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In 1975, dihydropteridine reductase (DHPR) deficiency was first recognized as a cause of tetrahydrobiopterin (BH(4)) deficiency, leading to hyperphenylalaninemia (HPA) and impaired biogenic amine deficiency. So far, more than 150 patients scattered worldwide have been reported and major progresses have been made in the understanding of physiopathology, screening, diagnosis, treatment, and molecular genetics of this inherited disease. Present knowledge on different aspects of DHPR deficiency, largely derived from authors' personal experience, is traced in this article., (Copyright 2003 Wiley Periodicals, Inc.)

Pseudohypoparathyroidism (PHP) is a heterogeneous disease characterized by PTH resistance and classified as types Ia, Ib, Ic, and II, according to its different pathogenesis and phenotype. PHP-Ia patients show Gsalpha protein deficiency, PTH resistance, and typical Albright hereditary osteodystrophy (AHO). Heterozygous mutations in the GNAS1 gene encoding the Gsalpha protein have been identified both in PHP-Ia and in pseudopseudohypoparathyroidism (PPHP), a disorder with isolated AHO. A single GNAS1 mutation may be responsible for both PHP-Ia and PPHP in the same family when inherited from the maternal and the paternal allele, respectively, suggesting that GNAS1 is an imprinted gene. To evaluate whether molecular diagnosis is a useful tool to characterize AHO and PHP when testing for Gsalpha activity and PTH resistance is not available, we have performed GNAS1 mutational analysis in 43 patients with PTH resistance and/or AHO. Sequencing of the whole coding region of the GNAS1 gene identified 11 mutations in 18 PHP patients, eight of which have not been reported previously. Inheritance was ascertained in 13 cases, all of whom had PHP-Ia: the mutated alleles were inherited from the mothers, who had AHO (PPHP), consistent with the proposed imprinting mechanism. GNAS1 molecular analysis confirmed the diagnosis of PHP-Ia and PPHP in the mutated patients. Our results stress the usefulness of this approach to obtain a complete diagnosis, expand the GNAS1 mutation spectrum, and illustrate the wide mutation heterogeneity of PHP and PHP-Ia.

Gain or loss of function mutations of the GNAS1 gene lead to McCune-Albright syndrome (MAS) or pseudohypoparathyroidism Ia (PHP-Ia), respectively. Patients with MAS, caused by a post-zygotic missense mutation leading to constitutive activation of Gs alpha, suffer from gonadotropin-independent precocious puberty, and delayed or incomplete sexual development and reproductive dysfunction is found in women with PHP-Ia, an inherited disorder caused by deficient expression or function of the Gs alpha protein. In females with MAS, 50% developed precocious puberty by the age of 4 years, the remaining between 4 and 8 years. Peripheral precocious puberty is often atypical and characterized by alternate periods of rapid progression and regression of pubertal development; menstrual bleeding may occur before breast development. Ovarian cyst growth and regression is often described as a sign of ovarian follicle hyperactivation. Notwithstanding this clinical heterogeneity, a subset of patients with MAS develop relentlessly progressive precocious puberty ultimately resulting in premature epiphyseal fusion and reduced adult stature. Long-term information on reproductive function has been obtained in females: some patients had regular menses without ovarian cysts on pelvic US scan, whereas others were oligomenorrheic and showed recurrent ovarian cysts. In males with MAS, precocious puberty occurred in three patients between 4 and 9 years of age. In one patient, long-term follow-up demonstrated normal plasma testosterone and gonadotropin values at the age of 17 years. On testicular sonography, multiple hyperechogeneic spots were found in both testicles (snow-storm appearance). Female patients with PHP-Ia were oligomenorrheic or amenorrheic; more than half had delayed or incomplete sexual development, They were mildly hypoestrogenic with normal to slightly elevated serum gonadotropin levels. These clinical and biochemical findings indicate partial resistance of the theca and granulosa cells of the ovary to gonadotropins due to deficient Gs alpha activity. Responsiveness might be sufficient to promote some degree of follicular development and steroid secretion, but insufficient to induce ovulation

