A mild phenotype of sensorineural hearing loss and palmoplantar keratoderma caused by a novel GJB2 dominant mutation.

Dominant GJB2 mutations are known to cause a syndromic form of sensorineural hearing loss associated with palmo-plantar skin manifestations. We present the genotype/phenotype correlations of a new GJB2 mutation identified in three generations of an Italian family (proband, mother and grandfather) whose members are affected by sensorineural hearing impairment associated with adult-onset palmoplantar keratoderma. In all affected members we identified a new heterozygous GJB2 mutation (c.66G > T, p.Lys22Asn) whose segregation, population frequency and in silico prediction analysis have suggested a pathogenic role. The p.Lys22Asn GJB2 mutation causes a dominant form of hearing loss associated with variable expression of palmoplantar keratoderma, representing a model of full penetrance, with an age-dependent effect on the phenotype.

Left-right differences in the proximal femur's strength of post-menopausal women: a multicentric finite element study.

UNLABELLED: The strength of both femurs was estimated in 198 post-menopausal women through subject-specific finite element models. Important random differences between contralateral femurs were found in a significant number of subjects, pointing to the usefulness of further studies to understand if strength-based classification of patients at risk of fracture can be affected by laterality issues. INTRODUCTION: Significant, although small, differences exist in mineral density and anatomy of contralateral proximal femurs. These differences, and their combined effect, may result in a side difference in femurs' strength. However, this has never been tested on a large sample of a homogenous population. METHODS: The strength of both femurs was estimated in 198 post-menopausal women through CT-derived finite element models, built using a validated procedure, in sideways fall conditions. The impact of the resulting asymmetry on the classification of subjects at risk of fracture was analysed. RESULTS: The small difference observed between sides (the right femur on average 4 % stronger than the left) was statistically significant but mechanically negligible. In contrast, higher random differences (absolute difference between sides with respect to mean value) were found: on average close to 15 % (compared to 9.2 % for areal bone mineral density (aBMD) alone), with high scatter among the subjects. When using a threshold-based classification, the right and left femurs were discordant up to over 20 % of cases (K always lower than 0.60) but the left femur was concordant (mean K = 0.84) with the minimum strength between right and left. CONCLUSION: Considering both femurs may be important when trying to classify subjects at risk of failure with strength estimates. Future studies including fracture assessment would be necessary to quantify the real impact.

Non-invasive first trimester fetal gender assignment in pregnancies at risk for X-linked recessive diseases.

OBJECTIVES: Prenatal diagnosis in families affected by X-linked recessive disorders should ideally be limited to the subjects at increased risk, i.e. male fetuses, in order to avoid the risk of fetal loss due to the invasive procedure in healthy female fetuses. The aim of the study was to assess the fetal sex within the first trimester of gestation by two non-invasive approaches, using ultrasonography and a molecular analysis of fetal DNA extracted from whole maternal blood with specific markers, in order to avoid invasive sampling in female fetuses. METHODS: A total number of 18 fetuses at risk for an X-linked recessive disease were included in the present investigation. Maternal peripheral blood was analysed between 7 and 12 weeks of gestation by nested PCR for the detection of fetal DNA and the prediction of fetal gender. In addition, when the biparietal diameter (BPD) was between 21 and 23 mm, an ultrasonographic examination was carried out to assess the fetal gender. CVS was then performed in male fetuses only. RESULTS: Fetal gender was correctly assigned by ultrasonography between 21 and 23 mm of BPD in all the cases studied, whereas DNA extracted from whole maternal blood accurately predicted the gender in all the female cases (10), but failed in 4 out of 8 male fetuses, erroneously assigned as females. CONCLUSION: The present study shows that sonography is able to accurately predict the fetal gender within the first trimester of pregnancy, whereas the molecular analysis of DNA extracted from whole maternal blood is biased by false-Y-negative results in 50% of the cases.

Prenatal diagnosis of female pseudohermaphroditism associated with bilateral luteoma of pregnancy: case report.

