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CLINICAL REPORT

Endocrine Phenotype of 6q16.1–q21 DeletionInvolving SIM1 and Prader–Willi Syndrome-LikeFeatures

Kosuke Izumi,1 Ryan Housam,2 Chirag Kapadia,3 Virginia A. Stallings,4,5 Livija Medne,6

Tamim H. Shaikh,7 Bassil M. Kublaoui,2,5 Elaine H. Zackai,1,5 and Adda Grimberg2,5*1Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania2Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania3Division of Endocrinology, Phoenix Children’s Hospital, Phoenix, Arizona4Division of Gastroenterology, Hepatology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania5Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania6Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania7Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado

Manuscript Received: 16 August 2012; Manuscript Accepted: 30 May 2013

How to Cite this Article:Izumi K, Housam R, Kapadia C, Stallings

VA, Medne L, Shaikh TH, Kublaoui BM,

Zackai EH, Grimberg A. 2013. Endocrine

phenotype of 6q16.1–q21 deletion involving

SIM1 and Prader–Willi syndrome-like

features.

Am J Med Genet Part A. 161A:3137–3143.

Conflict of interest: none.

K. Izumi, R. Housam and C. Kapadia are the first authors who

contributed equally to this work.�

Proximal interstitial 6q deletion involving Single-minded 1 (SIM1)

gene causes a syndromic form of obesity mimicking Prader–Willi

syndrome. In addition to obesity, Prader–Willi syndrome includes

several other endocrinopathies, such as hypothyroidism, growth

hormone deficiency, and hypogonadotropic hypogonadism. The

endocrine phenotype of interstitial 6q deletion remains largely

unknown, although clinical similarities between Prader–Willi syn-

drome and interstitial 6q deletion suggest endocrine abnormalities

also may contribute to the interstitial 6q deletion phenotype. This

report describes the endocrine phenotype in a propositus with the

Prader–Willi-like syndrome associated with an interstitial 6q dele-

tion including the SIM1 gene. Detailed endocrine evaluation of the

propositus during childhood and adolescence revealed hypopitu-

itarism, though initial endocrine evaluations during infancy were

unremarkable. Our patient raises the possibility that hypopituita-

rismmay be part of the phenotype, especially short stature, caused

by interstitial 6q deletion. SIM1 plays an important role in the

development of neuroendocrine lineage cells, implicating SIM1

haploinsufficiency in the pathophysiology of hypopituitarism seen

in our propositus. Early identification of endocrine abnormalities

can improve clinical outcome by allowing timely introduction of

hormone replacement therapy. Hence, we suggest that detailed

endocrine evaluation and longitudinal endocrine follow up be

performed in individuals with proximal interstitial 6q deletion

involving SIM1. � 2013 Wiley Periodicals, Inc.

Key words: SIM1; POU3F2; hypothyroidism; hypopituitarism

Correspondence to:

Adda Grimberg, M.D., Division of Endocrinology and Diabetes, The

Children’s Hospital of Philadelphia, Philadelphia, 3601 Civic Center

Boulevard, Philadelphia, PA 19104. E-mail: grimberg@email.chop.edu

Article first published online in Wiley Online Library

(wileyonlinelibrary.com): 16 August 2013

DOI 10.1002/ajmg.a.36149

INTRODUCTION

While classic Prader–Willi syndrome (PWS) is due to the absence of

paternally expressed imprinted genes at 15q11.2q13, there is well-

2013 Wiley Periodicals, Inc.

known clinical overlap with proximal interstitial 6q deletions,

occasionally referred to as Prader–Willi-like syndrome [Villa

et al., 1995; Cassidy et al., 2012]. One of the common clinical

features is obesity in the setting of developmental delays. In cases

with an interstitial 6q deletion, haploinsufficiency of the Single-

minded 1 (SIM1) gene at 6q16.2 represents the most probable

candidate gene for the obesity seen in Prader–Willi-like syndrome

[Holder et al., 2000; Michaud et al., 2001].

