Ingo Hansmann

Homologous sequences at human chromosome 9 bands p12 and q13-21.1 are involved in different patterns of pericentric rearrangements

A thorough study of the heterochromatin organisation in the pericentromeric region and the proximal long (q) and short (p) arms of human chromsome 9 (HSA 9) revealed homology between 9p12 and 9q13-21.1, two regions that are usually not distinguishable by molecular cytogenetic techniques. Furthermore, the chromosomal regions 9p12 and 9q13-21.1 showed some level of homology with the short arms of the human acrocentric chromosomes. We studied five normal controls and 51 clinical cases: 48 with chromosome 9 heteromorphisms, one with an exceptionally large inversion and two with an additional derivative chromosome 9. Using fluorescence in situ hybridisation (FISH) with three differentially labelled chromosome 9-specific probes we were able to distinguish 12 heteromorphic patterns in addition to the most frequent pattern (defined as normal). In addition, we studied one inversion 9 case with the recently described multicolour banding (MCB) technique. Our results, and previously published findings, suggest several hotspots for recombination in the pericentromeric heterochromatin of HSA 9. They also demonstrate that constitutional inversions affecting the pericentromeric region of chromosome 9 carry breakpoints located preferentially in 9p12 or 9q13-21.1 and less frequently in 9q12.

An excess of chromosome 1 breakpoints in male infertility.

In a search for potential infertility loci, which might be revealed by clustering of chromosomal breakpoints, we compiled 464 infertile males with a balanced rearrangement from Mendelian Cytogenetics Network database (MCNdb) and compared their karyotypes with those of a Danish nation-wide cohort. We excluded Robertsonian translocations, rearrangements involving sex chromosomes and common variants. We identified 10 autosomal bands, five of which were on chromosome 1, with a large excess of breakpoints in the infertility group. Some of these could potentially harbour a male-specific infertility locus. However, a general excess of breakpoints almost everywhere on chromosome 1 was observed among the infertile males: 26.5 versus 14.5% in the cohort. This excess was observed both for translocation and inversion carriers, especially pericentric inversions, both for published and unpublished cases, and was significantly associated with azoospermia. The largest number of breakpoints was reported in 1q21; FISH mapping of four of these breakpoints revealed that they did not involve the same region at the molecular level. We suggest that chromosome 1 harbours a critical domain whose integrity is essential for male fertility.

Duplication of Xq26.2-q27.1, including SOX3, in a mother and daughter with short stature and dyslalia

Duplications of the distal long arm of the X chromosome are rare and carrier females are usually phenotypically normal. We report on a 14‐year‐old short statured (height and weight <3rd centile) girl with dup(X)(q26.2q27.1) inherited from a short mother. The proband has minor dysmorphic features, lordosis, lack of menarche, late signs of puberty, low prepuberal levels of gonadotrophins and steroids, but borderline low IGF‐1 and normal IGF‐Bp3 serum levels. Both the proposita and her mother have severe speech problems with stuttering and dyslalia. The 44‐year‐old mother with a strikingly aged face and a prominent nose, had menarche at 15 years. Both maternal sisters and the grandmother of the proposita are also short. Karyotyping revealed an additional band at Xq26 in all metaphases from the proband, her mother, and two maternal aunts. Molecular cytogenetic investigations revealed an Xq26.2–q27.1 direct duplication of ∼7.5 Mb that encompasses or disrupts the SOX3 gene, which maps at the distal border of the duplicated segment. A similar chromosomal duplication was reported recently in five families and in each was associated with an abnormal phenotype in males with short stature [Hol et al., 2000 ; Solomon et al., 2002 , 2004 ]. Using an androgen‐receptor (HUMARA) gene methylation assay and FISH, we show that despite preferential inactivation of the dup(Xq) chromosome a significant proportion of lymphocytes in both mother and daughter carry an active duplicated X chromosome. Our findings further suggest that a dosage effect of SOX3 may to be responsible for a speech disorder in addition to short stature secondary to hypopituitarism.

