Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome

Abstract

Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency.
We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.
In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts, absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization.
Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.

Full text

R E S E A R C H Open Access
Impaired osteoblast and osteoclast function
characterize the osteoporosis of Snyder - Robinson
syndrome
Jessica S Albert
1,2
, Nisan Bhattacharyya
3
, Lynne A Wolfe
1,2
, William P Bone
1
, Valerie Maduro
1
, John Accardi
1
,
David R Adams
1,2
, Charles E Schwartz
4
, Joy Norris
3
, Tim Wood
4
, Rachel I Gafni
3
, Michael T Collins
3
, Laura L Tosi
5,6
,
Thomas C Markello
1,2
, William A Gahl
1,2*
and Cornelius F Boerkoel
1
Abstract
Background: Snyder-Robinson Syndrome (SRS) is an X-linked intellectual disability disorder also characterized by
osteoporosis, scoliosis, and dysmorphic facial features. It is caused by mutations in SMS, a ubiquitously expressed
gene encoding the polyamine biosynthetic enzyme spermine synthase. We hypothesized that the tissue specificity
of SRS arises from differential sensitivity to spermidine toxicity or spermine deficiency.
Methods: We performed detailed clinical, endocrine, histopathologic, and morphometric studies on two affected
brothers with a spermine synthase loss of function mutation (NM_004595.4:c.443A > G, p.Gln148Arg). We also
measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts
using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.
Results: In addition to the known tissue-specific features of SRS, the propositi manifested retinal pigmentary
changes, recurrent episodes of hyper- and hypoglycemia, nephrocalcinosis, renal cysts, and frequent respiratory
infections. Bone histopathology and morphometry identified a profound depletion of osteoblasts and osteoclasts,
absence of a trabecular meshwork, a low bone volume and a thin cortex. Comparison of cultured fibroblasts from
affected and unaffected individuals showed relatively small changes in polyamine content, whereas comparison of
cultured osteoblasts identified marked differences in spermidine and spermine content. Osteogenic differentiation
of the SRS-derived hBMSCs identified a severe deficiency of calcium phosphate mineralization.
Conclusions: Our findings support the hypothesis that cell specific alterations in polyamine metabolism contribute
to the tissue specificity of SRS features, and that the low bone density arises from a failure of mineralization.
Keywords: Spermine, Snyder-Robinson syndrome, Osteoblast, Osteoclast, Osteoporosis
Background
Polyamines are ubiquitous, aliphatic, positively charged
molecules that interact with anionic compounds such as
DNA, RNA, and ATP [1,2]. Homeostasis of the poly-
amines putrescine, spermidine, and spermine is essential
to cell growth and survival [3]. By addition of a propyla-
mine moiety, spermidine synthase (SRM) converts pu-
trescine into spermidine, and spermine synthase (SMS)
converts spermidine into spermine [4]. The balance of
spermine and spermidine is crucial for ion channel regu-
lation, transcription and translation [5-9].
Mutations of SMS, the gene encoding spermine synthase,
cause Snyder-Robinson syndrome (SRS), an X-linked
disorder first reported in 1969 [10]. The clinical features
of SRS include intellectual disability, dysmorphic facies,
speech and gait abnormalities, seizures, muscle hypopla-
sia, kyphoscoliosis, and osteoporosis [11-17]. All affected
males have hemizygous mutations in SMS that result in
reduced SMS activity and a decreased spermine:spermi-
dine ratio.
* Correspondence: gahlw@helix.nih.gov
1
Undiagnosed Diseases Program, Common Fund, Office of the Director,
National Institutes of Health, Bethesda, MD 20814, USA
2
Medical Genetics Branch, National Human Genome Research Institute,
Bethesda, MD, USA
Full list of author information is available at the end of the article
© 2015 Albert et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27
DOI 10.1186/s13023-015-0235-8

Atraumatic osteoporotic fractures commonly occur in
individuals with SRS, leading to significantly impaired
quality of life. Osteoporosis arises from disruption of the
equilibrium between osteoclastic bone resorption and
osteoblastic bone formation [18], which is regulated by
mechanical and endocrine stimuli [19]. This general un-
derstanding of osteoporosis has led to established thera-
peutic interventions, but further insights are required to
address the osteoporosis of SRS in a disease-specific
manner.
Here we define the osteoporotic disease of SRS in two
brothers with a missense mutation in SMS [20] and report
depletion of osteoblasts and osteoclasts, reduced cancel-
lous and cortical bone, reduced calcium-phosphate
mineralization in vitro, and markedly abnormal polyamine
content in human bone marrow stromal cells (hBMSCs).
These data offer new insights into the role of polyamines
in bone formation.
Clinical reports
Patient II-1
The propositus (II-1, Figure 1) is the 18-year-old son of
non-consanguineous healthy parents with no family his-
tory of intellectual disability or skeletal problems.
He was born by cesarean section at 40 weeks following
a gestation complicated by poor maternal weight gain and,
at 8 months, atypical fetal movements suggestive of in
utero seizures. His birth weight, length and occipitofrontal
circumference (OFC) were 2.38 kg (3%), 47 cm (13%) and
34.5 cm (26%), respectively. Apgar scores were 8 and 9 at
one and five minutes. He had saggy skin but no other
dysmorphic features. Following a perinatal intraven-
tricular hemorrhage associated with thrombocytopenia
(7×10
3
cells/μl) that corrected after 3 platelet transfusions,
he developed seizures, apnea with cyanosis, temperature
instability and hypoglycemia. His recurrent episodes of
hyperglycemia and hypoglycemia resolved with age and
the placement of a gastric tube that allowed more frequent
feedings.
Patient II-1 had tracheomalacia, upper airway obstruc-
tion, increased respiratory secretions, frequent aspira-
tions, and pulmonary infections from infancy. By age 7
years, he had chronic Pseudomonas aeruginosa respira-
tory infection. At 6 years, he developed proximal renal
tubular acidosis (RTA), nephrocalcinosis and nephro-
lithiasis. His renal stones were composed of carbonate
apatite and calcium oxalate. His RTA has been managed
with fluid and electrolyte replacement, spironolactone
and hydrochlorothiazide.
Although initially controlled with phenobarbital, his
seizures progressed to infantile spasms by 15 months.
Adrenocorticotropic hormone (ACTH) treatment transi-
ently reduced seizure frequency. His current anticonvul-
sant therapy includes rufinamide, felbamate, clonazepam,
and topiramate. He had delayed development with regres-
sion of several developmental milestones. He smiled and
laughed by 4 months, reached for toys by 6 months, had
vocalizations by 7 months, and could hold his head up
and roll onto his side by 8 months. He achieved his pincer
grasp at 18 months but lost it by 23 months. He never
walked. He lost many motor skills and all vocalization by
26 months of age. A cranial MRI at 14 years revealed cys-
tic encephalomalacia and a low parenchymal T2 signal in
the right temporal and right occipital lobes. These findings
were considered consequences of the perinatal intraven-
tricular hemorrhage.
When first evaluated at NIH at 15 years, Patient II-1
was awake but not interactive and could not sit inde-
pendently or hold up his head; he withdrew from nox-
ious stimuli. His height, weight and OFC were 129 cm
(<3%ile), 30.8kg (<3%ile) and 50.3 cm (<3%ile), respect-
ively. Facial dysmorphisms included a long, oval, asym-
metric face, midface hypoplasia, down-slanting palpebral
fissures, large, cupped ears, smooth philtrum, high-
arched palate and prognathia (Figure 1). His dental en-
amel was hypoplastic and secondary teeth 2, 6, 10, and
11 were absent. He had excessive drooling, sluggish
pupillary reflexes and left-sided hearing loss. He had fre-
quent seizures, severe hypotonia, decreased muscle bulk,
hypoactive deep tendon reflexes, kyphoscoliosis and flexion
contractures of most large and small joints (Figure 1). He
had one prepubertal (1-2 mL) testis that was undescended
but palpable in the inguinal canal and one undescended
testis, Tanner stage III pubic hair and a prepubertal
phallus. His skin was remarkable for excretions of carbon-
ate apatite, reflecting calcium/phosphate dysregulation.
His ophthalmologic exam revealed retinitis pigmentosa
and cortical blindness.
Previous skeletal problems included congenital bilateral
hip dislocation and fractures of his distal fibula (2 years),
right humerus (5 years) and spine (6 years). Kyphoscoliosis
developed between 6 and 12 years of age. A dual-energy
x-ray absorptiometry (DEXA) scan performed at age 15
years demonstrated a bone density of 0.341 gm/cm
2
BMD
(height adjusted Z-Score: -2.9 [21]) for the anteroposterior
Spine (L1-L4) and 0.342 gm/cm
2
bone mineral density
(height adjusted Z-Score: -6.5 [21]) for the right forearm.
Skeletal radiographs at 18 years revealed a 60° convex
right scoliosis, gracile bones with reduced mineral density,
and evidence of previous fractures (Figure 2E-J).
Patient II-3
Patient II-3, the brother of Patient II-1, was born at 37
weeks by cesarean section following an uncomplicated
pregnancy. His birth weight was 2.81 kg (25%). During
the immediate neonatal period his platelet count de-
creased from 90 to 45 k/μL, and he was admitted to
the neonatal intensive care unit for treatment with
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 2 of 13

Figure 1 Clinical and radiographic features. A. Face of patient II-1. B. Face of patient II-3. C. Palate of patient II-1. D. Hands of patient II-3. E-J.
Skeletal radiographs of Patient II-1 showing the left humerus (E), left forearm (F), left hand (G), pelvis (H), left femur (I) and left lower leg (J). Note
the gracile bones and undermineralization as well as the healing humeral fracture (E).
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 3 of 13

Figure 2 (See legend on next page.)
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 4 of 13

