Consanguinity and the Risk of Hashimoto's Thyroiditis

Introduction

Hypothyroidism is a common condition that is associated with significant morbidity, resulting in a significant health and economic burden. It can even be life-threatening (1 –3). Hashimoto's thyroiditis (HT) remains the most common cause of hypothyroidism in iodine-sufficient parts of the world. The incidence of autoimmune thyroiditis varies among different countries. The trend over the last 50 years remains controversial, but some studies suggest an increase in incidence (4).

The pathogenesis of HT has been studied extensively, and multiple theories have been suggested. The main etiology of the disease lies in a complex interaction between genetic and environmental factors, resulting in dysregulated immunity. The pathogenesis of HT involves T and B white blood cells (5), apoptotic pathways (6), autoantibodies against thyroid antigens, and complement-mediated thyroid cellular injury (7), all leading to destruction of the thyroid follicles.

Autoantibodies targeting thyroid antigens are important in both the pathogenesis and the diagnostic approach of HT. The two most important antibodies are antithyroid peroxidase antibodies (TPOAb) and antithyroglobulin (anti-Tg) antibodies. Previous studies indicate that TPOAb are present in >90% of HT patients (8,9). In addition, animal studies support the role of TPOAb and anti-Tg antibodies in autoimmune thyroiditis (10,11). The mechanism of inducing such effect in humans involves triggering an inflammatory reaction, as well as blocking the functionality of these antigens in the thyroid synthesis pathway (7,12).

Consanguineous marriage is a common practice in many countries and stems from religious and cultural beliefs. It is also believed to be more favorable for the women's status due to better support from her in-laws in difficult situations (13).Overall, one fifth of all marriages in the world are consanguineous, and in North Africa, the Middle East, and West Asia, intra-familial marriages can collectively account for up to 50% of all marriages (13,14). Studies show consanguineous unions comprised 27% of marriages in Jordan (15). Despite the social benefits suggested by a consanguineous marriage, it has adverse health effects on the offspring. For example, it increases the occurrence of recessive diseases by expressing the recessive deleterious alleles. It also increases the prevalence of diseases caused by rare mutations (13,16).

Several studies assessed the possible genetic etiology of autoimmune thyroid diseases, including HT. The candidate gene approach has identified several autoimmune thyroiditis susceptibility genes, such as human leukocyte antigen (HLA-DR) (17 –20), T-cell antigens (21,22), and thyroid-specific genes such as the thyroglobulin gene (23,24). In addition, whole-genome linkage studies identified at least seven autoimmune thyroiditis susceptibility loci on seven different chromosomes (25). All these suggest that the tendency to develop HT might be genetically inherited. In several genetically inherited diseases, consanguinity has been suggested as an etiological factor (26). No study has investigated the effect of consanguinity on the risk of HT. This work aimed to study such an association.

Materials and Methods

This case-control study was conducted at the Endocrine Division of Jordan University Hospital, a tertiary referral center in Amman, Jordan. The study was approved by the Institutional Review Board of the hospital. All subjects provided written consent for their participation in the study. The participants were not compensated for taking part in this study.

The required sample size for a significance threshold of 0.05 (α = 0.05, z-test) and statistical power of 90% (β = 0.10) was calculated using the following formula; \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} n = \frac { 2 [ { \rm P } 1 ( 1 - { \rm P } 1 ) + { \rm P } 2 ( 1 - { \rm P } 2 ) ] \times f ( \alpha , \beta ) } { ( { \rm P } 1 - { \rm P } 2 ) ^2 } \end{align*} \end{document}

Where n is the sample size, P1 is the prevalence of consanguinity in HT patients, P2 is the prevalence of consanguinity in the general population, α is a P (type 1 error), β is a P (type II error), and f(α,β) is a value calculated from α and β. Since the prevalence of consanguinity in HT patients was unknown, a pilot study was performed during the 10-month period from January 2015 to October 2015, in which 198 HT patients were recruited. Detailed consanguinity history among their parents was obtained. Ninety-two (46.5%) patients had consanguineous parents. The general prevalence of consanguinity in Jordan is 27% (15). The analysis indicated that a sample size of 247 (for each study group) would be required.

A total of 895 participants were enrolled in the study. They were classified into three groups: HT group, comprising 298 patients (49 males) who had primary hypothyroidism and who tested positive for TPOAb; non-HT hypothyroidism group, comprising 299 patients (47 males) who had primary hypothyroidism but were negative for TPOAb; and a healthy control group, comprising 298 healthy participants (48 males) who were negative for TPOAb and had normal thyroid function tests. Subjects in all studied groups were not blood related. Subjects in the three groups were adults (≥18 years old) and were sex and age matched. The presence of consanguinity was compared between the three study groups.

Socioeconomic factors, including number of siblings, number of pregnancies in female participants, maternal and paternal education level, and smoking status of both parents, were studied in all participants in the three groups (Table 3).

In this study, consanguinity was defined as marriages between second cousins or closer (26), and included the marriage of first cousins, second cousins, and first cousins once removed. Detailed family history from the participants was used to determine the relation status between the parents.

Primary hypothyroidism was defined as serum thyrotropin (TSH) >4.94 μIU/mL with a normal or low serum free thyroxine (fT4) level. HT was defined as serum TPOAb level ≥5.61 IU/mL. Since only about 1% of patients with HT have only anti-Tg antibodies (TgAb) without TPOAb (27), the participants were not tested for TgAb.

The TPOAb, TSH, and fT4 levels were measured by assays (Abbott Architect, Abbott Park, IL) that were based on a two-step immunoassay using a chemiluminescent magnetic immunoassay (CMIA) technology. TPOAb levels <5.61 IU/mL were considered negative. The functional sensitivity of the TPOAb assay was 0.5 IU/mL with precision of ≤10% total CV for samples ≥5.61 IU/mL, and the analytical sensitivity was ≤1.0 IU/mL. The reference range for the TSH assay was 0.35–4.94 μIU/mL (99% confidence interval). Its functional sensitivity was ≤0.0038 μIU/mL (upper 95% confidence limit of 0.0042 μIU/mL) with a precision ≤10% total CV and analytical sensitivity of ≤0.0025 μIU/mL. The fT4 reference range was 0.7–1.48 ng/dL (central 99% interval). It had a limit of quantitation (LoQ) of ≤0.4 ng/dL with a precision of ≤10% total CV for concentrations in the range of the low control (0.65 ng/dL), medium control (1.2 ng/dL), and high control (2.8 ng/dL).

More details of the methods are described in the Supplementary Data (Supplementary Data are available online at www.liebertpub.com/thy).

Results

There was no statistically significant difference in age (p = 0.40) or sex (p = 0.97) distribution among the three groups. Consanguinity varied between the three groups: HT group, n = 86 (28.9%); non-HT hypothyroidism group, n = 38 (12.7%); and the healthy control group, n = 33 (11.1%). The degree of consanguinity was also different between the three groups (Table 1).

Comparison of the HT and healthy control groups revealed consanguinity as a HT risk factor (OR = 3.3 [CI 2.1–5.1]; p < 0.0001); subjects with consanguineous parents were 3.3 times more likely to have HT. Upon stratification of different degrees of consanguinity, marriages between first cousins once removed had the strongest association with HT (OR = 5.6 [CI 1.9–17]; p = 0.002), followed by first-cousin marriages (OR = 3.7 [CI 1.9–7.2]; p < 0.0001) and second-cousin marriages (OR = 2.4 [CI 1.4–4.5]; p = 0.005; Table 2). In addition, consanguinity was more prevalent in HT patients than it was in non-HT hypothyroidism patients (OR = 2.8 [CI 1.8–4.3]; p < 0.0001), suggesting that consanguinity is a risk factor for developing HT, probably independent of hypothyroidism. In this comparison, second-cousin marriages had the strongest association with HT (OR = 5.1 [CI 2.3–11.2], p < 0.0001), followed by marriages between first cousins once removed (OR = 2.5 [CI 1.1–5.6]; p = 0.031) and first-cousin marriages (OR = 2.1 [CI 1.2–3.6]. p = 0.014; Table 2). The results did not reveal a significant association between consanguinity and the risk of non-HT hypothyroidism (OR = 1.2 [CI 0.7–1.9], p = 0.54; Table 2).

The three groups were compared for a set of socioeconomic parameters (Table 3). The number of siblings was largest among the healthy control group, followed by the non-HT group and the HT group, with means of 4.6, 4.1, and 3.2, respectively (p < 0.001). Female participants of the healthy control group had the highest number of pregnancies, followed by the non-HT and the HT group, with means of 4.3, 3.6, and 2.8, respectively (p < 0.001). The level of paternal and maternal education among participants of the three groups was also assessed. Participants among the three groups did not differ significantly for their paternal university education (p = 0.63). However, the HT group had significantly higher maternal university education (47.3%) compared with 40.1% and 28.2% for the non-HT and the healthy control groups, respectively (p < 0.001). There was no statistically significant difference of paternal or maternal smoking status of participants between the three groups.

Discussion

The etiology of HT remains unknown. However, studies have suggested that female sex (28) and several environmental factors can increase the risk of HT (29 –31). Multiple family and twin studies have indicated an increased risk of the disease in siblings and children of patients with HT (32 –34). The sibling risk ratio for HT has been reported to be as high as 28 (35). Twin studies showed a TPOAb concordance rate of 64% in monozygotic twins compared with 35% in dizygotic twins (36) and 9% in the general population (37).When both parents had antithyroid antibodies, 42% of the male offspring and 33% of female offspring were also positive for antibodies, whereas if only one parent had antithyroid antibodies, these percentages were 28.9% for the male offspring and 16.7% for the female offspring (34). All these findings support a significant role of genetic susceptibility for HT. Multiple genetic loci have been identified associated with HT, such as human leukocyte antigens (HLA) (17), cytotoxic T-cell associated antigen (38), vitamin D receptor (39), and heat shock chaperone proteins (40).

The results suggest that consanguinity is associated with an increased risk for HT (OR = 3.3). Consanguinity was more prevalent in HT patients compared with the non-HT hypothyroidism patients (OR = 2.8). These findings support the role of genetic factors in autoimmune inflammation and destruction of the thyroid. Several studies have already suggested a link between consanguinity and diseases caused by dysregulation of the immune system (41,42). In contrast, the findings of this study did not indicate consanguinity as a risk factor for non-HT hypothyroidism, suggesting that in non-autoimmune hypothyroidism, complex environmental and dietary factors may play a more important role than genetics do.

First cousins have more alleles in common than the second cousins or first cousins once removed. Therefore, one would expect that first-cousin marriages would increase the risk of HT more than the unions of more distant relatives. Surprisingly, a higher risk of HT was observed in children of first cousins once removed (when compared with healthy controls) and second cousins (when compared with non-HT hypothyroidism; Table 2). One possible explanation is that a complex set of genes may contribute to autoimmune thyroiditis, of which some might be protective, and different combination of alleles in these genes increase or decrease the risk of HT.

Since consanguinity may be more prevalent among lower socioeconomic strata of the population, the three groups were evaluated from this perspective. The results identified that participants with HT were more commonly in a higher social class compared with the other two groups. This suggests that consanguinity is less likely to be a surrogate marker for other confounding socioeconomic parameters. In addition, pregnancy was suggested to be a trigger for autoimmune thyroid disease, especially in the postpartum period (43,44). The results indicate that pregnancy is unlikely to be a confounding factor, since females with HT had the fewest number of pregnancies compared with the other groups.

The prevalence of TPOAb among the healthy control group was 13% (93/716), which is concordant with previous studies (45,46). The prevalence of HT in Jordan has not been previously studied. A recent study showed that 9.04% of Jordanian patients who underwent total thyroidectomy had HT (47).

This study compared the HT patients with two control groups (healthy controls and non-HT hypothyroidism subjects). This design allowed the impact of consanguinity on HT risk to be evaluated independent of hypothyroidism. Moreover, the study inquired about consanguinity only after setting up the age- and sex-matched control groups, which prevented any selection bias. Since a small number of related subjects could potentially amplify the prevalence of consanguinity, subjects who had a relative in any of the groups were excluded from the analysis. The moderate sample size is another strength of this study.

This study has a number of limitations. Even though the subject groups were carefully matched to reduce potential confounders, a caveat of retrospective studies is inherent bias. Furthermore, previous studies showed that about 0–10% of patients with HT may not have TPOAb (8,9). As a result, those patients who tested negative for TPOAb in the present study might actually have HT but were erroneously placed in the control groups. Moreover, despite its importance, a family history of HT among participants was not obtained because it was subject to recall bias. Finally, an association between the degree of consanguinity and the HT age of onset was not included. This is because the presence of TPOAb might have preceded the time of its discovery.

In conclusion, for the first time, this study indicates that parental consanguinity is associated with increased risk of HT. Larger epidemiologic and family-based genetic studies are needed to confirm this association and to provide more details of the complex cause of HT. These findings highlight the health impact of consanguinity and may have applications in empiric risk estimations in genetic counseling, particularly in countries with high rates of consanguineous marriages.