Genetic variation analysis in a Chinese Maffucci syndrome patient

Accepted Manuscript Genetic variation analysis in a Chinese Maffucci syndrome patient Yang Xue, MD, Jinwen Ni, MS, Mi Zhou, MS, Weiqi Wang, MS, Yuan Liu, MD, Yaowu Yang, MD, Professor, Xiaohong Duan, PHD, Professor PII:

S1010-5182(15)00160-2

DOI:

10.1016/j.jcms.2015.05.017

Reference:

YJCMS 2069

To appear in:

Journal of Cranio-Maxillo-Facial Surgery

Received Date: 7 January 2015 Revised Date:

25 May 2015

Accepted Date: 26 May 2015

Please cite this article as: Xue Y, Ni J, Zhou M, Wang W, Liu Y, Yang Y, Duan X, Genetic variation analysis in a Chinese Maffucci syndrome patient, Journal of Cranio-Maxillofacial Surgery (2015), doi: 10.1016/j.jcms.2015.05.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Genetic variation analysis in a Chinese Maffucci syndrome patient Yang Xuea MD, Jinwen Nia MS, Mi Zhoua MS, Weiqi Wangb MS, Yuan Liuc MD, Yaowu Yangb,* MD, Xiaohong Duana,* PHD a: State Key Laboratory of Military Stomatology, Department of Oral Biology, Clinic

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of Oral Rare and Genetic Diseases, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, P. R. China

b: State Key Laboratory of Military Stomatology, Department of Oral and

145 West Changle Road, Xi’an 710032, P. R. China

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Maxillofacial Surgery, School of Stomatology, the Fourth Military Medical University,

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c: State Key Laboratory of Military Stomatology, Department of Oral Histology and Pathology, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, P. R. China *Corresponding authors:

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Xiaohong Duan, Ph.D, Professor

State Key Laboratory of Military Stomatology, Department of Oral Biology, Clinic of Oral Rare and Genetic Diseases, School of Stomatology, the Fourth Military Medical

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University, 145 West Changle Road, Xi’an 710032, P. R. China

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Tel: 86-29-84776169；Fax: 86-29-84776169；E-mail: [email protected] Yaowu Yang, MD, Professor State Key Laboratory of Military Stomatology, Department of Oral and Maxillofacial Surgery, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi’an 710032, P. R. China Tel: 86-29-84772503；Fax: 86-29-84772503；E-mail: [email protected] Sources of grants: This work was supported in part by grants of the National Natural Science Foundation

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of China [81470728 (XD), 81271116 (XD), 81470757(YY)].

ACCEPTED MANUSCRIPT Abstract Objective: To report on the molecular genetic analysis of a Chinese patient with Maffucci syndrome. Methods: Using the genomic DNA extracted from the patient’s hemangioma sample,

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the coding exons and exon/intron splice junctions of the IDH1 and IDH2 genes were amplified by polymerase chain reaction (PCR) and then sequenced. Genomic DNA

was extracted from blood and a hemangioma sample from the patient, and also from

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her mother’s blood, for chromosome microarray analysis (CMA) by Affymetrix CytoScan HD array.

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Results: None of the known pathogenic mutations in the whole IDH1 or IDH2 genes was found in the patient’s hemangioma sample. CMA detected 40 tumor-specific copy number variations (CNVs), and one copy number neutral loss of heterozygosity (LOH) region. Among the 73 known genes included in the 40 CNV regions, only 2 genes,

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CHEK2 (604373) located in 22q12.1 and EP300 (602700) located in 22q13.2, were found to be related to tumorigenesis. We did not find any CNVs at the IDH1 and IDH2 loci.

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Conclusions: This is the first molecular genetic analysis report on a Chinese patient

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with Maffucci syndrome and our data enrich the understanding of the genetic background of Maffucci syndrome in different ethnic groups. The relationship between CHEK2, EP300 and Maffucci syndrome needs to be further explored. Keywords: copy number variation, IDH1, IDH2, Maffucci syndrome, microarray analysis

ACCEPTED MANUSCRIPT Introduction Maffucci syndrome is a rare, nonhereditary and congenital mesodermal dysplasia, characterized by a combination of multiple enchondromas and hemangiomas (Pansuriya et al., 2011a). It was first described in 1881 by Maffucci, and about 200

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cases have been described in the literature to date (Cai et al., 2013; Ono et al., 2012). There is neither sex preponderance nor a difference in incidence among races (Amary et al., 2011; Gao et al., 2013; Jermann et al., 2001).

