PMC7491304_01
a
Family pedigree. The proband is indicated by an arrow (P). The segregation was validated by Sanger Sequencing in the main family. b
ISG15 consists of two ubl domains (blue) that are linked by a proline residue. ISG15 is synthesized as a 17-kDa precursor that is proteolytically processed into a mature form of 15 kDa. This processing exposes a carboxy-terminal LRLRGG motif, required for ISGylation (yellow). The c.285delC variant produces a frameshift after Threonine 95 which results in a stop codon downstream. The c.299_312del produces a frameshift after Leucine 100 that has no stop codon within the sequence of the original gene. c
Crystallographic structure of human ISG15. The area disabled by the mutations is marked in green. Thr95 marks the beginning of the 285_del frameshift. Leu100 marks the beginning of the 299_312del frameshift. The C-terminus of the structure corresponds to the first 3 residues of the conjugation domain. d
Partial sequence chromatograms for ISG15 in the patient. DNA sequences of the WT (wild-type) and mutant ISG15 alleles are shown. Red rectangle denotes the sites of heterozygous sequence deletions
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Fig1
The second heterozygous variant, c.299_312del (NM_005101.4, GRCh37(hg19)), also known as c.297_313del, corresponds to a 14 nucleotide deletion in exon 2 which is also predicted to produce a frameshift below residue Leu100; p.Leu100Argfs (Fig. 1b). Sanger sequencing and familial segregation were performed confirming that the patient is a compound heterozygous for the above described mutations in ISG15 gene (Fig. 1d). AR mode of inheritance of ISG15 deficiency (Fig. 1a). According to ACMG criteria, both variants follow the pathogenic criterion: pathogenic very strong (PVS1), since they are both null variants, pathogenic moderate 2 (PM2) due to their low frequencies (with no reports or very low allele frequency in population databases such as GnomAD); and pathogenic moderate 3 (PM3), since we confirmed by Sanger Sequencing that the patient inherited both variants in trans, each from one parent. ISGylation site and thus triggering the loss of function of the entire protein (Fig. 1b, c). Compared with ubiquitin, which has very high (close to 100% identity) cross-species conservation, ISG15 protein has relatively low cross-species conservation. Its absence in many eukaryotic species suggests that it is not an essential housekeeping gene, leaving more room for its diversification during evolution.
a Chest CT scan. Bilateral focal areas of parenchymal consolidations, some of them with air bronchograms and with nodular appearance, distributed in the peripheral portions of the lower zones. Bilateral widespread septal thickening and peripheral tree in-bud changes in both lungs is observed too. Lobar air-trapping (ILL) on expiration was present. b Brain CT scan. Calcifications at the level of the caudate nuclei, lenticular nuclei and subcortical region of both frontal lobes are shown in the images
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Fig2
Lung and chest computed tomography (CT) scanning (Fig. 2a) showed some non specific and residual lesions, while brain CT evidenced basal ganglia calcifications (Fig. 2b).
PMC7491304_02
a
Family pedigree. The proband is indicated by an arrow (P). The segregation was validated by Sanger Sequencing in the main family. b
ISG15 consists of two ubl domains (blue) that are linked by a proline residue. ISG15 is synthesized as a 17-kDa precursor that is proteolytically processed into a mature form of 15 kDa. This processing exposes a carboxy-terminal LRLRGG motif, required for ISGylation (yellow). The c.285delC variant produces a frameshift after Threonine 95 which results in a stop codon downstream. The c.299_312del produces a frameshift after Leucine 100 that has no stop codon within the sequence of the original gene. c
Crystallographic structure of human ISG15. The area disabled by the mutations is marked in green. Thr95 marks the beginning of the 285_del frameshift. Leu100 marks the beginning of the 299_312del frameshift. The C-terminus of the structure corresponds to the first 3 residues of the conjugation domain. d
Partial sequence chromatograms for ISG15 in the patient. DNA sequences of the WT (wild-type) and mutant ISG15 alleles are shown. Red rectangle denotes the sites of heterozygous sequence deletions
13223_2020_473_Fig1_HTML.jpg
PMC7491304_02_13223_2020_473_Fig1_HTML.jpg
Fig1
The second heterozygous variant, c.299_312del (NM_005101.4, GRCh37(hg19)), also known as c.297_313del, corresponds to a 14 nucleotide deletion in exon 2 which is also predicted to produce a frameshift below residue Leu100; p.Leu100Argfs (Fig. 1b). Sanger sequencing and familial segregation were performed confirming that the patient is a compound heterozygous for the above described mutations in ISG15 gene (Fig. 1d). AR mode of inheritance of ISG15 deficiency (Fig. 1a). According to ACMG criteria, both variants follow the pathogenic criterion: pathogenic very strong (PVS1), since they are both null variants, pathogenic moderate 2 (PM2) due to their low frequencies (with no reports or very low allele frequency in population databases such as GnomAD); and pathogenic moderate 3 (PM3), since we confirmed by Sanger Sequencing that the patient inherited both variants in trans, each from one parent. ISGylation site and thus triggering the loss of function of the entire protein (Fig. 1b, c). Compared with ubiquitin, which has very high (close to 100% identity) cross-species conservation, ISG15 protein has relatively low cross-species conservation. Its absence in many eukaryotic species suggests that it is not an essential housekeeping gene, leaving more room for its diversification during evolution.