dexamethasone and a platelet transfusion. He also had
transient hypoglycemia, poor feeding and sensitivity to
light. By 4 days of life, his condition had stabilized and
he was discharged. Over the subsequent months, he
manifested moderate laryngomalacia, mild tracheobro-
chomalacia, severe torticollis and abnormally pigmen-
ted retinas. Like his brother, he has also had episodic
hyper- and hypoglycemia. He was diagnosed with
nephrocalcinosis at 1 year and RTA by 2 years. The
composition of his renal stones was carbonate apatite
and calcium oxalate.
Patient II-3 had global developmental delay, severe
hypotonia, and regression of milestones; he lost vocaliza-
tions and most motor skills by 15 months. An EEG at 6
months showed generalized slowing and disorganization;
at 18 months he manifested seizures and hypsarrhyth-
mia. An MRI performed at 2 years of age showed a mild
increase in ventricular size but no other abnormalities.
There was no evidence of hemorrhage or malformation.
Since age 5 years, he has required a vagal nerve stimula-
tor and bi-pap for adequate respiration. He also had re-
peated Pseudomonas aeruginosa pulmonary infections.
His infections are more frequent and severe than are
those of his brother.
Patient II-3 has had multiple atraumatic fractures in-
volving the clavicle, tibia, femur and humerus. He also
had congenital bilateral hip dislocation and, by 4 years
of age scoliosis.
Illness prevented Patient II-3 from traveling to the
NIH for evaluation, but review of his medical records
and photographs showed that he was alert and non-
ambulatory at age 10 years. He had facial features similar
to those of his brother (Figure 1), superficial skin excre-
tions, hypotonia, large joint contractures and muscle
atrophy.
Laboratory studies
The propositi had extensive laboratory testing. This
identified an elevated antibody titer to myelin basic pro-
tein and a mild intermittent anemia with low iron satur-
ation (7%; normal, 15-62) and ferritin (20 mcg/L; normal,
26 -388) for Patient II-1 and an elevated blood lactate level
of 7.8 mmol/L (0.5-2.2) and mild elevations of urine carni-
tine esters for Patient II-3. Testing for abnormalities in or-
ganic acids, amino acids, acylcarnitines, very long chain
fatty acids, lysosomal enzymes, biotinidase and copper
were unremarkable. Molecular testing of MECP2,mito-
chondrial DNA, a panel of lysosomal storage disease-
associated genes and the X-Linked Mental Retardation
9 Gene Panel (Greenwood Clinic, 2008), which did not
include SMS and FRMPD4, did not detect pathogenic
mutations. Patient II-1 had a normal karyotype (46,XY),
and clinical and research copy number variant analysis
did not detect any pathogenic variants (Additional file 1:
Table S 1 ) .
Methods
Patients
The propositi were accepted into the NIH Undiagnosed
Diseases Program (UDP) and enrolled in clinical proto-
col 76-HG-0238, approved by the Institutional Review
Board of the National Human Genome Research
Institute. Their parents gave written, informed consent.
CNV analysis
The NHGRI Genomics Core lab performed SNP determina-
tionsusingtheIlluminaBeadArrayPlatform(HumanOm-
niExpress, Illumina Corp., San Diego, CA, USA). Genome-
wide fluorescent intensities and genotype calls were analyzed
using Bead Studio and Genome Studio (Illumina Corp.).
Analysis of copy-number variations was performed using
PennCNV software, [22] and visual inspection using Gen-
ome Studio version 2010v3 build37/hg19 [23].
Exome sequence analysis
Genomic DNA was extracted from whole blood using
the Gentra Puregene Blood kit (Qiagen, Valencia, CA)
according to the manufacturer’s specifications. Exome
sequencing and analysis were performed as described
[24-26]. The potential pathogenicity of identified vari-
ants was predicted using CDPred, SIFT, PolyPhen2
[27-33] or the BLOSUM62 scoring matrix [34].
(See figure on previous page.)
Figure 2 Segregation, mutational analysis, and functional consequences of a novel SMS variant. A. Pedigree of the family of the propositi.
Affected males are shown by black squares. B. Sanger sequencing chromatograms showing the segregation of the SMS mutation NM_004595.4:c.443A
> G from the carrier mother to the affected boys. The unaffected father did not have this mutation. C. Conservation of the p.Gln148 (p.Q148) residue
across species. D. Drawing of the human SMS protein crystal complexed with spermidine and 5-methylthioadenosine. The mutated amino acid
(Gln148) is highlighted in yellow [Mac PyMOL [23]]. E-J. Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E, F),
Patient II-1 (G, H) and Patient II-3 (I, J) skin fibroblasts. SMS protein is shown in red and the nucleus is shown in blue. K. Immunoblot of skin fibroblast
lysates showing reduced SMS protein levels in the patients (II-1, II-3) compared to an unaffected control (cnt). Tubulin is shown as a loading control. L.
Graph showing steady state SMS protein levels in the patient and control fibroblasts relative to ß-tubulin levels. The data are based on 3 independent
experiments for each cell line. M. Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and
control fibroblasts. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. The data are
based on 2 independent experiments for each cell line. N. SMS enzyme activity (spermidine d8 peak per hour) in lymphoblasts of unaffected individuals
(Cnt), a cohort of 4 individuals with SRS (SRS) and patient II-1, * p < 0.05.
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 5 of 13

