Marshall-Smith syndrome: Novel pathogenic variant and previously unreported associations with precocious puberty and aortic root dilatation

Accepted Manuscript Marshall-Smith Syndrome: Novel pathogenic variant and previously unreported associations with precocious puberty and aortic root dilatation Anjali Aggarwal, Joanne Nguyen, Michelle Rivera-Davila, David Rodriguez-Buritica, MD PII:

S1769-7212(16)30326-3

DOI:

10.1016/j.ejmg.2017.04.012

Reference:

EJMG 3287

To appear in:

European Journal of Medical Genetics

Received Date: 13 September 2016 Revised Date:

23 February 2017

Accepted Date: 12 April 2017

Please cite this article as: A. Aggarwal, J. Nguyen, M. Rivera-Davila, D. Rodriguez-Buritica, MarshallSmith Syndrome: Novel pathogenic variant and previously unreported associations with precocious puberty and aortic root dilatation, European Journal of Medical Genetics (2017), doi: 10.1016/ j.ejmg.2017.04.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1 1

Marshall-Smith Syndrome: Novel Pathogenic Variant and Previously Unreported

2

Associations with Precocious Puberty and Aortic Root Dilatation

RI PT

3 4

Authors: Anjali Aggarwal2, Joanne Nguyen2, Michelle Rivera-Davila1, David Rodriguez-

5

Buritica2

SC

6

Divisions of 1Endocrinology and 2Genetics, Department of Pediatrics, University of Texas

8

Health Science Center at Houston, 6431 Fannin Street, Houston, Texas 77030

M AN U

7

9

12 13

TE D

11

Running title: Marshall-Smith syndrome

Conflict of interest disclosures: None.

EP

10

Correspondence to: David Rodriguez-Buritica, MD. 6431 Fannin Street, MSB 3.142, Houston,

15

TX 77030. Ph: 713-500-5755. Fax: 713-383-1475. Email:

16

[email protected]

17 18 19

AC C

14

ACCEPTED MANUSCRIPT 2 20 ABSTRACT

22

Marshall-Smith Syndrome (MRSHSS) is a very rare genetic disorder characterized by failure to

23

thrive and characteristic dysmorphic features associated with accelerated osseous maturation. We

24

present a nine-year-old girl who was diagnosed with MRSHSS based on characteristic clinical

25

features supported by the identification of a novel de novo pathogenic variant in the NFIX gene.

26

The patient also presented with precocious puberty diagnosed at five years of age and had an

27

abnormal GnRH stimulation test indicative of central precocious puberty. Central precocious

28

puberty has not been described in association with MRSHSS previously in the medical literature

29

and broadens our knowledge of the natural history of MRSHSS. The causes of advanced bone

30

age in this syndrome are also reviewed. Additionally, the patient showed progressive dilatation

31

of the aortic root. Although connective tissue abnormalities have been described in association

32

with MRSHSS, aortic root dilatation has not. Understanding the mechanism of comorbidities

33

such as advanced bone age and aortic root dilatation in MRSHSS patients enables future

34

development of anticipatory guidance, preventative care measures, and treatment guidelines.

36 37 38 39 40

SC

M AN U

TE D

EP AC C

35

RI PT

21

ACCEPTED MANUSCRIPT 3 41 42 KEY WORDS: Marshall-Smith syndrome; advanced bone age; precocious puberty; novel

44

pathogenic variant; aortic root dilatation

RI PT

43

45

SC

46

M AN U

47 48 49 50

54 55 56 57 58 59 60

EP

53

AC C

52

TE D

51

ACCEPTED MANUSCRIPT 4 61 62 TEXT

64

INTRODUCTION

65

Marshall-Smith Syndrome (MRSHSS) is a rare disorder first described in 1971 by Marshall et al

66

[1971]. It is characterized by dysmorphic features, failure to thrive, developmental delay, and

67

abnormal osseous maturation. In the literature, there are less than 100 patients described

68

worldwide but identification of patients is increasing with the availability of next-generation

69

sequencing. MRSHSS appears to be present among all populations, affecting males and females

70

equally, typically arising from a de novo occurrence. Historically, reported patients often died in

71

the neonatal period or early infancy, most commonly from respiratory compromise, but long

72

term survivors have been reported. As the number of known children and adults are small, many

73

aspects of the syndrome remain unknown. We discuss an unreported association of precocious

74

puberty in a patient with MRSHSS. Additionally, the currently reported patient showed

75

progressive dilatation of the aortic root. Connective tissue abnormalities have been described in

76

MRSHSS, but there is no report of aortic root dilatation to the best of our knowledge. Given that

77

mortality is decreasing and health outcomes are improving with modern medical care, further

78

natural history studies should continue to track the progression of puberty in affected individuals.

