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SUPPLEMENTARY MATERIAL
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DETAILED CLINICAL COURSE
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Details on clinical features and diagnostic procedures
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Birth weight (BW), length (L) and head circumference (HC) was below the 3rd percentile in
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both twins (twin A: BW 2230g, L 45.5cm, HC 31cm/ twin B: BW 1930g, L 45cm, HC
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30.5cm). In twin A, the neonatal period was complicated by a mild respiratory distress
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syndrome, hypoglycemia of 1.5 mmol/l and hyperbilirubinemia. Cranial ultrasound was
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normal in both. Weight and length showed continuous progress on the 50th and 75th percentile,
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respectively, while the head circumference was -2.9 SD in both with plateauing of head
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growth since the age of 18 months. Dysmorphic features became more apparent during the
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second year of life.
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Magnetic resonance imaging of the brain at age 13 months (twin A) and 14 months (twin B)
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respectively, showed a thin corpus callosum and slight cerebral atrophy in twin A, while twin
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B had isolated bilateral pallidal hyperintensity. Screening tests for metabolic diseases
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including amino acids in plasma and CSF, ammonia and pipecolic acid in plasma,
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acylcarnitine profile in blood, organic acids in urine, transferrin electrophoresis in serum
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(twin A), lactate in plasma and CSF (twin A), and neurotransmitter analysis in CSF in twin A
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were all normal.
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Development
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Early development was delayed in both boys: social smile was present by five months of age;
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laughter and babbles were noticed by five months in twin A and ten months in twin B, but
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active speech development remained absent in both. Griffiths mental developmental scales at
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age ten months corresponded to five to eight months. Neurological examination at the age of
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ten months revealed axial hypotonia and muscular hypertonia of the lower extremities. The
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last clinical exam at the age of 29 months revealed axial hypotonia and hypertonia of the
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lower extremities in both twins and a persistent choreo-athetoid movement disorder in twin A.
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Active sitting and grasping was absent in both. Griffiths mental developmental scales rated
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gross motor skills at five to six months, whereas social, fine motor, visual and cognitive skills
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were rated at three to four months.
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Course of epilepsy
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At the age of 12 months, recurrent irregular myoclonic jerks were observed in both twins and
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serial infantile spasms in twin A. EEG revealed rhythmic theta background activity and
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irregular multifocal and generalized spike waves. Twin B showed photosensitivity with
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myoclonic jerks, twin A had hypsarrhythmia in sleep and serial spasms at awakening. The
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latter was successfully treated with a four week regime of prednisolone, but myoclonic jerks
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were drug-refractory in both. At age 14 months, both twins developed atonic seizures with
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distinct cyanosis, starring and postictal sleep. Recurrent respiratory infections required several
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hospitalizations at age 15 and 16 months. EEG and seizures simultaneously deteriorated with
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frequent myoclonic jerks, atypical absences, atonic and tonic seizures. Along the recurrent
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infections and worsening of their epilepsy both twins had developmental regression with
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reduced social and visual interaction, aggravation of muscular hypotonia and loss of grasping
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in twin B. Several anticonvulsive drugs (levetiracetam, vigabatrine, topiramate, pyridoxine,
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clobazam, sultiam) as well as ketogenic diet failed to control seizures. Finally mesuximide
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efficiently controlled atypical absences and valproic acid mitigated the intensity and
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frequency of tonic seizures. In the further course, both boys suffered from generalized
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myoclonic jerks and asymmetric tonic seizures often initiated by a myoclonic jerk. The EEG
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was still severely abnormal with a continuous multifocal and generalized slow-spike-wave
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pattern, which in twin A was sometimes interrupted by an irregular theta activity of 20-30
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seconds duration.
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MATERIAL AND METHODS
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Genetic studies
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High-resolution microarray testing for copy number profiling was performed using the
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Cytoscan HD microarray (Affymetrix Inc., Inc., Santa Clara, CA, USA) targeting 2.65 million
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SNP and copy number markers. Copy number variants were analysed at a resolution of 20 kb
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minimum size and filtered against copy number variants detected in 1038 healthy European
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and American controls. Variants observed in at least 16% of reads with sufficient quality level
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were filtered against the dbSNP database. All previously unreported non-silent variants with
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potential deleterious effects as assessed by SIFT, PhyloPhen, Mutation Taster, Mutation
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assessor and FATHMM prediction were manually assessed for known associations of the
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affected gene with epilepsy or intellectual disability.
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Mutation modeling
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Structural analyses were based on the crystal structure of human spermine synthase (PDB:
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3C6M; (Wu, Min et al. 2008). SWISS-MODEL (Guex and Peitsch 1997) and RASMOL were
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used for structure analysis and visualization (Sayle and Milner-White 1995) (Figure S3).
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Western Blot
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Total cell lysates were obtained from cultured lymphoblasts by lysis of 2.4 x 106 cells with
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modified RIPA buffer (150 mMNaCl, 1 mM EDTA, 50 mMTris-HCl pH 7.4, 0.5 % sodium
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deoxycholate, 0.1 % SDS) containing protease inhibitor cocktail and phosphatase inhibitor
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cocktail 2 (Sigma-Aldrich, Buchs SG, Switzerland). Lysates were centrifuged at 20,000 x g
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for 15 minutes, supernatant was collected, and total protein concentration was determined by
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BCA as specified by the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL,
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USA). Whole cell lysate from HeLa cells was obtained from Abcam (Cambridge, MA, USA)
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as a positive control for detection of spermine synthase, as spermine synthase expression in
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HeLa cells was demonstrated on the antibody datasheet.
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Total cell lysates (10 µg per lane) were resolved by 10% SDS-PAGE and transferred to PVDF
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membrane. Membrane was blocked for 1 h in 3% BSA in Tris-buffered saline/Tween 20,
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followed by incubation with SMS antibody (1:1000 dilution; Abcam) or β-Actin antibody
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(1:5000 dilution; Abcam). Detection was performed using the appropriate peroxidase-
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conjugated secondary antibody (1:5000 dilution, Abcam) and Clarity Western ECL substrate
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(Biorad, Cressier, Switzerland). Images were obtained using the G:BoxChemiXL (Syngene,
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Cambridge, United Kingdom).
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Metabolomics data acquisition and preprocessing
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Mass spectrometer settings for full-MS were as follows: In-source CID 0.0 eV, µscans = 1,
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resolution = 70,000, AGC target 1e6, max IT = 35 ms, scan range 67 to 1000 m/z, spectrum
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data type, profile. Detector setting for dd-MS2 were: µscans = 1, resolution = 17,500, AGC
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target 1e5, max IT = 80 ms, loop count = 5, isolation window 4.0 m/z, NCE 30.0, intensity
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threshold 1.3e4, apex trigger 2 to 4s, spectrum data type, profile.
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RESULTS
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Metabolomics analysis
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Initial XCMS analysis gave 78903 total peaks (approx. 1012 per sample) over a time range of
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0-11.8 minutes and mass range of 68.981- 992.6799 m/z. CAMERA processing annotated
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1049 aligned data features into 446 peak groups based on isotopic and adduct patterns.
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Genetic analysis
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The crystal structure of human SMS (Wu, Min et al. 2008) revealed that the site of mutation
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(Arg130) is located close to the spermine binding site and is part of the dimer interface
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(Figure S3). In addition to hydrophobic interactions of its methylene groups, Arg130 also
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forms polar interactions with residues Glu133 and Gln157 of the second subunit. In the
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Arg130Cys mutant, the respective cysteine cannot form these interactions due to its shorter
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and uncharged side chain. Therefore, the mutation is expected to decrease dimer stability and
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to affect the structure of the adjacent spermidine binding site. Since dimerization was shown
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to be required for enzymatic activity (Wu, Min et al. 2008), the Arg130Cys mutation is
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expected to cause a significant decrease of enzyme function.
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FIGURE LEGENDS
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Figure S1. Untargeted metabolomics data PCA-plot showing significant difference between
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the SRS cohort (green; n=9) and control cohort (blue; n=19) plasma profiles. Data for each
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sample was acquired in technical triplicate with three longitudinal samples (July/December
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2014, February 2015) collected for SRS twins.
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Figure S2. Receiver operating characteristic (ROC) curve for differential expression of N8-
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acetylspermidine in A) control vs SRS twins July 2014 (area = 0.9), and B) controls vs all
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other SRS plasma samples (area = 1.0). False positive rate is defined as 1-specificity.
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Figure S3. Structure of human spermine synthase showing the site of the Arg130Cys
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mutation. (A) Structure of the dimeric enzyme. The N-terminal domain (residues 1-129) and
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the C-terminal domain (residues 130-366) are colored in cyan and blue, respectively. For the
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second subunit of the dimer the respective domains are colored orange and red. Arg130 is
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shown in space-fill and spermine (SPM) and 5’-deoxy-5’methylthioadenosine (MTA) are
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shown in stick presentation. (B) Same view as in (A) but with the second subunit shown in
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space-filled presentation. Arg130 (marked by an arrow) is located directly at the interface of
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the subunits. (C) Detailed view of the subunit interactions. Residues 125-135 of the first
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subunit are shown with Arg130 in space-filled presentation (atom type coloring). The second
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subunit is shown in space-filled presentation (orange/red) and the interacting residues
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Glu133/Gln157 are highlighted in green. (D) Same view as in (C) for the Arg130Cys mutant.
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Regions of poor packing and missing interactions are marked by a dotted circle.
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Figure S4. Western blot analysis of spermine synthase from control patients lymphoblasts
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(lanes 1 and 2), lymphoblast lysates from SRS twins A and B (lanes 3 and 4), blank (lane 5)
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and control Hela cell lysate (lane 6). Spermine synthase is clearly diminished in the SRS
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twins. β-actin is shown as a control.
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Figure S5. Mass spectral fragmentation pattern for N8-acetylspermidine used to confirm
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metabolite ID combined with accurate mass measurement. Top, reference compound; bottom,
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SRS patient plasma raw data.
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Figure S6. Mass spectral fragmentation pattern for spermidine used to confirm metabolite ID
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combined with accurate mass measurement. Top, reference compound; bottom, SRS patient
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plasma raw data.
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Table S1. Endogenous metabolites with highest discriminant values in a two class OLPS-DA
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model (control vs SRS). Fold-change values were calculated using area under curve values.
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REFERENCES
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Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for
comparative protein modeling. Electrophoresis 18(15): 2714-2723.
Sayle RA, Milner-White EJ (1995) RASMOL: biomolecular graphics for all. Trends Biochem
Sci 20(9): 374.
Wu H, Min J, Zeng H, et al. (2008) Crystal structure of human spermine synthase:
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implications of substrate binding and catalytic mechanism. J Biol Chem 283(23):
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16135-16146.
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