A novel PAX1 null homozygous mutation in autosomal recessive otofaciocervical syndrome associated with severe combined immunodeficiency.

Otofaciocervical syndrome (OFCS) is a rare disorder characterized by facial anomalies, cup-shaped low-set ears, preauricular fistulas, hearing loss, branchial defects, skeletal anomalies, and mild intellectual disability. Autosomal dominant cases are caused by deletions or point mutations of EYA1. A single family with an autosomal recessive form of OFCS and a homozygous missense mutation in PAX1 gene has been described. We report whole exome sequencing of 4 members of a consanguineous family in which 2 children, showing features of OFCS, expired from severe combined immunodeficiency (SCID). To date, the co-occurrence of OFCS and SCID has never been reported. We found a nonsense homozygous mutation in PAX1 gene in the 2 affected children. In mice, Pax1 is required for the formation of specific skeletal structures as well as for the development of a fully functional thymus. The mouse model strongly supports the hypothesis that PAX1 depletion in our patients caused thymus aplasia responsible for SCID. This report provides evidence that bi-allelic null PAX1 mutations may lead to a multi-system autosomal recessive disorders, where SCID might represent the main feature.

Fibroblast growth factor and endothelin-1 receptors mediate the response of human striatal precursor cells to hypoxia.

Fetal striatal transplantation has emerged as a new therapeutic strategy in Huntington's disease (HD). Hypoxia is one of the microenvironmental stress conditions to which fetal tissue is exposed as soon as it is isolated and transplanted into the diseased host brain. Mechanisms that support neuroblast survival and replenishment of damaged cells within the HD brain in the hypoxic condition have yet to be fully elucidated. This study is aimed at investigating the molecular pathways associated with the hypoxic condition in human fetal striatal neuroblasts (human striatal precursor (HSP) cells), using the hypoxia-mimetic agent cobalt chloride (CoCl2). We analyzed the effect of CoCl2 on HSP cell proliferation and on the expression of hypoxia-related proteins, such as hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Moreover, we evaluated fibroblast growth factor 2 (FGF2; 50ng/ml) and endothelin-1 (ET-1; 100nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. Dose-response experiments using increasing concentrations of CoCl2 (50-750µM) showed that the HSP cell growth was unaffected after 24h, while it increased at 48h, with the maximal effect observed at 400µM. In contrast, cell survival was impaired at 72h. Hypoxic conditions determined HIF-1α protein accumulation and increased gene and protein expression of VEGF, while FGF2 and ET-1 significantly stimulated HSP cell proliferation both in normoxic and hypoxic conditions, thus counteracting the apoptotic CoCl2 effect at 72h. The incubation with selective receptor (FGFR1, endothelin receptor A (ETA) and endothelin receptor B (ETB)) inhibitors abolished the FGF2 and ET-1 neuroprotective effect. In particular, ET-1 stimulated HSP cell survival through ETA in normoxic conditions and through ETB during hypoxia. Accordingly, ETA expression was down-regulated, while ETB expression was up-regulated by CoCl2 treatment. Overall, our results support the idea that HSP cells possess the machinery for their adaptation to hypoxic conditions and that neurotrophic factors, such as FGF2 and ET-1, may sustain neurogenesis and long-term survival through complex receptor-mediated mechanisms.

The distribution of KIR-HLA functional blocks is different from north to south of Italy.

