Case report Genetic variation analysis in a Chinese Maffucci syndrome patient Yang Xue a , Jinwen Ni a , Mi Zhou a , Weiqi Wang b , Yuan Liu c , Yaowu Yang b, ** , Xiaohong Duan a, * a State Key Laboratory of Military Stomatology, Department of Oral Biology, Clinic of Oral Rare and Genetic Diseases, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi'an 710032, PR China b 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, PR China 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, PR China article info Article history: Paper received 7 January 2015 Accepted 26 May 2015 Available online 3 June 2015 Keywords: Copy number variation IDH1 IDH2 Maffucci syndrome Microarray analysis 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, the coding exons and exon/intron splice junctions of the IDH1 and IDH2 genes were amplified by polymerase chain re- action (PCR) and then sequenced. Genomic DNA was extracted from blood and a hemangioma sample from the patient, and also from her mother's blood, for chromosome microarray analysis (CMA) by Affymetrix CytoScan HD array. 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, 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. Conclusions: This is the first molecular genetic analysis report on a Chinese patient 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. © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. 1. 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 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). In recent years, the genetic background of Maffucci syndrome has drawn great attention. It has been reported that somatic mosaic isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are associ- ated with enchondromas and spindle cell hemangiomas in Maffucci syndrome (Amaryet 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 muta- tions found in a hemangioma sample. A 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). 2. Case report 2.1. Patient data 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 * Corresponding author. Tel./fax: þ86 29 84776169. ** Corresponding author. Tel./fax: þ86 29 84772503. E-mail addresses: [email protected] (X. Duan), [email protected] (Y. Yang) Contents lists available at ScienceDirect Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com http://dx.doi.org/10.1016/j.jcms.2015.05.017 1010-5182/© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e1255
Genetic variation analysis in a Chinese Maffucci syndrome patient
Dec 26, 2022
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Genetic variation analysis in a Chinese Maffucci syndrome patientContents lists avai
Genetic variation analysis in a Chinese Maffucci syndrome patient
Yang Xue a, Jinwen Ni a, Mi Zhou a, Weiqi Wang b, Yuan Liu c, Yaowu Yang b, **, Xiaohong Duan a, *
a State Key Laboratory of Military Stomatology, Department of Oral Biology, Clinic of Oral Rare and Genetic Diseases, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi'an 710032, PR China b 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, PR China 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, PR China
a r t i c l e i n f o
Article history: Paper received 7 January 2015 Accepted 26 May 2015 Available online 3 June 2015
Keywords: Copy number variation IDH1 IDH2 Maffucci syndrome Microarray analysis
* Corresponding author. Tel./fax: þ86 29 84776169 ** Corresponding author. Tel./fax: þ86 29 84772503
E-mail addresses: [email protected] (X. Duan (Y. Yang)
http://dx.doi.org/10.1016/j.jcms.2015.05.017 1010-5182/© 2015 European Association for Cranio-M
a b s t r a c t
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, the coding exons and exon/intron splice junctions of the IDH1 and IDH2 genes were amplified by polymerase chain re- action (PCR) and then sequenced. Genomic DNA was extracted from blood and a hemangioma sample from the patient, and also from her mother's blood, for chromosome microarray analysis (CMA) by Affymetrix CytoScan HD array. 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, 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. Conclusions: This is thefirstmolecular genetic analysis report on aChinese patientwithMaffucci 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.
© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
1. 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 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).
In recent years, the genetic background of Maffucci syndrome has drawn great attention. It has been reported that somatic mosaic
. . ), [email protected]
axillo-Facial Surgery. Published by
isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are associ- atedwith enchondromas and spindle cell hemangiomas inMaffucci 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 muta- tions found in a hemangioma sample. A 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).
2. Case report
2.1. Patient data
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
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Y. Xue et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e1255 1249
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 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.
Radiographs showed multiple, irregularly shaped radiolucent areas with stippled calcification on bilateral pelvis and the left femur, humerus, ulna, radius, and index finger (Fig. 2AeD). The long bones on the left side of her body (including the femur, tibia, fibula, humerus, ulna and radius) were significantly shorter than those on the right. Computed tomographic angiography (CTA) with three dimensional reconstruction displayed the worm-eaten appearance of bone destruction involving the sternum, occipital
Fig. 1. Clinical features of the patient: The patient had obvious submandibular swelling on buccal mucosa (F) and tongue (G); unequal leg lengths (H); and multiple vascular malform
bone, bilateral pelvis, scapula, ribs, humerus, radius and thumb, and left femur and index finger (Fig. 2EeG). Both computed to- mography (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 bone involvement of the chin could also be seen (Fig. 2F and H).
The subcutaneous lesions in her hands and feet were diagnosed as venous malformation by physical examination and Doppler ul- trasonography. No obvious changewas found in the comprehensive metabolic panel (including general tests, electrolytes, and assess- ment 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. 3A); and a biopsy showed that the lesion in the left humerus was an enchondroma (Fig. 3B). On the basis of these findings e the development of multiple enchondromas in the extremities and the presence of subcutaneous vascular lesions e the diagnosis of Maffucci syndrome was established.
anterior and lateral views (AeC); small vascular malformations on her ears (D and E), ations on her hands and feet (IeN).
Fig. 2. Radiological features of the patient: (AeD) X-ray examination showed multiple, irregularly shaped radiolucent areas with stippled calcification on bilateral 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 radius) were significantly shorter than on the right side. (EeG) 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 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.
Y. Xue et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e12551250
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.
2.2. Genomic DNA preparation
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).
2.3. IDH1 and IDH2 mutation analysis
Using the gDNA extracted from the patient's hemangioma sample, the coding 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 with an ABI 377 Sequencer (PerkineElmer Corp., Norwalk, Conneticut, USA). Sequence variants were identi- fied using DNAStar, MegAlign 5.01 (Demonstration System DNAS- TAR, Inc., Madison, USA).
2.4. Single-nucleotide polymorphism (SNP) array analysis
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 Data- base of Genomic Variants (DGV, http://projects.tcag.ca/variation/).
3. Results
3.1. 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.
3.2. Results of CytoSan HD array
Both the analyzed tumor and the blood samples had chromo- somal aberrations. A summary of the CNVs in different categories and detailed information are presented in Tables 1 and 2, respec- tively. We found 15 regions with copy number gain and 25 regions with copy number loss in the patient's hemangioma tissue when compared with the patient's or her mother's blood samples. Dis- tribution of the CNVs in chromosomes was shown in Fig. 4. Chro- mosome 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 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
Fig. 3. Histologic examination of the tumors: (A) Histological pattern of the hem- angioma 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.
Y. Xue et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e1255 1251
22q13.2 may play an important role in osteosarcoma (259500) and colorectal cancer (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). Three of the 22 known genes in this region are related to human disease.
Table 1 Distribution of copy number variations (CNVs) in different categories.
Category of CNVs No. of CNVs (gain/loss)
Total CNVs from the patient's tumor and blood samples
50 (21/29)
CNVs from the patient's tumor sample 45 (18/27) CNVs from the patient's blood sample 5 (3/2) The patient's tumor-specific CNVsa 40 (15/25)
a The patient's tumor-specific CNVs means tumor CNVs not found in the patient's blood sample.
4. Discussion
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 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 attractedmuch attention. Parathyroid hormone receptor type 1 (PTHR1) gene mutationwas screened for in leukocyte and/or tumor DNA samples from 30 Maffucci syndrome patients, but none was detected (Couvineau et al., 2008; Pansuriya et al., 2011a; Rozeman et al., 2004). Other genes involved in the IHHePTHLH (Indian hedgehogeparathyroid hormone-like hormone) pathway, including parathyroid hormone related protein (PTHrP); Indian hedgehog (IHH); and guanine nucleotide binding protein, alpha stimulating activity polypeptide 1 (GNAS1); were tested in one Maffucci syndrome patient, but no mutationwas found (Couvineau et al., 2008). Recently, somatic mosaic mutations in the gene encoding IDH1 and IDH2 were found to be associated with enchondromas and spindle cell hemangiomas in Ollier disease and Maffucci 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 IDH1mutation 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 pa- tients (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 total, 10 out of 14 patients (71.4%) carried it (Table 5). We analyzed all the coding exons and introneexon 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 determine whether or not the IDH1-R132C mutation in hem- angiomas from Mafucci syndrome patients has male predomi- nance, because the number of tested patients is still limited.
