Fetal RHD detection from circulating cell-free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA.

OBJECTIVE: This study aimed to validate non-invasive RHD genotyping of cell-free fetal DNA (cff-DNA) using different DNA extraction methods and of fresh and frozen extracted cff-DNA. BACKGROUND: Non-invasive RHD genotyping of cff-DNA predicts fetal RhD phenotype, allowing for the rational implementation of antenatal immunoprophylaxis and representing a big step forward in the management of RhD-immunised women. Validation of a diagnostic method is mandatory before its clinical application. METHODS: RhD-negative pregnant women were recruited at different gestational ages. The cff-DNA extraction was carried out using manual and automatic methods in order to improve cff-DNA yield and optimise the extraction. Fetal RHD genotyping was performed using a commercial real-time polymerase chain reaction (PCR) kit, and the results were compared with postnatal serological RhD determination on cord blood. RESULTS: Overall, 133 plasma samples were examined for the validation process, and a total of 423 tests were performed. No differences have been observed between the two extraction methods or between fresh or frozen cff-DNA regarding cff-DNA stability and quality parameters. There was 100% concordance between fetal RHD genotyping of cff-DNA and RhD phenotype on cord blood for both extraction methods on both fresh and frozen cff-DNA. CONCLUSION: Our study shows the reliability of automatic and manual cff-DNA extraction methods and the possibility of freezing extracted cff-DNA when performing RHD genotyping. This result might be relevant for improving laboratory work and organisation through the development of a standardised procedure for fetal RHD genotyping on cff-DNA, laying the foundations for evidence-based use of anti-D Ig prophylaxis in RhD pregnant women.

Characterization of multi-locus imprinting disturbances and underlying genetic defects in patients with chromosome 11p15.5 related imprinting disorders.

The identification of multilocus imprinting disturbances (MLID) appears fundamental to uncover molecular pathways underlying imprinting disorders (IDs) and to complete clinical diagnosis of patients. However, MLID genetic associated mechanisms remain largely unknown. To characterize MLID in Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, we profiled by MassARRAY the methylation of 12 imprinted differentially methylated regions (iDMRs) in 21 BWS and 7 SRS patients with chromosome 11p15.5 epimutations. MLID was identified in 50% of BWS and 29% of SRS patients as a maternal hypomethylation syndrome. By next-generation sequencing, we searched for putative MLID-causative mutations in genes involved in methylation establishment/maintenance and found two novel missense mutations possibly causative of MLID: one in NLRP2, affecting ADP binding and protein activity, and one in ZFP42, likely leading to loss of DNA binding specificity. Both variants were paternally inherited. In silico protein modelling allowed to define the functional effect of these mutations. We found that MLID is very frequent in BWS/SRS. In addition, since MLID-BWS patients in our cohort show a peculiar pattern of BWS-associated clinical signs, MLID test could be important for a comprehensive clinical assessment. Finally, we highlighted the possible involvement of ZFP42 variants in MLID development and confirmed NLRP2 as causative locus in BWS-MLID.

A complete duplication of X chromosome resulting in a tricentric isochromosome originated by centromere repositioning.

BACKGROUND: Neocentromeres are rare and considered chromosomal aberrations, because a non-centromeric region evolves in an active centromere by mutation. The literature reported several structural anomalies of X chromosome and they influence the female reproductive capacity or are associated to Turner syndrome in the presence of monosomy X cell line. CASE PRESENTATION: We report a case of chromosome X complex rearrangement found in a prenatal diagnosis. The fetal karyotype showed a mosaicism with a 45,X cell line and a 46 chromosomes second line with a big marker, instead of a sex chromosome. The marker morphology and fluorescence in situ hybridization (FISH) characterization allowed us to identify a tricentric X chromosome constituted by two complete X chromosome fused at the p arms telomere and an active neocentromere in the middle, at the union of the two Xp arms, where usually are the telomeric regions. FISH also showed the presence of a paracentric inversion of both Xp arms. Furthermore, fragility figures were found in 56% of metaphases from peripheral blood lymphocytes culture at birth: a shorter marker chromosome and an apparently acentric fragment frequently lost. CONCLUSIONS: At our knowledge, this is the first isochromosome of an entire non-acrocentric chromosome. The neocentromere is constituted by canonical sequences but localized in an unusual position and the original centromeres are inactivated. We speculated that marker chromosome was the result of a double rearrangement: firstly, a paracentric inversion which involved the Xp arm, shifting a part of the centromere at the p end and subsequently a duplication of the entire X chromosome, which gave rise to an isochromosome. It is possible to suppose that the first event could be a result of a non-allelic homologous recombination mediated by inverted low-copy repeats. As expected, our case shows a Turner phenotype with mild facial features and no major skeletal deformity, normal psychomotor development and a spontaneous development of puberty and menarche, although with irregular menses since the last follow-up.

The Klinefelter syndrome is associated with high recurrence of copy number variations on the X chromosome with a potential role in the clinical phenotype.

The Klinefelter syndrome (KS) is the most frequent sex chromosomal disorder in males, characterized by at least one supernumerary X chromosome (most frequent karyotype 47,XXY). This syndrome presents with a broad range of phenotypes. The common characteristics include small testes and infertility, but KS subjects are at increased risk of hypogonadism, cognitive dysfunction, obesity, diabetes, metabolic syndrome, osteoporosis, and autoimmune disorders, which are present in variable proportion. Although part of the clinical variability might be linked to a different degree of testicular function observed in KS patients, genetic mechanisms of the supernumerary X chromosome might contribute. Gene-dosage effects and parental origin of the supernumerary X chromosome have been suggested to this regard. No study has been performed analyzing the genetic constitution of the X chromosome in terms of copy number variations (CNVs) and their possible involvement in phenotype of KS. To this aim, we performed a SNP arrays analysis on 94 KS and 85 controls. We found that KS subjects have more frequently than controls X-linked CNVs (39/94, [41.5%] with respect to 12/42, [28.6%] of females, and 8/43, [18.6%] of males, p < 0.01). The number of X-linked CNVs in KS patients was 4.58 ± 1.92 CNVs/subject, significantly higher with respect to that found in control females (1.50 ± 1.29 CNVs/subject) and males (1.14 ± 0.37 CNVs/subject). Importantly, 94.4% X-linked CNVs in KS subjects were duplications, higher with respect to control males (50.0%, p < 0.001) and females (83.3%, p = 0.1). Half of the X-linked CNVs fell within regions encompassing genes and most of them (90%) included genes escaping X-inactivation in the regions of X-Y homology, particularly in the pseudoautosomal region 1 (PAR1) and Xq21.31. This study described for the first time the genetic properties of the X chromosome in KS and suggests that X-linked CNVs (especially duplications) might contribute to the clinical phenotype.

Phylloid pattern of hypomelanosis closely related to chromosomal abnormalities in the 13q detected by SNP array analysis.

Phylloid hypomelanosis is a distinct type of pigmentary mosaicism characterized by congenital hypochromic macules resembling a floral ornament with various elements such as round or oval patches, asymmetrical macules similar to begonia leaves, or oblong lesions. It has been found to be predominantly associated with abnormalities in chromosome 13 and sometimes as-sociated with different extracutaneous abnormalities. Here, we report 2 new cases of phylloid hypomelanosis due to mosaicism involving chromosome 13. The first one is a mosaicism for a supernumerary marker belonging to chromosome 13 and the second one is the first report of phylloid hypomelanosis associated with a mosaic deletion of 13q. Because of the extremely low level of mosaicism in these 2 cases, SNP array analysis on skin fibroblasts was carried out, showing a 13q21.33-q34 duplication (71,024,411-115,103,529) and a 13q13.3-q34 (38,368,012-115,103,529) deletion. Both cases underline on the one hand the strict connection between phylloid hypomelanosis and anomalies of chromosome 13, and on the other hand the relevance of the SNP array analysis on skin fibroblasts in the detection of low-level mosaicism.

Microarray application in prenatal diagnosis: a position statement from the cytogenetics working group of the Italian Society of Human Genetics (SIGU), November 2011.

A precise guideline establishing chromosomal microarray analysis (CMA) applications and platforms in the prenatal setting does not exist. The controversial question is whether CMA technologies can or should soon replace standard karyotyping in prenatal diagnostic practice. A review of the recent literature and survey of the knowledge and experience of all members of the Italian Society of Human Genetics (SIGU) Committee were carried out in order to propose recommendations for the use of CMA in prenatal testing. The analysis of datasets reported in the medical literature showed a considerable 6.4% incidence of pathogenic copy number variations (CNVs) in the group of pregnancies with sonographically detected fetal abnormalities and normal karyotype. The reported CNVs are likely to have a relevant role in terms of nosology for the fetus and in the assessment of reproductive risk for the couple. Estimation of the frequency of copy number variations of uncertain significance (VOUS) varied depending on the different CMA platforms used, ranging from 0-4%, obtained using targeted arrays, to 9-12%, obtained using high-resolution whole genome single nucleotide polymorphism (SNP) arrays. CMA analysis can be considered a second-tier diagnostic test to be used after standard karyotyping in selected groups of pregnancies, namely those with single (apparently isolated) or multiple ultrasound fetal abnormalities, those with chromosomal rearrangements, even if apparently balanced, and those with supernumerary marker chromosomes.

De novo 911 Kb interstitial deletion on chromosome 1q43 in a boy with mental retardation and short stature.

Patients with distal deletions of chromosome 1q have a recognizable syndrome that includes microcephaly, hypoplasia or agenesis of the corpus callosum, and psychomotor retardation. Although these symptoms have been attributed to deletions of 1q42-1q44, the minimal chromosomal region involved has not yet defined. In this report, we describe a 7 years old male with mental retardation, cryptorchid testes, short stature and alopecia carrying only an interstitial de novo deletion of 911 Kb in the 1q43 region (239,597,095-240,508,817) encompassing three genes CHRM3, RPS7P5 and FMN2.

Premature birth with complicated perinatal course delaying diagnosis of prader-willi syndrome.

Prader-Willi syndrome in the newborn is essentially characterized by marked hypotonia, feeding difficulties, hypogonadism, and possible characteristic facial features. However, diagnosis at this age may be particularly difficult, and dysmorphic features may be subtle or absent. Prematurity can furthermore delay clinical features recognition and typical complications due to preterm birth may contribute to divert the diagnosis. We describe a preterm baby with a complicated perinatal course later diagnosed as PWS.

Genomic organization and chromosome mapping of the human homeobox gene HHEX.

In the present study, we report the genomic reconstruction of the human homeobox-containing gene HHEX by the use of the data available in public databases. This analysis allowed characterization of the gene organization showing that it is very similar to the mouse gene. Moreover the gene was mapped using FISH to 10q24.

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues.

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.

MFASAT: a new alphoid DNA sequence isolated from Macaca fascicularis (Cercopithecidae, Primates).

A new highly repeated DNA fragment isolated from Macaca fascicularis (MFASAT) is described. Our findings obtained by sequencing, Southern blot analysis, and fluorescent in situ hybridization (FISH) on metaphasic chromosomes strongly suggest that MFASAT can be considered as a member of the alphoid DNA family characteristic of Old World monkeys. The chromosomal localization of MFASAT, obtained by FISH, showed that this alphoid DNA is present in the peri-centromeric area of all the chromosomes. MFASAT showed a high degree of conservation when compared, by sequence alignment, to other Macaca species and Papio papio as expected for species with considerable genome conservation. A low degree of homology has been found comparing M. fascicularis alphoid DNA with a more distantly related Cercopithecidae species such as Cercopithecus aethiops.

Screening for fetal anomalies by ultrasound at 14 and 21 weeks.

A prospective study of screening for fetal abnormalities and chromosomal defects was carried out by ultrasound examination at 13-15 weeks of gestation and 20-22 weeks; the first scan was performed transvaginally and the second transabdominally. During a 4-year period (1991-95), 3490 unselected consecutive pregnancies with a total of 3514 fetuses were examined. There were 21 chromosomally abnormal fetuses, including ten with trisomy 21, and, in 19 (90.5%), fetal defects were detected at the first and/or second ultrasound examination. The most effective marker for chromosomal defects was increased nuchal translucency thickness (> or = 4 mm) at the 13-15-week scan, which was present in seven of the ten fetuses with trisomy 21 and in six of the 11 with other chromosomal abnormalities.

[The indications for the management of fetuses with choroid plexus cysts]. / Indicazioni per il management dei feti portatori di cisti dei plessi corioidei.

Choroid plexus cysts (CPC) in the fetus are still the subject of considerable debate in the literature. Because of their association with aneuploidy, and especially with trisomy 18, of which they are an ultrasonographic marker, the detection of fetal CPC now poses the problem of how these cases should be managed, since most occur in young women (there being no correlation between CPC and age), and since the incidence among the general population is fairly high (around 1%). With the aim of contributing further to the debate, a retrospective study was performed of all cases of fetal CPC diagnosed in our Centre between January 1984 and August 1994, together with a review of the relevant literature. There were 95 cases of fetal CPC with complete neonatal and necroptic data available. These cases included women recruited in the course of routine screening for congenital malformations carried out in our Centre at 14 and 21 weeks gestation, women referred to us from other Centres, and women recruited in the course of amniocentesis indicated for those aged over 35. In all cases in which fetal CPC was detected, a careful ultrasonographic examination was performed to exclude the presence of even the smallest morphologic anomaly. Whenever the fetus was found to have an anomaly karyotyping was done. If the CPC was not associated with any morphologic anomaly, karyotyping was proposed only to those women at risk of aneuploidy because of their age. There were 11 cases of trisomy 18, all of which presented morphologic anomalies associated with CPC. Some of these anomalies where "minor", however, and therefore difficult to assess even when a careful ultrasonographic examination was performed by an experienced operator (Intra ventricular septal defect, single umbilical artery). In 2 cases, CPC was associated with trisomy 21. Both women were aged over 35. All the other cases of CPC not associated with morphologic anomaly were normal on postnatal examination. From a meta-analysis of the literature, two distinct management protocols emerge for the problem of "isolated CPC detected at ultrasonographic examination". One group of authors recommends karyotyping for all women with fetal CPC, considering the presence of CPC in itself a risk factor for aneuploidy. The second group, to which we ourselves belong, believes it is sufficient to perform a careful ultrasonographic examination so as to exclude the presence of other morphologic anomalies associated with the CPC. Karyotyping should be proposed only to women at risk of aneuploidy because of their age (> 35). A review of the biggest series reported in the literature shows that, of a total of 1670 fetuses with CPC, 94 were trisomy 18. None of the cases of CPC "in isolation" emerged as being associated with this aneuploidy. However, numerous cases of trisomy 18 have been described in which CPC is associated with "minor" morphologic anomalies in the fetus which may be difficult to detect. It is therefore essential to perform a careful ultrasonographic examination in all cases of CPC, preferably in a Centre with specialist knowledge of ultrasonography. If this option is not available, then karyotyping of all women with fetal CPC is clearly advisable.

Screening for fra(x) mutation and Klinefelter syndrome in mental institutions.

A total of 9153 male subjects in mental institutions from six different Italian districts were screened for the presence of bilateral microorchidism, in order to detect Klinefelter syndrome. Among the 212 microorchidic patients found, 33 had an XXY karyotype (15.5%). Ninety-one Klinefelter patients with normal intelligence were also examined as a control group. Cytogenetical and clinical findings were compared in these two groups of patients and no difference was found. Two fra(x)positive subjects were found, one for each group of XXY patients; the influence of fra(x) mutation on their phenotype is discussed.

Fetal echocardiography: the role of the screening procedure.

1841 pregnant women without any risk factor for heart malformation of the fetus underwent an echocardiographic screening at an average gestational age of 23 weeks. Seven cases of heart diseases were diagnosed (0.38%), of which one died in utero at 28 weeks and another one was interrupted. In four cases the karyotype was carried out prenatally, while in the remaining three it was performed only after birth. There were 11 false-negative (0.59%), mainly concerning anomalies of minor clinical significance. Within this group, two newborns with interatrial defect of the ostium secundum type appeared to be affected by trisomy 21. Therefore, the rate of congenital heart diseases in live births not at risk (0.86%) was in line with the data reported in paediatric literature. On the other hand, the association with chromosomal anomalies was surprisingly frequent (4 out of the 9 karyotypes performed were anomalous). Such finding should be verified by studying a larger non-selected population. However, a fetal karyotype should be offered, for its practical consequences, also in those cases of cardiac anomalies of lesser clinical significance.

Deficiency of distal 8p--report of two cases and review of the literature.

A terminal deletion in the short arm of chromosome 8 was found in a 2.5-year-old boy: 46,XY,del(8) (p22.0) and in a 1-year-old girl: 46,XX,del(8) (p23.1) with dysmorphic craniofacial features and developmental retardation. Erythrocyte GSR activities of the boy and of his parents were within normal limits. Vitamin K dependent coagulation factors in the girl and her parents gave normal results. Clinical findings were compared with previously reported cases and suggested a recognizable syndrome.

Treatment of Ph+ chronic myeloid leukemia by gamma interferon.

The clinical, hematologic and cytogenetic effects of human recombinant gamma interferon (IFN) were investigated in 14 patients with Ph+ chronic myeloid leukemia (CML). Gamma-IFN was given at a daily dosage of 0.50 mg (= 10 x 10(6) U)/m2 from the 3rd week of treatment on, but the dosage had to be reduced to 0.25 mg/m2 in 10 cases and to 0.35 mg/m2 in 2 cases, because of the severity and persistence of side effects (mainly fever, fatigue, headache and pain). Only 2 patients tolerated the full dosage. The overall response rate was 64% (1 complete and 8 partial hematologic responses). Only patients in stable chronic phase responded. Two out of two patients in unstable chronic phase and two out of two patients in accelerated phase failed to respond. Eight out of nine responding patients remained in remission throughout the duration of treatment (30 to 35 weeks). No karyotypic conversion was detected. These data show that gamma IFN alone is effective in Ph+ CML, but that side effects can limit substantially the dosage and duration of treatment.

Fragile-X mutation and Klinefelter syndrome: a reappraisal.

To date the concurrent presence of the fragile-X and the Klinefelter syndromes in the same individual has been found at least 8 times either in the course of screening for the fra(X) condition in mentally retarded males or among the relatives of fra(X) propositi. Given the high frequency of both events in the general population and the heterogeneous approaches with which the above cases were ascertained, it has not been possible to determine unequivocally so far whether the finding is purely coincidental or the expression of some underlying biological relationship. To evaluate the issue, we have screened a large population of institutionalized mentally retarded males for microorchidism, and submitted to a full karyotype analysis and fra(X) testing the patients that were found to have marked bilateral microorchidism. Thus, in a total of 32 microorchidism patients identified among 1115 mentally retarded males, we found 6 to have a 47,XXY chromosome complement in all (or in most) of their cells, with one of them having also the fra(X) marker in 9% of the metaphases examined. In addition, another bearer of the fra(X) marker (but only in 4% of his metaphases) was found among 26 47,XXY mentally normal males ascertained throughout routine cytogenetic analysis of males with microorchidism referred to our genetic counseling unit during the last 10 years. In our laboratory the fra(X) marker has never been observed with such a frequency in a total of several hundred normal XY males and XX females studied as control cases in the course of previously reported family and population studies.(ABSTRACT TRUNCATED AT 250 WORDS)