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Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

Background A considerable minority of patients on waiting lists for kidney transplantation either have no diagnosis (and fall into the subset of undiagnosed cases) because kidney biopsy was not performed or histological findings were non-specific, or do not fall into any well-defined clinical category. Some of these patients might be affected by a previously unrecognised monogenic disease. Methods Through a multidisciplinary cooperative effort, we built an analytical pipeline to identify patients with chronic kidney disease (CKD) with a clinical suspicion of a monogenic condition or without a well-defined diagnosis. Following the stringent phenotypical and clinical characterization required by the flowchart, candidates meeting these criteria were further investigated by clinical exome sequencing followed by in silico analysis of 225 kidney-disease-related genes. Results By using an ad hoc web-based platform, we enrolled 160 patients from 13 different Nephrology and Genetics Units located across the Piedmont region over 15 months. A preliminary “remote” evaluation based on well-defined inclusion criteria allowed us to define eligibility for NGS analysis. Among the 138 recruited patients, 52 (37.7%) were children and 86 (62.3%) were adults. Up to 48% of them had a positive family history for kidney disease. Overall, applying this workflow led to the identification of genetic variants potentially explaining the phenotype in 78 (56.5%) cases. Conclusions These results underline the importance of clinical exome sequencing as a versatile and highly useful, non-invasive tool for genetic diagnosis of kidney diseases. Identifying patients who can benefit from targeted therapies, and improving the management of organ transplantation are further expected applications.

Background and Aims Autosomal dominant PKD determines formation of multiple cysts predominantly in the kidneys and usually becomes symptomatic during adulthood and can lead to renal failure. In contrast, in autosomal recessive PKD cysts occur in both the kidneys and the liver and usually presents an earlier onset. Obtaining genetic diagnosis is important to confirm clinical diagnosis and is required before treating with vasopressin 2 receptor blockers, which are the only drugs known to slow down the disease. Furthermore, in the case of kidney transplant from a living family member it is essential to exclude the presence of the mutation in the donor. We used clinical exome sequencing to provide genetic diagnosis to a cohort of patients with a clinical suspicion of PKD. Method 175 patients were referred to the Immunogenetics and Transplant Biology Service of the Turin University Hospital through a network of nephrology centers operating in the Piedmont region. Some patients were referred following genetic counseling. All patients signed an informed consent and the referring physicians provided relevant clinical data. DNA from eligible patients was extracted, checked for integrity, quantified and used for library preparation. A clinical exome sequencing (CES) kit by Illumina was used, allowing the analysis of 6,700 clinically relevant genes. Results Out of the 175 recruited patients eligible for CES, 38 (21.7%) had a clinical suspicion or diagnosis of PKD, with 50% of them presenting family history. The majority of the cohort was represented by male subjects (60.5%) and included both children (34.2%) and adults. The analytical approach was based on initial analysis of genes responsible for PKD (PKD1, PKD2 and PKHD1). If no mutation could be identified, analysis was then extended to a panel of 99 genes responsible for ciliopathies. This approach led to the identification of causative variants in 33/38 (86.8%) of the PKD cohort, while no variant could be identified in 5/38 patients. In 5/33 (15.2%) patients, mutations were inconclusive as found in heterozygosity in genes known to have an autosomal recessive mode of inheritance, while 27/33 (81.8%) were in line with the initial clinical suspicion/diagnosis. Of these, the majority was represented by missense mutations (12), followed by frameshift and nonsense mutations (6 each) and 3 splicing variants. As expected, the majority of mutations were found in PKD1 17/27 (63%), PKD2 3/27 (11.1%) and PKHD1 2/27 (7.4%). In these two latter patients, variants were found as compound heterozygosity. We also found mutations in other genes known to cause cysts, including TSC2 and CPT2. Of note, in 7 patients carrying PKD1 mutations, we found a second variant in PKD1 or PKHD1. Interestingly, when looking at patients characterized by kidney failure but lacking a clinical suspicion at recruitment or diagnosed with other phenotypes (66/175), we found variants in PKD1 and in PKD2 in 11 patients (9 and 2, respectively). Of all identified variants in PKD1, PKD2 and PKHD1 genes, 17.6% were annotated as pathogenic (C5), 41.2% were likely pathogenic (C4) and 41.2% were variants of unknown significance (C3). 19 variants in these genes were not previously reported. All the variants found in genes responsible for PKD were validated and confirmed by Sanger sequencing. Family segregation studies are ongoing. Finally, it is worth mentioning that in a portion of cases (5/38) with clinical and phenotypic features of PKD, supported also by a positive family history, we could not detect mutations in causative genes. These results may be explained by the presence of intronic variants, in line with data reported in literature. Conclusion These results demonstrate that CES may be applied to PKD patients to identify causative variants during their routine diagnostic flow. Furthermore, CES may be a useful tool to detect mutations in PKD-related genes in patients with undiagnosed diseases, considering its rapidly decreasing costs.