Clinical and Molecular Analysis in 2 Families With Novel Compound Heterozygous SBP2 (SECISBP2) Mutations

Abstract

Context

Selenocysteine insertion sequence binding protein 2 (SECISBP2, SBP2) is an essential factor for selenoprotein synthesis. Individuals with SBP2 defects have characteristic thyroid function test (TFT) abnormalities resulting from deficiencies in the selenoenzymes deiodinases. Eight families with recessive SBP2 gene mutations have been reported to date. We report 2 families with inherited defect in thyroid hormone metabolism caused by 4 novel compound heterozygous mutations in the SBP2 gene.

Case Descriptions

Probands 1 and 2 presented with growth and developmental delay. Both had characteristic TFT with high T4, low T3, high reverse T3, and normal or slightly elevated TSH. The coding region of the SBP2 gene was sequenced and analysis of in vitro translated wild-type and mutant SBP2 proteins was performed. Sequencing of the SBP2 gene identified novel compound heterozygous mutations resulting in mutant SBP2 proteins E679D and R197* in proband 1, and K682Tfs*2 and Q782* in proband 2. In vitro translation of the missense E679D demonstrated all four isoforms, whereas R197* had only 2 shorter isoforms translated from downstream ATGs, and Q782*, K682Tfs*2 expressed isoforms with truncated C-terminus. Reduction in serum glutathione peroxidase enzymatic activity was also demonstrated in both probands.

Conclusions

We report 2 additional families with mutations in the SBP2 gene, a rare inherited condition manifesting global selenoprotein deficiencies. Report of additional families with SBP2 deficiency and their evaluation over time is needed to determine the full spectrum of clinical manifestations in SBP2 deficiency and increase our understanding of the role played by SBP2 and selenoproteins in health and disease.

Selenocysteine insertion sequence binding protein 2 (SECISBP2 or SBP2) is an essential factor for selenoprotein synthesis (1). Among the 25 selenoproteins identified in the human genome, the iodothyronine deiodinases are well-characterized selenoenzymes that metabolize thyroid hormone. We first reported mutations in the SBP2 gene in patients with delayed growth and characteristic thyroid function test (TFT) abnormalities. These abnormalities were low serum T3, high serum T4, high serum rT3, and normal or slightly elevated serum TSH (2). Other clinical features were subsequently observed in the 8 families with SBP2 deficiency reported to date, axial muscular dystrophy, azoospermia, cutaneous photosensitivity, impaired immune function, and increased insulin sensitivity (2–7), indicating a multisystem disorder resulting from defective biosynthesis of multiple selenoproteins. We report herein 2 additional families with novel compound heterozygous SBP2 gene mutations and assess their SBP2 isoforms by in vitro translation.

Case description

Proband 1 (Fig. 1A, II-2) is an Argentinian girl born to nonconsanguineous parents after an uneventful pregnancy. She had normal neonatal screening for congenital hypothyroidism. At 1 year, she presented with failure to thrive, weight of 7.1 kg (-3.0 SD score [SDS]) and length of 70.6 cm (-1.3 SDS). She also had delayed developmental milestones, was able to sit without support at 9 months, and walk at 18 months. No other thyroid-related symptoms were present and no goiter was noted. She was referred for genetic evaluation when she was 5.4 years old. At that time, she still had growth delay with a low weight of 13.7 kg (-2.7 SDS) and height of 98 cm (-2.7 SDS) and delayed bone maturation with a bone age of 2 years. Serum TFT revealed a pattern similar to that previously characterized in SBP2 deficiency except that T3 was in the low normal range (Fig. 1A). Circulating growth hormone and IGF-1 levels were reportedly normal for age and sex.

Figure 1.

Pedigrees of the families (A and B, respectively) with corresponding serum thyroid function tests and GPx enzymatic activities. Subjects are identified by generation (roman numerals) and by a number on the right of each symbol. Results are aligned with each symbol representing a family member. Values outside the normal range are indicated in bold numbers. Abbreviations: GPx, glutathione peroxidase; TGab, antibodies against thyroglobulin; TPOab, antibodies against thyroid peroxidase.

Proband 2 (Fig. 1B, II-2) is a Turkish girl born to nonconsanguineous parents after an uneventful pregnancy. She had a history of developmental delay, started to walk and talk at the age of 2.5 years, and was evaluated at the age of 11 years for delayed growth, with weight and height in the third to 10th percentile. She was in early puberty with Tanner stage II breasts. Serum TFT revealed low T3, elevated T4 and rT3, and slightly elevated TSH levels (Fig. 1B). No other thyroid-related symptoms were apparent. Cutaneous photosensitivity, hypoglycemic episodes, or recurrent infections were not present and a hearing test was normal.

The clinical presentation and TFT abnormalities of the 2 probands raised the possibility of SBP2 deficiency. The study was approved by the institutional review boards of The University of Chicago and written informed consents were obtained.