Recent developments in the genetics and physiology of cystinuria do not support the traditional classification, which is based on the excretion of cystine and dibasic amino acids in obligate heterozygotes. Mutations of only two genes (SLC3A1 and SLC7A9), identified by the International Cystinuria Consortium (ICC), have been found to be responsible for all three types of the disease. The ICC set up a multinational database and collected genetic and clinical data from 224 patients affected by cystinuria, 125 with full genotype definition. Amino acid urinary excretion patterns of 189 heterozygotes with genetic definition and of 83 healthy controls were also included. All SLC3A1 carriers and 14% of SLC7A9 carriers showed a normal amino acid urinary pattern (i.e., type I phenotype). The rest of the SLC7A9 carriers showed phenotype non-I (type III, 80.5%; type II, 5.5%). This makes the traditional classification imprecise. A new classification is needed: type A, due to two mutations of SLC3A1 (rBAT) on chromosome 2 (45.2% in our database); type B, due to two mutations of SLC7A9 on chromosome 19 (53.2% in this series); and a possible third type, AB (1.6%), with one mutation on each of the above-mentioned genes. Clinical data show that cystinuria is more severe in males than in females. The two types of cystinuria (A and B) had a similar outcome in this retrospective study, but the effect of the treatment could not be analyzed. Stone events do not correlate with amino acid urinary excretion. Renal function was clearly impaired in 17% of the patients.

McCune-Albright syndrome (MAS) is a rare disease caused by somatic postzygotic mutations at Arg201 in the GNAS1 gene that encodes for the Gsalpha protein. Arg201 mutations are gain-of-function mutations in affected tissues (including bone, skin, endocrine glands and other tissues) that result in the activation of cAMP. We used a polymerase chain reaction(PCR)-based technique for the selective enrichment and analysis of the Arg201 mutant allele in 27 different tissues from 24 Italian patients with one or more signs of MAS. Arg201 mutations were identified in 13 different tissues (48.1%) from 11 patients (45.8%). Mutation detection rates differed across the various types of tissue samples, and the mutation was not always found in every tissue sample from the same patient. To overcome problems in the analysis of mutations in somatic mosaicism, as occurs in MAS, a highly sensitive molecular technique should be applied, the most appropriate tissue source selected, and various affected tissues from the same patient analyzed.

Background: Cystinuria is an inherited disorder of cystine and dibasic amino acids transport that results in urolithiasis because of poor cystine solubility. Three cystinuria phenotypes, differentiated according to urinary amino acid excretion in obligate heterozygotes, were regarded as allelic variants of a monogenic disease. Two mutated amino acid transporter genes, however, have been recently identified as responsible for cystinuria. Mutations in the SLC3A1 gene. encoding for the heavy subunit of the transporter protein rBAT, were associated with type I cystinuria, whereas type II and III cystinuria were associated with mutations in the SLC7A9 gene, encoding for a light subunit of rBAT. Lysine and arginine metabolism have, therefore, been evaluated in cystinuria homozygotes and heterozygotes to better define the cystinuria phenotypes and their correlations with these emerging genotypes., Patients and Methods: Lysine and arginine intestinal absorption and renal excretion were assessed by oral loading and compared to normal controls. Seven cystinuria homozygotes and 7 obligate heterozygotes belonging to the different types received alternately an oral dose of 0.5 mmol/kg body weight lysine or arginine. Plasma concentrations of lysine, arginine, ornithine (derived from rapid arginine conversion) were measured 0, 1, 2, and 3 hours after loading. Their urinary concentrations were measured in morning urine and in urine collected 0-6 hours after loading., Results: Gut lysine absorption was deficient in type II and III, and normal in type I cystinuria homozygotes. Impaired arginine intestinal absorption, as well as massive lysine, arginine, and ornithine hyperexcretion were shared by all homozygotes, irrespective of the type. All heterozygotes shared normal lysine absorption, whereas arginine absorption was slightly impaired in type II and III heterozygotes, which also displayed high lysine, arginine, and ornithine urinary excretion after loading., Conclusions: Two cystinuria phenotypes, type I and non-type I, can be identified in both homozygous and heterozygous cystinuric subjects by oral loading tests with lysine and arginine. In agreement with recent molecular findings, non-type I cystinuria comprises mentioned type II and type III, which constitute allelic variants of a cystine and dibasic amino acid transport disorder distinct from type I cystinuria.