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In recent years, the genetic background of Maffucci syndrome has drawn great

attention. It has been reported that somatic mosaic isocitrate dehydrogenase 1 (IDH1)

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and IDH2 mutations are associated with enchondromas and spindle cell hemangiomas in Maffucci syndrome (Amary et al., 2011; Amyere et al., 2014; Pansuriya et al., 2011b). Here, we report on a Chinese female patient affected by Maffucci syndrome, with no somatic mosaic IDH1 or IDH2 mutations found in a hemangioma sample. A

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high-resolution Affymetrix CytoScan HD Array was used to detect the possible related copy number variation (CNV) as well as copy number neutral loss of heterozygosity (LOH).

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Patient data

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Materials and methods

At first presentation, the patient was 18 years old; she weighed 40 kg and was 126

cm tall. Her left leg was 20 cm shorter than her right leg, while her left arm was 10 cm shorter than her right arm. She was born to non-consanguineous and healthy parents. Her mother had a history of two spontaneous abortions, and the patient was born at 28 weeks with no specified cause for the premature birth. She had a healthy younger brother. She complained of a progressive but painless mass in the submental area for more than 2 years. The patient’s leg lengths had been unequal since she was

ACCEPTED MANUSCRIPT one year old, and she had significantly enlarged limbs and joints and multiple purple lesions on her hands and feet since she was 3 years old (Fig.1). The symptoms gradually worsened with age. Her first menstrual period was at 18 years old; her periods were irregular with small amounts of bleeding.

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Radiographs showed multiple, irregularly shaped radiolucent areas with stippled calcification on bilateral pelvis and the left femur, humerus, ulna, radius, and index

finger (Fig.2 A–D). The long bones on the left side of her body (including the femur,

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tibia, fibula, humerus, ulna and radius) were significantly shorter than those on the right. Computed tomographic angiography (CTA) with three dimensional

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reconstruction displayed the worm-eaten appearance of bone destruction involving the sternum, occipital bone, bilateral pelvis, scapula, ribs, humerus, radius and thumb, and left femur and index finger (Fig.2 E–G). Both computed tomography (CT) and CTA showed an extensive soft tissue mass of about 6.4 × 4.5 × 7.0 cm in the submental

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area. Multiple punctate calcification in the mass and bone involvement of the chin could also be seen (Fig.2 F and H).

The subcutaneous lesions in her hands and feet were diagnosed as venous

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malformation by physical examination and Doppler ultrasonography. No obvious

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change was found in the comprehensive metabolic panel (including general tests, electrolytes, and assessment of renal and hepatic function). Pathological examination after surgical resection of part of the lesions showed that the lesions in the tongue and submental area were venous malformations (Fig.3 A); and a biopsy showed that the lesion in the left humerus was an enchondroma (Fig.3 B). On the basis of these findings – the development of multiple enchondromas in the extremities and the presence of subcutaneous vascular lesions – the diagnosis of Maffucci syndrome was established.

ACCEPTED MANUSCRIPT With written informed consent, the patient and her mother were included in this study. The study was authorized by the Ethics Committee, School of Stomatology, the Fourth Military Medical University, Xi’an, China. Genomic DNA preparation

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Blood and fresh hemangioma samples were obtained from the patient. Genomic DNA (gDNA) was extracted using QIAamp DNA blood and a tissue mini kit, respectively (Qiagen Inc., Chatsworth, CA, USA).

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IDH1 and IDH2 mutation analysis

Using the gDNA extracted from the patient’s hemangioma sample, the coding

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exons and exon/intron splice junctions of the IDH1 and IDH2 genes were amplified by polymerase chain reaction (PCR). Primers and PCR amplification conditions were previously described (Amary et al., 2011). PCR products were purified with DNA Fragment Quick Purification/Recover Kit (DingGuo, Beijing, China) and sequenced

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with an ABI 377 Sequencer (Perkin-Elmer Corp., Norwalk, Conneticut, USA). Sequence variants were identified using DNAStar, MegAlign 5.01 (Demonstration System DNASTAR, Inc., Madison, USA).

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Single-nucleotide polymorphism (SNP) array analysis

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Genomic DNA was extracted from the patient’s blood and hemangiomas for chromosome microarray analysis (CMA) by Affymetrix CytoScan HD array (Affymetrix, Inc., Santa Clara, CA, USA). Hybridizations were performed according to the manufacturer’s protocols. The data obtained were analyzed using the Chromosome Analysis Suite software package (Affymetrix, Inc., Santa Clara, CA, USA). Gene annotation and gene overlap were determined using the human genome build 19 (hg19). All the identified CNVs were compared with those reported in the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation/).

