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Inside out Behavioral phenotyping in genetic syndromes Mulder, P.A.

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Citation for published version (APA): Mulder, P. A. (2020). Inside out: Behavioral phenotyping in genetic syndromes.

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Download date:25 Sep 2021

6 Further delineation of Malan syndrome

Authors: Priolo M, Schanze D, Tatton-Brown K, Mulder PA, Tenorio J, Kooblall K, Acero IH, Alkuraya FS, Arias P, Bernardini L, Bijlsma EK, Cole T, Coubes C, Dapia I, Davies S, Di Donato N, Elcioglu NH, Fahrner JA, Foster A, González NG, Huber I, Iascone M, Kaiser AS, Kamath A, Liebelt J, Lynch SA, Maas SM, Mammì C, Mathijssen IB, McKee S, Menke LA, Mirzaa GM, Montgomery T, Neubauer D, Neumann TE, Pintomalli L, Pisanti MA, Plomp AS, Price S, Salter C, Santos-Simarro F, Sarda P, Segovia M, Shaw-Smith C, Smithson S, Suri M, Valdez RM, Van Haeringen A, Van Hagen JM, Zollino M, Lapunzina P, Thakker RV, Zenker M, Hennekam RC.

Human Mutation 2018; 7: 1-12 6 — 142 but rarely there is some overlap. Differentiation from Sotos and Sotos from Differentiation overlap. Weaver syndrome can bemadeby clinical evaluation only. some is there rarely but that usually can bediscernedeasily fromMarshall–Smith syndrome We concludethatMalansyndromehasawell recognizablephenotype site ofstopcodonformation. and Marshall-Smith syndrome can be discerned by differences in the in one family inwhich mother was mosaic. Variants causing Malan epilepsy with19p13.2microdeletions. Variants arosedenovo, except is nogenotype-phenotypecorrelationexcept foranincreasedrisk deletions orpointmutationsofNFIX behaviourally anxietyischaracteristic. Malansyndromeiscaused by lip, and prominent chin.Intellectual disability isuniversally present, triangular face, macrocephaly, prominent forehead, everted lower only half of affected adults. Cardinal facial characteristics include long, Results indicate that height is and collaboration aninternational compared datatothe35previously reportedindividuals. amolecularly through with diagnosis individuals affected confirmed 45 on data gathered We large groupofaffectedindividuals. number ofindividuals. We aimtodelineate the entity by studying a Malan syndrome is an overgrowth disorder described in a limited ABSTRACT > 2 SDS in infancy and childhood but in clustered mostly inexon 2. There 6 — 143 variants variants located at located Overgrowth Overgrowth NFIX [1]

[2],[21] The syndrome is The [22],[23] has been proposed been has characterized by a by characterized [2],[7],[10] NFIX [14],[21],[22] the others unpublished), provide detailed provide unpublished), the others [24] Haploinsufficiency of [3-20] Since the first description the entity has been described described been has entity the description first the Since [2] dysostosis, postnatal failure to thrive, short stature, unusual face, respiratory respiratory face, unusual short stature, to thrive, failure postnatal dysostosis, to severe developmental delay. and moderate compromise, Malan syndrome (MIM# 614753; also called as Sotos syndrome 2) is an 614753; also called Malan syndrome (MIM# phenotype, an unusual facial overgrowth, by characterized disorder, overgrowth heterozygous by problems. It is caused and behavioral disability, intellectual (NFIX;MIM# 164005), X I factor nuclear gene the of deletions or variants syndromes form a group of genetically determined disorders in which at least in which at least determined disorders form a group of genetically syndromes mean, the deviations (SDS) above greater than two standard height is equal to or and the growth be increased, may head circumference but also weight and not be or may may The overgrowth present from birth on. pattern is typically or dysplasias. associated with malformations chromosome 19p13.2. allelic to Marshall-Smith syndrome (MIM# 602535), to Marshall-Smith allelic Patients M.Z.) or molecular (M.P., M.P) (R.C.H., were referred for clinical Patients diagnosis, management advices (R.C.H.), or specifically for this study.Patients exome using targeted their referring physicians recognized by were typically three patients, disability. In for intellectual sequencing aimed at detecting causes a clinical diagnosis of Malan syndrome was molecularly confirmedby targeted and used to gather clinical was questionnaire Sanger sequencing. A dedicated The on every patient. were available photographs and clinical molecular data, METHODS Here, we report on 45 individuals with Malan syndrome (14 being mentioned we report on 45 individuals Here, data, in a table without clinical INTRODUCTION Overgrowth has been defined as “global or regional excess growth compared group”. peer age-related the part or body equivalent an to either in 35 individuals, to date. to individuals, 35 in clinical descriptions and their genotypes, review earlier reported patients, and review earlier reported patients, descriptions and their genotypes, clinical syndrome and to Sotos syndrome and compare data to those of Marshall–Smith in correlations genotype-phenotype refine We 277590). (MIM# syndrome Weaver NFIX variants. individuals with disease-causing as leading causative mechanism in Malan syndrome. mechanism in Malan as leading causative in Marshall-Smith syndrome were predicted to lead to abnormal proteins with proteins abnormal to lead to predicted were syndrome Marshall-Smith in binding and dimerization domain an abnormal C-terminus while their DNA (NMD), nonsense-mediated mRNA decay escape preserved. Some variants was effect of mutant NFIX proteins. suggesting a dominant-negative 6 — 144 90 andalignmentofparaloges andorthologes tohumanNFIX. protein isoforms encoded by transcripts reported in Ensembl Genome Browser NFIX human of Omega Clustal by alignment silico in performed We syndrome. tions located in exons 6, 7, and 8 and in variants associated with Marshall-Smith (v1.2.4; https://www.ebi.ac.uk/Tools/msa/clustalo/)forshortinsertions/dele ExPASy translation andalignmentwithwild-typeproteinby ClustalOmega neet alwn dsrmnto bten different between discrimination allowing interest, Specific Fisher). oligonucleotide primercombinationswereusedtoamplifycDNAfragments of (Thermo System using Synthesis was generated First-Strand SuperScriptTM cDNA the and cultures fibroblast skin from extracted was RNA Total array. SNP or sufficiently CGH array thought by identified were were was Microdeletions reliable. results sequencing exome bySanger unless patients, most results in performed exome of Confirmation protocols. local Sanger sequencingandexome sequencingwereperformedaccording to Molecular studies case ofinconsistenciesinscoringthesewerediscussedtill consensus. facial characteristics werescoredindependently by twoauthors (M.P., R.C.H.).In Hospital Bianchi-Melacrino-Morelli inReggioCalabria(N°200approval). study was approved by themedical ethicscommittee ofGreatMetropolitan analyses andpublication ofclinical andgenotypedatafacialphotographs. All participantsortheirlegalguardians gave writteninformedconsentformolecular Ethics phenotypes were accepted unless photographic evidence of differences was clear. scored independently by twoauthors (M.P., R.C.H.).Available descriptions of confounding genes. Facial phenotypes of patients depicted in publications were centromerically for furtherreferences. We excluded reports describingdeletionsextending Portuguese. Referencelistsofthusobtainedmanuscriptswere handsearched publication inalanguageotherthanEnglish, French, Dutch, Italian, Spanish, or OR “deletion19p13.2"“microdeletion19p13.2".Exclusion criterion was overgrowth” “NFIX OR II” type “Sotos OR 2" type “Sotos OR overgrowth” “Malan PubMed was searched forpublications using asterms“Malan syndrome” OR Literature search all variants gs.washington.edu/) scoreswereusedastoolforscoringdeleteriousnessof Combined AnnotationDependentDepletion(CADD;version 1.3;https://cadd. In silicoanalysisofmutantNFIXproteins by Sanger sequencing. (Supporting InformationFigure1).Analysis ofthesefragments was performed [25] . We performed in silico analysis of predicted mutant cDNAsafter > 1 MbfromNFIX because ofthelarge numberofpotentially NFIX transcript isoforms The 6 — 145 Sotos [22] variants. A summary of main main of summary A variants. NFIX were not different from the phenotype of phenotype the from different not were , is provided in Table 1, and illustrated in and illustrated 1, Table in is provided , [26] domains. Additional noteworthy data include noteworthy domains. Additional and Weaver syndrome Weaver and [24] Phenotype 1; facial features of Malan syndrome (Figure with typical patients presented All no patient had facial features typical Figure 7). Notably, Supporting Information The phenotypes of the four patients (patients syndromes. Weaver for Sotos or in the 3'end of NFIX 39–42) with variants patients with variants in other NFIX patients with variants two patients (patient 1 and 21) conceived via Artificial Reproduction Technique; 41, 40, 37, 35, 29, 26, 24, 23, 21, 15, 7, 6, nerves in patients optic underdeveloped arched 23); highly 7 and (patients and 42; periventricular nodular heterotopias hair (patients and 44); sparse 37, 33, 29, (patients 5, palate and dental crowding soft skin of their soles (patients 25 and 39); and facial and 38); loose and 25, 24, observed in individual patients were an asymmetry (patients 2 and 6). Problems deformity and cox bone morphology (patient 3); right coxa valga abnormal pelvic earlobes (patient prolapse (patient 5); absent valve and mitral cataract, arthritis, (patient 13); camptodactyly 6); mild restriction of elbow and kneemovements 19); marked (patient adulthood of his hands in early (patient 14); contractures plagiocephaly 21); (patient gliosis periventricular 20); (patient brachycephaly atrial septal vision, poor peripheral of the hamstrings, (patient 22); contractures but normally and increased susceptibility for infections (patient 23); large defect, hemangioma and cavernous 24); a supraorbital (patient functioning kidneys choroid fissure cyst (patient 26); curved tibiae (patient 28); central obstructive apnea, a flaccid larynx, talipes and cortical pineal gland, microcystic optic chiasm, a small hemangiomas, and bilateral hydroceles (patient(patient 37); headaches migraine vision impairment (patient 35); hemiplegic three 34); to bilateral due hydronephosis IV (patient 38); grade hypotension postural and brittle toenails (patient 39); an inguinal hernia, stenosis, proximal ureteral and stenosis, pulmonary valve intestinal malrotation, bladder agenesis, gall severe dilatation of the pulmonary trunk (patient 42); and a of sarcoma the fifth (patient 43). rib at age 9 years RESULTS We collected 45 patients with molecularly confirmed Malan syndrome. Three in 42 had variants 19p13.2 and microdeletions involving sized had variably involving patients with microdeletions yielded 21 searches NFIX. Literature intragenic with patients 14 and 19p13.2 clinical characteristics compared to those in Marshall-Smith syndrome, in Marshall-Smith compared to those characteristics clinical syndrome Figure 6.1 and Supporting Information Figure 6.7. Detailed individual patient Information Figure 6.7. Detailed Figure 6.1 and Supporting 6.1 Tables Supporting Materials (Supporting Information in data are provided We discuss 6.2. Table are summarized in Figure 6.2 and and 6.2). Molecular data in the tables. are not available that those results here only 6 — 146

Table 6.1 Comparison of Characteristics of Malan Syndrome Caused by NFIX Mutations and by Deletions of the Complete Gene, to those of Marshall-Smith Syndrome, Sotos Syndrome and Weaver Syndrome.

Malan syndrome Marshall-Smith Sotos Weaver

syndrome syndrome syndrome

NFIX NFIX All

mutations deletions

Growth Prenatal: Weight at birth<3rd centile 0 0 0 11/55 - -

a Prenatal: Weight at birth >98th centile 5/52 6/23 11/75 8/55 +++ ++ Postnatal: Height<3rd centile 0 0 0 38/39 - - Postnatal: Height>98th centile 32/54 13/24 45/78 0/18 +++ +++

Development Intellectual disability 55/55 24/24 79/79 39/39 +++b +++d

c Autism (A)/anxieties (F) A 18/51;F A 6/23;F A 24/74;F Na ? (F) +

33/53 6/19 39/72

Neurology Hypotonia (Ho)/hypertonia (He) Ho 35/50; Ho 21/24; Ho 56/74 Ho12/28 He4/28 Ho ++ Ho ++, He ++

He n.a He n.a Seizures/EEG anomalies 9/52 11/24 20/76 4/38 ++ - Brain anomalies: 19/42 11/20 30/62 12/39 WV++, C-; + BA-; CM- WV/C/BA/CM 12WV; 9C; 4WV; 6C; 2BA; 16WV; 15C; C 8; SC 1; 3CM 3CM 2BA; 6CM WV Craniofacial Macrocephaly 43/53 16/24 59/77 0/39 +++ ++ morphology Long/triangular face 47/55 20/24 67/79 0/57 +++ -

Prominent forehead 53/55 24/24 77/79 53/54 +++ +++

Proptosis 0 1/23 1/78 55/56 - - Underdeveloped midface 0 0 0 38/42 - - Short nose 24/53 13/21 37/74 43/50 - +++

Anteverted nares 28/53 15/22 43/75 44/53 - +++

Everted lips 39/52 14/21 53/73 13/19 + -

Small chin 1/55 1/24 2/79 55/57 - +++ Eyes Vision impaired 42/54 18/24 60/78 9/18 + + Blue sclerae 16/50 4/14 20/64 34/41 - -

Respiratory tract Airway obstructions 1/55 0 1/78 45/55 - -

Musculo-skeletal Abnormal bone maturation 28/37 11/12 39/49 57/57 +++ +++

anomalies Slender habitus 33/51 11/23 44/74 0/57 ++ - Kyphoscoliois 17/55 5/16 22/71 17/37 + +

Pectus excavatum/carinatum 18/52 11/17 29/69 3/37 + +

Other Hypertrichosis 0 0 0 29/35 - -

Gum hypertrophy 1/55 1/24 2/79 7/17 - - Cadiac defects 3/55 1/24 4/79 6/35 + - Umbilical hernia n.a n.a n.a 10/32 + ++ a weight to length normal as typically length >P98; b typically mild (IQ 50-69) or borderline (IQ 70-75); c typically mild (IQ 50-69) or moderate (IQ 35-50); d no unbiased series of molecularly confirmed patients available; +++ 75-100%; ++ 25-75%; + 5-25%; - 0-5% WV, wide ventricles; C, Corpus callosum underdevelopment; BA, brain atrophy; CM, chiari malformation.

6 — 147

underdevelopment; BA, brain atrophy; CM, chiari malformation. chiari CM, atrophy; brain BA, underdevelopment; callosum Corpus C, ventricles; wide WV,

onfirmed patients available; +++ 75 +++ available; patients onfirmed c molecularly of series unbiased no 5% - 0 - 25%; - 5 + 75%; - 25 ++ 100%; -

50); - 35 (IQ moderate or 69) - 50 (IQ mild typically

75); - 70 (IQ borderline or 69) - 50 (IQ mild typically

weight to length normal as typically length >P98; >P98; length typically as normal length to weight

hernia Umbilical n.a n.a n.a ++ + 10/32

defects Cadiac 4/79 1/24 3/55 - + 6/35

hypertrophy Gum 2/79 1/24 1/55 - - 7/17

Other

Hypertrichosis 0 0 0 - - 29/35

Pectus excavatum/carinatum Pectus 18/52 11/17 29/69 + 3/37 +

hoscoliois Kyp 17/55 5/16 22/71 + 17/37 +

Slender habitus Slender 33/51 11/23 44/74 ++ 0/57 -

anomalies

skeletal skeletal - Musculo Abnormal bone maturation bone Abnormal 28/37 11/12 39/49 +++ 57/57 +++

Respiratory tract Respiratory

Airway obstructions Airway

0 1/55 1/78 45/55 - -

sclerae Blue 16/50 4/14 20/64 34/41 - -

Eyes

Vision impaired Vision

42/54 4 18/2 60/78 9/18 + +

Small chin Small

1/55

1/24

2/79

55/57

- +++

Everted lips Everted

39/52

14/21

53/73

13/19

+ -

Anteverted nares Anteverted

28/53

15/22

43/75

44/53

+++

nose Short 24/53

13/21

37/74

43/50

+++

Underdeveloped midface Underdeveloped

0 0 0 38/42

- -

Proptosis

0 1/23

1/78

/56 55

- -

Prominent forehead Prominent

53/55 24/24 77/79 53/54 +++ +++

Long/triangular face Long/triangular

47/55 20/24 67/79 0/57 +++ -

morphology

Craniofacial Craniofacial Macrocephaly 43/53 16/24 59/77 +++ 0/39 ++

3CM 3CM WV 6CM 2BA;

12WV; 9C; 9C; 12WV; WV/C/BA/CM 4WV; 6C; 2BA; 2BA; 6C; 4WV; ; 15C; 15C; ; 16WV 1; SC 8; C

- CM ; - BA

Brain anomalies: anomalies: Brain 19/42 11/20 30/62 ; - C WV++, 12/39 +

Seizures/EEG anomalies Seizures/EEG

11/24 9/52 20/76 ++ 4/38 -

n.a He n.a He

Neurology

Hypotonia (Ho)/hypertonia (He) (Ho)/hypertonia Hypotonia 56/74 Ho 21/24; Ho 35/50; Ho ++ He , ++ Ho ++ Ho He4/28 Ho12/28

39/72 6/19 33/53

(F) (A)/anxieties Autism A 24/74;F 24/74;F A 6/23;F A 18/51;F A + (F) ? Na

Development

disability Intellectual 79/79 24/24 55/55 +++ +++ 39/39

b d

Postnatal: Height>98th centile Height>98th Postnatal: 45/78 13/24 32/54 +++ +++ 0/18

centile Height<3rd Postnatal: 0 0 0 - - 38/39

Prenatal: Weight at birth >98th centile >98th birth at Weight Prenatal: 11/75 6/23 5/52 ++ +++ 8/55

Growth

Prenatal: Weight at birth<3rd centile birth<3rd at Weight Prenatal: 0 0 0 - - 11/55

deletions mutations

All NFIX NFIX

syndrome syndrome syndrome

Smith Smith - Marshall syndrome Malan Weaver Weaver Sotos

6 — 148

Table 6.2 Spectrum of NFIX mutations in 42 presently reported patients and those reported in literature Patient / cDNA Protein Location Type of mutation Inheritance CADD Reference PHRED Scoreb P1 c.[28-1G>A;28-12T>A;28-13T>A] p.Asp10Profs*5 Intron 1 Splicing De novo 25 P2 c.43_49dup p.(Glu17Valfs*31) Exon 2 Frameshift De novo 31 P3 c.59T>C p.(Leu20Pro) Exon 2 Missense ND 26 Klaassens 2015 c.90_99del p.(Trp30Cysfs*24) Exon 2 Frameshift De novo 32 P4 c.92T>C p.(Phe31Ser) Exon 2 Missense De novo 26 Martinez 2015 c.112C>T p.(Arg38Cys) Exon 2 Missense De novo 34 1published as c.136C>T 1published as p.Arg46Cys P5 c.112C>T p.(Arg38Cys) Exon 2 Missense De novo 34 P6 c.113G>T p.(Arg38Leu) Exon 2 Missense ND 32 P7 c.142del p.(Met48Cysfs*9) Exon 2 Frameshift De novo 28 P8 c.154_155insT p.(Glu52Valfs*67) Exon 2 Frameshift De novo 33 Priolo 2012 c.157_177del p.(Glu53_Glu59del) Exon 2 In frame deletion De novo 16 P9 c.157_177del p.(Glu53_Glu59del) Exon 2 In frame deletion De novo 16 Martinez 2015 c.161G>C p.(Arg54Pro) Exon 2 Missense De novo 31 1published as c.185G>C 1published as p.Arg62Pro Yoneda 2012 c.179T>C p.(Leu60Pro) Exon 2 Missense De novo 26 P10 c.182T>C p.(Leu61Pro) Exon 2 Missense De novo 26 P11 c.187G>T p.(Glu63*) Exon 2 Nonsense De novo 39 Gurrieri 2015 c.191del p.(Lys64Serfs*30) Exon 2 Frameshift De novo 27 P12 c.200_201dup p.(Lys68Serfs*27) Exon 2 Frameshift De novo 31 P13 c.248T>G p.(Ile83Ser) Exon 2 Missense De novo 28 Oshima 2017 c.269dup p.(Asp90Glufs*29) Exon 2 Frameshift De novo 31 1published as c.290_291insA 1published as p.Asp98GlyfsX29 P14 c.280A>C p.(Thr94Pro) Exon 2 Missense ND 25 P15 c.294del p.(Phe99Serfs*3) Exon 2 Frameshift De Novo 28 P16 c.315_316insGGT p.(Leu105_Ser106insGly) Exon 2 In frame insertion De novo 16 P17 c.317C>T p.(Ser106Phe) Exon 2 Missense ND 28 P18 c.322_323delinsA p.(Pro108Thrfs*27) Exon 2 Frameshift De novo 33 P19 c.325G>T p.(Asp109Tyr) Exon 2 Missense De novo 29 P20 c.328C>T p.(Gln110*) Exon 2 Nonsense De novo 37 P21 c.337°>G p.(Lys113Glu) Exon2 Missense De novo 26 P22 c.343C>T p.(Arg115Trp) Exon 2 Missense De novo 34 Jezela-Stanek 2016 c.343C>Ta p.(Arg115Trp)a Exon 2 Missense De novo 34 published as c.367C>T published as p.Arg123Trp P23 c.346C>G p.(Arg116Gly) Exon 2 Missense De novo 27 P24 c.347G>A p.(Arg116Gln) Exon 2 Missense ND 30 P25 c.347G>A p.(Arg116Gln) Exon 2 Missense De novo 30 Gurrieri 2015 c.347G>C p.(Arg116Pro) Exon 2 Missense De novo 29 P26 c.358delC p.(Leu120Cysfs*15) Exon 2 Frameshift De novo 33 Yoneda 2012 c.362G>C p.(Arg121Pro) Exon 2 Missense Maternal 30 Gurrieri 2015 c.373A>G p.(Lys125Glu) Exon 2 Missense De novo 26 P27 c.383G>A p.(Arg128Gln) Exon 2 Missense ND 32 P28 c.408delinsTT p.(Lys136Phefs*3) Exon 2 Frameshift ND 31 P29 c.412_413delinsG p.(Lys138Glyfs*73) Exon 2 Frameshift De novo 28 P30 c.444G>C p.(Glu148Asp) Exon 2 Missense De novo 25 P31 c.463C>T p.(Gln155*) Exon 2 Nonsense De novo 37 p32 c.499C>A p.(His167Asn) Exon 2 Missense De novo 27 p33 c.520G>T p.(Glu174*) Exon 2 Nonsense De novo 38 p34 c.542dup p.(Tyr181*) Exon 2 Frameshift De novo 34 Malan 2010 c.568C>T p.(Gln190*) Exon 3 Nonsense De novo 40 p35 c.694C>T p.(Gln232*) Exon 4 Nonsense Maternal mosaic 40 p36 c.694C>T p.(Gln232*) Exon 4 Nonsense De novo 40 p37 c.759C>G p.(Tyr253*) Exon 5 Nonsense De novo 37 p38 c.779C>G p.(Thr260Ser) Exon 5 Missense De novo 13 p39 c.859dup p.(Glu287Glyfs*5) Exon 6 Frameshift De novo 35 Klaassens 2015 c.1012C>T p.(Gln338*) Exon 7 Nonsense De novo 42 P40 c.1055_1064del p.(Pro352Leufs*46) Exon 7 Frameshift De novo 36 P41 c.1116del p.(Ser373Profs*28) Exon 8 Frameshift De novo 34 P42 c.1117del p.(Ser373Profs*28) Exon 8 Frameshift De novo 35 All the variants refer to main transcript and major isoform of NFIX gene) (NM_002501.3) (Q14938). Nomenclature was checked using Mutalyzer website: https://www.mutalyzer.nl/ ; ND, not determined (due to unavailability of material from both parents). aMutations previously published with reference to another isoform (NM_001271043.2) that encodes a protein with 8 additional amino acids at the N-terminus. bCADD v1.3 PHRED-like (-10*log10(rank/total)) scaled C-score: ranking a variant relative to all possible substitutions of the human genome (8.6x10^9). A scaled C-score of greater of equal 10 indicates that these are predicted to be the 10% most deleterious substitutions that you can do to the human genome, a score of greater or equal 20 indicates the 1% most deleterious.

6 — 149

human genome, a score of greater or equal 20 indicates the 1% most deleterious most 1% the indicates 20 equal or greater of score a genome, human the to do can you that substitutions .

score of greater of equal 10 indicates that these are predicted to be the 10% most deleterious deleterious most 10% the be to predicted are these that indicates 10 equal of greater of score - C scaled A (8.6x10^9). genome

score: ranking a variant relative to all possible substitution possible all to relative variant a ranking score: - C scaled total)) 10*log10(rank/ - ( like - PHRED v1.3 CADD s of the human human the of s

terminus. - N the at acids

ously published with reference to another isoform (NM_ isoform another to reference with published ously previ Mutations 001271043.2) that encodes a protein with 8 additional amino amino additional 8 with protein a encodes that 001271043.2)

ermined (due to unavailability of material from both parents) both from material of unavailability to (due ermined det not ND, ; https://www.mutalyzer.nl/ website: Mutalyzer .

