We have described subjects, identified on the basis of a distinctive biochemical profile (elevated T4 and low selenoprotein levels), with compound heterozygous SECISBP2 defects that result in diminished synthesis of most known selenoproteins. The resulting phenotype is complex, but probably reflects 2 major pathogenic processes: first, tissue-specific effects caused by particular selenoprotein deficiencies in target organs; second, consequences of more generalized antioxidant selenoenzyme deficiencies with excess cellular ROS.
The testis is highly selenium enriched, with deficiency of this trace element in rodents being associated with infertility (25
). We have documented azoospermia with deficiency of 3 testis-enriched selenoproteins, mGPx4, TGR, and SELV, in our adult proband (Figure ). mGPx4, forming cross-links with other proteins, is a major structural component of the mitochondrial capsule in the midpiece of spermatozoa (26
). Murine mGPx4 inactivation causes male infertility (27
), and reduced human seminal mGPx4 activity correlates with oligospermia (28
). TGR, also highly enriched in spermatids, may catalyze protein disulfide bond isomerization in sperm development (29
). Human SELV expression is known to be testis restricted, but its function is unknown (3
). Consistent with the known importance of mGPx4 and TGR in latter spermatogenic stages, testicular histology in P1 showed maturation arrest, with preservation of early cell types (e.g., spermatogonia and spermatocytes) but lack of mature spermatids and spermatozoa (Figure G). SEPP, acting as a source of selenium to meet tissue demand, has also been implicated in spermatogenesis, with SEPP
-null mice showing abnormal sperm morphology and diminished testicular selenium content (30
). Thus, markedly reduced circulating SEPP levels in P1 (Figure B) may be an added factor contributing to his azoospermia. Although P2 was prepubertal, his serum SEPP (Figure C) and PBMC mGPx4 protein levels (Supplemental Figure 2A) were also low, suggesting that future spermatogenesis may also be compromised.
SELN is expressed in skeletal muscle, and mutations in either the coding region or 3′–untranslated region SECIS element of this gene are associated with a spectrum of myopathic disorders of varying clinical severity and age of onset, including RSMD1, desmin-related myopathy with Mallory body–like inclusions (MB-DRM), and congenital fiber type disproportion (CFTD) (32
). The musculoskeletal phenotype of our subjects showed a marked resemblance to SEPN1
myopathies: the pattern of muscle weakness (axial) and involvement (adductors, sartorius) was very similar to RSMD1 (Figure , B and C); muscle histology (type 1 fiber predominance and occasional minicores; Figure , B and C) and reduced fibroblast SELN protein levels (Figure G) were comparable to SEPN1
myopathy cases. A congenital myopathy resembling that seen in SEPN1
mutation cases was also noted in a childhood case described very recently (35
). Collectively, these results suggest that a skeletal myopathy, likely mediated by SELN deficiency, is a feature of this disorder.
Exposure of normal human skin to ultraviolet radiation (UVR) is known to generate free radicals and high millimolar levels of H2
). Furthermore, UVR exposure upregulates GPx (37
) and TrxR (38
) activity and MSRB levels (39
), protecting cells from oxidatively derived damage. We have documented cutaneous deficiency of GPx1, TrxR, and MSRB (Figure , B–D), together with increased cellular ROS (Figure A), membrane lipid peroxidation (Figure A), and oxidative DNA damage (Figure , C–E) in dermal fibroblasts from P1. These findings, together with excess lipid peroxidation and DNA damage (8-oxoGua and γH2AX foci) seen in SBP2-depleted cell lines (40
) and enhanced lipid peroxidation in murine GPx4 deficiency (41
), substantiate a link between selenoprotein deficiency and reduced cellular antioxidant defence. Furthermore, our observation of markedly enhanced oxidative damage following UV exposure in skin fibroblasts from P1 (Figure , A and B) provides a potential pathogenic basis for his photosensitivity. P2 was not abnormally photosensitive by the completion of the present study, but his cells were less susceptible to UV-mediated DNA damage (Figure D) and generated less H2
(Figure F). It is well recognized that cellular accumulation of ROS can itself damage enzymes (e.g., catalase) that mediate its removal (43
) or pathways (e.g., MSRB) that repair oxidized proteins (44
). As P2 is still very young (age 5 years at completion of the present study), it is conceivable that such cumulative ROS-mediated damage to antioxidant defence pathways could expose future photosensitivity.
The defective T cell proliferation we observed in P1 (Figure B) mirrors similar findings in murine T cell–specific selenoprotein knockout cells in which excess cellular ROS generated by T cell activation inhibits proliferation (45
). Having documented deficiency of antioxidant selenoenzymes in T cells and increased PBMC ROS levels in P1 (Figure , C and E), we suggest that a similar mechanism operates in the human context. Markedly shortened telomere length seen in primary cells from both cases (P1, Figure G; P2, Supplemental Figure 2D) correlated with reduced TRF length seen in SBP2 knockdown in human cell lines (46
) and may contribute to the slightly reduced rbc number and lymphopenia seen in P1 (Figure A). Thus, the degree of telomere shortening in his PBMCs is comparable to that seen in subjects with TERT
mutations, which is associated with aplastic anemia (47
); similarly, mice engineered to have short telomeres exhibit hematologic abnormalities, including low blood counts and impaired lymphocyte proliferation (48
). Intriguingly, shortened telomere length in T cells has also been specifically associated with connective tissue disorders, including lupus, RA, and scleroderma (49
); in this context, we note that P1 has marked Raynaud disease, albeit without other clinical or serological features of a connective tissue disorder. Although neither proband was overtly immunodeficient, telomere erosion is most likely to limit self-renewal of highly proliferative cell compartments (e.g., bone marrow), and it is conceivable that added insults (e.g., viral infection and drug toxicity) could compromise hemopoiesis or immune function in these cases.
LPS-induced TNF-α secretion from macrophages cultured in selenium-depleted medium is markedly enhanced (51
). A population study has correlated circulating TNF-α/IL-6 levels with a SNP in the SELS
gene and siRNA knockdown of SELS
in macrophages enhances LPS-induced TNF-α and IL-6 secretion (52
). We therefore suggest that the observed reduction in SELS levels (Figure B) accounted for the excess TNF-α and IL-6 production by PBMCs in P1 (Figure A). Whether particular selenoprotein deficiencies mediate defective IFN-γ production by his immune cells remains to be elucidated.
Based on previous observations of insulin resistance in subjects with SEPN1
-related myopathy (53
), we were surprised that both probands were markedly insulin sensitive, particularly in association with enhanced fat mass. Interestingly, increased fat mass was also a feature in the recently described childhood case (35
). Both probands exhibited deficiencies of antioxidant selenoenzymes (e.g., GPx1; Table and Figure B) and elevated cellular ROS. In this context, GPx1
-null mice with elevated ROS levels have recently been shown to be more insulin sensitive (54
). Elevated ROS levels enhance ERK phosphorylation in a murine Sirt-3
deficiency model (55
), and a similar mechanism could account for the increased pERK levels observed in fibroblasts from P1 (Figure , G and H). There is also evidence for cross-talk between ERK and Akt insulin signaling pathways (56
). It is therefore plausible that increased ROS and elevated pERK levels, mediating greater tissue insulin responsiveness, contribute to the enhanced insulin sensitivity in both probands.
Although hearing loss is a feature in dio2
-null mice (57
), the thyroid hormone profile in both P1 and P2 and other published cases is more congruent with combined partial deficiency of all deiodinases (12
). ROS are known to induce cochlear damage, which can be limited by antioxidant treatment (59
), and GPx1
-null mice exhibit susceptibility to noise-induced hearing loss (60
). ROS-mediated damage can also be cumulative (61
), and this alternative mechanism for hearing loss might account for our observation that auditory deficit was more severe in P1 (Supplemental Figure 3A), who is older than P2 (Supplemental Figure 3B).
Overall, we suggest that some phenotypes (e.g., azoospermia and myopathy) in this disorder are mediated by tissue-specific selenoprotein deficiencies; other features (e.g., cutaneous photosensitivity and impaired T cell proliferation) are likely mediated by impaired cellular antioxidant defense, reflecting the high oxidative stress to which these tissues are subjected with sun exposure and antigenic stimulation, respectively. Some phenotypes (e.g., photosensitivity and hearing loss) may be age-dependent, reflecting cumulative ROS-mediated damage; it is therefore conceivable that other pathologies linked to oxidative damage (e.g., neoplasia, neurodegeneration, and premature aging) may only manifest with time. Future identification of other affected subjects, by biochemical screening of cases of idiopathic male infertility, SEPN1
-like myopathies, or photosensitivity, for example, may delineate phenotypes that are more variably expressed. Thus, it is tempting to speculate that the biopsy-proven colitis of unknown etiology in P2 could be related to ileocolonic inflammation seen in compound GPx1/GPx2
knockout mice (62
), although P1 has no gastrointestinal problems. Conversely, whereas selective depletion of selenoproteins in murine osteochondroprogenitor cells results in epiphyseal abnormalities and chondronecrosis (63
), radiologic skeletal survey and bone mineral density in P1 were normal (data not shown). Further cases may also reveal additional phenotypes linked to deficiencies of selenoproteins of unknown function.
With regard to possible therapies, T3 treatment is beneficial in childhood cases with subnormal T3 levels, as has been documented by others (12
) and us in the case of P2, although a plateau in his height and weight suggests that growth retardation may not be solely related to thyroid status (Supplemental Figure 4). Although trials of selenium supplementation of previous cases raised circulating levels of the trace element, defective selenoprotein synthesis was not corrected (64
). As excess lipid peroxidation in cells from our cases was ameliorated by α-tocopherol exposure (Figure , A and C), we suggest that, as has been advocated in SEPN1
-related myopathy (65
), a trial of antioxidant treatment (e.g., n
-acetyl cysteine, vitamin E/α-tocopherol, and ascorbic acid) in this disorder might represent a rational alternative.