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Whole-exome sequencing identifies germline mutation in TP53 and ATRX in a child with ge- nomically aberrant AT/RT and her mother with anaplastic astrocytoma

Kristiina Nordfors1,2, Joonas Haapasalo3, Ebrahim Afyounian4, Joonas Tuominen4, Matti Annala4, Ser- gei Häyrynen4, Ritva Karhu5, Pauli Helén3, Olli Lohi1,2, Matti Nykter4,6, Hannu Haapasalo7, Kirsi J. Granberg4,6

Correspondence: Kristiina Nordfors Department of Pediatrics Tampere University Hospital Teiskontie 35, FI-33720 Tampere, Finland [email protected]

Short running title: Exome sequencing of AT/RT and astrocytoma cases from the same family.

Affiliations: 1 Department of Pediatrics, Tampere University Hospital, FI-33521 Tampere, Finland; 2 Tampere Cen- ter for Child Health Research, University of Tampere, FI-33014 Tampere, Finland; 3 Unit of Neurosur- gery, Tampere University Hospital, FI-33521 Tampere, Finland; 4 BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, FI-33520 Tampere, Finland; 5 Laboratory of Can- cer Genetics, University of Tampere and Tampere University Hospital, FI-33521 Tampere, Finland; 6 Science Center, Tampere University Hospital, FI-33521 Tampere, Finland; 7 Fimlab Laboratories Ltd., Tampere University Hospital, FI-33520 Tampere, Finland

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Abstract Brain tumors typically arise sporadically and do not affect several family members simultaneously. In the present study, we describe clinical and genetic data from two patients, a mother and her daughter, with familial brain tumors. Exome sequencing revealed a germline missense mutation in the TP53 and ATRX in both cases, and a somatic copy-neutral loss of heterozygosity (LOH) in TP53 in both atypical teratoid/rhabdoid tumor (AT/RT) and astrocytoma tumors. ATRX mutation was associated with the loss of ATRX expression. In the astrocytoma case, R132C missense mutation was found in the known hotspot site in isocitrate dehydrogenase 1 (IDH1) and loss of heterozygosity (LOH) was detected in TP53. The mother carried few other somatic alterations, suggesting that the IDH1 mutation and LOH in TP53 were sufficient to drive tumor development. The genome in the AT/RT tumor was atypically aneuploid: Most had experienced copy-neutral LOH or whole- gains. Only Chromosome 18 had normal diploid status. INI1/hSNF5/SMARCB1 was homozygously deleted in the AT/RT tumor. This report provides further information about tumor development in a predisposed genetic background and describes two special Li-Fraumeni cases with a familial brain tumor.

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INTRODUCTION An atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant embryonal tumor of the brain occur- ring most often in infants. Regardless of intensive therapy, the outcome of the patients remains poor: the 5-yr overall survival rate is ~30% (Fischer-Valuck et al. 2016). A mutation or deletion in the INI1/hSNF5/SMARCB1 occurs in the majority of AT/RT tumors, and this genetic alteration is the key finding in the diagnostic procedure (Louis et al. 2016). Recently, Johann et al. (2016) reported the existence of three epigenetic AT/RT subgroups. In this study, whole-genome DNA and RNA sequenc- ing did not reveal any recurrent genomic aberrations beyond biallelic deletion of INI1. Thus, different genetic abnormalities were not able to explain the differences between the AT/RT subgroups (Johann et al. 2016). Diffusely infiltrating astrocytomas are the most common primary brain tumors in adult populations. They are subdivided into low-grade diffuse astrocytomas (Grade II), anaplastic astrocytomas (Grade III), and glioblastomas (Grade IV). Anaplastic astrocytomas are the rarest type of diffusely infiltrating astrocytoma (Louis et al. 2016). The standard treatment modality for diffusely infiltrating astrocytomas is surgery combined with radiation and/or chemotherapy, but tumors acquire resistance rapidly. Muta- tions in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in >95% of Grade II-III gliomas and are associated with better patient prognosis than in IDH wild-type tumors (Louis et al. 2016). Howev- er, the 5-yr survival rate of IDH-mutated anaplastic astrocytomas is ~ 55-60% (Yan et al. 2009;Haapasalo et al. 2014). Most brain tumors are sporadic and occur in patients with no known predisposing germline variants. Several genetic variants are known to cause predisposition to brain tumors, but these variants also increase the risk of other tumor types, and are rare in the population. Such variants include hemizygous alterations of the neurofibromatosis 1 (NF1) (Gutmann et al. 2003), TP53 (Bougeard et al. 2015), as well as alterations in multiple DNA repair genes, such as mismatch repair (MMR) genes (Wei et al. 1997), X-ray cross-complementary genes (XRCC) (Kiuru et al. 2008), and O6- methylguanine-DNA methyltransferase (MGMT) genes (Felini et al. 2007). Germline mutations of the TP53 on Chromosome 17p13.1 cause Li-Fraumeni syndrome (Malkin et al. 1990). The TP53 gene encodes a transcription factor (p53) that normally regulates the and prevents damaged cells from dividing and forming tumors (Malkin et al. 1990). The Li-Fraumeni muta- tions can be inherited, or they can be de novo mutations arising early during embryogenesis or the development of germ cells in the parent. This syndrome has been shown to be associated with malig- nancies in a variety of tissue types (Iavarone et al. 1992; Sidransky et al. 1992; Nichols et al. 2001; Malkin 2011; Sorrell et al. 2013). Brain tumors, such as astrocytic tumors are possible (Guha and Malkin 2017), but there are no previous reports on AT/RT with Li-Fraumeni syndrome. Here, we re- port the exome sequences and detailed clinical history from two patients from the same family that have Li-Fraumeni syndrome. An anaplastic astrocytoma was diagnosed from the mother, whereas the child suffered from a rare AT/RT with complex copy-number alterations, including homozygous loss of INI1/hSNF5/SMARCB1 locus.

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RESULTS Clinical Presentation and Family History The first case is a 2-yr-old girl, born at a gestational age of 33 wk with a birth weight of 1.8 kg. She exhibited slow motor development and left side weakness after birth and received regular physiother- apy for the first months of her life. The first symptom was asymmetrical convulsion during a fever at the age of 2. Two weeks later, the girl suffered from right-sided weakness, irritation, and tiredness in the mornings. Central nervous system (CNS) magnetic resonance imaging (MRI) revealed a cerebral, cystic, large tumor, but there were no signs of obstruction of the cerebrospinal fluid (CSF) flow (Fig. 1A,B). A left-sided craniotomy was performed under general anesthesia, and a microneurosurgical radical resection was performed. A postoperative cranial MRI (1 wk later) showed that most of the tumor was resected, and there was a 2-cm wide residual tumor in the anterior border of the resection cavity, which was resected. There were no new neurological deficiencies after the procedure. Neuro- pathological diagnosis was highly malignant AT/RT. The cellular marker for proliferation MIB-1, an antibody against Ki-67 antigen, index was 20%, immunohistochemical INI1 staining was negative (i.e. pathological) (Fig. 1C).The patient was treated with induction chemotherapy (methotrexate, vincris- tine, etoposide, cyclophosphamide, and cisplatin) followed by three cycles of high-dose chemotherapy (thiotepa, carboplatin) with autologous stem cell transplantation. New cranial and spinal MRI was performed 3 mo after the surgeries, but they did not reveal any evident residual or new tumors. Nine months after diagnosis, a control cranial MRI revealed three recurrences in the upper medial and parasagittal region of the resection cavity (Fig. 1A). A recraniotomy and microneurosurgical tumor resection was performed under general anesthesia (Supplemental Fig. 1). A postoperative MRI showed a small residual tumor with contrast enhancement, and an immediate third tumor resection was performed. Local radiotherapy was given 50,4/1,8 Gy after the third surgery. The patient suffered from headaches and nausea, and two new extra-axial occipital tumors appeared a month after radia- tion as seen in the MRI. The patient received palliative care and died a little more than 1 yr after the diagnosis. The second case (mother) was ~30 yr old and was previously considered a healthy woman. Her mother had been diagnosed with carcinoma of the uterus in her 40s. The case had previously suf- fered from an episode of seizures years earlier, and valproic acid was initiated as an anticonvulsant medication. Two years later, she had another episode of symmetrical convulsions. An MRI of the CNS revealed a left-sided, frontal, cystic tumor, which was edemic and compressed the left lateral ventri- cle, but there were no signs of obstruction of the CNS fluid flow (Fig. 1D). Craniotomy with microneu- rosurgical radical resection was performed. Histopathological diagnosis was diffusively infiltrating astrocytoma Grade III (i.e., anaplastic astrocytoma). The tumor was weakly positive in IDH1 R132H- immunostaining (Fig. 1E) and the MIB-1 proliferation index was 5%. The mother received postopera- tive radiotherapy to tumor bed 59,4/1,8 Gy. Six control cranial MRIs during the 3-yr follow-up revealed no residuals or recidives. Six years after diagnosis, the patient was asymptomatic. As the child was diagnosed with AT/RT, genetic analyses were done due to family history. The results showed a germline TP53 mutation in both cases, which is known to cause Li-Fraumeni syndrome.

Predisposing Germline Variants Exome sequencing data were generated from blood-derived DNA samples from both patients (mother and child) to characterize additional predisposing germline mutations. As already observed with Sanger sequencing, both patients had a germline nonsynonymous single-nucleotide variant (SNV) in the TP53 gene (Table 1; Fig. 2A; Supplemental Table 1). It was a G>A substitution in the coding strand, leading to glycine>serine substitution (exon7:c.G733A, p.G245S in the transcript NM_000546). This variant has been previously linked to Li-Fraumeni, at least in sarcoma patients (Toguchida et al. 1992), and it is one of the most frequently reported TP53 missense mutations (7.4% of all) in the COSMIC database (http://cancer.sanger.ac.uk/cosmic). Quite surprisingly, both cases carried an ATRX SNV in their germline (Table 1; Fig. 2B; Supplemental Table 1). This was an A>G conversion in the coding strand, leading to asparagine>serine conversion in the amino acid sequence (exon23:c.A5579G, p.N1860S in the transcript NM_000489). The variant has been linked to X-linked α-thalassemia/mental retardation syndrome (Gibbons et al. 1995). The allele frequency of the A>G variant is 0.011 in the Finnish cohort in the ExAC database (Supple- mental Table 1) (http://exac.broadinstitute.org/). Both tumors were ATRX-negative in the immuno- Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

histochemistry analysis (Fig. 1C,E).

