Prenatal genetic counseling referrals for advanced maternal age: still room for improvement.

OBJECTIVE: The objective of this study is to evaluate genetic risks already present before pregnancy in a cohort of pregnant women referred for prenatal genetic counseling exclusively for advanced maternal age (AMA). METHOD: We retrospectively reviewed the records of 1353 women referred over 1 year (2010) for pre-test genetic counseling with the only indication of AMA at three Italian Clinical Genetic Services. RESULTS: Of the 1353 women fulfilling the inclusion criteria of the study, 87 (6.4%) had cumulatively 94 genetic risk factors not previously identified (one risk factor in 80 patients and two risk factors in seven). Twenty-six risk factors (27.7%) concerned heterogeneous or multifactorial conditions and 68 (72.3%) Mendelian or chromosomal disorders and consanguinity.In nine out of these 87 women, the estimated risk for the offspring of a genetic disease or a significant structural anomaly was >5%. Additional testing according to the identified risks was performed in 36 of these 87 women/families. CONCLUSIONS: The proportion of cases with additional risk factors is smaller than reported in previous studies, but it remains substantial and confirms the need for strategies to increase awareness of the public and health professionals responsible for the care of women in childbearing age.

Anorectal malformations and pregnancy-related disorders: a registry-based case-control study in 17 European regions.

OBJECTIVE: To identify pregnancy-related risk factors for different manifestations of congenital anorectal malformations (ARMs). DESIGN: A population-based case-control study. SETTING: Seventeen EUROCAT (European Surveillance of Congenital Anomalies) registries, 1980-2008. POPULATION: The study population consisted of 1417 cases with ARM, including 648 cases of isolated ARM, 601 cases of ARM with additional congenital anomalies, and 168 cases of ARM-VACTERL (vertebral, anal, cardiac, tracheo-esophageal, renal, and limb defects), along with 13 371 controls with recognised syndromes or chromosomal abnormalities. METHODS: Multiple logistic regression analyses were used to calculate adjusted odds ratios (ORs) for potential risk factors for ARM, such as fertility treatment, multiple pregnancy, primiparity, maternal illnesses during pregnancy, and pregnancy-related complications. MAIN OUTCOME MEASURES: Adjusted ORs for pregnancy-related risk factors for ARM. RESULTS: The ARM cases were more likely to be firstborn than the controls (OR 1.6, 95% CI 1.4-1.8). Fertility treatment and being one of twins or triplets seemed to increase the risk of ARM in cases with additional congenital anomalies or VACTERL (ORs ranging from 1.6 to 2.5). Maternal fever during pregnancy and pre-eclampsia were only associated with ARM when additional congenital anomalies were present (OR 3.9, 95% CI 1.3-11.6; OR 3.4, 95% CI 1.6-7.1, respectively), whereas maternal epilepsy during pregnancy resulted in a five-fold elevated risk of all manifestations of ARM (OR 5.1, 95% CI 1.7-15.6). CONCLUSIONS: This large European study identified maternal epilepsy, fertility treatment, multiple pregnancy, primiparity, pre-eclampsia, and maternal fever during pregnancy as potential risk factors primarily for complex manifestations of ARM with additional congenital anomalies and VACTERL.

An epidemiological survey of Mycoplasma hominis and Ureaplasma urealyticum in gynaecological outpatients, Rome, Italy.

The objective of this study was to assess the prevalence of Ureaplasma urealyticum and Mycoplasma hominis infections and to investigate associations between their presence in the lower female genital tract and lifestyle characteristics. The study was performed on a population of 3115 women, comparing the demographic and behavioural characteristics of 872 women with U. urealyticum infection and 142 women with M. hominis with uninfected women, using univariate and multiple logistic regression analysis. The prevalence of infection with U. urealyticum was 28% and M. hominis was 4.6%. In multivariate logistic regression analysis, intrauterine device, number of sexual partners and age (<35 years) were significantly associated with U. urealyticum while previous induced abortion, condom use and young age at first intercourse (<16 years) were associated with M. hominis infection. U. urealyticum infection presents the same demographic and behavioural characteristics of a sexually transmitted disease. The unprotective role of condom use suggests a non-sexual mode of transmission of M. hominis infection.

Epidemiology of Chlamydia trachomatis endocervical infection in a previously unscreened population in Rome, Italy, 2000 to 2009.