In childhood the traditional diagnostic approach to thyroid nodules consists of clinical, laboratory, and imaging evaluations. A safe and accurate procedure is needed to promptly identify patients who require surgery. In regard to the usefulness of fine needle aspiration biopsy, the data in the literature concerning children and adolescents are scanty. The aim of this study was to evaluate and compare the diagnostic accuracies of clinical, laboratory, and imaging data collected retrospectively in a group of pediatric patients with thyroid nodules submitted to fine needle aspiration biopsy. Forty-two patients who underwent surgery for thyroid nodules, recruited in 9 Italian pediatric endocrine units, were retrospectively studied. According to histological diagnosis, they were divided into 2 groups, 22 patients with benign lesions and 20 patients with malignant lesions. From clinical records we obtained data about 1) symptoms of neck compression; 2) cervical adenopathy; 3) thyroid function, calcitonin level, and antithyroid antibody titers; 4) ultrasonography; 5) (99m)Tc scintiscanning; and 6) cytology obtained with fine needle aspiration biopsy. Patients and nodule characteristics were analyzed statistically for associations with the presence of thyroid cancer. Among clinical findings, only adenopathy was significantly higher in the group with cancer (8 of 22 benign lesions vs. 16 of 20 malignant lesions; P = 0.006). Thyroid function and antibody titers were similar in the 2 groups, whereas the serum calcitonin level was elevated only in 1 patient with malignant lesions. Among ultrasonography findings, no significant statistical difference was found between the 2 groups with regard to number, dimensions, growth progression, or hypoechogenic pattern of the nodules. Regarding scintigraphic findings, no significant difference was found between the 2 groups. However, a positive correlation (r = 0.90; P < 0.0001) was found between fine needle aspiration biopsy cytological findings and histological diagnoses. The sensitivity, specificity, and accuracy of fine needle aspiration biopsy were 95%, 86.3%, and 90.4%, respectively. A multiple regression analysis showed that only fine needle aspiration biopsy (beta coefficient = 0.963; P < 0.0001) significantly contributed to detecting malignancy (multiple r = 0.973; P < 0.0001). This study provides strong evidence that fine needle aspiration biopsy is a safe technique even in childhood and adolescence, offering the best sensitivity, specificity, and accuracy in detecting malignancy compared with conventional approaches.

Three hundred sixty-five patients who underwent cadaver donor kidney transplantation between 1993 and 1998 were divided into four groups: 40 immunized patients with at least one peak panel-reactive antibody (PRA) value more than 50%, 11 hyperimmunized patients with more than three peak PRA values over 50%, 10 retransplanted patients and 304 control patients. Before transplantation, we ascertained the antibody specificities against individual HLA antigens (Prastat Sangstat ELISA method for HLA typing of first donor, husbands of multiparous women and potential donors against whom candidates gave positive cross-matches); thus, patients underwent transplantation excluding the presence of the HLA antigens previously detected and looking for high HLA (class I and II) compatibility. Actuarial graft survival after 12 months was satisfactory in all groups: 87% immunized, 81% hyperimmunized and 80% retransplanted vs 92% controls. Renal function at the end of the first year was similar and the number of rejection episodes in the first 3 months did not significantly differ.

