Supplementary Information s36

Supplementary Information

Case Reports

Patient II:3 (mother of proband)

Patient II:3 is one of five children born to non-consanguineous unaffected parents. There is no family history of neurological problems. Patient II:3 started to show symptoms at age 2 years old. Poor balance and coordination with ataxic movements, slurring of speech, mild learning difficulties were reported. However, patient II:3 first presented to clinical services as an adult when her son was diagnosed with ataxia. Examination at age 27 years showed that she had speech dyspraxia and moderate dysarthria. Her gait was mildly ataxic with slightly broadened base and tandem gait was moderately impaired. Power, tone, sensation and plantar reflexes were normal. She has a high nasal bridge, deep set eyes, low hairline and a wide neck (Fig. 1A). Klippel-Feil syndrome was excluded by cervical Xray. On neurological examination she had mild bilateral ptosis and consistently small pinpoint pupils. Eye movements were normal with a full range of movement and no nystagmus. There was no evidence of ocular apraxia. She had a corneal graft for keratoconus which developed in her teens.

Patient II:3 was extensively investigated by the metabolic, neurological and genetic teams. Serum lactate, alpha feto protein, serum protein, metabolic screen and vitamin E levels were all normal. Sequencing of ATXN1 (SCA1), ATXN2 (SCA3) ATXN3 (SCA3) and CACNA1A (SCA6) was negative. Mutations in POLG and mtDNA mutations and rearrangements were also excluded. Computed tomography (CT) of brain aged 32 years showed areas of low density in the occipital parenchyma with accompanying loss of grey-white matter delineation. Magnetic resonance imaging (MRI) of brain was normal with no signs of cerebellar atrophy. Growth parameters at 33 years of age were head circumference 59 cm (99.6th centile), height 167.5 cm (75th centile) and weight 109.9 kg (>99.6th centile). Patient II:3 was subsequently diagnosed with an early-onset dominant ataxic syndrome of unknown genetic cause. The patient reports a gradual improvement in symptoms over time. Currently her condition is static but is worsened by anxiety.

Patient III:1 (proband)

Patient III:1 was born at term by vaginal delivery weighing 4.05 kg. Concerns arose at one year of age when he was delayed in reaching his milestones; he sat up at age 12 months, started crawling at age 15 months and started walking and talking at age 24 months. He was unsteady on his feet and there was some evidence of gait ataxia. Dysmorphic features including epicanthic folds and hypoplastic teeth were noted. He was diagnosed with an atrial septal defect (ASD) measuring 6mm at age 4 years which was managed conservatively. He was noted to have fine and gross motor delay, ataxia, speech and language delay and poor pronunciation. He had a wide-based and high-stepping gait with raised arches and some toe-walking. His lower limbs were externally rotated and he had broad feet. He has difficulty in walking heel-to-toe. He had a very mild activity-evoked tremor but this was not present in later years. Ophthalmic examination aged 5.1 years identified bilateral pinpoint pupils similar to his mother’s but they reacted normally to light. He had full eye movements and no nystagmus. Fundal examination was normal. Keratoconus was not present. Neurological examination showed normal strength and power with normal reflexes. CT and MRI brain at age 10 years was normal with no evidence of cerebellar atrophy.

At 4.8 years, he had marked midline truncal ataxia, gross and fine motor delay, and speech delay. Tendon reflexes were present. Review at 9 years of age showed persistence of truncal ataxia and a mild degree of dysarthria. His balance has improved over time. He does not have a tremor. Fine and gross motor skills improved, though he is still delayed. Language has improved to within normal limits. Overall, improvements were observed until age 9 years, at which point his condition plateaued. Growth parameters at age 10.8 years were head circumference 55 cm (50-75th centile centile), height 139.2 cm (50th centile) and weight 23.2 kg (50th centile). Similar to his affected mother, patient III:1 has a high nasal bridge and deep set eyes (Fig. 1B).

Serum amino acids, metabolic screen, immunoglobulins, TFT, LFT, creatinine kinase, urea and electrolytes and blood lactate were all normal. Karyotype and array comparative genomic hybridisation analysis were normal. Genetic testing of ATM, FMR1 and PTEN excluded ataxia-telangiectasia, Fragile-X and Bannayan-Riley-Ruvalcaba syndrome respectively.