Parental mosaicism of JAG1 mutations in families with Alagille syndrome

The Alagille syndrome (AGS), a congenital disorder affecting liver, heart, skeleton and eye in association with a typical face, is an autosomal dominant disease with nearly complete penetrance and variable expression. AGS is caused by mutations in the developmentally important JAG1 gene. In our mutation screening, where 61 mutations in JAG1 were detected, we identified five cases where mosaicism is present. Our results point to a significant frequency of mosaicism for JAG1 mutations in AGS of more than 8.2%. Because mosaicism may be associated with a very mild phenotype, the appropriate diagnosis of AGS and consequently the determination of the recurrence risk can be complicated.

Minimal phenotype in a girl with trisomy 15q due to t(X;15)(q22.3;q11.2) translocation

Few cases of de novo unbalanced X;autosome translocations associated with a normal or mild dysmorphic phenotype have been described. We report a 3‐year‐old dizygotic female twin with prenatally ascertained increased nuchal translucency. Prenatal chromosome studies revealed nearly complete trisomy 15 due to a de novo unbalanced translocation t(X;15)(q22;q11.2) confirmed postnatally. A mild phenotype was observed with normal birth measurements, minor facial dysmorphic features (hypertelorism, short broad nose, and a relatively long philtrum), and moderate developmental delay at the age of 3 years in comparison to her male fraternal twin. Replication timing utilizing BrdU and acridine‐orange staining showed that the der(X) chromosome was late‐replicating with variable spreading of inactivation into the translocated 15q segment. The der(X) was determined to be of paternal origin by analyses of polymorphic markers and CGG‐repeat at FMR1. Methylation analysis at the SNRPN locus and analysis of microsatellites on 15q revealed paternal isodisomy with double dosage for all markers and the unmethylated SNRPN gene. The Xq breakpoint was mapped within two overlapping BAC clones RP11‐575K24 and RP13‐483F6 at Xq22.3 and the 15q breakpoint to 15q11.2, within overlapping clones RP11‐509A17 and RP11‐382A4 that are all significantly enriched for LINE‐1 elements (36.6%, 43.0%, 26.6%, 22.0%, respectively). We speculate that the attenuated phenotype may be due to inactivation spreading into 15q, potentially facilitated by the enrichment of LINE‐1 elements at the breakpoints. In silico analysis of breakpoint regions revealed the presence of highly identical low‐copy repeats (LCRs) at both breakpoints, potentially involved in generating the translocation.

Paramutation-like effects at the mouse scapinin (Phactr3) locus.

Paramutation-like phenomena have been extensively studied in plants and so far described for a very few engineered loci in the mouse. Here we report an allele-specific expression analysis of the Phactr3 (phosphatase and actin regulator 3) locus by identifying the first internal mouse transcripts with a paramutation-like effect not associated with transgenic or knockout mice. In our previous work, we showed that the Phactr3 gene was mainly transcribed in the brain, exhibiting a complex genomic organisation with four alternatively spliced leader exons. Due to the location of the Phactr3 gene in the distal imprinting region on mouse chromosome 2, we generated a mouse model to investigate the possible parental influence on the allelic expression pattern by reciprocally mating NMRI mice and Mus musculus castaneus. We were able to identify a single-nucleotide polymorphism in leader exon 1C representing restriction fragment length polymorphism. After reverse transcription PCR, NMRI and M. musculus castaneus showed a homozygous restriction pattern according to their genotype. Unlike this, reverse transcription PCR products of the F1 hybrids of both crosses were transcribed from the NMRI allele only. Therefore, the Phactr3 exon 1C splice variant is potentially strain specific regulated, leading to the expression of only one allele of the reciprocal crosses. So far, this has not yet been described for an internal mouse gene. These results potentially provide new insight into non-Mendelian inheritance in mammals and may serve also as a model for investigating the regulation of allele-specific expression.

Genomic organization and expression pattern of scapinin (PHACTR3) in mouse and human.

Scapinin has been found to bind to cytoplasmic actin and is also a putative regulatory subunit of protein phosphatase-1 (PP1). It is found attached to the nuclear matrix-intermediate filament (NM-IF) and is down-regulated by differentiation of tumor cells. We have analyzed the genomic structure and tissue-specific expression pattern of both the human scapinin gene (PHACTR3) and the orthologous mouse gene. Both genes showed a highly conserved complex genomic organization with four different leader exons. Alternative splicing of exon 5 was found to be limited to human and variable polyadenylation in mouse transcripts only. In both species expression seems to occur predominantly in the brain. By Northern blot analysis two major transcripts in human and three transcripts in mouse were detected. Expression analysis in the mouse revealed a tissue-specific complex transcription pattern in the brain and a specific pattern was observed during prenatal development. Based on the transcriptional data we therefore assume scapinin to have a distinct biological function in the mammalian brain.