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, somatic CNVs can be used to identify re- gions 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 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 somatic alterations were localized in 2p22.3, 2q24.3 and 14q11.2 (Amyere et al., 2014). Here, we report the first genome-wide analysis using Affymetrix CytoScan HDArray 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 500e600 bases (Wang et al., 2014). In addi- tion, this chip provides allelic imbalance information from SNPs. It has great power to detect known and novel chromosome aberra- tions across the entire human genome and features unbiased
Table 2 Regions with the patient's tumor-specific copy number variations (CNVs).
Location Start End Size (kbp) CN state Confidencea Genes OMIM number Phenotype (MIM number)
1p36.32 3318713 3540374 221.661 3Gainb 0.8713892 PRDM16 605557 Cardiomyopathy, dilated, 1LL (615373) Left ventricular noncompaction 8 (615373)
ARHGEF16 MEGF6 604266 MIR551A 615148
2q21.2-21.3c 135013377 135130039 116.662 3Gain 0.87145376 MGAT5 601774 4p16.1 10145295 10285183 139.888 3Gain 0.8776457 6q27 168207950 168353004 145.054 3Gain 0.87163925 C6ORF124 7q36.3 155439095 155568696 129.601 3Gain 0.8785316 RBM33 8p23.3 1797152 1885919 88.767 3Gain 0.87061596 ARHGEF10 608136 Slowed nerve conduction velocity (608236) 8p11.22 39247097 39386952 139.855 3Gain 0.878297 ADAM5P
ADAM3A 10q11.22 49882699 50010018 127.319 3Gain 0.8694517 WDFY4 613316 10q26.3 131362146 131465557 103.411 3Gain 0.8731973 MGMT 156569 11p15.5 2641128 2696586 55.458 3Gain 0.8749602 KCNQ1 607542 Atrial fibrillation, familial, 3 (607554)
Jervell and Lange-Nielsen syndrome (220400) Long QT syndrome-1 (192500) Short QT syndrome-2 (609621)
KCNQ1OT1 604115 BeckwitheWiedemann syndrome (130650) 12p13.33 2425429 2510942 85.513 3Gain 0.86910707 CACNA1C 114205 Timothy syndrome (601005)
Brugada syndrome 3 (611875) 12q13.13 52685758 52837735 151.977 3Gain 0.86786515 KRT86 601928 Monilethrix (158000)
KRT83 602765 Monilethrix (158000) KRT85 602767 Ectodermal dysplasia 4, hair/nail type (602032) KRT84 602766 KRT82 601078 KRT75 609025 Pseudofolliculitis barbae, susceptibility to (612318)
13q34 112712835 112823735 110.9 3Gain 0.8745371 SOX1 602148 18q12.2 34954712 35140658 185.946 3Gain 0.8710344 CELF4 612679 Xp22.13 17712240 17768581 56.341 3Gain 0.86663646 NHS 300457 NanceeHoran syndrome (302350)
Cataract 40, X-linked (302200) 2p15c 61717371 61832387 115.016 1Lossd 0.85956013 XPO1 602559 2q33.2c 204090886 204212172 121.286 1Loss 0.8628381 CYP20A1
ABI2 606442 3p14.3 57508034 57642045 134.011 1Loss 0.86074513 DNAH12 603340
PDE12 ARF4 601177 FAM116A
5p11- q11.1 46116069 49552685 3436.616 1Loss 0.88519186 5p13.2 37230368 37414508 184.14 1Loss 0.8529884 C5ORF42 614571 Joubert syndrome 17 (614615)
NUP155 606694 WDR70 MLLT4 159559
7q11.23 77257686 77349918 92.232 1Loss 0.8564842 PTPN12 600079 Colon cancer (114500) RSBN1L
7q22.3 104948315 105095529 147.214 1Loss 0.85920143 SRPK2 602980 8p12 30518798 30593771 74.973 1Loss 0.86142105 GSR 138300 Hemolytic anemia due to glutathione reductase deficiency 11p11.2 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 TRIM48 12q11-12 37959560 38428116 468.556 1Loss 0.8734972 12p13.31 7824993 7937572 112.579 1Loss 0.8595156 GDF3 606522 KlippeleFeil syndrome 3 (613702)
Microphthalmia with coloboma 6 (613703) Microphthalmia, isolated 7 (613704)
DPPA3 608408 CLEC4C 606677 NANOGNB
12p13.33 1019260 1073052 53.792 1Loss 0.8710307 WNK1 605232 Neuropathy, hereditary sensory and autonomic, type II (201300) Pseudohypoaldosteronism, type IIC (614492)
RAD52 600392 14q13.1e 34930992 35067437 136.445 1Loss 0.862398 C14OFR147 613540
EAPP 609486 SNX6 606098
17q21.33 49193876 49265359 71.483 1Loss 0.85486627 SPAG9 605430 NME1 156490 Neuroblastoma (256700) NME2 156491 MBTD1
17q23.2 59933393 59998268 64.875 1Loss 0.8602244 BRIP1 605882 Fanconi anemia, complementation group J (609054) Breast cancer, early onset (114480)
