Frequent COL4 mutations in familial microhematuria accompanied by later-onset Alport nephropathy due to focal segmental glomerulosclerosis.

Familial microscopic hematuria (FMH) is associated with a genetically heterogeneous group of conditions including the collagen-IV nephropathies, the heritable C3/CFHR5 nephropathy and the glomerulopathy with fibronectin deposits. The clinical course varies widely, ranging from isolated benign familial hematuria to end-stage renal disease (ESRD) later in life. We investigated 24 families using next generation sequencing (NGS) for 5 genes: COL4A3, COL4A4, COL4A5, CFHR5 and FN1. In 17 families (71%), we found 15 pathogenic mutations in COL4A3/A4/A5, 9 of them novel. In 5 families patients inherited classical AS with hemizygous X-linked COL4A5 mutations. Even more patients developed later-onset Alport-related nephropathy having inherited heterozygous COL4A3/A4 mutations that cause thin basement membranes. Amongst 62 heterozygous or hemizygous patients, 8 (13%) reached ESRD, while 25% of patients with heterozygous COL4A3/A4 mutations, aged >50-years, reached ESRD. In conclusion, COL4A mutations comprise a frequent cause of FMH. Heterozygous COL4A3/A4 mutations predispose to renal function impairment, supporting that thin basement membrane nephropathy is not always benign. The molecular diagnosis is essential for differentiating the X-linked from the autosomal recessive and dominant inheritance. Finally, NGS technology is established as the gold standard for the diagnosis of FMH and associated collagen-IV glomerulopathies, frequently averting the need for invasive renal biopsies.

Pan-cytokeratin markers for rapid frozen section immunocytochemistry from head and facial Mohs cases of basal cell carcinoma: a comparison and evaluation to determine the marker of choice.

The application of immunocytochemistry in the field of Mohs micrographic surgery (MMS) is well established. This study evaluates the use of pan-cytokeratins (AE1/AE3, MNF116 and AE1/AE3+PCK26) in the assessment of basal cell carcinoma (BCC) on frozen tissue debulk specimens. Fifty-five cases of BCC, all from head and facial sites, were assessed in the study. In addition to staining all cases for the three cytokeratin antibodies under investigation, sections were also stained with haematoxylin and eosin (H&E) to demonstrate tumour architecture and morphology. All sections for immunocytochemistry were stained on a Roche Ventana BenchMark Ultra automated platform employing a rapid frozen section protocol. Results were assessed based on the intensity of staining of keratinocytes (scale: 0-100%), as well as sensitivity of staining determined by the total percentage of keratinocytes stained within the tissue section. AE1/AE3 demonstrated the most consistent staining both in terms of intensity of staining and sensitivity, with a mean of 99.1% and 99.9%, respectively. AE1/AE3+PCK26 average results indicated scores of 70.6% for intensity and 87.2% for sensitivity, with MNF116 scoring 92.9% for intensity but only 57.3% for sensitivity. The data indicate that AE1/AE3 is the best pan-cytokeratin antibody to use in the assessment of BCC in MMS. The use of cytokeratin immunocytochemistry is justified in morphologically complex cases of BCC, or in cases where dense inflammatory infiltrate surrounding any suspicious cells make identification of small numbers of tumour cells difficult to determine with just an H&E stain. The significant rationale is that cytokeratin staining is a valuable adjunct in the study of tumour cell assessment in cases of MMS for BCC. In addition, the use of anti-AE1/AE3 cytokeratin antibodies provides the most consistent staining results for such cases.

X-linked Alport syndrome in Hellenic families: phenotypic heterogeneity and mutations near interruptions of the collagen domain in COL4A5.

The X-linked Alport syndrome (ATS) is caused by mutations in COL4A5 and exhibits a widely variable expression. Usually ATS is heralded with continuous microhematuria which rapidly progresses to proteinuria, hypertension and chronic or end-stage renal disease (ESRD) by adolescence, frequently accompanied by sensorineural deafness and ocular complications. Milder forms of ATS also exist. We studied 42 patients (19M, 23F) of nine Hellenic families suspected clinically of X-linked ATS who presented with marked phenotypic heterogeneity. We identified mutations in COL4A5 in six families. Two males with nonsense mutation E228X reached ESRD by ages 14 and 18. Frameshift mutation 2946delT followed the same course with early onset renal involvement and deafness. However, two males with the milder missense mutation G624D, reached ESRD after 39 years and one patient showed thin basement membrane nephropathy (TBMN). Another 5/8 affected males with missense mutation P628L also developed ESRD between 30 and 57 years, while three exhibit only mild chronic renal failure (CRF). The data support previous findings that certain mutations are associated with milder phenotypes and confirm that mutation G624D may be expressed as TBMN with familial hematuria. Similar conclusions apply for missense mutation P628L. Interestingly, mutations G624D and P628L are near the 12th natural interruption of COL4A5 triple helical domain, which may explain the milder phenotype.

Oil refinery sludge and green waste simulated windrow composting.

Oil refinery sludge (ORS) was mixed with shredded green wastes (GW) at ratios of 1:1 v/v (RI) and 1:3 v/v (RII). The mixtures, of approximately volumes of 1,020 l and 990 l respectively, were introduced in metal cubic containers of 1.0 m(3) volume, opened at the top and with small holes punctured in the bottom and the side. The containers were additionally insulated with a layer of rockwool (20 mm). The boxes were emptied, the mixtures were turned and water was added occasionally, in one to two weeks intervals, simulating a windrow composting system. Temperature, physiochemical characteristics, mineral oil and grease (MOG) concentration, polycyclic aromatic hydrocarbons (PAHs) concentration, carbon dioxide emission, methane emission and microorganisms presence were recorded either daily or every time the mixtures were turned, for a period of 120 days. RII recorded temperatures as high as 62 degrees C, reaching 56 degrees C in Day 6 and retained temperatures above 50 degrees C for more than 40 days. RI recorded its highest temperature of 53 degrees C in Day 77. The reason why the two mixtures behaved so differently can be explained by: (i) extended co-digestion phenomena by the microorganisms decomposing the GW in RII, (ii) toxic effect of ORS in RI due to the far larger amounts used (840 kg in RI in comparison with the 460 kg in RII). After Day 36 temperature increased gradually in RI and MOG and PAHs reduction was first noted. At the end of the experiment MOG concentration in RI was 57.2 mg/kg dry weight (dw) (52.1% reduction) where in RII was 34.3 mg/kg dw (62.1% reduction). Emissions of methane and carbon dioxide support the concept of the toxic effect and the delay ignition of the decomposing process in RI. In total, CO(2) and CH(4) emissions from RI were recorded to be 30.8 kg and 18.5 g, respectively, where from RII 59.6 kg of CO(2) and 6.4 g of CH(4) were emitted. An effort was made to determine the effect of temperature alone (as an abiotic treating parameter) in both mixtures. It can be supported that about least 15% of the MOG and PAHs removal.

Skin biopsy for the diagnosis of Alport syndrome.

Alport syndrome (AS) is the most common hereditary nephritis often associated with extrarenal manifestations. It was first described by Alport on 1927. There is a primary disorder in collagen type IV which is the main component of the basement membranes. Alport syndrome is more frequently inherited as an X-linked and less commonly as an autosomal dominant or autosomal recessive trait. We describe the case of a 3-year-old boy with the X-linked variant of AS. The diagnosis was at first speculated from the child's detailed family history and was finally confirmed by a skin biopsy. Skin biopsy is an efficient and less invasive method for the X-linked variant of the AS diagnosis.

Bone density in thalassemic children during the course of the disease.

The study was carried out on 50 thalassemic children and 20 control subjects aged 5--15 years. The cortical thickness in thalassemic children was less than in control subjects. The bone loss was more obvious in girls (P less than 0.0005) than in boys (P less than 0.05). The greater bone loss in girls could not be explained by the level of blood hemoglobin because in both sexes the mean value was the same (7.3 +/- 0.5 g/dl). Of the 12 thalassemic children observed for 9--24 months, 9 showed no signficant bone change, 2 showed an improvement of cortical thickness and only 1 showed bone loss. Of the 11 thalassemic children observed for 25--48 months, 4 showed significant bone loss and 7 no change of the bone density. In both groups the mean value of pretransfusion blood Hb was 7.3 g/dl and the sex distribution was equal. A relationship was found in both control and thalassemic children between bone density and urine phosphorus to urine creatinine ratio.