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The discovery of circulating fetal DNA in the plasma of pregnant women has greatly promoted advances in noninvasive prenatal testing. Screening performance is enhanced with higher fetal fraction and analysis of samples whose fetal DNA fraction is lower than 4% are unreliable. Although current approaches to fetal fraction measurement are accurate, most of them are expensive and time consuming. Here we present a simple and cost-effective solution that provides a quick and reasonably accurate fetal fraction by directly evaluating the size distribution of circulating DNA fragments in the extracted maternal cell-free DNA. The presented approach could be useful in the presequencing stage of noninvasive prenatal testing to evaluate whether the sample is suitable for the test or a repeat blood draw is recommended.

We investigated 52 cases of de novo unbalanced translocations, consisting in a terminally deleted or inverted-duplicated deleted (inv-dup del) 46th chromosome to which the distal portion of another chromosome or its opposite end was transposed. Array CGH, whole-genome sequencing, qPCR, FISH, and trio genotyping were applied. A biparental origin of the deletion and duplication was detected in 6 cases, whereas in 46, both imbalances have the same parental origin. Moreover, the duplicated region was of maternal origin in more than half of the cases, with 25% of them showing two maternal and one paternal haplotype. In all these cases, maternal age was increased. These findings indicate that the primary driver for the occurrence of the de novo unbalanced translocations is a maternal meiotic non-disjunction, followed by partial trisomy rescue of the supernumerary chromosome present in the trisomic zygote. In contrast, asymmetric breakage of a dicentric chromosome, originated either at the meiosis or postzygotically, in which the two resulting chromosomes, one being deleted and the other one inv-dup del, are repaired by telomere capture, appears at the basis of all inv-dup del translocations. Notably, this mechanism also fits with the origin of some simple translocations in which the duplicated region was of paternal origin. In all cases, the signature at the translocation junctions was that of non-homologous end joining (NHEJ) rather than non-allelic homologous recombination (NAHR). Our data imply that there is no risk of recurrence in the following pregnancies for any of the de novo unbalanced translocations we discuss here.

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.

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.

Syndromes caused by copy number variations are described as reciprocal when they result from deletions or duplications of the same chromosomal region. When comparing the phenotypes of these syndromes, various clinical features could be described as reversed, probably due to the opposite effect of these imbalances on the expression of genes located at this locus. The NFIX gene codes for a transcription factor implicated in neurogenesis and chondrocyte differentiation. Microdeletions and loss of function variants of NFIX are responsible for Sotos syndrome-2 (also described as Malan syndrome), a syndromic form of intellectual disability associated with overgrowth and macrocephaly. Here, we report a cohort of nine patients harboring microduplications encompassing NFIX. These patients exhibit variable intellectual disability, short stature and small head circumference, which can be described as a reversed Sotos syndrome-2 phenotype. Strikingly, such a reversed phenotype has already been described in patients harboring microduplications encompassing NSD1, the gene whose deletions and loss-of-function variants are responsible for classical Sotos syndrome. Even though the type/contre-type concept has been criticized, this model seems to give a plausible explanation for the pathogenicity of 19p13 microduplications, and the common phenotype observed in our cohort.

Thiamine metabolism dysfunction syndrome-4 (THMD4) includes episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy. Until now only two mutations (G125S and S194P) have been reported in the SLC25A19 gene as causative for this disease and a third mutation (G177A) as related to the Amish lethal microcephaly. In this work, we describe the clinical and molecular features of a patient carrying a novel mutation (c.576G>C; Q192H) on SLC25A19 gene. Functional studies on this mutation were performed explaining the pathogenetic role of c.576G>C in affecting the translational efficiency and/or stability of hMTPPT protein instead of the mRNA expression. These findings support the pathogenetic role of Q192H (c.576G>C) mutation on SLC25A19 gene. Moreover, despite in other patients the thiamine supplementation leaded to a substantial improvement of peripheral neuropathy, our patient did not show a clinical improvement.

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.

