Gemma Bullich

Clinical Utility of Genetic Testing in Children and Adults with Steroid-Resistant Nephrotic Syndrome

BACKGROUND AND OBJECTIVES The increasing number of podocyte-expressed genes implicated in steroid-resistant nephrotic syndrome (SRNS), the phenotypic variability, and the uncharacterized relative frequency of mutations in these genes in pediatric and adult patients with SRNS complicate their routine genetic analysis. Our aim was to compile the clinical and genetic data of eight podocyte genes analyzed in 110 cases (125 patients) with SRNS (ranging from congenital to adult onset) to provide a genetic testing approach. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Mutation analysis was performed by sequencing the NPHS1, NPHS2, TRPC6, CD2AP, PLCE1, INF2, WT1 (exons 8 and 9), and ACTN4 (exons 1 to 10) genes. RESULTS We identified causing mutations in 34% (37/110) of SRNS patients, representing 67% (16/24) familial and 25% (21/86) sporadic cases. Mutations were detected in 100% of congenital-onset, 57% of infantile-onset, 24 and 36% of early and late childhood-onset, 25% of adolescent-onset, and 14% of adult-onset patients. The most frequently mutated gene was NPHS1 in congenital onset and NPHS2 in the other groups. A partial remission was observed in 7 of 26 mutation carriers treated with immunosuppressive agents and/or angiotensin-converting enzyme inhibitors. Patients with NPHS1 mutations showed a faster progression to ESRD than patients with NPHS2 mutations. None of these mutation carriers relapsed after kidney transplantation. CONCLUSIONS We propose a genetic testing algorithm for SRNS based on the age at onset and the familial/sporadic status. Mutation analysis of specific podocyte-genes has a clinical value in all age groups, especially in children.

Targeted next-generation sequencing in steroid-resistant nephrotic syndrome: mutations in multiple glomerular genes may influence disease severity

Genetic diagnosis of steroid-resistant nephrotic syndrome (SRNS) using Sanger sequencing is complicated by the high genetic heterogeneity and phenotypic variability of this disease. We aimed to improve the genetic diagnosis of SRNS by simultaneously sequencing 26 glomerular genes using massive parallel sequencing and to study whether mutations in multiple genes increase disease severity. High-throughput mutation analysis was performed in 50 SRNS and/or focal segmental glomerulosclerosis (FSGS) patients, a validation cohort of 25 patients with known pathogenic mutations, and a discovery cohort of 25 uncharacterized patients with probable genetic etiology. In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes. In the discovery cohort, disease-causing mutations in SRNS/FSGS genes were found in nine patients. We detected three patients with mutations in an SRNS/FSGS gene and COL4A3. Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene. In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS. Our results indicate that patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 gene have increased disease severity.

Diagnosis of autosomal dominant polycystic kidney disease using efficient PKD1 and PKD2 targeted next-generation sequencing

Molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) relies on mutation screening of PKD1 and PKD2, which is complicated by extensive allelic heterogeneity and the presence of six highly homologous sequences of PKD1. To date, specific sequencing of PKD1 requires laborious long‐range amplifications. The high cost and long turnaround time of PKD1 and PKD2 mutation analysis using conventional techniques limits its widespread application in clinical settings. We performed targeted next‐generation sequencing (NGS) of PKD1 and PKD2. Pooled barcoded DNA patient libraries were enriched by in‐solution hybridization with PKD1 and PKD2 capture probes. Bioinformatics analysis was performed using an in‐house developed pipeline. We validated the assay in a cohort of 36 patients with previously known PKD1 and PKD2 mutations and five control individuals. Then, we used the same assay and bioinformatics analysis in a discovery cohort of 12 uncharacterized patients. We detected 35 out of 36 known definitely, highly likely, and likely pathogenic mutations in the validation cohort, including two large deletions. In the discovery cohort, we detected 11 different pathogenic mutations in 10 out of 12 patients. This study demonstrates that laborious long‐range PCRs of the repeated PKD1 region can be avoided by in‐solution enrichment of PKD1 and PKD2 and NGS. This strategy significantly reduces the cost and time for simultaneous PKD1 and PKD2 sequence analysis, facilitating routine genetic diagnostics of ADPKD.

HLA-DQA1 and PLA2R1 Polymorphisms and Risk of Idiopathic Membranous Nephropathy

BACKGROUND AND OBJECTIVES Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. RESULTS This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. CONCLUSION This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

Insight into response to mTOR inhibition when PKD1 and TSC2 are mutated

BackgroundMutations in TSC1 or TSC2 cause the tuberous sclerosis complex (TSC), while mutations in PKD1 or PKD2 cause autosomal dominant polycystic kidney disease (ADPKD). PKD1 lays immediately adjacent to TSC2 and deletions involving both genes, the PKD1/TSC2 contiguous gene syndrome (CGS), are characterized by severe ADPKD, plus TSC. mTOR inhibitors have proven effective in reducing angiomyolipoma (AML) in TSC and total kidney volume in ADPKD but without a positive effect on renal function.Methods and resultsWe describe a patient with independent truncating PKD1 and TSC2 mutations who has the expected phenotype for both diseases independently instead of the severe one described in PKD1/TSC2-CGS. Treatment with mTOR inhibitors reduced the AML and kidney volume for 2 years but thereafter they resumed growth; no positive effect on renal function was seen throughout. This is the first case addressing the response to mTOR treatment when independent truncating mutations in PKD1 and TSC2 are present.ConclusionsThis case reveals that although PKD1 and TSC2 are adjacent genes and there is likely cross-talk between the PKD1 and TSC2 signalling pathways regulating mTOR, having independent TSC2 and PKD1 mutations can give rise to a milder kidney phenotype than is typical in PKD1/TSC2-CGS cases. A short-term beneficial effect of mTOR inhibition on AML and total kidney volume was not reflected in improved renal function.

A kidney-disease gene panel allows a comprehensive genetic diagnosis of cystic and glomerular inherited kidney diseases

Molecular diagnosis of inherited kidney diseases remains a challenge due to their expanding phenotypic spectra as well as the constantly growing list of disease-causing genes. Here we develop a comprehensive approach for genetic diagnosis of inherited cystic and glomerular nephropathies. Targeted next generation sequencing of 140 genes causative of or associated with cystic or glomerular nephropathies was performed in 421 patients, a validation cohort of 116 patients with previously known mutations, and a diagnostic cohort of 207 patients with suspected inherited cystic disease and 98 patients with glomerular disease. In the validation cohort, a sensitivity of 99% was achieved. In the diagnostic cohort, causative mutations were found in 78% of patients with cystic disease and 62% of patients with glomerular disease, mostly familial cases, including copy number variants. Results depict the distribution of different cystic and glomerular inherited diseases showing the most likely diagnosis according to perinatal, pediatric and adult disease onset. Of all the genetically diagnosed patients, 15% were referred with an unspecified clinical diagnosis and in 2% genetic testing changed the clinical diagnosis. Therefore, in 17% of cases our genetic analysis was crucial to establish the correct diagnosis. Complex inheritance patterns in autosomal dominant polycystic kidney disease and Alport syndrome were suspected in seven and six patients, respectively. Thus, our kidney-disease gene panel is a comprehensive, noninvasive, and cost-effective tool for genetic diagnosis of cystic and glomerular inherited kidney diseases. This allows etiologic diagnosis in three-quarters of patients and is especially valuable in patients with unspecific or atypical phenotypes.

Autosomal Dominant Polycystic Kidney Disease: Clinical Assessment of Rapid Progression

Background: Autosomal dominant polycystic kidney disease (ADPKD) causes the development of renal cysts and leads to a decline in renal function. Limited guidance exists in clinical practice on the use of tolvaptan. A decision algorithm from the European Renal Association-European Dialysis and Transplant Association (ERA-EDTA) Working Groups of Inherited Kidney Disorders and European Renal Best Practice (WGIKD/ERBP) has been proposed to identify candidates for tolvaptan treatment; however, this algorithm has not been assessed in clinical practice. Methods: Eighteen-month cross-sectional, unicenter, observational study assessing 305 consecutive ADPKD patients. The ERA-EDTA WGIKD/ERBP algorithm with a stepwise approach was used to assess rapid progression (RP). Subsequently, expanded criteria based on the REPRISE trial were applied to evaluate the impact of extended age (≤55 years) and estimated glomerular filtration rate (eGFR; ≥25 mL/min/1.73 m2). Results: Historical eGFR decline, indicative of RP, was fulfilled in 26% of 73 patients who were candidates for RP assessment, mostly aged 31–55 years. Further tests including ultrasound and MRI measurements of kidney volume plus genetic testing enabled the evaluation of the remaining patients. Overall, 15.7% of patients met the criteria for rapid or likely RP using the algorithm, and the percentage increased to 27% when extending age and eGFR. Conclusions: The ERA-EDTA WGIKD/ERBP algorithm provides a valuable means of identifying in routine clinical practice patients who may be eligible for treatment with tolvaptan. The impact of a new threshold for age and eGFR may increase the percentage of patients to be treated.

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS)

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing.

Integration-free induced pluripotent stem cells derived from a patient with autosomal recessive Alport syndrome (ARAS)

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing.