Early Macrophage Infiltration and Sustained Inflammation in Kidneys From Deceased Donors Are Associated With Long-Term Renal Function.

Kidney transplants from living donors (LDs) have a better outcome than those from deceased donors (DDs). Different factors have been suggested to justify the different outcome. In this study, we analyzed the infiltration and phenotype of monocytes/macrophages and the expression of inflammatory and fibrotic markers in renal biopsy specimens from 94 kidney recipients (60 DDs and 34 LDs) at baseline and 4 months after transplantation. We evaluated their association with medium- and long-term renal function. At baseline, inflammatory gene expression was higher in DDs than in LDs. These results were confirmed by the high number of CD68-positive cells in DD kidneys, which correlated negatively with long-term renal function. Expression of the fibrotic markers vimentin, fibronectin, and α-smooth muscle actin was more elevated in biopsy specimens from DDs at 4 months than in those from LDs. Gene expression of inflammatory and fibrotic markers at 4 months and difference between 4 months and baseline correlated negatively with medium- and long-term renal function in DDs. Multivariate analysis point to transforming growth factor-ß1 as the best predictor of long-term renal function in DDs. We conclude that early macrophage infiltration, sustained inflammation, and transforming growth factor-ß1 expression, at least for the first 4 months, contribute significantly to the difference in DD and LD transplant outcome.

Recurrent X chromosome-linked deletions: discovery of new genetic factors in male infertility.

BACKGROUND: The role of X-linked genes and copy-number variations (CNVs) in male infertility remains poorly explored. Our previous array-CGH analyses showed three recurrent deletions in Xq exclusively (CNV67) and prevalently (CNV64, CNV69) found in patients. Molecular and clinical characterisation of these CNVs was performed in this study. METHODS: 627 idiopathic infertile patients and 628 controls were tested for each deletion with PCR+/-. We used PCR+/- to map deletion junctions and long-range PCR and direct sequencing to define breakpoints. RESULTS: CNV64 was found in 5.7% of patients and in 3.1% of controls (p=0.013; OR=1.89; 95% CI 1.1 to 3.3) and CNV69 was found in 3.5% of patients and 1.6% of controls (p=0.023; OR=2.204; 95% CI 1.05 to 4.62). For CNV69 we identified two breakpoints, types A and B, with the latter being significantly more frequent in patients than controls (p=0.011; OR=9.19; 95% CI 1.16 to 72.8). CNV67 was detected exclusively in patients (1.1%) and was maternally transmitted. The semen phenotype of one carrier (11-041) versus his normozoospermic non-carrier brother strongly indicates a pathogenic effect of the deletion on spermatogenesis. MAGEA9, an ampliconic gene reported as independently acquired on the human X chromosome with exclusive physiological expression in the testis, is likely to be involved in CNV67. CONCLUSIONS: We provide the first evidence for X chromosome-linked recurrent deletions associated with spermatogenic impairment. CNV67, specific to spermatogenic anomaly and with a frequency of 1.1% in oligo/azoospermic men, resembles the AZF regions on the Y chromosome with potential clinical implications.

Monocyte implication in renal allograft dysfunction.

Macrophages are involved in the development and progression of kidney fibrosis. The aim of this study was to analyse the phenotype of circulating monocytes and their ability to predict kidney allograft dysfunction in living kidney transplant recipients. Whole blood samples from 25 kidney recipients and 17 donors were collected at five time-points. Monocyte phenotype was analysed by flow cytometry, and interleukin (IL)-10 and soluble CD163 by enzyme-linked immunosorbent assay. One week after transplantation, surface CD163 and IL-10 levels increased significantly from baseline [2·99 ± 1·38 mean fluorescence intensity (MFI) to 5·18 ± 2·42 MFI for CD163; 4·5 ± 1·46 pg/ml to 6·7 ± 2·5 pg/ml for IL-10]. This CD163 increase correlated with 4-month creatinine levels (r = 0·4394, P = 0·04). However, soluble CD163 decreased significantly from baseline at 1 week (797·11 ± 340·45 ng/ml to 576·50 ± 293·60 ng/ml). CD14(+) CD16(-) monocytes increased at 4 months and correlated positively with creatinine levels at 12 and 24 months (r = 0·6348, P = 0·002 and r = 0·467, P = 0·028, respectively) and negatively with Modification of Diet in Renal Disease (MDRD) at 12 months (r = 0·6056, P = 0·003). At 4 months, IL-10 decreased significantly (P = 0·008) and correlated positively with creatinine at 2 years (r = 0·68, P = 0·010) and with CD14(+) CD16(-) monocytes at 4 months (r = 0·732, P = 0·004). At 24 h, levels of human leucocyte antigen D-related declined from 12·12 ± 5·99 to 5·21 ± 3·84 and CD86 expression decreased from 2·76 ± 1·08 to 1·87 ± 0·95. Both markers recovered progressively until 12 months, when they decreased again. These results indicate that monitoring monocytes could be a promising new prognostic tool of graft dysfunction in renal transplant patients.

Y-chromosome microdeletions are not associated with SHOX haploinsufficiency.

STUDY QUESTION: Are Y-chromosome microdeletions associated with SHOX haploinsufficiency, thus representing a risk of skeletal anomalies for the carriers and their male descendents? SUMMARY ANSWER: The present study shows that SHOX haploinsufficiency is unlikely to be associated with Y-chromosome microdeletions. WHAT IS KNOWN ALREADY: Y-chromosome microdeletions are not commonly known as a major molecular genetic cause of any pathological condition except spermatogenic failure. However, it has been recently proposed that they are associated not only with infertility but also with anomalies in the pseudoautosomal regions (PAR), among which SHOX haploinsufficiency stands out with a frequency of 5.4% in microdeletion carriers bearing a normal karyotype. This finding implies that sons fathered by men with Y-chromosome defects will not only exhibit fertility problems, but might also suffer from SHOX-related conditions. STUDY DESIGN: Five European laboratories (Florence, Münster, Barcelona, Padova and Ancona), routinely performing Y-chromosome microdeletion screening, were enrolled in a multicenter study. PARTICIPANTS/MATERIALS, SETTING, METHODS: PAR-linked and SHOX copy number variations (CNVs) were analyzed in 224 patients carrying Y-chromosome microdeletions and 112 controls with an intact Y chromosome, using customized X-chromosome-specific array-CGH platforms and/or qPCR assays for SHOX and SRY genes. MAIN RESULTS AND THE ROLE OF CHANCE: Our data show that 220 out of 224 (98.2%) microdeletion carriers had a normal SHOX copy number, as did all the controls. No SHOX deletions were found in any of the examined subjects (patients as well as controls), thus excluding an association with SHOX haploinsufficiency. SHOX duplications were detected in 1.78% of patients (n = 4), of whom two had an abnormal and two a normal karyotype. This might suggest that Y-chromosome microdeletions have a higher incidence for SHOX duplications, irrespective of the patient's karyotype. However, the only clinical condition observed in our four SHOX-duplicated patients was infertility. LIMITATIONS, REASONS FOR CAUTION: The number of controls analyzed is rather low to assess whether the SHOX duplications found in the two men with Y-chromosome microdeletions and a normal karyotype represent a neutral polymorphism or are actually associated with the presence of the microdeletion. WIDER IMPLICATIONS OF THE FINDINGS: Men suffering from infertility due to the presence of Y-chromosome microdeletions can resort to artificial reproductive technology (ART) to father their biological children. However, infertile couples must be aware of the risks implied and this makes genetic counseling a crucial step in the patient's management. This study does not confirm previous alarming data that showed an association between Y-chromosome microdeletions and SHOX haploinsufficiency. Our results imply that deletion carriers have no augmented risk of SHOX-related pathologies (short stature and skeletal anomalies) and indicate that there is no need for radical changes in genetic counseling of Yq microdeletion carriers attempting ART, since the only risk established so far for their male offspring remains impaired spermatogenesis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Italian Ministry of University (grant PRIN 2010-2012 to C.K.), Tuscan Regional Health Research Program ('Progetto Salute 2009') to G.F., the Spanish Ministry of Health (grant FIS-11/02254) and the European Union 'Reprotrain' Marie Curie Network (project number: 289880 to C.K.). The authors declare that no conflicting interests exist.

