Bert H.J. Eussen

Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome–lamina interactions occur through more than 1,300 sharply defined large domains 0.1–10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.

High-Throughput Analysis of Subtelomeric Chromosome Rearrangements by Use of Array-Based Comparative Genomic Hybridization

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, and miscarriages. Automated detection of subtle deletions or duplications involving telomeres is essential for high-throughput diagnosis, but impossible when conventional cytogenetic methods are used. Array-based comparative genomic hybridization (CGH) allows high-resolution screening of copy number abnormalities by hybridizing differentially labeled test and reference genomes to arrays of robotically spotted clones. To assess the applicability of this technique in the diagnosis of (sub)telomeric imbalances, we here describe a blinded study, in which DNA from 20 patients with known cytogenetic abnormalities involving one or more telomeres was hybridized to an array containing a validated set of human-chromosome-specific (sub)telomere probes. Single-copy-number gains and losses were accurately detected on these arrays, and an excellent concordance between the original cytogenetic diagnosis and the array-based CGH diagnosis was obtained by use of a single hybridization. In addition to the previously identified cytogenetic changes, array-based CGH revealed additional telomere rearrangements in 3 of the 20 patients studied. The robustness and simplicity of this array-based telomere copy-number screening make it highly suited for introduction into the clinic as a rapid and sensitive automated diagnostic procedure.

Cntnap2 is disrupted in a family with gilles de la tourette syndrome and obsessive compulsive disorder

Gilles de la Tourette syndrome (GTS) is a sporadic or inherited complex neuropsychiatric disorder characterized by involuntary motor and vocal tics. There is comorbidity with disorders like obsessive compulsive disorder and attention deficit hyperactivity disorder. Until now linkage analysis has pointed to a number of chromosomal locations, but has failed to identify a clear candidate gene(s). We have investigated a GTS family with a complex chromosomal insertion/translocation involving chromosomes 2 and 7. The affected father [46,XY,inv(2) (p23q22),ins(7;2) (q35-q36;p21p23)] and two affected children [46,XX,der(7)ins(7;2)(q35-q36;p21p23) and 46,XY,der(7)ins(7;2)(q35-q36;p213p23)] share a chromosome 2p21-p23 insertion on chromosome 7q35-q36, thereby interrupting the contactin-associated protein 2 gene (CNTNAP2). This gene encodes a membrane protein located in a specific compartment at the nodes of Ranvier of axons. We hypothesize that disruption or decreased expression of CNTNAP2 could lead to a disturbed distribution of the K(+) channels in the nervous system, thereby influencing conduction and/or repolarization of action potentials, causing unwanted actions or movements in GTS.

Molecular cytogenetic analysis of eight inversion duplications of human chromosome 13q that each contain a neocentromere.

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.

An unbalanced submicroscopic translocation t(8;16)(q24.3;p13.3)pat associated with tuberous sclerosis complex, adult polycystic kidney disease, and hypomelanosis of Ito

We report on a familial submicroscopic translocation involving chromosomes 8 and 16. The proband of the family had a clinical picture suggestive of a large deletion in the chromosome 16p13.3 area, as he was affected with tuberous sclerosis complex (TSC) and had α thalassaemia trait, and his half brother, who also had TSC, may have suffered additionally from polycystic kidney disease (PKD). FISH studies provided evidence for a familial translocation t(8;16)(q24.3;p13.3) with an unbalanced form in the proband and a balanced form in the father and in a paternal aunt.  The unbalanced translocation caused the index patient to be deleted for the chromosome 16p13.3-pter region, with the most proximal breakpoint described to date for terminal 16p deletions. In addition, FISH analysis showed a duplication for the distal 8q region. Since the index patient also had hypomelanosis of Ito (HI), either of the chromosomal areas involved in the translocation may be a candidate region for an HI determining gene. Furthermore, it is noteworthy that both carriers of the balanced translocation showed a nodular goitre, while the proband has hypothyroidism.

Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRK1A

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1–q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1–q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

A genome-wide copy number association study of osteoporotic fractures points to the 6p25.1 locus

Background Osteoporosis is a systemic skeletal disease characterised by reduced bone mineral density and increased susceptibility to fracture; these traits are highly heritable. Both common and rare copy number variants (CNVs) potentially affect the function of genes and may influence disease risk. Aim To identify CNVs associated with osteoporotic bone fracture risk. Method We performed a genome-wide CNV association study in 5178 individuals from a prospective cohort in the Netherlands, including 809 osteoporotic fracture cases, and performed in silico lookups and de novo genotyping to replicate in several independent studies. Results A rare (population prevalence 0.14%, 95% CI 0.03% to 0.24%) 210 kb deletion located on chromosome 6p25.1 was associated with the risk of fracture (OR 32.58, 95% CI 3.95 to 1488.89; p=8.69×10−5). We performed an in silico meta-analysis in four studies with CNV microarray data and the association with fracture risk was replicated (OR 3.11, 95% CI 1.01 to 8.22; p=0.02). The prevalence of this deletion showed geographic diversity, being absent in additional samples from Australia, Canada, Poland, Iceland, Denmark, and Sweden, but present in the Netherlands (0.34%), Spain (0.33%), USA (0.23%), England (0.15%), Scotland (0.10%), and Ireland (0.06%), with insufficient evidence for association with fracture risk. Conclusions These results suggest that deletions in the 6p25.1 locus may predispose to higher risk of fracture in a subset of populations of European origin; larger and geographically restricted studies will be needed to confirm this regional association. This is a first step towards the evaluation of the role of rare CNVs in osteoporosis.

5q11.2 deletion in a patient with tracheal agenesis

Tracheal agenesis (TA) is a rare congenital anomaly of the respiratory tract. Many patients have associated anomalies, suggesting a syndromal phenotype. In a cohort of 12 patients, we aimed to detect copy number variations. In addition to routine cytogenetic analysis, we applied oligonucleotide array comparative genomic hybridization. Our patient cohort showed various copy number variations, of which many were parentally inherited variants. One patient had, in addition to an inherited 16p12.1 deletion, a 3.6 Mb deletion on chromosomal locus 5q11.2. This patient had a syndromic phenotype, including vertebral, anal, cardiovascular and tracheo-oesophageal associated anomalies, and other foregut-related anomalies, such as cartilage rings in the oesophagus and an aberrant right bronchus. No common deletions or duplications are found in our cohort, suggesting that TA is a genetically heterogeneous disorder.

Identification of a gene on human chromosome 8q11 that is differentially expressed during prostate-cancer progression.

Using differential‐display RT‐PCR analysis between androgen‐dependent LNCaP‐FGC and androgen‐independent LNCaP‐LNO human prostate‐cancer cells, we have identified a gene not previously described as being expressed in prostate. The gene is more highly expressed in androgen‐independent than in androgen‐dependent LNCaP prostate‐cancer cells. Sequence analysis showed that the gene has already been cloned as a transcript present in embryonic brain, with unknown functions. Expression of the gene was found not to be restricted to the prostate, and not regulated by androgens in androgen‐independent prostate‐cancer cells. In concert with the cell‐culture system, Northern‐blot analysis of gene expression in vivo, using a panel of human prostate‐cancer xenografts, demonstrated that the gene is more highly expressed in androgen‐independent than in androgen‐dependent prostate‐cancer xenografts. The gene could be mapped on human chromosome 8q11. The 8q arm is known to be frequently amplified during prostate‐cancer progression and harbors several proto‐oncogenes potentially involved in cancer development. Since expression of the gene is positively correlated with prostate‐cancer progression and its 8q11 chromosomal localization, we hypothesize that the gene may be involved in the development and progression of prostate cancer. Int. J. Cancer 83:506–511, 1999.

Complex MAX Rearrangement in a Family With Malignant Pheochromocytoma, Renal Oncocytoma, and Erythrocytosis.

CONTEXT Familial pheochromocytoma (PCC) has been associated with germline mutations in 16 genes. Here we investigated three siblings presenting with bilateral pheochromocytomas. In addition, the index patient also exhibited renal oncocytoma and erythrocytosis, whereas the second sibling presented with a lymph node metastasis. DESIGN First, single-nucleotide polymorphism array and exome sequencing were performed on germline and PCC-derived DNA to identify genomic alterations in the index patient. Second, alterations were confirmed and validated by Sanger sequencing, analyzed by (multiplexed) PCR to determine the loss of the wild-type allele, and investigated by immunohistochemistry in the tumors of the three siblings. RESULTS The index patients germline DNA revealed a large complex genomic alteration encompassing the intragenic and promoter regions of Myc-associated factor X (MAX) and alpha-(1,6)-fucosyltransferase (FUT8). In all three siblings the MAX alteration was confirmed, and the loss of the wild-type MAX and FUT8 alleles was demonstrated in all tumors. Uniparental disomy of chromosome 14q, previously demonstrated as a hallmark for MAX-related PCC, was shown in the index patients PCC by single-nucleotide polymorphism array. Loss of MAX and FUT8 protein expression was demonstrated by immunohistochemistry in the tumors from the three siblings. CONCLUSIONS Our results indicate that large genomic deletions of MAX should be considered in familial and bilateral PCC with prior negative testing for gene mutations. In addition, our results confirm that MAX is a tumor suppressor gene for renal oncocytomas.