Somatic Alterations Tumor samples from both mother and child were analyzed with exome sequencing to reveal genetic alterations involved in tumor development. One freshly frozen sample was obtained from the mother during the first surgery and two tumor samples were obtained from the child: FFPE sample from the primary surgery and a freshly frozen sample from a relapse taken at 9 mo after initial diagnosis. Tu- mor purities were very high (>90%) in the child’s tumor samples, whereas the predicted purity was <60% in the mother’s tumor sample. The mother had an R132C somatic IDH1 mutation, which is the second most common IDH mutation in diffuse gliomas (Supplemental Table 2; Hartmann et al. 2009). Allele fraction for this mutation was only 0.24, suggesting also rather low (~50%) tumor purity. Additional missense mutations were de- tected in six other genes in the mother’s tumor, but none of them have been shown to have any on- cogenic role in diffuse gliomas. The proportion of the TP53 germline variant reads was increased in the mother’s tumor sample (Fig. 2A; Supplemental Table 1), suggesting copy-neutral loss of the wild- type variant, which is commonly observed in Grade II-III diffuse gliomas (Hattori et al. 2016). This was indeed the case according to the copy-number analysis that utilized both sequence coverage and heterozygous allele frequencies in the germline (Fig. 3A, Supplemental Fig. 2). In addition to loss of heterozygosity (LOH) in TP53 and the surrounding region in 17p13, only a hemizygously deleted re- gion was observed in the beginning of Chromosome X (Fig. 3A, Supplemental Fig. 2). No deletions in 1p or 19q were detected. The daughter’s tumor exhibited biallelic loss of INI1/hSNF5/SMARCB1 on Chromosome 22 (Fig. 3A,B), supported by negative INI1 staining (Fig. 1C). Biallelic loss of INI1 is the only known recurrent alteration in AT/RT and is used as major criteria for AT/RT diagnosis (Louis et al. 2016). Surprisingly, the child’s tumor harbored several additional copy-number alterations. Most of her tumor genome was occupied by whole-chromosome losses and/or gains (Fig. 3A; Supplemental Fig. 2). Copy-neutral LOH was observed in Chromosomes 3-6, 8-17, 19, 22, and X with a few exceptions: In the sample taken at diagnosis, Chr 12 was tetraploid, and there was a subclonal population of cells with tetraploid Chr 11. Furthermore, there was a third copy gain in the end 4q in the sample taken upon relapse. Both samples showed focal biallelic loss of INI1 locus. In addition to LOH regions, Chromosomes 1, 2, 7, 20, and 21 were either tetraploid or there was a combination of subclonal tetraploid and triploid chromosome counts. In the FFPE sample taken at diagnosis, the proportion of tetraploid subclone was ~30% for all the other chromosomes, except Chr 11, where the tetraploid proportion was ~20% of the tumor content. In the relapse sample, the proportion of tetraploid subclone was significantly small- er: ~3% of the tumor content. Surprisingly, Chr 18 was the only chromosome with a normal diploid chromosome count in the child’s tumors, which clearly differs from the typical AT/RT tumors that lack copy-number alterations outside Chr 22 (Louis et al. 2016). In addition, 13 chromosomes (Chromo- somes 3, 4, 6, 8, 9, 10, 12, 13, 15, 17, 19, 22, and X) of 17 chromosomes with LOH were maternal; only Chromosomes 5, 11, 14, and 16 with LOH were inherited from the father (Supplemental Table 1). Furthermore, the child had 28 nonsynonymous mutations in her tumors: nine mutations were present in both tumor specimens, one only in the primary tumor, and 18 only in the recurrent tumor (Supple- mental Table 2). None of the mutations had existing COSMIC IDs. Our literature search revealed two genes, namely DOK3 and SH3GLB2, which were mutated in both AT/RT tumors and have been previously linked to tumor suppression in and breast cancer, re- spectively (Berger et al. 2010; Zhang et al. 2011). Furthermore, two putative tumor suppressor genes, NEK4 and KLF14, were mutated only in the recurrent AT/RT sample. NEK4 has been reported to regulate the entry into replicative senescence and DNA-damage-induced cell cycle arrest in fibro- blasts (Nguyen et al. 2012), whereas KLF14 inhibits KRAS signaling and has demonstrated tumor suppressor activity (Tetreault et al. 2013; Fan et al. 2015).

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DISCUSSION Our results show that the mother and the child had a germline nonsynonymous SNV in the gene TP53. In addition, they both carried an ATRX SNV in their germline. As suspected, the mother had a somatic IDH1 mutation R132C and the daughter’s tumor exhibited biallelic loss of INI1/hSNF5/SMARCB1. The transcriptional regulator ATRX (α-thalassemia/mental retardation syndrome X-linked) protein is encoded by the ATRX gene, located in Xq21.1. ATRX contains an ATPase/helicase domain, and it belongs to the SWI/SNF family of remodeling (Gibbons et al. 2000). ATRX directly interacts with DNA as well as with several functional partners, presumably by assembling with differ- ent chromatin remodeling or transcriptional regulatory complexes. In addition, reduced ATRX levels induce chromosome segregation defects (Mayes et al. 2014). Similarly, INI1 is a member of the SWI/SNF family and plays a role as a tumor suppressor in malignant rhabdoid tumors (Versteege et al. 1998). Furthermore, alternative lengthening of telomeres (ALT) is a mechanism by which ~10%- 15% of cancer cells can extend their telomeres without the enzyme telomerase. Loss of ATRX/DAXX function appears to be required for ALT (Amorim et al. 2016). Approximately 10%-15% of human can- cers are suspected to use the ALT pathway, including a subset of nervous system tumors (e.g. me- dulloblastoma, low-grade diffuse gliomas, and ganglioneuroblastoma) (Lovejoy et al. 2012). By lengthening the telomere, ALT contributes to the unrestricted cell division that is a hallmark of cancer and likely makes cancer cells less vulnerable to chemotherapy or radiation therapy. These ALT- positive tumors use recombination to sustain immortality (Dilley and Greenberg 2015). Recent find- ings suggest that hampering ALT might be a potential new cancer treatment modality. ALT is disrupt- ed by the inhibition of protein kinase ATR leading to cell apoptosis (Lovejoy et al. 2012). ATRX is located in Chromosome X, and thus one of its alleles is epigenetically silenced. Both our patients had ATRX variants in their germline. There are discordant reports about the pathogenicity of the detected ATRX variant in ClinVar, and the variant frequency was 1.1% in a Finnish cohort, which also suggests a lower pathogenicity. However, this variant was associated with the loss of ATRX protein expression in both tumors. In addition, ATRX staining was negative in the endovascular cells in the mother and in all the cells surrounding the blood vessels in the child, suggesting that the normal ATRX allele has been imprinted in these cells. Other germline mutations that are not covered by exome sequencing might also be responsible for the ATRX loss in the variant-containing locus. Nevertheless, our data shows that ATRX loss or defect can be present also in nonmalignant cells, which might lead to unde- sired outcome after ATR inhibitor based treatments. Germline status and ATRX protein detection will be informative for ATRX-mutated cases if these treatment modalities become part of the clinical prac- tice in the future. The IDH mutation is frequently observed with TP53 and ATRX mutations in astrocytic tumors (Cai et al. 2014). IDH mutation is widely used as a clinical tool to sort glial tumors (Louis et al. 2016), and patients with IDH mutation have better outcomes than IDH wild-type diffuse glioma patients (Ichimura 2012). The mother carried a somatic IDH R132C mutation in her tumor. Antibodies raised against IDH1 R132H mutation gave a weakly positive signal in immunohistochemistry, which suggest cross- reactivity against R132C mutation. The mother’s outcome remains stable after 6 yr, which is better than the average prognosis. The lack of other oncogenic alterations is somewhat surprising, but as both TP53 and ATRX harbored a pathogenic variant already in the germline, the IDH1 hotspot muta- tion might have been enough to trigger the development of a diffuse glioma. These three genes are the most commonly altered genes in 1p19q nondeleted IDH-mutated diffuse gliomas, such as ana- plastic astrocytoma (Cancer Genome Atlas Research Network et al. 2015). The complexity of copy number changes observed in the child’s tumor cells is striking. How could this kind of genome have arisen? Most likely there was a loss of one pair for most chromosomes, followed by genome-wide duplication of chromosome counts. The loss of one chromosome to generate triploid counts could have happened soon after in Chr 2 and Chr 20, and possibly in Chr 1p. The fact that there was a combination of both tetraploid and hemizygously deleted diploid Chr 11 chromosome counts in the child’s first sample (child FFPE) suggest that the observed gains and losses took place sequentially, rather than simultaneously in all the chromosomes. The hemizygous loss of Chr 17 has generated a homozygous missense mutation in TP53, which has likely damaged the p53-dependent cell cycle checkpoint mechanisms (Vaziri et al. 2003). The fact that homozygous loss in Chr 22 has similar boundaries in both chromosomes suggests that the INI1 locus was fully deleted before the suggested genome-wide gain in chromosome counts. It is difficult to determine whether Chr 18 has remained diploid throughout all these genome rearrangements or whether one copy of both maternal Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

and paternal chromosomes has been deleted after a tetraploid stage. Li-Fraumeni is known to predispose to cancer but the question remains as to why both the mother and the child developed a brain tumor. It seems that the diffuse gliomas need other genetic alterations, such as IDH and ATRX mutations, for their development. The mother obtained somatic IDH1 mutation, and she carried an ATRX mutation in her germline. In AT/RT, the literature has re- vealed only one recurrent genetic mutation-namely, homozygous loss of INI1, which was also ob- served in the tumor of the child. To our knowledge, this is the first study to show that Li-Fraumeni syndrome with accompanying so- matic LOH in a patient with AT/RT. This highlights the fact that even in rare brain tumors, the anam- nesis of family history is essential, and modern genetic analyses of tumors and heredity should be performed.

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METHODS Immunohistochemical Analysis and Pathological Evaluation The tumor samples were fixed in 4% phosphate-buffered formaldehyde and processed into paraffin blocks. The sections were stained with hematoxylin and eosin (H&E). An experienced neuropa- thologist evaluated the tumor samples and determined the histopathological type and grade according to the criteria presented by the World Health Organization (WHO) (Louis et al. 2007). The tumor im- munostaining for IDH1 was performed using R132H point mutation-specific mouse monoclonal anti- body (Dianova GmbH) as previously described (Mäkelä et al. 2014). ATRX and INI1 immunostainings were performed using Ventana Benchmark GX immunoautomate (Ventana Medical Systems). DIS- COVERY EZ Prep solution (# 950-102, Ventana Medical Systems) was used for deparaffinization (72°C). Epitope retrieval was performed at 95°C in Cell Conditioning Solution CC1 (# 950-124), for 92 min for ATRX detection and 32 min for INI1 detection. Antibody incubation duration was 60 min with a rabbit polyclonal anti-ATRX antibody (dilution 1:500, HPA0001906, Atlas antibodies) and 28 min with a mouse monoclonal antibody anti-INI-1 (dilution 1:200, Clone: 25/BAF47, 612110, B&D Laborato- ries), both at 36°C. Both immunostainings were detected and visualized using OptiView DAB IHC Detection Kit (# 760-7000, Ventana Medical Systems) together with Optiview Amplification kit (# 760- 099, Ventana Medical Systems). All the washes were performed using Tris-based reaction buffer solution (# 950-300, Ventana Medical Systems). Nuclei were visualized with Hematoxylin II (#790- 2208, Ventana Medical Systems). Exome Sequencing DNA was isolated from the blood samples using a QIAGEN isolation kit, from the frozen samples with a QIAamp DNA Mini kit (QIAGEN) with RNAse treatment, and from the FFPE sample with a turX- TRAC FFPE DNA kit (Covaris). Library construction and exome sequencing were performed at Bei- jing Genomics Institute (BGI), Hong Kong. A SureSelect Human All V5 (target size 50M) kit (Agilent) was used for library preparation, and samples were sequenced with Illumina HiSeq2000 technology. The pursued sequencing depth was 50× for blood-derived samples and 100× for tumor samples (Supplemental Table 3).

Genome Alignment

The raw reads from the whole-exome sequencing experiment for each sample were aligned to the human reference genome (hg19) using Bowtie2 (version 2.2.5) (Langmead and Salzberg 2012) and converted to BAM format using SAMtools (version 0.1.19) (Li et al. 2009). Duplicate reads were marked and removed from the resulting BAM files using SAMBLASTER (version 0.1.21) (Faust and Hall 2014).

Somatic Copy-Number Aberration Analysis and Tumor Purity Estimation

Aligned reads were counted in 1000-bp windows across the entire genome using BEDTools-2.25.0 (Quinlan and Hall 2010). Coverage log ratios were calculated relative to a median reference derived from the two normal blood samples. GC fractions were calculated for all 1000-bp windows, and Loess regression was applied to coverage log ratios to correct for GC content bias. After GC bias correction, samples were corrected for differences in overall coverage using median-of-ratios normalization.

Heterozygous single-nucleotide polymorphisms (SNPs) were identified for each patient by searching their normal blood sample for genomic sites that displayed an allele fraction close to 50%. The alter- nate allele read count r was required to satisfy 0.05 ≤ BinCDF (r; n, 0.5) ≤ 0.95, and the total read count had to be at least 50. The list of heterozygous SNPs was further filtered to only include genomic sites annotated in the KAVIAR (Glusman et al. 2011) or ExAC databases (Lek et al. 2016). Polymor- phic indels and SNPs within 10 bp of indels (both somatic and germline) were discarded from the list of heterozygous SNPs to mitigate alignment artifacts.

Tumor purities were estimated based on the observed allele fractions for the detected heterozygous germline SNPs located in regions with copy-neutral LOH in each tumor sample.

