In a new article in European Neuropsychopharmacology, researchers Mark Horowitz and David Taylor provide guidance for tapering psychiatric drugs, whether for full discontinuation or to reduce the dose. They suggest a slow, individualized taper to minimize withdrawal effects.
“The general principle when reducing or stopping psychiatric medications is as follows. Make a small reduction, monitor for withdrawal effects or destabilization of the patient, then ensure stability before making further reductions. Reductions should probably be made in smaller and smaller increments because of the pharmacology of the drugs; the final dose before completely stopping will need to be very small.”
Horowitz and Taylor have previously written about this approach for antidepressants in Lancet Psychiatry and for antipsychotics in JAMA Psychiatry (with Sir Robin Murray).
A 2018 survey found that 84.6% of people who tried to discontinue an antidepressant experienced withdrawal symptoms, which lasted for over a year for 47% of them. Antidepressant withdrawal can include anxiety, tearfulness, dread, numbness, brain zaps (described as similar to “electric shocks”), nausea, vomiting, diarrhea, dizziness, fatigue, insomnia, nightmares, sexual problems, confusion, and amnesia.
Long-term use of psychiatric drugs causes the body to adapt to the presence of these drugs; when the drugs are removed from the system, the adaptations remain. This causes withdrawal.
“There is no reason to think that the brain or body can return to its pre-drug state in a manner of weeks after adaptation to years or decades of medication exposure,” write Horowitz and Taylor. They add, “Reports from patients of long-lasting effects are often dismissed because the drug is ‘out of the system.’ However, it is adaptations to the drug which persist, causing the brain to register a lack of the anticipated input from psychiatric drugs, which manifests as withdrawal effects.”
Some people may require months or even years to slowly decrease their dose before eventually stopping the drug. The researchers write:
“Withdrawal effects (and relapse) might be minimized by stopping psychiatric drugs over a period long enough for underlying adaptations to the drug to resolve.”
According to the researchers, based on studies of the drugs’ effects on the brain, psychiatric drugs impact the brain along with a hyperbolic relationship. That is, at low doses, small adjustments have huge impacts—but at high doses, even large adjustments have less of an impact.
“The relationship between dose of a psychiatric drug and its effects is hyperbolic,” they write. “This is a consequence of the law of mass action: when there are few molecules of a drug present at the site of action, every additional molecule has a large incremental effect, but when higher concentrations are present each additional molecule has less and less effect, as receptors become saturated.”
This means that dose reductions should not be linear (reduced by the same amount each time, e.g., 40, 30, 20, 10, 0 mg). Instead, one strategy is to reduce the current dose by 10% each time, especially ensuring that the last adjustment—to full discontinuation—is very small.
“For most psychiatric drugs, this means that the final dose required before completely stopping will be very small, much smaller than commonly used doses, and in many cases much smaller than available tablet formulations,” they write.
If the final doses are smaller than those available, then what are patients to do? Horowitz and Taylor suggest liquid formulations and tapering strips can fill that void. Many psychiatric drugs are already available in liquid form, which enables very small doses. However, tapering strips are just beginning to become more widely used.
Horowitz MA, & Taylor D. (2021). How to reduce and stop psychiatric medication. European Neuropsychopharmacology, 55, 4-7. https://doi.org/10.1016/j.euroneuro.2021.10.001 (Link)
I came off a cocktail including 2000mg (no typo) of Seroquel per day. I take nothing now.
In my experience, the slower the better. It can take years for your brain to come right.
For those going through this, be strong.
Dang, 2000 mg! That really is torture!
I was prescribed 25mg Seroquel per day and I consumed one quarter of this (about 6mg per day) because I found the 25mg per day too strong.
So you were on 320 times per day what I was on (according to my calculations).
Above was in the 2000’s. I was just taking a little Dollop.
(I came off the heavy Neuroleptics in the 1980s)
The Researchers recommend very careful withdrawal from Psychiatric Drugs, especially at lower levels, and they are 100% right about this (IMO).
The Researchers also draw attention to “Withdrawal Anxiety”. I withdrew very carefully from Neuroleptics but I suffered from Dreadful ‘High Anxiety’ in the process. This High Anxiety was almost disabling and would have driven me back onto the Neuroleptics had I not found a means of adjusting to it.
I think this is the Crux of the situation.
People withdrawing from Neuroleptics need also to be shown how to accommodate the longterm chemical withdrawal effects of these drugs – to survive.
There ARE Psychological Remedies Available for Withdrawal Anxiety (and Longterm Withdrawal Anxiety).
There appears to be some good news for advocates of a biochemical cause for those who are diagnosed with ADHD. Nature published some data which identified pathology within CDH2, a mutatation affecting N-cadherin function
CDH2 mutation affecting N-cadherin function
causes attention-deficit hyperactivity disorder in
humans and mice
We demonstrate that a novel mutation in CDH2 is
associated with familial ADHD, through impaired presynaptic
vesicle clustering, attenuated evoked transmitter release,
decreased spontaneous release, and reduction in dopaminergic
distribution within limbic pathways.
Symptoms were modified by methylphenidate, the most commonly prescribed therapeutic for ADHD. The mutated mice exhibited impaired presynaptic vesicle clustering, attenuated evoked transmitter release and decreased spontaneous release. Specific downstream molecular pathways were affected in both the ventral midbrain and prefrontal cortex, with reduced tyrosine hydroxylase expression and dopamine levels. We thus delineate roles for CDH2-related pathways in the pathophysiology of ADHD.
Birk, O.S. Following MD studies at Tel Aviv University, military service as a medical officer (Major) in the IDF and residency in Pediatrics at Sheba Medical Center, Birk did his PhD at the Weizmann Institute with Irun Cohen, delineating hsp60 as a crucial autoantigen in type 1 diabetes and allograft rejection, effective in their prevention. He then went on to do his training in clinical human genetics and post-doctorate with Heiner Westphal at the NIH, unraveling LHX9 as a gene critical for mammalian gonad formation.
Ohad Birk, M.D., Ph.D.
Director, Genetics Institute, Soroka Medical Center
Scientific Director, National Institute for Biotechnology in the Negev
Head, Morris Kahn Lab. of Human Genetics,
Faculty of Health Sciences,
Ben-Gurion University, Beer-Sheva, ISRAEL
Professor of Computer Science, Tel Aviv University
As usual, you are identifying an “association.” Nothing in your article speaks of a “cause,” nor does the degree of “association” appear anywhere here. There is no comparison stating what percentage of people who have this mutation who are “diagnosed” with “ADHD” vs those who are not, nor what percentage of “ADHD” sufferers actually have this particular mutation. To claim “biochemical cause” as you imply in your first sentence, we’d need to know that almost all “ADHD” sufferers had the mutation and almost no one who has the mutation is NOT diagnosed with “ADHD.”
The article also avoids the very important question of whether or not this is simply a normal genetic variation that has been pathologized because the associated behavior is annoying to adults.
It is tiresome to keep hearing of these “breakthroughs” which don’t address the most basic scientific questions yet claim “proof of biological causation.”
“CDH2 mutation affecting N-cadherin function
causes attention-deficit hyperactivity disorder in
humans and mice.”
This is a fact. It is proven through published research.
Nonsense. We would need to see that all or almost all cases of “ADHD” have this same mutation, and that no or almost no person with this mutation does NOT have the “ADHD” features. Given that “ADHD” is defined by subjective behavior and sociological variables, the odds that EVERY case of “ADHD” so “diagnosed” is caused by this mutation is exceedingly questionable. In any case, this question does not begin to be answered by a handful of mice, or even a very small number of “ADHD” cases studied. We also know that, for instance, waiting one year before enrolling a child in school leads to a 30% reduction in “ADHD” diagnoses (a number of studies confirm this), that sociological stress such as neglect and domestic abuse increases the rate of “diagnosis,” that placing such children in “open classrooms” makes it practically impossible for professionals to distinguish them from “normal” children, and that prior comparisons of “genetic markers” have proven extremely heterogeneous, that is, that many “diagnosed” with “ADHD” did NOT have the markers, and many who were NOT “diagnosed” did in fact have the same markers.
And of course, the entire line of inquiry ignores the rather obvious question of why a particular challenge in paying attention to boring activities is considered a “disorder” in the first place. I recall an article (which I can’t seem to locate now) that showed employer satisfaction with historically “ADHD” employees did not differ from those not so diagnosed. The explanation offered was that those with this “diagnosis” simply chose employment opportunities that fit with their personalities, rather than being forced, as they are in schools, to do what they are ordered to do with no ability to choose the activities they prefer. This is strongly supported by investigations in the 70s indicating that “ADHD” children are virtually indistinguishable from “normal” children in an open classroom environment.
In short, there is a huge number of variables involved in this analysis that are ignored, most particularly the problem of “selecting” subjects based on a subjective list of behavioral characteristics that are not truly measurable in any objective manner. This particular study does nothing to answer the bulk of questions that the diagnosis itself raises, yet even within the context of the accepted nomenclature, there is no way to draw the conclusion you claim from the extremely limited study you present.
They have nothing more to prove. It is done. They found what causes ADHD.
It is very simple.
Contact Dr. Birk if you disagree.
“CDH2 mutation affecting N-cadherin function
causes attention-deficit hyperactivity disorder in
humans and mice.”
I have made my points, and you appear to have no response other than “Dr. Birk said so.” The fact that Dr. Birk said so is of absolutely no consequence to me or to Science as a whole.
I don’t need to contact Dr. Birk – s/he has overstated the case by any level of scientific analysis. It appears we will be unable to agree on these points. You are free to believe Dr. Birk’s analysis if you wish, but it is still important for any real scientific discussion to point out the limitations of the research presented. Since you appear to have no interest in responding to the points I have raised, it appears that no further discussion is possible.
Best to you in the future.
My responses aren’t directled solely to you but are intended to benefit all who are interested in this fascinating topic. I encourage anyone to raise questions. You are reaching. Your “arguments” are far removed from what is now known: “CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice.”
This is a fact. It is proven through published research. Attack the research, vigorously, not matters that don’t acknowledge that fact. It is foundational. It may bear repeating dozens of times to allow it to sink in. This isn’t theoretical. It is not a hypothesis or a best guess.
You miss the point. Your concerns/objections have nothing to do with the facts which he proved. If you find fault in his testing protocol, in his math, calculations, etc., it is up to you to demonstrate what he/they did wrong. He established that 2 plus 2 equals 4. 1 plus 3 equals 4, but that is not what he prooved and it has nothing to do with his conclusion. He published peer reviewed, detailed explanations regarding each step (and more) he took to reach his conclusion. This reseacher proved “CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice.” You have failed utterly to dispute that. Not one of your objections has anything whatsoever to do with this: “CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice.” Discussing anything/everything else besides what he prooved has no bearing on that fact.
Trying desperately to deny reality doesn’t change a thing. He did it. He and his colleagues found the cancer. It is right there in black and white, you see. It does exist. It is real. He found it. It is an astounding, marvelous achievement. Many accepted it was only matter of time. Others, for some inexplicable reasons, assured everyone it could not be done; they were positive it was all explained away by poor parenting, poor diets, luke warm educators, greedy doctors and bigpharma.
This is a significant breakthrough and soon will be accepted throughout the scientific commuity and around the world. It is huge and I for one am delighted that such a dedicated and respected scholar, scientist, PhD, and a medical docotor without conflicts of interest, who studied this matter exhaustively, found the answer. It wasn’t easy. They worked extremely long and hard and deserve our gratitude.
I encourage you once again to take your concerns to him. You have nothing to lose.
Sorry, can’t agree with you here. I can’t argue with the result that a certain very small subset of people diagnosed with “ADHD” had the genetic mutation that Birk has located. I don’t want to argue with that. But science is limited to answering the question asked. This study showed that a small sample of humans diagnosed with “ADHD” had a certain genetic anomaly. That is ALL that it proved, whatever the researchers theorize it means. Science is inherently SKEPTICAL. It is supposed to doubt its own conclusions and try to come up with alternative explanations and to DISPROVE anything that it wants to prove. No real scientist has the attitude that one study, particularly with the small sample size involved, can “prove” that “ADHD” is biological! It’s an outrageous assertion scientifically. Consider that we all “knew” for a certainty the formulas for acceleration and force and the gravitational constant and so forth for CENTURIES before Einstein showed them to be an approximation rather than a precise description of reality. Science is always working to improve itself, and that means questioning assumptions and conclusions that are not established beyond a reasonable doubt, and even those that ARE established as “laws” are STILL subject to revision when contradictory data arrives on the scene.
Replication is the core of scientific verity, moreover, replication in the face of efforts to establish alternative explanations. You can’t take one study and extrapolate it to apply to the entire population in question, ESEPCIALLY when this population is defined by ridiculously subjective criteria like “displays poor listening skills” or “appears to be ‘driven by a motor’ or is often on the go.” Seriously, “Often on the go?” You think that every child who is “often on the go” would not be “on the go” except for some genetic anomaly? It is absurd to so readily believe that one study, which establishes nothing more than an ASSOCIATION with these “ADHD” traits, proves causality.
Anyway, you seem to have convinced yourself and are not really interested in discussion. I’m not sure why it is so important to you to make more of this study than it offers, but maybe you’ll be right and I’ll be wrong in the long run. But I certainly will not concede that a single study on a tiny subsection of this population proves “causality” of “ADHD.” Come back to me when you have at least three studies from different, non-biased sources that indicate that over 90% of those “diagnosed” with “ADHD” have this anomaly, and that less than 10% of the general population have the same anomaly, then maybe we can talk.
OK, I’m going to try one more thing here and then that’s it for this discussion for me. I will keep it very simple.
Let’s say we did a study and found that poison ivy causes skin rashes. We had a large sample of people and found that 90% of those exposed to poison ivy got a skin rash.
Can we now conclude that skin rashes are caused by poison ivy?
Or can we conclude that a certain subset of skin rashes are caused by poison ivy?
What would we need to do to demonstrate that ALL skin rashes are caused by poison ivy? Would this one study be sufficient to show that?
This is what you’re doing here. You are saying that people who have this mutation are likely to fit the description of “ADHD.” This is not something I’m arguing with. I’m asking you how you could possibly conclude from this one study that ALL cases of “ADHD” have this cause?
The answer is, you can’t. If you can’t see that, you can’t try talking science to me. It’s a fundamental tenet of science – correlation does not imply causation. There could be 500 different, distinct causes of the syndrome called “ADHD,” and this could be one of them. We could do the same with abused children – take 100 abused children and 25 of them are diagnosed with “ADHD.” Does this mean child abuse causes “ADHD?” No. It means at most that SOME “ADHD” behaviors are caused or increased by child abuse. It could even mean that abused kids are more likely to be taken to a psychiatrist for a diagnosis. Or that foster care placement makes kids more anxious and that this creates more “ADHD” symptomology. And there are many people who are diagnosed with “ADHD” that have no child abuse in their background. But it is a factor that contributes. That’s all we can say.
Like I said, you could be right, maybe every single case of “ADHD” has this mutation. But this study certainly doesn’t come anywhere CLOSE to proving such a thing. It shows that people who have this genetic mutation are likely to show “ADHD” symptoms. And that is ALL it shows. The rest will require further study.
A few highlights of their research
The p.H150Y mutation we identified is within the recognition domain of the protease responsible for this endogenous modification, and as we demonstrate through biochemical studies, impacts this tightly regulated post-processing. Also, long-term expression of an aberrant N-cadherin was demonstrated to reduce synapse connectivity. Accordingly, our in-vitro results support deficits in synaptic formation as an intriguing mechanism underlying the patients’ clinical manifestations.
This stuff is beyond fascinating. What they are discussing are the very building blocks, miniscule, tiny, itsy-bitsy molecules that constitute human thoughts!
Thus, our data support the postulation that replacing histidine with tyrosine debilitates the anchoring of the peptide within the catalytic pocket of furin protease and putatively impairs N-cadherin protein maturation.
Goodness gracious! The depth of their testing and understanding of the microscpic inner workings of the most complex structure in the universe is phenomenal.
BTW, N-cadherin a calcium-dependent cell adhesion molecule, is essential for normal CNS development. Homophilic binding of N-cadherin depends upon a specific conformation assumed by the molecule when it binds calcium. N-cadherin is a substrate for a specific zinc-dependent protease. The reliance of N-cadherin on two cations for proper function makes it a potential target for toxicants which act by replacing or modifying calcium or zinc at ion-binding sites. Exposure of the developing brain to lead, an ubiquitous toxicant known to interact with calcium, disturbs neural tube closure and subsequent maturation of the nervous system. Preliminary data indicates that lead may induce these effects by direct interaction with N-cadherin. Numerous common toxicants, including metals and solvents, also perturb cadherins and cause defective CNS development. These data indicate that changes in the spatio-temporal expression of cadherin can result in profound alterations in neural structure and function, and may underlie CNS malformations caused by numerous toxic agents
If P, then Q
Q, therefore P.
This is a classic logical fallacy taught in symbolic logic.
In other words: If it rains, the grass gets wet.
The grass is wet.
Therefore, it rained. This actually is the example in the text book.
One effect may have many causes. Finding one possible cause does not change that.
Remember that phrase attached to the conclusion of almost all studies: “Clearly more research is needed”?
Well this is why it is needed. One cause does not a conclusion make. Well, other than “this is one possible cause”.
CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice
Published: 26 October 2021
CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice
D. Halperin, A. Stavsky, R. Kadir, M. Drabkin, O. Wormser, Y. Yogev, V. Dolgin, R. Proskorovski-Ohayon, Y. Perez, H. Nudelman, O. Stoler, B. Rotblat, T. Lifschytz, A. Lotan, G. Meiri, D. Gitler & O. S. Birk
Nature Communications volume 12, Article number: 6187 (2021) Cite this article
Attention-deficit hyperactivity disorder (ADHD) is a common childhood-onset psychiatric disorder characterized by inattention, impulsivity and hyperactivity. ADHD exhibits substantial heritability, with rare monogenic variants contributing to its pathogenesis. Here we demonstrate familial ADHD caused by a missense mutation in CDH2, which encodes the adhesion protein N-cadherin, known to play a significant role in synaptogenesis; the mutation affects maturation of the protein. In line with the human phenotype, CRISPR/Cas9-mutated knock-in mice harboring the human mutation in the mouse ortholog recapitulated core behavioral features of hyperactivity. Symptoms were modified by methylphenidate, the most commonly prescribed therapeutic for ADHD. The mutated mice exhibited impaired presynaptic vesicle clustering, attenuated evoked transmitter release and decreased spontaneous release. Specific downstream molecular pathways were affected in both the ventral midbrain and prefrontal cortex, with reduced tyrosine hydroxylase expression and dopamine levels. We thus delineate roles for CDH2-related pathways in the pathophysiology of ADH
Attention-deficit hyperactivity disorder (ADHD) is one of the most common childhood-onset neuropsychiatric conditions, characterized by a persistent pattern of inattention, impulsivity, and hyperactivity, with complications often continuing into adulthood1. Affected individuals have difficulties in higher-level executive functions, which are mediated by late-developing frontal-striatal-parietal and frontal-cerebellar neuronal networks. These mainly include motor and interference inhibition, working memory, sustained attention, and temporal information processing2. Although its etiology is not well defined, ADHD appears to have substantial heritability, and as such, it has been the focus of considerable genetic research, with growing evidence that rare monogenic variants may possess an essential role in its pathogenesis3.
Here we describe three siblings of a consanguineous kindred presenting with severe ADHD, apparent as of early childhood. Through linkage analysis, whole-exome sequencing (WES), and biochemical studies, we identified a disease-associated homozygous missense mutation in CDH2, affecting proteolysis and maturation of the encoded N-cadherin adhesion protein, which is known to play a significant role in synaptogenesis, plasticity-induced long-term spine stabilization, and neurite outgrowth4,5. Notably, CDH2 has an essential role in regulating the proliferation of dopaminergic progenitors within the limbic system, primarily the ventral midbrain and prefrontal cortex6. Through generation and analysis of mice homozygous for the human mutation in the mouse CDH2 ortholog, we demonstrated hyperactivity and deficient sensorimotor integration in the mutant mice and delineated downstream physiological and molecular pathways mediating the phenotype, mainly alterations in synaptic properties and defects in dopamine neurotransmission. Thus, we identify the role of CDH2 and its downstream pathways in the pathophysiology of ADHD.
Three siblings of consanguineous Bedouin pedigree (Fig. 1a) presented with severe ADHD, diagnosed as of early childhood. Patient II:6 was born at term following an uneventful pregnancy, whereas the non-identical twin patients II:3 and II:4 were born prematurely at 32 weeks (weight appropriate for gestational age; 1900 and 2200, respectively). By the age of three, all siblings presented with a similar manifestation of severe hyperactivity behavior, predominantly hyperactive/impulsive. By the age of four, all patients met the criteria for ADHD, as outlined in the DSM-5. Concisely, information about ADHD manifestations was collected from semi-structured interviews conducted with both parents. Additional information was obtained from observations, questionnaires, and supplementary assessments: Clinical Evaluation of Language Fundamentals 5th Edition7 and Conner’s Parent Rating Scales-Revised8. The following were excluded in all three patients: scoring below 80 on both the performance and the verbal scales of the WISC-III9, psychosis, bipolar affective disorder, Tourette syndrome, multiple chronic tics, and a first-degree relative diagnosed with bipolar affective disorder and schizophrenia. All affected siblings were medication-free for 24 h prior to assessment and cognitive testing. Patients II:3 and II:6 (ages 11 and 7, respectively) reached normal developmental milestones, had no other comorbidities, and are completely normal in terms of intellect. Patient II:4 demonstrated mild developmental delay with autism spectrum disorder manifestations. Brain MRI (patient II:6, at 21 months) was normal. Blood pH, lactate, pyruvate, creatine, phosphokinase, and amino acids, as well as urinary organic acids, were within normal limits. Screening for congenital glycosylation defects, karyotype, and chromosomal microarrays were normal. All three patients were treated with stimulants, neuroleptics, and 3-Quinuclidinyl Benzilate. Notably, both parents and other siblings (including II:1 and II:7) were normal in terms of hyperactivity, intellect, and general health.
Fig. 1: Pedigree and CDH2 mutation.
a Pedigree of the consanguineous kindred studied. Beneath each individual is the allele corresponding to the CDH2 mutation. C and T denote the WT or mutant nucleotide, respectively. b Homozygosity scores. Genome-wide single nucleotide polymorphism (SNP) distributions were collected for all nine family members by bead-chip (>750 k/sample). The distribution of homozygous regions in the genome was determined using HomozygosityMapper (http://www.homozygositymapper.org/). Genomic regions are ordered by chromosome (green numbers). The blue arrow indicates the single homozygous locus on chromosome 18 shared by affected individuals. c Sanger sequencing. Through whole-exome sequencing, a single homozygous variant was found within the segregating locus: c.355 C > T in CDH2. CDH2 sequencing results of an unaffected individual (II:2), an obligatory carrier (I:1), and an affected individual (II:3) are shown. d Protein MSA. To demonstrate evolutionary conservation within the vicinity of mutated p.H150Y residue, eight representative CDH2 orthologs were selected for MSA. The recognition motif RXK/R-R is located approximately five residues downstream of the identified site of mutation. MSA, multiple sequence alignment.
Full size image
Linkage analysis, testing all nine family members, identified only one locus shared by the affected siblings: a ~11 Mb homozygous segment on chromosome 18 between SNPs rs11082423 and rs1480438 (Fig. 1b). Homozygosity mapping delineated this segment as the only homozygous disease-associated locus segregating as expected for autosomal recessive heredity within the studied kindred. WES data of individual II:3 were filtered for normal variants as described in Methods. A single homozygous variant was found within the locus: c.355 C > T; p.H150Y in CDH2 (transcript variant 1; NM_001792.4). This variant, validated by Sanger sequencing (Fig. 1c), was found to segregate within the family as expected for autosomal recessive heredity; neither compound heterozygous nor other homozygous mutations were found to co-segregate within this locus. Screening of the variant in 400 ethnically matched controls identified a single carrier and no homozygous mutants. This mutation has not been reported in the Genome Aggregation Database (gnomAD), with only fifteen CDH2 loss-of-function (LoF) variants (stop gain, frameshift, or essential splice site mutations) reported to date, none in a homozygous state. Multiple sequence alignment demonstrated the p.H150 residue to be highly conserved (Fig. 1d).
CDH2 protein structural analysis
CDH2 encodes a 906 amino acid protein, neuronal cadherin (N-cadherin), that is broadly expressed in the brain. It is known to play an essential role in synaptogenesis, synapse function, plasticity-induced long-term spine stabilization, and cortical organization. Classical cadherins are initially synthesized bearing a prodomain, thought to limit adhesion during the early stages of biosynthesis, which is then endogenously cleaved within the Golgi apparatus10. N-cadherin protein modeling (Fig. 2a) revealed that the sequence linking the prodomain to the outermost extracellular cadherin domain is unstructured and can be found in variable conformational loops, enabling the anchoring of proteolytic enzymes, mainly furin protease11. Computational studies (Fig. 2b) demonstrated that the furin protease consensus cleavage site contains approximately 20 residues, harboring the recognition motif RXK/R-R12, located five residues downstream to the p.H150Y mutation site. Using prediction tools for protein-peptide interactions, we demonstrated that the wildtype (WT) p.H150 residue is anchored within the catalytic pocket of the furin protease active-site, putatively contributing to its stable docking. In contrast, the mutant tyrosine sidechain, in a right rotamer conformation, is predicted to project perpendicularly, away from the catalytic pocket due to its non-polar, uncharged sidechain. Therefore, the mutation is predicted to interfere with the proteolysis and maturation of the protein.
Fig. 2: Structural analysis and disrupted cleavage of CDH2-mutated peptides.
a In-silico protein modeling. Ribbon representation of N-cadherin extracellular domains allows assessing the location of the identified mutation. Red, prodomain; green, extracellular cadherin domains (CADs 1-5); Blue, unstructured linker. The identified p.H150Y mutation resides within the unstructured region. Modeling was predicted using the SWISS-MODEL server (https://swissmodel.expasy.org/) based on the crystal structure of protocadherin GAmmaB4 extracellular domain (PDB ID 6E6B). b Electrostatic density representation map of furin protease (PDB ID 4Z2A). Furin cleavage site harbors the recognition motif RXK/R-R, which resides in proximity to the identified p.H150Y mutation site. Modeling was done using the HPEPDOCK server (https://omictools.com/hpepdock-tool), predicting protein-peptide interactions. The WT and CDH2-mutated sequences are denoted in pink and light blue, respectively; WT p.H150 residue is denoted with magenta, mutant p.Y150 residue with cyan. c Illustration of furin protease digestion assay; WT and mutant 22 amino-acid peptides, harboring the RXK/R-R recognition motif, were synthesized. Both peptides (10 µg) were digested by 2U enzyme furin protease followed by 30 °C incubation. Reaction mixtures were deactivated and subjected to LC-MS analysis. Predicted molecular weight is shown beneath each sequence. d Chromatogram separation and detection. Decreased absorbance of the digested full-length WT peptide at four-time intervals (0, 30, 60, 180 min) with a concurrent increase in absorbance of a fragmented small peptide (same was done for the mutant peptide). Peptides were separated by LC with subsequent tandem MS analysis. X axis: retention time (min), Y axis: relative absorbance (uAU). e Normalized cleavage efficacy after 180 min. The full-length peptide area under the curve (AUC) from MS analysis was calculated based on the desired spectral match. The ratio between AUC over time and the initial AUC at t=0 was extrapolated to calculate cleavage percentage. Left: percentage of peptide cleaved at t = 30, 60, and 180 min. (n = 3 for t = 30 and 60, n = 4 for t = 180; n represents an independent peptide digestion experiment subjected to LC-MS analysis (see Methods); mean ± SEM data were acquired, two-sided Student’s t-test, at t = 30 ns p = 0.08; t = 60 ns p = 0.44; t = 180 ns p = 0.052). Right: normalized cleavage efficacy at t = 180. Cleavage disruption of the mutated peptide is demonstrated with digestion >20% weaker in comparison with the WT peptide. UV, Ultraviolet; LC, liquid chromatography; MS, mass spectrometry.
Full size image
Decreased cleavage efficacy of the mutated p.H150Y peptide
The proprotein convertase family of enzymes plays an important role in activating other proteins13. To test whether the p.H150Y mutation interferes with protein processing, we performed a biochemical peptide cleavage assay using furin protease, the prototypical proprotein convertase; WT and mutant 22 amino-acid peptides were synthesized (GL Biochem, Shanghai), harboring the aforementioned RXK/R-R recognition and cleavage motif, conjugated with FITC and biotin at their N and C-terminus, respectively (WT: FITC-SKHSGHLQRQKRDW-K-biotin, Mutant: FITC-SKYSGHLQRQKRDW-K-biotin; Fig. 2c). Following digestion by furin (Fig. 2d)14, peptides were cleaved into two fragments based on the recognition preference of the protease (Fig. S1). Liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that proteolytic cleavage of the mutated peptide was substantially decreased compared with that of the WT peptide (Fig. 2e). Thus, our data support the postulation that replacing histidine with tyrosine debilitates the anchoring of the peptide within the catalytic pocket of furin protease and putatively impairs N-cadherin protein maturation.
CRISPR/Cas9-mutated knock-in mice
As the human CDH2 shares a high degree of similarity with its murine ortholog (Fig. 1d), with notable evolutionary conservation in the vicinity of the consensus prodomain cleavage motif, we generated two founder lines of CRISPR/Cas9-mutated mice harboring the specific human p.H150Y substitution in the Cdh2 mouse ortholog; Selected KI F0 mice were bred with C57BL/6JRcc WT mice for two cycles to generate non-chimeric F1 KI heterozygotes. Heterozygote F1 offspring were then bred and F2 offspring of WT and mutant origin (Cdh2H150T and Cdh2H150Y(2) founder lines) were used for all further experiments (details in Methods). Thorough studies of the Cdh2 homozygous knock-in (KI) 10-week-old mutant mice, with a specific focus on the brain, demonstrated no anatomical or histological abnormalities Fig. S2).
Behavioral and cognitive phenotypes in Cdh2-mutant mice
To assess possible in-vivo effects of the Cdh2 mutation, we performed behavioral studies of the mutant mice. 10-week-old WT and homozygous Cdh2H150Y KI mutant male mice (n = 18, nine mice per group) underwent a 3-week cassette of extensive phenotypic assessment (see Methods). Cdh2H150Y mice exhibited significantly greater traveling distance, increased velocity, and prolonged mobility time in the open-field exploratory test (OFT), recapitulating motor-associated features of hyperactivity (Fig. 3a-f). No differences were observed when examining the duration mice spent in the center of the open-field arena, a measure inversely related to neophobic behavior, nor in the amount of center/border crossings. In addition, no difference was evident in the rotarod test (Fig. 3m), implying no significant effect on primary motor control. Regarding cognitive evaluation, a trend was observed in the spontaneous alteration test (Y-maze, Fig. 3h), suggesting a potential effect on executive functions and working memory. A significant difference was observed in the acoustic startle reflex test (ASR, Fig. 3j, k), demonstrating an elevated startle amplitude of the Cdh2H150Y(1) mice, commonly associated with differences in sensorimotor integration. A similar pattern was observed in the pre-pulse inhibition test (Fig. 3l), in agreement with deficits in early-stage information processing, although no difference was evident in the pre-pulse attenuation fraction itself. Also, as part of the anxiety-domain assessment, no difference was demonstrated in the elevated plus-maze test (Fig. 3g). Lastly, the results of social interaction tests were inconclusive; although a significantly shorter interaction time in the resident-intruder test may imply a propensity for aggression (Fig. 3i), no differences were observed in the three-chamber sociability test followed by the social novelty test (Fig. 3n).
Fig. 3: Behavioral experiments in Cdh2 knock-in mice.
Behavioral test results of Cdh2H150Y(1) and WT mice following cassette of motor, anxiety, cognitive and social interactions domains assessment. a–f Exploratory OFT consists of a square arena measuring 50X50X33cm. Mice explored the arena for 6 min, while their location was recorded. a OFT tracking visualization. Cdh2H150Y mice exhibited (b) significantly greater traveling distance (*p = 0.04), (c) increased velocity (*p = 0.04) and (f) prolonged mobility time (*p = 0.03). d, e No differences were observed examining the duration mice spent in the center of the arena nor at the amount of center/border crossings. g Elevated plus-maze test consisting of two sets of opposing arms extending from a central platform. One set of arms was enclosed by a 15 cm wall, while the other was open. No differences were observed examining the duration mice spent in each set of arms (ns p = 0.4). h Spontaneous alteration test (Y-maze) consisting of three arms in 1200 Y-shaped maze. An alteration was defined as a complete cycle of consecutive entrances into each of the three arms. WT mice exhibited more alterations compared to Cdh2H150Y(1) mice (ns p = 0.06). i Resident-intruder test assesses the time for aggressive social interaction. Cdh2H150Y(1) mice demonstrated significantly shorter interaction time (*p = 0.03). j–l ASR test consists of a single noise burst (120 dB, 40 ms); thereafter, the amplitude of the animal flinch is recorded. Cdh2H150Y mice demonstrated a significantly elevated startle amplitude (*p = 0.03). This pattern was consistent with the pre-pulse inhibition test. m Rotarod test, assessing motor abilities, demonstrated no difference between the groups. n No differences were observed in the three-chamber sociability test, followed by a social novelty test. For all experiments, mean ± SEM data were acquired; n = 18, 9 mice per group, two-sided Student’s t-test. OFT, open-field test; ASR, acoustic startle reflex.
Contact Dr. Ohad Birk for further information
So you’re really talking about THREE cases here, all related to each other? With similar juvenile environments? No comparison subjects/control subjects?
Seems pretty weak to me.
The balance of the data included in this publication exceeded the size permitted by this cite. Dr. Ohad Birk’s contact information is available.
My efforts to add data from the research appears to be rejected for excessive size.
Behavioral experiments – detailed
The apparatus consisted of a square arena measuring 50X50X33cm under 15 lux illumination. The outer walls were wrapped with white paper to limit external stimuli and light gradients. Mice from both genotypes were allowed to explore the arena for 6 min, while their location was tracked and recorded by a video camera positioned overhead. The time spent in the central zone of the arena (10 × 10 cm) was extracted.
The apparatus consisted of four arms (30 × 5 cm each) extending from a central platform (5 × 5 cm). One set of arms, opposing one another, was enclosed by a 15-cm wall (‘closed arms’), while the other set was open with a 1-cm ledge on either side (‘open arms’). The maze was elevated 75 cm above the ground with illumination set 15 lux. Mice from both genotypes were placed in the central platform and allowed to explore the maze freely for 6 min. Entry into an arm was scored when the center of mass of the animal had entered an arm. The time spent and entries into each arm were extracted for analysis.
A spontaneous alternation test was performed to assess executive functions. The Y-shaped maze consisted of 3 plastic arms placed at 120° angles to each other. Mice were placed at the end of one arm and were allowed to explore the maze freely for 6 min without training, reward, or punishment. An alternation was defined as a complete cycle of consecutive entrances into each of the three arms. Percent alternation (PA) was calculated as follows: PA = number of alternations/(total number of entries into each arm – 2).
A test for aggressive social interaction. The intruder mouse is introduced into the cage of the test resident mouse following habituation. The observation starts when the resident first sniffs the intruder. The observation stops when the first attack (by either mouse) occurs, or when no attack has occurred by 5 min observation71.
Acoustic startle reflex (ASR)
This test evaluates an animal’s level of stress/arousal by measuring the extent of audible tone-induced flinching after acclimatization to background noise. Startle trials consist of a single noise burst (120 dB, 40 ms), thereafter the amplitude of animal flinch is recorded. Mean startle amplitude is calculated in a fully computerized, blinded, and unbiased measurement.
Pre-pulse inhibition (PPI) of the ASR
PPI is a measure of sensorimotor gating used to identify deficits in early-stage information processing. PPI trials consist of a pre-pulse of intensity 2, 4, 8, or 16 dB above background noise followed 100 ms later by a startling pulse (120 dB, 40 ms). Sessions are designed to include acclimation, pre-pulses, and pulse trials.
A test of motor abilities, which requires mice to balance on a rotating cylinder. The test consisted of three 4-minute trials. During each trial, rod rotation gradually increased up to 40 rotations/minute. The duration mice were balanced on the rod in each trial was measured. Trials were divided by at least 20-minute breaks, to avoid mice exhausting.
Three-chamber sociability test and social novelty test
Aims to evaluate animal levels of sociability (preference of an unfamiliar mouse over an object) and preference for social novelty (preference of a novel stranger over a familiar one). The device used in this test consists of three chambers (left, right, and central); the middle chamber is connected to the others via doors. In the habituation phase, the animal is allowed to explore the device freely for 10 min. In the second sociability phase, a stranger mouse is placed in one of the lateral chambers (inside a specially devised cup with bars). In the social novelty phase, another stranger is similarly introduced into the other lateral chamber. During phases 2 and 3, time spent in each chamber, the number of approaches to each stranger mouse and their frequency are tracked and recorded72.
Methylphenidate hydrochloride administration
Mice were administered with Methylphenidate hydrochloride (Biotechne 2 A/248099, UK)73; Methylphenidate (MPH) was dissolved in 0.9% normal saline and diluted to 1 mg/ml. Male 14-week-old C57BL/6JRcc WT (Envigo, Israel) and Cdh2H150Y(1/2) mice (n = 39) were weighed and injected with MPH or vehicle (0.9% saline solution) via intraperitoneal injection at 10 mg/kg body weight, 30 min before the initiation of tests. MPH dosages were chosen based on previous studies in rodents suggesting that these MPH dosages mirror those that are used in clinical practice15. MPH experiments were approved by the Hebrew University Ethics Committee on Animal Care and Use (Applications MD-20-16347-3).
Primary dense hippocampal cultures
Primary dense hippocampal cultures from P0-P2 pups of either sex were generated. Briefly, postnatal day 0–2 pups from WT and Cdh2H150Y littermates were decapitated and their brains quickly removed; hippocampi were dissected, sliced manually, and kept on ice in Hank’s Balanced Salt Solution (Biological Industries, Bet-Haemek, Israel) supplemented with 20 mM HEPES (termed HBSS; Biological Industries, Beit-Haemek, Israel) at pH 7.4. Hippocampus pieces were incubated for 20 min at room temperature (RT) within a digestion solution consisting of 5 ml HBSS, CaCl2 1.5 mM, EDTA 0.5 mM and 100 units of Papain (Worthington, Lakewood, NJ) activated with Cysteine (Sigma-Aldrich, Rehovot, Israel). Brain fragments were then gently triturated twice. Cells were seeded at a density of 80,000–100,000 cells per well on 12 mm #1 glass coverslips (CS; Bar-Naor Ltd, Ramat-Gan, Israel) coated with poly-D-Lysine (Sigma-Aldrich, Rehovot, Israel). Initially, cells were plated in a plating medium consisting of Neurobasal-A medium supplemented with 2% B27, 2 mM Glutamax I (Thermo-Fisher Scientific, Waltham, MA), 5% defined FBS and 1 μg/ml gentamicin (Biological Industries, Beit-Haemek, Israel). After 24 h, the plating medium was replaced by a serum-free culture medium consisted of Neurobasal-A, 2 mM Glutamax I, and 2% B27. Cultures were maintained at 37 °C in a 5% CO2 humidified incubator for about 12-15 days prior to staining and imaging.
Immunocytochemistry of hippocampal cultures
Days in-vitro (DIV) 8 and DIV 14 hippocampal neurons were fixed with 4% paraformaldehyde (EMS, Hatfield, PA) in PBS for 10 min, rinsed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 2 min, blocked with 5% powdered skim-milk (Sigma-Aldrich, Rehovot, Israel) in PBS for 1 h, rinsed, incubated with the primary antibody for 1 h, rinsed, incubated with the secondary antibody for 1 h, rinsed, and mounted in immumount (Thermo Fisher Scientific, Waltham, MA). All steps were performed at RT. Primary antibodies used: rabbit polyclonal anti-Synaptobrevin 2 (1:1000, Synaptic Systems), goat polyclonal anti-vesicular glutamate transporter-1 (vGlut1, 1:1000, Synaptic Systems), mouse monoclonal anti-Glutamic acid decarboxylase-6 (GAD-6, 1:1000, developed by D.I. Gottlieb, obtained from the Developmental Studies Hybridoma Bank, DSHB). Secondary antibodies used: Donkey anti-mouse IgG and donkey anti-rabbit IgG, labeled with Northern Lights 637 or 557, respectively (1:1000, R&D Systems), and donkey anti-goat IgG, labeled with AlexaFluor 647 (1:1000, Abcam).
Semi-quantitative synaptic immunofluorescence
To allow for a semi-quantitative comparison of immunostaining intensity of synapses, WT, and mutated Cdh2H150Y hippocampal neurons were processed under identical conditions. Synapses were detected semi-automatically using an in-house iterative algorithm based on serially decreasing to thresholds implemented in NIS-elements software (Nikon). Fluorescence values for each synapse were obtained from an area of 3 × 3 pixels located on its center of mass and an image average was generated. Because intensity values can vary from session to session, a normalization value was determined from the WT experiments of each session and further used to normalize all images acquired during that session, both WT and mutants. The normalized values were either averaged or used to compute cumulative distributions. Puncta that were positive for GAD65 were deemed GABAergic since GAD (Gamma-Amino Decarboxylase) is a vesicular enzyme mediating convergence of glutamate to GABA74; otherwise, they were considered glutamatergic.
Fluorescence microscopy of neuronal cultures
Fluorescence measurements were performed on a Nikon TiE inverted microscope driven by the NIS-elements software package (Nikon). The microscope was equipped with an Andor Neo 5.5 sCMOS 12 bit camera (Oxford Instruments), a 40 × 0.75 NA Super Fluor objective, a 60 × 1.4 NA oil-immersion apochromatic objective (Nikon), a perfect-focus mechanism (Nikon), and EGFP and Cy3 TE-series optical filter sets (Chroma), as well Cy5 filter set (Semrock).
Synapse width analyses
Synapse width was measured by drawing a line starting in the axon and through the synapse punctum, and fitting it using a Gaussian function as follows75:
Where xc is the center of the punctum maximum, y0 is the fluorescence of the axon, w2 is the variance of the Gaussian, and A is its amplitude. The full width at half-maximum (FWHM) fluorescence intensity is calculated as follows:
Fitting was performed by the least-squares error method using Origin 2020 (OriginLab). Independent images were acquired from cells grown on various coverslips obtained from a minimum of three different cultures. A mean FWHM was determined for each image, and then a grand average was calculated for WT and mutant cultures.
FM dye loading and unloading
Neurons were initially placed in normal saline (in mM: 150 NaCl, 3 KCl, 10 HEPES, 2 CaCl2, 2 MgCl2, 20 Glucose, pH adjusted to 7.35 with NaOH), then loaded with FM1-43 at a final concentration of 10 μM by depolarizing them for 2 min using hyperkalemic saline (90 KCl, 63 NaCl, otherwise as above) in the presence of the dye. The neurons were exposed to the dye in normal saline for an additional 5 min after depolarization, to allow labeling related to endocytosis to be completed. Afterward, neurons were washed with normal saline for 5 min, followed by 5 min washing with 1 mM ADVASEP-776 in normal saline. Finally, the cells were washed in normal saline and imaged. The cells were stimulated twice, once for 2 s at 20 Hz to access the size of the RRP and a second time for 120 sec to access the RcP, following a minute of recovery. The degree of unloading was calculated as the change in fluorescence (ΔF) normalized by baseline background-corrected fluorescence F0. Experiments were performed at RT in the presence of APV (50 μM) and DNQX (10 μM) to reduce destaining due to spontaneous network activity77.
Measuring vesicle cycling using sypHy
SypHy is a probe based on the internal fusion of a pH-sensitive GFP called pHluorin78 (pKa = 7.6), with the vesicular protein Synaptophysin I (SypI), so that pHluorin is located in the lumen of the synaptic vesicles (SVs) and allows the reportage of presynaptic activity31,79. Briefly, pHluorin fluorescence is quenched by the acidic pH of the intact vesicle lumen (pH~5.5). Upon stimulation, SVs fuse with the plasma membrane, thus exposing their lumen to the neutral pH of the extracellular environment (pH~7.3), causing an increase in the fluorescence of pHluorin. Following endocytosis, pHluorin is re-quenched due to the reacidification of the lumen by the vesicular proton pump (vATPase). Therefore, an increase in fluorescence reflects the exocytosis phase, and the subsequent decrease in fluorescence indicates the endocytosis phase of the vesicle cycle. The kinetics of exocytosis is determined by examining the time course of an upsurge in fluorescence upon stimulation in the presence of bafilomycin A, an inhibitor of the vATPase80, which inhibits neurotransmitter loading but not vesicle recycling81,82. In our experiments, 12-14 DIV neurons were field-stimulated in the presence of APV (50 µM) and DNQX (10 µM), in the presence or absence of bafilomycin A (Fig. 6a-c). After stimulation, the bath was perfused with saline in which 50 mM NaCl was replaced with NH4Cl. Ammonium ions are in equilibrium with aqueous ammonia, which is membrane-permeable and can diffuse into SVs to neutralize their lumen. Therefore, the combination of sypHy and NH4Cl reveals intact SVs within the terminals, and the size of the total SVs pool is measurable. The recycling vesicle pool (RcP) size is estimated by measuring the plateau of fluorescence intensity obtained during exhaustive stimulation (Fig. 6c), while the resting pool is that which completes the RcP to the total pool26. The baseline fluorescence intensity of sypHy (F0) in each synapse of interest was the average value measured in five successive images acquired before stimulation. The change in fluorescence (ΔF) at time t was calculated as F(t)−F0. Values for each synapse were normalized by the maximal fluorescence intensity (Fmax) measured after the treatment with NH4Cl (ΔF/Fmax).
Acute hippocampal slices
P18-P21 littermate mice for field-potential recordings and P28-P31 mice for whole-cell recordings from either sex were anesthetized with Isoflurane and decapitated. The brains were rapidly removed and placed in an ice-cold oxygenated cutting solution that contains (in mM): 252 Sucrose, 5 KCl, 1 CaCl2, 3 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 Glucose; pH 7.3 when bubbled with 95% O2/CO2. Transverse slices (300 µm) were cut on a vibratome (Leica 3000) and placed into a holding chamber containing oxygenated aCSF at RT for at least an hour prior to the recordings. ACSF solution contains (in mM): 124 NaCl, 3 KCl, 2 CaCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose; pH 7.4 when bubbled with 95% O2/CO2.
Slices were viewed through 40X water-immersion lenses (Olympus) in a BX51WI microscope (Olympus) mounted on an X–Y translation stage (Luigs and Neumann). Somatic whole-cell recordings were made using patch pipettes pulled from thick-walled borosilicate glass capillaries (1.5 mm outer diameter; Science Products). Pipettes had resistances of 5–7 MΩ when filled with a whole-cell voltage-clamp solution that contains (in mM): 135 CsCl, 4 NaCl, 2 MgCl2, and 10 HEPES (cesium salt), pH adjusted to 7.3 with CsOH. Voltage-clamp recordings were made with a MultiClamp 700B amplifier (Molecular Devices). Data were sampled at 10 kHz, amplified (gain 5), filtered at 3 kHz then digitized and analyzed using Clampfit. Membrane access resistance was maintained as low as possible (5-10 MΩ) and was compensated at 80%. Recordings were not corrected for liquid junction potential. Recordings were performed at 30 °C in the presence of Tetrodotoxin (TTX, 1 nM, Alomone Labs) and Bicuculline (GABA A antagonist. 10 µM, Alomone Labs). Inter-event intervals (IEIs) were calculated per recording (per cell) and averaged across slices (up to 3 slices per mouse).
Two-photon reconstruction of neuronal morphology
At the end of physiological recordings brain slices from WT and Cdh2H150Y littermates, the slice that contained the cells filled with SBFI fluorescent dye (0.5 µM, Thermo Fisher) was placed in an aCSF perfused chamber. Slices were scanned with an Ultima IV two-photon microscope (Bruker) equipped with a Mai Tai DeepSee pulsed laser (Spectra-Physics) and Olympus water-immersion lens ×60. Using two-photon excitation at 740 nm, we focused on the cell and scanned 30–40 slices of dendrites, soma, and axon at 0.5-μm depth intervals. The resulting z-stacks were imported into ImageJ (US National Institutes of Health) for reconstruction and image processing.
My objections remain unanswered. This is a speculative effort to establish a correlation between “ADHD diagnosis” and a certain genetic pattern. It is performed on a very small number of MICE. It does not address questions of rearing variables, does not establish probability of the “error” being found in the “ADHD-diagnose” population, nor in the general population vs. “ADHD-diagnosed.” It does nothing to establish any kind of “abnormality” of this genetic combination to distinguish it from normal genetic variation. One more “promise” of a “biological cause” which is nothing of the sort.
To conclude, we demonstrate that a novel mutation in CDH2 is associated with familial ADHD, through impaired presynaptic vesicle clustering, attenuated evoked transmitter release, decreased spontaneous release, and reduction in dopaminergic distribution within limbic pathways. We thus delineate the role of CDH2-related pathways in the pathophysiology of ADHD.
Finally, let me encourage you again to study in its entirety the documented data published regarding this research.
Res ipsa loquitur
“CDH2 mutation affecting N-cadherin function causes attention-deficit hyperactivity disorder in humans and mice”
A summary of the data
Again, claiming “cause” requires establishing that all or almost all “ADHD” sufferers HAVE the CDH2 mutation, and that no or almost no “normal” people have this mutation. To claim this from the data presented is pretty laughable. They’ve shown that a very small sample of people/mice subjectively judged to “have ADHD” without objective measures happen to have this particular mutation. Does EVERYONE with this mutation have “ADHD?” Or is it 10%, 20%, 50%? Does EVERYONE diagnosed with “ADHD” have this mutation? Do some have the mutation and NOT develop “ADHD symptoms?” Is the presence/absence of this mutation determinative of a person’s long-term success in life? Does “treatment” for this putative problem actually do anything to improve long-term outcomes for those so “diagnosed?” Is there even an objective way to determine who “has” or “does not have ADHD?”
It is scientifically absurd to declare that this mutation “causes” ADHD when the “disorder” itself is not objectively discernible, nor is there any indication that those so “diagnosed” have any kind of physiological problem or are just a normal variation of behavior that doesn’t work so well in modern society. After all, genetic diversity is central to species survival. Why is a genetic variation, even if it is 100% associated with this behavioral syndrome, automatically a “disorder” when our species depends on a wide variation of genotypes and phenotypes to survive?
Ah, now we get down to it. It is ASSOCIATED with “familial ADHD.” Associated means it occurs with a higher percentage in the “ADHD” population than in the general population. It does not mean it is a cause, as there may be many, many cases of the same mutation in people who don’t fit the “ADHD” criteria. “Correlation is not causation.”
We know for certain that familial domestic abuse is highly associated with “ADHD.” It occurs more frequently in the “ADHD” population than in the general population. Does that mean that familial domestic abuse causes “ADHD?” What about the many children who are exposed to the same kind of parental behavior who do NOT develop “ADHD” symptoms? What about the large number of “ADHD” diagnosed children who don’t have domestic abuse in their history?
Other associations are low iron, sleep apnea, younger age of starting school, being in a traditional classroom vs. an open classroom, abuse/neglect at an early age, etc. Are all of these “causes” of “ADHD” because they are “associated” with “familial” ADHD? Does a traditional classroom “cause” ADHD because more “ADHD” behavior is associated with it? Or does a traditional classroom structure simply not WORK for a certain percentage of children, for a wide variety of reasons?
Correlation is not causation. And you continue to avoid the most important question of why this particular set of subjective behaviors are automatically a “disease state” even if a consistent physiological cause were found.
Comments I made before MIA approved other comments I made, have not been adjudicated. What is the reason for this, may I ask?
COMMENTING AS MODERATOR:
All comments you have made should be approved at this point. They are not always approved in order they are submitted, as more than one person works on approving comments, and sometimes certain comments require more analysis than others, so their approval may be delayed in some cases. If there are any comments I have not approved, please let me know the thread and some of the content and I will see what has happened to the comment in question.
Hey, Thanks for the explanation. I appreciate it.