Efficient Targeted Next Generation Sequencing-Based Workflow for Differential Diagnosis of Alport-Related Disorders.

Alport syndrome (AS) is an inherited type IV collagen nephropathies characterized by microscopic hematuria during early childhood, the development of proteinuria and progression to end-stage renal disease. Since choosing the right therapy, even before the onset of proteinuria, can delay the onset of end-stage renal failure and improve life expectancy, the earliest possible differential diagnosis is desired. Practically, this means the identification of mutation(s) in COL4A3-A4-A5 genes. We used an efficient, next generation sequencing based workflow for simultaneous analysis of all three COL4A genes in three individuals and fourteen families involved by AS or showing different level of Alport-related symptoms. We successfully identified mutations in all investigated cases, including 14 unpublished mutations in our Hungarian cohort. We present an easy to use unified clinical/diagnostic terminology and workflow not only for X-linked but for autosomal AS, but also for Alport-related diseases. In families where a diagnosis has been established by molecular genetic analysis, the renal biopsy may be rendered unnecessary.

Collagen type IV nephropathy: genetic heterogeneity examinations in affected Hungarian families.

The Col4A3, Col4A4 and Col4A5 collagen type IV genes are found to be mutated in Col IV nephropathy. In males with a mutation in the Col4A5 gene (X-linked Alport syndrome: XL-AS), progressive renal disease always develops. Female carriers with a mutation in the Col4A5 gene can develop thin basement membrane nephropathy (TBMN). Males and females who carry 1 Col4A3 or Col4A4 mutation usually manifest TBMN with nonprogressive hematuria. In the event of 2 Col4A3 or Col4A4 gene mutations, the autosomal recessive AS will develop. We examined the cosegregation pattern of hematuria in 20 families. The renal biopsies led to diagnoses of AS in 7 families, and of TBMN in 6 families. In 7 others, the diagnosis of familial hematuria (FHU) was based on the clinical symptoms. Markers of the ColA3/Col4A4 and Col4A5 loci (Col4A3: CA11 and D2S401; Col4A4: HaeIII/RFLP; and Col4A5: DXS456, 2B6 and 2B20) were used to assess their linkage to the clinical symptoms and morphological alterations. Maximum likelihood and the FASTLINK version of the linkage program were applied to compute logarithm of the odds (LOD) scores. A linkage to the Col4A3/Col4A4 genes was identified in 5 families (FHU in 3, AS in 2 families, 25%, LOD score range: 0.20-3.51). The XL-AS pattern of inheritance seemed likely with Col4A5 in 9 families (45%, LOD: 0.43-4.20); we found 4 disease-causative mutations by high-resolution melting curve analysis (LC480) and sequencing in this group. In 2 FHU families, the linkage to chromosomes 2 and X was precluded. Knowledge of the genetic background of Col IV nephropathy is essential to avoid the misdiagnosis of FHU and early AS. The allele frequencies, heterozygosity content and polymorphism information content of the applied STR markers on unrelated Hungarian normal and affected chromosomes 2 and X were also calculated.

[Liver support systems today]. / A májpótló kezelés mai lehetoségei.

Liver failure carries a high mortality, both the acute type with no pre-existing liver disease (acute liver failure) and the acute decompensation superimposed on a chronic liver disorder (acute on chronic liver failure). Today, liver transplantation still represents the only curative treatment for liver failure due to end-stage liver diseases. Donor organ shortage is still the major limitation and many patients die while awaiting transplantation. Due to the scarcity of donor organs, liver support technologies are being developed to support patients with severe liver failure until either an organ becomes available for transplantation or their livers recover from injury. Early devices including hemodialysis, hemoperfusion, exchange transfusion, cross-hemodialysis, cross-circulation and plasmapheresis appeared inefficient. In the present day, liver support systems' designs fall into two main categories: cell-based, so-called bioartificial and non-cell-based, also known as artificial systems. Bioartificial liver support systems use either porcine hepatocytes or human hepatoma cell lines housed within a hollow-fiber bioreactor. The system perfuses the patient's whole blood or separated plasma through the luminal space in the bioreactor. Theoretically, these methods should optimally resemble normal hepatic tissue structure and function. However, the existing bioartificial systems are far from ideal solution in terms of immunological, infectological, oncological and financial problems and must still be thought of as experimental. The artificial systems are already available for the clinicians in limited quantities. These non-cell-based devices are intended to remove protein-bound and water-soluble toxins without providing synthetic function, which can be partially replaced with substitution of the failing substances (plasma proteins, coagulation factors). These systems include the hemodiabsorption (Liver Dialysis Unit) which is commercially available in the United States, the albumin dialysis which is available in Europe and the newly developed fractionated plasma separation and adsorption (FPSA) system. The simple method of albumin dialysis is "single pass albumin dialysis" (SPAD), which evolved into the so-called "molecular adsorbent recirculating system"(MARS). Prometheus system combines the FPSA method with high-flux hemodialysis. Although the results of many experimental and clinical trials prove the efficacy of the above mentioned methods, large randomized controlled trials are mandatory to establish the impact on survival benefit of artificial and bioartificial support systems versus standard therapy.

[Therapeutic effect of the latest extracorporal elimination procedure (Prometheus treatment) in acute liver failure caused by intoxication]. / A legújabb extracorporalis eliminációs eljárás (Prometheus-kezelés) terápiás hatékonysága mérgezés okozta akut májelégtelenségben.

INTRODUCTION: Despite intensive therapy the mortality of acute liver failure without organ transplantation is 60-90%. Because of organ shortage in liver transplantation, a significant number of patients dies while being on the waiting list. In order to diminish the mortality, various trials were introduced to remove the albumin-bound and water-soluble toxins in liver failure with the aim to support the spontaneous regeneration of the liver and maintaining the patients alive until liver transplantation. Prometheus treatment is a relatively new technique combining Fractionated Plasma Separation and Adsorption (FPSA) with a high-flux dialysis. During the procedure the patient's own separated albumin-rich plasma passes through special adsorbents making possible the elimination of albumin-bound toxins, while hemodialysis gets rid of water-soluble toxins. AIM: The authors' intention was to demonstrate the efficiency of Prometheus treatment in acute liver failure caused by intoxication. PATIENTS AND METHOD: Prometheus treatment was indicated in three patients who suffered from severe intoxication with paracetamol, potassium permanganate and Amanita phalloides, which resulted in a hepatic failure incurable with conservative therapy. RESULTS: Ten treatments were performed in the three female patients. No serious complication was observed. Due to the treatment the albumin-bound (indirect bilirubin p = 0.048; bile acid p = 0.001) and water-soluble (direct bilirubin p = 0.002; creatinine p = 0.007) toxins were significantly decreased. The level of ammonia, urea nitrogen, fibrinogen and antithrombin III did not change significantly. All the three patients were cured without liver transplantation. CONCLUSION: Prometheus treatment removes efficiently the accumulating toxins in acute liver failure. It is a safe elimination technique. In cases untreatable with conservative therapy it makes possible maintaining the patients alive until the liver regenerates spontaneously, or liver transplantation is feasible.