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Background Pallister-Killian syndrome is a rare, sporadic condition caused by mosaic tetrasomy of the short arm of chromosome 12 (12p). The main features are intellectual disability, seizures, dysmorphic features and a variety of congenital malformations. Most available information comes from individual case reports. We report the results of a British study into Pallister-Killian syndrome, which is the first to provide comprehensive data on a population-based sample. Method A detailed phenotypical study was carried out in Great Britain. All individuals with Pallister-Killian syndrome were eligible to participate. Each participant underwent a structured history, developmental assessment and clinical examination. Buccal mucosal samples were analysed by interphase fluorescence in situ hybridization (FISH) and blood samples by array comparative genomic hybridization (CGH). Genotype-phenotype correlations were sought in these tissues and existing skin biopsy reports. Results Twenty-two patients with Pallister-Killian syndrome, ranging from 4 months to 31 years were recruited and comprehensive data on each obtained. The birth incidence was 5.1 per million live births. Array CGH only suggested the diagnosis in 15.8% but buccal FISH could have made the diagnosis in 75.0%. There was no genotype-phenotype correlation in any of the tissues studied. This study shows that the high birth weights and profound intellectual disability classically described in Pallister-Killian syndrome are not universal. Mild or moderate intellectual disability was present in 27.6% of this cohort and all birth weights were within 2.67SD of the mean. New features which have not previously been recognised as part of Pallister-Killian syndrome include anhydrosis/ hypohydrosis and episodic hyperventilation, suggesting involvement of the autonomic system.

Blepharoptosis (ptosis) is defined as the abnormal drooping of the upper eyelid and is a feature of many conditions. It can be in isolated or syndromic form, bilateral or unilateral and congenital or acquired. Previously we have carried out linkage analysis on a family with dominantly inherited congenital bilateral isolated ptosis and found the condition to be linked to a region of approximately 20 megabases of chromosome Xq24-Xq27.1 with a cumulative LOD score of 5.89. We now describe further analysis using array comparative genomic hybridisation (array CGH), fluorescence in situ hybridisation (FISH), long range PCR and sequencing. This has enabled us to identify and characterise at the level of DNA sequence an insertional duplication and rearrangement involving chromosomes 1p21.3 and a small quasipalindromic sequence in Xq27.1, disruption of which has been associated with other phenotypes but which is cosegregating with X-linked congenital bilateral isolated ptosis in this family. This work highlights the significance of the small quasipalindromic sequence in genomic rearrangements involving Xq27.1 and the importance of comprehensive molecular and molecular cytogenetic investigations to fully characterise genomic structural complexity.

Chromosomal microarrays (CMA) are routinely used in postnatal genetic diagnosis. CMA is technically applicable in prenatal diagnosis. Pros and cons of routine use are discussed; technical aspects and dseign of array, yield, interpretation of CNV and variances of unknown significance (VOUS), quality control regimens.

Our aim was to construct a streamlined technical workflow to facilitate a prospective, multi-centre evaluation of array comparative genomic hybridisation (array-CGH) in the prenatal diagnostic context. A collection of commercially available DNA extraction and quantification techniques were evaluated and compared using minimal quantities of amniotic fluid, chorionic villi and cultured cells. When prenatal DNA of suitable quality and quantity was obtained, array-CGH was performed using Oxford Gene Technology's (OGT, Oxford, UK) CytoSure™ ISCA 8 × 60 K oligo array platform. With starting quantities of 2-4 ml amniotic fluid, 2-5 mg chorionic villi or under 150,000 cultured cells the following optimised technical workflow was identified: DNA extraction using the iGENatal™ kit (igenbiotech, Madrid, Spain) and quantification by the Qubit® 2.0 Fluorometer with the Qubit® dsDNA BR assay kit (Invitrogen™, Eugene, OR, USA). In addition, it was elucidated that array-CGH can be successfully performed with as little as 125 ng DNA in the experiment using the OGT CytoSure™ ISCA 8 × 60 K oligo array platform. Amidst an on-going debate on whether array-CGH should be applied in the prenatal diagnostic setting, by following the technical recommendations described here genetics laboratories can now gain exposure to prenatal array-CGH testing without compromising the conventional karyotype result.

The 8p23.1 duplication syndrome is a relatively rare genomic condition that has been confirmed with molecular cytogenetic methods in only 11 probands and five family members. Here, we describe another prenatal and five postnatal patients with de novo 8p23.1 duplications analyzed with oligonucleotide array comparative genomic hybridization (oaCGH). Of the common features, mild or moderate developmental delays and/or learning difficulties have been found in 11/12 postnatal probands, a variable degree of mild dysmorphism in 8/12 and congenital heart disease (CHD) in 4/5 prenatal and 3/12 postnatal probands. Behavioral problems, cleft lip and/or palate, macrocephaly, and seizures were confirmed as additional features among the new patients, and novel features included neonatal respiratory distress, attention deficit hyperactivity disorder (ADHD), ocular anomalies, balance problems, hypotonia, and hydrocele. The core duplication of 3.68 Mb contains 31 genes and microRNAs of which only GATA4, TNKS, SOX7, and XKR6 are likely to be dosage sensitive genes and MIR124-1 and MIR598 have been implicated in neurocognitive phenotypes. A combination of the duplication of GATA4, SOX7, and related genes may account for the variable penetrance of CHD. Two of the duplications were maternal and intrachromosomal in origin with maternal heterozygosity for the common inversion between the repeats in 8p23.1. These additional patients and the absence of the 8p23.1 duplications in published controls, indicate that the 8p23.1 duplication syndrome may now be considered a pathogenic copy number variation (pCNV) with an estimated population prevalence of 1 in 58,000. © 2013 Wiley Periodicals, Inc.

