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 We successfully identified mutations in all investigated cases, including 14 unpublished mutations in our Hungarian cohort.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.

Long-term follow-up after cyclophosphamide and cyclosporine-A therapy in steroid-dependent and -resistant nephrotic syndrome.

A retrospective study was made on 37 children with idiopathic nephrotic syndrome (INS). At the beginning, all patients were steroid sensitive but received more than one steroid course (median 4). Following several relapses, they became steroid dependent or steroid resistant. Group 1 consisted of 22 children [3 focal segmental glomerulosclerosis (FSGS), 19 minimal-change NS (MCNS)] who received cyclophosphamide (CP) orally for 2.5 +/- 0.5 months. Group 2 consisted of 15 children (7 FSGS, 8 MCNS) who received cyclosporine-A (CSA) for 28 +/- 15 months. The level of proteinuria decreased significantly and remained low during the follow-up. The relapse-free period was significantly longer in the CP group (CP 30 +/- 21.5; CSA 26.2 +/- 18 months, p < 0.001). The relapse rate decreased significantly in both groups and remained in this lower level during the follow-up (from 3.4 +/- 2.8 to 0.1 +/- 0.2/year in group 1, and from 3.7 +/- 3.1 to 0.6 +/- 0.8/year in group 2). At the end of the 5-year follow-up, 20/22 patients (90.9%) and 10/15 patients (66.6%) were in remission in groups 1 and 2 respectively, with or without treatment (p < 0.05). In the long term, both CP and CSA is effective second-line therapy following steroid monotherapy in INS patients, but the relapse rate was lower and the relapse free period was significantly longer in the CP-treated group.

Roles of paraoxonase and oxidative stress in adolescents with uraemic, essential or obesity-induced hypertension.

BACKGROUND/AIMS: Paraoxonase 1 (PON1) is associated with high-density lipoproteins in the plasma, and is capable of hydrolysing oxidized lipids and preventing the oxidation of low-density lipoproteins. Oxidative stress and the PON1 (activity and Q192R polymorphism) were analysed in adolescent patients with essential (n = 49) or obesity-induced hypertension (n = 79), uraemic patients (n = 20), and also in obese normotensive patients (n = 60) and age-matched controls (n = 57). METHODS: The PON1 activity was measured via paraoxon hydrolysis. The PON1 genotype was determined by real-time PCR. The levels of oxidized and reduced glutathione, the end-products of nitric oxide, cysteine, homocysteine and lipid peroxidation in the plasma were measured and related to the PON1 status. RESULTS: There were no significant differences between the patient groups and the control group in the genotype distributions and the allele frequencies of the Q192R polymorphism. The PON activity was significantly lower (p < 0.001) in the uraemic hypertensive group than in the controls. The MDA concentration was significantly higher in the uraemic hypertensive (p < 0.001) and obese hypertensive (p < 0.05) patients. The plasma NOx concentrations were significantly lower (p < 0.001) and the ratio MDA/NOx were significantly higher in all four patient groups. The GSH levels were significantly lower in the patients with hypertension (p < 0.001) and obesity-induced hypertension (p < 0.05) than in the controls, while the GSSG level (p < 0.01) and the ratio GSSG/GSH (p < 0.05) was significantly higher in the uraemic hypertensive group. The plasma homocysteine level was significantly higher (p < 0.001) in the uraemic hypertensive patients as compared with the controls. CONCLUSIONS: We found no significant correlation between the biochemical parameters and neither genotypes nor enzyme activities. The PON1 status and the levels of certain biochemical parameters are independently associated with the hypertension in hypertensive and obese hypertensive patients, and the elevated levels of lipid peroxides and plasma homocysteine may contribute to the increased risk of cardiovascular complications in patients on haemodialysis.

Different pathomechanisms of essential and obesity-associated hypertension in adolescents.

Obesity-induced hypertension and essential hypertension in lean patients are two different forms of hypertension. The main goal of this study was to test whether there are differences in biochemical parameters between subjects with obesity-associated hypertension and those with essential hypertension. We examined whether the biochemical responses to angiotensin-converting enzyme inhibitor (ACEI) ramipril therapy reveal properties of these two conditions that might explain the differences in clinical outcome. Before ramipril therapy, the hypertensive group exhibited increases in ACE activity (p<0.05), plasma malondialdehyde (MDA) concentration and the malondialdehyde/nitric oxide end-product ratio (MDA/NO(x)) (p<0.05), and decreases in xanthine oxidase (XO) activity (p<0.05) and plasma nitric oxide end-product (NO(x)) level (p<0.01). Before medication, plasma endothelin-1 (ET-1), plasma leptin, and leptin receptor levels were normal. Following ramipril treatment, ACE activity normalized. Before ACE inhibitor treatment, the obese-hypertensive group exhibited elevated levels of plasma ET-1 (p<0.05), plasma leptin (p<0.01), XO activity (p<0.05), plasma MDA and MDA/NO(x) (p<0.05), and reduced levels of plasma NO(x)(p<0.01) and leptin receptors (p<0.001). Following medication, the plasma NO(x) level, MDA/NO(x), and XO activity returned to normal while ACE activity decreased (p<0.001). In patients with essential hypertension, NO availability and ACE activity, and in those with obesity-associated hypertension, hyperleptinemic effects, NO level, endothelin-1 concentration and XO activity, may be important factors in the pathology.

[Collagen type IV nephropathy: from thin basement membrane nephropathy to Alport syndrome]. / IV-es típusú kollagén nephropathiák: a vékony bazális membrán nephropathiától az Alport-szindrómáig.

INTRODUCTION: Collagen type IV nephropathy includes the Goodpasture syndrome, thin basement membrane nephropathy and the Alport syndrome. Mutations in the coding Col(IV)A3/A4 and Col(IV)A5 genes are probable causes of the latter two. Thin basement membrane nephropathy is mostly familial and has an autosomal dominant inheritance, at least 40% of the families have hematuria that co-segregates with the Col(IV)A3 and/or Col(IV)A4 loci. 85% of Alport syndrome cases are transmitted as an X-linked semidominant form due to Col(IV)A5 mutations. About 14% of Alport syndrome cases exhibit autosomal recessive, and 1% autosomal dominant inheritance, both caused by mutations in the Col(IV)A3 or Col(IV)A4 genes in boys and in girls. AIM: The co-segregation pattern of hematuria was examined in two families with thin basement membrane nephropathy and one family with the Alport syndrome, using short tandem repeat markers, spanning the Col(IV)A3/A4 and Col(IV)A5 loci to assess their linkage to the clinical symptoms and morphological alterations in the renal biopsy specimens. METHODS: Markers: Col(IV)A3: CAll and D2S401; Col(IV)A4: HaeIII/RFLP; and Col(IV)A5: DXS456, 2B6 and 2B20. RESULTS: The hematuria displayed autosomal dominant inheritance and co-segregated with Col(IV)A3 markers in one of the thin basement membrane nephropathy families. In the second, the hematuria did not segregate with the Col(IV)A3/A4 or Col(IV)A5 loci, suggesting the possibility of another genetic locus for the disease. The Alport syndrome exhibited autosomal recessive inheritance and did not link to Col(IV)A5 markers, and the Col(IV)A3/A4 markers were informative only in part. CONCLUSION: Knowledge of the inheritance and genetic background of collagen type IV nephropathy will be very important in the diagnostics and genetic counseling in the future.

Long-term effects of levamisole treatment in childhood nephrotic syndrome.

The effects of levamisole treatment on long-term outcome were evaluated in a retrospective study of frequently-relapsing (FRNS, n=15), steroid-dependent (SDNS, n=13), and steroid-resistant (SRNS, n=6) nephrotic syndrome in 34 children (21 boys, 13 girls, mean age 5.0+/-3.4 years) during a 60-month follow-up period. The definition of frequent relapses was > or = 4 relapses per year. The current relapse was treated with prednisolone 60 mg/m2 per day for 4 weeks, then with 40 mg/m2 every other day for 4 weeks, after which the dose was tapered by 10 mg weekly. From the beginning of the 5th week, levamisole was introduced at a dose of 2 mg/kg per day. The duration of levamisole treatment was 17+/-7 months. Before starting levamisole treatment the mean level of proteinuria was 2.17+/-1.34 g/day and the relapse rate was 4.41/year. By the end of levamisole therapy, proteinuria had fallen to 0.142+/-0.211 g/day and the relapse rate to 0.41/year. No relapse occurred in 23 of the 34 patients during levamisole treatment. In the 24-month follow-up period after the discontinuation of levamisole, 28 children remained in total remission, while 6 had relapses. The cumulative steroid dose before levamisole therapy was 7,564.4+/-3,497.1 mg/year and following the introduction of levamisole 1,472.9+/-1,729.9 mg/year (P<0.0001). We observed reversible neutropenia in 5 patients, but no other side effects were seen. Our findings suggest that in FRNS and SDNS levamisole significantly reduces both the relapse rate and the cumulative steroid dose; therefore, it could be recommended for these patients. In SRNS patients it has also some benefit because proteinuria and the cumulative steroid dose could be reduced significantly.

Platelet aggregation, blood viscosity and serum lipids in hypertensive and obese children.

UNLABELLED: A group of 35 patients (median age 15.5 years, range 8-17 years) with juvenile essential hypertension, 15 with body mass index (BMI kg/m(2)) <25 and 20 with BMI >25, as well as 35 age and sex matched controls (BMI <25 n=20; BMI >25 n=15) were investigated to study the role of hypertension and obesity, separately and in combination, on in vitro platelet aggregation, blood and plasma viscosity, plasma lipid concentrations and lipid peroxidation as well as nitric oxide (NO) production. Obese children (hypertensive and controls) had significantly higher concentrations of total cholesterol and triglycerides. The levels of high density lipoprotein (HDL)-cholesterol were lower in obese hypertensive children than their non-obese counterparts. There was a significant increase in platelet aggregation and a decrease in NO levels in hypertensive patients (obese and non-obese) reflecting a significant negative correlation (r=-0.553 and -0.530, n=35; P<0.01, respectively). However, an increased tendency to aggregation was also evident in obese normotensive patients. A significant positive correlation was observed between the platelet aggregation and BMI (r=0.501, n=35; P<0.01). Plasma free thiols were decreased in hypertensive children independent of their BMI. An increased lipid peroxidation and higher blood and plasma viscosity were found only in obese patients with hypertension. Multivariate analysis revealed significant interactions in the effects of obesity and hypertension on platelet aggregation and thiol oxidation. CONCLUSION: in obese children an increased platelet aggregation and oxidative insult contribute to the development of hypertension and to the promotion of vascular damage.

Renin-angiotensin gene polymorphism in children with uremia and essential hypertension.

The M235T polymorphism of the angiotensinogen (ANG) gene, the I/D polymorphism of the angiotensin converting enzyme (ACE) gene, and the A1166C polymorphism of the angiotensin II type 1 receptor (AT1R) gene were identified in 70 patients with end-stage renal disease [20 pediatric ESRD, aged 14.9+/-3.1, years blood pressure (BP) 139+/-14/91+/-13 mmHg, 50 adult ESRD, aged 48.7+/-18.7 years, BP 149.1+/-24/96.9+/-12 mmHg], 35 with juvenile essential hypertension (JEHT, aged 14.4+/-2.7 years, 24-h mean BP 135.37+/-7.37/72.4+/-7.68 mmHg), 130 adult healthy normotensive controls (aged 34.9+/-8.1 years, BP 117.8+/-8.7/78.7+/-8.5 mmHg), and 20 pediatric controls (aged 13.2+/-1.2 years, BP 109+/-6.5/71+/-5.9 mmHg). The ACE gene polymorphism was determined by polymerase chain reaction and the ANG and AT1R gene polymorphisms by single-step LightCycler technology. The ACE gene distribution of the Hungarian controls did not differ from the results of the other Caucasian populations. In JEHT and pediatric ESRD patients, the MT genotype of ANG was more frequent than in controls (JEHT 80%, pediatric ESRD 74% versus controls 50%, P<0.02). The DD genotype of ACE was over-represented in pediatric ESRD compared with controls (ESRD 45% versus controls 22%, P<0.05). There was a non-significant increase in the CC genotype frequency of AT1R in adult patients with ESRD compared with controls. In conclusion, there was an increased frequency of the ACE DD genotype in pediatric ESRD, which could be a genetic risk factor for the development of ESRD. Furthermore, there was a significant increase in MT genotype frequency of ANG M235T polymorphism in pediatric ESRD and JEHT. The role of AT1R gene polymorphism needs further investigation.

ACE gene polymorphism and renal scarring in primary vesicoureteric reflux.

The objective of this study was to investigate whether mutations of the renin-angiotensin system genes are involved in primary vesicoureteric reflux (VUR) and VUR-associated renal scarring. The M235T polymorphism of the angiotensinogen ( ATG) gene, the I/D polymorphism of the angiotensin converting enzyme ( ACE) gene, and the A1166C polymorphism of the angiotensin II type 1 receptor ( AT1) gene were identified in 77 patients with primary VUR (aged 6.9+/-3.2 years, mean+/-SD) and 80 healthy controls (aged 33+/-7 years). Thirty-eight of the 77 VUR patients had low-grade VUR (grade I-III) and 39 had high-grade VUR (grade IV and V). Renal scarring was found in 43 VUR patients, while 34 patients had normal kidneys on dimercaptosuccinic acid scan. The ACE gene polymorphism was determined by polymerase chain reaction and the ATG and AT1 gene polymorphisms were determined by single-step LightCycler technology. We found significant over-representation of the DD genotype in patients with renal scarring (44 %) compared with normal controls (23%, P<0.05) and patients with no scar formation (21%, P<0.05). Significantly higher D and significantly lower I allele frequencies were present in VUR patients with scarred kidneys (D allele 0.64 and I allele 0.36) compared with controls (D allele 0.53 and I allele 0.47, P<0.05) and patients with unscarred kidneys (D allele 0.4 and I allele 0.6, P<0.05). No differences in the ATG and AT1 genotype distributions and allele frequencies were observed in VUR patients compared with the normal population. The DD genotype and D allele of ACE may be a genetic susceptibility factor contributing to scar formation in VUR. We detected no linkage of genetic polymorphisms of ATG and AT1 to VUR and VUR-associated renal scarring.