PMC5552663_01
Clinical presentation; proband is 16 years old boy with poor general growth, short stature postnatal microcephaly, lean cachectic face, and disproportionately large hands and ears. Inset images show large hand and evidences of cutaneous photosensitivity.
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F1
A 16-year-old boy was referred to Genetic Counseling Center in Shiraz Welfare organization (Shiraz, Iran) with chief complaint of general growth and developmental delay, including poor weight gain and short stature (Figure 1).
The proband (marked by arrow and confirmed using Sanger sequencing as homozygote for novel mutation) is a boy with cutaneous photosensitivity, severe growth retardation and intellectual disability. Parents have consanguineous marriage, and two of his uncles from mother side had similar phenotype and died around 16 and 17 years. The proband sister is a known case of Down syndrome. By using Sanger sequencing, parents and grandparents from mother side were confirmed as heterozygous carriers of the identified ERCC8 gene mutation and are marked with solid circles with corresponding sequencing chromatograms. Het, heterozygote; Hom, homozygote.
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F2
In addition, there is one case of documented trisomy 21 (Down syndrome) in the family (Figure 2). Sanger sequencing reagent from Applied Biosystems . Sanger sequencing data were analyzed using 4Peaks free software, which confirmed homozygote mutation in the proband and heterozygote in his parents and grandparents (Figure 2). (1) Whole exome sequencing only identified this homozygous mutation in ERCC8 gene (not in another gene involved in this syndrome) to be the main cause of CS in the proband. (2) As shown in Figure 2, using Sanger sequencing, the mutation was confirmed in the proband, and based on the heterozygote mutation in his parents and grandparents, the inheritance pattern must be an autosomal recessive mode. (3)
Prediction of splice site changes using MutationTaster with the use of NNSplice database. As can be seen in this figure, due to the mutation in the position of 57634 in gDNA sequence, the lost of donor sequence motif has occurred and an increase for a donor site in position of 57629 has been predicted, and due to these changes, normal splicing may be disturbed. MutationTaster determines the position of splice site change relative to intron/exon borders: if a loss/decrease of a splice site occurs at an intron/exon border or exon/intron border, this will be taken for a "real" splice site change. A gain of a completely new splice site is displayed, if the confidence score of the newly created splice site is > 0.3. An increase in an already existing splice site will be displayed if the change in the confidence score is >10%. All changes are displayed with the effect, genomic position of the splice site, the prediction score for wild-type (wt) and/or mutated (mu) splice site as generated by NNSplice, the (wt) detection sequence itself and very short sequence information about the splice site, with a pipe (|) indicating the border between intron and exon.
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F3
MutationTaster, an important online software designed for prediction of damaging effects of alternative amino acids, predicted that this variation will be damaging. (4) Distance to splice site is one nucleotide, and on the basis of information obtained from MutationTaster program, splice site may be changed due to this nucleotide change (Figure 3), and alteration within used splice site is likely to disturb normal splicing (Figure 3). MutationTaster uses a locally installed third-party splice site prediction program, namely NNSplice from the Berkeley Drosophila Genome Project (a web-based version is available at ) to analyze possible changes in splice sites. (5) Using I-Mutant v2.0 (Predictor of Protein Stability Changes upon Mutations), it was revealed that protein stability will be decreased upon the change of Glu 374 to Asp (Figure 4A).
(A) By using I-Mutant v2.0, it was revealed that with the substitution from Glu 374 to Asp, the stability of the protein will be decreased. (B) All alterations of stability for all other amino acids in this residue were also predicted by this program. As can be seen, most of the substitutions in this region can decrease the stability of the protein. (C) Comparative amino acids alignment of ERCC8 protein across Kingdoms shows that the Glu374 residue is highly conserved during evolution. The conserved Glu (E) residue is shown in the rectangular box. Protein sequences were obtained from National Center for Biotechnology. Symbols: (*):identical amino acids; (:):just similar amino acids.
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F4
MutationTaster, an important online software designed for prediction of damaging effects of alternative amino acids, predicted that this variation will be damaging. (4) Distance to splice site is one nucleotide, and on the basis of information obtained from MutationTaster program, splice site may be changed due to this nucleotide change (Figure 3), and alteration within used splice site is likely to disturb normal splicing (Figure 3). MutationTaster uses a locally installed third-party splice site prediction program, namely NNSplice from the Berkeley Drosophila Genome Project (a web-based version is available at ) to analyze possible changes in splice sites. (5) Using I-Mutant v2.0 (Predictor of Protein Stability Changes upon Mutations), it was revealed that protein stability will be decreased upon the change of Glu 374 to Asp (Figure 4A). Other substitutions in this residue were also predicted in term of protein stability (Figure 4B). (6) As can be seen in Figure 4C, the comparative amino acids alignment of ERCC8 protein across Kingdoms using T-Coffee Multiple Sequence Alignment Program revealed that this amino acid is highly conserved during evolution. (7) Genomic Evolutionary Rate Profiling (GERP) conservation score predicted a score 4.350 for this residue and as determined by this program that its score ranges from -12.3 to 6.17, with 6.17 being the most conserved, can confirm the conservation of this amino acid. (8)