Background: In cultured, dividing transformed T lymphocytes and in dividing bone marrow cells from normal men and those with a haematological malignancy, sex chromosome aneuploidy has been found to increase in prevalence and degree with age. This has rarely been investigated in non-dividing uncultured blood samples. The loss and gain of the X chromosome in dividing transformed lymphocytes in women with age is much more frequent than that of the Y chromosome in males. However, paradoxically X chromosome aneuploidy is rarely seen in the dividing cells of bone marrow of females. Methods: In blood samples from 565 men with breast cancer and 54 control men from the England and Wales general population, 80 cell nuclei per sample were scored for presence of X and Y chromosomes using fluorescent centromeric probes. Results: Sex chromosome aneuploidy, largely Y chromosome loss, was present in 63% of cases and 57% of controls, with the prevalence and degree of aneuploidy increasingly sharply and highly significantly with age. At ages 65–80 years, 71% of cases and 85% of controls showed aneuploidy and 15% and 25%, respectively, had ⩾10% of cells aneuploid. Allowing for age, aneuploidy was less prevalent (P=0.03) in cases than controls. Conclusion: Sex chromosome aneuploidy in non-dividing nuclei of peripheral blood cells is frequent in adult men, the prevalence and degree increasing sharply with age. The possible relation of sex chromosome aneuploidy to breast cancer risk in men, and to cancer risk generally, needs further investigation, ideally in cohort studies.

We report a large series of 173 patients with physical and/or neurological abnormalities and a de novo imbalance identified by array CGH. Breakpoint intervals were screened for the presence of low copy repeats (LCRs) to distinguish between rearrangements formed by non-allelic homologous recombination (NAHR) and rearrangements formed by other mechanisms. We identified significant differences in size and parental origin between the LCR-mediated and non-LCR groups. Non-LCR imbalances were evenly distributed among the four size intervals we defined, whereas LCR-mediated rearrangements had a narrow size distribution, predominantly between 1 and 5 Mb (P=0.001). Among the LCR-mediated rearrangements there were equal numbers of maternally and paternally derived cases. In contrast, for the non-LCR rearrangements there was a significant excess of paternal cases (P=0.024) over a wide size range including below 1 Mb. Our results provide novel evidence that unbalanced chromosome rearrangements are not only more frequent in males, but may also arise through different mechanisms than those seen in females. Although the paternal imbalances identified in our study are evenly distributed throughout the four size groups, there are very few maternal imbalances either 10 Mb. Furthermore, a lower proportion of paternal imbalances are LCR mediated (13/71) compared with the maternal imbalances (12/30). We hypothesise that imbalances of maternal origin arise predominantly through NAHR during meiosis, while the majority of imbalances of paternal origin arise through male-specific mechanisms other than NAHR. Our data suggest that mitotic mechanisms could be important for the formation of chromosome imbalances; however, we found no association with increased paternal age.

Purpose: Copy number variants have emerged as a major cause of human disease such as autism and intellectual disabilities. Because copy number variants are common in normal individuals, determining the functional and clinical significance of rare copy number variants in patients remains challenging. The adoption of whole-genome chromosomal microarray analysis as a first-tier diagnostic test for individuals with unexplained developmental disabilities provides a unique opportunity to obtain large copy number variant datasets generated through routine patient care. Methods: A consortium of diagnostic laboratories was established (the International Standards for Cytogenomic Arrays consortium) to share copy number variant and phenotypic data in a central, public database. We present the largest copy number variant case-control study to date comprising 15,749 International Standards for Cytogenomic Arrays cases and 10,118 published controls, focusing our initial analysis on recurrent deletions and duplications involving 14 copy number variant regions. Results: Compared with controls, 14 deletions and seven duplications were significantly overrepresented in cases, providing a clinical diagnosis as pathogenic. Conclusion: Given the rapid expansion of clinical chromosomal microarray analysis testing, very large datasets will be available to determine the functional significance of increasingly rare copy number variants. This data will provide an evidencebased guide to clinicians across many disciplines involved in the diagnosis, management, and care of these patients and their families. Genet Med 2011:13(9):777–784.

SOX2 is an early developmental transcription factor and marker of stem cells that has recently been implicated in the development of the pituitary gland. Heterozygous SOX2 mutations have been described in patients with hypopituitarism and severe ocular abnormalities. In the majority of published cases, the pituitary gland is either small or normal in size. Here, we report two unrelated patients with SOX2 haploinsufficiency (a heterozygous gene deletion and a novel c.143TC>AA/p.F48X mutation) who developed nonprogressive pituitary tumors of early onset, suggesting a congenital etiology. The truncating mutation resulted in significant loss of function and impaired nuclear localization of the mutant protein, in addition to a failure to repress β-catenin transcriptional activity in vitro. This is the first indication that SOX2 haploinsufficiency is implicated in the generation of pituitary tumors with distinct clinical characteristics, possibly mediated via its effects on the Wnt signaling pathway. 32:1376–1380, 2011. ©2011 Wiley Periodicals, Inc.

The availability of microarray technology has led to the recent recognition of copy number abnormalities of distal chromosome 22q11.2 that are distinct from the better-characterized deletions and duplications of the proximal region. This report describes five unrelated individuals with copy number abnormalities affecting distal chromosome 22q11.2. We report on novel phenotypic features including diaphragmatic hernia and uterine didelphys associated with the distal microdeletion syndrome; and frontomedial polymicrogyria and callosal agenesis associated with the distal microduplication syndrome. We describe the third distal chromosome 22q11.2 microdeletion patient with Goldenhar syndrome. Patients with distal chromosome 22q11.2 copy number abnormalities exhibit inter- and intra-familial phenotypic variability, and challenge our ability to draw meaningful genotype-phenotype correlations.

We report six patients with array deletions encompassing 12q14. Out of a total of 2538 array investigations carried out on children with developmental delay and dysmorphism in three diagnostic testing centres, six positive cases yielded a frequency of 1 in 423 for this deletion syndrome. The deleted region in each of the six cases overlaps significantly with previously reported cases with microdeletions of this region. The chromosomal range of the deletions extends from 12q13.3q15. In the current study, we report overlapping deletions of variable extent and size but primarily comprising chromosomal bands 12q13.3q14.1. Four of the six deletions were confirmed as de novo events. Two cases had deletions that included HMGA2, and both children had significant short stature. Neither case had osteopoikilosis despite both being deleted for LEMD3. Four cases had deletions that ended proximal to HMGA2 and all of these had much better growth. Five cases had congenital heart defects, including two with atrial septal defects, one each with pulmonary stenosis, sub-aortic stenosis and a patent ductus. Four cases had moderate delay, two had severe developmental delay and a further two had a diagnosis of autism. All six cases had significant speech delay with subtle facial dysmorphism.

We present an infant with a de novo cytogenetically visible interstitial deletion of approximately 21.9 Mb involving chromosome bands 7p15.1–7p12.1, with the loss of 119 genes confirmed by array CGH. The infant had a ventricular septal defect, hand and skull anomalies, and hyperglycaemia compatible with haploinsufficiency of TBX20, GLI3, and GCK genes, respectively. In addition, the infant had some features reminiscent of Beckwith Wiedemann syndrome including macroglossia, umbilical hernia, and a relatively large birth weight and we speculate that this is due to the deletion of GRB10, an imprinted gene on chromosome 7. This report illustrates how knowledge of genes within a deleted interval facilitates optimal medical management, can explain observed phenotypes, and stimulates research questions.

Background: Congenital chromosome abnormalities are relatively common in our species and among structural abnormalities the most common class is balanced reciprocal translocations. Determining the parental origin of de novo balanced translocations may provide insights into how and when they arise. While there is a general paternal bias in the origin of non-recurrent unbalanced rearrangements, there are few data on parental origin of non-recurrent balanced rearrangements. Methods: The parental origin of a series of de novo balanced reciprocal translocations was determined using DNA from flow sorted derivative chromosomes and linkage analysis. Results: Of 27 translocations, we found 26 to be of paternal origin and only one of maternal origin. We also found the paternally derived translocations to be associated with a significantly increased paternal age (p

Chromosome microdeletions or duplications are detected in 10–20% of patients with mental impairment and normal karyotypes. A few cases have been reported of mental impairment with microdeletions comprising tumor suppressor genes. By array-CGH we detected 4 mentally impaired individuals carrying de novo microdeletions sharing an overlapping segment of ∼180 kb in 17p13.1. This segment encompasses 18 genes, including 3 involved in cancer, namely KCTD11/REN, DLG4/PSD95, and GPS2. Furthermore, in 2 of the patients, the deletions also included TP53, the most frequently inactivated gene in human cancers. The 3 tumor suppressor genes KCTD11, DLG4, and GPS2, in addition to the GABARAP gene, have a known or suspected function in neuronal development and are candidates for causing mental impairment in our patients. Among our 4 patients with deletions in 17p13.1, 3 were part of a Brazilian cohort of 300 mentally retarded individuals, suggesting that this segment may be particularly prone to rearrangements and appears to be an important cause (∼1%) of mental retardation. Further, the constitutive deletion of tumor suppressor genes in these patients, particularly TP53, probably confers a significantly increased lifetime risk for cancer and warrants careful oncological surveillance of these patients. Constitutional chromosome deletions containing tumor suppressor genes in patients with mental impairment or congenital abnormalities may represent an important mechanism linking abnormal phenotypes with increased risks of cancer.

Severe ocular malformations, including anophthalmia-microphthalmia (AM), are responsible for around 25% of severe visual impairment in childhood. Recurrent interstitial deletions of 14q22–23 are associated with AM and a wide range of extra-ocular phenotypes including brain anomalies. The homeobox gene OTX2 is located at 14q22.3 and has recently been identified as mutated in AM patients. Eight human OTX2 mutations have been reported in subjects with severe eye malformations, including AM, and variable developmental delay. We screened a novel AM cohort for mutations and deletions in OTX2, and identified four new mutations in six individuals and two cases of whole gene deletions. Our data suggest that OTX2 mutations and deletions account for 2–3% of AM cases. © 2008 Wiley-Liss, Inc.

Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus.Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues.Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye.Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

We report the analyses of breakpoints in 31 phenotypically normal and 14 abnormal carriers of balanced translocations. Our study assesses the differences between balanced translocations in normal carriers and those in abnormal carriers, focusing on the presence of genomic imbalances at the breakpoints or elsewhere in the genome, presence of cryptic chromosome rearrangements, and gene disruption. Our hypothesis is that all four features will be associated with phenotypic abnormalities and absent or much less frequent in a normal population. In the normal cohort, we identified neither genomic imbalances at the breakpoints or elsewhere in the genome nor cryptic chromosome rearrangements. In contrast, we identified candidate disease-causing imbalances in 4/14 abnormal patients. These were three breakpoint associated deletions and three deletions unrelated to the breakpoints. All six de novo deletions originated on the paternally inherited chromosome. Additional complexity was also present in one of these cases. Gene disruption by the breakpoints was present in 16/31 phenotypically normal individuals and in 5/14 phenotypically abnormal patients. Our results show that translocations in phenotypically abnormal patients are molecularly distinct from those in normal individuals: the former are more likely to be associated with genomic imbalances at the breakpoints or elsewhere and with chromosomal complexity, whereas the frequency of gene disruption is similar in both normal and abnormal translocation carriers.

We report a male patient born at 37-weeks gestation, weighing 1.885 kg (

Objective: The aim of this study was to determine if there is a significant difference in the risk of developing Wilms tumour between patients with submicroscopic and those with visible deletions of the WT1 tumour suppressor gene. Methods: To determine which subjects had WT1 deletions, high-resolution chromosomal deletion analysis of the 11p13 region was carried out in 193 people with aniridia. The rationale for this was that aniridia is caused by loss of function of one copy of the PAX6 gene, and although most patients with aniridia have intragenic mutations, a proportion has deletions that also include the nearby WT1 gene. Fluorescence in situ hybridisation (FISH) analysis of patients with aniridia identifies people with WT1 deletions regardless of whether they have Wilms tumour, allowing the deletion size to be correlated with clinical outcome. Results: Wilms tumour was not observed in any case without a WT1 deletion. Of subjects in whom WT1 was deleted, 77% with submicroscopic deletions (detectable only by high-resolution FISH analysis) presented with Wilms tumour compared with 42.5% with visible deletions (detectable by microscopy). This difference was significant. Conclusions: High-resolution deletion analysis is a useful tool for assessing the risk of Wilms tumour in neonates with aniridia. People with submicroscopic WT1 deletions have a significantly increased risk of Wilms tumour, and a high level of vigilance should be maintained in such cases.

Background: Developmental eye anomalies, which include anophthalmia (absent eye) or microphthalmia (small eye) are an important cause of severe visual impairment in infants and young children. Heterozygous mutations in SOX2, a SOX1B-HMG box transcription factor, have been found in up to 10% of individuals with severe microphthalmia or anophthalmia and such mutations could also be associated with a range of non-ocular abnormalities. Methods: We performed mutation analysis on a new cohort of 120 patients with congenital eye abnormalities, mainly anophthalmia, microphthalmia and coloboma. Multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridisation (FISH) were used to detect whole gene deletion. Results: We identified four novel intragenic SOX2 mutations (one single base deletion, one single base duplication and two point mutations generating premature translational termination codons) and two further cases with the previously reported c.70del20 mutation. Of 52 patients with severe microphthalmia or anophthalmia analysed by MLPA, 5 were found to be deleted for the whole SOX2 gene and 1 had a partial deletion. In two of these, FISH studies identified sub-microscopic deletions involving a minimum of 328 Kb and 550 Kb. The SOX2 phenotypes include a patient with anophthalmia, oesophageal abnormalities and horseshoe kidney, and a patient with a retinal dystrophy implicating SOX2 in retinal development. Conclusion: Our results provide further evidence that SOX2 haploinsufficiency is a common cause of severe developmental ocular malformations and that background genetic variation determines the varying phenotypes. Given the high incidence of whole gene deletion we recommend that all patients with severe microphthalmia or anophthalmia, including unilateral cases be screened by MLPA and FISH for SOX2 deletions.

Using fluorescent in situ hybridization (FISH) with the probe p15 (D15Z1), we investigated the distribution of the polymorphic 15p signal which has been reported to occur on acrocentric chromosomes in addition to chromosome 15. The short arm of chromosome 15 has a characteristic signal pattern when hybridized with the FISH probe D15Z1. However, the D15Z1 signal can occasionally be seen on the short arm of other acrocentric chromosomes. We studied the distribution of the D15Z1 probe in 1657 patients consisting both of individuals with a normal karyotype and those with a variety of chromosome abnormalities involving the acrocentric chromosomes. Our results show that one in six individuals, regardless of their patient ascertainment category or karyotypic status, had one or more additional D15Z1 signals, and that there were no significant differences in the distribution of extra signals among the patient groups.

The ability to identify the clinical nature of the recurrent duplication of chromosome 17q12 has been limited by its rarity and the diverse range of phenotypes associated with this genomic change. In order to further define the clinical features of affected patients, detailed clinical information was collected in the largest series to date (30 patients and 2 of their siblings) through a multi-institutional collaborative effort. The majority of patients presented with developmental delays varying from mild to severe. Though dysmorphic features were commonly reported, patients do not have consistent and recognizable features. Cardiac, ophthalmologic, growth, behavioral, and other abnormalities were each present in a subset of patients. The newly associated features potentially resulting from 17q12 duplication include height and weight above the 95th percentile, cataracts, microphthalmia, coloboma, astigmatism, tracheomalacia, cutaneous mosaicism, pectus excavatum, scoliosis, hypermobility, hypospadias, diverticulum of Kommerell, pyloric stenosis, and pseudohypoparathryoidism. The majority of duplications were inherited with some carrier parents reporting learning disabilities or microcephaly. We identified additional, potentially contributory copy number changes in a subset of patients, including one patient each with 16p11.2 deletion and 15q13.3 deletion. Our data further define and expand the clinical spectrum associated with duplications of 17q12 and provide support for the role of genomic modifiers contributing to phenotypic variability.

A two-year-old boy presenting with bilateral aniridia and psychomotor retardation had a de novo (2;3;11) highly complex rearrangement which was characterized as far as possible by means of G-banding and FISH assays with multiple probes including cosmids for the Wilms, Aniridia, Genital anomalies and Retardation (WAGR) region, alphoid repeats for chromosomes 2, 3 and 11, subtelomere probes for 2p/2q, 3p/3q and 11q and BACs for 2q32 and 3q13. We identified ∼15 breakpoints with at least three interchromosomal and three intrachromosome anomalies involving chromosome 11. Both parents had normal karyotypes and no cryptic 11p rearrangements revealed by the chromosome 11 cosmid panel. The lack of a deletion of PAX6 pointed to the direct insertion of an ∼300-kb segment involving the cosmids FO2121 and AO4160, and more specifically the insertion’s proximal breakpoint in the ∼150-kb segment between FO2121 and FAT5 (PAX6), as the responsible factor for the patient’s aniridia via a position effect resulting in functional haploinsufficiency of the PAX6 gene. This case illustrates the importance of recognizing that de novo complex chromosomal rearrangements found in patients with diverse clinical features may contribute to the phenotype, but that multiple mechanisms and higher levels of complexity may be unmasked by high resolution molecular cytogenetic studies.

Leri-Weill dyschondrosteosis (LWD) is a pseudoautosomal dominant disorder characterized by disproportionate short stature and a characteristic curving of the radius, known as the "Madelung deformity." SHOX mutations resulting in SHOX haploinsufficiency have been found in LWD and in a variable proportion of patients with idiopathic short stature (ISS), whereas homozygous loss of SHOX results in the more severe Langer mesomelic dysplasia (LMD). Defects in SHOX have been identified in approximately 60% of LWD cases, whereas, in the remaining approximately 40%, the molecular basis is unknown. This suggests either genetic heterogeneity or the presence of mutations in unanalyzed regions of SHOX, such as the upstream, intragenic, or downstream regulatory sequences. Therefore, the pseudoautosomal region 1 (PAR1) of 80 patients with LWD, in whom SHOX deletions and mutations had been excluded, was screened for deletions by use of a new panel of microsatellite markers. We identified 12 patients with LWD who presented with a novel class of PAR1 deletions that did not include SHOX. The deletions were of variable size and mapped at least approximately 30-530 kb downstream of SHOX. In our cohort, this type of deletion accounted for 15% of cases. In all cases, the deletions cosegregated with the phenotype. No apparent phenotypic differences were observed between patients with SHOX deletions and those with this new class of PAR1 deletions. Thus, we present here the identification of a second PAR1 region implicated in the etiopathogenesis of LWD. Our findings suggest the presence of distal regulatory elements of SHOX transcription in PAR1 or, alternatively, the existence of an additional locus apparently involved in the control of skeletal development. Deletion analysis of this newly identified region should be included in the mutation screening of patients with LWD, LMD, and ISS.