INTS2 611346 22q13.2 41019892 41078204 58.312 1Loss 0.86675566 MKL1 606078 Megakaryoblastic leukemia, acute
MCHR1
Y. Xue et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e12551252
Table 2 (continued )
Location Start End Size (kbp) CN state Confidencea Genes OMIM number Phenotype (MIM number)
22q12.3 36776985 36867803 90.818 1Loss 0.8657487 MYH9 160775 Macrothrombocytopenia and progressive sensorineural deafness (600208) MayeHegglin anomaly (155100) Fechtner syndrome (153640) Deafness, autosomal dominant 17 (603622) Sebastian syndrome (605249) Epstein syndrome (153650)
TXN2 609063 22q11.21 21197828 21254889 57.061 1Loss 0.86194324 SNAP29 604202 Cerebral dysgenesis, neuropathy, ichthyosis,
and palmoplantar keratoderma syndrome (609528) PI4KA 600286
22q12.1 29014907 29252732 237.825 1Loss 0.8577769 CHEK2f 604373 Breast and colorectal cancer, susceptibility to Prostate cancer, familial, susceptibility to (176807) Breast cancer, susceptibility to (114480) Osteosarcoma, somatic (259500) LieFraumeni syndrome (609265)
XBP1 194355 Major affective disorder-7, susceptibility to (612371) HSCB 608142 TTC28 615098 CCDC117
22q13.2 41288061 41499651 211.59 1Loss 0.8607409 EP300f 602700 Colorectal cancer, somatic (114500) RubinsteineTaybi syndrome (613684)
XPNPEP3 613553 Nephronophthisis-like nephropathy 1 (613159) RBX1 603814 MIR128-1 611774 SCML1 300227
Xp11.1 58428644 61923701 3495.057 1Loss 0.8884171 Xp22.33 1211761 1358900 147.139 1Loss 0.8741655 CRLF2 300357 Xq25 122899948 123020078 120.13 1Loss 0.8740075 XIAP 300079 Lymphoproliferative syndrome X-linked 2 (300635)
OMIM: Online Mendelian Inheritance in Man. a 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 the data fits the assigned
state for that marker. b 3Gain means heterozygous duplication. c Somatic alterations localized in chromosome 2, however far away from 2p22.3 and 2q24.3, reported by Amyere et al., 2014. d 1Loss means heterozygous loss. e Somatic alteration localized in chromosome 14 (14q13.1) nearby 14q11.2, reported by Amyere et al., 2014. f Somatic mutations in these genes may play a role in tumorigenesis.
Y. Xue et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e1255 1253
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…
Genetic variation analysis in a Chinese Maffucci syndrome patient
Yang Xue a, Jinwen Ni a, Mi Zhou a, Weiqi Wang b, Yuan Liu c, Yaowu Yang b, **, Xiaohong Duan a, *
a State Key Laboratory of Military Stomatology, Department of Oral Biology, Clinic of Oral Rare and Genetic Diseases, School of Stomatology, the Fourth Military Medical University, 145 West Changle Road, Xi'an 710032, PR China b 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, PR China 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, PR China
a r t i c l e i n f o
Article history: Paper received 7 January 2015 Accepted 26 May 2015 Available online 3 June 2015
Keywords: Copy number variation IDH1 IDH2 Maffucci syndrome Microarray analysis
* Corresponding author. Tel./fax: þ86 29 84776169 ** Corresponding author. Tel./fax: þ86 29 84772503
E-mail addresses: [email protected] (X. Duan (Y. Yang)
http://dx.doi.org/10.1016/j.jcms.2015.05.017 1010-5182/© 2015 European Association for Cranio-M
a b s t r a c t
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, the coding exons and exon/intron splice junctions of the IDH1 and IDH2 genes were amplified by polymerase chain re- action (PCR) and then sequenced. Genomic DNA was extracted from blood and a hemangioma sample from the patient, and also from her mother's blood, for chromosome microarray analysis (CMA) by Affymetrix CytoScan HD array. 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, 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. Conclusions: This is thefirstmolecular genetic analysis report on aChinese patientwithMaffucci 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.
© 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
1. 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 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).
In recent years, the genetic background of Maffucci syndrome has drawn great attention. It has been reported that somatic mosaic
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isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are associ- atedwith enchondromas and spindle cell hemangiomas inMaffucci 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 muta- tions found in a hemangioma sample. A 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).
2. Case report
2.1. Patient data
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
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Y. Xue et al. / Journal of Cranio-Maxillo-Facial Surgery 43 (2015) 1248e1255 1249
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 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.
Radiographs showed multiple, irregularly shaped radiolucent areas with stippled calcification on bilateral pelvis and the left femur, humerus, ulna, radius, and index finger (Fig. 2AeD). The long bones on the left side of her body (including the femur, tibia, fibula, humerus, ulna and radius) were significantly shorter than those on the right. Computed tomographic angiography (CTA) with three dimensional reconstruction displayed the worm-eaten appearance of bone destruction involving the sternum, occipital
Fig. 1. Clinical features of the patient: The patient had obvious submandibular swelling on buccal mucosa (F) and tongue (G); unequal leg lengths (H); and multiple vascular malform
bone, bilateral pelvis, scapula, ribs, humerus, radius and thumb, and left femur and index finger (Fig. 2EeG). Both computed to- mography (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 bone involvement of the chin could also be seen (Fig. 2F and H).
The subcutaneous lesions in her hands and feet were diagnosed as venous malformation by physical examination and Doppler ul- trasonography. No obvious changewas found in the comprehensive metabolic panel (including general tests, electrolytes, and assess- ment 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. 3A); and a biopsy showed that the lesion in the left humerus was an enchondroma (Fig. 3B). On the basis of these findings e the development of multiple enchondromas in the extremities and the presence of subcutaneous vascular lesions e the diagnosis of Maffucci syndrome was established.
anterior and lateral views (AeC); small vascular malformations on her ears (D and E), ations on her hands and feet (IeN).
Fig. 2. Radiological features of the patient: (AeD) X-ray examination showed multiple, irregularly shaped radiolucent areas with stippled calcification on bilateral 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 radius) were significantly shorter than on the right side. (EeG) 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 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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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.
2.2. Genomic DNA preparation
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).
2.3. IDH1 and IDH2 mutation analysis
Using the gDNA extracted from the patient's hemangioma sample, the coding 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 with an ABI 377 Sequencer (PerkineElmer Corp., Norwalk, Conneticut, USA). Sequence variants were identi- fied using DNAStar, MegAlign 5.01 (Demonstration System DNAS- TAR, Inc., Madison, USA).
2.4. Single-nucleotide polymorphism (SNP) array analysis
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 Data- base of Genomic Variants (DGV, http://projects.tcag.ca/variation/).
3. Results
3.1. 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.
3.2. Results of CytoSan HD array
Both the analyzed tumor and the blood samples had chromo- somal aberrations. A summary of the CNVs in different categories and detailed information are presented in Tables 1 and 2, respec- tively. We found 15 regions with copy number gain and 25 regions with copy number loss in the patient's hemangioma tissue when compared with the patient's or her mother's blood samples. Dis- tribution of the CNVs in chromosomes was shown in Fig. 4. Chro- mosome 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 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
Fig. 3. Histologic examination of the tumors: (A) Histological pattern of the hem- angioma 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.
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22q13.2 may play an important role in osteosarcoma (259500) and colorectal cancer (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). Three of the 22 known genes in this region are related to human disease.
Table 1 Distribution of copy number variations (CNVs) in different categories.
Category of CNVs No. of CNVs (gain/loss)
Total CNVs from the patient's tumor and blood samples
50 (21/29)
CNVs from the patient's tumor sample 45 (18/27) CNVs from the patient's blood sample 5 (3/2) The patient's tumor-specific CNVsa 40 (15/25)
a The patient's tumor-specific CNVs means tumor CNVs not found in the patient's blood sample.