All the variants refer to main transcript and major i major and transcript main to refer variants the All Nomenclature was checked using using checked was Nomenclature (Q14938). (NM_002501.3) gene) NFIX of soform

P42 novo De Frameshift 8 Exon p.(Ser373Profs*28) c.1117del 35

P41

novo De Frameshift 8 Exon p.(Ser373Profs*28) c.1116del 34

P40 novo De Frameshift 7 Exon p.(Pro352Leufs*46) c.1055_1064del 36

c.1012C>T 2015 Klaassens 7 Exon Gln338*) p.( 42 novo De Nonsense

p39 6

59dup c.8 Exon Exon p.(Glu287Glyfs*5) 35 novo De Frameshift

p38 c.779C>G 5 Exon p.(Thr260Ser) 13 novo De Missense

p37

c.759C>G 5 Exon p.(Tyr253*) 37 novo De Nonsense

p36 c.694C>T 4 Exon p.(Gln232*) 40 novo De Nonsense

p35

c.694C>T 4 Exon p.(Gln232*) 40 mosaic Maternal Nonsense

c.568C>T 2010 Malan 3 xon E Gln190*) p.( 40 novo De Nonsense

p34 c.542dup 2 Exon p.(Tyr181*) 34 novo De Frameshift

p33

c.520G>T 2 Exon p.(Glu174*) 38 novo De Nonsense

p32 c.499C>A 2 Exon p.(His167Asn) 27 novo De Missense

P31 c.463C>T 2 Exon p.(Gln155*) 37 novo De Nonsense

P30

c.444G>C 2 Exon p.(Glu148Asp) 25 vo no De Missense

P29

c.412_413delinsG 2 Exon p.(Lys138Glyfs*73) 28 novo De Frameshift

P28

c.408delinsTT 2 Exon p.(Lys136Phefs*3) 31 ND Frameshift

P27 c.383G>A p.(Arg128Gln) Missense 2 Exon ND 32

Gurrieri 2015 Gurrieri c.373A>G Lys125Glu) p.( Missense 2 xon E novo De 26

Yoneda 2012 Yoneda c.362G>C Arg121Pro) p.( Missense 2 Exon Maternal 30

P26

c.358delC p.(Leu120Cysfs*15) Frameshift 2 Exon novo De 33

eri 2015 eri Gurri c.347G>C Arg116Pro) p.( Missense 2 Exon novo De 29

P25

c.347G>A p.(Arg116Gln) Missense 2 on Ex novo De 30

P24

c.347G>A p.(Arg116Gln) Missense 2 Exon ND 30

P23 c.346C>G

p.(Arg116Gly)

Exon 2 Exon Missense

De novo De 27

published as c.367C>T as published

published as p.Arg123Trp as published

2016 Stanek - Jezela c.343C>T Arg115Trp) p.( Exon 2 Exon Missense

De novo De 34

a a

P22 c.343C>T

p.(Arg115Trp)

2 Exon Missense De novo De 4 3

P21

c.337°>G

p.(Lys113Glu)

Exon2

Missense

De novo De

P20

c.328C>T

p.(Gln110*)

Exon 2 Exon

Nonsense De novo De

P19

c.325G>T

p.(Asp109Tyr)

Exon 2 Exon

Missense

De novo De

P18

c.322_323delinsA

08Thrfs*27) p.(Pro1

Exon 2 Exon

Frameshift

D novo e 33

P17

c.317C>T

p.(Ser106Phe)

Exon 2 Exon

Missense

ND 28

P16

c.315_316insGGT

p.(Leu105_Ser106insGly)

Exon 2 Exon In frame insertion frame In De novo De

P15 c.294del

p.(Phe99Serfs*3)

Exon 2 Exon Frameshift

De Novo De 28

P14

c.280A>C

p.(Thr94Pro)

Exon 2 Exon Missense ND 25

published as c.290_291insA as published

p.Asp98GlyfsX29 as published

1 1

Oshima 2017 Oshima

c.269dup

p.(Asp90Glufs*29)

Exon 2 Exon Frameshift

De novo De 31

P13

c.248T>G p.(Ile83Ser) Missense 2 Exon novo De 28

P12

c.200_201dup

p.(Lys68Serfs*27) Frameshift 2 Exon novo De 31

Gurrieri 2015 Gurrieri c.191del Lys64Serfs*30) p.( Frameshift 2 Exon novo De 27

P11

c.187G>T p.(Glu63*) Nonsense 2 Exon novo De 39

P10 c.182T>C p.(Leu61Pro) Missense 2 Exon novo De 26

Yoneda 2012 Yoneda T>C c.179 Leu60Pro) p.( Missense 2 Exon novo De 26

published as c.185G>C as published

published as p.Arg62Pro as published

1 1

c.161G>C 2015 Martinez 2 Exon Arg54Pro) p.( 31 novo De e Missens

c.157_177del 2 Exon p.(Glu53_Glu59del) 16 novo De deletion frame In

c.157_177del 2012 riolo P 2 Exon .(Glu53_Glu59del) p 16 novo De deletion frame In

P8 c.154_155insT 2 Exon p.(Glu52Valfs*67) 33 novo De Frameshift

P7 c.142del 2 Exon p.(Met48Cysfs*9) 28 novo De Frameshift

P6 c.113G>T 2 Exon p.(Arg38Leu) 32 ND Missense

c.112C>T 2 Exon p.(Arg38Cys) 34 novo De Missense

c.136C>T as published rg46Cys p.A as published

1 1

c.112C>T 2015 Martinez 2 Exon Arg38Cys) p.( 34 novo De Missense

c.92T>C 2 Exon p.(Phe31Ser) 26 novo De Missense

c.90_99del 2015 Klaassens 2 Exon Trp30Cysfs*24) p.( 32 novo De Frameshift

P3 c.59T>C 2 Exon p.(Leu20Pro) 26 ND Missense

P2 c.43_49dup 2 Exon p.(Glu17Valfs*31) 31 vo no De Frameshift

P1 13T>A] - 12T>A;28 - 1G>A;28 - c.[28 novo De Splicing 1 Intron p.Asp10Profs*5 25

core S

Reference PHRED

/ Patient Inheritance mutation of Type Location Protein cDNA CADD

6 — 150 the mRNA, thereby indicating degradation by NMD(SupportingInformation Figure4). different protein-coding isoforms showed that the mutant allele was almost absent in c.1116del in exon 8, cDNA sequence analysis using oligonucleotide primers stop codon (p.Asp10Profs*5) (Supporting Information Figure 3). In patient 41 carrying specific for 10 nucleotidesofintron1intothe mRNAthatpredictedaframeshift andpremature leads toutilizationofanectopic acceptor splicesite, whichresulted in aninclusionof investigated by sequencingofcDNA. Thereby itcouldbedemonstrated thatthevariant phenotype (SupportingInformation Table 1;Figure1a4, a5, b3, c1, e3, e6;) and fourareframeshift variants. These patientspresentedall atypical Malan binding andimerizationdomain.Ofthese, oneisamissense, two arenonsense, variants arein exons 5–8outsideoftheregioncodingforN-terminalDNA that all variants aredeleterious(Table 2).Only sevenMalansyndrome-associated In silicoanalysis ofNFIX 2). All variants wereabsentfrom1000genomes, ExAC, andgnomADdatabases. Figure Information (Supporting only NFIX of domain CTF-NFI the in conserved is orthologous and paralogous NFI proteins except fora change of Thr-260, which changes (c.28-1G> change atthespliceacceptorregionofexon 2consistingofthreenucleotide termination codons, twoin-frame insertions/deletions, andacomplex denovo small deletions and insertions/duplications causing frameshift and premature illustrated inFigure2. We found18missenseandsevennonsensevariants,14 Molecular results arelistedin Table 2, andtheirdistribution over thegeneis Genotype > two of fibroblasts individuals. Inpatient1harboringthecomplex denovo variant c.[28-1G> from extracted RNA on performed were experiments RT-PCR RNA Analysis [7] 24, and 25). A different missense change was reported previously (p.Arg116Pro) previously reported was change missense different A 25). and 24, involving residueArg116 (p.Arg116Gly; p.Arg116Glu) inthreepatients(Patients 23, As a child she had learning difficulties requiring individualized educational individualized requiring program andasanadult shehadananxietydisorder. difficulties learning had she child a As was phenotypically normal, heightwas 165.1cm, andhadnormalintelligence. blood using WES (allele countabnormalinsixofthetotal44reads[14%]).She patient 35, inwhomthemother was foundtobemosaicfortheNFIX All variants forwhichparentswerechecked(n reported. and p.Arg115Trp; inframe deletionp.Glu53_Glu59del)hadbeenpreviously Most variants were inexon 2.Only threevariants (missensevariants p.Arg38Cys all threevariants andpaternitywas confirmed. . All observed missense variants affected amino acids, highly conserved in conserved highly acids, amino affected variants missense observed All . A;28-13T [8],[14],[18] > A] at the splice acceptor of exon 2 the consequence of this change was A] at the splice acceptor of exon 2 the consequence of this change was The remainingvariants arenovel. We foundtwomissensevariants A, c.28-12T > variants using CADDv1.3PHREDlikeC-scoresuggested A andc.28-13T > = 35)weredenovo except for A). Parents werenegative for variant in A;28-12T A;28-12T [10] . 6 — 151 Figure 6.1 A facial view of presently reported 42 individuals with Malan reported 6.1 A facial view of presently Figure in numbers to correspond panels the in individuals of Numbers syndrome. detailed descriptions For mentioned below each picture. Ages are the Tables. and text please see Tables 6 — 152 common butadult height isabove 2SDSinonly one-third ofindividuals. the growthpattern inSotossyndrome which initialovergrowth isvery those whoshowovergrowth. The growthpatterninMalan syndromeresembles patients, with likely ascertainmentbiasasthisismorefrequently determinedin patients inwhomthiswas evaluated butdataare available fromonly 50out of79 remained with age as of 13 adults only six still have aheight Postnatal height andheadsize are typically > the meanin~90%ofpatients.Headcircumference is> weight isabove the2SDSinonly aminorityofpatients, butbirthweightisabove data frompreviously publishedandpresently reportedcases revealsthatbirth Malan syndromeisknownasanovergrowth syndrome.However, thecombined refine themajorcharacteristics ofMalansyndrome. confirmed molecularly with individuals (SupportingInformation Tables 6.1–6.3). This hasallowed usto individuals reported 45 previously 35 of those of to findings the series compare and syndrome, Malan a on here report We DISCUSSION domain; orange: CAAT-box transcription factor–nuclearfactorI(CTF-NFI) domain it; green: MH1 (MAD homology 1) domain and the N-terminal DNA binding (DNAbd) and 2.Blue:putative DNA bindinganddimerization domain ofthegeneand inside introns1 except introns scale areareand circles.in reported Exons additional with variants missense, Recurring shown. for is deletions intragenic legend and splicing, ins/dels, color nonsense, The syndrome. gene) the (above Marshall-Smith and Figure 6.2NFIX > 2SD in10/13adults. Boneageis advanced 1year ormorein80%of cartoon with all reported variants in Malan (underneath the gene) the (underneath Malan in variants reported all with cartoon 2 SDSbut height may decrease > 2 SDS. Head circumference 2 SDSin37%ofnewborns. [26] 6 — 153 and a and NFIX containing microdeletion demonstrated seizures and seizures containing microdeletion demonstrated These individuals had both point variants and deletions deletions and variants point both had individuals These [5],[18] point mutations to the 24 individuals with deletions of deletions with individuals the 24 to mutations point Skeletal anomalies are frequent, especially a slender habitus a especially anomalies are frequent, Skeletal Seizures and/or EEG abnormalities occurred in 18% of the presently in 18% of the presently Seizures and/or EEG abnormalities occurred [22] NFIX [10] . Underdeveloped optic nerves have also been noticed in Marshall-Smith Marshall-Smith in noticed been also have nerves optic Underdeveloped NFIX. When comparing characteristics of the 56 individuals with Malan syndrome of the 56 individuals with When comparing characteristics by caused (59%) together with long hands (60%) (Supporting Information Figure 7b), (Supporting Information Figure 7b), (59%) together with long hands (60%) syndrome. Other signs such as sometimes described as resembling Marfan 1). frequencies (Table occur at varying or excavatum pectus carinatum, scoliosis, The most characteristic facial findings include a long or triangular face, prominent face, triangular or long a include findings facial characteristic most The down-slanting palpebral set eyes, deeply depressed nasal bridge, forehead, fissures, short nose with anteverted naresa cupid in upper vermillion with a thin open, is often held that mouth small and upturned tip, philtrum, long With age the and a prominent chin (Figure 1). an everted lower lip, bow shape, skin the chin becomes more prominent, face seems to become more elongated, open (Supporting Information and the mouth tends to become more folds deeper, Figure 7c). Visual problems are common (76%) but often limited to nonspecific is and nystagmus and hypermetropia, abnormalities such as strabismus,myopia, degrees variable had individuals reported presently the of Thirteen uncommon. which has also been reported in three earlier optic nerves, of underdeveloped patients. reported reported patients and 27% of all known patients. reported patients and 27% of all Neuro-imaging yielded normal results in most patients. If abnormalities were If abnormalities patients. in most results yielded normal Neuro-imaging ventricles enlarged as such findings nonspecific usually were these then present, periventricular and dysplasias cortical However, body. callosal and a small previously as has been as well, occasionally do occur nodular heterotopias reported. Intellectual disability is invariably present, but detailed studies are lacking. detailed studies are but present, disability is invariably Intellectual of group reported presently the in studies ongoing of results Preliminary and severe, to moderate from varying severity is that indicate individuals several Independently, is present. disability intellectual mild exceptionally and anxieties demonstrate patients their that indicated have physicians referring behaviour. anxious their worsen to seems which noise, to sensitive extremely are referring clinicians in by anxieties were mentioned spontaneously In our series, may The behaviour in 52% of individuals. characteristics” the item “Behavioural to themselves become aggressive which patients may culminate in crises during are in progress to studies cognitive and behavioural More detailed and to others. instruments. psychological using dedicated substantiate the behaviour of syndrome. variable number variable of there other were genes, no differencessignificant in growth or skeletal manifestations. facial characteristics, cognitive impairment, pattern, Individuals with an NFIX 10/56 in (11/24 in deleted patients vs. EEG abnormalities more frequently 6 — 154 of thedysostosis inthisdisorder. seemingly advanced boneageofthedistalextremities, whichisamanifestation of Marshall-Smith syndrome as an overgrowth disorder is caused by the resemblances but also many differences. The growth pattern in these entities these in pattern growth The differences. many also but resemblances Sotos syndrome. We comparedthethree entities(Table 6.1) andfoundsome Malan syndromehasbeen suggested toresemble Weaver syndromeand still befound. remains small andwithadditional reportscluesforacontinuousspectrummay seem tobetwoseparate entities.However, thenumberofreportedindividuals this occurredinpatient43.MalansyndromeandMarshall-Smith syndrome with eitherentityonehasconsideredtheotherdiagnosis.In thepresentseries, completely limited to individuals with this entity. In only very few individuals occurs only inMarshall-Smith syndrome, andgingiva hypertrophy isalmost likely duetothemarkedosteopenia(RCH, personal observation). Hypertrichosis Marshall-Smith syndromethescoliosisremainsprogressivealsointoadulthood, occasionally. Scoliosisiscommon inbothentities, butonly inindividualswith in Marshall-Smith andsternumabnormalitieshave beenrecorded only Smith syndromearemorepronounced.Aslenderhabitus isuncommon are presentinbothentities, butgenerally thefacialabnormalitiesinMarshall- as a prominent forehead, short nose with anteverted nares, and everted lower lip midface and small chin in Marshall-Smith syndrome. Some facial findings such due totheabsenceofmacrocephaly andpresenceofproptosis, underdeveloped similar results in both entities. There are major differences in facial morphology all knownindividualswithMarshall-Smith syndrome. growth parameters belowthe3 birth weightbelowthe3rd centile, nonehave macrocephaly, andalmostall have syndromes, 20%ofnewbornswithMarshall-Smith syndromehave infacta and postnatally. Although theentityisoftencategorized among theovergrowth bothprenatally pattern, in growth markedly differ syndrome Marshall-Smith to result from adominant-negative action(Table 1;Figure2).Individualswith CACNA1A be explained by thepresenceofacontiguousgenedisorder. Deletionsinvolving patients with point variants). The increased prevalence of seizures/epilepsy may the deletiondidnotincludeCACNA1A. seizures. Incontrast, only onepatienthasbeenreportedwithseizuresinwhom 14 individualsinwhomthedeletedregionincludedCACNA1A, syndrome, We compared the findings inMalan syndrome to those reported in Marshall-Smith with NFIXdeletionsshowedthesesymptoms. patient whohadepisodicataxiaatagenineyears. Noneofthepresentpatients a singlepatientwithcyclical vomitingresponsivetopizotifenandaliterature that islocated 109kbfromNFIX [22],[23] anallelic disorder caused by variants inNFIX rd centilepostnatally. The wrongfulassignment [22] Cognitiveimpairmenthasbeenpresentin [8] Klaassensandcoworkers seem toplay aparticularrole.Ofthe [23] Neuro-imagingyields that ispostulated 10 developed [10] reported 6 — 155