The killer cell immunoglobulin-like receptor (KIR)-human leukocyte antigen (HLA) interaction represents an example of genetic epistasis, where the concomitant presence of specific genes or alleles encoding receptor-ligand units is necessary for the activity of natural killer (NK) cells. Although KIR and HLA genes segregate independently, they co-evolved under environmental pressures to maintain particular KIR-HLA functional blocks for species survival. We investigated, in 270 Italian healthy individuals, the distribution of KIR and HLA polymorphisms in three climatic areas (from cold north to warm south), to verify their possible geographical stratification. We analyzed the presence of 13 KIR genes and genotyped KIR ligands belonging to HLA class I: HLA-C, HLA-B and HLA-A. We did not observe any genetic stratification for KIR genes and HLA-C ligands in Italy. By contrast, in a north-to-south direction, we found a decreasing trend for the HLA-A3 and HLA-A11 ligands (P = 0.012) and an increasing trend for the HLA-B ligands carrying the Bw4 epitope (P = 0.0003) and the Bw4 Ile80 epitope (P = 0.0005). The HLA-A and HLA-B KIR ligands were in negative linkage disequilibrium (correlation coefficient -0.1211), possibly as a consequence of their similar function in inhibiting NK cells. The distribution of the KIR-HLA functional blocks was different along Italy, as we observed a north-to-south ascending trend for KIR3DL1, when coupled with HLA-B Bw4 ligands (P = 0.0067) and with HLA-B Bw4 Ile80 (P = 0.0027), and a descending trend for KIR3DL2 when coupled with HLA-A3 and HLA-A11 ligands (P = 0.0044). Overall, people from South Italy preferentially use the KIR3DL1-HLA-B Bw4 functional unit, while those from the North Italy equally use both the KIR3DL2-HLA-A3/A11 and the KIR3DL1-HLA-B Bw4 functional units to fight infections. Thus, only KIR3DL receptors, which exert the unique role of microbial sensors through the specific D0 domain, and their cognate HLA-A and HLA-B ligands are selectively pressured in Italy according to geographical north-to-south distribution.

Progress in restorative neurosurgery: human fetal striatal transplantation in Huntington's disease. Review.

The purpose of this paper was to offer a review of the rationale, methods, biological and clinical results of human fetal striatal transplantation (HFST) in the treatment of Huntington's disease (HD). HD is a heritable neurodegenerative disease in which degeneration of neurons in the striatum leads to motor, psychiatric and cognitive deficits. The disease is progressive and inexorably lethal. At present there are no curative treatments for HD. A restorative therapy based on the intrastriatal transplantation of striatal neuroblasts taken from human fetus is currently being explored as potential treatment in selected HD patients. Pilot clinical trials of HFST have been started in few neurosurgery restorative centres. Results demonstrated that HFST is feasible and safe without relevant adverse effects; grafted neuroblasts survive, grow without evidence of neoplasia or teratoma, build new tissue with striatal-like imaging features, and move into the host brain towards short and long-distance cortical and sub-cortical targets. HFST delays disease progression and provides a period of improvement and stability. Even though larger-scale studies are still necessary to establish the true value of such a treatment, at this time, HFST represents a promising experimental therapy for patients with HD and one of the most interesting clinical application of restorative neurosurgery.

The conundrum of HLA-DRB1*14:01/*14:54 and HLA-DRB3*02:01/*02:02 mismatches in unrelated hematopoietic SCT.

Uncertainty still exists on the role of polymorphisms outside the HLA-DRB1 binding site or inside the HLA-DRB3 binding groove in unrelated hematopoietic SCT (HSCT). The ideal model to solve the conundrum consists of the transplants mismatched for HLA-DRB1*14:01/*14:54 and/or for HLA-DRB3*02:01/*02:02. A task force was set up in Italy to recruit transplanted pairs defined as HLA-DRB1*14:01 before 2006, the year crucial for the proper definition of the HLA-DRB1*14:54 allele in molecular biology. Out of 2723 unrelated pairs, 189 transplanted in Italy from 1995 to 2006 were HLA-DRB1*14:01 positive; 103/189 pairs with good historical DNA were retyped for HLA-DRB1*14 and HLA-DRB3 at-high resolution level; 31/103 pairs had HLA-DRB1*14 and/or HLA-DRB3 mismatched; 99/103, having complete clinical data, underwent statistical analysis for OS, TRM, disease-free survival and acute and chronic GvHD. No significant involvement of HLA-DRB1*14:01/*14:54 or HLA-DRB3*02:01/*02:02 mismatches was found, either alone or combined. Our findings suggest that disparities at exon 3 of the HLA-DRB1 gene seem unlikely to influence the outcome after HSCT. The same may be envisaged for HLA-DRB3(*)02:01 and (*)02:02 alleles which, although differing in the Ag binding site, seem unable to modulate an appreciable immune response in an HSCT setting.