As reliable data on Chlamydia trachomatis infection in Italy are lacking and as there is no Italian screening policy, epidemiological analyses are needed to optimise effective strategies for surveillance of the infection in the country. We collected data from 6,969 sexually active women aged 15 to 55 years who underwent testing for endocervical C. trachomatis infection at the Cervico-Vaginal Pathology Unit in the Department of Gynaecology and Obstetrics of Sapienza University in Rome between 2000 and 2009. The mean prevalence of C. trachomatis endocervical infection during this period was 5.2%. Prevalence over time did not show a linear trend. Univariate analysis demonstrated a significant association of infection with multiple lifetime sexual partners, younger age (<40 years), never having been pregnant, smoking, use of oral contraceptives, and human papillomavirus and Trichomonas vaginalis infections. Multivariate stepwise logistic regression showed that T. vaginalis infection, age under 20 years and more than one lifetime sexual partner remained significantly associated with C. trachomatis infection in the final model. Prevalence of C. trachomatis in this study was high, even among women aged 25­39 years (5.1%): our data would suggest that a C. trachomatis screening policy in Italy is warranted, which could lead to a more extensive testing strategy.

The Italian external quality assessment scheme in classical cytogenetics: four years of activity.

BACKGROUND: The Italian external quality assessment scheme in classical cytogenetics was started in 2001 as an activity funded by the National Health System and coordinated by the Italian Public Institute of Health. OBJECTIVES: The aim of our work is to present data from the first 4 years of activity, 2001-2004. METHODS: Italian cytogenetics public laboratories were enrolled on a voluntary basis, and this nationwide program covered prenatal, postnatal and oncological diagnosis. The scheme is annual and retrospective; a panel of experts reviewed the quality of images and reports in order to assess technical, analytical and interpretative performance. RESULTS: Over the 4-year period, the number of participating laboratories increased: from 36 in 2001, 46 in 2002, 49 in 2003 to 51 in 2004. The overall technical performance was satisfactory. Inadequacy or lack of information in reporting was the most frequent analytical inaccuracy identified in all parts of the scheme. However, the percentage of complete reports increased significantly during the period: by 36% in postnatal diagnosis between 2001 and 2004 (p < 0.001) and by 42% in oncological diagnosis between 2002 and 2004 (p = 0.003). CONCLUSIONS: Our experience reveals that participation in external quality assessment programs has significant advantages, helping to standardize and to assure quality in cytogenetic testing.

Prenatal diagnosis of a de novo satellited chromosome 18 (18ps) associated with 18p deletion.

De novo satellited non-acrocentric chromosomes are very rare findings in prenatal diagnosis. Here we report the first case of a de novo 18ps, associated with del(18p), detected at prenatal diagnosis. A 37 years old woman underwent Chorionic Villus Sampling (CVS) for advanced maternal age. Cytogenetic analysis on direct CVS preparation (CVSc) revealed a male karyotype with a nonfamilial satellited 18ps and a reciprocal translocation t(17;19)(P11.1;q11) of maternal origin. The mesenchimal CVS culture (CVSm) showed a mosaic of cell lines with various involvement of chromosome 18: 18ps [36/70]/ r(18) [25/70]/ del(18p) [3/70]/ -18 [6/70]. Amniotic fluid cells (AFC) confirmed the homogeneous karyotype found at CVSc. The molecular cytogenetic characterization, performed on AFC, allowed the following diagnosis: 46,XY, +15, dic(15;18)(p11.1;p11.2), t(17;19)(p11.1;q11)mat. ish dic(15;18)(tel 18p-, D15Z1+, wcp18-, wcp 18+, D18Z1+, tel 18q+). The foetal autopsy disclosed subtle facial dysmorphisms and corpus callosum hypoplasia. In case of prenatal detection of de novo terminal ectopic NORs an accurate cytogenetic and molecular analysis should be performed in order to rule out subtle unbalancements.

Paracentric inversion of Yq and review of the literature.

We report on the second prenatal diagnosis of familial paracentric inversion of the long arm of Y chromosome [46, X, inv(Y)(q11.2q12)]. The anomaly was detected through an amniocentesis performed because of advanced maternal age. The inversion has been detected by standard GTG banding methods and better characterized by FISH with painting probe and specific satellite probes DYZ1 and DYZ3. The inversion derived from phenotypically normal father. Pregnancy was uneventful and an healthy child was born. We discuss the issue concerning genetic prenatal counselling of this rare condition and we report the clinical follow up of the child.

Intronic breakpoint definition and transcription analysis in DMD/BMD patients with deletion/duplication at the 5' mutation hot spot of the dystrophin gene.

Dystrophin mutations occurring at the 5' end of the gene frequently behave as exceptions to the "frame rule," their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5' dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3-7, 3-6 and duplication of exons 2-4. Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3-7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3-7 deletion. Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2-4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5' dystrophin mutations, including recoding mechanisms and unusual splicing choices.