79

CASE REPORT

80

We report a nine-year-old girl who initially presented to our Medical Genetics clinic for

81

evaluation when she was four years old. She was born to a 19-year-old primigravida mother and

82

nonconsanguineous 24-year-old father. There is no significant family history. There were no

AC C

EP

TE D

M AN U

SC

RI PT

63

ACCEPTED MANUSCRIPT 5 known exposures during pregnancy. Detection of fetal hydrocephalus during a routine antenatal

84

ultrasound led to amniocentesis which revealed a 46,XX karyotype. At 38 weeks gestation,

85

Cesarean section was performed secondary to fetal hydrocephalus. Birth weight was 2.863 kg

86

(25-50th percentile), length was 50 cm (50-75th percentile), and head circumference was 34.5 cm

87

(50-75th percentile). APGAR scores were 8 and 9, at 1 and 5 minutes, respectively. She remained

88

in the neonatal intensive care unit for approximately one month due to poor feeding requiring

89

gastrostomy tube placement, which was eventually removed at the age of 12 months. Post-natal

90

brain MRI showed an absent septum pellucidum and communicating hydrocephalus which was

91

noted to improve on serial brain imaging and did not require surgical intervention.

SC

M AN U

92

RI PT

83

Development was globally delayed. She sat at one year, crawled at three years, and walked without assistance at four years. She has intellectual disability and attends special

94

education classes. She has three words and approximately 60 signs to indicate her needs. She is

95

toilet trained but has nocturnal incontinence. Past medical history also includes recurrent ear

96

infections requiring bilateral myringotomy and pneumatic equalization tube placement, hearing

97

loss, myopia, and astigmatism. The initial eye exam and imaging suggested small optic nerves

98

and subsequent detailed evaluations revealed optic atrophy.

EP

99

TE D

93

At eight years of age, weight was 21.4 kg (3-10th percentile), height 124.5 cm (10-25th percentile), and head circumference 53.5 cm (90-95th percentile). Physical examination

101

demonstrates a triangular face, wide forehead, large, folded and low set ears, prominent eyes,

102

depressed nasal bridge, anteverted nares, short philtrum, everted lips and microretrognathia (Fig

103

1a, 1b). Additional features included hirsutism, thin extremities with decreased muscle bulk,

104

hyperextensible joints, long fingers, clinodactyly, kyphosis, and bilateral pes plano valgus. She

105

was noted to have thelarche (Tanner stage 2) at the age of 4-½ years. Growth velocity increased

AC C

100

ACCEPTED MANUSCRIPT 6 (8 cm/year) and pubertal changes progressed over the ensuing four months with the appearance

107

of fine downy hair on the mons pubis and Tanner stage 3 breast development for which she

108

underwent detailed hormonal evaluation.

109

RESULTS

110

Endocrine studies: Initial hormonal evaluation at 4-½ years old was normal. This included free

111

thyroxine (FT4): 1.15 ng/dL (0.76-1.46 ng/dL), thyroid stimulating hormone (TSH), 3rd

112

generation: 2.490 mIU/L (0.360-3.740 mIU/L), estradiol: 12.9 pg/ml (<15 pg/ml), follicle-

113

stimulating hormone (FSH): 2.5 mIU/mL (1.0-4.2), luteinizing hormone (LH): 0.04 mIU/mL

114

(0.02-0.3), adrenocorticotrophic hormone (ACTH): 16 pg/mL (0-46 pg/mL), and cortisol: 9.3

115

mcg/dL (3.0-23.0 mcg/dL ).

SC

M AN U

116

RI PT

106

Provocative testing was performed as she advanced with puberty clinically. GnRH analog was administered at a dose of 20 mcg/kg during which LH, FSH, and estradiol levels were

118

obtained at baseline and at 180 minutes with additional LH and FSH levels every 60 minutes

119

until 180 minutes. Results are shown in Supplementary Table I (supplementary material is

120

available online). The GnRH stimulation test confirmed central precocious puberty and the

121

patient was started on depot GnRH analog at the age of five years. Serial monitoring of FSH,

122

LH and bone age on therapy are shown in Fig 2.