[Spontaneous regression of lung metastases from renal cell carcinoma: the importance of immunogenetic factors and a review of the literature]. / Regressione spontanea delle metastasi polmonari da carcinoma renale. Ipotesi immunogenetiche e revisione della letteratura.

Spontaneous regression of renal cell cancer (RCC) metastases most frequently occurs after nephrectomy, since reducing target size makes possible a more effective immune response. RCC is immunotherapy-sensitive, as are RCC metastases, especially metastases to the lungs (organs are rich in immune cells and continually exposed to antigens), which have an antigenic effect. Immunotherapy could be perfected if a renal antigen that could be incorporated into a vaccine were identified. This would have played an important role in treatment and follow-up. Vaccine therapy for RCC is no longer far out of reach. The characteristics of RCC make it a fertile field for the study of prometastatic and endogenous antiangiogenic factors.

Radiation-induced cutaneous carcinoma of the head and neck: is there an early role for p53 mutations?

BACKGROUND: Little is known about the molecular mechanisms underlying ionizing radiation-induced carcinogenesis of the skin. AIMS: To investigate the possible role of p53 in radiodermatitis and in the development of radiation-induced cutaneous carcinomas. METHODS: The study group comprised six patients affected by cutaneous carcinomas arising in radiodermatitis (one squamous cell carcinoma and five basal cell carcinomas), and seven patients presenting only chronic radiodermatitis. Skin specimens were evaluated for p53 immunohistochemical expression. Using laser-assisted microdissection, areas with different p53 immunoreactivity were separately submitted to DNA isolation and p53 gene analysis. RESULTS: In the majority of cases (9/12, 75%), p53 immunoreactivity was detected in radiation-damaged epidermis. In carcinomas p53 oncoprotein was expressed by several neoplastic cells in one case (16.7%%), or by nearly all neoplastic cells in four (66.7%). SSCP band shifts were detected in 9/25 samples (36%) microdissected from irradiated epidermis and in 3/6 (50%) carcinomas. DNA sequencing demonstrated two repeatedly found mutations: a G deletion at codon 244 and an A-->G transition at codon 205, as well as hallmarks of ultraviolet mutagenic action, including a C-->T transition occurring at a dipyrimidine site and a CC-->TT tandem double-base transition. CONCLUSION: Our data indicate that irradiation induces significant p53 alterations that may be relevant in the modification of epithelial maturation processes and may be responsible for the high risk for development of carcinomas in radiodermatitis.

Shared-epitope HLA-DRB1 alleles and sex ratio in Italian patients with rheumatoid arthritis.

OBJECTIVE: To investigate the association between the HLA-DRB1 alleles sharing the epitope (Q/R)(K/R)RAA and rheumatoid arthritis (RA) in a large sample of Italian patients (N = 264) recruited from a single centre over the last 5 years. METHODS: Patients' classification according to the ACR criteria. DNA typing of HLA-DRB1 alleles by conventional polymerase chain reaction sequence specific oligonucleotide probing techniques. RESULTS: Low-resolution DRB1 "generic" typing showed a significantly higher frequency of DR4+ RA patients as compared to normal controls. Both DR1 and DR10 specificities were over-represented in our patients, but neither reached the statistically significant P level of 0.05 after Bonferroni's correction. However, direct search of Q(K/R)RAA epitopes, which are present in most DR4+ and DRl+ samples, demonstrated that these motifs were found at increased frequencies in RA patients. Stratification according to gender did not show differences in the proportion of disease-associated HLA alleles. CONCLUSIONS: Our study confirms the association of HLA-DR4, and -DR1 alleles, and more generally speaking of the shared epitopes Q(K/R)RAA, with disease susceptibility in Italian patients.