[2] A putative The domains [31] [29],[30] missense variants involving involving missense variants 2 SDS in the first fewyears of life, but Based on these suggestions it is plausible that the [29] Facially, individuals with Malan, Sotos, and Weaver Weaver and Sotos, individuals with Malan, Facially, [27] Experimental evidence for this mechanism has previously has previously Experimental evidence for this mechanism [28] We found a clustering of NFIX Three Malan syndrome-associated missense variants are localized at are localized Three Malan syndrome-associated missense variants [7],[8],[12],[14],[18] [31] variants have been reported in 56 unrelated patients with Malan syndrome been reported in 56 unrelated patients with Malan syndrome have variants positively charged amino acids (at positions Lys 113, Arg115, Arg116, Arg121, Arg121, Arg116, Arg115, 113, amino acids (at positions Lys charged positively lysine and arginine Both residues. 15 of region small a in Arg128) and Lys125, and their charge or binding sites, in protein-activating located are frequently groups. charged distribution is ideal for binding negatively or immediately adjacent tothe putative NLS. The NLS regions are usually located located The NLS regions are usually adjacent tothe putative NLS. or immediately domains. near DNA-binding been provided for a small number of variants causing Malan syndrome. Malan causing variants of number small a for provided been is similar: individuals with Sotos and Weaver syndrome are also frequently are also frequently syndrome Weaver and individuals with Sotos is similar: grow > age at birth, for gestational large adult height falls typically within two SDS of the mean. Developmental delay is delay of the mean. Developmental within two SDS typically falls height adult moderate typically is there syndrome inMalan but entities three in all common typically have patients andWeaver Sotos while disability, intellectual severe to of cognitive distribution a wide is there although disability, to moderate mild and functional abilities. nuclear localization (NLS) domain encompassing amino acids 36–50 of (url: nls-mapper.iab.keio. cNLS mapper is predicted by (RKRKYFKKHEKRMSK) NFIX NFIA NLS1 in the paralogous with overlapping This domain is completely ac.jp/). NLS2 are and both NLS1 that demonstrated NFIA on studies Previous protein. nucleus. the to protein the of translocation full the for required mutated proteins harboring missense variants may either fail to shuttle into the may variants mutated proteins harboring missense haploinsufficiency. thus explaining nucleus or bind DNA, are highly conserved in all NFI proteins and are located near the DNA binding DNA the near located are and proteins NFI all in conserved highly are domain. (Table 6.2). Twenty-seven of the variants observed in Malan syndrome were observed in Malan syndrome of the variants Twenty-seven 6.2). (Table stop premature predicting variants site splice and frameshift nonsense, that these mutated in the 5' part of the mRNA. It is likely codons mostly leading to thereby (NMD), nonsense-mediated decay mRNAs are cleared by haploinsufficiency. Together with the patient cohort presented here, a total of 51 different intragenic intragenic different 51 of total a here, presented cohort patient the with Together NFIX syndrome share macrocephaly and a prominent forehead, and, in Sotos and and, and a prominent forehead, syndrome share macrocephaly Malan syndrome, a long face. The other facial signs differ betweenentities. We the conclude three that usually differentiating the three entities is possible by clinical evaluation alone, and that this differentiation is of major importance We propose that of patients and their families. and counseling for adequate care of 2" because as "Sotos syndrome type not be indicated Malan syndrome should between the two entities. the clear differences Twenty-three different missense variants have been identified, most of these (22 these of most identified, been have variants missense different Twenty-three There is a in the DNA binding and dimerization domain. out of 23) are located of basic residues (in 12 of the 23 missense variants), predominant involvement Arg116; Arg115; Arg38; positions (at once than more altered been have some and Lys125). 6 — 156 clinical dataprovided) variantwas inheritedfromamotherwhopresentedwithtall stature(noother reports ofvariants thathave beeninherited.Inone, thep.Arg121Pro missense behavioral issues that may point to a very mild presentation. There are two other mosaic motherwhowas phenotypically normalbutwhohadsomelearningand However, oneoftheindividualspresentedherehadavariant inheritedfroma Malan syndromeistypically caused by adenovo mutational event(Table 2). 6). and 5 Figures Information (Supporting proteins NFIX mutant associated suggests thatthereisnomotifsharedby all Marshall-Smith syndrome- functions mediatedby theiraltered Cterminus.However, thepresentanalysis DNA-bindingcapacities,conserved havespecific with also might proteins these variants that aside fromtheirabilitytogenerate expressed mutantproteins syndrome. Itcannot beexcluded forMarshall-Smith syndrome-associated confirming the rule that haploinsufficiency is the mechanism underlying Malan the most3'position(c.1116del)thatmutantmRNAisalmostabsent, thus have alsoshownhereforaMalansyndrome-associatedframeshift variant with Malan syndrome-associated variants (Supporting Information Figures 5–6). We translational stopcodonstrictly separates Marshall-Smith syndrome—from despite theoverlap inthegenomicpositionsofvariants, thepositionof that predicts syndrome Marshall-Smith and syndrome Malan in proteins NFIX other individualswithMalansyndrome.Alignment ofthecorrespondingmutated (P39, P40, P41, P42 and Klaassens patient) do not differ in any way from those of families withMalansyndrome. of careful evaluation ofparentsandpropercounselingaboutrecurrence risks in was notdetectedintheir lymphocytes. These observations stressthe importance in oneoftheparents.Bothparents werephenotypically normal andthedeletion Malan syndromeandapartial NFIX has beendemonstrated thatmutantmRNA doesnotundergoNMD. in exons 9 and 10 (Figure 2). For several Marshall-Smith-associated variants it DNA binding and dimerization domain, especially in exons 6–8 and sometimes syndrome have exclusively beenfoundin regions outside of those coding forthe and Marshall-Smith syndrome. Variants associatedwithMarshall-Smith There arestrikinggenotype-phenotypecorrelationsbetweenMalansyndrome reported previously inanindividualwith Malansyndrome. exons 6, 7, and 8). Another variant in this part of the gene, p.Gln338*, has been (in gene the of part 3' the within located were that syndrome) Malan by affected individuals five (in variants four identified however,we contrast, In 2). (Figure domain. This was indeedtrueformostofthenovel variants reportedinourstudy restricted tothe5'partofgeneencodingforDNAbindinganddimerization evidence suggestedthatvariants associatedwithMalansyndromeareinstead [20] andNimmakayalu etal. deletion, suggesting germlinemosaicism [16] reported twosisters with [10] Their phenotypes Their [2],[21] Previous 6 — 157 signaling. variants that variants The true frequency, spontaneous The true frequency, [34] the protein mutated in Shprintzen-Goldberg mutated in Shprintzen-Goldberg the protein [32] and one individual with Marshall-Smith syndrome syndrome Marshall-Smith with individual and one [16],[17] and those with intragenic mutations. NFIX and those with intragenic the mechanisms leading to haploinsufficiency rather than NFIX The SKI protein in turn regulates SMAD-dependent TGF-β SMAD-dependent in turn regulates The SKI protein 2 SDS in half of them. Macrocephaly remains present in 75%. There remains present in 75%. 2 SDS in half of them. Macrocephaly [33] Consistent with a possible relationship of Malan syndrome with Marfan-like relationship of Malan syndrome Consistent with a possible connective tissue disorders, one individual in the present series had a marked individual in the present series had one connective tissue disorders, from the present and two of the 15 individuals pulmonary artery dilatation, at age 3 (one aorta an enlarged had aortic evaluation, who underwent series (one with other individuals with Malan syndrome two In the literature, years). been reported a whole gene deletion) have and one with variant a frameshift dilatation, aortic with syndrome. are significant and recognizable differences between Malan syndrome and other and syndrome Malan between differences recognizable and significant are and we therefore syndrome, Weaver including Sotos and syndromes, overgrowth There syndrome. Malan for 2" type syndrome “Sotos of designation the challenge but be an increased risk to develop aortic dilatation in Malan syndrome, may is indicated. surveillance long-term whether determine to needed are data more There may be a specific in progress. detailed studies that are presently but this requires further anxieties, behavioral phenotype,characterized especially by there , CACNA1A due to deletion of likely for the occurrence of epilepsy, Except is no difference in phenotype between individuals with 19p13.2 microdeletions encompassing CONCLUSION syndrome that presents overgrowth We conclude that Malan syndrome is an as adult is less marked in adulthood but overgrowth in infancy and childhood, height is > [12] Individuals with Malan syndrome exhibit some symptoms reminiscent of symptoms reminiscent some exhibit with Malan syndrome Individuals TGF-β of the dysregulation by caused Marfan-like syndromes Marfan or pathway (slender body build, long fingers, scoliosis, and pectus formation). of NFI proteinswith interaction link could be the A possible pathophysiological oncogene SKI, Sloan-Kettering has been reported with aortic root dilatation. has been reported with course course and clinical significance of aortic dilatations prudent seems it but uncertain, remains currently syndrome Marshall-Smith in Malan syndrome and of the large with either syndromes for enlargement individuals all to evaluate or not. indicated this is truly whether until reliable data prove vessels, cause Malan syndrome are typically located in exons coding for the DNA binding in exons located syndrome are typically Malan cause variants As for truncating few exceptions. with and dimerization domain, elsewhere in NFIX, dominant-negative effects for Malan syndrome-associated frameshiftvariants related to the position of the translational of the gene are likely in the 3' exons NMD. of the mutant mRNA by stop codon and clearance 6 — 158 Senior InvestigatorAward (toR.V.T.). (NIHR) Oxford Biomedical Research Centre Program (toK.K., R.V.T.), andNIHR Foundation (toK.K., R.V.T., R.C.H.), theNational InstituteofHealth Research and Jordan's Guardian Angles(toG.M.M.), by theMarshall-Smith Research National Institute of Neurological Disorders and Strokes (grant K08N092898) study was fundedinpartby ISCIII, FederFunds FISP115/01481(toJ.T.), by the GeneDx forproviding theinformationonmosaicisminoneofparents. This for MendelianGenomicsandtheDDDstudyhelpinmolecularanalyses, and study.We thankDrs. StefanieBeck-Wödl, Tobias B.Haack, Baylor-Hopkins Center We thankthepatientsandtheirfamiliesforgenerousparticipationto ACKNOWLEDGEMENTS 6 — 159 patients series. Eur J Med Genet Med Genet Eur J series. patients 58:488–491. - Fala M, Kucharczyk A, Jezela-Stanek Wicher D, M, Mlynek D, Jurkiewicz na K, Cieslikowska Kugaudo M, M, Rydzanicz Krajewska-Walasek Ploski R, E, Ciara A, (Sotos syn- Malan syndrome 2016. M. with 19p13.2 drome 2) in two patients NFIX gene and deletion encompassing Biomed NFIX sequence variant. novel Pap Med Fac Univ Palacky Olomouc Czech Repub 160:161-167. S, Doria S, Agueda C, Silva R, Jorge disabili- Intellectual 2015. Leao M. of - A new case ty and overgrowth syndrome 19p13.13 microdeletion Am J Med with digital abnormalities. Genet 167:2839–2843. Rosser EM, D, Morrogh Klaassens M, Jaffer F, Vreeburg M, Bok LA, Segboer Kumar RM, Quinlivan Belzen M, van T, Malan syn- 2015. Scott RH. JA, Hurst A, with de drome: Sotos-like overgrowth - and dele variants NFIX sequence novo tions in six new patients and a review of Eur J Hum Genet 162:1–6. the literature. Kuroda Mizuno Y, Mimaki Y, M, Oka A, Sato Y, Ogawa S, Kurosawa K. 2017. patients with 19p13.2 deletion Two NFIX and (Malan syndrome) involving develop- overgrowth, with CACNA1A - Clin Dys and epilepsy. mental delay, morphol 26:224-227. Ski and SnoN: Negative 2004. Luo K. Curr regulators of TGF-β signaling. Opin Genet Dev 14:65–70. S, Wustefeld M, Ravoet PA, Lysy E, Wyns Nassogne MC, P, Bernard of syn- A new case 2009. C. Sibille with cryptic dromic craniosynostosis Am J Med 19p13.2-p13.13 deletion. Genet 149A:2564-2568. San- Marin-Reina P, F, Martinez chis-Calvo A, Perez-Aytes A, Oltra S, Pantoja Monfort S, S, Mayo M, Rosello J, Orellana C. 2015. Novel mutations Marshall-Smith of NFIX gene causing syndrome or Sotos-like syndrome: Res Pediatr two phenotypes. one gene, 78:533–539.

