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 DMRT2 promoter. Long range effects on gene expression cannot be excluded so far.

20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

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.

High frequency of mosaicism among patients with neurofibromatosis type 1 (NF1) with microdeletions caused by somatic recombination of the JJAZ1 gene.

Detailed analyses of 20 patients with sporadic neurofibromatosis type 1 (NF1) microdeletions revealed an unexpected high frequency of somatic mosaicism (8/20 [40%]). This proportion of mosaic deletions is much higher than previously anticipated. Of these deletions, 16 were identified by a screen of unselected patients with NF1. None of the eight patients with mosaic deletions exhibited the mental retardation and facial dysmorphism usually associated with NF1 microdeletions. Our study demonstrates the importance of a general screening for NF1 deletions, regardless of a special phenotype, because of a high estimated number of otherwise undetected mosaic NF1 microdeletions. In patients with mosaicism, the proportion of cells with the deletion was 91%-100% in peripheral leukocytes but was much lower (51%-80%) in buccal smears or peripheral skin fibroblasts. Therefore, the analysis of other tissues than blood is recommended, to exclude mosaicism with normal cells in patients with NF1 microdeletions. Furthermore, our study reveals breakpoint heterogeneity. The classic 1.4-Mb deletion was found in 13 patients. These type I deletions encompass 14 genes and have breakpoints in the NF1 low-copy repeats. However, we identified a second major type of NF1 microdeletion, which spans 1.2 Mb and affects 13 genes. This type II deletion was found in 8 (38%) of 21 patients and is mediated by recombination between the JJAZ1 gene and its pseudogene. The JJAZ1 gene, which is completely deleted in patients with type I NF1 microdeletions and is disrupted in deletions of type II, is highly expressed in brain structures associated with learning and memory. Thus, its haploinsufficiency might contribute to mental impairment in patients with constitutional NF1 microdeletions. Conspicuously, seven of the eight mosaic deletions are of type II, whereas only one was a classic type I deletion. Therefore, the JJAZ1 gene is a preferred target of strand exchange during mitotic nonallelic homologous recombination. Although type I NF1 microdeletions occur by interchromosomal recombination during meiosis, our findings imply that type II deletions are mediated by intrachromosomal recombination during mitosis. Thus, NF1 microdeletions acquired during mitotic cell divisions differ from those occurring in meiosis and are caused by different mechanisms.

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.

Alagille syndrome associated with a paracentric inversion 20p12.2p13 disrupting the JAG1 gene.

Mutations in the human gene Jagged1 (JAG1) localized in 20p12 have been recently identified as causal for the anomalies found in patients with Alagille syndrome (AGS). This gene encodes a ligand for the Notch1 transmembrane receptor, which plays a key role in cell-to-cell signaling during differentiation and is conserved from C. elegans to human. We report a paracentric inversion (PAI) of chromosome 20p12.2p13 in an individual with AGS who also had alpha-1-antitrypsin deficiency. To our knowledge, this is the first published case of PAI involving the short arm of chromosome 20. Using FISH, fiberFISH, and molecular studies with a approximately 40 kb cosmid clone encompassing the entire 36 kb JAG1 gene, we demonstrate that the gene was disrupted by the inversion breakpoint between exons 5 and 6. An unusual association between two most common causes of chronic liver disease in childhood, AGS and alpha-1-antitrypsin deficiency, as well as their influence on the proband's abnormal phenotype are discussed.

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.

Localization of Alagille syndrome to 20p11.2-p12 by linkage analysis of a three-generation family.

Alagille syndrome (AGS) or arteriohepatic dysplasia is a rare but well-defined clinical entity that is usually inherited as an autosomal dominant trait. A limited number of patients carry a deletion in chromosome 20p, with 20p11.23-p12.2 as the area of minimal overlap. Recently, a family has been identified in which a balanced translocation with a breakpoint in 20p12 co-segregates with the AGS phenotype. Here, we report a three-generation family with AGS and in which the affected members have a normal karyotype. Linkage analysis was performed with markers from the 20p candidate region. A lod score of Z = 2.96 was obtained with D20S27 at no recombination. Combining D20S27 and D20S61 to a single highly informative locus resulted in a maximum lod score of Z = +3.56 at theta = 0.0. Haplotype analysis positioned AGS between D20S59 and D20S65, markers that define an interval of about 40 cM. Allelic loss was not observed for the tested markers and no abnormalities in the PAX1 candidate gene were detected. These findings demonstrate that the locus on chromosome 20p could be responsible for AGS in cytogenetically normal patients and argues for a general role of this locus in the aetiology of AGS.

Molecular structure and chromosomal mapping of the human homolog of the agouti gene.