To test the hypothesis that translocation breakpoints in normal individuals are simple and do not disrupt genes, we characterised the breakpoints in 13 phenotypically normal individuals incidentally ascertained with an apparently balanced reciprocal translocation. Cases were karyotyped, and the breakpoints were refined by fluorescence in situ hybridisation until breakpoint-spanning clones were identified. 1 Mb array-CGH was performed as a whole genome analysis tool to detect any imbalances in chromatin not directly involved in the breakpoints. Breakpoint-associated imbalances were not found in any of the patients analysed in this study. However, breakpoints which disrupted known genes were identified in two patients, with RYR2 disrupted in one patient and COL13A1 in the other. In a further eight patients, Ensembl mapping data suggested that a gene might be disrupted by a breakpoint. In one further patient, the translocation was shown to be nonreciprocal. This study shows that apparently balanced reciprocal translocations in phenotypically normal patients do not have imbalances at the breakpoints, in contrast to phenotypically abnormal patients where the translocation breakpoints are often associated with cryptic imbalances. However, phenotypically normal individuals, and phenotypically abnormal individuals may have genes disrupted and therefore inactivated by one of the breakpoints. The significance of these disruptions remains to be determined.

Summary Background In 2004, the UK National Screening Committee (UKNSC) recommended that new screening programmes for Down's syndrome need not include karyotyping and can offer prenatal diagnosis for the syndrome with FISH (fluorescence in-situ hybridisation) or PCR as rapid diagnostic tests. The UKNSC also recommended that FISH or PCR tests should only include trisomies 13, 18, and 21. We undertook a retrospective cytogenetic audit to assess the probable clinical effect of these proposed policy changes. Methods 23 prenatal cytogenetic laboratories from the UK public sector submitted data for amniotic fluid or chorionic villus samples referred from April, 1999, to March, 2004. We obtained data for the details of all abnormal karyotypes by reason for referral and assessed the efficiency of FISH and PCR rapid tests for the detection of chromosome abnormalities. Findings Of 119 528 amniotic fluid and 23 077 chorionic villus samples, rapid aneuploidy testing replacement of karyotyping would have resulted in about one in 100 and one in 40 samples having an undetected abnormal karyotype, respectively. Of these missed results, 293 (30%) of 1006 amniotic fluid samples and 152 (45%) of 327 chorionic villus samples were associated with a substantial risk of an abnormal phenotypic outcome. Of 34 995 amniotic fluid and 3049 chorionic villus samples that had karyotyping and a rapid test on the same sample, none of the three technologies was completely reliable to detect an abnormal karyotype, but the best protocol for an interpretable result was PCR and karyotyping or FISH and karyotyping. Interpretation Replacement of full karyotyping with rapid testing for trisomies 13, 18, and 21 after a positive screen for Down's syndrome will result in substantial numbers of liveborn children with hitherto preventable mental or physical handicaps, and represents a substantial change in the outcome quality of prenatal testing offered to couples in the UK.

The WAGR contiguous gene deletion syndrome is a combination of Wilms tumor, Aniridia, Genito-urinary abnormalities, and growth and mental retardation which is invariably associated with an 11p13 deletion. We report two monozygotic twins and a third, unrelated patient with WAGR syndrome and additional clinical features not usually associated with WAGR. Both twins had developmental delay, growth deficiency, severe ocular involvement (nystagmus, aniridia, cataracts), atrial septal defect and two uncommon findings: agenesis of the corpus callosum and duplication of the halluces. One twin developed Wilms tumors aged 19 months while her sister remained tumor free by the age of 6.5 years. The singleton patient showed typical WAGR syndrome and preaxial hallucal polydactyly. Molecular cytogenetic studies refined the identification of the extent of the deleted segments, which were not identical in the two families. The two deletions included the PAX6 and WT1 genes as previously reported in typical WAGR patients. The unusual anomalies described in this report, may represent the expression of low penetrant traits associated with haploinsufficency of one or more of the genes present in the deletion (PAX6 is expressed in CNS) or may indicate epistatic influences of modifier genes on the expression of gene(s) present in the WAGR region

Aniridia, Wilms tumor, genitourinary abnormalities, growth and mental retardation are the cardinal features of the WAGR 11p13 deletion syndrome. The Potocki-Schaffer syndrome or proximal 11p deletion syndrome (previously DEFECT11 syndrome) is a contiguous gene syndrome associated with deletions in 11p11.2, principal features of which are multiple exostoses and enlarged parietal foramina. Mental handicap, facial dysmorphism and craniosynostosis may also be associated. We report a patient with combined WAGR and Potocki-Shaffer syndromes, and obesity. She presented with aniridia, cataract, nystagmus, corneal ulcers and bilateral congenital ptosis. A left nephroblastoma was detected at 15 months. Other features included moderate developmental delay, growth deficiency, facial dysmorphism, multiple exostoses and cranial lacunae. High-resolution and molecular cytogenetics confirmed a del(11)(p11.2p14.1) deletion with a proximal breakpoint between the cosmid DO8153 and the BAC RP11-104M24 to a distal breakpoint between cosmids CO8160 (D11S151) and F1238 (D11S1446). The deletion therefore includes EXT2, ALX4, WT1 and PAX6. This case appears to be the second patient reported with this combined deletion syndrome and confirms the association of obesity in the WAGR spectrum, a feature previously reported in four cases, and for which the acronym WAGRO has been suggested. Molecular and follow-up data on the original WAGRO case are briefly presented.

