Archives of Dentistry and Oral Health ISSN: 2638-4809 Volume 3, Issue 1, 2020, PP: 01-05 Preliminary Investigations on Distinct Proteomics Profiles for Different Subtypes of Non-Syndromic Cleft Palate Based on SELDI-TOF-MS Hong He1,2#, Xueting Wang1, Xinyue Zhang1, Ying Wang1, Guoyun Wang3#*, Feiyun Ping3* 1

2

The Affiliated3 Stomatology Hospital, Zhejiang University School of Medicine, Hangzhou, China. Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou China. *Co-Corresponding Authors: Guoyun Wang, Feiyun Ping. Affiliated Second Hospital, School of Medicine, Zhejiang University, China. #Co-first Authors: Hong He, Guoyun Wang.

Abstract Cleft lip and palate usually affect patients in speech, hearing, appearance and psychology, and they need multidisciplinary care from birth to adulthood. However, it is still difficult to make a clear and early diagnosis of cleft palate during pregnancy. The study of differential proteomics will increase our understanding for the pathogenesis and manifestations of cleft palate, and help find methods to assist in early screening, prevent or even in treating cleft palate. Thirty clinical patients with cleft lip and palate and thirty normal controls were collected, the expression between different groups was detected by SELDI-TOF-MS technology, and the differentially expressed were analyzed. In between the cleft palate only (CPO) group and the normal control group, the differently expressed protein peaks observed were 4180.3542 and 5639.2742. In between the CPO group and the complete cleft palate and lip group, the peaks were 3291.2347, 4812.2454, 4969.911 and 7791.0462. And in between the unilateral cleft palate and lip group and the bilateral cleft palate and lip group, the peaks were 2529.6336 and 5069.8178. These results may benefit the research on the etiology and early detection of non-syndromic cleft palate. Keywords: Non-syndromic cleft palate; SELDI-TOF-MS; Proteomics; Etiology