123

Imaging: Brain MRI at four years and nine months old revealed diffuse paucity of white matter

124

with compensatory dilation of the ventricles with associated colpocephaly, absent septum

125

pellucidum, small optic nerves and tracts, and dysmorphic corpus callosum. No abnormalities

126

were detected in the pituitary. Major intracranial flow voids were maintained. Repeat MRI at the

127

age of eight years showed no significant change.

AC C

EP

TE D

117

ACCEPTED MANUSCRIPT 7 128

The Greulich and Pyle (GP) bone age at the chronological age of four years ten months was seven years. Treatment was started at the age of five years. Bone age (BA) at five years five

130

months old was eight years ten months and at eight years one month old was between 10-11

131

years. BA/CA (bone age/chronological age) ratio progressively decreased on treatment (Fig 2b).

132

Echocardiogram (Echo): Initial Echo at the age of 7 years 11 months showed mild dilatation of

133

aortic root at the sinotubular junction (Ao ST Jx) measuring 20.95 mm (Z score: +2.16).

134

Diameter of the aorta at the level of the sinus of Valsalva (Ao-Sinus of Valsalva) was 23.52 mm

135

(Z score: +1.68) and aortic valve annulus (AoV ann) measured 14.93 mm (Z score: 0.36). A

136

follow-up Echo at the age of 9 years 6 months showed progressive dilatation of the aortic root.

137

Ao ST Jx measured 23 mm (Z score: +3.3), Ao-Sinus of Valsalva 26 mm (Z score: +2.7), AoV

138

ann 19 mm (Z score: +2.7). Other than aortic root dilatation, the heart was structurally and

139

functionally normal.

140

Genetic testing: Chromosomal analysis showed an inversion on chromosome 9, which was

141

considered a benign variant. Chromosomal microarray was normal. Whole exome sequencing

142

(WES) at the age of six years revealed a Sanger validated novel de novo frameshift pathogenic

143

variant: NM_002501:c.990_997del (p.D330fs) in exon 7 of NFIX located on Chr19:13189459.

144

Paternal sample was not included. Mother tested negative. WES also revealed variants of

145

unknown clinical significance (VUS) for genes related to the clinical phenotype. These included,

146

TREX1 (Aicardi-Goutieres syndrome 1), LAMC3 (cortical malformations, occipital), WDR62

147

(microcephaly 2, primary, autosomal recessive, with cortical malformations) and COL18A1

148

(Knobloch syndrome, type 1). The associated syndromes were considered less likely given their

149

pattern of inheritance, phenotypic features and in-silico prediction. These are detailed in

AC C

EP

TE D

M AN U

SC

RI PT

129

ACCEPTED MANUSCRIPT 8 Supplementary Table 2. WES re-analysis at the age of 9 years revealed no additional findings or

151

variant re-classification.

152

DISCUSSION

153

The diagnosis of MRSHSS is established through a careful history and physical examination

154

with confirmatory molecular testing of NFIX. Currently, de novo pathogenic variants in NFIX are

155

the only known cause of MRSHSS [Malan et al, 2010]. Nuclear factor I, X-type (NFIX) is a

156

ubiquitous 47-kD dimeric DNA-binding protein, belonging to a family of transcription factors

157

whose gene was mapped to 19p13 by Seisenberger et al [1993]. Pathogenic variants in exons 2

158

and 3 of NFIX, that result in a dominant negative effect, have also been reported as causative of

159

Malan syndrome (Sotos syndrome 2; MIM: 614753) which is, in contrast to MRSHSS, an

160

overgrowth syndrome that resembles Sotos syndrome [Klaassens M et al, 2015]. Malan et al.

161

demonstrated ubiquitous expression of NFIX in all human tissues and cell types tested, including

162

the cerebral cortex, hippocampus, faintly in the thalamus, mandibular arch, cartilage primordium

163

of the humerus, scapula, and vertebrae, perichondrium, proliferating zone of the digit. The

164

pattern of expression of NFIX correlates with several of the clinical features of this syndrome.

SC

M AN U

TE D

EP

165

RI PT

150

A total of 19 NFIX point pathogenic variants associated with MRSHSS have been reported thus far [Martinez et al, 2015]. Previously reported spectrum of pathogenic variants

167

associated with MRSHSS includes 16 small insertions/deletions (61%), three splice site

168

pathogenic variants (12%), and seven larger deletions in exons (27%) [Schanze et al, 2014].

169

Pathogenic variants causing MRSHSS are distributed along exons 6 to 10. Most pathogenic

170

variants causing MRSHSS are clustered in exon 7 (as was present for the currently reported

171

patient), which is considered a pathogenic variant hotspot [Martinez et al, 2015]. MRSHSS-

AC C

166

ACCEPTED MANUSCRIPT 9 172

associated pathogenic variants encode dysfunctional proteins that act in a dominant negative

173

manner and result in a severe phenotype [Martinez et al, 2015]. Symptoms of the syndrome are usually present before birth, just as in the current patient

RI PT

174

where hydrocephalus was detected prenatally. Clinical features that have been described in

176

patients with MRSHSS [Shaw et al, 2010] include developmental delay, failure to thrive, unusual

177

craniofacial features including a high forehead, proptosis, midface hypoplasia, short nose,

178

anteverted nares, prominent premaxilla, short philtrum, everted lips, irregular dentition, gum

179

hypertrophy, retrognathia, narrow ear canals, abnormal pinnae and low set ears. Eye

180

abnormalities include astigmatism, myopia, blue sclerae, glaucoma, shallow orbits, and optic

181

nerve atrophy. Respiratory problems include obstructive sleep apnea and choanal stenosis.

182

Neurological features include hypertonia, hypotonia, cervical cord compression, dilated cerebral

183

ventricles, hypoplastic callosal body, pachygyria, polymicrogyria, and septo-optic dysplasia.

M AN U

SC

175

Cardiac features include ventricular septal defect and pulmonary hypertension. Aortic

185

root dilatation has not been described previously in patients with MRSHSS or other conditions

186

for which VUS were detected by WES. Though, connective tissue abnormalities such as easy

187

bruising, abnormal scarring, skin and joint hyperextensibility have been described in patients

188

with MRHSS before [Adam MP et al, 2005]. Thus, periodic echocardiograms may be considered

189

to screen individuals with MRSHSS for aortic abnormalities. Other features include hirsutism,

190

cryptorchidism and pes planus. The current patient demonstrates many of these clinical features.

191

Fortunately, she was not affected with early respiratory complications, a major cause of

192

morbidity and mortality in MRSHSS patients, which may contribute to her better survival and

193

health. She has since developed moderate persistent asthma at eight years of age which is well-

194

controlled with medication management.

AC C

EP

TE D

184

ACCEPTED MANUSCRIPT 10 195

Musculoskeletal features associated with MRSHSS include craniosynostosis, kypohoscoliosis, osteopenia, and bone fractures. Bone age is invariably reported as advanced at

197

birth and during childhood. Detailed medical records for the current patient including previous

198

imaging and bone age (prior to the age of four years) were not available. Full skeletal surveys of

199

individuals with MRSHSS do not show an advanced bone age elsewhere, and only mild

200

abnormal bone maturation in the long tubular bones (wide epiphyses). In the hand, the carpus

201

appears more advanced in age than the phalanges [Shaw et al, 2010]. Mesenchymal ossification

202

defects as described previously in the skeletal findings of MRSHSS most likely represent an

203

osteochondrodysplasia involving anarchic ossification of epiphyseal centers [Eich et al, 1991;

204

Hassan et al, 1976, Marshall et al, 1976]. Interestingly, advanced skeletal age has also been

205

described in Malan syndrome [Klaassens M et al, 2015].

SC

M AN U

206

RI PT

196

Central precocious puberty (CPP) is an interesting finding in the currently reported patient. CPP is defined as early activation of the hypothalamic-pituitary-gonadal (HPG) axis

208

before the age of nine years in boys and eight years in girls. Increase in linear growth, thelarche

209

and advanced skeletal age are clinical findings of precocious puberty in females. CPP is a known

210

cause of advanced BA. However, reports of MRSHSS patients with consistent advanced BA

211

have not demonstrated consistent clinical findings of precocious puberty in general, and CPP in

212

particular. A consideration may be that earlier reported patients often died prematurely before

213

they could have demonstrated clinical features of precocious puberty. Two case reports have

214

described detailed hormonal evaluations to test the HPG axis. The first patient [Matustik et al,

215

1985] was noted at birth to have a bone age of two years. At age eight months, the bone age was

216

six years. The following were normal for age: T4, FSH, LH, somatomedin-C, and testosterone.

217

17-hydroxyprogesterone (17-OHP) was elevated (840 ng/dl; nl 30-100 ng/dl) as was

AC C

EP

TE D

207

ACCEPTED MANUSCRIPT 11 androstenedione (67 ng/dl; n1<50 ng/dl). An ACTH stimulation test suggested an adrenal

219

enzymatic defect. ACTH stimulation tests were performed on both parents. The 30 minute

220

increase in 17-OHP was elevated in both (father-13.8 ng/dl/min; mother-8.9 ng/dl/min; nl<6.5

221

ng/dl/min) suggesting a heterozygous state for congenital adrenal hyperplasia. The authors

222

proposed that an inherited abnormality in androgen production may be contributing to the

223

osseous maturation seen in MRSHSS. The second patient, [Gomez-Santos, 2014] showed a bone

224

maturity of 14–15 months in her wrists at birth. Hormonal evaluation including ACTH, cortisol,

225

LH, FSH, TRH, TSH, growth hormone, testosterone, 17-OHP, aldosterone, and prolactin was

226

normal.

SC

M AN U

227

RI PT

218

Given that the GP BA/CA ratio improved in the current patient while on treatment with GnRH analogs suggests that the advanced bone age may be partly caused by premature HPG

229

activation. NFIX is known to be expressed in brain and the currently reported patient had no

230

identifiable known causes for CPP. We thus hypothesize that the etiology of CPP in this patient

231

could be related to her pathogenic NFIX pathogenic variant. Stature in MRSHSS patients is

232

typically normal in infancy and early childhood. By the second decade, height progressively

233

diverges from normal so that the final height is less than the third percentile. This is compounded

234

to some degree by kyphoscoliosis. Advanced bone age can lead to premature closure of the

235

epiphysis and ultimately result in short stature. Therefore, we suggest that MRSHSS patients be

236

monitored for clinical signs of precocious puberty. The current patient was managed with GnRH

237

analog therapy and has shown appropriate response clinically. This has not only stopped

238

progression of her pubertal characteristics but also decreased the gap between her CA and BA.

239

The CPP in MRSHSS patients can be challenging for endocrinologists who routinely utilize

AC C

EP

TE D

228

ACCEPTED MANUSCRIPT 12 240

serial bone age interpretations using wrist and hand radiographs as a treatment guide. It may be

241

more prudent to monitor the physical pubertal changes to guide therapy in these patients. With advancements in healthcare, there are now reports of adults with MRSHSS [Shaw et

RI PT

242

al, 2010]. We are in an era of medicine whereby it is no longer uncommon to encounter patients

244

with rare disorders growing into adulthood. Therefore, expanding our knowledge of these

245

conditions will benefit individuals with such rare disorders and their families by allowing

246

identification of the correct diagnosis, elimination of misdiagnoses, and individualized treatment

247

based on the underlying pathophysiology and molecular mechanism of the disease.

248

ACKNOWLEDGEMENT

249

We would like to thank the family for their participation in this study.

250

REFERENCES

M AN U

1. Adam MP, Hennekam RC, Keppen LD, Bull MJ, Clericuzio CL, Burke LW, Ormond

TE D

251

SC

243

KE, Hoyme EH. Marshall-Smith syndrome: natural history and evidence of an

253

osteochondrodysplasia with connective tissue abnormalities. Am J Med Genet

254

A. 2005 Aug 30;137(2):117-24.

256 257 258

2. Eich GF, Silver MM, Weksberg R, Daneman A, Costa T. Marshall-Smith syndrome: new radiographic, clinical, and

AC C

255

EP

252

pathologic observations. Radiology. 1991 Oct;

181(1):183–188.

3. Gómez-Santos E1, Lloreda-García JM, Fernández-Fructuoso JR, Martínez-Ferrández

259

C, Leante-Castellanos JL, Fuentes-Gutiérrez C. Neonatal Marshall-Smith syndrome.

260

Clin Dysmorphol. 2014 Apr; 23(2):42-44.

ACCEPTED MANUSCRIPT 13 261

4. Hassan M, Sutton T, Mage K, Limal JM, Rappaport R. The syndrome of accelerated bone maturation in the newborn infant with dysmorphism and congenital

263

malformations. (The so-called Marshall–Smith syndrome.) Pediatr Radiol. 1976:

264

5:53–57.

RI PT

262

5. Klaassens M, Morrogh D, Rosser EM, Jaffer F, Vreeburg M, Bok LA5, Segboer T,

266

van Belzen M, Quinlivan RM, Kumar A, Hurst JA, Scott RH. Malan syndrome:

267

Sotos-like overgrowth with de novo NFIX sequence variants and deletions in six new

268

patients and a review of the literature. Eur J Hum Genet. 2015 May; 23(5):610-615.

270 271

6. Matustik, C, Munson, D, Fenton, L. Marshall-Smith syndrome: androgen

M AN U

269

SC

265

abnormalities. Pediatric research (1985) 19, 188a–188a.

7. Malan V, Rajan D, Thomas S, Shaw AC, Louis Dit Picard H, Layet V, Till M, van Haeringen A, Mortier G, Nampoothiri S, Puseljić S, Legeai-Mallet L, Carter

273

NP,Vekemans M, Munnich A, Hennekam RC, Colleaux L, Cormier-Daire V. Distinct

274

effects of allelic NFIX mutations on nonsense-mediated mRNA decay engender

275

either a Sotos-like or a Marshall-Smith syndrome. Am J Hum Genet. 2010 Aug 13;

276

87(2):189-198.

278 279 280

EP

8. Marshall RE, Graham CB, Scott CR, Smith DW. Syndrome of accelerated skeletal maturation and relative failure to thrive: a newly recognized clinical growth disorder.

AC C

277

TE D

272

J Pediatr. 1971 Jan; 78(1):95-101.

9. Martinez F, Marín-Reina P, Sanchis-Calvo A, Perez-Aytés A, Oltra S, Roselló M,

281

Mayo S, Monfort S, Pantoja J, Orellana C. Novel mutations of NFIX gene causing

282

Marshall-Smith syndrome or Sotos-like syndrome: one gene, two phenotypes. Pediatr

283

Res. 2015 Nov; 78(5):533-539.

ACCEPTED MANUSCRIPT 14 284

10. Schanze D, Neubauer D, Cormier-Daire V, Delrue MA, Dieux-Coeslier A, Hasegawa T, Holmberg EE, Koenig R, Krueger G, Schanze I, Seemanova E, Shaw

286

AC, Vogt J, Volleth M, Reis A, Meinecke P, Hennekam RC, Zenker M. Deletions in

287

the 3' part of

288

the NFIX gene including a recurrent Alumediated deletion of exon 6 and 7 account fo

289

rpreviously unexplained cases of Marshall-Smith syndrome. Hum Mutat. 2014 Sep;

290

35(9):1092-1100.

SC

291

RI PT

285

11. Seisenberger, C, Winnacker, E.-L, Scherthan, H. Localisation of the human nuclear factor I/X (NFI/X) gene to chromosome 19p13 and detection of five other related loci

293

at 1p21-22, 1q42-43, 5q15, 11p13 and 20q13 by FISH. Hum. Genet. 1993; 91: 535-

294

537.

295

12.

M AN U

292

Shaw AC, van Balkom ID, Bauer M, Delrue MA, Van Haeringen A, Holmberg E, Knight SJ, Mortier G, Nampoothiri S, Pušeljić S, Zenker M, Cormier-Daire

297

V, Hennekam RC. Phenotype and natural history in Marshall-Smith syndrome. Am J

298

Med Genet A. 2010 Nov; 152A (11):2714-2726.

TE D

296

FIGURE LEGENDS

300

Fig 1: Front (a) and side profile (b) at the age of 5 years showing large forehead, proptosis,

301

abnormal pinnae and low set ears, depressed nasal bridge, anteverted nares, short philtrum,

302

everted lips, microretrognathia,

303

1(c): Left hand radiograph (at 8 years 1 month old) showing broad, rectangular proximal

304

phalanges and advanced bone age (10-11 years).

305

Fig 2 (a): Line diagram showing serial hormone levels on treatment with GnRH analogs.

AC C

EP

299

ACCEPTED MANUSCRIPT 15 (b): Bar graph showing a sustained decrease in Greulich and Pyle bone age/chronological age

307

ratio (BA/CA) while on treatment. Arrow indicates time of initiation of treatment.

308

SUPPLEMENTAL DATA DESCRIPTION

309

Supplementary Table I: Results of GnRH stimulation test

310

Supplementary Table 2: Variants of Unknown Clinical Significance in Disease Genes related to

311

Clinical Phenotype detected by the Whole Exome Sequencing.

312

EJMG signed publication permit

313

Declaration of new/rare genetic and genomic variant to public database:

314

Novel NFIX variant has been submitted to LOVD. Individual ID# 00100339

M AN U

SC

RI PT

306

315

AC C

EP

TE D

316

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT