ASSOCIATION OF ASMT GENE WITH ASD IN SOUTH INDIAN POPULATION

ANUJA GANESAN, 2011305003, BTECH INDUSTRIAL BIOTECHNOLOGY, ANNA UNIVERSITYMAY 2015 1

INTRODUCTION TO AUTISMoAutism is a complex neurodevelopmental disability that manifests itself within the first three years of life of an affected individual.

oCommon symptoms include impaired verbal communication, social interaction and restricted, repetitive behaviour.

oASD includes Autistic disorder, Rett syndrome, Asperger syndrome, Childhood disintegrative disorder, pervasive developmental disorder, not otherwise specified. Each such syndrome differ in the degree of impairment.

oBeing a collection of complex disorders, numerous gene-gene and gene-environment interactions are found to be risk factors for developing ASD.

oThe most recent estimate for all autism spectrum disorders, taken together, is 1 in 68 children aged 8 years in the US.

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CIRCADIAN RHYTHM, MELATONINoCircadian rhythm is an internal clock that oscillates for approximately 24 hours, and is entrainable in response to external stimuli such as light and temperature.

oThe rhythmic release of melatonin is regulated by the master clock, the suprachiasmatic nucleus (SCN) of the anterior hypothalamus in response to light. Body’s internal melatonin rhythm exhibits a close association with the internal circadian component of the sleep propensity rhythm.

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ASMT GENEoASMT gene is located in the pseudoautosomal region (PAR) at the end of the short arms of the X and Y chromosomes precisely in the position Xp22.3 and Yp11.3. oThe human ASMT gene is approximately 47 kb in length and contains 9 coding exons. oAcetylserotonin O-methyltransferase or hydroxyindole O-methyltransferase enzyme catalyses the final reaction in the synthesis of melatonin from tryptophan and is rate limiting. This enzyme is abundant in the pineal gland.

TRP-OH L-AAAD AANAT ASMT

TRYPTOPHAN --> 5-HYDROXY TRYPTOPHAN -->SEROTONIN -->N-ACETYLSEROTONIN -->MELATONIN

ASMT

N-ACETYLSEROTONIN + S-ADENOSYL METHIONINES-ADENOSYL L-HOMOCYSTEINE + MELATONIN

+H+

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SCOPE OF THE STUDYHypothesis:

Researchers estimate that between 40% and 80% of children with autism have difficulty sleeping. Concurring to the findings, 63-65% of ASD patients also have pineal hypofunction, low plasma melatonin concentrations due to low ASMT enzyme activity. The low ASMT activity observed in autism have been partly mapped to variations within the ASMT gene.

Aim:

To screen exon 6 and 9 of ASMT gene for single nucleotide variants in ASD patients and normal control samples, also to test the association of such variants to ASD in South Indian population.

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METHODS USEDPERIPHERAL BLOOD : 50 cases and 50 controls.

• DNA Isolation-Salting out method

• Quantification-UV Spectrophotometer

POLYMERASE CHAIN REACTION(PCR)

• Gradient PCR

•Amplification PCR

GENOTYPING BY SEQUENCING

•Sequencing PCR (Sanger’s method)

•Sequencing cleanup

•SNP analysis by sequence scanner software

STATISTICAL ANALYSIS

•Chi square test

•Hardy Weinberg equilibrium

•1000 Genome Database

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EXON 6 49.8° 50.2° 51.1° 52.5° 54.3° 56.2° 58.3° 60.2 ° 62° 63.5° 64.0° 65.1°

479bp

GRADIENT IMAGE AMPLIFICATION IMAGE

500bp

100bp

100bp

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EXON 6 AGCTTGCAGTGAGCGGAAATCGTGCCACTGCACTCCAGCCTGGGCGACAGAGCCAGACTCTGTCTCAAAAATAAATAAATTAATAAATAAAAGGATGCGTTCATGTCCTTTGCAGGGACAYGGATAAAGCTGGAAACCATCATTCTCAGC

rs6588809 AAACTATCGCAAGGACAGAAAACCAAACACCGCGTGTTCTCACTCATAGGTGGGAACTGAACAATGAGACCACRTG

rs28613362

GACACAGGGAGGGGAAYGTCACACACTGGGGCCTGTCAGGGCCTGGGGGGCTGGGAGGCTGGGGGAGGGAGAGC rs7471973 ACTAGGACAAATACCTAATGTAAATGACGAGTCGATGGGTGCAGCAAACCACCATGGCACATGGATACCTATGTAACAAAACCGCATGTTCTGCACATGTACCCTAGAACTTAAAGTATAATAAAAAGAAAAAAAAGACCAGACATGGCGGAAGGACCCAAGTTCCTGGGTTGGACCCT

Note: Sequence highlighted in brown is primer sequence. Sequence highlighted in blue is exon region. Common SNPs are highlighted in red. Y denotes allele change from C to T. R denotes allele change from A to G.

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Electropherogram showing different genotypes of rs28613362(A/G)

SNP Alleles MAF F-score Position Functional significancers6588809 T/C 0.45 0 Exon Protein coding(W to R)

rs28613362 A/G 0.38 NA Intron Not validated rs7471973 C/T 0.13 NA Intron Not validated

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Electropherogram showing different genotypes in rs6588809(T/C)

Electropherogram showing different genotypes in rs7471973(C/T)

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STATISTICAL ANALYSIS rsID TT CT CC p-value T C p-value

rs6588809

Cases 15(0.3) 21(0.42) 14(0.28) 0.305 51(0.51) 49(0.49) 0.1166Controls 20(0.4) 22(0.44) 8(0.16) 62(0.62) 38(0.38)

rs28613362

AA AG GG p-value A G p-value

Cases 9(0.18) 8(0.16) 33(0.66) 0.027 26(0.26) 74(0.74) 0.001Controls 19(0.38) 10(0.2) 21(0.42) 48(0.48) 52(0.52)

rs7471973

CC CT TT p-value C T p-value

Cases 31(0.62) 15(0.3) 4(0.08) 0.9 77(0.77) 23(0.23) 0.622Controls 29(0.58) 16(0.32) 5(0.1) 74(0.74) 26(0.26)

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MXL JPT CLM KER CEU FIN ASW YRI PUR CHB LWK CHS GBR TSI IBS0

0.2

0.4

0.6

0.8

1

1.2

Comparison of allelic frequency of rs6588809 with world population

C T

CHS TSI CHB YRI ASW FIN CLM JPT MXL KER LWK PUR CEU GBR IBS0

0.2

0.4

0.6

0.8

1

1.2

Comparison of genotypic frequency of rs6588809 with world population

CC CT TTKER-Keralites in South India CHS-Southern Han Chinese

IBS-Iberian population in Spain GBR-British in England and Scotland

CHB-Han Chinese in Beijing, China TSI-Tuscans in Italy

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CEU IBS GBR CLM TSI KER MXL PUR FIN ASW JPT LWK CHS CHB YRI0

0.2

0.4

0.6

0.8

1

1.2

Comparison of allelic frequency of rs28613362 with world population

A G

CEU IBS GBR CLM TSI KER MXL PUR FIN ASW JPT LWK CHS CHB YRI0

0.2

0.4

0.6

0.8

1

1.2

Comparison of genotypic frequency of rs28613362 with world population

AA AG GG

YRI-Yoruba in Ibadan, Nigera CLM-Colombian from Medellian, Colombia

ASW-American's of African Ancestry in SW USA JPT-Japanese in Tokyo, Japan

FIN-Finnish in Finland MXL-Mexican Ancestry from Los Angeles USA

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IBS KER GBR TSI CEU CLM PUR LWK FIN ASW YRI JPT MXL CHB CHS0

0.2

0.4

0.6

0.8

1

1.2

Comparison of genotypic frequency of rs7471973 with world population

CC CT TT

KER IBS TSI GBR CEU CLM PUR LWK FIN ASW YRI JPT MXL CHB CHS0

0.2

0.4

0.6

0.8

1

1.2

Comparison of allelic frequency of rs7471973 with world population

C T

CEU-Utah Residents (CEPH) with Northern andWestern European ancestry LWK-Luhya in Webuye, Kenya

PUR- Puerto Rican from Puerto Rica

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EXON 9

52° 54° 56° 58° 60° 62°

507bp

GRADIENT IMAGE AMPLIFICATION IMAGE

500bp

100bp 100bp

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EXON 9CGGTGCCCTGACTGTCCTCTGAGGTCTGGCTGTCCTTATGGTTCACTGGGACTTTGGCACAGTCTGTCTGTGTGTTTGTGTGTGATG

TGGACTGTGCCCCTCCCTTTCTAGGTGGTGGCATTCTGGTAATTGAAAGCCTCCTGGATGAAGACAGGCGAGGTCCTCTGCTCACGC

AGCTCTACTCTCTGAACATGCTTGTGCAGACGGAAGGGCAGGAGAGGACCCCCACCCACTACCACATGCTCCTCTCTTCTGCTGGCTT

CAGAGACTTCCAGTTTAAGAAAACAGGAGCCATTTATGATGCCATTTTAGCCAGGAAATAACTGTTTCTTGTGACCTGGAACTAACGTCA

AAGCACACAAGACATAATAATAAAGACATGTACCTCCAGTGGCTTCTTGTTCTTGGTGTTCACATGATAAGTGACTGGAGGCTTCTGTGG

ACAGGGTTGGACTGCTTCTACTTTGTAGATTATTTCCCAGGCTTTACTGAGGACCACGCTGATGG

Known variants in exon 9:Intronic variants-5; Missense variants-9; Synonymous variants-3; 3’UTR variant-2Note: Sequence highlighted in brown is primer sequence. Sequence highlighted in blue is exon region.

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CONCLUSIONoExons 6 and 9 were chosen based on functional significance. They were amplified, sequenced and statistically analysed for variants.

oRs28613362 was found to be statistically and significantly associated with ASD in South Indian population at both genotypic and allelic level.

oRs7471973 and rs6588809 do not show significant association to ASD in this population.

oIn order to draw conclusion about ASMT gene association with ASD in South Indian population, extensive screening of the entire gene is necessary. Such a study should be done in an increased sample size so that random sampling error is minimised.

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