Molecular Genetics of Epilepsy and Mental Retardation Limited to Females (EFMR)

A thesis submitted for the degree of Doctor of Philosophy to the University of Adelaide

By Kim Hynes

School of Molecular and Biomedical Science, Division of Genetics, University of Adelaide

December 2009

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Appendix - A

Dibbens, L.M. et al. (2008) X-linked protocadherin 19 mutations cause female- limited epilepsy and cognitive impairment. Nature Genetics, v. 40 (6), pp. 776 - 781, June2008

NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1038/ng.149

Appendix - B

Epilepsy and mental retardation limited to females with PCDH19 mutations can present de novo or in single generation families

Kim Hynes, Patrick Tarpey, Leanne M Dibbens, et al.

J Med Genet 2010 47: 211-216 originally published online September 14, 2009 doi: 10.1136/jmg.2009.068817

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To subscribe to Journal of Medical Genetics go to: http://jmg.bmj.com/subscriptions Mutation report Epilepsy and mental retardation limited to females with PCDH19 mutations can present de novo or in single generation families Kim Hynes,1,2 Patrick Tarpey,3 Leanne M Dibbens,1,4 Marta A Bayly,1 Samuel F Berkovic,5 Raffaella Smith,3 Zahyia Al Raisi,1 Samantha J Turner,5 Natasha J Brown,6,7 Tarishi D Desai,5 Eric Haan,1,4 Gillian Turner,8 John Christodoulou,9 Helen Leonard,10 Deepak Gill,11 Michael R Stratton,3 Jozef Gecz,1,2,4 Ingrid E Scheffer5,6

< Supplementary table 1 is ABSTRACT smaller pedigrees, EFMR due to mutations in published online only at http:// Background Epilepsy and mental retardation limited to PCDH19 may well be an under-recognised cause of jmg.bmj.com/content/vol47/ females (EFMR) is an intriguing X-linked disorder seizures and intellectual disability (ID), since the issue3 affecting heterozygous females and sparing hemizygous characteristic unique inheritance pattern is not 1 SA Pathology at the Women’s males. Mutations in the protocadherin 19 (PCDH19) obvious in the pedigrees. This prediction has recently and Children’s Hospital, North fi PCDH19 Adelaide, SA, Australia gene have been identified in seven unrelated families with been validated by the identi cation of 2School of Molecular and EFMR. mutations in 12 unrelated individuals (11 females Biomedical Science, University Methods and results Here, we assessed the frequency and one mosaic male) with severe epileptic enceph- of Adelaide, Adelaide, Australia of PCDH19 mutations in individuals with clinical features alopathies resembling Dravet syndrome that were 3Wellcome Trust Sanger which overlap those of EFMR. We analysed 185 females proven de novo in six, and familial in five, with four Institute, Hinxton, UK 4 4School of Paediatrics and from three cohorts: 42 with Rett syndrome who were unaffected carrier fathers and one with mild ID. Reproductive Health, University negative for MECP2 and CDKL5 mutations, 57 with Our assessment of 27 females with EFMR iden- of Adelaide, Adelaide, Australia autism spectrum disorders, and 86 with epilepsy with or fi 5 ti ed that while seizures beginning by age 3 years Epilepsy Research Centre and without intellectual disability. No mutations were were the hallmark of this disorder, there were Department of Medicine, University of Melbourne, Austin identified in the Rett syndrome and autism spectrum several additional clinical features. Sixty seven per Health, Heidelberg, Victoria, disorders cohorts suggesting that despite sharing similar cent of affected females had ID or were of borderline Australia clinical characteristics with EFMR, PCDH19 mutations are intellect. Early development varied from normal to 6 Department of Paediatrics, not generally associated with these disorders. Among the abnormal; developmental regression commonly University of Melbourne, Royal 86 females with epilepsy (of whom 51 had seizure onset occurred with seizure onset. A number of psychi- Children’s Hospital, Melbourne, fi Australia before 3 years), with or without intellectual disability, we atric features were identi ed in affected females; 7Barwon Health, Geelong, identified two (2.3%) missense changes. One obsessive features were present in 33%, aggressive Victoria, Australia (c.1671C/G, p.N557K), reported previously without 8 behaviour in 26% and autism spectrum disorders GOLD Service, Hunter Genetics, clinical data, was found in two affected sisters, the first (ASD) in 22%.2 Waratah, New South Wales 2298, Australia EFMR family without a multigenerational family history of Here, we aimed to ascertain if there is a broader 9Western Sydney Genetics affected females. The second, reported here, is a novel de spectrum of phenotypes caused by PCDH19 Program, The Children’s Hospital novo missense change identified in a sporadic female. mutations, by performing mutation screening in at Westmead and Disciplines of The change, p.S276P, is predicted to result in functional three cohorts of females with clinical features that Paediatrics and Child Health & disturbance of PCDH19 as it affects a highly conserved Genetic Medicine, University of overlap those of EFMR. These included Rett Sydney, Sydney, Australia residue adjacent to the adhesion interface of EC3 of syndrome (RS) females, females with ASD, and 10Telethon Institute for Child PCDH19. females with epilepsy with or without ID. Health Research, Centre for Conclusions This de novo PCDH19 mutation in Child Health Research, a sporadic female highlights that mutational analysis University of Western Australia, SUBJECTS AND METHODS Perth, Australia should be considered in isolated instances of girls with RS patient cohort 11TY Nelson Department of infantile onset seizures and developmental delay, in Females with EFMR often have normal early Neurology, The Children’s addition to those with the characteristic family history of developmental milestones and regress in infancy. Hospital at Westmead, EFMR. Westmead, New South Wales, They have later autistic features and ID. Thus the Australia course of EFMR resembles that of RS, including RS Epilepsy and mental retardation limited to females girls with early onset seizures. RS females from the Correspondence to (EFMR; MIM 300088) is remarkable as an X-linked Australian Rett database,5 who did not have Professor Jozef Gecz, SA disorder due to its unusual inheritance pattern, mutations in MECP2 or CDKL5/STK9, were Pathology, Women’s and 1 2 ¼ Children’s Hospital, 72 King displaying sex limited expression. Typically selected (n 42). William Road, North Adelaide, X-linked disorders are characterised by the presence SA 5006, Australia; of affected males and unaffected carrier females. Autism spectrum disorder patient cohort [email protected] PCDH19, the gene encoding protocadherin 19, was In view of the prominent autistic features observed Received 21 April 2009 recognised as the causative gene for EFMR in seven in girls with EFMR, a cohort of girls with ASD was Revised 14 July 2009 unrelated families, with each family carrying selected from the Autism Genetic Resource Accepted 20 August 2009 a unique mutation.3 EFMR is easily identified by Exchange (AGRE) database of the Cure Autism inspection of large pedigrees where the phenotype Now Foundation, USA (n¼50) and from our occurs in multiple affected females connected patient cohort (n¼7). Individuals screened were through unaffected male relatives. However, in selected based on the pedigree information; each

J Med Genet 2010;47:211e216. doi:10.1136/jmg.2009.068817 211 Mutation report proband was selected from a pedigree with multiple affected (figure 1C). Serine p.276 is equivalent, based on alignment to the females across one or more generations and no affected males. EC1 domains of classical cadherins and protocadherin a4, to residues Y37 and F38 respectively, which are involved in the Females with epilepsy, with or without ID formation of the bC strand and adjacent to a highly conserved In 51/86 cases, seizure onset was under 3 years, in 6/86 cases residue important for the formation of a hydrophobic cluster seizure onset was after 3 years. The age of seizure onset was (figure 1D).6 7 Based on the serine p.276 conservation, location unknown in the remaining 29/86 cases. No other cause for within the extracellular domain of PCDH19 and its absence from epilepsy was identified. control chromosomes, we postulate that this change has func- tional significance and thus represents a novel PCDH19 mutation. PCDH19 mutation screening Nucleotide variation of PCDH19 Mutation screening was performed on genomic DNA extracted PCDH19 fi by standard methods. Primers were designed in intronic and Sequence analysis of also identi ed six silent and one fi exonic sequences such that coding sequence and a minimum of missense variants ( gure 2A table 1). Three silent changes; / / / 40 base pairs (bp) of flanking intronic sequence could be analysed c.402C A (p.I134I), c.1137C T (p.G379G) and c.1627C T fi by direct sequencing (supplementary table 1). High throughput (p.L543L) had previously been identi ed as part of the HapMap e project, while the remaining variants are novel. re-sequencing of exons 1 6, which encompasses the entire d coding region of PCDH19 (GenBank Accession number Analysis of the conservation of PCDH19 among the proto- NM_001105243), was performed. Due to the high GC content of cadherins demonstrates that the extracellular domain of fi PCDH19 1 affecting high throughput sequencing quality, PCDH19 in particular is highly conserved ( gure 2B). The level of this exon was re-analysed for mutations by denaturing high conservation is much lower for the cytoplasmic domain. This performance liquid chromatography for the cohort of epilepsy, had previously been noted for the protocadherin family in 8 fi with or without ID patients, and by manual sequencing for the general. To date all missense changes identi ed in the extracel- cohort of RS and ASD patients. Sequence changes were verified lular domain of PCDH19 cause EFMR or PCDH19 related fi 34 on the original DNA sample and segregation analysis was Dravet-like syndrome ( gure 1E). The missense change / performed on all available members of a pedigree. Polymerase c.3319C G (p.R1107G), which occurs in the cytoplasmic fi chain reaction (PCR), cleanup and sequencing was performed as domain, had previously been identi ed in healthy female indi- 4 fi previously reported.3 viduals as a rare variant. We also identi ed a missense change c.2873G/A (p.R958Q) in the cytoplasmic domain of PCDH19 RESULTS in a female with ASD; however, this is unlikely to be a causative PCDH19 screening mutation because as part of our large scale X chromosome exon 9 fi RS cohort re-sequencing effort, we identi ed this sequence variant in an Sequencing of PCDH19 in 42 females with RS identified no unrelated family with X-linked ID (Patrick Tarpey, March, 2009). mutations. The RS female cohort had previously been screened The p.R958Q change did not segregate with the intellectual for mutations in MECP2 and CDKL5/STK9 and all individuals disability and was present in three unaffected females. It appears were negative for mutations in either gene. This RS cohort was that while missense changes in the extracellular domain of therefore a highly enriched cohort for finding additional causative PCDH19 cause EFMR or PCDH19-related Dravet-like syndrome, genes for RS. this particular missense change in the cytoplasmic domain of PCDH19 is tolerated in normal individuals. Autism spectrum disorder cohort Clinical description of the PCDH19 positive cases PCDH19 We did not identify any mutations in in 57 index Small family (family 7 in Dibbens et al 20083) females with ASD from families with multiple affected females and no affected males. This suggests that PCDH19 is unlikely to Proband be a significant contributor to ASD alone in females. The 25-year-old librarian was selected for screening because she had a history of infantile onset seizures. At 18 months, she had Epilepsy, with or without ID cohort two simple febrile generalised tonic-clonic seizures. At 3 years Mutation screening in this cohort of 86 females identified two 1 month, she started having afebrile right hemiclonic seizures missense changes in PCDH19 (2/86 or 2.3%). Both changes were lasting up to 5 min. Following an allergic reaction to carbama- identified in the cohort of 51 females with documented seizure zepine, she was treated with valproate and later required the onset before 3 years. One of the changes, c.1671C/G (p. addition of clonazepam. At 8.5 years, she presented with atonic N557K), and the associated pedigree have previously been drop attacks in clusters and received clobazam with valproate, as reported (family 7).3 Here we report detailed clinical data on this her behaviour had deteriorated on clonazepam. Her last seizure family (see below). occurred at 12.5 years and she was weaned off antiepileptic The second change identified was a novel heterozygous treatment at 13.5 years. A postictal electroencephalogram (EEG) missense change c.826T/C (p.S276P). The patient’s parents did study at 4 years showed biposterior high voltage slowing more not carry the change (figure 1B) and biparental testing using six pronounced on the left without epileptiform activity. Magnetic microsatellite markers confirmed that the pedigree structure was resonance imaging (MRI) was not performed. correct (figure 1A). Thus this girl carries a de novo change; At 10 years, she was diagnosed with Asperger syndrome. She however, the possibility of gonadal mosaicism in one of the had obsessional features such as crawling out of bed very care- parents cannot be excluded. Unfortunately, neither of the unaf- fully in the morning so that her bed would not be messy and her fected sisters was available for genetic analysis. bedroom was very tidy. Her full scale IQ showed that she was of The c.826T/C nucleotide change is predicted to cause average intellect; however, she had significant difficulties with replacement of the serine (S) at position p.276 with vocabulary and visuospatial tasks. Despite these findings, she a proline (P) (figure 1). Serine p.276 is highly conserved across coped well academically at school but experienced social diffi- a range of protocadherins and PCDH19 of various species culties. She completed a university degree.

212 J Med Genet 2010;47:211e216. doi:10.1136/jmg.2009.068817 Mutation report

Figure 1 De novo missense mutation in an affected female. (A) Pedigree of the family; black symbol denotes the affected girl. (B) Sequence chromatograms of PCDH19 exon 1 illustrating the c.826T/C mutation identified in the proband (II-2, bottom panel) but absent in the parents (I-1 and I-2); the two unaffected sisters (II-1 and II-3) were unavailable for genetic analysis. (C) Partial alignment of human protocadherins (PCDHs) and orthologs of PCDH19 from available species shows a high degree of conservation of the serine 276 residue. (D) Clustal W alignment of the extracellular (EC) 1 domain of classical cadherins (C, E, and N) and PCDH a4 with the EC3 domain of PCDH19 which contains the S276 residue mutated in the de novo case (circled). Arrows indicate residues involved in the formation of the bC sheet, gray for cadherins and black for PCDHs. Residues involved in the adhesion interface of cadherins and the formation of a hydrophobic cluster in PCDHs are illustrated in bold.67p.S276 is adjacent to a highly conserved residue involved in the formation of a hydrophobic cluster (p.Y275). (E) Schematic representation of the PCDH19 protein with all PCDH19 mutations identified to date. Mutations identified in EFMR patients are illustrated above the protein schematic,3 with the mutation identified in this investigation illustrated in bold. Mutations identified in the Dravet-like (DS) patients4 are illustrated below the schematic.

Sister of the proband changed to valproate. Seizures were controlled by 6 years and Her 23-year-old sister presented with right hemiclonic seizures medication was weaned at 10 years. Seizures recurred after at 2 years. Her seizures began with a scream, then a fixed gaze 8 months, and she was commenced on valproate with the later and clonic activity of the right arm and face, which sometimes addition of lamotrigine. By 12 years, seizures began with a 10 s involved her leg. After 6 months on carbamazepine, she was cephalic aura of feeling strange, nauseated and dizzy without Table 1 Variation identified in PCDH19 through true vertigo. Her head and eyes would deviate to the right and sequencing of 186 females sometimes she would have right hemiclonic activity. Seizures Nucleotide Protein Type of Frequency would cluster with 20 in 1 day. Seizures remained highly change consequence change (%) refractory until they came under control at 16 years when leve- tiracetam was added to carbamazepine and lamotrigine. c.6 G/A p.E2E Silent 1.23 / Her EEG at 11 years showed a photoparoxysmal response with c.402 C A p.I134I Silent 7.80 fl / high voltage polyspike wave at a number of ash frequencies. c.655 C T p.L219L Silent 0.44 e c.1137 C/T p.G379G Silent 3.43 Video EEG monitoring at 14 years captured eight seizures which c.1627 C/T p.L543L Silent 20.62 began by the patient calling out to her mother, staring blankly, c.1683 G/A p.P561P Silent 0.43 followed by head and eye deviation to the left and oral autom- atisms. The EEG showed an ictal rhythm which began with right c.2873 G/A p.R958Q Missense 0.46 frontotemporal slowing evolving to sharply contoured theta The line dictates the distinction between the extracellular domain maximal at T4, which spread to involve the right hemisphere. of PCDH19 (above the line) and the cytoplasmic domain (below the line). MRI of the brain at 13 years was normal apart from mild PCDH19, protocadherin 19. ventriculomegaly.

J Med Genet 2010;47:211e216. doi:10.1136/jmg.2009.068817 213 Mutation report

Figure 2 Population variation and conservation of protocadherin 19 (PCDH19). (A) Schematic representation of the PCDH19 protein with all non-disease causing variants identified to date. Silent variants are illustrated above and missense variants below the protein schematic. (B) Conservation score of PCDH19 relative to all other d-protocadherins. Conservation scores were obtained from Scoreconsdhttp://www.ebi.ac. uk. Each bar on the graph represents the conservation score of a single amino acid in PCDH19. Grey bars illustrate amino acids in the extracellular domain of PCDH19 and black the cytoplasmic domain. Missense mutations (n¼7) are illustrated as black lines on top of the graph. Missense variants (n¼2) are illustrated as grey lines. The PCDH19 protein schematic under the graph illustrates the position of functional domains of PCDH19. EFMR missense mutations are indicated with a hash (#), Dravet-like syndrome mutations are indicated with a star (*), and variant missense changes in grey.

Her early motor developmental milestones were normal but neurological signs or dysmorphic features. MRI of the brain was she spoke single words late at 15 months and put two words normal. She was the second child of unrelated Vietnamese together after 2 years. She did not regress. She was diagnosed parents and had two normal sisters, aged 3 and 9 years. There with attention deficit hyperactivity disorder at 6 years and was was no family history of seizures or ID. treated with dexamphetamine into adult life. Neuropsychological assessment at 13 years showed that she was of borderline intel- DISCUSSION lect with good preservation of visuospatial function and average We sought to ascertain the frequency of PCDH19 mutations in performances in general knowledge and non-verbal concept females who share clinical characteristics with those that occur formation. She had difficulties in both verbal and visual domains in EFMR.1 2 The most typical phenotype seen in EFMR females of memory function. She also showed planning and organisation comprises a normally developing infant whose seizures begin problems. She attended a special school and then completed under 3 years (mean age of 14 months), sometimes with devel- a certificate in caring for the elderly as a young adult. There was opmental regression at seizure onset. Two thirds of affected no further family history of seizures or ID. females have borderline intellect or ID, which varies from mild to profound; autistic features may be prominent.2 We selected three De novo case cohorts based on specific phenotypic features to ascertain This 7-year-old girl presented with her first seizure at 1 year of whether PCDH19 was a significant cause of these phenotypes. age, 5 days after receiving her measles, mumps and rubella First, a number of phenotypic similarities exist between RS immunisation. She had clusters of afebrile generalised tonic- and EFMR. RS (RTT, MIM 312750) is a severe neuro- clonic seizures and later had seizures with fever. Her EEG at developmental disorder which predominantly affects females. 14 months showed left central and frontal epileptiform activity, RS is characterised by developmental regression between 6 and but was normal at 4 and 6 years. By 7 years, she had episodes of 18 months of age and the development of other features convulsive status epilepticus, which responded to phenytoin in including stereotypic hand movements and hyperventilation. combination with lamotrigine and clobazam. She had previously Additional characteristics include seizures 10 and autistic tried valproate. Early developmental milestones were normal, but features.11 12 Mutations in either MECP213 or CDKL5 10 14 15 are by 4 years she had moderate ID. At 7 years, she received a diag- found in the majority of girls with RS; however, approximately nosis of an autism spectrum disorder; she had not shown 25% of patients with RS do not carry a mutation of either gene.16 neurodevelopmental regression. Examination showed no focal Therefore it is likely that additional genes contribute to RS.11 We

214 J Med Genet 2010;47:211e216. doi:10.1136/jmg.2009.068817 Mutation report used an enriched RS cohort, where mutations of MECP2 and epilepticus, myoclonic and absence seizures, and a lesser degree of CDKL5 had been excluded, to screen for PCDH19 mutations. ID than cases with classical Dravet syndrome with a mutation in Despite the phenotypic similarities shared between females with the sodium channel gene SCN1A.4 These PCDH19 mutation RS and EFMR, we did not identify any mutations in PCDH19. positive cases closely resemble our severe EFMR cases. Indeed, This suggests that PCDH19 is unlikely to make a significant our experience is that the severe EFMR phenotypes, while contribution to RS. The possibility remains that PCDH19 could sharing some features with Dravet syndrome, lack the charac- be a rare cause of RS. While there are a number of phenotypic teristic temporal evolution of Dravet syndrome.21 In Dravet similarities between EFMR and RS females, the mutant males in syndrome seizure onset occurs at around 6 months, followed by these two disorders show opposite phenotypes. EFMR trans- frequent convulsive seizures over the next year typically mitting males do not have seizures or ID while the rare males involving episodes of febrile status epilepticus. Between who have RS are more severely affected than their affected 1e4 years, other seizure types emerge and include partial, sisters.17 Thus, the absence of PCDH19 mutations in the RS absence, myoclonic and atonic seizures. Seizures typically remain cohort, together with the opposing phenotypes in hemizygous refractory. Children with Dravet syndrome develop normally in males carrying mutations, strongly suggests that unrelated the first year of life, then their development slows and cognitive molecular mechanisms are responsible for these two disorders. impairment is usual. This is quite different from the temporal Second, psychiatric features are prominent in females with evolution of EFMR. EFMR. In particular, ASD and/or autistic features have been The three new EFMR positive girls described here do not have identified in 22% of EFMR females.2 ASD has a significant genetic Dravet syndrome. The sisters with the familial mutation had component with at least 76 genes or chromosomal loci proposed seizure onset at 18e24 months of age with hemiclonic seizures to be associated with ASD (reviewed in Abrahams and Gesch- in both and febrile generalised tonic-clonic seizures in one. wind18). None of the associated genes account for more than Seizures were brought under control in both by mid-adolescence 1e2% of cases to date (reviewed in Abrahams and Geschwind18). and they were of normal intellect. Our third patient with The PCDH19 gene has not previously been associated with a de novo PCDH19 mutation presented at 1 year with afebrile autism; however, two close sequence relatives PCDH9 and generalised tonic-clonic seizures, which is rather late for Dravet PCDH10 (also d protocadherins) have recently been implicated as syndrome. She only had convulsive attacks and her episodes of potential candidate genes for autism.19 20 Despite this, we found status epilepticus occurred late at 7 years. Thus detailed pheno- no evidence that PCDH19 causes isolated ASD within the - typic information shows that her picture is not that of Dravet tively small cohort that we screened. We did nevertheless find that syndrome although her outcome is similarly severe. the PCDH19 mutations identified in our third cohort occurred in The p.S276P mutation affects a residue in EC3 of PCDH19, association with Asperger syndrome in one proband and ASD in which is highly conserved across a number of orthologs and the other, confirming the association between ASD and EFMR. paralogs. The p.S276 is predicted, based on the crystal structure Third, we screened 86 females with epilepsy with or without of the extracellular (EC) 1 domain of cadherins7 and proto- ID where the age of seizure onset was known to be <3 years in cadherin-a,6 to be involved in the formation of the bC strand 51 cases. Half the affected females had a family history of (figure 1D). The EC repeat domains of cadherins and proto- seizures; however, their families did not show the characteristic cadherins are believed to be composed of two b sheets packed face inheritance pattern of EFMR over multiple generations. This to face. One b sheet is composed of four b strands (bA, bC, bF and screen allowed us to identify the smallest EFMR family to date bG) and the second b sheet is composed of the remaining three with just two affected girls in a single generation and a normal b strands (bB, bD and bE).67The p.Y275 residue, which is adja- transmitting father who carried a novel de novo PCDH19 cent to the mutated p.S276 residue, is equivalent to residues p. mutation.3 Taken together, our previous3 and current screening Y377 and p.F386 in EC1 of cadherins and protocadherins, respec- ascertained two PCDH19 mutations out of 86 females from small tively (figure 1D). p.Y37/p.F38 is an important residue involved in kindreds and sporadic cases. Thus EFMR is not a common cause the adhesion interface of cadherins and the complementary of seizures and ID in females; however, a frequency of 2.3% hydrophobic core of protocadherins. We predict that the p.S276P means that EFMR must be considered part of the differential mutation will disrupt the formation of the bC strand destroying diagnosis given its major genetic counselling implications. Each the hydrophobic core of the EC3 domain. While the EC1 domain female with a PCDH19 mutation has a 50% risk of having of classical cadherins was originally thought to be entirely affected daughters who may have a more or less severe pheno- responsible for the homophilic adhesive activity of cadherins, type, and a 50% risk of having transmitting sons. Furthermore in there is increasing evidence highlighting the importance of the most cases the mutation will have been inherited from a parent. other EC domains including EC3.22 23 We therefore believe that This could have implications for other branches of the family. this mutation results in a functional knockout of PCDH19. A novel de novo p.S276P missense mutation was detected in The EC domains of protocadherins appear to be crucial for a sporadic female with refractory tonic-clonic seizures and ID. normal function. The amino acid sequence of the EC domains of This girl’s phenotype poses a striking contrast to the proband of protocadherins is highly conserved, while the sequence of the the small EFMR family who had relatively easily controlled cytoplasmic domains shows more variation between the infantile seizures and was of normal intellect. Previously cases of different protocadherin subclasses.8 Our screening of PCDH19 in EFMR have been recognised based on its distinctive inheritance 186 females, analysed as part of this investigation, failed to pattern of affected females through several generations with identify any normal missense variation in its EC domain, while normal transmitting males. PCDH19 testing should therefore be two missense changes have been identified in the cytoplasmic considered as one of the options in females with seizure onset domain.4 The high level of conservation and the absence of within the first 3 years with developmental delay or ID. normal missense variation in the EC domain of PCDH19 suggest Recently Depienne and co-authors emphasised that females strong evolutionary constraints acting on this part of the protein. presenting with a severe encephalopathy resembling Dravet This finding also has implications for PCDH19 diagnostic testing, syndrome may have PCDH19 mutations. They noted that the which should be prioritised to the EC and the transmembrane PCDH19 cases had a later age of onset, less frequent status domains. However, truncation mutations in the cytoplasmic