In addition to obesity, other endocrine features associated with

PWS include hypothyroidism, growth hormone (GH) deficiency,

and hypogonadotropic hypogonadism [Diene et al., 2010; Vaiani

3137

3138 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

et al., 2010]. Thus, GH treatment has become standard therapy for

children with PWS [McCandless, 2011; Cassidy et al., 2012]. Clini-

cal similarities between PWS and interstitial 6q deletion suggest

endocrine abnormalities also may contribute to the interstitial 6q

deletion phenotype. The endocrine phenotype of interstitial 6q

deletion remains largely unknown, except for hypothyroidism

reported in several cases [Zherebtsov et al., 2007; Bonaglia

et al., 2008; Rosenfeld et al., 2012]. This report describes the

endocrine phenotype, specifically hypopituitarism, in a propositus

with Prader–Willi-like syndrome associated with an interstitial 6q

deletion including SIM1 gene.

CLINICAL REPORT

The propositus was originally referred to the Genetics clinic at age

7months.Hewasbornat term(38weeks) via cesareandue tobreech

presentation and fetal heart rate decelerations. The pregnancy had

been complicated by clinically significant emesis and dehydration

throughout gestation. The infant was small for gestational age

(SGA) with head sparing: birth weight was 2.02 kg and length

43.8 cm (<10th centile for 38 weeks gestation, both 50th centile

for 34weeks gestation), while head circumferencewas 31 cm(10th–

25th centile for 38 weeks gestation). Due to his SGA status and

hypotonia, the infant remained in the hospital for 2 weeks

postnatally.

Physical features at age 7 months included a flat nasal bridge,

squared off helix on the right ear,mildly short upper limbs, bilateral

5th finger clinodactyly, and a very tapered and small middle

phalanx. Hand length was 25th centile for age bilaterally. Genital

exam revealed a penile chordee. Neurological examwas notable for

axial hypotonia. The diagnosis of PWS was considered because of

short stature and hypotonia. However, fluorescent in situ hybrid-

ization (FISH) analysis targeting SNRPN region did not reveal the

typical deletion, and normal Southern blot methylation analysis

indicated normal biparental inheritance. G-banded chromosome

analysis was normal.

FIG. 1. Facial appearances and hands/feet of the patient. A,B: At age 5 yea

and small palpebral fissures, flat nasal bridge, short hands and feet, bilater

Follow-up at age 5 years found a short, obese boy with a round

facewith full cheeks, downslanting and small palpebral fissures, and

normal ears (Fig. 1). On exam at age 121/2 years, he had significant

abdominal adiposity with mild non-violaceous striae. Skin exam

showed no acanthosis nigricans, signs of skin picking, or self-

mutilatory behavior. Extremity exam included acromicria (hand

length of 13.0 cm and foot length of 16.5 cm, both <5th percentile

for age), syndactyly of toes 2–4, and pes planus. Pubertal exam

showed microphallus, no axillary hair, no pubic hair, and

descended 2-ml testes.

Cognitive development was significantly delayed. Developmen-

tal evaluation at age 12 years demonstrated that the propositus was

functioning at the first grade level with gradual upward progress

(detailed developmental assessment is described in the Supplemen-

tal Document). Temper tantrums when frustrated were reported,

but no self-mutilatory behaviors were observed.

At this time, copy-number variation analysis using a SNP-based

microarray revealed a 9.8Mb interstitial deletion at 6q16.1–q21

(chr6:98,119,288–107,977,239 (hg18)) (Fig. 2), that included SIM1

and 30 other genes. FISH analysis using RP1-60O19 probe con-

firmed the presence of the deletion. As neither of his parents had the

deletion, the deletion had occurred de novo.

ENDOCRINE EVALUATION

ObesityAlthough initially SGA, excessive weight gain began after

18 months of age despite attempts at dietary restriction

(Fig. 3). For weightmanagement, he was started on a hypocaloric

diet for age and size, but this did not remedy his obesity. Thyroid

function panel, GH, insulin-like growth factor-1 (IGF-1),

and IGF binding protein-3 (IGFBP-3) levels were within the

reference range at age 2 years. By age 5 years, height was 94.4 cm

(<5th centile, 50th centile for 3 years), weight was 25.5 kg

(>97th centile), with a body mass index (BMI) of 28.6 kg/m2

(>97th centile). His resting energy expenditure, measured by

rs. C: At age 11 years. Note round face with full cheeks, downslanting

al 5th finger clinodactyly, and syndactyly of toes 2–4.

