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1 Supplementary Materials Supplementary Table S1 Supplementary Table S2 Supplementary Table S3 Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure S3 Supplementary Reference S1 Supplementary Reference S2 Temple syndrome: comprehensive molecular and clinical findings in 32 Japanese patients Masayo Kagami, MD, PhD, Keisuke Nagasaki, MD, PhD, Rika Kosaki, MD, PhD, Reiko Horikawa, MD, PhD, Yasuhiro Naiki, MD, PhD, Shinji Saito, MD, PhD, Toshihiro Tajima, MD, PhD, Tohru Yorifuji, MD, PhD, Chikahiko Numakura, MD, PhD, Seiji Mizuno, MD, PhD, Akie Nakamura, MD, PhD, Keiko Matsubara, MD, PhD, Maki Fukami, MD, PhD and Tsutomu Ogata, MD, PhD
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Page 1: media.nature.com · 1 Supplementary Materials Supplementary Table S1 Supplementary Table S2 Supplementary Table S3 Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure

1

Supplementary Materials

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Reference S1

Supplementary Reference S2

Temple syndrome: comprehensive molecular and clinical findings

in 32 Japanese patients

Masayo Kagami, MD, PhD, Keisuke Nagasaki, MD, PhD, Rika Kosaki, MD, PhD,

Reiko Horikawa, MD, PhD, Yasuhiro Naiki, MD, PhD, Shinji Saito, MD, PhD,

Toshihiro Tajima, MD, PhD, Tohru Yorifuji, MD, PhD, Chikahiko Numakura, MD, PhD,

Seiji Mizuno, MD, PhD, Akie Nakamura, MD, PhD, Keiko Matsubara, MD, PhD,

Maki Fukami, MD, PhD and Tsutomu Ogata, MD, PhD

Page 2: media.nature.com · 1 Supplementary Materials Supplementary Table S1 Supplementary Table S2 Supplementary Table S3 Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure

Table S1. Clinical manifestations in 32 patients with Temple syndrome.Patient Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10(Epi)genetic cause UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GCSex Male Male Male Male Male Female Female Male Male FemaleAge at the last examination (y) 8/12 9/12 1 2/12 1 3/12 1 11/12 2 5/12 2 7/12 2 9/12 2 10/12 8 4/12Karyotype … 46,XY … … 46,XY 46,XY 46,XX 46,XY 46,XY 46,XX<Pregnancy and delivery>Gestational age (w) 38 40 34 36 39 38 39 37 39 30Premature delivery (≤ 36 w) – – + + – – – – – +Delivery (caesarean : vaginal) Caesarean Vaginal Caesarean Caesarean Vaginal Caesarean Vaginal Caesarean Vaginal CaesareanPlacental weight g (%) 350 (82) 332 (62) … … … 300 (57) 390 (74) … … …Hypoplastic placenta (≤ 80%) – + … … … + + … … …Medically assisted reproduction – – – – – – – – … –Paternal age at childbirth (y) 31 43 30 36 29 34 40 29 48 35Maternal age at childbirth (y) 39 42 28 42 29 34 36 29 40 27<Craniofacial appearance>Relative macrocephaly at birth* + + + + – + – + + –Relative macrocephaly at present* + … – … + – – … … …Prominent forehead (1–3 y) + + + + + + – – + –Triangular face + + – – – + – – – –Ear anomalies – … – – – – – – + –Recurrent otitis media – – – – – – – – – +High arched palate + – + … – + + – + +Irregular teeth … … … … + – – – + +PWS-like appearance† ± – + – – – – – – ±<Growth and maturation>Prenatal growth failure‡ + + + + + + + + + +Birth length cm (SDS) 45.0 (–1.5) 46.4 (–1.9) 42.3 (–2.0) 41.5 (–2.0) 44.9 (–2.2) 43.0 (–2.5) 45.0 (–2.2) 44.0 (–1.4) 41.8 (–3.2) 36.3 (–1.4)Birth weight kg (SDS) 2.06 (–2.3) 2.32 (–2.8) 1.53 (–2.0) 1.65 (–2.7) 2.15 (–2.9) 1.78 (–3.5) 2.04 (–3.0) 2.00 (–2.0) 1.65 (–4.3) 0.98 (–2.0)Birth OFC cm (SDS) 34.0 (+0.7) 34.2 (+0.6) 31.0 (+0.1) 32.0 (–0.2) 30.5 (–2.1) 31.5 (–1.2) 30.0 (–2.5) 33.0 (+0.2) 30.0 (–2.3) 25.0 (–1.3)Post-natal growth failure‡ + + + + + + + + + +Present height cm (SDS)§ 51.4 (–8.0) 67.3 (–1.7) 67.9 (–3.5) 61.2 (–3.9) 71.5 (–4.3) 80.0 (–2.5) 79.3 (–3.0) 82.8 (–2.6) 72.8 (–5.7) 121.0 (–1.0)Present weight kg (SDS)§ 2.88 (–5.7) 6.11 (–2.7) 6.55 (–3.2) 6.02 (–2.6) 7.50 (–3.2) 8.35 (–2.6) 9.30 (–1.6) 9.15 (–2.9) 6.91 (–4.6) 22.4 (–0.8)Present OFC cm (SDS) 37.8 (–4.9) 42.4 (–1.7) 43.1 (–2.2) … 45.5 (–1.8) 44.0 (–2.4) 45.0 (–2.4) … … …GH secretion … … … … … … … Normal … NormalGH treatment – – – – – – – – – –Precocious puberty … … … … … … … … … +Treatment for precocious puberty … … … … … … … … … +Menarche (y) … … … … … … … … … …<Developmental status>Age at head control (m) – 7 4 10 8 4 5 6 5 7Age at sitting without support (m) – 9 10 11 13 7 … 10 7 12Age at walking without support (m) – … … … – 18 15 15 17 34IQ/DQ … … 91 … 73 … … … 90 …Special class for delayed children … … … … … … … … … +**Neurological and/or emotional problems<Other findings>Hypotonia or its history (with poor suck) + + – + + – – + + +Small hands and/or feet + – – + + – + + + +Clinodactyly – – – – + + + + + +Simian crease – … + – – – … + + …Joint hypermobility + – – + + – – + – –Body asymmetry – – – – – – – – – –Scoliosis – + – – – – – – – +Feeding difficulties and/or low BMI + – + + + – – + + +Undermasculinized genitalia – – + (MP) + (HS) … – … + (CO) – …Hypercholesterolemia – - + … – – … … – –Diabetes mellitus (type 2) – - - … – – – … – –<Clinical diagnosis>Initial diagnosis for genetic analysis PWS SRS SRS PWS SRS SRS SGA-SS PWS SRS PWSRe-assessed diagnosis in infancy PWS & SRS PWS & SRS SRS PWS & SRS PWS & SRS SRS SGA-SS PWS & SRS PWS & SRS PWSRe-assessed diagnosis from puberty … … … … … … … … … TS14<PWS: salient features prompting genetic testing>Hypotonia (with poor suck) + + – + + – – + + +Global developmental delay (≥ 2 y) … … … … … – – – – +Excessive eating with central obesity (≥ 6 y) … … … … … … … … … –Cognitive impairment (≥ 13 y) … … … … … … … … … …Hypothalamic hypogonadism (≥ 13 y) … … … … … … … … … …Behavior problems (≥ 13 y) … … … … … … … … … …<SRS: Netchine-Harbison scoring system features>Number of positive features (4/5) (3/5) (4/5) (4/5) (3/5) 4/6 2/6 4/6 5/6 3/6Birth length and/or weight ≤ –2 SDS + + + + + + + + + +Relative macrocephaly at birth* + + + + – + – + + –Postnatal height (at ~ 2 years) ≤ –2 SDS … … … … … + + + + +Prominent forehead (1–3 y) + + + + + + – – + –Body asymmetry – – – – – – – – – –Feeding difficulties and/or low BMI + – + + + – – + + +<TS14: salient features >Pre- and/or post-natal growth failure + + + + + + + + + +Precocious puberty … … … … … … … … … +Treatment for precocious puberty … … … … … … … … … +Reference 1

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Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Patient 16 Patient 17 Patient 18 Patient 19 Patient 20 Patient 21 Patient 22UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-TR/GC UPD-MR/PE UPD-MR/PE UPD-MR/PE UPD-MR/PE UPD-PE¶ UPD-PE¶

Male Female Female Male Male Male Female Male Male Female Female Male9 11/12 11 5/12 12 3/12 12 6/12 15 0/12 20 4/12 1 8/12 3 1/12 3 7/12 10 3/12 5 10/12 16 1/12

… 46,XX/47,XX,+mar 46,XX 46,XY 46,XY 46,XY 46,XX 46,XY 46,XY … 46,XX 46,XY

40 40 37 36 40 38 39 36 38 40 39 37– – – + – – – + – – – –

Vaginal Caesarean Caesarean Caesarean Vaginal Vaginal Vaginal Caesarean Caesarean Caesarean Vaginal Vaginal… … … … … … … … 294 (56) … 635 (120) …… … … … … … … … + … – …– – – – – – – ICSI+FET – – … –36 36 42 28 32 39 32 30 36 27 … …34 36 41 24 30 37 30 30 36 23 31 35

… – – + + … – + – + – –… – … … … … – + … … … …– – + + – … + + + – – –– – – + … … – – + – + –– – – – … … – – + – – –– – – – – + – – – – – ++ – – + + … + + + – – –+ + – – … … – – – + – –– – + + – – – ± – – – +

+ + + – + + + + + + + –46.8 (–1.6) 48.0 (–2.4) 40.6 (–2.4) 45.7 (–0.3) 45.0 (–2.4) … 45.0 (–2.2) 40.5 (–3.2) 45.0 (–1.5) 45.0 (–4.0) 43.3 (–3.0) 50.0 (+1.4)2.17 (–3.2) 2.14 (–0.4) 1.68 (–2.7) 1.96 (–1.6) 1.82 (–4.5) 1.92 (–2.7) 1.92 (–3.4) 1.43 (–2.2) 1.81 (–3.0) 1.86 (–2.8) 2.36 (–2.1) 3.80 (+3.8)

… 28.0 (–3.9) 30.4 (–1.6) 32.0 (–0.1) 31.0 (–1.8) … 30.5 (–2.1) 30.8 (–0.9) 31 (–1.8) 32 (–1.2) 31.0 (–1.8) 34.5 (+1.4)– + + + + + + + + + + –

135.9 (+0.2) 134.5 (–1.8) 136.0 (–2.4) 148.5 (–0.5) 154.3 (–2.1) 163.2 (–1.3) 76.7 (–1.6) 80.0 (–4.0) 88.0 (–2.6) 127.2 (–1.7) 100.3 (–1.5) 163.0 (–1.1)28.4 (–0.5) 28.2 (–1.4) 41.4 (–0.3) 34.9 (–1.0) 45.3 (–1.2) 64.0 (+0.1) 7.97 (–2.2) 9.00 (–3.1) 12.0 (–1.6) 20.2 (–1.9) 16.2 (–0.6) 99.3 (+4.3)

