7q35 Microdeletion and 15q13.3 and Xp22.33 Microduplications in a Patient with Severe Myoclonic Epilepsy, Microcephaly, Dysmorphisms, Severe Psychomotor Delay and Intellectual Disability.

Copy number variations (CNVs) play a key role in the pathogenesis of several diseases, including a wide range of neurodevelopmental disorders. Here, we describe the detection of three CNVs simultaneously in a female patient with evidence of severe myoclonic epilepsy, microcephaly, hypertelorism, dimorphisms as well as severe psychomotor delay and intellectual disability. Array-CGH analysis revealed a ∼240 kb microdeletion at the 7q35 inherited from her father, a ∼538 kb microduplication at the 15q13.3 region and a ∼178 kb microduplication at Xp22.33 region, both transmitted from her mother. The microdeletion in 7q35 was included within an intragenic region of the contactin associated protein-like 2 (CNTNAP2) gene, whereas the microduplications at 15q13.3 and Xp22.33 involved the cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and the cytokine receptor-like factor 2 (CRLF2) genes, respectively. Here, we describe a female patient harbouring three CNVs whose additive contribution could be responsible for her clinical phenotypes.

Clinical update on sensorineural hearing loss in Turner syndrome and the X-chromosome.

Hearing loss is one of the major medical concerns in girls and women with Turner syndrome (TS) and has a negative effect on well-being and quality of everyday life. Sensorineural hearing loss is the most common type of hearing loss, affecting more than half of adults with TS. Karyotypes with a loss of the short p-arm on the X-chromosome are more prone to ear and hearing problems. The importance of detecting, investigating, and treating hearing loss with hearing aids cannot be emphasized enough. The pathophysiology of the sensorineural hearing loss in TS is not known, but theories regarding estrogen deficiency, the cell cycle delay hypothesis, IGF-1 deficiency and the possible role of the KDM6A gene are discussed. Due to the diversity of symptoms and conditions within the same karyotype, a combination of genetic factors altered by epigenetic and/or hormonal effects is probable. Further research is needed regarding the pathophysiology of ear and hearing problems in TS to develop new treatment methods.

X-linked ichthyosis: Clinical and molecular findings in 35 Italian patients.

Recessive X-linked ichthyosis (XLI), the second most common ichthyosis, is caused by mutations in the STS gene encoding the steroid sulfatase enzyme. A complete deletion of the STS gene is found in 85%-90% of cases. Rarely, larger deletions involving contiguous genes are detected in syndromic patients. We report the clinical and molecular genetic findings in a series of 35 consecutive Italian male patients. All patients underwent molecular testing by MLPA or aCGH, followed, in case of negative results, by next-generation sequencing analysis. Neuropsychiatric, ophthalmological and paediatric evaluations were also performed. Our survey showed a frequent presence of disease manifestations at birth (42.8%). Fold and palmoplantar surfaces were involved in 18 (51%) and 7 (20%) patients, respectively. Fourteen patients (42%) presented neuropsychiatric symptoms, including attention-deficit hyperactivity disorder and motor disabilities. In addition, two patients with mental retardation were shown to be affected by a contiguous gene syndrome. Twenty-seven patients had a complete STS deletion, one a partial deletion and 7 carried missense mutations, two of which previously unreported. In addition, a de novo STS deletion was identified in a sporadic case. The frequent presence of palmoplantar and fold involvement in XLI should be taken into account when considering the differential diagnosis with ichthyosis vulgaris. Our findings also underline the relevance of involving the neuropsychiatrist in the multidisciplinary management of XLI. Finally, we report for the first time a de novo mutation which shows that STS deletion can also occur in oogenesis.

[Turner syndrome in the hôpital du Mali, a case]. / Un cas de Syndrome de Turner a L'Hopital du Mali.

Turner syndrome is a chromosomal aberration linked to the complete or partial absence of an X chromosome. Its prevalence is 1/2500 female newborns. We report a case in the department of internal medicine and endocrinology of the hospital of MALI. This was a 14-year old girl who consulted for stunting and puberty. She presented clinically a dysmorphic syndrome, TANNER stage1 secondary sexual characteristics, weight and height at -3 DS and a bone age estimated to 9½ years old on the X-ray of the hand. The hormonal assessment showed an elevation of FSH and LH and the genetic study showed an iso chromosome Xq. This result was part of a Turner syndrome with Xq iso chromosome.

Le syndrome de Turner est une aberration chromosomique liée à l'absence complète ou partielle d'un chromosome X. Sa prévalence est de 1/2500 nouveaux nés de sexe féminin. Nous rapportons un cas dans le service de médecine interne et d'endocrinologie de l'hôpital du MALI. Il s'agissait d'une fille de 14 ans, qui a consulté pour retard de croissance et de puberté. Elle présentait cliniquement un syndrome dysmorphique, les caractères sexuels secondaires au stade1 de TANNER, le poids et la taille à −3 DS et un âge osseux estimé à 9 ans et demi à la radiographie de la main. Le bilan hormonal montrait une élévation de la FSH et la LH et l'étude génétique montrait un iso chromosome Xq. Ce résultat entrait dans le cadre d'un syndrome de Turner avec iso chromosome Xq.

Three-dimensional genome structures of single diploid human cells.

Three-dimensional genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for haploid cells but has remained a challenge for diploid cells. We developed a single-cell chromatin conformation capture method, termed Dip-C, that combines a transposon-based whole-genome amplification method to detect many chromatin contacts, called META (multiplex end-tagging amplification), and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions.

G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing.

Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing.

Familial X/Y Translocation Encompassing ARSE in Two Moroccan Siblings with Sensorineural Deafness.

Unbalanced translocations involving X and Y chromosomes are rare and associated with a contiguous gene syndrome. The clinical phenotype is heterogeneous including mainly short stature, chondrodysplasia punctata, ichthyosis, hypogonadism, and intellectual disability. Here, we report 2 brothers with peculiar gestalt, short stature, and hearing loss, who harbor an X/Y translocation. Physical examination, brainstem acoustic potential evaluation, bone age, hormonal assessment, and X-ray investigations were performed. Because of their dysmorphic features, karyotyping, FISH, and aCGH were carried out. The probands had short stature, hypertelorism, midface hypoplasia, sensorineural hearing loss, normal intelligence as well as slight radial and ulnar bowing with brachytelephalangy. R-banding identified a derivative X chromosome with an abnormally expanded short arm. The mother was detected as a carrier of the same aberrant X chromosome. aCGH disclosed a 3.1-Mb distal deletion of chromosome region Xp22.33pter. This interval encompasses several genes, especially the short stature homeobox (SHOX) and arylsulfatase (ARSE) genes. The final karyotype of the probands was: 46,Y,der(X),t(X;Y)(p22;q12).ish der(X)(DXYS129-,DXYS153-)mat.arr[hg19] Xp22.33(61091_2689408)×1mat,Xp22.33(2701273_3258404)×0mat,Yq11.222q12 (21412851_59310245)×2. Herein, we describe a Moroccan family with a maternally inherited X/Y translocation and discuss the genotype-phenotype correlations according to the deleted genes.

[Method for the Molecular Cytogenetic Visualization of Fragile Site FRAXA].

Fragile X syndrome is one of the most common reasons for human hereditary mental retardation. It is associated with the expansion of CGG repeats in the 5'-untranslated region of the FMR1 gene, which results in the suppression of its expression and the development of the disease. At present, methods based on PCR and Southern blot analysis are used for diagnostics of the fragile X syndrome. The presence of a fragile site FRAXA on the X chromosome is typical for patients with this pathology. We developed a method of visualizing this site in cell cultures obtained from patients using the fluorescent in situ hybridization (FISH) and the combination of two probes. The method allows one to detect five types of signals on the X chromosome, three of which are normal, while two are associated with the emergence of fragile site FRAXA. An analysis of the distribution of all signal types in cell lines from healthy individuals and patients with fragile X syndrome demonstrated that the method allows one to determine differences between lines with a high statistical significance and that it is applicable to detecting cells that are carriers of the syndrome.

Structural aspects of the inactive X chromosome.

A striking difference between male and female nuclei was recognized early on by the presence of a condensed chromatin body only in female cells. Mary Lyon proposed that X inactivation or silencing of one X chromosome at random in females caused this structural difference. Subsequent studies have shown that the inactive X chromosome (Xi) does indeed have a very distinctive structure compared to its active counterpart and all autosomes in female mammals. In this review, we will recap the discovery of this fascinating biological phenomenon and seminal studies in the field. We will summarize imaging studies using traditional microscopy and super-resolution technology, which revealed uneven compaction of the Xi. We will then discuss recent findings based on high-throughput sequencing techniques, which uncovered the distinct three-dimensional bipartite configuration of the Xi and the role of specific long non-coding RNAs in eliciting and maintaining this structure. The relative position of specific genomic elements, including genes that escape X inactivation, repeat elements and chromatin features, will be reviewed. Finally, we will discuss the position of the Xi, either near the nuclear periphery or the nucleolus, and the elements implicated in this positioning.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.

The phenotype spectrum of X-linked ichthyosis identified by chromosomal microarray.

BACKGROUND: Steroid sulfatase (STS) gene disruption causes X-linked ichthyosis (XLI). Interrogating the entire genome through chromosomal microarray (CMA), a test primarily used to screen patients with noncutaneous congenital anomalies, may detect STS deletions incidentally. OBJECTIVE: We sought to determine the variability of skin features associated with STS deletions diagnosed through CMA and to compare these findings with XLI cases reported in the literature and recognized in a dermatology clinic. METHODS: Male patients with an STS deletion were identified from 23,172 consecutive postnatal blood samples tested with CMA at Mayo Clinic. A comparison group of male patients with biochemically confirmed XLI was ascertained in the dermatology clinic. The available patient medical records, skin histopathology, and photographs were evaluated and a literature search of patients with XLI was conducted. RESULTS: Children whose diagnosis was made incidentally through CMA had milder skin phenotypes, including dryness or eczema, or both, and did not manifest the polygonal or "dirty" scale described as typical of XLI in the literature. LIMITATIONS: The small sample size, limited clinical information, and assessment by nondermatologists in a subset of cases may have influenced the results. CONCLUSION: STS deletions may cause a milder skin phenotype than the typical presentation of XLI.

Quantitatively imaging chromosomes by correlated cryo-fluorescence and soft x-ray tomographies.

Soft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant.

Variable escape from X-chromosome inactivation: identifying factors that tip the scales towards expression.

In humans over 15% of X-linked genes have been shown to 'escape' from X-chromosome inactivation (XCI): they continue to be expressed to some extent from the inactive X chromosome. Mono-allelic expression is anticipated within a cell for genes subject to XCI, but random XCI usually results in expression of both alleles in a cell population. Using a study of allelic expression from cultured lymphoblasts and fibroblasts, many of which showed substantial skewing of XCI, we recently reported that the expression of genes lies on a contiunuum between those that are subject to inactivation, and those that escape. We now review allelic expression studies from mouse, and discuss the variability in escape seen in both humans and mice in genic expression levels, between X chromosomes and between tissues. We also discuss current knowledge of the heterochromatic features, DNA elements and three-dimensional topology of the inactive X that contribute to the balance of expression from the otherwise inactive X chromosome.

Anterior positioning of sex chromosomes on the head of human sperm sorted using visible wavelengths.