FIG. 2. Graphic representation of copy-number variation analysis data using a SNP-based microarray. This image shows the deletion of a

9.8 Mb fragment in 6q16.1–q21 (shown by red-boxed area). Chromosome 6 is shown as an ideogram in the bottom panel going from left to

right with the centromere shown in green. Log2Ratio of the signal from the propositus versus control sample is shown on the right. The top

panel shows the Log2Ratio data from each individual probe (red dots) and the middle panel shows the data normalized over 50 probes (blue

line). The probes that are deleted cluster around Log2Ratio of �1.0 and the normal copy number BACs cluster around Log2Ratio of 0.00. The

green lines below the ideogram show the heterozygous SNPs across the chromosome. The deleted fragment is hemizygous (single allele) and

therefore devoid of heterozygous SNPs.

FIG. 3. Growth chart of the patient. Arrow indicates the age at initiation of GH supplementation, and dashed arrow indicates the age of GH

discontinuation. Arrowhead indicates the midparental height of 165.3 cm.

IZUMI ET AL. 3139

TABLE II. Growth Hormone Stimulation Test at the Age of 12.5 Years

Time Growth hormone (ng/ml)

Baseline 0.097

þ15min 0.64

þ30min 1.9

þ45min 4.3

þ60min 6.5

þ90min 4.3

3140 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

indirect calorimetry, was 60% of normal for age and sex. His

weight at 18 years was 83.3 kg (75–90th centile), height was

152 cm (<5th centile, 50th centile for 12 years) with a BMI of

36 kg/m2 (>98th centile). Waist circumference was 114 cm.

He continues to have hyperphagia but can be distracted easily

from further intake; there is no food-scavenging behavior. The

propositus was screened routinely for diabetes mellitus, and his

most recentHbA1cwas in the high-normal range (5.7%).He also

passed an oral glucose tolerance test with a normal fasting

glucose of 93mg/dl and normal glucose of 99mg/dl 2 hr after

glucose load.

GrowthLinear growth velocity was normal or near normal until 2 years of

age, after which time he developed marked, progressive linear

growth failure (Fig. 3). Hismother’s height of 154.9 cm and father’s

height of 162.6 cm lead to a gender adjustedmid-parental height of

165.2 cm (7.4th centile) [Tanner et al., 1970]. At age 121/2 years,

at his initial evaluation in our endocrine clinic, height measured

119 cm (�4.5 SD), and weight was 51.8 kg (90th centile), with a

BMI of 36.6 kg/m2 (þ2.6 SD). Head circumference was 53.5 cm

(25th–50th centile). The combination of poor linear growth, short

hands and feet, delayedbone age (bone age 10 years at chronological

age 12–1/3 years), and low IGF-1 concentration (Table I), suggested

the possibility of GH deficiency. The propositus underwent GH

stimulation testing with growth-hormone-releasing hormone

(GHRH) (sermorelin acetate, 1mcg/kg), and arginine hydrochlo-

ride (5ml/kg), in tandem.Results indicatedGHdeficiency, since his

peakGHlevel of 6.53 ng/ml fell below thegenerally acceptednormal

cut-off of at least 10 ng/ml (Table II). He was subsequently treated

with GH from age 121/2 years until age 17, when epiphyseal fusion

was documented. Despite growing 32 cm in the 4.5 years on GH

therapy (annualized growth velocity of 7 cm/year), his adult height

was 151 cm, still 2 SD below his mid-parental target height and

3.6 SD below mean.

TABLE I. Initial Endocrine Evaluation at 12 Years and 6 Months

Assay Value Reference range

Serum IGF-1 (ng/dl) 105 139–395

Serum IGFBP-3 (mg/dl) 6.2 2.7–8.9

Serum TSH (mIU/ml) 2.6 0.5–4.5

Serum free T4 (ng/dl) 0.93 0.8–2

Serum LH (mIU/ml) 0.35 0.02–0.3

Serum testosterone (ng/dl) 9.9 <10 (prepubertal status)

8 am serum cortisol (mcg/dl) 6.4 9–22

8 am serum osmolality (mOsm/L) 289 270–300

8 am urine osmolality (mOsm/L) 258

IGF-I, insulin-like growth factor 1; IGFBP-3, insulin-like growth factor binding protein 3; TSH, thyroidstimulating hormone; free T4, free thyroxine; LH, luteinizing hormone. IGF-1 and LH weremeasuredat Esoterix Laboratory Services, CalabasasHills, CA. IGFBP-3, TSH, free T4, testosterone,cortisol, and serum and urine osmolalities were measured at Laboratory Corporation of America,Burlington, NC.

PubertyOn initial exam at 121/2 years, the propositus was Tanner stage I

for pubic hair and testicular volume (2ml bilaterally). He also

had microphallus and no axillary hair. Initial labs confirmed

prepubertal status, with testosterone 7 ng/dl and luteinizing

hormone (LH; measured by immunochemiluminometric assay)

0.03mIU/ml. However, he spontaneously entered puberty at age

13 years, and is currently, at the age of 18 years, Tanner stageV for

pubic hair and testicular volume (18ml) with penile length of

10 cm, without receiving testosterone therapy. Repeat hormone

measurements at 18 years showed LH 1.43 mIU/ml, FSH

2.48mIU/ml, and testosterone 350 ng/dl (all levels were in the

normal reference range).

Posterior Pituitary EvaluationAt age 121/2 years, the parents reported that the propositus drank

large quantities of water, with frequent urination of copious

volumes. He was unable to sleep through the night, awakening

to drink and to urinate several times nightly. Initial outpatient

morning labs could neither diagnose nor exclude diabetes insip-

idus, so he was admitted to the hospital for a water deprivation test

(Table I). Results showed that although he was able to concentrate

his urine, serum osmolality reached higher than normal levels,

suggesting he had a reset osmostat (Table III). In other words,

neurohypophyseal secretionof arginine-vasopressin (AVP)wasnot

triggered in the usual fashion to prevent serum hyperosmolality.

This likely explained his chronic polyuria with secondary polydip-

sia. MRI with and without contrast showed that the anterior

TABLE III. Water Deprivation Study at the age of 12.5 Years

Time

Serum sodium

(mmol/L)

Serum osmolality

(mOsm/kg)

Urine osmolality

(mOsm/kg)

0820 142 298 260

0915 143 294 457

1015 141 294 554

1115 144 300 702

1215 145 301 770

1315a 143 302 763

1415 144 308 801

1505 146 302 790

aSubcutaneous DDAVP given with decrease in thirst and polydipsia.

IZUMI ET AL. 3141

pituitary was at the lower end of normal size and the posterior

pituitary bright spot was absent with no other abnormalities noted.

He was started on intranasal desmopressin with improvement in

symptoms.

Thyroid AxisThyroid profile was normal at age 7 months. At age 13 years, in

evaluation of his poor growth and obesity, he had repeatedly

borderline low free thyroxine (T4) values with normal TSH levels,

indicative of central hypothyroidism. He was started on levothyr-

oxine 50mcg daily at the age of 14–3/12 years after a free T4

measurement of 1.0 ng/dl (Reference range: 1.1–2.1 ng/dl) twice

within a5-monthperiod.Hewas thenmaintainedon levothyroxine

75mcg daily with the most recent free T4 value of 1.4 ng/dl

(reference range: 0.9–1.4) at age 17 years.

Adrenal AxisInitial evaluation via low-dose (1mcg) cortrosyn stimulation test

showed a normal peak cortisol of 25.7mcg/dl, suggesting normal

adrenal axis function at the age of 12 years. He subsequently had a

low 8 am cortisol of 4.8mcg/dl [Forest, 2003]. Corticotropin-

releasing hormone (CRH) stimulation testing at age 13 years

showed a sub-normal peak cortisol of 16.8mcg/dl, indicating

partial adrenal insufficiency. Since that time, he has not required

maintenance hydrocortisone replacement but does receive stress

dose steroids for acute illness and procedures.