Female pseudohermaphroditism associated with luteoma of pregnancy (LP) is a rare condition characterized by varying degrees of masculinization of a female fetus. We describe a case, diagnosed at 13 weeks gestation. Transvaginal ultrasound at 5 weeks of gestation revealed a normal intrauterine gestational sac and an enlarged maternal right ovary. Re-examination at 13 weeks showed a fetus with male external genitalia. Cytogenetic investigation on amniotic fluid revealed a normal female karyotype 46,XX. Follow-up sonography confirmed the previous assignment of male external genitalia and a second amniocentesis was negative for the SRY gene. High levels of androgens were found in the maternal blood. A diagnosis of female pseudohermaphroditism associated with bilateral LP was made. A healthy girl was born by Caesarean section with complete masculinization of external genitalia (Prader V). Histology confirmed a bilateral LP. To the best of our knowledge this represents the first case of prenatal diagnosis of female pseudohermaphroditism associated with LP and demonstrates the feasibility of diagnosis by sonography from 13 weeks gestation. This is also the first case described of Prader V masculinization associated with LP.

Sonographic early fetal gender assignment: a longitudinal study in pregnancies after in vitro fertilization.

OBJECTIVES: A longitudinal evaluation by sonography of external genitalia in human embryos/early fetuses with a known time from fertilization is lacking. Our aim was to assign by sonographic evaluation of external genitalia the early fetal gender in a cohort of pregnancies after in vitro fertilization. Sonographic examinations were performed in each case in three subsequent sessions over a period of time early in gestation in order to establish a temporal threshold, expressed in terms of days from fertilization, at which absolute accuracy in gender prediction is achievable. METHODS: Thirty-two fetuses were included in this prospective longitudinal study. Each was examined three times for gender assignment. The first observation was performed between 65 and 69 days from fertilization, the second between 70 and 74 days and the third between 75 and 79 days. Transvaginal and/or transabdominal sonography was used to detect the 'sagittal sign' as a marker of fetal gender. The results of ultrasound examinations were compared with gender at birth or with karyotype obtained from amniotic fluid cells or chorionic villus sampling. RESULTS: Fetal gender assignment was feasible in 29 out of 32 fetuses (90%) at the first examination and in all cases at the second and third examinations. Fetal gender prediction was correct in 76% of cases in which fetal gender was assigned (22/29) at the first examination; accuracy for males was 46% (6/13) and for females 100% (16/16). At the second and third examinations, accuracy for gender prediction achieved 100% for both genders. Concerning the temporal threshold, absolute accuracy in gender prediction was achieved at 69 days from fertilization, corresponding to 11+6 weeks based on the last menstrual period. CONCLUSION: This study provides important information about the earliest stage, expressed in terms of days from fertilization, at which it is possible to make a certain diagnosis of fetal gender by sonography.

First-trimester fetal sex prediction by deoxyribonucleic acid analysis of maternal peripheral blood.

OBJECTIVES: We investigated whether the number of weeks of gestation influences the accuracy of first-trimester fetal sex prediction by analysis of deoxyribonucleic acid extracted from whole maternal blood. A comparison was also made to determine whether a difference exists between this approach and the deoxyribonucleic acid analysis of transcervical cells performed on the same group of subjects. STUDY DESIGN: Deoxyribonucleic acid was isolated from 50 maternal blood samples taken between gestational weeks 7 and 11. The sex of the fetus was assessed by nested polymerase chain reaction specific for the amelogenin gene. A receiver-operating characteristic curve analysis was used to correlate the accuracy of fetal gender prediction with the gestational age and also to compare the goodness of the 2 methods under investigation. RESULTS: Analysis of the receiver-operating characteristic curve provided a cutoff value of 9 weeks 4 days of gestation for both tests, indicating that a higher degree of accuracy in the sex assignment was obtained in those samples taken before or at this time. However, this difference was statistically significant only for analysis of deoxyribonucleic acid from maternal blood. The comparison between tests of deoxyribonucleic acid from maternal blood and from transcervical cells showed that the first approach is better, although a statistically significant difference was not found. CONCLUSION: Analysis of maternal blood deoxyribonucleic acid is a better approach than analysis of trans-cervical cell deoxyribonucleic acid in fetal sex prediction. The highest degree of accuracy is obtained when blood is drawn before 10 weeks of gestation. This can be important when sampling of chorionic villi should be avoided because of the risk of an X-linked disease when the fetal sex is female.