ACCEPTED MANUSCRIPT Results IDH1 and IDH2 mutation analysis Neither IDH1-R132C/IDH2-R172S nor other mutations in the whole of the IDH1 or IDH2 genes were found in the patient’s hemangioma sample.

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Results of CytoSan HD array

Both the analyzed tumor and the blood samples had chromosomal aberrations. A summary of the CNVs in different categories and detailed information are presented

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in Tables 1 and 2, respectively. We found 15 regions with copy number gain and 25 regions with copy number loss in the patient’s hemangioma tissue when compared

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with the patient’s or her mother’s blood samples. Distribution of the CNVs in chromosomes was shown in Fig 4. Chromosome 22 exhibited the most frequent losses (20%); and chromosome X represented 6.7% of all gains and 12% of all losses. Most of the identified regions were known to have common CNV in the Database of

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Genomic Variants. Seventy-three known genes were included in the 40 regions, while 25 were found to be related to human disease (Table 2). In particular, somatic mutations in CHEK2 (604373) located in 22q12.1 and EP300 (602700) located in

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22q13.2 may play an important role in osteosarcoma (259500) and colorectal cancer

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(114500). We did not see any CNVs at the IDH1 and IDH2 loci. A summary of the LOH in different categories is presented in Table 3. We also

found an LOH region (Xq22.3 104232592-107407242) positive in the patient’s tumor tissue but negative in the patient’s blood sample (Table 4). After analysis, we found that the patient’s tumor-specific copy number neutral LOH located in Xq22.3 was caused by maternal uniparental disomy (data was not shown). Two of the 22 known genes in this region are related to human disease. Discussion

ACCEPTED MANUSCRIPT The typical clinical presentation of Maffucci syndrome includes multiple enchondromas and vascular lesions, which are commonly associated with phleboliths (Biber et al., 2004; Cai et al., 2013). It can be diagnosed relatively easily solely on clinical grounds (Gao et al., 2013). The diagnosis of Maffucci syndrome was

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established for the present patient on the basis of her typical presentation and pathological examination.

In recent years, the genetic background of Maffucci syndrome has attracted much

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attention. Parathyroid hormone receptor type 1 (PTHR1) gene mutation was screened for in leukocyte and/or tumor DNA samples from 30 Maffucci syndrome patients, but

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none was detected (Couvineau et al., 2008; Pansuriya et al., 2011a; Rozeman et al., 2004). Other genes involved in the IHH–PTHLH (Indian hedgehog–parathyroid hormone-like hormone) pathway, including parathyroid hormone related protein (PTHrP); Indian hedgehog (IHH); and guanine nucleotide binding protein, alpha

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stimulating activity polypeptide 1 (GNAS1); were tested in one Maffucci syndrome patient, but no mutation was found (Couvineau et al., 2008). Recently, somatic mosaic mutations in the gene encoding IDH1 and IDH2 were found to be associated with

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enchondromas and spindle cell hemangiomas in Ollier disease and Maffucci

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syndrome (Amary et al., 2011; Amyere et al., 2014; Pansuriya et al., 2011b). According to these reports, 18 out of 24 tested patients (75.0%) carried IDH1 mutation rather than IDH2, and IDH1-R132C was the only hotspot mutation (Table 5). The mutation was absent in the DNA from patients' blood, muscle or saliva (Pansuriya et al., 2011b). With an analysis of the published data on the tested hemangioma DNA samples of Maffucci syndrome patients (Amary et al., 2011; Amyere et al., 2014; Pansuriya et al., 2011b), we found that 4 out of 7 female patients (57.1%) and 6 out of 7 male patients (85.7%) carried an IDH1-R132C mutation. In

ACCEPTED MANUSCRIPT total, 10 out of 14 patients (71.4%) carried it (Table 5). We analyzed all the coding exons and intron–exon junctions of the IDH1 and IDH2 genes in the hemangioma DNA sample from our patient; however, neither the hotspot mutation nor any other known pathogenic mutation could be detected. Further research is needed to

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determine whether or not the IDH1-R132C mutation in hemangiomas from Mafucci syndrome patients has male predominance, because the number of tested patients is still limited.

