Missense splice variant (g.20746A>G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions

  • Agnieszka Bińczak-Kuleta Department of Clinical and Molecular Biochemistry, Pomeranian Medical University in Szczecin, Szczecin, Poland
  • Aleksander Szwed Department of Orthopedics and Child Traumatology, Pomeranian Medical University in Szczecin, Szczecin, Poland
  • Mark R. Walter Department of Microbiology, Bevill Biomedical Research Building, University of Alabama at Birmingham, Birmingham, AL, USA
  • Maciej Kołban Department of Orthopedics and Child Traumatology, Pomeranian Medical University in Szczecin, Szczecin, Poland
  • Andrzej Ciechanowicz Department of Clinical and Molecular Biochemistry, Pomeranian Medical University in Szczecin, Szczecin, Poland
  • Jeremy S. C. Clark ​Department of Clinical and Molecular Biochemistry, Pomeranian Medical University in Szczecin, Szczecin, Poland
Keywords: Bacilli strain Calmette-Guerin, environmental mycobacteria, interferon gamma receptor 1, Mendelian susceptibility to mycobacterial disease, Mycobacterium bovis bacillus Calmette-Guérin, osteoarticular lesions

Abstract

Previously, dominant partial interferon-gamma receptor 1 (IFN-g-R1) susceptibility to environmental mycobacteria was found with IFNGR1 deletions or premature stop. Our aim was to search for IFNGR1 variants in patients with mycobacterial osteoarticular lesions. Biopsies from the patients were examined for acid-fast bacilli, inflammatory cell infiltration, and mycobacterial niacin. Mycobacterial rRNA was analyzed using a target-amplified rRNA probe test. Peripheral-blood-leukocyte genomic DNA was isolated from 19 patients using the QIAamp DNA Mini Kit, and all IFNGR1 exons were sequenced using an ABIPRISM 3130 device. After the discovery of an exon 5 variant, a Polish newborn population sample (n = 100) was assayed for the discovered variant. Splice sites and putative amino acid interactions were analyzed. All patients tested were positive for mycobacteria; one was heterozygous for the IFNGR1 exon 5 single-nucleotide-missense substitution (g.20746A>G, p.Ile183Val). No other variant was found. The splice analysis indicated the creation of an exonic splicing silencer, and alternatively, molecular graphics indicated that the p.Ile183Val might alter beta-strand packing (loss of van der Waals contacts; Val183/Pro205), possibly altering the IFN-g-R1/IFN-g-R2 interaction. The probability of non-deleterious variant was estimated as <10%. Heterozygous IFNGR1:p.Ile183Val (frequency 0.003%) was found to be coincidental with mycobacterial osteomyelitis. The small amount of variation detected in the patients with osteoarticular lesions indicates that screens should not yet be restricted: Intronic variants should be analyzed as well as the other genes affecting Type 1 T-helper-cell-mediated immunity.

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Author Biographies

Agnieszka Bińczak-Kuleta, Department of Clinical and Molecular Biochemistry, Pomeranian Medical University in Szczecin, Szczecin, Poland

Department of Clinical and Molecular Biochemistry

Aleksander Szwed, Department of Orthopedics and Child Traumatology, Pomeranian Medical University in Szczecin, Szczecin, Poland

Department of Orthopedics and Child Traumatology

Mark R. Walter, Department of Microbiology, Bevill Biomedical Research Building, University of Alabama at Birmingham, Birmingham, AL, USA
Department of Microbiology
Maciej Kołban, Department of Orthopedics and Child Traumatology, Pomeranian Medical University in Szczecin, Szczecin, Poland

Department of Orthopedics and Child Traumatology

Andrzej Ciechanowicz, Department of Clinical and Molecular Biochemistry, Pomeranian Medical University in Szczecin, Szczecin, Poland

Department of Clinical and Molecular Biochemistry

Jeremy S. C. Clark, ​Department of Clinical and Molecular Biochemistry, Pomeranian Medical University in Szczecin, Szczecin, Poland

Department of Clinical and Molecular Biochemistry

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Missense splice variant (g.20746A>G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions
Published
2016-08-02
How to Cite
1.
Bińczak-Kuleta A, Szwed A, Walter MR, Kołban M, Ciechanowicz A, Clark JSC. Missense splice variant (g.20746A&gt;G, p.Ile183Val) of interferon gamma receptor 1 (IFNGR1) coincidental with mycobacterial osteomyelitis - a screen of osteoarticular lesions. Bosn J of Basic Med Sci [Internet]. 2016Aug.2 [cited 2021Apr.22];16(3):215-21. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/1232
Section
Molecular Biology

INTRODUCTION

Susceptibilities to mycobacterial osteoarticular infections are thought to result from disorders in Type 1 T-helper-cell-mediated immune inflammation, resulting in deficient production and/or action of interleukin 12 (IL12) and interferon gamma (IFN-γ) [1]. Genetic disorders associated with inborn errors in IFN-γ-dependent immunity include those which impair production of IFN-γ (IL12B, IL12RB1, IRF8, ISG15, NEMO) and those which impair response to IFN-γ (IFNGR1, IFNGR2, STAT1, IRF8, CYBB) [2]. Note that, perhaps “Familial” (rather than “Mendelian”) “susceptibility to mycobacterial disease” might be a more appropriate term to describe many of these disorders, as “most of these inborn errors do not show complete clinical penetrance for the case-definition phenotype of Mendelian susceptibility to mycobacterial diseases” [2].

Variants in IFNGR1 often result in susceptibility to environmental mycobacteria (e.g., Mycobacterium avium; immunodeficiency 27a), M. bovis bacilli strain Calmette-Guerin (BCG), as well as to Helicobacter pylori [3], Salmonella (~5%), Listeria, herpes virus, cytomegalovirus, and Histoplasma [4]. Further, diseases/infections associated with missense variants in IFNGR1 include Epstein-Barr virus infection; disseminated M. tuberculosis infection (p.Gly219Arg [5]); allergies [6], dermatitis, and lymphadenitis; and variants of IFNGR1 are thought to be associated with susceptibility to Schistosoma mansori and M. tuberculosis [7]. Promoter variants have been associated with atopic cataracts, protection against cerebral malaria [8], protection from tuberculosis [9], and with the clinical outcome of HBV infection in Chinese adults [10].

The signaling complex which binds IFN-γ is formed from a dimer of IFN-γ-receptor 1 (IFN-γ-R1) which then binds two IFN-γ-R2 molecules (note that the official short names for these proteins, P15260 and P38484, respectively, both have two hyphens according to UniProtKB, e.g., http://www.uniprot.org/uniprot/P15260); IFN-γ-R1 also interacts with JAK1, JAK2, and STAT1 (minimum evidence level 2 [11]). On binding to the signaling complex, IFN-γ induces antiviral activity, increases expression of the class II major histocompatibility complex, and causes B cell maturation and release of mediators of inflammation ([12]; IFN-γ is ubiquitously expressed and highly expressed in plasma/pancreas). IFN-γ itself is highly conserved: No variants of IFN-γ have ever been found [13]. It is, therefore, perhaps not surprising that the receptor gene IFNGR1 is also highly conserved, illustrated by the fact that only one missense polymorphism (defined as having a frequency >1% worldwide; p.Leu467Pro; frequency 5%) was found by the Exome Aggregation Consortium (ExAC) in 60,706 unrelated individuals [14]. In this study, three missense low-frequency (defined as frequency <1%) variants were found with world frequencies 0.1-1%: p.Val14Met, p.Gly180Arg, and p.His335Pro; and nine with 0.01-0.1%: p.Met11Leu, p.Thr31Ile, p.Val46Ile, p.Val61Ile, p.Asn79Ser, p.Pro148Arg, p.Glu197Lys, p.Thr189Lys, and p.Pro431Leu. The variant subject of this paper, p.Ile183Val, was found at 0.003%, only in the European (non-Finnish) population.

Tolerance to missense variants with frequencies <1% should be questioned, even if predicted to be “benign” or “tolerated” in silico, because of the likelihood of negative selection maintaining low frequency. Note that even the polymorphism p.Leu467Pro (which is probably a marker for another frequent causal variant [15]) is associated with allergies [6] and Helicobacter susceptibility [3] (p.Leu467Pro is listed in this reference as Leu450Pro, i.e., minus the signal peptide).

