Supplementary Appendix 1

High yield of pathogenic germline mutations causative or likely causative of the cancer phenotype in selected children with cancer

Illja J. Diets1,2, Esmé Waanders1,2,3, Marjolijn Ligtenberg1,2,4, Diede A.G. van Bladel1,2, Eveline J. Kamping1,2, Peter Hoogerbrugge3, Saskia Hopman5, Maran Olderode-Berends6, Erica Gerkes6, David A. Koolen1, Carlo Marcelis1, Gijs Santen7, Martine J. van Belzen7, Dylan Mordaunt8, Lesley McGregor8, Elizabeth Thompson8, Antonis Kattamis9, Agata Pastorczak10, Wojciech Mlynarski10, Denisa Ilencikova11, Anneke Vulto-van Silfhout1, Thatjana Gardeitchik1, Eveline de Bont12, Jan Loeffen13, Anja Wagner14, Arjen Mensenkamp1, Roland P. Kuiper1,2,3, *, Nicoline Hoogerbrugge1,2, *, Marjolijn Jongmans1,2,3,5, *

¹ Department of Human Genetics, Radboud university medical center, Nijmegen, The Netherlands 2 Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands 3 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands 4 Department of Pathology, Radboud university medical center, Nijmegen, The Netherlands 5 Department of Genetics, University Medical Center Utrecht, Utrecht, The Netherlands 6 Department of Genetic, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 7 Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands 8 Department of Genetics and Molecular Pathology, SA Pathology, Women's and Children's Hospital, North Adelaide, Australia 9 First Department of Pediatrics, Athens University Medical School, Athens, Greece 10 Department of Pediatrics, Oncology, Hematology and Diabetology, Medical University of Lodz, Poland 11 2nd Pediatric Department, Children's University Hospital, Comenius University, Bratislava, Slovakia. 12 Department of Pediatric Oncology and Hematology, Beatrix Children's Hospital, University Medical Center Groningen, Groningen, the Netherlands 13 Department of Pediatric Oncology and Hematology, Sophia Children’s Hospital, Erasmus Medical Center, Rotterdam, The Netherlands 14 Department of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands * These authors contributed equally

1 Table of contents Supplementary Tables ...... 5 Supplementary Table S1 – Cancer gene panel ...... 5 Supplementary Table S2 – Patients with multiple malignancies ...... 5 Supplementary Table S3 – Tumor types of index patients ...... 6 Supplementary Table S4 – Candidate genes ...... 7 Supplementary Table S5 – Overview of clinical features and mutations found in category 1 (ID and/or congenital anomalies) ...... 8 Supplementary Table S5 – Overview of clinical features and mutations found in category 1 (ID and/or congenital anomalies - Continued) ...... 9 Supplementary Table S6 – Overview of clinical features and mutations found in category 2 (cancer twice) ...... 10 Supplementary Table S7 – Overview of clinical features and mutations found in category 3 (family history) ...... 10 Supplementary Table S7 – Overview of clinical features and mutations found in category 3 (family history - continued) ...... 11 Supplementary Table S7 – Overview of clinical features and mutations found in category 3 (family history - continued) ...... 12 Supplementary Table S8 – Overview of clinical features and mutations found in category 4 (adult type of cancer) ...... 12 Supplementary Table S9 – Overview of clinical features and mutations found in category 5 (multiple reasons for inclusion) ...... 13 Clinical descriptions of patients ...... 14 Case #01 ...... 14 Case #02 ...... 14 Supplementary Figure S1 – Detection of mosaic mutation in ARID1A ...... 15 Case #03 ...... 16 Case #04 ...... 16 Supplementary Figure S2 – Family tree of case #04 ...... 17 Case #05 ...... 17 Supplementary Figure S3 – Family tree of case #05 ...... 18 Case #06 ...... 19 Case #07 ...... 19 Supplementary Figure S4 – Family tree of case #07 ...... 19 Case #08 ...... 20 Case #09 ...... 20

2 Case #10 ...... 21 Supplementary Figure S5 – Family tree of case #10 ...... 21 Case #11 ...... 22 Case #12 ...... 22 Supplementary Figure S6 – Family tree of case #12 ...... 23 Case #13 ...... 23 Case #14 ...... 24 Case #15 ...... 24 Case #16 ...... 24 Case #17 ...... 25 Supplementary Figure S7 – Family tree of case #17 ...... 25 Case #18 ...... 26 Case #19 ...... 26 Case #20 ...... 27 Case #21 ...... 27 Supplementary Figure S8 – Family tree of case #21 ...... 28 Case #22 ...... 28 Supplementary Figure S9 – Family tree of case #22 ...... 29 Case #23 ...... 29 Case #24 ...... 30 Supplementary Figure S10 – Family tree of case #24 ...... 30 Case #25 ...... 31 Case #26 ...... 31 Case #27 ...... 32 Supplementary Figure S11 – Family tree of case #27 ...... 32 Case #28 ...... 33 Supplementary Figure S12 – Family tree of case #28 ...... 33 Case #29 ...... 33 Supplementary Figure S13 – Family tree of case #29 ...... 34 Case #30 ...... 34 Case #31 ...... 35 Case #32 ...... 35 Case #33 ...... 36 Case #34 ...... 37

3 Supplementary Figure S14 – Family tree of case #34 ...... 37 Case #35 ...... 38 Case #36 ...... 38 Supplementary Figure S15 – Family tree of case #36 ...... 39 Case #37 ...... 39 Case #38 ...... 40 Case #39 ...... 41 Supplementary Figure S16 – Family tree of case #39 ...... 41 Case #40 ...... 42 References ...... 43

4 Supplementary Tables

Supplementary Table S1 – Cancer gene panel

ALK CENPJ EXT1 GREM1 MUTYH RAD50 SBDS TMEM127 APC CHEK2 EXT2 HABP2 NBN RAD51C SDHA TNFRSF11A ARMC5 CREBBP FAN1 HAX1 NF1 RAD51D SDHAF2 TP53 ATM CTC1 FANCA HNF1A NF2 RB1 SDHB TSC1 ATR CTNNA1 FANCB HOXB13 NHP2 RECQL SDHC TSC2 BAP1 CYLD FANCC KIF1B NOP10 RECQL4 SDHD VHL BARD1 DDB2 FANCD2 KIT NSD1 RET SEMA4A WAS BLM DDX11 FANCE KLLN OGG1 RINT1 SFTPA1 WRAP53 BMPR1A DICER1 FANCF LZTR1 PALB2 RPL11 SFTPA2 WRN BRCA1 DKC1 FANCG MAX PARK2 RPL35A SLX4 WT1 BRCA2 EGFR FANCI MDH2 PHOX2B RPL5 SMAD4 XPA BRIP1 ELANE FANCL MEN1 PMS2 RPS10 SMAD9 XPC BUB1 ERCC1 FANCM MET POLD1 RPS17 SMARCA4 XRCC2 BUB1B ERCC2 FH MLH1 POLE RPS19 SMARCB1 BUB3 ERCC3 FLCN MPL PRF1 RPS24 SQSTM1 CDC73 ERCC4 G6PC3 MRE11A PRKAR1A RPS26 STK11 CDH1 ERCC5 GDNF MSH2 PTCH1 RPS7 SUFU CDK4 ERCC6 GFI1 MSH6 PTEN RTEL1 TERT CDKN2A EXO1 GPC3 MUC5B PTPRJ RUNX1 TINF2

The cancer gene panel is based on the hereditary cancer gene panel used in diagnostics in the Radboudumc (https://www.radboudumc.nl/Informatievoorverwijzers/Genoomdiagnostiek/en/Pages/ Hereditarycancer.aspx), version DG 2.4x.

Supplementary Table S2 – Patients with multiple malignancies

Patient ID First malignancy Age in Second malignancy Age in years years 5 Precursor B-cell acute 3 Common B-cell acute lymphoblastic 8 lymphoblastic leukemia leukemia (hyperdiploid) 7 Neuroblastoma 3 Embryonal rhabdomyosarcoma 4 14 Low-grade glioma 14 Acute myeloid leukemia 15 16 Ganglioneuroblastoma 10 Precursor B-cell acute lymphoblastic 10 leukemia 18 Anaplastic glioma 3 Renal cell carcinoma 9 32 Basal cell carcinoma 18 Multiple basal cell carcinomas (>10) 18-21 34 Medulloblastoma 8 Pancreatoblastoma 10 38 Burkitt type Non-Hodgkin 13 Melanoma 44 lymphoma Solitary fibrous tumor 45

5

Supplementary Table S3 – Tumor types of index patients

Tumor Type No. of tumors Leukemia 13 Acute lymphoblastic leukemia 10 Acute myeloid leukemia 3 Lymphoma 9 Hodgkin lymphoma 4 Non-Hodgkin lymphoma 2 Burkitt lymphoma 3 Brain tumors 10 Medulloblastoma 3 Glioma/glioblastoma 3 Tumor of unknown origin 2 Pineoblastoma 1 Anaplastic ependymoma 1 Solid tumors 14 Neuroblastoma 4 Wilms tumor 1 Neuroendocrine tumor appendix 1 Pleuropulmonary blastoma 1 Thyroid carcinoma 1 Hepatocellular carcinoma 1 Hepatoblastoma 1 Embryonal rhabdomyosarcoma 1 Renal cell carcinoma 1 Pancreatoblastoma 1 Solitary fibrous tumor 1 Skin cancer 2 Basal cell carcinoma (>10) 1 Melanoma 1

Total 48

The total number of tumors exceeds the total number of included index patients (40), because some patients developed more than one tumor.

6

Supplementary Table S4 – Candidate genes

Gene & mutation Patient ID Inheritance Tumor type (age) Additional medical history and family history

NCR3LG1 10 Paternal Precursor B-cell acute Paternal uncle developed a common-ALL as a child. Both patients carry the c.268C>T (p.Arg90*) lymphoblastic leukemia (1.5) variant. E4F1 11 De novo Diffuse large B-cell lymphoma Congenital multicystic kidney, congenital uterus anomaly c.169C>T (p.Arg557*) (15)

KDM3B 25 De novo Hodgkin lymphoma (14) Intellectual disability, feeding difficulties, umbilical and inguinal hernia c.3095A>T (p.Asp1032Val) KDM3B 33 Maternal Acute myeloid leukemia (13) Congenital hypothyroidism, congenital hip dysplasia, mild intellectual c.277G>T (p.Glu93*) disability. Mother mild intellectual disability. LATS1 32 Unknown Multiple basal cell carcinomas No additional features c.728C>T (p.Arg243Lys) (18) MAD1L1 17 Maternal T-lymphoblastic lymphoma Maternal cousin T-cell Non-Hodgkin lymphoma (carries variant); father c.2118_2133del (7) juvenile astrocytoma age 12; paternal cousin leptomeningeal gliomatosis at (p.Leu707fs) age 20 TRIM28 22 Unknown Wilms tumor (0.5) Esophageal atresia, congenital heart defect. c.246_247del (p.Cys82fs) Sister bilateral Wilms tumor at age 2 (both carry the variant) TUBB1 28 Maternal Precursor B-cell acute Maternal cousin with acute myeloid leukemia at age 1.5 (both carry the c.151insTG (p.Tyr51fs) lymphoblastic leukemia (1.5) variant) TYK2 5 Paternal Acute lymphoblastic leukemia Paternal family history of breast- and ovarian cancer due to BRCA2 mutation c.2279C>T (p.Pro760Leu) twice (cytogenetically which this boy carries as well 1. different) (3, 8)

Genes were selected as candidate genes if they have at least two of the following items: a known association with tumorigenesis in general and/or the specific tumor type of the patient, interaction with known cancer genes or presence of variants in a gene in multiple patients. In the section with patient descriptions these genes are discussed in more detail.

Abbreviations: ALL = acute lymphoblastic leukemia

7 Supplementary Table S5 – Overview of clinical features and mutations found in category 1 (ID and/or congenital anomalies)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome ID diagnosis sequencingǂ (years) 1 Male ALL 8 ID, trigonocephaly ACTB (p.V209L) – De novo 2 Male ALL 16 ID, slow dentition, hypoplastic toe nails ARID1A (c.4993+1G>A) – De novo 3 Female ALL 2 ID, congenital hip dysplasia SCN2A (p.R856*) – De novo 6 Male Medulloblastoma 2 ID, generalized hypotonia, craniosynostosis 8 Male Medulloblastoma 4 ID, macro-scaphocephaly, coarse facial features, phimosis. 9 Male Hodgkin lymphoma 10 PDD-NOS, congenital testicular abnormalities (testicular seminiferous tubule dysgenesis with severe hypoplasia) 11 Female Non-Hodgkin 15 Congenital multicystic and dysplastic kidney, congenital E4F1 (p.R557*) – De novo lymphoma uterus didelphys with occlusive vaginal septum 13 Female Pleuropulmonary 1 Rib- and skin anomalies blastoma 15 Male Burkitt lymphoma 7 ID, bilateral inguinal hernia, facial dysmorphisms, cardiac anomalies 19 Female AML 8 Delayed psychomotor development, microcephaly EP300 (p.P1877fs) – De novo 20 Male Hodgkin lymphoma 9 Asperger’s syndrome, psoriasis, testicular hydrocele 23 Female Tumor thalamus 6 Developmental delay, dysmorphic features, short stature, high myopia 25 Male Hodgkin lymphoma 14 ID, feeding difficulties, umbilical and inguinal hernia, short KDM3B (p.D1032V) – De novo stature 26 Male Hepatoblastoma 10 Microcephaly, multiple hyper- and hypopigmentations of the skin, recurrent infections, mitral valve stenosis, primary hyperparathyroidism and micropenis 30 Male Hodgkin lymphoma 17 Hypospadia, spondylolysis of L5 and S1 vertebrae, large hyperpigmented lesion

8 Supplementary Table S5 – Overview of clinical features and mutations found in category 1 (ID and/or congenital anomalies - Continued)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome no. diagnosis sequencingǂ (years) 31 Male Glioblastoma 10 ID, imperforate anus, epilepsy, mitochondrial complex I + III deficiency 33 Female AML 13 Mild ID, congenital hip dysplasia, congenital hypothyroidism CHEK2 (p.T367fs) BAP1 (p.I706V) – De novo KDM3B (p.E93*) 37 Male Burkitt lymphoma 3 Horseshoe kidney, tall stature (>+2.5 SD), recurrent infections EZH2 (p.A682T) – De novo 40 Male Tumor 1 ID, cerebral paresis, ptosis mesencephalon

Abbreviations: ID = intellectual disability; ALL = acute lymphoblastic leukemia; PDD-NOS = pervasive developmental disorder, not otherwise specified; AML = acute myeloid leukemia; SD = standard deviations ǂ In this column the variants from Table 2 and 3 and Supplementary Table S4 are shown. Candidate genes are shown in light-grey.

9 Supplementary Table S6 – Overview of clinical features and mutations found in category 2 (cancer twice)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome no. diagnosis sequencingǂ (years) 5 Male Precursor B-cell ALL 3 Hyperdiploid common B-ALL at age 7 (cytogenetically different) TYK2 (p.P760L) BRCA2 (p.E1511fs) 7 Female Neuroblastoma 3 Embryonal rhabdomyosarcoma at age 4 DICER1 (p.L805P) 14 Male Low-grade glioma 14 AML at age 16 16 Female Neuroblastoma 10 ALL at age 10, synchronous diagnosis with neuroblastoma 18 Male Low-grade glioma 3 Renal cell carcinoma at age 9 TP53 (p.G245S) – De novo 38 Female Burkitt lymphoma 13 Melanoma at age 44, solitary fibrous tumor at age 45, multiple duodenal tubular adenomas (low-high grade dysplasia)

Abbreviations: ALL = acute lymphoblastic leukemia; AML = acute myeloid leukemia ǂ In this column the variants from Table 2 and 3 and Supplementary Table S4 are shown. Candidate genes are shown in light-grey.

Supplementary Table S7 – Overview of clinical features and mutations found in category 3 (family history)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome no. diagnosis sequencingǂ (years) 10-I Male ALL 1.5 Uncle with ALL (10-II). B7H6 (p.R90*) 10-II Male ALL 6 Cousin with ALL (10-I). B7H6 (p.R90*) 12-I Male Neuroendocrine 10 Sister with neuroendocrine tumor of the appendix (12-II) tumor appendix 12-II Female Neuroendocrine 11 Brother with neuroendocrine tumor of the appendix (12-I) tumor appendix

10 Supplementary Table S7 – Overview of clinical features and mutations found in category 3 (family history - continued)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome no. diagnosis sequencingǂ (years) 17-I Male Non-Hodgkin 7 Maternal cousin NHL (17-II), father brain tumor (17-III) and MAD1L1 in pt. 17-I and 17- lymphoma paternal cousin leptomeningeal gliomatosis (17-IV). II (p.L707fs) 17-II Male Non-Hodgkin 6 History of pancytopenia and hepatosplenomegaly due to MAD1L1 in pt. 17-I and 17- lymphoma congenital toxoplasmosis. Cousin with NHL (17-I). II (p.L707fs) 17-III Male Juvenile astrocytoma 12 Son NHL (17-I), unrelated cousin NHL (17-II), cousin grade II leptomeningeal gliomatosis (17-IV). 17-IV Male Leptomeningeal 20 Uncle brain tumor (17-III), cousin NHL (17-I). gliomatosis 21-I Male Papillary thyroid 17 Sister with follicular thyroid carcinoma at age 13 (21-II) DICER1 (p.V455fs) carcinoma 21-II Female Follicular thyroid 13 Brother with papillary thyroid carcinoma at age 17 (21-I) DICER1 (p.V455fs) carcinoma 22-I Female Wilms tumor 1.5 Esophageal atresia, complex congenital heart defect, retinopathy TRIM28 (p.Cys82fs) left eye. Sister with bilateral Wilms tumor (22-II). 22-II Female Wilms tumor 0.5 Sister with multiple congenital anomalies and Wilms tumor (22-I) TRIM28 (p.Cys82fs) (bilateral) 24-I Female ALL 4 Sister with ALL at age 4 (24-II) 24-II Female ALL 4 Sister with ALL at age 4 (24-I) 27 Male Anaplastic 6 ADHD, maternal aunt with brain astrocytoma at age 3 CHEK2 (p.T367fs) ependymoma 28-I Female ALL 1.5 Cousin with AML at age 1.5y (28-II) TUBB1 (p.Tyr51fs) 28-II Female AML 1.5 Cousin with ALL at age 1.5y (28-I) TUBB1 (p.Tyr51fs) 29-I Male Neuroblastoma 1 Father with ALL at age 3 (29-II) 29-II Male ALL 3 Son with neuroblastoma at age 3 (29-I)