FIG. 4. Schematic diagram of 6q deletion seen in our patient and the pre

reported overlapping 6q deletions.

DISCUSSION

Here we describe the results of detailed endocrine phenotyping of a

propositus with interstitial 6q16.1–q21 deletion causing Prader–

Willi-like syndrome features. Of particular importance, despite

unremarkable initial endocrine evaluations in infancy, he devel-

oped hypopituitarism during childhood and adolescence. Longi-

tudinal clinical follow-up enabled us to monitor the development

and progress of each symptom. This observation underscores the

importance of ongoing monitoring of endocrine phenotype in

individuals with interstitial 6q deletion.

In proximal interstitial 6q deletion syndrome, deletion of SIM1

has been implicated in the development of obesity [Faivre

et al., 2002; Varela et al., 2006]. SIM1 is a homologue of Drosophila

single-minded gene, which encodes a basic helix–loop–helix PAS

transcription factor [Chrast et al., 1997]. SIM1 deficient mice are

obese [Michaud et al., 2001; Holder et al., 2004], and there has been

one reported human subject with a balanced translocation disrupt-

ing SIM1 who was also obese [Holder et al., 2000]. Among

previously reported cases over 2 years of age with 6q deletion

encompassing SIM1 and overlapping with that of our patient, 10

out of 12 patients had obesity. This suggests a major role of SIM1

haploinsufficiency in the pathogenesis of obesity in these subjects

[Grati et al., 2005; Le Caignec et al., 2005; Klein et al., 2007;

Zherebtsov et al., 2007; Bonaglia et al., 2008; Spreiz et al., 2010;

Woo et al., 2010; Rosenfeld et al., 2012] (Fig. 4). Interestingly, the

resting energy expenditure of the propositus was decreased, which

is distinctly different from that of the patient with a translocation

sence or absence of endocrine phenotype in relation to the previously

3142 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

disrupting SIM1 and Sim1 heterozygous mice [Holder et al., 2000;

Michaud et al., 2001].

WhileSIM1deletion is anattractive candidate to explain theobesity

seenwithin the Prader–Willi-like syndromephenotype of 6q deletion,

haploinsufficiencyofSIM1byitself likelydoesnotexplainourpatient’s

short stature. The previously reported patient with balanced translo-

cation disrupting SIM1was actually tall during her pre-pubertal years

[Holder et al., 2000]. Our propositus with short stature is clearly

different from the phenotype of SIM1 deficiency or non-genetic

obesity in children where tall stature is the norm. Hence, the contri-

butionofother gene(s) located in6q isproposed in thepathogenesisof

short stature. Deficiency of one or multiple pituitary hormones,

including GH, TSH, and gonadotropins, can stunt statural growth.

As our propositus showed, such deficiencies can develop over time in

patients with Prader–Willi-like syndrome. Thus, undiagnosed and

untreated hormone deficiencies can construe a possiblemechanismof

short stature in interstitial 6q deletion.

Among the previously reported cases of interstitial 6q deletion,

short stature was occasionally observed (Fig. 4). Our endocrine

evaluation identifiedGHdeficiency as a possible explanation for his

short stature, at least in part. Another patient with interstitial 6q

deletion was reported to have GH deficiency, suggesting GH

deficiency may be part of the phenotype [Bonaglia et al., 2008].

There may be a genetic (non-hormonal) component to the short

stature as well. However, we were not able to determine the critical

genomic region for short stature in 6q, because there was no

commonly deleted chromosomal region seen only in individuals

with short stature (Fig. 4).

In addition to GH deficiency, other hormonal deficiencies

reported with 6q deletions include hypothyroidism and diabetes

insipidus, both of which were found in our propositus [Klein

et al., 2007; Zherebtsov et al., 2007; Bonaglia et al., 2008; Rosenfeld

et al., 2012] (Fig. 4). Three previous cases with interstitial 6q

deletion were found to have hypothyroidism [Zherebtsov

et al., 2007; Bonaglia et al., 2008; Rosenfeld et al., 2012] (Fig. 4).

In case 10ofRosenfeld et al. [2012], the patientwas reported tohave

a small pituitary gland as well as central hypothyroidism. Bonaglia

et al. [2008] described a case with mild primary hypothyroidism

alongwithGHdeficiency. Furthermore, Patient 3 reported byKlein

et al. [2007] had central diabetes insipidus. Therefore, although

occurrence of hypopituitarism has never been described in associa-

tion with 6q deletion, pituitary hormone deficiencies have been

reported in the past. Such pituitary hormone deficiencies may

contribute to the resultant phenotype of interstitial 6q deletion

syndrome including short stature.

The exact etiology of hypopituitarism, which manifested during

childhood, remains unclear in our patient. Although such late-

onset symptoms seem unlikely to be related to underlying genetic

alterations, genetic mutations can cause late-onset hypopituita-

rism, as exemplified by patients with PROP1 genemutations [Fluck

et al., 1998; Mendonca et al., 1999; Pavel et al., 2003]. Further, our

propositus’ pituitarywas small and lacking the posterior bright spot

on MRI. Therefore, we speculate that haploinsufficiency of a gene

or genes in the 6q16.1q21 region may play a role in regulating

hypothalamic–pituitary development and/or function.

Among the genes located in 6q16.1q21, SIM1may be involved in

the pathogenesis of hypopituitarism in conjunction with haploin-

sufficiency of other genes. In our propositus, we hypothesize

that SIM1 might be playing a major role in the pathogenesis

of reset osmostat, central hypothyroidism, and central adrenal

insufficiency based on the following supportive evidence. SIM1

has been demonstrated to be required for the development of

neuroendocrine lineage cells, specifically,CRH, thyrotropin-releas-

ing hormone (TRH), AVP, oxytocin (OXT) and somatostatin

neurons [Michaud et al., 1998]. In Sim1 heterozygous knockout

mice, hypothalamicmRNA levels ofAVP, which encodes vasopres-

sin, were reduced by 41% compared to wild type mice, and the

number of vasopressin producing cells was reduced by 50%

[Kublaoui et al., 2008; Duplan et al., 2009]. Further, conditional

Sim1 homozygous knockoutmice were found to drink significantly

more water than their wild type littermates, recapitulating the

diabetes insipidus/reset osmostat phenotype [Dr. Andrew Zinn,

personal communication]. Sim1 heterozygous mice also exhibited

a reduction in hypothalamic CRH and TRH mRNA expression

by 30% and 42%, respectively. It is notable that Sim1 is not

co-expressed with GHRH or gonadotropin-releasing hormone

(GnRH) neurons. GHRH is synthesized by neurons in the arcuate

nucleus and ventromedial nucleus and GnRH is synthesized in

neurons in the preoptic area; Sim1 is not expressed in these nuclei.

Based simply on Sim1 expression patterns, haploinsufficiency of

SIM1 in our propositus would not be expected to account for GH

deficiency.

Another candidate gene whose haploinsufficiency could be

related to hypopituitarism is POU3F2 (BRN2) [Castro et al.,

2006]. SIM1 and POU3F2 act in a cascade of transcription factors

essential for AVP, oxytocin, thyrotropin-releasing hormone, and

CRH neuron differentiation in the supraoptic nucleus and para-

ventricular hypothalamic nucleus [Michaud et al., 1998]. The

combination of SIM1 and POU3F2 haploinsufficiency in our

patient may explain the observed hypopituitarism affecting the

hypothalamic–pituitary–adrenal axis, hypothalamic–pituitary–

thyroid axis, and vasopressin dysregulation.

Here we report on a propositus with Prader–Willi-like pheno-

type and interstitial 6q deletion including SIM1 associated with

obesity, short stature, and hypopituitarism. Our case suggests the

possibility that hypopituitarism may be part of the phenotype

caused by interstitial 6q deletion. Early identification of endocrine

abnormalities can lead to a better clinical outcome by the early

introduction of hormone replacement therapy. Hence, we suggest

that detailed endocrine evaluation and longitudinal endocrine

follow up be performed in individuals with interstitial 6q deletion.

ACKNOWLEDGMENTS

The authors greatly appreciate the patient’s parents for their

support of research and disseminating knowledge about their

son’s condition. They are key team members in his care, as we

all learn from him.

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