J Med Genet 2010;47:211e216. doi:10.1136/jmg.2009.068817 215 Mutation report domain, which are subject to nonsense-mediated decay, are likely 6. Morishita H, Umitsu M, Murata Y, Shibata N, Udaka K, Higuchi Y, Akutsu H, to result in EFMR, therefore the cytoplasmic domain should still Yamaguchi T, Yagi T, Ikegami T. Structure of the cadherin-related neuronal receptor/ protocadherin-alpha first extracellular cadherin domain reveals diversity across be screened for mutations. cadherin families. J Biol Chem 2006;281:33650e63. Further to this, Mateus et al24 identified that missense muta- 7. Patel SD, Ciatto C, Chen CP, Bahna F, Rajebhosale M, Arkus N, Schieren I, Jessell tions in the EC domain or the juxtamembrane domain (which TM, Honig B, Price SR, Shapiro L. Type II cadherin ectodomain structures: implications for classical cadherin specificity. Cell 2006;124:1255e68. affect the integrity of the EC domain) of Cadherin 1 increase cell 8. Sano K, Tanihara H, Heimark RL, Obata S, Davidson M, St John T, Taketani S, motility and EGFR activation, while missense mutations in the Suzuki S. Protocadherins: a large family of cadherin-related molecules in central cytoplasmic domain have no effect on EGFR activation or cell nervous system. EMBO J 1993;12:2249e56. Xenopus laevis 9. Tarpey PS, Smith R, Pleasance E, Whibley A, Edkins S, Hardy C, O’Meara S, Latimer motility. An interesting correlate is also seen in the C, Dicks E, Menzies A, Stephens P, Blow M, Greenman C, Xue Y, Tyler-Smith C, paraxial protocadherin, where the EC and transmembrane Thompson D, Gray K, Andrews J, Barthorpe S, Buck G, Cole J, Dunmore R, Jones D, domains are required for its cell sorting function, while the Maddison M, Mironenko T, Turner R, Turrell K, Varian J, West S, Widaa S, Wray P, cytoplasmic domain is not.25 Whether the EFMR phenotype may Teague J, Butler A, Jenkinson A, Jia M, Richardson D, Shepherd R, Wooster R, Tejada MI, Martinez F, Carvill G, Goliath R, de Brouwer AP, van Bokhoven H, Van Esch H, therefore occur as a result of defects in cell sorting and/or Chelly J, Raynaud M, Ropers HH, Abidi FE, Srivastava AK, Cox J, Luo Y, Mallya U, motility is yet to be investigated. Moon J, Parnau J, Mohammed S, Tolmie JL, Shoubridge C, Corbett M, Gardner A, This report describes a sporadic female with EFMR with Haan E, Rujirabanjerd S, Shaw M, Vandeleur L, Fullston T, Easton DF, Boyle J, PCDH19 Partington M, Hackett A, Field M, Skinner C, Stevenson RE, Bobrow M, Turner G, a mutation in . Our recent report of six new families led Schwartz CE, Gecz J, Raymond FL, Futreal PA, Stratton MR. A systematic, large-scale us to conjecture that EFMR could be an under-diagnosed disorder resequencing screen of the X chromosome coding exons in mental retardation. due to the lack of appreciation of the unusual inheritance pattern Nat Genet 2009;41:535e43. with unaffected transmitting males as a major recognisable 10. Scala E, Ariani F, Mari F, Caselli R, Pescucci C, Longo I, Meloni I, Giachino D, Bruttini 2 3 M, Hayek G, Zappella M, Renieri A. CDKL5/STK9 is mutated in Rett syndrome variant feature, which is not visible in small families. Here we relied with infantile spasms. J Med Genet 2005;42:103e7. solely on phenotypic attributes to select the cohort from which 11. Hagberg B, Aicardi J, Dias K, Ramos O. A progressive syndrome of autism, dementia, we identified the first sporadic affected female with EFMR. In ataxia, and loss of purposeful hand use in girls: Rett’s syndrome: report of 35 cases. fi PCDH19 Ann Neurol 1983;14:471e9. light of this nding, mutational analysis of should be 12. Young DJ, Bebbington A, Anderson A, Ravine D, Ellaway C, Kulkarni A, de Klerk N, considered in sporadic females with seizures beginning under Kaufmann WE, Leonard H. The diagnosis of autism in a female: could it be Rett 3 years of age, in particular in the context of developmental delay syndrome? Eur J Pediatr 2008;167:661e9. 13. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome with or without ID. is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet e Acknowledgements We are grateful to the families for their support and 1999;23:185 8. participation in this study. We thank Associate Professor John Mulley for useful 14. Tao J, Van Esch H, Hagedorn-Greiwe M, Hoffmann K, Moser B, Raynaud M, ´ discussion and critical assessment of the manuscript, Ms Linda Turelli and Dr Kathryn Sperner J, Fryns JP, Schwinger E, Gecz J, Ropers HH, Kalscheuer VM. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5/STK9) gene are associated Friend for performing the biparental testing. We gratefully acknowledge the with severe neurodevelopmental retardation. Am J Hum Genet resources provided by the AGRE Consortium and the participating AGRE families. This 2004;75:1149e54. work has been supported by funding from the Wellcome Trust, National Health and 15. Weaving LS, Christodoulou J, Williamson SL, Friend KL, McKenzie OL, Archer H, Medial Research Council of Australia, Thyne-Reid Charitable Trusts and The Jack Evans J, Clarke A, Pelka GJ, Tam PP, Watson C, Lahooti H, Ellaway CJ, Bennetts B, Brockhoff Foundation and NIH for the RS cohort. Leonard H, Ge´cz J. Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation. Am J Hum Genet Funding Other Funders: Wellcome Trust; National Health and Medical Research e Council of Australia. 2004;75:1079 93. 16. Downs J, Bebbington A, Woodhead H, Jacoby P, Jian L, Jefferson A, Leonard H. Competing interests None. Early determinants of fractures in Rett syndrome. Pediatrics 2008;121:540e6. 17. Hardwick SA, Reuter K, Williamson SL, Vasudevan V, Donald J, Slater K, Ethics approval This study was conducted with the approval of the Child Youth and Bennetts B, Bebbington A, Leonard H, Williams SR, Smith RL, Cloosterman D, Women’s Health Service, Adelaide, Australia. Christodoulou J. Delineation of large deletions of the MECP2 gene in Rett syndrome Eur J Hum Genet Provenance and peer review Not commissioned; externally peer reviewed. patients, including a familial case with a male proband. 2007;15:1218e29. 18. Abrahams BS, Geschwind DH. Advances in autism genetics: on the threshold of REFERENCES a new neurobiology. Nat Rev Genet 2008;9:341e55. 1. Ryan SG, Chance PF, Zou CH, Spinner NB, Golden JA, Smietana S. Epilepsy and 19. Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, Shago M, Moessner R, mental retardation limited to females: an X-linked dominant disorder with male Pinto D, Ren Y, Thiruvahindrapduram B, Fiebig A, Schreiber S, Friedman J, sparing. Nat Genet 1997;17:92e5. Ketelaars CE, Vos YJ, Ficicioglu C, Kirkpatrick S, Nicolson R, Sloman L, Summers A, 2. Scheffer IE, Turner SJ, Dibbens LM, Bayly MA, Friend K, Hodgson B, Burrows L, Gibbons CA, Teebi A, Chitayat D, Weksberg R, Thompson A, Vardy C, Crosbie V, Shaw M, Wei C, Ullmann R, Ropers HH, Szepetowski P, Haan E, Mazarib A, Afawi Z, Luscombe S, Baatjes R, Zwaigenbaum L, Roberts W, Fernandez B, Szatmari P, Scherer Neufeld MY, Andrews PI, Wallace G, Kivity S, Lev D, Lerman-Sagie T, Derry CP, SW. Structural variation of chromosomes in autism spectrum disorder. Am J Hum Korczyn AD, Gecz J, Mulley JC, Berkovic SF. Epilepsy and mental retardation limited to Genet 2008;82:477e88. females: an under-recognized disorder. Brain 2008;131(Pt 4):918e27. 20. Morrow EM, Yoo SY, Flavell SW, Kim TK, Lin Y, Hill RS, Mukaddes NM, Balkhy S, 3. Dibbens LM, Tarpey PS, Hynes K, Bayly MA, Scheffer IE, Smith R, Bomar J, Sutton E, Gascon G, Hashmi A, Al-Saad S, Ware J, Joseph RM, Greenblatt R, Gleason D, Ertelt Vandeleur L, Shoubridge C, Edkins S, Turner SJ, Stevens C, O’Meara S, Tofts C, JA, Apse KA, Bodell A, Partlow JN, Barry B, Yao H, Markianos K, Ferland RJ, Barthorpe S, Buck G, Cole J, Halliday K, Jones D, Lee R, Madison M, Mironenko T, Varian Greenberg ME, Walsh CA. Identifying autism loci and genes by tracing recent shared J, West S, Widaa S, Wray P, Teague J, Dicks E, Butler A, Menzies A, Jenkinson A, ancestry. Science 2008;321:218e23. Shepherd R, Gusella JF, Afawi Z, Mazarib A, Neufeld MY, Kivity S, Lev D, Lerman-Sagie 21. Carranza D, Dibbens LM, Beroric SF, Scheffer IE. Do PCDH19 mutations cause Dravet T, Korczyn AD, Derry CP, Sutherland GR, Friend K, Shaw M, Corbett M, Kim HG, syndrome or a ’pseudo-Dravet’ phenotype? Online comment 2009 Apr 12. Sporadic Geschwind DH, Thomas P, Haan E, Ryan S, McKee S, Berkovic SF, Futreal PA, Stratton infantile epileptic encephalopathy caused by mutations in PCDH19 resembles MR, Mulley JC, Ge´cz J. X-linked protocadherin 19 mutations cause female-limited Dravet syndrome but mainly affects females. Depienne et al. PLoS Genet 2009. epilepsy and cognitive impairment. Nat Genet 2008;40:776e81. 22. Chien YH, Jiang N, Li F, Zhang F, Zhu C, Leckband D. Two stage cadherin kinetics 4. Depienne C, Bouteiller D, Keren B, Cheuret E, Poirier K, Trouillard O, Benyahia B, require multiple extracellular domains but not the cytoplasmic region. J Biol Chem Quelin C, Carpentier W, Julia S, Afenjar A, Gautier A, Rivier F, Meyer S, Berquin P, 2008;283:1848e56. He´lias M, Py I, Rivera S, Bahi-Buisson N, Gourfinkel-An I, Cazeneuve C, Ruberg M, 23. Tsuiji H, Xu L, Schwartz K, Gumbiner BM. Cadherin conformations associated with Brice A, Nabbout R, Leguern E. 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216 J Med Genet 2010;47:211e216. doi:10.1136/jmg.2009.068817 Supplementary table 1: PCDH19 specific primers used for PCR amplification and sequencing.

Primer Name Sequence (5’to 3’) Size Product Annealing (bp) (bp) Temperature (°C) PCDH19ex1-F1 TCGGAGGGGTGTGGAGAGG 19 428 64 PCDH19ex1-R1 CTGAACGACAATGCGCCCA 19 PCDH19ex1-F2 ATTGACCGTGATCTGCTGTGC 21 507 64 PCDH19ex1-R2 AAAACTCGCCTCCCAACACA 20 PCDH19ex1-F3 TGACCGACTCCAATGACAACAACC 24 551 62 PCDH19ex1-R3 ACTATTCTGGTGGACGGACGGCTG 24 PCDH19ex1-F4 GCCTTGGTGCGGGTGTCTGAT 21 480 60 PCDH19ex1-R4 TAACCACGAGCAGACCAAGGCG 22 PCDH19ex1-F5 TACCAGGTCATTGTGCAGGAGAAC 24 629 60 PCDH19ex1-R5 AATCTACTTGTCCCCTGCTCTCGA 24 PCDH19ex1-F6 CAGGTCAATGGCGAAGTCAGA 21 383 60 PCDH19ex1-R6 AATCCTGGTGCTTTTGTTTCCTAC 24 PCDH19ex2-F CTGAATGGAGAAGGATGCTGAA 22 352 62 PCDH19ex2-R GTTTTGGACTGGGGTAGGAG 20 PCDH19ex3-F GGAGAAACCCGATGATGTAATG 22 529 60 PCDH19ex3-R GCGAGCAGCTAAAGAAGGAGA 21 PCDH19ex4-F CTTCTAAGTAGGGCAGTTC 19 317 60 PCDH19ex4-R ATTACCAGTGATGTTTATTTAC 22 PCDH19ex5-F TCATTACAGAGCCGACATCATC 22 296 60 PCDH19ex5-R ACATTTTGGGTTCTTTGGAGT 21 PCDH19ex6-F1 TGGGGAGTAACAGTGAATC 19 476 60 PCDH19ex6-R1 CAGGGCAATGGTGTAAGAC 19 PCDH19ex6-F2 CTGAAAGGCAAGAGGACTG 19 429 60 PCDH19ex6-R2 TGGTGAGCAATTAAAACAAGA 21

Appendix - C

Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) CFB 3835726 0.000144333 -2.079 SBNO2 2946353 0.000179034 -1.622 EFNA5 3442514 0.000180141 -2.908 GPR133 3709838 0.000198681 -12.123 TNFAIP8 3633578 0.000266549 -1.968 DLG7 2886174 0.000311588 2.351 MET 2818454 0.000323544 3.899 GAS7 3335070 0.000407765 -1.946 KREMEN1 2392945 0.000655602 -1.635 TMTC1 3881443 0.000723578 -11.618 NDC80 2438282 0.000767773 1.531 ENPP1 2636319 0.000830242 9.905 LAMB1 3744800 0.000882644 2.191 POLA2 2346934 0.000957987 1.708 SLC39A8 3610110 0.00111788 -4.432 HDAC9 3138204 0.00118715 1.787 LMCD1 2740067 0.00128545 -2.208 CBLN3 3240987 0.00130265 -1.758 FAM83D 3918535 0.00137482 1.603 NTF3 3757970 0.00140864 1.917 SPAG5 3630736 0.00177854 1.713 HRSP12 2902804 0.00185486 -4.174 APOD 3219215 0.00187127 -11.384 CXCR7 3263944 0.00196225 -2.491 GALNT6 2611056 0.0019718 1.988 NXN 2717101 0.00204499 1.892 DDIT3 3888522 0.00209703 2.212 EXO1 2469213 0.00231903 1.596 S1PR2 2991395 0.00247827 -2.725 HIST1H3F 4022925 0.00254486 2.094 UHRF1 2333136 0.00262331 1.852 NR2F2 3408831 0.002718 1.931 PRR11 2859667 0.002749 2.198 A4GALT 3438061 0.0027625 -2.158 LOH3CR2A 2779095 0.00297637 -2.745 ITGA6 3954879 0.00301935 6.752 SECTM1 3507710 0.00303331 -2.793 MAP3K8 3589697 0.00309126 -2.324 PDGFC 3607510 0.00312614 3.885 TTK 2646818 0.0032121 1.634 DAPK2 2507896 0.00324919 -2.523 MGC50273 2410522 0.00351667 -1.574 CDON 3813297 0.0035283 -5.464 PRC1 3588658 0.00355747 2.215 TACC3 3653072 0.00358376 1.687 ENDOD1 3140213 0.00383857 2.262 TCF7L2 3639031 0.00393235 -1.652 TCF19 3458587 0.00394341 1.986 LPCAT2 3309602 0.00403128 2.574 ITGA3 2435735 0.0041971 4.153 NPAL3 2592005 0.00428215 2.239 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) RNASEH2A 2775909 0.00444925 1.606 CEP97 3886532 0.00449382 1.651 C1RL 3214451 0.00451782 -1.831 FANCI 3096271 0.00456019 1.650 CENPI 3309112 0.00473583 1.698 3261886 0.00480534 -3.925 RASSF8 2548617 0.00487114 -1.946 TOP2A 3756193 0.00529975 2.626 RAB43 3110272 0.00555269 2.173 RPL39L 2827185 0.00567115 1.518 SAR1A 2604254 0.00570971 -1.634 SLC7A1 2664209 0.0057896 1.853 OXTR 3463112 0.00582871 7.496 FZD6 3917938 0.00586388 1.715 EN1 3969422 0.00627971 -1.832 SOD2 3980560 0.00628844 -2.184 C1R 3592511 0.00635134 -3.213 TNFAIP2 3061319 0.00640823 -1.732 TJP2 2769182 0.00648476 2.028 CDC6 3371003 0.00649997 1.974 PLK1 2485636 0.00654411 2.190 RPS15A 2555252 0.00690641 -2.505 C8orf40 3772279 0.00692185 -1.742 C3 3308864 0.0070344 -2.871 RFC3 3636562 0.00703787 1.714 MSC 2957126 0.00712518 -1.794 CCNB2 3286776 0.00718261 1.939 ABCA6 4054204 0.00720254 -2.795 UBE2T 3631397 0.0072077 2.239 KIAA1524 3373675 0.00722977 1.562 KIF4A 2628785 0.00728368 2.014 CDC42EP3 2609347 0.00732741 1.740 BCL3 3442475 0.00736729 -2.104 ADH1B 3031624 0.00742888 -4.172 LMNB1 3415668 0.00760431 1.766 NRBF2 3590014 0.00772067 -1.676 NDST2 3442322 0.00777123 -1.627 MMP16 2772566 0.00779049 1.959 ABCA9 3232979 0.00786924 -1.512 MDK 3872398 0.00790572 1.680 KIF2C 3123541 0.0081744 1.920 MCM5 2709606 0.00819643 1.753 CPZ 2825629 0.00846528 -3.198 AXUD1 2709631 0.00848959 -1.602 AKR1C3 2779231 0.00876774 -5.469 IL10RB 2394608 0.00897028 -1.753 SESN1 3312490 0.00917771 1.509 TP53I11 2813414 0.00919847 -2.204 C10orf26 2701109 0.00924152 -1.535 PPFIBP1 3367338 0.00929616 1.877 POMGNT1 2416218 0.00939436 1.532 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) KIF23 2916716 0.00941001 1.803 HMGB2 3721658 0.00948578 1.883 PPL 2702395 0.00957097 -2.197 RRM2 3091797 0.00970319 3.707 FAM110B 3623948 0.00975692 -3.089 VEPH1 2786322 0.00975714 1.694 FAM127B 3356417 0.00980213 -1.637 SACS 3599811 0.00983489 2.033 BCAT1 3886576 0.0100792 2.603 RACGAP1 3175494 0.0101026 1.966 BUB1B 3821893 0.0101138 1.502 TGFBR1 2451200 0.0101757 -1.729 KIF18A 2640449 0.010214 1.692 AKR1C2 3145980 0.0102684 -6.194 GRK5 3385307 0.0104556 -1.831 CEBPD 2401333 0.0105308 -1.728 PLAC8 2477073 0.0107269 1.758 PPARG 3821263 0.0109845 -1.684 STAT3 2838201 0.0109989 -1.690 NCAPG 3910785 0.0111063 2.217 OR10G9 2914777 0.0111153 -1.726 AKR1C1 2779823 0.0111198 -9.188 COL12A1 3247757 0.011672 1.729 HNMT 3294816 0.0117739 -1.922 HMMR 3084950 0.0119043 1.661 BUB1 3234760 0.0119644 1.961 RGS4 2681114 0.0120625 13.967 FOXD1 3339167 0.012078 1.531 SERPING1 3409211 0.0120825 -4.053 RAD18 2881413 0.0121936 1.568 TBC1D16 3728964 0.012352 -1.835 PFN2 3400586 0.012425 3.407 RIPK3 3944147 0.0124522 -2.493 CASC5 3190242 0.0127965 1.598 PENK 3644340 0.0128686 -1.568 TMEM176B 3363266 0.0130605 -7.371 RAB11FIP2 3485074 0.0132067 -2.473 FLJ14154 3181728 0.0132176 -1.543 MITF 2571457 0.0138376 -1.537 TPX2 3350830 0.0140158 2.387 XRCC4 2791197 0.0141255 1.528 MTF2 2466002 0.0141723 1.688 HIST1H2AB 3736636 0.014403 1.862 IGSF10 2669930 0.0144485 -2.837 FEZ1 3250699 0.0146392 -1.579 NETO2 2473284 0.0146455 2.050 ALDH1B1 3108859 0.0146843 -1.770 CENPK 3962578 0.0146843 1.699 SLC3A2 3595979 0.0146907 1.645 GLIPR1 2923939 0.0147976 1.963 CEBPB 2982319 0.0148626 -1.609 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) HIST1H1D 3770944 0.0149175 1.845 DAZAP2 3415229 0.0149661 -1.732 FAM133B 3706659 0.0149774 -2.199 CPXM2 4004853 0.0150137 3.402 OSR1 3331903 0.0154197 -1.943 TES 2720251 0.0163287 4.739 ADH1A 3274758 0.0164996 -5.302 CSPG4 3408733 0.0166679 2.416 C14orf4 3850166 0.0167326 -1.532 CDCA3 3266408 0.0167721 1.555 LOC339803 2933175 0.0168226 1.773 MFHAS1 3776139 0.0168936 -1.538 SLC38A1 2334098 0.0171338 -2.094 LRPAP1 3060332 0.0172455 1.770 CHST13 3452231 0.0172976 -1.542 NUF2 2525989 0.0173142 1.914 UBL3 2991150 0.0173691 2.036 LCE3D 3768703 0.0175167 -1.559 TNFSF4 2928690 0.0176428 5.148 ZNF436 2925953 0.0176474 -2.171 NTN1 3821908 0.0178958 -1.660 STEAP4 3061438 0.0179291 -1.495 CCDC23 3186491 0.0179343 -1.726 E2F7 2572909 0.0181689 1.641 ALDH3A2 3331355 0.0183593 -1.531 TRIP13 3638204 0.0184934 1.921 C10orf72 3768791 0.0186969 -1.710 ASPM 3593931 0.0187078 1.813 CXCL12 2542420 0.0189346 -3.084 LNX2 2359483 0.0190485 1.502 GLDN 3815116 0.0190959 -3.654 ME3 2559494 0.0192793 -1.653 IGF1R 3481543 0.0192924 -2.126 GCNT1 3248897 0.0194033 1.737 SGK1 2793951 0.0196832 -3.044 DEPDC1 3550278 0.0198377 2.744 CXCL1 3321361 0.0198453 -3.752 ZFP36L2 2515627 0.0205234 -1.997 FANCI 3398241 0.020578 1.641 OCIAD2 2534354 0.0207422 1.778 SLC7A11 3143660 0.0207611 3.324 TNXB 3447694 0.0208623 -1.871 PKIG 2838656 0.021068 -2.123 GPR153 3069366 0.0212365 -1.595 PSMC3IP 3335029 0.0212657 1.603 STEAP2 2378937 0.0212825 -2.506 MELK 3078435 0.0213076 1.649 HJURP 3293244 0.0213645 1.820 ARHGAP20 2731381 0.0214619 -1.549 C13orf33 2609414 0.0215403 -2.499 FGF7 2530539 0.02162 -3.963 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) GPX3 2595252 0.0219728 -1.537 FAM86C 3595691 0.0219977 -1.713 TGIF1 3345427 0.0220187 -1.535 CDK6 3678462 0.0220781 2.392 AIG1 2779199 0.0223521 2.558 C3orf64 3838094 0.0224123 1.741 C10orf104 3848039 0.0224308 -1.545 C1QTNF1 3403168 0.0224761 -1.644 KIAA1305 4004575 0.0225886 -1.824 STX8 3689880 0.0227551 -1.532 FGF9 3553531 0.022926 -3.336 SELENBP1 2946219 0.0229945 -1.963 CKAP2L 3168385 0.0231074 1.533 UACA 2806468 0.0231907 2.887 EIF4EBP2 3267036 0.0233213 -1.737 ZDHHC14 3726154 0.0237302 -1.501 KIAA1324L 3645901 0.0238599 -3.724 GJC1 2676854 0.0240108 2.216 LCE1F 3714068 0.0241222 -1.839 KRT33A 3572869 0.0241482 -1.867 FLJ42875 3353876 0.0242117 -1.533 ZFP36 3020192 0.0242434 -3.485 CYB5A 3661152 0.0242871 -2.194 BNC1 3286602 0.0245187 1.740 ZBTB44 3020343 0.0246826 -1.743 PRDX6 3984655 0.0250494 1.740 AADACL1 3449068 0.0254149 2.176 LIPC 2955673 0.0254541 -1.752 SPRR4 3505319 0.0254772 -1.505 TNFRSF10A 2809128 0.0255198 1.682 TNKS1BP1 3661718 0.0255419 -1.542 TMEM176A 3565663 0.0255534 -5.953 JUNB 2803329 0.0256169 -2.959 VPREB3 3310953 0.0256674 -1.558 SPATA13 2751066 0.0257097 -1.604 C2 3011861 0.0257838 -1.621 ITGA1 3136271 0.0261505 2.514 ITGB3BP 3473378 0.0262557 1.690 PALM 3355733 0.0263375 -1.664 SRPX 3690154 0.0263614 -1.980 CYP7B1 2536071 0.0265017 -1.650 PRLHR 2899298 0.0265337 -2.164 ITGA11 3289235 0.0271901 2.023 C1S 3628832 0.0274148 -2.557 FAM111B 3558270 0.0276023 1.640 CCNB1 2451693 0.0277572 2.635 SPON1 3173880 0.0279479 -1.943 CDC42EP2 2805078 0.0280437 -1.683 LRRFIP1 2397948 0.0281064 1.618 WNT5B 2469252 0.0282393 1.657 CCND1 2422722 0.0283645 1.537 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) BOC 2975014 0.0285829 -1.790 CNN1 2779271 0.0285956 1.691 DOCK10 3416344 0.0286442 2.473 PDGFRL 2634027 0.0290396 -2.596 LOC440104 3510858 0.0293146 -1.647 SAMD9 2449559 0.0294544 2.151 SH3BP5 3327057 0.0296048 1.939 FMOD 3040363 0.0297819 -2.289 DKK3 3756880 0.0301248 2.384 SERINC5 2626802 0.030209 1.981 BIRC5 3099566 0.0302512 1.651 TGFBR3 2325410 0.0307456 -2.281 TWIST1 2417528 0.0309176 -1.721 HOXC9 3844978 0.0312264 -1.835 SIM2 2364438 0.0313593 -1.698 CHST2 2902844 0.0314223 -1.603 SSPN 2549565 0.0317076 1.592 HIST1H3J 3941793 0.0319314 1.896 CDH6 3247818 0.0323841 -2.254 PNRC1 2714955 0.0324983 -1.540 ABL1 3079172 0.0325087 -1.570 FTO 3757840 0.0325873 -1.922 SLIT3 3454223 0.0326355 -1.843 C2orf7 2533999 0.0326509 1.522 BDKRB2 2717857 0.0328578 -1.874 BASP1 3948640 0.0328851 -1.546 FTL 3920171 0.0329281 -2.458 NCAM1 2420521 0.0329773 2.686 HMCN1 3907524 0.0329978 3.565 SLC1A4 3402150 0.0332329 1.675 FLJ14213 3134013 0.0333443 1.892 NFIL3 2687979 0.0335938 -2.526 CENPO 3610958 0.0339255 1.558 SQRDL 3507798 0.0343638 -2.555 PALLD 3448152 0.0343725 2.027 DTL 3773241 0.0344908 2.114 HRK 3349858 0.0348409 -1.553 NNMT 2662020 0.0349293 -1.622 MFGE8 2862317 0.0351287 2.683 ZFP36L1 2444283 0.0351721 -2.101 RAB9A 2961177 0.0352972 -1.804 EPHA2 3169331 0.0353157 1.497 CUGBP2 3744965 0.0355978 -2.982 CLDN23 3315675 0.0358241 -1.628 ZNF671 2364381 0.0358764 1.520 NR4A1 2388219 0.0361551 -1.912 C20orf19 3683018 0.0362203 -1.767 FLI1 3414846 0.0363815 1.643 RGS10 2367743 0.0364865 -1.496 FBLN1 3781980 0.036539 -3.269 EXTL3 3879372 0.0366776 1.786 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) CHDH 3569754 0.0367117 -1.675 MKI67 3338192 0.0372003 1.879 AURKA 3832978 0.0372202 1.551 SMPDL3A 3720896 0.0372212 -1.662 FMNL2 2409069 0.0373217 1.511 CDC20 3561039 0.0373638 3.282 PTPRG 2367843 0.0375304 1.870 C2orf43 3558226 0.0375977 1.564 UBE2D1 2449104 0.0379042 -2.009 MCM3 2510713 0.0379185 1.541 ADH1C 3233049 0.0380399 -2.033 C9orf19 3147508 0.038137 -2.070 PAPPA 3333711 0.0386603 -2.315 TENC1 3759335 0.0386699 -1.506 ASPA 3059942 0.0390487 -1.753 CPS1 2550522 0.0390995 1.606 C15orf41 3087703 0.0391053 1.695 ANK2 3593408 0.039459 -1.506 DNM1 2359431 0.0397299 -2.328 C11orf44 3774906 0.0398042 -1.755 KIAA0232 3251353 0.040205 1.610 ITPR2 3390641 0.0405861 1.642 TNFAIP8L3 2601648 0.0406184 -1.761 KLF10 2835715 0.0409664 -2.355 HIBCH 3416996 0.0409742 1.586 SNED1 2902178 0.0413415 -1.922 SOCS3 3454892 0.0414684 -2.434 SLC38A4 2798915 0.0415091 -2.581 OSR2 3607537 0.041664 -4.140 SSX2IP 4012154 0.0422927 1.551 LTBR 3507003 0.0425149 -1.610 SGMS1 3739867 0.042541 -1.562 IQGAP3 3191724 0.0426045 1.553 C2orf33 3484117 0.0427105 -1.735 OR2M7 3468743 0.0427127 -1.779 C10orf10 2869880 0.0427347 -2.288 CRIM1 2435005 0.0427609 1.672 PLTP 3329343 0.0430075 -1.629 RPS4X 2768654 0.0431855 2.077 B3GALT2 3480885 0.0432971 2.776 HUNK 3402444 0.0434849 -1.685 SUMO1 2705748 0.0440333 -1.653 WISP2 2700585 0.0440871 -3.240 WNT2 2371873 0.0443603 -1.655 BTN3A2 3746574 0.0445299 1.698 IL15RA 3396593 0.0446539 -1.540 EMX2 3529951 0.044934 -2.202 TSPAN13 3349293 0.0451233 2.963 C18orf17 3452417 0.0457119 -1.737 DHRS3 2949622 0.0458198 -1.918 SNAI1 2864449 0.0458869 -1.875 Appendix Table C1

EFMR female / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Unaffected) ADH5 3396770 0.0459776 -1.497 SLC8A1 2570616 0.0460634 2.068 PTTG1 3750785 0.0461586 2.077 ZIC1 2661992 0.0464511 2.699 SLC9A3R2 3422855 0.0465817 1.652 KIF20A 3275690 0.0468067 2.386 PMP22 3127775 0.0468991 -2.995 IL7R 2576608 0.0472184 2.775 NT5DC3 3884892 0.0473479 1.638 H3F3B 3264621 0.0474247 -1.647 MASP1 2968652 0.0478165 -3.362 RPS14 2758298 0.0478744 1.738 SH3BP5L 2947095 0.0479048 1.547 KLF11 3776504 0.0479788 -1.769 PDIA4 3736290 0.0481283 1.528 DARS2 2397025 0.0483155 1.708 NFKBIA 2543066 0.0484472 -2.272 ENPP5 2465902 0.0484758 -1.508 SCFD2 3817698 0.0486249 2.262 STAT5A 2946364 0.0486254 -1.628 IFITM1 3888613 0.0486565 -2.045 PDCD4 3168508 0.0493248 -1.933 FOXO1 3288518 0.0493751 -1.499 TMEM47 2830638 0.0493839 -1.545 IGJ 2646125 0.0497838 1.537 TAGLN 3067302 0.0498871 2.652 Appendix Table C2

Transmitting male / control male results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Transmitting vs. Control) NFX1 3166880 9.34E-05 1.496 N6AMT2 3504392 0.000542238 1.711 JMJD4 2459296 0.000653534 -1.504 LOC400590 3717034 0.000685502 1.683 UQCC 3903889 0.000924353 1.563 TRIM22 3318443 0.00145782 2.229 ARHGAP12 3283920 0.00173839 1.570 HIST1H2AH 2899772 0.00203518 -1.704 HMG2L1 3944046 0.00215696 1.548 PARN 3681377 0.00260115 1.504 TMEM136 3352485 0.00260206 1.676 PYGL 3564210 0.00267381 1.523 ABTB1 2640916 0.00327271 -1.621 LOC147804 3840857 0.00337753 -2.586 STAT3 3757840 0.00340748 1.536 EPB41L3 3797032 0.00341221 1.663 TOP2B 2666478 0.00360563 1.517 CCDC56 3758148 0.00384809 1.785 CCDC59 3464000 0.00441927 1.703 EPHA2 2397948 0.00479116 -1.517 PIAS1 3599280 0.00503791 1.523 UVRAG 3340697 0.00553045 1.579 ESCO1 3800779 0.0063103 1.644 PATL1 3374746 0.00648501 1.740 TRIM5 3360622 0.00715986 1.556 EWSR1 3941907 0.00763759 1.580 DENND4C 3164221 0.00791718 1.519 ZCCHC6 3212976 0.00826616 1.611 GFPT2 2890660 0.0082905 1.593 SETX 3228007 0.00875389 1.634 JARID1A 3439603 0.00875551 1.496 LTA4H 3466740 0.00889094 1.828 CENTA1 3034987 0.00903216 -1.619 TIMM9 3566652 0.00911823 1.572 ZBTB41 2449619 0.00927917 1.520 ISCU 3430776 0.00964445 1.506 UBE2L3 3938175 0.0102405 1.575 SAMHD1 3904691 0.0103613 2.522 MRPL10 3760945 0.0109506 1.552 NAE1 3695268 0.0110794 1.503 TROVE2 2372924 0.0110806 1.644 C11orf1 3348748 0.011091 1.497 TEX10 3217807 0.011481 1.502 SPG11 3621948 0.011483 1.608 MGC16169 2780734 0.0120577 1.620 MAN1A1 2971801 0.0122405 1.779 C1orf78 2406662 0.0126673 -1.551 USPL1 3484005 0.0127152 1.588 TMEM2 3209384 0.012779 1.844 ARHGAP21 3281621 0.012829 2.009 UQCRC2 3652218 0.0130186 1.576 ZNF507 3828887 0.0132677 1.637 IFIT2 3257192 0.0136824 1.925 Appendix Table C2

Transmitting male / control male results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Transmitting vs. Control) ESF1 3898224 0.0137552 1.565 SESN3 3387259 0.0139994 3.009 TPP2 3499453 0.0142032 1.610 LOC152217 2659676 0.0142871 -1.506 MCCC1 2707824 0.0147184 1.649 FAM118B 3355021 0.0148583 1.539 GLT8D2 3468888 0.0148649 1.679 ZBTB33 3989089 0.0149521 1.540 TST 3959918 0.0152167 -2.050 CSF2RB 3944564 0.0153545 -1.564 OPCML 3399004 0.015601 -1.700 KTI12 2412690 0.0157804 1.553 TMEM126A 3343008 0.0158439 1.864 HSDL2 3185205 0.0159304 1.760 OBFC2A 2520533 0.0160804 1.694 LIPA 3299585 0.016171 2.162 C11orf48 3375999 0.0162109 2.266 FTO 3661152 0.016493 1.523 SELL 2443450 0.0172633 -1.738 VPS35 3689922 0.0176977 1.607 XRN2 3879467 0.0179792 1.556 UBE2F 2534564 0.0184649 1.914 PGD 2319802 0.0185541 1.782 DTX3L 2638962 0.0186412 1.956 UNC5B 3250990 0.0191776 -1.775 NSMAF 3136782 0.0191911 1.699 FIG4 2920962 0.0192706 1.567 MRPL15 3098454 0.019328 -2.524 NCOA3 3887635 0.0193561 1.558 STAT1 2592268 0.0193965 1.979 ACO1 3166477 0.020037 1.640 IFI16 2362394 0.0200736 1.928 IPO8 3449304 0.0201772 1.554 2339328 0.0202563 1.645 ATM 3347658 0.0204868 1.685 CTR9 3320301 0.0208405 1.667 PSMD5 3223646 0.0210303 1.676 STARD4 2870828 0.0212093 1.880 BTAF1 3257938 0.0212901 1.576 EEA1 3465593 0.0213964 1.840 EIF2AK1 3037193 0.0215023 1.538 SLTM 3626704 0.0219294 1.537 EHBP1 2484970 0.0225107 1.634 LYRM5 3408573 0.0225977 2.318 PBLD 3292590 0.0227243 1.621 NOL5A 3873874 0.0229909 -1.655 PARP9 2692060 0.0233101 2.052 BNC2 3200040 0.0239224 1.797 SBF2 3362468 0.0246953 1.672 LOXL3 2560286 0.0247208 -2.579 AK3L1 2340315 0.0247874 -1.858 SCARA3 3091475 0.0248025 1.816 NAP1L1 3462843 0.0251459 1.622 Appendix Table C2

Transmitting male / control male results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Transmitting vs. Control) COMP 3855218 0.0253724 -3.131 MGST3 2365119 0.0258111 1.626 ACOX1 3771215 0.0259135 1.671 TMEM135 3343546 0.0259349 1.687 URM1 3190394 0.0259363 1.496 MRPL50 3218067 0.0261884 1.550 2357193 0.0265663 3.233 MGC72080 3062665 0.0269627 -1.948 WDR45 4007774 0.0270975 -1.505 DKFZP434P211 3939952 0.0279223 -2.306 ZNF417 3843662 0.0280726 1.906 3893716 0.0283497 1.517 BRE 2474791 0.028569 1.593 UBR5 3147321 0.0285856 1.518 LOC285636 2807862 0.0296148 1.633 GPR177 2417390 0.0296343 2.855 TFPI 2591421 0.0297131 1.580 EPB41L2 2973995 0.0300779 1.741 TDG 3429365 0.0301093 1.766 USP33 2419113 0.03035 1.498 CTNNAL1 3219621 0.0310194 1.676 C7orf24 3044129 0.0312739 1.539 HOXB3 3761313 0.0316707 -1.628 IL1A 2571483 0.0317077 -1.695 C18orf17 3781980 0.0320697 1.639 PPM2C 3107342 0.0328501 1.539 IFIT1 3257246 0.0332179 2.334 CENPA 2473991 0.0332256 -1.498 ATF5 3839103 0.0334389 -2.292 HOXD4 2516912 0.0334479 -1.576 AKR1C3 3233049 0.0337528 2.199 SH3BP5 2664209 0.0339706 1.496 BBS9 2996321 0.0341617 1.508 ABCC9 3446919 0.0345437 4.574 CYP4A22 2335048 0.0350868 -1.728 SNORA44 2403557 0.0351099 1.639 KRTAP5-6 3316987 0.0354643 -2.015 CYB5A 3813297 0.0355387 2.307 C14orf108 3537557 0.0356187 1.574 PPP1R7 2536183 0.035734 1.578 BLOC1S3 3836135 0.0362499 -1.565 USP8 3593652 0.0363073 1.567 CACNG8 3841157 0.0364972 -1.514 SSTR1 3533184 0.0367379 -1.801 IFIT3 3257204 0.0369911 2.182 IFIT5 3257268 0.0372414 1.641 RBM38 3890597 0.0375678 -1.657 MAP3K8 3240987 0.0375775 1.503 SLC7A5P1 3684100 0.0376383 -1.745 MSI2 3728147 0.0378274 -1.697 SNAP23 3591006 0.0379103 1.550 TMEM50B 3929664 0.0381312 1.551 PAPD1 3283378 0.038525 1.588 Appendix Table C2

Transmitting male / control male results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Transmitting vs. Control) VPS13B 3108901 0.0385557 1.578 PCMTD2 3894047 0.0386923 1.603 LOC157740 3124360 0.0390263 -1.517 IL32 3645626 0.0391672 -1.581 PAQR4 3645477 0.0400818 -1.713 BTN3A3 2899413 0.0402298 2.230 TRAF3IP2 2969810 0.0402767 1.704 MID1 3999395 0.0404388 1.760 REV3L 2969677 0.0405306 2.722 C14orf162 3570218 0.0405886 -1.577 RPL17 3807487 0.0408302 1.869 CCL2 7385547 0.0416219 2.135 ZNF367 3216319 0.0417263 -1.498 EDN1 2895244 0.0418852 -1.652 ADRA1B 2837970 0.0419381 -1.695 ISL2 3602634 0.0426696 -1.543 BLOC1S1 3416909 0.0427077 -2.046 HSPA2 3540136 0.0427323 -2.487 UBE2D1 3247757 0.0429564 1.711 NUP37 3468261 0.0430644 1.800 UBL5 3820161 0.0433763 -1.584 EMILIN2 3776279 0.0439531 -1.642 LOC116349 2845342 0.0447462 -1.675 MGC34796 2339109 0.0454492 -1.804 SLC35A1 2916345 0.0457038 1.552 PTBP2 2348060 0.0460825 1.616 RABGAP1 3188299 0.0466788 1.794 SDCBP 3099750 0.0468848 1.582 SPATA18 2726910 0.0468942 1.550 CALB2 3667508 0.0470496 -1.862 PLSCR1 2699726 0.0470877 1.791 PIK3R1 2813060 0.0472201 1.726 3551120 0.047344 -3.218 MYO1E 3626826 0.0474929 2.257 KIAA1826 3389529 0.0475693 1.637 POPDC3 2967276 0.0476137 1.540 HTR3D 2655308 0.0476616 -1.524 HIST1H4E 2899216 0.0478489 -1.581 MGC27348 2992936 0.048102 -1.619 PAX3 2600881 0.0481901 -1.604 EIF1AX 4002148 0.0485908 1.806 ZNF468 3869847 0.0489033 1.591 LYRM4 2939892 0.0491781 1.585 GTF2B 2421753 0.0492796 1.916 PSMA2 3047963 0.0496745 1.506 MGST1 3406589 0.0497923 1.790 Appendix Table C3

EFMR female / all control (n=3 female and n=3 male) results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Control) T1560 3099561 0.000910698 -1.710 OR10G9 3353876 0.00100223 -1.562 IFI44 2343511 0.00127293 2.102 SACS 3505319 0.00130662 1.618 VDAC1 2875954 0.001613 -1.720 3724917 0.00162084 -1.625 BCL3 3835726 0.0016926 -1.530 LOC142937 3233686 0.00196301 -1.551 XRCC4 2818454 0.00196924 1.516 TCF7L2 3264621 0.00251706 -1.576 CLIC6 3919278 0.00262026 -1.620 LCE1F 2359431 0.00291481 -1.851 C2orf43 2543066 0.00318606 1.549 A4GALT 3962578 0.00350144 -1.528 GPR19 3445028 0.00446837 -2.871 HHLA3 2341645 0.00454418 -1.584 ADRB3 4049835 0.00508465 -1.575 LOC339803 2555252 0.00509234 1.869 FAM111B 3331903 0.00633063 1.621 RGS4 2364381 0.00638692 5.356 PDGFC 2791197 0.00715098 2.064 SH3BP5 2664209 0.00755751 1.605 DPPA3 3403414 0.00777364 -1.531 2845417 0.00845196 -1.550 APOBEC3B 3945545 0.00894806 1.660 NEDD8 2344450 0.00919384 -2.799 NEU2 2532681 0.00968077 -1.696 CPZ 2717857 0.00972393 -1.848 GPR133 3438061 0.0104755 -3.627 PCK1 3890640 0.0106633 -1.529 ZBTB12 2949570 0.0109488 -1.528 RFC3 3485074 0.0111802 1.592 OCIAD2 2768654 0.0113261 1.564 SPRR1A 2359492 0.0113963 -1.624 OR6N2 2439508 0.0115582 -1.825 TRIM2 2748061 0.0116125 1.522 OR2AT4 3382175 0.0121367 -1.654 OR1L8 3224333 0.0124608 -1.799 KCNQ1DN 3317482 0.0124729 -1.840 C20orf91 3881045 0.0124957 -1.760 GZMM 3815005 0.0133313 -1.517 PALLD 2751066 0.0136003 1.729 LOC220594 3748400 0.0141778 1.848 SIM2 3920171 0.014318 -1.557 3959185 0.0143798 -1.519 RPL6 3472089 0.014691 -1.753 GAS7 3744965 0.0150009 -1.602 KRTAP3-1 3756668 0.0153822 -1.803 SOD3 2721633 0.0156568 -1.567 LRRFIP1 2534456 0.0163243 1.723 Appendix Table C3

EFMR female / all control (n=3 female and n=3 male) results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Control) LCE5A 2359282 0.0164104 -1.516 DAPK2 3628832 0.0178112 -1.732 SNAI1 3888522 0.0179721 -2.020 PRKY 4029152 0.0180676 -1.717 HUNK 3917938 0.0181047 -1.734 HMGB2 2793951 0.0182767 1.607 SAMD9 3061438 0.0187522 1.770 NCAM1 3349293 0.020411 2.056 C14orf178 3545550 0.020697 -1.522 AIG1 2928690 0.0208191 1.595 PRKAR2B 3018375 0.0213603 1.518 SPINT3 3907311 0.022635 -2.077 LPCAT2 3661718 0.0227291 1.722 EFNB2 3524570 0.0229814 2.499 EMX2 3266408 0.0234597 -1.743 OR10G7 3395817 0.02358 -1.766 IGJ 2772566 0.0242317 1.540 CX3CR1 2669979 0.0244163 -1.643 ENPP4 2909020 0.024509 -1.691 FAM26D 2922732 0.0246811 -2.221 GLRB 2749191 0.0246914 1.603 RTP4 2657025 0.0247527 -1.677 DEPDC1 2417528 0.0250279 2.011 B4GALT1 3203413 0.0253888 -1.678 OR2M7 2465902 0.0259166 -1.804 CXCL2 2773434 0.02607 -1.571 MBL1P1 3254091 0.0263732 1.513 CENPK 2859667 0.0264495 1.736 ARP11 3078948 0.0268526 -1.524 NETO2 3690154 0.0276919 1.745 FAM18B 3713539 0.0277875 -1.677 CXCL6 2731350 0.029091 -1.673 2766532 0.0295247 -1.515 ARSK 2820865 0.0297346 1.656 C3orf14 2627080 0.0299273 1.649 C3 3848039 0.03037 -1.942 TFRC 2712632 0.0307907 1.557 KIAA0101 3629103 0.0308168 1.550 OR2V2 2844987 0.0314177 -1.714 HGF 3058944 0.0323519 1.934 POMZP3 3057755 0.0324585 -1.546 NUF2 2364438 0.0325146 1.711 DLG7 3565663 0.032821 1.570 HIST1H3J 2947095 0.0330062 1.536 FAM110B 3099566 0.0338126 -1.667 IFITM3 3357840 0.0341074 -1.808 CLEC14A 3561868 0.0341465 1.741 ZFY 4028568 0.0344891 -2.132 CYP2A6 3862873 0.0346733 2.311 ENPP1 2925953 0.0348632 2.949 Appendix Table C3

EFMR female / all control (n=3 female and n=3 male) results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Status) Fold-Change(Affected vs. Control) WFS1 2716953 0.035254 -1.575 CDC6 3720896 0.0355591 1.570 HMMR 2838656 0.0363601 1.571 SELI 2473784 0.0364902 1.574 RAB42 2327572 0.0373365 -1.590 MGC34796 2339109 0.0374659 -1.598 CSH2 3766453 0.0376662 -1.688 PPL 3678462 0.0379456 -1.523 OR4N4 3583382 0.0384048 1.642 IFNA2 3201319 0.0389999 -1.794 A26C1A 2505993 0.0392176 -2.031 TJP2 3173880 0.0399409 1.513 VWA1 2315894 0.0402292 -1.522 LOC286161 3082590 0.0416354 -1.733 MET 3020343 0.0420184 1.795 SLC38A5 4007437 0.0425189 -1.525 IGFBP5 2598828 0.0431064 4.621 C8orf13 3124388 0.0440673 -1.514 DDX3Y 4030162 0.0447391 -4.087 C8orf77 3121002 0.0449023 -1.501 BCGF1 3663393 0.0449897 1.700 WNT2 3069366 0.045124 -1.524 RAB43 3689880 0.0453066 1.556 RPS4Y1 4028512 0.0453398 -8.954 BUB1 2570616 0.045374 1.527 ACCN3 3031800 0.0454387 -1.506 CXCL12 3286602 0.0455067 -1.823 AKR1C2 3274758 0.0464347 -2.718 PFN2 2700585 0.0470573 1.822 HIBCH 2592005 0.0474983 1.583 TTK 2914777 0.0477382 1.524 UQCRFS1 3857691 0.0478125 -1.510 DCBLD1 2922972 0.0479219 1.630 ABCC3 3726691 0.0479551 -1.524 UNQ1829 3498476 0.0486064 1.847 C21orf99 2576788 0.049078 -1.631 LOC4951 3062738 0.0493403 -1.809 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) NLGN4Y 4030371 2.71E-06 3.561 USP9Y 4030063 2.28E-05 3.586 RPS4Y1 4028512 2.59E-05 74.835 EPHA2 2397948 0.000107194 1.711 STAT1 2592268 0.000139913 -2.117 TMEM126B 3342983 0.000210038 -1.805 KLF4 3219215 0.000239021 -4.470 DDX3Y 4030162 0.00033042 18.679 OSR2 3108859 0.000472157 -10.282 TNFAIP8 2825629 0.000558926 -2.018 ZFY 4028568 0.000719864 2.519 CYorf15A 4031068 0.000845549 1.947 ITPR2 3448152 0.000947717 1.543 UTY 4035017 0.000974345 2.284 TMTC1 3449068 0.00102152 -11.796 N6AMT2 3504392 0.00105834 -1.765 RYBP 2682436 0.00110127 -2.232 FABP3 2404418 0.00121643 -3.968 SPC25 2585933 0.00124507 1.587 KRT19 3757108 0.00130048 3.267 PMP22 3746574 0.00141736 -3.416 GPR177 2417390 0.00147625 -3.297 C1R 3442475 0.0016634 -3.018 EIF1AY 4031136 0.00171164 6.377 RRM2 2469252 0.00174792 3.242 ABBA-1 3697183 0.00185594 -1.630 ADAMTSL1 3163982 0.00190831 2.201 CCBL2 2421782 0.00195557 -1.902 ADAMTS5 3927480 0.00205209 -3.134 DCN 3465274 0.00209972 -2.491 CAT 3326400 0.00214991 -1.639 AKR1C3 3233049 0.0022922 -4.534 HDAC9 2991395 0.00238267 1.546 ENPP1 2925953 0.00255388 6.593 RGS4 2364381 0.00267105 3.165 GAB1 2745547 0.0026994 -1.570 LAMB1 3067302 0.00272439 1.795 ZNF313 3888474 0.00272966 -1.607 HIST1H3J 2947095 0.00293117 1.947 CXCR7 2533999 0.00335555 -2.564 RAB9A 3969422 0.00349398 -2.438 EIF2S3 3971877 0.00374606 -1.592 MITD1 2566645 0.00418361 -1.661 LYRM5 3408573 0.00446336 -1.874 C1S 3403168 0.00452818 -2.672 ITGA3 3726154 0.00481506 3.670 EIF3H 3149754 0.00512634 -1.675 DAZAP2 3414846 0.00520945 -1.778 STAT3 3757840 0.00538839 -1.575 EML1 3551485 0.00571306 -1.921 CYB5A 3813297 0.00577089 -2.904 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) CDON 3396770 0.00581547 -5.456 CYP1B1 2548699 0.00582809 -2.748 KRT18 3415576 0.00586147 5.292 HTR3D 2655308 0.0058632 1.513 DOK5 3889833 0.00595001 -4.908 SMAD2 3806905 0.00603265 -1.515 CST6 3335894 0.00603599 2.228 CYBRD1 2515240 0.0061245 -1.637 CDC14B 3216356 0.00613392 -1.672 ADAMTSL1 3164086 0.00623306 2.336 TP53I11 3371003 0.00648618 -2.099 ADAM33 3895552 0.0066063 -1.617 MMP16 3143660 0.00697037 2.022 LMCD1 2609347 0.00710048 -2.348 BOC 2636319 0.00720732 -1.675 TRHDE 3422458 0.00726719 2.253 CCDC56 3758148 0.00743544 -1.577 C15orf41 3588658 0.00755501 1.569 MET 3020343 0.00762705 3.149 TMEM97 3715489 0.00767951 -2.158 CFB 2902844 0.00772908 -2.163 C2orf33 2530539 0.00773054 -2.544 UACA 3631397 0.00780411 2.211 SNX9 2933331 0.00782761 -2.387 ANGPTL2 3225855 0.0082248 -3.327 KIF5A 3418298 0.00837411 -1.509 PYGL 3564210 0.00837877 -1.752 CBX6 3960930 0.00838864 -1.664 HSDL2 3185205 0.0083973 -1.829 C10orf72 3288518 0.00853703 -1.799 RSPO3 2924851 0.00873472 -11.011 KREMEN1 3941793 0.00881676 -1.562 NTF3 3402150 0.00884083 2.167 LNX1 2769346 0.00904578 1.510 FBLN1 3948640 0.00906513 -3.502 KLF13 3587015 0.00914106 -1.589 ZNF436 2401333 0.00914135 -2.533 EFNA5 2869880 0.00917373 -2.489 LOH3CR2A 2609414 0.00930861 -3.351 CAMK2N1 2400177 0.00947539 -2.487 PAPPA 3186491 0.00949155 -2.844 ITGA6 2515627 0.00965705 6.301 RABGAP1 3188299 0.00977795 -1.631 ZNF367 3216319 0.00998584 1.503 HIST1H2AL 2900091 0.0102802 3.249 TMEM135 3343546 0.0104426 -1.652 ZNF185 3995392 0.0105402 1.663 TBC1D16 3773241 0.0108614 -1.994 LNX2 3507003 0.0110553 1.555 SYAP1 3970130 0.0110779 -2.341 FAM118A 3948543 0.0113685 -2.377 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) SLC9A9 2699145 0.0115078 -3.567 GALNT6 3454892 0.0116145 2.391 ZNF234 3835467 0.0116575 -1.634 WTAP 2934089 0.0118674 -1.570 FTH1 3375648 0.0125336 -1.989 MAGOH 2413180 0.0127997 -1.954 MLLT3 3200982 0.0128155 -1.561 C1RL 3442514 0.0128164 -1.790 DST 2958325 0.0128336 1.600 TGFBR3 2422722 0.0130168 -2.612 AK3L1 2340315 0.013112 1.864 NUDT11 4008427 0.0137182 -1.583 FAM118B 3355021 0.0138185 -1.917 GALNT5 2511603 0.0138877 1.741 C7orf41 2995254 0.0139472 -1.609 EWSR1 3941907 0.0139595 -1.634 HOMER2 3636391 0.0140404 1.544 SFRS5 3542207 0.0140796 -1.496 BIRC5 3736290 0.0142518 1.532 CHMP4B 3882681 0.0143034 -1.594 DNAJC8 2403470 0.014483 -1.607 PPP2R3C 3560864 0.0148497 -1.501 TBC1D8 2567447 0.0151679 -1.709 PHF11 3489481 0.0155265 -1.725 UBE2E3 3969604 0.0155444 1.658 SRGN 3250146 0.0158775 4.736 GK 3972929 0.0159581 -1.818 HIST1H4B 2946208 0.0160906 1.633 CTNS 3706700 0.0161136 -1.627 SPG20 3509719 0.0162897 -1.884 EIF1AX 4002148 0.0166536 -2.808 LSM6 2746269 0.0168156 -2.010 SLC7A5P1 3684100 0.0168996 1.679 UNQ1887 3474697 0.0170189 -1.543 MAN1A1 2971801 0.0172126 -2.240 ZFP36L2 2550522 0.0173176 -1.992 SDPR 2592532 0.0175741 1.703 OPCML 3399004 0.017623 1.626 PPFIBP1 3409211 0.0177961 1.512 NXN 3739867 0.0179153 1.732 PMAIP1 3790704 0.0179245 1.559 IQGAP3 2438282 0.0179296 1.562 BRE 2474791 0.0180482 -1.579 ABCA9 3768703 0.0180532 -1.750 CPXM2 3310953 0.0180625 3.698 DDHD1 3564997 0.0181162 -1.660 C18orf17 3781980 0.018546 -2.090 MT4 3662086 0.018803 1.517 ACO1 3166477 0.0188361 -1.592 DOCK5 3090512 0.0188413 1.826 PSG4 3863929 0.0189657 -2.861 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) ZFP36L1 3569754 0.0191059 -1.726 DOCK10 2601648 0.0193497 1.962 FAM44B 2887633 0.0193715 -1.573 APOD 4054204 0.0194137 -8.670 CDC42EP3 2548617 0.0194241 2.245 SIRT2 3861689 0.0197655 -1.791 RAB8B 3597476 0.0199584 -1.606 C1orf21 2371547 0.0204665 -1.676 BNC1 3636562 0.020582 2.130 XG 3966891 0.0207266 -2.323 ABCC9 3446919 0.0208022 -5.370 FEZ1 3396593 0.0211835 -1.573 VGLL3 2684851 0.0212443 -2.558 TM4SF1 2700365 0.0213269 3.129 LRIG1 2680591 0.0215717 -1.685 GEMIN8 4000269 0.0218504 -1.643 ASPA 3706659 0.0219988 -2.034 CXorf15 3970166 0.0220609 -1.721 IFT52 3886179 0.0222284 -1.548 DPT 2443120 0.0223755 -4.895 SUMO2 3770632 0.0224585 -1.683 PPAP2B 2414366 0.0224776 -1.571 GRK5 3267036 0.0227194 -1.631 AHR 2991233 0.0229258 -1.810 MCM5 3944147 0.0230664 1.739 IL1R1 2496962 0.0230722 -3.369 GAS7 3744965 0.0232244 -1.541 OPTN 3235726 0.0232617 -1.621 SEMA3C 3058759 0.0234683 -2.282 PPP1R7 2536183 0.024007 -1.583 CACHD1 2340078 0.0240316 -1.849 AVPI1 3302495 0.0242858 -1.870 YPEL5 2475628 0.0242904 -1.824 FLJ14213 3327057 0.0243402 1.826 EI24 3354731 0.0245249 -1.818 KCNQ5 2913277 0.0250414 3.926 PRELP 4053085 0.0250856 -2.893 MCCC1 2707824 0.0251346 -1.518 GLIPR1 3422855 0.0252565 2.142 TBC1D15 3422326 0.0254838 -1.680 NTAN1 3681674 0.0255026 -1.565 PLEKHM1 3759849 0.0256773 -1.667 AGT 2460296 0.0259411 1.939 BLOC1S1 3416909 0.0259709 2.104 ACADM 2342576 0.0262247 -1.599 FAM72A 4052881 0.0263297 1.578 HOXD4 2516912 0.0263498 1.615 PSME1 3529609 0.0265392 -1.651 LOC554174 3963913 0.0266408 1.773 TPCN1 3432678 0.0267493 -1.657 RPS19 3834465 0.0269202 1.771 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) UBE2F 2534564 0.0270454 -1.902 HRSP12 3145980 0.027105 -3.983 PLOD1 2320581 0.0272262 1.810 CDC20 2333136 0.0274275 4.294 ABCA6 3768791 0.0274346 -2.674 HIST1H1A 2946194 0.0277095 3.274 ATP8B4 3623552 0.027769 -1.577 PPARG 2611056 0.0283399 -1.617 SLC39A8 2779823 0.0284563 -3.793 SLC9A3R2 3644340 0.0286519 2.244 SLC7A1 3507710 0.0287461 2.242 EIF3E 3148582 0.028981 -2.175 HNMT 2507896 0.0292035 -2.039 DLG7 3565663 0.0292996 1.906 SQRDL 3592511 0.0294231 -3.381 CAB39L 3513752 0.0294463 -1.726 SSPN 3408831 0.0294841 1.830 ZNF238 2388794 0.0296829 1.574 KIF18A 3367338 0.0297937 1.840 BAG1 3203482 0.0300668 -1.564 CSPG4 3633578 0.030153 1.898 SUB1 2805581 0.0301699 -1.721 PRDX6 2367743 0.0302885 1.538 NFIL3 3214451 0.0303451 -2.705 PFKFB3 3233605 0.0304714 1.919 IGF1R 3610958 0.0305461 -2.056 VAMP4 2443989 0.0306354 -1.525 FAM92A1 3107151 0.030722 -1.693 KIAA1305 3529951 0.0310432 -2.114 KIAA0196 3152220 0.0312237 -1.953 TWIST1 3040363 0.0315256 -1.567 CA5B 3969855 0.0318259 -1.699 COL8A1 2633390 0.0319735 2.762 3775147 0.0322613 1.617 REV3L 2969677 0.0324732 -2.658 WBP5 3985523 0.0326873 -2.143 NOV 3113202 0.0327697 -4.253 MITF 2628785 0.0328189 -1.579 FAT 2797393 0.0328664 1.725 ZBTB44 3398241 0.0328878 -1.577 DNAJB4 2343289 0.033004 2.449 PTRF 3757917 0.033409 1.518 ITGA1 2809128 0.0336578 2.351 PATL1 3374746 0.0338084 -1.658 CCND1 3338192 0.0339143 1.839 NFKB1 2737717 0.0339399 -1.599 LPCAT2 3661718 0.0340953 1.832 C5orf30 2822492 0.0341347 2.181 STS 3967689 0.0343942 -2.344 SOD2 2982319 0.0344817 -2.398 MASP1 2709631 0.0350725 -2.189 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) SGCE 3061805 0.0351759 -2.189 RAB7L1 2452667 0.0355441 -2.058 CBLN3 3558270 0.0359162 -1.549 PTPN14 2455418 0.0360136 1.550 NCK1 2644155 0.0362506 -1.763 SLC9A7 4006841 0.0362757 1.739 BCAT1 3447694 0.0362813 2.012 GJC1 3759335 0.0365153 2.051 SESTD1 2589929 0.0366015 -1.658 SPATA13 3481543 0.0368828 -1.593 THRB 2666147 0.0369525 -2.037 TPST1 3005444 0.0369588 -2.161 SPAG4 3883441 0.036997 1.517 GNG2 3535628 0.037174 2.555 C22orf9 3963676 0.0372711 -1.981 KCNK2 2379974 0.0374222 -1.703 EGR3 3127584 0.0378761 1.551 ANK2 2740067 0.0379427 -1.955 PRR12 3838624 0.0379832 -1.569 FAM133B 3247818 0.0383563 -2.038 HSD17B11 2777070 0.038573 -2.238 WNK4 3722084 0.0386525 2.069 PSMD10 4017519 0.0387304 -1.681 C15orf15 3625234 0.0389814 -1.572 ADH1A 2779199 0.0393858 -6.068 KRTAP2-4 3756723 0.0394002 4.740 DNM1 3190242 0.0394847 -2.113 KLF5 3493543 0.0395406 1.704 KLHDC2 3534923 0.0397196 -1.533 TLE1 3211579 0.0399181 -1.533 IL7R 2806468 0.0400319 2.872 AKR1C2 3274758 0.0402226 -4.721 GPR133 3438061 0.0402329 -5.255 CHURC1 3540353 0.0406285 -1.545 FLJ34077 3301609 0.0408708 -1.682 DCBLD2 2686023 0.0409146 1.617 SLC43A1 3373893 0.041083 -1.588 NEGR1 2418078 0.0414658 -2.205 TACC3 2714955 0.0416609 1.791 MAP2K1IP1 2779408 0.0420786 -1.763 ATP13A2 2398736 0.0421845 1.654 GSTT1 3955102 0.0423268 -3.925 TRAM1 3139882 0.042525 -1.573 GABRE 4026010 0.0425327 -1.777 SEMA3A 3059464 0.042715 -2.675 ADH1B 2779231 0.042756 -4.511 SERPING1 3331355 0.042756 -2.883 ALDH3A2 3714068 0.0427707 -1.777 SLC12A8 2693014 0.0428927 1.590 PARP9 2692060 0.0429518 -2.317 CXCL12 3286602 0.0431537 -2.264 Appendix Table C4

Control male / control female results P<0.05 and fold change greater that +/- 1.5 Gene Symbol Transcript ID p-value(Sex) Fold-Change(Male vs. Female) CD24 4035833 0.0432393 1.991 LMNB1 2827185 0.0433302 1.547 UBE2D4 2999640 0.0434227 -1.818 C10orf104 3251353 0.0434236 -1.798 APCDD1L 3911485 0.0434479 1.986 SNRPD1 3781082 0.0436992 -1.558 NPAL3 2325410 0.0437863 2.230 EDN1 2895244 0.0439678 1.508 C1orf123 2413153 0.0440587 -1.496 NPBWR2 3914346 0.0442308 1.586 SECTM1 3774906 0.0443955 -2.717 SNX2 2826295 0.0444744 -2.059 HSPA2 3540136 0.0445114 3.065 LOC130576 2509988 0.0445644 1.617 MAP3K8 3240987 0.0447617 -1.909 C1GALT1 2989435 0.0450315 -2.074 SDCBP 3099750 0.04508 -1.985 CCDC109B 2739160 0.0452107 -2.508 COL12A1 2961177 0.0452237 1.596 TBC1D2 3217194 0.0452842 1.804 TRAF3IP2 2969810 0.0457029 -1.877 C2orf12 2583602 0.0457606 -1.775 ARL4C 2604390 0.0463122 -2.440 SAR1A 3293244 0.046431 -1.651 C13orf33 3484117 0.0464358 -2.726 ASPM 2449559 0.0472672 1.532 PTTG1 2838201 0.0473813 2.131 DDX3X 3974838 0.0474239 -1.665 KIAA0746 2764192 0.0482268 2.316 DAPK2 3628832 0.0487678 -1.814 CCDC23 2409069 0.0488538 -1.641 MYO1E 3626826 0.0491481 -2.058 RPS27L 3628469 0.0493446 -1.645 RIMS1 2913123 0.049545 1.503 TMEM98 3717870 0.0495608 -1.574 OXTR 2661992 0.0496543 8.289 PRKY 4029152 0.0498144 1.566 GABPA 3916576 0.0498245 -1.773 Appendix Table C5

Genes identified in EFMR/control female and male control/female control P<0.05 EFMR/Control females Male controls/female controls Gene Symbol P-value Fold change P-value Fold change ABCA6 0.0072 -2.80 0.0274 -2.67 ABCA9 0.0079 -1.51 0.0181 -1.75 ADH1A 0.0165 -5.30 0.0394 -6.07 ADH1B 0.0074 -4.17 0.0428 -4.51 AKR1C2 0.0103 -6.19 0.0402 -4.72 AKR1C3 0.0088 -5.47 0.0023 -4.53 ALDH3A2 0.0184 -1.53 0.0428 -1.78 ANK2 0.0395 -1.51 0.0379 -1.95 APOD 0.0019 -11.38 0.0194 -8.67 ASPA 0.0390 -1.75 0.0220 -2.03 ASPM 0.0187 1.81 0.0473 1.53 BCAT1 0.0101 2.60 0.0363 2.01 BNC1 0.0245 1.74 0.0206 2.13 BOC 0.0286 -1.79 0.0072 -1.68 C10orf104 0.0224 -1.54 0.0434 -1.80 C10orf72 0.0187 -1.71 0.0085 -1.80 C13orf33 0.0215 -2.50 0.0464 -2.73 C15orf41 0.0391 1.70 0.0076 1.57 C18orf17 0.0457 -1.74 0.0185 -2.09 C1R 0.0064 -3.21 0.0017 -3.02 C1RL 0.0045 -1.83 0.0128 -1.79 C1S 0.0274 -2.56 0.0045 -2.67 C2orf33 0.0427 -1.73 0.0077 -2.54 CBLN3 0.0013 -1.76 0.0359 -1.55 CCDC23 0.0179 -1.73 0.0489 -1.64 CCND1 0.0284 1.54 0.0339 1.84 CDC20 0.0374 3.28 0.0274 4.29 CDC42EP3 0.0073 1.74 0.0194 2.25 CDON 0.0035 -5.46 0.0058 -5.46 CFB 0.0001 -2.08 0.0077 -2.16 COL12A1 0.0117 1.73 0.0452 1.60 CPXM2 0.0150 3.40 0.0181 3.70 CSPG4 0.0167 2.42 0.0302 1.90 CXCL12 0.0189 -3.08 0.0432 -2.26 CXCR7 0.0020 -2.49 0.0034 -2.56 CYB5A 0.0243 -2.19 0.0061 -1.64 DAPK2 0.0032 -2.52 0.0488 -1.81 DAZAP2 0.0150 -1.73 0.0052 -1.78 DLG7 0.0003 2.35 0.0293 1.91 DNM1 0.0397 -2.33 0.0395 -2.11 DOCK10 0.0286 2.47 0.0193 1.96 EFNA5 0.0002 -2.91 0.0092 -2.49 ENPP1 0.0008 9.90 0.0026 6.59 EPHA2 0.0353 1.50 0.0001 1.71 FBLN1 0.0365 -3.27 0.0091 -3.50 FEZ1 0.0146 -1.58 0.0212 -1.57 FLJ14213 0.0333 1.89 0.0243 1.83 GALNT6 0.0020 1.99 0.0116 2.39 GAS7 0.0004 -1.95 0.0232 -1.54 GJC1 0.0240 2.22 0.0365 2.05 Appendix Table C5

Genes identified in EFMR/control female and male control/female control P<0.05 EFMR/Control females Male controls/female controls Gene Symbol P-value Fold change P-value Fold change GLIPR1 0.0148 1.96 0.0253 2.14 GPR133 0.0002 -12.12 0.0402 -5.26 GRK5 0.0105 -1.83 0.0227 -1.63 HDAC9 0.0012 1.79 0.0024 1.55 HIST1H3J 0.0319 1.90 0.0029 1.95 HNMT 0.0118 -1.92 0.0292 -2.04 HRSP12 0.0019 -4.17 0.0271 -3.98 IL7R 0.0472 2.78 0.0400 2.87 IQGAP3 0.0426 1.55 0.0179 1.56 ITGA1 0.0262 2.51 0.0337 2.35 ITGA3 0.0042 4.15 0.0048 3.67 ITGA6 0.0030 6.75 0.0097 6.30 ITPR2 0.0406 1.64 0.0009 1.54 KIAA1305 0.0226 -1.82 0.0310 -2.11 KIF18A 0.0102 1.69 0.0298 1.84 KLF4 0.0048 -3.92 0.0002 -4.47 KREMEN1 0.0007 -1.64 0.0088 -1.56 LAMB1 0.0009 2.19 0.0027 1.80 LMCD1 0.0013 -2.21 0.0071 -2.35 LMNB1 0.0076 1.77 0.0433 1.55 LNX2 0.0190 1.50 0.0111 1.56 LOH3CR2A 0.0030 -2.75 0.0093 -3.35 LPCAT2 0.0040 2.57 0.0341 1.83 MAP3K8 0.0031 -2.32 0.0448 -1.91 MASP1 0.0478 -3.36 0.0351 -2.19 MCM5 0.0082 1.75 0.0231 1.74 MET 0.0003 3.90 0.0076 3.15 MITF 0.0138 -1.54 0.0328 -1.58 MMP16 0.0078 1.96 0.0070 2.02 NFIL3 0.0336 -2.53 0.0303 -2.71 NFKBIA 0.0484 -2.27 0.0339 -1.60 NPAL3 0.0043 2.24 0.0438 2.23 NTF3 0.0014 1.92 0.0088 2.17 NXN 0.0020 1.89 0.0179 1.73 OSR2 0.0417 -4.14 0.0005 -10.28 OXTR 0.0058 7.50 0.0497 8.29 PAPPA 0.0387 -2.31 0.0095 -2.84 PMP22 0.0469 -2.99 0.0014 -3.42 PPARG 0.0110 -1.68 0.0225 -1.57 PPFIBP1 0.0093 1.88 0.0178 1.51 PRDX6 0.0250 1.74 0.0303 1.54 PTTG1 0.0462 2.08 0.0474 2.13 RAB9A 0.0353 -1.80 0.0035 -2.44 RACGAP1 0.0101 1.97 0.0098 -1.63 RGS4 0.0121 13.97 0.0027 3.16 RRM2 0.0097 3.71 0.0017 3.24 SECTM1 0.0030 -2.79 0.0444 -2.72 SERPING1 0.0121 -4.05 0.0428 -2.88 SLC7A1 0.0058 1.85 0.0287 2.24 SLC9A3R2 0.0466 1.65 0.0287 2.24 Appendix Table C5

Genes identified in EFMR/control female and male control/female control P<0.05 EFMR/Control females Male controls/female controls Gene Symbol P-value Fold change P-value Fold change SOD2 0.0063 -2.18 0.0345 -2.40 SPATA13 0.0257 -1.60 0.0369 -1.59 STAT3 0.0110 -1.69 0.0054 -1.58 TACC3 0.0036 1.69 0.0417 1.79 TBC1D16 0.0124 -1.84 0.0109 -1.99 TGFBR3 0.0307 -2.28 0.0130 -2.61 TMTC1 0.0007 -11.62 0.0010 -11.80 TNFAIP8 0.0003 -1.97 0.0006 -2.02 TP53I11 0.0092 -2.20 0.0065 -2.10 TWIST1 0.0309 -1.72 0.0315 -1.57 UACA 0.0232 2.89 0.0078 2.21 ZFP36L1 0.0352 -2.10 0.0191 -1.73 ZFP36L2 0.0205 -2.00 0.0173 -1.99 ZNF436 0.0176 -2.17 0.0091 -2.53