First-trimester euploid miscarriages analysed by array-CGH.

It is estimated that 10-15 % of all clinically recognised pregnancies results in a miscarriage, most of which occur during the first trimester. Large-scale chromosomal abnormalities have been found in up to 50 % of first-trimester spontaneous abortions and, for several decades, standard cytogenetic analysis has been used for their identification. Recent studies have proven that array comparative genomic hybridisation (array-CGH) is a useful tool for the detection of genome imbalances in miscarriages, showing a higher resolution, a significantly higher detection rate and overcoming problems of culture failures, maternal contamination and poor chromosome morphology. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in euploid miscarriages and could be causative for the spontaneous abortion. We analysed with array-CGH technology 40 foetal tissue samples derived by first-trimester miscarriages with a normal karyotype. A whole-genome microarray with a 100-Kb resolution was used for the analysis. Forty-five copy number variants (CNVs), ranging in size between 120 Kb and 4.3 Mb, were identified in 31 samples (24 gains and 21 losses). Ten samples (10/31, 32 %) have more than one CNV. Thirty-one CNVs (68 %) were defined as common CNVs and 14 were classified as unique. Six genes and five microRNAs contained within these CNVs will be discussed. This study shows that array-CGH is useful for detecting submicroscopic CNVs and identifying candidate genes which could account for euploid miscarriages.

Array-CGH and clinical characterization in a patient with subtelomeric 6p deletion without ocular dysgenesis.

Subtelomeric terminal 6p deletion has been recognized as a clinically identifiable syndrome including facial dysmorphism, malformation of the anterior eye chamber, hearing loss, heart defects, and developmental delay. Genotype-phenotype correlations of previously published patients have strongly suggested anterior eye segment anomalies as one of the major malformations of the syndrome if the critical 6p25 region contains the FOXC 1 gene. In addition, the presence in this region of one or more genes involved in hearing loss has been hypothesized. We report a patient with a 47,XYY karyotype and submicroscopic terminal 6p deletion. Further characterization of the deletion with array comparative genome hybridization also revealed a cryptic microduplication on chromosome 19. The patient showed dysmorphic features, neuromotor retardation, and profound language impairment, in absence of hearing loss and structural eye anomalies. As far as we know this is the first reported terminal 6p25.1 deletion case without eye dysgenesis precisely characterized by array-CGH. Our result suggests that the genes in this region may not be obvious candidates for hearing loss and demonstrate the need for further elucidation of the function of the genes involved in eye developmental processes.

Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth.

Sotos syndrome is characterized by pre- and post-natal overgrowth, typical craniofacial features, advanced bone age, and developmental delay. Some degree of phenotypic overlap exists with other overgrowth syndromes, in particular with Weaver syndrome. Sotos syndrome is caused by haploinsufficiency of the NSD1 (nuclear receptor SET domain containing gene 1) gene. Microdeletions involving the gene are the major cause of the syndrome in Japanese patients, whereas intragenic mutations are more frequent in non-Japanese patients. NSD1 aberrations have also been described in some patients diagnosed as Weaver syndrome. Some authors have suggested a certain degree of genotype-phenotype correlation, with a milder degree of overgrowth, a more severe mental retardation, and a higher frequency of congenital anomalies in microdeleted patients. Data on larger series are needed to confirm this suggestion. We report here on microdeletion and mutation analysis of NSD1 in 59 patients with congenital overgrowth. Fourteen novel mutations, two previously described and one microdeletion were identified. All patients with a NSD1 mutation had been clinically classified as "classical Sotos," although their phenotype analysis demonstrated that some major criteria, such as overgrowth and macrocephaly, could be absent. All patients with confirmed mutations shared the typical Sotos facial gestalt. A high frequency of congenital heart defects was present in patients with intragenic mutations, supporting the relevance of the NSD1 gene in the pathogenesis of this particular defect.

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.

Cryptic 1p36.3/6q25.2 translocation in three generations ascertained through a foetus with IUGR and cerebral malformations.

Here we describe a foetus with intrauterine growth retardation (IUGR), cerebral malformations and a 46,XY,der(1),t(1;6)(p36.3;q25.2) karyotype owing to a familial cryptic translocation segregating in three generations. A balanced translocation was present in the mother, the maternal uncle, the aunt and the grandmother. A female first cousin with dysmorphisms, hydrocephalus and mental retardation was a carrier of a partial trisomy 1p and a partial monosomy 6q. Multiple miscarriages were present in the family pedigree. Parents of the foetus had three other pregnancies: a male with a balanced translocation, and two foetuses with 1p36.3-pter monosomy and 6q25.2-qter trisomy.

