When to suspect infantile hypercalcemia-1?

PURPOSE: The screening test to suspect infantile hypercalcemia-1 (HCINF1) is the measure of 25(OH)D3/24,25(OH)2D3 ratio at mass spectroscopy (MS). When the ratio is > 80, the gold standard for the diagnosis is genetic analysis. Given its limited availability, MS may not represent a screening test and most cases of HCINF1 remain undiagnosed. Aim of the study is to identify cut-offs of serum calcium and PTH useful to suspect patients with HCINF1. METHODS: We compared the levels of total serum calcium and PTH of 6 patients with HCINF1 harboring biallelic CYP24A1 pathogenic variants with 3 different control groups: (1) 12 subjects wild type for CYP24A1; (2) 12 subjects matched for age and sex; (3) 12 subjects matched for vitamin D levels. We validated the cut-offs, testing the number of adult patients affected by HCINF1 reported in the literature that could be identified using these cut-offs. RESULTS: A serum calcium level > 9.6 mg/dL showed the highest sensitivity (100%) and specificity (91%) in the comparison between homozygous and wild-type subjects. A serum PTH index < 0.315 showed the highest sensitivity (100%) and specificity (83.3%). A serum calcium level > 9.6 mg/dL was able to identify all adult HCINF1 patients whereas a PTH ratio < 0.315 identified 89.8% of the cases. Superimposable results were obtained using the other control groups. CONCLUSION: Patients with serum calcium levels higher than 9.6 mg/dL and a PTH index lower than 0.315 are likely to be affected by HCINF1. Their diagnosis may be confirmed using MS and genetic analysis.

Disorders of sexual development with XY karyotype and female phenotype: clinical findings and genetic background in a cohort from a single centre.

PURPOSE: 46, XY disorders (or differences) of sex development (DSD) are a group of clinical conditions with variable genetic background; correct diagnosis is often difficult, but it permits to optimize the management. The aim of this study is to identify clinical and genetics features of a group of women with 46, XY DSD to define some issues characterizing people with 46, XY DSD in Italy. METHODS: Retrospective analysis of girls and women with 46, XY DSD and female phenotype evaluated between year 2000 and 2016, performed by anonymised database, focusing on the clinical features and management, including presentation, first diagnostic suspect, gonadal surgery and molecular diagnostic delay. RESULTS: A total of 84 records were collected (mean age at clinical presentation: 9.1 ± 7.9 years; mean age at definitive diagnosis: 20.1 ± 15.0 years). Complete androgen insensitivity syndrome was the most common diagnosis (60%). Only 12 patients (14.3%) did not receive a molecular diagnosis. Early misdiagnoses frequently occurred; diagnostic delay was 10.2 ± 11.2 years, being reduced in patients presenting from 2007 to 2016. The discordance between genotypic and phenotypic sex during pregnancy or at birth determined early reason for referral in a considerable percentage (4.9%). CONCLUSION: Misdiagnosis and long diagnostic delays are present in females with 46, XY DSD in Italy, but the new genetic techniques permit faster right diagnoses in the last years. The centralization in dedicated third level units permits to reduce the number of patients without a molecular diagnosis, allowing better clinical management and appropriate genetic counselling.

17ß-hydroxysteroid dehydrogenase type 3 deficiency: female sex assignment and follow-up.

BACKGROUND: Deficiency of 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is a rare autosomal recessive 46,XY disorder of sex development (DSD). It is due to pathogenetic variants in the HSD17B3 gene. Mutated genes encode an abnormal enzyme with absent or reduced ability to convert Δ4-androstenedione (Δ4-A) to testosterone (T) in the fetal testis. Affected individuals are usually raised as females and diagnosis is made at puberty, when they show virilization. METHODS: A girl with a presumptive diagnosis of complete androgen insensitivity syndrome underwent endocrine and genetic assessment. Long-term follow-up was reported. RESULTS: The diagnosis of 17ß-HSD3 deficiency was made (stimulated T/Δ4-A ratio: 0.15; HSD17B3 gene analysis: c.277+4A>T in intron 3/c.640_645del (p.Glu214_Glu215del) in exon 9. After extensive information, parents decided to maintain female sex. Gonadal removal was performed and histological evaluation demonstrated deep fibrosis of testicular tissue. Follow-up till 8.5 years of age showed somatic and neuro-psychological development fitting with the female sex. CONCLUSIONS: Management of a child with the rare 17ß-HSD3 deficiency remains challenging. Any decision must be carefully evaluated with parents. Long-term follow-up must be warranted by a multidisciplinary DSD team to evaluate the adequacy of the choices made on quality of life in later life.

Clinical and genetic findings in a series of Italian children with pure hereditary spastic paraplegia.

BACKGROUND: hereditary spastic paraplegias (HSP) are a group of neurodegenerative disorders characterized by progressive lower extremity spastic weakness. SPG7, SPG4 and SPG3A are some of the autosomal genes recently found as mutated in recessive or dominant forms of HSP in childhood. SPG31 is more often associated with a pure spastic paraplegia phenotype, but genotype-phenotype correlation is still unclear. The aims of the current study was: (i) to verify the mutational frequency of SPG4, SPG3A, SPG31 and SPG7 genes in our very-well-selected childhood sample, and (ii) to improve our knowledge about the clinical and electrophysiological HSP phenotypes and their possible correlation with a specific mutation. METHODS: a sample of 14 Italian children affected by pure HSP (mean age at diagnosis 5.9 years) was extensively investigated with electrophysiological, neuroradiological and genetic tests. RESULTS: three SPG4 mutations were identified in three patients: two novel missense mutations, both sporadic, and one multiexonic deletion already reported. A novel large deletion in SPG31 gene involving exons 2-5 was also detected in one young patient. No mutations in the SPG7 and in the SPG3A genes were found. CONCLUSIONS: our data confirm that HSP represent a heterogeneous group of genetic neurodegenerative disorders, also in sporadic or autosomal recessive early onset forms. Multiplex Ligation-dependent Probe Amplification-based mutation screening for SPG4 and SPG31 genes would be added to sequencing-based screening of SPG4, SPG31 and SPG3A genes in the routine diagnosis of HSP children.

A girl with tomboy behavior: lesson from misdiagnosis in a baby with ambiguous genitalia.

5alpha-Reductase-2 deficiency is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alpha-reductase type 2 gene. It presents at birth with variable degree of undervirilization. Here, a baby with 5alpha-reductase-2 deficiency and misdiagnosis of complete androgen insensitivity syndrome, female sex assignment and early gonadectomy is described. During primary school, the girl developed tomboy behavior. Molecular analysis demonstrated compound heterozygosity for 5alpha-reductase type 2 gene mutations (exon 2: Q126R; exon 4: H230P). This child underlines the need for adequate endocrine and genetic testing for a definite diagnosis before gender is assigned in children with ambiguous genitalia and surgical interventions are carried out. Inadequate work-up may result in inappropriate gender assignment in infancy with possible inferences on outcome.

Body composition and metabolic profile in women with complete androgen insensitivity syndrome.

Clinical and experimental data suggest that androgen receptor (AR) signaling plays a role on body composition, glucose homeostasis and lipid metabolism. The effect of AR disruption on such parameters was not extensively investigated in human people. A group of young to middle-age adult women with complete androgen insensitivity syndrome (CAIS, n = 18, age 32.2 +/- 9.3 years; women with testes removed n = 14) was investigated for body mass index (BMI), body composition (dual energy X-ray absorptiometry), serum glucose levels, insulin sensitivity (HOMA-IR) and lipid profile. Mean BMI (24.2 +/- 7.4 kg/m(2)) was not significantly increased (T-score 1.0 +/- 2.5, p = NS vs Italian female reference values), but prevalence of obesity was higher in women with CAIS than that reported in age-related Italian females (16.7% vs 3.6%, respectively). The majority of obese individuals with CAIS was in the subgroup with intact testes (3/4). DXA assessment (n = 15) demonstrated values of total free fat mass similar to that of 46,XX female controls. Increased body fat was found in CAIS women in comparison with both female and male controls. Abnormal values of cholesterol (total and LDL) and HOMA-IR were present in a large subset of patients. Our data suggest that in women with CAIS disruption of AR signaling may increase body fat and affect some metabolic parameters. Assessment of body composition, metabolic profile and, likely, cardiovascular risk seems to be advisable with ageing in these individuals.

Molecular characterization of 6 unrelated Italian patients with 5alpha-reductase type 2 deficiency.

Steroid 5alpha-reductase (5alphaR) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alphaR type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5alphaR deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5alphaR deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.

Early diagnosis of 5alpha-reductase deficiency in newborns.

5Alpha-reductase-2 deficiency is a rare autosomal recessive form of 46,XY disorders of sex differentiation (DSD), caused by mutations in the steroid 5alpha-reductase type 2 gene (SRD5A2), presenting at birth with variable degrees of undervirilization. We report on three Italian newborns with 46,XY DSD in whom the evaluation of testosterone, dihydrotestosterone, testosterone/dihydrotestosterone (T/DHT) ratio and molecular analysis of the 5alpha-reductase type 2 gene was made in their first month of life. Baseline T/DHT ratio suggested 5alpha-reductase-2 deficiency; the diagnosis was confirmed by molecular genetics (homozygous mutation in exon 4 [G196S], heterozygous mutation in exon 1 and 5 [W35X/Y235F], heterozygous mutation plus polymorphism in exon 1 [G34W/A49T]). Proper investigation permitted early reassignment to male sex in two babies, assigned to female sex just after birth. In infancy, the T/DHT ratio, assessed by suitable assay methods and evaluated by age-appropriate reference values, seems to be able to select newborns affected by 5alpha-reductase-2 deficiency. Molecular analysis of the SRD5A2 gene should be warranted in newborns with abnormal ratio before sex assignment.

Nonhomologous Robertsonian translocations (NHRTs) and uniparental disomy (UPD) risk: an Italian multicentric prenatal survey.

OBJECTIVES: The risk of uniparental disomy (UPD) occurrence associated with the prenatal finding of balanced nonhomologous Robertsonian translocations (NHRTs) has been estimated only on limited empirical data. The aim of the study was to verify the estimate of the general risk, to get narrower confidence intervals by cumulating the data and to obtain risk estimates for specific translocation types. METHODS: We tested for UPD 160 prenatal specimens referred to the participant centers after the cytogenetic finding of NHRT. RESULTS: One case of upd(14)mat was found, associated with a 45,XX,der(14;22)mat fetal karyotype. The general empirical risk of UPD occurrence in NHRT carrier fetuses, corrected for the actual number of chromosomes analyzed, was 0.76% (95% CI 0.02-4.25%). Cumulative data with previous studies gives a general risk of UPD associated with NHRT of 0.80% (95% CI 0.17-2.34%). The UPD risk for the specific NHRT der(13;14) did not significantly differ from that of the other NHRTs taken together. CONCLUSION: The present survey confirms the previously estimated risk of occurrence of UPD in offspring of NHRT carriers as a low, but not negligible risk, worth being investigated in prenatal diagnosis.

Identification of Fas (APO-1/CD95) and p53 gene mutations in non-small cell lung cancer.

Fas (APO-1/CD95) is a broadly expressed death receptor involved in a series of physiological and pathological apoptotic processes. One of the possible mechanisms for resistance to apoptosis signaling in the immune system as well as in the pathogenesis of non-lymphoid malignancies is the presence of Fas mutations within the entire gene. We investigated, in 79 non-small cell lung cancer (NSCLC) samples, the promoter and the entire coding region of the Fas gene by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing in order to detect putative alterations. Sixteen of 79 tumor samples (20.2%) were found to have Fas alterations, either in promoter or exon region. Since the loss of Fas apoptotic function might be linked to p53 alterations, which are often involved in the development of NSCLC, we analyzed p53 status in 40 of the 79 NSCLC samples. p53 mutations were found to be more frequently present than Fas gene alterations (25 out of 40 cases, 62.5%). These data increase the knowledge regarding mutations of apoptosis-genes involved in the pathogenesis of NSCLC, and give benefits for the clinical management of this type of tumor.

Alterations of Fas (APO-1/CD 95) gene and its relationship with p53 in non small cell lung cancer.

The Fas (APO-1/CD95) system regulates a number of physiological and pathological processes of cell death. The ligand for Fas induces apoptosis by interacting with a transmembrane cell surface Fas receptor. The key role of the Fas system has been studied mostly in the immune system, but Fas mutations, one of the possible mechanisms for resistance to apoptosis signaling, may be involved in the pathogenesis of non-lymphoid malignancies as well. To better understand the potential involvement of Fas system in non-small cell lung cancer (NSCLC) we evaluated Fas and Fas-ligand mRNA expression by polymerase chain reaction in 102 tumor samples and in 44 normal surrounding tissues. Although over 60% of the human NSCLC analysed expressed both genes, they seem to be unable to induce apoptosis in vivo by autocrine suicide. In this regard, we investigated in 79 cases, the promoter and the entire coding region of the Fas gene by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing for detecting putative alterations. Sixteen tumors (20.25 %) were found to have Fas alterations, in promoter and/or exon region. In all cases samples carried heterozygous alterations and mostly showed simultaneous mutations of p53 gene. Moreover, the quantitative analysis of Fas mRNA expression showed high levels of Fas messenger associated with p53 wild-type status alone. Taken together, these findings point to an involvement of Fas/Fas-ligand system in the development of NSCLC, suggesting that the loss of its apoptotic function might be linked to p53 alterations which contribute to the self-maintenance of cancer cells.

Renal involvement in feline immunodeficiency virus infection: p24 antigen detection, virus isolation and PCR analysis.

Renal alterations characterized morphologically by glomerular and tubulo-interstitial lesions and clinically by a heavy proteinuria and sometimes by renal failure are frequent in feline immunodeficiency virus (FIV) infected cats. To investigate the possible role of local FIV replication in the genesis of this renal damage, renal tissues of 15 consecutive naturally infected and five non-infected cats were examined for traces of the virus by immunohistochemistry, using a monoclonal anti-p24 antibody in a streptavidin-biotin peroxidase labeled system, cultivation and polymerase chain reaction (PCR). Tubular epithelial cells as well as scattered interstitial inflammatory and glomerular cells were positive for p24 antigen in 13 cats. Viral isolation was successful in seven cats, and FIV gag DNA and RNA sequences were detected in 14 and five cats, respectively. Control cats were constantly negative. Although not conclusive, these results suggest that a direct role of FIV in the induction of the renal damage observed in infected animals is possible.

FIV infection of macrophages: in vitro and in vivo inhibition by dideoxycytidine 5'-triphosphate.

We have evaluated in vitro and in vivo whether it is possible to protect cat macrophages from feline immunodeficiency virus (FIV) infection by the administration of dideoxycytidine 5'-triphosphate (DDCTP). Since cell membranes are impermeable to phosphorylated drugs we have encapsulated DDCTP into autologous erythrocytes and modified erythrocyte membranes to target these drug-loaded cells to macrophages. DDCTP-loaded erythrocytes reduced FIV production by macrophages infected in vitro or obtained from naturally or experimentally infected cats. The same treatment protected the majority of peritoneal macrophages during a 7 month experimental FIV infection and reduced the percentage of circulating lymphocytes stained with an anti-p24 antibody. These results suggest that the administration of nucleoside analogues in phosphorylated form is feasible and their targeting to macrophages reduces FIV infection in vitro and in vivo.

The feline lymphoid cell line MBM and its use for feline immunodeficiency virus isolation and quantitation.

We report on the development of a feline T lymphoblastoid cell line obtained from the peripheral blood mononuclear cells (PBMC) of a specific pathogen free cat and designated MBM. The cells are pan-T+, CD4- and CD8- and remained interleukin-2-dependent and concanavalin A-dependent throughout the period of observation. MBM cells have proved at least as sensitive as fresh blasts to infection with cell-free stocks of three feline immunodeficiency virus (FIV) isolates. Upon infection, they exhibit a lytic cytopathic effect. Repeated attempts to establish a chronic infection have failed. Using a limiting cell dilution method, it has been shown that MBM cells may be more sensitive than fresh blasts as substrate for isolating FIV from the PBMC of infected cats. These studies have also shown that considerable individual variations exist in the virus loads present in the PBMC of naturally infected cats, and that load size does not appear to correlate with cat age, clinical status, CD4/CD8 ratio and titer of serum neutralizing antibody.

A neutralizing antibody-inducing peptide of the V3 domain of feline immunodeficiency virus envelope glycoprotein does not induce protective immunity.

Specific-pathogen-free cats, immunized with a 22-amino-acid synthetic peptide designated V3.3 and derived from the third variable region of the envelope glycoprotein of the Petaluma isolate of feline immunodeficiency virus (FIV), developed high antibody titers to the V3.3 peptide and to purified virus, as assayed by enzyme-linked immunoassays, as well as neutralizing antibodies, as assayed by the inhibition of syncytium formation in Crandell feline kidney cells. V3.3-immunized animals and control cats were challenged with FIV and then monitored for 12 months; V3.3 immunization failed to prevent FIV infection, as shown by virus isolation, anti-whole virus and anti-p24 immunoglobulin G antibody responses, and positive PCRs for gag and env gene fragments. Sequence analysis of the V3 region showed no evidence for the emergence of escape mutants that might have contributed to the lack of protection. The sera of the V3.3-hyperimmunized cats and two anti-V3.3 monoclonal antibodies neutralized FIV infectivity for Crandell feline kidney cells at high antibody dilutions but paradoxically failed to completely neutralize FIV infectivity at low dilutions. Moreover, following FIV challenge, V3.3-immunized animals developed a faster and higher antiviral antibody response than control cats. This was probably due to enhanced virus replication, as also suggested by quantitative PCR data.

Feline immunodeficiency virus infection of macrophages: in vitro and in vivo inhibition by dideoxycytidine-5'-triphosphate-loaded erythrocytes.

Although HIV-1 and other mammalian lentiviruses infect macrophages, they are not cytopathic. Consequently, these infected long-lived cells serve as major virus reservoirs with a key role in the propagation of the virus throughout the body as well as in the pathogenesis of AIDS. Furthermore, well-differentiated macrophages possess low abilities to phosphorylate the most common reverse transcriptase inhibitors of the nucleoside analog family. In an attempt to overcome these problems we have evaluated in vitro and in vivo in a feline immunodeficiency animal model whether it is possible to protect macrophages from FIV infection by direct administration of dideoxycytidine-5'-triphosphate (ddCTP). Because the cell membranes are impermeable to phosphorylated drugs we have encapsulated ddCTP into autologous erythrocytes. The drug-loaded erythrocyte membranes were then modified to target these carrier cells to macrophages. ddCTP-loaded erythrocytes were able to reduce FIV production by macrophages infected in vitro or obtained from naturally or experimentally infected cats. Furthermore, the administration of ddCTP-loaded erythrocytes protected the majority of peritoneal macrophages during a 7-month experimental FIV infection and reduced the percentage of circulating lymphocytes stained by an anti-p24 antibody. These results suggest that the administration of nucleoside analogs in phosphorylate form is feasible and their targeting to macrophages reduces FIV infection both in vitro and in vivo.

Serum neutralization of feline immunodeficiency virus is markedly dependent on passage history of the virus and host system.

Sera from feline immunodeficiency virus (FIV)-infected cats exhibited extremely low levels of neutralizing antibodies against virus passaged a few times in vitro (low passage), when residual infectivity was assayed in the CD3+ CD4- CD8- MBM lymphoid cell line or mitogen-activated peripheral blood mononuclear cells. By sharp contrast, elevated titers of highly efficient neutralizing activity against FIV were measured, by use of high-passage virus, in assays on either the fibroblastoid CrFK or MBM cell line. However, high-passage virus behaved the same as low-passage virus after one in vivo passage in a specific-pathogen-free cat and reisolation. Subneutralizing concentrations of infected cat sera enhanced the production of low-passage virus by MBM cells, an effect not seen with high-passage virus in CrFK cells. These qualitative and quantitative discrepancies could not be attributed to differences in the amount of immunoreactive viral material, to the amount of infectious virus present in the viral stocks, or to the presence of anti-cell antibodies. The observed effects were most likely due to the different passage history of the viral preparations used. The observation that neutralizing antibodies detected with high-passage virus were broadly cross-reactive in assays with CrFK cells but isolate specific in MBM cells suggests also that the cell substrate can influence the result of FIV neutralization assays. This possibility could not be tested directly because FIV adapted to grow in CrFK cells had little infectivity for lymphoid cells and vice versa. In vitro exposure to infected cat sera had little or no effect on the ability of in vivo-passaged FIV to infect cats. These data reveal no obvious relationship between titers against high-passage virus and ability to block infectivity of FIV in cats and suggest caution in the use of such assays to measure vaccine efficacy. In conclusion, by contrast with what has been previously reported for the use of CrFK cells and high-passage virus, both natural and experimental infections of cats with FIV generate poor neutralizing antibody responses with regard to in vivo protection.