11 Supplementary Table S7 – Overview of clinical features and mutations found in category 3 (family history - continued)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome no. diagnosis sequencingǂ (years) 36-I Female ALL 5 Brother with ALL at age 2 (36-II) ETV6 (p.R433C) – De novo 36-II Male ALL 2 Sister with ALL at age 5 (36-I) 39-I Female Neuroblastoma 2 Cousin with neuroblastoma (39-II) at age 2, cousin with Wilms tumor (39-III) 39-II Female Neuroblastoma 2 Cousin with neuroblastoma (39-I) at age 2, cousin with Wilms tumor (39-III) 39-III Male Wilms tumor 3.5 Two cousins with neuroblastoma at age 2 (39-I and 39-II)

Abbreviations: ALL = acute lymphoblastic leukemia; NHL = Non-Hodgkin lymphoma; AML = acute myeloid leukemia; ADHD = Attention Deficit Hyperactivity Disorder; ǂ In this column the variants from Table 2 and 3 and Supplementary Table S4 are shown. Candidate genes are shown in light-grey.

Supplementary Table S8 – Overview of clinical features and mutations found in category 4 (adult type of cancer)

Patient Gender Malignancy Age at Additional malignancies or other clinical features Outcome of whole exome no. diagnosis sequencingǂ (years) 35 Male Hepatocellular 5 No additional features BUB1 (p.V991I) - De novo carcinoma APOB (p.Y981*)

ǂ In this column the variants from Table 2 and 3 and Supplementary Table S4 are shown. Candidate genes are shown in light-grey.

12 Supplementary Table S9 – Overview of clinical features and mutations found in category 5 (multiple reasons for inclusion)

Patient Gender Inclusion Malignancy Age at Additional malignancies or other clinical features Outcome of whole no. reasons diagnosis exome (years) sequencingǂ 4 Male 1, 3 Pineoblastoma 1.5 Complicated neonatal period with unexplained pulmonary DICER1 (p.S1470fs) hypertension (after revision lung biopsy based on pleuropulmonary blastoma); distant cousin with brain tumor (who also carries DICER1 mutation). 32 Male 2, 4 Basal cell carcinoma 18 Multiple basal cell carcinomas SPRED1 (p.R16*) LATS1 (p.R243K) 34-I Female 2, 3 Medulloblastoma 8 Pancreatoblastoma, half sister with medulloblastoma (34-II) APC (p.Ile1060fs) – De novo 34-II Female 2, 3 Medulloblastoma 8 Half sister with medulloblastoma (34-I)

ǂ In this column the variants from Table 2 and 3 and Supplementary Table S4 are shown. Candidate genes are shown in light-grey.

13 Clinical descriptions of patients

Case #01 General information Gender Male Malignancy Precursor B-cell acute lymphoblastic leukemia at age 8 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study SNP array showed homozygous areas; KMT2D, MLL2 and FMR1 testing without abnormalities

Additional description This boy was born after an uncomplicated pregnancy and delivery. He had developmental delay from the start with an estimated Intelligence Quotient (IQ) of 55 at the age of 13. He has a trigono- and microcephaly, low-set ears, telecanthus with prominent epicanthal folds, wide set nipples and short thumbs and 5th digits on both hands. He has had complex partial seizures from the age of 15 years. A brain MRI did not show lissencephaly or polymicrogyria, but it did show diffuse subtle malformations of the cerebral development with a thin cortex.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Whole exome analysis revealed a de novo mutation in the actin-encoding gene ACTB (p.Val209Leu). Heterozygous missense mutations in this gene cause Baraitser-Winter syndrome [OMIM: 243310] 2. Our patient was included in a genotype-phenotype study (patient B25) which included one other patient who developed cancer, a cutaneous lymphoma 3. In addition, a third patient with ACTB-based Baraitser-Winter syndrome who developed AML was identified recently 4. ACTB is deregulated in multiple malignancies, amongst which also leukemia and lymphoma 5. Furthermore, somatic mutations in ACTB in diffuse large B-cell lymphoma have been described 6. This indicates that actinopathies may be low-penetrant cancer-predisposing disorders and that the ACTB mutation in this boy might have contributed to the development of leukemia. Case #02 General information Gender Male Malignancy Common B-cell acute lymphoblastic leukemia at age 16 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP analysis and FMR1 sequencing without abnormalities

Additional description This boy was born after a pregnancy complicated by polyhydramnios due to fetal hydronephrosis for which no cause was found. He had recurrent urinary tract infections. He had a slow dentition with the first teeth appearing at 2 years of age. His nails grow slow as well and he has hypoplastic toe nails, most explicit on his 5th toe. His development was delayed; he started walking at the age of 2,5 years and started talking at the age of 3 years. An IQ-test showed a verbal IQ of 80 and a performance IQ of 55. He has epicanthal folds, full lips, a broad mouth and hirsutism. He had a height of 176 cm (-1.5 SD) and a weight of 51.6 kg (-0.5 SD). From the age of 18 onwards he had recurrent nephrolithiasis.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Whole exome analysis revealed a de novo splice site mutation in ARID1A (c.4993+1G>A). Heterozygous mutations in this

14 gene cause Coffin-Siris syndrome [OMIM: 614607] 7, which is a rare congenital anomaly syndrome characterized by intellectual disability, microcephaly, coarse facial features, hypertrichosis and hypoplastic nails of the fifth finger and/or toe. The splice site mutation found in this boy was missed initially by whole exome analysis. Because this boy presented with features resembling Coffin-Siris syndrome we performed targeted sequencing of ARID1A, which showed a mosaic splice site mutation in this gene (Supplementary Figure S1). When looking back at the exome data, this mosaic mutation was also present in the exome data, but not in enough reads to pass our threshold (Supplementary Figure S1). There is evidence that ARID1A mutations occur mostly mosaic8.

ARID1A is a member of the SWI/SNF family, a family of genes involved in transcription regulation by chromatin remodeling 9. ARID1A is commonly mutated in gastric cancers, ovarian clear cell carcinoma and pancreatic cancer. Several case reports of malignancies in patients with Coffin-Siris syndrome are available, including a patient with an ARID1A mutation who developed a hepatoblastoma 7,10,11. Furthermore, both somatic and germline mutations in other SWI/SNF genes were previously described in cancer 12. All together this suggests that the germline ARID1A mutation may have contributed to the leukemia that this boy developed. Sanger sequencing of ARID1A in leukemia derived DNA of the boy did not reveal a second mutation nor loss of heterozygosity of the germline mutation, but confirmed the presence of the germline mutation in the tumor.

Supplementary Figure S1 – Detection of mosaic mutation in ARID1A

Screenshot from Integrative Genomics Viewer version 2.3.14 (IGV, Broad Institute, Cambridge, UK). The splice site mutation in this boy was called in only 2 out of 23 reads, confirming the mosaic state of this mutation.

15 Case #03 General information Gender Female Malignancy Precursor B-cell acute lymphoblastic leukemia at age 2 (ETV6-RUNX1 translocation, 9p13.2 deletion containing PAX5). Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP analysis and FMR1 testing without abnormalities

Additional description This girl was born after an uncomplicated pregnancy and delivery. She was born with congenital hip dysplasia. Both her motor- and psychological development are delayed and has an autism spectrum disorder (ASD). She has no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and her parents. With whole exome analysis we identified a de novo truncating mutation in SCN2A (p.Arg856*). Heterozygous mutations in this gene cause early infantile epileptic encephalopathy and benign familial infantile seizures [OMIM: 613721 and OMIM: 607745] 13. In addition, mutations in SCN2A have also been described in patients with ID, but without epilepsy 14,15. SCN2A encodes a subunit of a voltage-gated sodium channel which generates and propagates action potentials in neurons 13. This mutation explains her delayed development, but we do not expect that this mutation in SCN2A played a role in the development of leukemia in this girl. Case #04 General information Gender Male Malignancy Pineoblastoma at age 16 months (MSI negative) Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP analysis, PTEN, MLH1, MSH2, MSH6 and PMS2 sequencing were without abnormalities

Additional description This boy was born after an uncomplicated pregnancy and delivery. During pregnancy, a hydronephrosis of the left kidney was detected on ultrasound. In the first day postpartum, he developed acute respiratory and circulatory failure due to an unexplained pulmonary hypertension, for which he was treated with extracorporeal membrane oxygenation (ECMO). During this period, he was also diagnosed with an euthyroid sick syndrome, a cholangitis for which he had a cholecystectomy, and a right sided cerebral hemorrhage of the occipital lobe. A lung biopsy at the age of 5 months showed congenital bullous emphysema in his lungs. He had no dysmorphic features other than a prominent forehead.