Genetic analysis

Sequencing of the coding region of the SBP2 gene identified 4 novel compound heterozygous mutations in the affected individuals (Fig. 1, Table 1). Proband 1 harbored a paternally inherited missense SBP2 gene mutation, c.2037G > T, leading to the substitution of the conserved glutamic acid 679 with aspartic acid, p.Glu679Asp, E679D, located in the RNA-binding domain and predicted to be damaging by PolyPhen-2 algorithm (8). She also harbored a maternally inherited nonsense SBP2 gene mutation, c.589C > T, leading to a premature stop codon, p.Arg197*, R197*. Her siblings inherited the wild-type (WT) parental alleles (Fig. 1A).

Table 1.

Reported Mutations in the SBP2 Gene

Family	Mutations	Protein Change	Comments on Putative Defect	No. of Affected	Status	Ref.
1	c.1619G > A	R540Q	Predicted damaging (PolyPhen-2 score 1)	3	Homozygous	(2)
2	c.1312A > T	K438*	Truncated functional domain	1	Compound heterozygous	(2)
	c.1283 + 29G > A abnormal splicing	Frameshift	Truncated functional domain
3	c.382C > T	R128*	Shorter isoformsa	1	Homozygous	(5)
4	c.358C > T	R120*	Shorter isoformsa	1	Compound heterozygous	(3)
	c.2308C > T	R770*	Truncated functional domain
5	c.668delT	F223Ffs*32	Shorter isoforma	1	Compound heterozygous	(7)
	c.881-155T > A abnormal splicing	Frameshift	Shorter isoforma
6	c.2071T > C	C691R	Predicted damaging (PolyPhen-2 score 1)	1	Compound heterozygous	(7)
	Intronic SNPs (?) abnormal splicing	Frameshift	Transcripts lacking exons 2–4 or exons 3–4
			Shorter isoformsa
7	c.1529_1541dup CCAGCGCCCCACT	M515Qfs*48	Truncated functional domain	1	Compound heterozygous	(6)
	c.235C > T	Q79*	Shorter isoformsa
8	c.800_801insA	K267Kfs*2	Shorter isoforma	1	Homozygous	(4)
9	c.589C > T	R197*	Shorter isoformsa	1	Compound heterozygous	This report
	c.2037G > T	E679D	Predicted damaging (PolyPhen-2 score 1)
10	c.2344C > T	Q782*	Truncated after functional domain	1	Compound heterozygous	This report
	c.2045_2048delAACA	K682Tfs*2	Truncated functional domain

The nomenclature as transcript ID ENST00000375807.7 (854-amino acid long).

^aShorter isoform(s) generated from downstream ATG(s) (M139, M233, M300) containing C-terminal functional domain.

Proband 2 harbored a paternally inherited SBP2 gene 4-nucleotide deletion, c.2045_2048delAACA, leading to frameshift with a premature stop codon truncating the C-terminal functional domains, p. Lys682Thr fs*2, K682Tfs*2. She also harbored a maternally inherited nonsense SBP2 gene mutation c.2344C > T, leading to a premature stop codon, p.Glu782* (Q782*), located just after the minimal functional domain. Her brother inherited the maternal mutation Q782* and the normal paternal allele, whereas her sister inherited both WT parental alleles (Fig. 1B).

Serum glutathione peroxidase enzymatic activity

The enzymatic activity of glutathione peroxidase (GPx) in serum was measured in all members of the 2 families and was significantly decreased in the affected individuals compared with the unaffected family members, whose genotypes were heterozygous for 1 of the mutant SBP2 allele and a normal allele (parents of each proband and the brother of proband 2) or had 2 normal SBP2 alleles (both sisters of proband 1 and the sister of proband 2). In proband 1, the serum GPx enzymatic activity was low at 44 vs average of 124 mU/mL in the unaffected family members and, similarly, in proband 2, it was 15 vs average of 126 mU/mL in the unaffected family members (individual values are included in Fig. 1A and B).

Analysis of in vitro translated WT and mutant SBP2

Human SBP2 encodes a full-length protein of 854 amino acids and smaller isoforms that are translated from downstream ATGs (9). To assess the effect of the mutations on the full-length SBP2, we synthesized in vitro SBP2 proteins containing the novel mutations identified (10).

WT full-length SBP2 (Fig. 2A, lane 1) and that of the missense SBP2 E679D (Fig. 2A, lane 3) expressed all 4 isoforms translated from the 4 ATG start codons, Met1, Met139, Met233, and Met300 (Fig. 2B). In vitro translation of R197* demonstrated only the presence of 2 shorter isoforms translated from downstream ATGs Met233 and Met300 (Fig. 2A, lane 2), whereas the in vitro translation of the 2 mutant SBP2 proteins, Q782* and K682Tfs*2, showed isoforms of reduced molecular weight by Western blot, consistent with a truncated C-terminus (Fig. 2A, lanes 4 and 5, respectively).

Figure 2.

Analysis of mutant SBP2 proteins. (A) Western blot analysis of in vitro translated wild-type and mutant SBP2 showing different isoforms. Full-length wild-type and mutant human SBP2 were translated in vitro using the T7 TnT Quick Coupled Transcription/Translation System. A total of 1 μL of the in vitro translated protein was resolved by SDS/PAGE, and probed with SECISBP2 polyclonal antibody targeted against the N-terminal of the protein. (B) Schematic representation of human SBP2 showing the location of all reported SBP2 mutations including those identified in this report. Region of minimal functional protein encompassing codon 399-774 is showed in gray. The positions of methionine used as alternative translational initiation sites (M1, M139, M233, M300) are indicated with arrowheads. The mutations reported herein are represented in boxed text. Abbreviation: Abn.splic., abnormal splicing.

Discussion

SBP2 deficiency is an autosomal recessive disease manifesting a multisystem disorder resulting from defective biosynthesis of the selenoproteins. The inheritance is autosomal recessive and the incidence is unknown. Although the clinical manifestations are variable, the chief complaints present in all cases of SBP2 deficiency in children are growth delay with delayed bone maturation. On further evaluation, this is accompanied by the characteristic TFT abnormalities. This combination of clinical signs and biochemical markers has been pathognomonic for SBP2 deficiency and finding biallelic mutations in the SBP2 gene establishes the diagnosis. Patients of different ethnicities have been identified (2–7); however, this remains an underdiagnosed syndrome. More recently, patients with biallelic SBP2 defects have been identified through whole-exome sequencing performed as part of a broader genetic investigation in children with developmental delay that included growth delay among multiple other clinical features (personal observation); subsequent TFT measurements confirmed the characteristic serum pattern.

We previously demonstrated in the fibroblasts of the SBP2-deficient patients and in a mouse model of Sbp2 deficiency that decreased enzymatic activities of the selenoenzymes deiodinases contribute to the observed TFT (2, 11). Both cases in this report have the TFT pattern of SBP2 deficiency. Although serum T3 in the proband of family 1 was in the low normal range (Fig. 1B), her T3/T4 ratio was 5.9 vs average T3/T4 ratio of 19.2 in the unaffected family members. This is due to the decreased T3 generation caused by decreased 5′ deiodination in SBP2 defects and was similar to T3/T4 ratios in other SBP2-deficient patients. Proband 2 had a T3/T4 ratio of 3.5 vs average T3/T4 ratio of 15.5 in the unaffected family members.

All affected patients with SBP2 deficiency reported to date have preserved partial SBP2 activity (2–7) (Table 1), with the shorter functional isoforms being able to rescue the SBP2 deficiency in early truncation mutations (5). The region encompassing codons 399-774 of the SBP2 protein contains the minimal functional domains required for all known SBP2 functions, including SECIS binding, Sec incorporation, and ribosome binding (Fig. 2B) (9). For proband 1, the E679D mutation located in the RNA binding domain is predicted to be deleterious by functional prediction algorithms, whereas the mutant R197* SBP2 protein preserves 2 of the shorter SBP2 isoforms translated from the downstream ATGs (Met 233 and Met 300) that contain the intact functional domain. For proband 2, the K682Tfs*2 frameshift mutation results in the truncation of the protein within the region of minimal functional domain and is presumed to be devoid of function. The Q782* mutation, located right after the minimal functional domain, is expected to preserve partial SBP2 activity. In terms of genotype–phenotype correlation, from the cases reported so far, there seems to be a more severe clinical presentation if the defect truncates or inactivates the C-terminal domain. A case homozygous for an early termination R128* manifested a relatively mild phenotype because the defect was partially rescued by the smaller isoforms containing the intact C-terminal functional domain (5). Whereas a patient compound heterozygous for a similar early termination, R120*, and a mutation truncating the functional domain R770* had a severe phenotype as all the isoforms translated from the R770* were putatively inactive (3).

In summary, we report new cases of SBP2 deficiency in 2 families. Genetic analysis of the SBP2 gene confirmed the diagnosis with the identification of 4 novel compound heterozygous mutations in the affected individuals resulting in partial SBP2 deficiency. Report of additional families with SBP2 deficiency and their evaluation over time is needed to increase our understanding of the full spectrum of clinical manifestations in SBP2 deficiency.

Glossary

Abbreviations

GPx

glutathione peroxidase

SECISBP2 and SBP2

selenocysteine insertion sequence binding protein 2

SDS

SD score

TFT

thyroid function test

WT

wild-type

Additional Information

Disclosure Summary: The authors have nothing to disclose.

Data Availability: All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.