Mitotic recombination effects homozygosity for NF1 germline mutations in neurofibromas.

Pure populations of neurofibroma-derived Schwann cells bearing both NF1 mutated alleles (NF1-/-) have been isolated from different neurofibromas showing loss of heterozygosity of nearly the entire 17q chromosome. By comparing molecular and fluorescent in situ hybridization analysis of these cells, we demonstrate mitotic recombination is the mechanism underlying this type of loss of heterozygosity leading to reduction to homozygosity of NF1 germline mutation.

Further insights into the role of T222P variant of RXFP2 in non-syndromic cryptorchidism in two Mediterranean populations.

The aetiopathogenesis of isolated cryptorchidism remains largely unknown. Mutation screenings in the most relevant candidate genes for testicular maldescent lead to controversial data in the literature. In particular, the role of the T222P genetic variant of the RXFP2 gene is still debated. Given the controversies, the aim of this study was to provide further data on this genetic variant in two Mediterranean populations. A total of 577 subjects from Spain and 550 from Italy (with and without a history of cryptorchidism) were analysed. The T222P substitution was found in both unilateral and bilateral cases and in a total of 12 controls. These data exclude a clear-cut cause-effect relationship between T222P variant and testicular maldescent. The T222P variant was found at a similar frequency in both cases and controls in the Spanish population, whereas in Italy, the frequency of T222P resulted significantly higher in the cryptorchid group (p = 0.031). The observed difference between the two countries and the highly variable phenotypic expression of the T222P variant may depend on the genetic background or on environmental conditions. The haplotype analysis of the RXFP2 gene in T222P carriers and their parents showed that this variant is linked to the previously inferred C-C-G-A-13 haplotype and consequently provides further support to the 'founder effect' hypothesis. In conclusion, our data indicate that T222P is a frequent variant in the Spanish population with no pathogenic effect. Although in Italy it seems to confer a mild risk (odds ratio = 3.17, 95% confidence interval: 1.07-9.34) to cryptorchidism, the screening for this variant for diagnostic purposes is not advised because of the relatively high frequency of control carriers (1.4% of Italian men without a history of cryptorchidism).

WT1 mutations may be a cause of severe renal failure due to nephroblastomatosis in Wilms' tumor patients.

Wilms' tumor suppressor gene (WT1) encodes a transcription factor required for normal development of the genitourinary system. Germline WT1 mutations have been described in a wide spectrum of pathological conditions, including kidney diseases, genital abnormalities and Wilms' tumor. Here we report a 4-year-old male patient who presented with bilateral cryptorchidism, Wilms' tumor, nephroblastomatosis and renal failure without nephrotic proteinuria. Sequence analysis of the WT1 gene demonstrated a constitutional heterozygous nonsense mutation in exon 7, which leads to a truncation of the WT1 protein at the zinc-finger 1. In the DNA of the tumor, we observed the same mutation in homo/hemizygosity. Given the requirement of WT1 for normal development, the WT1 mutation is likely to be responsible for the nephroblastomatosis and, in consequence, for the severe renal failure observed in our patient. This finding extends the spectrum of kidney diseases related to WT1 mutations and points to the need to screen for this gene in children with genitourinary abnormalities and Wilms' tumor because of the associated risk of nephroblastomatosis and renal failure in those carrying WT1 mutations.

Preservation of renal function in a patient with Fabry nephropathy on enzyme replacement therapy.

Fabry disease is an X-linked recessive inborn error of glycosphingolipid metabolism caused by the deficient activity of the lysosomal enzyme, alpha-galactosidase A. Enzyme replacement therapy (ERT) for this disorder has been available in Europe since 2001. However, its effect on advanced renal failure remains controversial. We report the case of a patient whose decline in renal function was reduced by the administration of ERT (agalsidase-alpha). This reduction was more pronounced after doubling the dose of the enzyme. The rate of deterioration of eGFR went from 6.3 ml/min/year prior to the start of ERT (0.2 mg/kg) to 2 ml/min/year (0.4 mg/kg). To our knowledge, this is the first reported case of a patient with moderately impaired renal function treated with high doses of ERT and follow-up of 6 years. The data shown here suggest that ERT may have a very positive impact on renal function even in advanced stages. The role of proteinuria and its control seem to have a clear responsibility for this favorable outcome.