The human ejaculate contains subpopulations of sperm with distinct properties. Human X- and Y-bearing sperm were separated with fluorescence activated cell sorting. To avoid the use of UV light the quantitative DNA dyes DRAQ5® and Dyecycle™ Vybrant® Violet were used. Sorting efficiency was similar for both dyes, but lower than what is usually obtained with the classical method involving Hoechst 33342 and UV light (60-70% enrichment, versus 80-90%). A total of 2,739 spermatozoa were evaluated, from seven distinct samples using fluorescence in situ hybridization (FISH) chromosomal probes. No differences were found in sorted and unsorted populations in terms of chromosome positioning, and numeric chromosomal anomalies were not more evident following cell sorting. Furthermore in both sorted and unsorted populations the sex chromosomes were clearly located in the anterior portion of the sperm head, while a control autosome (chromosome 18) showed no such tendency, confirming previous findings. These results suggest that other quantitative DNA dyes may be used for sex chromosome-based human sperm sorting, but with lower efficiency than the standard UV-Hoechst based assay.

A top-down analysis of Xa- and Xi-territories reveals differences of higher order structure at ≥ 20 Mb genomic length scales.

The active and inactive X (Xa;Xi) territory with its seemingly highly compacted Barr body in nuclei of female mammalian cells provide a key example for studies of structure/function relationships in homologous chromosomes with different functional properties. Here we used about 300 human X-specific large insert clones to generate probe sets, which target physically or functionally defined sub-chromosomal segments. We combined 3D multicolor FISH with quantitative 3D image analysis in order to compare the higher order organization in Xi-and Xa-territories in human diploid fibroblasts (HDFs) at various length scales ranging from about 50 Mb down to 1 Mb. Xi-territories were characterized by a rounder shape as compared to the flatter and more extended shape of Xa-territories. The overall compaction of the entire Xi-territory, including the Barr body, was only 1.2-fold higher than the Xa-territory. Significant differences, however, were noted between distinct subchromosomal segments: At 20 Mb length scales higher compaction in Xi-territories was restricted to specific segments, but higher compaction in these segments was not correlated with gene density, transcriptional activity, LINE content or histone markers locally enriched in Xi-territories. Notably, higher compaction in Xi-territories observed for 20 Mb segments was not reflected accordingly by inclosed segments of 1-4 Mb. We conclude that compaction differences result mainly from a regrouping of ~1 Mb chromatin domains rather than from an increased condensation of individual domains. In contrast to a previous report, genes subject to inactivation as well as escaping from inactivation were not excluded from the interior of the Barr body.

Meiotic recombination errors, the origin of sperm aneuploidy and clinical recommendations.

Since the early 1990s male infertility has successfully been treated by intracytoplasmic sperm injection (ICSI), nevertheless concerns have been raised regarding the genetic risk of ICSI. Chromosome aneuploidy (the presence of extra or missing chromosomes) is the leading cause of pregnancy loss and mental retardation in humans. While the majority of chromosome aneuploidies are maternal in origin, the paternal contribution to aneuploidy is clinically relevant particularly for the sex chromosomes. Given that it is difficult to study female gametes investigations are predominantly conducted in male meiotic recombination and sperm aneuploidy. Research suggests that infertile men have increased levels of sperm aneuploidy and that this is likely due to increased errors in meiotic recombination and chromosome synapsis within these individuals. It is perhaps counterintuitive but there appears to be no selection against chromosomally aneuploid sperm at fertilization. In fact the frequency of aneuploidy in sperm appears to be mirrored in conceptions. Given this information this review will cover our current understanding of errors in meiotic recombination and chromosome synapsis and how these may contribute to increased sperm aneuploidy. Frequencies of sperm aneuploidy in infertile men and individuals with constitutional karyotypic abnormalities are reviewed, and based on these findings, indications for clinical testing of sperm aneuploidy are discussed. In addition, the application of single nucleotide arrays for the analysis of meiotic recombination and identification of parental origin of aneuploidy are considered.

A novel spliced fusion of MLL with CT45A2 in a pediatric biphenotypic acute leukemia.

BACKGROUND: Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia. METHODS: Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia. RESULTS: Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences. CONCLUSION: We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first Cancer/Testis antigen family gene found fused with MLL in acute leukemia, future studies addressing its biologic relevance for leukemogenesis are warranted.