18q-syndrome and ectodermal dysplasia syndrome: description of a child and his family.

The 18q- syndrome [MIM #601808] is a terminal deletion of the long arm of chromosome 18. The most common deletion extends from region q21 to qter. We report here a nine-year-old boy possessing a simple 18q- deletion who had abnormalities of the brain, skull, face, tooth, hair, bone, and skin, plus joint laxity, tongue palsy, subtle sensoneural deafness, mental and speech delay, attention deficit hyperactivity disorder (ADHD), tic, and restless legs syndromes. His karyotype was 46, XY, del (18)(q21.31-qter). The size of the deletion was approximately 45 cM. Most of these abnormalities were not explained by the 18q- deletion. The family pedigree suggested the presence of a subtle involvement of ectodermal and/or mesodermal structures. Karyotypes of the other family members were normal.

Model based sensitivity analysis of arterial pressure response to hemodialysis induced hypovolemia.

The role of hemodynamic and regulatory factors in the arterial pressure response to hemodialysis induced hypovolemia was investigated by means of a computer model of the cardiovascular system, including the main short-term pressure regulatory mechanisms. The model mimics the arterial and venous systemic circulation, Starling's law and inotropic heart regulation, arterial and cardiopulmonary baroreflex controls of resistance, and capacitance vessels. All of the model parameters have a clear physiologic meaning: 10 represent the systemic circulation, 4 describe cardiac pump performance, and 3 characterize baroreflex regulation. Sensitivity analysis is performed to determine the effect of each parameter on the pressure response to mild hypovolemia (a 10% blood volume reduction after 4 hours). The results demonstrate that circulatory parameters, such as resistances and compliances, have no relevant effect upon the pressure response. Conversely, regulation of venous capacity seems to play a pivotal role in sustaining arterial pressure during hemodialysis induced hypovolemia. Regulation of systemic peripheral resistance exerts a compensatory action only as long as the blood volume reduction is < 5%, but it is inadequate to compensate for a larger blood volume reduction when venous capacity regulation is absent. A paradoxical arterial pressure increase during hypovolemia can be referred to a prevalence of cardiopulmonary afferences in the regulatory process.

Lack of evidence of a genetic origin in the impaired spermatogenesis of a patient cohort with low-grade varicocele.

Varicocele is the most common clinical finding in infertile men but controversy continues to surround the utility of its treatment. An increased response of FSH to gonadotrophin-releasing hormone testing has been described in patients with varicocele, while the co-influence of Yq chromosome microdeletions in the infertility associated to this pathology is still under investigation. We studied 30 patients with first- and second-grade varicocele, 15 idiopathic oligozoospermic men and 21 age-matched healthy controls. All subjects underwent testicular Doppler ultrasonography, semen analysis, gonadotrophin-releasing hormone testing and baseline blood sampling for total and free testosterone, PRL, 17beta-estradiol, SHBG evaluation and Yq chromosome analysis. Apart from FSH, no difference in baseline hormonal levels was found between the groups. The patients with varicocele showed both an increased basal (p=0.007) and GnRH-induced FSH response (peak and AUC) (p=0.004) in comparison with the controls, while the idiopathic oligozoospermic men had only higher GnRH-induced FSH AUC (p=0.04). In the varicocele group, FSH peaks after GnRH testing correlated positively with the grade of disease (r=0.42, p=0.02) and negatively with sperm count (r=-0.50, p=0.005) and bilateral testis volume (r=-0.52, p=0.005). Sperm count and sperm motility were similarly significantly reduced both in patients with varicocele and in patients with idiopathic oligozoospermia in comparison with healthy controls. Yq chromosome analysis by sequence-tagged site PCR revealed no microdeletion in the AZF regions in any subject studied. Given the quite small number of subjects studied, our overall findings can only prompt us to suggest a possible causal role of varicocele in the impairment of spermatogenesis in our patients. Furthermore, although a genetic co-influence (i.e. Yq microdeletions) does not seem to be involved in the pathogenesis of infertility in men with varicocele and mild to moderate oligozoospermia, genetic screening seems to be advisable, especially in those patients who present a severe impairment of sperm count, as has been suggested by recent literature data.