The details of all cytogenetic abnormalities diagnosed in the Wessex Regional Genetics Laboratory (WRGL) since 1967 to the present day have been recorded in the Salisbury Treasury of Interesting Chromosomes (STOIC). From this resource, we identified 137 patients with constitutional autosomal supernumerary marker chromosomes (SMC) ascertained in four principal groups: (i) 37% with abnormal phenotypes; (ii) 7% couples with reproductive difficulties; (iii) 47% antenatal diagnoses and (iv) 9% miscellaneous. Overall, 81 (59%) SMCs were mosaics and 56 (41%) nonmosaics. Of the 109 cases with known parental origins, 70% were de novo, 19% maternally and 11% paternally inherited. The chromosomal origins of 112/137 (82%) of the SMCs have been determined by fluorescence in situ hybridization (FISH). In all, 36/112 (32%) were derived from nonacrocentric autosomes, and 76/112 (68%) from the acrocentric autosomes 13/21, 14, 15 and 22. Of these acrocentric SMCs, 39 (51%) were derived from chromosome 15, so that SMC(15) constituted 39/112 (35%) of all SMCs with known chromosomal origins. The frequencies with which mosaicism was observed varied considerably according to the chromosomal origin of the SMCs and accounted for 8/39 (20%) SMC(15), 13/37 (35%) SMCs from other acrocentrics and 25/36 (69%) of nonacrocentric SMCs. The data were analysed for parental age effects, and only de novo SMC(15)s were found to be associated with a significantly increased maternal age.

We report the clinical, cytogenetic, and molecular findings in a family in which a t(Y;15)(p11.2;q12) is segregating. The Y chromosome breakpoint disrupts the DYZ5 sequence containing the TSPY genes that are exclusively expressed in the testes while the chromosome 15 breakpoint is within the GABRG3 gene. The father and his son who both carried the balanced form of the translocation are clinically normal. A daughter who carried the der Y had the clinical features of Prader-Willi syndrome while a son who carries the der 15 has mild developmental delay and hypogonadism. The relationship of the translocation to the clinical phenotypes is discussed.

Fifty chromosomally normal couples with three or more miscarriages were examined using fluorescent in situ hybridisation (FISH) and a library of subtelomere-specific probes together with alphoid repeats mapping to the acrocentric centromeres. Six abnormalities were found. Firstly, a cryptic reciprocal subtelomere translocation between the long arm of a chromosome 3 and the short arm of a chromosome 10. The other five cryptic abnormalities involved the acrocentric chromosome pericentromeric regions and in one case also Yp. Two patients had a rearranged chromosome 13, where the centromeric region was found to be derived from the short arm, centromere and proximal long arm of chromosome 15. Another two patients had a derived chromosome 22, where the centromere was replaced by two other centromeres, one derived from chromosome 14 and the other from either chromosome 13 or 21, while one patient had the subtelomere region of Yp translocated onto the short arm of a chromosome 21. These abnormalities may be the underlying cause of the recurrent miscarriages, because they may result in abnormal pairing configurations at meiosis leading to non-disjunction of whole chromosomes at metaphase I. The frequency of rearrangements seen in the recurrent miscarriage patient population was significantly different from that in the control group ( P=0.0096, Fisher's exact test) due to the acrocentric pericentromeric abnormalities.

Seventy-seven patients with aniridia, referred for cytogenetic analysis predominantly to assess Wilms tumor risk, were studied by fluorescence in situ hybridization (FISH), through use of a panel of cosmids encompassing the aniridia-associated PAX6 gene, the Wilms tumor predisposition gene WT1, and flanking markers, in distal chromosome 11p13. Thirty patients were found to be chromosomally abnormal. Cytogenetically visible interstitial deletions involving 11p13 were found in 13 patients, 11 of which included WT1. A further 13 patients had cryptic deletions detectable only by FISH, 3 of which included WT1. Six of these, with deletions500 kb, share a similar proximal breakpoint within a cosmid containing the last 10 exons of PAX6 and part of the neighboring gene, ELP4. Two of these six patients were mosaic for the deletion. The remaining four had chromosomal rearrangements: an unbalanced translocation, t(11;13), with a deletion including the WAGR (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation) region, and three balanced rearrangements with what appear to be position effect breakpoints 3' of PAX6: (a) a t(7;11) with the 11p13 breakpoint approximately 30 kb downstream of PAX6, (b) a dir ins(12;11) with a breakpoint50 kb from PAX6, and (c) an inv(11)(p13q13) with a breakpoint75 kb downstream of PAX6. The proportion and spectrum of chromosome anomalies in familial (4/14, or 28.5%) and sporadic (26/63, or 41%) cases are not significantly different. An unexpectedly high frequency of chromosomal rearrangements is associated with both sporadic and familial aniridia in this cohort.

Using comparative genomic hybridisation, we have analysed genetic imbalance in a series of 86 ependymomas from children and adults. Tumours were derived from intracranial and spinal sites, and classified histologically as classic, anaplastic or myxopapillary. Ependymomas showing a balanced profile were significantly (P

The WAGR contiguous gene deletion syndrome is a combination of Wilms tumor, aniridia, genito-urinary abnormalities, and mental retardation. An 8.5-year-old girl was initially investigated at the age of 18 months for congenital bilateral aniridia, cataracts, glaucoma and epicantus. The ultrasound (US) scan showed polycystic kidney disease. FISH study revealed deletion of the WT1 and PAX6 gene in the 11p13 WAGR region. Forty days after the first kidney US, the second US revealed a 3 cm tumor in the right kidney: a Wilms tumour, treated successfully with the Wilm's tumor protocol. The authors conclude that the identification of the deletions in the WAGR region in patients with aniridia should definitely be done. In addition, Wilms tumor can have a very rapid growth, which, per se requires frequent and careful ultrasound kidney controls. Polycystic kidneys can be part of the WAGR presentation.

Interstitial microdeletions of 20q chromosome are rare, only 17 patients have been reported in the literature to date. Among them, only six carried a proximal 20q11.21-q11.23 deletion, with a size ranging from 2.6 to 6.8 Mb. The existence of a 20q11.2 microdeletion syndrome has been proposed, based on five previously reported cases that displayed anomalies of the extremities, intellectual disability, feeding difficulties, craniofacial dysmorphism and variable malformations. To further characterize this syndrome, we report on six new patients with 20q11.2 microdeletions diagnosed by whole-genome array-based comparative genomic hybridization. These patient reports more precisely refined the phenotype and narrowed the minimal critical region involved in this syndrome. Careful clinical assessment confirms the distinctive clinical phenotype. The craniofacial dysmorphism consists of high forehead, frontal bossing, enophthalmos, and midface hypoplasia. We have identified a 1.62 megabase minimal critical region involved in this syndrome encompassing three genes - GDF5, EPB41L1, andSAMHD1– which are strong candidates for different aspects of the phenotype. These results support that 20q11.2 microdeletion syndrome is a new contiguous gene deletion syndrome with a recognizable phenotype.

We report on a man with mental retardation and a complex karyotype with cells containing up to three morphologically distinct supernumerary marker chromosomes (SMCs) in most metaphases. Fluorescence in situ hybridisation studies using chromosome 15-specific probes characterised the presence of seven SMCs all derived from chromosome 15. The results suggest that the patient originally had a large inv dup(15) containing two copies of the Prader-Willi/Angelman critical region which became mitotically unstable, and by a process of dynamic mosaicism various morphologically distinct SMCs arose.

Point mutations in the SYNGAP1gene were described recently as arelativelyfrequentcauseofmentalretardation,presentinabout3%of nonsyndromic individuals with mental retardation [Hamdanet al., 2009]. We ascertained a carrier of a deletion encompassingSYNGAP1 among 300 patients with mental retardation studied by1Mb array-comparative genomic hybridization (a-CGH). Data onthe patient were deposited in the DECIPHER database (Databaseof Chromosomal Imbalances and Phenotype in Humans usingEnsembl Resources, http://www.sanger.ac.uk/PostGenomics/decipher).Informed consent for publishing results and photos was obtainedfrom the patient’s legal guardians.The boy (DECIPHER USP002265) was the only child of non-consaguineous healthy parents, and no other cases of mentalimpairment were known among first- or second-degree relatives.He was born at term by caesarean; his birth weight was 3,315 g(between 25th and 50th centiles) and length 47cm ( 3rd centile).Motor development was considered slightly delayed: he sat upwithout support at 6

Editor—We report a case in which a termination of pregnancy for fetal abnormality at 18 weeks' gestation showed a supernumerary marker chromosome. This extra chromosome did not hybridise to any alphoid probes and was found to have a chromosome 3 origin when investigated by M-FISH. An anomaly ultrasound scan was performed because of raised alphafetoprotein and beta HCG levels at 17 weeks' gestation in a 32 year old, primigravida mother. The scan showed a large and cystic left kidney, banana sign, and absent cisterna magna, and signs of an open sacral spina bifida. The pregnancy was terminated and necropsy showed a male fetus consistent with 18 weeks' gestation with no dysmorphic facial features. A high arched palate with a small amount of postnuchal oedema was noted as well as a single transverse palmar crease on the right hand. Inspection of the back showed a 1.3 cm long lumbosacral myelomeningocele with protruding lower lumbar spinal cord. On internal examination the cerebral hemispheres were fully cleaved and appeared fluctuant suggesting the possibility of internal hydrocephalus. The posterior fossa of the brain was reduced in anteroposterior diameter as well as appearing deep and funnel shaped, and the extension of the cerebellar tonsils was below the level of the foramen magnum. These findings are consistent with Arnold-Chiari malformation. There was marked asymmetry of the kidneys; the right kidney showed normal fetal lobation and shape but the left kidney was very large and had thin, translucent, subcapsular cysts, especially at the lower pole. The cut surface showed a poor demarcation between the cortex and medulla and the presence of cysts in most of the renal parenchyma. These findings are consistent with cystic renal dysplasia. The placenta was unremarkable and the cord had three normal blood vessels. The chromosomes of the abortus were examined …

Neocentromeres are fully functional centromeres that have arisen in previously noncentromeric chromosomal locations on rearranged chromosomes. The formation of neocentromeres results in the mitotic stability of chromosomal fragments that do not contain endogenous centromeres and that would normally be lost. Here we describe a unique collection of eight independent patient-derived cell lines, each of which contains a neocentromere on a supernumerary inversion duplication of a portion of human chromosome 13q. Findings in these patients reveal insight into the clinical manifestations associated with polysomy for portions of chromosome 13q. The results of FISH and immunofluorescent analysis of the neocentromeres in these chromosomes confirm the lack of alpha-satellite DNA and the presence of CENtromere proteins (CENP)-C, -E, and hMAD2. The positions of the inversion breakpoints in these chromosomes have been placed onto the physical map of chromosome 13, by means of FISH mapping with cosmid probes. These cell lines define, within chromosome 13q, at least three distinct locations where neocentromeres have formed, with five independent neocentromeres in band 13q32, two in band 13q21, and one in band 13q31. The results of examination of the set of 40 neocentromere-containing chromosomes that have thus far been described, including the 8 neocentromere-containing chromosomes from chromosome 13q that are described in the present study, suggest that chromosome 13q has an increased propensity for neocentromere formation, relative to some other human chromosomes. These neocentromeres will provide the means for testing hypotheses about sequence requirements for human centromere formation.

Using fluorescence in situ hybridization (FISH), supernumerary marker chromosomes (SMC) from all the human autosomes except chromosome 5, have now been described, most being derived from the acrocentric autosomes. This review summarizes the results of 168 cases of autosomal SMC excluding those from chromosome 15 where FISH has been used to define the chromosomal origin of the SMC and from which phenotypic information is available. Although the number of reported cases from some of the chromosomal SMC groups remains small, the pooled data suggest that the risk of an abnormal phenotype associated with a randomly ascertained de novo SMC derived from the acrocentric autosomes (excluding 15s) is ∼7% compared with ∼28% for SMCs derived from the nonacrocentric autosomes. Am. J. Med. Genet. 75:367-381, 1998. © 1998 Wiley-Liss, Inc.

