Molecular Diagnosis of Xeroderma Pigmentosum using Next-Generation Sequencing

Abstract

Xeroderma pigmentosum (XP) is a genetic disease characterized by a decreased capability to repair DNA damages. It follows autosomal recessive mode of inheritance. It presents a range of symptoms including severe sunburn, photophobia, alterations in skin pigmentation, ocular and neurological abnormalities, dry skin, and freckling in sun exposed areas. The disease prevalence estimates at 1/1,000,000 in the Europe and USA, however, the figures are far higher in countries having high consanguineous marriages. Clinical severity and age of onset are variable in different complementation groups of XP. Pathogenic sequence variation in 8 genes involved in DNA repair underlying the disease etiology. The study was designed to explore the genetic basis of XP in six Pakistani families (Family A, B, C, D, E, F) using whole exome and Sanger sequencing. Genomic DNA was extracted from peripheral blood of the recruited families. Whole exome paired-end sequencing was performed by making 51 Mb Sure Select V4 libraries. DNA shearing, hybridization, and bridged amplification were performed. The extension and imaging were attained in automated cycles through Illumina HiSeq 2000/2500 sequencer. Standard bioinformatics pipeline was used to analyze the data. Rare pathogenic variants not present in controls were filtered out and their cosegregation with the XP phenotype in the family was confirmed by using Sanger sequencing. In Family A and B, probands and healthy individuals were subjected to whole exome sequencing and Sanger sequencing. A novel pathogenic sequence variation, c.1723dupG; p. Val575Glyfs*4 in family A and a previously reported variation c.437dupA; p.Tyr146* in family B were observed in POLH gene. The change [GTC (val) to GGTC (gly-C)] in family A resulted in the frameshift whereby introducing an early stop codon (V-Gfs*4) whereas the variant c.437dupA changed tyrosine (Y) amino acid at 146 into a stop codon which is destined to truncate the POLH gene product. We also investigated in family A the effect of mutation ex-vivo in HEK (Human Embryonic Kidney) 293T cells. We then performed in silico analysis of POLH gene and other POLZ complex genes expression in publicly available single cell mRNAseq datasets from adult human healthy and aging skin. We found overlapping expression of POLH, REV3L and POLD2 in multiple cell types including differentiated and undifferentiated keratinocytes, pericytes and melanocytes in DRSML QAU healthy skin. However, in aging human skin, POLH expression is less in comparison with its POLZ complex partners. The variants were analyzed to have pathogenic effect on protein structure and hence function. These POLH variants were identified to be cosegregating with XP phenotype. The proband of the Family C affected with autosomal recessive XP was subjected to whole exome sequencing. We identified a novel c.889G>T; p.Glu297* sequence variation in XPC gene on exon seven. Sanger sequencing revealed full segregation of the variant with XP phenotype in the family. This Glu – stop change in XPC gene also has been observed first time in XP patients. We conclude that XPC c.889G>T; p.Glu297* variation caused the XP disease in the studied family. In Family D, the proband was subjected to whole exome sequencing. It found a reported pathogenic variant, c.2251-1G>C; rs754673606, in XPC gene. It is a splice site variant that affects the splicing of the intron. The variant fully segregated in the family. The results support the previous finding and identify it as the cause of the XP disease in the recruited family. Whole exome sequencing of the probands of the Family E and Family F affected with XP was performed. No variant was found in the genes previously linked to XP. In the hunt of new causative pathogenic variant in the family, we adopted standard bioinformatics pipeline, however, none of the variants segregated with the XP phenotype in the families. These results support the evidence of further genetic heterogeneity in XP disease. We believe that an unidentified genetic or epigenetic factor might be the cause of the disease in our both families. Overall, in silico analysis of all four variants identified, either in POLH or XPC gene, in this study shows a disruption in the protein structures which ultimately has the potential to cause detrimental consequence on the overall function of protein. In conclusion, this study highlights the clinical impact of whole exome sequencing and the molecular diagnosis of heterogeneous XP disorder. The data obtained from the study may also help in the better genetic counselling and prenatal diagnosis of the families.