Chromosomal anomalies are well known to be an important cause of infertility, sterility and pregnancy loss. Balanced Reciprocal Translocation Mosaicism (BRTM) is an extremely rare phenomenon, mainly observed in subjects with a normal phenotype accompanied by reproductive failure. To date the mechanism of origin and the incidence of BRTM are poorly defined. Here we describe 10 new cases of BRTM. In 9 cases chromosome analysis revealed the presence of two different cell lines, one with a normal karyotype and the second with an apparently balanced reciprocal translocation. In the remaining case, both cell lines showed two different, but apparently balanced, reciprocal translocations. We document the clinical implications of BRTM, discuss its frequency in our referred population and suggest that carrier individuals might be more frequent than expected.

Biallelic mutations in RECQL4 gene, a caretaker of the genome, cause Rothmund-Thomson type-II syndrome (RTS-II) and confer increased cancer risk if they damage the helicase domain. We describe five families exemplifying clinical and allelic heterogeneity of RTS-II, and report the effect of pathogenic RECQL4 variants by in silico predictions and transcripts analyses. Complete phenotype of patients #39 and #42 whose affected siblings developed osteosarcoma correlates with their c.[1048_1049del], c.[1878+32_1878+55del] and c.[1568G>C;1573delT], c.[3021_3022del] variants which damage the helicase domain. Literature survey highlights enrichment of these variants affecting the helicase domain in patients with cancer outcome raising the issue of strict oncological surveillance. Conversely, patients #29 and #19 have a mild phenotype and carry, respectively, the unreported homozygous c.3265G>T and c.3054A>G variants, both sparing the helicase domain. Finally, despite matching several criteria for RTS clinical diagnosis, patient #38 is heterozygous for c.2412_2414del; no pathogenic CNVs out of those evidenced by high-resolution CGH-array, emerged as contributors to her phenotype.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

Neurodevelopmental disorders are a group of diseases characterized by either structural or functional alterations. The clinical spectrum can vary from isolated intellectual disability to more complex syndromes. Molecular karyotyping can explain 14%-18% of cases due to the presence of large pathogenic CNVs. Moreover, small CNVs involving single genes might result in a monogenic disease. In this article we report two cases of intragenic CTNND2 deletion, detected by molecular karyotyping, in patients with isolated intellectual disability.

Does the 1.5 Mb Microduplication in Chromosome Band Xp22.31 Have a Pathogenetic Role? New Contribution and a Review of the Literature Flavio Faletra,* Adamo Pio D’Adamo, Maria Santa Rocca, Marco Carrozzi, Maria Dolores Perrone, Vanna Pecile, and Paolo Gasparini Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy Institute for Maternal and Child Health, IRCCS ‘‘Burlo Garofolo’’, University of Trieste, Trieste, Italy

Osteogenesis imperfecta (OI) is a hereditary bone disease characterized by decreased bone density and multiple fractures, usually inherited in an autosomal dominant manner. Several gene encoding proteins related to collagen metabolism have been described in some cases of autosomal recessive OI (including CRTAP, LEPRE1, PPIB, FKBP65, SERPINF1, BMP1, WNT1, FKBP10). Recently, TMEM38B, a gene that encodes TRIC-B, a monovalent cation-specific channel involved in calcium flux from intracellular stores and in cell differentiation, has been associated with autosomal recessive OI. Here, we describe the second deletion-mutation involving the TMEM38B gene in an 11 year-old Albanian female with a clinical phenotype of OI, born to parents with suspected consanguinity. SNP array analysis revealed a homozygous region larger than 2 Mb that overlapped with the TMEM38B locus and was characterized by a 35 kb homozygous deletion involving exons 1 and 2 of TMEM38B gene.

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.

In this paper, is reported the identification of two chimeric patients, a rare finding if sexual abnormalities are absent. However, their chimeric condition is responsible at least for the Silver–Russell phenotype observed in one of the two patients. By single nucleotide polymorphism‐array analyses, it was possible to clearly define the mechanism responsible for this unusual finding, underlining the importance of this technique in bringing out the perhaps submerged world of chimeras.

Deletions of the short arm of chromosome 2 are exceedingly rare and only nine cases involving regions from 2p23 to 2pter have been reported to date. Most of these deletions had only been analysed by GTG banding. Here, we report an interstitial de novo deletion resulting in a microdeletion of 3.9 Mb involving 2p23.2-p23.3 segment, detected by SNP-array analysis, in a 5 year-old boy showing hypotonia, over- weight, dysmorphic facial features and cryptorchidism. We compared the clinical features of the present case to previously described patients with deletions within this chromosomal region. Our case adds new information to the deletion of the distal part of chromosome 2p improving the knowledge on this rearrangement.

The phenotypic manifestations of microdeletions in the 19q13.32 region are still poorly known. In this paper we report a patient who presented with hypotonia, developmental delay, facial dysmorphism, micrognathia, kyphoscoliosis, and buried penis. Chromosomal microarray revealed an interstitial 327 kb de novo microdeletion in the 19q13.32 region comprising eight genes (ARGHAP35, NPAS1, TMEM160, ZC3H4, SAE1, BBC3, MIR3190, and MIR3191). Previously reported cases of microdeletions in the 19q13.32 region were reviewed and compared to our patient, highlighting the common features of a possible 19q13.32 microdeletion syndrome.

The identification of causes underlying intellectual disability (ID) is one of the most demanding challenges for clinical Geneticists and Researchers. Despite molecular diagnostics improvements, the vast majority of patients still remain without genetic diagnosis. Here, we report the results obtained using Whole Exome and Target Sequencing on nine patients affected by isolated ID without pathological copy number variations, which were accurately selected from an initial cohort of 236 patients. Three patterns of inheritance were used to search for: (1) de novo, (2) X-linked, and (3) autosomal recessive variants. In three of the nine proband-parent trios analyzed, we identified and validated two de novo and one X-linked potentially causative mutations located in three ID-related genes. We proposed three genes as ID candidate, carrying one de novo and three X-linked mutations. Overall, this systematic proband-parent trio approach using next generation sequencing could explain a consistent percentage of patients with isolated ID, thus increasing our knowledge on the molecular bases of this disease and opening new perspectives for a better diagnosis, counseling, and treatment.

Lipoblastoma is a benign tumor that often has rearrangements of the 8q11-13 region targeting the PLAG1 gene, which is involved in the process of tumourigenesis. We described a signature of chromotripsis in a thigh lipoblastoma, as first report of chromotripsis in a benign tumor.

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.

Deletion of the terminal part of long arm of chromosome 4 is a condition characterized by facial dysmorphisms, cardiac and limb defects, and developmental delay. Deletions usually involve the terminal part of the chromosome and most frequently are interstitial. Here, we report a de novo interstitial deletion resulting in a microdeletion of 6.9 Mb involving 4q31.3-q32.1 segment, detected by SNPs-Array technique in a 4-year-old female showing severe speech delay, mild facial dysmorphisms, and joint laxity. Phenotype–genotype relationships looking at the genes involved in this part of the chromosome were also carried out and data compared with those previously described. © 2012 Wiley Periodicals, Inc.

Deletions of the short arm of chromosome 2 are exceedingly rare, having been reported in few patients. Fur- thermore most cases with deletion in 2p11.2–p12 have been studied using standard karyotype and so it is not possible to delineate the precise size of deletions. Here, we describe a 9-year-old girl with a 9.4 Mb de novo interstitial deletion of region 2p11.2–p12 identified by SNP array analysis. The deleted region encompasses over 40 known genes, including LRRTM1, CTNNA2 and REEP1, haploinsuffi- ciency of which could explain some clinical features of this patient such as mental retardation, speech delay and gait abnormalities. A comparison of our case with previously reported patients who present deletions in 2p11.2–p12 was carried out. Our case adds new information to the deletion of 2p11.2–p12, improving the knowledge on this rearrangement.

The deletion of the locus 17p13 was associated with Miller–Dieker lissencephaly syndrome (MDLS). Deletion (Delatycki and Leventer 2009) or mutations (Lo Nigro et al. 1997) in the PAFAH1B1 (601545) gene cause lissencephaly (Gu and Lupski 2008), but facial dysmorphology and other abnormalities in patients with MDLS seem to be the result of the deletion of additional genes distal to PAFAH1B1 (Cardoso et al. 2003; Saillour et al. 2009). The gene responsible for the greater severity of MDLS compared to isolated lissencephaly is the YWHAE (Haverfield et al. 2009) (605066) gene, encoding 14-3-3-epsilon. It was very recently demonstrated that the duplication in 17p13 is a new clinical entity (OMIM 613215), characterized by mental retardation and other variable clinical and radiological findings (Roos et al. 2009; Spalice et al. 2009). There are three different areas within the MDS region containing dosage-sensitive genes. One containing the PAFAH1B1 gene and the other, more distal, which includes the TUSC5, YWHAE, CRK, and MYO1C genes. Until now, few cases have been described with duplication in the MDS region (Mei et al. 2008; Bi et al. 2009) and only two of them show a duplication including only the PAFAH1B1 area. All patients with duplication in the MDS region have common clinical and phenotypic features. Nevertheless, there are several important differences between those with only duplication in the PAFAH1B1 area as compared with those who have also (or only) a duplication in the distal area. Clinical report