Germline and Somatic Variant Calling

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Variants were called using an inhouse variant calling pipeline based on SAMtools mpileup output (Li et al. 2009). Variants were called heterozygous if, at a given locus, at least 20% of all reads showed the alternate allele and homozygous alternate or if, at a given locus, at least 80% of all reads showed the alternate allele. Annotation for both germline and somatic variants was performed using ANNO- VAR (Wang et al. 2010) in combination with custom scripts. Variants were further annotated with im- pact on protein coding, Cosmic ID (Bamford et al. 2004), and the SNP database (dbSNP) ID (Sherry et al. 2001). Furthermore, annotations included allele frequencies reported in 1000 Genomes (The 1000 Genomes Project Consortium 2010), the National Heart, Lung, and Blood Institute (NHLBI) Ex- ome Sequencing Project (ESP6500, unpublished, http://evs.gs.washington.edu/EVS/), SISu, and ExAC (Lek et al. 2016). To select somatic variants, we discarded the variants that were also detected in the respective germline sample. The rest of the somatic variants were manually inspected to re- move sequencing artifacts.

We discarded germline variants if the population frequency of alternate alleles was >0.01 in either of the three following cohorts: all five populations of 1000 Genomes (The 1000 Genomes Project Con- sortium 2010), the Sequencing Initiative Suomi cohort (SISu, unpublished, http://www.sisuproject.fi/) that consists of exomes of 10,000 Finnish individuals, and all seven populations of the ExAC cohort (Lek et al. 2016). In addition, we devised a Bayesian scoring scheme that assigns each variant locus a score using the total, reference, and alternate allele read count information. The score represents the confidence that at least 1 (Bayes_score_1; Supplemental Table 1) or 2 (Bayes_score_2; Supple- mental Table 1) of the samples have variation (i.e. heterozygous or homozygous alternate) in a given locus. All the germline variants with the Bayesian score of <0.9999999 or lack of allele frequency information in all the above reference cohorts were discarded.

ADDITIONAL INFORMATION Data Deposition and Access The variants described in this study have been submitted to ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) under accession numbers SCV000693709 - SCV000693712. Ethics Statement Patient consent was obtained in writing. The study was approved by the Finnish Committee of Ethics of Tampere University Hospital and by the National Supervisory Authority for Welfare and Health. Acknowledgments We thank Mr. Erkki Karttunen, Mrs. Satu Ranta, Mrs. Marita Nieminen, Ph.D. Juha Kesseli, and Ms. Paula Kosonen for their skillful technical assistance and Ph.D. Joanna Ilvesaro for her expertise in immunohistochemical staining protocols. We acknowledge the personnel at Tampere University Hos- pital and Fimlab Laboratories for their contribution to sample collection and handling. The authors would also like to acknowledge the CSC — IT Center for Science, Ltd. (https://www.csc.fi/csc) for providing the computational resources. Author Contributions KN created all the consent forms and gathered the clinical data along with JH, OL, and PH. HH has done the neuropathological assessments. EA, MA, and SH performed the computational analyses. KG, JT, JH and KN manually confirmed and interpreted the obtained results. KN and MN have pro- vided financial support for the study. KG, OL, and MN supervised the project. All contributors took part in the writing and approved the manuscript. Funding This study was financially supported by grants from the Nona and Kullervo Väre Foundation (KN), Lea and Arvo Ylppö Foundation (KN), the Finnish Pediatric Research Foundation (KN), Tellervo Edessalo Foundation (OL), the Academy of Finland (grant 259038 to KG, 269474 to MN), the Emil Aaltonen Foundation (MA), Päivikki ja Sakari Sohlberg Foundation (KG), Competitive State Research Financ- ing of the Expert Responsibility area of Tampere University Hospital (JH, HH, grants 9T042 and 9UO41 to MN), and Pirkanmaa Cancer Society.

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Figure legends

Figure 1. (A) Timeline of the clinical courses and patient treatments. Chemotherapy: induction therapy using HD-MTX (high-dose metothrexate), VCR (vincristine), cisplatin, cyclophosphamide, and etopo- side. (B) MRI of the primary AT/RT of the child. (L) left side, (R) right side. (C) Histology of the primary tumor of the child (200× magnification). Scale bar, 20 µm. (D) MRI of the primary tumor of the mother. (L) left side, (R) right side. (E) Histology of the primary anaplastic astrocytoma of the mother (200× magnification). Scale bar, 20 µm. Figure 2. Both the mother and the child carried a germline mutation in both TP53 and ATRX genes. (A) The sequencing coverage and TP53 allele fraction information in blood and tumor samples as visualized in integrative genomics viewer (IGV). The green color represents the variant allele. Copy- neutral LOH was observed in all the tumor samples. The sequencing coverage range is indicated in the square brackets. The exon, transcript base and amino acid information in the major transcript (NM_000546) are marked above the figure. (Bottom) TP53 mutation is located at the DNA binding domain. (B) The sequencing coverage and ATRX allele fraction information in blood and tumor sam- ples as visualized in IGV. The orange color represents the variant allele. Copy-neutral LOH was ob- served in both AT/RT tumor samples. The exon, transcript base and amino acid information in the major transcript (NM_000489) are marked above the figure. (Bottom) ATRX mutation is located at the SNF2N domain. Figure 3. Somatic copy-number alterations in mother’s and child’s tumors. (A) Copy-neutral LOH was observed in most chromosomes in the child’s tumor, but chromosome gains were also prevalent. In the mother, there was a copy-neutral LOH in Chr 17p.3 and a hemizygous deletion in Chr X. In the chromosomal counts figure for the child, A represents a genomic region inherited from the mother and B a genomic region inherited from the father. Normal chromosomal count is thus marked with AB and, for example, copy-neutral LOH in either paternal or maternal chromosomal region is marked with AA/BB. If a tumor contains two subclones with different counts for a genomic region, the chromoso- mal counts for each subclone are separated with a + sign. (B) A region in Chr 22q11.23 was homozy- gously deleted in both tumor samples obtained from the child. Genes falling within this region, includ- ing SMARCB1/hSNF5/INI1, are visualized in the figure.

Table 1. Variant information for ATRX and TP53 germline variants, which were present in both cases.