… … … … … … 45.6 (–0.7) 48.2 (–0.9) … … … …… Normal … Normal … GHD … Normal … Normal Normal …– + (SGA-SS) – + (SGA-SS) – + (GHD) – + (SGA-SS) – + (SGA-SS) + (SGA-SS) –+ + + + + + … … … + … –+ + + + – + … … … + … –… 9 10/12 – … … … … … … – … …

6 5 8 9 … … 9 9 6 6 3 79 12 8 10 … … 12 15 10 10 9 1218 27 22 26 14 … – 24 19 … … 18… … 114 98 … 100 56 … 74 83 … 90– + – – – – … … … – … –

West syndrome Footnote-1 Footnote-2

+ + + + – – + + + – – ++ + + + + + + + + + + +– – – – … … – + … – – –– – – – … + – – … – – –– – + + – – – + – – – –– – – – – … + + + – – –+ + – – – – – – – + – –+ + + + + … + + – – – –– … … – – – … + (MP) – … … + (CO)– – – – – … – – – – – +– – – – – … – – – – – +

TS14 PWS PWS PWS TS14 TS14 SRS PWS PWS SGA-SS SGA-SS PWSPWS PWS PWS & SRS PWS & SRS SRS Uncertain PWS & SRS PWS & SRS PWS & SRS SGA-SS SGA-SS PWSTS14 TS14 TS14 TS14 TS14 TS14 … … … TS14 … –

+ + + + – – + + + – – +– + – – – – … + + – – –– – – – – – … … … – … +… … … … – – … … … … … –… … … … – – … … … … … –… … … … – – … … … … … –

(2/5) 3/6 4/6 4/6 4/6 (2/2) (4/5) 6/6 4/6 3/6 2/6 0/6+ + + – + + + + + + + –… – – + + … – + – + – –– + + + + + … + + + + –– – + + – … + + + – – –– – – – – … + + + – – –+ + + + + … + + – – – –

+ + + + + + + + + + + –+ + + + + + … … … + … –+ + + + – + … … … + … –

2 3 3 3

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Patient 23 Patient 24 Patient 25 Patient 26 Patient 27 Patient 28 Patient 29 Patient 30 Patient 31 Patient 32UPD-PE# Epimutation Epimutation Epimutation Epimutation Epimutation Epimutation Deletion (DLK1 ) Deletion (DLK1 ) Deletion (DLK1 & RTL1 )

Female Male Female Female Female Male Male Female Male Female20 8/12 3 2/12 9 2/12 9 5/12 11 1/12 11 10/12 33 49 62 3846,XX … 46,XX 46,XX 46,XX 46,XY 46,XY 46,XX 46,XY 46,XX

36 40 37 37 41 41 39 39 … 40+ – – – – – – – … –

Caesarean Vaginal Caesarean Caesarean Caesarean Vaginal Vaginal Vaginal … Vaginal… … … … … … … … … …… … … … … … … … … …– – IVF-ET – – – – – – –37 22 38 34 32 31 25 33 … 3127 22 36 29 28 27 25 36 … 29

+ – + – – + – … … …+ … – … … – – … … ++ + + + + + – + … –– – + + – + – – … –+ – – – – – – – … –+ – + – + – + + … +… – + – – + – … … –– – – + + + + – … ++ – – – ± – ± – … –

+ – + + – + – + … +39.0 (–2.7) 47.5 (–1.1) 36.5 (–3.9) 43.0 (–1.8) 50.0 (±0.0) 46.5 (–2.1) 47.5 (+0.8) … … 45.3 (–2.4)1.43 (–3.0) 2.57 (–1.6) 1.20 (–4.6) 1.78 (–2.4) 3.00 (–0.2) 2.20 (–2.7) 2.69 (–1.1) 2.10 (–2.5) … 2.30 (–2.2)30.0 (–1.4) 34.0 (+0.5) 30.0 (–2.0) 31.0 (–1.1) 34.5 (+0.6) 32.5 (–0.7) 33.2 (±0.0) … … …

+ + + + + + + + + +129.1 (–5.5) 81.6 (–3.5) 125.5 (–1.0) 120.4 (–2.1) 127.3 (–2.5) 143.5 (–0.6) 159 (–2.0) 146.0 (–2.2) 145.0 (–2.9) 135.0 (–4.4)33.2 (–2.5) 10.7 (–1.7) 22.3 (–1.2) 23.5 (–1.1) 35.0 (–0.4) 37.6 (–0.4) 49.9 (–1.2) 45.0 (–1.3) 61.0 (–0.1) 38.0 (–1.9)51.5 (–3.7) … 50.3 (–1.5) … … 51.5 (–0.9) 56.5 (–0.7) … … 52.8 (–2.9)

GHD Normal Normal … Normal Normal Normal … … …+ (GHD) – + (SGA-SS) – – + (SGA-SS) – – – –

– … + + + – + + … –– … + + + – – – – –

13 9/12 … 8 6/12 – – … … 10 3/12 … 12 1/12

7 5 … 3 8 7 … … … …9 9 … 6 11 10 … … … …36 15 … 16 19 24 24 … … …53 … … … … … 106 … … …+ … – – – – – – – –

Footnote-3 Footnote-4

+ + – – + + + – … ++ + + + + + + + + ++ – + + – + – – … –– + – – – + + – … –– – ... – + + + – … –+ – + – + + – – … –+ – – – – – – – – –+ – – + + – + – … +… – … … … – – … … …– – – + + – + + … –– – – - - – + +‡‡ … –

PWS PWS SRS SRS PWS SRS PWS KOS family KOS family KOS familyPWS & SRS PWS SRS SRS PWS & SRS PWS & SRS PWS SRS Uncertain PWS & SRS