4. Discussion
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 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 attractedmuch attention. Parathyroid hormone receptor type 1 (PTHR1) gene mutationwas screened for in leukocyte and/or tumor DNA samples from 30 Maffucci syndrome patients, but none was detected (Couvineau et al., 2008; Pansuriya et al., 2011a; Rozeman et al., 2004). Other genes involved in the IHHePTHLH (Indian hedgehogeparathyroid hormone-like hormone) pathway, including parathyroid hormone related protein (PTHrP); Indian hedgehog (IHH); and guanine nucleotide binding protein, alpha stimulating activity polypeptide 1 (GNAS1); were tested in one Maffucci syndrome patient, but no mutationwas found (Couvineau et al., 2008). Recently, somatic mosaic mutations in the gene encoding IDH1 and IDH2 were found to be associated with enchondromas and spindle cell hemangiomas in Ollier disease and Maffucci 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 IDH1mutation 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 pa- tients (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 total, 10 out of 14 patients (71.4%) carried it (Table 5). We analyzed all the coding exons and introneexon 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 determine whether or not the IDH1-R132C mutation in hem- angiomas from Mafucci syndrome patients has male predomi- nance, because the number of tested patients is still limited.
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, somatic CNVs can be used to identify re- gions 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 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 somatic alterations were localized in 2p22.3, 2q24.3 and 14q11.2 (Amyere et al., 2014). Here, we report the first genome-wide analysis using Affymetrix CytoScan HDArray 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 500e600 bases (Wang et al., 2014). In addi- tion, this chip provides allelic imbalance information from SNPs. It has great power to detect known and novel chromosome aberra- tions across the entire human genome and features unbiased
Table 2 Regions with the patient's tumor-specific copy number variations (CNVs).
Location Start End Size (kbp) CN state Confidencea Genes OMIM number Phenotype (MIM number)
1p36.32 3318713 3540374 221.661 3Gainb 0.8713892 PRDM16 605557 Cardiomyopathy, dilated, 1LL (615373) Left ventricular noncompaction 8 (615373)
ARHGEF16 MEGF6 604266 MIR551A 615148
2q21.2-21.3c 135013377 135130039 116.662 3Gain 0.87145376 MGAT5 601774 4p16.1 10145295 10285183 139.888 3Gain 0.8776457 6q27 168207950 168353004 145.054 3Gain 0.87163925 C6ORF124 7q36.3 155439095 155568696 129.601 3Gain 0.8785316 RBM33 8p23.3 1797152 1885919 88.767 3Gain 0.87061596 ARHGEF10 608136 Slowed nerve conduction velocity (608236) 8p11.22 39247097 39386952 139.855 3Gain 0.878297 ADAM5P
ADAM3A 10q11.22 49882699 50010018 127.319 3Gain 0.8694517 WDFY4 613316 10q26.3 131362146 131465557 103.411 3Gain 0.8731973 MGMT 156569 11p15.5 2641128 2696586 55.458 3Gain 0.8749602 KCNQ1 607542 Atrial fibrillation, familial, 3 (607554)
Jervell and Lange-Nielsen syndrome (220400) Long QT syndrome-1 (192500) Short QT syndrome-2 (609621)
KCNQ1OT1 604115 BeckwitheWiedemann syndrome (130650) 12p13.33 2425429 2510942 85.513 3Gain 0.86910707 CACNA1C 114205 Timothy syndrome (601005)
Brugada syndrome 3 (611875) 12q13.13 52685758 52837735 151.977 3Gain 0.86786515 KRT86 601928 Monilethrix (158000)
KRT83 602765 Monilethrix (158000) KRT85 602767 Ectodermal dysplasia 4, hair/nail type (602032) KRT84 602766 KRT82 601078 KRT75 609025 Pseudofolliculitis barbae, susceptibility to (612318)
13q34 112712835 112823735 110.9 3Gain 0.8745371 SOX1 602148 18q12.2 34954712 35140658 185.946 3Gain 0.8710344 CELF4 612679 Xp22.13 17712240 17768581 56.341 3Gain 0.86663646 NHS 300457 NanceeHoran syndrome (302350)