Background: During haemodialysis blood membrane contact causes the release of the content of platelet alpha-granules, which contain platelet-derived growth factor (PDGF). In view of its possible role in accelerated atherosclerotic processes, we evaluated the intra- and post-dialytic changes in PDGF-AB serum levels during haemodialysis sessions performed using a cellulosic membrane., Methods: Using the ELISA method, PDGF-AB, platelet factor-4 (PF4) and beta-thromboglobulin (beta-TG) levels were determined in peripheral blood, as well as in arterial and venous haemodialyser lines, in 10 patients each of whom underwent five consecutive dialysis sessions with a CU membrane. Blood samples were taken at 0, 15, 30, 60, 120, 180 and 240 min during dialysis and at 1, 4 and 20 h after the end of the session. In the same group of patients the levels of the same molecules were also determined after a heparin bolus injection of 4500 IU, blood samples were taken at 0, 15 and 30 min after injection of the bolus., Results: PDGF-AB serum levels increased, remained consistently high during the haemodialysis session (in particular +134+/-20% after 30 min, P<0.001, and +140+/-5% after 240 min, P<0.001) and returned to basal values only after 20 h following the end of the session. PF4 and beta-TG showed a similar trend to PDGF. The heparin bolus injection caused only a small increase (+15+/-5% at 30 min) in PDGF-AB serum levels., Conclusions: PDGF-AB is released during dialysis mainly as consequence of the blood-membrane contact and it returns only slowly to basal values.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Blood-membrane contact in the extracorporeal circuit affects the activation of many biological systems. Among these, phagocytizing activity has been reported to be influenced by the nature of the hemodialysis membrane used, whether cellulosic or synthetic. This work reports on an ex-vivo, comparative test between cellulosics and synthetics concerning the effects of blood-membrane contact on the polymorphonucleate and monocyte phagocytizing function, both during and after the hemodialysis session. By means of flow cytometry, we evaluated the capacity for phagosoma formation and oxidative burst both in polymorphonucleates and monocytes. Ten hemodialysis patients were included in the study. Six separate dialysis procedures for each patient were considered, one per dialyzer (3 cellulosic and 3 synthetic membranes). Tests were performed at 15', 60', 210' and 4 hours after the session end. Comparative evaluation was made according to Student's t test. Polymorphonucleate phagocytosis and oxidative burst activation were globally more marked for synthetic than cellulosic membranes, tending to level out in the post-dialysis. This result could be affected by their functional exhaustion following pulmonary sequestration. Monocyte intradialytic phagocytosis and oxidative burst proved more activated by cellulosic membrane. All differences tended to vanish in the post-dialysis.

Five novel mutations are described which result in the rare hyperphenylalaninemia DHPR-deficiency. Three of these are located at different intron/exon boundaries within the DHPR gene, and disrupt the maturation of the DHPR transcript such that little full-length mRNA can be detected by RT-PCR. Each mutation alters a conserved nucleotide within the splice site consensus sequence, and results in the skipping of an exon and, in one case, the activation of an inappropriate splicing signal. Two further mutations are missense mutations resulting in a non-conservative amino acid change within the DHPR protein (L14P and G17V) and are associated with a severe phenotype.

Aortic transplantation has progressively gained interest over the last few years and it is becoming a first choice indication in the substitution of infected prostheses. The most frequent complication in long-term vascular outcome (wall thickening, aneurysmatic dilation, stenosis), may occur through an immunological mechanism. In this study we investigated nine recipients, aged 48 to 65 years, of aorta segment replacement for anti-HLA antibody production (specificity and Ig class), CD3-CD4-CD8 T cell subpopulation dynamics and aorta wall thickness. Mismatch-specific IgG antibodies to HLA class I and HLA class II antigens were detected 1, 3 and 6 months after transplantation and persisted at a high concentration for at least 1 year. Furthermore, the absolute number of CD3, CD4 and CD8 positive lymphocytes increased progressively after aorta allograft. Tomography scanning showed a progressive thickness of the aorta wall. We can speculate that these anti-HLA antibodies in the recipients have the potential to harm the implant; therefore, aorta allograft should involve the induction of immunological tolerance by appropriate immunosuppressants.