Clinical and molecular characterisation of 80 patients with 5p deletion: genotype-phenotype correlation.

The majority of deletions of the short arm of chromosome 5 are associated with cri du chat syndrome (CdCS) and patients show phenotypic and cytogenetic variability. To perform a genotype-phenotype correlation, 80 patients from the Italian CdCS Register were analysed. Molecular cytogenetic analysis showed that 62 patients (77.50%) had a 5p terminal deletion characterised by breakpoint intervals ranging from p13 (D5S763) to p15.2 (D5S18). Seven patients (8.75%) had a 5p interstitial deletion, four (5%) a de novo translocation, and three (3.75%) a familial translocation. Of the remaining four patients, three (3.75%) had de novo 5p anomalies involving two rearranged cell lines and one (1.25%) had a 5p deletion originating from a paternal inversion. The origin of the deleted chromosome 5 was paternal in 55 out of 61 patients (90.2%). Genotype-phenotype correlation in 62 patients with terminal deletions highlighted a progressive severity of clinical manifestation and psychomotor retardation related to the size of the deletion. The analysis of seven patients with interstitial deletions and one with a small terminal deletion confirmed the existence of two critical regions, one for dysmorphism and mental retardation in p15.2 and the other for the cat cry in p15.3. Results from one patient permitted the cat cry region to be distally narrowed from D5S13 to D5S731. Furthermore, this study lends support to the hypothesis of a separate region in p15.3 for the speech delay.

Plasma levels of amyloid beta 40 and 42 are independent from ApoE genotype and mental retardation in Down syndrome.

In Down syndrome (DS) brain an early, selective accumulation of amyloid beta (Abeta) peptides ending at residue 42 (Abeta42) occurs. Whether this event depends on an altered processing of amyloid beta precursor protein (APP) or on defective clearance is uncertain. To investigate this issue, we measured Abeta species 40 and 42 in plasma from 61 patients with DS, 77 age-matched normal controls, and 55 mentally retarded subjects without chromosomal abnormalities. The Abeta 40 and 42 plasma levels were then correlated with apolipoprotein E (apoE) genotypes in all groups of cases, and with I. Q. and Mini Mental Status Examination values in DS subjects. Both Abeta species were significantly elevated in DS compared to control groups, and the extent of their increase reflects that expected from APP gene overexpression. Plasma levels of Abeta 40 and 42 did not correlate with apoE genotypes in DS and control cases, and with the extent of mental retardation in DS subjects. The results indicate that accumulation and clearance of plasma and cerebral Abeta are regulated by different and independent factors.

Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients.

The hypothesis that sperm aneuploidy and diploidy increase as a function of spermatogenesis impairment was addressed. Ejaculated semen samples from a series of men (n = 22) with very low total normal motile count (1 x 10(6)) was analysed in terms of sperm aneuploidy and diploidy by in-situ hybridization and compared with controls (n = 10). Germ cell aneuploidy was also analysed in an additional series of infertile patients presenting unexplained infertility (n = 3), congenital absence of the vas deferens (CAVD) (n = 6) and non-obstructive azoospermia (n = 3) undergoing IVF, microsurgical epididymal sperm aspiration (MESA)/ICSI and testicular sperm extraction (TESE)/ICSI cycles respectively. In-situ hybridization for chromosomes 1, 17, X and Y was performed on ejaculate, epididymal and testicular spermatozoa. Significantly higher sperm aneuploidy and diploidy rates where found (for the four chromosomes analysed) in spermatozoa from oligoasthenoteratozoospermia (OAT) over controls (18 versus 2.28% and 2.8 versus 0.13% respectively; P < 0.001). Testicular germ cells had even higher rates of sperm aneuploidy and diploidy. However, in this group it was difficult to determine whether the cells analysed were dysmorphic spermatozoa or spermatids. The data warrant further investigation on the cytogenetic abnormalities found in most germ cells identified in testicular tissue biopsies of azoospermic patients.

FRAXE mutation in a mentally retarded subject and in his phenotypically normal twin brother.