Family history showed that the parents were consanguineous in the third degree. A cousin of his father died of leukemia at the age of 4. A cousin of his mother died of a meningeal sarcoma at the age of 3 years old.

16

Supplementary Figure S2 – Family tree of case #04

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Cancer gene panel analysis revealed a pathogenic frameshift mutation in DICER1 (p.S1470fs), which he inherited from his mother 16. Heterozygous mutations in this gene cause the DICER1 tumor predisposition syndrome [MIM: 601200] 17. DICER1 mutation carriers are at risk for a wide spectrum of malignancies, including pleuropulmonary blastomas, pineoblastomas, cystic nephromas and ovarian Sertoli-Leydig cell tumors 17. In retrospect with knowledge of this mutation, the congenital bullous emphysema in his lungs was reclassified as a pleuropulmonary blastoma. Furthermore, by asking specifically for features characteristic of the DICER1 tumor predisposition syndrome, we learned that mother was diagnosed with multinodular goiter at 15 years of age and that a cousin of his mother had a Sertoli- Leydig cell tumor. Segregation analysis revealed that the cousin who died because of meningeal fibrosarcoma had inherited the same DICER1 mutation. Case #05 General information Gender Male Malignancy Precursor B-cell acute lymphoblastic leukemia at age 3. Common B-cell acute lymphoblastic leukemia at age 8 (hyperdiploid, cytogenetically different from first leukemia). Inclusion category Category 2 - Cancer twice Testing before study 250k SNP array and TP53 analysis were without abnormalities. BRCA2 analysis showed a known familial BRCA2 mutation, a mitomycin-C test showed no abnormalities

17 Additional information This boy was born after an uncomplicated pregnancy and delivery. His early psychomotor development was normal and he has no dysmorphic features. He has a hypopigmentation in the groin area of 13 by 7 cm. After his second leukemia his cognition was tested, which revealed an IQ of 98 and a disorder in the autism-spectrum.

Family history showed a BRCA2 mutation in the mother’s family and consequently numerous family members with breast- and ovarian cancer. A brother of the father had a benign fibrous histiocytoma removed from his knee at 12 years of age.

Supplementary Figure S3 – Family tree of case #05

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. The BRCA2 mutation that is present in the mother’s family was also found in this patient. A higher incidence of childhood cancer amongst BRCA mutation carriers has been described 18,19, but this mutation alone likely does not explain the phenotype of this boy. Therefore, we performed whole exome analysis. We found a paternally inherited heterozygous missense variant in TYK2 (c.2279C>T; p.P760L). In a parallel study, we also found a germline variant in this gene (c.2282G>T; p.G761V) in a boy who developed two independent T-lineage ALL occurrences 1. Functional studies showed that both variants result in increased auto-phosphorylation and phosphorylation of several downstream STAT proteins. TYK2 is a family member of the Janus Kinases JAK1, JAK2, and JAK3, which are all somatically activated by variants and translocations in various hematologic malignancies 20.

18 Case #06 General information Gender Male Malignancy Classic medulloblastoma grade IV at age 22 months Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP array and ACTB, SMARCB1 and PTCH1 testing were without abnormalities Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a craniosynostosis of the sutura metopica and a head circumference of 52cm (+2 SD). He had muscle hypotonia since birth with hyperlaxity of his joints. He was diagnosed with congenital hip dysplasia. He has hypertelorism and upturned ear lobes. He has a severe developmental delay.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #07 General information Gender Female Malignancy Neuroblastoma stage IV at age 3, embryonal rhabdomyosarcoma of the botryoid type of the cervix at age 4 Inclusion category Category 2 - Cancer twice Testing before study TP53 and NRAS testing without abnormalities

Additional information This girl was born after an uncomplicated pregnancy and delivery. She has a normal development and there are no additional comorbidities or facial dysmorphisms. A paternal aunt developed leukemia at age 2.5.

Supplementary Figure S4 – Family tree of case #07

Results of whole exome sequencing WES was performed on germline DNA of the index patient and her parents. Cancer gene panel analysis identified a missense mutation in DICER1, which this girl inherited from her father. Mutations in this gene cause the DICER1 tumor predisposition syndrome [OMIM: 606241] 16. DICER1

19 mutation carriers are at risk for a wide spectrum of malignancies, including pleuropulmonary blastomas, pineoblastomas, cystic nephromas and ovarian Sertoli-Leydig cell tumors 17. In addition, we identified a somatic DICER1 p.Glu1813Asp mutation in DNA derived from the embryonal rhabdomyosarcoma by Sanger sequencing. This mutation is located in the RNAse IIIb domain and is a hotspot mutation in DICER1-associated tumors 21. Identification of this well-known ‘second hit’ mutation underscores the pathogenicity of the germline missense mutation that we have found. Neuroblastoma is not among the malignancies frequently identified in children with germline DICER1 mutations 17. In DNA derived from the neuroblastoma, we did not identify a somatic mutation in DICER1 nor signs of loss-of-heterozygosity of the germline variant. Oncoscan array did not reveal a somatic deletion of the DICER1 gene. Hence, we did not confirm a direct correlation between the germline mutation in DICER1 and the neuroblastoma in this patient, but we cannot exlude such a relationship. Recently, a second patient with a mutation in DICER1 and neuroblastoma has been identified 22. Strikingly, somatic mutations in the tumor of this patient were also not present. Case #08 General information Gender Male Malignancy Medulloblastoma grade IV at age 4.5 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study PTCH1- and SUFU testing were without abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a delayed motor- and speech development; he could lift his head at 8 months old and walk at 2 years old. He has a macro- and scaphocephaly, a coarse facial appearance, phimosis and 1 skin depigmentation. His length was 105 cm (-1.5 SD) and his head circumference was 55 cm (+2 SD). His medulloblastoma was initially classified as a desmoplastic medulloblastoma. Later this was re-classified as a classic subtype medulloblastoma.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. In this boy a de novo variant in HNRNPU was found (p.Pro272del). Truncating mutations in this gene have been described in multiple papers in 4 patients with intellectual disability and epilepsy 23-27. None of these patients developed cancer. HNRNPU is a heterogeneous nuclear ribonucleoprotein (hnRNP) that binds RNAs and participates in pre-mRNA processing 28. Our patient does show some overlapping features with the patients described, but he is less severely affected and does not have epilepsy. Functional studies will need to be performed to determine the effect of this variant. It is currently unknown if this variant is causative of the phenotype of this boy. Case #09 General information Gender Male Malignancy Nodular lymphocyte predominant Hodgkin lymphoma at age 10 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP array without abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He was diagnosed with PDD-NOS, but otherwise he had a normal development. There were no facial dysmorphic features. By evaluation for tumor localizations at diagnosis a testicular biopsy was performed to evaluate the abnormalities that were seen with ultrasound. The biopsy showed he had a testicular seminiferous tubule dysgenesis based on severe germinal hypoplasia.

20

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #10 General information Gender Male Malignancy Precursor B-cell acute lymphoblastic leukemia at age 18 months (hyperdiploid subtype with TEL/AML1 fusion gene) Inclusion category Category 3 - Family history Testing before study 250k SNP array without abnormalities

Additional information A maternal uncle was diagnosed with a common B-cell acute lymphoblastic leukemia at age 6.

Supplementary Figure S5 – Family tree of case #10

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his affected uncle. In both patients, whole exome analysis identified a variant in the NCR3LG1 gene (p.Arg90*). NCR3LG1 codes for the B6H7 protein, which is the ligand for the activation of the NKp30 receptor of NK cells and induces cell cytotoxicity and cytokine secretion 29. B7H6 is detected on the cell surface of many tumor cells and has a role in tumor immune surveillance and in the inflammatory response to infectious diseases 30. A lack of functional B7H6 protein as a result of the truncating variant may result in reduced NK cell killing of the tumor cells, which may allow early tumor cells to evade detection and grow out to full leukemia.