Confirmatory sequencing
For the SMS variant, Qiagen HotStarTaq master mix
(Qiagen, Valencia, CA) was used to amplify the putative
variant and 200 flanking nucleotides using the primers 5′-
TGTGGCTTTCTTTTGCACAC-3′and 5′-TGCATCT
CAAAAACCAGCAG-3′. Unincorporated primers and
nucleotides were removed using ExoSAP-IT reagent
(USB, Cleveland, OH, USA). Sanger capillary sequencing
was used to sequence the PCR products (Macrogen,
Rockville, MD), and the sequences were aligned and ana-
lyzed using Sequencher v.4.10.1 (Gene Codes, Ann Arbor,
MI, USA). Mutation interpretation was conducted using
Alamut 2.0 (Interactive Biosoftware, San Diego, CA, USA).
Bone biopsy and histomorphometry
Patient II-1 was given two courses of demeclocycline
prior to the biopsy so that dynamic histomorphometry,
including measurement of bone turnover, could be per-
formed [35]. A bicortical transiliac crest core biopsy was
performed and immediately split. The sample was placed
in 70% ethanol and sent to The Johns Hopkins School of
Medicine for histomorphometry, which was performed
as described [35].
Western blot analysis
Protein was extracted from fibroblast and osteoblast cell
lines with RIPA buffer (Thermo Scientific, Waltham, MA)
or used directly from Clontech human protein library (Clon-
tech Laboratories, Mountain View, CA). Lysates (30ug) were
electrophoresed on a 4-10% SDS-polyacrylamide gel and
transferred to a polyvinylidene fluoride (PVDF) membrane.
Using gentle agitation, the membrane was blocked overnight
at 4ºC with casein blocking buffer (Thermo Scientific,
Waltham, MA) and 10% horse serum. Anti-SMS (1:1000)
(Novus Biologicals, Littleton, CO), anti-β-tubulin (1:1000,
AbCam, Cambridge, UK), and anti-GAPDH (1:1000, Gene
Tex, Irvine, CA) were used as primary antibodies. Horsera-
dish peroxidase-conjugated secondary antibodies (1:10000,
Bio-rad Laboratories, Hurcules, CA) were used to detect the
primaryantibodies.Theantibody-enzyme complexes were
detected by chemiluminescence using Amersham ECL west-
ern blotting detection reagent (GE Life Sciences, Pittsburgh,
PA) or WesternSure Premium Chemiluminescent Substrate
(LI-COR, Lincoln, NE) according to the manufacturer’s
specifications. β-tubulin or GAPDH was detected as a load-
ing control.
Cellular fractionation
Cellular fractionation was performed using NE-PER Nuclear
and Cytoplasmic Extraction Reagents (Thermo Scientific,
Waltham, MA) per the manufacturer’s recommendations.
Immunoblotting was performed as described above. β-tubu-
lin and p84 (GeneTex, Irvine, CA) served as loading con-
trols for the cytoplasmic and nuclear fractions, respectively.
qRT-PCR analysis
RNA was extracted from cells using the RNeasy Mini Kit
(Qiagen, Valencia, CA) per the manufacturer’s specifica-
tions. RNA was converted to cDNA using the VILO cDNA
synthesis kit (Life Technologies, Grand Island, NY) per the
manufacturer’s protocol. 100ng of cDNA was amplified
using Sso Advanced SYBR Green supermix (Bio-Rad
Laboratories, Richmond, CA) per the manufacturer’sspeci-
fication. qRT-PCR for measuring SMS steady state mRNA
levels was performed on the Bio-Rad CFX96 Real-Time
system (Bio-Rad Laboratories, Richmond, CA) using
primers 5′-gattggtgttgctggacctt-3′and 5′-tgactcaattcttt
cattctttcct-3′. PCR was cycled 33 times and annealing
temperature was 58 degrees, melt curve was incremented
at 0.5 degrees from 65-95 degrees. mRNA levels were nor-
malized to GAPDH mRNA levels, a house-keeping gene.
Cell culture
Epstein-Barr virus (EBV)-transformed lymphoblast cells
were cultured as described previously [36]. Fibroblast cells
were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 4.5 g/L D-glucose, L-glutam-
ine, sodium pyruvate (Life Technologies, Grand Island,
NY), 10% fetal bovine serum (Life Technologies, Grand
Island, NY) and 1 X antibiotic (Life Technologies, Grand
Island, NY). Fibroblasts were grown at 37°C with 5% CO2.
Human bone marrow stromal cells (hBMSC) were iso-
lated and grown in culture as previously described [37].
Briefly, bone biopsy samples from patient(s) were used as
the starting material. Cells were grown in α-minimal es-
sential medium (α-MEM) containing 20% fetal bovine
serum (FBS, Atlanta Biologicals, Lawrenceville, GA, USA),
L-glutamine (Glutamax, GIBCO, Carlsbad, CA, USA), and
penicillin-streptomycin mix. When noted, cells were also
grown in the above medium containing Dexamethasone
(10-8 M dexamethasone and 10-4 M ascorbate). Cells
were cryopreserved in α-MEM containing 50% FBS and
5% dimethyl sulphoxide (DMSO). Control hBMSC were
obtained from the rib of a 51 year-old Caucasian male.
Osteogenic differentiation assay
Control and experimental BMSCs were plated (6x104/12-
well plate) in triplicate. Cells were kept either untreated (α-
MEM containing 20% FBS, L-glutamine, antibiotics) or
treated with an osteogenic differentiation media (α-MEM,
20% FBS, L-glutamine, antibiotics, 5x10-3M β-glycerophos-
phate, 1x10-4M Ascorbic Acid Phosphate and 1x10-8M
dexamethasone). Media was changed every 3-4 days. After
18 days, cells were rinsed with HBSS (Hanks’Balanced Salt
Solution, Invitrogen, Grand Island, NY) and were fixed at
room temperature using 4% paraformaldehyde solution in
1X phosphate buffered saline (PBS). Cells were washed
with distilled water, and were stained with Alizarin Red so-
lution at room temperature for 20 minutes. Finally, the cells
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 6 of 13

