Investigation

of

autism

and

GABA

receptor

subunit

genes

in

multiple

ethnic

groups

Autism

is

a

neurodevelopmental

disorder

of

complex

genetics

characterized

by

impairment

in

social

interaction

and

communication

as

well

as

repetitive

behavior

Multiple

lines

of

evidence

including

alterations

in

levels

of

GABA

and

GABA

receptors

in

autistic

patients

indicate

that

the

GABAergic

system

which

is

responsible

for

synaptic

inhibition

in

the

adult

brain

may

be

involved

in

autism

Previous

studies

in

our

lab

indicated

association

of

noncoding

single

nucleotide

polymorphisms

-LRB-

SNPs

-RRB-

within

a

GABA

receptor

subunit

gene

on

chromosome

and

interaction

between

SNPs

in

and

-LRB-

also

on

chromosome

-RRB-

within

Caucasian

autism

patients

Studies

of

genetic

variation

in

African-American

autism

families

are

rare

Analysis

of

Caucasian

and

an

independent

population

of

African-American

families

with

SNPs

within

and

strengthened

the

evidence

for

involvement

of

in

autism

risk

in

Caucasians

-LRB-

p

p

and

p

-RRB-

and

gave

evidence

of

significant

association

in

African-Americans

-LRB-

p

and

p

-RRB-

The

and

interaction

was

also

confirmed

in

the

Caucasian

dataset

-LRB-

most

significant

pair

and

p

-RRB-

Analysis

of

the

subset

of

families

with

a

positive

history

of

seizure

activity

in

at

least

one

autism

patient

revealed

no

association

to

however

three

SNPs

within

showed

significant

allelic

association

-LRB-

p

-RRB-

-LRB-

p

-RRB-

and

-LRB-

p

-RRB-

These

results

confirmed

our

earlier

findings

indicating

and

as

genes

contributing

to

autism

susceptibility

extending

the

effect

to

multiple

ethnic

groups

and

suggesting

seizures

as

a

stratifying

phenotype

Introduction

Autism

is

a

neurodevelopmental

disorder

characterized

by

severe

impairment

in

social

interaction

and

communication

as

well

as

repetitive

behavior

Autism

is

part

of

a

spectrum

of

disorders

denoted

autism

spectrum

disorders

-LRB-

ASD

-RRB-

which

in

addition

to

autism

include

Asperger

syndrome

childhood

disintegrative

disorder

and

pervasive

developmental

disorder

not

otherwise

specified

-LRB-

PDD-NOS

-RRB-

Onset

of

this

disorder

is

typically

before

years

of

age

The

incidence

of

autism

is

estimated

at

approximately

one

in

individuals

with

an

increased

incidence

in

males

-LSB-

-RSB-

Incidences

of

approximately

two

to

three

in

are

reported

when

a

broader

diagnosis

includes

the

entire

spectrum

of

pervasive

developmental

disorders

-LSB-

-RSB-

Evidence

indicates

that

this

disease

has

a

strong

genetic

component

Twin

studies

have

shown

that

monozygote

twins

have

a

higher

recurrence

rate

than

dizygotic

twins

While

the

actual

percentages

have

varied

recurrence

rates

have

been

measured

as

high

as

in

monozygote

twins

and

in

dizygotic

twins

-LSB-

-RSB-

Despite

this

strong

evidence

for

a

genetic

component

the

inheritance

appears

to

be

complex

with

estimates

that

more

than

genes

may

be

involved

in

its

inheritance

-LSB-

-RSB-

with

different

families

carrying

different

combinations

of

contributing

genes

The

genes

may

act

independently

or

interactively

adding

to

the

complexity

of

determining

the

genetic

contribution

to

this

disorder

There

are

two

approaches

to

identifying

genetic

contributors

to

disease

The

first

is

a

genome

wide

search

in

which

linkage

or

association

analysis

is

used

to

identify

regions

of

the

genome

that

may

contain

autism

susceptibility

genes

The

second

is

the

candidate

gene

approach

which

investigates

a

specific

gene

or

genes

for

involvement

in

autism

risk

In

the

candidate

gene

approach

genes

are

chosen

for

study

based

on

either

what

is

known

about

the

gene

's

function

its

location

-LRB-

for

example

in

a

recognized

linkage

peak

-RRB-

or

a

combination

of

both

Several

candidates

are

hypothesized

to

be

involved

in

autism

however

no

single

candidate

gene

has

consistently

emerged

as

involved

in

autism

risk

One

candidate

pathway

that

is

hypothesized

to

be

involved

in

autism

is

the

GABAergic

system

Hussman

-LSB-

-RSB-

suggested

that

autism

is

the

result

of

an

imbalance

of

the

excitatory

glutamatergic

and

inhibitory

GABAergic

pathways

resulting

in

overstimulation

in

the

brain

and

inability

to

filter

out

excess

stimuli

from

environmental

and

intrinsic

sources

This

theory

is

supported

by

multiple

lines

of

evidence

First

histological

biochemical

and

molecular

approaches

have

demonstrated

altered

levels

and

distribution

of

GABA

and

GABA

receptors

in

peripheral

blood

and

plasma

as

well

as

in

the

brain

including

decreased

GABA-A

receptors

and

benzodiazepine

binding

sites

in

the

hippocampal

formation

-LSB-

-RSB-

There

are

also

reported

alterations

in

GABAergic

neurons

as

demonstrated

by

the

increased

packing

density

of

GABAergic

interneurons

in

the

and

subfields

and

by

the

decreased

numbers

and

reduced

size

of

cerebellar

GABAergic

Purkinje

cells

-LSB-

-RSB-

In

addition

duplications

and

isodicentric

chromosomes

in

the

region

containing

the

three

clustered

GABA

receptor

subunits

and

on

chromosome

have

been

associated

with

autism

-LSB-

-RSB-

As

well

evidence

for

both

linkage

and

allelic

association

have

been

reported

for

this

same

GABA

gene

cluster

although

the

findings

have

not

been

consistent

across

datasets

-LSB-

-RSB-

Investigation

of

association

of

GABA

receptor

subunits

outside

of

the

chromosome

region

has

been

limited

-LSB-

-RSB-

Lastly

mutations

have

been

reported

in

multiple

GABA

receptor

genes

in

families

with

epilepsy

-LSB-

-RSB-

Given

the

high

comorbidity

of

autism

with

epilepsy

and

seizures

these

data

suggest

that

a

similar

molecular

etiology

could

exist

between

the

disorders

Signaling

in

the

GABAergic

system

is

mediated

by

receptors

for

the

neurotransmitter

GABA

There

are

known

GABA

receptor

subunits

arranged

in

clusters

throughout

the

genome

Functional

pentamers

formed

by

various

combinations

of

these

subunits

result

in

receptors

of

varying

properties

and

sensitivities

The

amounts

and

functional

capabilities

of

individual

receptor

subunits

that

form

a

specific

pentamer

can

affect

the

amount

and

quality

of

signaling

in

different

parts

of

the

brain

Previously

published

work

from

our

laboratory

analyzed

autosomal

GABA

receptor

genes

on

four

different

chromosomes

using

SNPs

in

a

Caucasian

dataset

of

families

This

analysis

revealed

allelic

association

to

-LRB-

p

-RRB-

within

gamma-aminobutyric

acid

-LRB-

GABA

-RRB-

A

receptor

-LRB-

-RRB-

on

chromosome

-LSB-

-RSB-

and

a

significant

interaction

with

within

the

neighboring

gene

gamma-aminobutyric

acid

-LRB-

GABA

-RRB-

A

receptor

-LRB-

-RRB-

Despite

similar

prevalence

rates

between

Caucasian

and

African-Americans

-LSB-

-RSB-

autism

studies

in

African-Americans

are

rare

Risk

alleles

may

be

different

between

ethnic

groups

or

the

same

risk

alleles

may

have

differential

effects

in

each

ethnic

group

warranting

studies

in

multiple

groups

Evidence

that

phenotypic

factors

including

indicators

of

language

development

may

be

more

severe

in

African-Americans

compared

to

non-Hispanic

Caucasians

-LSB-

-RSB-

is

consistent

with

these

possibilities

and

underscores

the

need

to

investigate

autism

in

different

ethnic

groups

In

this

study

we

present

a

validation

of

our

previous

report

in

an

independent

dataset

of

African-American

families

as

well

as

confirmation

of

our

previous

results

in

an

expanded

Caucasian

sample

of

autism

families

Materials

and

methods

Samples

All

families

were

drawn

from

a

large

multisite

study

of

autism

genetics

conducted

in

the

southeastern

United

States

These

families

are

recruited

through

the

Center

for

Human

Genetics

-LRB-

CHG

-RRB-

at

Duke

University

Medical

Center

-LRB-

DUMC

-RRB-

the

University

of

South

Carolina

and

the

Center

for

Human

Genetic

Research

at

Vanderbilt

-LRB-

N

African-American

and

Non-Hispanic

Caucasian

families

-RRB-

through

support

groups

advertisements

and

clinical

and

educational

settings

All

sites

recruited

enrolled

and

sampled

individuals

with

autism

and

family

members

per

study

protocols

approved

by

their

respective

institutional

review

boards

-LRB-

IRBs

-RRB-

Written

informed

consent

was

obtained

from

parents

and

from

children

who

were

able

to

give

informed

consent

Families

were

enrolled

based

on

probands

meeting

the

following

core

inclusion

criteria

of

-LRB-

-RRB-

probands

ranging

from

three

to

years

of

age

-LRB-

-RRB-

a

presumptive

clinical

diagnosis

of

autism

and

-LRB-

-RRB-

an

expert

clinical

diagnosis

of

autism

using

DSM-IV

criteria

-LSB-

-RSB-

supported

by

the

Autism

Diagnostic

Interview-Revised

-LRB-

ADI-R

-RRB-

-LSB-

-RSB-

and

in

some

cases

the

Autism

Diagnostic

Observation

Schedule

-LRB-

ADOS

-RRB-

-LSB-

-RSB-

Our

original

ascertainment

protocol

relied

on

clinical

expertise

and

the

ADI-R

The

ADOS

was

subsequently

added

as

a

required

diagnostic

method

and

families

that

were

missing

ADOS

assessments

were

updated

as

available

To

assure

valid

ADI-R

results

all

participants

who

met

current

diagnostic

criteria

for

autism

were

included

only

if

they

had

a

minimal

developmental

level

of

months

as

extrapolated

from

the

Vineland

Adaptive

Behavior

Scale

score

-LSB-

-RSB-

or

had

an

IQ

equivalent

greater

than

based

on

scores

from

a

standardized

measure

of

cognitive

ability

such

as

an

the

Wechsler

Scale

of

Intelligence

for

Children

edition

-LSB-

-RSB-

Differential

Abilities

Scale

-LSB-

-RSB-

Mullen

Scales

of

Early

Learning

-LSB-

-RSB-

or

Leiter

Intelligence

Scale-Revised

-LSB-

-RSB-

IQ

data

was

derived

from

medical

records

or

direct

assessment

Exclusion

criteria

for

participation

in

the

larger

genetics

study

included

severe

sensory

problems

-LRB-

e.g.

visual

impairment

or

hearing

loss

-RRB-

significant

motor

impairments

-LRB-

e.g.

failure

to

sit

by

months

or

walk

by

months

-RRB-

or

identified

metabolic

genetic

or

progressive

neurological

disorders

based

on

screening

by

clinical

staff

Additional

samples

are

from

the

Autism

Genetic

Research

Exchange

-LRB-

AGRE

-RRB-

These

individuals

were

qualified

using

similar

methods

including

ADI-R

ADOS

VABS

and

a

standardized

measure

of

IQ

Thirty-nine

African-American

families

were

used

in

an

initial

GABA

receptor

screen

Follow-up

analysis

of

significant

findings

was

performed

in

African-American

families

Analysis

of

the

extended

Caucasian

dataset

included

non-Hispanic

Caucasian

families

One-hundred

and

five

new

non-Hispanic

Caucasian

families

were

added

to

the

analysis

-LRB-

families

previously

analyzed

by

Ma

et

al.

-LSB-

-RSB-

were

newly

identified

as

Hispanic

and

were

omitted

from

the

current

study

in

an

effort

decrease

heterogeneity

in

the

Caucasian

dataset

-RRB-

Classification

of

history

of

seizure

activity

in

autism

patients

was

based

on

question

from

the

ADI-R

which

queries

for

both

current

and

lifetime

presence

of

convulsions

seizures

and

epilepsy

Caregiver

responses

to

question

are

coded

to

indicate

no

seizure

activity

seizure

activity

with

no

definitive

diagnosis

of

epilepsy

and

seizures

with

a

definite

diagnosis

of

epilepsy

Using

lifetime

ratings

two

groups

of

families

were

defined

those

in

which

no

seizure

activity

was

reported

and

those

in

which

seizure

activity

was

present

in

at

least

one

autism

patient

In

addition

question

allows

for

coding

of

febrile

seizures

Families

with

only

febrile

seizures

were

classified

as

negative

for

seizure

activity

and

not

included

in

the

seizure

subset

analysis

Both

families

with

positive

and

negative

history

of

seizure

activity

were

included

in

our

overall

dataset

This

resulted

in

a

dataset

of

Caucasian

families

with

a

positive

history

for

seizures

Molecular

analyses

and

genotyping

The

analysis

of

GABA

receptor

subunit

genes

was

performed

in

African-American

families

as

previously

described

-LSB-

-RSB-

Briefly

between

three

and

seven

intronic

UTR

and

synonymous

coding

SNPs

within

each

gene

were

identified

from

Applied

Biosystems

-LRB-

ABI

Foster

City

CA

USA

-RRB-

Assay

on

Demand

-LRB-

AoD

-RRB-

products

resulting

in

SNPs

within

GABA

receptor

genes

on

four

autosomes

Genes

analyzed

were

and

GABRP

from

chromosome

and

from

chromosome

and

from

chromosome

and

and

from

chromosome

Additional

SNPs

within

and

were

analyzed

in

the

extended

African-American

-LRB-

N

-RRB-

and

Caucasian

-LRB-

N

-RRB-

datasets

to

expand

the

coverage

of

variation

across

this

region

Thirty-five

SNPs

representative

of

different

linkage

disequilibrium

-LRB-

LD

-RRB-

blocks

across

the

two

genes

-LRB-

in

and

in

-RRB-

were

genotyped

-LRB-

Fig.

-RRB-

SNP

's

for

genotyping

were

selected

from

online

databases

-LRB-

University

of

California

Santa

Cruz

and

NCBI

dbSNP

-RRB-

and

from

resequencing

of

exons

and

surrounding

areas

of

both

and

genes

Fig.

locations

of

SNPs

for

extended

analysis

within

and

Horizontal

arrows

represent

transcriptional

direction

Vertical

arrows

represent

SNPs

as

numbered

and

identified

below

the

diagram

Pter

indicates

the

P-terminal

end

of

the

chromosome

and

cen

indicates

the

centromere

SNP

genotyping

was

performed

using

Taqman

allelic

discrimination

assays

-LRB-

Applied

Biosystems

-RRB-

DNA

was

extracted

from

whole

blood

according

to

established

protocols

-LSB-

-RSB-

and

ng

of

genomic

DNA

was

used

per

reaction

Amplification

was

performed

on

GeneAmp

PCR

Systems

thermocyclers

with

cycling

conditions

as

recommended

by

Applied

Biosystems

Fluorescence

was

measured

using

Applied

Biosystem

's

Genotype

discrimination

was

conducted

using

ABI

Prism

SDS

software

Quality

control

to

ensure

accurate

genotyping

involved

two

different

CEPH

DNAs

in

quadruplicate

on

each

well

plate

as

well

as

the

presence

of

samples

which

were

replicated

elsewhere

in

the

sample

list

Additionally

genotyping

efficiency

is

required

Statistical

analysis

To

ensure

genotyping

quality

Pedcheck

was

run

for

detection

of

Mendelian

inheritance

inconsistency

One

affected

and

one

unaffected

individual

from

each

family

were

selected

randomly

for

tests

of

Hardy

Weinberg

equilibrium

-LRB-

HWE

-RRB-

which

was

assessed

using

exact

tests

from

the

Genetic

Data

Analysis

program

-LSB-

-RSB-

Pairwise

LD

between

markers

was

calculated

using

graphical

overview

of

linkage

disequilibrium

-LRB-

GOLD

-RRB-

-LSB-

-RSB-

in

the

parents

of

autism

cases

for

both

the

African-American

and

Caucasian

samples

LD

was

evaluated

in

parents

to

increase

the

available

sample

size

for

analysis

and

comparison

between

the

two

ethnic

groups

The

pedigree

disequilibrium

test

-LRB-

PDT

-RRB-

and

its

extension

the

genotypic

pedigree

disequilibrium

test

-LRB-

genoPDT

-RRB-

-LSB-

-RSB-

were

used

to

test

for

association

to

autism

susceptibility

The

EMDR

-LSB-

-RSB-

an

extension

of

the

MDR

-LSB-

-RSB-

was

used

to

test

for

potential

gene

gene

interaction

to

identify

specific

locus

combinations

of

interest

for

further

investigation

and

validation

of

previous

results

EMDR

analysis

was

performed

using

seven

SNPs

the

four

in

found

to

show

significant

allelic

or

genotypic

association

in

the

Caucasian

sample-set

and

the

three

in

found

to

be

significant

in

the

seizure

subgroup

One

two

and

three-way

analysis

was

performed

on

the

Caucasian

dataset

For

case-control

pairs

used

in

EMDR

the

proband

-LRB-

or

most

completely

genotyped

affected

child

-RRB-

from

each

multiplex

and

triad

family

was

selected

-LRB-

n

total

-RRB-

as

a

case

Controls

were

generated

using

the

untransmitted

alleles

from

parental

genotypes

In

this

study

a

cross-validation

option

was

not

utilized

The

sample

size

of

the

African-American

dataset

is

too

small

to

provide

power

for

EMDR

analysis

not

allowing

us

to

test

for

validation

of

the

interaction

seen

in

the

paper

of

Ma

et

al.

within

the

African-American

dataset

The

haplotype

family-based

association

test

-LRB-

HBAT

-LSB-

-RSB-

-RRB-

was

used

for

haplotype

association

analysis

using

the

significant

SNPs

in

Results

Allelic

association

studies

of

SNPs

across

the

GABA

receptor

subunit

genes

in

the

African-American

screen

set

of

families

revealed

association

in

-LRB-

p

-RRB-

and

-LRB-

p

-RRB-

the

same

two

genes

identified

through

allelic

association

and

interaction

analysis

in

the

Caucasian

dataset

-LSB-

-RSB-

Genotypic

association

analysis

revealed

the

same

SNP

-LRB-

p

-RRB-

and

marginal

significance

within

GABRP

-LRB-

p

-RRB-

The

remaining

SNPs

showed

no

significant

association

-LRB-

data

not

shown

-RRB-

Analysis

of

the

screening

SNPs

and

newly

identified

SNPs

within

and

in

the

Caucasian

population

-LRB-

N

-RRB-

and

within

the

extended

African-American

population

-LRB-

N

-RRB-

-LRB-

Table

-RRB-

revealed

new

SNPs

with

significant

association

In

the

Caucasian

dataset

also

significant

in

the

study

of

Ma

et

al.

-LRB-

p

-RRB-

showed

significant

allelic

association

-LRB-

p

-RRB-

Additional

significant

SNPs

were

identified

in

as

well

-LRB-

p

-RRB-

and

-LRB-

p

-RRB-

Genotypic

association

was

also

seen

in

these

SNPs

-LRB-

p

and

respectively

-RRB-

as

well

as

in

a

fourth

SNP

also

in

-LRB-

p

-RRB-

In

the

African-American

dataset

-LRB-

p

-RRB-

identified

in

the

smaller

African-American

dataset

above

and

-LRB-

p

-RRB-

were

found

to

be

associated

with

the

allele

based

test

Genotypic

association

was

also

identified

in

-LRB-

p

-RRB-

No

SNPs

within

were

found

to

be

associated

with

autism

in

either

ethnic

group

Table

of

and

in

extended

Caucasian

and

African-American

PDTaPDTaGeno

PDTaPDTGeno

PDTPDTGeno

-LRB-

G/G

-RRB-

-LRB-

T

-RRB-

-LRB-

T/T

-RRB-

-LRB-

C

-RRB-

-LRB-

C/C

-RRB-

-LRB-

G

-RRB-

-LRB-

A

-RRB-

-LRB-

A/A

-RRB-

-LRB-

A

-RRB-

-LRB-

A/A

-RRB-

Bold

indicates

significant

valuesaAssociated

allele/genotype

shown

in

parenthesis

The

majority

of

pairwise

values

between

the

significant

SNPs

were

less

than

in

both

ethnic

groups

-LRB-

Table

-RRB-

However

a

few

SNPs

have

values

between

and

SNPs

and

have

values

of

in

African-Americans

and

in

Caucasians

and

and

have

a

pairwise

of

in

Caucasians

and

in

African-Americans

Allele

frequencies

are

similar

yet

not

identical

between

the

two

groups

One

SNP

however

did

have

large

differences

in

minor

allele

frequencies

-LRB-

MAFs

-RRB-

between

the

two

groups

with

a

MAF

of

in

African-Americans

but

only

in

Caucasians

-LRB-

Table

-RRB-

Haplotype

analysis

using

the

four

SNPs

with

significant

allelic

or

genotypic

association

in

the

Caucasian

families

revealed

a

significant

global

test

-LRB-

p

-RRB-

in

the

Caucasian

population

further

supporting

the

involvement

of

these

SNPs

or

another

variant

on

the

haplotype

background

The

haplotype

composed

of

all

risk

alleles

trends

towards

significance

with

autism

as

a

risk

haplotype

-LRB-

p

-RRB-

while

the

haplotype

of

all

protective

alleles

resulted

in

being

highly

significantly

protective

-LRB-

p

Table

-RRB-

Table

allele

frequencies

and

linkage

disequilibrium

in

Caucasian

and

African-American

datasetsBold

indicates

significant

valuesaMAF

minor

allele

frequencyTable

frequency

and

association

in

Caucasian

Bold

indicates

significant

values

Subsetting

of

the

and

data

to

analyze

all

families

with

positive

history

for

seizures

revealed

no

association

to

However

three

SNPs

within

were

found

to

be

both

allelically

and

genotypically

associated

with

autism

-LRB-

p

and

p

for

PDT

and

genoPDT

respectively

-RRB-

-LRB-

p

and

p

-RRB-

and

-LRB-

p

and

p

-RRB-

These

three

SNPs

all

had

pairwise

values

less

than

-LRB-

data

not

shown

-RRB-

In

the

Caucasian

population

EMDR

verified

the

single

locus

effect

identified

through

PDT

analysis

in

-LRB-

p

-RRB-

and

identified

two

different

significant

two-locus

gene

gene

effects

between

and

with

-LRB-

p

-RRB-

and

with

-LRB-

p

-RRB-

Several

three

locus

effects

were

also

significant

-LSB-

and

-LRB-

p

-RRB-

and

-LRB-

p

-RRB-

and

-LRB-

p

-RRB-

and

and

-LRB-

p

-RRB-

-RSB-

-LRB-

Table

-RRB-

Table

results

in

Caucasian

dataset

between

and

SNPsSignificant

interactionsGeneSNP

Discussion

We

have

confirmed

the

involvement

of

in

autism

through

identification

of

significantly

associated

SNPs

within

an

independent

African-American

population

Furthermore

we

have

strengthened

our

original

findings

including

identification

of

additional

associated

SNPs

and

a

significant

interaction

between

and

and

in

an

extended

dataset

-LRB-

N

-RRB-

of

Caucasian

autism

families

The

AA

dataset

contains

only

families

and

in

general

power

can

be

a

problem

in

small

sample

sets

if

an

effect

is

not

seen

However

for

the

gene

we

did

find

significant

results

in

the

AA

dataset

Furthermore

studies

in

small

datasets

such

as

the

AA

dataset

can

also

be

less

robust

than

in

larger

datasets

However

the

fact

that

we

find

the

same

gene

significant

in

two

different

ethnic

groups

is

supportive

of

the

role

of

in

autism

risk

The

identification

of

two

different

two-way

interactions

between

and

provides

additional

evidence

of

the

complex

interaction

of

these

two

genes

in

autism

The

-LRB-

-RRB-

with

-LRB-

-RRB-

interaction

is

between

the

same

two

SNPs

previously

reported

in

the

study

of

Ma

et

al.

and

is

still

significant

in

our

larger

dataset

A

second

significant

two-way

interaction

was

found

also

including

in

as

well

as

in

further

supporting

that

interactions

between

these

two

genes

are

involved

in

autism

risk

Although

does

not

have

a

significant

PDT

result

it

is

found

in

both

significant

interaction

pairs

and

in

each

of

the

significant

three-way

interactions

The

MDR

approach

used

in

these

analyses

is

specifically

designed

to

detect

interaction

effects

both

in

the

presence

and

absence

of

main

effects

It

is

possible

that

both

of

the

two-way

interactions

are

being

identified

due

to

LD

between

the

two

SNPs

-LRB-

and

-RRB-

There

is

significant

correlation

between

the

two

SNPs

although

the

value

-LRB-

Table

-RRB-

between

them

is

not

large

-LRB-

-RRB-

Given

that

these

do

not

appear

to

be

causative

variants

it

is

likely

that

the

true

variant

yet

to

be

identified

is

in

LD

with

these

SNPs

Examination

of

interaction

in

the

independent

dataset

of

African-American

families

was

not

possible

due

to

the

limited

sample

size

We

also

identified

variants

within

as

associated

within

the

autistic

population

with

seizures

One

of

these

variants

also

shows

a

significant

interaction

with

SNPs

in

-LRB-

Table

-RRB-

While

no

effect

was

seen

in

for

the

seizure

subset

the

sample

size

may

be

too

small

to

conclusively

determine

its

role

in

seizure

status

in

autism

However

the

enhanced

findings

in

in

the

seizure

subset

implicate

as

a

contributor

to

genetic

risk

in

these

patients

Despite

the

identification

of

in

both

ethnic

groups

different

SNPs

were

found

to

be

associated

The

identification

of

distinct

SNPs

within

these

populations

may

indicate

differences

in

allele

frequency

and

linkage

disequilibrium

within

the

two

racial

groups

differences

in

the

haplotypic

background

in

which

identical

causative

variations

originated

or

differences

in

the

causative

variation

SNP

for

example

which

is

significant

in

the

African-American

group

shows

little

variation

in

the

Caucasian

dataset

therefore

providing

no

power

for

detection

of

an

effect

in

this

group

Other

SNPs

however

show

similar

allele

frequencies

between

the

two

populations

Some

differences

in

LD

do

exist

between

the

two

ethnic

groups

as

well

however

the

majority

of

the

differences

are

small

The

largest

differences

in

LD

are

in

pairwise

values

with

which

appear

to

mostly

be

due

to

the

fact

that

the

SNP

is

practically

monoallelic

in

the

Caucasian

population

The

Caucasian

dataset

suggests

that

there

is

a

significant

association

of

SNP

haplotypes

with

autism

risk

while

the

African-American

set

does

not

However

this

difference

may

be

due

to

the

small

size

of

the

African-American

dataset

Therefore

while

it

appears

that

minor

allele

frequency

differences

can

account

for

the

lack

of

association

of

in

the

Caucasian

dataset

additional

studies

are

needed

to

determine

whether

the

other

differences

in

results

between

the

two

ethnic

groups

are

due

to

sample

size

differences

differential

LD

with

the

causative

variation

in

the

two

populations

or

are

population-specific

risk

alleles

While

we

have

identified

several

associated

SNPs

we

do

not

predict

any

of

the

ones

in

to

have

functional

consequences

therefore

it

is

unlikely

that

these

are

primary

variants

leading

to

the

autism

susceptibility

One

of

the

SNPs

identified

in

in

the

seizure

subset

however

is

in

the

untranslated

region

-LRB-

UTR

-RRB-

Given

that

multiple

GABA

receptor

subunits

combine

in

varying

combinations

to

form

a

functional

GABA

receptor

even

minor

changes

in

levels

of

a

particular

subunit

may

alter

the

makeup

of

receptors

within

a

particular

cell

type

and

alter

the

GABAergic

signaling

Therefore

variations

within

potential

regulatory

regions

such

as

untranslated

regions

and

promoters

could

play

an

important

role

It

will

be

important

to

look

at

potential

changes

that

may

result

from

this

and

other

potential

UTR

variations

as

well

as

sequence

coding

and

potential

regulatory

regions

to

identify

the

primary

variation

or

variations

leading

to

altered

autism

susceptibility

In

summary

these

data

show

that

the

GABA

receptors

are

implicated

in

the

etiology

of

autism

in

two

different

ethnic

populations

and

suggest

seizures

as

a

stratifying

phenotype

Furthermore

these

results

support

our

earlier

findings

indicating

and

as

genes

contributing

to

autism

susceptibility

independently

in

the

case

of

and

through

complex

interactions

with

each

other

O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O


Effects of risedronate on fracture risk in postmenopausal women with osteopenia Summary This posthoc analysis of four trials studied the efficacy of risedronate to reduce fragility fractures in postmenopausal women with osteopenia -LRB- i.e. , T-scores between − 1 and − 2.5 -RRB- . Risedronate reduced the fracture risk by 73 % -LRB- = 0.023 -RRB- in this population of women with low femoral neck bone mass and no prevalent vertebral fractures . Introduction Low bone mass , defined by bone mineral density -LRB- BMD -RRB- values in the osteopenic range , can progress to osteoporosis if left untreated . Additionally , it has recently become apparent that osteopenia is a significant risk factor for fragility fractures in older women . Studies using peripheral and central dual x-ray absorptiometry -LRB- DXA -RRB- to determine BMD have demonstrated that approximately half of all fragility fractures occur in women with osteopenic BMD -LSB- 1 -- 5 -RSB- . As the number of women with low bone mass is projected to increase to 30.4 million in 2020 , this condition presents a significant medical problem and socioeconomic burden -LSB- 6 -RSB- . Prevalence projections for 2020 are significantly higher than current rates -LRB- 21.8 million women in 2002 -RRB- due to expected population growth and aging -LSB- 7 -RSB- . Based on BMD testing , the World Health Organization -LRB- WHO -RRB- clinically defines women with osteopenia as those whose BMD T-score is between 1 and 2.5 standard deviations -LRB- SD -RRB- below the mean of peak bone mass in healthy , young normal women -LRB- i.e. , − 1 to − 2.5 SD -RRB- ; women with osteoporosis include all those at or below − 2.5 SD -LSB- 8 -- 10 -RSB- . However , these definitions provide a diagnostic categorization of low bone mass rather than an intervention threshold that could be used to determine the need for the initiation of treatment to reduce fracture risk . Physicians are often faced with making treatment decisions for postmenopausal patients with T-scores in the osteopenic range , increasing the need for evidence-based guidance to define populations at high risk for fracture -LSB- 11 -RSB- . The National Osteoporosis Foundation -LRB- NOF -RRB- currently recommends an algorithm for initiation of therapy to reduce the risk of fracture -LSB- 12 -RSB- . According to this algorithm , treatment should be initiated in osteopenic women with T-scores < − 2.0 by central DXA with no risk factors and in women with T-scores < − 1.5 by central DXA with one or more risk factors -LSB- 9 , 10 , 12 , 13 -RSB- . Despite the existence of NOF guidelines for the treatment of postmenopausal women with low bone mass , limited clinical data exist as a foundation for supporting therapeutic intervention in this group . Although it is possible to identify osteopenic women at higher or lower short-term risk for fracture -LSB- 11 -RSB- , there are limited data regarding the effectiveness of osteoporosis treatments to lower fracture risk in these women . This is in part due to the inherent difficulty in collecting fracture data from osteoporosis trials , which usually exclude women with higher T-scores who may fall into the osteopenic range . The assembly of large data sets is , therefore , difficult and the feasibility of prospective osteopenic fracture trials is limited due the lower incidence of fracture in this population compared with patients with osteoporosis -LSB- 14 -RSB- . The use of post hoc analysis is one way to overcome some of these obstacles and provide information about effectiveness of treatments in non-osteoporotic populations . We have analyzed data from four Phase III risedronate trials : BMD Multinational -LRB- BMD-MN -RRB- -LSB- 15 -RSB- , BMD-North America -LRB- NA -RRB- -LSB- 16 -RSB- , Vertebral Efficacy with Risedronate Therapy-Multinational -LRB- VERT-MN -RRB- -LSB- 17 -RSB- , and Vertebral Efficacy with Risedronate Therapy-North America -LRB- VERT-NA -RRB- -LSB- 18 -RSB- in which efficacy and safety of risedronate in the prevention and treatment of postmenopausal osteoporosis have been demonstrated . Using data only from osteopenic women included in these trials , this study evaluated the effect of risedronate in reducing the risk of fragility fractures in women with femoral neck T-scores in the osteopenic range and without prevalent vertebral fracture . Methods Study design and patients This retrospective analysis included the subsets of postmenopausal women with osteopenia in four risedronate placebo-controlled osteoporosis trials -LSB- 15 -- 18 -RSB- of 1.5 to 3 years ' duration . Patients included in the current analysis were postmenopausal women who had no radiographic vertebral fractures at baseline and had a femoral neck T-score between − 1 and − 2.5 SD as measured by DXA -LRB- NHANES III -RRB- . These selection criteria were similar to those in previously published studies looking at similar populations -LSB- 19 , 20 -RSB- . Although inclusion criteria for the VERT trials required patients to have prevalent vertebral fractures , re-adjudication conducted by central radiographic assessment after enrollment showed that between 10 % and 15 % of enrolled patients had no prevalent vertebral fracture and could , therefore , be included in the present study . Our analysis was based on BMD assessment at the femoral neck rather than the lumbar spine , due to its greater reliability and predictive value and wide acceptance internationally for clinical diagnosis -LSB- 2 , 13 -RSB- . Patients treated with estrogen and estrogen-related drugs within 3 months of study entry or for more than 1 month within 6 months of study entry were excluded from the original studies . All patients received 1,000 mg calcium and , if needed , 500 IU vitamin D daily . Adverse event information was collected at the study visits made every 3 months during the trials . The investigator recorded adverse events reported by the patients , as well as adverse events observed on examination of the patient . Identification of fragility fractures Fragility fractures were defined as a composite of a patient 's incident morphometric vertebral and osteoporosis-related nonvertebral fractures -LRB- i.e. , six fracture types including clavicle , humerus , wrist , pelvis , hip or leg fractures -RRB- . This endpoint was chosen to include all radiographically confirmed fractures . With respect to the nonvertebral fractures , the selection of the nonvertebral fracture sites -LRB- clavicle , humerus , wrist , pelvis , hip or leg -RRB- followed the pre-specified definition of osteoporosis-related nonvertebral fractures of the risedronate trial program , which has been reported in all regulatory and previous scientific reports from this program . All osteoporosis related nonvertebral fractures required radiographic confirmation . The term `` leg '' fracture as used in the risedronate trials is a summary term , including the treatment emergent adverse event codes for fibula , tibia , subtrochanteric fracture , and leg , but not those for ankle or distal tibia fracture , the terms by which ankle fractures are usually reported . Statistical analyses The primary analysis focused on investigating the effect of risedronate to reduce fragility fractures -LRB- both vertebral and nonvertebral -RRB- in postmenopausal women who had no radiographic vertebral fractures at baseline and were considered osteopenic based on a femoral neck T-score ranging between − 1 SD and − 2.5 SD . For consistency with previous risedronate studies , the statistical analysis used time-to-first-event methodology . Cumulative fracture incidence was estimated using the Kaplan -- Meier method , and a Cox proportional hazards regression model was used to estimate the relative fracture risk reduction , via the hazard ratio -LRB- HR -RRB- , between the risedronate 5 mg and placebo groups . The regression model was stratified for each trial to allow for separate underlying hazard functions . In order to determine the acceptability of combining data from all trials , potential heterogeneity assessments were performed using the Cox regression model to assess the trial-by-treatment , age-by-treatment and baseline femoral neck BMD-by-treatment interactions . Fracture efficacy for nonvertebral osteoporosis-related fractures and new vertebral fractures were also investigated . A sensitivity analysis was conducted to investigate whether the exclusion of patients with lumbar spine T-score < − 2.5 SD would influence the outcome of the primary analyses . As the inclusion criteria for the primary analysis were low bone mass at the femoral neck -LRB- BMD T-score between − 1 and − 2.5 SD -RRB- , we felt it was important to test whether the exclusion of patients with an osteoporotic BMD at another site would significantly affect the results . Adverse events , serious adverse events , withdrawals , deaths , and upper gastrointestinal adverse events were summarized by treatment group . Differences in the proportions of patients between the treatment groups were tested using the Fisher 's exact test . Results Patients A total of 620 postmenopausal women met the criteria for low bone mass and were included in the current analysis . Patients received either placebo -LRB- = 309 -RRB- or risedronate 5 mg -LRB- = 311 -RRB- . Baseline characteristics were similar for each patient group -LRB- Table 1 -RRB- . The mean age was 64 years for both groups and mean baseline femoral neck T-scores were − 1.84 in the placebo group and − 1.85 in the risedronate group , respectively . Table 1Baseline characteristics of patients with a femoral neck T-Score ≥ − 2.5 SD and ≤ − 1 SD without prevalent vertebral fractures All patientsExcluding lumbar spine T-score < − 2.5 SDPlacebon n = 309Risedronate 5 mg n = 311Placebon n = 146Risedronate 5 mg n = 147Age -LRB- years -RRB- a64 -LRB- 7.5 -RRB- 64 -LRB- 7.5 -RRB- 63 -LRB- 8.3 -RRB- 65 -LRB- 7.0 -RRB- Femoral neck T-scorea , b − 1.84 -LRB- 0.396 -RRB- − 1.85 -LRB- 0.406 -RRB- − 1.77 -LRB- 0.418 -RRB- − 1.76 -LRB- 0.398 -RRB- Height -LRB- cm -RRB- a159 .8 -LRB- 6.92 -RRB- 159.8 -LRB- 6.05 -RRB- 160.6 -LRB- 6.14 -RRB- 160.4 -LRB- 6.32 -RRB- Weight -LRB- kg -RRB- a65 .9 -LRB- 11.92 -RRB- 66.8 -LRB- 11.19 -RRB- 69.8 -LRB- 12.91 -RRB- 69.6 -LRB- 12.27 -RRB- BMI -LRB- kg/m2 -RRB- a25 .9 -LRB- 4.54 -RRB- 26.2 -LRB- 4.36 -RRB- 27.2 -LRB- 5.1 -RRB- 27.1 -LRB- 4.78 -RRB- Patients with prevalent OP-related nonvertebral fractures56 -LRB- 18 % -RRB- 59 -LRB- 19 % -RRB- 34 -LRB- 23 % -RRB- 31 -LRB- 21 % -RRB- a Mean -LRB- standard deviation -RRB- b Based on National Health and Nutrition Examination Survey III reference database Potential heterogeneity assessments revealed that there were no significant interactions for trial-by-treatment -LRB- = 0.495 -RRB- , age-by-treatment -LRB- = 0.981 -RRB- or baseline femoral neck BMD-by-treatment -LRB- = 0.188 -RRB- Effect on fractures Risedronate treatment was found to significantly reduce the risk of combined morphometric vertebral and nonvertebral fractures by 73 % in women with a femoral neck T-score between − 1 and − 2.5 and no prevalent vertebral fracture ; HR = 0.27 , 95 % CI = 0.09 -- 0.83 , = 0.023 -LRB- Fig. 1 -RRB- . The cumulative fragility fracture incidence was 6.9 % and 2.2 % respectively for placebo and risedronate . Fig. 1Reduction of fragility fracture risk in patients with femoral neck T-score between − 1 and − 2.5 SD and no prevalent vertebral fractures When separated according to nonvertebral and vertebral fractures , the cumulative nonvertebral fracture incidence was 5.4 % and 0.4 % , respectively , for placebo and risedronate -LRB- HR = 0.09 , 95 % CI = 0.01 -- 0.71 -LRB- = 0.022 -RRB- , and the cumulative vertebral fracture incidence was 4.2 % and 1.8 % , respectively , for placebo and risedronate -LRB- HR = 0.44 , 95 % CI = 0.11 -- 1.78 -LRB- = 0.249 -RRB- . Sensitivity analyses When patients with baseline LS spine T-scores below − 2.5 SD were excluded , results consistent with those from the primary analysis population were observed , although the reduced sample size widened the confidence intervals and led to a loss of statistical significance . For the 293 patients who had both femoral neck and LS spine T-scores between − 2.5 and − 1 SD , the hazard ratio for fragility fractures was 0.22 -LRB- 95 % CI = 0.03 -- 2.02 , = 0.182 -RRB- , a magnitude of effect similar to that in the primary analysis population . Safety In the subgroup investigated for this analysis , no statistically significant differences in adverse events between the risedronate treated and the placebo group were recorded during the trial -LRB- Table 2 -RRB- . The observation in the subgroup analysis reported here is consistent with the findings in the overall population of the original trials -LSB- 15 -- 18 -RSB- . Table 2Summary of adverse events -LRB- AEs -RRB- for patients with a femoral neck T-score ≥ − 2.5 SD and ≤ − 1 SD and noprevalent vertebral fractures -LRB- BMD and VERT Trials -RRB- CategoryNumber of patients -LRB- % -RRB- - valuePlacebo -LRB- = 309 -RRB- Risedronate 5 mg -LRB- = 311 -RRB- AEs290 -LRB- 93.9 % -RRB- 293 -LRB- 94.2 % -RRB- 0.87 Serious AEs40 -LRB- 12.9 % -RRB- 56 -LRB- 18.0 % -RRB- 0.10 AEs associated with death 2 -LRB- 0.6 % -RRB- 0 -LRB- 0.0 % -RRB- 0.25 UGI AEs 77 -LRB- 24.9 % -RRB- 81 -LRB- 26.0 % -RRB- 0.78 Withdrawn due to AEs 38 -LRB- 12.3 % -RRB- 26 -LRB- 8.4 % -RRB- 0.11-values were from Fisher 's exact test.UGI : upper gastrointestinal Discussion Osteopenia is common in postmenopausal women and contributes to the increased risk of fracture in this population . There is a need for clinical data examining both fracture risk in women with T-scores in the osteopenic range and the evidence for an effect of treatment on reducing that risk . Data from the National Osteoporosis Risk Assessment -LRB- NORA -RRB- study reported that more than half of women experiencing self-reported fractures of the hip , vertebrae , rib and wrist/forearm at one year fell into the osteopenic category based on peripheral BMD T-scores between − 1 and − 2.5 -LSB- 21 -RSB- . Results from the present study show that risedronate treatment significantly reduced the overall risk of fragility fractures by 73 % over 3 years in patients with a femoral neck T-score between − 1 and − 2.5 and no prevalent vertebral fracture . A similar reduction in risk -- that did not reach statistical significance due to low numbers -- was observed when patients with lumbar spine T-scores below − 2.5 were excluded from the analysis . Previous studies in women with osteoporosis have shown that risedronate reduces the risk of morphometric vertebral fractures by up to 65 % in the first year of treatment -LSB- 17 , 18 -RSB- , with significant reductions in clinical vertebral and nonvertebral fractures as early as 6 months -LSB- 22 , 23 -RSB- . Moreover , a study in osteoporotic women without prevalent fracture demonstrated that treatment with risedronate reduced the risk of first vertebral fracture by 75 % -LRB- i.e. , a relative risk of 0.25 -RRB- -LSB- 24 -RSB- . Previously reported studies have examined the efficacy of alendronate -LSB- 20 , 25 -RSB- and raloxifene -LSB- 19 -RSB- on fracture risk reduction in osteopenic populations . A retrospective re-analysis of data from the Fracture Intervention Trial -LRB- FIT -RRB- showed a 60 % reduction of vertebral fractures -LSB- RR 0.40 -LRB- 0.19 -- 0.76 95 % CI -RRB- -RSB- in osteopenic women defined as having a femoral neck BMD T-score between − 1.6 and − 2.5 -LSB- 20 -RSB- . The overall analysis included patients with or without prevalent vertebral fractures , however , and since prevalent vertebral fractures are an important risk factor for further fractures and a criterion for severe osteoporosis -LSB- 8 , 13 -RSB- , classifying such women as osteopenic is subject to debate . When a subset of this population , those without prevalent fractures , was analyzed separately the results were similar , but did not reach statistical significance -LSB- RR 0.46 -LRB- 0.16 − 1.17 95 % CI -RRB- -RSB- . Results in patients with both hip and spine BMD in the osteopenic range are not reported in this paper . A re-analysis of the Multiple Outcomes of Raloxifene Evaluation -LRB- MORE -RRB- study reported similar rates of vertebral fracture risk reduction in raloxifene-treated women with osteopenia -- defined as a total hip T-score > − 2.5 without a prevalent vertebral fracture - compared with those with osteoporosis at 3 years -LSB- 19 -RSB- . The relative risk reduction for vertebral fractures with raloxifene compared with placebo was 0.53 -LRB- 0.32 -- 0.88 , 95 % CI -RRB- in osteopenic women ; the relative risk for clinical vertebral fractures in osteopenic women was 0.25 -LRB- 0.04 -- 0.63 -RRB- . Information about reduction of nonvertebral fractures has not been provided in either the alendronate or raloxifene analyses . Conjugated equine estrogens significantly reduced the risk of clinical vertebral , hip and total fractures in postmenopausal women in the Women 's Health Initiative , the vast majority of whom did not have bone density testing but who were not selected based on having diagnosed osteoporosis -LSB- 26 -RSB- . Finally , a study of women ≥ 75 years of age in the UK reported that oral daily clodronate 800 mg for 3 years reduced any clinical fracture by 20 % , but not hip fractures , in women unselected for having osteoporosis -LSB- 27 -RSB- . This reduction was independent of baseline BMD , but the number needed to treat was greater in the non-osteoporotic subjects . The fracture benefit observed with risedronate in patients with low bone mass in our analysis may result in part from the effect of this compound on the preservation of bone microarchitecture . In previous studies of both early postmenopausal women and osteoporotic patients risedronate was observed to preserve trabecular architecture , including trabecular number , thickness and separation , compared to placebo -LSB- 28 , 29 -RSB- , which is likely to contribute to a maintenance of bone strength -LSB- 30 -RSB- . This beneficial effect on trabecular architecture has also been observed with long-term treatment with risedronate , as shown by recent data from a study in postmenopausal osteoporotic women -LSB- 31 -RSB- . Bone microarchitecture , assessed by three-dimensional micro-computed tomography , was maintained at similar levels over a five-year treatment period -LSB- 31 -RSB- . The sustained preservation of bone microarchitecture equates to a long-term beneficial effect on bone strength and may contribute to the decrease in fracture risk observed with risedronate treatment . Limitations of the current study include the selection of initial populations in the VERT and BMD trials based on the presence of risk factors for osteoporosis -LRB- including low BMD and prevalent vertebral fractures -RRB- . Because the original trials were designed to examine the efficacy of osteoporosis treatments in an osteoporotic population , rather than to determine representative incidence of fracture in a general osteopenic population , the data provided in the present analysis may reflect an overestimation of the fracture risk in the general postmenopausal population . It is likely that in the relatively near future fracture risk assessment using a combination of femoral neck BMD , age , and other risk fractures will be utilized to determine absolute fracture risk and also serve as a basis for determining treatment intervention thresholds . In many older postmenopausal women , osteopenia will be an important risk factor for fracture , placing some of them at high absolute risk of a fracture event over a 10 year interval -LSB- 32 -RSB- , and treatment with a pharmacologic agent will be indicated to lower risk . The data presented here demonstrate that in addition to the established efficacy in preventing fracture in osteoporotic women with or without prevalent fractures , risedronate significantly reduces the risk of fragility fractures in this population of postmenopausal women with osteopenic BMD values at the femoral neck and no prevalent vertebral fractures .

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B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O

fragility fracture
nonvertebral fracture
osteopenia
risedronate
vertebral fracture