The 9p24.3 Breakpoint of a Constitutional t(6;9)(p12;p24) in a Patient with Chronic Lymphocytic Leukemia Maps Close to the Putative Promoter Region of the DMRT2 Gene

B-cell chronic lymphocytic leukemia (B-CLL) is a very common hematological malignancy. Although several alterations in different loci have been identified and established as prognostic factors the pathogenetic cascade remains obscure. Here we give an account on a 71-year-old man with B-CLL and a translocation t(6;9) in his diagnostic bone marrow. Subsequent chromosome analysis of his blood lymphocytes revealed a constitutional karyotype 46,XY,t(6;9) (p12;p24) that has not been previously reported. Seeking for gene disruption correlated with the B-CLL we precisely mapped both breakpoints by fluorescence in situ hybridization (FISH) analysis with chromosome-specific bacterial artificial chromosome (BAC) clones and their long-range polymerase chain reaction (LRPCR) subfragments. An 11-kb LRPCR subfragment derived from RP11-399A15 was found to span the breakpoint at 6p12.1. FISH analysis with a 12-kb LRPCR fragment derived from RP11-147I11 which overlaps with RP11-110M16 as well as with a cDNA for DMRT2 (doublesex and mab-3 related transcription factor 2) maps the 9p24.3 breakpoint maximum 10 kb upstream from DMRT2. In silico analysis of the transcripts within the vicinity of the breakpoints revealed that the translocation does not disrupt any known genes but could affect the putative DMRT2promoter. Long range effects on gene expression cannot be excluded so far.

Paternal isodisomy 7q secondary to monosomy 7 at recurrence in a Down syndrome child with acute myelogenous leukemia

We report a boy with Down syndrome and leukemia who acquired uniparental isodisomy of chromosome 7q as a secondary chromosomal change during recurrence of the disease. His karyotype before therapy was 46,XY,der(1)t(1;1)(p36;q32),-7,+21c/46,idem,del(9)(p22), whereas at recurrence it was 46,XY,der(1)t(1;1)(p36;q32,-7,der(7)(qter-->p22 through pter::q10-->qter),del(9)(p22),+21c/47,XY,+21c. By using polymerase chain reaction amplification of D7S493 and D7S527 markers, we identified the loss of the maternal chromosome 7 with a consequent paternal isodisomy in the clone with dup7q. This rearrangement could be implicated in the progression of the disease by causing (1) nullisomy for a gene or genes located on 7p22-->pter, (2) functional double doses of exclusively paternal expressed genes, and (3) restoration of the effects produced by haploinsufficiency of biparental expressed genes.

Molecular cytogenetic characterization of a familial pericentric inversion 3 associated with short stature.

Short stature refers to the height of an individual which is below expected. The causes are heterogenous and influenced by several genetic and environmental factors. Chromosomal abnormalities are a major cause of diseases and cytogenetic mapping is one of the powerful tools for the identification of novel disease genes. Here we report a three generation family with a heterozygous pericentric inversion of 46, XX, inv(3) (p24.1q26.1) associated with Short stature. Positional cloning strategy was used to physically map the breakpoint regions by Fluorescence in situ hybridization (FISH). Fine mapping was performed with Bacterial Artificial Chromosome (BAC) clones spanning the breakpoint regions. In order to further characterize the breakpoint regions extensive molecular mapping was carried out with the breakpoint spanning BACs which narrowed down the breakpoint region to 2.9 kb and 5.3 kb regions on p and q arm respectively. Although these breakpoints did not disrupt any validated genes, we had identified a novel putative gene in the vicinity of 3q26.1 breakpoint region by in silico analysis. Trying to find the presence of any transcripts of this putative gene we analyzed human total RNA by RT-PCR and identified transcripts containing three new exons confirming the existence of a so far unknown gene close to the 3q breakpoint.