were washed with distilled water for 6 times and were
photographed.
Spermine synthase activity assay
To test SMS activity in lymphoblastoid cell lines, we
measured the production of deuterated spermine
(spermine d8) from deuterated spermidine (spermidine
d8). Briefly, cells were harvested by centrifugation and
washed 2X with PBS and suspended in 50mM sodium
phosphate buffer pH 7.2 with protease inhibitor (Sigma,
St. Louis, MO). Samples were frozen at -80ºC. Upon
thawing, the samples were subjected to 2 freeze-thaw cy-
cles using an ethanol and dry ice bath. After centrifuga-
tion, protein in the supernatant was quantified using the
Lowry assay. To test for SMS activity, 70 μg of protein
was incubated with 0.1M sodium phosphate buffer 7.5,
10μM spermidine d8 (Sigma, St. Louis, MO), protease
inhibitor (Sigma, St. Louis, MO), 100μM dcSAM, and
50uM 4-MCHA (Sigma, St. Louis, MO) in a total vol-
ume of 100 μL. Baseline reactions were stopped immedi-
ately with 100μL acetonitrile/0.1% formic acid; other
samples were incubated at 37ºC for 24 h and then
stopped by addition of acetonitrile/0.1% formic acid.
Spermine d8 was quantified by LC/MS/MS. Enzymatic
activity was represented as the area of the spermidine d8
peak per hour.
Measurement of polyamine content
The spermine/spermidine ratio was determined in the
lymphoblastoid cell lines using LC/MS/MS as described
[36]. Fibroblast and hBMSC polyamines were measured
using the Biochrom 30 polyamine protocol. Briefly, hu-
man fibroblasts and hBMSCs were cultured as described
above. Cells were harvested and pelleted and polyamines
were extracted with a volume of 10% PCA equivalent to
4-fold the weight of the cells in milligrams. After 1 h of
incubation on ice, cells were centrifuged at 13,000 rpm
for 10 min at 4 degrees. The polyamines were fraction-
ated and quantitated on the Biochrom 30 using an ion
exchange polyamine column, compatible with sodium
chemistry.
Immunofluorescent localization of SMS
Immunofluorescent detection of SMS was modified from a
previously described protocol [38]. Briefly, 2 x 104 cells were
grown overnight on a coverslip in a 6-well plate. The cells
were fixed with 3.7% paraformaldehyde (PFA) for 25 min at
room temperature and permeabilized with 0.1% Triton X-
100, 2mg/ml BSA and 1mM NaN3 for 5 min at room
temperature. All cells were blocked for 2 h with casein
blocking buffer with 10% horse serum and then incubated
at 4°C overnight with anti-SMS (1:100) (Sigma, St. Louis,
MO) diluted in blocking buffer. They were then gently
washed 3 times with PBS and incubated with secondary
antibodies conjugated with Alexa 488 or Alexa 555 (2ug/ml,
Life Technologies, Grand Island, NY) for 2 h at room
temperature. Cells were then washed 3 times with PBS and
mounted in Vectashield containing 4′, 6-diamidino-2-phe-
nylindole (DAPI, Vector Laboratories, Burlington, ON,
Canada). Images were acquired using a 63X Zeiss plan-apo-
chromat oil, 1.4 NA, DIC objective lens on a Zeiss LSM 780
confocal microscope using Zen 2011 acquisition software.
Alkaline phosphatase activity
Upon reaching confluence, hBMSCs were plated in
6- well plates (100,000 cells/well). After 10 days of
growth in MEM Alpha with 20% FBS, the hBMSCs
were stimulated with 10mM β-glycerophosphate (Sigma
St. Louis, MO), 50μM ascorbic acid 2-phosphate
(Sigma, St. Louis, MO) and 100nM Dexamethasone
(Sigma, St. Louis, MO) for 5 days. Alkaline phosphatase
activity was quantified using StemTAG Alkaline Phos-
phatase Colorimetric Kit (Cell Biolabs, San Diego, CA).
Results
Exome sequencing identifies a novel SMS mutation
diagnostic of SRS
The propositi had a maternally inherited hemizygous
transition (NM_004595.4:c.443A > G) in SMS identified
by exome sequencing (Additional file 1: Table S2; full
VCF file is available on request) and confirmed by
Sanger sequencing (Figure 2A-B). This mutation en-
codes the missense mutation p.Gln148Arg (CDPred
score: -9; SIFT score: 0; PolyPhen2: probably damaging
(0.998, Sensitivity 0.27, Specificity 0.99)). The Gln148
residue is conserved to S. cerevisiae (Figure 2C) and re-
sides in the central β-strand domain that functions as a
cap for the carboxyl terminal catalytic domain. It is one
of 8 residues involved in the binding of 5’-methylthioa-
denosine (MTA) (Figure 2D) [39,40], which functions as
an amine acceptor.
Similar to other SRS-associated SMS mutations [11-17],
the p.Gln148Arg variant decreased the steady state level of
SMS protein detectable in cultured fibroblasts by immuno-
fluorescence and immunoblotting (Figure 2E-K). It reduced
total SMS steady state levels 2.6-fold (Figure 2L); nuclear
SMS was reduced 5.8-fold and soluble cytosolic SMS 2.7-
fold (Figure 2M).
Spermine d8 generation by lymphoblastoid lysates ex-
pressing p.Gln148Arg SMS was 37- fold less than that
for unaffected controls (Figure 2N). Additionally, as
measured by LC/MS/MS, the ratio of spermine: spermi-
dine in the lymphoblastoid lysates was reduced 10-fold
compared to unaffected controls.
The propositi had features previously not reported with
SRS, including more profound intellectual disability, retinal
pigmentary changes, renal dysfunction, frequent pulmonary
infections and hyper- and hypoglycemia (Table 1). Although
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 7 of 13

we hypothesized that rare mutations in other genes contrib-
uted to these features, only a maternally inherited X-linked
variant in FRMPD4 (c.583A > G, p.K195E) segregated with
the disease and had a predicted pathogenicity of at least
probably damaging in Polyphen [27]; SIFT and CDPred pre-
dict the variant as tolerated. FRMPD4, which has been asso-
ciated with autism and schizophrenia [41], encodes a
product that regulates dendritic spine morphogenesis [42].
No pathogenic mutations were observed in genes associated
with retinal pigmentary changes.
Bone formation is decreased in patient II-1
SMS is widely expressed (Additional file 1: Figure S1);
consequently, we questioned why specific tissues such as
bone are particularly affected in SRS patients. To define
better the osteoporosis of SRS, we performed a bicortical
transiliac crest core biopsy of Patient II-1. The specimen
was soft and fragmented when removed from the tre-
phine (Figure 3A). Histomorphometry revealed an ab-
sence of a trabecular meshwork, a low bone volume and
a thin cortex. Cancellous bone volume was markedly de-
creased at 4.7%, compared with 23% ± 4.4 in healthy
controls. The cortical mean width measured only 238
microns compared to a mean of 1202 microns in con-
trols. Osteoblastic activity was markedly reduced with
osteoblasts occupying only 1% of the osteoid surface
(normal range 12.1% ± 4.6). There was no observable
osteoclastic activity in the patient’sspecimen,dem-
onstrated by an eroded surface of 0% (normal range
4.1% ± 2.3) and an osteoclast surface of 0% (normal
range 0.7% ± 0.6). Surface bone formation rate was
11.6 μm
3
/μm
2
/y (normal range: 35.8 μm
3
/μm
2
/y ± 8.9).
CSF neurotransmitter levels do not suggest sympathetic
nervous system dysfunction in Patient II-1
Because bone density and turnover are controlled in part
by the sympathetic nervous system [43-49], we measured
CSF neurotransmitters in Patient II-1 for evidence of in-
creased sympathetic activity. The 5-methyltetrahydrofolate
(methyl donor), 5-hydroxyindoleacetic acid (serotonin me-
tabolite), 3-O-methyldopa (metabolite of L-Dopa), and tet-
rahydrobiopterin (cofactor for the synthesis of serotonin,
dopamine, norepinephrine) were all within normal limits.
Although homovanillic acid (catecholamine metabolite)
(459 nmol/L) and neopterin (catabolic product of GTP)
(30 nmol/L) were slightly above normal limits (324 and 28
nmol/L, respectively), these slight elevations were consid-
ered clinically insignificant.
Endocrine evaluation in patient II-1
Because bone density is also modulated by multiple
endocrine regulators [19], we assessed these in Patient
II-1. Intact PTH, Vitamin D, magnesium, cortisol, insulin-
like growth factor 1, thyroxine, thyroid stimulating hor-
mone, prolactin and ACTH were unremarkable (Table 2).
Also, manual inspection of the exome results did not
identify any predicted deleterious mutations of IGF1 or
IGF1R despite >20 fold depth of short read coverage
across the length of each exon. Prior to admission to the
NIH, patients II-1 and II-3 had fluctuations in blood cal-
cium and phosphate levels (Table 3). These fluctuations
had no apparent pattern or mediating factor based on diet,
fluid intake, or clinical status, and at the time of admission
to NIH, the blood levels of calcium and phosphorus were
normal. Tubular reabsorption of phosphate was normal,
but, the calculated 24-hr urine calcium excretion (derived
from an 18-hr collection) was elevated. Osteocalcin levels
were low for pubertal stage, consistent with decreased
bone formation. At age 15 years, patient II-1 had undes-
cended testes, remarkably delayed puberty, low LH and
FSH levels and undetectable testosterone. By age 18 years,
he had entered early puberty as evidenced by an increase
in LH and FSH, and testosterone levels in the Tanner II
range. Testicular ultrasound at that time identified both tes-
tes in the inguinal canal, with volumes of 5.7 mL and 1.7
mL. At a chronological age of 18 years and 4 months, his
hone age was 12 years and 6 months.
Table 1 Comparison of clinical features of all reported
SRS patients
Features Reported
patients
Patient II-1 Patient II-3
Cognitive impairment 15/15 + +
Seizures 8/15 + +
Myopia 2/13 + * + *
High, narrow palate 3/13 + +
Prominent lower lip 12/15 + -
Speech abnormalities 15/15 + ** + **
Diminished body bulk 15/15 + +
Kyphoscoliosis 13/15 + +
Osteoporosis 11/11 + +
Long finger/toes 12/15 + *** #
Unsteady gait 10/15 + **** + ****
Renal abnormalities 3/13 + +
Nephrocalcinosis
a
0/13 + +
Frequent infections 0/13 + +
Retinal pigment changes 0/13 + +
Hypo-/Hyper-glycemia 1/13 + +
Muscle fiber
abnormalities
1/13 + +
#Not assessed.
*Partial blindness.
**No vocalizations.
***Contractures of fingers.
****Non-ambulatory.
a
Note: Nephrocalcinosis in these patients previously recorded in
GeneReviews [20].
The phenotype data used to generate this table was collected from [11-17].
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 8 of 13

Figure 3 (See legend on next page.)
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 9 of 13

