of the conversion of RAS from the inactive to the active form. Mutations of KRAS and BRAF also

Noonan syndrome, for example, NRAS, BRAF, and MAP2K1 [14, 55, 62–66].

determine RAS/MAPK up-regulation [35, 62–66].

and higher order cognitive domain [14, 16, 39, 48, 49, 57–59].

signaling [38, 47, 48, 56–58].

34 Learning Disabilities - An International Perspective

learning disabilities [39, 60, 61].

[15, 54, 56, 58, 61].

**3.2. Noonan syndrome**

Legius syndrome is an autosomal dominant genetic disorder caused by germline mutations in the SPRED1 which induce loss-of-function in the product protein. It is typically characterized by multiple café au lait macules without neurofibromas or other tumors, intertriginous freckling, lipomas, macrocephaly, and learning disabilities/ADHD/developmental delays [57, 69].

SPRED1 is a negative regulator of the Ras/MAPK pathway, being a substrate of SHP2, and its mutation leads to a hyperactivation of this cascade, and in animal models, it also has been seen that mice have some deficits in hippocampus-dependent spatial learning and in several phases of visual discrimination learning [35, 70].

#### **3.4. LEOPARD syndrome**

LEOPARD syndrome (Noonan syndrome with multiple lentigines) is an autosomal dominant genetic disorder, caused by mutation in *PTPN11* (p.Y279C and p.T468P) and *RAF1*. Phenotypically, they have the same features of Noonan syndrome patient but with multiple lentigines, electrocardiogram abnormalities, pulmonary valve stenosis, abnormal genitalia, growth retardation, and ocular hypertelorism [14, 16, 17, 35, 71].

Several studies conducted in vitro have demonstrated that mutation in PTPN11 leads to a reduced catalytic activity in SHP2 causing a loss-of-function, despite studies conducted in animal models have demonstrated that this residual activity is sufficient to generate a gain-of-function like phenotype in the cascade that leads to a hyperactivation of Ras/MAPK pathway [14, 16, 35, 71].

In this syndrome, learning disability are reported in the 30% of cases [54] and are more evident in verbal recall memory performance but relative sparing of visual and spatial recognition memory [16, 66, 71].

#### **3.5. Costello syndrome**

Costello syndrome is one of the rare syndromes of the group of RASopathies. It is caused by heterozygous activating germline mutations in *HRAS*. Typically, it is a missense mutation that induced the reduction of the intrinsic GTPase activity of RAS, which remains in the active form facilitating the synaptic trafficking of AMPA receptors. Besides, it has been seen that its action occurs in the spine dendritic structures too, which presents an increased density [14, 16, 35, 72].

This syndrome is phenotypically characterized by failure to thrive; short stature; developmental delay or intellectual disability; coarse facial features, curly, or sparse fine hair; loose, soft skin with deep palmar and plantar creases; papillomatosis of the face and perianal region; diffuse hypotonia and joint laxity; cardiac involvement (cardiac hypertrophy, valvar pulmonic stenosis, arrhythmia); relative or absolute macrocephaly Chiari I malformation with associated anomalies including hydrocephalus or syringomyelia. Moreover, they present an increased risk, approximately 15%, to malignant tumors [14, 16, 72].

Particularly, in these patients, it has been observed that verbal learning and memory are impaired but are better than the nonverbal cognitive abilities, while the visual associative memory is performed in the mildly disabled, but the related data are not completely clear [35, 73].

#### **3.6. CFC syndrome**

Cardiofaciocutaneous (CFC) syndrome is a very rare RASopathy. It is caused by heterozygous activating mutations in *BRAF* (~75%), *MAP2K1,* and *MAP2K2* (~25%), *KRAS* (<2%) that cause a deregulation of the RAS-MAPKinase cascade in a positive way. It is inherited in an autosomal dominant manner [14, 16, 74, 75].

CFC is characterized by craniofacial dysmorphology, congenital heart disease (pulmonic stenosis and other valve dysplasias, septal defects, hypertrophic cardiomyopathy, and rhythm disturbances), dermatologic abnormalities (xerosis, hyperkeratosis, ichthyosis, keratosis pilaris, ulerythema ophryogenes, eczema, pigmented moles, hemangiomas, and palmoplantar hyperkeratosis), growth retardation, and intellectual and learning disability [14, 16, 74, 75].

Assessing the learning ability, CFC patients present significant delay in adaptive skills, impaired spatial learning, and hippocampal long-term potentiation. It has been evidenced disability in verbal skills, especially the communication abilities were more impaired than the comprehension and in spatial learning [35, 75].

Mutations in MAP2K1, which are frequently associated with neurological complications and intellectual disability, can be associated with a milder clinical and neurocognitive profile more typical of individuals with Noonan syndrome. Variability of expression may arise from a complex interplay between RAS/MAPK pathway genotype, epigenetics, medical and obstetric factors, and environmental influences [76].