Introduction including genetic factors with highly genetically heterogeneous and environmental risk factors [5]. Cleft palate and lip are the most common congenital deformities of the oral and maxillofacial region, arising In recent years, with the popularization of routine in approximately 1.7 per 1000 live births worldwide examinations during pregnancy and the improvement with ethnic and geographic variation [1]. Cleft palate of the performance of ultrasound imaging technology, can occur alone or concurrently with cleft lip and can fetuses with cleft lip can be detected through early pregnancy tests, and the pregnancy can be terminated only (CPO), unilateral in time. However, it’s still a challenge to make a cleft palate and lip, and bilateral cleft palate and lip be classified into cleft palate clear diagnosis of cleft palate during pregnancy with reporting errors range from 10-60%, because palate problems with speaking, feeding, hearing, appearance [2]. Children suffering these disorders usually have is an internal structure which can be easily shadowed and psychology, and need multidisciplinary care by the surrounding bones and it’s largely associated from birth to adulthood [1,3]. While some cleft palate with characterization of the secondary palate [5,6]. is part of genetic syndrome with others systemic Nevertheless, the detecting accuracy is improving diseases or malformations such as congenital heart with newer technologies, such as three-dimensional defects (31.1%), deformations (22.4%), hydrocephaly ultrasound using modern information technology [7]. (11.2%) [4], the 50-70% of orofacial clefts are non- Moreover, the recurrence of cleft is quite common and syndromic clefts, which are caused by multiple factors unpredictable. 1 Archives of Dentistry and Oral Health V3 . I1. 2020 Preliminary Investigations on Distinct Proteomics Profiles for Different Subtypes of Non-Syndromic Cleft Palate Based on SELDI-TOF-MS Palatal development is a complex process involving cleft palate and lip, and bilateral cleft palate and lip. Meanwhile, 30 children (18 males, 12 females), pathways, transcription factors and cell-surface from healthy children outpatients for regular oral receptors,different whichgene are expression, manifested a via network proteins. of Lulin signaling examination were recruited and set as healthy control /loci associated there is no biological kinship among them. All patients analysis,Huang et al. including [8] identified IRF6 several, PAX9, DLK1, POMGNT2, andgroup. healthy All the controls subjects participating aged between in 0.3-6the experiment years, and WHSC1,to non-syndromic DOCK9, FOXC2-FOXL1, orofacial cleft MAU2 (NSOFC) for CPO, by mate-IRF6, have understood the relevant content and obtained a MYCN, VAX1 and MAFB for cleft palate with cleft signed, written, informed consent. lip and revealed that developmental transcription Collection and Disposition of Serum Samples of orofacial cleft. We drew 4mL fasting peripheral blood of all the indicatedfactors (TFs) some played molecular important pathways roles relevant in the regulation to proper Several animal model studies, have were put at room temperature for 0.5-1h. Then the subjects in the early morning, and the blood specimens signaling pathways [2]. sera was separated by 20-30min centrifugation (3000 closure of the palate, such as BMP, TGF-β SHH, FGF r/ and With the development and application of proteomics placed in -20°C refrigerator. technology, it’s possible to analyze the proteins min), then drawn into an EP tube with label Laboratory Procedures of Serum Samples screening, and discover the relevant characteristic After thawing the serum samples at room temperature expressed differentially by large-scale high-throughput proteins in the process of cleft palate, which can and 5min centrifugation (3000r/min, 4°C), 10 increase our understanding about the etiology of non-syndromic cleft palate and improve prevention, mol/ (3-[(3-cholamidopropyl)μL diagnosis, treatment, and prognosis for patients with dimethylammonio]-1-propanesulfonate),sample was diluted with 90μL 0.5% U9 buffer pH7.14), (9 orofacial cleft. The proteomics technology, surface L urea, 0.15% CHAPS enhanced laser desorption/ 60minthen added at 4°C. to After albumin 5min affinity centrifugation resin (CibacronBlue (2000r/min, the molecular weight of ionizedionization protein time-of-flight and draw 4°C3GA,) Sigma) , shaking at 600r/min and reacting for amass mass spectrometry spectrum with(SELDI-TOF-MS) high sensitivity. [9,10], Comparing can detect may Bioprocessor, 50μL supernatant with H4 chip was installed diluted (Ciphergen) to 200μL with for HEPES (20mmol/L, pH7.14), and added to a 96-well the protein profiles of different groups, we find 60min oscillatory reaction (600r/min, 4°C), then specific proteins related to the disease. patientsTherefore, and in our normal research, human we used controls, SELDI-TOF-MS which may to time.discarded Wash the the supernatant. chip with deionized Wash the water chip 2with times 200μL and screen the differential serum protein between cleft HEPES (20mmol/L, pH7.14) 3 times and 5min each cleft palate. CHCA (Ciphergen) to the chip after drying and repeat. benefit the research on the etiology of non-syndromic 1min each time. Add 0.15μL energy absorbing molecule Methods SELDI-TOF-MS Biotechnology Clinical Date Finally dry the chip ready for later experiment. Test the equipment operation and detector sensitivity. Thirty cleft pediatric patients (18 males, 12 females) Correct the accuracy and make the error rate less than 0.1% using the peptide molecular mass standard who were presented and treated at Department of the maximum protein molecular weight was 30000, fromOral and March Maxillofacial 2005 to January Surgery, 2006, the Second were recruited, Affiliated the(Ciphergen range Biosystems). of peaks detected Set parameters automatically as follows: was excludingHospital ofpatients Zhejiang with University syndromes School as we of know, Medicine such 2000~30000 m/z, the laser intensity was 135 and 65 as Van der Woude syndrome, Robin’s syndrome, laser shots for per sample. Meckel syndrome, velo-cardiofacial syndrome ). The study group was divided into 4 subtypes, The protein chip plate containing the detection including cleft lip only, cleft palate only, unilateral samples was put into the mass spectrometer, and (VCFs Archives of Dentistry and Oral Health V3 . I1 . 2020 2 Preliminary Investigations on Distinct Proteomics Profiles for Different Subtypes of Non-Syndromic Cleft Palate Based on SELDI-TOF-MS was used to collect the data (VersionSELDI Protein 3.0, Ciphergen Biology System Biosystems) II plus (PBSwas used II plus) to peaksResu withlts differential expression between groups. correct the original data to make. ProteinChip the total ion Softwareintensity and molecular weight of the data uniform. Then filter mass to charge ratio (m/z) were observed (table 1). The statistically differential protein peak values of noise by In between the cleft palate only group and the normal the secondary to 1.0 Biomarker Wizard (Ciphergen, setting the initial noise filter value to 2.2 and In between , which m/z peaks existed in more control group, the differently expressed protein peaks USA) software was used for preliminary screening of the cleft palate only group and the cleft palate and than 10% of samples with the deviation of the same observed were 4180.3542 and 5639.2742. differential proteins In between the unilateral lip group, the peaks were 3291.2347, 4812.2454, proteinStatistical m/z Analysispeak in different samples less than 0.3%. cleft palate and lip group and the bilateral cleft 4969.911 and 7791.0462. Kruskal-Wallis multivariate analysis of variance used palate and s lip group, the peaks were 2529.6336 and toTable1 analyze. Differential the data ofprotein different peak groupvalues to find protein 5069.8178. Groups Differential protein peak values (p<0.01) Cleft palate only vs Normal controls Cleft palate only vs Cleft palate and lip nilateral cleft palate and lip vs Bilateral cleft palate and lip 4180.3542, 5639.2742 3291.2347, 4812.2454, 4969.911, 7791.0462 DiscussionU of cleft2529.6336, palate. After 5069.8178 the emergence of proteomics In our study, t or combined with have providedtechnology, new differential technical protein support screening for the proteomics, functional or more groups,he and peak P intensityvalue indicates reflects the the degree content of studysuch asof disease SELID-TOF genes, proteins, cell functions,LCMS, clinical difference ofT protein with the same PSR between two manifestations. peptidesdifference. between he differential protein peaks suggest the existence of differential functional proteins or surface-enhanced adsorption of special chips. The chip by various genetic genes. different Lulin Huanggroups, et al.and [8] thathave the The ly principle undergoes of special SELDI treatment technology to isdirectly based drop on the subtypes of non-syndromic/ cleft palate are affected sample to be tested on the chip surface (the sample to genes/loci (IRF6, PAX9, DLK1, POMGNT2, WHSC1, befirst tested can be derived from serum, urine, secretion, DOCK9,identified FOXC2-FOXL1, 18 genes loci MAU2 responsible) were for associated NSOFC, withnine CPO, seven genes/loci (IRF6, DLK1, MYCN, VAX1, MAFB, GRM5, ALX1) were associated with cleft lip only (CLO), surfacecell lysate, of the tissue, solid etc.),phase then matrix, specific the impurity proteins protein in the and four genes/loci (IRF6, MYCN, VAX1, MAFB) were andsample other are contaminants adsorbed to on the the chip chip by are affinity. washed On away the associated with cleft lip and palate, from which we can

iswith dried, a buffer it is bombardedsolution, and with then a an laser energy in aabsorbing vacuum find IRF6 and DLK1 were related to both CPO and CLO. cleft palate with cleft lip and tube.molecular After (EAM) absorbing is added energy, to thethe chip.protein After is resolvedthe chip So, our results can be the supporting evidence for the and ionized. It is detached from the surface of the chip proteins.different mechanisms of CPO on the protein pathway and different structural the cathode, where it is detected by an ion detector. risk factor for CPO but is protective for CLO oppositely under the force of an electric field and flew toward/z) of The down-regulated expression dosage of IRF6 is a the pathogenesis of CLO/CPO. And it’s necessary to do Due to the different mass-to-charge ratios (m [11,12]. The developmental TFs play critical roles in these ions, the flight time in the vacuum tube is also role of proteins on the occurrence and development isdifferent. detected. Among them, the small flight time is short, further identification of the protein and confirm the the fastest flight and the first to reach the ion detector 3 Archives of Dentistry and Oral Health V3 . I1. 2020 Preliminary Investigations on Distinct Proteomics Profiles for Different Subtypes of Non-Syndromic Cleft Palate Based on SELDI-TOF-MS with cleft lip, unilateral and bilateral cleft palate and information quickly. By comparing the maps of normal lip, hopefully providing some data for the study on peopleSELDI software with those can ofprocess patients and withanalyze a certain mass spectrum disease, pathogenesis of cleft.

In the past, chromatography, two-dimensional Acknowledgement electrophoresis,we can find and capturespectroscopy, the disease-specific mass spectrometry proteins. and The work was supported by the Teaching Reform other techniques were used in protein research. These technologies are not suitable for large-scale population yxyb20172030. National and provincial Health screening and clinical examination because of their Project of Zhejiang University School of - Medicine high requirements, expensive instruments, tedious ste Commission Co-construction Fund WKJ ZJ-1623. National Health Commission Public Welfare Fund andps mass and spectrometer. long time-consuming. The sample does SELDI-TOF-MS not need to (201502018). National Key R&D Program of China technology combines the specific protein chip system Baoshi Oversea 2016YFC0902702. 2016 Fund for 151 talents second of serum, urine, tissue and cell lysate to the chip for level Zhejiang Province 2014-2017. be purified in advance. It can directly add the samples detection. The obtained map is simple and repeatable. Scholarship zups1507 2013-2015. China Scholarship It can detect the protein with molecular weight of 200- Council [2009]3004. Scientific Research Foundation 500kd, especially for the detection of low abundance for the Returned Oversea Chinese Scholar J20120036 protein or polypeptide. The analysis of a sample can fromConsent HR Office and of EthicalZhejiang Province. Approval be completed in tens of minutes, and the amount of information processed is far greater than that of two- According to international standard, patients’ written dimensional electrophoresis. This technology can be consent and ethical approval have been collected and used for protein quantitative analysis which can not preserved by the authors. References number of samples without pretreatment with high be carried out by MALDI [3]. It can analyze a small [1] Mossey PA, Little J, Munger RG, et al. Cleft lip suitable for basic medical research, clinical diagnosis andthroughput, large-scale high population efficiency screeningand high sensitivity, [4]. and is 1773-85. and palate. Lancet. 2009 Nov 21; 374 (9703): Children aged 0.3-6 years were selected, avoiding [2] content after puberty [13]. Meanwhile familial cleft Burg ML, Chai Y, Yao CA, et al. Epidemiology, lipsignificant and palate changes patients in the hormone were excluded, and body reducing protein Etiology, and Treatment of Isolated Cleft Palate. [3] genetic interference and improving the reliability Front Physiol. 2016; 7: 67. Provided that the Leslie EJ, Marazita ML. Genetics of cleft lip and relevant characteristic proteins can be discovered in cleft palate. Am J Med Genet C Semin Med Genet. of differential protein research. the process of cleft lip and palate, the treatment and [4] 2013 Nov;163C(4):246-258. interference of cleft lip and palate can be guided on the protein pathway. Through the reconstruction RegistryP. Mossey, MeetingE. Catilla. Global on Craniofacial Registry and Anomalies. Database of protein function, the clinical performance of the on Craniofacial Anomalies: Report of a WHO cleft lip and palate can stay or turn to the normal apps.who.int/iris/handle/10665/42840 sequence. Geneva: World Health Organization. 2003. https:// [5] However, due to the limited sample size, there’s a VueTutschek rendering B, Blaas technology HK. 3D in ultrasound assessment and of fetal the fetal palate. Re: Qualitative evaluation of Crystal furtherlack of intensive statistical studies significance are needed. for the comparison of protein spectrum differences. More samples and lip and palate. Ultrasound Obstet Gynecol 2017; Conclusion [6] 50: 274–277 Steinberg JP, Gosain AK. Thirty Years of Prenatal between cleft palate and normal, cleft palate only and This paper reports the differential serum proteome Cleft Diagnosis: What Have We Learned? Plast 4 Archives of Dentistry and Oral Health V3 . I1 . 2020 Reconstr Surg. 2015 Sep;136(3):550-7. Preliminary Investigations on Distinct Proteomics Profiles for Different Subtypes of Non-Syndromic Cleft Palate Based on SELDI-TOF-MS [7] W . current overview. Arch Physiol Biochem. 2010

weeksójtowicz of gestation A, Wójtowicz based on an W, analysis Jurek J,of etstatic al [11] Oct-Dec;116(4-5):188-96. ultrasoundEvaluation ofimages the fetalusing palate modern at 11IT techniques. to 13 (+6) VariantsZucchero and TM, the Cooper Risk of ME, Isolated Maher Cleft BS, Lip et al.or Interferon Regulatory Factor 6 (IRF6) [8] PrenatH Diagn. 2018 May;38(6):414-421.ic factors [12] , Ling W, Guli, et al. Association of single- uang L, Jia Z, Shi Y, et al. Genet Palate. N Engl J Med 2004; 351:769-780 define CPO and CLO subtypes of nonsyndro non-syndromicMijiti A cleft lip with or without cleft micorofacial cleft. PLoS Genet. 2019 Oct 14; nucleotide polymorphisms in the IRF6 gene with 15(10):e1008357. enhanced laser desorption ionization time-of- palate in the Xinjiang Uyghur population. British [9] Seibert V, Wiesner A, Buschmann T, et al. Surface- Journal of Oral and Maxillofacial Surgery, 2015, [13] ProteinChip technology in proteomics research. 53(3):268-274. flight mass spectrometry (SELDI TOF-MS) and NonsyndromicHe H, Zhang MY, Cleft Wang Lip with Y, et or al. without Differencial Cleft Serum Proteomes between Subphenotypes of [10] Pathol Res Pract. 2004;200(2):83-94. , Gemoll T, Roblick UJ, Auer G, et al. SELDI-TOF Palate. Archives of Dentistry and Oral Health V2. serum proteomics and colorectal cancer: a I2. 2019 2(2):24-28

Citation: Hong He#, Xueting Wang, Xinyue Zhang, Ying Wang, Guoyun Wang#*, Feiyun Ping*. ­­­­­Preliminary Investigations on Distinct Proteomics Profiles for Different Subtypes of Non-Syndromic Cleft Palate Based on SELDI-TOF-MS. Archives of Dentistry and Oral Health. 2020; 3(1): 01-05. Copyright: © 2020 Hong He, Xueting Wang, Xinyue Zhang, Ying Wang, Guoyun Wang, Feiyun Ping. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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