Compared to cultured SRS fibroblasts, SRS hBMSC have
comparable SMS mRNA and protein levels but pronounced
disturbances of polyamine levels
While absolute polyamine levels are not substantially
abnormal in cultured fibroblasts and lymphoblastoid cells
of SRS individuals, spermidine/spermine ratios reflected
the SMS enzyme deficiency [12-17]. Neither lymphoblas-
toid cells nor skin fibroblasts, however, are affected tissues
in SRS. We hypothesized therefore that cells from affected
tissues such as bone had differences in SMS expression or
polyamine metabolism accounting for their clinical mani-
festations. To test this, we compared SMS expression and
polyamine levels among cultured skin fibroblasts and
hBMSC. Cultured skin fibroblasts and hBMSC had com-
parable steady state SMS mRNA levels (Figure 3B) and
SMS protein levels (data not shown), suggesting that differ-
ential SMS expression is an unlikely basis for expression of
SRS features. Additionally, excluding differential degrad-
ation of the mutant SMS protein as the basis for expression
of SRS features, the cultured hBMSC derived from Patient
II-1 had SMS protein levels only 1.7-fold lower than for
control hBMSC (Figure 3C-K), whereas his fibroblasts had
2.6-fold less SMS protein compared to control fibroblasts.
To test if differences in polyamine metabolism might
contribute to the differential expression of SRS features,
we compared polyamine levels in cultured skin fibro-
blasts and hBMSCs. Skin fibroblast lysates derived from
Patients II-1 and II-3 had mean spermidine levels 1.78-
fold higher than control fibroblasts (p = 0.28) and mean
spermine levels 7.40-fold lower than control fibroblasts
(p = 0.21). In contrast, lysates of cultured hBMSCs from
Patient II-1 contained mean spermidine levels 5.07-fold
higher (SEM = 22.8, p = 0.001) than control hBMSCs
(Figure 3L) and had no detectable spermine.
SRS hBMSCs have decreased osteogenic activity
To determine the differences in osteogenic potential,
hBMSCs isolated from an unaffected control and Patient
II-1 were treated with osteogenic differentiation media
and stained with Alizarin Red S, an anthraquinone dye
that stains the calcium deposits indicative of mature os-
teocytes [50,51]. Based on the intensity of Alizarin Red S
Table 2 Markers of bone and endocrine function in
Patient II-1 at 18 years of age
Metabolite Patient II-1
values
Reference
range
Intact parathyroid hormone (pg/mL) 28.9 (15-65)
1,25-dihydroxycholecalciferol (pg/mL) 70 (18-64)
25-dihydroxycholecalciferol (ng/mL) 39 (33-100)
Osteocalcin (ng/mL) 35.7 (7.3-38.5)
1
,
(49-167)
2
Free thyroxine (ng/dL, direct dialysis) 1.6 (1-2.4)
Thyroid stimulating hormone (μIU/mL) 1.8 (0.4-4)
Testosterone (ng/mL) 43.6 (100-740)
1
,
(8-418)
2
Free testosterone (ng/dL) 0.5 (7.4-22.6)
1
Sex hormone binding
globulin (nmol/L)
63 (10-60)
1
,
(44-160)
2
Follicle stimulating hormone (U/L) 8.7 (1-11)
1
Luteinizing hormone (U/L) 2.5 (1-8)
1
Prolactin (mcg/dL) 9.4 (2-25)
Cortisol (mcg/dL, morning) 16.5 (5-25)
Adrenocorticotropic hormone (pg/mL) 12.7 (0.0-46.0)
Insulin-like growth factor (ng/mL) 347 (75-420)
2
pH Urine 8.5 (5-8)
18-hr urine volume (mL/24 h) 2885 (600-1800)
Calculated urine calcium excretion
(mmol/kg/24 h)
0.134 (<0.1)
Tubular reabsorption of
phosphorus (%TRP)
93.5 (85-95)
1
Reference ranges for age,
2
Reference range for pubertal stage.
(See figure on previous page.)
Figure 3 Characterization of the bone and osteoblast pathology. A. Photograph of the bone biopsy. B. Steady state SMS mRNA levels
relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were
derived by qRT-PCR analysis of 3 independent extractions of total RNA. C. Immunoblot showing steady state SMS protein expression in patient
and control osteoblasts. ß-tubulin is shown as a loading control. D. Graph showing steady state SMS protein levels in the patient and control
hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line.
E-J. Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown
in red and the nucleus is shown in blue. K. Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei
of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression.
L. Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine
levels than do the patient fibroblasts, * p < 0.05, *** p < 0.005. M. Osteogenic potential of bone marrow stromal cells (hBMSCs) isolated from Patient II-1
sample is markedly lower than that of an unaffected control (cnt). The hBMSCs were seeded in triplicates (6x10
4
/12-well) and either kept untreated (-)
or treated (+) with osteogenic differentiation media (see Methods) for 18 days. After the treatment, cells were fixed and were stained with Alizarin Red
S to check for calcium deposition, a marker of osteogenic differentiation.
Table 3 Calcium phosphate levels in patients II-1 and II-3
Serum Metabolite Patient II-1 Patient II-3 Reference range
Calcium (mg/dL) 7.8-10.6 6.6-10.1 8.78-10.5
Phosphorus (mg/dL) 2.4-5.1 3.5-5.3 3.1-5.1
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 10 of 13

staining, the differentiated hBMSCs from Patient II-1
produced markedly fewer calcium deposits than did
those from the control (Figure 3M). Addition of 1μM
spermine did not alter the Alizarin Red S staining (data
not shown).
Discussion
We report two brothers with SRS in whom we identified
aSMS mutation (NM_004595.4:c.443A > G, p.Gln148Arg)
resulting in near absence of enzyme activity and decreased
steady state SMS protein levels. To better delineate the
tissue-specificity of SRS features, we investigated the
low bone density of SMS and observed functional osteo-
blast and osteoclast deficiencies, a marked spermidine
and spermine imbalance in hBMSCs and poor calcium
phosphate mineralization by differentiated hBMSCs.
We therefore speculate that polyamines play a critical
role in osteoblasts that is not required by other cells,
such as lymphoblasts and fibroblasts.
The p.Gln148Arg mutation represents the first SMS
mutation to alter the MTA binding site [52]. Disturbance
of MTA homeostasis could contribute to the more severe
phenotype of the propositi relative to that of other SRS
patients. MTA is needed for the transfer of the aminopro-
pyl group from decarboxylated S-adenosylmethionine
(dcAdoMet) to spermidine; it is also an inhibitor of SRM
and SMS [53], and consequently decreases cellular sperm-
ine concentrations [54]. Indeed, the spermine:spermidine
ratio of our patients’cells was reduced 3-fold more than
the ratio for cells tested from any other SRS patient (C.S.,
unpublished data).
The severity of the phenotype in the propositi relative to
other individuals with SRS might alternatively be attribut-
able to differing insults or genetic backgrounds. For ex-
ample, the perinatal intraventricular hemorrhage of patient
II-1 might contribute to the severity of his neurological fea-
tures, although the presence of similar neurological features
in his brother, who did not have an ischemic or
hemorrhagic brain insult, suggests this is not a substantial
contributor. On the other hand, the variant in FRMPD4
might modify the genetic background and thereby contrib-
ute to the intellectual disability of the propositi. We did not
identify other environmental insults or appropriately segre-
gating, pathogenic variants to explain the additional fea-
tures of the propositi. Consequently, if other genetic
contributors have a significant role in modifying the pheno-
type of SRS, they were either not detected by our exome se-
quencing or were common polymorphisms excluded by
our analyses. It remains possible that epigenetic and sto-
chastic factors also modulate the expressivity of SRS.
Central neuroendocrine signaling does not appear to be
impaired in SRS; however, peripheral neuroendocrine sig-
naling including hypogonadism and altered calcium and
phosphate homeostasis might contribute as might other
issues such as immobilization, renal tubular acidosis, low
muscle mass and medications. It is thought that male
hypogonadism decreases bone mineral density because
androgens promote osteoblast differentiation and prolifer-
ation and decrease the activity of osteoclasts [55]. Add-
itionally, impaired calcium and phosphate homeostasis
impede osteoclast function [56], and hypercalciuria is as-
sociated with decreased bone density, as well as the
nephrocalcinosis seen in this patient. Several anticonvul-
sants, including clonazepam and topiramate, are also asso-
ciated with decreases in bone mineral density [57]. These
factors alone are unlikely to fully account for the de-
creased bone mineral density observed in our patients,
since other SRS individuals have had low bone density in
the absence of these issues [11]. Rather, the predominant
mechanism of osteoporosis in SRS is likely related to im-
paired polyamine metabolism.
The disease mechanisms and phenotypic expansion re-
ported herein provide some insight for the management of
SRS. Optimal control of the metabolic abnormalities, lim-
ited use of medications known to affect bone, and appro-
priate physical therapy should be part of the management
plan in any chronically ill, immobilized individual. Since
analysis of the bone did not detect increased osteoclastic
activity, bisphosphonate therapy would likely be of minimal
effectiveness unless further studies refute our observations.
In addition, if study of additional patients establishes the
association of renal disease, retinal pigmentary changes,
and perturbations of glucose homeostasis with SRS, then
screening for and symptomatic management of these prob-
lems has the potential to improve patient care.
Conclusions
This report identifies a novel SRS-associated SMS muta-
tion, p. Gln148Arg, and expands the SRS phenotype. It
also provides the first evidence that SRS patients have a
loss of osteoblast and osteoclast activity and that the low
bone density of SRS likely arises by a cell intrinsic process.
Consent
The patients’parents gave written, informed consent for
publication of this case report and any accompanying
photographs.
Additional file
Additional file 1: Table S1. CNVs detected in the propositi. Table S2.
Exome variants meeting rarity and predicted deleteriousness requirements
and segregating with disease. Figure S1. Expression profile of SMS mRNA
and protein. (A) Graph showing qRT-PCR detection of SMS mRNA levels in
total RNA extracted from the respective tissues. SMS mRNA levels
were normalized to the mRNA levels of GAPDH. (B) Immunoblot
showing SMS protein levels in lysates from the respective tissues. (C)
SMS protein expression in each tissue plotted relative to GAPDH protein
expression.
Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 11 of 13

Competing interests
The authors declare that they have no competing interests.
Authors’contributions
JSA designed, performed and interpreted all of thepolyamine assays, the
protein and RNA expression and imaging assays and drafted the manuscript.
Coordinated the re-admission of the patient so, bone biopsy and further
endocrinology studies could be obtained. NB cultured human bone marrow
stromal cells and performed the differentiation experiments and the calcification
assays. LW was the primary nurse practitioner involved with thepatients. She
coordinated the clinical sample testing and performed history and physical
exam. WB aided in the culturing human bone marrow stromal cells and
performed the differentiation experiments and the calcification assay and
aided in the analysis of the NGS and SNP chip data. VM cultured human
bone marrow stromal cells and performed the differentiation experiments
and the calcification assays. JA did the sanger confirmation. DA was the
attending physician when the patient was admitted, and coordinated
clinical care while at the NIH. CS helped with coordination of the enzyme
assay and provided samples from other individuals with SRS and expertise
about the disorder. JN performed the spermine synthase enzymatic assay.
TW helped with interpretation of the enzymatic assays. RG Was the
endocrinologist involved with the case that helped partition the bone
biopsy sample in the operating room and coordinated bone marrow stem
cell culture. MC provided consultation for endocrinology findings. LT performed
the bone biopsy. TM performed the NGS analysis that identified the variant in
SMS. WG coordinated the admission of these individuals into the UDP, curated
the chart, provided insights into interpretation of data and obtained funding for
the studies. All authors read and approved the final manuscript.
Acknowledgements
We thank Drs. Paul Lee, May Christine Malicdan and Grace Zhai for critical
review of the manuscript. We would like to thank Dr. Edward McCarthy at
The Johns Hopkins School of Medicine for performing the bone
histomorphometry, Dr. Camilo Toro for imaging interpretations and Shira
Ziegler for technical guidance for the alkaline phosphatase assay. This work
was supported by the Intramural Research Programs of the National Human
Genome Research Institute and National Institute of Dental and Craniofacial
Research and the Common Fund of the NIH Office of the Director, National
Institutes of Health. This work as also supported by extramural funding from
NINDS (NS073854) and South Carolina Department of Disabilities and Special
Needs (SCDDSN).
Author details
1
Undiagnosed Diseases Program, Common Fund, Office of the Director,
National Institutes of Health, Bethesda, MD 20814, USA.
2
Medical Genetics
Branch, National Human Genome Research Institute, Bethesda, MD, USA.
3
Skeletal Clinical Studies Unit, Craniofacial and Skeletal Disease Branch,
National Institute of Dental and Craniofacial Research, National Institutes of
Health, Bethesda, MD 20892, USA.
4
J.C. Self Research Institute, Greenwood
Genetics Centre, Greenwood, SC 29646, USA.
5
George Washington University
School of Medicine, Washington, DC, USA.
6
Children’s National Medical
Center, Washington, DC, USA.
Received: 9 July 2014 Accepted: 28 January 2015
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Albert et al. Orphanet Journal of Rare Diseases (2015) 10:27 Page 13 of 13