A MEASUREMENT OF PERSONALITY TRAITS ASSOCIATED WITH RASOPATHY SYNDROMES. A Project Presented to the Faculty of California State University, Stanislaus

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1 A MEASUREMENT OF PERSONALITY TRAITS ASSOCIATED WITH RASOPATHY SYNDROMES A Project Presented to the Faculty of California State University, Stanislaus In Partial Fulfillment of the Requirements for the Degree of Master of Science in Genetic Counseling By Jessica Gage July 2014

2 CERTIFICATION OF APPROVAL A MEASUREMENT OF PERSONALITY TRAITS ASSOCIATED WITH RASOPATHY SYNDROMES by Jessica Gage Signed Certification of Approval Page is On File with the University Library Lauren Weiss, Ph.D. Assistant Professor Department of Psychiatry University of California, San Francisco Date Katherine Rauen, Ph.D. Professor Department of Pediatrics University of California, Davis Date Janey Youngblom, Ph.D. Professor of Genetics California State University, Stanislaus Date

3 2014 Jessica Gage ALL RIGHTS RESERVED

4 ACKNOWLEDGEMENTS Acknowledgements go to the co-authors Lauren Weiss, Janey Youngblom, and Katherine Rauen for their insights and guidance on this study. Thanks go to Brigid Adviento for her assistance and support through all aspects of this project. Thank you to Jessica Dow, Charles Dow, Harold Stanislaw, and Edward Gage for their knowledge of and assistance with statistical analyses. Thank you to Carol Mathews for her help with selecting a questionnaire for the project. Thank you to the CSUS Biology Research Committee for funding. iv

5 TABLE OF CONTENTS PAGE Acknowledgements... List of Tables... List of Figures... iv vi vii Abstract... viii Introduction... 1 Methods and Materials Participants Questionnaire Data Analysis Results Study Population Questionnaire Data Discussion Limitations Future Studies Conclusion References Appendix A. Questionnaire B. Questionnaire Scoring Guide v

6 LIST OF TABLES TABLE PAGE 1. Characteristics of subject groups Inverse-variance weighted means for each subject group Inverse-variance weighted means for RASopathies and Controls vi

7 LIST OF FIGURES FIGURE PAGE 1a. Box plots for Agreeableness b. Inverse-variance weighted means for Agreeableness a. Box plots for Energy/Extraversion b. Inverse-variance weighted means for Energy/Extraversion a. Box plots for Conscientiousness b. Inverse-variance weighted means for Conscientiousness a. Box plots for Emotional Instability b. Inverse-variance weighted means for Emotional Instability a. Box plots for Intellect/Openness b. Inverse-variance weighted means for Intellect/Openness a. Box plots for Sense of Humor b. Inverse-variance weighted means for Sense of Humor Histograms showing percent distributions of means vii

8 ABSTRACT The RASopathies are a category of genetically inherited disorders that involve mutations in the Ras/mitogen activated protein kinase pathway. Neurofibromatosis type 1 (NF1), Noonan syndrome, Costello syndrome, and Cardiofaciocutaneous (CFC) syndrome are among these, and the clinical phenotypes include physical dysmorphologies, growth problems, developmental delay, and elevated cancer risks. Specific personality traits in individuals with RASopathies have been noted by clinicians and research investigators, however no systematic study has been done to date that scientifically correlates these traits to establish personality phenotypes and no systematic comparisons have been conducted between the four syndromes. A modified version of the Big Five Questionnaire for Children by Barbaranelli et al. (2003) was administered to parents of children diagnosed with a RASopathy at the Third International Meeting on the Genetic Syndromes of the Ras/MAPK Pathway and at a Neurofibromatosis Symposium held at University of California-San Francisco. The results showed that as a group, the individuals diagnosed with a RASopathy differed significantly when compared to unaffected sibling Controls in personality traits measured, scoring lower on Agreeableness, Energy/Extraversion, Conscientiousness, Intellect/Openness, and higher on Emotional Instability. Sense of Humor was not significantly different between the two groups. There were also differences found within the individual RASopathy groups for each trait when compared to each other as well as the Control group. With a clearer definition of these personality traits and differences, parents, teachers, and clinicians can better diagnose, understand, and provide for these individuals. The results from this study could be used as a stepping stone to the possible identification and discovery of a genetic predisposition to certain personality traits. viii

9 INTRODUCTION The RASopathies are a category of genetically inherited syndromes that involve germline mutations in the Ras/mitogen activated protein kinase (MAPK) pathway (Tidyman & Rauen, 2009). This pathway is normally involved in cell growth and development; therefore, mutations are associated with syndromes that exhibit physical dysmorphologies, growth problems, developmental delay, and elevated cancer risks. Eight autosomal dominantly-inherited syndromes are known to be caused by specific mutations in this signaling cascade: Noonan syndrome, Neurofibromatosis type 1 (NF1), LEOPARD syndrome (also known as multiple lentigines syndrome and considered as a subset of Noonan syndrome), Hereditary gingival fibromatosis (HGF), Capillary malformation arteriovenous malformation syndrome (CM-AVM), Costello syndrome, Cardiofaciocutaneous (CFC) syndrome, and Legius syndrome (Tidyman & Rauen, 2009). Collectively, the RASopathies affect about 1 in 1,000 individuals (Rauen, 2013). The current study focuses specifically on NF1, Noonan syndrome, Costello syndrome, and CFC syndrome. NF1 involves mutations in the NF1 gene which cause dis-inhibition of the Ras proteins and an increased signaling cascade, leading to abnormal cell growth. About half of all NF1 mutations are inherited from an affected parent, and half are de novo mutations (Friedman, 2012). Clinical features include café-au-lait macules, skinfold freckling, lisch nodules, neurofibromas (benign Schwann cell tumors) and other tumors. Often, a clinical diagnosis is sufficient and genetic testing is not required to 1

10 2 diagnose individuals with NF1 (Friedman, 2012). Individuals with this condition are usually of short stature, and may have cardiovascular abnormalities and developmental delays (Williams et al., 2009); learning disabilities occur in about half of all affected individuals (Friedman, 2012). NF1 is a common RASopathy, with a prevalence of about 1 in 3,000 (Rauen, 2013). Noonan syndrome is a disorder which involves mutations in PTPN11, SOS1, KRAS, NRAS, RAF1, BRAF, SHOC2, CBL, RRAS, and RIT1 genes, with PTPN11 mutations contributing to about half of all reported cases (Roberts et al., 2013; Flex et al., 2014). These genes encode proteins in the Ras/MAPK pathway and mutations in these genes cause constant activation of the proteins (Roberts, Allanson, Tartaglia, & Gelb, 2013). Thirty to 75 percent of individuals with Noonan syndrome inherit the mutation from an affected parent and the prevalence of this syndrome is reportedly 1 in 1000 to 1 in 2500 individuals (Allanson & Roberts, 2011). Clinical features include dysmorphic facial features, short stature, bleeding disorders, and mild intellectual disability (Tidyman & Rauen, 2009); congenital cardiovascular anomalies occur in about 50 to 80 percent of affected individuals (Allanson & Roberts, 2011). Costello syndrome is most commonly caused by missense mutations in a single proto-oncogene, HRAS; this is the only known gene associated with Costello syndrome. About 300 cases of Costello syndrome have been reported worldwide (Gripp & Lin, 2012). Clinical features include course facial features, curly or fine hair, short stature, intellectual disability, and possible cardiovascular abnormalities. These individuals also have abnormal skin findings such as papillomata, wart-like

11 3 skin lesions, which develop in early childhood (Gripp & Lin, 2011). Individuals with Costello syndrome have a 15 percent lifetime risk of developing certain cancers including rhabdomyosarcoma, neuroblastoma, and transitional cell carcinoma of the bladder (Gripp & Lin, 2012). CFC syndrome involves mutations in BRAF, MAP2K1 and MAP2K2, and KRAS. To date, few cases of inherited CFC syndrome have been reported with mutations in MAP2K2 and KRAS; the majority of cases are caused by de novo mutations. Over 100 individuals worldwide have been reported to have CFC syndrome, but the prevalence of this syndrome is estimated to be about 200 to 300 individuals (Rauen, 2012). Clinical features of individuals with CFC syndrome overlap with those of NF1, Noonan syndrome, and Costello syndrome; although, these individuals may not have elevated cancer risks (Tidyman & Rauen, 2009). The phenotype of CFC syndrome presents as Noonan syndrome-like facial features, mild to severe cognitive delay, and abnormalities of the heart, skin, eyes, gastrointestinal tract, and central nervous system (Pavithra, Mallya, & Pai, 2012). Genotypephenotype correlations have been reported in CFC syndrome, presenting with a range in severity of this syndrome (Rauen, 2012). The origin and development of one s personality is not well understood; a multifactorial model has been proposed which includes environmental and genetic factors collaboratively influencing personality traits in an individual. Genetic factors may contribute to about 30 to 60 percent of variance in personality characteristics

12 4 (Samochowiec et al., 2001). However, some genetic disorders involve distinct behavioral and personality traits as components of the clinical phenotype. Williams syndrome is perhaps the most notable genetic syndrome with a welldefined personality phenotype (Järvinen-Pasley et al., 2008). Williams syndrome is a genetic disorder caused by a chromosomal microdeletion of about 24 genes, including the elastin gene, at 7q Affected individuals usually have distinct facial features, short stature, heart problems, and developmental delay ranging from mild to severe (Doyle, Bellugi, Korenberg, & Graham et al., 2004). Independent of IQ, individuals with Williams syndrome have strong verbal short-term memory and language skills. Unique personality traits include overfriendliness and excessive empathy (Morris, 2013). A study by Lieshout et al. (1998) measured personality traits of children and adolescents with Prader-Willi syndrome, Fragile-X syndrome, and Williams syndrome and also aimed to relate the family context and parental behaviors to the personality profiles of the affected individuals. The California Child Q-Set (CCQ) was administered to parents of children with these three genetic disorders including 28 children with Williams syndrome. Results showed that individuals with Williams syndrome were similar to the control group in Extraversion and Agreeableness, but scored lower in Conscientiousness, Emotional Stability, and Openness. These personality traits were shown to be unaffected by and independent of parent behaviors and family context (Lieshout et al., 1998). The social phenotype of these individuals is described as highly gregarious and people-oriented, with no stranger anxiety (Mervis & Klein-Tasman, 2000). Individuals with Williams syndrome also have a

13 5 particular interest in music and tend to be musically creative. Research seeking a genotype-phenotype correlation for these personality traits is ongoing and some hypotheses have been proposed, however no single gene within the 7q11.23 region has been conclusively associated with the social profile of Williams syndrome (Tassabehji, 2003). Researchers and clinicians have noted distinctive personality traits in children who have been diagnosed with a RASopathy, but no studies have formally been conducted to measure these traits in Noonan syndrome, Costello syndrome, and CFC syndrome, and determine how they differ between the four syndromes. In contrast to Williams syndrome, the RASopathies result from single-gene mutations as opposed to a multi-gene deletion. A recently published paper by Alfieri et al. (2014) studied neurobehavioral features and mental health disorders in Noonan syndrome, LEOPARD syndrome, Costello syndrome, Noonan-like syndrome with loose anagen hair, and Cardiofaciocutaneous syndrome. Parents of individuals affected by these syndromes completed the Child Behavior Checklist (CBCL) which measures behavior problems in children, Social Communication Questionnaire version lifetime (SCQ-L) which measures movement, language, gestures, and interactions, and the Modified Checklist for Autism in toddlers (M-CHAT). Their findings showed that the SCQ-L and M- CHAT scores were significantly different between all of the syndromes. Attention problems and social problems were higher in individuals with CFC syndrome when compared to Noonan syndrome and Costello syndrome; Noonan syndrome was

14 6 shown to be associated with anxiety and depression issues. Overall, when compared to an unaffected control group, individuals affected by a RASopathy had higher rates of internalizing and externalizing problems (Alfieri et al., 2014). Another study by Adviento et al. (2013) measured social characteristics in NF1, Noonan syndrome, Costello syndrome, and CFC syndrome, to establish the prevalence of autism spectrum disorders (ASDs) in RASopathy. Two questionnaires were administered to parents of individuals with these syndromes- the SCQ and the Social Responsive Scale (SRS). The results showed that 54% of CFC syndrome subjects, 26% of Costello syndrome subjects, 21% of Noonan syndrome subjects, and 11% of NF1 subjects reached the threshold for significant autism features and were significantly higher than sibling controls. As a whole, individuals with RASopathies were shown to have less clinically significant social impairments when compared to individuals with idiopathic ASDs, but had more social impairments compared to sibling controls (Adviento et al., 2013). The current study focuses on personality traits in RASopathies, building on previously studied personality phenotypes and systematically comparing personality traits in NF1, Noonan syndrome, Costello syndrome, and CFC syndrome. Personality traits in NF1 have been studied and reported in the literature. When compared to unaffected children, a study by Prinzie et al. (2013) which used the California Child Q-set (CCQ), concluded that individuals with NF1 were shown to score similarly in Agreeableness, lower in Conscientiousness, Emotional Stability, and Openness, and higher in Extraversion. They also had less motor activity and were more dependent

15 7 and irritable when compared to a control group (Prinzie et al., 2003). Individuals with NF1have been noted to have difficulties forming friendships and are more often rejected by their peers (Barton & North, 2004). These individuals have also been described as more sensitive and isolated than their peers and as having poorer social competence (Lehtonen, Howie, Trump, & Huson, 2013). Personality traits in Noonan syndrome have been mentioned in the literature but have not been formally studied. In a study by Collins and Turner (1973), which looked at 27 cases of Noonan syndrome, the children were described as confident, happy, and talkative, and appeared to act with a cocktail party manner. In another study on psychological and psychiatric aspects of Noonan syndrome, the investigators mentioned that the children were remarkably friendly, cooperative and very willing to please.all aspects of a social desirable attitude (Verhoeven, Wingbermühle, Egger, Van der Burgt, & Tuinier, 2008); however, it was noted that this was merely an observation, and not a result from the study. In regards to Costello syndrome, Philip and Sigaudy (1998) stated in a Syndrome of the Month feature that because the majority of patients with Costello syndrome have a happy, outgoing personality, this behaviour seems consistent enough to be proposed as a diagnostic criterion. Interestingly, this has been mentioned in other papers including one that retrospectively looked at behavioral traits in 10 individuals with Costello syndrome and observed gradual happy and social characteristics after age four (Kawame et al., 2003). Another paper that studied temperament in children with Costello syndrome found significantly higher

16 8 emotionality in affected children versus the control subjects (Galéra et al., 2006). To date, no studies have been conducted that systematically evaluate personality traits and establish a personality phenotype of these individuals. The Costello syndrome GeneReviews includes sociable, outgoing personality in the clinical diagnosis section and also addresses the friendly personality of these individuals as an overlap in phenotype with Williams syndrome (Gripp & Lin, 2012). Mentions of particular personality traits in individuals with CFC syndrome are rare, although one paper by Armour and Allanson (2008) compares the engaging, warm and friendly personality of these individuals to those affected by Costello syndrome. The personality observations in Costello syndrome were the main drive for the current study and led to an addition of a sense of humor measurement to the study. A modified version of the Big Five Questionnaire for Children by Barbaranelli et al. (2003) was administered to parents of children diagnosed with a RASopathy as well as unaffected siblings. Unique personality phenotypes were predicted among the four main RASopathies when compared to each other. Differences between the individuals diagnosed with a RASopathy and unaffected individuals were also predicted, since it is hypothesized that specific personality traits are unique to the syndrome itself. Noonan and Costello syndrome may have similar results, scoring high on Agreeableness and Sense of Humor, as the literature notes that children with these syndromes are particularly friendly and outgoing. These results may differ from the NF1results, since personality traits in these individuals are more understood

17 9 and individuals with NF1 have been said to be somewhat less sociable (Lehtonen et al., 2013). With a clearer definition of these personality traits and possible differences, parents, teachers, and clinicians can better diagnose, understand, and provide for these individuals. The results from this study could be used as a stepping stone to the possible discovery of a genetic predisposition to certain personality traits. Each RASopathy is caused by single-gene mutation and can directly point to a gene of interest; association with a personality profile would be informative about specific genes that can influence personality.

18 MATERIALS AND METHODS Participants Parents of children who have been diagnosed with a RASopathy were asked to participate in the current study and complete a questionnaire. Inclusion criteria were genetic diagnosis of Noonan syndrome, Costello syndrome, or CFC syndrome, and genetic or clinical diagnosis of NF1. Individuals of all age were recruited for the study, although parents of adults were asked to complete a questionnaire in terms of their child s personality traits when they were school-aged. The first set of participants was recruited at the Third International Meeting on the Genetic Syndromes of the Ras/MAPK Pathway, held on June 30 to August 4, 2013 at the Renaissance Orlando at SeaWorld in Orlando, Florida. Since NF1 is typically underrepresented at the international meeting, the second set of participants was recruited at the University of California, San Francisco (UCSF) Neurofibromatosis Symposium on February 8, At the international meeting in Orlando, Florida, participants were consented and recruited through the UCSF Genetics of Autism Research Program (UCSF Institutional Review Board Protocol #: ). Parents were asked to complete a paper questionnaire for their affected child, and also one for an unaffected child, if applicable. Questionnaires were completed and returned at the meeting. At the UCSF Neurofibromatosis Symposium, participants signed a Consent to be Contacted for Future Research form. After the symposium, UCSF Genetics of Autism Research 10

19 11 Program consent forms and a paper questionnaire were sent by mail to the participants. Parents of children with NF1 were asked to complete the questionnaire for their affected child and also for an unaffected child, if applicable, and return the questionnaire by mail. The unaffected children were considered healthy controls. These sibling data provided a control population with which to compare the results from individuals diagnosed with a RASopathy. By using siblings as controls, they were well-matched to the affected individuals in background genetics and environment as opposed to using a randomized, unaffected population as controls. This study was approved by the California State University, Stanislaus Institutional Review Board (Protocol #: ). Questionnaire The Big Five Questionnaire for Children by Barbaranelli et al. (2003) was created by a team of investigators in Rome, Italy to adequately measure Big Five personality factors in late childhood. The questionnaire was published in an article titled A questionnaire for measuring the Big Five in late childhood from the journal Personality and Individual differences. In this article, the authors described the phrase-based, self-report personality questionnaire that was generated to measure five factors: Energy/Extraversion, Agreeableness, Conscientiousness, Emotional Instability, and Intellect/Openness. These were chosen after pilot studies found them to be the most accurate in describing personality characteristics in children, starting with a pool of 285 trait adjectives. According to Barbaranelli et al., Energy/Extraversion refers to aspects such as activity, enthusiasm, assertiveness, and

20 12 self-confidence, Agreeableness is a concern and sensitivity towards others and their needs, Conscientiousness assesses dependability, orderliness, precision, and the fulfilling of commitments, Emotional Instability assesses feelings of anxiety, depression, discontent, and anger, and Intellect/Openness measures intellect, especially in the school domain, broadness or narrowness of cultural interests, fantasy/creativity, and interest in other people (2003, p.649). Each trait is measured by scoring 13 statements, with a total of 65 items. The Barbaranelli aimed to validate the measure by administering the questionnaire to children ages eight to 13, in grades four and five (elementary school) and six through eight (junior high school), and also to mothers and teachers to establish concurrent validity between self-reported answers and observations of the children. Elementary school children used a 3-point Likert scale to score each statement, while junior high school children, mothers, and teachers used a 5-point Likert scale with 1 scoring as Almost never to 5 scoring Almost always. Results showed that scale validity coefficients, which measured internal validity of the Big Five factors, were high for all groups. Specifically, for self-reports in elementary school and junior high school children, the scale validity mean was 0.90 with a standard deviation of 0.06, for mother ratings the mean was 0.94 with a standard deviation of 0.02, and for teacher ratings the mean was 0.94 with a standard deviation of Results also showed that convergent correlations among self-report elementary school children and mothers were statistically significant (p<0.001) for all factors except Conscientiousness. Convergent correlations among junior high school children and mothers were statistically significant (p<0.001) for all

21 13 five factors. Correlations between the questionnaire and other personality measurements (Academic Achievement, Externalization and Internalization behavior problems, and the Junior Personality Questionnaire (Esenck, 1995) which measured Psychoticism, Extraversion, and Neuroticism) were high and significant for each factor, indicating construct and concurrent validity of the Big Five Questionnaire for Children (Barbaranelli et al., 2003). Several published studies have used the Big Five Questionnaire for Children as a personality measure, including a study by Carrasco Ortiz and del Barrio Gandara (2007) which aimed to measure the relationship between temperament and personality factors and depression in children and adolescents. This study reported the Reliability of the questionnaire, using Cronbach s Alpha, to be: 0.87 for Conscientiousness, 0.77 for Extraversion, 0.82 for Openness, 0.77 for Emotional Instability, and 0.71 for agreeableness (Carrasco Ortiz & del Barrio Gandara, 2007). A study by Muris et al. (2005) titled Psychometric properties of the Big Five Questionnaire for Children (BFQ-C) in a Dutch sample of young adolescents studied the reliability and validity of the Big Five Questionnaire for Children. They administered the Big Five Questionnaire for Children, the Junior version of the Eysenck Personality Questionnaire, and the Strengths and Difficulties Questionnaire, a measure that looks at domains of child psychopathology and personal strengths, to 222 Dutch adolescents (120 females, 102 males; mean age years). In terms of reliability, results showed that the internal consistency of the Big Five Questionnaire for children was satisfactory with Cronbach s alphas ranging from 0.71 to 0.83.

22 14 The results also showed that the questionnaire had high validity as it properly correlated with the comparison questionnaires. The findings from this study were similar to findings in the original paper on the Big Five Questionnaire for Children (Barbaranelli et al., 2003), providing further evidence that this measure is reliable and valid (Muris, Meesters, &Diederen, (2005). The questionnaire used in the current study was a modified version of the Big Five Questionnaire for Children. Since the questionnaire was administered as a parent-report measure, and the original Big Five Questionnaire for Children was written as a child self-report measure, the first person statements were changed to third person statements (for example, I easily get angry was changed to My child easily gets angry ). The questionnaire was originally written in Italian and translated to English; because of this, some statements were confusing and were slightly altered to better interpret (for example, I understand immediately was modified to My child understands things quickly ). Another modification of the questionnaire was adding a Sense of Humor scale. This scale consisted of five statements, including one statement in the original questionnaire, My child likes to joke. Four statements were added to the questionnaire; these were created on the basis that sense of humor consists of five components, according to the book Sense of Humor: Explorations of a Personality Characteristic (Ruch, 2007). These components were described as Motivational, Cognitive, Emotional, Social, and Behavioral (Ruch, 2007). Statements added were: My child laughs at other s jokes, My child has the ability to make others laugh through his/her own humor, and My child displays a good

23 15 sense of humor, in an attempt to measure the five sense of humor components. Each statement in the questionnaire was rated on a 5-point Likert scale where 1= Hardly ever, 2= Rarely, 3= Sometimes, 4= Often, 5= Almost always. Another option for each statement was Too young, which was marked when the parent felt their child was too young to evaluate on the particular statement. Since this questionnaire measures personality traits in children and adolescents, parents of adults were asked to answer statements based on personality traits of their children when they were school-age. The questionnaire also included demographic questions. Parents were asked to write their child s name, the age of their child, and whether their child is in or was in special education classes. Data Analysis Quantitative statistical analyses were performed with statistical programming software S.A.S. 9.3 (Statistic Analysis System, version 9.3, Copyright , SAS institute, Inc., Cary, NC, USA) as well as Excel Microsoft Office Values for each statement in each questionnaire were entered as 1, 2, 3, 4, or 5 and for statements reported as Too young, no value was entered. Data management was performed to prepare the data for analysis in S.A.S. and Excel; this included separating the data based on which statements in the questionnaire corresponded to each trait (13 statements for Agreeableness, Energy/Extraversion, Conscientiousness, Emotional Instability and Intellect/Openness, and 5 statements for Sense of Humor). This data was separated further by which subject group it corresponded to.

24 16 Individuals who had less than three responses for statements that corresponded to a particular trait were not used for analysis of that trait (one individual with Costello syndrome for Agreeableness, one individual with Costello syndrome and one with CFC syndrome for Conscientiousness, two individuals with CFC syndrome and one individual with NF1 for Sense of Humor). Mean trait values were calculated for each individual. To account for missing data and some individuals having more answers than others, inverse-variance weighted group means were calculated for each syndrome within each trait. Standard error of the group means were also calculated for each syndrome in each trait. Similarly, inverse-variance weighted group means and standard error of the means were calculated for the RASopathies group as one. Unpaired t tests were calculated to determine significant differences between the RASopathies group as one versus the control group, for each trait, and to determine significant differences between each RASopathy as well as each RASopathy compared to the sibling control group. The t tests incorporated the inverse-variance weighted means as well as the standard errors of the means using GraphPad (GraphPad Software, Inc., Copyright 2014). To account for multiple testing, p 0.01 was considered to be criteria for significance. A table was created in Excel to summarize the inverse-variance weighted means and standard error of the means for trait by subject type - NF1, Noonan syndrome, Costello syndrome, CFC syndrome, and Controls- as well as indicate significance of t tests. Bar graphs were created in Excel to show the inverse-variance weighted means for each syndrome in each trait, as well as the standard error of the group mean (in error bars). Box plot graphs were

25 17 created in S.A.S. for each trait, displaying the spread of the un-weighted trait means for each individual in each particular syndrome group. Histograms were created in using the Analysis TookPak in Excel for the RASopathies as one group as well as the control sibling subject group. These display the percent distributions of the unweighted means of each individual in the groups by bin number along the x-axis (corresponding to the 1 to 5 Likert scale).

26 RESULTS Study Population A total of 106 questionnaires were completed by parents of children with RASopathies. As shown in Table 1, this included 62 questionnaires for affected children and 44 questionnaires for unaffected children. At the Third International Meeting on the Genetic Syndromes of the Ras/MAPK Pathway, parents of 11 children with Noonan syndrome (7 females, 4 males; age range 3.5 to 24 years, mean age years), 20 children with Costello syndrome (8 females, 12 males; age range 2.5 to 36 years, mean age years), and 19 children with CFC syndrome (5 females, 14 males; age range 3 to 18 years, mean age 9.10 years) participated. After the UCSF Neurofibromatosis Symposium, parents of 12 individuals (7 females, 5 males; age range 2 to 37 years, mean age years) with NF1 returned questionnaires. Data from both conferences were collected from a total of 44 unaffected children (18 females, 26 males; age range 3 to 41, mean age years), the control group. Of the 62 participants diagnosed with a RASopathy, 37 (59.7%) were reportedly in Special Education classes. This included 3 children with NF1 4 reported no, 5 did not report), 7 children with Noonan syndrome (2 reported no, 2 did not report), 14 children with Costello syndrome (3 reported no, 2 did not report), and 13 children with CFC syndrome (3 reported no, 2 did not report). In the healthy control group, 3 individuals were reportedly in Special Education classes and 41 individuals were not in Special Education classes. 18