Biometrical threshold of biparietal diameter for certain fetal sex assignment by ultrasound.

OBJECTIVES: The aim of this study was to establish the biometric threshold of biparietal diameter (BPD), assumed to be an independent variable of gestational age, at which 100% accuracy in the assessment of fetal sex by ultrasonography is achievable. METHODS: Transvaginal and/or transabdominal sonography was used for detecting the 'sagittal sign' as a marker of fetal sex in 385 fetuses with BPD between 18 and 29 mm. The results of ultrasound examination were compared with sex at birth or with karyotype obtained from amniotic fluid cells or chorionic villus sampling. RESULTS: Fetal sex assignment was feasible in 337 of 385 cases (87.5%). Of the 312 fetuses with known fetal sex outcome, 164 were males and 148 were females. An accuracy rate of 100% was achieved when a BPD of > or = 23 mm was obtained. CONCLUSION: This study provides important information about the earliest stage of fetal development, expressed in terms of BPD, at which a diagnosis of fetal sex can be made with 100% accuracy.

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro.

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.

Analysis of fetal sex in TCC sample DNA: a contribution to the validation of this approach.

We compared two methods of collection of transcervical cell samples, mucus aspiration and cytobrush, with respect to the efficiency in determining fetal sex and we correlated the results with the week of gestation (7-11 weeks) to evaluate if the age of gestation influenced the success of the analysis. DNA extracted from TCC samples recovered by mucus aspiration (n = 27) and cytobrush (n = 36) were analysed by nested PCR to predict fetal sex. The statistical indices of sensitivity, specificity, positive predictive value and negative predictive value were determined, and compared with those of other studies previously performed. No statistically significant difference was found between the two methods of TCC sampling concerning the success of fetal sex prediction which was high for both methods (78 per cent and 89 per cent) and no correlation with the week of gestation was found. Transcervical cell sampling represents an encouraging prospect for first-trimester prenatal diagnosis even when the least invasive techniques are used.

Enrichment of fetal nucleated red blood cells from the maternal circulation for prenatal diagnosis: experiences with triple density gradient and MACS based on more than 600 cases.

OBJECTIVE: We wanted to obtain statistically relevant data about the efficiency of our method for the isolation of fetal nucleated red blood cells (NRBCs) from the maternal circulation. METHODS: More than 600 samples were investigated using a triple density gradient followed by magnetic separation of anti-CD71-labeled cells, and yields and purities of recovered NRBCs were determined. RESULTS: The enrichment effectivity as well as the morphological condition of cells was reproducibly good, if blood samples were enriched within 48 h after sampling. The efficacy was independent of various methodological parameters and our technique was superior to other magnetic cell-sorting techniques. Mean yields and purities of NRBCs increased with increasing gestational age, ranging from 100 to 1,000 cells per 40-ml blood sample and from 0.1 to 1%, respectively, from the 6th week of gestation to term. In pregnancies with preeclampsia NRBCs were increased by a factor of 10. CONCLUSION: Our enrichment technique proved to be optimized with respect to various methodological parameters, which were compared in the present study, and it is efficient and reproducible for the enrichment of NRBCs from the maternal circulation in all three gestational trimesters.

Absence of human papillomavirus type 16 E6 transcripts in HPV 16-infected, cytologically normal cervical scrapings.

By a combination of reversed transcription and subsequent polymerase chain reaction (RNA-PCR), 23 cytologically normal cervical scrapings, positive for the presence of human papillomavirus type 16 (HPV 16) DNA, were analyzed for the presence of transcripts originating from the E6 region of the viral genome. This region is thought to be involved in transformational, tumorigenic events. No mRNAs of the E6 region were detectable using the most sensitive PCR-mediated procedure currently available. Since it was previously shown that in cytological abnormal cervical scrapings about one-half of the samples positive for HPV 16 DNA express mRNAs of the E6 region, a difference between normal and abnormal cervical scrapings, when the HPV 16 is present, exists. The observed difference between cytologically normal and abnormal, HPV-DNA-positive cervical scrapes may eventually be used as a prognostic marker for screening of women at risk for the development of cervical carcinoma. However, firm establishment of the putative correlation between tumor progression and the presence of E6 transcripts requires extensive follow-up analysis of HPV-positive patients.

Human papilloma virus infection and Ki-ras oncogene in paraffin-embedded squamous carcinomas of the cervix.

42 paraffin-embedded squamous cervical carcinomas were screened for the presence of human papilloma virus (HPV; 6b, 11, 16, 18) and for activation of the Ki-ras oncogene family by polymerase chain reaction. In 72% of cases we found one or more HPV types, but no mutations of the Ki-ras gene (codon 12-1 and 61-1, 61-2 and 61-3) were found. We conclude that mutations of the Ki-ras oncogene, at the positions analyzed, are not likely to be involved in the events leading to cervical carcinogenesis.

Detection of the human papilloma virus type 16 mRNA-transcripts in cytological abnormal scrapings.

The RNA-polymerase chain reaction (PCR) was carried out on cervical scrapings to detect and analyze transcripts from the E6-E7 open reading frames (ORF) of human papilloma virus type 16 (HPV16). The method, described previously for cervical squamous carcinomas and cervical intraepithelial neoplasias, was adapted to cervical scrapings. A primer set and two different probes specific for the E6-E7 ORFs were selected. One of the probes was able to detect the amplification products from the full length, the major, and the minor transcripts whereas the other was specific for the major transcript only. To check the quality of the mRNA in the cervical scrapings, a primer set and a probe specific for the human keratin mRNA were selected. A group of 17 abnormal cytological cervical scrapes, which were positive for HPV16 DNA, was analyzed. In this group the human papilloma virus was not always transcriptionally active, as HPV16 mRNA transcripts were detected only in about one-half (8/17) of the samples. These findings suggest that the RNA-PCR method on cervical scrapings may be very useful for epidemiological studies on the role of transcriptionally active/inactive HPV16 genes in the pathogenesis of an HPV16 infected lesion.

Perinatal AIDS.

AIDS is the major public health concern today. The short-term history of the disease adds considerable emphasis to the problem in that it has literally exploded in a few years. The pathophysiology and natural history and, what is most important, the treatment, remain enigmatic. Management of women at risk of HIV infection is colored by the nature of the illness, the subgroups of the people first infected, the mode of transmission, and the implication of vertical transmission.

Semiquantitative PCR in the diagnosis of HIV infection: correlation to clinical stage and serological status.

Authors utilized the polymerase chain reaction (PCR) technique to detect the presence of HIV DNA in 31 subjects (12 seropositive patients, 4 seronegative, at risk persons and 15 seronegative, not at risk controls). PCR was highly sensitive (enabling the detection of as few as 10 proviral genomes) and specific. By comparison to known amounts of HIV DNA, it was possible to obtain semiquantitative evaluation. No correlation was found between the proviral amount and the clinical stage of the disease or the p24 antigenemia.

Multiple primer pairs polymerase chain reaction for the detection of human papillomavirus types.

A polymerase chain reaction (PCR) based on the use of multiple primers enabling the simultaneous detection of HPV-6b, -11, -16 and -18 in a single tube reaction was developed and validated on cervico-vaginal specimens, including tissues embedded in paraffin. This PCR setting proved to be specific and sensitive, allowing the detection of as few as 200 viral particles per specimen.