21 Case #11 General information Gender Female Malignancy Diffuse large B-cell lymphoma at age 15 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study HNF1β and BCHE testing were without abnormalities

Additional information This girl was born after an uncomplicated pregnancy and delivery. After birth, a congenital multicystic dysplastic kidney was diagnosed and this kidney was removed at age 2. In puberty, a congenital uterus didelphys with an occlusive vaginal septum was diagnosed. Her psychomotor development was normal and there are no dysmorphic features. Her father was diagnosed with prostate cancer at the age of 49.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and her parents. Whole exome analysis revealed a de novo truncating variant in E4F1 (p.Arg557*). E4F1 is proposed as a new CAKUT-gene (congenital anomalies of the kidney and urinary tract) since it has an interaction with the known CAKUT-gene HNF1B 31. Furthermore, E4F1 functions as an ubiquitin E3 ligase for TP53, which is a known tumor predisposition gene 32. Ubiquitination and modification of TP53 by E4F1 leads to the activation of certain target genes and in cell cycle arrest 32. If E4F1 is unable to perform this ubiquitination, the target genes will not be activated and cell cycle arrest will not occur, which allows cells to divide uncontrollably. We performed LOH-analysis on lymphoma derived DNA, which showed that the mutated allele was lost in the tumor. This does not exclude a cancer predisposing role for the E4F1 variant. Variants that are cancer predisposing are not necessarily beneficial for a full-blown malignancy and can be lost based on selection advantage and clonal outgrowth. Case #12 General information Gender Male Malignancy Neuroendocrine tumor (NET) of the appendix at age 10 Inclusion category Category 3 - Family history Testing before study 250k SNP array and APC testing were without abnormalities

Additional information This boy was born with congenital hip dysplasia after an uncomplicated pregnancy and delivery. His sister was diagnosed with a neuroendocrine tumor of the appendix as well at the age of 11 years. She had congenital hip dysplasia and club feet and was born after an uncomplicated pregnancy and delivery. Both siblings have a normal development and no dysmorphic features.

22

Supplementary Figure S6 – Family tree of case #12

Results of whole exome sequencing WES was performed on germline DNA of both siblings and their parents. Our analysis focused on shared variants between the siblings. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #13 General information Gender Female Malignancy Pleuropulmonary blastoma at age 1 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study DICER1 and SNP array analysis without abnormalities

Additional information This girl was born with a peculiar rosette-shaped skin hamartoma at the left part of the thorax. An X- ray of the thorax at the age of 1 month showed rib- and soft tissue anomalies with no specific origin. She has thin third and fourth ribs on the left side and her fifth to eighth rib are strongly aberrant, mainly on the ventral end. There were no other skeletal anomalies found. At the age of 1 year she was diagnosed with a pleuropulmonary blastoma of the left chest wall with metastases in the right lung. She has multiple molar teeth with an abnormal shape. She had a antrachoanal polyp of the right maxillary sinus, which was removed at age 11 and showed no signs of a malignancy

Results of whole exome sequencing WES was performed on skin-biopsy derived DNA of the index patient and blood derived DNA of her parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis.

23 Case #14 General information Gender Male Malignancy Low grade glioma of the brainstem (BRAF+) at age 14 and acute myeloid leukemia with characteristics of a myeloid sarcoma (translocation (11;17) with MLL rearrangement) at age 15. Inclusion category Category 2 - Cancer twice Testing before study Not performed

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a normal psychomotor development and no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #15 General information Gender Male Malignancy Burkitt type B-cell non-Hodgkin lymphoma at age 7 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP array and MED12 analysis were without abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. His development was delayed and he has a mild intellectual disability. As a child, he had a bilateral inguinal hernia and a thyroglossal duct cyst in his neck, which was surgically removed. On physical examination, several dysmorphic features stood out. These were retrognathia, plagiocephaly, full eyebrows, ptosis, a large nose with a broad tip, open mouth behavior, large teeth, a brachydactyly of his hands and feet and the presence of fetal fingerpads. On a CT-scan performed at diagnosis of the malignancy an arteria lusoria, a persistent left superior vena cava and a double inferior vena cava were found. His length was 165.5 cm (-1.5 SD) and his head circumference was 55 cm (-0.5 SD). His mother has the same facial appearance in addition to anal atresia.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his mother. Paternal DNA was unavailable. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #16 General information Gender Female Malignancy Thoracal ganglioneuroblastoma (NMYC and LOH negative) and precursor B- cell acute lymphoblastic leukemia at age 10. Inclusion category Category 2 - Cancer twice Testing before study 250k SNP array without abnormalities

Additional information This girl was born after an uncomplicated pregnancy and delivery. She had a normal development and no dysmorphic features.

24

Results of whole exome sequencing WES was performed on germline DNA of the index patient. Parental DNA was unavailable. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #17 General information Gender Male Malignancy T-cell lymphoblastic lymphoma at age 7 Inclusion category Category 3 - Family history Testing before study 250k SNP array and TP53 analysis were without abnormalities

Additional information This boy was born prematurely at 34 weeks of gestation. He had a normal psychomotor development. He had a cutaneous syndactyly of the second and third toes on both sides and clinodactyly of the third and fourth toes on both sides. In his family, multiple relatives developed cancer at a young age. His father was diagnosed with a brain tumor at age 12: a juvenile astrocytoma grade II for which he received radiotherapy. One cousin on mother’s side of the family was diagnosed with a T-cell Non-Hodgkin lymphoma at age 6 and had a history of pancytopenia and hepatosplenomegaly due to a congenital toxoplasmosis infection and an epimembranous glomerulonephritis. The second cousin was diagnosed with leptomeningeal gliomatosis at age 20 and died shortly after this diagnosis.

Supplementary Figure S7 – Family tree of case #17

Results of whole exome sequencing WES was performed on germline DNA of the index patient and the three affected family members. A frameshift variant was identified in MAD1L1 (p.Leu707fs)) in the two cousins with non-Hodgkin lymphoma (NHL). This gene encodes a component of the mitotic spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate. The C-terminal domain of MAD1L1, which is significantly altered by the variant, targets the protein to the kinetochores 33,34, but karyotype analysis of lymphocyte metaphases from the index patient did

25 not show an increase in chromosome miss-segregations. Further cell biological studies are needed to assess the functional consequences of this variant.

In addition, a variant in SAMD9 (p.Gln1445del) was identified in the index patient, his father and his cousin with leptomeningeal gliomatosis. Mutations in SAMD9 were recently described to cause MIRAGE syndrome, which is an acronym for myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy 35. This phenotype was not present in our family. Furthermore, the mutations found by Narumi et al are all missense mutations expected to cause a gain-of-function. The contribution of the SAMD9 variant to cancer development in our patients needs to be studied further. Case #18 General information Gender Male Malignancy Anaplastic glioma at age 3 and renal cell carcinoma at age 9 Inclusion category Category 2 - Cancer twice Testing before study NF1 analysis was without abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He has a normal psychomotor development and no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Cancer gene panel analysis revealed a de novo mutation in TP53 (p.Gly245Ser). Heterozygous germline mutations in this gene cause Li-Fraumeni syndrome (LFS, OMIM: 151623), a cancer predisposition syndrome characterized by a wide tumor spectrum with onset both in children and adults. In childhood, the LFS tumor spectrum is mostly characterized by osteosarcomas, adrenocortical carcinomas, central nervous system tumors and soft tissue sarcomas. Renal cancer in childhood is a rare presentation of LFS. A recent study showed that only 3% percent of carriers of a germline TP53 mutation who developed cancer were affected by renal cancer, with a median age of tumor onset of 49 years 36. The missense mutation identified in this boy affects the DNA-binding domain of TP53 and is a known pathogenic mutation (http://p53.iarc.fr/TP53GermlineMutations.aspx) 37. Case #19 General information Gender Female Malignancy Acute myeloid leukemia at age 8 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study Unknown

Additional information This girl was born after an uncomplicated pregnancy and delivery. She has a delayed psychomotor development and a microcephaly. She has no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and her parents. Whole exome analysis revealed a de novo frameshift mutation in EP300 (p.Pro1877fs). Heterozygous mutations in this gene are known to cause Rubinstein-Taybi syndrome (RSTS, OMIM: 180849). RSTS is an autosomal dominant neurodevelopmental disorder characterized by growth deficiency, broad thumbs and toes, intellectual disability and characteristic craniofacial appearance. Mutations in EP300 only account for 3-8% of the RSTS-cases 38-40 and the typical dysmorphic features of RSTS are variably present in

26 patients with an EP300 mutation 41. EP300 encodes the p300 transcriptional co-activator protein, which shares 63% homology with CREBBP at the amino acid level 42. It functions as a histone acetyltransferase that regulates transcription via chromatin remodeling and is important in processes of cell proliferation and differentiation 43. Somatic loss of function mutations in EP300 have been reported in multiple malignancies, whereas no patients with a germline EP300 mutation affected by cancer have been reported so far. In contrast, multiple malignancies have been reported in CREBBP related RSTS patients 38. These tumors are mainly of neural and developmental origin. Case #20 General information Gender Male Malignancy Nodular sclerosing Hodgkin lymphoma at age 9 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study Not performed

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a normal development and a normal intelligence. He was diagnosed with Asperger’s syndrome for which he was treated with Risperidone. His medical history shows psoriasis. He has no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #21 General information Gender Male Malignancy Papillary thyroid cancer at age 17 Inclusion category Category 3 - Family history Testing before study Not performed

Additional information This boy was born after an uncomplicated pregnancy and delivery. His sister was diagnosed with follicular thyroid carcinoma at age 13. Both have a normal psychomotor development and no dysmorphic features. Their father died at the age of 47 due to a ruptured aortic aneurysm.

