Abstract
Context
Genetic risk factors play a major role in the pathoetiology of autoimmune thyroid diseases (AITD). So far, only common risk variants have been identified in AITD susceptibility genes. Recently, rare genetic variants have emerged as important contributors to complex diseases, and we hypothesized that rare variants play a key role in the genetic susceptibility to AITD.
Objective
We aimed to identify new rare variants that are associated with familial AITD.
Methods
We performed deep sequencing of 3 previously mapped AITD-linked loci (10q, 12q, and 14q) in a dataset of 34 families in which AITD clustered (familial AITD).
Results
We identified 13 rare variants, located in the inositol polyphosphate multikinase (IPMK) gene, that were associated with AITD (ie, both Graves’ disease [GD] and Hashimoto’s thyroiditis [HT]); 2 rare variants, within the dihydrolipoamide S-succinyltransferase (DLST) and zinc-finger FYVE domain-containing protein (ZFYVE1) genes, that were associated with GD only; and 3 rare variants, within the phosphoglycerate mutase 1 pseudogene 5 (PGAM1P5), LOC105369879, and methionine aminopeptidase 2 (METAP2) genes, that were associated with HT only.
Conclusion
Our study demonstrates that, in addition to common variants, rare variants also contribute to the genetic susceptibility to AITD. We identified new rare variants in 6 AITD susceptibility genes that predispose to familial AITD. Of these, 3 genes, IPMK, ZFYVE1, and METAP2, are mechanistically involved in immune pathways and have been previously shown to be associated with autoimmunity. These genes predispose to thyroid autoimmunity and may serve as potential therapeutic targets in the future.
Keywords: Graves’ disease, Hashimoto’s thyroiditis, genes, rare variants, single nucleotide polymorphisms, Thyroid
Autoimmune thyroid diseases (AITD) are the most prevalent autoimmune diseases in the United States and have been estimated to affect 5% to 10% of the population (1-3). AITD encompass a clinical spectrum (4) comprising Graves’ disease (GD), which manifests by thyrotoxicosis (5), Hashimoto’s thyroiditis (HT) (6) manifesting by hypothyroidism, and subclinical autoimmune thyroiditis manifesting by positive thyroid autoantibodies without clinical disease (7). Both GD and HT are characterized by the formation of autoreactive T cells targeting thyroid antigens that infiltrate the gland and can either cause thyroid cell death and destruction of the thyroid in HT (6) or generation of TSH receptor stimulating antibodies inducing overproduction of thyroid hormones in GD (8). Treatment of AITD can be challenging (9-13), and the burden of disease associated with AITD is very high (1). Therefore, better therapies targeting the disease-causing mechanisms are needed.
AITD are complex disease caused by epigenetic interactions between susceptibility genes and environmental triggers (14). Studies using whole genome approaches and candidate gene analyses have mapped 6 major AITD susceptibility genes that include 4 immune regulatory genes (HLA-DRb1-Arg74, CTLA-4, CD40, and PTPN22) and 2 thyroid-specific genes (thyroglobulin [Tg] and TSH receptor [TSHR]) (15) (reviewed in (16)). So far, only common variants have been identified in these 6 gene-loci, and these common variants, due to their small effect size, explain only part of the overall heritability of AITD (17). Therefore, we still have no explanation for the “missing heritability” in AITD, that is, what other genetic factors predispose to AITD. One potential explanation is the existence of rare variants in multiple genes. Indeed, rare variants (defined as having minor allele frequency <1%) have been proposed to account for some of the “missing heritability” in complex diseases (17-20). However, identifying rare variants in the general population by case-control studies is challenging, as it is difficult to determine whether a newly discovered variant is a true variant or a sequencing artifact; moreover, it is even more challenging to determine which of the true rare variants identified in affected individuals are causative. Unlike case-control studies, family-based studies are ideal for identifying causative rare variants because they co-segregate with disease (ie, are genetically linked) within families (21-25). Moreover, selecting families who are known to be linked with a specific gene-locus for deep sequencing increases the likelihood of identifying causative variants because, by virtue of showing linkage to the disease, the region has been proven to harbor a susceptibility gene (22).
