Genomic profiling in rare kidney disease

Rare diseases (RD), generally defined by an incidence of less than 1:2000, affect about 3-6% of the population. To date, over 600 different genetic kidney diseases have been identified. Most of them with the exception of autosomal dominant polycystic kidney disease (ADPKD) are rare to ultrarare disorders. Due to their rarity and often genetic heterogeneity, analysis is difficult and diagnosis is frequently delayed. Clinically, (rare) kidney diseases (RKD) are mainly divided into the following categories: Congenital or developmental kidney and urogenital tract malformations, electrolytes or metabolic disorder, glomerular disease, secondary renal, hereditary renal cancer syndromes, or tubulointerstitial kidney disease. Steroid-resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS), are leading causes of end-stage renal disease (ESRD) in children, adolescents, and adults. Although several SRNS genes could be identified mostly in younger children, the genetic basis of SRNS/FSGS in adolescents and adults is far from being completely understood. Reliable discrimination of genetic versus non-genetic forms is an imperative as the identification of monogenic rare kidney disease has numerous implications in a precision medicine setting. Currently, the predominant application of short-read based sequencing techniques results in preferential detection of point mutations and small-sized deletions/insertions while larger structural aberrations (large deletions/insertions), gene rearrangements, and mutations in homologous or repetitive regions frequently escape detection. This project combines short-read based high throughput next-generation sequencing (GPS/WES/WGS) with unparalleled structural variant analyses to overcome previous limitations in genetic analyses. Clinically relevant examples, that such structural variants (SV) are important in rare kidney diseases, are complement activation gene cluster (RCA; chromosome1q32) in atypical haemolytic uremic syndrome (aHUS) as well as the deletion of the chloride channel ClC-Kb associated with Bartter syndrome type 3. The major goal of this project is to unravel the genetic basis of genetic forms of RKD like SRNS/FSGS, aHUS, and tubulopathies and to define a pipeline for the molecular genetic analyses of rare kidney diseases as a best-practice clinical routine at the University Hospital of Cologne. Specifically, my aim was to perform profound genome analyses using biosamples from a cohort of paediatric and adult SRNS/FSGS patients, that have been collected over the last years and integrate this with already existing genetic data. By a comprehensive genomic approach, we aim to improve the diagnostics of chronic kidney disease, enhance our IV understanding of the underlying pathomechanisms, and contribute to practice-changing discoveries by precision diagnostics, that allow individualized therapies

Long-read sequencing identifies a common transposition haplotype predisposing for CLCNKB deletions

BACKGROUND: Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. METHODS: Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. RESULTS: We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16×10-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. CONCLUSIONS: The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general

Unraveling structural rearrangements of the CFH gene cluster in atypical hemolytic uremic syndrome patients using molecular combing and long-fragment targeted sequencing

Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging due to its high degree of sequence homology. Following first-line NGS gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology, to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome (aHUS) and known SVs, and 18 patients with aHUS or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to NGS was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease

Germline C1GALT1C1 mutation causes a multisystem chaperonopathy

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients

Unstable TTTTA/TTTCA expansions in MARCH6 are associated with Familial Adult Myoclonic Epilepsy type 3

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements