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Prenatal diagnosis plays a crucial role in clinical genetics. Non-invasive prenatal diagnosis using fetal cells circulating in maternal peripheral blood has become the goal of prenatal diagnosis, to obtain complete fetal genetic information and avoid risks to mother and fetus. The development of high-efficiency separation technologies is necessary to obtain the scarce fetal cells from the maternal circulation. Over the years, multiple approaches have been applied, including choice of the ideal cell targets, different cell recovering technologies, and refined cell isolation yield procedures. In order to provide a useful tool and to give insights about limitations and advantages of the technologies available today, we review the genetic research on the creation and validation of non-invasive prenatal diagnostic testing protocols based on the rare and labile circulating fetal cells during pregnancy.

Background Chromosomal microarray analysis (CMA) is nowadays widely used in the diagnostic path of patients with clinical phenotypes. However, there is no ascertained evidence to date on how to assemble single/combined clinical categories of developmental phenotypic findings to improve the array‐based detection rate. Methods The Italian Society of Human Genetics coordinated a retrospective study which included CMA results of 5,110 Italian patients referred to 17 genetics laboratories for variable combined clinical phenotypes. Results Non‐polymorphic copy number variants (CNVs) were identified in 1512 patients (30%) and 615 (32%) present in 552 patients (11%) were classified as pathogenic. CNVs were analysed according to type, size, inheritance pattern, distribution among chromosomes, and association to known syndromes. In addition, the evaluation of the detection rate of clinical subgroups of patients allowed to associate dysmorphisms and/or congenital malformations combined with any other single clinical sign to an increased detection rate, whereas non‐syndromic neurodevelopmental signs and non‐syndromic congenital malformations to a decreased detection rate. Conclusions Our retrospective study resulted in confirming the high detection rate of CMA and indicated new clinical markers useful to optimize their inclusion in the diagnostic and rehabilitative path of patients with developmental phenotypes., This study provides a retrospective study coordinated by the Italian Society of Human Genetics (SIGU) of 5,110 patients referred to CMA for variable combined clinical phenotypes, collected by 17 Italian laboratories. The data extrapolated by the present cohort are compared to those of previously reported studies. In addition, the detection rate of single/combined clinical categories of patients sorted out from the overall cohort is evaluated to correctly assess the inclusion of the CMA in the diagnostic path of patients with the developmental clinical phenotypes.

VACTERL association is defined by the occurrence of congenital malformations: vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula with esophageal atresia, radial and renal dysplasia, and limb defects. No genetic alterations have been discovered except for some sporadic chromosomal rearrangements and gene mutations. We report a boy with VACTERL association and shawl scrotum with bifid scrotum who presented with a de novo Yq11.223q11.23 microdeletion identified by array CGH. The deletion spans 3.1 Mb and encompasses several genes in the AZFc region, frequently deleted in infertile men with severe oligozoospermia or azoospermia. Herein, we discuss the possible explanation for this unusual genotype-phenotype correlation. We suggest that the deletion of the BPY2 (previously VCY2) gene, located in the AZFc region and involved in spermatogenesis, contributed to the genesis of the phenotype. In fact, BPY2 interacts with a ubiquitin-protein ligase, involved in the SHH pathway which is known to be implicated in the genesis of VACTERL association.

Over the past two decades, there has been a rapid evolution in prenatal screening for fetal chromosome abnormalities. Initially, testing was focused on the identification of affected pregnancies in either the first, or, the second trimester (e.g. the Combined test or the triple test). This was replaced by sequential modalities (e.g. contingent screening) that have enhanced detection while reducing the need for invasive testing. More recently, the introduction of technologies based on cell-free DNA (cfDNA) in maternal plasma and enrichment of fetal cells in maternal circulation have further refined the concept of sequential screening. In this review, we document our experience with serum and ultrasound-based contingent screening where we were able to achieve a detection rate of 96.8%, a false-positive rate of 2.8% and an odds of being affected given a positive result of 1:11. We also describe our initial experience with a novel sequential protocol that includes the analysis of fetal cells in maternal blood.Methods for enrichment for fetal cells cfDNA and cfDNA technologies offer the possibility of greater sensitivity and specificity as well as expansion in the scope of genetic disorders detectable. As costs decline, these technologies will become increasingly used as primary screening tools. In the meantime, sequential use offers a practical approach to maximizing the benefits of prenatal testing. Keywords: Maternal serum, Ultrasound, Screening, Chromosome abnormality, Fetal cells, Cell-free DNA

Background A long sought goal in medical genetics has been the replacement of invasive procedures for the detection of chromosomal aneuploidies by isolating and analyzing fetal cells or free fetal DNA from maternal blood, avoiding risk to the fetus. However, a rapid, simple, consistent, and low-cost procedure suitable for routine clinical practice has not yet been achieved. The purpose of this study was to assess the feasibility of predicting fetal aneuploidy by applying our recently established dual-probe FISH protocol to fetal cells isolated and enriched from maternal blood. Methods A total of 172 pregnant women underwent prospective testing for fetal aneuploidy by FISH analysis of fetal cells isolated from maternal blood. Results were compared with the karyotype determined through invasive procedures or at birth. Results Seven of the samples exhibited fetal aneuploidy, which was confirmed by invasive prenatal diagnosis procedures. After enrichment for fetal cells, the frequency of trisomic cells was at least double in samples from aneuploid pregnancies (range 0.38–0.90%) compared to samples from normal pregnancies (≤0.18%). One false negative result was also obtained. Conclusions Noninvasive prenatal aneuploidy screening using fetal cells isolated from maternal blood is feasible and could substantially reduce the need for invasive procedures.

Interstitial 16p13.3 microduplication, encompassing the CREBBP gene, is now considered a well recognizable syndrome. To date, 28 patients have been reported with a 16p13.3 microduplication. The majority of the patients share a similar phenotype which is mainly characterized by typical facial dysmorphisms and variable intellectual disability. Other features include microcephaly, growth retardation, limb anomalies and defects of the brain, heart, genitalia, palate and eyes. We report on a de novo microduplication of chromosome 16p13.3 revealed using array-comparative genomic hybridization (array-CGH) technology in a patient presenting with variable congenital anomalies and typical facial dysmorphisms, but with no evidence of developmental delay. This case highlights the importance of an accurate clinical examination and the utility of array-CGH in pediatric patients with a characteristic phenotype but without intellectual disability.

A prenatal case of a de novo interstitial deletion distal to 8q24 was reported. Ultrasound examination and postmortem evaluation demonstrated no apparent phenotypic alterations. Array CGH showed an 11.4-Mb loss in chromosome 8 ranging from 8q24.13 to 8q24.23. This case partially overlaps the 2 cases previously described in the literature.

We describe a newborn female with a de novo duplication of chromosomes 2q31.2 and 2q37.3, and a de novo monosomy 9p24.3. The clinical findings of this patient include congenital heart defects, dysmorphic facial features, hypotonia, feeding difficulties and microcephaly. Ultrasonographic prenatal findings were negative for foetal malformations. Only a mild pyelectasis was reported. This is the first report of molecular cytogenetic characterization of a partial trisomy 2q31.2–37.3 with monosomy 9p24.3.

From January 1st 2013 to August 31st 2016, 24408 pregnant women received the first trimester Combined test and contingently offered second trimester maternal serum screening to identify those women who would most benefit from invasive prenatal diagnosis (IPD). The screening was based on first trimester cut-offs of ≥1:30 (IPD indicated), 1:31 to 1:899 (second trimester screening indicated) and ≤1:900 (no further action), and a second trimester cut-off of ≥1:250. From January 2014, analysis of fetal cells from peripheral maternal blood was also offered to women with positive screening results. For fetal Down syndrome, the overall detection rate was 96.8% for a false-positive rate of 2.8% resulting in an odds of being affected given a positive result (OAPR) of 1:11, equivalent to a positive predictive value (PPV) of 8.1%. Additional chromosome abnormalities were also identified resulting in an OAPR for any chromosome abnormality of 1:6.6 (PPV 11.9%). For a sub-set of cases with positive contingent test results, FISH analysis of circulating fetal cells in maternal circulation identified 7 abnormal and 39 as normal cases with 100% specificity and 100% sensitivity. We conclude that contingent screening using conventional Combined and second trimester screening tests is effective but can potentially be considerably enhanced through the addition of fetal cell analysis.