In a cytogenetic, molecular, and clinical study of patients with autosomal supernumerary marker chromosomes (SMC), 6 out of 72 (8.3%) were shown by fluorescence in situ hybridisation (FISH) to be derived from chromosome 22. PCR microsatellite analysis and FISH using primers and cosmids from proximal 22q showed 3 of the 6 to contain euchromatin. The first, a de novo nonmosaic bisatellited, dicentric SMC, was acsertained in a patient with cat eye syndrome and Duane anomaly. Microsatellite analysis showed the SMC was maternal in origin with euchromatin extending to D22S427, i.e., proximal to the DiGeorge syndrome critical region (DGSCR). The second, a nonmosaic bisatellited, dicentric marker, was found in a child with severe hypotonia and developmental delay and had been inherited from the patient's phenotypically normal father. FISH showed the SMC to contain euchromatin extending into the DGSCR. The third, a de novo SMC, was ascertained antenatally and was shown to contain 22q euchromatin extending distal to the DGSCR. The 19-week terminated fetus was phenotypically normal at autopsy. Two of the three SMC(22)s not containing detectable proximal 22q euchromatin were ascertained coincidentally in phenotypically normal individuals, whereas the third, the only mosaic with a minority euploid cell line, was found in a patient with mild developmental delay. These results suggest that SMC(22)s devoid of proximal 22q euchromatin are not associated with adverse phenotypic effects whereas SMC(22)s containing euchromatin may be found in individuals with phenotypes ranging from cat eye syndrome to normal.

In this report we describe a male patient with a rare de novo interstitial deletion of chromosome 2q14.1-q22.1. His karyotype was reported as 46,XY,del(2)(q13q21) but subsequent array comparative genomic hybridization (array CGH) analysis redefined the deletion breakpoints as 2q14.1 and 2q22.1. Eight patients have been reported with deletions either within or spanning the region 2q13 or 2q14 to 2q22.1. In five patients the diagnosis was made by karyotype analysis alone and in three reported patients and the proband array CGH analysis was also performed. When the proband was compared with the eight previously reported patients it was apparent that they shared many clinical findings suggesting that patients with a de novo interstitial deletion involving 2q13 or 2q14 to 2q21 or 2q22 may have a recognizable phenotype. There are 14 known disease-associated genes in the deleted region of 2q14.1-q22.1 and their possible phenotypic effects on the proband and the eight previously reported patients are discussed.

Inv dup(15) is the most common supernumerary marker chromosome in humans. To investigate the mechanism responsible for this frequent chromosome rearrangement, we characterized the breakpoints in 18 individuals with small inv dup(15) chromosomes [i.e., negative for the Prader-Willi (PWS)/Angelman syndrome (AS) critical region]. Since two proximal breakpoint regions ("hotspots") for PWS/AS deletions have been previously identified with the most proximal 15q markers D15S541/S542 and S543, we hypothesized that formation of the small inv dup(15) chromosomes may involve one or both of these breakpoint hotspots. By analysis with S542, both breakpoint regions were found to be involved in approximately equal frequencies. In ten cases, the inv dup(15) was negative for S542 (Class I), indicating the breakpoint is between the centromere and the most proximal marker on chromosome 15. For the other eight cases, S542 was positive by fluorescence in situ hybridization (5/5) and/or microsatellite analysis (7/7), but S543 was negative (Class II). These two breakpoint regions appear to be the same as the two proximal breakpoints reported in the common PWS/AS deletions. To initiate cloning and sequencing of the Class II breakpoint, the gap in the yeast artificial chromosome (YAC) contig between S541/S542 and S543 was filled by screening the CEPH YAC and mega-YAC libraries. YACs 705C2 and 368H3 were found to bridge this gap, and therefore contain the more distal breakpoint region. The finding of consistent breakpoints in small inv dup(15), like that found in PWS/AS deletions, provides strong evidence for hotspots for chromosome breakage in this region. In addition, our results show that two extra copies (tetrasomy) of the region from 15cen to the euchromatic region containing S542 are present in individuals with Class II breakpoints. Since most individuals carrying a small inv dup(15) are phenotypically normal, the euchromatin region included in the small inv dup(15) chromosomes does not appear to contain genes with clinically significant dosage effects.

OBJECTIVES: To assess the incidence of microdeletions of chromosomal region 22q11 in a population of infants coming to a regional pediatric cardiac center with selected abnormalities of the ventricular outflow tracts and aortic arch and, further, to provide phenotypic/genetic correlations to determine whether patients with 22q11 deletions can be clinically recognized in infancy. BACKGROUND: DiGeorge syndrome and velocardiofacial syndrome are frequently associated with malformations of the ventricular outflow tracts and aortic arch. Both are usually caused by microdeletions of chromosomal region 22q11. The overall importance of such deletions as a cause of these cardiac malformations remains to be established. STUDY DESIGN: All infants with the candidate cardiac phenotypes during a 34-month period were studied. Dysmorphic features, type of cardiac defect, serum calcium concentration, and thymic status were recorded. Cytogenetic studies, including high-resolution karyotyping and fluorescence in situ hybridization using cosmids (cEO or cH748) from the DiGeorge critical region, were performed after clinical assessment. RESULTS: Fifty infants (including 36 with tetralogy of Fallot with or without pulmonary atresia) were seen during the study period. Twenty-six infants (52%) were dysmorphic, including 19 who were considered to have a phenotypic appearance consistent with 22q11 deletion. Genetic analysis confirmed hemizygosity for 22q11 in 8 of these 19 cases. Results of fluorescence in situ hybridization studies were normal in 22 infants without dysmorphic features and in 5 infants with dysmorphic features not suggestive of a 22q11 deletion. CONCLUSIONS: Microdeletions of chromosomal region 22q11 are an important cause of selected malformations of the ventricular outflow tracts and aortic arch and account for about 15% to 20% of cases. These deletions may be clinically recognized in early infancy and can be rapidly confirmed by fluorescence in situ hybridization. (J PEDIATR 1996;129:26-32)

Thirty perinatal solid tissue samples were used in a pilot study to test the efficacy of collagenase disaggregation onto coverslips, open system culture under low O 2 conditions, pre-harvest incubation in bromodeoxyuridine (BrdU) and colcemid, and in situ automated harvesting. Following the success of the pilot study, the new method was applied to a further 126 consecutive diagnostic samples making a total of 156. The method reduced average tissue culture times from 17 to 8.7 days (range 2-17), improved success rates from 76 to 88 per cent, and simultaneously increased the resolution of cytogenetic analysis.