Abstract
LAY ABSTRACT
The diagnosis of autism is based solely on behavioral characteristics. There is currently no laboratory test that can be done to identify autism. In this study, we investigated a molecule called brain derived neurotrophic factor (BDNF) as a possible early biologic marker for autism. BDNF is a small protein found throughout the central nervous system and in circulating blood. We measured the level of BDNF in blood collected from women during pregnancy and from their babies at birth. We found that the concentration of BDNF in the maternal mid-pregnancy and newborn blood specimens was similar for children with autism, children with mental retardation, and children with typical development. The results of this study suggest that BDNF is unlikely to be a useful early biologic marker for autism.
SCIENTIFIC ABSTRACT
Objective
To investigate levels of brain-derived neurotrophic factor (BDNF) in mid-pregnancy and neonatal blood specimens as early biologic markers for autism.
Methods
We conducted a population-based case-control study nested within the cohort of infants born from July 2000 – September 2001 to women who participated in the prenatal screening program in Orange County, California. Cases (n=84) were all children receiving services for autism at the Regional Center of Orange County. Two comparison groups from the same study population were included: children with mental retardation or developmental delay (n=49) receiving services at the same regional center, and children not receiving services for developmental disabilities, randomly sampled from the California birth certificate files (n=159), and frequency-matched to autism cases on sex, birth year, and birth month. BDNF concentrations were measured in archived mid-pregnancy and neonatal blood specimens drawn during routine prenatal and newborn screening using a highly sensitive bead-based assay (Luminex).
Results
The concentration of BDNF in maternal mid-pregnancy and neonatal specimens was similar across all three study groups.
Conclusion
These data do not support previous findings of an association between BDNF and autism and suggest that the concentration of BDNF during critical periods of early neurodevelopment is not likely to be a useful biomarker for autism susceptibility.
Keywords: biologic markers, neurotrophin, autism, BDNF, prenatal
Introduction
Autism Spectrum Disorders (ASD) are characterized by impairments in social interaction, verbal and nonverbal communication, and stereotyped behaviors and interests (Lord et al. 2000), and diagnosis is based solely on behavioral criteria (Willemsen-Swinkels and Buitelaar 2002). The etiology of ASD is not well understood, though it likely involves genetic, immunologic, and environmental factors (Volkmar et al. 2004). The dramatic increase in reported prevalence has spurred an intense effort to identify early biological markers (Chakrabarti and Fombonne 2005). Such markers could allow for earlier identification and therapeutic intervention, contributing to improved prognosis (Aman 2005).
Brain-derived neurotrophic factor (BDNF) is a small protein found throughout the central nervous system (CNS) and peripheral blood. BDNF is involved in the survival and differentiation of dopaminergic neurons in the developing brain (Hyman et al. 1991), and plays an important role in the formation and plasticity of synaptic connections (Binder and Scharfman 2004). BDNF is trophic for serotonergic neurons, and abnormalities in serotonin levels are the most common biochemical findings in autism (Anderson 2002;Tsai 2005). Animal studies suggest that concentrations of BDNF in the CNS and serum are closely correlated (Karege et al. 2002), offering the possibility that concentrations in peripheral blood may be useful as a possible biologic marker for autism.
Skewed expression of BDNF has been linked to neurologic and psychiatric disorders including Fragile X syndrome, epilepsy, Parkinson’s disease, Alzheimer’s disease, schizophrenia and depression (Pezet and Malcangio 2004;Binder and Scharfman 2004;Hashimoto et al. 2004;Angelucci et al. 2005). Elevated levels of BDNF were found in postmortem brain tissue from adults with autism (Perry et al. 2001) and concentrations different from those in controls have been found in peripheral blood of adults and children diagnosed with autism (Miyazaki et al. 2004;Connolly et al. 2006; Hashimoto et al. 2006;Enstrom et al. 2008). The two studies that have examined BDNF levels in neonatal specimens from individuals later diagnosed with autism have yielded inconsistent results (Nelson et al. 2001;Nelson et al. 2006).
In the present study we sought to examine the association between ASD and levels of BDNF in maternal mid-pregnancy serum and newborn blood specimens.
Methods
Subjects
The study sample was based on the Early Markers for Autism (EMA) Study, a population-based, nested case-control study designed to evaluate biologic markers of susceptibility and exposure in archived maternal mid-pregnancy and neonatal blood specimens from the same mother-baby pairs. The study population was drawn from the cohort of children born in California from July 2000 –September 2001 to women who were pregnant in Orange County, CA, and who participated in the State’s prenatal expanded alpha-fetoprotein screening program (XAFP). Only mother-child pairs for whom both a maternal mid-pregnancy and neonatal specimen was found in the archive (described below) were eligible for inclusion. All study procedures were approved by the institutional review boards of the California Health and Human Services Agency and Kaiser Permanente Northern California.
Three groups of children were identified: children with autism, children with mental retardation (MR) but not autism, and general population controls (GP). Children with autism or MR were ascertained from the California Department of Developmental Services (DDS), which operates a system of 21 Regional Centers (RC) that coordinate services for persons with autism, mental retardation, and other developmental disabilities. The system is widely utilized across socio-economic levels and racial/ethnic groups. DDS maintains a centralized electronic database of diagnostic and identifying information on all clients, derived from information provided by the RCs on a standardized, statewide reporting form. DDS and the RCs are mandated to provide services to individuals with autistic disorder and children with other Pervasive Developmental Disorders (PDDs) who have MR (IQ <70) or are substantially handicapped, as well as children with MR. GP controls were randomly sampled from the birth certificate files after excluding all past or current DDS/RC clients and were frequency matched to autism cases by sex, birth month and birth year at a 2:1 ratio.
Diagnostic verification
Following a protocol initially developed by the Metropolitan Atlanta Developmental Disabilities Surveillance Program (Yeargin-Allsopp et al. 2003), trained medical record abstractors reviewed and abstracted detailed diagnostic and clinical data from RC records for all subjects with autism or MR. Expert clinical review was conducted of abstracted data to classify children with autism by subtype based on DSM-IV-TR criteria, by onset type, and by cognitive status. Onset type was based on parental report or clinical observations and categorized as “early” (no statement of loss of social and/or language skills, or statement of early and sustained delays or plateauing of skills without actual loss), “regressive” (clear loss of previously acquired language and/or social skills), or unknown. MR determination was based on composite scores on standardized cognitive and functional tests (MR: composite score < 70; no MR: all scores > 70 or some scores <70 and others >70; unknown: no standardized scores in chart). The final analytic sample consisted of 84 children with autism (autistic disorder [DSM-IV-TR 299.0] (n=55), Asperger’s disorder [299.8] (n=0), PDD-NOS [299.8] (n=5), unknown (n=22); early onset (n=64), regressive (n=17), unknown (n=3); with MR (n=34), without MR (n=30), MR status unknown (n=20)); 49 children with MR but not autism (mild [DSM-IV-TR 317] (n=20), moderate [318.0] (n=12); severe [318.1] (n=11); profound [318.2] (n=3); unspecified [319] (n=3); known etiology (n=29), unknown etiology (n=20)); and 159 GP controls.
Specimen Collection
Maternal mid-pregnancy and neonatal blood specimens were retrieved from the prenatal and newborn screening specimen archives maintained by the California Department of Public Health. The prenatal specimen archive contains nearly 200,000 serum and blood cell pellet specimens remaining from women enrolled in the State’s XAFP screening program between November 1999 and December 2002 and living in three Southern California counties. As part of the screening program, venous blood was collected at 15–19 weeks gestation in serum separator tubes by obstetrical care service providers and underwent XAFP testing at a single regional laboratory, typically within seven days of collection (median time = 3 days). During transit via US Postal Service to the regional screening laboratory, no effort was made to control the temperature of the specimens. After testing, leftover specimens were kept under refrigeration for 1–2 days and then stored at −20°C.
The neonatal specimen archive contains dried bloodspots collected for screening purposes on nearly every infant born in California (~500,000 per year) since 1980. All newborn blood specimens for children included in this study were obtained by the heel-stick method, usually within 24–48 hours of birth. The blood specimens were collected at the nursery on a special S&S filter paper and allowed to dry at room temperature prior to transport to the regional laboratory for routine screening for metabolic and other disorders. During transport, usually by courier from the hospital to a regional screening laboratory, the temperature of the specimens was not controlled. Blood spots remaining after routine testing were catalogued and stored at −20°C.
BDNF measurements
We examined the concentration of BDNF in both maternal mid-pregnancy and neonatal specimens. For the neonatal determination, two 3mm punches of each neonatal bloodspot were placed in 96 well plates and eluted with 180μl of a buffer composed of 25ml of 10mM phosphate buffered saline with pH 7.4 and a protease inhibitor tablet (Roche Complete Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN)). The plates were then placed on a shaker for 1 hour at room temperature to thoroughly extract the blood sample from the paper spot. After incubation, eluates were transferred into cryogenic tubes and stored at −25°C until analyzed. Prior to analysis, serum and bloodspot eluates were thawed and centrifuged for 10 minutes.
BDNF concentration in undiluted bloodspot eluates and maternal serum was measured using a commercially available bead based Luminex kit (Biosource Human BDNF Antibody Bead Kit, Invitrogen-Biosource, Carlsbad, CA) in accordance with the kit protocols. Briefly, 50μl of sample was incubated with anti-BDNF-conjugated beads on a plate shaker for two hours, washed, and detection antibody was added for 1-hour incubation. Following washing, streptavidin-PE was added for 30 minutes. The plates were then read on a Bio-Plex 100 (BioRad, Hercules, CA), and analyzed using Bio-Plex Manager software with a 5-point curve.
Total protein for each bloodspot sample was measured with a commercially available kit (Pierce BCA protein assay, Pierce Biotechnology, Rockford, IL). The bloodspots were normalized for total protein concentration using the Bradford protein assay. This normalized for differences due to original spotting and elution. There was no need to normalize the maternal specimens for protein content as these were undiluted, whole blood serum. Internal standards were run to standardize the tests between plates.
Statistical Analysis
The distributions of BDNF levels in mother-baby pairs in the autism group were compared to the distributions in the GP and MR groups using two-sample t-tests (means) and Kruskal-Wallis tests (medians). To explore patterns of association between BDNF concentrations and autism risk, we compared categories of BDNF level [0–33% (reference), 34%-66%, 67%-90%, >90%)] across study groups with a chi-square statistic. Finally, to examine the association of autism with BDNF levels after adjustment for possible confounders, we fit separate logistic regression models for autism vs. GP controls and autism vs. MR controls. Autism vs. control status was regressed on log10-transformed BDNF levels with adjustment for several covariates related to the blood draw or associated with autism in previous epidemiologic studies. Separate models were run for maternal mid-pregnancy and neonatal specimens. Total protein was included as a covariate in neonatal specimen models to normalize the BDNF levels with respect to total protein concentration of the eluted bloodspot. For all newborn BDNF values that were below the limit of detection (LOD), we assigned a value of LOD/2; all maternal values were above the detection limit.
Results
Compared to the MR and GP control children, children with autism were significantly less likely to have a mother who was Hispanic or born in Mexico and more likely to have an older mother. Children with autism were significantly more likely than children with MR to be male and born in 2000 (Table 1).
Table 1.
Demographic Characteristics of Participants in the Early Markers for Autism (EMA) Study
| Autism (N=84)
|
GP Control (N=159)
|
MR Control (N=49)
|
Autism vs. GP control
|
Autism vs. MR control
|
||||
|---|---|---|---|---|---|---|---|---|
| N | (%) | N | (%) | N | (%) | P-value* | P-value* | |
| Gender | 0.91 | 0.0003 | ||||||
| Male | 73 | (86.9) | 139 | (87.4) | 29 | (59.2) | ||
| Female | 11 | (13.1) | 20 | (12.6) | 20 | (40.8) | ||
| Birth Year | 0.16 | 0.003 | ||||||
| 2000 | 23 | (27.4) | 31 | (19.5) | 26 | (53.1) | ||
| 2001 | 61 | (72.6) | 128 | (80.5) | 23 | (46.9) | ||
| Plurality | 0.42 | 0.88 | ||||||
| Singleton | 81 | (96.4) | 156 | (98.1) | 47 | (95.9) | ||
| Multiple | 3 | (3.6) | 7 | (1.9) | 2 | (4.1) | ||
| Mother’s Race | 0.17 | 0.76 | ||||||
| White | 57 | (69.5) | 126 | (79.2) | 37 | (75.5) | ||
| Asian | 19 | (23.2) | 28 | (17.6) | 9 | (18.4) | ||
| Other | 6 | (7.3) | 5 | (3.1) | 3 | (6.1) | ||
| Mother’s Ethnicity | 0.0007 | <0.0001 | ||||||
| Hispanic | 20 | (23.8) | 73 | (45.6) | 28 | (57.1) | ||
| Non-Hispanic | 64 | (76.2) | 86 | (54.1) | 21 | (42.9) | ||
| Mother’s Birth Country | <0.0001 | <0.0001 | ||||||
| US | 45 | (53.6) | 71 | (44.7) | 16 | (32.7) | ||
| Mexico | 9 | (10.7) | 58 | (36.5) | 22 | (44.9) | ||
| Other | 30 | (35.7) | 30 | (18.9) | 11 | (22.4) | ||
| Maternal Age (years) | 30.9 | (5.2) | 28.2 | (5.5) | 28.3 | (5.3) | 0.0003 | 0.007 |
| Maternal Weight at XAFP blood draw (lbs) | 145.1 | (26.7) | 146.9 | (33.8) | 150.3 | (39.3) | 0.65 | 0.42 |
| Gestational Age at XAFP blood draw (days) | 119.8 | (7.9) | 118.8 | (7.4) | 118.4 | (8.6) | 0.35 | 0.36 |
| Gestational Age at birth(days) | 272.1 | (18.5) | 271.0 | (14.3) | 266.3 | (28.1) | 0.63 | 0.20 |
|
| ||||||||
| Median | (25–75%) | Median | (25–75%) | Median | (25–75%) | P-value* | P-value* | |
|
| ||||||||
| Baby Age at NBS Blood Draw (days) Median | 1.2 | (1.0–1.8) | 1.2 | (1.0–1.6) | 1.4 | (1.1–2.2) | 0.37 | 0.25 |
SD is standard deviation
Based on chi-square test for categorical variables, two-sample T-test for means, Kruskal-Wallis for medians
Using Luminex technology and the same antibodies for maternal and newborn specimens, the concentration of BDNF was substantially higher (~100-fold) in maternal mid-pregnancy specimens than neonatal specimens (Table 2). BDNF levels in the two specimen types were not correlated (r=−0.15, P=0.08). Significant differences in mean BDNF concentrations, measured as either pg/ml or pg/ug, between the three diagnostic groups were not detected in either specimen type (Table 2). Comparisons of percentile categories of BDNF concentrations between autism vs. GP controls and autism vs. MR also failed to yield any significant differences for maternal or neonatal specimens (data not shown). BDNF concentrations in maternal and newborn specimens (BDNF measured as a continuous variable) were not associated with risk of autism in either crude or adjusted models (relative to the GP controls or MR controls) (Table 3). No differences in maternal or newborn BDNF levels were observed between autism subgroups (regression vs. early onset; autism with MR vs. autism without MR), or between autism subgroups and GP controls (data not shown).
Table 2.
BDNF concentration measured by Luminex in mid-pregnancy serum and newborn dried bloodspot eluates from autistic subjects compared to general population (GP) and MR control subjects in the Early Markers for Autism (EMA) Study
| N | Mean | SD | P-value* | Median | Range | P-value* | |
|---|---|---|---|---|---|---|---|
| Maternal Mid-Pregnancy BDNF (pg/ml)** | |||||||
| Autism | 34 | 3860.9 | 1345.7 | 3923.8 | 549.4-8468.9 | ||
| GP Control | 78 | 3917.6 | 1312.7 | 0.83 | 4080.9 | 863.3-6881.9 | 0.57 |
| MR Control | 20 | 4337.6 | 1391.6 | 0.22 | 4350.2 | 1525.3-7416.3 | 0.12 |
| Neonatal BDNF (pg/ml) | |||||||
| Autism | 84 | 54.1 | 40.0 | 54.4 | 6.1-187.0 | ||
| GP Control | 159 | 53.7 | 47.7 | 0.95 | 49.5 | 6.1-309.1 | 0.53 |
| MR Control | 49 | 46.1 | 37.4 | 0.26 | 36.1 | 6.1-131.1 | 0.26 |
| Neonatal BDNF/protein (pg/ug)*** | |||||||
| Autism | 84 | 0.0083 | 0.0062 | 0.0081 | 0.0007-0.0275 | ||
| GP Control | 159 | 0.0082 | 0.0073 | 0.91 | 0.0074 | 0.0005-0.0485 | 0.46 |
| MR Control | 49 | 0.0077 | 0.0068 | 0.57 | 0.0059 | 0.0006-0.0315 | 0.33 |
SD is standard deviation.
All comparisons are to the autism group. Means compared with a two-sample t-test; medians compared with a Kruskal-Wallis test.
BDNF assay run on only a random sample of maternal specimens.
Table 3.
Risk of autism associated with a log10-unit increase in the concentration of BDNF measured in mid-pregnancy serum and newborn dried bloodspot eluates, Early Markers for Autism (EMA) Study.
| Autism vs. GP Control | Autism vs. MR Control | |||
|---|---|---|---|---|
| ORcrude(95% CI) | ORadj (95% CI) | ORcrude (95% CI) | ORadj (95% CI) | |
| Prenatal BDNF | 0.8 (0.28–2.1) | 0.74a (0.22–2.5) | 0.8 (0.29–2.1) | 1.4c (0.19–10.7) |
| Neonatal BDNF | 1.1 (0.83–1.4) | 1.1b (0.78–1.7) | 1.2 (0.85–1.6) | 0.81d (0.40–1.6) |
Adjusted for maternal age, race, ethnicity, birth place, weight at prenatal blood draw, gestational age at prenatal blood draw
Adjusted for maternal age, race, ethnicity, birth place, gestational age, baby’s age at newborn screening, total protein, and plate
Adjusted for maternal age, race, ethnicity, birth place, weight at prenatal blood draw, gestational age at prenatal blood draw, sex, birth year
Adjusted for maternal age, race, ethnicity, birth place, gestational age, baby’s age at newborn screening, total protein, plate, sex, birth year
Note: individuals with race=‘other’ or race=‘missing’ excluded from table
Discussion
In our evaluation of BDNF concentrations in maternal mid-pregnancy and newborn specimens using Luminex technology, we found no differences between subjects with autism compared to subjects with MR or to general population controls. BDNF was initially identified as a possible biologic marker for autism in an early study using newborn blood specimens and a single antibody immunoassay (Nelson, et al. 2001). In that study, increased levels of BDNF were found in specimens from children with ASD or mental retardation compared to children with cerebral palsy or controls (Nelson et al. 2001). A subsequent study using both a Luminex double-antibody immunoaffinity bead-based assay and ELISA technology found no case-control differences in neonatal BDNF levels compared to typically developing control children (Nelson et al. 2006).
Evaluations of BDNF in older subjects have consistently found differences between those with autism and controls, with higher levels associated with autism in most studies. Miyazaki et al measured serum BDNF concentrations using commercial ELISA kits and found BDNF concentrations elevated in the ASD (mean age 7.6 years) and MR (mean age 11 years) groups compared to the controls (mean age 23.3 years); however, differences in age between study groups makes interpretation of these data difficult (Miyazaki et al. 2004). Also using commercial ELISA kits, Hashimoto et al reported significantly lower serum BDNF levels in autistic males (age 18–26) compared to age- and sex-matched healthy controls (Hashimoto et al. 2006). Connolly et al measured concentrations of BDNF and antibodies to BDNF in children with ASD, Landau-Kleffner Syndrome, Epilepsy, pervasive developmental disorder-not otherwise specified (PDD-NOS), childhood disintegrative disorder (CDD), healthy normal controls, and non-neurological illness (NNI) (Connolly et al. 2006). Using ELISA technology, BDNF levels were higher in children with ASD than healthy control and NNI children, but not significantly distinguishable from the other groups; higher titers of antibodies to BDNF were also found in children with ASD or CDD compared to healthy controls. Finally, using Luminex technology, Enstrom et al reported significantly elevated BDNF levels in the plasma of 2–5 year old children with autism compared to age-matched non-autistic controls (Enstrom et al. 2008). A trend toward higher BDNF levels in children with early onset autism compared to children with regressive autism was noted (studies summarized in Table 4).
Table 4.
Published studies on BDNF levels in peripheral blood and autism.
| Reference (location) | ASD Group(s), Specimen Collection Age Range, Mean (SD) | Control Group(s), Specimen Collection Age Range, Mean (SD) | Specimen Type | Assay Method | BDNF Concentration Mean (SD) | Results |
|---|---|---|---|---|---|---|
| Nelson et al, 2001 (California) | ASD: n=69, Children born 1983–1985, 2.2 (1.2) days |
MR: n=60, 1983–1985 CA births, 2.5 (1.7) days
CP: n=63, 1983–1985 CA births, 5.4 (5.9) days GP: n=54, 1983–1985 CA births, 2.6 (1.6) days |
Dried newborn bloodspots | Recycling Affinity Chromatography |
ASD: 37.4 (19.9) pg/ml
MR: 36.8 (18.6) pg/ml CP: 16.3 (9.7) pg/ml GP: 13.3 (5.0) pg/ml |
Neonatal concentration significantly higher in ASD and MR groups compared to CP and GP controls; no difference between ASD and MR groups |
| Miyazaki et al, 2004 (Japan) | ASD: n=18, 3–27 years, 7.6 (6.1) years |
MR: n=20, 1–20 years, 11.0 (5.9) years
HC: n=16, 22–24 years, 23.3 (0.9) years |
Serum | ELISA |
ASD: 25.2 (2.5) ng/ml
MR: 29.0 (2.3) ng/ml Healthy: 17.5 (2.0) ng/ml |
Mean concentration higher in ASD and MR groups than HC: no difference between ASD and MR groups |
| Connolly et al, 2005 (Missouri) |
Autism: n=37, 5.9 (3.9) years
CDD: n=19, 7.0 (2.4) years PDD-NOS: n=14, 4.8 (3.9) years |
Landau Kleffner: n=9, 7.4 (2.3) years
Epilepsy: n=33, 5.9 (3.9) years HC: n=17, 4.3 (2.0) years NNI: n=16, 4.0 (3.2) years |
Serum | ELISA |
Autism: 32,279 pg/ml
CDD: 36,872 pg/ml HC: 8,708 pg/ml NNI: 8,964 pg/ml |
Mean concentration significantly higher in Autism and CDD than HC and NNI; Autism, CDD, and epilepsy had anti-BDNF IgG and IgM |
| Nelson et al, 2006 (California) | ASD: n=27, Children born in 1995, 1.2 (0.89) days |
DS: n=26, 1995 CA births, 1.2 (0.9) days
GP: n=20, 1995 CA births, 1.2 (0.9) days |
Dried newborn bloodspots | Luminex |
ASD: 3404 (1131) pg/ml, 27.4 (9.4) pg/mg
DS: 3211 (1312) pg/ml, 23.1 (1.0) pg/mg GP: 3299 (844) pg/ml, 26.4 (8.2) pg/mg |
No difference in mean concentration between groups |
| Hashimoto et al, 2006 (Japan) | Autism: n=18, 18–26 years, 21.2 (2.1) years | HC: n=18, 18–26 years, 22.2 (2.2) years | Serum | ELISA |
Autism: 25.6 (2.2) ng/ml
HC: 61.6 (10.9) ng/ml |
Mean concentration significantly lower in Autism than HC group |
| Katoh- Semba et al, 2007 (Japan) | Autism: n=56, 0–59 years | HC: n=218, 0–59 years | Serum | Bead-based two- site sandwich enzyme immunoassay | 0–9 year olds: Autism: 174.1 (190.4) pg/ml
HC: 400.6 (148.8) pg/ml 10–19 year olds: Autism: 552.5 (354.3) pg/ml HC: 323.4 (118.8) pg/ml |
Compared to HC group, mean concentration in Autism significantly lower in 0–9 year olds, and significantly higher in teenagers |
| Enstrom et al, 2008 (California) | Autism: n=37, 2.1–5.9 years, 3.58 years (median) |
DD: n=6, 2.0–3.4 years, 2.71 years (median)
TD: n=27, 2.2–4.6 years, 3.1 years (median) SIB: n=9, 2.3–4.6 years, 3.8 years (median) |
Plasma | Luminex |
Autism: 2512.1 (327.9) pg/ml
DD: 1457.6 (211.1) pg/ml TD: 1463.1 (174.6) pg/ml SIB: 1444.7 (164.8) pg/ml |
Mean concentration significantly higher in Autism compared to all control groups; early onset group higher than regressive group |
ASD: autism spectrum disorder; CDD: childhood disintegrative disorder; CP: cerebral palsy; PDD-NOS: pervasive developmental disorder-not otherwise specified; MR: mental retardation without autism; TD: typically developing; GP: general population; HC: healthy controls; SIB: sibling of autism; DD: developmental delay; NNI: non-neurological illness; DS: Down syndrome
Differences in the analytic platforms used, assay methods, and subject populations may explain the inconsistencies among studies. The antibodies used for capture and detection in the various assays may recognize different forms of the BDNF molecule and differences in dilution factors or developmental age of subjects at specimen collection are likely to influence results (Nelson et al. 2006;Katoh-Semba et al. 2007). The Luminex bead-based assay used for BDNF determinations in our study is highly sensitive (<10pg/mL), and serum levels in maternal mid-pregnancy specimens were similar to those reported previously (Lommatzsch et al. 2006), whereas the standard ELISA assay only measures down to the ng/ml range. If elevated BDNF levels characterize a specific autism endophenotype, inconsistent findings across studies may be related to differences in the phenotypic composition of the populations studied. As noted by Nelson, et al (2006), resolution of differences across studies would be aided by definition, using mass spectroscopy or a micro-chromatographic method, of the molecular nature of the material captured by different immunoassay methods.
In conclusion, BDNF concentrations in maternal mid-pregnancy and newborn blood specimens did not differentiate children with autism from control children. Larger studies adequately powered to investigate differences by autism phenotypes are needed.
Acknowledgments
National Institute of Mental Health, R01- MH72565, L. Croen, PI; the National Alliance for Autism Research, 824/LC/01-201-004-00-00, L. Croen, PI; and the California Tobacco-related Disease Research Program, 8RT-0115, M. Kharrazi, PI.
Funding was provided by grants from the National Institute of Mental Health (R01-MH72565, L. Croen, PI), the National Alliance for Autism Research (824/LC/01-201-004-00-00, L. Croen, PI), and the California Tobacco-Related Disease Research Program (8RT-0115, M. Kharrazi, PI). We thank Ron Torres for DNA amplification; Jack Collins, Roxana Odouli and Tiffany Wong for project coordination; Julie Ruedaflores for record review and abstraction; Meredith Anderson and Daniel Najjar for assistance with data management and analysis; and Steve Graham and Debbie Hildebrandt for record linkage and specimen retrieval.
References
- Aman MG. Treatment planning for patients with autism spectrum disorders. J Clin Psychiatry. 2005;66(Suppl 10):38–45. [PubMed] [Google Scholar]
- Anderson GM. Genetics of childhood disorders: XLV. Autism, part 4: serotonin in autism. J Am Acad Child Adolesc Psychiatry. 2002;41:1513–1516. doi: 10.1097/00004583-200212000-00025. [DOI] [PubMed] [Google Scholar]
- Angelucci F, Brene S, Mathe AA. BDNF in schizophrenia, depression and corresponding animal models. Mol Psychiatry. 2005;10:345–352. doi: 10.1038/sj.mp.4001637. [DOI] [PubMed] [Google Scholar]
- Binder DK, Scharfman HE. Brain-derived neurotrophic factor. Growth Factors. 2004;22:123–131. doi: 10.1080/08977190410001723308. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chakrabarti S, Fombonne E. Pervasive Developmental Disorders in Preschool Children: Confirmation of High Prevalence. Am J Psychiatry. 2005;162:1133–1141. doi: 10.1176/appi.ajp.162.6.1133. [DOI] [PubMed] [Google Scholar]
- Connolly AM, Chez M, Streif EM, Keeling RM, Golumbek PT, Kwon JM, Riviello JJ, Robinson RG, Neuman RJ, Deuel RM. Brain-derived neurotrophic factor and autoantibodies to neural antigens in sera of children with autistic spectrum disorders, Landau-Kleffner syndrome, and epilepsy. Biol Psychiatry. 2006;59:354–363. doi: 10.1016/j.biopsych.2005.07.004. [DOI] [PubMed] [Google Scholar]
- Enstrom A, Onore C, Tarver A, Hertz-picciotto I, Hansen R, Croen L, Van de Water J, Ashwood P. Peripheral blood leukocyte production of BDNF following mitogen stimulation in early onset and regressive autism. Am J Biochem Biotechnol. 2008;4(2):121–129. doi: 10.3844/ajbbsp.2008.121.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hashimoto K, Iwata Y, Nakamura K, Tsujii M, Tsuchiya KJ, Sekine Y, Suzuki K, Minabe Y, Takei N, Iyo M, Mori N. Reduced serum levels of brain-derived neurotrophic factor in adult male patients with autism. Prog Neuropsychopharmacol Biol Psychiatry. 2006;30:1529–1531. doi: 10.1016/j.pnpbp.2006.06.018. [DOI] [PubMed] [Google Scholar]
- Hashimoto K, Shimizu E, Iyo M. Critical role of brain-derived neurotrophic factor in mood disorders. Brain Res Brain Res Rev. 2004;45:104–114. doi: 10.1016/j.brainresrev.2004.02.003. [DOI] [PubMed] [Google Scholar]
- Hyman C, Hofer M, Barde YA, Juhasz M, Yancopoulos GD, Squinto SP, Lindsay RM. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra. Nature. 1991;350:230–232. doi: 10.1038/350230a0. [DOI] [PubMed] [Google Scholar]
- Karege F, Schwald M, Cisse M. Postnatal developmental profile of brain-derived neurotrophic factor in rat brain and platelets. Neurosci Lett. 2002;328:261–264. doi: 10.1016/s0304-3940(02)00529-3. [DOI] [PubMed] [Google Scholar]
- Katoh-Semba R, Wakako R, Komori t, Shigemi H, Miyazaki N, Ito H, Kumagai T, Tsuzuki M, Shigemi K, Yoshida F, Nakayama A. Age-related changes in BDNF protein levels in human serum: differences between autism cases and normal controls. Int J Devl Neuroscience. 2007 doi: 10.1016/j.ijdevneu.2007.07.002. [DOI] [PubMed] [Google Scholar]
- Lommatzsch M, Hornych K, Zingler C, Schuff-Werner P, Hoppner J, Virchow JC. Maternal serum concentrations of BDNF and depression in the perinatal period. Psychoneuroendocrinology. 2006;31:388–394. doi: 10.1016/j.psyneuen.2005.09.003. [DOI] [PubMed] [Google Scholar]
- Lord C, Cook EH, Leventhal BL, Amaral DG. Autism spectrum disorders. Neuron. 2000;28:355–363. doi: 10.1016/s0896-6273(00)00115-x. [DOI] [PubMed] [Google Scholar]
- Miyazaki K, Narita N, Sakuta R, Miyahara T, Naruse H, Okado N, Narita M. Serum neurotrophin concentrations in autism and mental retardation: a pilot study. Brain Dev. 2004;26:292–295. doi: 10.1016/S0387-7604(03)00168-2. [DOI] [PubMed] [Google Scholar]
- Nelson KB, Grether JK, Croen LA, Dambrosia JM, Dickens BF, Jelliffe LL, Hansen RL, Phillips TM. Neuropeptides and neurotrophins in neonatal blood of children with autism or mental retardation. Ann Neurol. 2001;49:597–606. [PubMed] [Google Scholar]
- Nelson PG, Kuddo T, Song EY, Dambrosia JM, Kohler S, Satyanarayana G, Vandunk C, Grether JK, Nelson KB. Selected neurotrophins, neuropeptides, and cytokines: developmental trajectory and concentrations in neonatal blood of children with autism or Down syndrome. Int J Dev Neurosci. 2006;24:73–80. doi: 10.1016/j.ijdevneu.2005.10.003. [DOI] [PubMed] [Google Scholar]
- Perry EK, Lee ML, Martin-Ruiz CM, Court JA, Volsen SG, Merrit J, Folly E, Iversen PE, Bauman ML, Perry RH, Wenk GL. Cholinergic activity in autism: abnormalities in the cerebral cortex and basal forebrain. Am J Psychiatry. 2001;158:1058–1066. doi: 10.1176/appi.ajp.158.7.1058. [DOI] [PubMed] [Google Scholar]
- Pezet S, Malcangio M. Brain-derived neurotrophic factor as a drug target for CNS disorders. Expert Opin Ther Targets. 2004;8:391–399. doi: 10.1517/14728222.8.5.391. [DOI] [PubMed] [Google Scholar]
- Tsai SJ. Is autism caused by early hyperactivity of brain-derived neurotrophic factor? Med Hypotheses. 2005;65:79–82. doi: 10.1016/j.mehy.2005.01.034. [DOI] [PubMed] [Google Scholar]
- Volkmar FR, Lord C, Bailey A, Schultz RT, Klin A. Autism and pervasive developmental disorders. J Child Psychol Psychiatry. 2004;45:135–170. doi: 10.1046/j.0021-9630.2003.00317.x. [DOI] [PubMed] [Google Scholar]
- Willemsen-Swinkels SH, Buitelaar JK. The autistic spectrum: subgroups, boundaries, and treatment. Psychiatr Clin North Am. 2002;25:811–836. doi: 10.1016/s0193-953x(02)00020-5. [DOI] [PubMed] [Google Scholar]
- Yeargin-Allsopp M, Rice C, Karapurkar T, Doernberg N, Boyle C, Murphy C. Prevalence of autism in a US metropolitan area. JAMA. 2003;289:49–55. doi: 10.1001/jama.289.1.49. [DOI] [PubMed] [Google Scholar]