The aim of this study was to identify rare variants that are associated with AITD using deep sequencing of known AITD-linked loci. We performed deep sequencing of 3 loci that we have previously shown to be linked with AITD: a locus on chromosome 14q linked with GD, a locus on 12q linked with HT, and a locus on 10q linked with both GD and HT (ie, AITD) (26, 27). We sequenced these loci in a dataset of AITD families, and report here the identification of new rare variants in these loci that are associated with AITD.
Methods
Families
The project was approved by the Albert Einstein College of Medicine institutional review board. For this study we analyzed 34 families, which represents a subset of a dataset of families previously described in detail (26). These 34 families were selected because they showed strong linkage to the 3 loci sequenced (10q, 12q, and 14q). All families analyzed were multiplex for AITD (ie, they had at least 2 affected individuals). The 34 families comprised 196 individuals and each family had on the average 5.8 members. Of the 196 family members, 42 (21.4%) had GD; 49 (25%) had HT; 104 (53.1%) were unaffected; and 1 member (0.5%) had unknown phenotype. GD was diagnosed by (1) clinical and biochemical thyrotoxicosis requiring treatment; (2) positive TSH receptor antibodies; and/or (3) diffusely increased radioactive iodine uptake in the thyroid gland. HT was diagnosed by (1) clinical and biochemical hypothyroidism requiring levothyroxine replacement; and (2) positive thyroid peroxidase (TPO) antibodies. None of the family members had type 1 diabetes, Addison’s disease, or other autoimmune diseases. Each participant gave a written informed consent before participating.
AITD-Linked Loci Information
Based on our previous linkage studies, we selected 3 loci to be sequenced: the chromosome 10q locus that is linked with AITD (ie, linked with both GD and HT), the 14q locus that is linked with GD only, and the 12q locus that is linked with HT only (26). We determined the size and location of the regions to be sequenced based on our multipoint linkage analysis that defined the microsatellite boundaries of the linked loci (26). Table 1 shows the boundaries of the loci we sequenced.
Table 1.
Loci boundaries (hg38 coordinates)
| Linked phenotype | Locus | Microsatellite boundaries | Base boundaries (hg38 coordinates) |
|---|---|---|---|
| AITD | 10q | D10S1659 – D10S1743 | chr10:57,302,483 – 65,667,738 |
| GD | 14q | D14S251 – D14S1000 | chr14:70,658,864 – 81,639,922 |
| HT | 12q | D12S88 – D12S346 | chr12:85,977,605 – 99,134,752 |
Sequencing of the AITD-Linked Loci
Library preparation was performed using the KAPA HTP Library Preparation Kit KR0426-v4.15 (Kappa Biosystems, Wilmington, MA, USA) according to the manufacturer’s instructions, and the target regions were captured using a custom design with the SeqCap EZ Library SR kit version 5.0 (Roche NimbleGen, Inc, Madison, WI, USA). Sequencing was performed using the Illumina NextSeq 500 Sequencing System (Illumina, San Diego, CA, USA). Sequenced reads were mapped to the 10q, 12q, and 14q genomic regions and variants were called based on mismatch analyses with the reference genome (hg38 assembly). Known single nucleotide polymorphisms (SNPs) were identified and removed, and only new single nucleotide variants were analyzed for association with disease.
