Whole-Genome Sequencing In A Familial Focal Segmental Golmerulosclerosis

Objective: Focal and segmental glomerulosclerosis(FSGS) is an common type of primary glomerular diseases in adults and children. The major clinical manifestations are proteinuria, nephritic syndrome, rapid progressive renal insufficiency, hematures and hypertention. The therapeutic agents available for FSGS are not very effective and only a small percentage of affected individuals will achieve complete remission. In western countries,primary glomerular disease is the third cause of end-stage renal disease(ESRD). FSGS is responsible for 12-35% of primary nephritic syndrome(NS),and the incidence is rising. While in our country, FSGS accounts for 3.2-5.8%.The pathogensis of FSGS has not been fully elucidated. It can occur as a primary disorder(called primary FSGS), as a consequence of genetic mutations in podocyte-specific proteins(also called familial FSGS) or as a secondary disorder. The inheritance pattern of FFSGS appears as autosomal dominant or autosomal recessive. Recently, it had found that more than 18%primary FSGS were FFSGS. Up to now, using linkage analysis, positional cloning or high-throughput sequencing people found many disease-causing gene, such as NPHS2, ACTN4, TRPC6, CD2 AP, PLCE1, LMX1 B, LAMβ2,INF2 and MYO1 E. Their encoded proteins are expressed in the podocyte and slit diaphragm, which plays a key role in the pathogenetic progress of FFSGS.The functional or structural alteration in mutant proteins damage glomerular filtration barrier through affect cytoskeletal structure, signal transduction and cellular homeostasis. Therefore, to analyze the mutations of these podocyte molecules in FSGS families and screening the genes will help to clarify the pathogenesis of FSGS and to achieve early diagnosis and effective treatment.With the human genome project(HGP) and the international Hap Map project complete, next-generation sequencing(NGS) developed rapidly.Recently the whole genome sequencing(WGS) technology is used in mendelian inheritance disease, complex disease, rare disease and cancer of humans, which has gradually become a very important tool for finding pathogenic genes and exploring the development of disease through sequencing of individual genome, analysis of the differences in individuals and groups, concurrently compared the sequences with known sequence. WGS enables recognize the identification of single nucleotide variations(SNVs),insertions and deletions(In Dels), copy number variations(CNVs) and structural variations(SVs) more comprehensive than whole exome sequencing(WES). The WGS techonology can either analysis the base sequence or find genetic characteristics comprehensively and accurately. With the emergence of Hiseq X ten, the cost is decreasing, which is not obstacles at all. Therefore WGS technology has been applied to genetic screening, targeted therapy and cancer risk assessment to achieve early detection, early diagnosis, early intervention and accuate treatment.Whole genome sequencing was preceded in 2 patients and their mother from a family with FSGS, which has been identified by clinical, laboratory and pathological examination, then multi-step screening was made for the data from sequencing to choose the candidate genes, provide the basis for confirm the pathogenic gene.Methods: We chose 2 patients who had been identified as FSGS and their mother from a family. The peripheral blood was obtained to extract DNA from the three using the QIAquick Gel Extraction kit and then whole genome sequencing using the Illumina Hi Seq X Ten. The result were filtered against the human databases of HAPMAP, db SNP138 and 1000 Genome Project, and common variations which had been reported were wiped out, then non-synonymous variants in exonic and splicing regions were retained. Using SIFT and Polyphen-2 software to predict the influence in protein function of the variations and candidate genes was selected initially. And then query OMIM, GO, KEGG pathway databases to analyze its biological characteristics and the potential mechanism.Results: The number of SNVs are 3038061, 3132594 and 3037609 SNVs,the number of In Dels are 401259, 432406 and 398040, the number of SVs are2917, 2211 and 3088, the number of CNVs are 135, 211 and 91. And all the data of the two patients and their mother were acquired by sequencing. After multi-step screening, SNVs are 106, 94 and 104 respectively, with 54 SNVs are shared by the two patients, while In Dels are 604, 590 and 628 respectively,with 455 In Dels are shared by the two patients. After combining the mutual non-synonymous variation from the three,19 genes were screened out.Conclusion:The 19 genes(KDM4A, CF7L1, ADRA2 B, KIF1 A, TOP2 B,GPR115, AK9, KCNT1, WDR96, ZNF384, KRT3, DDX55, ADCK1, TIPIN,JMJD8, STUB1, FAM83 G, SLC5A10, SH3BGR) might be the candidate genes of FFSGS, but we need do the sanger sequencing to excluding false positive, and more investigation is necessary.