The effects of vessel joint where the both sides have different wall properties on the heart pulse propagation are investigated. Such a local disturbance can influence post-transplantation pathology and evolution of the organ inconsistency. Using a mathematical model, developed in a previous article, we perform analytical analysis and present some qualitative and quantitative estimations. The effects of jointed vessels with different thicknesses and radii on the local concentration of the pressure, radial wall displacement, bending moment and shear force are analyzed in detail. In particular, it is obtained that the bending and shear stresses at a joint sharply and strongly increase in comparison with the uniform vessel ones.

Dihydropteridine reductase (DHPR) is an enzyme involved in recycling of tetrahydrobiopterin (BH4), the cofactor of the aromatic amino acid hydroxylases. Its deficiency is characterized by hyperphenylalaninemia due to the secondary defect of phenylalanine hydroxylase and depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases. The DHPR cDNA has been cloned and mapped on 4p15.3. In the present study we report the genomic structure of the DHPR gene (QDPR). This gene includes seven exons within a range of 84-564 bp; the corresponding introns are flanked by canonic splice junctions. We also present a panel of PCR primers complementary to intronic sequences that greatly facilitates amplification of the gene and provides a genomic DNA approach for mutation detection. We have used this approach to study six patients with DHPR deficiency. Four known mutations (G23D, H158Y, IVS5G+ 1A, R221X) and two new mutations (Y150C and G218ins9bp) were found. The Y150C mutation was found in compound heterozygosity with G23D, a mutation always associated with a severe phenotype in homozygous patients. This patient has an intermediate phenotype (good response to monotherapy with BH4). The mutant enzyme for Y150C was expressed in an E. coli system. Comparison of its kinetic parameters with those of the G23D mutant enzyme showed that it is not as effective as the wild-type enzyme, but is more active than the G23D mutant. This patient's intermediate phenotype is thus due to the mild DHPR mutation Y150C. Correlations between genotypes and phenotypes were also found for the other mutations.

Objective: To define the accuracy of preoperative evaluation of prognostic factors in detecting patients with low risk of node metastasis in which different surgical approaches can be proposed., Subjects: Seventy-five patients with a histologically proven endometrial carcinoma were considered in this study., Methods: All the patients underwent a preoperative evaluation of grading (G), and myometrial invasion (M) by endometrial biopsy and transvaginal ultrasound (TVS). In 41 patients preoperative ploidy of carcinoma cells (P) was determined by flow cytometry. Pre-surgical G, M and P were then compared with surgical specimens. We considered 'low risk', patients with no or moderate myometrial invasion, well-differentiated histological grading and diploid DNA., Results: We were able to identify 19/23 (82.6%) low risk cases. Correct identification of high risk patients was obtained in 49/52 (94%) patients. In three low risk patients, correctly diagnosed preoperatively, the final FIGO stage was IIIA (two for adnexal involvement and one for positive peritoneal washing)., Conclusions: Our findings suggest that it is possible to detect preoperatively patients with a low risk of node metastasis. Alternative surgical approaches, i.e. vaginal surgery, can be taken into account in such patients.

We assessed the efficacy of GH treatment in 25 GH deficient patients irradiated for brain tumors (eight with glioma cranio-irradiated, eleven with medulloblastoma and six with ependymoma craniospinal-irradiated). We administered GH at doses of 0.6-0.9 IU/kg/week for one to three years at least two years after diagnosis of the tumor. We assessed the efficacy of the treatment each year by comparing the values of height velocity over bone age and change in the ratios progression of chronological age/progression of bone age and progression of statural age/progression of bone age. The treatment promoted satisfactory growth; better results were obtained in patients with glioma, who received cranial irradiation only, than in those with medulloblastoma or ependymoma, who received spinal irradiation as well. Moreover, the growth prognosis improved, especially in the cranio-irradiated patients. In our series of patients four presented tumor recurrence; these results did not differ significantly from those in irradiated patients with cerebral tumors who were not treated with GH.

A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria chromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26).

We report on a kidney transplant recipient experiencing an unexpected early acute vascular graft rejection. Retrospective analysis of patient serum samples, utilizing a new ELISA HLA screening technique, revealed that the rejection crisis and the subsequent graft loss were due to a pretransplant donor-specific pre-sensitization caused by a non-complement-fixing antibody of IgG2 class. The case illustrates the clinical significance of non-complement-fixing anti-HLA antibodies. In addition it is shown that ELISA methods are suitable for detecting potentially harmful donor pre-sensitization in waiting-list patients not detectable by standard lymphocytotoxicity techniques. Hence ELISA could be an alternative to flow cytometry for this purpose. It is concluded that screening and cross-matching techniques which detect non-complement-fixing anti-HLA antibodies could improve graft outcome, and should form part of the immunological monitoring of kidney transplant waiting-list patients.

Intradialytic coagulative and platelet activation, one of the main consequences of blood-membrane contact, was studied in a group of 5 RDT patients with a comparative evaluation of 3 different dialytic membranes: Cuprophan (CU), Polysulfone (PS) and Cellulose Triacetate (CT). Each patient underwent 5 consecutive dialysis sessions with the above mentioned membranes. Intradialytic platelet activation was studied through a morpho-functional evaluation between the mean platelet volume (MPV) and Serotonin (S), beta-Thromboglobulin (beta-TG) and Platelet Factor 4 (PF4) serum levels. These determinations were made before HD (time 0) and after 30', 120', and 240'. We also checked the intradialytic status of thrombogenesis and fibrinolysis determining aPTT, thrombin time, fibrinogen, antithrombin III (AT III), alpha-2 antiplasmin and plasminogen, at the same time intervals. All membranes tested (CU, PS, CT) caused appreciable intradialytic platelet activation, above all after 15' and at the end of dialysis sessions, more marked for CU than PS or CT. In particular MPV showed a decrease throughout the session (-5% at 30' and -9% at 240') while S, beta TG and PF4 peripheral blood levels showed a significant increase at the same intervals with CU membrane. Lastly coagulative and fibrinolytic parameters showed no significant differences among any of the membranes tested.

Ultraviolet (UV)-induced repair and mutational spectra were analyzed in an inducible marker gene, the metallothionein-l/guamine-xanthine phosphoribosyl transferase (gpt) fusion gene, carried by an Epstein-Barr virus-derived shuttle vector episomically maintained in human cells. The repair rate of UV photodimers from the shuttle-vector molecules was typical of transcriptionally active sequences, 70% of the dimers being removed within 8 h after irradiation. The spectrum obtained under basal gene transcription was compared with that obtained under induced transcription. In both cases, base substitutions at dipyrimidine sequences predominated. Multiple mutations and deletions probably due to recombinational events induced by UV damage were also observed. Most of the UV-mutated dipyrimidine sites were located in the transcribed strand and were independent of the transcriptional activity of the target gene. In contrast, the distribution of mutations throughout the coding region of the gpt gene was affected by transcription, with a preferential clustering of mutations occurring in the 3' half of the gene after transcription induction. The strand bias observed in the UV spectra most likely reflects selection for nonfunctional gpt protein.

Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described (I, II, and III). An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. To assess whether mutations in SLC3A1 are involved in different cystinuria phenotypes, linkage with this gene and its nearest marker (D2S119) was analyzed in 22 families with type I and/or type III cystinuria. Linkage with heterogeneity was proved (alpha = 0.45; P < 0.008). Type I/I families showed homogeneous linkage to SLC3A1 (Zmax > 3.0 at theta = 0.00; alpha = 1), whereas types I/III and III/III were not linked. Our data suggest that type I cystinuria is due to mutations in the SLC3A1 gene, whereas another locus is responsible for type III. This result establishes genetic heterogeneity for cystinuria, classically considered as a multiallelic monogenic disease.

In order to identify the molecular basis of phenylketonuria (PKU) in Italy, we screened the entire coding sequence of the phenylalanine hydroxylase gene in 20 Italian PKU patients, whose origins are scattered throughout Italy. The frequency of each identified mutation and of 5 other European mutations was determined within a panel of 92 Italian PKU patients. This approach allowed us to identify 20 different PKU mutations and characterize 64% of the Italian PKU chromosomes. Eleven mutations (IVS10nt546, L48S, R158Q, R261Q, P281L, R261X, R252W, delta T55, IVS7nt1, IVS12nt1, Y414C) represent 55.4% of the Italian PKU alleles, the most common mutations being IVS10nt546 (12.4%) and L48S (9%). All the other mutations are very rare. These data confirm the great heterogeneity expected from previous RFLP haplotype studies. Genotype/phenotype correlation allowed for assessment of the clinical impact of the 20 identified mutations.

We evaluated the growth pattern, bone age, insulin-like growth factor I (IGF I) secretion and thyroid function in 24 perinatally infected children: 9 asymptomatic or paucisymptomatic (group 1) and 15 with a more advanced disease state and treated with zidovudine (group 2). Statural and ponderal growth were compared with those of 37 at-risk children who seroreverted. During the two-year follow-up, 22% of children in group 1 had impaired growth, 33% bone age delay, 45% reduced IGF I levels but none had thyroid dysfunction. In group 2, 53% had growth failure, 53% bone age delay, 86% reduced IGF I levels and 40% thyroid dysfunction. Among seroreverters, none showed growth impairment; statistically significant differences were found for height, weight and height velocity between perinatally infected children and seroreverters. Since auxological and hormonal evaluations run parallel to the clinical course of infection, these indices may be useful in monitoring disease progression.

Hemoperfusion is a blood purification technique involving direct contact between blood and adsorbent substances (sorbents). There are three basic kinds of sorbent: activated charcoal, immunoadsorbents, resins. Following our previous experience on charcoal hemoperfusion, a new coated anionic exchange resin for blood purification specifically designed to remove phosphates was experimentally employed in animals. 3 pigs, in which uremia had been surgically induced, underwent 6 extracorporeal hemoperfusion sessions (2 per pig) with a cartridge containing 100 gr of resin. The phosphate clearance proved satisfactory, values being 120 ml/min after 10' and around 80 ml/min after 2 hours. The biocompatibility of the resin and of the coating membrane was satisfactory. The negligible variation in pH and plasma bicarbonate during all sessions confirmed the low absorption by the tested resin of other blood anions competing with phosphate.

Flow cytometry (FC) may be considered as a fundamental technique in studying cell biology and pathology. It combines the quantitative character of biochemical methods with the multiparametric capacities of microscope analysis in a high-precision process for rapid analysis of individual cell characteristics. Three original FC techniques routinely applied in the field of renal transplantation are reported in the present study. They concern the donor-recipient cross-match test, the morphological analysis of urinary sediment and the modulation of the density of various membrane antigens on the lymphocyte surface. A common factor underlies all these methods: they aim to provide the physician with a reliable diagnostic tool in clinical renal transplantation.

A search for antibodies against hepatitis C virus (HCV) was performed in 185 patients on chronic hemodialysis by means of 1st and 2nd generation ELISA tests. Immunoblot assays were performed on positive sera. This study shows a 38% prevalence of HCV-positive patients in our dialysis population according to the 2nd generation ELISA test which shows a higher specificity and sensitivity when compared to the 1st generation one (38 vs. 20%). A correlation was found between the prevalence of HCV-positive patients and how long they had been on dialysis and how many blood transfusions they had received.

Diagnosis in patients with ambiguous genitalia is based on various investigations. Simple genital examination is not sufficient to attribute sex. Scarce androgenization in a male patient or marked androgenization in a female may both lead to the same stages of genital ambiguity according to Prader. It is important to get information about genital ambiguity in the family, drug consumption during gestation and signs of virilization in pregnant mothers. External genital findings must be integrated by visualization of urogenital sinus by X-rays, ultrasounds and endoscopy. Furthermore, hormonal evaluations on plasma and 24 hours urines allow diagnosis of some disorders of adrenal and gonadal steroidogenesis. To define genetic sex, chromosomal examination is required to integrate X chromatine investigation and fluorescent staining of Y chromosome. Evaluation of psychosexuality in patients who have already got gender identity is mandatory.