MICA gene polymorphisms in an Italian paediatric series of juvenile Behçet disease.

The objective of this study was to investigate MICA (major histocompatibility complex MHC class I chain-related genes) polymorphisms in an Italian series of patients with juvenile Behcet disease (jBD) and to compare these genetic findings with the high prevalence of inflammatory mucosal disease, which occurs in Western populations. Ten families which included at least 1 affected patient were studied. We genotyped 18 patients (13 children and 5 adults) affected with the complete or incomplete form of jBD comparing the results to those found in a population of 20 apparently healthy individuals. The MICA transmembrane polymorphism was analysed by PCR and polyacrylamide gel electrophoresis. HLA typing was assessed by SSP-PCR technique. Statistical analysis was performed using chi2 based methods. In our series the prevalence of gastrointestinal disease was high (41%). Seven of 10 patients were HLA-B51 positive. MICA A6 allele was present in 70% of probands as compared to 25% of an ethnically matched control population. On the other hand, MICA A5.1 was present in 20% of probands as compared to 60% in controls. Out of 5 A6 homozygotes, 2 probands and 2 affected relatives developed a severe gut inflammatory disease. The study of MICA gene polymorphisms disclosed an independent association with genetic risk for jBD. The combination of MICA A6 and HLA-B51 is the strongest genetic marker for this disease. Homozygous A6 patients seem to develop more severe mucosal gut involvement. This finding sheds light on the role of a receptor for MICA, named NKG2D, presented by natural killer cells, and CD8+, alphabetaT cells and gammadeltaT cells, usually localised in gut mucosa.

Cumulative prognostic value of p53 mutations and bcl-2 protein expression in head-and-neck cancer treated by radiotherapy.

We investigated the prognostic significance of p53-gene mutation (exon 5-9) and bcl-2-protein expression in primary squamous-cell carcinoma of the head and neck (HNSCC) treated by curative radiotherapy (RT). Primary squamous-cell carcinomas for analysis were obtained from 85 consecutive head-and-neck-cancer patients, with complete follow-up data. We detected bcl-2 protein in 24% (20/85) of HNSCC studied; 38 (45%) of the 85 tumours had cells bearing p53 mutations. A strong association was observed between tobacco exposure and bcl-2-protein expression (p = 0.003), an association also evident in those patients who had a p53-mutated carcinoma (p = 0.049). Moreover, we found that most of the bcl-2-positive cancers (70%) were also mutated in the p53 gene (p = 0.010). In univariate and in multivariate analyses, the simultaneous detection of bcl-2 expression and a p53-gene mutation in a tumour biopsy specimen was associated with greater risk of locoregional failure (p = 0.002 and 0.001 respectively) and worse survival (p = 0. 045 and 0.033) within 5 years in HNSCC patients treated by RT. The present study shows a cumulative prognostic value of simultaneous detection of bcl-2 over-expression and p53-gene aberration in some primary HNSCC treated with conventional RT, and provides further evidence for cross-talk between p53 and bcl-2, suggesting that these genes are important determinants of radiation-induced apoptosis, thereby modulating resistance to RT. Int. J. Cancer (Pred. Oncol.) 84:573-579, 1999.

Epstein-Barr virus infection and P53 expression in HIV-related oral large B cell lymphoma.

BACKGROUND: Head and neck non-Hodgkin's lymphomas in HIV positive patients are highly related with Epstein-Barr virus (EBV) infection. In general, viral agents can alter p53 protein levels by enhancing degradation of cellular p53 or by increasing its half-life by viral protein-p53 interaction. Moreover, it has been reported that modifications of p53 gene can modulate tumor cells' response to radio- and chemotherapy. METHODS: To assess a possible role of EBV infection, p53 protein deregulation, and p53 gene alterations in exons 5 to 8, we have studied six cases of HIV-related primary oral large B-cell lymphoma. We used in situ hybridization (ISH) for EBV-DNA and EBV-encoded nuclear RNA-1 (EBER-1), immunohistochemistry (IHC) for EBV latent membrane protein -1 (LMP-1) and p53 proteins expression, and single strand conformational polymorphism (SSCP) analysis to screen p53 gene mutations in exons 5 to 8. RESULTS: The EBV-DNA was present in all specimens, according to conventional DNA-ISH. No evidence for EBER-1 was found by ISH. The presence of EBV-DNA was correlated with the LMP-1 expression in all but one case. Moreover, p53 protein expression was negative in three cases and strongly positive in the others. However, mutational analysis of p53 gene in exons 5-8 showed no alteration. CONCLUSIONS: Our data may suggest that both EBV infection and LMP-1 expression may cause p53 loss of function even in the absence of p53 gene mutations, as assessed by SSCP. We speculate that the presence of EBV-infection and p53 protein deregulation may be responsible for radio- and chemotherapy resistance, by influencing apoptosis of cancer cells.

Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy.

Autosomal recessive nemaline (rod) myopathy is clinically and genetically heterogeneous. A clinically distinct, typical form, with onset in infancy and a non-progressive or slowly progressive course, has been assigned to a region on chromosome 2q22 harbouring the nebulin gene Mutations have now been found in this gene, confirming its causative role. The gene for slow tropomyosin TPM3 on chromosome 1q21, previously found to cause a dominantly inherited form, has recently been found to be homozygously mutated in one severe consanguineous case. Here we wished to determine the degree of genetic homogeneity or heterogeneity of autosomal recessive nemaline myopathy by linkage analysis of 45 families from 10 countries. Forty-one of the families showed linkage results compatible with linkage to markers in the nebulin region, the highest combined lod scores at zero recombination being 14.13 for the marker D2S2236. We found no indication of genetic heterogeneity for the typical form of nemaline myopathy. In four families with more severe forms of nemaline myopathy, however, linkage to both the nebulin and the TPM3 locus was excluded. Our results indicate that at least three genetic loci exist for autosomal recessive nemaline myopathy. Studies of additional families are needed to localise the as yet unknown causative genes, and to fully elucidate genotype-phenotype correlations.

A method for point mutation analysis that links SSCP and dye primer fluorescent sequencing.

A method is presented for mutation detection directly from single-strand conformational polymorphism (SSCP) variants. This approach is based on: (i) amplification of the exons to be analysed by SSCP using the forward primer with an eight-base tail to form a universal SSCP cassette; (ii) excision from the gel of the shifted silver-stained bands; (iii) reamplification of the eluted DNAs using, as the forward primer, a 26-base universal adaptor primer corresponding to the 18-base-21M13 sequence plus the eight nucleotides of the universal SSCP cassette; and (iv) direct sequencing of the purified products using the standard-21M13 fluorescent primer. This procedure presents several advantages including the avoidance of a cloning step which leads to significant time reduction, while maintaining comparable accuracy at relatively low costs. In conclusion, the presence of the universal SSCP cassette and subsequent reamplification with the same adaptor primer for direct sequencing makes the method universal for scanning and identification of gene alterations.

Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

Variable response to the diepoxybutane test in two dizygotic twins with Fanconi's anemia and flow cytometry for diagnosis confirmation.

Fanconi's anemia (FA) is a rare, genetically heterogeneous, autosomal recessive disorder characterized by bone marrow failure, congenital abnormalities, chromosome instability, and increased susceptibility to neoplasia. Congenital abnormalities vary in location and in severity and not all patients are affected. Although the primary defect of FA is unknown, hypersensitivity to the clastogenic effect of agents that introduce cross-links in the DNA, such as diepoxybutane (DEB), is a marker of the FA phenotype in patients suffering from aplastic anemia without the physical characteristics of the syndrome and, conversely, in cases with abnormalities in the preanemic phase. We report the case of two dizygotic twins suffering from FA with discordant hematologic data. The DEB test repeated several times in various laboratories yielded conflicting results, whereas cell cycle studies by flow cytometry revealed a pattern typical of FA patients. Moreover, the flow cytometric pattern was correlated with the clinical severity of the disease.

Potential biomarkers in predicting progression of epithelial hyperplastic lesions of the larynx.

Factors contributing to malignant transformation of laryngeal pre-neoplastic lesions remain largely unknown. Potential etiologic factors may be related to a genetically controlled sensitivity to environmental carcinogens. In this study, we investigated bleomycin-induced chromosome fragility in 15 patients with laryngeal keratoses who experienced a malignant transformation of pre-neoplastic lesions during follow-up, as compared with chromosome fragility in 15 historical controls with no progression of laryngeal keratoses during a 10-year follow-up, in a match-paired analysis. Chromosomal analysis demonstrated a higher sensitivity to clastogens in patients with malignant progression of laryngeal pre-neoplastic lesions than that of control patients with no evolution of their original laryngeal keratoses (p < 0.01). Furthermore, in the attempt to identify possible prognostic markers we studied proliferative activity (MIB-1 expression) and p53 gene aberration in biopsy samples from non-invasive and invasive laryngeal lesions in both groups. p53 immunostaining was observed in 10/15 (66.7%) of pre-neoplastic lesions and in 11/15 (73.3%) of metachronous laryngeal cancers. No differences in terms of p53 expression were noted between transformed and not-transformed lesions. Mutations at p53 gene were observed in 3/15 (20%) of pre-invasive biopsies and in 4/5 (80%) of the laryngeal cancers analyzed. Our data suggest that p53 alteration is an early event in the genesis of a subset of laryngeal carcinomas and that there is no conclusive data about the possible clonal development of metachronous laryngeal carcinoma from a p53 mutated pre-invasive disease in the same patient. MIB-1 expression was found to progressively increase with degree of epithelial hyperplasia and dysplasia in both transformed (p = 0.007) and not-transformed (p < 0.1) lesions. Surprisingly, pre-invasive lesions with tumor evolution showed a lower proliferative activity when compared with laryngeal lesions without malignant transformation (p = 0.013). These data suggests that subjects with pre-neoplastic laryngeal lesion showing an increased susceptibility to carcinogens and with less proliferative disease could be at a higher risk for development of laryngeal carcinoma.

Immunohistochemical vs molecular biology methods. Complementary techniques for effective screening of p53 alterations in head and neck cancer.

The purpose of this study was to correlate p53 gene alterations and their expression in 85 head and neck squamous cell carcinomas. Genomic p53 was amplified with the polymerase chain reaction (PCR) from formalin-fixed, paraffin-embedded tissues. Exons 5 through 8 were screened for mutations by means of non-isotopic single-strand conformation polymorphism (SSCP) analysis. p53 expression was detected by means of immunohistochemistry (IHC) with the p53 monoclonal antibody DO-7. Twenty-three lesions (27%) showed both SSCP variants and DO-7 immunostaining, whereas 37 (44%) demonstrated both SSCP normal patterns and negative staining. Twenty-five lesions (29%) were discordant, including 12 IHC-positive (14%) and 13 SSCP-positive (15%) lesions. However, discordant IHC-negative, SSCP-positive lesions could have been easily interpreted after sequencing of the abnormal samples. Had these been screened with IHC only, all nonsense or frameshift p53 mutations would have been missed. IHC-positive, SSCP-negative lesions were interpreted in light of current models of p53 immunodetection. Had these been screened with SSCP or sequencing only, a sizeable number of tumors expressing p53 protein abnormalities would have been undetected. Therefore, simultaneous use of both methods increases the number of p53 abnormalities detected, and is warranted.