Introduction

Schizophrenia is a chronic and disabling disorder with an approximate 1% incidence in the population, and 120 million afflicted individuals worldwide [1]. Hallucinations, delusions, and emotional, cognitive, and motor deficits are common symptoms that appear usually in late adolescence or early adulthood [2, 3]. Many studies detected a genetic link to the disorder, although no single or small number of genes accounts for the majority of cases [4, 5]. Systematic analyses of different pedigrees segregating schizophrenia suggested multiple genes with a complex inheritance pattern [6, 7]. Child adoption studies have supported a polygenic and multifactorial aetiology [8].

Environmental factors linked to schizophrenia include winter parturition [9], maternal stress during pregnancy [10, 11], second trimester and postnatal infection, labour and perinatal traumas, immigration, residency in urban areas and so on [12, 13], and paternal occupation and age [14]. Paternal age has been linked to schizophrenia since 1958 [15]. Most studies including a recent meta-analysis have confirmed paternal age as a risk factor for schizophrenia [16–24], however negative results also exist in the literature [25]. The link between paternal age and schizophrenia, argues that de novo changes to DNA can lead to schizophrenia, because the sole biological contribution of fathers to progeny is DNA. De novo mutations are linked to neurodevelopmental disorders and cancers [26, 27] and may explain the high prevalence of schizophrenia despite the decreased fertility of schizophrenia patient [14]

Malaspina suggested that single base pair mutations, trinucleotide repeat expansion, or genetic imprinting changes might be responsible for the observed effect [28]. However, DNA replication and epigenetic changes are closely linked at both the macromolecules level (for example, DNA replication and global DNA methylation), and the metabolic level (for example, synthesis of purines and deoxythymidine triphosphate (dTTP) and S-adenosyl methionine, the cofactor that donates a methyl group). Hence, it would not be surprising that paternal age influences both of these as well as other overlapping processes.

In 2002, Malaspina et al. reported that the paternal age at birth of sporadic cases averaged 4.7 years older than familial cases of schizophrenia, and attributed approximately 27% of the sporadic cases to paternal age, and likely due to de novo mutations [14]. However, Pulver et al. observed that paternal age is not different in (1) familial versus sporadic cases, and (2) first-degree and second-degree paternal relatives of probands, and argued that these results did not support the linkage of paternal age to spontaneous forms of schizophrenia [29]. It is quite clear that studies on large families and pre/post-index generations are important to distinguish genetic from sporadic cases. However, it should be noted that, if advanced paternal age is a risk factor for schizophrenia, the frequency and coincidence of disease would be more in children of fathers with higher age and could be misinterpreted as genetic inheritance. This may obscure the detection of sporadic versus familial cases that could be overcome in part by the examining the effects of higher birth rank, accompanied with advanced paternal age, on schizophrenia pathogenesis in large families.

This study examined the relationship between paternal age and schizophrenia in an Iranian population, a distinct cultural group not previously studied. Typical Iranian families are large with a wide variation in paternal age not typically seen in Western families. The study of large families provides us with an opportunity to examine the relationship between birth order, paternal age and schizophrenia. If advanced paternal age is an important risk factor for schizophrenia, disease frequency would be greater in children with higher birth rank as well (that is, higher paternal age). Hence, we examined the effects of higher birth rank and advanced paternal age on schizophrenia.

Methods

This case-control study was performed in Tehran in 2005 and 2006 recruiting 220 patients (case group) with a diagnosis of schizophrenia from both psychiatric hospitals and private clinics. The diagnosis was made based on Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) criteria in a semistructured interview by two psychiatrists. The control group was made up of 220 individuals from non-psychiatric hospital wards of the same district, matched for sex (P = 0.84, χ2 test) and age (P = 0.37, t test) versus the case group. Individuals with any neurological problem, substance abuse, mental retardation or mood disorder were excluded from the case and control groups. Patients in an acute psychotic phase were excluded because of their inability to give consent. No organic problems were identified in any patients with schizophrenia by medical examinations. All cases and controls completed consent forms and data was collected on each subjected by trained medical students completing two instruments. The demographic questionnaire recorded sex, employment, education, paternal age, maternal age, paternal and maternal age at parturition, family history of psychotic disorders, number of children, birth rank within siblings, and age of disease onset. Both the patients and their carers were asked about the presence of similar disorders in first-degree and second-degree relatives. Those with evidence for positive family history underwent for further evolution by contacting their psychiatrist, inspecting their medical records or structured interview of the subjects and their carers in order to confirm or rule out the presence of mental diseases. The second diagnostic instrument was the Structured Clinical Interview for DSM Disorders (SCID), previously validated for diagnosis of schizophrenia in the Iranian population.

Differences between study groups were examined using the χ2 test for categorical variables, and an independent sample t test (or Mann-Whitney U test) for continuous variables. Probability values of P < 0.05 were considered statistically significant. Multiple logistic regression analysis was performed with adjustment for independent association of all factors with schizophrenia as a dependent variable. One model (included all independent variables at one stage) was developed to adjust for covariates. The analysis was performed using SPSS software (V.11.5; SPSS, Chicago, IL, USA).

Results

A greater number of cases (Table 1) had a birth rank of ≥3 versus controls (35% versus 24%, P = 0.01, OD = 1.7, 95% CI 1.12 to 2.5). The difference in the occurrence of psychiatric disorders, including schizophrenia, schizoaffective disorder and major mood disorders (unipolar and bipolar disorder) in first-degree relatives was significant in case versus control groups (P = 0.04): 20.5% versus 13.2%, respectively (Table 1).

Table 1 Demographic characteristics of cases and controls
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Paternal age at parturition of the proband was significantly different between the case and control groups. For instance, the mean paternal age at birth was 30 ± 6.26 versus 26.45 ± 5.64, in case versus control groups, respectively (P = 0.0001) (Tables 1 and 2). A significantly higher number of fathers with an age of ≥32 were in the case group versus the control group (Table 3). Analysis of the paternal age distribution (Figure 1) revealed that the fathers of the case group tended to have children at an earlier age, but that a greater number (76, 35%) of cases' fathers age were ≥32, than those in control group (n = 33, 15%). Individuals with fathers' ages at birth ≥32 were 2.77 times at higher risk for schizophrenia (P < 0.0001), OR = 3.7 (95% CI 1.9 to 7.3). The mean age of mothers at birth was marginally different (P = 0.02) between the case and control groups (26 versus 25.07, respectively). Logistic regression analysis (Table 4), adjusting for the effect of maternal age, birth rank and family history, indicated that paternal age is an independent predisposing factor to schizophrenia; its OR was 3.78 (95% CI 1.9 to 7.3).

Table 2 Analysis of the effect of paternal age on cases (n = 220) and controls (n = 220)
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Table 3 Sample classes as a function of parental or maternal age at birth
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Figure 1
figure 1

Distribution of paternal age in case and control subjects.

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Table 4 Stepwise analysis of the effect of paternal age on cases, controls
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This case-control study providing data on a previously unexplored Iranian population supports the hypothesis that advanced paternal age and a birth rank of higher than 3 (that is, higher age of father at birth) are linked to schizophrenia pathogenesis.

Discussion

Our results linking advanced paternal age to schizophrenia are comparable to observations in other populations. Further, logistic regression analysis has suggested that higher birth rank along with advanced paternal age is more of a risk factor for schizophrenia than maternal age. Birth rank and short birth interval were previously found to be risk factors for schizophrenia [30–32]. The effect of higher birth order/rank may reflect the costs of childbearing on a mother's long-term health, as Christensen et al. analysing twin data found support for the proverb 'a tooth, a child' [33]. Higher birth rank effects may also be a function of advanced paternal and/or maternal age. Along these lines, our study of large families provides evidence for the first time that the relationship between birth order/rank and schizophrenia may be due to paternal age. In fact, as advanced paternal age was a risk factor for schizophrenia, the disease frequency was greater in children with higher birth rank as well (that is, higher paternal age). Higher birth rank (and advanced paternal age) may account for the coincidence of disease in multiple siblings, whereas such coincidences are taken as evidence for genetic inheritance. However, paternal age effects on schizophrenia pathogenesis may be mediated through both genetic and/or epigenetic mechanisms [34, 35].

Epigenetics refers to heritable but potentially reversible changes in DNA methylation, RNA (editing and interference), and protein (for example, histone) modification and provide mechanisms for the environment to interact with the genome [36]. The multiplicity of targets and modifications linked to epigenetic programming suggests that perturbations to such pathways are likely to account for a large amount of phenotypic variations. Advanced paternal age is associated with an elevated incidence of environmental exposures that may affect the spermatozoid epigenetic memory. These epigenetic changes could be the underlying mechanisms of paternal age effects on schizophrenia pathogenesis.

About 50% of monozygotic (aka identical) twins afflicted by disease and starting life with identical genomes are discordant for schizophrenia, and when concordant they do not have the same psychiatric phenotypes [1]. Additionally, all children of monozygotic twins concordant or discordant for schizophrenia have the same elevated probability of becoming ill [37, 38]. These observations suggest that genetic susceptibility is transmitted to offspring, but environmental factors also contribute in disease occurrence and presentation. An elevated somatic mutation rate in twins discordant for schizophrenia was detected around simple trinucleotide repeat sequences, for example, (CAG)n [39]. It is of note that this study was limited to (CAG)n repeat occurrences, and we do not know whether the increased mutation rate extends to other sequences. However, other studies have also detected an increase in neuronal aneuploidies in schizophrenic brains [40, 41]. Several groups have suggested that the genetic-environmental linkage reflects the need for 'two-hits' (genetic plus somatic changes) to DNA. Other studies have shown that genetic and epigenetic changes occur with age [42, 43], and epigenetic differences exist between monozygotic twins [44, 45].

In sperm, dominant and codominant base change mutations will impact progeny and subsequent generations in a simple Mendelian fashion. A recessive mutation will not be detected in progeny (except occasionally when a second mutated allele is present) unless the target gene is maternally imprinted and silenced. Epigenetic changes at imprinted genes could lead to inappropriate expression or lack of expression. The effects on subsequent generations will be dependent on the parental origin of the change. Further, epigenetic DNA methylation is reset during passage through the germline and during embryonic and post natal development; hence, it is not clear what changes will be passed to subsequent generations [36].

The dissection of the paternal age effect in large families is advantageous given the plethora of potential DNA changes and the consequences for subsequent generations. The paternal age effect may be due to inadequate nutrition and/or exposure to environmental toxins during spermatogenesis. For instance, it is estimated that 30% of American diets are deficient in folate [46, 47], a nutrient required for the biosynthesis of TTP and DNA synthesis as well as purine nucleotides that are necessary for DNA and RNA synthesis and other cellular processes (for example, epigenetic changes to RNA and proteins, energy transduction, and signally). The success of genetic studies on rare human disease has prompted research on complex common disease to focus on genetic approaches while largely ignoring environmental factors that have the potential to change not only DNA but also cause epigenetic changes to DNA, RNA and proteins. For instance, paternal age and occupations linked to schizophrenia in progeny may involve exposures that induce oxidative stress and impact folate metabolism [48]. A comprehensive understanding of schizophrenia and other common complex diseases requires studies on the interaction of the environment with specific genotypes, some predisposed, to severe illness. Such studies are required as several preventable known environmental and nutritional factors (for example, folic acid deficiency) can induce epigenetic alterations that may impact the quality of human life in general.