Screening, Genetic Variants, and Bipolar Disorders: Can Useful Hypotheses Arise from the Sum of Partial Failures?

Round 1

Reviewer 1 Report

CACNA1C gene polymorphism (rs1006737) is a susceptibility factor for both schizophrenia (SCZ) and bipolar disorder (BP). The cause of cognitive dysfunction in bipolar disorder (BD) is not well understood. BDNF and CACNA1C are two susceptibility genes for the disorder that have also been reported to be associated with cognitive deficits in the disorder, but the studies have been small and with conflicting results.Such as  S. Rolstad et al found that the common genetic risk variants in rs6265 and rs1006737 are not associated with cognitive dysfunction.(DOI:10.1080/13546805.2016.1185405).

The sample size included in this study is too small,Only 21 patients with bipolar disorde.In addition, the included research samples are all over 60 years old. Whether they have hypertension, diabetes, Sleep disorder, Alzheimer's disease, smoking and drinking are not shown in the clinical data.

Author Response

Referee 1.

Q1: CACNA1C gene polymorphism (rs1006737) is a susceptibility factor for both schizophrenia (SCZ) and bipolar disorder (BP). The cause of cognitive dysfunction in bipolar disorder (BD) is not well understood. BDNF and CACNA1C are two susceptibility genes for the disorder that have also been reported to be associated with cognitive deficits in the disorder, but the studies have been small and with conflicting results. Such as  S. Rolstad et al. found that the common genetic risk variants in rs6265 and rs1006737 are not associated with cognitive dysfunction. (DOI:10.1080/13546805.2016.1185405).

R1: Thanks for the comment. The genetic variant and its links with bipolar disorder have been better described in the discussion session of the manuscript, reporting also the recent associated literature. Furthermore, the article mentioned by Rolstad et al., has been revisited by other authors, reporting a possible association between the SNP (rs1006737) in CACAN1C and cognitive functioning in BD, i.e., some publications described possible biomolecular mechanisms responsible for the mentioned SNP and mood disorders (Baker et al., 2023, doi: 10.1080/19336950.2023.2176984). On the other hand, following the associated genetic clinical database SNPedia, this mutation has been assigned an odds ratio of 1,7 for bipolar disorder. At the same time, the database has assigned it, until now, a trait for autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, and schizophrenia, reporting, in particular, different publications through genome-wide association studies (GWAS).

 

Q2: The sample size included in this study is too small, only 21 patients with bipolar disorder. In addition, the included research samples are all over 60 years old.

R2: Thanks for the comment. It was explained that it is a study that is useful for generating hypotheses through an almost qualitative methodology and mixed methods. Using elderly people in this type of studies is an advantage because it allows to exclude "prospective" false negatives, i.e. people that are currently not affected by bipolar disorders, but who might develop it in the future [as reported in the discussion]. This theme has been one of the critical elements in the debate about the accuracy of screeners for BD in the past.

 

Q3: Whether they have hypertension, diabetes, Sleep disorder, Alzheimer's disease, smoking and drinking are not shown in the clinical data.

R3: Thanks for the comment. In the methods, we have added “The exclusion criteria of the above cited previous study excluded cognitive deficits, substance abuse disorders, and severe chronic disorders. Mean inflammatory indices were normal at the end of the RCT [41]. Of the total sample of people without bipolar disorder, 10% had type 2 diabetes; 40% had mild-medium hypertension, 10% had had a diagnosis of a neoplastic disease, which was in remission [42]. 10% had at least grade I obesity [43]. Of the 21 patients with bipolar disorders, 7 (33%) suffered from at least I degree obesity (BMI ≥30), 3 (14.%) from type II diabetes and 6 (29%) from moderate or mild hypertension”.

 

Reviewer 2 Report

According to the definition of the scope and aims of the journal, Clinics and Practice  is an open access journal that provides an advanced forum for studies related to clinical medicine. The scope is broad including  many clinically oriented specialists. Therefore, the papers should be clear and understandable. This is not the case with reviewed article.

Concrete remarks

Introduction

In the subsection Introduction the current situation dealing with usage of genetic markers should be summarized. The aim is vaguely defined (… to verify whether the MDQ can be an accurate screening tool for bipolar disorder and whether the presence of the CACNA1C gene and the  genetic variant RS1006737 can be considered as an accurate screener for BD).

Materials and Methods

Methods are insufficiently described  - abbreviations are not explained, no reason for using CACNA1C gene (rs1006737) is given. What is the function of  CACNA1C gene (rs1006737), what does this gene encode?

 Questionnaires in psychiatry are used as a screening tool, however without a subsequent psychiatric examination has a low value of the evidence. Classification of neuropsychiatric disorders is phenomenological, based on presence of symptoms. Psychiatrists hope, that in near future the biological markers, e.g., objective measurement of pathophysiological  processes involved in disease development will be used  and enable prediction of disease course, prognosis, and treatment efficacy. Due to progress in genetics , genetic markers are very promising.

Results

The results are not clinically relevant, and negative results could be easily predicted.

Discussion and Conclusions

A confrontation with current literature dealing with the topic (… whether the MDQ can be an accurate screening tool for bipolar disorder and whether the presence of the CACNA1C gene and the  genetic variant RS1006737 can be considered as an accurate screener for BD ) is lacking (for example  Gordovez FJA et al , Mol Psychiatry 2020 ;25(3):544-559, Scotti-Muzzi E. et al, Eur Neuropsychopharmacol. 2022; 59:26-35).

Using Genome-Wide Association Studies (GWAS) many single nucleotide polymorphisms (SNPs) have been identified to be associated with BD. A few higher-risk associations have also been identified, such as a rare copy number variant on some chromosomes. Despite the fact, that SNPs in the CACNA1C gene have emerged as the most significantly associated with the disease, there is  an overlap of genes  associated with bipolar disorder with that of schizophrenia, major depression, and other disorders. So, it is not probable that existence of a special polymorphism of one gene could be sufficient for screening and risk evaluation.

Author Response

Referee 2.

Q1: According to the definition of the scope and aims of the journal, Clinics and Practice  is an open access journal that provides an advanced forum for studies related to clinical medicine. The scope is broad including  many clinically oriented specialists. Therefore, the papers should be clear and understandable. This is not the case with reviewed article.

R1: Thanks for the comment. We believe we have better clarified the hypotheses and conclusions. The fact that two case-finding tools present complementary results (in case of a positive genetic test, the probability of having the disorder is very high, whereas, in case of a negative score on the paper and pencil test, the probability of not having the disorder is very high) and that these tools are absolutely unreliable suggests that they measure risk factors different and probably complementary. This has strong pathogenetic implications and suggests interpretative hypotheses, but also potential future developments for synergy screeners.

 

Concrete remarks

Q2: Introduction

In the subsection Introduction the current situation dealing with usage of genetic markers should be summarized. The aim is vaguely defined (… to verify whether the MDQ can be an accurate screening tool for bipolar disorder and whether the presence of the CACNA1C gene and the  genetic variant RS1006737 can be considered as an accurate screener for BD).

R2: Thanks for the comment. The hypothesis was better specified and linked to the contradictory results of the genetic tests, in particular to the MDQ / genetic risk association. It was also well clarified that our intent was not to test for a specific risk by adopting only the (one) gene under test. Rather, since it had been verified that that gene was associated with bipolar disorder but was also found in healthy people without bipolar disorder, but with adaptive hyperactivity (therefore positive tests could result in bipolar disorder or being healthy), we wanted to verify if this could be synergistic with what was identified by the MDQ test (which did not only identify people with bipolar disorder, but also people with other disorders).

 

Q3: Materials and Methods

Methods are insufficiently described  - abbreviations are not explained, no reason for using CACNA1C gene (rs1006737) is given. What is the function of  CACNA1C gene (rs1006737), what does this gene encode?

R3: Thanks for the comment. Abbreviations in this part are now explained. Considering what our objectives were, we explained these aspects better in the discussion:

“Gene variant identification has yielded promising results in BD biology, particularly in the identification of genes coding for calcium channel subunits such as CACNA1C [48]. The Gene CACNA1C (Calcium Voltage-Gated Channel Subunit Alpha1 C) is located in locus 12p13.33, it Encodes an alpha-1 subunit of a voltage-dependent calcium channel, the Suspect SNPs associated with BD is the rs1006737 allele A [49]. An association was found between calcium channel genetics (linked with variants of CACNA1C) and increased glutamatergic metabolites in BD, possibly playing a synergic role in intracellular Ca2+ overload and excitotoxicity [50]. However, the same variants have also been found to be associated with schizophrenia and not just BD [51] and current methods estimate that the cumulative impact of many frequent alleles may explain only 38% of the BD phenotypic variance [52]”

 

Q4: Questionnaires in psychiatry are used as a screening tool, however without a subsequent psychiatric examination has a low value of the evidence.

R4: The questionnaires (especially the MDQ) proved to be inaccurate precisely in relation to the clinical diagnosis assumed to be the golden standard. This is probably because clinicians analyze what patients say, what clinicians see themselves, and what key informants say with the patient's consent, such as the wife or husband, used as a source of information (this is important, especially in Bipolar II Disorder where there is often little awareness by the patient). Of these three sources, the questionnaires use only the first one. Nonetheless, the questionnaires have shown some accuracy, albeit not sufficient. [Reported in discussion].

 

Q5: Classification of neuropsychiatric disorders is phenomenological, based on presence of symptoms. Psychiatrists hope, that in near future the biological markers, e.g., objective measurement of pathophysiological  processes involved in disease development will be used  and enable prediction of disease course, prognosis, and treatment efficacy. Due to progress in genetics , genetic markers are very promising.

R5: It was addressed in the Discussion:

Psychiatrists hope that biological markers, e.g., objective measurement of pathophysiological processes involved in disease development, will be used soon and will enable the prediction of disease course, prognosis, and treatment efficacy. However, considering the decades of "promising" genetic research, the results are disappointing and hopes for the future do not allow us to address a current problem of great concern in terms of public health, since bipolar disorder is often diagnosed years late. Perhaps it could be useful to generate pathogenetic hypotheses about how different factors can interact on complex pathogenic pathways. Since we start from a descriptive classification, it is possible that the same syndrome is the result of different biological mechanisms or that some conditions can identify potential synergistic risk factors in producing the disorder in question. From this point of view, an analysis with mixed methods and on a small sample can produce pathogenetic hypotheses without which the search for biomarkers can be reduced to throwing fishing nets. In this framework and only from a heuristic perspective, which obviously requires all the necessary verifications, this small study seems to suggest the hypothesis that a condition of hyperactivation common to many disorders and identified by the positivity to the MDQ (which is common to bipolar disorder, PTSD and anxiety disorders [9-12]) and whose genetic basis has not yet been clarified) can trigger bipolar disorder in people with a predisposition to hyperactivity (i.e. in people with the condition identified by the genetic variant analyzed).

 

Q6: Results

The results are not clinically relevant, and negative results could be easily predicted.

R6: It seems that both screeners show a certain accuracy, but with a perfectly specular result, i.e. one with perfect sensitivity (if the genetic test is positive, that is certainly a case), the other with excellent specificity (if the MDQ is negative, then it is almost certainly a non-case), which seems a result that is anything but insignificant and certainly not predictable. This is important both for the possibility of developing a mixed test and, especially for the speculative/pathogenic interpretations that this result can represent if confirmed [reported in the Discussion]

 

Q7: Discussion and Conclusions

A confrontation with current literature dealing with the topic (… whether the MDQ can be an accurate screening tool for bipolar disorder and whether the presence of the CACNA1C gene and the  genetic variant RS1006737 can be considered as an accurate screener for BD) is lacking (for example  Gordovez FJA et al , Mol Psychiatry 2020 ;25(3):544-559, Scotti-Muzzi E. et al, Eur Neuropsychopharmacol. 2022; 59:26-35).

Using Genome-Wide Association Studies (GWAS) many single nucleotide polymorphisms (SNPs) have been identified to be associated with BD. A few higher-risk associations have also been identified, such as a rare copy number variant on some chromosomes. Despite the fact, that SNPs in the CACNA1C gene have emerged as the most significantly associated with the disease, there is  an overlap of genes associated with bipolar disorder with that of schizophrenia, major depression, and other disorders. So, it is not probable that existence of a special polymorphism of one gene could be sufficient for screening and risk evaluation.

R7:Thanks for the comment. Our intent was not to test for a specific risk by adopting only the gene under test. Rather, since it had been verified that that gene was associated with bipolar disorder but was also found in healthy people without bipolar disorder but with adaptive hyperactivity (therefore positive tests could result in bipolar disorder or being healthy), we wanted to verify if this could be synergistic with what was identified by the MDQ test (which did not only identify people with bipolar disorder, but also people with other disorders).

Round 2

Reviewer 2 Report

The manuscript have been improved, all ratings "low" according to my opinion the rating in now  "average".  I recommend it accept inpresent form.