Patient III:2 (sibling of proband)

Patient III:2, the younger sister of patient III:1, was born at term weighing 2.9 kg. She was diagnosed with cystic fibrosis through neonatal screening and is compound heterozygous for two pathogenic CFTR variants; ΔF508 and p.(Gly551Asp). Neonatal history was unremarkable. Examination at age 6 months showed age-appropriate development but there were signs of mild motor delay. She was not rolling over or sitting in the tripod position. She also had decreased central tone, macroglossia and overlapping 3rd toes on both feet. A supra-umbilical hernia and an epigastric defect were also identified. Follow up showed delay in gross and fine motor development. She started to show signs of ataxia by age 2 years. Speech was difficult to understand but she had many words. Ophthalmic examination at 2.5 years identified a blocked tear duct but showed that eye movements were full and refraction and fundi normal. Review at 2.9 years identified dysmorphic features including a depressed nasal bridge, epicanthic folds bilaterally, mild frontal bossing and mild hypertelorism (Fig. 1C). There was evidence of metatarsus valgus. Her growth parameters were head circumference 48 cm (25th-50th centile), height 93.5 cm (25th-50th centile) and weight 12.5 kg (10-25th centile).

Supplementary Methods

Plasmid construction

ITPR1 variant (c.106C>T; p.(Arg36Cys)) was constructed from the full-length human ITPR1 (SI-, SII-, SIII- [GenBank ID : D26070.1] (Yamada et al., 1994) that was subcloned into pBluescriptII KS+ (pBKS-hIP3R1). To generate pBKS-hIP3R1-Arg36Cys, we digested pBKS-hIP3R1 with SacII and promoted self-ligation to subclone the 4869 bp SacII fragment of pBKS-hIP3R1. Then, site-directed mutagenesis of the plasmid was performed using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies CA, US) with primers containing the appropriate substitutions (5’-gggcctggttgatgattgttgtgttgtacagcc-3’ and 5’-ggctgtacaacacaacaatcatcaaccaggccc-3’). Then, the 6851 bp SacII fragment from pBKS-hIP3R1 was inserted in the SacII site of the mutated plasmid to obtain pBKS-hIP3R1-Arg36Cys. For use in cell transfection, the SalI fragment of pBKS-hIP3R1-Arg36Cys was subcloned into the SalI site of pBact-S to construct pBactS-hIP3R1-Arg36Cys (Furuichi et al., 1989).

The plasmid encoding the GST fusion protein with the NH2-terminal 589 amino acids of human ITPR1 (pcold GST-589hIP3R1-Arg36Cys or -WT) was constructed as follows. The NH2-terminal fragment was amplified by PCR using the sense primer (5’-tcggatccatgtctgacaaaatg-3’ containing a BamHI site [underlined]), the antisense primer (5’-ggaagcttcatttccgattattgtg-3’ containing a HindIII site [underlined]), and pBactS-hIP3R1-Arg36Cys or – WT as the template. The resultant PCR fragment was digested with BamHI and HindIII, and subcloned into the BamHI and HindIII sites of pcold GST DNA (Takara, Shiga, Japan).

Expression and preparation of the NH2-terminal 589 amino acids of human ITPR1

The E. coli BL21Codon Plus (DE3) RIL cells (Takara, Shiga, Japan) containing pcold GST-589hIP3R1-Arg36Cys or GST-589hIP3R1-WT plasmid were grown in 100 mL of Luria-Bertani (LB) medium containing ampicillin (LA) at 37°C overnight. Then, the culture was transferred into 1000 mL of fresh LA medium, and incubated at 37°C. When OD600 of the culture reached 0.4-0.8 measured by V-630Bio UV-VIS Spectrophotometer (Jasca, Tokyo, Japan), the culture was quickly cooled to 15℃ for 30 min. After isopropyl-b-D-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 100 mM, the culture was incubated for 6 hours at 15°C.

Preparation of expression protein was carried out as described previously with minor modification (Yoshikawa et al., 1999). After freezing and thawing cycle, the cell suspended in a binding buffer (1 mM 2-mercaptoethanol, 1 mM EDTA, and 50 mM Tris–HCl [pH 8.0 at 4°C]) was sonicated by 9 cycles of 10 sec on and 10 sec off at 4°C with Astrason XL2020 (Misonix, NY, US). Total cell sonicate was centrifuged at 42,500 × g for 20 min at 4°C, and the resultant supernatant was followed by batch purification with glutathione-Sepharose 4B (GE Healthcare, Buckinghamshire, UK). The cleavage of GST tag with PreScssion Protease (GE Healthcare, Buckinghamshire, UK) was performed according to the manufacturer’s instruction, and the purified 589hIP3R1 proteins were dialyzed to the binding buffer. Protein concentrations were determined by a Bio-Rad protein assay kit using BSA as a reference.

IP3 binding assay

IP3 binding assay was performed as described previously (Yoshikawa et al., 1999). 589hIP3R1 (0.4 mg) was diluted to 50 ml with the binding buffer, and incubated with 2.6-10.4 nM [3H]IP3 (PerkinElmer, MA, US) and various concentration (0-300 nM) of unlabeled IP3 (Dojindo Laboratories, Kumamoto, Japan) for 10 min on ice. After adding 2 mL of 50 mg/mL g-globulin and 50 mL of 30% (w/v) polyethylene glycol 6000, the mixture was incubated for 5 min on ice. The protein-PEG complex was pelleted by centrifugation at 18,000 × g for 5 min at 4°C. The resulting pellet was solubilized with 90 mL of Solvable (PerkinElmer, MA, US) for 30 min at 42°C, and neutralized by adding 18 mL of acetic acid. After mixing 10 mL of Pico-Fluor Plus (PerkinElmer, MA, US), radioactivity of the neutralized samples was measured with TRI-CARB 2900TR Liquid Scintillation Analyzer (Packard, MA, US). Specific IP3 binding activity was assessed as described elsewhere (Yoshikawa et al., 1999; Uchiyama et al., 2002; Bosanac et al., 2005).

Cell culture and transfection

R23-11 cells (Sugawara et al., 1997) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% chicken serum, 50 mM 2-mercaptoethanol, 4 mM glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 39.5 ºC in 5% CO2 (Uchida et al., 2003). Transfection of R23-11 cells was performed by Amaxa Nucleofector (Lonza, Basel, Switzerland) according to instructions.

Single cell Ca2+ imaging

Single cell Ca2+ imaging was performed using the method with a slight modification (Yamazaki et al., 2011). Briefly, R23-11 cells were collected at 24-28 hours after transfection and loaded with 3 mM Fura-2AM (Dojindo Laboratories, Kumamoto, Japan) for 30 min at 30°C in balanced salt solution (BSS; 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 10 mM glucose, and 20 mM HEPES-NaOH, pH7.4) containing 2 mM CaCl2 and 0.025% bovine serum albumin (BSA) (BSS+Ca2++BSA). After washed with BSS+Ca2++BSA, cells were resuspended with BSS+Ca2++BSA and placed into a homemade glass bottom 3.5 cm dish. Floating cells were removed three times by aspirating the supernatant and resupplying BSS+Ca2+ (not containing BSA). Fura-2 fluorescent signals were acquired with an inverted microscope ECLIPSE TE300 (Nikon, Tokyo, Japan), a cooled CCD camera ORCA-ER (Hamamatsu Photonics, Shizuoka, Japan), and an objective lens SFluor (20x, NA : 0.75 [Nikon, Tokyo, Japan]) with excitation filters at 340 ± 10 and 380 ± 10 nm, a beam splitter at 400 nm, and a bandpass emission filter at 510–550 nm. Ca2+ signals were elicited by bath application of anti-B cell receptor (BCR) antibody M4 (final concentration at 1.0 mg/mL) at RT (Sugawara et al., 1997). The fluorescent signals were recorded every 5 sec at an emission wavelength of 510 nm with alternating excitation at 340 and 380 nm (F340 and F380, respectively). The imaging data acquisition were performed with Aquacosmos (Hamamatsu Photonics, Shizuoka, Japan), and off-line analyses were performed with self-making JAVA program. The emission ratio, F340/F380, was defined as R, and ΔR was defined as R - Rresting, where Rresting is the average value of R measured for 1 min before M4 addition. The peak amplitude in arbitrary units (a.u.) was defined as a maximum value of ΔR for 10 min in each cell. The total Ca2+ signal in a.u. was determined as the summation of ΔR/Rbase for 10 min in each cell.

Western blotting

Cells (7.0 × 105) were lysed with 300 mL SDS-PAGE sample buffer after 14 hours of transfection, and the cell lysates were boiled at 95 ºC for 3 min. The cell lysates (15 mL) were separated by 6.5 % SDS-PAGE, and transferred to a membrane at 12 V for 1 hour. The membrane was blocked for 1 hour in PBS with 0.05% Tween-20 (PBS-T) containing 5% skim milk, and then incubated with primary antibodies against ITPR1 (KM1112 (Sugiyama et al., 1994)) (1:1000), and GAPDH (Novus Biological, CO, US) (1:2000) in PBS-T containing 5% skim milk for 1 hour at room temperature. The membrane was washed three times for 15 min in total with PBS-T and incubated with sheep anti-mouse IgG-horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) (1:5000) for 30 min at room temperature. After washed three times for 15 min in total, the membrane was incubated with immobilon western HRP Substrate (Millipore, MA, US), and protein bands were visualized by LAS 4000 (Fuji film, Tokyo, Japan). GAPDH was used for loading control of whole cell protein extracts.

Supp. Table S1. Prioritisation of exome variants

Prioritisation criteria / III:1
(proband) / II:2 (affected mother)
Variants and indels identified / 370,980 / 316,634
+ Autosomal / 365,143 / 308,830
+ Frequency <1% in dbSNP145, 1000 Genomes and NHLBI EVS databases / 47,037 / 39,769
+ Missense, nonsense, splice site or indel / 1,569 / 1,489
+ Heterozygous (dominant inheritance) / 1,314 / 1,251
+ Shared by proband and his mother but not by the two unaffected relatives who underwent exome sequencing / 97
+ Absent in 60 Irish control exomes / 21
+ In a gene associated with ataxia (HP:000251) / 1

Whole exome sequencing was performed for the proband and his affected mother. Variant prioritisation was performed assuming an autosomal dominant model.

Table 2. Rare heterozygous variants remaining after variant filtering

Gene / Transcript ID / cDNA change / Protein change / dbSNP144 ID (minor allele frequency) / OMIM disease association / Previous association with ataxia
TTN / NM_001267550.1 / c.33513_33515dup / p.(Glu11172dup) / novel / Cardiomyopathy and/or muscular dystrophy / -
NUP35 / NM_001287585.1 / c.438T>A / p.(Phe146Leu) / rs61756082 (0.04%) / - / -
ITPR1 / NM_001168272.1 / c.106C>T / p.(Arg36Cys) / novel / AD Spinocerebellar ataxia type 15 and 29 / Yes
FSIP2 / NM_173651.2 / c.10006A>G / p.(Arg3336Gly) / rs142306380 (0.1%) / - / -
SPATS2L / NM_001282744.1 / c.1097G>A / p.(Arg366Gln) / rs200772751 (no maf) / - / -
TMEM151B / NM_001137560.1 / c.54_56delCGG / p.(Gly21del) / novel / - / -
ADGB / NM_024694.3 / c.3160C>T / p.(Leu1054Phe) / novel / - / -
SYNE1 / NM_182961.3 / c.3188T>C / p.(Val1063Ala) / rs141464488 (0.08%) / AD Emery-Dreifuss muscular dystrophy 4; AR spinocerebellar ataxia 8 / Recessive ataxia only
MYCT1 / NM_025107.2 / c.302C>T / p.(Ala101Val) / rs145687943 (no maf) / - / -
MAD1L1 / NM_001013836.1 / c.1322T>C / p.(Met441Thr) / novel / Somatic lymphoma and prostate cancer / -
VPS13B / NM_152564.4 / c.8903A>G / p.(Asn2968Ser) / rs28940272 (0.14%) / AR Cohen syndrome / -
GLT6D1 / NM_182974.2 / c.740T>C / p.(Leu247Pro) / novel / - / -
OASL / NM_003733.3 / c.118G>A / p.(Val40Met) / rs201649356 (0.02%) / - / -
DAPK2 / NM_014326.3 / c.1066G>A / p.(Ala356Thr) / rs140635820 (0.02%) / - / -
ZBTB7C / NM_001039360.2 / c.1816G>A / p.(Ala606Thr) / novel / - / -
ARHGEF18 / NM_015318.3 / c.1332C>A / p.(Asp444Glu) / novel / - / -
ZNF69 / NM_021915.2 / c.440C>T / p.(Pro147Leu) / rs147439765 (0.32%) / - / -
PGPEP1 / NM_017712.3 / c.583C>T / p.(Leu195Phe) / rs144146557 (0.06%) / - / -
ZNF43 / NM_001256649.1 / c.1574C>T / p.(Thr525Ile) / novel / - / -
IRGC / NM_019612.3 / c.470T>C / p.(Leu157Pro) / rs142490244 (no maf) / - / -
VPS16 / NM_022575.3 / c.1840G>A / p.(Glu614Lys) / rs61729230 (0.1%) / -
TRIOBP / NM_001039141.2 / c.2803delG / p.(Ala935Hisfs*70) / novel / AR deafness type 28 / -

Variant prioritisation identified 21 rare heterozygous variants which were shared by the proband and his affected mother. Only 1 of the 21 genes has previously been associated with autosomal dominant ataxia; ITPR1. Abbreviations: AD: autosomal dominant, AR: autosomal recessive