The agouti (a) locus in mouse chromosome 2 normally regulates coat color pigmentation. The mouse agouti gene was recently cloned and shown to encode a distinctive 131-amino acid protein with a consensus signal peptide. Here we describe the cloning of the human homolog of the mouse agouti gene using an interspecies DNA-hybridization approach. Sequence analysis revealed that the coding region of the human agouti gene is 85% identical to the mouse gene and has the potential to encode a protein of 132 amino acids with a consensus signal peptide. Chromosomal assignment using somatic-cell-hybrid mapping panels and fluorescence in situ hybridization demonstrated that the human agouti gene maps to chromosome band 20q11.2. This result revealed that the human agouti gene is closely linked to several traits, including a locus called MODY (for maturity onset diabetes of the young) and another region that is associated with the development of myeloid leukemia. Initial expression studies with RNA from several adult human tissues showed that the human agouti gene is expressed in adipose tissue and testis.

Cystatin C (CST3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA), and other members of the cystatin gene family are clustered on chromosome 20p11.2.

The cystatin C gene (CST3) encodes a low-molecular-weight cysteine proteinase inhibitor belonging to family II of the cystatin superfamily and is mutated in cases of hereditary cystatin C amyloid angiopathy (HCCAA). CST3, which along with other family II cystatin genes is a member of the cystatin gene family, has been assigned to chromosome 20. To investigate the genomic organization on chromosome 20, the CST3 gene and related sequences were regionally mapped by fluorescence in situ hybridization (FISH), Southern blot, and pulsed-field gel electrophoresis (PFGE) analysis using the cDNA cystatin C probe C6a and three genomic probes, C3E1, C3E2, and C3E2-2. Probe C3E2-2, which like probe C3E2 is specific for CST3, hybridized to only one HindIII and one XbaI fragment on Southern blots and to a 300-kb BssHII PFGE fragment. FISH with probe C3E2 mapped this locus to chromosome 20p11.2, with an FL-pter value of 0.37 +/- 0.07 on the physical map. Probe C3E1 containing the most conserved cystatin gene exon (exon 1) and its flanking sequences hybridized with more fragments, e.g., to eight XbaI and nine HindIII fragments on conventional Southern blots and to eight SmaI, two BssHII (900 and 300 kb), and two NotI fragments after PFGE. FISH with C3E1 revealed only one single site at 20p11.2 with an FL-pter value of 0.37 +/- 0.04, identical to that obtained with C3E2.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal diagnosis of the Pallister-Killian mosaic aneuploidy syndrome by CVS.

Prenatal cytogenetic analysis at 11 weeks of gestation revealed an abnormal karyotype 47,XX,+mar in all metaphases obtained from a chorionic villi sample after 24 h culture. Karyotyping of amniotic fluid cells in the second trimester showed mosaicism 47,XX,+i(12p)/46,XX with 10% aneuploid cells. The pregnancy was terminated at 20 weeks of gestation on the patient's request. The aborted fetus showed typical manifestations of the Pallister-Killian mosaic aneuploidy syndrome. The identity of the supernumerary isochromosome 12p was proven by LDH isozyme electrophoresis using cultured fibroblasts and by nonradioactive in situ hybridization using a biotinylated set of chromosome 12-specific DNA probes.

G protein Gs alpha (GNAS 1), the probable candidate gene for Albright hereditary osteodystrophy, is assigned to human chromosome 20q12-q13.2.

Guanine nucleotide-binding proteins, also known as G proteins, mediate intracellular responses to a wide variety of extracellular stimuli. A variety of genes that specify the synthesis of the components of guanine nucleotide proteins have been identified. One of these proteins, termed Gs alpha (GNAS1), is the G protein component of the olfactory signal transduction cascade. Mutations in the GNAS1 gene leading to Gs alpha protein deficiency are known to be associated with pseudohypoparathyroidism Ia (Albright hereditary osteodystrophy) and certain pituitary tumors with acromegaly. Studies on the human--mouse somatic cell hybrids provisionally assigned this gene to chromosome 20. We have now confirmed this localization on chromosome 20 and regionally assigned the GNAS1 gene to 20q12-q13.2 by in situ hybridization.

Down syndrome at birth not detected by first-trimester chorionic villus sampling.

A case of a false-negative first-trimester diagnosis following chorionic villus sampling is reported that ended with the birth of a child with Down syndrome. Chromosome analysis of 30 metaphases from 24 h-cultured chorionic villi obtained in week 12 of gestation showed a normal chromosome constitution. However, the newborn showed manifestations of Down syndrome, and 99 of 100 metaphases analysed from cultured lymphocytes showed 47, XY, + 21. The remaining metaphase was normal (46, XY).

The predictive value of chorionic villus histology for identifying chromosomally normal and abnormal spontaneous abortions.

To evaluate the significance of placental histology, a collaborative histological and cytogenetic study on the products of 123 spontaneous abortions of 6-19 week pregnancies was performed. From each sample, 2-3 g was dissected randomly and analysed histologically with no prior knowledge of the karyotype. Chromosomes were prepared from the remaining chorionic villi after an overnight incubation in culture medium; they were analysed by Q- or G-banding. The frequency and type of chromosome anomalies detected are comparable to those seen in other studies. Altogether, 49.6% were chromosomally abnormal, trisomies predominating (49.2%), followed by polyploidy (27.9%) and monosomy X (14.5%). The histological classification failed for technical reasons in 11 cases (8.9%). To classify the remaining cases, 18 different histological criteria were considered independently by two pathologists. A definite diagnosis was not possible for 27 abortions (22.0%), and 50 were classified as being chromosomally abnormal. This proved to be correct in 36 (72.0%), but incorrect in as many as 14 (28.0%). No evidence for an abnormal karyotype was found histologically in samples from 35 abortuses. Nevertheless, 9 of them (25.7%) had an abnormal karyotype. The predictive value of chorionic villus histology seems to be inadequate, as only 62 of 112 samples were correctly classified by histology (55.4%). The value differs little from the a priori probability of an abnormal or normal karyotype in abortuses of corresponding gestational ages.

MECHANISMS OF NONDISJUNCTION AND IMPLICATIONS FOR TESTING ANEUGENS.

THE HUMAN SPECIES IS AFFECTED BY A HIGH FREQUENCY OF CONSTITUTIONAL ANEUPLOIDY WHICH IS MAINLY DERIVED FROM NONDISJUNCTION DURING PARENTAL MEIOSIS AND PREFERENTIALLY DURING MATERNAL MEIOSIS I. IN UNRAVELLING THE MECHANISMS INVOLVED WE STUDIED VARIOUS CONDITIONS CAUSING AND ASSOCIATED WITH NONDISJUNCTION IN OOCYTES FROM DJUNGARIAN HAMSTERS. CONSIDERING OUR OBSERVATIONS WE SUGGEST THAT THE ORDERLY SEGREGATION OF CHROMOSOMES AT MEIOSIS APPEARS TO RESULT FROM A CASCADE OF DIFFERENTIATION PROCESSES REQUIRING THE INTERACTION OF AN EXTRA-, TRANS- AND INTRACELLULAR SIGNALLING. FAILURES MAY OCCUR AT ANY LEVEL ALTERING E.G.: (1) THE CONTROL OF FOLLICULAR MATURATION/FUNCTION (OR GENERALLY THE FUNCTION OF THE SOMATIC COMPARTMENT WITHIN THE GONADS) (2) THE SIGNALLING BETWEEN SOMA AND MEIOCYTE (3) THE FUNCTION OF THE CYTOPLASM AND CYTOPLASMIC ORGANELLES SUCH AS THE SPINDLE AND MITOCHONDRIA (BEERMANN ET AL. 1988) (3) THE RATE OF MEIOSIS (5) CHROMOSOME BEHAVIOR AND THEIR INTERACTION WITH MICROTUBULI. NONDISJUNCTION APPEARS TO BE ONE CONSEQUENCE OF A DISTURBED CONTROL OF MEIOCYTE PROLIFERATION. THE CAUSES MIGHT, IN ANALOGY, BE AS DIVERSE AS THOSE LEADING TO NEOPLASTIC GROWTH OF SOMATIC CELLS. NOTWITHSTANDING THE NECESSITY FOR SIMPLIFICATION IN MUTAGENICITY TESTING ONE HAS TO REMIND THAT THE MAJORITY OF TRISOMIES ORIGINATE FROM A MEIOSIS I ERROR. SO FAR WE CAN NOT BE SURE THAT SOMATIC CELL TESTING MAY INDICATE PUTATIVE EVENTS DURING THE ABOVE DESCRIBED COMPLICATED INTERACTIVE MATURATION PROCESS OF GERM CELLS.

Partial deletion of 4p in fetal cells not present in chorionic villi.

A case of a prenatal diagnosis at the second trimester is presented showing a normal karyotype in 12 metaphases from chorionic villi. In all cultured amniotic cells, however, and also in all fetal fibroblasts analyzed after abortion a structural anomaly (46,XY;del 4(pter----p15.2) was detected. Prenatal diagnosis was performed because of intrauterine growth retardation, cleft lip and esophagus atresia by ultrasound. The fetal stigmata are compatible with the Wolf Hirschhorn syndrome. We conclude that amniocentesis may be indicated notwithstanding a normal CV-diagnosis in those rare pregnancies with a characteristically abnormal ultrasound.

Allocation of a transgenic c-myc integration site to mouse chromosome 8B3-C1.

A recombinant plasmid containing the mouse c-myc gene was injected into mouse pronuclei. The transgenic line 478 contains about 100 copies of the transgene integrated into one chromosome site. By in situ hybridization, the integration site was localized to chromosome 8B3-C1.