Molecular staging of bladder cancer with RT-PCR assay for CK20 in peripheral blood, bone marrow and lymph nodes: comparison with standard histological staging.

BACKGROUND: The aim of this study was to analyze whether the CK20 reverse transcriptase polymerase chain reaction (RT-PCR) is suitable for detecting circulating tumor cells and residual tumor cells in lymph nodes, in patients with muscle invasive transitional cell carcinoma (TCC) of the bladder, and to compare these results with standard histological staging. PATIENTS AND METHODS: The nested RT-PCR assay was used to analyze the CK20 transcript in the peripheral blood, bone marrow, lymph nodes, the tumor and normal biopsies of bladder from 57 patients with invasive TCC of the bladder, who underwent radical cystectomy, and from 9 patients with noninvasive TCC. RESULTS: Lymph node pathological status was positive in 24 out of the 57 patients studied and all of them except I showed expression of CK20, with a correlation between histological technique and RT-PCR of 95.8%. A statistically significant correlation of lymph node CK20 RT-PCR with the standard risk factor of pathological stage (p = 0.04) was observed Blood and bone marrow CK20 RT-PCR showed no correlation with pathological stage. CONCLUSION: Lymph node CK 20 RT-PCR correlates with pathological stage in bladder cancer. The CK20 RT-PCR assay appears to be a highly sensitive and specific method for detecting circulating tumor cells and residual disease in lymph nodes in patients with invasive bladder cancer. Further evaluation of the significance of CK20 as a molecular marker for staging and follow-up in these patients is necessary.

Cold shock induces the insertion of a cryptic exon in the neurofibromatosis type 1 (NF1) mRNA.

Alternative splicing is a regulatory process of gene expression based on the flexibility in the selection of splice sites. In this manuscript we present the characterisation of an alternative splicing of the NF1 pre-mRNA induced by cold-shock conditions. We demonstrate that the accuracy of the splicing mechanism was perturbed after keeping samples for a short period of time at room temperature, resulting in the insertion of a 31-bp cryptic exon between exons 4a and 4b of the NF1 mRNA. This alternative splicing is not cell type specific and is not induced by other stress conditions such as heat shock or hyper-osmolarity. The alternative spliced mRNA is efficiently transported to the cytoplasm and it is proven to belong to the poly A(+)mRNA fraction. Previous misleading interpretations about this transcript, together with our finding relating its presence to cold shock and not to the NF1 disease, strongly indicate that this phenomenon should be taken into account in genetic testing when RNA methodology is used for mutation detection. This is the first description of an alternative splicing induced by cold shock in a human pre-mRNA and should provide further insights into the factors that control alternative splicing.

[Collagen IV (alpha3-alpha4) nephropathy]. / La nefropatía del coláigeno IV (aalpha3-alpha4).

Recent evidence has shown that the COL4A3, COL4A4 and COL4A5 genes are involved in different renal manifestations. Mutations in these collagen type IV genes affect the glomerular basement membrane (GBM) giving rise to a nephropathy whose symptoms range from isolated hematuria to severe renal failure. This disorder has been traditionally considered to be different entities: Autosomal Dominant Alport syndrome, Familial Benign Hematuria, Autosomal Recessive Alport Syndrome carriers. But the increased knowledge of the molecular basis of this clinical diversity prompted us to agglutinate these entities under the name of "collagen type IV nephropathy". This fact has relevant implications in diagnosis, prognosis and management.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Gene expression profiles in prostate cancer: identification of candidate non-invasive diagnostic markers.

OBJECTIVE: To analyze gene expression profiles of prostate cancer (PCa) with the aim of determining the relevant differentially expressed genes and subsequently ascertain whether this differential expression is maintained in post-prostatic massage (PPM) urine samples. MATERIAL AND METHODS: Forty-six tissue specimens (36 from PCa patients and 10 controls) and 158 urine PPM-urines (113 from PCa patients and 45 controls) were collected between December 2003 and May 2007. DNA microarrays were used to identify genes differentially expressed between tumour and control samples. Ten genes were technically validated in the same tissue samples by quantitative RT-PCR (RT-qPCR). Forty two selected differentially expressed genes were validated in an independent set of PPM-urines by qRT-PCR. RESULTS: Multidimensional scaling plot according to the expression of all the microarray genes showed a clear distinction between control and tumour samples. A total of 1047 differentially expressed genes (FDR≤.1) were indentified between both groups of samples. We found a high correlation in the comparison of microarray and RT-qPCR gene expression levels (r=.928, P<.001). Thirteen genes maintained the same fold change direction when analyzed in PPM-urine samples and in four of them (HOXC6, PCA3, PDK4 and TMPRSS2-ERG), these differences were statistically significant (P<.05). CONCLUSION: The analysis of PCa by DNA microarrays provides new putative mRNA markers for PCa diagnosis that, with caution, can be extrapolated to PPM-urines.

Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders in humans and is caused by mutations in the NF1 gene. To date, the majority of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the mRNA level. We have applied a whole NF1 cDNA screening methodology to the study of 80 unrelated NF1 patients and have identified 44 different mutations, 32 being novel, in 52 of these patients. Mutations were detected in 87% of the familial cases, but in 51% of the sporadic ones. At least 15 of the 80 NF1 patients (19%) had recurrent mutations. The study shows that in 50% of the patients in whom the mutations were identified, these resulted in splicing alterations. Most of the splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites. One frameshift, two nonsense and two missense mutations were also responsible for alterations in mRNA splicing. The location and type of mutation within the NF1 gene, and its putative effect at the protein level, do not indicate any relationship to any specific clinical feature of NF1. The high proportion of aberrant spliced transcripts detected in NF1 patients stresses the importance of studying mutations at both the genomic and RNA level. It is possible that part of the clinical variability in NF1 could be due to mutations affecting mRNA splicing, which is the most common molecular defect in NF1.

Prenatal diagnosis of sporadic neurofibromatosis type 1 (NF1) by RNA and DNA analysis of a splicing mutation.

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans with an incidence of 1 in 3500. Most of the NF1 mutations reported so far (over 240 mutations) are unique. Specific prenatal diagnosis can only be provided to familial cases by an indirect linkage analysis or to families with a previously identified mutation. Here we report the first prenatal diagnosis in sporadic NF1 by direct characterization of the mutation. We first identified the skipping of exon 10b of NF1 in the mRNA from a woman affected by NF1 and without familial history of the disease. The analysis of genomic DNA identified mutation IVS10b+1G-->A as the cause of the skipping of exon 10b. Chorionic villus sampling (CVS) was performed at 10 weeks of gestation and total RNA was directly extracted from the sample. After reverse transcription (RT) and polymerase chain reaction (PCR) of the cDNA, the skipping of exon 10b was not identified in the CVS upon agarose gel electrophoresis. The fetal origin of the CVS was confirmed via polymorphic markers and the absence of the IVS10b+1G-->A mutation was confirmed by genomic analysis.

Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a prevalence of around 1 in 3500, affecting all ethnic groups. The clinical manifestations of the disease are variable, even among members of the same family, and affect a variety of tissues and cell types, including skin, iris, central and peripheral nervous systems and skeletal system. It has been reported that the majority of sporadic mutations in NF1 arise in paternally inherited alleles. We present here a collaborative study of the parental origin and type of mutation in individuals with de novo NF1, who account for up to a half of all cases of clinically diagnosed NF1. We have studied intragenic and extragenic markers in 470 NF1 families. In 32 of these families it was possible to assess the parental origin of a de novo NF1 mutation either by linkage analysis (in families with three generations) or by the detection of an intragenic deletion in a sporadic NF1 case. Eleven of these 32 families have three generations (the second and third generation being affected), with the mutation (not a large deletion) being of paternal origin in 82% of them (P < 0.05). In the other 21 families an intragenic deletion was detected, in 76% being in the maternal chromosome and in 24% in the paternal one (P < 0.05). Our results suggest that in NF1 the majority of deletions occur in oogenesis, while other types of mutations should account for the paternally derived NF1 mutations.