Non-Genetic Risk Factors for Holoprosencephaly

American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 154C:73–85 (2010)

A R T I C L E

Non-Genetic Risk Factors for HoloprosencephalyCANDICE Y. JOHNSON AND SONJA A. RASMUSSEN*

Holoprosencephaly (HPE) is a congenital defect of the brain characterized by incomplete cleavage of theembryonic forebrain into left and right hemispheres. Although a substantial proportion of cases of HPE can beattributed to genetic abnormalities, the etiology in many cases remains unknown, with non-genetic risk factorsbelieved to be important contributors. Due to the low birth prevalence of this defect, it has proven difficultto conduct studies of sufficient size to identify risk factors with certainty. This article provides a summary of non-genetic risk factors for HPE that have been investigated in case reports and case series, animal studies, andepidemiologic studies, including maternal illnesses, therapeutic and non-therapeutic exposures, nutritionalfactors, and sociodemographic factors. The article also highlights challenges in study design and further areas forresearch to better understand the etiology of HPE. Published 2010 Wiley-Liss, Inc.{

KEY WORDS: holoprosencephaly; risk factors; teratogens; diabetes; salicylates; cholesterol

How to cite this article: Johnson CY, Rasmussen SA. 2010. Non-genetic risk factors forholoprosencephaly. Am J Med Genet Part C Semin Med Genet 154C:73–85.

INTRODUCTION

Holoprosencephaly (HPE) is a congen-

ital defect of the brain characterized by

incomplete cleavage of the embryonic

forebrain into left and right hemispheres.

Affected persons often have accompany-

ing facial dysmorphology, ranging

from severe (e.g., cyclopia) to more mild

(e.g., mild hypotelorism or a single

central maxillary incisor) abnormalities,

although 10–20% of affected persons

have no facial abnormalities [Cohen,

1989a]. The prevalence at birth of

this condition is about 1 per 10,000

live births [Croen et al., 1996;

The prevalence at birth of this

condition is about 1 per

10,000 live births.

Rasmussen et al., 1996]. However, the

defect appears to occur much more

frequently earlier in pregnancy, with an

estimated rate of 4 per 1,000 conceptuses

[Matsunaga and Shiota, 1977], which

suggests a high rate of fetal loss.

However, the defect

appears to occur much

more frequently earlier in

pregnancy, with an

estimated rate of 4 per

1,000 conceptuses, which

suggests a high rate of

fetal loss.

In most cases, the causes of HPE

are unknown. Genetic causes include

chromosome abnormalities and single

gene disorders [Munke et al., 1988;

Cohen, 2006; Roessler and Muenke,

2010]. Among persons with HPE, about

25–50% have chromosome abnormal-

ities and about 18–25% have a mutation

in a single gene that causes syndromic

HPE [Muenke and Gropman, 2008].

The most commonly mutated gene in

persons with HPE is the sonic hedgehog

(SHH) gene, which codes for the sonic

hedgehog protein, a secreted protein

that plays a critical role in patterning of

the ventral neural tube, the anterior–

posterior limb axis, and ventral somites

[Muenke and Gropman, 2008]. In

recent years, much progress has been

made in understanding genetic factors

involved in the etiology of HPE

[Bendavid et al., 2009; Bendavid et al.,

2010]. However, even among families in

whom a single gene has been shown

to increase the risk for HPE, the

phenotype varies significantly among

family members who carry the gene,

The findings and conclusions in this reportare those of the authors and do notnecessarily represent the official position ofthe Centers for Disease Control and Preven-tion.

Candice Y. Johnson, M.Sc., is a doctoralstudent in the Department of Epidemiologyat Emory University and Guest Researcher atthe National Center on Birth Defects andDevelopmental Disabilities at the Centers forDisease Control and Prevention in Atlanta.She is interested in the epidemiology of birthdefects and the contributions of chronicillness during pregnancy to birth defectetiology.

Sonja A. Rasmussen, M.D., M.S., is apediatrician and clinical geneticist and cur-rently serves as Senior Scientist at theNational Center on Birth Defects andDevelopmental Disabilities at the Centersfor Disease Control and Prevention inAtlanta. Her research interests include theidentification of risk factors for birth defects,morbidity and mortality associated with birthdefects and genetic conditions, and theimpact of infections on the pregnant womanand her embryo or fetus.

*Correspondence to: Sonja A. Rasmussen,M.D., M.S., 1600 Clifton Road, MS E-86,CDC, Atlanta, GA 30333.E-mail: [email protected]

DOI 10.1002/ajmg.c.30242Published online 26 January 2010 in Wiley

InterScience (www.interscience.wiley.com)

Published 2010 Wiley-Liss, Inc.{This article is a US Government work and, as such, is inthe public domain in the United States of America.

including some family members with a

normal brain [Roessler et al., 1996;

Solomon et al., 2010], a finding that

supports a role for other genetic and

non-genetic factors [Ming and Muenke,

2002].

Evidence that supports the involve-

ment of certain non-genetic risk factors

is available from several sources, includ-

ing case reports/series, experimental

studies using animal models, and epi-

demiologic studies. Each of these has an

important role to play in better under-

standing non-genetic risk factors for

HPE, but each has its own strengths

and limitations [Rasmussen et al., 2007].

Case reports can alert investigators to

potential adverse effects of a particular

exposure so that the exposure can be

further investigated in an epidemiologic

study or experimental animal model.

However, in an individual case report or

case series, it is difficult to know if the

adverse effects are caused by the partic-

ular exposure or if the observed relation

is coincidental. Studies using an animal

model allow experimental manipulation

of the exposure while other factors (such

as genetic background) are held con-

stant; however, animal studies may not

always predict results in humans [Scialli

et al., 2004]. Epidemiologic studies of

HPE are challenging, given its low birth

prevalence, and only a few studies have

focused on identifying risk factors for

this defect. These studies have been

retrospective and have utilized a case–

control study design. The studies are

often dependent on maternal recall of

exposures, which can be incomplete and

possibly biased because of knowledge of

the outcome (i.e., recall bias) [Kallen,

2005]. In addition, it may sometimes be

challenging to determine if a particular

exposure is the risk factor or whether

another (confounding) factor may be

responsible for the observed increased

risk. In this manuscript, we will review

the evidence for certain non-genetic risk

factors, including data from case reports/

series, animal models, and epidemio-

logic studies. In addition, we will discuss

key issues for consideration in future

studies in order to improve available

information on non-genetic risk factors

for HPE.

EPIDEMIOLOGIC STUDIESOF HOLOPROSENCEPHALY

We are aware of four case–control

studies that have focused on a spectrum

of non-genetic risk factors for HPE.

Because these studies have been used to

address a large number of risk factors,

and thus will be referred to repeatedly

below, information about them is briefly

summarized here. In addition to these

studies, a few other epidemiologic

studies of specific risk factors (e.g.,

diabetes and obesity) have also some-

times included HPE as one of many

defects studied.

Human Embryo Center for

Teratological Studies

(Kyoto Embryo Study)

Between 1962 and 1974, a total of

36,380 conceptuses (resulting in 3,411

undamaged embryos) were collected by

Kyoto University in Japan from physi-

cians performing pregnancy termina-

tions [Matsunaga and Shiota, 1977].

Among the 3,411 embryos, 150 with

HPE were identified. A matched case–

control study was conducted where

two non-cases were selected from the

undamaged embryos as controls and

matched to each HPE case on parity

and month of last menstrual period.

Karyotyping was not performed, and the

proportion of cases with chromosomal

abnormalities is unknown. Physicians

performing pregnancy terminations

were asked to collect information on

the reproductive and exposure histories

of the mothers.

California Birth Defects

Monitoring Program (CBDMP)

Forty-eight cases of non-syndromic

HPE (live births, fetal deaths

�20 weeks’ gestation or pregnancy

terminations) were identified between

1993 and 1996 using data from the

CBDMP, a birth defects surveillance

system that uses active case ascertain-

ment methods that include review of

medical records at hospitals and genetics

clinics in 11 California counties [Croen

et al., 2000]. Infants with a chromosome

abnormality were excluded. Controls

were liveborn infants without birth

defects randomly selected from births

in the hospitals in the area. Mothers were

interviewed by telephone. Another epi-

demiologic study of HPE using data

from the CBDMP focused on cases

identified between 1983 and 1988 and

included 53 non-syndromic cases

[Croen et al., 1996].

Latin American Study of

Congenital

Malformations (ECLAMC)

Between 1967 and 2000, 281 liveborn or

stillborn (birthweight �500 g) infants

with non-syndromic HPE were identi-

fied through the ECLAMC birth defects

surveillance system, a hospital-based

system that includes births in all South

American countries (except Guyanas)

[Orioli and Castilla, 2007]. Cases with

chromosome abnormalities or other

syndromes were excluded. Controls

were selected as the next non-mal-

formed child born in the same hospital

as the case, matched on sex. Mothers

were interviewed by the pediatrician

shortly after the birth.

National Birth Defects Prevention

Study (NBDPS)

The NBDPS is an ongoing, population-

based case–control study that ascertains

liveborn infants, fetal deaths (�20

weeks’ gestation), and pregnancy termi-

nations with major birth defects through

birth defect surveillance systems in

10 regions of the United States [Yoon

et al., 2001; Miller et al., 2010]. Cases

with recognized single gene disorders

and chromosome abnormalities are

excluded [Rasmussen et al., 2003].

Controls are liveborn infants without

birth defects selected from study catch-

ment areas. Mothers of case and control

infants are interviewed up to 2 years

after delivery. From 1997 through

2004, 74 cases of non-syndromic HPE

were identified [Miller et al., 2010].

Mothers with pre-existing diabetes were

excluded from the analysis of HPE

risk factors. Analyses were adjusted

for potential confounders including

74 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

maternal age, education, race/ethnicity,

number of previous pregnancies, and

periconceptional use of aspirin and folic

acid.

MATERNAL ILLNESSES

Diabetes

Maternal diabetes is a well-recognized

risk factor for birth defects [Becerra

et al., 1990; Correa et al., 2008] and is

one of the most extensively studied

risk factors for HPE. HPE is estimated

to occur in �1% of births to

diabetic women [Barr et al., 1983].

Maternal diabetes is a

well-recognized risk factor for

birth defects and is one of the

most extensively studied risk

factors for HPE. HPE is

estimated to occur in �1% of

births to diabetic women.

Surveillance data from the New York

State Congenital Malformations Regis-

try and data from a case series collected

by the Carter Centers for Brain

Research have noted the prevalence of

pre-existing diabetes among mothers of

children with HPE to be 6% and 9%,

respectively [Olsen et al., 1997; Sta-

shinko et al., 2004].

Results from a mouse model are

consistent with the observations from

these case series. In one study, pregnant

mice were given streptozotocin intra-

peritoneally on gestational day 2 to

induce diabetes, and the rate of several

congenital anomalies (including HPE)

among offspring was observed. Diabetic

mice had an increased risk of delivering

offspring with HPE; this risk was

reduced among mice treated with insu-

lin. To observe the interaction between

diabetes and ethanol, the authors also

exposed the pregnant diabetic mice to

ethanol, and a decrease in the rate of

HPE was observed. This led the authors

to hypothesize that ethanol exposure

might result in increased pregnancy

loss of severely malformed embryos

[Padmanabhan and Shafiullah, 2004].

Four epidemiologic studies of pre-

existing diabetes [Croen et al., 2000;

Anderson et al., 2005; Orioli and

Castilla, 2007; Correa et al., 2008] and

three of four studies of gestational

diabetes [Martinez-Frias et al., 1998;

Croen et al., 2000; Anderson et al.,

2005; Correa et al., 2008] have observed

the prevalence of diabetes among moth-

ers of infants with HPE to be at least

twice as high as among control mothers

(Table I). In the study using CBDMP

data, the increased risk associated

with ever having diabetes was mainly

observed among mothers who reported

taking insulin to treat their diabetes

(odds ratio [OR] 10.2, 95% confidence

interval [CI] 1.9–39.4 for mother taking

insulin, OR 0.8, 95% CI 0.2–2.8 for

those not taking insulin) [Croen et al.,

2000]. A study using data from the

NBDPS noted that the association

between HPE and pregestational diabe-

tes to be stronger when HPE occurred

with other unrelated birth defects (OR

16.2; 95% CI 1.6–163.9), compared to

when HPE occurred as an isolated defect

(OR 6.0, 95% CI 0.7–49.8) [Correa

et al., 2008]. However, CIs were wide

because the number of cases with HPE

was small.

Despite the fact that diabetes is a

well-recognized risk factor for HPE and

other birth defects, its mechanism of

action as a teratogen is not well under-

stood. Hyperglycemia, oxidative stress,

and other metabolic disturbances

have been investigated as potential

mechanisms for diabetic teratogenesis

[Kousseff, 1999; Ornoy, 2007]. Better

understanding of the mechanism

could lead to improved therapeutic

options for pregnant women with dia-

betes to reduce their risk for birth

defects; although reduced risks of

birth defects appear to be achievable

through better preconceptional care and

glycemic control of diabetes during

pregnancy, diabetic women continue

to have an increased risk for having

children with birth defects [Suhonen

et al., 2000; Ray et al., 2001; Platt et al.,

2002].

Infections

Avarietyof infections have been reported

among mothers of infants with HPE [see

Cohen and Shiota, 2002 for a review].

Congenital cytomegalovirus (CMV)

infection has been described as a possible

cause of HPE in several case reports and

case series [Barr et al., 1983; Byrne et al.,

1987; Rasmussen et al., 1996; Kilic and

Yazici, 2005]. However, documentation

of congenital CMV was incomplete in

several of these cases. Other infections

noted in mothers who have had a child

with HPE include one case where rubella

was suspected in the mother, who had a

rash and high fever during the first weeks

of fetal development [Khudr and Olding,

1973], and two infants with HPE and

trisomy 13 who showed evidence of

herpes simplex virus and of congenital

syphilis [Rasmussen et al., 1996]. As with

CMV, documentation of these congenital

infections was incomplete.

Three epidemiologic studies have

investigated risk of HPE among mothers

with respiratory illnesses, respiratory

infections, or influenza during preg-

nancy. While the study from ECLAMC

identified an increased risk among

mothers with influenza (OR 2.8, 95%

CI 1.0–7.9) and an increased risk among

mothers with respiratory illness was

reported using data from CBDMP (OR

2.0, 95% CI 0.9–4.5) [Croen et al., 2000;

Orioli and Castilla, 2007], a subsequent

study from NBDPS found no association

with respiratory infection or with fever

during pregnancy [Miller et al., 2010].

Studies using data from NBDPS

and CBDMP have each suggested an

increased risk of HPE among mothers

with sexually transmitted infections.

NBDPS investigators reported an OR

of 2.1 (95% CI 0.9–5.1) for any sexually

transmitted infection [Miller et al.,

2010], while CBDMP researchers

reported a prevalence of genital herpes

or chlamydia of 4/48 among HPE case

mothers and 2/107 among control

mothers [Croen et al., 2000].

High Blood Pressure

Associations between HPE and mater-

nal high blood pressure have been

ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 75

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investigated in epidemiologic studies

using data from the NBDPS and

CBDMP, neither of which identified

an association [Croen et al., 2000; Miller

et al., 2010].

Anemia

An increased risk of HPE to mothers

with anemia was reported using data

from the CBDMP (OR 3.4, 95% CI

1.0–11.8) [Croen et al., 2000], although

a subsequent study using data from

ECLAMC found no such association

(OR 0.9, 95% CI 0.1–5.9) [Orioli and

Castilla, 2007] The ECLAMC study also

examined HPE and use of iron supple-

ments during pregnancy, and no associ-

ation was seen [Orioli and Castilla,

2007].

Other Maternal Illnesses

and Injuries

Researchers used CBDMP data to

investigate associations between HPE

and skin problems, allergies, urinary

tract infection, and injury. No associa-

tions were identified [Croen et al.,

2000]. A single case of lobar HPE has

been reported born to a mother with

phenylketonuria who had high phenyl-

alanine levels throughout the first 8–

10 weeks of pregnancy [Keller et al.,

2000].

MATERNAL THERAPEUTICEXPOSURES

Salicylates, Including Aspirin

Three case reports of children with HPE

among mothers taking large doses of

aspirin during early pregnancy have

been described, and two additional cases

have been described born to mothers

taking sodium salicylate and sulfasalazine

(Table II) [Khudr and Olding, 1973;

Benawra et al., 1980; Agapitos et al.,

1986; Koyama et al., 1996; Sezgin et al.,

2002]. In these cases, the mothers had

additional potential risk factors such as

parental consanguinity [Sezgin et al.,

2002], smoking [Benawra et al., 1980],

and use of other medications [Khudr and

Olding, 1973; Koyama et al., 1996]

(Table II).

Salicylate use has been investigated

in three epidemiologic studies with

inconsistent results. Findings from the

CBDMP and NBDPS showed that

mothers who have had a child with

HPE were over twice as likely to have

taken aspirin or salicylates during early

pregnancy (Table III) [Croen et al.,

2000; Miller et al., 2010], whereas the

study from ECLAMC showed no asso-

ciation [Orioli and Castilla, 2007].

Findings from the CBDMP

and NBDPS showed that

mothers who have had a child

with HPE were over twice as

likely to have taken aspirin or

salicylates during early

pregnancy (Table III), whereas

the study from ECLAMC

showed no association.

As with all studies showing a relation

between use of a medication and birth

defects, it is important to consider

whether the observed association

between salicylates and HPE observed

in some studies is related to the medi-

cation itself or to the indication for

which the medication was used.

Other Analgesics

One case report has described HPE in an

infant born to a mother who took

acetaminophen in her second trimester

of pregnancy, but the mother had also

TABLE II. Case Reports of Holoprosencephaly Where Maternal Salicylate Use Was Reported

Study Maternal characteristics Salicylate exposure Diagnosis

Other reported exposures

or risk factors

Sezgin et al. [2002] 38-year-old with 2

healthy children

Aspirin 500 g twice daily for

1 week during first trimester

Cyclopia, astomia, agnathia

(46, XX)

Consanguinity (parents first

cousins)

Agapitos et al. [1986] 41-year-old G2P1 Aspirin up to 4 g/day during

first trimester

Cyclopia, proboscis, multiple

other birth defects, no

chromosomal studies available

Advanced age of mother (41)

and father (56)

Benawra et al. [1980] 20-year-old G2P0 Aspirin 3–4.5 g/day during first

trimester

Cyclopia, polypoid proboscis,

multiple other birth defects,

normal chromosomes

Acetaminophen 6–10 tablets/

day during second trimester,

smoking 2 packs/day

throughout pregnancy

Khudr and

Olding [1973]

22-year-old G2P1 Sodium salicylate 3.6 g/day for

7 days during first trimester

Cyclopia, proboscis,

low-set ears

Viremia during week 3 of fetal

development, treatment with

high-dose penicillin G

procaine, cortisone

Koyama et al. [1996] 32-year-old G0P0 with

Crohn’s disease

Sulfasalazine 3 g/day before and

during pregnancy

Semilobar holoprosencephaly

(46, XX)

Ferrostatin (Fe) 100 mg/day,

kernac (plant extract)

240 mg/day, protective drug

for gastritis or gastric ulcer,

artificial insemination


taken aspirin in the first trimester

and smoked throughout pregnancy

[Benawra et al., 1980]. Results from

the NBDPS found a weak association

between acetaminophen use during

pregnancy and HPE (OR 1.5, 95% CI

0.9–2.5), but no association with

ibuprofen use [Miller et al., 2010].

Antiepileptic Medications

Kotzot et al. [1993] reported an infant

with HPE whose mother was treated

with phenytoin and primidone. In

addition to HPE, the infant had features

of fetal hydantoin syndrome, including

hypertelorism, midfacial hypoplasia, and

hypoplastic distal phalanges and nails of

the fingers and toes [Kotzot et al., 1993].

To address this possible association, a

review of infants born at Boston area

hospitals who were exposed to antiepi-

leptic medications was performed, and

one case of HPE was recorded among

112 antiepileptic-exposed pregnancies.

However, in a subsequent analysis of 453

antiepileptic-exposed pregnancies, no

cases with HPE were identified [Holmes

and Harvey, 1994]. Rosa subsequently

reviewed reports of adverse events to the

U.S. Food and Drug Administration

(FDA). He identified seven cases of

suspected HPE including the case

reported by Kotzot et al. [1993]

among mothers taking antiepileptic

drugs during pregnancy, including car-

bamazepine, valproate, phenytoin, and

primidone (used alone or in combina-

tion) [Rosa, 1995]. The proportion of

infants with HPE was higher among

antiepileptic users than in mothers

exposed to medications other than

anticonvulsants.

Retinoic Acid

In a series of 154 mothers with prenatal

exposure to isotretinoin, a retinoic

acid derivative used to treat severe acne,

21 had a child with a birth defect, one of

whom had HPE [Lammer et al., 1985].

Between 1969 and 1993, 25 cases of

birth defects delivered following first-

trimester exposure to topical tretinoin

(all-trans-retinoic acid), a medication

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used for treatment of acne and of

aging and sun-damaged skin, were

reported to the FDA. Five of these

25 had HPE, compared to 19 cases of

HPE occurring in the 8,700 reports

where the exposure was not retinoic acid

[Rosa, 1994].

Animal models also support a pos-

sible relation between prenatal exposure

to retinoic acid and HPE. Oral admin-

istration of all-trans-retinoic acid in

early pregnancy (gestational day 7) to

inbred mice has produced a range of

craniofacial phenotypes consistent with

HPE [Kalter, 1992; Sulik et al., 1995;

Lipinski et al., 2010].

Antibiotics

Case reports have described use of

antibiotics among mothers who have

had a child with HPE, including

penicillin procaine [Khudr and Olding,

1973], ampicillin [Garzozi and Barkay,

1985], and trimethoprim–sulfamethox-

azole [Ronen, 1992]. In the Kyoto

embryo study, 0.8% of case mothers

and 1.2% of control mothers reported

using antibiotics during pregnancy

[Matsunaga and Shiota, 1977].

Hormones

Case reports have described the use

of several different hormones during

early pregnancy among mothers who

have had a child with HPE, including

allyloestrenol and dihydrogesterone

[Stabile et al., 1985], oral contraceptives

[Ronen and Andrews, 1991], and

cortisone [Khudr and Olding, 1973],

although most mothers taking these hor-

mones were taking other medications.

Use of sex hormones was associated

with an increased risk of HPE in the

study using data from ECLAMC (OR

3.7, 95% CI 1.3–11.0) [Orioli and

Castilla, 2007]. An elevated risk was also

seen in NBOPS among mothers using

progesterone before or during the first

trimester of programming, although the

insert was not statistically significant

(OR 2.3, 75%, CI 0.7–7.3) [Miller et

al., 2010]. In the Kyoto embryo study,

23% of case mothers had taken proges-

tagens during early pregnancy compared

to 15% of control mothers [Matsunaga

and Shiota, 1977], while investigators

from CBDMP found no association

between HPE and use of birth control

pills [Croen et al., 2000].

Alkaloids Including Cyclopamine

and Jervine

In sheep and several other animal

species, consumption of the corn lily

Veratum californicum in early pregnancy

was associated with malformations

characteristic of HPE in the offspring

[Coventry et al., 1998; Hovhannisyan

et al., 2009; Welch et al., 2009].

In sheep and several other

animal species, consumption

of the corn lily Veratum

californicum in early pregnancy

was associated with

malformations characteristic of

HPE in the offspring.

Cyclopamine and jervine have since

been identified as the primary terato-

genic components of the corn lily plant.

These alkaloids are structural analogs of

cholesterol and act by inhibiting hedge-

hog signaling [Hovhannisyan et al.,

2009]. These components and related

compounds are under investigation as

therapeutic agents for treatment of

psoriasis and several malignancies [Tas

and Avci, 2004; Feldmann et al., 2008;

Garber, 2008; Von Hoff et al., 2009]. We

are not aware of any reports of human

exposures to these alkaloids during

pregnancy.

Statins

Several pieces of evidence have led to

concerns that maternal exposure to

statins, which are used to lower choles-

terol levels, might raise the risk for HPE.

These include the fact that some genetic

conditions with defects in cholesterol

metabolism include HPE as a feature in

about 5% of cases [Kelley et al., 1996;

Incardona and Roelink, 2000], inhib-

ition of cholesterol biosynthesis has

previously been shown to result in

HPE in animal studies [Kolf-Clauw

et al., 1997; Roux et al., 2000], and

cholesterol is required for hedgehog

signaling, a pathway which is critical in

embryonic development and has

been implicated in HPE [Roessler and

Muenke, 2003; Edison and Muenke,

2004b]. One report of 52 statin-exposed

pregnancies reported to the FDA iden-

tified 20 mothers who had a child with a

major malformation, including 1 with

HPE [Edison and Muenke, 2004a,b,

2005]. However, among 22 mothers

participating in the NBDPS and the

Slone Epidemiology Center Birth

Defects Study who were exposed to

statins during pregnancy and had a child

with a major birth defect, none had an

infant with HPE [Petersen et al., 2008].

Analyses using a case–control design

have not been possible because of the

low frequency of exposure to statins

during pregnancy.

Other Medications

Case reports and case series have

described a wide variety of medication

use during pregnancy among mothers

who have had a child with HPE,

including methotrexate (used for cancer

treatment and as an abortifacient) [Seid-

ahmed et al., 2006], tranexamic acid

(used for the treatment of excessive

bleeding) [Garzozi and Barkay, 1985],

chlordiazepoxide (a benzodiazepine

derivative used for the treatment of

anxiety) [Ronen and Andrews, 1991],

imipramine (a tricyclic antidepressant)

[Ronen and Andrews, 1991], lithium

(used in the treatment of bipolar

disorder) [Rasmussen et al., 1996], and

dimenhydrinate (an over-the-counter

medication used to treat nausea and

motion sickness) [Ronen, 1992]. A

higher prevalence of medication use

overall among case mothers compared

to control mothers was observed in the

Kyoto embryo study [Matsunaga and

Shiota, 1977].

Fertility Treatments

Use of artificial insemination has been

reported in a case of HPE, although the


mother had other potential risk factors

such as use of salicylates and other

medications during pregnancy [Koyama

et al., 1996]. Data from the NBDPS

suggest an increased risk of HPE among

mothers who have undergone assisted

reproductive technologies (OR 4.2,

95% CI 1.3–13.7), but not for fertility

treatments overall (OR 0.9, 95% CI

0.3–2.9) [Miller et al., 2010].

Medical Exposures

No association between HPE and

X-rays or scans during pregnancy was

observed by NBDPS or CBDMP inves-

tigators [Croen et al., 2000; Miller et al.,

2010]. Data from CBDMP were also

used to investigate whether HPE was

associated with surgery during preg-

nancy, and no association was seen


MATERNALNON-THERAPEUTICEXPOSURES

Alcohol

Several case reports have implicated

heavy alcohol consumption during the

first trimester of pregnancy as a cause of

HPE, although many of the reported

mothers had other potential risk factors

such as smoking and medication use

[Bonnemann and Meinecke, 1990;

Ronen and Andrews, 1991; Coulter

et al., 1993; Rasmussen et al., 1996].

Animal studies have supported a

role for maternal alcohol exposure in the

etiology of HPE. Acute maternal etha-

nol exposure at an early stage (gastrula-

tion) in embryonic development in mice

has been shown to induce a spectrum of

facial abnormalities consistent with HPE

[Sulik and Johnston, 1982; Webster

et al., 1983]. In a study of weekly

Animal studies have supported

a role for maternal alcohol

exposure in the etiology of

HPE. Acute maternal ethanol

exposure at an early stage

(gastrulation) in embryonic

development in mice has been

shown to induce a spectrum of

facial abnormalities consistent

with HPE.

administration of ethanol throughout

gestation to pregnant pigtail macaques,

HPE was observed in a single fetus

[Siebert et al., 1991]. Brief ethanol

exposure of zebrafish during early gas-

trulation has been shown to induce

cyclopia [Blader and Strahle, 1998].

Animal models have also been used to

provide insight into the mechanism by

which alcohol produces these abnor-

malities in animals. These studies suggest

that ethanol exposure results in pertur-

bation of expression of certain devel-

opmental genes at a critical period in

development [Blader and Strahle, 1998;

Higashiyama et al., 2007].

However, results of epidemiologic

studies of maternal alcohol use during

pregnancy have been inconsistent. An

elevated risk (OR 2.0, 95% CI 0.9–4.5)

was seen in the study using CBDMP data

[Croen et al., 2000], but researchers

using data from NBDPS and the

Kyoto embryo study found no associa-

tion between alcohol use and HPE

[Matsunaga and Shiota, 1977; Miller

et al., 2010]. Using data from CBDMP,

investigators noted an increased risk of

HPE when mothers consumed alcohol

and also smoked during pregnancy (OR

5.4, 95% CI 1.4–20.0) [Croen et al.,

2000].

Smoking

Several case reports have described

heavy smoking during pregnancy

among mothers who had children with

HPE, although most mothers had other

potential risk factors such as alcohol

intake and medication use [Benawra

et al., 1980; Burck et al., 1982; Ronen

and Andrews, 1991]. Three epidemio-

logic studies have investigated the role of

smoking as a potential risk factor for

HPE, with conflicting results. CBDMP

researchers found a fourfold increased

risk (OR 4.1, 95% CI 1.4–12.0)

[Croen et al., 2000], whereas investiga-

tors from the NBDPS and the Kyoto

embryo study saw no association

[Matsunaga and Shiota, 1977; Miller

et al., 2010].

Illicit Drugs

Among mothers of infants with HPE

identified through the New York State

Congenital Malformations Registry,

medical records indicated that 3 of 82

used cocaine or marijuana during preg-

nancy [Olsen et al., 1997]. Maternal

marijuana use has been described in

a case report of HPE, although this

mother had also consumed alcohol

heavily during pregnancy [Coulter

et al., 1993]. No association between

HPE and maternal illicit drug use has

been seen in epidemiologic studies using

data from NBPDS and CBDMP [Croen

et al., 2000; Miller et al., 2010].

NUTRITIONAL FACTORS

Body Mass Index

Three studies have investigated associa-

tions between body mass index (BMI)

and HPE. Increased risks were observed

in studies using data from CBDMP (OR

1.5, 95% CI 0.6–4.0 for BMI >29 kg/

m2 vs. BMI �29 kg/m2) and from the

Texas Birth Defects Registry (OR 1.4,

95% CI 0.5–3.8 for BMI �30.0 kg/m2

vs. BMI 18.5–24.9 kg/m2, adjusted for

ethnicity, age, education, smoking, alco-

hol use, and folic acid use and excluding

mothers with pre-existing diabetes),

although CIs were wide and included

the null value [Croen et al., 2000;

Anderson et al., 2005]. The study from

NBDPS, which also excluded women

with diabetes from the analysis, found no

association between maternal pre-preg-

nancy obesity and HPE (OR 0.8, 95%

CI 0.4–1.6 for BMI �30.0 kg/m2 vs.

BMI 18.5–24.9 kg/m2, adjusted for

age, race/ethnicity, education, previous

pregnancies, folic acid use, and aspirin

use) [Miller et al., 2010]. The

Texas study also investigated interactions

between gestational diabetes and obesity

and observed that obesity alone

(BMI �30.0 kg/m2 compared to BMI


<30.0 kg/m2) had no effect, but among

obese women who also had gestational

diabetes, the risk of HPE was increased

over sixfold (OR 6.5, 95% CI 1.3–31)

[Anderson et al., 2005].

Diet

A case of HPE that occurred in a mother

who adhered to a low-calorie diet

during early pregnancy has been

reported, although the mother had other

potential risk factors for HPE such as use

of antibiotics during pregnancy [Ronen,

1992]. NBDPS investigators noted that

quality of diet may be associated with

HPE, with lower risks observed for

mothers who had high consumption of

skim or low-fat milk, yogurt, butter,

string beans, broccoli, chicken, and fish

[Miller et al., 2010].

Cholesterol

Much attention has been given to the

involvement of cholesterol in the etiol-

ogy of HPE (see discussion in the Statins

Section). However, NBDPS is the only

epidemiologic study to date that has

investigated dietary cholesterol intake

in relation to HPE risk, and no asso-

ciation was observed (OR 1.0, 95%

Much attention has been given

to the involvement of cholesterol

in the etiology of HPE (see

discussion in the Statins

Section). However, NBDPS is

the only epidemiologic study to

date that has investigated

dietary cholesterol intake in

relation to HPE risk, and no

association was observed.

CI 0.5–2.0 for highest vs. lowest

quartiles of intake) [Miller et al., 2010].

Folic Acid and Multivitamin Use

Based on data from the NBDPS, women

who used folic acid or multivitamins

during pregnancy were at a decreased

risk of having an infant with HPE (OR

0.4, 95% CI 0.2–1.0) [Miller et al.,

2010]; however, this association was not

observed in analysis of data from

CBDMP (OR 1.4, 95% CI 0.6–3.3)


PREGNANCYCHARACTERISTICS ANDREPRODUCTIVE HISTORY

Twinning

HPE has been reported to occur more

frequently among twins. Studies have

consistently found approximately 6% of

HPE cases being a member of twins or

multiple births, a higher percentage than

expected [Odent et al., 1998; Bullen

et al., 2001; Miller et al., 2010]. In a case

series in Indiana which collected pedi-

gree information on each proband with

HPE, the twinning rate was elevated in

the probands’ and the parents’ gener-

ations compared to the expected rate

among families in Indiana [Roach et al.,

1975]. Epidemiologic studies from

ECLAMC and CBDMP observed no

excess of cases among twins, but power

to detect this finding was low [Croen

et al., 1996; Orioli and Castilla, 2007].

Sex Ratio

Sex ratio may be a potential risk factor

for HPE since an excess of females with

HPE has been consistently reported

in the literature [Kallen et al., 1992;

Rasmussen et al., 1996; Whiteford and

Tolmie, 1996; Olsen et al., 1997; Odent

et al., 1998; Croen et al., 2000; Forrester

and Merz, 2000; Stashinko et al., 2004;

Orioli and Castilla, 2007], although

some studies have found a more even

ratio of males and females affected

[Bullen et al., 2001; Miller et al.,

2010]. The sex ratio has been

Sex ratio may be a potential risk

factor for HPE since an excess of

females with HPE has been

consistently reported in the

literature, although some

studies have found a more even

ratio of males and

females affected.

reported to differ based on the presence

or absence of other birth defects

[Orioli and Castilla, 2007] and for

subtypes of HPE [Roach et al., 1975;

Whiteford and Tolmie, 1996; Odent

et al., 1998]. More research is needed to

determine the reasons for the altered sex

ratio among HPE cases and within

subtypes.

Vaginal Bleeding

During Pregnancy

Significant associations were identified

between vaginal bleeding during preg-

nancy and HPE in the study from

ECLAMC (OR 2.2, 95% CI 1.1–4.6)

and from the Kyoto embryo study,

where 70% of case mothers reported

bleeding, compared to 26% of control

mothers [Matsunaga and Shiota, 1977;

Orioli and Castilla, 2007].

Menstrual Cycles

No difference in menstrual cycle irreg-

ularity between case and control moth-

ers was observed in the Kyoto embryo

study [Matsunaga and Shiota, 1977].

Researchers from CBDMP suggested a

twofold increased risk for HPE among

mothers who experienced menarche at

age 11 or younger, compared to mothers

with menarche between ages of 12 and

14 (OR 2.3, 95% CI 0.9–5.7) [Croen

et al., 2000].

Parity

No association was observed between

HPE and number of previous live births

from studies from CBMDP and the

Kyoto embryo study, although the

Kyoto embryo study did observe an

increased risk with lower parity when

parity was treated as a categorical

as opposed to a continuous variable


[Matsunaga and Shiota, 1977; Croen

et al., 2000].

Gravidity

Increased risk among mothers with high

gravidity has been observed by CBDMP

(OR 2.1, 95% CI 0.7–6.4 for gravidity 4

vs. 1) and ECLAMC (OR 1.71, 95% CI

0.94–3.10 for gravidity 4 or more vs.

3 or less) investigators [Croen et al.,

2000; Orioli and Castilla, 2007]. Based

on data from NBDPS, no statistically

significant association was observed

between gravidity and HPE, although

risk tended to increase with increasing

gravidity [Miller et al., 2010].

Previous Pregnancy Loss

No association was observed between

HPE and previous pregnancy loss in

epidemiologic studies from CBDMP,

ECLAMC, and NBDPS [Croen et al.,

2000; Orioli and Castilla, 2007; Miller

et al., 2010]. The Kyoto embryo study

observed more miscarriages among case

mothers compared to control mothers

(29.5% vs. 21.0%), although the differ-

ence was not statistically significant

[Matsunaga and Shiota, 1977].

Pregnancy Intention

NBDPS investigators identified an

increased risk for mothers whose pre-

gnancies were unintended compared to

those that were intended (OR 1.9, 95%

CI 1.0–3.6), but not for mothers whose

pregnancies were mistimed [Miller et al.,

2010].

Season of Conception

Month of conception of HPE cases

appeared to peak in March and has a

low in September in the Kyoto embryo

study, although this finding was not

statistically significant [Matsunaga and

Shiota, 1977].

SOCIODEMOGRAPHICFACTORS

Age

Maternal age has been inconsistently

associated with HPE, with some studies

finding lower risks among young

mothers and higher risks among older

mothers [Kallen et al., 1992; Orioli and

Castilla, 2007], others finding increased

risk among both younger and older

mothers [Rasmussen et al., 1996; For-

rester and Merz, 2000], and other studies

finding no association with age [Matsu-

naga and Shiota, 1977; Croen et al.,

2000; Miller et al., 2010]. Some of this

variation may be due to inclusion of

infants with chromosome abnormalities

(including trisomies) in some studies of

HPE. No association between HPE and

paternal age was seen in the three

studies that investigated this association

[Matsunaga and Shiota, 1977; Croen

et al., 1996; Orioli and Castilla, 2007].

Race/Ethnicity

Higher risks of HPE among non-white

compared to white mothers have been

observed in four studies conducted in

the United States and United Kingdom

[Rasmussen et al., 1996; Olsen et al.,

1997; Croen et al., 2000; Ong et al.,

2007], while in a study in Hawaii, a

greater risk among Far East Asians was

seen, compared to Pacific Islanders (RR

3.01, 95% CI 1.30–5.94) [Forrester and

Merz, 2000]. No association was iden-

tified between paternal race/ethnicity

and HPE in the one study that investi-

gated this association [Croen et al.,

2000].

CBDMP investigators observed an

elevated risk among mothers born out-

side the United States or Mexico (OR

3.1, 95% CI 1.1–8.6) [Croen et al.,

2000], while the analysis using data from

the NBDPS found no increased risk to

mothers born outside the United States

[Miller et al., 2010].

Socioeconomic Status

Mothers with less than a high school

education had a higher risk of HPE

compared to mothers with a high school

education by investigators using data

from CBDMP (OR 2.1, 95% CI 0.8–

6.0) and from NBDPS (OR 2.5, 95% CI

1.1–5.6) [Croen et al., 2000; Miller

et al., 2010]. NBDPS investigators also

noted a slightly increased risk for

mothers with lower income (OR 1.5,

95% CI 0.8–2.8), whereas the study

using ECLAMC data found no asso-

ciation between HPE and socioeco-

nomic status [Orioli and Castilla, 2007;

Miller et al., 2010].

CONCLUSIONS

Few non-genetic risk factors for HPE

have been definitively identified to date,

with diabetes being among the most

commonly studied and the risk factor

that has resulted in the strongest associ-

ations, with fairly consistent results

between studies. Other potential risk

Few non-genetic risk factors for

HPE have been definitively

identified to date, with diabetes

being among the most

commonly studied and the risk

factor that has resulted in the

strongest associations, with

fairly consistent results

between studies.

factors that may warrant further study

include salicylate use, respiratory and

sexually transmitted infections, and use

of assisted reproductive technologies,

which have been identified in epide-

miologic studies as possibly associated

with increased risk of HPE, and quality

of diet and use of multivitamin supple-

ments, which may be associated with a

decreased risk.

Caution is needed, however, in

interpreting the results of epidemiologic

studies of HPE. Since the prevalence of

HPE is much higher among embryos (4

per 1,000) [Matsunaga and Shiota, 1977]

than among live births (1 per 10,000)

[Croen et al., 1996; Rasmussen et al.,

1996], studying potential risk factors

among live births or fetuses at 20 weeks

or greater gestational age, as is com-

monly done, may result in the identi-

fication of exposures which are in fact

not risk factors for HPE, but risk factors


for survival of the fetus long enough for

detection of the anomaly.

Small sample size leading to low

power to detect an effect is another

consequence of the low birth prevalence

of HPE, which results in unstable effect

estimates and wide CIs. These wide CIs,

which frequently contain the null value

(OR¼ 1), make it difficult to identify

which exposures may be true non-

genetic risk factors for HPE as opposed

to merely findings observed by chance.

In this article some associations whose

CIs include the null are referred to as

potential risk factors when the effect

estimate was fairly large or when more

than one study found similar results;

however, further research will be needed

to determine whether these are true risk

factors or not.

Failure to exclude infants with

conditions of genetic etiology (e.g.,

chromosome abnormalities and single

gene disorders) presents another

challenge to studying non-genetic risk

factors for HPE. Studies using etiolog-

ically heterogeneous case groups may

hamper identification of potential non-

genetic risk factors [Rasmussen et al.,

2003]. For example, inclusion of differ-

ent proportions of cases with chromo-

somal anomalies may be responsible for

the variability seen in studies of the

association between maternal age and

HPE. Similarly, given that maternal

diabetes has been identified as a strong

risk factor for HPE, studies aiming to

identify other risk factors for HPE may

benefit from exclusion of infants with

diabetic mothers from the analysis.

Another key factor in these studies

is ensuring that all infants actually have

HPE. Older studies often depended on

facial phenotype for diagnosis of HPE,

but some infants with HPE have normal

facies [Cohen, 1989a] and some infants

with mild facial features consistent with

HPE may not have HPE [Cohen,

1989a,b]. Given the current widespread

availability of neuroimaging, future

studies should require that imaging

studies (or pathologic analysis in

deceased infants) documenting HPE be

available. However, even when neuro-

imaging is available, other brain defects

may be mistaken for HPE: 19% of scans

sent to the Carter Centers for Brain

Research were found to not have

HPE after review by radiologists with

expertise in HPE [Stashinko et al.,

2004].

One consequence of a stricter case

definition is further reduction in num-

berof cases available for study. Analysis of

the interaction between genetic and

non-genetic risk factors will also be

important, since some genotypes may

increase the risk for HPE after certain

exposures, but these studies will require

even larger sample sizes. Future studies

will likely require collaboration between

multiple investigators to reach a sample

size large enough to identify non-

genetic risk factors for HPE.

ACKNOWLEDGMENTS

We would like to thank Dr. Jaime Fr ı́as

for his helpful comments on an earlier

draft of the manuscript.

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Non-Genetic Risk Factors for Holoprosencephaly