#3639 WHEN WHOLE EXOME SEQUENCING IS NOT GOOD ENOUGH: BACK TO THE ROOTS OR FORWARD TO THE FUTURE

Abstract Background and Aims Over the last few years, massively parallel sequencing (MPS) has been established in genetic diagnostics, where large virtual panels of candidate genes can be screened on a whole exome basis. This has enabled large analyses for most medical indications, including renal genetic diseases. The 25 year old, index female patient was referred for disease clarification. Both non-related parents suffered from end-stage renal disease, with kidney failure in mid adulthood and familiar cystic disease in both the maternal and paternal lines. An external genetic analysis applying MPS on a restricted panel of genes retrieved no conclusive result, apart from two variants in the UMOD gene, classified as class 3 and 4 following ACMG criteria. Method Whole exome sequencing (WES), linkage analysis, haplotype reconstruction, whole genome sequencing (WGS), long-range PCR (LR-PCR). Results The detailed pedigree of the family displayed an autosomal dominant disease with several family members reaching end-stage kidney disease on both the maternal and paternal sides. The maternal family is characterized by polycystic disease of the kidney and most profoundly the liver, with two female members already liver transplanted and one on the waiting list. The paternal family shows more unspecific chronic kidney disease (CKD) with multiple cysts in the kidney, but not in the liver. Two male members reported gout attacks, before the knowledge of CKD. We consented 12 family members for research studies and collected blood for DNA. Contemporary MPS of a virtual panel for cystic diseases (> 60 genes, i.e. PKD1 and PKD2, all known genes for cystic liver disease) in the index patient did not yield a conclusive result, apart from the previously reported UMOD variants [c.464G>A, p.(Cys155Tyr) and c.907G>C, p.(Asp303His)]. These variants both segregated in the paternal family, defining them as lying in cis on one allele. WES was performed on a trio of three affected family members of the maternal family, yielding no further result. Linkage analysis showed 9 peaks with a LOD score above 2.0. One peak is situated on chromosome 16, containing the candidate genes PKD1 and IFT140. WGS revealed the heterozygous variant c.2180T>C, p.(Leu727Pro) in exon 11 of PKD1, which has been reported as causative numerous times in the past. Conventional LR-PCR, which is designed to acknowledge the highly homologous regions of the 6 pseudogenes of PKD1, also retrieved the PKD1 variant, which was not called by MPS. Comparing the sequence reads of the WES with the WGS, the PKD1 variant was detected in 12% in the former, whereas complete detection was achieved in the latter. Since the cutoff for calling variants in WES was set to 20%, the PKD1 variant was not visible to the genetic analyst. The index patient indeed inherited both diseases from her parents, giving her the simultaneous diagnoses of ADPKD and ADTKD(-UMOD). Conclusion ADPKD is one of the most frequent genetic diseases of the human, with PKD1 being involved in > 80% of cases. With the apparent ease of modern technology, increasingly ADPKD families are presented for genetic consultation, also to non-specialized centers. It is tempting to use MPS considering the number of candidate genes and the size of PKD1 (46 exons). However, a negative result should be supported either by LR-PCR or WGS, taking the profound difficulties with the 6 pseudogenes of PKD1 into consideration. It is not known whether the predominance of liver disease in this family with the PKD1 c.2180T>C variant implicates a genotype-phenotype correlation, or whether other (genetic) modifiers may press for a liver phenotype. The renal prognosis of the index case is feared to be worse baring both diseases, even with the pathogeneses being different with a ciliopathy (ADPKD) and a toxic proteinopathy (ADTKD-UMOD), respectively. Since both affected genes are situated on the respective chromosomes 16, the patient bares a 100% risk of receiving children with an adult onset kidney disease, where pre-implantation technology will not solve this specific problem.