Patient Liver Heart Vertebra Facies Eye
140 + + - - -
227 + + + - -
311 + - - + +
406 + + + - -
Alagille Syndrome
Autosomal Dominant
Disease
Occurs once every 100000
live births
Diagnosed clinically by the presence of three of five crucial
symptoms
Paucity of bile duct, vertebral deformities,
posterior embryotoxon, pulmonary defects,
typical facial appearance
Approximately 90% of all patients reported to carry a
mutated JAG1 gene
JAG1 gene36 kb, 26 exons,
vast exon and intron lengths
JAG-1 Protein
Implicated in tissue patterning, cell fate determination and
morphogenesis
Acts as ligand for NOTCH1 receptor
in the NOTCH signalling pathway
Consists of evolutionary conserved domains: EGF repeats and DSL regions
essential for ligand receptor interaction
Variable expressivity of
disease
Misdiagnosis using standard clinical
procedure due to patients manifesting
mild end of phenotypic spectrum of disease
High Resolution Melt Analysis
DNA Sequencing
PolyPhen Analysis
Patient 406’s SNP in evolutionary conserved DSL does not affect production of full
length protein or change conformationMutations in other non coding regions can be screened to confirm diagnosis
Uncovering the molecular basis of syndromic intrahepatic bile duct paucity in local patients
Glen Yeo1, Tan Bee Leng2, Marion Aw2, Quak Seng Hock2, Lai Poh San2
1SRP Student, Hwa Chong Institution, Singapore 269734 2Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228
Results and Discussion
Alagille Syndrome (AGS) is an autosomal dominant rare disease caused by mutations in JAG1 gene which codes for a protein regulating cell fate determination. It is characterised by cholestasis with paucity of interlobular bile ducts and anomalies of the cardiovascular system, skeleton, eyes, and face. This disease is caused by mutations in JAG1 gene which produces a dysfunctional protein. There is wide variation in clinical symptoms making clinical diagnosis challenging. Due to the rarity of this disease and the high incidence of new mutations, uncovering the molecular basis of patients with such clinical symptoms is important to provide a definite basis for the disease. In this study, four patients with AGS presenting signs were analyzed and mutations were found in three of four patients, with another being a reported polymorphism. Two of these mutations are novel, while other mutation had previously been reported at least thrice. PolyPhen analysis of the missense mutation screened predicts it to be severely damaging. The results of this study confirms the diagnosis of AGS in three out of four patients screened, confirming diagnosis and offering possible carrier screening for the family. The main objective of this proposal is to identify JAG1 mutations in patients and determine the predicted effect on the protein. DNA analysis was carried out by melt curve mutation screening. One of the detection mutations, a missense mutation, was analyzed by in silico tool, PolyPhen. The effect of these mutations on the clinical presentations in the patients was analyzed against reported phenotypes and known mutation databases for this disease.
Materials and Methods
Abstract Introduction Aims and Objectives
Conclusion
In this study, three mutations and one polymorphism were identified in the four patients suspected of AGS. The three mutations, missense, deletion and nonsense, of patients 311, 140 and 227 respectively, are predicted to be disease-causing and affect the important EGF repeat region of the JAG1 protein.
AcknowledgementsReferences1) Krantz, I.D. et al. (1997) Deletions of 20p12 in Alagille syndrome: frequency and molecular characterization. Am. J. Med. Genet. 70: 80-862) Li L, Krantz ID, Deng Y, et al. (1997) Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet 16:243–51.3)Lu F, Morrissette JJ & Spinner NB (2003) Conditional JAG1 mutation shows the developing heart is more sensitive than developing liver to JAG1 dosage. Am J Hum Genet 72: 1065–1070.4) Oda T, Elkahloun AG, Pike BL, Okajima K, Krantz ID, Genin A, Piccoli DA, Meltzer PS, Spinner NB, Collins FS, Chandrasekharappa SC. (1997) Mutations in the human Jagged1 gene are responsible for Alagille syndrome. Nature Genet 16:235–242.5) Heritage ML, MacMillan JC, Colliton RP, Genin A, Spinner NB, Anderson GJ. (2000) Jagged1 (JAG1) mutation detection in an Australian Alagille syndrome population. Hum Mutat 16:408–416.
I would like to thank Professor Lai Poh San for her guidance through this project and the laboratory staff of the Department of Paediatrics for their support in this study. I am grateful to National University of Singapore (NUS) for providing me this opportunity to participate in this project under the Science Research Programme (SRP 10/11).
Alagille Syndrome is a rare disease with an incidence of one in every 100,000 live births. It can be diagnosed by the paucity of bile duct on liver biopsy and the presence of at least three out of five major clinical signs.
AGS is caused by mutations in the large 26 exon JAG1 gene located on chromosome 20p12, which encodes a 135 kDa protein. The JAG1 protein consists of several evolutionary conserved domains; which includes a signal peptide, the conserved Delta-Serrate-Lag2 Domain (DSL), a 16 epidermal growth factor (EGF)-like repeats, a cysteine rich region and a transmembrane domain. The protein acts as a ligand for the NOTCH1 receptor involved in the evolutionary conserved signaling pathway, which controls cell differentiation. Due to the considerable variable expression of the disease, its true incidence may be under-estimated due to difficulties in accurate clinical diagnosis.
1) Study the molecular basis of four local Chinese patients who showed symptoms of intra-hepatic bile duct paucity and syndromic features of Alagille Syndrome.
2) Utilize rapid and cost-effective methods for mutation screening.
Fig 2 General experimental design of project to achieve stated experimental objectives
Fig 1 Summary flowchart of literature of AGS, which acts as basis for experimentation
PCR and Primer Optimization
Preparation of Master Mix and HRM Protocol
HRM Analysis
DNA Sequencing
PolyPhen Analysis
Pre existing lab primers for PCR were checked via Primer3, mFOLD, Primer-BLAST and MeltSim to determine its
suitability as melt primers. Gel electrophoresis was also carried out to test Post-PCR products.
DNA sequencing was carried out for melt curves that did not cluster together after HRM
analysis. The Big Dye Terminator Sequencing Kit was utilized, and Chromas software was used to
conduct analysis of results.
PCR reactions were carried out in 96 well plates using BioRad’s CFX96 RT PCR system. Melt Analysis was conducted via
Precision Melt Analysis software. Melt curves were generated and
plotted onto difference curves, and compared with normal controls.
Reaction volumes of primers, DNA, PCR buffer, MgCl2, dNTPs, EvaGreen dye and Platinum Taq polymerase were prepared and
carried out in accordance to PCR cycling conditions. Melt analysis
was then carried out on Post-PCR product on same plate.
In silico analysis of mutation data was carried out by comparing the
known reference sequence of JAG1, and translated to the
positions of these codons. The FASTA sequence of JAG-1 was then
input into PolyPhen software for prediction of severity.
Exon 10 (c.1753G>A)
EXON 4 (C.588C>T)
Exon 6 (c.1213delC)
Exon 18 (c.2643C>T)
Fig 3 Results of gel electrophoresis of testing amplification with lab primers
Fig 4 PCR and HRM protocol used as seen on computer screen
Fig 5 Ilustration of the principle of HRM (High Resolution Melt) Analysis
Fig 6 Illustration of the principle of automated sequencing
Fig 7 FASTA sequence of JAG-1 as inputted into PolyPhen
Fig E & F - Melt curve (left) and difference curve (right) of patient 311
Fig A & B - Melt curve (left) and difference curve (right) of patient 140
Fig C & D - Melt curve (left) and difference curve (right) of patient 227
Fig G & H - Melt curve (left) and difference curve (right) of patient 406
Fig 8 Sequencing result of patient 140
Fig 9 Sequencing result for patient 227
Fig 11 Sequencing result for patient 406
Table 2 Clinical phenotypes of patients in this study (affected organs)
Fig 12 Mapping identified mutations to regions of the JAG-1 protein
C>T
Table 1 Summary table of mutations and affected codons found in all four patients
Patient 140 with deletion mutation manifests only two symptoms diagnostic of Alagille SyndromeSuggests possibility of presence of other genetic modifiers of disease
Image obtained from http://dna.utah.edu/Hi-Res/TOP_Hi-Res%20Melting.html
Image obtained from http://www.dnassequencing.com/2011/01/07/automated-dna-sequencing-6/
1) Precise identification and investigation of mutations in the JAG1 gene would confirm clinical diagnosis of suspected Alagille Syndrome patients, thus providing options of carrier
screening and genetic counseling for affected families.
2) Rapid and cost effective mutation screening methods could circumvent the problems posed by the lack of mutation hotspots and the large 26 exon size of the JAG1 gene.
Rationale
Fig 10 Sequencing and PolyPhen result of patient 311
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