Knoers, N. V. A. M. | bioRxiv Science

bioRxiv2023

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

Leukemias are genetically heterogeneous, and knowing the type of genetic aberration is clinically important for progression prognosis and choice of treatment. The standard-of-care (SOC) methods in leukemia diagnostics are karyotyping, SNP-array and FISH. Optical genome mapping (OGM) has emerged as their replacement as it can detect different types of structural aberrations simultaneously. However, although OGM can additionally detect much smaller aberrations (500 bp vs. 5-10 Mb with karyotyping), its resolution is too low to define the location of aberrations between two labels and the breakpoints are disputable when labels are not distinct enough. Here, we test whether Cas9-directed long-read sequencing (LRS) can fill this gap. From an internal Bionano implementation study we selected ten OGM calls either located in low resolution areas or with disputable breakpoints that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION / GridION device. We could confirm all the OGM aberrations with Cas9-directed LRS and the actual breakpoints of the OGM calls were located between 0.2-5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. We show that Cas9-directed LRS can fill the gap of low resolution OGM areas thereby improving the prediction of clinical significance. We envisage that this technique can be an relevant secondary technique in diagnostic workflows including OGM.

genetics↗

bioRxiv2021

mTOR-activating mutations in RRAGD cause kidney tubulopathy and cardiomyopathy (KICA) syndrome

BackgroundOver the last decaces, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, {+/-}20% of all tubulopathy patients remain without genetic diagnosis. Here, we explore a large multicentric patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia and dilated cardiomyopathy (DCM). MethodsWhole exome and genome sequencings were performed with various subsequent functional analyses of identified RRAGD variants in vitro. ResultsIn 8 children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt-wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients additionally suffered from DCM requiring heart transplantation in 3 of them. An additional dominant variant in RRAGD was simultaneously identified in eight members of a large family with a similar renal phenotype. RRAGD encodes GTPase RagD mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron include the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro, ConclusionsOur findings establish a novel disease phenotype combining kidney tubulopathy and cardiomyopathy (KICA) caused by an activation of mTOR signaling suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function. SIGNIFICANCE STATEMENTHere, we report on heterozygous variants in RRAGD in patients with profound hypomagnesemia, renal salt wasting, nephrocalcinosis, and dilated cardiomyopathy. The identified RagD variants induce a constitutive activation of mTOR signaling in vitro. These findings not only establish a novel monogenic disorder of the kidney tubule, but demonstrate the essential role of mTOR signaling for distal tubular electrolyte handling and cardiac function.

genetics↗