FGFR3 mutation in thanatophoric dysplasia type 1 with bilateral cystic renal dysplasia: coincidence or a new association?

Thanatophoric dysplasia (TD) is a lethal dwarfism condition due to missense mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Examination of TD patients reveals mainly the involvement of the skeletal system and the brain, but also renal and cardiovascular anomalies have been described. We report the prenatal detection of TD type 1 (TD1) associated with bilateral cystic renal dysplasia (CRD) Potter's type II, in which the molecular analysis reveals the typical Arg248Cys substitution in the FGFR3 gene. CRD has not been previously described in TD or other conditions due to FGFR3 mutations, but occurs in Apert syndrome (due to FGFR2 mutations). The possible involvement of renal developmental defect in FGFR3 mutations is discussed.

International cooperation and networking in genetic health care provision: issues arising from the genetic services plan for the Emilia-Romagna region, Italy.

The aims of this report are to describe the genetic plan for Emilia-Romagna, a region in Italy, and to contribute to the international exchange of information on developing and applying policy frameworks to provide high-quality and comprehensive genetic health care in the publicly funded health systems. At the present time there is no national policy for genetic medicine in Italy, and only two regions, Emilia-Romagna and Liguria, have formally agreed to a strategic plan for health care in genetics. The current provision of genetic services in Emilia-Romagna is described focusing on the intra- and inter-organizational linkages to ensure a comprehensive system of coordinated activities. Strengths and implementation areas are highlighted. Points that must be solved within the regional or national context are the definition of the level of assistance required in genetic medicine, the formal professional recognition of the genetic counselor and the adjustment of the billing mechanisms to the complexities of clinical genetic services. Issues that need to be addressed at a wider level include full assessment of genetic tests before their introduction into clinical practice, networking to provide tests for the rarest genetic diseases, consensus on fundamental terminology and clinical and administrative data sets to promote a cohesive framework for the flow of information throughout the health care systems with respect to genetics.

Evidence of kinesin heavy chain (KIF5A) involvement in pure hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) are characterized by progressive lower extremity spasticity due to an axonal degeneration of motor and sensory neurons. We report a four-generation pedigree segregating an autosomal dominant phenotype for HSP and showing a linkage to the SPG10 locus, coding for Kinesin family member 5A. Subsequent to a denaturing high performance liquid chromatography (dHPLC) mutation screening we found a new missense mutation 838C>T (R280C) at an invariant arginine residue in a region involved in the microtubule binding activity.

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.

Occurrence of del(GIB6-D13S1830) mutation in Italian non-syndromic hearing loss patients carrying a single GJB2 mutated allele.

Molecular screening for GJB2 (connexin 26) mutations represents the standard diagnostic approach for the genotype definition of non-syndromic deafness. Nevertheless, a single GJB2 pathogenic mutation is detectable in a relevant number of cases, therefore failing to explain the phenotype. We aimed at assessing the occurrence of the recently described del(GIB6-D13S1830) mutation, occurring in the connexin 30 gene, in a group of Italian hearing-impaired patients carrying a single GJB2 mutated allele. A total of 59 non-syndromic hearing loss (NSHL) patients were screened for GJB2 mutations. Among these, nine NSHL patients were found to be heterozygous for a single GJB2 mutation. These patients, heterozygotes for different GJB2 mutated alleles (35delG, L90P, M34T, V153I), together with 11 additional 35delG/neg cases previously described, were studied for the presence of the del(GIB6-D13S1830) mutation. Two double heterozygotes del(GIB6-D13S1830)/35delG were identified. In both cases the degree of hearing loss was profound. Furthermore, GJB2 molecular screening led to the identification of a novel change (T55G) occurring in compound heterozygosity with the V37I mutation. In conclusion, our data suggest a significant frequency of del(GIB6-D13S1830) mutation in Italian hearing-impaired subjects (10% of unexplained GJB2 heterozygotes) similar to that reported in other European countries.

Prenatal diagnosis of a complete mole coexisting with a dichorionic twin pregnancy: case report.

A complete mole coexisting with dichorionic twins was diagnosed by the combined use of sonography and chorionic villus sampling at 10 weeks gestation. The pregnancy resulted in the death of one fetus at 31 weeks from presumed feto-maternal haemorrhage, while the other fetus survived in good condition. A summary of the available literature, combined with this report, reveals a total of seven pregnancies with twins and a coexistent complete mole. Only two out of 14 fetuses survived. Maternal complications included one case of pre-eclampsia and one persistent trophoblastic tumour. Accurate diagnosis of complete mole is possible by genetic analysis of chorionic villi obtained with standard transabdominal sampling. Twins with a coexistent complete mole will usually undergo miscarriage. However, fetal survival is possible and the maternal risks seem limited. A concomitance between gestational trophoblastic disease and the occurrence of feto-maternal haemorrhage is observed.

Congenital heart defects: 15 years of experience of the Emilia-Romagna Registry (Italy).

OBJECTIVES: Collection and assessment of data from the Emilia-Romagna Region on the occurrence of congenital heart defects in order to identify an homogeneous group of patients for further aetiologic and genetic studies. MATERIALS AND METHODS: The present study is based on 1549 stillborn and live born babies affected by congenital heart defect out of 330,017 consecutive births (4.7 per 1000). RESULTS: The frequency and type of congenital heart defects have been identified together with the sex ratio, associated extracardiac anomalies, chromosomal anomalies and the risk of precurrence in relatives. The impact of prenatal diagnosis on prevalence was low during the study period. CONCLUSIONS: The study has provided epidemiological data for public health surveillance of congenital heart defects in the Emilia-Romagna region. The creation of a system for the nationwide recording of congenital heart defects designed with regard to the sources of ascertainment, the diagnostic criteria, and the system of classification is emphasised.

Exploring the clinical and epidemiological complexity of GJB2-linked deafness.

GJB2 mutation analysis was performed in 179 unrelated subjects with sporadic or familial hearing loss (HL). Among 57 families, 18 showed a vertical transmission of HL, the disease being present in two or three generations. Besides 155 nonsyndromic cases, 24 patients presenting with extra-auditory clinical signs were included in the molecular study. GJB2 mutation analysis was also performed in 19 subjects with an anamnestic history of perinatal risks factors for acquired HL. The 35delG mutation accounted for 22.1% of analyzed chromosomes in sporadic cases and 39.4% in familial cases; 35delG prevalence reached 41% in autosomal recessive and 44.4% in pseudodominant pedigrees. Two novel GJB2 mutations were identified in compound heterozygosity with 35delG allele (D159V, 284ins/dup[CACGT]). Two 35delG homozygous subjects were identified among HL cases classified as environmental in origin. Four patients 35delG heterozygous (35delG/V95M, 35delG/L90P, 35delG/167delT, and 35delG/?) and two homozygous presented with extra-auditory clinical signs involving different organs (skin, vascular system, hemopoietic lineages, and thyroid). In a high proportion of 35delG heterozygous HL patients (52%), no second GJB2 mutation was detected. The reported data highlight the complexity of the genetic epidemiology of GJB2-linked deafness, further enlarging the spectrum of situations in which GJB2 mutation analysis should be performed. The presence of extra-auditory signs in a significant portion of GJB2-mutated patients suggests the possibility that GJB2 loss of function could contribute to clinical phenotypes presenting in association with deafness. This hypothesis deserves further investigation. The failure to identify a presumed partnering GJB2 mutation in a high proportion of deaf patients remains a challenging problem to be clarified.

MECP2 gene mutation analysis in the British and Italian Rett Syndrome patients: hot spot map of the most recurrent mutations and bioinformatic analysis of a new MECP2 conserved region.

Rett syndrome (RTT) is an X-linked dominant neurological disorder, which appears to be the most common genetic cause of profound combined intellectual and physical disability in Caucasian females. This syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of unknown target genes. We report a detailed mutational analysis of a large cohort of RTT patients from the UK and Italy. This study has permitted us to produce a hot spot map of the mutations identified. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, conserved among brain-specific regulatory factors.

Risk of birth defects in a population exposed to environmental lead pollution.

To investigate the relation between environmental lead and risk of birth defects in humans, we examined the prevalence at birth of congenital anomalies in an industrial area of northern Italy heavily polluted with lead. Through a population-based registry of birth defects, we identified anomalies diagnosed during three consecutive periods characterized by decreasing environmental lead exposure, 1982-1986, 1987-1990 and 1991-1995. In the lead-polluted area, we observed an excess risk of cardiovascular defects which decreased from 2.59 [95% confidence interval (CI) 1.68-3.82] in the first period to 1.18 (95% CI 0.62-2.06) and 0.97 (95% CI 0.57-1.54) in the subsequent periods. We also found an excess risk of oral clefts and musculoskeletal anomalies, with decreasing trends over time. We could not identify homogeneous patterns of temporal variation for other congenital anomalies, neither did we detect cases of neural tube defects. These results appear to support an association between severe parental lead exposure and specific birth defects.

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR.

In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single-stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase-chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele-specific 10- and 12-mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25 degrees C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10-mer W1282X probes. By contrast, when the 12-mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real-time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis.