X-linked, COL4A5 hypomorphic Alport mutations such as G624D and P628L may only exhibit thin basement membrane nephropathy with microhematuria and late onset kidney failure.

Alport syndrome (ATS) results from X-linked, COL4A5 mutations (85%) or from autosomal recessive homozygous or compound heterozygous COL4A3/A4 mutations (15%), associated with alternate thinning and thickening as well as splitting and lamellation of the glomerular basement membranes. In contrast, familial microhematuria with thin basement membranes is thought to result from heterozygous COL4A3/A4 mutations. This absolute separation may not always be true. Renal biopsies and molecular genetics were used to study microhematuric families in the Hellenic population we serve. The COL4A5 gene was studied by PCR and direct re-sequencing for new mutations, while PCR-RFLP was used to identify more carriers of known COL4A5 and COL4A3/A4 mutations. Molecular genetics in two undiagnosed microhematuric Cypriot families, revealed COL4A5 mutation P628L indicating X-linked ATS. Of nine males, seven developed end stage kidney disease (ESKD) between 31 and 56, while two are well at 51 and 57, exhibiting microhematuria and thin basement membrane nephropathy (TBMN). COL4A5 mutation G624D was also identified in six Greek families. Seventy five members had DNA tests and 37 proved positive. Four positive males developed ESKD at 61, 51, 50 and 39 years, while the remaining and all females showed only microhematuria. A literature search revealed eight papers with six similar hypomorphic COL4A5 mutations presenting as phenocopies of TBMN. In conclusion, X-linked COL4A5 ATS mutations produce a phenotypic spectrum with a) classical ATS with early onset ESKD, neurosensory deafness and ocular defects b) males with only ESKD and late deafness and c) males due to missense mutations, such as G624D and P628L that may only exhibit microhematuria, TBMN, mild chronic renal failure (CRF) or late onset ESKD. Consequently when investigating "benign familial hematuria" these and other similar X-linked COL4A5 mutations should also be searched for.

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.

Transfusional iron overload and chelation therapy with deferoxamine and deferiprone (L1).

Iron is essential for all living organisms. Under normal conditions there is no regulatory and rapid iron excretion in humans and body iron levels are mainly regulated from the absorption of iron from the gut. Regular blood transfusions in thalassaemia and other chronic refractory anaemias can result in excessive iron deposition in tissues and organs. This excess iron is toxic, resulting in tissue and organ damage and unless it is removed it can be fatal to those chronically transfused. Iron removal in transfusional iron overload is achieved using chelation therapy with the chelating drugs deferoxamine (DF) and deferiprone (L1). Effective chelation therapy in chronically transfused patients can only be achieved if iron chelators can remove sufficient amounts of iron, equivalent to those accumulated in the body from transfusions, maintaining body iron load at a non-toxic level. In order to maintain a negative iron balance, both chelating drugs have to be administered almost daily and at high doses. This form of administration also requires that a chelator has low toxicity, good compliance and low cost. DF has been a life-saving drug for thousands of patients in the last 40 years. It is mostly administered by subcutaneous infusion (40-60 mg/kg, 8-12 h, 5 days per week), is effective in iron removal and has low toxicity. However, less than 10% of the patients requiring iron chelation therapy worldwide are able to receive DF because of its high cost, low compliance and in some cases toxicity. In the last 10 years we have witnessed the emergence of oral chelation therapy, which could potentially change the prognosis of all transfusional iron-loaded patients. The only clinically available oral iron chelator is L1, which has so far been taken by over 6000 patients worldwide, in some cases daily for over 10 years, with very promising results. L1 was able to bring patients to a negative iron balance at doses of 50-120 mg/kg/day. It increases urinary iron excretion, decreases serum ferritin levels and reduces liver iron in the majority of chronically transfused iron-loaded patients. Despite earlier concerns of possible increased risk of toxicity, all the toxic side effects of L1 are currently considered reversible, controllable and manageable. These include agranulocytosis (0.6%), musculoskeletal and joint pains (15%), gastrointestinal complaints (6%) and zinc deficiency (1%). The incidence of these toxic side effects could in general be reduced by using lower doses of L1 or combination therapy with DF. Combination therapy could also benefit patients experiencing toxicity with DF and those not responding to either chelator alone. The overall efficacy and toxicity of L1 is comparable to that of DF in both animals and humans. Despite the steady progress in iron chelation therapy with DF and L1, further investigations are required for optimising their use in patients by selecting improved dose protocols, by minimising their toxicity and by identifying new applications in other diseases of iron imbalance.

Loss of heterozygosity in polycystic kidney disease with a missense mutation in the repeated region of PKD1.

Loss of heterozygosity (LOH) is a molecular phenomenon that denotes the loss of one of the two alleles at a specific locus. It is frequently associated with tumour suppressor genes in various cancers and also with hyperproliferative disorders, although not exclusively. Interestingly, in conditions where there is an inherited germline mutation, the lost allele is always the functional one, thereby rendering a phenotypically dominant disease of recessive character at the cellular level. A disease more recently shown to be associated with LOH is polycystic kidney disease type 1, a systemic disorder characterized by significant pleiotropy. The main pathology is from renal cyst formation that eventually leads to end-stage renal failure during adult life. We describe the identification of a missense mutation in the repeated part of the PKD1 gene, exon 31, that substitutes valine for methionine. The mutation, M3375V, cosegregates with the disease phenotype in a large Cypriot family. During transplantation of one patient, one of the polycystic kidneys was removed and DNA was isolated from cystic epithelial cells. In 3 of 17 cysts examined with intragenic and flanking polymorphic markers on chromosome 16 we detected LOH, since the wild-type allele was lost, thereby rendering the affected kidneys of mosaic character. The degree of LOH was extensive and varied among the three cysts, supporting the multiplicity of expression of the phenomenon on different occasions. No LOH was detected for other selected loci examined. Our work further supports the hypothesis that the rate-limiting step in cyst formation may be the occurrence of a second somatic hit, although other factors may be also involved. The high frequency of mutations at this locus may, to a great extent, explain the variability in phenotype observed among patients in the same families, and the relatively high frequency of the disease worldwide.

Serum/ascites albumin gradient: its value as a rational approach to the differential diagnosis of ascites.

BACKGROUND: The utility of differentiating ascites into 'transudate' and 'exudate' has recently been challenged. The aim of the present study was to compare the diagnostic accuracy of the serum/ascites albumin gradient, proposed as a new biochemical criterion for the differential diagnosis of ascites, with the markers traditionally used for the classification of peritoneal fluid into transudate and exudate. METHODS: Paired ascitic fluid and serum samples from 51 patients were examined with an established method for the diagnosis of the cause of ascitic fluid collection. Included in the study were 32 patients with ascites related to portal hypertension (cirrhosis, n = 28; 'cardiac' ascites, n = 2; Budd-Chiari, n = 2) and 19 patients with ascites not related to portal hypertension (peritoneal carcinomatosis, n = 17; tuberculous peritonitis, n = I; secondary bacterial peritonitis, n = 1). Specimens were collected during an episode of spontaneous bacterial peritonitis in 7 of 28 patients with cirrhosis. The serum/ascites albumin gradient was compared with ascitic fluid total protein, ascites/serum total protein ratio, ascites lactic dehydrogenase concentration, and ascites/serum lactic dehydrogenase ratio. RESULTS: The diagnostic accuracy was 98% for the serum/ascites albumin gradient compared with only 52%-80% for the four other markers tested. In patients with infected ascites, diagnostic accuracy was 89% for the albumin gradient and < or = 50% for the four other markers. CONCLUSIONS: The classification of ascites into transudate and exudate appears to be based on markers with low diagnostic accuracy. Differential diagnosis of ascites should be based on the serum/ascites albumin gradient, which is a reliable marker distinguishing ascites related to portal hypertension from all other causes of ascitic fluid collection, regardless of the presence of bacterial infection.

Short-term, high-dose muzolimine treatment in patients with chronic renal failure and acute fluid retention.

Muzolimine, the new sulphonamide-free loop-diuretic with both high ceiling and long-lasting activities, was tested in 21 adult patients with chronic renal failure (CRF) (creatinine clearance ranging from 30 to 5 ml/min) and acute fluid overload. Low-protein diet and individual drug therapy were unchanged throughout the study. All patients received a single oral dose of 240 mg of muzolimine for 4 or 6 consecutive days depending on individual response. Clinical status, diuresis, body weight, blood and urine chemistry were recorded daily. In 19 out of 21 patients muzolimine treatment induced reversal of edema and congestive heart failure and a satisfactory fluid balance was achieved. Only two patients did not respond to diuretic treatment and required dialysis to control fluid balance and azotemia. In responsive patients diuresis increased by 50-100% and no rebound antidiuresis was observed after drug withdrawal. Body weight decreased meanly by 9%. No significant change occurred in serum concentration of K throughout the study, even in the 11 patients on digoxin. Except for a slight decrease of serum Cl by the end of treatment, no significant change in serum electrolytes was recorded. No effect was observed on blood glucose, urea and creatinine clearance whereas a slight increase of serum uric acid was recorded. Urinary lysozyme and gamma-GT were similar before and after the trial. Apart from a single case of muscle cramps, no significant side-effects were recorded. In conclusion, the present results indicate that short-term, high-dose oral muzolimine treatment is effective and safe in most patients with advanced CRF and acute fluid retention.