27 19 Table 1 Characteristics of Subject Groups NF1 Noonan Costello CFC Control Total Number of Subjects Number of Females Number of Males Age Range Mean Age % Special Education Yes % Special Education No % Special Education No Answer Questionnaire Data The inverse-variance weighted mean trait score was calculated for each syndrome as well as standard error of each group mean, and is displayed in Table 2. This table shows that for each trait, each RASopathy subject group scored significantly different than the Control group at a p 0.01 level except for Costello syndrome for Agreeableness, NF1 for Emotional Instability, and Noonan syndrome for Sense of Humor. The Costello syndrome group scored significantly higher than all other RASopathy groups as well as the control group for Emotional Instability; Noonan syndrome scored the next highest, and the weighted mean score was significantly different than all other subject groups. CFC syndrome scored significantly lower than all subject groups on Intellect/Openness and Noonan

28 20 syndrome scored the highest of the RASopathy groups, but lower than the control group. The NF1 subject group scored significantly lower than all other subject groups on Sense of Humor. Table 2 Inverse-variance weighted trait means for each subject group RASopathy Subjects CFC Syndrome Costello Syndrome NF1 Noonan Syndrome Agreeableness 3.82 b, c, e 4.35 a, d 4.14 a, d, e 3.68 b, c, e (0.05) (0.05) (0.04) (0.06) Control Subjects 4.45 a, c, d (0.022) Energy/Extraversion 3.55 c, e (0.06) 3.81 e (0.40) 3.89 a, d, e (0.08) 3.41 c, e (0.07) 4.53 a, b, c, d (0.02) Conscientiousness 2.89 b, d, e (0.07) 3.22 a, e (0.06) 3.07 e (0.08) 3.28 a, e (0.07) 4.00 a, b, c, d (0.03) Emotional Instability 2.55 b, d, e (0.06) 3.32 a, c, d, e (0.05) 2.47 b, d (0.06) 2.91 a, b, c, e (0.07) 2.35 a, b, d (0.03) Intellect/Openness 2.60 b, c, d, e (0.06) 3.08 a, d, e (0.07) 3.21 a, d, e (0.04) 3.42 a, b, c, e (0.07) 4.01 a, b, c, d (0.04) Sense of Humor 4.52 c, e (0.03) 4.57 c, e (0.02) 3.92 a, b, d, e (0.05) 4.46 c (0.05) 4.43 a, b, c (0.02) Note. Standard error of the mean is noted in parenthesis below the mean. a p 0.01 compared to CFC syndrome, b p 0.01 compared to Costello syndrome, c p 0.01 compared to NF1, d p 0.01 compared to Noonan syndrome, and e p 0.01 compared to control subjects for t-tests. Box plots and bar graphs for each trait show the spread of individual means as well as the inverse-variance weighted group means for each syndrome in each trait, in Figures 1 through 6.

29 21 Figure 1a. Box plots for Agreeableness CFC Costello NF1 Noonan Control Figure 1b. Inverse-variance weighted means for Agreeableness. Error bars show standard error of the means.

30 22 There was overlap in individual means for all subject groups on Agreeableness, as shown in Figure 1a; CFC syndrome and NF1 had the largest range in individual average scores. The Noonan syndrome group scored the lowest on Agreeableness, but was not significantly different than CFC syndrome (Figure 1b). Costello syndrome scored the highest of the RASopathy groups, but was not significantly different than NF1. Figure 2a. Box plots for Energy/Extraversion. Figure 2a shows that for the individual means for each syndrome for Energy/Extraversion, the ranges all have fairly similar spreads. Also, the first and third quartile ranges inside of the boxes have substantial overlap for all of the RASopathy groups.

31 CFC Costello NF1 Noonan Control Figure 2b. Inverse-variance weighted means for Energy/Extraversion. Error bars show standard error of the means. As seen in Figure 2b, Costello syndrome has the largest standard error for Energy/Extraversion. The Noonan syndrome group scored the lowest on Energy/Extraversion, but was not significantly different from CFC syndrome or Costello syndrome. NF1 syndrome scored the highest of the RASopathy groups; Costello syndrome scored the next highest and was only significantly different from the control group.

32 24 Figure 3a. Box plots for Conscientiousness CFC Costello NF1 Noonan Control Figure 3b. Inverse-variance weighted means for Conscientiousness. Error bars show standard error of the means.

33 25 Figure 3a shows that NF1 has the largest range of individual means for Conscientiousness. CFC syndrome scored the lowest on Conscientiousness, and was significantly different than all other subject groups except NF1 (Figure 3b). Noonan syndrome scored the highest of all RASopathy groups, but was not significantly different from Costello syndrome or NF1. Figure 4a. Box Plots for Emotional Instability. Similar to Conscientiousness, NF1 has the largest range in individual means for Emotional Instability (Figure 4a).

34 CFC Costello NF1 Noonan Control Figure 4b. Inverse-variance weighted means for Emotional Instability. Error bars show standard error of the means. Figure 4b shows that Costello syndrome scored the highest for Emotional Instability and the Control group scored the lowest. Costello syndrome and Noonan syndrome groups scored significantly different from all other groups.

35 27 Figure 5a. Box plots for Intellect/Openness CFC Costello NF1 Noonan Control Figure 5b. Inverse-variance weighted means for Emotional Instability. Error bars show standard error of the means.

36 28 CFC syndrome scored significantly lower on Intellect/Openness than all other subject groups (Figure 5b). Noonan syndrome scored significantly higher than all other RASopathy groups. Each RASopathy group scored significantly lower than the control subject group. Figure 6a. Box plots for Sense of Humor.

37 CFC Costello NF1 Noonan Control Figure 6b. Inverse-variance weighted means for Sense of Humor. Error bars show standard error of the means. The NF1 subject group scored significantly lower than all other groups for Sense of Humor (Figure 6b) and also had the smallest range of individual means (Figure 6a). Costello syndrome scored the highest of all subject groups for Sense of Humor, and was not significantly different from CFC syndrome or Noonan syndrome. The control subject group scored significantly lower than Costello syndrome and CFC syndrome and significantly higher than NF1, but did not score significantly different than Noonan syndrome. When combined as a single group, the RASopathies have fairly different distributions of individual means for each trait, as do the control group (Figure 7). For the RASopathies group, Agreeableness and Energy/Extraversion have leftskewed distributions with peaks near 4.0 and 4.5, respectively, meaning that the highest percent of individual means fell into these bins.

38 Agreeableness * Energy/Extraversion * 40.0% 40.0% 30.0% 20.0% 10.0% RASopathies Control 30.0% 20.0% 10.0% RASopathies Control 0.0% % % 30.0% 20.0% 10.0% 0.0% Conscientiousness Emotional Instability * * 40.0% 30.0% RASopathies 20.0% RASopathies Control 10.0% Control 0.0% Intellect/Openness * Sense of Humor 60.0% 60.0% 40.0% 20.0% 0.0% RASopathies Control Figure 7. Histograms showing percent distributions of means for RASopathy subjects compared to Control subjects, for each trait. X-axis displays bins for answers on the Likert scale. * p < 0.01 for unpaired t-tests using inverse-variance weighted means and standard error of the means. 40.0% 20.0% 0.0% RASopathies Control 30

39 Distributions for the RASopathy group for Conscientiousness, Emotional Instability, Intellect/Openness, and Sense of Humor were more normal distributions. The peak for Conscientiousness was near 3.5, the peak for Emotional Instability was near 2.5, the peak for Intellect/Openness was near 3.5, and the peak for Sense of Humor was near 4.0. Distributions for the Control group for Energy/Extraversion and Intellect/Openness were skewed left. Distributions for the other four traits were more normal. Similar to the RASopathy groups, the peak for Energy/Extraversion was 4.5 and the peak for Emotional Instability was 2.5. For the other traits, the peaks for the control group were in higher bins. T tests for the RASopathy group compared to the Control group showed that inverse-variance weighted means for each trait were significantly different between the groups, except for Sense of Humor (Table 3). The RASopathy group scored lower on Agreeableness, Energy/Extraversion, Conscientiousness, and Intellect/Openness, and scored higher on Emotional Instability when compared to the Control group. They scored slightly higher than the Control group on Sense of Humor, but the weighted means were not significantly different. 31

40 32 Table 3 Inverse-variance weighted trait means for RASopathies and Controls RASopathy Group Control Group Agreeableness 4.05* (0.02) 4.45 (0.02) Energy/Extraversion 3.66* (0.03) Conscientiousness 3.12* (0.03) Emotional Instability 2.87* (0.03) Intellect/Openness 3.11* (0.03) 4.53 (0.02) 4.00 (0.03) 2.35 (0.03) 4.00 (0.04) Sense of Humor 4.47 (0.02) 4.43 (0.01) Note. Standard error of the mean is noted in parenthesis below the mean.* p 0.01 compared to the Control group for t-tests.

41 33 DISCUSSION This study provides new information regarding personality phenotypes and personality profile differences in RASopathies and also supports some previous studies. Compared to unaffected siblings, individuals diagnosed with a RASopathy were found to display personality traits that were lower in Agreeableness, Energy/Extraversion, Conscientiousness, and Intellect/Openness and higher in Emotional Instability. Interestingly, the RASopathy group scored slightly higher than the control group on Sense of Humor. These findings support the study by Alfieri et al. (2014) which found that individuals diagnosed with a RASopathy had more Emotional, Externalizing and Internalizing problems when compared to a control group. In the original paper by Barbaranelli et al. (2003) where the Big Five Questionnaire for Children was first published, it was noted that Externalizing problems inversely correlated with Conscientiousness, and Internalizing problems and Emotional problems correlated with Emotional Instability. The original paper also found high correlations of Conscientiousness and Intellect/Openness with Academic Achievement, which they measured by looking at school records and scores from standardized tests. Intellect/Openness was ultimately chosen as a factor for the original Big Five Questionnaire for Children because the authors felt that children and young adolescents do not generally have exposure to the range of intellectual experience, cultural opportunities, and social issues that go into the multifaceted measure of openness among adults (Barbaranelli et al., 2003). The authors were creating a questionnaire for typical

42 34 school-aged children and in the population of the current study, most individuals had some sort of intellectual disability since mild to severe intellectual disability is part of the clinical phenotype in RASopathies (Alfieri et al., 2014). In each RASopathy group in the current study, parents reported that 25-70% were either currently or previously in special education and 10-41% did not provide an answer to that particular question. Therefore, it is not surprising that in this study, individuals diagnosed with a RASopathy scored lower than the control group on Intellect/Openness. The results from this study show that each RASopathy has a somewhat unique personality phenotype. Individuals with NF1 scored second highest of the RASopathy subjects on Agreeableness, scoring lower than the Control subjects and then Costello syndrome. These results differ from those in a previous study by Prinzie et al. (2013) which found that individuals with NF1 scored similar to the control group in Agreeableness; in this study, the score for NF1 on Agreeableness was significantly lower than the control group. However, the previous study reported that individuals with NF1 scored lower than the control group in Conscientiousness and Openness, which was consistent with this study. In the current study, NF1 scored the second lowest of all of the subject groups for Conscientiousness, just scoring higher than CFC syndrome but not significantly. NF1 scored the second highest of the RASopathy on Intellect/Openness, scoring significantly lower than the Control subjects as well as Noonan syndrome. The study by Prinzie at el. (2013) reported that individuals with NF1 scored higher than the control group for Emotional Instability and Extraversion. In the current study, NF1 was found to score higher on Emotional Instability when compared to the control group, but

43 35 scored the lowest of all of the RASopathy groups on this factor. However, NF1 was found to score significantly lower than the Control subjects for Energy/Extraversion, conflicting with the results of the previous study (Prinzie et al., 2013). In this study, NF1 scored significantly lower than all other groups for Sense of Humor. It was predicted that individuals with Noonan syndrome would score high on Agreeableness and Sense of Humor. The results from this study showed that Noonan syndrome scored the lowest on Agreeableness when compared to all of the other subject groups; however they did not score significantly lower than CFC syndrome. These results do not support the hypothesis, although Noonan syndrome did score higher than the Control group and NF1 for Sense of Humor. Noonan syndrome scored the lowest of all other subject groups for Energy/Extraversion, and the highest of all of the other RASopathy groups on Conscientiousness and Intellect/Openness. They also scored the second highest on Emotional Instability, just below NF1. These results are consistent with the study by Alfieri et al. (2014) which found that individuals with Noonan syndrome had behavioral and emotional problems, and also stated that they tended to have less severe intellectual disability. As predicted in the hypothesis that started this study, Costello syndrome scored the highest of all subject groups on Sense of Humor. These individuals scored significantly higher than NF1 and the Control subjects, and only slightly higher than Noonan syndrome and CFC syndrome. This result is particularly interesting because unlike Noonan syndrome and CFC syndrome, mutations in only one gene, HRAS, have been reported to cause Costello syndrome. Costello syndrome also scored the highest of all of the

44 36 RASopathy groups on Agreeableness. These individuals scored the second highest on Energy/Extraversion and Conscientiousness compared to the other RASopathies, and the second lowest on Intellect/Openness, scoring significantly higher than CFC syndrome. Costello syndrome scored significantly higher than all of the subject groups for Emotional Instability. Individuals with CFC syndrome scored the lowest of all other subject groups on Conscientiousness and Intellect/Openness. Since these individuals tend to have more severe intellectual disability, and Barbaranelli et al. (2003) report correlations of Conscientiousness and Intellect/Openness to Academic Achievement, these findings are not surprising. CFC syndrome scored the second lowest of the RASopathies on Energy/Extraversion, Agreeableness, and Emotional Instability. This group scored the second highest of the RASopathies on Sense of Humor. These results are similar to Costello syndrome and are consistent with reports from a paper by Armour and Allanson (2008) which compare CFC syndrome to Costello syndrome in terms of friendliness and outgoingness. Limitations One limitation of this study is the questionnaire that was used. As mentioned previously, this questionnaire was not designed for, nor validated in populations with intellectual disability. Therefore, many of the statements were marked as too young meaning the individual was too young for the parent to evaluate on that particular statement, or possibly the child was too intellectually disabled to evaluate on that particular statement. Because another option was available that was not part of the 1

45 37 through 5 Likert scale, the data were missing quite a bit of data. To account for this, individuals who had less than three responses for a particular trait were excluded from the analysis of that particular trait. Another limitation of the questionnaire was that the Big Five Questionnaire for Children was originally designed as a self-report measure for children. Because the IRB for the current study did not allow direct participation from the affected individuals, and because many of these individuals have intellectual disability, the questionnaire was modified for parents to score statements about their children. However, the original paper by Barbaranelli et al. (2003 reports that the self-report questionnaire results significantly converged with mother-report questionnaire results. Therefore, the authors of the current study felt that it was appropriate to distribute a modified version of the Big Five Questionnaire to parents as an observational-report measure only. Since parent-reports were used, there may have been potential circumstances of over- or underestimating children s personality traits. Another limitation of this study is the number of participants in each syndrome group. NF1 and Noonan syndrome are more common syndromes with prevalence rates of 1 in 3,000 and 1 in 1,000 to 1 in 2,500, respectively (Rauen, 2013; Allanson & Roberts, 2011). A total of 12 individuals with NF1 and 11 individuals with Noonan syndrome were a part of this study, whereas 20 individuals with Costello syndrome and 19 individuals with CFC syndrome participated. Costello syndrome and CFC syndrome are rare syndromes with only about 300 individuals worldwide having been reportedly diagnosed with each of these conditions (Gripp & Lin, 2012; Rauen, 2012). One reason for having more participants with Costello syndrome and CFC syndrome is that