The FRAXE fragile site, 600 kb distal to the more common FRAXA, has been reported to be expressed in subjects with mild non-syndromal mental retardation (MR). Amplification of more than 200 GCC repeats, associated with methylation of the adjacent CpG island at Xq28, leads to the expression of the fragile site. In 1996 a large gene, FMR2, transcribed distally from the CpG island and downregulated by repeat expansion and methylation, was identified. Among 232 mentally retarded patients, tested FRAXA negative, we identified an Italian family segregating a hypermethylated expansion at the FRAXE locus in two dizygotic twin brothers, their sister and their mother. The index case was referred at 23 years of age with severe MR, epilepsy, a dysmorphic face with a high arched palate, marfanoid habitus and hyperreflexia of the lower limbs. His brother was referred to as normal and psychometric tests confirmed he is not mentally retarded. All members of the family underwent FRAXE molecular analysis, after cytogenetic expression of the fraX site and negative FRAXA test. Interestingly, an expansion and a hypermethylation at the FRAXE locus were found in all of them. Fibroblasts from the clinically normal brother were assayed for FMR2 expression and the transcription of the gene was found to be silenced. The presence of a phenotypically normal male with absent FMR2 expression in fibroblasts suggests that the relationship between the FRAXE mutation, FMR2 expression and MR needs to be further investigated.

Effects of ventilator resetting on indirect calorimetry measurement in the critically ill surgical patient.

OBJECTIVE: To evaluate the effect of acute changes in minute ventilation (VE) on oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient, and energy expenditure during volume-controlled mechanical ventilation in the critically ill surgical patient. The effects on some oxygen transport variables were assessed as well. DESIGN: Prospective, randomized clinical study SETTING: Adult surgical intensive care unit of a university teaching hospital. PATIENTS: Twenty adult critically ill surgical patients were studied during volume-controlled mechanical ventilation. INTERVENTIONS: After a basal period of stability (no changes over time in body temperature, energy expenditure, blood gases, acid-base status, cardiac output, and ventilatory parameters), VE was then randomly either increased or reduced (+/-35%) by a change in tidal volume (VT), while respiratory rate and inspiratory/expiratory ratio were kept constant. Settings were then maintained for 120 mins. During the study, patients were sedated and paralyzed. MEASUREMENTS AND MAIN RESULTS: VO2, VCO2, and respiratory quotient were measured continuously by a Nellcor Puritan Bennett 7250 metabolic monitor (Nellcor Puritan Bennett, Carlsbad, CA). Hemodynamic and oxygen transport parameters were obtained every 15 mins during the study. Despite large changes in VE, VO2 and energy expenditure did not change significantly either in the increased or in the reduced VE groups. After 15 mins, VCO2 and respiratory quotient changed significantly after ventilator resetting. VCO2 increased by 10.5 +/- 1.1% (from 2.5 +/- 0.10 to 2.8 +/- 0.12 mL/min/kg, p< .01) in the increased VE group and decreased by 12.4 +/- 2.1% (from 2.7 +/- 0.17 to 2.4 +/- 0.16 mL/min/kg, p< .01) in the reduced VE group. Similarly, respiratory quotient increased by 16.2% +/- 2.2% (from 0.87 +/- 0.02 to 1.02 +/- 0.02, p< .01) and decreased by 17.2% +/- 1.8% (from 0.88 +/- 0.02 to 0.73 +/- 0.02, p< .01). VCO2 normalized in the reduced VE group, but remained higher than baseline in the increased VE group. Respiratory quotient did not normalize in both groups and remained significantly different from baseline at the end of the study. Cardiac index, oxygen delivery, and mixed venous oxygen saturation increased, while oxygen extraction index decreased significantly in the reduced VE group. Neither of the mentioned parameters changed significantly in the increased VE group. CONCLUSIONS: We conclude that, during controlled mechanical ventilation, the time course and the magnitude of the effect on gas exchange and energy expenditure measurements caused by acute changes in VE suggest that VO2 and energy expenditure measurements can be used reliably to evaluate and quantify metabolic events and that VCO2 and respiratory quotient measurements are useless for metabolic purposes at least for 120 mins after ventilator resetting.

[Gonadotropin response to GnRH and seminal parameters in low grade varicocele]. / Risposta delle gonadotropine al GnRH e parametri seminali nel varicocele di basso grado.

The pathogenetic role of varicocele in male infertility is still controversial. Although epidemiological data have clearly shown a higher incidence of varicocele in the population of subfertile and infertile patients, the real effectiveness of the surgical repair of varicocele, expressed as increase in the pregnancy rate, is still debated. The presurgical gonadotropin releasing hormone (GnRH) test is the most reliable predictive index of successful surgical outcome in terms of fertility. Only patients with an increased gonadotropin response (in particular FSH) to GnRH will benefit from the surgery. The aim of the present study was to evaluate the gonadotropin response to GnRH 50 micrograms i.v. in a group of patients with low-medium grade varicocele. At the beginning of the test, a fine needle was inserted into the forearm and kept patent by a saline solution. Blood samples were collected at the following experimental times: 0, +15, +30, +60, +90, +120 min. The stimulus was administered i.v. as bolus at time 0. The gonadotropin response to the stimulus and baseline levels of testosterone, PRL, 17 beta oestradiol and SHBG were compared with those of a control group. Moreover, all the patients underwent semen analysis after 3-7 days' abstinence and to ultrasound-doppler of the testis. Finally, we preliminarily looked for the presence of microdeletions on the Yq chromosome by polymerase chain reaction. No difference in baseline hormonal levels was found between the patients with varicocele and the controls; the LH response to GnRH was also similar in the two groups. The patients with varicocele showed a significantly (p = 0.03) higher FSH response (13.6 +/- 5.9 mUI/ml) to GnRH than controls (3.8 +/- 0.5 mUI/ml). A significant positive correlation (r = 0.6, p = 0.05) was found between LH peaks after GnRH testing and varicocele grade. Nine of 11 patients with varicocele showed significant seminal abnormalities (i.e., oligoasthenospermia): all patients showed a normal karyotype and no microdeletions were detected on the Yq chromosome. The authors underline the importance of presurgical GnRH testing in patients with low grade varicocele, given the close correlation between gonadotropin-stimulated peaks and varicocele grade found in the study. The presence of significant seminal abnormalities, even in patients with low grade varicocele, suggests the use of molecular genetic techniques to detect possible microdeletions on the Yq chromosome, which may be responsible for the infertility.

Cytogenetic and molecular study of 32 Down syndrome families: potential leukaemia predisposing role of the most proximal segment of chromosome 21q.

Down syndrome (DS) children have a 10-20-fold increased risk of developing ALL or AML compared to non-DS children. An increased disomic homozygosity of the polymorphic DNA markers in the pericentromeric region of chromosome 21q (21q11) has repeatedly been found in DS patients with ANLL-M7 and DS-specific transient abnormal myelopoiesis (TAM), compared to the majority of DS subjects without leukaemia. Analysis of cytogenetic heteromorphisms and 26 polymorphic DNA markers from chromosome 21q showed an increased number of pericentromeric crossovers between the non-disjoined chromosomes in DS-ANLL cases (3/11), compared to DS-ALL (0/9) and DS-nonleukaemic cases (0/12). These findings are compatible with the model of disomic homozygosity of the predisposing allele of a putative pericentromeric gene, as an explanation for the high prevalence of ANLL in DS.

Enantioselective determination of the hydroxylated metabolites of mexiletine in human plasma.

Pre-column derivatization with o-phthaldialdehyde and N-acetyl-l-cysteine was used for liquid-chromatographic diastereomeric resolution of p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM), metabolites of mexiletine formed by aromatic and aliphatic hydroxylation, respectively. The resulting diastereomeric derivatives were resolved on a C18 column and monitored by fluorescence detection. The diastereomeric elution order for both metabolites was determined on the basis of the circular dichroism spectra of each eluted fraction. Plasma samples (500 microliters) showed recoveries greater than 75% for both the metabolites. Calibration curves in plasma samples were linear over the concentration ranges 10-500 and 20-1,000 ng/ml for each enantiomer of PHM and HMM, respectively. The limits of quantitation were found to be 10.0 and 5.0 ng/ml for both enantiomers of PHM and HMM. The within-day and between-day coefficients of variation were less than 10%. The assay was shown to be suitable for a pharmacokinetic study performed in a patient with ventricular arrhythmias following the short-term oral treatment of 200 mg t.i.d. of racemic mexiletine hydrochloride.

An improved method for the detection of Down's syndrome aneuploidy in uncultured amniocytes.

We report a modified method for the rapid detection of aneuploidies directly on human uncultured amniocytes that simplifies and shortens the entire experimental procedure, yielding signals which allow correct diagnosis of trisomy 21 in 97% of cases. The improvement is based on two points: 1) use of cosmid pockets specific for the Down's syndrome minimal region as FISH probes, and 2) a modified protocol for the fixation and preparation of amniocytes.