4p Monosomy and 12p trisomy have been discussed and redefined along with recently reviewed chromosomal syndromes. 12p Trisomy syndrome is characterized by normal or increased birth weight, developmental delay with early hypotonia, psychomotor delay, and typical facial appearance. Most likely, the observed phenotypic variability depends on the type and extent of the associated partial monosomy. Partial deletions of the short arm of one chromosome 4 cause the Wolf-Hirschhorn syndrome (WHS). Affected patients present Greek helmet face, growth and mental retardation, hypotonia, and seizures. The combination of these characteristics constitutes the phenotypic core of WHS. We present a clinical and molecular cytogenetic characterization of a 4-year old mentally retarded girl with macrosomy, facial dysmorphisms, and epilepsy, in whom an unbalanced t(4;12)(p16.3;p13.3) translocation was detected, giving rise to partial 4p monosomy and partial 12p trisomy. Because the patient shows most of the phenotypic characteristics of 12p trisomy, this case could contribute to a better definition of the duplicate critical region that determines the phenotype of the 12p trisomy syndrome.

Objective We surveyed the datasheets of 29 laboratories concerning prenatal diagnosis of de novo apparently balanced chromosome rearrangements to assess the involvement of specific chromosomes, the breakpoints distribution and the impact on the pregnancy outcome. Method By means of a questionnaire, data on 269.371 analyses performed from 1983 to 2006 on amniotic fluid, chorionic villus and fetal blood samples were collected. Results A total of 246 balanced anomalies were detected at frequencies of 72% for reciprocal translocations, 18% for Robertsonian translocations, 7% for inversions and 3% for complex chromosome rearrangements. The total frequencies of balanced rearrangements were 0.09%, 0.08% and 0.05% on amniotic fluid, chorionic villus and fetal blood samples. Conclusion A preferential involvement of chromosomes 22, 7, 21, 3, 9 and 11 and a less involvement of chromosomes X, 19, 12, 6 and 1 was observed. A nonrandom distribution of the breakpoints across chromosomes was noticed. Association in the location of recurrent breakpoints and fragile sites was observed for chromosomes 11, 7, 10 and 22, while it was not recorded for chromosome 3. The rate of pregnancy termination was about 20%, with frequencies decreasing from complex chromosomal rearrangements (33%), reciprocal translocations (24%) to inversions (11%) and Robertsonian translocations (3%). Copyright © 2009 John Wiley & Sons, Ltd.

Subtelomeric rearrangements are one of the main causes of multiple congenital anomalies and mental retardation, and they are detected in 5% of patients. We report on a 6.5-year-old boy with mental retardation, dysmorphic features, and behavioral problems, who revealed 1q44-qter trisomy and 22q13.3-qter monosomy due to a maternal cryptic translocation t(1;22). We compared the clinical and cytogenetic data of our patient with those of another case presenting a pure 22qter monosomy and with those of all 1qter trisomy cases reported in the international literature. To the best of our knowledge, the subterminal 1q trisomy found in the present case has been reported in only 12 patients to date (including five familial cases). This report aims to contribute to our understanding of 1q44-qter trisomy.

A young girl with 46, XX, r(18)/46, XX/45, XX, –18 chromosome constitution is reported. She displays a slight degree of mental retardation. The line with the ring chromosome predominates in blood lymphocytes. In skin fibroblast culture the ring(18) line showed a constant decreasing trend, from 45% at the first passage, down to its disappearance at the 19th passage, where only 46, XX cells were observed. The child/mid parents' ratio of Peptidase A activity in red cells was 0.36. The Peptidase A activity in a fibroblast clone 46, XX, r(l 8) was 0.55 compared to the 46, XX line. These data suggest that the PEP A locus was lost in ring formation.

Introduction. Developmental delay and dysmorphic features affect 1 - 3 % of paediatric population. In the last few years molecular cytogenetic high resolution techniques (comparative genomic hybridization arrays and single-nucleotide polymorphism arrays) have been proven to be a first-tier choice for clinical diagnostics of developmental delay and dysmorphic features. Methods and results. In the present article we describe the clinical advantages of molecular cytogenetic approach (comparative genomic hybridization arrays and single nucleotide polymorphism arrays) in the diagnostic procedure of two children with developmental delay, dysmorphic features and additional morphological phenotypes. Additionally, we demonstrate the necessity of fluorescent in situ hybridization utilisation to identify the localisation and underlying mechanism of detected chromosomal rearrangement. Conclusions. Two types of chromosomal abnormalities were identified and confirmed using different molecular genetic approaches. Comparative genomic hybridization arrays and single nucleotide polymorphism arrays are hereby presented as important methods to identify chromosomal imbalances in patients with developmental delay and dysmorphic features. We emphasize the importance of molecular genetic testing in patients’ parents for the demonstration of the origin and clinical importance of the aberrations prior determined in the patients. The results obtained using molecular cytogenetic high resolution techniques methods are the cornerstone for proper genetic counselling to the affected families.

Fanconi anemia (FA) is a rare bone marrow failure disorder characterized by clinical and genetic heterogeneity with at least 17 genes involved, which make molecular diagnosis complex and time‐consuming. Since next‐generation sequencing technologies could greatly improve the genetic testing in FA, we sequenced DNA samples with known and unknown mutant alleles using the Ion PGM ™ system (IPGM). The molecular target of 74.2 kb in size covered 96% of the FA‐coding exons and their flanking regions. Quality control testing revealed high coverage. Comparing the IPGM and Sanger sequencing output of FANCA,FANCC, and FANCG we found no false‐positive and a few false‐negative variants, which led to high sensitivity (95.58%) and specificity (100%) at least for these two most frequently mutated genes. The analysis also identified novel mutant alleles, including those in rare complementation groups FANCF and FANCL. Moreover, quantitative evaluation allowed us to characterize large intragenic deletions of FANCA and FANCD2, suggesting that IPGM is suitable for identification of not only point mutations but also copy number variations.

Angelman Syndrome (AS) is due to the loss of function of the single UBE3A gene, mapping to chromosome 15q11-q13 and encoding the E6AP ubiquitin ligase. Expression of UBE3A is subject to genomic imprinting which is restricted to the brain, where only the maternal allele is transcribed. AS pathogenetic mechanisms include deletion of the maternal 15q11-13 chromosomal region, chromosome 15 paternal uniparental disomy (UPD), Imprinting Defects (ImpD) leading to silencing of the maternal allele and intragenic mutations of the maternal UBE3A allele. From our AS cohort we sorted out for detailed clinical-molecular characterization six mosaic cases, five with ImpD epimutations and one with patUPD15. This latter case referred for intellectual disability and fortuitously solved by SNP array, is, to our knowledge, the unique patient reported with mosaic patUPD of this imprinted region. Somatic epimutation mosaicism represents a challenge for both clinical and molecular diagnostics. The described patients, referred to our center either for uncertain AS or simply for intellectual disability, could be molecularly characterized by applying a multi-method approach including Methylation-Sensitive PCR and MS-MLPA without a strict cut off. The percentage of normal cells detected ranged up to 40%. We confirm the mild phenotype reported in mosaic AS ImpD and provide a detailed analysis of IQ. Mild mental retardation, with significant difficulties in language expression, but only mildly impaired performance skills, together with pathognomonic EEG, is a cue not to overlook in mosaic AS patients. Mosaic epimutations should be searched also in patients with minor AS features and presenting only with intellectual disability.

Purpose:The harmful effects of inbreeding are well known by geneticists, and several studies have already reported cases of intellectual disability caused by recessive variants in consanguineous families. Nevertheless, the effects of inbreeding on the degree of intellectual disability are still poorly investigated. Here, we present a detailed analysis of the homozygosity regions in a cohort of 612 patients with intellectual disabilities of different degrees.Methods:We investigated (i) the runs of homozygosity distribution between syndromic and nonsyndromic ID (ii) the effect of runs of homozygosity on the ID degree, using the intelligence quotient score.Results:Our data revealed no significant differences in the first analysis; instead we detected significantly larger runs of homozygosity stretches in severe ID compared to nonsevere ID cases (P = 0.007), together with an increase of the percentage of genome covered by runs of homozygosity (P = 0.03).Conclusion:In accord with the recent findings regarding autism and other neurological disorders, this study reveals the important role of autosomal recessive variants in intellectual disability. The amount of homozygosity seems to modulate the degree of cognitive impairment despite the intellectual disability cause.

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.

Background: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. Methods and Findings: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. Conclusions: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma. Stem Cells 2014;32:1239–1253

Phelan–McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan–McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan–McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan–McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region. © 2014 Wiley Periodicals, Inc.

Intellectual disability (ID) with autosomal recessive (AR) inheritance is believed to be common; however, very little is known about causative genes and genotype–phenotype correlations. The broad genetic heterogeneity of AR-ID, and its usually nonsyndromic nature make it difficult to pool multiple pedigrees with the same underlying genetic defect to achieve consistent nosology. Nearly all autosomal genes responsible for recessive cognitive disorders have been identified in large consanguineous families from the Middle East, and nonsense mutations in TRAPPC9 have been reported in a total of 5. Although several recurrent phenotypic abnormalities are described in some of these patients, the associated phenotype is usually referred to as nonsyndromic. By means of single-nucleotide polymorphism-array first and then by exome sequencing, we identified a new pathogenic mutation in TRAPPC9 in two Italian sisters born to healthy and apparently nonconsanguineous parents. It consists of a homozygous splice site mutation causing exon skipping with frameshift and premature termination, as confirmed by mRNA sequencing. By detailed phenotypic analysis of our patients, and by critical literature review, we found that homozygous TRAPPC9 loss-of-function mutations cause a distinctive phenotype, characterized by peculiar facial appearance, obesity, hypotonia (all signs resembling a Prader–Willi-like phenotype), moderate-to-severe ID, and consistent brain abnormalities.

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.

Introduction Opitz trigonocephaly C syndrome (OTCS) is a rare malformation syndrome with the following features: synostosis of metopic suture, craniofacial abnormalities, severe mental retardation and a multitude of pathological findings affecting almost every organ system. OTCS is associated with a high mortality rate. Case presentation We describe the case of a Caucasian male baby who died at five months of age during surgical correction of the craniofacial anomaly. Conclusion As previously reported, OTCS may have an increased mortality rate during craniofacial surgery. Careful evaluation of surgery risk-benefit ratio is warranted in such patients.

Background: Beckwith-Wiedemann syndrome (BWS) is a clinically variable and genetically heterogeneous disorder, providing evidence that imprinted genes play key roles in the control of fetal growth. Clinically, diagnostic criteria include macrosomia, macroglossia, abdominal wall defects, neonatal hypoglycaemia, visceromegalies and hemihyperplasia. Component clinical manifestations also include renal abnormalities, adrenocortical cytomegaly and a characteristic facial appearance, with midface hypoplasia and ear anomalies. Genetically, BWS is associated with disturbances within two different domains on 11p15 that are controlled by distinct imprinting control regions (ICR), ICR1 and ICR2. The majority of patients have abnormalities within ICR2. In particular, loss of maternal methylation accounts for 50-60% of cases, and is associated with reduction in the expression of the CDKN1C gene, a member of the cyclin dependent kinase inhibitor family acting as negative regulator of cell proliferation. Mutations in CDKN1C are detected in another 5-10% of subjects with sporadic BWS. Chromosome deletions affecting ICR2 are uncommon. Methods and findings: We report on a patient with BWS in which a de novo 11p15 deletion was detected by array comparative genomic hybridisation. Clinically, the patient presented with mild mental retardation and minor physical anomalies. The deletion, that was demonstrated to be maternal in origin by SNP array, encompassed ICR2 and several flanking genes, including CDKN1C. A normal methylation pattern of ICR1 was observed. Conclusions: This observation provides evidence that, among the genetic defects associated with BWS, a 11p15 microdeletion encompassing ICR2 identifies a peculiar clinical phenotype, with high recurrence risk in offspring of female carriers. It also supports the model of two independent domains within the BWS locus.

Based on genotype-phenotype correlation analysis of 80 Wolf-Hirschhorn syndrome (WHS) patients, as well as on review of relevant literature, we add further insights to the following aspects of WHS: (1) clinical delineation and phenotypic categories; (2) characterization of the basic genomic defect, mechanisms of origin and familiarity; (3) identification of prognostic factors for mental retardation; (4) chromosome mapping of the distinctive clinical signs, in an effort to identify pathogenic genes. Clinically, we consider that minimal diagnostic criteria for WHS, defining a "core" phenotype, are typical facial appearance, mental retardation, growth delay and seizures (or EEG anomalies). Three different categories of the WHS phenotype were defined, generally correlating with the extent of the 4p deletion. The first one comprises a small deletion not exceeding 3.5 Mb, that is usually associated with a mild phenotype, lacking major malformations. This category is likely under-diagnosed. The second and by far the more frequent category is identified by large deletions, averaging between 5 and 18 Mb, and causes the widely recognizable WHS phenotype. The third clinical category results from a very large deletion exceeding 22-25 Mb causing a severe phenotype, that can hardly be defined as typical WHS. Genetically, de novo chromosome abnormalities in WHS include pure deletions but also complex rearrangements, mainly unbalanced translocations. With the exception of t(4p;8p), WHS-associated chromosome abnormalities are neither mediated by segmental duplications, nor associated with a parental inversion polymorphism on 4p16.3. Factors involved in prediction of prognosis include the extent of the deletion, the occurrence of complex chromosome anomalies, and the severity of seizures. We found that the core phenotype maps within the terminal 1.9 Mb region of chromosome 4p. Therefore, WHSCR-2 should be considered the critical region for this condition. We also confirmed that the pathogenesis of WHS is multigenic. Specific and independent chromosome regions were characterized for growth delay and seizures, as well as for the additional clinical signs that characterize this condition. With the exception of parental balanced translocations, familial recurrence is uncommon.

Chromosomal abnormalities may cause growth failure before or since birth. 9q duplication is reported as a cause of intrauterine growth restriction, mild dysmporphism, and intellectual disabilities. We report a case of a maternally inherited 9q21.31q21.33 duplication causing prenatal and postnatal growth restriction with feeding refusal and mild facial dysmorphisms, prenatally diagnosed by single-nucleotide polymorphism array analysis. Hypothesis of the possible pathogenic mechanisms are discussed.

PURPOSE: We evaluated the experiences of 19 Italian laboratories concerning 241 small supernumerary marker chromosomes (sSMCs) with the aim of answering questions arising from their origin from any chromosome, their variable size and genetic content, and their impact on the carrier's phenotype. METHODS: Conventional protocols were used to set up the cultures and chromosome preparations. Both commercial and homemade probes were used for the fluorescent in situ hybridization analyses. RESULTS: A total of 113 of the 241 sSMCs were detected antenatally, and 128 were detected postnatally. There were 52 inherited and 172 de novo cases. Abnormal phenotype was present in 137 cases (57%), 38 of which were antenatally diagnosed. A mosaic condition was observed in 87 cases (36%). In terms of morphology, monocentric and dicentric bisatellited marker chromosomes were the most common, followed by monocentric rings and short-arm isochromosomes. The chromosomes generating the sSMCs were acrocentric in 132 cases (69%) and non-acrocentric chromosomes in 60 cases (31%); a neocentromere was hypothesized in three cases involving chromosomes 6, 8, and 15. CONCLUSION: The presented and published data still do not allow any definite conclusions to be drawn concerning karyotype-phenotype correlations. Only concerted efforts to characterize molecularly the sSMCs associated or not with a clinical phenotype can yield results suitable for addressing karyotype-phenotype correlations in support of genetic counseling.

We report on a 3-year-old male with developmental delay, autistic behavior, and minor abnormalities consistent with trisomy 8 syndrome whose cytogenetic analysis revealed mosaicism for a supernumerary ring chromosome (SRC). Fluorescence in situ hybridization (FISH) studies, using centromeric and yeast artificial chromosome (YAC) probes, were performed to characterize further the supernumerary chromosome. The ring origin has been detected from the short arm of chromosome 8, resulting in r(8)(p10p23.1). Moreover, uniparental disomy (UPD) using microsatellite analysis was excluded. To our knowledge a total of 25 cases, confirmed by FISH, have been reported with either supernumerary marker or ring chromosome 8. We present a detailed clinical and molecular cytogenetic characterization of this additional case in order to better define the genotype-phenotype correlation.