Legends for Supplementary Figures and Tables Supplemental Figure 1. The histology of the recurrent AT/RT of the child resected nine months after primary diagnosis. Supplemental Figure 2. The sequence coverage logratios and allele fractions the in child’s primary tumor (FFPE) and recidive (frozen sample) as well as in the mother’s tumor. This data was used to determine the chromosome counts for the child and DNA copy number alterations for the mother. Supplemental Table 1. Germline variants that are shared by the mother and the child as well as germline variants detected only in the child. All the variants with COSMIC ID or ClinVar accession number were included into the table. Supplemental Table 2. Somatic mutations in the anaplastic tumor of the mother and in the AT/RT tumors of the child. Supplemental Table 3. Sequencing experiment details, including aimed and observed average se- quencing coverage in the exomic regions that were targeted with sequencing, total read counts and the number and fraction of aligned reads Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Gene Chromosome HGVS coding DNA reference HGVS protein reference Variant type Predicted effect dbSNP/dbVar ID Genotype ClinVar ID ATRX Chr X:76856021 NM_000489:exon23:c.5579A>G NM_000489:exon23:p.N1860S (p.Asn1860Ser) nonsynonymous SNV Asn1860Ser rs45439799 Heterozygous VCV000011724.1 TP53 Chr 17:7577548 NM_000546:exon7:c.733G>A NM_000546:exon7:p.G245S (p.Gly245Ser) nonsynonymous SNV Gly245Ser rs28934575 Heterozygous VCV000012365.2 Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press bayes_score_1bayes_score_2 Chr Start End Ref Alt Func.refGeneGene.refGeneGeneDetail.refGeneExonicFunc.refGeneAAChange.refGeneavsnp147 cosmic70 SISU1000g2015aug_all1000g2015aug_eurExAC_ALLExAC_AFRExAC_AMRExAC_EAS ExAC_FIN ExAC_NFEExAC_OTHExAC_SASesp6500si_allCLINSIG CLNDBN CLNACC CLNDSDB CLNDSDBIDblood_childblood_mothertumor_FFPE_childtumor_frozen_childtumor_mother 1 1 chr1 45276025 45276025 C G exonic BTBD19 . nonsynonymousBTBD19:NM_001136537:exon2:c.227C>G:p.T76S SNV rs191977029ID=COSM1197177,COSM1197176;OCCURENCE=1(lung). 0.002396170.006 0.0062 0 0.0098 0 0.0019 0.0130 0 0.0016 0.006132 . . . . . 0/1:38:83 0/1:32:71 0/1:96:1460/1:62:99 0/1:47:88 1 1 chr1 200617625200617625C T exonic DDX59 . nonsynonymousDDX59:NM_001031725:exon7:c.1538G>A:p.S513N SNV rs142176836ID=COSM390003,COSM390004;OCCURENCE=1(lung)0.0182 0.0005990420.003 0.0023 0 8.642e-05 0.0027 0.0209 0.0014 0.0121 0.0004 0.000308 . . . . . 0/1:51:1010/1:45:1030/1:83:1610/1:78:1380/1:64:133 1 1 chr1 216062306216062306A G exonic USH2A . nonsynonymousUSH2A:NM_206933:exon41:c.7685T>C:p.V2562A SNV rs56385601. 0.006767 0.004792330.0139 0.0065 0.0024 0.0036 0 0.0054 0.0097 0.0044 0.0022 0.006535 Benign not_specifiedRCV000041915.3MedGen CN169374 0/1:52:91 0/1:45:85 0/1:91:1470/1:90:1360/1:62:125 Downloaded1 from molecularcasestudies.cshlp.org1 chr1 227071475 on October227071475 6, 2021 - PublishedC by ColdT Spring Harborexonic Laboratory PressPSEN2 . nonsynonymousPSEN2:NM_000447:exon5:c.211C>T:p.R71W,PSEN2:NM_012486:exon5:c.211C>T:p.R71W SNV rs140501902. 0.01865 0.001397760.004 0.0034 0.0007 0.0010 0 0.0195 0.0037 0.0033 0.0005 0.002768 Likely benignnot_specifiedRCV000172587.2MedGen CN169374 0/1:21:46 0/1:15:37 0/1:64:1360/1:47:78 0/1:20:56 1 1 chr1 247587343247587343G A exonic NLRP3 . nonsynonymousNLRP3:NM_001127462:exon3:c.598G>A:p.V200M,NLRP3:NM_001243133:exon3:c.592G>A:p.V198M,NLRP3:NM_004895:exon3:c.598G>A:p.V200M,NLRP3:NM_183395:exon3:c.598G>A:p.V200M,NLRP3:NM_001127461:exon4:c.598G>A:p.V200M,NLRP3:NM_001079821:exon5:c.598G>A:p.V200M SNV rs121908147. 0.02334 0.003993610.0119 0.0083 0.0013 0.0038 0.0001 0.0253 0.0099 0.0156 0.0060 0.006843 Pathogenic|LikelyFamilial_cold_urticaria|not_provided|not_specified|Familial_cold_autoinflammatory_syndrome benign|other|LikelyRCV000004619.5|RCV000224634.1|RCV000248492.1|RCV000312024.1MedGen:OMIM:Orphanet:SNOMED_CT|MedGen|MedGen|MedGen benignC0343068:120100:ORPHA47045:238687000|CN221809|CN169374|CN2307570/1:68:1250/1:57:1120/1:123:1710/1:98:1450/1:73:133 1 1 chr1 248344164248344164C T exonic OR2M2 . nonsynonymousOR2M2:NM_001004688:exon1:c.877C>T:p.R293C SNV rs139377253ID=COSM225993;OCCURENCE=1(NS)0.005888 0.002196490.002 0.0021 0.0033 0.0010 0 0.0054 0.0019 0.0044 0.0027 0.002537 . . . . . 0/1:34:65 0/1:21:55 0/1:56:1080/1:51:84 0/1:32:73 1 0,9999999974chr2 173355776173355776C T exonic ITGA6 . nonsynonymousITGA6:NM_000210:exon21:c.2704C>T:p.R902W,ITGA6:NM_001079818:exon21:c.2704C>T:p.R902W,ITGA6:NM_001316306:exon21:c.2347C>T:p.R783W SNV rs199587983. 0.02376 0.0001996810.001 0.0018 0 0 0 0.0242 0.0009 0.0022 6.061e-05 0.000154 Likely benignEpidermolysis_bullosa_with_pyloric_atresiaRCV000296803.1MedGen:OMIM:OrphanetC1856934:226730:ORPHA794030/1:10:26 0/1:11:22 0/1:11:16 0/1:24:38 0/1:13:27 1 1 chr2 187559048187559048- CAA exonic FAM171B . nonframeshiftFAM171B:NM_177454:exon1:c.148_149insCAA:p.Q50delinsPK insertion. ID=COSM1172829;OCCURENCE=2(oesophagus),1(biliary_tract)...... 0/1:33:1120/1:29:88 0/1:51:1540/1:34:86 :15:102 1 1 chr2 228564010228564010C T exonic SLC19A3 . nonsynonymousSLC19A3:NM_025243:exon3:c.421G>A:p.G141S SNV rs148144444. 0.01128 0.002196490.007 0.0038 0.0006 0.0020 0.0001 0.0107 0.0051 0.0077 0.0007 0.003921 other not_specifiedRCV000177348.5MedGen CN169374 0/1:48:94 0/1:47:83 0/1:63:1350/1:48:1210/1:57:101 1 1 chr3 14180731 14180731 C T exonic TMEM43 . nonsynonymousTMEM43:NM_024334:exon11:c.934C>T:p.R312W SNV rs113449357. 0.03188 0.003793930.0099 0.0090 0.0021 0.0030 0 0.0354 0.0113 0.0143 0.0023 0.009842 Benign|Benign|Benign|Benign|LikelyCardiomyopathy|not_specified|Arrhythmogenic_right_ventricular_cardiomyopathy,_type_5|Cardiovascular_phenotype|Cardiomyopathy,_ARVCRCV000030555.1|RCV000039394.5|RCV000233894.1|RCV000243225.1|RCV000336793.1MedGen:Orphanet:SNOMED_CT|MedGen|MedGen:OMIM|MedGen|MedGen benignC0878544:ORPHA167848:85898001|CN169374|C1858379:604400|CN230736|CN2391810/1:37:79 0/1:41:81 1/1:130:1361/1:85:93 0/1:60:117 1 1 chr3 105421032105421032C G exonic CBLB . nonsynonymousCBLB:NM_170662:exon12:c.1865G>C:p.S622T SNV rs41302192. 0.006919 0.003793930.0139 0.0060 0.0019 0.0012 0 0.0073 0.0085 0.0088 0.0041 0.005151 not providednot_specifiedRCV000120475.1MedGen CN169374 0/1:35:66 0/1:29:49 1/1:78:83 1/1:78:81 0/1:35:79 1 1 chr3 132427063132427063T C exonic NPHP3 . nonsynonymousNPHP3:NM_153240:exon7:c.1157A>G:p.N386S SNV rs142021049. 0.001805 0.0007987220.003 0.0017 0.0006 0.0020 0 0.0015 0.0025 0.0033 0 0.002922 Uncertain significance|Likelynot_provided|not_specifiedRCV000082660.6|RCV000259061.1 benignMedGen|MedGenCN221809|CN1693740/1:22:45 0/1:14:39 1/1:42:44 1/1:51:56 0/1:26:52 1 1 chr4 1936897 1936897 C A exonic WHSC1 . nonsynonymousWHSC1:NM_133334:exon6:c.1582C>A:p.H528N,WHSC1:NM_001042424:exon7:c.1582C>A:p.H528N,WHSC1:NM_133335:exon7:c.1582C>A:p.H528N,WHSC1:NM_133331:exon8:c.1582C>A:p.H528N,WHSC1:NM_007331:exon9:c.1582C>A:p.H528N,WHSC1:NM_133330:exon9:c.1582C>A:p.H528N SNV rs139753036. 0.02211 0.0005990420.003 0.0032 0 8.664e-05 0 0.0250 0.0032 0.0066 0 0.001384 Likely benign4p_partial_monosomy_syndromeRCV000366205.1MedGen:OMIM:Orphanet:SNOMED_CTC1956097:194190:ORPHA280:171220040/1:28:72 0/1:27:53 1/1:81:85 1/1:65:71 0/1:38:83 1 0,9999999995chr4 147560458147560458- GGC exonic POU4F2 . nonframeshiftPOU4F2:NM_004575:exon1:c.166_167insGGC:p.G56delinsGR insertion. ID=COSM1173024;OCCURENCE=1(oesophagus)...... 0/1:41:74 0/1:12:35 0/1:48:97 0/1:33:65 0/1:29:69 1 1 chr6 41754574 41754574 - CTT exonic PRICKLE4 . nonframeshiftPRICKLE4:NM_013397:exon8:c.862_863insCTT:p.L288delinsPF insertion. ID=COSM1173227,COSM1173226;OCCURENCE=2(oesophagus)...... 0/1:53:1310/1:53:113:15:170 :5:99 0/1:55:129 1 1 chr6 76124678 76124678 C T exonic FILIP1 . nonsynonymousFILIP1:NM_001300866:exon2:c.11G>A:p.R4Q,FILIP1:NM_015687:exon2:c.11G>A:p.R4Q,FILIP1:NM_001289987:exon3:c.20G>A:p.R7Q SNV rs145500939ID=COSM3430885,COSM3430884;OCCURENCE=1(large_intestine)0.003159 0.001996810.007 0.0029 0.0016 0.0027 0 0.0029 0.0041 0.0033 6.067e-05 0.003537 . . . . . 0/1:55:1110/1:49:92 1/1:127:1361/1:104:1130/1:71:136 1 1 chr6 152464786152464786G A exonic SYNE1 . nonsynonymousSYNE1:NM_033071:exon138:c.24947C>T:p.P8316L,SYNE1:NM_182961:exon138:c.25091C>T:p.P8364L SNV rs148376885. 0.004511 0.0001996810.001 0.0018 0.0002 0.0010 0 0.0039 0.0027 0.0022 0 0.002230 Likely benign|LikelyCerebellar_ataxia|not_specified|Emery-Dreifuss_muscular_dystrophy benign|LikelyRCV000286927.1|RCV000335833.1|RCV000341932.1.|MedGen|MedGen:Orphanet:SNOMED_CT benign .|CN169374|C0410189:ORPHA261:1115080040/1:20:42 0/1:28:42 1/1:73:78 1/1:62:67 0/1:37:66 1 1 chr7 147600763147600763C A exonic CNTNAP2 . nonsynonymousCNTNAP2:NM_014141:exon14:c.2205C>A:p.N735K SNV rs200610099. 0.006321 . . 0.0006 0 0 0 0.0073 0.0003 0.0023 0 0.000077 Uncertain significancenot_specifiedRCV000187203.1MedGen CN169374 0/1:25:43 0/1:14:38 0/1:60:1190/1:44:98 0/1:33:73 1 1 chr8 6302436 6302436 C T exonic MCPH1 . nonsynonymousMCPH1:NM_001172575:exon7:c.1049C>T:p.A350V,MCPH1:NM_001172574:exon8:c.1193C>T:p.A398V,MCPH1:NM_024596:exon8:c.1193C>T:p.A398V SNV rs202241113. 0.006466 . . 0.0007 0 0.0005 0 0.0076 0.0003 0 0.0007 . Uncertain significancenot_specifiedRCV000194732.1MedGen CN169374 0/1:53:97 0/1:38:77 1/1:88:95 1/1:95:99 0/1:63:103 1 1 chr8 10464796 10464796 G C exonic RP1L1 . nonsynonymousRP1L1:NM_178857:exon4:c.6812C>G:p.P2271R SNV rs183232880ID=COSM1159821;OCCURENCE=1(haematopoietic_and_lymphoid_tissue)0.01601 0.0007987220.004 0.0032 0.0007 0.0022 0 0.0151 0.0036 0.0089 0 0.001877 Likely benignOccult_macular_dystrophyRCV000358023.1MedGen:OMIM:OrphanetC3150833:613587:ORPHA2478340/1:37:74 0/1:26:50 1/1:78:81 1/1:58:62 0/1:43:90 1 0,9999999979chr9 21971137 21971137 T G exonic CDKN2A . nonsynonymousCDKN2A:NM_000077:exon2:c.221A>C:p.D74A,CDKN2A:NM_001195132:exon2:c.221A>C:p.D74A SNV rs200429615ID=COSM4163712,COSM4163711,COSM4163709,COSM4163710;OCCURENCE=2(thyroid). . . 0.0017 0.0003 0.0002 0 0.0106 0.0014 0 0.0031 ...... 0/1:10:22 0/1:8:14 0/1:16:33 0/1:6:19 0/1:4:14 1 0,9999999038chr9 21974753 21974753 A C exonic CDKN2A . nonsynonymousCDKN2A:NM_000077:exon1:c.74T>G:p.V25G,CDKN2A:NM_001195132:exon1:c.74T>G:p.V25G,CDKN2A:NM_058197:exon1:c.74T>G:p.V25G SNV rs748780473ID=COSM1645941;OCCURENCE=1(kidney). . . 0.0010 0.0001 0 0 0.0138 0.0005 0.0025 0 ...... 0/1:16:66 0/1:15:56 0/1:21:86 0/1:16:44 0/1:18:60 1 0,9999999973chr9 102590845102590845A C exonic NR4A3 . nonsynonymousNR4A3:NM_006981:exon3:c.521A>C:p.D174A,NR4A3:NM_173199:exon3:c.521A>C:p.D174A,NR4A3:NM_173200:exon4:c.554A>C:p.D185A SNV rs201802808ID=COSM4163159,COSM4163158;OCCURENCE=1(thyroid). . . 0.0022 0 0 0 0.5 0.0032 0 0.0014 ...... 0/1:13:33 0/1:7:9 0/1:24:56 0/1:8:20 0/1:10:21 1 0,9999999977chr9 109690578109690578A C exonic ZNF462 . nonsynonymousZNF462:NM_021224:exon3:c.4385A>C:p.N1462T SNV rs80154710ID=COSM4163190,COSM4163191;OCCURENCE=1(thyroid). . . 0.0002 0.0001 0 0.0003 0.0003 0.0002 0 0 ...... 0/1:25:89 0/1:14:49 0/1:25:90 0/1:17:74 0/1:22:92 1 1 chr11 6023849 6023849 C T exonic OR56A4 . nonsynonymousOR56A4:NM_001005179:exon1:c.530G>A:p.R177H SNV rs183652867ID=COSM1743999;OCCURENCE=1(biliary_tract)0.01594 0.001198080.005 0.0018 9.615e-05 0.0003 0 0.0180 0.0013 0.0044 0.0003 0.000077 . . . . . 0/1:39:72 0/1:21:59 0/1:32:111:6:83 0/1:44:89 1 1 chr11 7960151 7960151 C T exonic OR10A3 . nonsynonymousOR10A3:NM_001003745:exon1:c.917G>A:p.R306Q SNV rs190044689ID=COSM1218488;OCCURENCE=1(large_intestine),1(liver)0.001955 0.0001996810.001 0.0002 0 8.678e-05 0 0.0020 6.056e-05 0 0 ...... 0/1:38:65 0/1:41:77 :20:113 :4:101 0/1:51:98 1 1 chr11 55579400 55579400 C T exonic OR5L1 . nonsynonymousOR5L1:NM_001004738:exon1:c.458C>T:p.T153M SNV rs144467940ID=COSM1218792;OCCURENCE=2(large_intestine)0.0214 0.001198080.006 0.0018 0 8.643e-05 0.0001 0.0222 0.0009 0.0055 0.0001 ...... 0/1:66:1240/1:46:98 :25:132 :9:133 0/1:68:128 1 1 chr11 58125740 58125740 G C exonic OR5B17 . nonsynonymousOR5B17:NM_001005489:exon1:c.803C>G:p.T268R SNV rs144440324ID=COSM147243;OCCURENCE=1(stomach)0.003376 0.00179712. 0.0014 0 0 0.0139 0.0032 0.0002 0.0033 0.0009 0.000077 . . . . . 0/1:61:1220/1:47:89 0/1:34:130:8:125 0/1:64:125 1 1 chr12 6062708 6062708 G A exonic VWF . nonsynonymousVWF:NM_000552:exon48:c.7940C>T:p.T2647M SNV rs61751302ID=COSM357715;OCCURENCE=1(lung)0.01489 0.001397760.007 0.0027 0.0006 0.0002 0 0.0169 0.0031 0.0044 0.0002 0.001615 not providednot_providedRCV000086901.1MedGen CN221809 0/1:29:66 0/1:33:59 0/1:65:1311/1:83:86 0/1:36:81 1 1 chr12 52345597 52345597 T G exonic ACVR1B . nonsynonymousACVR1B:NM_004302:exon1:c.70T>G:p.S24A,ACVR1B:NM_020328:exon1:c.70T>G:p.S24A SNV rs781239014ID=COSM4147175;OCCURENCE=1(thyroid). . . 0.0001 0 0 0 0 0.0002 0 0.0005 ...... 0/1:16:36 0/1:11:19 0/1:26:90 0/1:8:22 0/1:12:29 1 1 chr12 53453163 53453163 G C exonic TNS2 . nonsynonymousTNS2:NM_015319:exon18:c.1768G>C:p.V590L,TNS2:NM_170754:exon18:c.1738G>C:p.V580L,TNS2:NM_198316:exon18:c.1366G>C:p.V456L SNV rs763260776ID=COSM3688298,COSM3688299;OCCURENCE=1(large_intestine). . . 0.0010 0 0.0007 0 0.0018 0.0013 0.0074 0 ...... 0/1:25:60 0/1:22:47 0/1:76:1541/1:63:71 0/1:41:83 1 1 chr12 101768586101768586C T exonic UTP20 . nonsynonymousUTP20:NM_014503:exon55:c.7132C>T:p.R2378C SNV rs760622502ID=COSM1232154;OCCURENCE=1(large_intestine)0.000451 . . 5.769e-05 9.61e-05 0 0.0001 0.0005 0 0.0011 6.062e-05 ...... 0/1:61:1130/1:39:98 0/1:93:1481/1:99:1040/1:55:108 1 1 chr13 36220430 36220430 C T exonic NBEA . nonsynonymousNBEA:NM_001204197:exon10:c.1031C>T:p.T344M,NBEA:NM_015678:exon50:c.7652C>T:p.T2551M SNV rs200250784ID=COSM357833;OCCURENCE=1(lung)0.006167 0.0001996810.001 0.0006 0 9.032e-05 0 0.0069 0.0003 0.0024 0 0.000080 . . . . . 0/1:45:77 0/1:29:57 1/1:95:1011/1:81:86 0/1:59:111 1 1 chr13 110830258110830258C T exonic COL4A1 . nonsynonymousCOL4A1:NM_001845:exon33:c.2647G>A:p.V883I SNV rs41275090ID=COSM1169345,COSM1169344;OCCURENCE=1(haematopoietic_and_lymphoid_tissue)0.004361 0.001597440.003 0.0045 0.0008 0.0394 0 0.0035 0.0009 0.0033 0 0.000615 Likely benign|LikelyAngiopathy,_hereditary,_with_nephropathy,_aneurysms,_and_muscle_cramps|Porencephaly|Brain_small_vessel_disease_with_hemorrhage benign|LikelyRCV000272999.1|RCV000303358.1|RCV000365013.1MedGen:OMIM:Orphanet|.|MedGen:OMIM benign C2673195:611773:ORPHA73229|.|C1843512:6075950/1:33:66 0/1:25:54 1/1:98:1011/1:66:68 0/1:36:86 1 1 chr14 92472186 92472186 C T exonic TRIP11 . nonsynonymousTRIP11:NM_004239:exon11:c.2134G>A:p.E712K SNV rs143524436. 0.03445 0.002995210.0109 0.0074 0.0023 0.0026 0 0.0318 0.0089 0.0022 0.0026 0.006843 Likely benignAchondrogenesisRCV000374983.1. . 0/1:67:1170/1:52:108:9:124 :4:133 0/1:64:138 1 1 chr15 42837298 42837298 G A exonic LRRC57 . stopgain LRRC57:NM_153260:exon5:c.655C>T:p.R219Xrs750150624ID=COSM961750;OCCURENCE=2(endometrium). . . 8.406e-06 0 0 0 0 0 0 6.72e-05 ...... 0/1:35:84 0/1:48:83 1/1:107:1111/1:92:1050/1:61:108 1 1 chr15 43816513 43816513 G A exonic MAP1A . nonsynonymousMAP1A:NM_002373:exon4:c.2842G>A:p.V948I SNV rs202112026ID=COSM339622;OCCURENCE=1(lung),1(endometrium)0.001654 0.0003993610.002 0.0011 0 0 0 0.0021 0.0017 0.0022 6.056e-05 0.000645 . . . . . 0/1:51:95 0/1:40:88 1/1:134:1391/1:103:1080/1:74:134 1 1 chr15 91549010 91549010 C T exonic VPS33B . nonsynonymousVPS33B:NM_001289148:exon12:c.863G>A:p.R288Q,VPS33B:NM_001289149:exon12:c.671G>A:p.R224Q,VPS33B:NM_018668:exon13:c.944G>A:p.R315Q SNV rs145303578. 0.002898 . . 0.0010 0.0002 0.0006 0 0.0038 0.0013 0 0.0004 0.001155 Uncertain significance|UncertainArthrogryposis,_Renal_Dysfunction,_and_Cholestasis_Syndrome|not_specifiedRCV000333016.1|RCV000400617.1MedGen|MedGen significanceCN239176|CN1693740/1:22:47 0/1:34:60 1/1:114:1221/1:56:60 0/1:42:91 1 1 chr16 4847824 4847824 C T exonic ROGDI . nonsynonymousROGDI:NM_024589:exon10:c.713G>A:p.R238H SNV rs138409264. 0.01388 0.003594250.004 0.0052 0.0049 0.0023 0 0.0144 0.0068 0.0045 0.0002 0.005695 Uncertain significanceKohlschutter's_syndromeRCV000389190.1MedGen:OMIM:SNOMED_CTC0406740:226750:1094780070/1:40:73 0/1:28:53 :12:126 :4:67 0/1:32:64 1 1 chr17 7577548 7577548 C T exonic TP53 . nonsynonymousTP53:NM_001126115:exon3:c.337G>A:p.G113S,TP53:NM_001126116:exon3:c.337G>A:p.G113S,TP53:NM_001126117:exon3:c.337G>A:p.G113S,TP53:NM_001276697:exon3:c.256G>A:p.G86S,TP53:NM_001276698:exon3:c.256G>A:p.G86S,TP53:NM_001276699:exon3:c.256G>A:p.G86S,TP53:NM_001126118:exon6:c.616G>A:p.G206S,TP53:NM_000546:exon7:c.733G>A:p.G245S,TP53:NM_001126112:exon7:c.733G>A:p.G245S,TP53:NM_001126113:exon7:c.733G>A:p.G245S,TP53:NM_001126114:exon7:c.733G>A:p.G245S,TP53:NM_001276695:exon7:c.616G>A:p.G206S,TP53:NM_001276696:exon7:c.616G>A:p.G206S,TP53:NM_001276760:exon7:c.616G>A:p.G206S,TP53:NM_001276761:exon7:c.616G>A:p.G206S SNV rs28934575ID=COSM1640833,COSM121036,COSM6932,COSM121035,COSM121037,COSM3356965;OCCURENCE=25(central_nervous_system),31(stomach),13(upper_aerodigestive_tract),2(vulva),20(oesophagus),7(lung),1(cervix),19(haematopoietic_and_lymphoid_tissue),1(soft_tissue),24(ovary),1(salivary_gland),2(endometrium),6(skin),30(breast),4(small_intestine),12(urinary_tract),9(pancreas),4(liver),3(prostate),115(large_intestine). . . 8.239e-06 0 0 0 0 1.498e-05 0 0 0.000077 Pathogenic|Pathogenic,Pathogenic|Pathogenic|Pathogenic|LikelyLi-Fraumeni_syndrome_1|not_provided,Li-Fraumeni-like_syndrome|Hereditary_cancer-predisposing_syndrome|Li-Fraumeni_syndrome_1|Adenocarcinoma|not_provided|Li-Fraumeni_syndromeRCV000013142.22|RCV000161025.2,RCV000013162.20|RCV000130147.5|RCV000144669.1|RCV000148909.1|RCV000154014.4|RCV000226657.1MedGen:OMIM|MedGen,MedGen|MedGen:SNOMED_CT|MedGen:OMIM|MedGen|MedGen|MedGen:Orphanet:SNOMED_CTC1835398:151623|CN221809,C2675080|C0027672:699346009|C1835398:151623|C0001418|CN221809|C0085390:ORPHA524:4288500010/1:23:51 0/1:18:41 pathogenic|Pathogenic|Pathogenic1/1:89:97 1/1:48:54 0/1:55:76 1 1 chr17 61559041 61559041 G A exonic ACE . nonsynonymousACE:NM_000789:exon7:c.1060G>A:p.G354R SNV rs56394458. 0.02708 0.003793930.0139 0.0078 0.0011 0.0033 0 0.0262 0.0105 0.0077 0.0015 0.006459 Likely benignRenal_dysplasiaRCV000382163.1MedGen:OMIM:SNOMED_CTC0266313:267430:2049490010/1:61:1030/1:50:87 1/1:136:1551/1:92:94 0/1:44:100 1 1 chr17 76136994 76136994 C T exonic TMC8 . nonsynonymousTMC8:NM_152468:exon16:c.1982C>T:p.P661L SNV rs139972217ID=COSM437652;OCCURENCE=1(breast). 0.002196490.004 0.0049 0.0009 0.0028 0 0.0159 0.0072 0.0021 0.0003 0.002465 . . . . . 0/1:41:66 0/1:27:48 1/1:113:1191/1:51:56 0/1:29:67 1 1 chr18 2926779 2926779 A G exonic LPIN2 . nonsynonymousLPIN2:NM_014646:exon13:c.1735T>C:p.S579P SNV rs150022314. . 0.0007987220.004 0.0027 0.0002 0.0011 0.0001 0.0013 0.0042 0.0023 0.0012 0.001692 Uncertain significance|Uncertainnot_specified|Majeed_syndromeRCV000222234.2|RCV000271785.1MedGen|MedGen:OMIM:Orphanet significanceCN169374|C1864997:609628:ORPHA772970/1:47:1010/1:43:83 0/1:63:1380/1:38:98 0/1:44:100 1 1 chr18 3215083 3215083 T C exonic MYOM1 . nonsynonymousMYOM1:NM_003803:exon2:c.139A>G:p.S47G,MYOM1:NM_019856:exon2:c.139A>G:p.S47G SNV rs202145133. 0.03824 0.002595850.0099 0.0054 0.0004 0.0012 0 0.0354 0.0059 0.0092 0 0.002009 Benign|Benignnot_specified|Hypertrophic_cardiomyopathyRCV000213229.1|RCV000228018.1MedGen|Human_Phenotype_Ontology:MedGen:OrphanetCN169374|HP:0001639:C0007194:ORPHA2175690/1:21:40 0/1:12:28 0/1:33:88 0/1:20:45 0/1:16:39 1 1 chr21 46875961 46875961 G A exonic COL18A1 . nonsynonymousCOL18A1:NM_030582:exon1:c.517G>A:p.G173S,COL18A1:NM_130444:exon1:c.517G>A:p.G173S SNV rs62000960. 0.007697 0.001597440.006 0.0031 0.0006 0.0016 0 0.0055 0.0042 0.0047 0.0018 0.002686 Uncertain significancenot_specifiedRCV000202786.1MedGen CN169374 0/1:49:1050/1:38:81 0/1:58:1450/1:36:1070/1:36:78 1 1 chr21 47721986 47721986 - TGG exonic C21orf58 . nonframeshiftC21orf58:NM_001286463:exon7:c.577_578insCCA:p.H193delinsPN,C21orf58:NM_001286462:exon8:c.577_578insCCA:p.H193delinsPN,C21orf58:NM_001286476:exon8:c.577_578insCCA:p.H193delinsPN,C21orf58:NM_001286477:exon8:c.577_578insCCA:p.H193delinsPN,C21orf58:NM_058180:exon8:c.895_896insCCA:p.H299delinsPN insertion. ID=COSM1172757;OCCURENCE=1(oesophagus)...... 0/1:15:43 0/1:26:42 0/1:29:121:6:46 0/1:28:45 1 1 chrX 32404572 32404572 T C exonic DMD . nonsynonymousDMD:NM_004011:exon5:c.506A>G:p.K169R,DMD:NM_004012:exon5:c.497A>G:p.K166R,DMD:NM_000109:exon33:c.4505A>G:p.K1502R,DMD:NM_004006:exon33:c.4529A>G:p.K1510R,DMD:NM_004009:exon33:c.4517A>G:p.K1506R,DMD:NM_004010:exon33:c.4160A>G:p.K1387R SNV rs72468638. . 0.005562910.0131 0.0054 0 0 0.0166 0.0410 0.0035 0.0084 0.0004 0.000189 Benign|Likelynot_specified|Dilated_cardiomyopathy_3B benign RCV000080622.5|RCV000372007.1MedGen|MedGen:OMIMCN169374|C3668940:3020450/1:39:64 0/1:26:52 1/1:58:65 1/1:59:66 0/1:30:63 1 1 chrX 76856021 76856021 T C exonic ATRX . nonsynonymousATRX:NM_138270:exon22:c.5465A>G:p.N1822S,ATRX:NM_000489:exon23:c.5579A>G:p.N1860S SNV rs45439799. . 0.002913910.0104 0.0063 0.0012 0.0013 0 0.0110 0.0093 0.0063 0.0030 0.006346 Pathogenic|BenignATR-X_syndrome|not_specifiedRCV000012490.16|RCV000078966.6MedGen:OMIM:Orphanet|MedGenC1845055:301040:ORPHA847|CN1693740/1:55:1320/1:59:1301/1:122:1381/1:119:1280/1:69:133 Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

CHROM POSITION REF ALT FUNCTION NEARBY_GENESEXONIC_FUNCTION AA_CHANGECOSMIC 1000G ESP6500 ExAC_ALL ExAC_FIN SISU tumor_childtumor_FFPE_childtumor_motherblood_childblood_motherGroup Comments How many Germtime thisline gene variant notes chr1 17952475 C T exonic ARHGEF10Lnonsynonymous SNV ARHGEF10L:NM_001011722:exon12:c.C1225T:p.R409C,ARHGEF10L:NM_018125:exon14:c.C1342T:p.R448C0/1:15:72 0/0:0:119 0/0:0:85 0/0:0:68 0/0:0:52 Child_frozen only 1 chr1 156288729 C T exonic CCT3 nonsynonymous SNV CCT3:NM_001008800:exon6:c.G575A:p.R192Q,CCT3:NM_005998:exon8:c.G689A:p.R230Q0,000077 3,30E-05 0 0/1:35:59 0/1:47:99 0/0:0:53 0/0:0:22 0/0:0:29 Child only 1 chr2 174829200 T G exonic SP3 nonsynonymous SNV SP3:NM_001172712:exon2:c.A89C:p.H30P,SP3:NM_003111:exon2:c.A89C:p.H30P0/1:4:16 0/0:0:96 0/0:0:37 0/0:1:35 0/0:0:17 Child_frozen only 1 chr2 209113113 G A exonic IDH1 nonsynonymous SNV IDH1:NM_005896:exon4:c.C394T:p.R132CID=COSM28747;OCCURENCE=155(bone),1(NS),180(haematopoietic_and_lymphoid_tissue),12(soft_tissue),1(autonomic_ganglia),3(skin),1(prostate),2(large_intestine),93(central_nervous_system),13(biliary_tract)0/0:0:72 0/0:0:67 0/1:17:71 0/0:0:55 0/0:0:45 Mother only 1 chr2 233745940 C T exonic NGEF nonsynonymous SNV NGEF:NM_001114090:exon12:c.G1582A:p.V528M,NGEF:NM_019850:exon14:c.G1858A:p.V620M0,000077 0/1:28:91 0/1:38:1560/0:0:85 0/0:0:81 0/0:0:59 Child only 1 chr3 52780770 C A exonic NEK4 nonsynonymous SNV NEK4:NM_001193533:exon8:c.G1390T:p.V464F,NEK4:NM_003157:exon9:c.G1657T:p.V553F0/1:25:46 0/0:0:59 0/0:0:34 0/0:0:39 0/0:0:36 Child_frozen only 1 chr4 55981126 C A exonic KDR nonsynonymous SNV KDR:NM_002253:exon5:c.G573T:p.M191I 0/1:45:95 0/0:0:115 0/0:0:101 0/0:0:77 0/0:0:69 Child_frozen only 1 chr5 140306677 G T exonic PCDHAC1 nonsynonymous SNV PCDHAC1:NM_018898:exon1:c.G200T:p.G67V,PCDHAC1:NM_031882:exon1:c.G200T:p.G67V0/1:52:1090/1:65:1440/0:0:114 0/0:0:125 0/0:0:112 Child only 1 chr5 176930173 A C exonic DOK3 nonsynonymous SNV DOK3:NM_001144876:exon5:c.T560G:p.L187R,DOK3:NM_001144875:exon6:c.T866G:p.L289R0/1:5:9 0/1:6:14 0/0:0:70 0/0:1:46 0/0:0:63 Child only adjacent bases 2 chr5 176930174 G A exonic DOK3 nonsynonymous SNV DOK3:NM_001144876:exon5:c.C559T:p.L187F,DOK3:NM_001144875:exon6:c.C865T:p.L289F0/1:7:11 0/1:7:15 0/0:0:70 0/0:1:47 0/0:0:62 Child only adjacent bases 2 chr6 17600839 T A exonic FAM8A1 nonsynonymous SNV FAM8A1:NM_016255:exon1:c.T199A:p.L67M 0/0:0:16 0/0:0:36 0/1:5:19 0/0:0:51 0/0:0:16 Mother only 1 chr6 52770616 T A exonic GSTA3 nonsynonymous SNV GSTA3:NM_000847:exon2:c.A17T:p.K6M 0/1:23:47 0/0:0:58 0/0:0:46 0/0:0:38 0/0:0:30 Child_frozen only 1 chr7 130418616 T G exonic KLF14 nonsynonymous SNV KLF14:NM_138693:exon1:c.A245C:p.H82P 0/1:4:19 0/0:0:39 0/0:0:14 0/0:0:21 0/0:0:15 Child_frozen only 1 chr8 42587616 T C exonic CHRNB3 nonsynonymous SNV CHRNB3:NM_000749:exon5:c.T1166C:p.V389A 0/0:0:63 0/0:0:55 0/1:17:76 0/0:0:61 0/0:0:44 Mother only 1 chr8 146033245 G T exonic ZNF517 nonsynonymous SNV ZNF517:NM_213605:exon5:c.G944T:p.R315L 0/1:15:59 0/0:0:109 0/0:0:67 0/0:0:65 0/0:0:36 Child_frozen only 1 chr9 131777052 A C exonic SH3GLB2 nonsynonymous SNV SH3GLB2:NM_020145:exon4:c.T466G:p.S156A 1/1:29:35 1/1:37:46 0/0:0:31 0/0:0:37 0/0:0:20 Child only 1 chr10 90484313 T A exonic LIPK nonsynonymous SNV LIPK:NM_001080518:exon1:c.T13A:p.L5I 1/1:74:78 0/1:67:91 0/0:0:71 0/0:0:57 0/0:0:59 Child only 1 chr11 428582 C T exonic ANO9 nonsynonymous SNV ANO9:NM_001012302:exon13:c.G1078A:p.A360T0,000154 0,0002 0 0/0:0:86 0/0:0:127 0/1:20:85 0/0:0:95 0/0:0:76 Mother only 1 chr11 6411941 C G exonic SMPD1 nonsynonymous SNV SMPD1:NM_000543:exon1:c.C113G:p.A38G,SMPD1:NM_001007593:exon1:c.C113G:p.A38G0/1:11:37 0/1:15:62 0/0:0:61 0/0:3:70 0/0:0:49 Child only 1 chr11 60620166 T G exonic PTGDR2 nonsynonymous SNV PTGDR2:NM_004778:exon2:c.A1030C:p.T344P 0/1:5:22 0/0:1:60 0/0:0:23 0/0:0:23 0/0:0:20 Child_frozen only 1 chr11 85406336 C A exonic SYTL2 stopgain SNV SYTL2:NM_032379:exon9:c.G1033T:p.E345X,SYTL2:NM_206930:exon9:c.G1000T:p.E334X,SYTL2:NM_001162952:exon10:c.G913T:p.E305X,SYTL2:NM_206928:exon12:c.G3673T:p.E1225X,SYTL2:NM_206929:exon12:c.G1033T:p.E345X,SYTL2:NM_206927:exon13:c.G3721T:p.E1241X,SYTL2:NM_001162951:exon18:c.G2707T:p.E903X,SYTL2:NM_001162953:exon18:c.G2710T:p.E904X,SYTL2:NM_032943:exon19:c.G2635T:p.E879X0/1:31:64 0/0:0:75 0/0:0:92 0/0:0:53 0/0:0:42 Child_frozen only adjacent bases 2 chr11 85406337 C A exonic SYTL2 nonsynonymous SNV SYTL2:NM_032379:exon9:c.G1032T:p.E344D,SYTL2:NM_206930:exon9:c.G999T:p.E333D,SYTL2:NM_001162952:exon10:c.G912T:p.E304D,SYTL2:NM_206928:exon12:c.G3672T:p.E1224D,SYTL2:NM_206929:exon12:c.G1032T:p.E344D,SYTL2:NM_206927:exon13:c.G3720T:p.E1240D,SYTL2:NM_001162951:exon18:c.G2706T:p.E902D,SYTL2:NM_001162953:exon18:c.G2709T:p.E903D,SYTL2:NM_032943:exon19:c.G2634T:p.E878D0/1:31:65 0/0:0:74 0/0:0:91 0/0:0:53 0/0:0:42 Child_frozen only adjacent bases 2 chr11 92087920 G A exonic FAT3 nonsynonymous SNV FAT3:NM_001008781:exon1:c.G2642A:p.S881N 0/0:0:137 0/0:0:134 0/1:31:1490/0:0:133 0/0:0:116 Mother only 1 nonsynonymous SNV heterozygous variants, large transcript chr12 43884206 T G exonic ADAMTS20 nonsynonymous SNV ADAMTS20:NM_025003:exon7:c.A1109C:p.N370T 0/0:0:13 0/1:5:24 0/0:0:30 0/0:0:27 0/0:0:21 Child_FFPE only 1 chr12 56495623 T - exonic ERBB3 frameshift deletion ERBB3:NM_001982:exon28:c.3813delT:p.G1271fs 0/1:32:75 0/0:0:145 0/0:0:99 0/0:0:69 0/0:0:64 Child_frozen only 1 chr12 111758401 C G exonic CUX2 nonsynonymous SNV CUX2:NM_015267:exon17:c.C2588G:p.A863G 0/1:4:17 0/0:0:65 0/0:0:9 0/0:0:20 0/0:0:8 Child_frozen only 1 chr13 41897310 T A exonic NAA16 nonsynonymous SNV NAA16:NM_001110798:exon5:c.T521A:p.F174Y,NAA16:NM_018527:exon5:c.T521A:p.F174Y,NAA16:NM_024561:exon5:c.T521A:p.F174Y0/1:37:96 0/1:36:92 0/0:1:100 0/0:0:87 0/0:0:70 Child only 1 chr15 78807391 G T exonic AGPHD1 nonsynonymous SNV AGPHD1:NM_001013619:exon3:c.G419T:p.S140I,AGPHD1:NM_001083612:exon3:c.G419T:p.S140I0/1:22:46 0/0:0:54 0/0:1:47 0/0:0:31 0/0:0:21 Child_frozen only 1 chr15 90793870 C A exonic TTLL13 nonsynonymous SNV TTLL13:NM_001029964:exon2:c.C8A:p.P3Q 0/1:21:42 0/0:0:60 0/0:0:49 0/0:0:35 0/0:0:29 Child_frozen only 1 chr16 11001718 C T exonic CIITA nonsynonymous SNV CIITA:NM_000246:exon11:c.C2369T:p.A790V 0/1:23:57 0/0:0:122 0/0:0:73 0/0:0:88 0/0:0:66 Child_frozen only 1 chr17 41738490 C T exonic MEOX1 nonsynonymous SNV MEOX1:NM_004527:exon1:c.G413A:p.G138E,MEOX1:NM_013999:exon1:c.G413A:p.G138E,MEOX1:NM_001040002:exon2:c.G68A:p.G23E0/1:40:1000/0:1:132 0/0:0:101 0/0:0:89 0/0:0:72 Child_frozen only 1 chr18 23854711 G A exonic TAF4B nonsynonymous SNV TAF4B:NM_005640:exon4:c.G682A:p.G228R 0/1:47:1200/0:0:136 0/0:0:135 0/0:1:103 0/0:0:97 Child_frozen only 1 chr19 18990180 T C exonic CERS1 nonsynonymous SNV CERS1:NM_021267:exon5:c.A770G:p.Y257C,CERS1:NM_198207:exon5:c.A770G:p.Y257C0/0:0:48 0/0:0:102 0/1:24:67 0/0:0:60 0/0:0:40 Mother only 1 chr19 46404989 A C exonic MYPOP nonsynonymous SNV MYPOP:NM_001012643:exon2:c.T43G:p.L15V 0/0:0:30 0/0:0:70 0/1:5:21 0/0:0:31 0/0:0:22 Mother only 1 chr20 60908189 A G exonic LAMA5 nonsynonymous SNV LAMA5:NM_005560:exon26:c.T3239C:p.L1080P 0/1:5:11 0/0:0:25 0/0:0:8 0/0:0:17 0/0:0:14 Child_frozen only 1 nonsynonymous SNV heterozygous c.A3251C:p.H1084P Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

bayes_score_1bayes_score_2 Chr Start End Ref Alt Func.refGeneGene.refGeneGeneDetail.refGeneExonicFunc.refGeneAAChange.refGeneavsnp147 cosmic70 SISU1000g2015aug_all1000g2015aug_eurExAC_ALLExAC_AFRExAC_AMRExAC_EAS ExAC_FIN ExAC_NFEExAC_OTHExAC_SASesp6500si_allCLINSIG CLNDBN CLNACC CLNDSDB CLNDSDBIDblood_childblood_mother tumor_FFPE_childtumor_child tumor_mother 1 0 chr1 154543919 154543919 C T exonic CHRNB2 . nonsynonymousCHRNB2:NM_000748:exon5:c.620C>T:p.A207V SNV rs201622476ID=COSM675853;OCCURENCE=1(lung)0.00015 . . 1.648e-05 0 0 0 0.0002 0 0.0011 0 . Uncertain significancenot_specifiedRCV000186988.1MedGen CN169374 0/1:54:113 0/0:0:89 0/1:98:173 0/1:46:130 0/0:0:118 1 0 chr1 160011511 160011511 C T exonic KCNJ10 . nonsynonymousKCNJ10:NM_002241:exon2:c.812G>A:p.R271H SNV rs3795339 . 0.006015 0.002995210.001 0.0019 0 0 0.0168 0.0068 0.0005 0.0044 0.0002 . Likely benignnot_specifiedRCV000117321.3MedGen CN169374 0/1:51:108 0/0:0:98 0/1:101:168 0/1:44:133 0/0:0:115 1 0 chr1 181702689 181702689 C T exonic CACNA1E . nonsynonymousCACNA1E:NM_001205294:exon20:c.3008C>T:p.T1003M,CACNA1E:NM_000721:exon21:c.3065C>T:p.T1022M,CACNA1E:NM_001205293:exon21:c.3065C>T:p.T1022M SNV rs74127835ID=COSM1336852,COSM1336851;OCCURENCE=1(large_intestine)0.01023 0.004992010.002 0.0032 0.0163 0.0026 0 0.0117 0.0015 0.0035 0.0004 0.005884 . . . . . 0/1:45:80 0/0:0:69 0/1:74:155 0/1:40:98 0/0:0:115 1 0,0000000001chr2 71829924 71829924 A G exonic DYSF . nonsynonymousDYSF:NM_001130976:exon35:c.3850A>G:p.I1284V,DYSF:NM_001130977:exon35:c.3850A>G:p.I1284V,DYSF:NM_001130984:exon35:c.3853A>G:p.I1285V,DYSF:NM_001130986:exon35:c.3853A>G:p.I1285V,DYSF:NM_001130455:exon36:c.3895A>G:p.I1299V,DYSF:NM_001130978:exon36:c.3892A>G:p.I1298V,DYSF:NM_001130980:exon36:c.3943A>G:p.I1315V,DYSF:NM_001130981:exon36:c.3943A>G:p.I1315V,DYSF:NM_001130983:exon36:c.3895A>G:p.I1299V,DYSF:NM_001130985:exon36:c.3946A>G:p.I1316V,DYSF:NM_001130987:exon36:c.3946A>G:p.I1316V,DYSF:NM_003494:exon36:c.3892A>G:p.I1298V,DYSF:NM_001130979:exon37:c.3985A>G:p.I1329V,DYSF:NM_001130982:exon37:c.3988A>G:p.I1330V SNV rs121908954. 0.007599 0.001797120.005 0.0047 0.0011 0.0018 0.0001 0.0073 0.0071 0.0037 0.0011 0.005228 Pathogenic|Pathogenic|otherMiyoshi_muscular_dystrophy_1|Limb-girdle_muscular_dystrophy,_type_2B|not_specifiedRCV000007049.5|RCV000007050.5|RCV000153183.5MedGen:OMIM:Orphanet|MedGen:OMIM:Orphanet|MedGenC1850808:254130:ORPHA45448|C1850889:253601:ORPHA268|CN1693740/1:17:40 0/0:0:32 0/1:58:119 0/1:36:61 0/0:0:60 1 0 chr2 179393691 179393691 G A exonic TTN . nonsynonymousTTN:NM_003319:exon188:c.79592C>T:p.T26531I,TTN:NM_133432:exon189:c.79967C>T:p.T26656I,TTN:NM_133437:exon189:c.80168C>T:p.T26723I,TTN:NM_133378:exon309:c.99083C>T:p.T33028I,TTN:NM_001256850:exon310:c.101864C>T:p.T33955I,TTN:NM_001267550:exon360:c.106787C>T:p.T35596I SNV rs55842557. 0.06724 0.003793930.0159 0.0098 0.0013 0.0012 0 0.0764 0.0094 0.0200 0.0004 0.004918 Benign|Benign|Benign|Benign|Likelynot_specified|Dilated_cardiomyopathy_1G|Limb-girdle_muscular_dystrophy,_type_2J|Cardiovascular_phenotype|Dilated_Cardiomyopathy,_Dominant|Limb-Girdle_Muscular_Dystrophy,_Recessive|Distal_myopathy_Markesbery-Griggs_type|Hereditary_myopathy_with_early_respiratory_failure|Hypertrophic_cardiomyopathy|Myopathy,_early-onset,_with_fatal_cardiomyopathyRCV000040981.7|RCV000231148.1|RCV000231148.1|RCV000253556.1|RCV000286567.1|RCV000287599.1|RCV000341504.1|RCV000347285.1|RCV000382062.1|RCV000395312.1MedGen|MedGen:OMIM|MedGen:OMIM:Orphanet|MedGen|MedGen|MedGen|MedGen:OMIM:Orphanet|MedGen:OMIM:Orphanet|Human_Phenotype_Ontology:MedGen:Orphanet|MedGen:OMIM:Orphanet benign|LikelyCN169374|C1858763:604145|C1837342:608807:ORPHA140922|CN230736|CN239310|CN239352|C1838244:600334:ORPHA609|C1863599:603689:ORPHA178464|HP:0001639:C0007194:ORPHA217569|C2673677:611705:ORPHA289377 benign|Likely0/1:60:103 0/0:0:99 benign|Likely 0/1:91:133benign|Likely benign|Likely0/1:88:140 benign0/0:0:125 1 0 chr2 220082505 220082505 G A exonic ABCB6 . nonsynonymousABCB6:NM_005689:exon2:c.574C>T:p.R192W SNV rs149202834. 0.003464 0.0005990420.002 0.0018 9.621e-05 0.0004 0 0.0018 0.0030 0 0.0003 0.001615 other Langereis_blood_groupRCV000201942.1MedGen:OMIMC3276339:1116000/1:28:68 0/0:0:44 0/1:85:139 0/1:49:80 0/0:0:87 1 0 chr2 242690726 242690726 G A exonic D2HGDH . nonsynonymousD2HGDH:NM_001287249:exon7:c.661G>A:p.G221S,D2HGDH:NM_152783:exon8:c.1063G>A:p.G355S SNV rs139321130. 0.0394 0.001397760.004 0.0095 0.0010 0.0025 0.0001 0.0383 0.0115 0.0212 0.0044 0.008232 Likely benign|Likelynot_specified|D-2-hydroxyglutaric_aciduria benignRCV000193545.1|RCV000399857.1MedGen|MedGenCN169374|CN2330400/1:30:60 0/0:0:49 0/1:67:123 0/1:53:88 :1:67 1 0 chr3 42251578 42251580 GGA - exonic TRAK1 . nonframeshiftTRAK1:NM_001265609:exon13:c.1842_1844del:p.614_615del,TRAK1:NM_014965:exon13:c.1890_1892del:p.630_631del,TRAK1:NM_001265608:exon14:c.2064_2066del:p.688_689del deletionrs753440774ID=COSM308433;OCCURENCE=1(haematopoietic_and_lymphoid_tissue),1(kidney)...... 0/1:44:92 0/0:0:48 :5:85 :5:69 :1:86 1 0 chr3 122288441 122288441 C T exonic DTX3L . nonsynonymousDTX3L:NM_138287:exon3:c.1505C>T:p.A502V SNV rs202191528ID=COSM1037173;OCCURENCE=1(endometrium)0.000459 0.0003993610.001 0.0006 0.0003 0.0003 0 0 0.0010 0 0.0001 0.000769 . . . . . 0/1:74:129 0/0:0:111 :16:145 :13:139 0/0:0:148 1 0 chr5 90087161 90087161 C G exonic ADGRV1 . nonsynonymousADGRV1:NM_032119:exon70:c.14515C>G:p.Q4839E SNV rs79464236. . 0.006389780.003 0.0036 0 0 0.0391 0.0112 0.0009 0.0088 0.0019 . other not_specifiedRCV000039527.5MedGen CN169374 0/1:29:52 0/0:0:47 1/1:69:75 1/1:60:63 0/0:0:79 1 0 chr6 129601231 129601231 C T exonic LAMA2 . nonsynonymousLAMA2:NM_000426:exon18:c.2476C>T:p.R826W,LAMA2:NM_001079823:exon18:c.2476C>T:p.R826W SNV rs118147866ID=COSM3702889;OCCURENCE=1(liver)0.02243 0.003194890.0089 0.0050 0.0012 0.0012 0.0002 0.0226 0.0062 0.0022 0.0012 0.004767 other|Likelynot_specified|not_provided|Congenital_Muscular_Dystrophy,_LAMA2-related benign|UncertainRCV000153434.4|RCV000224933.1|RCV000315412.1 significanceMedGen|MedGen|MedGenCN169374|CN221809|CN2393260/1:72:127 0/0:0:112 :13:148 :10:130 0/0:0:152 1 0 chr6 170592563 170592565 TGT - exonic DLL1 . nonframeshiftDLL1:NM_005618:exon9:c.1802_1804del:p.601_602del deletionrs573197356. . 0.001198080.004 0.0025 0 0.0007 0 0.0197 0.0024 0.0055 0.0002 0.000799 Uncertain significanceHoloprosencephalyRCV000223908.1Human_Phenotype_Ontology:MedGenHP:0001360:CN0012460/1:43:119 0/0:0:96 :9:159 :5:121 0/0:0:131 1 0 chr7 2552882 2552882 - GATG exonic LFNG . stopgain LFNG:NM_001166355:exon2:c.139_140insGATG:p.D47_G48delinsGX. ID=COSM1173311;OCCURENCE=1(oesophagus)...... 0/1:23:65 0/0:0:53 0/1:31:127 0/1:22:86 0/0:0:75 1 0 chr8 55540268 55540268 C T exonic RP1 . nonsynonymousRP1:NM_006269:exon4:c.3826C>T:p.P1276S SNV rs151316028. 0.005564 0.0003993610.002 0.0021 0.0002 0.0002 0 0.0050 0.0031 0.0033 0 0.001538 Uncertain significancenot_specifiedRCV000178266.1MedGen CN169374 0/1:73:133 0/0:0:127 :8:146 :8:157 0/0:0:162 1 0 chr8 143957788 143957788 A G exonic CYP11B1 . nonsynonymousCYP11B1:NM_000497:exon5:c.823T>C:p.Y275H,CYP11B1:NM_001026213:exon5:c.823T>C:p.Y275H SNV rs141368413. 0.01777 0.001198080.006 0.0018 0 0 0 0.0190 0.0014 0.0083 0 0.000077 Likely benign|LikelyCongenital_adrenal_hyperplasia|Hyperaldosteronism,_familial,_type_I benignRCV000282706.1|RCV000349385.1MedGen:Orphanet:SNOMED_CT|MedGen:OMIM:OrphanetC0001627:ORPHA418:237751000|C1260386:103900:ORPHA4030/1:34:78 0/0:0:45 :7:131 :4:68 0/0:0:82 1 0 chr9 75355093 75355093 C T exonic TMC1 . nonsynonymousTMC1:NM_138691:exon9:c.421C>T:p.R141W SNV rs11143384. 0.002105 0.002196490.0089 0.0070 0.0015 0.0054 0.0002 0.0026 0.0101 0.0011 0.0045 0.006843 Benign|Likelynot_specified|Nonsyndromic_Hearing_Loss,_Dominant|Nonsyndromic_Hearing_Loss,_Recessive benign|LikelyRCV000041143.3|RCV000274209.1|RCV000313087.1 benignMedGen|MedGen|MedGenCN169374|CN239435|CN2394390/1:50:97 0/0:0:81 :10:127 :9:106 0/0:0:124 1 0 chr9 130941316 130941316 C A exonic CIZ1 . nonsynonymousCIZ1:NM_001257976:exon7:c.867G>T:p.Q289H,CIZ1:NM_001131016:exon8:c.1170G>T:p.Q390H,CIZ1:NM_001257975:exon8:c.1260G>T:p.Q420H,CIZ1:NM_012127:exon8:c.1170G>T:p.Q390H SNV rs61740197. . 0.0009984030.004 0.0042 0.0012 0.0017 0 0.0045 0.0067 0.0022 0 0.005843 Benign Dystonia RCV000232078.1MedGen C0393593 0/1:42:84 0/0:0:48 :3:107 :3:62 0/0:0:77 1 0 chr9 138594163 138594163 G C exonic KCNT1 . nonsynonymousKCNT1:NM_001272003:exon1:c.59G>C:p.G20A,KCNT1:NM_020822:exon1:c.59G>C:p.G20A SNV rs146292575. 0.000453 0.004792330.006 0.0066 0.0012 0.0029 0.0017 0.0003 0.0073 0.0085 0.0144 0.005168 other|Benign|Benignnot_specified|Early_infantile_epileptic_encephalopathy_14|Epilepsy,_nocturnal_frontal_lobe,_5RCV000117372.4|RCV000226410.1|RCV000226410.1MedGen|MedGen:OMIM|MedGen:OMIMCN169374|C3554195:614959|C3554306:6150050/1:40:73 0/0:0:46 :14:106 :6:47 0/0:0:58 1 0 chr9 140444628 140444628 - T exonic PNPLA7 . frameshift PNPLA7:NM_001098537:exon1:c.22dupA:p.S8fs,PNPLA7:NM_152286:exon1:c.22dupA:p.S8fsinsertion . ID=COSM1683427;OCCURENCE=1(prostate)...... 0/1:31:58 0/0:0:43 :8:111 :5:53 0/0:0:66 1 0 chr10 50678884 50678884 T G exonic ERCC6 . nonsynonymousERCC6:NM_000124:exon18:c.3122A>C:p.Q1041P SNV rs139007661. 0.000923 0.0009984030.004 0.0016 0.0005 0.0038 0 0.0014 0.0020 0.0011 0.0001 0.001461 Uncertain significance|Likelynot_specified|Cockayne_syndrome|Macular_degeneration|Cerebrooculofacioskeletal_SyndromeRCV000170382.3|RCV000266566.1|RCV000323987.1|RCV000358951.1 benign|LikelyMedGen|MedGen:Orphanet:SNOMED_CT|Human_Phenotype_Ontology:MedGen|MedGenCN169374|C0009207:ORPHA191:21086008|HP:0000608:C1849131|CN239231 benign|Likely0/1:45:94 benign 0/0:0:83 :10:120 :11:116 :1:125 1 0 chr10 50690906 50690906 G A exonic ERCC6 . nonsynonymousERCC6:NM_000124:exon10:c.1996C>T:p.R666C SNV rs61760163. 0.000902 0.0009984030.004 0.0016 0.0005 0.0038 0 0.0014 0.0021 0.0011 0.0001 0.001461 Uncertain significance|Likelynot_specified|Cerebrooculofacioskeletal_Syndrome|Cockayne_syndrome|Macular_degenerationRCV000170374.3|RCV000289884.1|RCV000344840.1|RCV000384253.1 benign|LikelyMedGen|MedGen|MedGen:Orphanet:SNOMED_CT|Human_Phenotype_Ontology:MedGenCN169374|CN239231|C0009207:ORPHA191:21086008|HP:0000608:C1849131 benign|Likely0/1:36:65 benign 0/0:0:41 :8:95 :2:76 0/0:0:80 1 0 chr10 88451687 88451687 G A exonic LDB3 . nonsynonymousLDB3:NM_001080115:exon5:c.724G>A:p.A242T,LDB3:NM_007078:exon5:c.724G>A:p.A242T,LDB3:NM_001080114:exon6:c.583G>A:p.A195T,LDB3:NM_001080116:exon6:c.583G>A:p.A195T,LDB3:NM_001171611:exon6:c.928G>A:p.A310T,LDB3:NM_001171610:exon7:c.928G>A:p.A310T SNV rs759790522ID=COSM240477;OCCURENCE=1(prostate)0.00015 . . 4.123e-05 0 0 0 0.0002 1.5e-05 0 0.0002 ...... 0/1:43:84 0/0:0:63 :16:151 :5:87 0/0:0:102 1 0 chr11 113283437 113283437 T C exonic DRD2 . nonsynonymousDRD2:NM_016574:exon6:c.892A>G:p.K298E,DRD2:NM_000795:exon7:c.979A>G:p.K327E SNV rs71653614. 0.02226 0.002795530.0099 0.0038 0.0005 0.0005 0 0.0222 0.0039 0.0099 0.0024 0.002540 Benign Dystonia RCV000229349.1MedGen C0393593 0/1:34:79 0/0:0:74 0/1:94:123 1/1:73:80 0/0:0:98 1 0,0000040865chr14 74706508 74706508 G A exonic VSX2 . nonsynonymousVSX2:NM_182894:exon1:c.244G>A:p.G82R SNV rs375294678. . . . 0.0010 0 0 0 0 0.0026 0 0 0.000265 Uncertain significance|UncertainVSX2-related_Microphthalmia|Microphthalmia,_isolated_6|not_specifiedRCV000307977.1|RCV000346344.1|RCV000350100.1MedGen|MedGen:OMIM|MedGen significance|UncertainCN239429|C3150757:613517|CN1693740/1:13:29 significance0/0:0:16 1/1:40:43 1/1:22:23 0/0:0:24 0,99999999870,0000000025chr14 94754643 94754643 C T exonic SERPINA10. stopgain SERPINA10:NM_001100607:exon3:c.972G>A:p.W324X,SERPINA10:NM_016186:exon3:c.972G>A:p.W324Xrs61754487. 0.01564 0.001397760.007 0.0042 0.0011 0.0005 0 0.0159 0.0055 0.0088 0.0012 0.004229 Uncertain significanceVenous_thrombosis,_susceptibility_toRCV000005413.3MedGen CN068878 0/1:10:25 0/0:0:27 1/1:25:27 1/1:24:30 0/0:0:34 1 0 chr16 88495349 88495349 G A exonic ZNF469 . nonsynonymousZNF469:NM_001127464:exon1:c.1471G>A:p.A491T SNV rs117555121. . 0.009984030.0089 0.0014 0 0 0.0394 0 0.0008 0.0078 0.0005 . Likely benignCorneal_fragility_keratoglobus,_blue_sclerae_AND_joint_hypermobilityRCV000405817.1MedGen:OMIM:SNOMED_CTC0268344:229200:317980040/1:41:82 0/0:0:69 1/1:124:135 1/1:71:76 0/0:0:94 1 0,0000080096chr19 8369919 8369921 CTC - exonic CD320 . nonframeshiftCD320:NM_016579:exon2:c.262_264del:p.88_88del deletionrs150384171. . 0.006988820.0089 0.0088 0.0027 0.0144 0.0133 0.0028 0.0102 0.0102 0.0033 0.010144 Uncertain significance|Likelynot_specified|not_providedRCV000185812.2|RCV000224888.1 benignMedGen|MedGenCN169374|CN2218090/1:14:31 0/0:0:15 :2:42 :2:25 0/0:0:19 1 0 chr19 11210979 11210979 G T exonic LDLR . nonsynonymousLDLR:NM_000527:exon2:c.148G>T:p.A50S,LDLR:NM_001195798:exon2:c.148G>T:p.A50S,LDLR:NM_001195799:exon2:c.148G>T:p.A50S,LDLR:NM_001195800:exon2:c.148G>T:p.A50S,LDLR:NM_001195803:exon2:c.148G>T:p.A50S SNV rs137853960. . . . 0.0007 0.0003 0.0008 0 0 0.0010 0 0 0.000538 Likely benign,notFamilial_hypercholesterolemia,not_provided|Hypercholesterolaemia|Familial_hypercholesterolemia provided|UncertainRCV000238452.1,RCV000058916.1|RCV000148578.1|RCV000210234.2MedGen:OMIM:SNOMED_CT:SNOMED_CT,MedGen|MedGen|MedGen:OMIM:SNOMED_CT:SNOMED_CT significance|otherC0020445:143890:397915002:398036000,CN221809|C0020443|C0020445:143890:397915002:3980360000/1:55:82 0/0:0:62 :11:115 :7:86 0/0:0:91 1 0,0000000013chr19 49969403 49969403 C T exonic ALDH16A1 . nonsynonymousALDH16A1:NM_001145396:exon13:c.1648C>T:p.R550C,ALDH16A1:NM_153329:exon14:c.1801C>T:p.R601C SNV rs201175681ID=COSM3389210;OCCURENCE=1(pancreas). 0.0009984030.005 0.0016 0.0011 0 0 0.0526 0.0024 0.0060 0.0003 0.000079 . . . . . 0/1:14:26 0/0:0:28 :2:67 :2:25 0/0:0:34 1 0 chr22 37471208 37471208 G A exonic TMPRSS6 . nonsynonymousTMPRSS6:NM_001289000:exon11:c.1309C>T:p.R437W,TMPRSS6:NM_001289001:exon11:c.1309C>T:p.R437W,TMPRSS6:NM_153609:exon11:c.1336C>T:p.R446W SNV rs117576908. 0.04842 0.003594250.0119 0.0084 0.0014 0.0016 0 0.0523 0.0081 0.0155 0.0052 0.005382 Benign|Likelynot_provided|Microcytic_anemia benign RCV000224834.1|RCV000364393.1MedGen|MedGen:OMIM:Orphanet:SNOMED_CTCN221809|C0085576:206200:ORPHA209981:2343490070/1:42:94 0/0:0:65 :8:128 :5:74 0/0:0:92 Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Sample_name Sample_type Library_type Aimed_target_coverageAverage_target_coverage Tot al_reads Tot al_m apped_reads Mother blood Exome 50x 43x 73997330 73587399 (99.45%) Mother tumor Exome 100x 79x 145658968 144581977 (99.26%) Child blood Exome 50x 75x 103122034 102484161 (99.38%) Child tumor Exome 100x 69x 134697146 133652537 (99.22%) Child_FFPE tumor_FFPE Exome 100x 129x 243597842 237735683 (97.59%) Downloaded from molecularcasestudies.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Whole-exome sequencing identifies germline mutation in TP53 and ATRX in a child with ge-nomically aberrant AT/RT and her mother with anaplastic astrocytoma

Kristiina Nordfors, Joonas Haapasalo, Ebrahim Afyounian, et al.

Cold Spring Harb Mol Case Stud published online March 30, 2018 Access the most recent version at doi:10.1101/mcs.a002246

Supplementary http://molecularcasestudies.cshlp.org/content/suppl/2018/03/26/mcs.a00224 Material 6.DC1

Published online March 30, 2018 in advance of the full issue.

Accepted Peer-reviewed and accepted for publication but not copyedited or typeset; Manuscript accepted manuscript is likely to differ from the final, published version. Published onlineMarch 30, 2018in advance of the full issue.

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Published by Cold Spring Harbor Laboratory Press