Cataract 40, X-linked (302200) 2p15c 61717371 61832387 115.016 1Lossd 0.85956013 XPO1 602559 2q33.2c 204090886 204212172 121.286 1Loss 0.8628381 CYP20A1
ABI2 606442 3p14.3 57508034 57642045 134.011 1Loss 0.86074513 DNAH12 603340
PDE12 ARF4 601177 FAM116A
5p11- q11.1 46116069 49552685 3436.616 1Loss 0.88519186 5p13.2 37230368 37414508 184.14 1Loss 0.8529884 C5ORF42 614571 Joubert syndrome 17 (614615)
NUP155 606694 WDR70 MLLT4 159559
7q11.23 77257686 77349918 92.232 1Loss 0.8564842 PTPN12 600079 Colon cancer (114500) RSBN1L
7q22.3 104948315 105095529 147.214 1Loss 0.85920143 SRPK2 602980 8p12 30518798 30593771 74.973 1Loss 0.86142105 GSR 138300 Hemolytic anemia due to glutathione reductase deficiency 11p11.2 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 TRIM48 12q11-12 37959560 38428116 468.556 1Loss 0.8734972 12p13.31 7824993 7937572 112.579 1Loss 0.8595156 GDF3 606522 KlippeleFeil syndrome 3 (613702)
Microphthalmia with coloboma 6 (613703) Microphthalmia, isolated 7 (613704)
DPPA3 608408 CLEC4C 606677 NANOGNB
12p13.33 1019260 1073052 53.792 1Loss 0.8710307 WNK1 605232 Neuropathy, hereditary sensory and autonomic, type II (201300) Pseudohypoaldosteronism, type IIC (614492)
RAD52 600392 14q13.1e 34930992 35067437 136.445 1Loss 0.862398 C14OFR147 613540
EAPP 609486 SNX6 606098
17q21.33 49193876 49265359 71.483 1Loss 0.85486627 SPAG9 605430 NME1 156490 Neuroblastoma (256700) NME2 156491 MBTD1
17q23.2 59933393 59998268 64.875 1Loss 0.8602244 BRIP1 605882 Fanconi anemia, complementation group J (609054) Breast cancer, early onset (114480)
INTS2 611346 22q13.2 41019892 41078204 58.312 1Loss 0.86675566 MKL1 606078 Megakaryoblastic leukemia, acute
MCHR1
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Table 2 (continued )
Location Start End Size (kbp) CN state Confidencea Genes OMIM number Phenotype (MIM number)
22q12.3 36776985 36867803 90.818 1Loss 0.8657487 MYH9 160775 Macrothrombocytopenia and progressive sensorineural deafness (600208) MayeHegglin anomaly (155100) Fechtner syndrome (153640) Deafness, autosomal dominant 17 (603622) Sebastian syndrome (605249) Epstein syndrome (153650)
TXN2 609063 22q11.21 21197828 21254889 57.061 1Loss 0.86194324 SNAP29 604202 Cerebral dysgenesis, neuropathy, ichthyosis,
and palmoplantar keratoderma syndrome (609528) PI4KA 600286
22q12.1 29014907 29252732 237.825 1Loss 0.8577769 CHEK2f 604373 Breast and colorectal cancer, susceptibility to Prostate cancer, familial, susceptibility to (176807) Breast cancer, susceptibility to (114480) Osteosarcoma, somatic (259500) LieFraumeni syndrome (609265)
XBP1 194355 Major affective disorder-7, susceptibility to (612371) HSCB 608142 TTC28 615098 CCDC117
22q13.2 41288061 41499651 211.59 1Loss 0.8607409 EP300f 602700 Colorectal cancer, somatic (114500) RubinsteineTaybi syndrome (613684)
XPNPEP3 613553 Nephronophthisis-like nephropathy 1 (613159) RBX1 603814 MIR128-1 611774 SCML1 300227
Xp11.1 58428644 61923701 3495.057 1Loss 0.8884171 Xp22.33 1211761 1358900 147.139 1Loss 0.8741655 CRLF2 300357 Xq25 122899948 123020078 120.13 1Loss 0.8740075 XIAP 300079 Lymphoproliferative syndrome X-linked 2 (300635)
OMIM: Online Mendelian Inheritance in Man. a 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 the data fits the assigned
state for that marker. b 3Gain means heterozygous duplication. c Somatic alterations localized in chromosome 2, however far away from 2p22.3 and 2q24.3, reported by Amyere et al., 2014. d 1Loss means heterozygous loss. e Somatic alteration localized in chromosome 14 (14q13.1) nearby 14q11.2, reported by Amyere et al., 2014. f Somatic mutations in these genes may play a role in tumorigenesis.
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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…
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