Transmission Disequilibrium Test for Association
To analyze for association of rare variants with disease we performed family-based association analyses using the transmission disequilibrium test (TDT). The TDT test was performed using PLINK (28). The TDT compares the rate of transmission of parental variants to affected offspring with the rate expected if there was no preferential transmission (29). A significantly increased transmission of a variant to affected offspring indicates a risk variant that is associated with the disease; conversely, a significantly decreased transmission of a variant to affected offspring indicates a protective associated allele (30). Genotypes of single nucleotide variants were entered into PLINK, and the TDT test was performed assuming 3 affectedness statuses - HT, GD, or AITD (in these families both GD and HT were considered affected). The TDT was performed using the PLINK package (v1.07) (28) (http://zzz.bwh.harvard.edu/plink/fanal.shtml), and SNPs found to be significantly associated with one of the 3 phenotypes (P < 0.05) were then annotated with the biologically and clinically relevant information from various databases.
Results
Characteristics of the AITD Families
The characteristics of the AITD families we studied are summarized in Table 2. Of the 34 families; 10 (29.4%) had only GD-affected members, 9 (26.5%) had only HT-affected members; and 15 (44.1%) had both GD- and HT-affected members. All families were linked with at least one of the 3 loci with some families linked with more than one locus; 19 families (55.9%) showed linkage of AITD with 10q, 14 families (41.2%) showed linkage of HT with 12q, and 13 families (38.2%) showed linkage of GD with 14q. As expected, among GD-affected individuals (n = 42) 31 (73.8%) were female, and among HT-affected individuals (n = 49) 44 (89.8%) were female, consistent with the well-known female predisposition of AITD. In contrast, among unaffected family members (n = 104) 52 (50%) were female.
Table 2.
Characteristics of the families analyzed in the study
| Group | No. | % |
|---|---|---|
| Families: | 34 | 100 |
| GD-only families | 10 | 29.4 |
| HT-only families | 9 | 26.5 |
| Mixed (GD + HT) families | 15 | 44.1 |
| Families linked with 10qa | 19 | 55.9 |
| Families linked with 12q | 14 | 41.2 |
| Families linked with 14q | 13 | 38.2 |
| Individuals: | 196 | 100 |
| Affected individuals: | 91 | 46.4b |
| GD | 42 | 21.4b |
| • Female | 31 | 73.8c |
| • Male | 11 | 26.2c |
| HT | 49 | 25.0b |
| • Female | 44 | 89.8d |
| • Male | 5 | 10.2d |
| Unaffected individuals: | 104f | 53.1b |
| • Female | 52 | 50.0e |
| • Male | 52 | 50.0e |
a Note that the same family could be linked with more than one locus
b Percentage of total individuals
c Percentage of GD-affected individuals
d Percentage of HT-affected individuals
e Percentage of unaffected individuals
f One individual (female) had unknown phenotype (0.5%)
Rare Variant Sequencing of AITD Genes on Chromosome 10q
Deep sequencing of the chromosome 10q locus, which was previously shown to be linked with both GD and HT (AITD) (26, 27), identified 13 rare variants that were strongly associated with AITD (Table 3). In Table 3, the column labeled “T” shows the number of transmissions of the rare variant to an affected offspring, while the column labeled “U” indicates the number of times the rare variant was not transmitted to affected offspring. If there is no association of the rare variant with disease the rates of transmission and untransmission to affected offspring should be equal since the probabilities of transmission and untransmission are both 0.5. A significantly increased transmission rate indicates a rare variant that confers risk for disease. In contrast, a significantly increased untransmission rate indicates a protective rare variant. Intriguingly, all 13 rare variants associated with AITD on chromosome 10q were protective. Remarkably, all these variants were located in the gene inositol polyphosphate multikinase (IPMK).
Table 3.
Rare variants identified in the chromosome 10q locus, linked with AITD (GD + HT)
| SNV | Position | T | U | P value | Transcript | Location of SNV | Common allele | Rare variant |
|---|---|---|---|---|---|---|---|---|
| AITD10.1001 | 58,194,262 | 0 | 9 | 0.0027 | NM_152230:IPMK | exonic | C | T |
| AITD10.1002 | 58,191,598 | 0 | 6 | 0.01431 | NM_152230:IPMK | exonic | T | A |
| AITD10.1003 | 58,192,372 | 0 | 6 | 0.01431 | NM_152230:IPMK | exonic | A | G |
| AITD10.1004 | 58,192,884 | 0 | 6 | 0.01431 | NM_152230:IPMK | exonic | A | G |
| AITD10.1005 | 58,193,478 | 0 | 6 | 0.01431 | NM_152230:IPMK | exonic | C | A |
| AITD10.1006 | 58,195,625 | 0 | 6 | 0.01431 | NM_152230:IPMK | exonic | G | A |
| AITD10.1007 | 58,193,779 | 1 | 8 | 0.01963 | NM_152230:IPMK | exonic | C | T |
| AITD10.1008 | 58,193,436 | 2 | 10 | 0.02092 | NM_152230:IPMK | exonic | C | T |
| AITD10.1009 | 58,191,617 | 0 | 5 | 0.02535 | NM_152230:IPMK | exonic | A | G |
| AITD10.1010 | 58,191,894 | 0 | 5 | 0.02535 | NM_152230:IPMK | exonic | T | C |
| AITD10.1011 | 58,195,907 | 0 | 5 | 0.02535 | NM_152230:IPMK | exonic | G | _ |
| AITD10.1012 | 58,195,589 | 1 | 7 | 0.03389 | NM_152230:IPMK | exonic | GT | CC |
| AITD10.1013 | 58,195,995 | 1 | 7 | 0.03389 | NM_152230:IPMK | exonic | A | G |
Abbreviations: SNV, single nucleotide variant; T, transmitted to affected offspring; U, untransmitted to affected offspring.
Rare Variant Sequencing of GD Genes on Chromosome 14q
Deep sequencing of the chromosome 14q locus, which was previously shown to be linked with GD (26, 27), identified 2 rare variants that were strongly associated with GD (Table 4). Similar to the rare variants associated with AITD, both 14q variants showed a significantly decreased rate of transmission, suggesting that they were protective. These 2 rare variants are located in the genes dihydrolipoamide S-succinyltransferase (DLST) and zinc-finger FYVE domain-containing protein (ZFYVE1), respectively.
Table 4.
Rare variants identified in the chromosome 14q locus, linked with GD
| SNV | Position | T | U | P value | Transcript | Location of SNV | Common allele | Rare variant |
|---|---|---|---|---|---|---|---|---|
| GD14.1001 | 74,902,807 | 0 | 6 | 0.01431 | NM_001933:DLST | exonic | G | A |
| GD14.1002 | 72,969,587 | 0 | 5 | 0.02535 | NM_178441:ZFYVE1 | exonic | GG | CA |
Abbreviations: SNV, single nucleotide variant; T, transmitted to affected offspring; U, untransmitted to affected offspring.
Rare Variant Sequencing of HT Genes on Chromosome 12q
Deep sequencing of the chromosome 12q locus, which was previously shown to be linked with HT (26, 27), identified 3 rare variants that were associated with HT (Table 5). One of the rare variants is a risk variant, while the other 2 are protective. The risk A/C variant is located in phosphoglycerate mutase 1 pseudogene 5 (PGAM1P5). The 2 protective C/A and G/A variants are located in the methionine aminopeptidase 2 (METAP2) gene and LOC105369879 locus, respectively.
Table 5.
Rare variants identified in the chromosome 12q locus, linked with HT
| SNV | Position | T | U | P value | Transcript | Location of SNV | Common allele | Rare variant |
|---|---|---|---|---|---|---|---|---|
| HT12.1001 | 95,672,340 | 5 | 0 | 0.02535 | NR_077225:PGAM1P5* | exonic | A | C |
| HT12.1002 | 87,330,962 | 0 | 4 | 0.0455 | NR_135020:LOC105369879 | exonic | C | A |
| HT12.1003 | 95,514,978 | 0 | 4 | 0.0455 | NR_133673:METAP2 | exonic | G | A |
Abbreviations: SNV, single nucleotide variant; T, transmitted to affected offspring; U, untransmitted to affected offspring.
Discussion
Significant progress has been made in the past 3 decades in our understanding of the genetics of AITD. However, so far, all variants that have been identified to be associated with AITD are common variants (with minor allele frequency >5%). Here, for the first time, we report the mapping of rare variants that contribute to the genetic susceptibility to AITD. Our strategy was to analyze families in which GD and HT clustered (familial AITD) and which previously showed strong linkage with 3 loci on chromosomes 10q (AITD locus), 12q (HT locus), and 14q (GD locus) (26). This strategy proved successful, but our results are currently only applicable to familial AITD and it remains to be determined if they can also be extended to sporadic AITD. We have previously reported that common variants that are associated with GD had similar effects in familial and sporadic GD (31). However, it is not known whether the same applies for the new rare variants we identified, and it is possible that rare variants are unique to familial AITD. It also remains to be determined whether the rare variants we identified in our AITD families are also associated with other autoimmune diseases or whether they are unique to AITD.
Interestingly, most of the rare variants we identified were protective (Tables 3-5). Indeed, protective rare variants have been previously reported in several autoimmune diseases, such as inflammatory bowel disease (32), rheumatoid arthritis (33), psoriasis (34), and systemic lupus erythematosus (35). Several mechanisms can explain how a protective rare variant may be associated with disease. For example, a protective rare variant may protect individuals in a family with AITD who inherit common risk variants. Alternatively, rare variants may protect from environmental triggers of disease. In the case of autoimmunity, it has been suggested that the common variants may increase immune responsiveness to infections but at the expense of increased susceptibility to autoimmunity, and that the rare protective variants may decrease immune responsiveness, thus reducing the risk for autoimmunity (34, 35). The clinical relevance of the protective rare variants we identified is unclear. One could theoretically sequence these rare variants in offspring of families with familial AITD, and if the offspring carry the protective rare variants, that would indicate protection from developing AITD. However, this is not practical or useful in clinical practice. The main significance of the rare variants we identified is that they give us insight into the mechanisms underlying the development of AITD and identify potential new drug targets.
Our deep sequencing of 3 loci that were previously shown to be linked with AITD, GD, and HT, identified rare variants that were associated with each of these 3 phenotypes. The 10q locus is linked with AITD and we identified 13 AITD-associated rare variants in this locus that were all located within the IPMK gene. The 14q locus is linked with GD and we identified 2 GD-associated rare variants in this locus that were located within the dihydrolipoamide S-succinyltransferase (DLST) and zinc-finger FYVE domain-containing protein (ZFYVE1) genes, respectively. Similarly, we identified 3 rare variants that were associated with HT and were located at the HT-linked 12q locus within the phosphoglycerate mutase 1 pseudogene 5 (PGAM1P5), LOC105369879, and methionine aminopeptidase 2 (METAP2) genes, respectively.
The IPMK gene on 10q harbored 13 rare variants that were associated with AITD. IPMK is an enzyme that plays a catalytic role in inositol phosphate metabolism (36, 37). In addition, IPMK also has a noncatalytic role in regulating major signaling networks (38, 39), such as mediating the activation of mammalian target of rapamycin (mTOR) in response to essential amino acids (40). One of the noncatalytic roles of IPMK is enhancing toll-like receptor (TLR) signaling by stabilizing TRAF6 in macrophages, suggesting a function of IPMK in TLR-induced innate immunity (41). Furthermore, IPMK was found to have a noncatalytic role in activation of autophagy and liver regeneration (42), and potentially as a therapeutic target for autophagy-related diseases. Autophagy has been implicated as an important mechanism in autoimmunity (43-45), including thyroid autoimmunity (46-48). One study from our group detected increased expression levels of autophagy-related gene 5 (ATG5) and LC3 (which are key genes in autophagy pathways) in thyroid tissues from AITD patients (49). It is possible that activation of autophagy by several mechanisms including IPMK can lead to lysosomal degradation of thyroglobulin into pathogenic peptides, which may be presented by antigen presenting cells and trigger autoimmune thyroiditis (49).
The rare variants that were associated with GD on 14q were located within the DLST and ZFYVE1 genes. DLST encodes a mitochondrial protein that belongs to the 2-oxoacid dehydrogenase family. It is part of the E2 component of the 2-oxoglutarate hydrogenase complex that catalyzes the overall conversion of 2-oxoglutarate to succinyl-CoA and CO2 (50). DLST showed the strongest association with GD, although it does not have any known immune functions and has not been previously associated with autoimmunity. In contrast, ZFYVE1, which was also strongly associated with GD, has been shown to have an important role in the TLR-mediated innate immunity and inflammatory responses by promoting the ligand binding of TLR3 (51). Interestingly, similarly to IPMK, which plays a role in activation of autophagy, ZFYVE1 is involved in autophagosome biogenesis and initiation of autophagy (52-54). Thus, the new variants we identified within the IPMK and ZFYVE1 genes may be associated with AITD by modulating autophagic pathways.
Rare variants located within PGAM1P5, LOC105369879, and METAP2, located on 12q, showed association with HT. PGAM1P5 is a pseudogene (www.ncbi.nlm.nih.gov/gene/?term=PGAM1P5), and therefore, its importance in HT is unclear. LOC105369879 is a noncoding RNA gene and its function is not known (https://www.ncbi.nlm.nih.gov/gene/?term=LOC105369879). However, the METAP2 gene, which also showed association with HT, is an enzyme that regulates cellular protein synthesis and is highly expressed in T cells. An inhibitor of MetAP2, Lodamin, was found to suppress T cell receptor–mediated T cell proliferation and reduced proinflammatory cytokine production in autoimmune uveitis (55). Given that HT is a T cell–mediated disease these data suggest a role for MetAP2 in the etiology of HT. PPI-2458, another inhibitor of MetAP2, was found to ameliorate the pathophysiological disease process of rheumatoid arthritis (56), suggesting an important role of MetAP2 in the pathogenesis of rheumatoid arthritis. In another study, MetAP2 was found to be expressed in germinal center B cells (57). Blocking MetAP2 by PPI-2458 was also found to inhibit differentiation of B cells into plasma cells, as well as disrupting germinal center formation (56). This suggests a role of MetAP2 in regulating B-cell function, too.
In summary, by performing deep sequencing we identified rare variants that were associated with familial AITD. The rare variants we identified were located within genes that play important roles in immune pathways and may play a role in autoimmunity in general. Our data demonstrate that, in addition to common variants, rare variants also play an important role in the development of thyroid autoimmunity.
Acknowledgments
This work was supported in part by grants DK067555 and DK073681 from National Institute of Diabetes and Digestive and Kidney Diseases (to Y.T.), and an American Thyroid Association Grant 2019 (to C.W.L.).
Glossary
Abbreviations
- AITD
autoimmune thyroid disease(s)
- DLST
dihydrolipoamide S-succinyltransferase
- GD
Graves’ disease
- HT
Hasimoto’s thyroiditis
- IPMK
inositol polyphosphate multikinase
- METAP2
methionine aminopeptidase 2
- PGAM1P5
phosphoglycerate mutase 1 pseudogene 5
- SNP
single nucleotide polymorphism
- TDT
transmission disequilibrium test
- TLR
toll-like receptor
- ZFYVE1
zinc-finger FYVE domain-containing protein
Additional Information
Disclosures: Dr. Tomer declares that he has submitted 3 patent disclosures that are not related to the content of this manuscript. Dr. Tomer was previously (1/2015 to 6/2017) the principal investigator on a basic research project jointly funded by the Juvenile Diabetes Research Foundation and Pfizer. The current manuscript is not related to that research project. All other authors have no potential conflict of interest to declare.
Data Availability
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
