Anti-Metallothionein IgG and levels of metallothionein in autistic families

Metallothioneins (MTs) are a family of small proteins containing 61–68 amino acids with an unusually high concentration of cysteine. MT-1, the most functional and active MT in humans, has the ability to react with and enhance the detoxification of a number of metals including zinc, mercury, copper and cadmium. MT dysfunction may result, then, in many of the aetiological syndromes observed in autistic children, such as the leaky gut. It has been proposed that allergic autoimmune reactions occurring after exposure to heavy metals, may contribute to some symptoms associated with autism. Therefore abnormalities in MT concentration and/or structure, as well as the presence of anti-MT antibodies, may be associated with autism. We used direct ELISAs to quantitate the concentration of serum anti-metallothionein IgG in 66 individuals (parents and children) from 14 families with autistic children, as well as 11 controls from families with no history of autism. We measured the concentration of serum metallothionein in 39 of the above family members from 8 families. Our results indicate that a significantly high number (23 of 66) of autistic family members had high levels of anti-metallothionein IgG, when compared to controls (1 ) and the production of these antibodies correlated with levels of metallothionein, suggesting that the production of these antibodies is inherited. However, the presence of these antibodies does not correlate with autism, types of autism, including regression, or demographics such as allergies, respiratory problems or GI disease. This suggests that the presence of anti-metallothioein antibodies is not causative to autism and may be the result of other immunological pathology seen in many autistics.


Introduction
Control tissues provided by the National Disease Research Interchange, Philadelphia, PA.
Besides detoxification of heavy metals, functions of MT in the body include development of brain neurons, maturation of the GI tract, antioxidation, boosting immune function and delivery of zinc to cells [11,.Evidence also suggests that Autistic individuals are prone to developing autoimmune disorders and Autism appears to be more common in families with a history of autoimmune disorders [18,19].
MT dysfunction may result, then, in many of the issues seen with autistic children, such as the leaky gut syndrome, incomplete breakdown of casein/gluten protein by zinc dependent enzymes, disrupted ability to combat yeast, reduced production of stomach acid and impaired stimulation of the pancreas by secretin.It may also lead to inability to clear the body of heavy metals, a dysfunctional immune system, and ultimately to the neurological changes seen in ASD.It would also explain the male sex predominance (4:1) seen in autism, because MT synthesis is enhanced by oestrogen and progesterone.In a study of 53 autism-spectrum patients at The Pfeiffer Tr eatment Centre, scientists found abnormal levels of copper and zinc in blood (p <.1) indicating defective functioning of metallothionein (MT) proteins [2].
Metallothionein levels fluctuate in direct response to heavy metal levels such as zinc [21].The fluctuation of metallothionein levels and/or the production of abnormally structured metallothionein in autistic individuals may stimulate an auto-immune response and therefore, may be associated with anti-MT antibody production and the etiology of autism.
In this preliminary study we used ELISAs and Western Blotting to detect and measure serum antibodies to metallothionein in 33 autistic and 33 non-autistic individuals (parents and nonautistic siblings) from 14 families, as well as 11 age-matched control individuals with no family history of autism.
We also used direct ELISAs to quantitate the concentration of serum metallothionein in 21 autistic and 18 non-autistic individuals from 8 of the above autistic families.
Our results indicate that a significantly high number (23 of 66) of family members from families with autistic children have high levels of antimetallothionein IgG, when compared to controls (/11 age matched individuals from families with no family history of autism) and the production of these antibodies is likely inherited.However, the presence of these antibodies does not correlate with autism (parents and non-autistic siblings were just a likely to have anti-MT IgG), type of autism such as regression, or demographics such as allergies, respiratory problems or GI disease.

ELISA (Enzyme Linked Immunosorbent Assay) to measure auto-antibodies to metallothionein
Purified metallothionein-1 (Sigma), at a concentration of 5 ng/µl of bicarbonate buffer (pH 9.6), was fixed to wells of a 96 well polystyrene microculture plate (Corning) by incubation overnight at 4 degrees C. Excess metallothionein was dumped from wells and all wells were blocked by washing 3 ҂ with 3 microliters of blocking solution (Superblock, Pierce).One hundred microliters of primary antibody (Positive control -mouse Mab to metallothionein-1 diluted 1:5 with PBS; Negative control -PBS; Experimental serum from autistic and non-autistic individuals diluted 1:5 with PBS) was added to appropriate wells and plate was incubated for 2 hours at 3 degrees C. All wells were washed 3 times using PBS/tween.One hundred microliters of goat antimouse secondary antibody, conjugated with alkaline phosphatase (Biorad), diluted 1:5 with PBS to positive control; Goat anti-human (Biorad) diluted 1:5 with PBS to all other wells, added to appropriate wells and incubated for 45 minutes at 3 degrees C. All wells washed 5 times with PBS/tween.One hundred microliters of substrate for alkaline phosphatase added to all wells and plate was incubated at room temperature until significant colour change in positive control.Colour change measured using ELISA Reader (Biorad).

Direct ELISA to quantitate the concentration of serum metallothionein
Purified metallothionein-1 (Sigma) at concentrations of 1 µg, 5 ng, 5 ng and 5 ng per 1 µl of bicarbonate buffer (pH 9.6), and serum at a dilution of 1:5, were fixed to wells of 96 well polystyrene microculture plate (Corning) by incubation overnight at 4 degrees C. Excess metallothionein/serum was dumped from wells, and all wells were blocked by washing 3 ҂ with 3 microliters of blocking solution (Superblock, Pierce).One hundred microliters of primary antibody (mouse Mab to metallothionein-1, diluted 1:5 with PBS) was added to all wells and plate was incubated for 2 hours at 3 degrees C. All wells were washed 3 times using PBS/tween.One hundred microliters of secondary antibody, conjugated with alkaline phosphatase (goat anti mouse, Biorad, diluted 1:5 with PBS) added to all wells and incubated for 45 minutes at 3 degrees C. All wells washed 5 times with PBS/tween.One hundred microliters of substrate for alkaline phosphatase added to all wells and plate was incubated at room temperature until significant colour change in positive control.Colour change measured using ELISA Reader (Biorad).

Blot
Proteins were transferred to nitrocellulose at 3 volts, overnight at 4C. Nitrocellulose flooded with blocking solution (1% casein/PBS) and rocked gently overnight.

Immunoblot
Te n ml of primary antibody (Positive controlmouse Mab to metallothionein-1 diluted 1:5 with PBS; Negative control -PBS; Experimental serum from autistic and non-autistic individuals diluted 1:5 with PBS) was flooded over appropriate nitrocellulose sheets and incubated for 2 hours at room temperature.All sheets were washed 3 times using PBS/tween, with five minutes for each wash, rocking gently at room temperature.Te n ml of secondary antibody conjugated with Horse Radish Peroxidase (HrP) (goat anti-mouse [Biorad] diluted 1:5 with PBS to positive control; Goat anti-human [Biorad] diluted 1:5 with PBS to all other wells) added to appropriate sheets and incubated for 45 minutes at room temp.All sheets were washed 5 times with PBS/tween, with five minutes for each wash, rocking gently at room temperature.Te n ml of substrate for HrP added to all sheets at room temperature until significant colour change in positive control.

Results
The presence of anti-metallothionein IgG was confirmed using western blotting.In figure 1, antibodies from an autistic family member (93) are shown directed at metallothionein, compared to the same reactivity of monoclonal antibody to metallothionein (positive control).
We used direct ELISAs to quantitate the concentration of serum anti-metallothionein IgG in 66 individuals (parents and children) from 14 families (from Autism Genetic Resource Exchange) each family having at least one autistic child, as well as 11 age-matched individuals from families with no history of autism as controls (from the National Disease Research Interchange).Results are shown in table 1.A typical assay is shown in figure 2. For consistency, all assays were measured when + control OD reached 1. ± .1.
Three pairs of identical twins with autism were tested for anti-MT IgG.Individuals in each pair had almost identical levels of antibody (table 1).
In all families where anti-metallothionein antibodies were detected (/14), at least one parent had anti-MT IgG.In five of these families, both of the parents had anti-MT IgG.In the remaining families, none of the members had anti-MT antibodies.
Thirty-nine family members, from 8 of the 14 families with at least one autistic child, were tested for the serum concentration of metallothionein using a direct ELISA.A typical assay is shown in figure 4. Tw elve of 1 individuals with high anti-MT IgG also had high MT serum concentration (above 3 µg/ml), whereas only 4 of 22 with no anti-MT IgG had high MT concentration (p <.5) (table 1).
Tw o of the three pairs of identical twins were tested for MT concentration.Individuals in each pair also had similar concentrations of MT.
There was no significant difference in the concentration of anti-MT IgG when comparing affected children categorised by autistic type (verbal, non-verbal and non-verbal regression) to control individuals (individuals with no family Anti-metallothionein IgG in controls.Mean OD ± SD of Controls (agematched individuals with no family history of autism).each control serum assayed at least 3 times.

Figure 4
Metallothionein concentration in family members with autistic children.Typical elISA measuring the concentration of MT in serum from autistic family members compared to controls (+ controls 1-.001 µg/100 µl purified MT; -control PBS).
history of autism, parents and non-autistic siblings) (figure 5).We only assayed one PDD, Aspergers and verbal regression individual and they are not included in this comparison.
Using medical history reports (AGRE), we analysed the phenotypic characterisation of the probands of family members tested in this study (table 1).Thirty-five affected children were tested for anti-MT IgG.Answers to medical history questions were not available for thirteen of these children.Of the 22 remaining, 5 reported respiratory disease (23%).Of these five, none had high levels of anti-MT IgG.We assayed four of these for metallothionein serum concentration.
None of these had high levels of metallothionein (figure 6).Although this may suggest that these antibodies may be protective against respiratory disease, the levels of antibody were not significantly different from the amount of antibody in controls.
Only 2 of 22 affected children were reported to have allergies, compared to 9 of 18 parents, and anti-MT IgG levels in these 2 were not significantly different from controls.
Anti-MT antibodies were not significantly different from controls in five affected children with hyperbilirubinaemia and two autistic children with reported GI disease (figure 6).

Discussion
Autoimmune reactions after exposure to heavy metals such as mercury have been causally implicated in autism.As metallothionein (MT) is the primary metal-detoxifying protein in the body, we conducted a study of the MT-1 protein and antibodies to metallothionein (anti-MT IgG) in autistic children, normal children and parents from 14 families, as well as age matched control individuals (no family history of autism).
Metallothioneins (MT), a family of small proteins containing 61-68 amino acids with an un-usually high concentration of cysteine, function as intracellular distributors and mediators of heavy metals, including copper and zinc, and heavy metal detoxification.
Data indicates that copper to zinc ratio is abnormally high in individuals with autism [2].This might be explained by an altered function due to an altered conformation of the protein.
Measurements of MT levels, as well as zinc, have been used to demonstrate zinc deficiency.
MT increases rapidly after zinc supplementation and decreases if the diet is deficient in zinc [21].
Studies indicate that many children with autism have an immune abnormality of some type, including myeloperoxidase deficiency, severe combined immunodeficiency, and IgA deficiencies.Tw enty percent of autistics have IgG subclass deficiencies, and deficiencies in complement C4b.Concentrations of IL-12 and interferon gamma are much higher in autistic children than in normal children [22][23][24][25][26]. Autistic individuals are also susceptible to auto-immune disorders [2,28].
Using an ELISA and western blotting, we searched for serum antibodies to metallothionein-1 in autistic and non-autistic individuals from 14 families, as well as 11 age-matched individuals with no autistic family history.In all families where anti-metallothionein antibodies were detected (/14), at least one parent had anti-MT IgG.In five of these families, both of the parents had anti-MT IgG.In the remaining families, none of the members had anti-MT antibodies.Also, three sets of identical twins from three families were tested for MT serum concentration and anti-MT IgG.In all cases the concentration of MT and anti-MT IgG was virtually the same in each set.This suggests that the presence of these auto-antibodies is associated with autism, and the production of these antibodies is most likely inherited.
Using a direct ELISA we correlated the presence of anti-MT IgG and concentration of serum metallothionein.In all five families with anti-MT antibodies, the individuals with the highest level of antibodies also had the highest level of metallothionein.In 4 of 5 of these families, the concentration of metallothionein was significantly higher than the 3 families with no antibodies.This suggests that higher levels of metallothionein are associated with anti-MT IgG.
It has previously been reported that anti-MT antibody is present in the circulation of healthy persons, including both normal and autistic children [3,31].
Singh and Hanson reported in 26 [29] that serum levels of MT did not significantly differ between normal and autistic children.Furthermore, autistic children harboured normal levels of anti-MT, without any significant difference between normal and autistic children.They suggested that, because autistic children have a normal profile of MT and anti-MT, the mercury-induced autoimmunity to MT may not be implicated in the pathogenesis of autism.Although we found that a significant number of autistic family members (parents and children) had high levels of anti-MT, which corresponded with high levels of MT, the levels of MT and anti-MT did not correlate with autism (parents and non autistic children were just as likely to have high levels), or any type of autism (verbal, non-verbal and non-verbal regression) or demographic group (GI or respiratory disease).This supports the findings of Sing and Hanson.
We could not identify any factors, including immune dysfunction such as allergies, which could explain why only selected families had anti-MT IgG.The presence of anti-MT IgG in some autistic families could be associated with the fact that autistic children are more susceptible to immune dysfunction, including autoimmune disease.We gratefully acknowledge the resources provided by the Autism Genetic Resource Exchange (AGRE) Consortium and the participating AGRE families.The Autism Genetic Resource Exchange is a program of Cure Autism Now and is supported, in part, by grant MH6454 from the National Institute of Mental Health to Daniel H. Geschwind (PI).
C-55649 C-55648 C-55455 C-55458 C-55646 C-55457 C-55636 C-55637 C-55458 C-55638 controls (age matched indivuduals with no family history of autism) M/S/A/NV M/S/A/V F/P/NA M/P/NA autistic family members and controls

Figure 5
Figure 5Anti-MT IgG levels in affected children of different types compared to control groups.

Figure 6
Figure 6 Demographic groups with Anti-MT IgG.Anti-MT levels in affected children of different demographic groups compared to controls (age-matched individuals with no family history of autism).MT contents of normal human serum are typically well below 1 ng/ml.