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16:665-677. transcriptomes. NatRevMolCell Biol an intricate machinerythatshapes Nonsense-mediated mRNAdecay: Lykke-Andersen S, Jensen TH. 2015. 11:e0149189. drome: A systematic review. PLoS One nition andbehaviour inSotossyn- Lane C, MilneE, Freeth M. 2016. Cog- Am JMedGenet161A:2972-2980. tions: Clarifyingtheclinical phenotype. Weaver syndromeandEZH2muta- growth Consortium, Rahman N. 2013. Over Childhood S; T,Seal Cole IK, ple Titheradge H, Van Maldergem L, Tem- M, Strenger V, StuurmanK, Taylor C, M, RankinJ, Shears D, SimonM, Splitt McConnell V, Medeira A, Ozono K, Patton Joss S, KinningE, Lynch SA, MageeA, Cushing T, FlinterF, JacquemontML, LM, Clericuzio CL, Cormier-Daire V, Douglas J, ArmstrongR, Banka S, Bird Tatton-Brown K, Murray A, Hanks S, ic variants. NatGenet46:310-315. relative pathogenicityofhumangenet general framework forestimatingthe BJ, CooperGM, ShendureJ. 2014. A P,O’Roack Jain DM, Witten M, Kircher 15:264-271. Sotos syndrome. EurJHumGenet Tatton-Brown K, RahmanN. 2007. tism. JIntellect DisabilRes55:973-987. study ofcognition, phenotypeandau- shall-Smith syndrome: an exploratory Development andbehaviour inMar sens M, HoekHW, Hennekam RC. 2011. Van BalkomID, Shaw A, Vuijk PJ, Frans- et 152A:2714–2726. shall–Smith syndrome. AmJMedGen- Phenotype andnatural history inMar Cormier-Daire V, Hennekam RC. 2010. Nampoothiri S, PuseljicS, ZenkerM, A, Holmberg E, KnightSJ, MortierG, Cole TR, DelrueMA, Van Haeringen Shaw AC, van BalkomID, BauerM, 35:1092-1100. shall-Smith syndrome. Hum Mutat previously unexplained cases ofMar deletion ofexon 6and7accountfor - - - - - 6 — 161 - Doyle AJ, Doyle JJ, Bessling SL, SL, Bessling JJ, Doyle AJ, Doyle D, Schepers ME, Lindsay S, Maragh K, Sauls T, Homfray G, Mortier E, Gillis DW, Mohr D, Leahy Huso ND, Norris RA, Destrée Hen- A, Caulfield Scott MJ, AF, Laer Van CJ, Curry PH, Arn RC, nekam HC. Dietz BL, Loeys AS, McCallion L, repres- TGF-β the in Mutations 2012. Shprintzen-Goldberg cause SKI sor Nat aneurysm. syndrome with aortic Genet 44:1249-1254. Rivera-Davila Nguyen J, A, Aggarwal Mar 2017. D. Rodriguez-Buritica M, path- syndrome: Novel shall-Smith unre- and previously ogenic variant precocious ported associations with Eur dilatation. puberty and aortic root J Med Genet 60:391-394.

33. 34. :118-124. EBS Lett 484:118-124. Tarapore P, Richmond C, Zheng G, Co- G, Zheng C, Richmond P, Tarapore U, Kruse J, Kopchick B, Kelder SB, hen E. Stavnezer C, Colmenares AE, Sippel DNA binding and transcriptional 1997. the Ski oncoprotein me- by activation Nucleic with NFI. interaction diated by Res 25:3895–3903. Acids Imagawa Imagawa M, A, Sakaue Osa- R, Tanabe nuclear Two 2000. T. Nishihara S, da for signals are required localization of nuclear factor nuclear translocation 1-A. F Yao K, Wu Y, Chen Q, Zhang Z, Chen Zhang Z, Q, Chen Y, Wu K, Yao X, Zhang element within the Lysine-rich Y. 2016. for domain is essential The DNA-binding Arginine/ function of and nuclear localization resistance. pathogen intracellular the PLoS ONE 11: e0162832 - Nucle 1995. YA. Lefebvre LC, LaCasse or DNA- overlap signals ar localization in nucleic ac- domains RNA-binding Re- Acids Nucleic id-binding proteins. 23:1647-1656 search

32. 31. 30. 29. 6 — 162 those twotranscripts all changesinexons 1-8have theidentical denomination. a referenceinthemajorityofprevious publications onNFIXvariants. Duetothenatureof tissues investigatedincludingbrain (seeSuppl.Fig.S4).NM_002501.3has been usedas latter. According to our own experiments the Δexon9 isoforms are more abundant in most is identical toNM_002501.3except forthelackofalternatively splicedexon 9inthe function –ifthereisoneanyway –remainsunknown. The isoformENST00000592199.5 biological their and protein NFI a defining domains functional typical the contain not do database, butthesehave notbeen consideredhere, because thepredictedgeneproducts Threevery proteins. the encoded of short transcript isoformsarealsoannotatedasproteincodingin the ENSEMBL C-terminus different a in resulting frame reading spliced exon 7isinframe, whilealternative splicingofexon 9 leadstoshiftingofthe (printed in grey), whose expression and biological relevance is not clear. The alternatively by exons 2-4isinvariant inall isoformsexcept fortranscript ENST00000588228.5 of acids encoded by exon 1. The biological function and relevance of these variable N-termini amino N-terminal very the in differ may proteins resulting that demonstratesillustration first exon (red or pink color) or by changing the reading frame in exon 10 (green color). This Color coding illustrates sequences differing from this isoform by either using an alternative transcripts usingthe11exon transcript isoformENST00000592199.5 asreference(top). S1 Fig. Transcript isoformsofhumanNFIX.Graphical alignmentofvarious knownNFIX SUPPLEMENTAL MATERIALS NFIX proteinareunknown.Notably, theDNAbindinganddimerizationdomainencoded 6 — 163 S2 Fig. Alignment of orthologous and paralogous NFI proteins: This alignment NFI proteins: and paralogous Alignment of orthologous S2 Fig. syndrome-associated Malan by affected residues acid amino of conservation the illustrates the putative color shows alignment in S7 Fig.). Blue background missense mutations (full green color represents gene and inside it, DNA binding and dimerization domain of the N-terminal DNA binding (DNAbd) domain. the MH1 (MAD homology 1) domain and the factor – nuclear transcription part of the CAAT-box a small colored box shows The orange this in discovered mutation missense (CTF-NFI)the only I factor containing domain on top of the alignment and the missense changes are indicated The observed domain. in the by affected acids amino the while red, in printed is acid amino conserved respective signal The putative nuclear translocation are printed in blue. deletion E53_E59del frame as a black horizontal bar. sequence (NLS1) is indicated 6 — 164 The inserted10nucleotidesfromintron1intothemutantRNAare printedinredcolor. of thereverse strand areshown). The vertical linesindicate theexon 1–exon 2junction. sequencing fromanormalindividualandpatient1(electropherograms fromsequencing splice site ishighlighted by a violet frame. Exonic nucleotides are printed in capitals. (B) cDNA authentic splice acceptorishighlighted by agreeframe andthe predicted newacceptor NGS readdatausingtheIntegrative Genomics Viewer (IGV; datanotshown). The agatthe variants. The 1G>A;28-12T>A;28-13T>A] inintron1areshown.(A)Genomicsequenceshowingthethree Results ofgenomicandcDNAsequencinginpatient1carrying thedenovomutationc.[28- Fig.S3 Demonstration alteredof intronin mutation complex a by caused splicing 1: -phase of the three variants has been confirmed by visual evaluation of evaluation visual by confirmed been has variants three the of cis-phase 6 — 165 RT-PCR and NFIX RT-PCR cDNA sequencing on RNA derived from fibroblasts of patient 41 withc.1116del the variant. Four heterozygous cDNA samples derived from fibroblast cultures from this patient were available (cDNA1-4) and one Primer positions in control exon 7 (forward) fibroblastand 10 (reverse) allow cDNAthe amplification of was from the long NFIX afragment isoform cDNA and usedan isoform lacking (co-cDNA). exon 9 (Δe9 (A) isoform). The Δe9 the representing fragment that demonstrates electropherogram gel agarose resulting isoform is much more abundant in the fibroblast cDNA 9 junction (reverese) 8 – exon and at the exon compared 7 (forward) (B) Primer positions in exon to the long isoform. lead to specific amplification (C) Sequence size. the expected band at a distinct shows gel electropherogram agarose of fragmentthe fromthe of and patient the from DNA genomic of sequencing from obtained electropherograms the long isoform.The resulting the heterozygous Genomic DNA shows respectively. products shown in (A) and (B), RT-PCR long the from as well as isoform Δe9 the mainly containg cDNA in while variant, frameshift be may allele mutant the of traces Only visible. clearly is sequence type wild the only isoform and long the from both RNA, mutant that indicate findings These arrows). (blue appreciated (NMD). decay nonsensemediated by degraded completely almost is isoforms, Δe9 the from S4 Fig. The frameshift mutation S4 The frameshift c.1116del Fig. in exon 8 leads to NMD. 6 — 166 red: thisreport. violet: Martinezetal., PediatrRes2015;78:533-539. dark blue:Aggarwal etal., EurJMedGenet 2017;60:391-394. light blue:Malanetal., Am JHumGenet2010;87:189-198. green: Schanzeetal., HumMutat2014;35:1092-1100. Specific coloring of mutations names encodes the references for the respective mutations: abnormal functionconferredby auniformC-terminaldomainofmutantproteins. specific a of hypothesis a supporting not thus C-termini, different various predict +1/-2 tail oftheencodedmutantprotein, butmutationscreating ashiftofthereadingframe by mutations creatingashiftofthereadingframe by -1/+2predictacommonC-terminal stop codonoccurs beforeexon 9inthisisoform.Marshall-Smith syndrome-associated codons inexon 9, 10or11, whileinMalansyndrome-associatedframeshift mutationsthe appreciated, all Marshall-Smith syndrome-associated mutations createtranslational stop red asterisk marks the position of the predicted translational stop codon. As it can be of thereadingframe by +1/-2(asfor1bpdeletions)isindicated by yellow color. The of thereadingframe by -1/+2(asfor1bpinsertions)isindicated by bluecolor.Shifting of eachtranscript indicate thecodonpositionofshiftreadingframe. Shifting displayed. Deleted exons areindicated by ablackhorizontal bar. Numbers atthebottom syndrome-associated transcripts carrying frameshift mutationsinexons 6and8are and mutated transcripts (all ofthemcreateashiftthereadingframe) and(C)Malan normal of alignment ENST00000592199.5 (seeSuppl.Fig.S1).(B)Marshall-Smith syndrome-associated Clustal-Omega a of mutated NFIXtranscripts. (A) Wild typereferringtothe11exon transcript isoform presentation Graphical Fig. S5 6 — 167 ) Wild type referring to the shorter 10 exon Wild type referring to the shorter 10 exon (A) Schanze et al., Hum Mutat 2014; 35:1092-1100. green: Schanze et al., Am J Hum Genet 2010; 87:189-198. light blue: Malan et al., Eur J Med Genet 2017; 60:391-394. et al., dark blue: Aggarwal Pediatr Res 2015; 78:533-539. violet: Martinez et al., red: this report. Specific coloring of mutations names encodes the references for the respective mutations: respective the for references the encodes names mutations of coloring Specific transcript isoform (NM_002501.3; Q14938; Δe9 isoform) lacking exon 9 of the long isoform long the of 9 exon lacking isoform) Δe9 Q14938; (NM_002501.3; isoform transcript ) Marshall- (see Suppl. Fig. S1 and Suppl. Fig. S5 for comparison). (B ENST00000592199.5 and (C) Malan syndrome-associated Smith syndrome-associated mutated transcripts Deleted exons 6 and 8 are displayed. in exons mutations frameshift carrying transcripts indicate bottom of each transcript at the a black horizontal bar. Numbers by are indicated -1/+2 by Shifting of the reading frame frame. the codon position of the shift of the reading +1/-2 by reading frame blue color. Shifting of the by (as for 1bp insertions) is indicated the position The red asterisk marks color. yellow by (as for 1bp deletions) is indicated Marshall-Smith that all be appreciated stop codon. It can of the predicted translational of this stop codons in the last exon syndrome-associated mutations create translational mutations the stop of Malan syndrome-associated frameshift while in the case isoform, which has been found to be the most For this isoform, last exon. the before codon occurs abundant syndrome- one in itfibroblasts is (Suppl. evident Fig. that S4A), Marshall-Smith No uniform (NMD). decay nonsense-mediated activate associated mutations cannot syndrome-associated mutant proteins. C-terminal tail is predicted for Marshall-Smith S6 Fig. Graphical presentation isoform). (short transcripts NFIX mutated of a Clustal-Omega alignment of normal and 6 — 168

6 — 169

(F1R834), gallus gallus: nfix (Q90932), xenopus laevis: nfix (Q66IX4), and mus musculus: nfix (P70257). nfix musculus: mus and (Q66IX4), nfix laevis: xenopus (Q90932), nfix gallus: gallus (F1R834),

sapiens: NFIA isoform 1 (Q12857), NFIB isoform 1 (O00712), NFIC isoform 1 (P08651), NFIX isoform 1 (Q14938), danio rerio: nfixb nfixb rerio: danio (Q14938), 1 isoform NFIX (P08651), 1 isoform NFIC (O00712), 1 isoform NFIB (Q12857), 1 isoform NFIA sapiens:

number given in brakets) proteins from caenorhabditis elegans: nfi-1 (Q5H9N3), drosophila melanogaster: NfI (Q86P06), homo homo (Q86P06), NfI melanogaster: drosophila (Q5H9N3), nfi-1 elegans: caenorhabditis from proteins brakets) in given number

www.ebi.ac.uk/Tools/msa/clustalo/) alignment of orthologous and paralogous NFI Uniprot (http://www.uniprot.org/; accession accession (http://www.uniprot.org/; Uniprot NFI paralogous and orthologous of alignment www.ebi.ac.uk/Tools/msa/clustalo/)

S7 Fig. Clustal-Omega alignment of orthologous and paralogous NFI proteins: proteins: NFI paralogous and orthologous of alignment Clustal-Omega Fig. S7 Complete Clustsal-Omega v1.2.4 (https:// v1.2.4 Clustsal-Omega Complete

6 — 170 To becontinuedonthenextpage Table Ia.Newly ReportedPatients withMalanSyndrome and Variants inNFIX Eyes Craniofacial Development Postnatal growth Prenatal growth Age Gender Development Postnatal growth Prenatal growth Age Gender anomalies Seizures/EEG MRI Brain Skeletal

ID ID

Joint laxity boneage Advanced Long hands Pectus excavatum/carinatum Kyphoscoliosis Slender habitus Blue sclerae impaired:H/S/N/M/A/O Vision Prominent chin Everted lower lip (E) /open mouth appearance (O) Thin upper vermillion/ cupid bow mouth Small Long philtrum Upturned nasal Short nose nasalDepressed bridge Deeply set eyes Down Prominent forehead Long /triangular face Other behavioral anomalies Hypotonia retardationMotor Autistic/autistifom behavior Intell >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Autistic/autistifom behavior Intell disab/glob devdelay >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Nl/WV/C/BA/CM

disab/glob delay dev - slanting palpebral fissures

tip/anteverted nares

S,M 12y 14y CM 16 M M E 1 + + + + + + + + + + + + + + + + + + + + + + + + ? ? ------

S,N 16y 10y 17 F F 2 + + + + + + + + + + + + ? ? ? ? ? ? ------

CM,WV E/O 20y 17y 18 S F F 3 + + + + + + + + + + + + + + + + + ? ? ? ------

14y 24y 19 M M E S 4 + + + + + + + + + + + + + + + + + ------

13y

20 4y M M

+ + 5

+ + + + + + + + + + + + + + + + + ? ?

------

E/O 42y 21 2y M F + + 6 + + + ^ + + + + + + + ? ? ? ? ? ? ------

WV,* E/O 22 3y 4y M M + 7 + + + ^ + + + + + + + ------

E/O 31y 23 7y M H F + + + + + + + + + + + + + + + + + + 8 ? ? ? ------

E/O 12y 24 5y M M S + + + + + + + + + + + + + + + + 9 ? ? ? ? ? ------

E/O 15y 25 10 6y F F + + + + + + + + + + + + + + + + + ? ? ? ? ? ? ? ? ------

E/O 14y H,S 26 11 8y F F + + + + + + + + + + + + + + + + + + + ? ? ------

42y 27 12 9y M M N E + + + + + + + + + + + + ? ? ? ------

E/O 15y 12y 28 13 M S F + + + + + + + + + + + + + + + + + + + + ? ? ? ------

12y 14y 29 14 M E F + + + + + + + + + + + + + + + ? ? ------

H,O 11y 30 15 6y O F F + + + + + + + + + + + + + + + + + ? ? ------

6 — 171

------? ? + + + + + + + + + + + + + + + + + F F O 6y 30 15 11y H,O

------? ? + + + + + + + + + + + + + + + F E M 29 14 12y 14y

------? ? ? + + + + + + + + + + + + + + + + + + + + F S M 28 13 15y 12y E/O

------? ? ? + + + + + + + + + + + + E N M M 9y 27 12 42y

------? ? + + + + + + + + + + + + + + + + + + + F F 8y 26 11 H,S 14y E/O

------? ? ? ? ? ? ? ? + + + + + + + + + + + + + + + + + F F 6y 25 10 15y E/O

------? ? ? ? ? + + + + + + + + + + + + + + + + 9 S M M 5y 24 12y E/O

------? ? ? + + + + + + + + + + + + + + + + + + 8 F H M 7y 23 31y E/O

------+ + ^ + + + + + + + + + 7 M M 4y 3y 22 E/O WV,*

------? ? ? ? ? ? + + + + + ^ + + + + + + + 6 F M 2y 21 42y E/O

------? ? + 5 + + + + + + + + + + + + + + + + + + M M 4y 20 13y

------+ + + + + + + + + + + + + + + + + 4 S E M M 19 24y 14y

------

? ? ? + + + + + + + + + + + + + + + + + 3 F F S

18 17y

20y E/O

CM,WV

- -

- - -

- - - ? ?

? ? ?

? + +

+ + + + +

+ + +

+ + 2

F F 17 10y 16y S,N

------? ? + + + + + + + + + + + + + + + + + + + + + + + + 1 E M M 16 CM 14y 12y S,M

tip/anteverted nares tip/anteverted

slanting palpebral fissures palpebral slanting - /glob devdelay /glob disab

Nl/WV/C/BA/CM Weight>2SDS Length>2SDS OFC >2SDS >2SDS Height OFC >2SDS dev delay disab/glob Intell behavior Autistic/autistifom Depressed nasal bridge Depressed nasal nose Short nasal Upturned Long philtrum Small mouth bow cupid vermillion/ upper Thin (O) appearance mouth /open (E) lip lower Everted chin Prominent Vision impaired:H/S/N/M/A/O sclerae Blue habitus Slender Kyphoscoliosis excavatum/carinatum Pectus Long hands Advanced bone age laxity Joint Weight>2SDS Length>2SDS OFC >2SDS >2SDS Height OFC >2SDS Intell behavior Autistic/autistifom Motor retardation Hypotonia anomalies behavioral Other face Long /triangular forehead Prominent Down eyes set Deeply

ID ID

growth Postnatal Development Development Craniofacial Eyes Skeletal Brain MRI Seizures/EEG anomalies Gender Age growth Prenatal Gender Age growth Prenatal growth Postnatal 6 — 172 To becontinued onthenextpage Table Ia. continued Craniofacial Development Postnatal growth Prenatal growth Age Gender anomalies Seizures/EEG MRI Brain Skeletal Eyes Craniofacial Development Postnatal growth Prenatal growth Age Gender Craniofacial Development Postnatal growth Prenatal growth Age Gender anomalies Seizures/EEG MRI Brain Skeletal Eyes

ID ID ID

Long philtrum Upturned nasal Short nose nasalDepressed bridge Deeply set eyes Down Prominent forehead Long /triangular face Other behavioral anomalies Hypotonia retardationMotor Autistic/autistifom behavior Intell >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Blue sclerae impaired:H/S/N/M/A/O Vision Prominent chin Everted lower lip (E) /open mouth appearance (O) Thin upper vermillion/ cupid bow mouth Small Long philtrum Upturned nasal tip/anteverted nares Short nose nasalDepressed bridge Deeply set eyes Down Prominent forehead Long /triangular face Other behavioral anomalies Hypotonia retardationMotor Autistic/autistifom behavior Intell disab/glob devdelay >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Nl/WV/C/BA/CM Joint laxity boneage Advanced Long hands Pectus excavatum/carinatum Kyphoscoliosis Slender habitus Blue sclerae impaired:H/S/N/M/A/O Vision Prominent chin Everted lower lip (E) /open mouth appearance (O) Thin upper vermillion/ cupid bow mouth Small Prominent forehead Long /triangular face Other behavioral anomalies Hypotonia retardationMotor Autistic/autistifom behavior Intell disab/glob devdelay >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Nl/WV/C/BA/CM Joint laxity boneage Advanced Long hands Pectus excavatum/carinatum Kyphoscoliosis Slender habitus

disab/glob delay dev - - slanting palpebral fissures slanting palpebral fissures

tip/anteverted nares

S,M 14y 12y 11y CM 31 16 M M M E + + + + + + + + + + + + + + + + + + + + + + 1 + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ? ------

S,N 10y 16y 12y 32 17 F F F + + + + + + + + + + 2 + + + + + + + + + + + + + + ? ? ? ? ? ? ? ? ? ------

CM,WV E/O E/O 17y 20y 16y 33 18 M O S F F + + + + + + + + + + + + + + 3 + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ? ? ------

24y 14y 13y 19 34 M M M A E S + + + + + + + + + + + + + + 4 + + + + + + + + + + + + + + + + + + + ? ------

WV,C 3y8m

13y S,A

20 35 4y

M M

+ + + + + + + + + + + + + + + + + 5 + + + + + + + + + + + + + + + + + + + + + +

? ? ?

------

S,N,O 4y2m C,BA E/O E/O 42y 21 36 2y M M F + ^ + + + + + + + + + 6 + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ? ? ? ------

WV,* E/O 22y 22 37 4y 3y M M M E + + ^ + + + + + + + + 7 + + + + + + + + + + + + + + + + + + + ? ? ------

M,^ E/O E/O 21y 31y 38 23 7y +° M M H F + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 8 ? ? ? ? ? ------

E/O E/O 12y 39 24 3y 5y M M M

S + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 9 ? ? ? ? ? ? ? ? ? ------^

E/O 15y 40 25 10 3y 6y M F F + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ? ? ? ? ? ? ? ------

S,M E/O E/O 10y 14y H,S 41 26 11 8y M F F + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ------

21y 42y 42 27 12 9y M M M M N E + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ------

E/O E/O 15y 12y CM 28 13 M S S F + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ------

16F/26M TOTAL S,N,^ 40/42 35/42 26/41 25/38 32/37 16/3 42/42 34/42 21/41 12/25 5/28 4/40 E/O 12y 14y WV 29 14 M E F + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ------

H,O 11y H 30 15 6y O C F F + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? ? ------

6 — 173

------? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + F F C O 6y 15 30 H 11y H,O

------? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + F E M 14 29 WV 12y 14y E/O 4/40 5/28 12/25 21/41 34/42 42/42 16/3 32/37 25/38 26/41 35/42 40/42 S,N,^ TOTAL 16F/26M

------? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + F S S M 13 28 CM 15y 12y E/O E/O

------? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + E N M M M M 9y 42 12 27 21y 42y

------? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + F F M 8y 26 41 11 H,S 10y 14y E/O E/O S,M

------? ? ? ? ? ? ? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + F F M 3y 6y 25 40 10 15y E/O

^ ------? ? ? ? ? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 9 S

M M M 3y 5y 24 39 12y E/O E/O

------? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 8 F H M M +° 7y 23 38 21y 31y E/O E/O M,^

------? ? + + + + + + + + + + + + + + + + + + + + ^ + + + + + + + + + 7 E M M M 4y 3y 22 37 22y E/O WV,*

------? ? ? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + ^ + + + + + + + + + 6 F M M 2y 21 36 42y E/O E/O C,BA 4y2m S,N,O

------? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 5 F E M M 4y 20 35 S,A 13y 3y8m WV,C

------? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 4 S E A M M M 34 19 13y 24y 14y

------

? ? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 3 F F S

O M

33 18

16y 17y

20y E/O E/O CM,WV

- - -

- -

- - -

? ? ? ? ?

? ? ?

