Mutation in the type IV collagen genes (COL4A3, COL4A4 and COL4A5) have been reported in Alport syndrome (AS) and benign familial hematuria (BFH). AS is a progressive inherited nephropathy, characterized by irregular thinning, thickening and splitting of the glomerular basement membrane (GBM), often associated with hearing loss and ocular symptoms. BFH is a dominantly inherited nephropathy manifested by uniform thinning of the GBM on ultrastructural examination, lifelong glomerular hematuria, minimal proteinuria and usually normal renal function. Considering the similarities inGBM abnormalities, BFH cannot be clinically differentiated from initial stages of AS. We analyzed 112 patients from 43 unrelated families with suspected AS or BFH. A nonisotopic single stranded conformational analysis (SSCA) after amplification of 52, 47 and 51 exons with boundary intronic sequences of COL4A3, COL4A4 and COL4A5 genes was used for mutation screening. Six different mutations were found in COL4A5 gene in Alport syndrome suspectedpatients, comprising four missense mutations (G198E, G310R, G624D, K664N), a splice site mutation (1234+5 G>T) and a mutation causing frameshift (3615-3616del C). In COL4A3 gene three (G487C, G1015E, 3547-3548insGGA) and inCOL4A4 four mutations (3353 G>C + 3354-3358delACCAG, 3068+2T>G, 3497+1G>A), all in heterozygous state, were identified only in patients with benign familial hematuria. Ten of the mutations are to the best of our knowledge new and private. A mutation was detected in 80% of AS patients and in 22% patientsdiagnosed with BFH and we found our optimised non-isotopic SSCA as cost effective, simple, efficient and suitable method for mutation screening and diagnostics. Our study broadened the spectrum of mutations in COL4A3, COL4A4 and COL4A5 and demonstrated the involvement of the COL4A3 and COL4A4 genes in the pathogenesis of BFH. (Abstract truncated at 2000 characters).

Alport syndrome (AS) is hereditary progressive nephropathy, which is characterized by renal failure accompanied by hematuria and varying degrees ofproteinuria, sensorineural deafness, and eye lesions. Carrier females have avariable and generally milder clinical course of disease. In 95% of male patients the disease is characterized by complete renal failure. Pathognomonicfindings in glomerular basement membrane (GBM) such as thickeningand thinning with splitting of the lamina densa have been revealed at the ultrastructural level. In vast families, AS is inherited as a dominant X-chromosome-linked trait. Mutations in COL4A5 gen (Xq22), which codes for major structural component of GBM - alpha5 chain of collagen IV, have been found in majority (85%) of families with X-chromosome-linked AS . To date, close to 300 different mutations have been identified in COL4A5 gene which appear randomly along the gene. Mutations in COL4A3 and COL4A4 genes, located at 2q35, cause autosomal recesive and dominant type of AS. Approximately 420 bp apart form COL4A5 gene is located COL4A6 gene, which is believed to be involved in aetiology of a rare type of AS companied by diffuse leiomiomatosis. Here, we report the first systematic mutation screening of all51 exons with boundary intronic sequences of COL4A5 gene by SSCP analysis in 16 randomly collected AS suspected families in slovenian population. Seven different mutations were identified in 8 families by cycle sequencing. A previous described mutation G624D (GGT>GAT) has been identified in two families. We identified seven different mutations, six of them are to the bestof our knowlage newČ G669R (GGT>CGT), G325R (GGA>CGA), R266X (CGA>TGA), G811R (GGA>AGA), G319D (GGT>GAT) and 1234+17 T del. Intronic mutation 1234+17 T del most likely influences the splicing of mRNA. (Abstract truncated at 2000characters).

The multiple endocrine neoplasia type 2 (MEN 2) syndromes are dominantly inherited syndromes of tumor formation and disordered development that involvefour tissues: the "C"-cells of thyroid, the adrenal medulla, the parathyroid and the intestinal autonomic nerve plexuses. The inheritance has been estimated to be about 1/30000 of the general population. In 1961 Sipple described the high incidence of the carcinoma of the thyroid in patients with pheochromocytoma. Medulliary thyroid carcinoma (MTC) was first described by Hazard in 1959. Subsequently it was recognized that carcinoma of the thyroid that occurred with pheochromocytoma was always MTC. In 1968 Steiner et al. introduced term MEN 2 to distinguish new syndrome from MEN 1 where tumor of parathyroid, pituitary and endocrine pancreatic tumor emerge. There are three distinct clinical varieties of MEN 2, namely MEN 2A, MEN 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A accounts for 69% of cases, while MEN 2B and FMTC for 9% and 22% of cases of hereditary MTC respectively. About 95% of individuals that carry the mutation that predispose for MEN 2A develop MTC, 50% develop pheochromocytoma and about 10-30% develop parathyroid disease. In patients that carry MEN 2B specific mutations, develop MTC and pheochromocytoma at approximately same percentage of cases, while parathyroid disease appears uncommon. MTC emerge at younger age an tends to be more aggresive than observed in MEN 2A cases. Additional to endocrinopathies, several muscular and sceletal abnormalities are present. Disorganized peripheral nerve tissue gives rise to neuromas of tongue and lips. Along the intestinum, the number of entrinsic autonomic ganglia is increased, while hiperplasia of extrinsic autonomic nerve fibers in the wall of the intestine leads to disturbance of intestinal motility. (Abstract truncated at 2000 characters).

Cystic fibrosis (CF) is one of the most common fatal autosomal recessive hereditary disease in Caucasian population, with a carrier frequency of 1 in 25, and with a disease frequency of approximately 1 in 2000 live-born children. CF affects tissues that produce mucus, these are airway, gastriontestinal tract, pancreatic and hepatobiliary ducts, liver and male urogenital tract. Severe classical form of CF is characterized by elevated sweat electrolyte levels, chronic sinopulmonary disease, and pancreatic insufficiency. Approximately 95 % of men with CF are infertile due to the congenital bilateral absence of the vas deferens (CBAVD). The gene responsiblefor CF (Cystic Fibrosis Transmembrane conductance Regulator - CFTR)was cloned and characterized in 1989. Almost 900 different mutations havebeen identified in CFTR gene of patients affected with CF. The most commonmutation in CFTR protein is a deletion of phenylalanine at position 508 (F508). This deletion is present in 68 % of CF chromosomes worldwide. Due to many known diseases (neonatal meconium ileus, liver cirrhosis, bilateral and unilateral absence of the vas deferens, as well as diabetes, pancreatitis, and Crohn's disease), which share some clinical features with CF, researchers started to study the involvement of alterations in CFTR gene in these diseases. One of first such connections was discovered in 1992, when it was found out that aproximatelly 70 % of otherwise healthy men but infertile due to CBAVD had mutations in the CFTR gene. There were studies that confirmed butalso denied the involvement of CFTR gene in male infertility in the beginning of our research in 1997. In the Department of obstetrics and Gynecology in Ljubljana a ICSI (Intracytoplasmic Sperm Injection) methode was applied in 1995. So we wanted to clarify the involvement of CFTR gene in infertile men who were indicated for ICSI methode. (Abstract truncated at 2000characters).