27

Supplementary Figure S8 – Family tree of case #21

Results of whole exome sequencing WES was performed on germline DNA of both affected siblings. We focused on shared variants between the siblings and cancer gene panel analysis revealed a pathogenic frameshift mutation in DICER1 (p.Val455fs). Mutations in this gene cause the DICER1 tumor predisposition syndrome [MIM: 601200] 16. DICER1 mutation carriers are at risk for a wide spectrum of malignancies, including pleuropulmonary blastomas, pineoblastomas, cystic nephromas and ovarian Sertoli-Leydig cell tumors 17. Rare cases of differentiated thyroid cancer in patients with germline mutations in DICER1 have been reported as well 44,45. The presentation of two siblings with thyroid cancer and no other features related to the DICER1 tumor predisposition syndrome is rather unique. Segregation analysis showed that this mutation was not present in the mother. Analysis could not be performed on the father, but it is likely that he carried the DICER1 mutation. Case #22 General information Gender Female Malignancy Wilms tumor at age 1.5 years Inclusion category Category 1 - ID and/or congenital anomalies and Category 3 - Family history Testing before study Unknown

Additional information This girl was born to non-consanguineous parents. She was born prematurely at the gestational age of 34+0 weeks. She was born with a cesarean section because of polyhydramnios and reduced placental blood flow. After birth, an esophageal atresia and tracheoesophageal fistula (Vogt IIIb) were diagnosed. Also a complex congenital heart defect was diagnosed consisting of a coarctation of the aorta with a hypoplastic aortic arch, ventricular septal defect, patent ductus arteriosus, patent foramen ovale and insufficiency of the tricuspidal- and pulmonal valve. Her heart defect and esophageal atresia were both surgically treated. She had a retinopathy of her left eye. She has no dysmorphic features. At the age of 1.5 years old she was diagnosed with a Wilms tumor. Her sister was born after an uncomplicated pregnancy and delivery. She had a normal development, no

28 dysmorphic features and no medical history. Cardiologic screening showed a clinically insignificant foramen ovale apertum. This girl developed a bilateral Wilms tumor at the age of 6 months.

The mother of the siblings is known with goiter and a maternal aunt has a congenital heart defect. A maternal cousin of these girls had multiple tumors of the medulla of unknown origin.

Supplementary Figure S9 – Family tree of case #22

Results of whole exome sequencing WES was performed on germline DNA of both affected sisters. We focused on shared variants between the siblings. Whole exome analysis in these siblings revealed a frameshift aberration in TRIM28 (p.Cys82fs). TRIM28 is a protein involved in gene silencing, cell growth and differentiation, neoplastic transformation, DNA repair and maintenance of genomic integrity 46. TRIM28 has an interaction with the WTX tumor suppressor, a gene in which somatic inactivating mutations in Wilms tumor have been described frequently 47. Nuclear WTX co-immunoprecipitates with TRIM28, thereby modulating its chromatin binding and coregulating the expression of DNA repeat elements 48. It is possible that the variant in TRIM28 is causing the phenotype in these sisters. Case #23 General information Gender Female Malignancy Thalamic tumor of unknown origin, most likely a low-grade glioma (age 6) Inclusion category Category 1 - ID and/or congenital anomalies Testing before study Unknown

Additional information This girl was born after a pregnancy complicated by gestational diabetes. She was born 3,5 weeks premature and was admitted to the high care unit because of apneas. At the age of 2 it became apparent that she had a developmental delay, most notable in the speech- and language area. She has several dysmorphic features: frontal bossing, retrognathia, full lips, long philtrum, protruding

29 ears (which were surgically corrected), fetal fingerpads and a clinodactyly (mild on her hands, more prominent on her feet). Her head circumference is 50.5 cm (0 SD). She also has a high myopia (OD +8.5, OS +9), a lordosis and she has a short stature (length 107.5 cm; <- 2.5 SD), which appears mildly disproportionate. At the age of 6 years old a thalamic tumor was observed on MRI. Biopsies were taken, but were inconclusive. It is expected to be a low-grade glioma.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and her parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #24 General information Gender Female Malignancy Precursor B-cell acute lymphoblastic leukemia at age 3 Inclusion category Category 3 - Family history Testing before study Not performed

Additional information This girl was born to non-consanguineous parents after an uncomplicated pregnancy and delivery. She developed ALL at age 3. She was included in our study because her sister developed precursor B- cell acute lymphoblastic leukemia as well, at the age of 4 years old. The tumor of this second girl showed MLL rearrangement. This girl also has dyslexia, Friedreich’s foot and a myoclonus of the left eye. Both sisters have an otherwise normal development and no dysmorphic features.

Supplementary Figure S10 – Family tree of case #24

Results of whole exome sequencing WES was performed on germline DNA of both affected sisters. We focused on shared variants between the sisters. Whole exome analysis revealed several candidate genes. Both sisters carry a variant in SAMD9L (p.Arg406*). Somatic mutations in SAMD9L are associated with both myelodysplastic syndrome and leukemia 49,50. Recently, patients with germline missense mutations in SAMD9L and a varying phenotype with neurologic and hematologic abnormalities have been described 49. Our patients do not show overlapping features with this phenotype up to now. We performed WES on the tumor of the index patient, but no second hit mutations in SAMD9L were found.

30 Case #25 General information Gender Male Malignancy Nodular sclerosing Hodgkin lymphoma at age 14 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP array and PTPN11-, BRAF-, KRAS-, HRAS- and SOS1 analysis without abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had feeding problems in infancy and he was diagnosed with an umbilical- and inguinal hernia at the age of 2 months. He has a moderate intellectual disability with autistiform behavior. He has several dysmorphic features, including downslanted palpebral fissures, ptosis, large ears, large mouth, relatively long fingers and toes and a webbed neck. He has a short stature (length 165 cm; -2.5 SD) compared to his family members and generalized hypotonia.

Results of whole exome sequencing WES was perfomed on the index patient and his parents. Whole exome analysis revealed a de novo variant in KDM3B (p.Asp1032Val). KDM3B is a histone H3K9 demethylase involved in histone modification and chromatin remodeling 51,52. We did not find loss of heterozygosity in lymphoma- derived DNA. Both somatic and germline mutations in chromatin remodeling genes are associated with an increased risk of cancer and ID has also been linked to germline mutations in chromatin remodeling genes. The finding of variants in KDM3B in two patients (the other is Case #33) with a similar phenotype suggests a possible role in the development of both ID and cancer in these patients. Case #26 General information Gender Male Malignancy Hepatoblastoma at age 10 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study Array CGH and NF1, NBS1 and mismatch repair genes analysis showed no abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He has several dysmorphic features and comorbidities that suggest the presence of a genetic syndrome. He has a microcephaly (head circumference 50.5cm (-2SD)), small chin, prominent nasal root, anteverted nares and upslanted palpebral fissures. His skin shows multiple café-au-lait macules (>12 on his trunk, several on his legs) and vitiligo. As a child, he had recurrent infections. He was also diagnosed with a mitral valve stenosis, primary hypoparathyroidism, a micropenis and he has calcifications of the globus pallidus on a CT-scan. His psychomotor development is normal.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his mother. Paternal DNA was not available. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis.

31 Case #27 General information Gender Male Malignancy Anaplastic ependymoma, WHO grade III at age 6 Inclusion category Category 3 - Family history Testing before study SNP array and TP53 analysis without abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a normal psychomotor development. He was diagnosed with Attention Deficit Hyperactivity Disorder. He has downslanted palpebral fissures, long eyelashes and protruding ears. The sister of his mother had an astrocytoma at age 3 from which she died. The maternal grandmother and 3 of her sisters developed breast cancer at ages 49, 52, 54 and 60. In two sisters, BRCA1 and BRCA2 analysis showed no abnormalities.

Supplementary Figure S11 – Family tree of case #27

Results of whole exome sequencing WES was performed in the index patient only. DNA of his deceased aunt was not available. Cancer gene panel analysis revealed a mutation in CHEK2 (c.1100delC, p.Thr367Metfs*15), which was maternally inherited. CHEK2* 1100delC was first reported in Li-Fraumeni-like families 53 and the mutation is now an established breast cancer susceptibility allele 54,55. In the Netherlands, CHEK2*1100delC is common with a carrier frequency of 1.2% (95% CI, 0.7% to 1.8%) 56. Indications are available that CHEK2*1100delC is associated with increased risk for other cancers in addition to breast cancer 57,58. To our knowledge, no studies have been performed to specifically test the association between this variant and childhood cancer risk. Epidemiologic and family studies have suggested that the breast cancer risk associated with CHEK2*1100delC is modified by other genetic factors in a multiplicative fashion 59-61. Such a multi-gene model might also be involved in childhood cancer.

32 Case #28 General information Gender Female Malignancy Precursor B-cell acute lymphoblastic leukemia at age 1.5 years Inclusion category Category 3 - Family history Testing before study Not performed

Additional information This girl was born after an uncomplicated pregnancy and delivery. She was diagnosed with asthma and atopic dermatitis. Her cousin, also a girl born after an uncomplicated pregnancy and delivery, developed acute myeloid leukemia at age 1.5 years. Both girls have a normal development and no dysmorphic features.

Supplementary Figure S12 – Family tree of case #28

Results of whole exome sequencing WES was performed on germline DNA of both affected cousins. We focused on shared variants between the cousins and whole exome analysis revealed a variant in TUBB1 (p.Tyr51fs) in both girls. TUBB1 is the major beta-tubulin expressed in platelets and megakaryocytes and is required for optimal platelet assembly. Heterozygous mutations in this gene cause TUBB1-related macrothrombocytopenia 62. No patients with germline mutations in TUBB1 and cancer have been described. However, several other genes are described to cause both platelet abnormalities in addition to an increased risk of cancer, such as ETV6 and RUNX1 63-65. Laboratory analysis showed no signs of macrothrombocytopenia in these cousins however. Case #29 General information Gender Male Malignancy Neuroblastoma at age 9 months Inclusion category Category 3 - Family history Testing before study Not performed

33 Additional information This boy was born after an uncomplicated pregnancy and delivery. At the age of 9 months, he was diagnosed with neuroblastoma (without NMYC amplification). He is included in this study because his father was diagnosed with acute lymphoblastic leukemia at age 3. After inclusion in the study a paternal cousin developed Ewing sarcoma at age 12.

Supplementary Figure S13 – Family tree of case #29

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. We focused on shared variants between the index patient and his affected father, but no possibly causative variants were found. Case #30 General information Gender Male Malignancy Hodgkin lymphoma at age 17, nodular sclerosing subtype Inclusion category Category 1 - ID and/or congenital anomalies Testing before study Not performed

Additional information This boy was born after an uncomplicated pregnancy and delivery. After birth, a hypospadia was diagnosed for which he needed surgical correction. He had a normal psychomotor development and a normal intelligence. He has grade 1 spondylolysis of his L5 and S1 vertebrae. He has no dysmorphic features. He has a large hyperpigmented lesion on the left side of his trunk and back. The border is irregular and there are several darker macules within the hyperpigmented region.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis.

34 Case #31 General information Gender Male Malignancy High grade glioblastoma (without BRAF mutation) at age 10 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study SNP array, mitochondrial DNA analysis and MLPA of the telomeric regions were without abnormalities. Analysis of the OXPHOS-genes showed a NDUFA1 missense variant of which the clinical relevance is unknown.

Additional information This boy was born after a complicated delivery. He was in breech position and the umbilical cord was wrapped around his neck three times. Therefore, a caesarian section was performed. He was born with an imperforate anus for which he needed surgery. In the first months of his life, it became apparent that he had a delayed psychomotor development. He also had a strabismus, epilepsy and lactate acidosis. A muscle biopsy showed a mitochondrial complex I and III deficiency. A clinical diagnosis of Leigh syndrome was made based on his problems in combination with an abnormal MRI (symmetrical thalamic hyperintensities), but this could not be genetically confirmed. He has no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis. Case #32 General information Gender Male Malignancy Multiple basal cell carcinomas (>10) from age 18 onwards Inclusion category Category 2 – Cancer twice and Category 4 - Adult tumor as a child Testing before study PTCH-, SUFU and BAP1 analysis showed no abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a normal psychomotor development, a normal intelligence and no behavioral problems. He has no dysmorphic features, in particular no macrocephaly, palmar pits or frontal bossing. At the age of 18 years, he was diagnosed with a superficial basal cell carcinoma on his left shoulder. In the following year, he developed 5 more basal cell carcinomas. By the age of 22, he had developed 19 basal cell carcinomas in total and one MBAIT (melanocytic BAP1-associated intradermal tumor). This MBAIT showed loss of BAP1, but no germline BAP1 mutation was found.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Multiple interesting variants were identified.

1. With whole exome analysis we identified a previously reported pathogenic mutation in the SPRED1 gene (p.Arg16*) 66. This mutation was paternally inherited. Germline loss-of-function mutations in this gene cause Legius syndrome [OMIM: 611431], which is characterized by multiple café-au-lait macules, intertriginous freckling, lipomas, macrocephaly, learning disabilities and ADHD 66. These features are absent in both our patient and his father. A thorough review of the literature revealed one other example of non-penetrance of a presumed pathologic SPRED1 variant in a 60-year-old male with no café-au-lait macules or freckling 67. It has been suggested that individuals with Legius syndrome are at an increased risk for leukemia 66,68, but associations with other malignancies so far

35 have not been made. Professor Legius is not aware of patients with Legius syndrome who have developed BCCs (personal communication). Sequencing of the c.46C>T (p.(Arg16*) variant in tumor derived DNA of our proband excluded loss of the wild type SPRED1 allele in one of the BCCs of the patient.

2.This patient also carried a variant in LATS1 (p.Arg243Lys), which was paternally inherited. LATS1 is a known tumor-suppressor kinase involved in the Hippo/YAP signaling pathway and plays a role in restraining cell proliferation and promoting apoptosis. Recently, deleterious LATS1 mutations were recurrently found in BCCs 69,70. A role for LATS1 in the pathogenesis of BCC is further supported by the report of biallelic inactivation of LATS1 in an infiltrative BCC of a patient with Basal cell nevus syndrome [OMIM: 109400] 71. Using Sanger sequencing, we did not find indications for loss of the wild type allele in LATS1 in DNA derived from a BCC in this patient. Case #33 General information Gender Female Malignancy Acute myeloid leukemia at age 13 (FLT3-ITD and NPM1 mutation) Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP array showed no abnormalities

Additional information This girl was born after an uncomplicated pregnancy and delivery by cesarean section. She was born with congenital hip dysplasia and congenital hypothyroidism due to an ectopic thyroid gland. She has a mild intellectual disability. She has a mild proptosis, large ears and short and broad distal phalanges of both thumbs. Her mother has a mild intellectual disability as well in addition to hearing loss.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and her parents. Multiple possibly pathogenic variants were identified. Whole exome sequencing additionally was performed on tumor DNA, revealing no loss of heterozygosity or second hit mutations in any of the candidate genes.

1. Cancer gene panel analysis revealed a de novo change of unknown significance in BAP1 (p.Ile706Val). Mutations in this gene are correlated with predisposition to uveal and skin melanoma, mesothelioma and renal cancer 72,73. No patients with AML have been described.

2. Cancer gene panel analysis showed a paternally inherited mutation in CHEK2 (c.1100delC, p.Thr367Metfs*15). CHEK2* 1100delC was first reported in Li-Fraumeni-like families 53 and the mutation is now an established breast cancer susceptibility allele 54,55. In the Netherlands, the CHEK2*1100delC is common with a carrier frequency of 1.2% (95% CI, 0.7% to 1.8%) 56. Indications are available that CHEK2*1100delC is associated with increased risk for other cancers in addition to breast cancer 57,58. To our knowledge, no studies have been performed to specifically test the association between this variant and childhood cancer risk. Epidemiologic and family studies have suggested that the breast cancer risk associated with CHEK2*1100delC is modified by other genetic factors in a multiplicative fashion 59-61. Such a multi-gene model might also be involved in childhood cancer.

3. Whole exome analysis revealed a maternally inherited variant in KDM3B (p.Glu93*). KDM3B is a histone H3K9 demethylase involved in histone modification and chromatin remodeling 51,52. Both somatic and germline mutations in chromatin remodeling genes are associated with an increased risk of cancer and ID has also been linked to germline mutations in chromatin remodeling genes. The finding of mutations in KDM3B in two patients (the other is Case #25) with a similar phenotype

36 suggests a possible role in the development of both ID and cancer in these two patients. Functional studies are being performed to identify this role. Case #34 General information Gender Female Malignancy Medulloblastoma at age 8, polyposis and pancreatoblastoma Inclusion category Category 2 - Cancer twice and Category 3 - Family history Testing before study APC testing showed a pathogenic mutation (p.Ile1060fs)

Additional information This girl was born after an uncomplicated pregnancy and delivery. She developed medulloblastoma at the age of 8. Her half-sister also developed a medulloblastoma at 8 years of age. APC testing was performed and a pathogenic mutation was found in the index patient, but not in her half-sister. Both have a normal development and no dysmorphic features.

Supplementary Figure S14 – Family tree of case #34

Results of whole exome sequencing WES was performed on germline DNA of both affected half-siblings. We focused on the variants shared between the siblings. In the index patient, a previously identified mutation in APC was confirmed (p.Ile1060fs). This mutation was absent in both her half-sister and her parents, confirming a de novo occurrence of the mutation. No additional likely causative mutations were found by whole exome analysis. Mutations in APC cause familial adenomatous polyposis, a syndrome characterized by a predisposition to colon cancer [OMIM: 175100]. Mutations in APC are also known to cause brain tumors 74, specifically medulloblastoma. The phenotype of the index patient, including the pancreaticoblastoma, is explained by this mutation. We do not have an explanation for the medulloblastoma of the half-sister in this family, since she did not carry the APC mutation. It is possible that her cancer is attributable to the population cancer risk.

37 Case #35 General information Gender Male Malignancy Hepatocellular carcinoma at age 5 Inclusion category Category 4 - Adult tumor as a child Testing before study A mitomycin-C test was performed to exclude Fanconi anemia.

Additional information This boy was born after an uncomplicated pregnancy and delivery. He had a normal development, no medical history and no dysmorphic features.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Multiple interesting variants were identified.

1. With the cancer gene panel analysis a de novo missense variant in the BUB1 gene was found (p.Val991Ile). BUB1 encodes a protein involved in the spindle assembly checkpoint 75. We have demonstrated that heterozygous mutations in this gene can lead to mosaic aneuploidies in different tissues of patients with early-onset colorectal cancer 75, but a role of this variant in childhood cancer initiation has not yet been described. We tested blood samples for aneuploidies, but no excess of aneuploidies has been found in this boy (2%). Furthermore we looked for premature chromatid separation, which was not found.

2. With whole exome analysis a paternally inherited mutation in APOB was identified (p.Tyr981*). Mutations in APOB cause familial hypobetalipoproteinemia (FHBL), which is an autosomal codominant disorder characterized by hypocholesterolemia due to lost capacity to form plasma lipoproteins in liver and intestine and to export lipids from these organs 76,77. Both the boy and his father indeed have low to normal cholesterol levels, suggesting an effect of this truncating mutation. In FHBL, fatty liver is a characteristic feature, and there are several reports of associated cirrhosis and hepatocellular carcinoma 77-79, which may point toward a genetic susceptibility for cancer in APOB mutation carriers. No signs of fatty liver nor cirrhosis were observed in liver tissue of our patient, so a direct correlation between his hepatocellular carcinoma and the germline APOB mutation seems unlikely. However, one other patient with FHBL attributable to an APOB mutation and hepatocellular carcinoma without cirrhosis has been reported 79. The liver histology in this patient revealed a moderate degree of steatosis and fibrosis outside the hepatocarcinoma lesion. Case #36 General information Gender Female Malignancy Acute lymphoblastic leukemia at age 5 Inclusion category Category 3 - Family history Testing before study Not performed

Additional information This girl was born after an uncomplicated pregnancy and delivery. She was diagnosed with acute lymphoblastic leukemia at age 5. Her brother was diagnosed with acute lymphoblastic leukemia as well, at the age of 3 years.

38

Supplementary Figure S15 – Family tree of case #36

Results of whole exome sequencing WES was performed on germline DNA of both affected siblings and their parents. Multiple interesting variants were identified.

1. With cancer gene panel analysis we found that the girl harbored a de novo variant in ETV6 (p.R433C), which was not shared by her brother. ETV6 is a transcription factor required for hematopoiesis and is frequently somatically affected by translocations and copy number aberrations in ALL 80,81. In addition, germline mutations have been associated with thrombocytopenia and leukemia development 63,64,82. In the study of Moriyama et al 63, a germline variant at the same location (p.R433H) was found in a cohort of leukemia patients, though no functional testing was performed. The prediction programs we used all indicate this variant as pathogenic suggesting that this variant might have played a role in the leukemia development in this girl. Intriguingly, her brother did not share this variant, suggesting that yet other genes may contribute to leukemia susceptibility in this family or that the leukemia in de sibling was a sporadic leukemia.

2. With whole exome analysis we found that both siblings shared two variants in autism susceptibility candidate 2 (AUTS2; p.Glu204Gly and p.Arg986Gln) in a compound heterozygous state (i.e. inherited from each of the parents). AUTS2 is involved in neurological and early lymphoid development. Mutations have been associated with autism spectrum disorders 83 and copy number aberrations and mutations are found in an ID syndrome called the AUTS2-syndrome 84. PAX5-AUTS2 fusion genes are described in B-lineage ALL 85,86 and increased expression of AUTS2 is reported in T-lineage ALL 87. AUTS2 binds the polycomb repressive complex 1 (PRC1). This epigenetic regulator, like the name indicates, has a role in gene repression. However, after binding of AUTS2 this role is reverted to gene activation by recruitment of EP300, another well-known player in hematopoiesis and leukemogenesis 86-89. It is unclear whether the variants found in our study are activating or loss-of-function variants. Case #37 General information Gender Male Malignancy Burkitt type Non-Hodgkin lymphoma at age 3 Inclusion category Category 1 - ID and/or congenital anomalies Testing before study 250k SNP array and NSD1 analysis without abnormalities

39 Additional information This boy, 18 years old at time of inclusion, was born after an uncomplicated pregnancy and delivery. At the age of 3 weeks he had a bacterial sepsis. His umbilical cord fell off late at the age of 6 weeks. He had a normal psychomotor development. He had recurrent upper respiratory tract and ear infections. Laboratory investigation showed a low IgG and IgA. He has a horseshoe kidney on the right side. He has a tall stature, his height followed the +2.5SD line and he had a bone age of 1.5 years ahead of his biological age. He has some dysmorphic features; a high forehead, upslanted palpebral fissures, a white hair wisp above his left ear, a partial syndactyly of his 2nd and 3rd toes on both sides and a mild clinodactyly of his toes. He was diagnosed with Attention Deficit Disorder for which he receives medication.

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Whole exome analysis revealed a de novo missense mutation in EZH2 (p.Ala682Thr). Mutations in EZH2 cause Weaver syndrome [OMIM: 277590] 90, which is in accordance with his clinical phenotype. EZH2, a histone methyl transferase subunit of a Polycomb repressor complex, is recurrently mutated in several forms of cancer and is highly expressed in numerous others. Both gain-of-function and loss-of-function mutations occur in cancers, but are associated with distinct cancer types 91. Multiple patients with Weaver syndrome who developed a malignancy have been described 92-94. In a paper reporting 48 patients with Weaver syndrome, three patients developed cancer 93. One of these patients who developed leukemia and neuroblastoma carried the same mutation as our patient. The other two patients developed a pre-T-cell non-Hodgkin lymphoma at age 13 years and a neuroblastoma at age 4 years. Recently, a patient with Weaver syndrome and acute myeloid leukemia in adolescence has been reported 94. Case #38 General information Gender Female Malignancy Burkitt type non-Hodgkin lymphoma at age 13 Inclusion category Category 2 - Cancer twice Testing before study BRCA1 analysis no abnormalities (tested because of a known mutation in the family)

Additional information This girl was born after an uncomplicated pregnancy and delivery. She had a normal psychomotor development and no dysmorphic features. At the age of 13, she was diagnosed with a Burkitt type Non-Hodgkin lymphoma stage IV with localizations in the mediastinum, supraclavicular and with bone marrow involvement. On a PET-CT scan made for staging, multiple hepatic cysts were seen. At the age of 44, she developed a melanoma (not in the irradiated area), which was surgically removed completely. One year later at the age of 45, she was diagnosed with a hemangiopericytoma or solitary fibrous tumor in the left temporal lobe. From the ages 44 to 47, she had multiple duodenal tubular adenomas, one with high grade dysplasia.

Results of whole exome sequencing WES was performed on germline DNA of the index patient. No parental DNA was available. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis.

40 Case #39 General information Gender Female Malignancy Neuroblastoma at age 2 Inclusion category Category 3 - Family history Testing before study PHOX2B, KRAS, NRAS and BRAF no abnormalities

Additional information This girl was born after an uncomplicated pregnancy and delivery. She had a normal development and some dysmorphic features: frontal bossing, deep set eyes and a slight downslant. In her maternal family multiple children developed cancer: a cousin developed neuroblastoma at the age of 2.5 years and another cousin was diagnosed with a Wilms tumor at age 3.5 years. This boy had a slow development and some mild dysmorphisms, such as frontal bossing, downslanted palpebral fissures and open mouth behavior.

Supplementary Figure S16 – Family tree of case #39

Results of whole exome sequencing WES was performed on germline DNA of all three patients with childhood cancer. We focused on shared variants. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis.

41 Case #40 General information Gender Male Malignancy Cerebral tumor, suspected low-grade glioma (no biopsy taken) at age of 14 months Inclusion category Category 1 - ID and/or congenital anomalies Testing before study RASPN, CHRNE and MED16 analysis showed no abnormalities

Additional information This boy was born after an uncomplicated pregnancy and delivery. Since the age of 3 months he had a convergent strabismus was noted and he had a delayed motor development. At the age of 14 months, a cerebral tumor was found. No biopsies were taken, but it is suspected to be a low grade glioma. He has a congenital ptosis and he has some dysmorphic features: high arched eyebrows, broad nose, long philtrum and low-set and large ears. He has a short stature (-2.8SD).

Results of whole exome sequencing WES was performed on germline DNA of the index patient and his parents. Likely causative mutations were not found in the cancer gene panel nor with whole exome analysis.

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