Two of the three missense low-frequency variants found with the highest frequency >0.1% have been found to be associated with deleterious effects: p.His335Pro and p.Val14Met. Although the p.His335Pro transcript assessment by ExAC shows polyphen output “benign” (“most likely lacking any phenotypic effect”) and SIFT output “tolerated” (“not deleterious”), p.His335Pro is associated with susceptibility to H. pylori infection [3]. p.Val14Met (polyphen: “Possibly-damaging,” SIFT: “Tolerated”) is associated with the greatest risk for development of systemic lupus erythematosus in the Japanese population in heterozygous state [12]. From those with a frequency 0.1-0.01%, p.Val61Glu was found in a compound heterozygote together with a deletion, resulting in recessive complete susceptibility to mycobacterial infection ([4]; note here, this variant was incorrectly described as p.Val61Ile and corrected in the Leiden Open Variation Database [LOVD] to p.Val61Glu).

The LOVD (www.lovd.nl/IFNGR1; Fokkema et al. [16]) listed 9 missense low-frequency variants of IFNGR1 associated with disease (accessed November 2015; Table 1). All of these were associated with autosomal recessive immunodeficiency-27A, with susceptibility to mycobacteria (most often to M. avium).

IFNGR1 variants associated with mycobacterial infections were extensively studied by Dorman et al. [4] using a large cohort of patients worldwide, and clinical features of recessive and dominant IFNGR1 disorders were assessed. All dominant disorders found were associated with either c.819_822del, 818delT, or 832G>T (p.Glu278*); all producing truncated IFN-γ-R1 proteins. Most (79%) of the dominant partial patients had mycobacterial infections; one-third had osteomyelitis with no other organs apparently affected.

TABLE 1: Deleterious (and putative deleterious) missense variants of IFNGR1 with estimated frequencies and association with disease

The aim of our study was to search for IFNGR1 variants by sequencing all exons, in a cohort of Polish patients with osteoarticular lesions.

MATERIALS AND METHODS

Patients

From child patients hospitalized at the Clinic of Orthopedics and Child Traumatology at the Pomeranian Medical University in Szczecin, Poland, between 2005 and 2010, 20 consecutive patients (11 girls; 9 boys; mean admission age 12 years; age range 7-17 years) were diagnosed with osteoarticular lesions and enrolled at a children’s clinic. Written informed consent was obtained from all legal guardians. The study protocol and DNA screen were approved by the Pomeranian Medical University Bioethics Committee in compliance with the Helsinki Declaration (2013 revision). Note that all patients had been inoculated using the BCG vaccine before age one.

Methods

Biopsies (open or needle biopsies from bone or arthroscopic synovial biopsies) were examined by a histopathologist for inflammatory cell infiltration, acid-fast bacilli, and with the Niacin test. A target-amplified mycobacterial rRNA probe test (Amplified MTD Test, Hologic, Wiesbaden, Germany) was used. One patient was omitted from the sequencing study, but at a later date, mycolic acid high-performance liquid chromatography was used to identify M. fortuitum in this patient. All patients received multiple drug anti-mycobacterial treatment with good result (mean observation time: 4 years).

Peripheral-blood-leukocyte genomic DNA from 19 patients was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Using primer selection software (Lasergene v8.0, DNASTAR, Madison, Wisconsin, USA), primers were designed for all exons of IFNGR1:GenBank: NG_007394 (chromosome 6q23); sequencing was carried out using ABIPRISM 3130, Sequencing Analysis Software v5.4 (Applied Biosystems, Life Technologies Polska, Warsaw, Poland).

Following the discovery of an exon 5 variant, a population group (100 Polish newborns randomly chosen from the Newborn DNA Repository at the Department of Clinical and Molecular Biochemistry at the Pomeranian Medical University in Szczecin [17]) was screened by sequencing exon 5.

Database search included the ExAC Browser [18]; Leiden Open Variation Database (www.lovd.nl/IFNGR1; [16]); UCSC Genome Browser [19]; Seattle SNPs [20]; NCBI ClinVar database [21]; and ENSEMBL [22]. Probability of finding a non-deleterious missense variant (n = 87), i.e., not including deletions, frameshifts, stop codons or intronic splice region variants, at random in a European (non-Finnish) sample was estimated from ExAC Browser data by 1 - ((product (1 - frequency of each variant)) ^ 38) for 38 haploid genotypes (i.e., 19 patients).

An open-source molecular graphics system (Pymol, version 1.3rl [23]) was used to visualize amino acid interactions. Polyphen and SIFT designations for the variant found were taken from ENSEMBL. Splice variant analysis used the Human Splicing Finder [24].

RESULTS

All patients screened (n = 20) suffered from osteoarticular pathologies (Table 2). For all patients the analysis of biopsy specimens using the target-amplified nucleic acid probe test detected mycobacterial rRNA, indicating species from the M. tuberculosis complex (i.e., either M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. canetti, or M. pinnipedii). One patient (number 1, Table 2) tested positive with niacin and acid-fast bacilli tests, suggesting M. tuberculosis itself. The histopathological examination for acid-fast bacilli or inflammatory cell infiltration gave only several positive results (Table 2), indicating perhaps that in most patients mycobacterial infections were latent.

TABLE 2: Characteristics and treatment of the patients with osteoarticular lesions whose IFNGR1 exons were sequenced

The analysis of sequencing chromatograms (n = 19) identified the missense low-frequency variant IFNGR1:GenBank: NG_007394:g.20746A>G (corresponding to GenBank: NM_000416.2:c.547A>G; dbSNP: rs752756474) in exon 5 from one allele (male; patient number 12; admission age 17 years; Figure 1). This patient had: The right 2nd metatarsal bone inflamed (Figure 2); no other inflammatory lesions; no apparent autoimmune disease; was well-nourished; pain increased over 6 months with diagnosis of a fracture.

FIGURE 1: Chromatogram showing the interferon gamma receptor 1 (IFNGR1):p.Ile183Val low-frequency variant (antisense sequence of part of exon 5). Arrow indicates the NG_007394:g20746A>G (p.Ile183Val) variant.
FIGURE 2: Mycobacterial lesion. Right foot showing inflammatory lesion (X) of the 2nd metatarsal bone of the patient 12 (in which the low-frequency variant interferon gamma receptor 1 (IFNGR1):p.Ile183Val was identified). White bar length = 2.5 cm.

The low-frequency variant gave ATC>GTC (p.Ile183Val) at codon 183. No other variants were found in the patients or in the population group subsequently analyzed for this low-frequency variant.

This low-frequency variant was previously found in two European (non-Finnish) people from worldwide exome analysis of ~61,000 unrelated individuals [18], i.e., at 0.003% frequency. IFNGR1 is highly conserved, and it is possible to estimate the probability of finding a non-deleterious variant at random from the IFNGR1 entry in the ExAC browser [18]. Note that this analysis does not include other categories of mutation, e.g., intronic splice variants, frameshifts, stop codons, etc., and that no other missense exonic splice variants have been found. This estimate for finding any European (non-Finnish), non-deleterious, missense exonic variant (n = 87) at random in 19 patients was 0.095. However, we know that it is likely that many variants are held at low frequencies by negative selection (it is also likely that so-far undetected variants also have very low frequency), and many have not been investigated sufficiently to confirm the lack of disease association, and this is therefore likely an overestimate. We, therefore, submit that the p.Ile183Val low-frequency variant, with 0.003% frequency, is here associated with mycobacterial disease.

Polyphen and SIFT analyses for this variant gave “tolerated” and “benign” respectively (ENSEMBL). However, molecular graphics (Pymol [23]; Figure 3) indicated that, although structural changes would appear to be minor, p.Ile183Val could alter beta-strand packing due to loss of van der Waals contacts between Val183 and Pro205. It should also be noted that the variant is found at the first nucleotide of exon 5, and is, therefore, the only missense exonic splice variant to have been identified. Analysis via the Human Splicing Finder indicated that the variant alters an exonic splicing enhancer and creates an exonic splicing silencer, thereby potentially altering splicing - perhaps more likely for a possible deleterious effect.

FIGURE 3: Molecular interactions at site of interferon gamma receptor 1 (IFNGR1):p.Ile183Val low-frequency variant. (A) Ribbon diagram of the IFN-γ/IFN-γ-R1 complex. Ile183 is colored magenta; positioned near the putative binding site of the IFN-γ-R2 chain, (B) Close-up view of residues surrounding Ile183 in the crystal structure of IFN-γ-R1. The Ile183Val low-frequency variant could alter the packing of the beta-strands due to loss of van der Waals contacts between Val183 and Pro205 (black lines:p: 0.31 nm; q: 0.55 nm; r: 0.34 nm), possibly altering interaction with IFN-γ-R2.

DISCUSSION

Our study has identified a new low-frequency variant IFNGR1:c.547A>G (p.Ile183Val) found with mycobacterial osteomyelitis. The infective agent was very likely environmental mycobacteria or possibly M. bovis BCG (from tuberculosis vaccination).

While it was not possible to eliminate the possibility of mere coincidence of a non-deleterious variant with this disease, the chances of this are low (<10%), and note that, the low-frequency variant detected is very rare (with 0.003% world frequency) and might be deleterious. We therefore feel that, together with the creation of an exonic splicing silencer (or the loss of van der Waals contacts) and with the patient characteristics, this result should be presented to the scientific community. This is the first case of a non-synonymous exonic splice variant to be found, and would be the first case in which a missense low-frequency variant (rather than a deletion/premature stop codon) is associated with dominant susceptibility to mycobacterial osteomyelitis.

The limitations of the study were as follows:

  1. Biochemical data were not, unfortunately, obtained which could have provided evidence that disruption in IFN-γ-R1 action was the cause of susceptibility to mycobacteria.

  2. Only one case was found, in which the IFNGR1:c.547A>G variant was associated with mycobacterial susceptibility.

  3. The screen of the patients with osteoarticular lesions did not include adults (only children were enrolled at the clinic).

It is of some significance that our study, with all exons of IFNGR1 sequenced, did not find any other low-frequency variant previously associated with mycobacterial infection. A similar study in 93 South Korean patients [25] also did not identify previously reported low-frequency variants in IL12RB1 and IFNGR1, found with mycobacterial infection, despite sequencing all exons (although further changes: Three polymorphisms, one silent and two missense low-frequency variants were identified). As mentioned in the introduction, this indicates that further genes involved in the production or response to IFN-γ should be analyzed, and that, although the study of IFNGR1 and mycobacterial infection by Dorman et al. [4] was very comprehensive, it was not complete.

It is, therefore, of some importance that screening groups do not narrow the search for IFNGR1 low-frequency variants prematurely where cost allows (e.g. Quispel et al. [26] only studied exons 2, 3, and 6). A similar observation was made by Fraser et al. [7], who suggested that sequencing all exons and introns of IFNGR1 might identify previously “unidentified allelic variations in the IFNGR1 gene (that) might elevate or decrease the risk in this ethnic population (Croatian), as a part of the multigenic predisposition to tuberculosis.” (Also note that, IFN-γ-R1 has at least 11 splice variants and thorough analyses of these, and the consequences of variation, has yet to be achieved, although it is thought that only one splice variant predominates).

Dorman et al. [4] identified 38 patients worldwide with a dominant partial deficiency in IFN-γ-R1, 22 patients with recessive complete IFN-γ-R1 deficiency, and 2 patients with recessive partial deficiency. The mean age of those with dominant partial deficiency was 13.4 years, and 32% of patients had mycobacterial infection of bone with no other organs affected (as in our patient number 12 with p.Ile183Val; although note the median number of bone lesions was five, whereas the patient 12 only had one bone lesion). With dominant partial deficiency, the penetrance by environmental mycobacterial disease was 45% by age 10 years in the Dorman et al. [4] study. Of importance here is that, in this study, all dominant partial cases were heterozygous for IFNGR1 low-frequency variants c.819_822del, 818delT, or 832G>T - all producing premature translation stop, resulting in IFN-γ-R1 proteins truncated in the extracellular domain; lacking intracellular domains, JAK1, and STAT1 binding sites. In addition, lack of a recycling motif leads to the accumulation of non-functional IFN-γ-R1 proteins (at ~10x normal levels) on the cell membrane, which bind IFN-γ to the same extent as reference IFN-γ-R1 proteins [13,26] but do not transmit a signal. This is thought to result in impedance of reference proteins produced by the other allele (especially as inactive dimers of affected plus reference proteins will be formed), giving diminished cellular responsiveness to IFN-γ, i.e. dominant partial deficiency results from a dominant negative effect rather than from decrease in gene dosage, and only very high administered concentrations of IFN-γ can compensate for this deficiency.

In dominant partial deficiency very low plasma levels of IFN-γ are found, as in healthy patients, probably indicating sufficient clearance via (lower) levels of receptor binding. In dominant partial deficiencies, IFN-γ does not accumulate to the high levels found in recessive complete deficiency, where there is impaired clearance together with lack of receptor binding [27]. In the latter cases, severe infection with mycobacterial diseases often occurs [13], with a familial preponderance for high IFN-γ levels [27], both of which are not common with dominant partial deficiencies.

Note that in our case, the affected amino acid (183) is found in the extracellular domain and the p.Ile183Val low-frequency variant could alter beta-strand packing due to loss of van der Waals contacts between Val183 and Pro205, altering interactions with IFN-γ-R2 (but this idea would have to be validated experimentally). It would be interesting to now carry out an experiment similar to that by van de Wetering et al. [15], in which the IFN-γ-responsive cell line THP-1 was transduced with IFNGR1 gene variant p.Tyr66Cys (previously a IFN-γ-R1 deficient cell line transduced with IFNGR1:p.Tyr66Cys had given no STAT1 phosphorylation). This resulted in the expression of p.Tyr66Cys at the cell surface but no hampering of action of the reference IFN-γ-R1 with no reduction in response to IFN-γ (with a dominant p.Ile183Val low-frequency variant this would be expected to reduce response to IFN-γ).

In contrast to recessive complete forms, dominant partial deficiency is seldom fatal [27] and can usually be treated, as in our case. Fieschi et al. [27] recommended that, along with genetic counseling, “undetectable or low levels of IFN-γ should lead to the child being treated with subcutaneous IFN-γ while searching for mild low-frequency variants of IFNGR1 and IFNGR2, or null low-frequency variants of IL12B and IL12RB1”. Patients with such low-frequency variants should not be vaccinated with the M. bovis BCG vaccine.

It remains to be seen whether the low-frequency variant p.Ile183Val affects IFN-γ-R1 function (and whether/how this low-frequency variant affects the various IFN-γ-R1 splice variants). The cause of mycobacterial osteoarticular infection in most patients in this study could not be explained by exonic low-frequency variants of IFNGR1, and therefore, the other genes mentioned earlier should also be analyzed in order to evaluate further mechanisms for susceptibility to mycobacterial osteoarticular infections.

CONCLUSION

  1. Coincidence of heterozygous low frequency (0.003%) variant IFNGR1:g.20746A>G (p.Ile183Val) was found with susceptibility to mycobacterial osteomyelitis. This is the first missense exonic splice variant to be found and also is the first time that mycobacterial osteomyelitis has been found together with an IFNGR1 missense variant in dominant mode. The probability of detecting a non-deleterious variant was estimated at <10%.

  2. Lack of other low-frequency variants found with all exons of IFNGR1 sequenced, in the screen of patients with osteoarticular lesions, indicates that screening should not yet be restricted to those IFNGR1 exons, in which deleterious low-frequency variants have so far been found; plus other genes involved in Type 1 T-helper-cell-mediated immune inflammation should also be analyzed.

DECLARATION OF INTERESTS

The authors declare no conflict of interests.

Acknowledgements

ACKNOWLEDGMENTS

This study was supported by Research Grant No. N N403 583338 from the Polish Ministry of Science and Higher Education. This funding body had no role in design, in the collection, analysis, and interpretation of data; in the writing of the manuscript and in the decision to submit the manuscript for publication.

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