? + + + +

+ + +

+ + + + +

+ +

+ + +

2 + F

F F

32 17 12y 10y 16y S,N

------? ? ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 1 E M M M 31 16 CM 11y 14y 12y S,M

tip/anteverted nares tip/anteverted

slanting palpebral fissures palpebral slanting slanting palpebral fissures palpebral slanting - - /glob devdelay /glob disab

OFC >2SDS >2SDS Height OFC >2SDS dev delay disab/glob Intell behavior Autistic/autistifom Motor retardation Hypotonia anomalies behavioral Other face Long /triangular forehead Prominent Slender habitus Slender Kyphoscoliosis excavatum/carinatum Pectus Long hands Advanced bone age laxity Joint Nl/WV/C/BA/CM Weight>2SDS Length>2SDS Prominent forehead Prominent Down eyes set Deeply bridge Depressed nasal nose Short nares tip/anteverted nasal Upturned Long philtrum Small mouth bow cupid vermillion/ upper Thin (O) appearance mouth /open (E) lip lower Everted chin Prominent Vision impaired:H/S/N/M/A/O sclerae Blue Weight>2SDS Length>2SDS OFC >2SDS >2SDS Height OFC >2SDS dev delay disab/glob Intell behavior Autistic/autistifom Motor retardation Hypotonia anomalies behavioral Other face Long /triangular Small mouth bow cupid vermillion/ upper Thin (O) appearance mouth /open (E) lip lower Everted chin Prominent Vision impaired:H/S/N/M/A/O sclerae Blue habitus Slender Kyphoscoliosis excavatum/carinatum Pectus hands Long Advanced age bone laxity Joint Nl/WV/C/BA/CM Length>2SDS OFC >2SDS >2SDS Height OFC >2SDS Intell behavior Autistic/autistifom Motor retardation Hypotonia anomalies behavioral Other face Long /triangular forehead Prominent Down eyes set Deeply bridge Depressed nasal nose Short nasal Upturned Long philtrum Weight>2SDS

ID ID ID

Seizures/EEG anomalies Gender Age growth Prenatal growth Postnatal Development Craniofacial Development Craniofacial Eyes Skeletal Brain MRI Craniofacial Craniofacial Eyes Skeletal Brain MRI Seizures/EEG anomalies Gender Age growth Prenatal growth Postnatal Gender Age growth Prenatal growth Postnatal Development 6 — 174 Table Ia. continued Craniofacial Development Postnatal growth Prenatal growth Age Gender anomalies Seizures/EEG MRI Brain Skeletal Eyes Craniofacial malformation Chiari disk pallor; ventricles; Wide WV: C: Corpus callosum underdevelopment; BA: ? unknown;H: Hypermetropia; S:Strabismus; myopia; A:Astigmatism;O:opti M: N:Nystagmus; anomalies Seizures/EEG MRI Brain Skeletal Eyes subtle corticalsplasia dy leukomalacia, enlarged extra posterior lat

ID eral

ventricle

^ Underdeveloped optic nerve ° Reduced white volume matter posteriorly,

Ç tunnel vision Thin upper vermillion/ cupid bow mouth Small Long philtrum Upturned nasal tip/anteverted nares Short nose nasalDepressed bridge Deeply set eyes Down Prominent forehead Long /triangular face Other behavioral anomalies Hypotonia retardationMotor Prominent forehead Long /triangular face Other behavioral anomalies Hypotonia retardationMotor Autistic/autistifom behavior Intell disab/glob devdelay >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Nl/WV/C/BA/CM Joint laxity boneage Advanced Long hands Pectus excavatum/carinatum Kyphoscoliosis Slender habitus Blue sclerae impaired:H/S/N/M/A/O Vision Prominent chin Everted lower lip (E) /open mouth appearance (O) Vision impaired:H/S/N/M/A/O Vision Prominent chin Everted lower lip (E) /open mouth appearance (O) Thin upper vermillion/ cupid bow mouth Small Long philtrum Upturned nasal tip/anteverted nares Short nose nasalDepressed bridge Deeply set eyes Down Nl/WV/C/BA/CM Joint laxity boneage Advanced Long hands Pectus excavatum/carinatum Kyphoscoliosis Slender habitus Blue sclerae - axial space frontally, bilateral p - - slanting palpebral fissures slanting palpebral fissures

left papillar coloboma left papillar

eriventricular nodular heterotopia (R>L),

s mall area heterotopia at

* Mild periventricular

Brain atrophy; CM:

11y E/O 31 M + + + + + + + + + + + + + + + + + + + + + + + + + ? ? ? + + + + + + + + + ------? ------

12y E/O 32 M F + + + + + + + + + + + + ? ? ? + + + + + + ------

c E/O 16y E 33 M O + + + + + + + + + + + + + + + + + + + ? ? ? S + + + + + + + + + ------? /O ------

13y E/O 34 M A + + + + + + + + + + + + + + + + ? + + + + + + ------? ------

WV,C 3y8m

S,A

E/O WV

F + + + + + + + + + + + + + +

+ + + + + + ?

+ + + + + + + + + + + ------

- - ?

- - - - -

S,N,O 4y2m C,BA E/O 36 M + + + + + + + + + + + + + + + + + + + ? O + + + + + + + + + ------? ------

H,S,N,MA,O 22y 37 M E + + + + + + + + + + + + + + + + + + ? ? O C + + + + + + + + + + + ------

A,S,ç M,^ E/O 21y E/O 38 +° M + + + + + + + + + + + + + + + + + + + + ? ? + + + + + + + + + + ------

E/O E/O 39 3y M

+ + + + + + + + + + + + + + + + + + ? ? ? ? S + + + + + + + + + + + + ------^ ------

S, E 40 3y M + + + + + + + + + + + + + + + + + ? ? ? + + + ------? O^ /O ------

WV, C WV, S,M E/O 10y 41 O^ M + + + + + + + + + + + + + + + + + + + + + + + ? ? E + + + + + + + ------? ------

21y WV 42 M M + + + + + + + + + + + + + + + + + + + + ? O ^ + + + + ------? ------

E/O CM S + + + + + + + + + + ------

7WV 5C3CM 7WV

16F/26M O:28/42 E:30/42 TOTAL S,N,^ 40/42 35/42 26/41 25/38 32/37 16/3 42/42 34/42 21/41 12/25 13/33 12/38 19/27 19/3 12/42 12/42 25/42 32/42 30/42 26/42 26/42 13/42 19/42 17/42 12/42 28/42 28/42 5/28 4/40 8/42 9/44 E/O WV + + + + + + + + + + + + + + ------

C + + + + + + + + + + + + + + ? ? ------

6 — 175

------? ? + + + + + + + + + + + + + + C

------+ + + + + + + + + + + + + + WV E/O 8/42 9/44 4/40 5/28 13/33 28/42 28/42 12/42 17/42 19/42 13/42 26/42 26/42 30/42 32/42 25/42 12/42 12/42 19/3 19/27 12/38 34/42 42/42 16/3 32/37 25/38 26/41 35/42 40/42 12/25 21/41 S,N,^ TOTAL E:30/42 O:28/42 16F/26M

7WV 5C 3CM

------+ + + + + + + + + + S CM E/O

------? ------+ + + + ^ O ? + + + + + + + + + + + + + + + + + + + + M M 42 WV 21y

------? ------+ + + + + + + E ? ? + + + + + + + + + + + + + + + + + + + + + + + M O^ 41 10y E/O S,M WV, C

------/O O^ ? ------+ + + ? ? ? + + + + + + + + + + + + + + + + + M 3y 40 E S,

------^ ------+ + + + + + + + + + + + S ? ? ? ? + + + + + + + + + + + + + + + + + +

M 3y 39 E/O E/O

------+ + + + + + + + + + ? ? + + + + + + + + + + + + + + + + + + + + M +° 38 E/O 21y E/O M,^ A,S,ç

------+ + + + + + + + + + + C O ? ? + + + + + + + + + + + + + + + + + + E M 37 22y H,S,N,MA,O

------? ------+ + + + + + + + + O ? + + + + + + + + + + + + + + + + + + + M 36 E/O C,BA 4y2m S,N,O

- - - - - ? ------+ + + + + + + + + + + ? + + + + + + + + + + + + + + + + + + + + F E 35 S, WV E/O S,A 3y8m WV,C

------? ------+ + + + + + ? + + + + + + + + + + + + + + + + A M 34 E/O 13y

------/O ? ------+ + + + + + + + + S

? ? ? + + + + + + + + + + + + + + + + + + + O

M 33

E 16y E/O c

- -

- - -

- - - - -

- - -

+ + +

? ? ?

+ + +

F M

32 E/O 12y

------? ------+ + + + + + + + + ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + M 31 E/O 11y

Brain atrophy; CM: atrophy; Brain

* Mild periventricular periventricular Mild *

mall area heterotopia at at heterotopia area mall s

eriventricular nodular heterotopia (R>L), (R>L), heterotopia nodular eriventricular

left papillarleft coloboma

slanting palpebral fissures palpebral slanting slanting palpebral fissures palpebral slanting - - axial space frontally, bilateral p bilateral space frontally, axial - sclerae Blue habitus Slender Kyphoscoliosis excavatum/carinatum Pectus Long hands Advanced bone age laxity Joint Nl/WV/C/BA/CM Down eyes set Deeply bridge Depressed nasal nose Short nares rted tip/anteve nasal Upturned Long philtrum Small mouth bow cupid vermillion/ upper Thin (O) appearance mouth /open (E) lip lower Everted chin Prominent Vision impaired:H/S/N/M/A/O Prominent forehead Prominent Weight>2SDS Length>2SDS OFC >2SDS >2SDS Height OFC >2SDS dev delay disab/glob Intell behavior Autistic/autistifom Motor retardation Hypotonia anomalies behavioral Other face Long /triangular Everted lower lip (E) /open mouth appearance (O) appearance mouth /open (E) lip lower Everted chin Prominent Vision impaired:H/S/N/M/A/O sclerae Blue habitus Slender Kyphoscoliosis excavatum/carinatum Pectus hands Long Advanced age bone laxity Joint Nl/WV/C/BA/CM Motor retardation Hypotonia anomalies behavioral Other face Long /triangular forehead Prominent Down eyes set Deeply bridge Depressed nasal nose Short nares tip/anteverted nasal Upturned Long philtrum Small mouth bow cupid vermillion/ upper Thin Ç tunnel vision vision Ç tunnel

° Reduced volume white matter posteriorly, posteriorly, matter volume white Reduced ° nerve optic ^ Underdeveloped

ventricle

eral ID

posterior lat posterior leukomalacia, enlarged extra enlarged leukomalacia, dysplasia cortical subtle Brain MRI Seizures/EEG anomalies Eyes Skeletal ? unknown; H: Hypermetropia; S:Strabismus; N: Nystagmus; M: A: Astigmatism; O: opti myopia; S:Strabismus; Hypermetropia; ? unknown; H: BA: underdevelopment; callosum Corpus C: WV: Wide ventricles; pallor; disk Chiari malformation Development Craniofacial Eyes Skeletal Brain MRI Seizures/EEG anomalies Gender Age growth Prenatal growth Postnatal Craniofacial

6 — 176 Table Ib.Patients withMalanSyndrome and Variants inNFIXReportedLiterature Anomalies Seizures/EEG MRI Brain Skeletal Eyes Craniofacial signs Development Postnatal growth Prenatal growth mutation NFIX Gender per Underdeveloped optic nerve ventricles; Wide WV: C: Corpus callosum ? unknown;H: Hypermetropia; S:Strabismus; myopia;A:Astigmatism;O: optic M: N:Nystagmus; disk pallor; sistent pellucidum septum cavum

Advanced boneage Advanced Long hands ecavatum/carinatum Pectus Kyphoscoliosis Slender habitus Blue sclerae impaired:H/S/N/M/A/O Vision Prominent chin appearance (O) /open mouth lip (E) lower Everted bow vermillion/cupid Thin upper mouth Small Long philtrum tip/anteverted nares Upturned nasal Short nose nasalDepressed bridge Deeply set eyes palpebral fissures Down Prominent forehead Long /triangular face anomalies Other behavioral Hypotonia retardationMotor behavior Autistic/autistifom delay disability/glob dev Intellectual >2SDS OFC Height >2SDS >2SDS OFC Length>2SDS Weight>2SDS Nl/WV/C/BA/CM Joint laxity

- slanting slanting

;

# perivent

ricular white matter hyperintensity, possible heterotopias and a

nderdevelopment; BA: (Gln190*) Malan 2010 S,N E,O WV p. F + + + + + + + + + + + + + + + + ------

(Le60Pro) E,O p. S F + + + + + + + + + + + + + + + + + + + ? ? ? ? ------

Yoneda

2012

Brain atrophy; CM: Chiari malformation Chiari CM: atrophy; Brain p.(Arg121

Pro)

M O + + + + + + + + + + ? ? ? ? ? ------

Glu59del) p.(Glu53_ S,N, O S,N, Priolo 2012

E,O C, ^ C, F + + + + + + + + + + + + + + + + + + + + + + + + + + + + -

p.(Trp30C 24) ysfs* WV, # WV, E.O S F + + + + + + + + + + + + + + + + ? ? ? ------

Klaassen

2015

p.(Gln3 38*)

E,O M ; S + + + + + + + + + + ? ? ? ------

p.(Arg11

6Pro) WV,C E,O

F + + + + + + + + + + + + + + + + + + + + + + ------

Gurrieri p.(Lys125 2015

WV,C Glu) E,O O F + + + + + + + + + + + + + + + + + + + + + + + ------

p.(Lys64S erfs*30) E,O M O + + + + + + + + + + + + + + + + + + + + + + ------

p.(Arg38C E,O ys) M S + + + + + + + + + + + + + + + + + + + + + Martinez ------

2015 p.(Arg54P

ro) O S F + + + + + + + + + + + + + + + + ------

p.(Arg115 Jezela Stanek 2016 Trp) E,O WV S F + + + + + + + + + + + + + + + ? ? ------

p.(Asp90G Oshima lufs*29) 2017 M + + + + + + + + + + + + + + ? ? ? ? ? ? ? ? ? ? ? ------

p.(Lys125 Asn) 2017 M + + + + + ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Lu ------

O:12/12 E:10/12 5wv/3c 6 10/11 10/13 1 11/13 10/12 12/12 11/13 13/13 12/13 11/13 12/13 14/14 10/13 10/14 2/13 6/11 8/11 9/12 7/14 5/14 6/10 9/12 9/12 9/12 7/12 3/12 8/13 2/12 6/12 2/12 1/14 Total M/8F 0/14

6 — 177

0/14 M/8F Total 7/14 9/12 8/11 6/11 2/13 2/12 8/13 3/12 7/12 9/12 9/12 9/12 6/10 5/14 1/14 2/12 6/12 10/11 10/14 10/13 14/14 12/13 11/13 12/13 13/13 11/13 12/12 10/12 11/13 1 10/13 6 5wv/3c E:10/12 O:12/12

------Lu ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? + + + + + M 2017 Asn) p.(Lys125

------? ? ? ? ? ? ? ? ? ? ? + + + + + + + + + + + + + + M 2017 lufs*29) Oshima p.(Asp90G

------? ? + + + + + + + + + + + + + + + F S WV E,O Trp) 2016 Stanek Jezela p.(Arg115

------+ + + + + + + + + + + + + + + + F S O ro)

p.(Arg54P 2015

------Martinez + + + + + + + + + + + + + + + + + + + + + S M ys) E,O p.(Arg38C

------+ + + + + + + + + + + + + + + + + + + + + + O M E,O erfs*30) p.(Lys64S

------+ + + + + + + + + + + + + + + + + + + + + + + F O E,O Glu) WV,C

2015 p.(Lys125 Gurrieri

------+ + + + + + + + + + + + + + + + + + + + + + F E,O

WV,C 6Pro)

p.(Arg11

- -

? ?

? +

+ +

+ S

;

E,O

38*) p.(Gln3

2015

Klaassen

------? ? ? + + + + + + + + + + + + + + + + F S E.O WV, # ysfs*24) p.(Trp30C

- + + + + + + + + + + + + + + + + + + + + + + + + + + + + F C, ^ E,O

2012 Priolo S,N, O p.(Glu53_ Glu59del)

------? ? ? ? ? + + + + + + + + + + O M

Pro)

p.(Arg121 Brain atrophy; CM: Chiari malformation

2012

Yoneda

------? ? ? ? + + + + + + + + + + + + + + + + + + + F S p. E,O (Le60Pro)

------+ + + + + + + + + + + + + + + + F p. WV E,O S,N 2010 Malan (Gln190*) nderdevelopment; BA: nderdevelopment;

ricular white matter hyperintensity, possible heterotopias and a heterotopias possible hyperintensity, matter white ricular

# perivent

;

slanting -

Pectus Pectus ecavatum/carinatum Long hands Advanced age bone laxity Joint Nl/WV/C/BA/CM Height >2SDS Height OFC > 2SDS Intellectual dev disability/glob delay Autistic/autistifom behavior Motor retardation Hypotonia behavioral Other anomalies face Long /triangular forehead Prominent Down fissures palpebral eyes set Deeply bridge Depressed nasal nose Short nasal Upturned nares tip/anteverted Long philtrum Small mouth upper Thin vermillion/cupid bow Evertedlower lip(E) mouth /open (O) appearance chin Prominent Vision impaired:H/S/N/M/A/O sclerae Blue habitus Slender Kyphoscoliosis Weight>2SDS Length>2SDS OFC >2SDS

sistent cavum septum pellucidum sistent ? unknown; H: Hypermetropia; S:Strabismus; N: Nystagmus; M: myopia; A: Astigmatism; O: optic disk pallor; pallor; N: Nystagmus; disk M: myopia; A: Astigmatism; O: optic S:Strabismus; Hypermetropia; ? unknown; H: callosum Corpus C: WV: Wide ventricles; nerve optic Underdeveloped per Brain MRI Seizures/EEG Anomalies Postnatal growth Postnatal Development signs Craniofacial Eyes Skeletal Gender NFIX mutation growth Prenatal

6 — 178 Table II.Patients withDeletionsofCompleteNFIXinthePresentReportandReportedinLiterature mildly reduced of volume the white type1 matter, Cm Pz3 (°)WV; In Klaassens series. Pz1: S,N; Pz2: S; Pz3: S; (&) In Shimoima seri In Dolan series. Vision impaired pz1 anomalies Seizures/EEG MRI Brain Skeletal Eyes Craniofacial Development growth Postnatal growth Prenatal A Gender WV: Wide ventricles; Wide WV: C: corpus callosum underdevelopment; BA: brain atrophy; Chiari CM: malformation; (*) ? unknown;H: Hypermetropia; S:Strabismus; myopia;A:Astigmatism;O: optic M: N:Nystagmus; disk pallor and optic nerve underdevelopment; Pz2: mild Brain atrophy MRI of frontal lobes; pz3: C, cystic lesion; pz4: ge

Nl/WV/C/BA/CM Joint laxity boneag Advanced Large long hands Pectus excavatum/carinatum Kyphoscoliosis Slender habitus Blue sclerae impaired:H/S/N/M/A/O Vision Pointed/prominent chin appearancemouth (O) Everted lower lip (E) /open bow Thin upper vermillion/cupid mouth Small Long philtrum tip/anteverted nares Upturned Nasal Short nose Depressed set eyes Deep fissures Down Prominent forehead Long (L) /triangular (T) face Other behavioral anomalies Hypotonia retardationMotor Autistic/autistifom behavior dev delay disability Intellectual >2SDS OFC Height >2 (cm)>2SDS OFC Birth height (cm)>2SDS (g)>2SDS weight Birth

- slanting palpebral SDS

nasal bridge

/glob /glob : S,N; pz2: S,O and optic nerve underdevelopment; pz3: S, O, pz4: S,N,O

S,N,M, WV,C E,O 4 9y M O + + + + + + + + + + + + + + + + + + + + + + + + + - - - - 3

Present report

17y E,O

4 M + + + + + + + + + + + + + + + + + ? ? ? ? ------4

21y 4 O S F + + + + + + + + + + + + + + ------5

es. Pz1 CM type1; Pz2: Cm type1, type1; Pz2:Cm es. CM Pz1 Auvin Auvin 2009 E,O M + + + + + + + + + + + + + + + + ? ? ------

2/2H,S/H,A 1/2 WV,C 1/2 1 Malan Malan 2010 M/ E,O 1/2 1/2 1/2 0/2 2/2 2/2 2/2 2/2 1/2 2/2 1/2 2/2 2/2 1/2 1/2 0/2 2/2 2/2 1/2 1/2 1/2 2/2 2/2 2/2 2/2 1/2 2/2 2/2 2/2 2/2

3F/1M 4/4 (*) 4/4 Dolan 3/4(*) 2010 3/3 3/4 2/4 1/1 2/2 1/4 2/4 3/4 4/4 2/4 1/4 2/4 1/4 3/4 1/4 2/4 1/4 4/4 4/4 3/4 4/4 0/4 4/4 4/4 3/4 ? ? ? ? ?

Nimma kayalu 2/2S/S

1/2 C 2013 1/2 1/2 1/2 1/2 0/2 1/2 2/2 0/2 1/2 1/2 1/2 0/2 0/2 0/2 0/2 2/2 2/2 0/2 2/2 2/2 0/2 2/2 2/2 1/2 2 ? ? ? ? ? F

- Natiq 2014 E,O BA S F + + + + + + + + + + + + + + + + ------

jima 2014 Shimo- 2/3 (&) 1/3 0/3 0/3 2/3 2/3 0/3 1/3 1/3 3/3 2/3 2/3 3/3 2/3 1/3 2/3 2/3 3/3 3/3 0/3 3/3 3/3 0/3 3/3 1/3 1/3 3F ? ? ? ? ? ?

Klaassens 2M/2F 3/4 (°) 3/4 2015 E,O 0/4 0/4 1/4 1/4 1/1 1/1 3/4 1/4 0/4 2/2 2/2 2/2 2/2 2/2 2/2 2/2 1/2 1/2 3/4 4/4 2/4 1/4 2/4 4/4 1/4 4/4 1/4 2/4 ? ? ?

Jorge 2015 S,N F + + + + + + + + + + + + ? ? ? ? ? ? ? ? ? ? ? ? ? ? - - - - -

Jazela Stanek 2016 E,O S F + + + + + + + + + + + + + + + ? ------

- Dong Dong 2016 E,O M C S + + + + + + + + + + + + + + ? ? ? ------

Kuroda Kuroda 2017 WV O S F + + + + + + + + + + + + + ? ? ------

4WV/6c/2cm 8M/16 F 8M/16 O:16/21 E:14/21 1 11/20 11/12 10/15 11/17 11/23 18/24 16/21 16/21 10/21 14/22 15/22 13/20 13/21 11/21 24/24 20/24 21/24 23/24 24/24 16/24 13/24 Total 6/12 5/16 4/13 7/21 6/19 6/23 7/23 2/19 6/23 1/24

6 — 179

1/24 6/23 6/19 7/21 4/13 5/16 6/12 6/23 2/19 7/23 Total 1 13/24 16/24 24/24 23/24 21/24 20/24 24/24 11/21 13/21 13/20 15/22 14/22 10/21 16/21 16/21 18/24 11/23 11/17 10/15 11/12 11/20 E:14/21 O:16/21 8M/16 F 4WV/6c/2cm

------? ? + + + + + + + + + + + + + F S O WV 2017 Kuroda

------? ? ? + + + + + + + + + + + + + + S C M E,O 2016 Dong -

------? + + + + + + + + + + + + + + + F S E,O 2016 Stanek Stanek Jazela

- - - - - ? ? ? ? ? ? ? ? ? ? ? ? ? ? + + + + + + + + + + + + F S,N 2015 Jorge

? ? ? 0/4 1/4 3/4 1/1 1/1 1/4 4/4 3/4 1/2 1/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 1/4 0/4 0/4 2/4 1/4 4/4 1/4 4/4 2/4 1/4 2/4 E,O 2015 3/4 (°) 2M/2F Klaassens Klaassens

? ? ? ? ? ? 3F 2/3 1/3 1/3 0/3 2/3 1/3 2/3 3/3 2/3 2/3 3/3 3/3 3/3 0/3 3/3 3/3 2/3 2/3 0/3 0/3 1/3 1/3 1/3 3/3 0/3 2/3 (&) 2/3 - Shimo jima 2014 jima

------+ + + + + + + + + + + + + + + + F S BA E,O 2014 Natiq Natiq -

F ? ? ? ? ? 2 1/2 1/2 1/2 0/2 1/2 1/2 2/2 1/2 0/2 0/2 1/2 1/2 0/2 0/2 0/2 0/2 2/2 2/2 2/2 0/2 2/2 2/2 1/2 2/2 2013 1/2 C 1/2

2/2S/S kayalu Nimma

? ? ? ? ?

2/4

1/1 2/2

1/4 2/4

3/4 4/4

2/4 1/4

3/4 1/4

2/4 1/4

4/4 4/4

3/4 4/4

0/4 4/4

4/4 3/4

3/3

3/4

2010

3/4(*)

Dolan Dolan

4/4 (*) 3F/1M

0/2 2/2 2/2 2/2 2/2 1/2 2/2 1/2 2/2 2/2 1/2 1/2 0/2 2/2 2/2 1/2 1/2 1/2 2/2 2/2 2/2 2/2 2/2 1/2 2/2 2/2 1/2 2/2 1/2 1/2 E,O M/ 2010 Malan 1 1/2 WV,C 2/2H,S/H,A

------? ? + + + + + + + + + + + + + + + + M E,O 2009 Auvin es. Pz1es. CM Pz2: Cm type1; type1,

5 ------+ + + + + + + + + + + + + + F S O 4 21y

4 ------? ? ? ? + + + + + + + + + + + + + + + + + M 4

E,O 17y

Present reportPresent

3 - - - - + + + + + + + + + + + + + + + + + + + + + + + + + O M 9y 4 E,O WV,C S,N,M,

: S,N; pz2: S,O and optic nerve underdevelopment; pz3: S, O, pz4: S,N,O S,N,O pz4: O, S, pz3: underdevelopment; nerve optic and S,O pz2: S,N; : /glob

bridge nasal

SDS slanting palpebral palpebral slanting -

Height >2 Height OFC >2SDS Intellectual disability delay dev behavior Autistic/autistifom Motor retardation Hypotonia anomalies behavioral Other face (T) /triangular Long (L) forehead Prominent Down fissures Deep eyes set Depressed nose Short Nasal Upturned nares tip/anteverted Long philtrum Small mouth vermillion/cupid upper Thin bow /open (E) lip lower Everted (O) mouth appearance chin Pointed/prominent Vision impaired:H/S/N/M/A/O sclerae Blue habitus Slender Kyphoscoliosis excavatum/carinatum Pectus hands long Large Advanced bone ag laxity Joint Nl/WV/C/BA/CM Birth weight (g)>2SDS (cm)>2SDS height Birth OFC (cm)>2SDS

ge ? unknown; H: Hypermetropia; S:Strabismus; N: Nystagmus; M: myopia; A: Astigmatism; O: optic disk pallor pallor disk N: Nystagmus; M: myopia; A: Astigmatism; O: optic S:Strabismus; Hypermetropia; ? unknown; H: (*) malformation; CM: Chiari atrophy; brain BA: underdevelopment; callosum corpus C: WV: Wide ventricles; pz1 impaired Vision series. Dolan In pz4: lesion; cystic C, pz3: lobes; frontal of MRI atrophy Brain mild Pz2: underdevelopment; nerve and optic seri Shimoima In (&) S; Pz3: S; Pz2: S,N; Pz1: series. Klaassens In WV; (°) Pz3 Cm matter, type1 white the volume of reduced mildly Seizures/EEG anomalies Postnatal growth Development Craniofacial Eyes Skeletal Brain MRI Gender A Prenatal growth 6 — 180 Tables andtext. Ages are mentioned below each picture.Fordetailed descriptions please see Numbers ofindividualsinthepanelscorrespondtonumbers inthe Tables. with Malansyndrome Fig. S8a,S8b,S8c.Clinical phenotypeofpresently reported42individuals A. Sidefacialviewofalimitednumberpresently reportedindividuals. 6 — 181 Changes with age in a limited number of presently reported individuals C. Changes with age in a limited number of presently Distal limb characteristics and general body build in a limited number and general B. Distal limb characteristics reported individuals. of presently