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Recently, CNV has been recognized as one of the most important genomic

alterations that plays a role in cancer pathogenesis (Iafrate et al., 2004). In addition,

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somatic CNVs can be used to identify regions of the genome that are involved in disease phenotypes (Chen et al., 2013; Jasmine et al., 2012; Sanjmyatav et al., 2011). Though genome-wide analysis of CNV and LOH using Affymetrix SNP 6.0 array on enchondromas and chondrosarcomas from four Maffucci syndrome patients was

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reported (Pansuriya et al., 2011a), the causative gene was not identified. Using the Affymetrix SNP 6.0 array, recent research which included nine frozen tumor samples from patients with Maffucci syndrome found that the most frequently encountered

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somatic alterations were localized in 2p22.3, 2q24.3 and 14q11.2 (Amyere et al.,

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2014). Here, we report the first genome-wide analysis using Affymetrix CytoScan HD Array on hemangioma and blood samples from a Chinese patient with Maffucci syndrome. The Affymetrix CytoScan HD Array (a CNV-targeted array) is based on the validated Genome-Wide Human SNP Array 6.0 and characterized by 2.6 million CNV markers including approximately 750,000 genotype-able SNP probes and 1,900,000 non-polymorphism probes, with the median inter-marker distance of 500–600 bases (Wang et al., 2014). In addition, this chip provides allelic imbalance information from SNPs. It has great power to detect known and novel chromosome

ACCEPTED MANUSCRIPT aberrations across the entire human genome and features unbiased whole-genome coverage, with the highest physical coverage of the genome (Chen et al., 2013; Veerappa et al., 2013). After scan and analysis, we found 40 specific CNVs and one copy number neutral

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LOH region located in the X chromosome from the patient’s tumor tissue; however,

most of the identified regions were known to have common CNVs in the Database of Genomic Variants. There was no overlap between our result and the regions reported

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by Pansuriya et al. (2011a). Compared with the results of Amyere et al.(2014), we

found one somatic alteration localized in chromosome 14 (14q13.1) near to 14q11.2;

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and three somatic alterations localized in chromosome 2 (2q21.2-21.3, 2p15, 2q33.2) however, far away from 2p22.3 and 2q24.3. Ninety-five known genes were included in the CNV and LOH regions. According to the information published by OMIM, neither genes related to IDH1/IDH2, nor genes associated with angiogenesis,

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chondroma formation, growth or sexual characteristics were found, except for CHEK2 (604373) and EP300 (602700) , which have been reported to play important roles in tumorigenesis.

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CHK2, a protein kinase that is activated in response to DNA damage, is involved

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in cell cycle arrest. In addition to the relationship between somatic mutation of CHEK2 and osteosarcoma (259500), large sample clinical trials have found germline and somatic CHEK2 mutations in both sporadic and familial prostate cancer, suggesting that CHEK2 mutations may contribute to the development of prostate cancer through the reduction of CHK2 activation in response to DNA damage and/or oncogenic stress (Dong et al., 2003; Wu et al., 2006). Furthermore, Schutte et al. (2003) and Meijers-Heijboer et al. (2003) found the 1100delC in CHEK2 makes an appreciable contribution to breast and colorectal cancer susceptibility. The EP300

ACCEPTED MANUSCRIPT gene encodes p300, a histone acetyltransferase that regulates transcription via chromatin remodeling and is important in the processes of cell proliferation and differentiation (Gayther et al., 2000). Gayther et al. found that the EP300 gene had mutated in epithelial cancers (colorectal cancer and breast cancer) as well as cancer

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cell lines (colorectal, breast, and pancreatic) and provided the first evidence that it behaved as a classic tumor suppressor gene. Thereafter, Pasqualucci et al. (2011)

found that some patients with diffuse large B-cell lymphoma and follicular lymphoma

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displayed genomic deletions and/or somatic mutations in the EP300 gene. Recently, Le Gallo et al. (2012) identified somatic mutation in the EP300 gene in 13 primary

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serous endometrial tumors and 40 additional serous tumors. However, further research is needed to determine whether or not these two genes play a role in the development of enchondromas and hemangiomas. Conclusion

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No genetic study of an Asian patient with Maffucci syndrome had previously been reported. Here, for the first time, we detected genetic variations in hemangioma tissue and blood from a Chinese patient with Maffucci syndrome. No known pathogenic

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mutation was found in the IDH1 or IDH2 genes in the hemangioma sample.

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Additionally, we found 40 tumor-specific CNVs and 1 LOH region using Affymetrix CytoScan HD array. CHEK2 and EP300 might be the causative genes, this needs further confirmation. Our data enriches the understanding of the genetic background of Maffucci syndrome in different ethnic groups.

ACCEPTED MANUSCRIPT Competing interests The authors declare that they have no competing interests. Authors' contributions XD critically revised the manuscript, contributed to the study design, analysis, and

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supervised the research. YY coordinated clinical diagnosis, collected phenotype

information and contributed to the study design. YX drafted and critically revised the manuscript, performed the statistical analyses and the interpretation of the data. MZ

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performed the blood collection and DNA extraction. JN performed IDH1 and IDH2 mutation analysis. WW coordinated clinical diagnosis and collected phenotype

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information. YL coordinated histologic investigation and the description. All authors read and approved the final manuscript. Acknowledgments

We are thankful for the agreement of the patient and her family members for

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joining in this research.

ACCEPTED MANUSCRIPT References Amary MF, Damato S, Halai D, Eskandarpour M, Berisha F, Bonar F, et al.: Ollier disease and Maffucci syndrome are caused by somatic mosaic mutations of

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karyotyping. Mol Syndromol 5: 259-267, 2014

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Biber C, Ergun P, Turay UY, Erdogan Y, Hizel SB: A case of Maffucci 's syndrome with pleural effusion: ten-year follow-up. Ann Acad Med Singapore 33: 347-350, 2004

Cai Y, Wang R, Chen XM, Zhao YF, Sun ZJ, Zhao JH.: Maffucci syndrome with the

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spindle cell hemangiomas in the mucosa of the lower lip: a rare case report and literature review. J Cutan Pathol 40: 661-666, 2013 Chen W, Yuan L, Cai Y, Chen X, Chi Y, Wei P, et al.: Identification of chromosomal

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ACCEPTED MANUSCRIPT Gao H, Wang B, Zhang X, Liu F, Lu Y: Maffucci syndrome with unilateral limb: a case report and review of the literature. Chin J Cancer Res 25: 254-258, 2013 Gayther SA, Batley SJ, Linger L, Bannister A, Thorpe K, Chin SF, et al.: Mutations

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truncating the EP300 acetylase in human cancers. Nat Genet 24: 300-303, 2000 Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, et al.: Detection of large-scale variation in the human genome. Nat Genet 36: 949-951, 2004

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Jasmine F, Rahaman R, Dodsworth C, Roy S, Paul R, Raza M, et al.: A genome-wide

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study of cytogenetic changes in colorectal cancer using SNP microarrays: opportunities for future personalized treatment. PLoS One 7: e31968, 2012 Jermann M, Eid K, Pfammatter T, Stahel R: Maffucci's syndrome. Circulation 104: 1693, 2001

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Le Gallo M, O'Hara AJ, Rudd ML, Urick ME, Hansen NF, O'Neil NJ, et al.: Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Nat Genet 44:

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Ono S, Tanizaki H, Fujisawa A, Tanioka M, Miyachi Y: Maffucci syndrome complicated with meningioma and pituitary adenoma. Eur J Dermatol 22:130-131, 2012 Pansuriya TC, Oosting J, Verdegaal SH, Flanagan AM, Sciot R, Kindblom LG, et al.:

ACCEPTED MANUSCRIPT Maffucci syndrome: a genome-wide analysis using high resolution single nucleotide polymorphism and expression arrays on four cases. Genes Chromosomes Cancer 50: 673-679, 2011a

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Pansuriya TC, van Eijk R, d'Adamo P, van Ruler MA, Kuijjer ML, Oosting J, et al.: Somatic mosaic IDH1 and IDH2 mutations are associated with enchondroma and spindle cell hemangioma in Ollier disease and Maffucci syndrome. Nat Genet 43:

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1256-1261, 2011b

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Pasqualucci L, Dominguez-Sola D, Chiarenza A, Fabbri G, Grunn A, Trifonov V, et al.: Inactivating mutations of acetyltransferase genes in B-cell lymphoma. Nature 471: 189-195, 2011

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Cleton-Jansen AM, et al., Enchondromatosis (Ollier disease, Maffucci syndrome) is not caused by the PTHR1 mutation p.R150C. Hum Mutat 24: 466-473, 2004 Sanjmyatav J, Junker K, Matthes S, Muehr M, Sava D, Sternal M, et al.: Identification

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of genomic alterations associated with metastasis and cancer specific survival in

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clear cell renal cell carcinoma. J Urol 186: 2078-2083, 2011 Schutte M, Seal S, Barfoot R, Meijers-Heijboer H, Wasielewski M, Evans DG, et al.: Variants in CHEK2 other than 1100delC do not make a major contribution to breast cancer susceptibility. Am J Hum Genet 72: 1023-1028, 2003