– … TS14 TS14 TS14 – TS14 TS14 … –

+ + – – + + + – … ++ – – – – – – – – –– … – – – – + – – –– … … … … … – – – –– … … … … … – – – –– … … … … … – – – –

6/6 2/6 5/6 4/6 4/6 5/6 2/6 (3/5) (1/1) (3/5)+ – + + – + – + … ++ – + – – + – … … …+ + + + + + + + + ++ + + + + + – + … –+ – + – + + – – … –+ – – + + – + – … +

+ + + + + + + + + +– … + + + – + + … –– … + + + – – – – –3 4, 5 5 5, 6 4, 5 7 7 7

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Abbreviations: UPD-TR/GC: UPD(14)mat mediated by trisomy rescue or gamete complementation; UPD-MR/PE: UPD(14)mat mediated by monosomy rescue (MR)- orpostfertilization mitotic error; UPD-PE: UPD(14)mat mediated by postfertilization mitotic error; y, year; m, month, w, week; SDS, standard deviation score; OFC, occipitofrontalcircumference; GH, growth hormone; SGA-SS, small for gestational age-short stature; IQ/DQ, intellectual/developmental quotient; BMI, body mass index; ICSI, intracytoplasmic sperminjection; FET, frozen embryo transfer; IVF-ET, in vitro fertilization and embryo transfer; GHD, growth hormone deficiency; MP, micropenis; CO, cryptorchidism; and HS, hypospadias.* Birth OFC SDS ≥1.5 above birth length or weight SDS; present OFC SDS ≥1.5 above present height or weight SDS.† Including narrow forehead, almond-shaped eyes, and a triangular mouth.‡ Birth length and/or weight ≤ –2 SDS for gestational age; post-natal height (the latest height in childhood before the onset of pubertal growth spurt or the initiation of GH therapy, or in adulthood without GH therapy) ≤ –2 SDS for age).§ The actual latest present height data; thus, several patients show heights of the normal range (> –2 SDS) because of pubertal growth spurt or GH therapy, and this is not reflected by the assessment of post-natal growth failure.¶ Segmental isodisomy.# Mosaicism for 46,XX/46,XX,upd(14)mat.** Both of the parents were enrolled in special classes in childhood.†† Placed on levothyroxine supplementation therapy from 12 5/12 years of age.‡‡ Placed on hypoglycemic agent since adulthood.Footnote-1: weak integration of social, emotional, and communicative behaviors; difficulty in communication; being fascinated and stuck to details without noticing the whole; and unable to have a prospective view.Footnote-2: autistic tendency; abnormality of behavior and affection; compulsive adherence to specific matters; nonfunctional routines; and difficulty in social interactions.Footnote-3: excitement; self-injurious behaviors; aggressive behavior; restlessness; obstinate character; depression; and eating disorders.Footnote-4: stuck to details without noticing the whole; obstinate character; and poor communication skills.

References1. Goto M, Kagami M, Nishimura G, Yamagata T. A patient with Temple syndrome satisfying the clinical diagnostic criteria of Silver-Russell syndrome. Am J Med Genet A 2016;170:2483–2485.2. Tohyama J, Yamamoto T, Hosoki K et al. West syndrome associated with mosaic duplication of FOXG1 in a patient with maternal uniparental disomy of chromosome 14. Am J Med Genet A 2011;155A:2584–2588.3. Hosoki K, Kagami M, Tanaka T et al. Maternal uniparental disomy 14 syndrome demonstrates Prader-Willi syndrome-like phenotype. J Pediatr 2009;155:900–903.4. Kagami M, Mizuno S, Matsubara K, et al. Epimutations of the IG-DMR and the MEG3-DMR at the 14q32.2 imprinted region in two patients with Silver-Russell syndrome- compatible phenotype. Eur J Hum Genet 2015;23:1062–1067.5. Kagami M, Matsubara K, Nakabayashi K et al. Genome-wide multilocus imprinting disturbance analysis in Temple syndrome and Kagami-Ogata syndrome. Genet Med 2016 Sep 15. doi: 10.1038/gim.2016.123.6. Hosoki K, Ogata T, Kagami M, Tanaka T, Saitoh S. Epimutation (hypomethylation) affecting the chromosome14q32.2 imprinted region in a girl with upd(14)mat-like phenotype. Eur J Hum Genet 2008;16:1019–1023.7. Kagami M, Sekita Y, Nishimura G, et al. Deletions and epimutations affecting the human 14q32.2 imprinted region in individuals with paternal and maternal upd(14)-like phenotypes. Nat Genet 2008;40:237–242.

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Pt. 24 Pt. 25 Pt.26 Pt. 27 Pt. 28 Pt. 29 Median (Min ~ Max)*Initial Dx PWS SRS SRS PWS SRS PWSRe-assessed Dx in infancy PWS SRS SRS PWS & SRS PWS & SRS PWS<TS14- and KOS14-related DMRs>MEG3/DLK1 :IG-DMR CpG-1 24 34 25 31 22 22 58 (49 ~ 68)(Ch. 14q32.2) CpG-2 22 33 24 28 18 22 54 (40~ 62)

CpG-3 48 57 53 56 40 40 68 (54 ~ 78)CpG-4 17 25 22 22 13 21 53.5 (43 ~ 64)

MEG3 :TSS-DMR CpG-5 3 25 7 3 3 3 52 (43 ~ 56)(Ch. 14q32.2) CpG-6 3 25 8 4 3 4 55 (52 ~ 65)

CpG-7 4 26 5 4 4 3 37 (32 ~ 55)CpG-8 5 28 6 6 6 4 60 (44 ~ 74)CpG-9 2 22 4 3 2 2 36 (26 ~ 47)

<Other imprinting disease-related DMRs>PLAGL1 :alt-TSS-DMR CpG-10 49 51 51 47 48 48 47 (31 ~ 52)(Ch. 6q24.2) CpG-11 48 47 49 47 45 47 45 (27 ~ 51)

CpG-12 47 45 49 48 44 50 48 (40 ~ 56)CpG-13 41 37 43 38 37 44 39 (31 ~ 47)CpG-14 48 47 51 50 45 47 50 (40 ~ 58)CpG-15 43 45 46 45 43 51 49 (37 ~ 55)CpG-16 48 50 53 52 48 50 53 (41 ~ 58)

PEG10 :TSS-DMR CpG-17 56 60 59 55 53 51 56 (50 ~ 59)(Ch. 7q21.3) CpG-18 55 57 57 55 56 50 55 (50 ~ 59)

CpG-19 54 55 55 53 54 55 53 (48 ~ 59)CpG-20 56 57 57 56 56 52 54.5 (50 ~ 59)CpG-21 50 55 55 51 51 48 50 (45 ~ 55)

MEST :alt-TSS-DMR CpG-22 54 62 58 67 69 58 60 (56 ~ 70)(Ch. 7q32.2) CpG-23 55 63 57 66 68 57 59 (55 ~ 69)

CpG-24 52 59 55 62 66 54 58 (52 ~ 68)CpG-25 57 64 58 70 70 56 61 (42 ~ 73)CpG-26 53 59 55 61 65 56 57 (47 ~ 66)CpG-27 56 62 60 68 72 56 60 (54 ~ 70)

H19/IGF2 :IG-DMR CpG-28 41 41 49 51 51 57 48 (37 ~ 60)(Ch. 11p15.5) CpG-29 41 45 50 50 51 58 50 (39 ~ 64)

CpG-30 40 40 48 48 49 55 46 (36 ~ 57)CpG-31 40 40 41 44 49 55 45 (36 ~ 55)

KCNQ1OT1 :TSS-DMR CpG-32 48 54 50 57 49 51 58 (49 ~ 66)(Ch. 11p15.5) CpG-33 52 55 54 61 52 54 61 (52 ~ 68)

CpG-34 41 49 45 53 44 45 48 (41 ~ 54)CpG-35 46 52 45 55 46 47 48 (42 ~ 55)CpG-36 55 56 55 62 55 51 67 (55 ~ 72)CpG-37 56 58 55 62 52 50 64 (55 ~ 71)

SNURF :TSS-DMR CpG-38 43 41 45 40 39 42 42 (36 ~ 47)(Ch. 15q11.2) CpG-39 44 41 45 41 38 46 43 (36 ~ 48)

CpG-40 43 42 46 42 40 46 44 (36 ~ 50)CpG-41 42 42 46 42 39 42 44 (37 ~ 48)CpG-42 37 38 39 38 36 36 38 (32 ~ 42)CpG-43 41 42 43 42 40 40 42 (36 ~ 47)

GNAS -A/B :TSS-DMR CpG-44 44 44 44 43 42 41 41 (34 ~ 45)(Ch. 20q13.32) CpG-45 44 44 44 43 41 41 40 (33 ~ 45)

CpG-46 44 46 47 44 45 42 43 (35 ~ 47)CpG-47 39 42 42 41 40 39 38 (32 ~ 42)CpG-48 41 42 42 41 40 41 40 (33 ~ 45)CpG-49 36 40 40 39 39 37 38 (31 ~ 42)CpG-50 44 43 44 41 41 39 40 (32 ~ 44)

Reference This paper 1 (Pt. 2) 1 (Pt. 4) 1 (Pt.1) 1 (Pt. 3) This paper

* The reference data have been obtained from 50 control adults.Markedly low MIs highlighted with orange permit the diagnosis of Temples syndrome; the methylation indices, especially those for theMEG3:TSS-DMR , are relatively high in patient 25. Slightly low MIs and slightly high MIs are highlighted with yellow and blue, respectively.The values outside the normal ranges were confirmed by triplicated experiments, and the mean value of the three experiments are shown.The data of Pts. 25−28 have been adopted from Kagami et al.Reference1. Kagami M, Matsubara K, Nakabayashi K, et al. Genome-wide multilocus imprinting disturbance analysis in Temple syndrome and Kagami-Ogata syndrome. Genet Med 2016 Sep 15. doi: 10.1038/gim.2016.123. [Epub ahead of print]

Table S2. Methylation indices (MIs, %) for CpG dinucleotides determined by pyrosequencing analysis in patients with epimutations.

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Table S3. Endocrine data and pubertal findings in 18 patients with Temple syndrome.Patient 8 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Patient 16 Patient 18

Male Female Male Female Female Male Male Male MaleExamined age (years:months) 2:09 1:04 … … … 4:09 … 6:06 2:11Stimulus Arginine Arginine … … … Arginine … Arginine ArginineGH (basal / peak*) (μg/L) 0.16 / 11.4 1.02 / 6.76 … … … 0.23 / 3.90 … 0.17 / 3.58 0.05/10.4Examined age (years:months) … … … 4:00 … 4:09 … 6:06 …Stimulus … … … Arginine … Clonidine … Insulin …GH (basal / peak) (ng/mL) … … … Normal … 0.19 / 6.3 … 0.27 / 3.79 …Examined age (years:months) 2:09 7:02 6:05 … … 4:09 … 6:06 2:11IGF-1 (ng/mL) 82 330 280 … … 36 … 120 98GH therapy (indication) No No No Yes (SGA-SS) No Yes (SGA-SS) No Yes (GHD) Yes (SGA-SS)Examined age (years:months) 2:09 7:02 9:06 6:05 8:05 10:11 11:07 8:08 2:11Tanner stage G1, PH1 B2, PH2 G2, PH3 B2, PH1 B2-3, PH2 G2, PH2 G4, PH4 G2-3, PH1 G1, PH1LH (basal / peak†) (mIU/mL) 0.1/… 10.1 / … 2.7 / … <0.6 / … 5.9 / … 1.7 / … 0.7 / … 1.2 / 26.2 <0.2 / 4.7FSH (basal / peak†) (mIU/mL) 1.3/… 5.9 / … 6.3 / … 2.9 / … 4.0 / … 6.0 / … 3.0 / … 5.2 / 14.7 1.4 / 22.3Testosterone (ng/dL) <3 … 242 … … 10 112 11 N.E.Estradiol (pg/mL) … 34 … <10 12 … … … …Examined age (years:months) … … 9:11 10:09 8:09 12:09 15:00 …Tanner stage … … G2, PH3 B4, PH4 B3, PH3 G2, PH1‡ G5, PH5 … …Stimulus … … (–) (–) (–) (–) … … …LH (basal / peak†) (mIU/mL) … … <0.2‡/ … 2.8 / … 0.6‡ / … <0.2‡/ … 2.4 / … … …FSH (basal / peak†) (mIU/mL) … … <1.0‡ / … 5.8 / … 0.8‡ / … <1.0‡ / … 3.5 / … … …Testosterone (ng/dL) … … 6‡ … … 46‡ 395 … …Estradiol (pg/mL) … … … <25 <10‡ … … … …Menarche (years:months) 9:10GnRH analog therapy (age at start) No Yes (7:04) Yes (9:08) Yes (10:11) Yes (8:06) Yes (10:11) No Yes (8:08) No

Patient 20 Patient 21 Patient 23 Patient 24 Patient 25 Patient 26 Patient 27 Patient 28 Patient 29Female Female Female Male Female Female Female Male Male

Examined age (years:months) 1:01 3:01 5:05 3:02 3:07 … … 10:01 33:02Stimulus Arginine Arginine Arginine Arginine Arginine … … Arginine InsulinGH (basal / peak*) (μg/L) (–) / 14.1 0.5 / 2.7 2.3 / 3.4 6.59 / 9.78 2.04 / 18.8 … … 0.19 / 6.98 0.31/14.17Examined age (years:months) … 3:01 5:05 … 3:07 … … … …Stimulus … Insulin Insulin … Insulin … … … …GH (basal / peak) (ng/mL) … 0.40 / 8.10 1.9 /5.9 … 3.87 / 5.56 … … … …Examined age (years:months) 3:00 3:01 5:05 3:01 4:00 … 7:01 10:01 33:02IGF-1 (ng/mL) 36 90 56 52 85 … 170 90 91GH therapy (indication) Yes (SGA-SS) Yes (SGA-SS) Yes (GHD) No Yes (SGA-SS) No No Yes (SGA-SS) NoExamined age (years:months) 7:10 5:10 10:06 … 7:01 6:06 7:08 10:00 33:02Tanner stage B3, PH1 B1, PH1 B1, PH1 … B2, PH1 B2, PH1 B2-3, PH1 G2, PH1 G5, PH5LH (basal / peak†) (mIU/mL) 0.3 / 35.7 0.3 / … <0.1 / … … <0.5 / … 0.3 / … 3.3 / … 0.6 / … 2.5 / 37.8FSH (basal / peak†) (mIU/mL) 6.2 / 32.0 4.3 / … 7.3 / … … 2.3 / … 2.7 / … 4.5 / … 6.1 / … 5.3 / 14.6Testosterone (ng/dL) … … … N.E. … … … <3 266Estradiol (pg/mL) <25 <10 21.5 … <10 <10 <10 … …Examined age (years:months) 8:05 6:01 13:08 … 8:07 … 8:01 10:07 …Tanner stage B3, PH1‡ B1, PH1 B4 … B3, PH3 … B3, PH1 G3-4, PH2 …Stimulus (–) (–) (–) … GnRH … (–) (–) …LH (basal / peak†) (mIU/mL) 1.2‡ / … 0.3 / … 5.2/ … … 2.8 / 51.0 … 0.2‡ / … 1.3 / … …FSH (basal / peak†) (mIU/mL) 9.6‡ / … 6.4 / … 9.3 / … … 7.5 / 18.3 … 0.5‡ / … 8.1 / … …Testosterone (ng/dL) … … … … … … … 8.0 …Estradiol (pg/mL) <20‡ 10 No data … N.E. … <10‡ … …Menarche 13:9 8:08GnRH analog therapy (age at start) Yes (8:0) No No No Yes (8:07) Yes (6:10) Yes (7:09) No No

Abbreviations: GH, growth hormone; IGH-1, insulin-like growth factor-1; LH, luteinizing hormone; FSH, follicle stimulating hormone; GnRH, gonadotropin releasinghormone; SGA-SS, small for gestational age-short stature; GHD, GH deficiency; G, genitalia; B, breast, and PH, pubic hair.* The peak values during GH provocation tests: arginine 0.5 g/kg [max. 30 g] i.v. over 30 minutes, insulin 0.05–0.1 U/kg bolus i.v., or clonidine 0.1–0.15 mg/m² [max. 0.15 mg] p.o.; blood sampling at 0, 30, 60, 90, and 120 minutes.† The peak values during GnRH stimulation tests: GnRH 100 μg/m² [max. 100 μg] bolus i.v.; blood sampling at 0, 30, 60, 90, and 120 minutes.‡ On GnRH analog therapy.The hormone values above and below the age- and sex-matched reference ranges are boldfaced and italicized, respectively.Tanner pubertal development earlier than the normal tempo are boldfaced.The diagnosis of GHD is made when all the serum GH values during ≥ two provocation tests are below 6 ng/mL.GH therapy for SGA-SS is performed for patients who are born small for gestational age (birth length and weight below 10 percentile plus birth length or weight < –2 SD), and remain short at 3 years of age (height velocity < 0 SD and height < –2.5 SD).GH therapy using recombinant GH is performed at a single fixed dosage of ~0.175 mg/kg/week for GHD since 1986, and its cost is fully covered by the Japanese government, whereas that is performed at variable dosages between 0.23 and 0.47 mg/kg/week (sometimes smaller dosages) for SGA-SS since 2008, and its cost is usually covered by the local government depending on the patient ages (usually <15 years of age).GnRH analog therapy (s.c. every 4 weeks) was performed with the initial dosage of 30 μg/kg and the maximum dosage of 90 μg/kg until 2010, and with larger dosages up to 180 μg/kg since 2011.Conversion factors to the SI unit: 1.0 for GH, IGF-1, LH, and FSH; 0.0347 for testosterone; and 3.67 for estradiol.

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Classification of 32 patients with TS14 Group Subgroup Number UPD(14)mat TR/GC type 16 MR/PE type 4 PE type 3 Epimutations 6 Deletions DLK1 2 DLK1 & RTL1 1

Figure S1. Molecular approach and classification of identified TS14 patients. A. Schematic representation of the physical map of the chromosome 14q32.2 imprinted region.

PEGs are shown in blue, MEGs in red, a probably non-imprinted gene (DIO3) in black, and the DMRs in green.

B. Molecular approach for the identification of TS14 from 346 patients examined in this study. C. Classification of a total of 32 Japanese patients with TS14, consisting of 19 newly identified

patients and 13 previously reported patients. D. Methylation status of the MEG3/DLK1:IG-DMR and the MEG3:TSS-DMR, and expression

patterns of the imprinted genes. P: paternally derived chromosome; and M: maternally derived chromosome. Black and white circles represent methylated and unmethylated DMRs, respectively. The deleted regions are shown with stippled squares.

Upd(14)mat (n=23)

DLK1 0 X RTL1 0 X MEGs 2 X

M M

Epimutation (n=6)

DLK1 0 X RTL1 0 X MEGs 2 X

M M

Deletion (DLK1) (n=2) M

P DLK1 0 X RTL1 1 X MEGs 1 X

Deletion (DLK1 & RTL1)

(n=1) M P DLK1 0 X

RTL1 0 X MEGs 1 X

Control DLK1 1 X RTL1 1 X MEGs 1 X M

P

DLK1

MEG8 snoRNAs microRNAs MEG3 RTL1as

RTL1 DIO3

A

B

C

D

UPD(14)mat (n = 17)

Deletion analysis

Epimutation (n = 2) Microdeletion (n = 0)

Biparental

346 patients

Methylation analysis

Hypomethylation

Normal (n = 327)

Parental origin analysis

MEG3:TSS-DMR MEG3/DLK1:IG-DMR

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10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

SRS-boy: H19-DMR hypo UPD(7)mat

Pt. 29

Pt. 31

Pt. 32 Pt. 30

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

SRS-girl: H19-DMR hypo UPD(7)mat

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Pt. 22 (BMI = 36)

>18

TS14-girl: Pt.10 Pt.12 Pt.13 Pt.20 Pt.21 Pt.23 Pt.25 Pt.26 Pt.27

TS14-boy: Pt.11 Pt.14 Pt.15 Pt.16 Pt.28

>18

>18

>18

Figure S2. Body mass indices (BMIs) in Japanese patients with Temple syndrome and those with Silver-Russell syndrome. The data are plotted on the percentile BMI curves of Japanese children (3, 10, 25, 50, 75, 90, and 97 percentiles). The 50 percentile growth curves are boldfaced.

30

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DevelopmentPatient (Epi)genetic

causeAge(y:m)

DQ/IQ Schooleducation

Pt.1 UPD(14)mat 0:08 N.E. …Pt. 2 UPD(14)mat 0:09 N.E. …Pt. 3 UPD(14)mat 1:02 91 …Pt. 4 UPD(14)mat 1:03 N.E. …Pt. 5 UPD(14)mat 1:11 73 …Pt. 6 UPD(14)mat 2:05 N.E. …Pt. 7 UPD(14)mat 2:07 N.E. …Pt. 8 UPD(14)mat 2:09 N.E. …Pt. 9 UPD(14)mat 2:10 90 …Pt. 10 UPD(14)mat 8:04 N.E. Special classPt. 11 UPD(14)mat 9:11 N.E. Regular classPt. 12 UPD(14)mat 11:05 N.E. Special classPt. 13 UPD(14)mat 12:03 114 Regular classPt. 14 UPD(14)mat 12:06 98 Regular classPt. 15 UPD(14)mat 15:00 N.E. Regular classPt. 16 UPD(14)mat 20:04 100 Regular classPt. 17 UPD(14)mat 1:08 56 …Pt. 18 UPD(14)mat 3:01 N.E. …Pt. 19 UPD(14)mat 3:07 74 …Pt. 20 UPD(14)mat 10:03 83 Regular classPt. 21 UPD(14)mat 5:10 N.E. …Pt. 22 UPD(14)mat 16:01 90 Regular classPt. 23 UPD(14)mat 20:08 53 Special classPt. 24 Epimutation 3:02 N.E. …Pt. 25 Epimutation 9:02 N.E. Regular classPt. 26 Epimutation 9:05 N.E. Regular classPt. 27 Epimutation 11:01 N.E. Regular classPt. 28 Epimutation 11:10 N.E. Regular classPt. 29 Epimutation 33 106 Regular classPt. 30 Microdeletion 49 N.E. Regular classPt. 31 Microdeletion 62 N.E. Regular classPt. 32 Microdeletion 38 N.E. Regular class

210

Figure S3. The developmental status. The orange, green, yellow, and blue bars represent the period beforehead control, that after head control and before sitting without support, that after sitting without support andbefore walking without support, and that after walking without support. The gray bars denote the periodwith no information. DQ: developmental quotient; IQ: intellectual quotient; N.E.: not examined; Age: age atthe last examination (year: month).

876543

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Reference S1. A reference list of previously reported 65 patients with Temple syndrome

1. Ioannides Y, Lokulo-Sodipe K, Mackay DJ, Davies JH, Temple IK. Temple syndrome:

improving the recognition of an underdiagnosed chromosome 14 imprinting disorder: an analysis of 51 published cases. J Med Genet 2014;51:195–201. (The references for TS14 up to 2014 have been cited in this review article).

2. Azzi S, Salem J, Thibaud N, et al. A prospective study validating a clinical scoring system and demonstrating phenotypical-genotypical correlations in Silver-Russell syndrome. J Med Genet 2015;52:446–453.

3. Rosenfeld JA, Fox JE, Descartes M et al. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster. Am J Med Genet A 2015;167A:345–353.

4. Kagami M, Mizuno S, Matsubara K et al. Epimutations of the IG-DMR and the MEG3-DMR at the 14q32.2 imprinted region in two patients with Silver-Russell Syndrome-compatible phenotype. Eur J Hum Genet 2015;23:1062–1067.

5. Dworschak GC, Draaken M, Hilger AC et al. Genome-wide mapping of copy number variations in patients with both anorectal malformations and central nervous system abnormalities. Birth Defects Res A Clin Mol Teratol 2015;103:235–242.

6. Severi G, Bernardini L, Briuglia S et al. New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer. Am J Med Genet A 2016;170A:162–169.

7. Briggs TA, Lokulo-Sodipe K, Chandler KE, Mackay DJ, Temple IK. Temple syndrome as a result of isolated hypomethylation of the 14q32 imprinted DLK1/MEG3 region. Am J Med Genet A 2016;170A:170–175.

8. Goto M, Kagami M, Nishimura G, Yamagata T. A patient with Temple syndrome satisfying the clinical diagnostic criteria of Silver-Russell syndrome. Am J Med Genet A 2016;170A:2483–2485.

9. Sachwitz J, Strobl-Wildemann G, Fekete G et al. Examinations of maternal uniparental disomy and epimutations for chromosomes 6, 14, 16 and 20 in Silver-Russell syndrome-like phenotypes. BMC Med Genet 2016;17:20.

10. Shin EH, Cho E, Lee CG. Temple syndrome: A patient with maternal hetero-UPD14, mixed iso- and hetero-disomy detected by SNP microarray typing of patient-father duos. Brain Dev 2016;38:669–673.

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Reference S2. A reference list of previously reported 16 patients with microdeletions

involving the 14q32.2 imprinted region of paternal origin Patients 30–32: Kagami M, Sekita Y, Nishimura G et al. Deletions and epimutations affecting the human 14q32.2 imprinted region in individuals with paternal and maternal upd(14)-like phenotypes. Nat Genet 2008;40:237–242. Case 1: Mitter D, Buiting K, von Eggeling F et al. Is there a higher incidence of maternal uniparental disomy 14 [upd(14)mat]? Detection of 10 new patients by methylation-specific PCR. Am J Med Genet A 2006;140:2039–2049. Case 2: Béna F, Gimelli S, Migliavacca E et al. A recurrent 14q32.2 microdeletion mediated by expanded TGG repeats. Hum Mol Genet 2010;19:1967–1973. Case 3: Zada A, Mundhofir FE, Pfundt R et al. A Rare, Recurrent, De Novo 14q32.2q32.31 Microdeletion of 1.1 Mb in a 20-Year-Old Female Patient with a Maternal UPD(14)-Like Phenotype and Intellectual Disability. Case Rep Genet 2014;2014:530134. Case 4: Dworschak GC, Draaken M, Hilger AC et al. Genome-wide mapping of copy number variations in patients with both anorectal malformations and central nervous system abnormalities. Birth Defects Res A Clin Mol Teratol 2015;103:235–242. Case 5: Rosenfeld JA, Fox JE, Descartes M et al. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster. Am J Med Genet A 2015;167A:345–353. Case 6: Severi G, Bernardini L, Briuglia S et al. New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer. Am J Med Genet A 2016;170A:162–169. Case 7: Severi G, Bernardini L, Briuglia S et al. New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer. Am J Med Genet A 2016;170A:162–169. Case 8: Severi G, Bernardini L, Briuglia S et al. New patients with Temple syndrome caused by 14q32 deletion: Genotype-phenotype correlations and risk of thyroid cancer. Am J Med Genet A 2016;170A:162–169. Case 9: Rosenfeld JA, Fox JE, Descartes M et al. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster. Am J Med Genet A 2015;167A:345–353. Case 10: Kagami M, O'Sullivan MJ, Green AJ et al. The IG-DMR and the MEG3-DMR at human chromosome 14q32.2: hierarchical interaction and distinct functional properties as imprinting control centers. PLoS Genet 2010;6:e1000992. Case 11: Rosenfeld JA, Fox JE, Descartes M et al. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster. Am J Med Genet A 2015;167A:345–353. Case 12: Rosenfeld JA, Fox JE, Descartes M et al. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster. Am J Med Genet A 2015;167A:345–353. Case 13: Rosenfeld JA, Fox JE, Descartes M et al. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster. Am J Med Genet A 2015;167A:345–353.


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