PMC5677950_01
RNU4ATAC structural elements, and MOPD1 and Roifman syndrome causal variants. Stem I and Stem II are both elements at the 3' and 5' of U4atac, respectively, which base pair with U6atac. These elements are separated by an intramolecular 5' stem-loop. Another stem loop is located at the 3' end of U4atac. The Sm protein binding site is important for binding of the Sm proteins, which are important for the assembly of the complex and its import into the nucleus. The Stem II, Stem I, 5' stem-loop and Sm binding site are all highly conserved. Delineated in red is the Stem II domain, which is obligatory for the pathogenesis of Roifman syndrome. Adapted from refs.
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Fig1
A cluster analysis was performed to show that the minor intron retention levels perfectly discriminate affected subjects (with homozygous Stem II or compound heterozygous Roifman Syndrome mutations) from family-matched unaffected controls (Fig. 7); results are identical using two different distance metrics (Manhattan and Euclidean, see Fig. 7 and Supplementary Fig. 1).  
Skeletal features of Roifman syndrome in patients 1 and 2. a Pelvic X-ray of patient 1 featuring flattening of the humoral heads and shortening of the femoral necks, representing early stages of spondyloepiphyseal skeletal dysplasia. b Lateral spine X-ray of patient 1 featuring anterior vertebral notching of the lower thoracic vertebrae, and loss of lumbar lordosis. c Pelvic X-ray of patient 2 featuring bilateral small, flattened and slightly broadened femoral heads. d Lateral spine X-ray of patient 2 featuring loss of lumbar lordosis.
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Fig2
The RNA-seq-aligned reads in kindred 1 confirmed the presence of the homozygous mutation in patient 1 (Supplementary Fig. 2).
Facial features of the patient 2. Narrow palpebral fissures, a long philtrum and a thin upper lip are evident, in keeping with the classical facial features of Roifman syndrome patients. Informed consent was obtained for publication of patient images.
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Fig4
He was found to have characteristic dysmorphic features (Fig. 4), spondyloepiphyseal dysplasia (Fig. 2c, d), as well as retinal dystrophy.
Pedigree of Roifman syndrome patients. a Pedigree of kindred 1 (K1), the family of patient 1, showing a c.16 G > A homozygous mutation. b Sequencing analysis of patient 1 and her heterozygous carrier parents. c Pedigree of kindred 2 (K2), the family of patient 2, showing a compound heterozygous mutation c.116 A > G/17 G > A. d Sequencing analysis of patient 2 and his heterozygous carrier parents. e Sequencing analysis of wild type RNU4ATAC. Circles represent female subjects, while squares denote male subjects. The black colour represents an affected status. The grey colour represents individuals who were unavailable for sequencing. The white colour represents affected non carrier, and half black-half white colour represents non affected heterozygous carrier. [=] indicates no variant detected.
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Fig5
An analysis of other family members revealed another sister who is a carrier of the same mutation and two non-carrier brothers (Fig. 5a, b). The mother was found to be a carrier of the 17 G > A variant and the father of the 116 A > G variant (Fig. 5c, 5d).
Percentage intron retention (PIR) in patient 1 and unaffected family members. PIR across different introns is displayed as a boxplot: the affected patient 1 clearly displays greater minor intron retention than the unaffected wild-type siblings, whereas major intron retention levels are lower and similar across samples. Hom_RS-patient 1; Hom_CT1.nc-unaffected, non-carrier male sibling of patient 1, aged 3 months; Hom_CT2.nc-unaffected, non-carrier male sibling of patient 1, aged 9 years. Grey boxes-major intron retention levels, orange boxes-minor intron retention levels.
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Fig6
Using the vast-tools analysis pipeline, consistent minor intron retention was detected in affected compared to unaffected subjects, with no significant differences in major intron retention (Fig. 6).
Cluster analysis for minor intron retention of affected subjects vs. controls. Cluster analysis was performed using the euclidean distance metrics. A clear discrimination of minor intron retention levels is demonstrated between affected subjects of both heterozygous and homozygous mutations, and their unaffected controls. C.Het_CT1.c-Unaffected carrier mother of patient 2; Hom_CT1.nc-unaffected, non-carrier male sibling of patient 1, aged 3 months; Hom_CT2.nc-unaffected, non-carrier male sibling of patient 1, aged 9 years; Hom_RS-Patient 1; C.Het_RS-Patient 2.
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Fig7
A cluster analysis was performed to show that the minor intron retention levels perfectly discriminate affected subjects (with homozygous Stem II or compound heterozygous Roifman Syndrome mutations) from family-matched unaffected controls (Fig. 7); results are identical using two different distance metrics (Manhattan and Euclidean, see Fig. 7 and Supplementary Fig. 1).  