Veerappa AM, Vishweswaraiah S, Lingaiah K, Murthy M, Manjegowda DS, Nayaka R, et al.: Unravelling the complexity of human olfactory receptor repertoire by copy number analysis across population using high resolution arrays. PLoS One

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cancer. Hum Mutat 27: 742-747, 2006

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Wu X, Dong X, Liu W, Chen J: Characterization of CHEK2 mutations in prostate

ACCEPTED MANUSCRIPT Figure legends Fig. 1 Clinical features of the patient: The patient had obvious submandibular swelling on anterior and lateral views (A–C); small vascular malformations on her

multiple vascular malformations on her hands and feet (I–N).

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ears (D and E), buccal mucosa (F) and tongue (G); unequal leg lengths (H); and

Fig. 2 Radiological features of the patient: (A–D) X-ray examination showed

multiple, irregularly shaped radiolucent areas with stippled calcification on bilateral

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pelvis and the left femur, humerus, ulna, radius, and index finger. It also showed that the long bones on her left side (including the femur, tibia, fibula, humerus, ulna and

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radius) were significantly shorter than on the right side. (E–G) Computed tomographic angiography (CTA) with three dimensional reconstruction showing the worm-eaten appearance of bone destruction involving the sternum, occipital bone, bilateral pelvis, scapula, ribs, humerus, radius and thumb, and left femur and index

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finger. (F and H) Both computed tomography (CT) and CTA showed an extensive soft tissue mass of about 6.4 × 4.5 × 7.0 cm in the submental area. Multiple punctate calcification in the mass and chin bone involvement also could be seen.

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Fig. 3 Histologic examination of the tumors: (A) Histological pattern of the

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hemangioma showing broad, thin-walled blood vessels, lined by a single layer of flat endothelial cells and filled with blood, within the skin dermis. (B) The enchondromas consist of abundant hyaline cartilage matrix. The chondrocytes are situated within lacunar spaces, have uniform small round nuclei, and finely granular, often vacuolated, eosinophilic cytoplasm. Fig. 4 Distribution of the copy number variations (CNVs) in chromosomes

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Table 1 Distribution of copy number variations (CNVs) in different categories Category of CNVs

No. of CNVs (gain/loss) 50 (21/29)

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Total CNVs from the patient’s tumor and blood samples

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CNVs from the patient’s tumor sample CNVs from the patient’s blood sample The patient’s tumor-specific CNVsa

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The patient’s tumor-specific CNVs means tumor CNVs not found in the patient’s blood sample.

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a

45 (18/27) 5 (3/2) 40 (15/25)

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Size Location

Start

CN

OMIM

a

End

Confidence

Genes

state

3540374

221.661 3Gainb

135130039

116.662 3Gain

10145295

10285183

139.888 3Gain

6q27

168207950

168353004

7q36.3

155439095

155568696

8p23.3

1797152

1885919

0.87145376

Cardiomyopathy, dilated, 1LL (615373) 605557 Left ventricular noncompaction 8 (615373)

ARHGEF16 MEGF6

604266

MIR551A

615148

MGAT5

601774

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135013377

4p16.1

0.8713892

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2q21.2-21.3c

3318713

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PRDM16

1p36.32

Phenotype (MIM number)

number

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(kbp)

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Table 2 Regions with the patient’s tumor-specific copy number variations (CNVs)

0.8776457

145.054 3Gain

0.87163925

C6ORF124

129.601 3Gain

0.8785316

RBM33

88.767 3Gain

0.87061596

ARHGEF10

608136

Slowed nerve conduction velocity (608236)

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8p11.22

39247097

39386952

139.855 3Gain

0.878297

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ADAM5P ADAM3A 49882699

50010018

127.319 3Gain

0.8694517

WDFY4

10q26.3

131362146

131465557

103.411 3Gain

0.8731973

MGMT

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10q11.22

KCNQ1

2641128

2696586

55.458 3Gain

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2425429

52685758

2510942

52837735

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12p13.33

85.513 3Gain

151.977 3Gain

156569 Atrial fibrillation, familial, 3 (607554) Jervell and Lange-Nielsen syndrome (220400) 607542 Long QT syndrome-1 (192500)

0.8749602

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11p15.5

613316

0.86910707

Short QT syndrome-2 (609621)

KCNQ1OT1

CACNA1C

604115

Beckwith–Wiedemann syndrome (130650)

