Klotho Gene and Selective Serotonin Reuptake Inhibitors:
Response to Treatment in Late-Life Major Depressive Disorder
Giulia Paroni
1
& Davide Seripa
1
& Andrea Fontana
2
& Grazia D’Onofrio
1
&
Carolina Gravina
1
& Maria Urbano
1
& Filomena Addante
1
& Madia Lozupone
3
&
Massimiliano Copetti
2
& Alberto Pilotto
4
& Antonio Greco
1
& Francesco Panza
1,3
Received: 20 May 2015 /Accepted: 11 January 2016
#
Springer Science+Business Media New York 2016
Abstract Klotho protein, encoded by the Klotho gene (KL) at
locus 13q12, is an antiaging hormone-like protein playing a
pivotal role in cell metabolism homeostasis and associated to
longevity and age-related diseases. In particular, altered cell
metabolism in central nervous system may influence the be-
havior of serotoninergic neurons. The role of KL in the re-
sponse to treatment with selective serotonin reuptake inhibi-
tors (SSRIs) in late-life depressive syndromes and late-life
major depressive disorder (MDD) is unclear. We genotyped
three single-nucleotide polymorphisms (SNPs) of KL in 329
older patients with diagnosis of late-life MDD, treated with
SSRIs and evaluated with the Hamilton Rating Scale for
Depression 21-items (HRSD-2 1) at baseline and after
6 months. A reduction ≥50 and <10 % in HDRS-21 score
was considered as response or nonresponse to therapy, respec-
tively, and the values of reduction between 10 and 49 % as
poor responders. After 6 months of SSRI treatment, 176
patients responded, 54 patients did not respond and 99 patients
showed a poor response. Ordinal logistic models showed a
significant association between mutation of SNP rs1207568
and responders and, similarly, for each unitary risk allele in-
crease overlapping results were found. Conversely, a signifi-
cantly higher frequency of the minor genotype of SNP
rs9536314 was found in nonresponders. Considering the
pre-post differences of HRSD-21 scores as a continue vari-
able, we confirmed a significant improvement of depressive
symptoms after treatment in patients carrying at least one mi-
nor allele at rs1207568 and a worse response in patients ho-
mozygous for the minor allele at rs9536314. Our results were
the first that suggested a possible role of KL in the complex
pathway of SSRI response in late-life MDD.
Keywords Klotho
.
Late-life depression
.
Late-life major
depressive disorder
.
Selective serotonin reuptake inhibitors
.
Response to treatment
.
Older age
Introduction
Among neuropsychiatric disorders, depression is increasingly
becoming common and disabling in older age [1]. Late-life
depression (LLD) refers to depressive syndromes that arise in
adults older than 65 years [2, 3], encompassing both late-onset
as well as early-onset cases that recur or continue into later
years of life [4]. Prevalence estimates of clinically significant
LLD vary widely from 4.5 to 37.4 % according to the popu-
lation studied, sample size, and nonpsychiatric comorbidity
[5]. Furthermore, in LLD, there is a general consensus on a
syndromal approach to depression to identify symptom clus-
ters such as major depressive disorder (MDD) [2, 3]. Among
late-life depressive syndromes, clinically significant MDD is a
common disease in Western countr ies with a poole d
Electronic supplementary material The online version of this article
(doi:10.1007/s12035-016-9711-y) contains supplementary material,
which is available to authorized users.
* Giulia Paroni
giulia.paroni@operapadrepio.it
1
Gerontology-Geriatric Research Laboratory, Department of Medical
Sciences, I.R.C.C.S. BCasa Sollievo della Sofferenza^, 71013 San
Giovanni Rotondo, Foggia, Italy
2
Biostatistic Unit, I.R.C.C.S. BCasa Sollievo della Sofferenza^, 71013
San Giovanni Rotondo, Foggia, Italy
3
Neurodegenerative Disease Unit, Department of Basic Medicine,
Neuroscience, and Sense Organs, University of Bari Aldo Moro,
Bari, Italy
4
Geriatrics Unit, Department of OrthoGeriatrics, Rehabilitation and
Stabilitation, Frailty Area, Galliera Hospital NR-HS, Genoa, Italy
Mol Neurobiol
DOI 10.1007/s12035-016-9711-y
prevalence of 7.2 % [5] and an incidence rate of 0.2–14.1/100
person-years [6]. It has been also recently reported that LLD
may result in increased risks of comorbidity [7],
polypharmacy [8], frailty [9], an d also all-caus e mortality
[10], thus becoming a major public health concern for the next
future [1, 11, 12]. Despite these data, LLD is still under rec-
ognized in clinical practice, with less than 10 % of older sub-
jects that have formal evaluation and treatment for this condi-
tion [11–13].
According to a recent hypothesis, MDD may be the result
of a dysregulation of the stress system in response to a predis-
posing, stressful environment [14, 15]. Stress in life is un-
avoidable, affecting everyone on a daily basis. Psychological
stress in mammals triggers a rapidly organized response for
survival, but it may also cause a variety of behavioral disor-
ders and depression [16]. The same hypothesis strongly links
MDD to parainflammation and endoplasmatic reticulum (ER)
stress and to three potential interrelated modulators of this
process, the central nervous system (CNS) insulin, the perox-
isome proliferator-activated receptor-γ, and Klot ho.
Parainflammation is an adaptive response of the innate im-
mune system that occurs in the context of stressors to which
we were not exposed during our early evolution, including
overfeeding, underactivity, aging, and drugs [17, 18].
Multiple physiological or pathological conditions that affect
protein folding or overload the capacity of ER to fold proteins,
i.e., increased glutamate stimulation [18] or overfeeding [19],
can cause ER stress, an imbalance between the cellular de-
mand for ER function and ER capacity [20]. If the ER re-
sponse is overwhelmed, substantial amounts of calcium are
released into the cytoplasm, leading to apoptosis [18].
Therefore, ER stress is involved in disease states because it
can lead to cell death or compromise of cellular resilience [21,
22]. The stress response also plays important roles in numer-
ous human pathophysiological processes caused by protein
misfolding and retention in the ER, organismal responses to
malignancy, neurodegeneration, atherosclerosis, and type 2
diabetes [17, 18, 21–24]. Moreover, a failed ER stress re-
sponse may play an important role in the depressive syn-
drome, in part by leading to cell loss or decreased cellular
resiliency [20].
Klotho, originally identified as an antiaging protein in mice
[25], plays a major role in the biochemical pathways regulat-
ing ER stress response. Klotho protein ameliorates chemically
induced ER stress signaling [26], and Klotho gene (KL) may
be a potential translational target in ER stress in MDD.
Klotho-deficient mice showed an increased overall burden
of CNS oxidative stress [27],
suggesting that this protein
may exert brain-wide antioxidative and anti-inflammatory ef-
fects [28, 29]. Klotho decreases parainflammation and im-
proves the efficiency of the ER stress response induced by
an upregulation of KL that may be relevant in increasing neu-
ronal resilience in MDD patients [14, 15, 20], including
adaptive hippocampal neurogenesis, thus suggesting that KL
functional genetic variations may alter MDD resilience and
treatment. Currently, selective serotonin reuptake inhibitors
(SSRIs) are the first-line pharmacological treatment of mod-
erate and severe depression in older adults [30]. Several clin-
ical trials, however, reported a variable response rate to SSRI
up to 65 %, particularly in older patients [31, 32]. While major
evidences suggested an important contribution of pharmaco-
genetics in SSRI response in younger subjects, the role of
genetics in SSRI response in LLD is still under debate
[33–36]. The aim of the present study was to investigate the
KL gene polymorphisms to evaluate a potential involvement
of this gene in the response to SSRI treatment in patients with
late-life MDD.
Methods
Subjects
The present study was conducted fulfilling the Declaration of
Helsinki, the guidelines for Good Clinical Practice and the
Strengthening the Reporting of Observ ationa l Studies in
Epidemiology (STROBE) guidelines [ 37]. From January
2009 to May 2011, a total of 1311 older subjects who consec-
utively attended the Geriatric Unit of the Istituto di Ricovero e
Cura a Carattere Scientifico (IRCCS) Ca sa Sollievo della
Sofferenza and who self-reported depressive symptoms were
screened for possible study enrollment (Fig. 1). The approval
of the study for experiments using human subjects was obtain-
ed from the local Ethics Committee on human experimenta-
tion. Written informed consent for research was obtained from
each patient or from relatives/legal guardian in the case of
critically disabled demented patients. All the 329 older sub-
jects enrolled were Caucasians and did not include people of
Jewish, Eastern Europe, or Northern Africa descent, with most
individuals having Central and Southern Italy ancestry.
Inclusion/Exclusion Criteria
Inclusion criteria were as follows: (1) Caucasian race; (2) age
≥65 years; (3) diagnosis of MDD according to the criteria of
the Diagnostic and Statistical Manual of Mental Disorders,
Fourth Edition, Text Revision (DSM-IV-TR) [2]; (4) need
for the treatment with antidepressants of the SSRI class; (5)
Mini Mental State Examination (MMSE) [38]score≥15, be-
cause of the use of the 15-item Geriatric Depression Scale
(GDS-15) [39, 40] that has been validated within this
MMSE score limit [41]; and (6) written informed consent.
Patients were excluded from the study if (1) they had age at
onset of depression before 65 years, (2) they refuse to enter in
the study or to sign the informed consent, (3) they were al-
ready in treatment with SSRIs or other antidepressants, or had
Mol Neurobiol
a recent history (last year) of therapy with antidepressants or
other psychotropic drugs (benzodiazepines, antipsychotics,
mood stabilizers), (4) a severity of MDD not allowing to col-
lect adequate data, and (5) a MMSE score <15.
Study Protocol
At baseline, demographic and clinical characteristics were
collected by a structured interview and clinical evaluation.
All included patients with late-life MDD were initially treated
for 2 weeks with escitalopram 5 mg/daily, sertraline 25 mg/
daily, paroxetine 10 mg/daily, or citalopram 10 mg/daily on
the basis of the clinical judgment of the physician, who was
unaware of the genetic profile of the patient. Two weeks after
that dose titration was completed, 41 patients dropped out
because of adverse drug reactions (ADRs) (n =8)orbecause
of refusal to undergo the follow-up treatment (n = 33) (Fig. 1).
Patients who had followed the treatment with satisfactory or
good compliance and without clinically relevant ADRs
(n = 329) increased the dosage of escitalopram to 10 mg/daily,
sertraline to 50 mg/daily, paroxetine to 20 mg/daily, or
citalopram to 20 mg/daily for the following 22 weeks
(Fig. 1). After treatment, the clinical assessment was repeated,
including the evaluation of cognitive and functional status,
compliance, and drug-related adverse events.
Diagnosis of Depression and Response to Treatment
Diagnosis of late-life MDD was made according to the diag-
nostic criteria from the DSM-IV-TR [2] using the GDS-15 to
evaluate depressive symptoms. To define the response to treat-
ment with SSRIs, we used the Hamilton Rating Scale for
Depression with 21 items (HRSD-21) [42] that evaluates the
depressive symptoms. Initially, as previously reported [42,
43], we defined as responder (R) a patient who showed a
reduction of at least 50 % on the HRSD-21 score, as poor-
responder (PR) if the reduction was between 10 and 49 %, and
as nonresponder (NR) if the reduction is <10 % [10].
Afterward, we considered the HRSD-21 as a continue variable
to compare the variations between baseline and follow-up.
Genetic Analysis
Genomic DNA was purified from fresh/frozen blood samples
following salting-out method [44]. In the present study, we
analyzed the three single-nucleotide polymorphisms (SNPs)
rs1207568, rs9536314, and rs564481. The SNPs were chosen
for their functional role in altering protein product
(rs1207568) [45] or amino acid (rs9536314 and rs564481)
[46]. Overall, these SNPs span 45 kb block at the KL locus
(13p12) (33,590,184–33,634,983 bases from pter). The SNP
rs1207568 (33,590,184 bases from pter), i.e., the C
4,614
→ T
near the 5′UTR of the KL gene, was determined with
the allele discrimination assay using TaqMan technology.
Briefly, genomic DNA was analyzed with the ABI PRISM
7700 Sequence Detector system (Life Technologies
Corporation, Carlsbad, CA, USA) using the assay
c_7604792_10 according to manufacturer instructions. The
SNP rs9536314 (33,628,138 bases from pter), i.e., the G
42,
568
→ T in exon 2 of the KL gene causing the amino acid
change Phe
352
→ Val, was determined as follows. Briefly, ge-
nomic DNA was amplified by PCR with a hot-start at 94 °C
for 7′,followedby30cyclesat94°Cfor30″,50°Cfor30″,
and 72 °C for 30″. The PCR product (279 bp) was digested
with Mae III for 6 h at 55 °C. The analysis on a 3 % agarose
gel electrophoresis revealed the three genotypes T/T (232 +
47 bps), T/G (232 + 182 + 50 + 47 bps), and G/G (182 + 50 +
47 bps). The SNP rs564481 (33,634,983 bases from pter), i.e.,
the C
49,413
→ T causing the sense amino acid change
His
589
→ His in exon 4 of the KL gene was determined with
the allele discrimination assay using TaqMan technology.
Briefly, genomic DNA was analyzed with the ABI PRISM
7700 Sequence Detector system (Life Technologies
Corporation, Carlsbad, CA, USA) using the assay
c_592739_10 according to manufacturer instructions. No dif-
ferences wer e observ ed in respect to the expected Hardy-
Weinberg equilibrium for one locus. No differences were also
observed when the present genotype frequencies were com-
pared with those reported form the 1000 genomes project
(http://www.1000genomes.org). We also analyze d at the
solute carri er family 6 (neurotransmitter transporter,
serotonin), member 4 (SLC6A4) locus the polymorphism C,
also known as 5HTT gene linked polymorphic region
(5HTTLPR) and cytochrome P450 (CYP) 2D6 as previously
described [47, 48] to avoid the possibility that these two fac-
tors may influence our analysis.
Statistical Analysis
Patients’ baseline characteristics were reported as mean± stan-
dard deviation or frequencies and percentage for continuous
and categorical variables, respectively. Normal distribution
assumptions were checked by means of Q -Q plot and
Shapiro-Wilks and Kolmogorov-Smirnov tests. Comparisons
between continuous variables were performed using two-
sample t test or Mann-Whitney U test, in case of nonnormal
distributed data. Comparisons between categorical variables
were performed using Pearson chi-squared test or Fisher exact
test, as appropriate. Associations between KL genotypes and
the patient response to SSRI treatment were assessed using
both multivariable ordinal logistic regression models (which
assumes that the coefficients that describe the relationship
between R or PR vs. NR is the same as those that describe
the relationship between R vs. PR or NR), and multivariable
ANOVA models (which modeled the pre-post reduction of
HRSD-21 scores) for categorical and continuous SSRI
Mol Neurobiol
treatment response, respectively. Multivariable ordinal logistic
models were adjusted for age, sex, educational level, SSRI
treatment type, and 5-HTTLPR genotype, assuming different
genetic models of inheritance (i.e., dominant, recessive, and
additive) [49, 50]. Multivariable ANOVA models were adjust-
ed for HRSD-21 score at baseline, age, sex, educational level,
SSRI treatment type, and 5-HTTLPR genotype, assuming dif-
ferent genetic models of inheritance (i.e., dominant, recessive,
and additive). Risks were reported as odds ratio (OR) along
with their 95 % confidence interval (95 % CI). The greater the
HRSD-21 mean reduction, the better the treatment response.
Adjusted means of HRSD-21 reduction, along with stan-
dard errors, were eventually estimated for each KL geno-
type, within each SNP, separately. To control for multiple
comparisons, p values derived from both multivariable
models were further adjusted following Hochberg’smeth-
od (adj p). For sake of completeness, binary logistic regres-
sion multivariable models w ere further estimat ed wi thin R
vs. NR, PR vs. NR, and R vs. PR groups, separately. The
HaploView 4.2 genetic software package was used to esti-
mate the values of linkage disequilibrium (LD) coefficient
r
2
as well as to estimate and compare haplotype frequen-
cies at the KL locus, between the study groups [51]. p
Values <0.05 were considered for statistical significance.
All statistical analyses were performed using SAS, version
9.3 (SAS Institute, Cary, NC).
Power Calculation
To detect a statistical association between any KL genotype
(e.g., major/major vs. minor/major vs. minor/minor) and treat-
ment response (i.e., R vs. PR vs. NR), a sample size of 329
patients achieves 80 % power to detect a Cohen’seffectsize
(W) [52]of0.19usingaPearson’s chi-squared test evaluated
on a two-way contingency table with (3-1) × (3-1) = 4° of free-
dom, with a significance level of 0.05.
Results
Demographic and Clinical Characteristics of Patients
The analysis was carried out on 329 patients with late-life
MDD (95 men and 234 women, mean age 77.58 ± 6.63 years,
age range from 65 to 94 years) (Fig. 1). Demographic and
clinical characteristics of patients at baseline are summarized
in Table 1. No significant differences were observed between
men and women in age, MMSE score, and SSRI treatments.
Conversely, a significant difference between genders was ob-
served in educational level (p < 0.001), in the GDS-15 score
(p = 0.007), and in the HRSD-21 scores (p =0.002). Baseline
and follow-up clinical characteristic of patients according to
the status of response to SSRI treatment are summarized in
Table 2. No significant differences were observed between R,
PR, and NR patients with late-life MDD in gender distribu-
tion, age, educational level, HRSD-21, GDS-15, and MMSE
scores at baseline. Moreover, no significant differences were
observed in response distribution (R vs. PR vs. NR) in patients
treated with the four different SSRIs. As expected, at follow-
up, significant differences were observed in the HRSD-21
score and in GDS-15 score among the three groups of patients
(R vs. PR vs. NR). Moreover, MMSE score at follow-up was
significantly higher in R than PR (p = 0.017) and NR groups
(p =0.002).
Genotype Analyses
Genotype distribution at KL locus according to the status of
response to SSRI treatment is summarized in Table 3. There
was a significant difference of genotype distribution among all
of the three groups for the SNP rs9536314 (adj p = 0.033). The
associations between KL genotypes and the status of response
to SSRI treatment are summarized in Table 4. We found that
rs1207568 followed a dominant and an additive model of
inheritance, whereas rs9536314 followed a recessive model
of inheritance. Indeed, results from ordinal logistic models
showed that patients who carried at least one minor allele at
rs1207568 had a significantly better response to SSRI treat-
ment than those who carried both major alleles (OR = 2.29,
95 % CI 1.35–3.87, adj p = 0.006) and, similarly, for each
unitary risk allele increase (OR = 2.13, 95 % CI 1.33–3.40,
adj p = 0.005), overlapping results were found. Conversely,
patients who were homozygous for the mutation at
rs9536314 had a significantly worse response to SSRIs treat-
ment than those who carried at least one majo r allele
(OR = 0.14, 95 % CI 0.03–0.63, adj p =0.031).
Looking at the pre-post differences in HRSD-21 scores
(HRSD-21 reduction) as a continuous variable, the analysis
evidenced that patients carrying at least one minor allele at
rs1207568 achieved higher improved response to treatment,
independently of their baseline HRSD-21 and SSRIs treat-
ment type than patients carrying major alleles (adj
p = 0.021). Indeed, a mean reduction of 10.46 (95 % CI
9.11–11.81) and 8.69 (95 % CI 7.59–9.79) points of HRSD-
21 were estimated in both groups, respectively. Conversely,
patients homozygous for the minor allele at rs9536314
showed a worse response to SSRI treatment (adj p =0.026).
Indeed, a mean reduction of 4.11 (95 % CI 0.15–8.07) and
Fig. 1 Flowchart of patients with late-life major depressive disorder
(MDD) screened for possible study enrollment. CDR Clinical Dementia
Rating, MMSE Mini Mental State Examination, GDS-15 15-item Geriat-
ric Depression Scale, HRSD-21 Hamilton Rating Scale for Depression 21
items, MDD major depressive disorder, ADR adverse drug reaction,
ΔHRSD-21 pre-post differences of HRSD-21 scores from baseline to
the end of treatment with selective serotonin reuptake inhibitors
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