Study design A population of 10 156 pregnant women with singleton pregnancies were screened by the integrated test. Risks were retrospectively recalculated for contingent test strategies with first step intermediate risk groups defined by first trimester upper cut-offs of 1 : 10, 1 : 30, 1 : 50, and 1 : 70 and lower cut-offs 1 : 1500, 1 : 1200, 1 : 1100, and 1 : 900. The second trimester high risk group was based on a single cut-off of 1 : 250. Results In the first trimester, the detection rate (DR) ranged from 21% (6/29) to 52% (15/29) as the high risk first trimester cut-off was changed from 1 : 10 to 1 : 70. The corresponding first trimester false positive rate (FPR) increased from 0.2% to 1.4%. In the second trimester, an additional 21/29 (72%) to 12/29 (41%) affected pregnancies could be detected with an additional 1.6% to 2.7% false positives when lower first trimester cut-offs of 1 : 900 to 1 : 1500 were used. The best results were obtained with the upper first trimester cut-off of 1 : 30 and lower first trimester cut-off of 1 : 900, which yielded a rate of women requiring a second trimester test of only 12%, with overall DR and FPR of 93% and 2.8%, respectively. Conclusions Although the study population was relatively small, the results confirm the advantage of using contingent screening and suggest optimal first trimester cut-offs of 1 : 30 (lower cut-off) and 1 : 900 (upper cut-off). © 2012 John Wiley & Sons, Ltd.

We report the clinical and molecular investigations in a girl with 46,X,-X,+der(X)t(X;Y)(p22;q11) de novo karyotype who presented an intricate phenotype characterized by mental retardation and facial dysmorphisms in combination with short stature. The structure of the derivative X chromosome was studied using BAC array-CGH which disclosed the Xp22 breakpoint between the STS and the VCX3A gene and the presence of the Yq11.1qter chromosome. It is common that females with Xp;Yq translocations present only short stature and are normal in every other aspect. Thus, this would be the first case in which a girl with Xp;Yq translocation presents an unusual phenotype with intermediate male clinical features with Xp;Yq translocations. The risk of developing gonadoblastoma in females with Y chromosome material is also discussed and, to this effect, different explanations related to this apparent variation are also presented.

We report on a 10-year-old patient with childhood apraxia of speech (CAS) and mild dysmorphic features. Although multiple karyotypes were reported as normal, a bacterial artificial chromosome array comparative genomic hybridization revealed the presence of a de novo 14.8-Mb mosaic deletion of chromosome 7q31. The deleted region involved several genes, including FOXP2, which has been associated with CAS. Interestingly, the deletion reported here was observed in about 50% of cells, which is the first case of mosaicism in a 7q31 deletion. Despite the presence of the deletion in only 50% of cells, the phenotype of the patient was not milder than other published cases. To date, 6 cases with a deletion of 9.1-20 Mb involving the FOXP2 gene have been reported, suggesting a new contiguous gene deletion syndrome characterized mainly by CAS caused by haploinsufficiency of the genes encompassed in the 7q critical region. This report suggests that children found with a deletion involving the FOXP2 region should be evaluated for CAS and that analysis of the FOXP2 gene including array comparative genomic hybridization should be considered in selected patients with CAS. Mosaic deletions in this area may also be considered as causative of CAS.

We report on an 18-month-old boy conceived by assisted reproduction technology with developmental delay, hypotonia, microcephaly, frontal bossing, a mild convergent squint, malformed ears, and a short neck. Karyotype analysis revealed a de novo 7q21.1q22.3 duplication characterized by array comparative genomic hybridization (array-CGH) as a segment of 18.69 Mb. Duplications of the long arm of chromosome 7 are uncommon. There are 18 reported cases of different 7q segments with a pure duplication with no additional deletion of other chromosomes. As a consequence, duplications of chromosome 7q have been classified in 4 groups on the basis of the involved region. The present case is included in group 3 which involves interstitial duplications of different sizes. In the literature, only one case with an apparently smaller duplication of the same region has been described. Despite this, the phenotype is different. Moreover, the 2 patients share some phenotypic features, such as psychomotor delay, hypotonia, frontal bossing, short neck, and strabismus. However, the absence of physical characterization in most of the reported cases could justify the lacking phenotype-genotype correlation in patients with partial 7q duplication. Further studies using recent molecular approaches such as array-CGH might permit a more clinically useful grouping of 7q duplications.

Objective To compare the efficacy of combined, stepwise sequential, and contingent screening versus the integrated test in detecting fetal aneuploidies. Study design First trimester combined test, sequential second trimester, and contingent risks were retrospectively calculated for 7292 unselected pregnant women with singleton pregnancies who had received integrated screening. The first trimester testing was based on nuchal translucency, pregnancy-associated plasma protein-A, and free-beta-human chorionic gonadotrophin (free β-hCG) and the second trimester tests were alpha-fetoprotein, hCG, and unconjugated estriol. A second trimester risk of 1:250 defined a positive result for all protocols with the contingent protocol based on additional second trimester testing for those with risks between 1:30 and 1:1200. Results Among the population submitted for the integrated test, the detection rate was 19/21 (90%) for Down syndrome (DS) and 6/6 (100%) for Edwards syndrome (ES) and the DS false-positive rate (FPR) was 247/7271 (3.4%). Provision of the first trimester combined test alone would have resulted in a 17/21 (81%) detection rate for DS, that of 4/6 (67%) for ES and a DS FPR of 292/7271 (4.0%). The sequential and contingent approaches had the same final detection rates as the integrated test but potentially allowed a high proportion of the affected pregnancies to be detected in the first trimester. The lowest net DS FPR was seen with the contingent approach (2.6%) and using this protocol only 12.7% of women would have required second trimester testing. Conclusions Integrated, sequential, and contingent screenings are all more efficacious than the combined test. Overall, the contingent approach was the most efficient with a high-detection rate, the lowest FPR, and the least amount of testing. Copyright © 2011 John Wiley & Sons, Ltd.

Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.

Supernumerary invdup(15) chromosomes, now also reported as sSMC(15), containing two additional copies of Prader-Willi/Angelman critical region (PWACR) have been associated with distinct clinical phenotype that includes hypotonia, dysmorphisms, developmental delay/mental retardation, autistic behaviour, and epilepsy. We report on a healthy adult male carrying an sSMC(15) with two copies of PWACR in 20-50% of cells from different tissues. Molecular analyses showed the sSMC(15) as resulting from a PWACR-duplicated region spanning 8Mb which is larger than those in the only two other healthy PWACR-duplicated sSMC(15) carriers previously reported. Mosaicism level and mosaic cell line rate variation among different tissues observed in our case support mosaicism in critical tissues as of relevance for sSMC(15) phenotype-genotype correlations.

We report the first case of an 18p11.32 deletion, detected by array CGH, associated with a drug-resistant form of atypical absence epilepsy, global developmental delay and no signs of holoprosencephaly (HPE). In particular, this region encompasses 19 genes, and none of these genes have been strictly associated with epilepsy. Among these, TGIF1 is expressed in the fetal and adult nervous system, and its deletion has been related to central nervous system diseases. TGIF1 deletions have previously been reported in patients with a comparable phenotype as seen in our case and in children whose neurological signs and symptoms were considerable, but not epileptiform. Mutations and deletions involving the TGIF1 gene have been described in patients with HPE in an autosomal dominant model of inheritance. However, TGIF1 mutations have also been reported in normal individuals and in patients with mental retardation or showing a very mild phenotype, suggesting the characteristic of incomplete penetrance and variable expressivity. Therefore, a TGIF1 deletion may not be always related to HPE, and it may have a link to the development of epilepsy.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused in the majority of cases by deletions of the DMD gene and are readily detectable in affected males by multiplex polymerase chain reaction (PCR). However, different approaches must be used for the identification of female carriers, in which deletions are not detectable by PCR, because of the presence of a normal X chromosome. In this study, we used the multiple ligation probe amplification (MLPA) tool for the identification of female carriers of DMD deletions or duplications in 12 families with a single affected male, 10 of which were previously diagnosed as carriers of a DMD rearrangement, and the remaining two as having an unknown disease-causing mutation. In all the investigated affected males, MLPA analysis confirmed the presence of a DMD rearrangement, and in six of them allowed the refinement of the breakpoints. In 12 female relatives of the affected patients, MLPA analysis showed a DMD deletion or duplication, confirming their carrier status. Two of these were the mother and the sister of a patient whose disease-causing mutation was not known. MLPA analysis thus proved to be an useful tool for the analysis of both affected males and females carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known, providing useful information for the genetic counselling of the family.

Background SHOX alterations have been reported in 67% of patients affected by Léri-Weill dyschondrosteosis (LWD), with a larger prevalence of gene deletions than point mutations. It has been recently demonstrated that these deletions can involve the SHOX enhancer region, rather that the coding region, with variable phenotype of the affected patients. Here, we report a SHOX gene analysis carried out by MLPA in 14 LWD patients from 4 families with variable phenotype. Case presentation All patients presented a SHOX enhancer deletion. In particular, a patient with a severe bilateral Madelung deformity without short stature showed a homozygous alteration identical to the recently described 47.5 kb PAR1 deletion. Moreover, we identified, for the first time, in three related patients with a severe bilateral Madelung deformity, a smaller deletion than the 47.5 kb PAR1 deletion encompassing the same enhancer region (ECR1/CNE7). Conclusions Data reported in this study provide new information about the spectrum of phenotypic alterations showed by LWD patients with different deletions of the SHOX enhancer region.

We report a case of a two year-old girl with persistent thrombocytopenia, syndactyly, and mild psychomotor delay with speech delay. Proband chromosomal analysis from peripheral blood detected a mosaicism with two cell lines: the first with a ring chromosome 21 46,XX,r (21) (88%), and the second 45,XX,-21 (12%). In uncultured cells the level of mosaicism was 15% for the 46,XX,r(21) cell line and 85% for the 45,XX,-21 cell line. A different level of mosaicism was detected on buccal smear cells with a r(21) in 94%, and monosomy 21 in 6% of cells. The findings in the different cell lines are consistent with loss of the ring chromosome in the blood line.

A population of 10,156 pregnant women with singleton pregnancies were screened by the integrated test. Risks were retrospectively recalculated for contingent test strategies with first step intermediate risk groups defined by first trimester upper cut-offs of 1 : 10, 1 : 30, 1 : 50, and 1 : 70 and lower cut-offs 1 : 1500, 1 : 1200, 1 : 1100, and 1 : 900. The second trimester high risk group was based on a single cut-off of 1 : 250.In the first trimester, the detection rate (DR) ranged from 21% (6/29) to 52% (15/29) as the high risk first trimester cut-off was changed from 1 : 10 to 1 : 70. The corresponding first trimester false positive rate (FPR) increased from 0.2% to 1.4%. In the second trimester, an additional 21/29 (72%) to 12/29 (41%) affected pregnancies could be detected with an additional 1.6% to 2.7% false positives when lower first trimester cut-offs of 1 : 900 to 1 : 1500 were used. The best results were obtained with the upper first trimester cut-off of 1 : 30 and lower first trimester cut-off of 1 : 900, which yielded a rate of women requiring a second trimester test of only 12%, with overall DR and FPR of 93% and 2.8%, respectively.Although the study population was relatively small, the results confirm the advantage of using contingent screening and suggest optimal first trimester cut-offs of 1 : 30 (lower cut-off) and 1 : 900 (upper cut-off).

We describe the case of an adult patient affected by multiple exostoses, severe mental retardation, epilepsy and facial dysmorphisms with a deletion of ∼2.3 Mb on chromosome 11p11.21, correlated to Potocki-Shaffer syndrome (PSS). PSS is a rare contiguous gene deletion syndrome, mainly characterized by multiple exostoses and bilateral parietal foramina. Mental retardation and craniofacial dysmorphisms have often been reported, too. Although the patient showed many signs of PSS since early childhood, the diagnosis was suggested only when we examined her at adult age. This case highlights how frequently rare diseases remain undiagnosed till adulthood and is an excellent example of the need for a timely and correct diagnosis.

Departments of Pediatrics, Oral Sciences, Nano and Biotechnology, G. d’Annunzio University, Chieti and Medical Genetic Unit, Fondazione IRCCS C’a Granda Ospedale Maggiore Policlinico, Milan, Italy Correspondence to Dr Chiara Palka, MD, Department of Pediatrics, G. d’Annunzio University, Via dei Vestini, 15, 66100 Chieti, Italy Tel: + 39 0871 358015; fax: + 39 0871 574831; e-mail: chiarapalka@libero.it

We describe the case of a boy with psychomotor delay and dysmorphic features, with a germline 16q22.1 microdeletion identified by array-CGH. The deletion spans 0.24 Mb and encompasses three genes (ZFP90, CDH3 and CDH1). The deletion has been demonstrated to be inherited from his mother who was affected by lobular breast cancer (LBC) without any other apparently phenotypic features. We suppose that the microdeletion, in particular ZFP90 which is cerebrally expressed, is causative for the boy's phenotype. Mental retardation in the affected boy can recognize several mechanisms such as variable expressivity, non-penetrance, multifactorial/polygenic inheritance, recessive inheritance, a second rearrangement event and epigenetics. Furthermore, we suggest that the deletion of the CDH1, a tumor suppressor gene, involved in hereditary diffuse gastric cancer (HDGC) and LBC predisposed the mother to the carcinoma.

To compare the efficacy of combined, stepwise sequential, and contingent screening versus the integrated test in detecting fetal aneuploidies.First trimester combined test, sequential second trimester, and contingent risks were retrospectively calculated for 7292 unselected pregnant women with singleton pregnancies who had received integrated screening. The first trimester testing was based on nuchal translucency, pregnancy-associated plasma protein-A, and free-beta-human chorionic gonadotrophin (free β-hCG) and the second trimester tests were alpha-fetoprotein, hCG, and unconjugated estriol. A second trimester risk of 1:250 defined a positive result for all protocols with the contingent protocol based on additional second trimester testing for those with risks between 1:30 and 1:1200.Among the population submitted for the integrated test, the detection rate was 19/21 (90%) for Down syndrome (DS) and 6/6 (100%) for Edwards syndrome (ES) and the DS false-positive rate (FPR) was 247/7271 (3.4%). Provision of the first trimester combined test alone would have resulted in a 17/21 (81%) detection rate for DS, that of 4/6 (67%) for ES and a DS FPR of 292/7271 (4.0%). The sequential and contingent approaches had the same final detection rates as the integrated test but potentially allowed a high proportion of the affected pregnancies to be detected in the first trimester. The lowest net DS FPR was seen with the contingent approach (2.6%) and using this protocol only 12.7% of women would have required second trimester testing.Integrated, sequential, and contingent screenings are all more efficacious than the combined test. Overall, the contingent approach was the most efficient with a high-detection rate, the lowest FPR, and the least amount of testing.

We report on an apparently normal 5-month-old boy with a X;Y complex rearrangement identified first on prenatal diagnosis and found on array-CGH to have a 7.6 Mb duplication of Xp22.3 chromosome and a deletion of Yq chromosome, distal to the AZFa locus. Karyotype analysis on amniotic fluid cell cultures revealed a de novo homogenous chromosome marker that we interpreted as an isochromosome Yp. FISH analysis using SRY probe revealed only one signal on the derivative Y chromosome. The final karyotype was interpreted as 46,X,der(Y)t(X;Y)(p22.31;q11.22). Translocation Xp22;Yq11 in male are very rare event and only 4 cases have been published, all showing mental retardation and malformations. Herein we discussed some possible explanation for this apparent phenotypic variability.

Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR, which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation, or interaction with coactivators.

Complex chromosomal rearrangements with more than two breakpoints are rare. We report on a 5-year-old girl, evaluated because of psychomotor delay, ectrodactyly of right hand and feet, craniofacial dysmorphic features, cleft palate, deafness, and tetralogy of Fallot. A standard karyotype suggested a small intrachromosomal duplication of chromosome 7q. The chromosomal rearrangement was characterized by mBAND, which disclosed a reciprocal interstitial translocation t(7;8)(q21q22;q23q24). FISH analysis and array-CGH analysis showed a paracentric inversion of 7q and a microdeletion of 7q21.13. The parents had normal chromosomes. The deletion found in the present patient confirms that candidate region of ectrodactyly-deafness (OMIM 220600) maps to 7q21 and suggests new candidate genes for that disorder. This patient also had facial features reminiscent of tricho-rhino-phalangeal syndrome and one chromosome breakpoint involved band 8q24, a locus for this disorder. In addition, FOG1 gene maps to 8q23 and has been implicated in a subset of subjects with tretralogy of Fallot. We suggest that the aberration of 8q may have contributed to her facial and cardiac findings.

Objective: To investigate the relationship between maternal serum screening markers and pregnancy outcome in fetuses with cystic hygroma at 15–18 weeks of gestation. Study design: We retrospectively reviewed case-notes of 34 consecutive singleton fetuses with cystic hygroma referred at 15–18 weeks of gestation. All cases had maternal blood sampled for triple screening at the time of the ultrasound scan. Results In total, 62% of fetuses with cystic hygroma had abnormal chromosome complements and 80% had a poor outcome. Six fetuses presenting normal values of human chorionic gonadotropin (0.5–2.5 MoM [multiples of the median]), serum alpha-fetoprotein (0.5–2.5 MoM) and unconjugated estriol (>0.5 MoM), normal karyotype and absence of associated structural anomalies had an uneventful outcome. Conclusions Our data demonstrated that cystic hygroma at 15–18 weeks has a strong association with chromosomal abnormalities. In euploid fetuses, maternal serum screening results may have a role in the diagnostic work-up of the pregnancy.

Deletions of the SHOX gene (Xp22-Yp11.3) are associated with Leri–Weill dyschondrosteosys (LWD) and idiopathic short stature. It has been estimated that SHOX deletions occur in 1,000–2,000 individuals in the total population, suggesting that this alteration should be investigated in all cases with unexplained short stature. SHOX deletions are currently investigated using fluorescence in situ hybridization (FISH) or molecular analysis of intragenic CA repeats. However, both techniques show some limitations. In the present study, the use of the multiple ligation probe amplification (MLPA) assay for the identification and characterization of SHOX deletions in 15 LWD patients, 3 of which carriers of chromosome abnormalities involving the SHOX gene, is reported. MLPA analysis demonstrated the heterozygous deletion of SHOX in seven patients (46.6%), disclosing the presence of two different proximal breakpoints. In patients with abnormal karyotype, MLPA analysis was able to identify the chromosomal rearrangement, showing, in addition to the SHOX deletions, the gain or loss of other genes mapped on the X and Y chromosomes. Since MLPA analysis can be carried out on a simple buccal swab, avoiding invasive peripheral blood collection, this technique represents a fast, simple and high throughput approach in the screening of SHOX deletions, able to provide more information as compared to FISH and microsatellite analysis.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

No data have been so far reported on the relationship between polymorphisms of LEP gene and cardiovascular disease.We genotyped a tetranucleotide repeat mapped in the 3'UTR of the LEP gene (LEP-tet) in 109 subjects with cardiovascular events and in 109 control subjects.Univariate analysis and multivariate logistic regression analysis adjusted for age, gender, smoking status, history of hyperlipidemia, hypertension or diabetes showed not significant association between the genotype of the LEP-tet and cardiovascular diseases. Moreover, no differences were observed in the plasma leptin concentrations between cases and control subjects (22 +/- 19 vs 22 +/- 14 ng/ml, P = 0.52) and in relation to the LEP-tet classes or carriage of specific alleles (P = 0.76 for the association between LEP-tet classes and leptin levels in overall analysis).In conclusion, our data do not support an association between the LEP-tet microsatellite polymorphism of the human LEP gene and cardiovascular diseases.

Genetic testing of the cystic fibrosis transmembrane conductance (CFTR) gene is currently performed in couples undergoing assisted reproduction techniques (ART), because of the high prevalence of healthy carriers in the population and the pathogenic relationship with congenital bilateral absence of vas deferens (CBAVD). However, discordant data have been reported concerning the usefulness of this genetic test in couples with no family history of cystic fibrosis (CF). In this study, we report the results of CFTR molecular screening in 1195 couples entering ART. Genetic testing was initially carried out in a single partner of each couple. CFTR mutations were detected in 55 subjects (4.6%), a percentage that overlaps with the one reported in the general population. However, significantly higher frequencies of were found in CBAVD individuals (37.5%) and in males with nonobstructive azoospermia (6.6%). The 5T allele was found in 78 patients (6.5%). This figure was again significantly different in males with nonobstructive-azoospermia (9.9%) and in those with CBAVD (100%). All together, 139 subjects (11.6%) had either a CFTR mutation or the 5T allele. Subsequent molecular analysis of their partners disclosed a CFTR mutation or 5T allele in nine cases (6.5%). However, none of these couples had CFTR alterations in both members, a CFTR mutation being invariably present in one partner and the 5T allele in the other. In order to improve genetic counselling of these couples, the TG-M470V-5T association was analyzed, and a statistically significant relationship between 12TG-V470 and CBAVD was detected.

Objective Isolated elevations in midtrimester maternal serum human chorionic gonadotrophin concentrations (MShCG) have been reported to be associated with a substantially increased likelihood of fetal congenital malformations. The reported malformations included a wide range of organ systems, originating at different embryologic developmental stages. The purpose of our study was to determine the significance of an isolated elevated MShCG (>2.5 MoM) in midtrimester for the detection of fetal structural anomalies in a large population. Methods Among 10 144 women who underwent a biochemical triple screen at 15 to 18 weeks' gestation, 463 patients, who had an elevated MShCG, but normal α-fetoprotein (AFP) and unconjugated estriol (uE3) levels, were identified. Patients with an integrated calculated Down syndrome risk above 1:250 were excluded. Only nonsmokers, at ages

BACKGROUND: This study analyses the prevalence of karyotype changes and Yq11 microdeletions among couples referred for assisted reproduction techniques. METHODS: Prior to receiving either IVF or ICSI treatment, each partner of 2078 infertile couples was screened for karyotype changes by GTG-banding technique on peripheral lymphocytes. No subject presented with obvious phenotype of chromosomal rearrangement. All the oligo/azoospermic men with normal karyotype were further investigated by PCR for Yq11 microdeletions. RESULTS: Eighty-two out of 2078 couples (3.95%) had one partner carrying a chromosomal change, and 10 out of 202 (4.95%) men showed Yq11 microdeletions. The chromosomal rearrangements were 44 (2.1%) translocations, 23 (1.1%) gonosomal mosaics, six (0.3%) 47,XXY, five (0.24%) marker chromosomes, three (0.14%) inversions and one (0.05%) duplication. Frequency of anomalies in men and women were similar: 42 and 40 cases respectively. CONCLUSIONS: Partners of infertile couples requiring IVF or ICSI treatment appear to be affected by higher frequency of chromosomal rearrangements than the general population. Categories with greater risk were represented by men with sperm cell count < 20 3 10 6 sperm/ml, and women with history of pregnancy loss.