Lack of association of Lysyl oxidase (LOX) gene polymorphisms with intracranial aneurysm in a south Indian population

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Molecular Biology ReportsAn International Journal on Molecularand Cellular Biology ISSN 0301-4851 Mol Biol RepDOI 10.1007/s11033-013-2693-1

Lack of association of Lysyl oxidase (LOX)gene polymorphisms with intracranialaneurysm in a south Indian population

Sanish Sathyan, Linda Koshy,K. R. Sarada Lekshmi, H. V. Easwer,S. Premkumar, Jacob P. Alapatt, SureshNair, R. N. Bhattacharya, et al.

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Lack of association of Lysyl oxidase (LOX) gene polymorphismswith intracranial aneurysm in a south Indian population

Sanish Sathyan • Linda Koshy • K. R. Sarada Lekshmi •

H. V. Easwer • S. Premkumar • Jacob P. Alapatt •

Suresh Nair • R. N. Bhattacharya • Moinak Banerjee

Received: 13 December 2012 / Accepted: 14 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Intracranial aneurysm (IA) accounts for 85 %

of haemorrhagic stroke and is mainly caused due to

weakening of arterial wall. Lysyl oxidase (LOX) is a

cuproenzyme involved in cross linking structural proteins

collagen and elastin, thus providing structural stability to

artery. Using a case–control study design, we tested the

hypothesis whether the variants in LOX gene flanking the

two LD block, can increase risk of aSAH among South

Indian patients, either independently, or by interacting with

other risk factors of the disease. SNPs were genotyped by

fluorescence-based competitive allele-specific PCR (KAS-

Par) chemistry. We selected 200 radiologically confirmed

aneurysmal cases and 235 ethnically and age and gender

matched controls from the Dravidian Malayalam speaking

population of South India. We observed marked interethnic

differences in the genotype distribution of LOX variants

when compared to Japanese and African populations.

However, there was no significant association with any of

the LOX variants with IA. This study also could not

observe any significant role of LOX polymorphisms in

influencing IA either directly or indirectly through its

confounding factors such as hypertension and gender in

South Indian population.

Keywords Intracranial aneurysm � Subarachnoid

haemorrhage � Lysyl oxidase (LOX) � SNP � India

Introduction

Intracranial aneurysm (IA) accounts for 85 % of sub-

arachnoid hemorrhage (SAH), which contributes for

5–15 % of strokes, but occurs at a fairly young age [1].

Reports have shown worldwide incidence of aSAH

(aneurysmal subarachnoid hemorrhage) in 10.5 cases per

100,000 person-years [2]. In general population 2–3 %

individual harbor IA [3]. Prevalence of IA in India based

on autopsies ranges from 0.2 to 10.3 % from various parts

of India, with a mean prevalence of 5.3 % [4–6].

Involvement of genetic component with IA is well

documented. Familial clustering of IA shows four fold

higher risks of aneurysmal rupture in first degree relative

and identical aneurysm location in twins study supports

this cause [7, 8]. Various theories have been proposed for

the development of aneurysm, such as Congenital medial

defects theory, Acquired internal elastic lamina defects

theory [9, 10] and Hemodynamic Stress theory [11], but the

etiology of IA still remains controversial. Most of these

theories reflect on the imbalances in the Extracellular

matrix (ECM) remodeling. ECM remodeling plays an

important role in maintaining the structure and integrity of

the intracranial arteries. Elastin and collagen fibers form

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-013-2693-1) contains supplementarymaterial, which is available to authorized users.

S. Sathyan � L. Koshy � K. R. Sarada Lekshmi �M. Banerjee (&)

Human Molecular Genetics Laboratory, Rajiv Gandhi Centre for

Biotechnology, Thiruvananthapuram 695 014, Kerala, India

e-mail: [email protected]

H. V. Easwer

Department of Neurosurgery, Sree Chitra Tirunal Institute for

Medical Science and Technology, Thiruvananthapuram, India

S. Premkumar � J. P. Alapatt � S. Nair � R. N. Bhattacharya

Department of Neurosurgery, Calicut Medical College,

Calicut, India

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Mol Biol Rep

DOI 10.1007/s11033-013-2693-1

Author's personal copy

the framework for ECM providing strength and elasticity to

the artery. Lysyl oxidase (LOX) is an important class of

copper containing enzyme involved in covalent cross

linking of these fibrous proteins by formation of aldehydes

between lysine residues that insolubilize these extracellular

proteins namely collagen and elastin [12], thus giving

strength to intracranial arteries. Animal models for IA are

mainly based on the LOX inhibitor such as b-aminopro-

pionitrile(BAPN) [13, 14]. These observations strengthens

the role of LOX gene in pathophysiology of IA.

Interestingly, one of the earliest studies on genome wide

linkage analysis identified the loci 5q22-31 comprising of

the LOX gene to be associated with IA [15]. Reduced

extracellular matrix is a prominent feature of cerebral

aneurysms. Therefore, inspite of a strong hypothesis and

genetic linkage to the LOX this gene has not been inves-

tigated for its possible role in IA. The gene has 5 transcripts

and seven exons. The processed product of proteolytic

cleavage of Pro-LOX consist of a functional 32-kDa LOX

enzyme and an 18-kDa LOX propeptide (LOX-PP) [16]. A

non-synonymous SNP rs1800449 (G473A, Arg158Gln)

located in first exon of LOX causing an arginine to gluta-

mine change in the conserved region of LOX propeptide

has been implicated with coronary artery disease (CAD)

[17]. Based on these backgrounds the present study intends

to explore the involvement of the variants in the LOX with

IA in a genetically homogenous Indian population.

Materials and methods

Study population

The study populations consist of radiologically confirmed

200 aneurysmal cases and 235 ethnically age and gender

matched controls from the Dravidian Malayalam speaking

population of South India, as described in our earlier

publication [18]. All patients had angiographically docu-

mented saccular aneurysms with SAH confirmed by cranial

computed tomography (CT), magnetic resonance angiog-

raphy (MRA) and digital subtraction angiography (DSA).

The neurological grade was classified as per World Fed-

eration of Neurological Surgeons (WFNS) scale and all

grades of aSAH were eligible for inclusion. Exclusion

criteria included nonsaccular aneurysm, arteriovenous

malformations or hematological disorders. The control

group consisted of unrelated ethnically matched volunteers

recruited from individuals admitted to the hospitals for any

reason other than neurological diseases. Data were col-

lected by the investigators directly by interviewing the

patients and controls using a structured questionnaire to

obtain information regarding demographic characteristics.

The study was as per the ICMR guidelines and approved by

Institute ethical committee (IEC).

SNP selection and genotyping

SNP’s were selected based on the functional relevance,

tagging status and minor allele frequency in different

populations. Fours SNPs where selected includes a func-

tionally significant variant present in LOX propeptide,

rs1800449, G473A, Arg158Gln and three other SNPs

rs2956540, rs10519694 and rs3792801 which were selec-

ted based on tagging status using genotype data obtained

from Caucasian individuals in the HapMap project (Hap-

Map Data Rel 24/Phase II Nov08, on NCBI B36 assembly,

dbSNP b126) on the basis of pairwise linkage disequilib-

rium with a r2 threshold of 0.8 and minor allele fre-

quency C0.05 to capture all the common SNPs. SNPs

rs2956540, rs10519694 and rs1800449 were present in the

single LD block, while rs379801 and rs379802 formed the

second minor block in caucasian population (Supplemen-

tary Figure 1). Genotyping was performed by fluorescence-

based competitive allele-specific PCR (KASPar) chemistry

(Kbiosciences, UK).

Table 1 Clinical characteristics and demographic variable of cases

and controls

Characteristics Cases Controls

Mean age ± SD, years 51.8 ± 10.8 51.0 ± 14.1

Men, % 55.2 48.7

Women, % 44.8 51.3

History of hypertension, % 36.2 16

History of diabetes, % 5.5 14.7

Family history of aSAH, % 3.7 0.0

Cigarette smoking, % 42.9 18

Alcohol use, % 19.6 12

Intracranial aneurysm location (%)

Anterior communicating artery 40.95

Anterior cerebral artery 8.57

Middle cerebral arteries 22.86

Internal carotid artery 17.14

Posterior communicating artery 9.05

Posterior cerebral artery 0.48

Basilar artery 0.48

Vertebral arteries 0.48

Intracranial aneurysm type (%)

Single aneurysm 84.51

Multiple Aneurysm 8.41

Two position 5.75

Three position 1.33

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Statistical analysis

Genotype and allelic frequencies were computed and were

checked for deviation from Hardy–Weinberg equilibrium

(http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl).

Haplotypic association of samples was carried out using

the COCAPHASE program in the UNPHASED v3.1

package (http://www.mrc-bsu.cam.ac.uk/personal/frank/

software/unphased/) [19] with 10,000 permutations. To

estimate LD between pairs of loci in the patient and control

populations, standardized disequilibrium coefficient (D0)and squared correlation coefficient (r2) were calculated

using Haploview 4.1 (www.broad.mit.edu/mpg/haploview/)

[20]. LD blocks were defined in accordance with Gabriel’s

criteria [21]. Further stratification of the patients was done

to understand the role of LOX variant within the genders

and between the hypertensive and non-hypertensive

groups. However due to the exploratory nature of this

study, no adjustment for multiple testing was made, as not

to penalize our data by missing possibility of important

findings.

Results

The demographic and clinical characteristics of the patients

and controls are summarized in Table 1. All the four SNPs

selected for the study was in Hardy–Weinberg equilibrium

(p [ 0.05). Functionally significant non synonymous SNP

rs1800449 (G473A) was not found to be associated at

genotypic (p = 0.4165) and allelic level (p = 0.77,

OR = 0.9284, CI 0.6325–1.363) with IA in this population

(Table 2). Similarly while comparing the allele and geno-

typic frequencies of the other three SNPs among patients

and controls, no significant difference was observed

(Table 2). Thus none of the studied SNPs were found to be

associated with IA in this population at genotypic and

allelic level. Linkage disequilibrium (LD) plot using

Haploview 4.1 software too showed high similarity

between cases and control (Fig. 1). LD plot was found to

be similar to Caucasian population. However, when the LD

patterns were constructed for other world populations taken

from HapMap data, we observed marked differences with

African and Mongoloid populations (Fig. 2).

Table 2 Comparison of the genotype and allele frequencies of LOX gene variants between patients and controls

AA AG GG X2 p value A G OR (95 %Confidence interval) p value

rs1800449

Cases 5 45 150 55 345

2.66 22.34 75.00 1.752 0.417 13.75 86.25 0.9284(0.6325–1.363) 0.77

Controls 3 62 167 68 396

1.29 26.72 71.98 14.66 85.34

CC CG GG X2 p value C G OR (95 %Confidence interval) p value

rs2956540

Cases 77 103 20 257 143

38.46 51.65 9.89 0.432 0.806 64.25 35.75 1.078(0.8193–1.419) 0.62

Controls 90 125 29 305 183

36.89 51.23 11.89 62.50 37.50

CC CT TT X2 p value C T OR (95 %Confidence interval) p value

rs10519694

Cases 128 67 5 323 77

64.00 33.50 2.50 1.020 0.601 80.75 19.25 1.140(0.8191–1.587) 0.45

Controls 148 83 10 379 103

61.41 34.44 4.15 78.63 21.37

CC CT TT X2 p value C T OR (95 %Confidence interval) p value

rs3792801

Cases 137 57 6 331 69

68.50 28.50 3.00 0.426 0.808 82.75 17.25 1.005 (0.7050–1.432) 1.00

Controls 156 70 5 382 80

67.53 30.30 2.16 82.68 17.32

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We further investigated the role of these allelic and

genotypic variants in gender stratification and hypertension

to understand whether the genetics of these confounding

factors act as modifiable risk factor for IA. None of the

alleles or genotypes of LOX were found to influence gender

(Supplementary Table-1).and hypertension in causing IA

either directly or through modifiable risk factor (Supple-

mentary Table-2).

Discussion

In the present study the four SNPs rs1800449 (473 G/A),

rs2956540, rs10519694 and rs3792801 within the LOX

were selected based on tagging status comprising of two

LD blocks covering the complete gene based on the

genotype data of Caucasians from phase II HapMap Pro-

ject. LD pattern observed in our control population was

similar to Caucasians but distinctly different from Chinese,

Japanese and Africans. LOX is known to function as the

initiator of crosslinking of elastin and collagen and in the

repair of these ECM molecules during aging and disease.

Thus inactivation of the LOX would have debilitating

consequences. Differences in the LD patterns would sug-

gest that LOX variants might have a strong ethno-genetic

influence in modulating a debilitating consequence of

imbalances in ECM remodeling.

Though functionally and positionally based on linkage

studies, LOX has been implicated in development of IAs in

Japanese [15] but this could not be verified in a validation

study with LOX variants in the same Japanese population

comprising of 172 IA patients and 192 controls [22]. In the

present study too we report lack of association of LOX

variants with IA inspite of distinctly different LD patterns

between our study population and Japanese population.

Lack of association observed in the present study could be

explained with following reasons at positional and func-

tional level. Firstly even though LOX gene loci 5q22-31

was found to be positional candidate gene for IA in linkage

studies [15] this loci was never found to be associated in

Genome wide association studies carried out with higher

sample size [23–26].This conflicting result may be due to

genetic heterogeneity, power of study, difference in sample

size and difference in statistical method employed. Vali-

dating such an observation using a candidate gene

approach in a genetically well stratified population do

confirm that this gene may not play a significant role by

itself.

Secondly, at functional level the gene possesses an

important role in maintaining the structural integrity and

stability of vessel wall. Animal models were also generated

by LOX inhibitor BAPN [13]. The LOX expression was

found to be down regulated in IA arteries [14]. LOX having

a role in endothelial dysfunction [27], a major cause in

pathogenesis of IA, is shown to regulated by number of

factors including homocysteine level [28], low density

lipoprotein (LDLs) [29], TGF-b1 [30] and TNF alpha [31].

The results obtained in the present study suggest that LOX

may not to be a causal variant at genetic level. LOX

expression is known to be regulated by various extraneous

control cases

Fig. 1 Linkage disequilibrium pattern of studied Lysyl oxidase(LOX)

SNPs in cases and controls

Fig. 2 Linkage disequilibrium patterns in different HapMap populations of the world. (S1 South India, CEU European, JPT Japanese, CHB Han

Chinese, YRI Yoruba African)

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factors. Therefore, one cannot not downplay the impor-

tance of LOX in IA, without understanding the role of

confounding factors involved in regulation of LOX.

Increased LOX activity has been reported in hyperten-

sion which can result in alterations in the degree of cross-

linking of vascular collagens [32]. To address the role of

these confounding factors we further investigated the

association of LOX variants in hypertension and gender as

modifiable factors in influencing IA. Here too we could not

observe any significant role of LOX polymorphisms in

influencing IA either directly or indirectly through its

confounding factors. LOX variant rs1800449 (473AA) has

been reported to be associated with cardiovascular disease

including ischemic stroke [17]. Differences in cerebro-

vascular and cardiovascular stroke could be due to differ-

ence in structural architecture of vessel walls or could be

due to differences in the pathobiology of two strokes.

We conclude that allelic or genotypic variants of LOX

do not play a significant role in South Indian population

either directly or through modifiable factors to result in

IAs. However, differences in allelic and genotypic distri-

bution of LOX were observed in different ethnic popula-

tions of the world.

Acknowledgments SS acknowledges the Council of Scientific and

Industrial Research CSIR), Government of India for providing junior

research fellowship. We also acknowledge all the patients and their

family members who cooperated in this study.

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