Timothy syndrome (601005) 114205 Brugada syndrome 3 (611875)

0.86786515

KRT86

601928

Monilethrix (158000)

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KRT83

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KRT85

602765

KRT84

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KRT82

Xp22.13

2p15 c 2q33.2 c

112823735

110.9 3Gain

0.8745371

34954712

35140658

185.946 3Gain

0.8710344

17712240

17768581

61717371

61832387

204090886

204212172

EP

18q12.2

112712835

56.341 3Gain

AC C

13q34

TE D

KRT75

0.86663646

SOX1 CELF4

NHS

Monilethrix (158000) Ectodermal dysplasia 4, hair/nail type

602767

(602032)

602766 601078 Pseudofolliculitis barbae, susceptibility to 609025 (612318) 602148 612679 Nance–Horan syndrome (302350) 300457 Cataract 40, X-linked (302200)

115.016 1Lossd

0.85956013

XPO1

121.286 1Loss

0.8628381

CYP20A1

602559

ACCEPTED MANUSCRIPT

606442

RI PT

ABI2 DNAH12

3p14.3

57508034

57642045

134.011 1Loss

0.86074513

SC

PDE12

M AN U

ARF4

603340

601177

FAM116A

37230368

49552685

37414508

3436.616 1Loss

0.88519186

184.14 1Loss

7q22.3

77257686

104948315

77349918

105095529

AC C

7q11.23

92.232 1Loss

147.214 1Loss

C5ORF42

614571

NUP155

606694

Joubert syndrome 17 (614615)

0.8529884

EP

5p13.2

46116069

TE D

5p11- q11.1

WDR70 MLLT4

159559

PTPN12

600079

0.8564842 RSBN1L 0.85920143

SRPK2

602980

Colon cancer (114500)

ACCEPTED MANUSCRIPT

Hemolytic anemia due to glutathione 30593771

74.973 1Loss

0.86142105

48709714

48925789

216.075 1Loss

0.88030905

11p11.12

50434607

51377161

942.554 1Loss

0.868982

11p11.12- q11

51581310

55039996

3458.686 1Loss

0.8715354

12q11-12

37959560

38428116

468.556 1Loss

0.8734972

TE D

7937572

112.579 1Loss

reductase deficiency

TRIM48

GDF3

Klippel–Feil syndrome 3 (613702) 606522

Microphthalmia with coloboma 6 (613703) Microphthalmia, isolated 7 (613704)

0.8595156

EP

7824993

AC C

12p13.31

138300

M AN U

11p11.2

GSR

RI PT

30518798

SC

8p12

DPPA3

608408

CLEC4C

606677

NANOGNB

ACCEPTED MANUSCRIPT

WNK1 1019260

1073052

53.792 1Loss

0.8710307

SC

12p13.33

RI PT

Neuropathy, hereditary sensory and

RAD52

14q13.1e

34930992

35067437

136.445 1Loss

M AN U

C14OFR147

0.862398

EAPP

17q23.2

59933393

49265359

59998268

71.483 1Loss

0.85486627

EP

49193876

AC C

17q21.33

TE D

SNX6

64.875 1Loss

0.8602244

SPAG9 NME1 NME2

605232

autonomic, type II (201300) Pseudohypoaldosteronism, type IIC (614492)

600392 613540 609486 606098 605430 156490

Neuroblastoma (256700)

156491

MBTD1

BRIP1

Fanconi anemia, complementation group J 605882 (609054)

ACCEPTED MANUSCRIPT

INTS2

41019892

41078204

58.312 1Loss

0.86675566

606078

TE D

36867803

90.818 1Loss

MYH9

Macrothrombocytopenia and progressive sensorineural deafness (600208) May–Hegglin anomaly (155100) 160775

Fechtner syndrome (153640)

0.8657487 Deafness, autosomal dominant 17 (603622)

EP

36776985

AC C

22q12.3

Megakaryoblastic leukemia, acute

MCHR1

M AN U

22q13.2

611346

SC

MKL1

RI PT

Breast cancer, early onset (114480)

Sebastian syndrome (605249) Epstein syndrome (153650) TXN2

609063

ACCEPTED MANUSCRIPT

SNAP29 21197828

21254889

57.061 1Loss

0.86194324

SC

22q11.21

RI PT

Cerebral dysgenesis, neuropathy, ichthyosis,

M AN U

PI4KA

29252732

237.825 1Loss

(609528)

Breast and colorectal cancer, susceptibility to Prostate cancer, familial, susceptibility to (176807) 604373 Breast cancer, susceptibility to (114480) Osteosarcoma, somatic (259500)

0.8577769

EP

29014907

AC C

22q12.1

and palmoplantar keratoderma syndrome

600286

TE D

CHEK2f

604202

Li–Fraumeni syndrome (609265) XBP1

Major affective disorder-7, susceptibility to 194355 (612371)

HSCB

608142

ACCEPTED MANUSCRIPT

TTC28

RI PT

615098

CCDC117

M AN U

SC

EP300 f

XPNPEP3

22q13.2

41288061

41499651

211.59 1Loss

TE D 58428644

61923701

3495.057 1Loss

0.8884171

Xp22.33

1211761

1358900

AC C

EP

Xp11.1

0.8741655

Xq25

122899948

123020078

147.139 1Loss

120.13 1Loss

602700

Rubinstein–Taybi syndrome (613684) Nephronophthisis-like nephropathy 1 613553 (613159)

0.8607409

RBX1

0.8740075

MIR128-1 SCML1

CRLF2

XIAP

Colorectal cancer, somatic (114500)

603814 611774 300227

300357 Lymphoproliferative syndrome X-linked 2 300079 (300635)

ACCEPTED MANUSCRIPT

Confidence: An indicator of the likelihood that the segment represents a real change in that region of the genome and is a measure of how likely

RI PT

a

the data fits the assigned state for that marker; b3Gain means heterozygous duplication; cSomatic alterations localized in chromosome 2, however

SC

far away from 2p22.3 and 2q24.3, reported by Amyere et al., 2014; d1Loss means heterozygous loss; eSomatic alteration localized in

M AN U

chromosome 14 (14q13.1) nearby 14q11.2, reported by Amyere et al., 2014; fSomatic mutations in these genes may play a role in tumorigenesis.

AC C

EP

TE D

OMIM: Online Mendelian Inheritance in Man.

ACCEPTED MANUSCRIPT

No. of LOH

Total CNVs from the patient’s tumor and blood samples

11

LOH from the patient’s tumor sample

6

LOH from the patient’s blood sample

5

The patient’s tumor-specific LOHa

1

EP

M AN U

TE D

The patient’s tumor-specific LOH means tumor LOH not found in the patient’s blood samples.

AC C

a

SC

Category of LOH

RI PT

Table 3 Distribution of copy number neutral loss of heterozygosity (LOH) in different categories

ACCEPTED MANUSCRIPT

Size Start

End

Confidence

Genes

(kbp)

number 300277

M AN U

IL1RAPL2 TEX13A

300312

NRK

300791 314200

TE D

SERPINA7 MUM1L1

107407242

3174.65

1

CXORF57

EP

104232592

RNF128

AC C

Xq22.3

300439

TBC1D8B RIPPLY1 CLDN2

Phenotype (MIM number)

SC

Location

OMIM

RI PT

Table 4 Regions with the patient’s tumor-specific copy number neutral loss of heterozygosity (LOH)

300575 300520

Thyroxine-binding globulin deficiency

ACCEPTED MANUSCRIPT

RI PT

MORC4 RBM41

M AN U

CXORF41

SC

NUP62CL

AC C

TSC22D3 MID2 TEX13B

Deafness X-linked 1 (304500) Arts syndrome (301835)

311850

Gout, PRPS-related (300661) Phosphoribosylpyrophosphate synthetase superactivity

EP

TE D

PRPS1

Charcot-Marie-Tooth disease, X-linked recessive, 5 (311070)

(300661) 300506 300204 300313

ACCEPTED MANUSCRIPT

ATG4A

300663 303631

AC C

EP

TE D

M AN U

COL4A6

300880

RI PT

PSMD10

300620

SC

VSIG1

Leiomyomatosis, diffuse, with Alport syndrome (308940)

ACCEPTED MANUSCRIPT

No. of patients tested

mutation positive

with hemangioma

(%)

sample

10

6 (60.0%)

Male

14

12 (85.7%)

Total

24

18 (75.0%)

M AN U

Female

IDH1-R132C

mutation positive

SC

IDH1-R132C

(%)

7

4 (57.1%)

7

6 (85.7)

14

10 (71.4%)

TE D

No. of patients

RI PT

Table 5 The relationship between IDH1-R132C mutation state and gender of patients with Maffucci syndrome

AC C

EP

The information was summarized from the following literature: Amary et al., 2011; Amyere et al., 2014; Pansuriya et al., 2011b

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT