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.

AcknowledgementsReferences

1) 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