Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder which maps to chromosome 4qter, distal to the D4S139 locus. The cosmid clone 13E, isolated in a search for homeobox genes, was subsequently mapped to 4q35, also distal to D4S139. A subclone, p13E-11, detects in normal individuals a polymorphic EcoRI fragment usually larger than 28 kilobases (kb). Surprisingly, using the same probe we detected de novo DNA rearrangements, characterized by shorter EcoRI fragments (14-28 kb), in 5 out of 6 new FSHD cases. In 10 Dutch families analysed, a specific shorter fragment between 14-28 kb cosegregates with FSHD. Both observations indicate that FSHD is caused by independent de novo DNA rearrangements in the EcoRI fragment detected by p13E-11.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Genomic imbalances in mental retardation.

INTRODUCTION: It has been estimated that cytogenetically visible rearrangements are present in approximately 1% of newborns. These chromosomal changes can cause a wide range of deleterious developmental effects, including mental retardation (MR). It is assumed that many other cases exist where the cause is a submicroscopic deletion or duplication. To facilitate the detection of such cases, different techniques have been developed, which have differing efficiency as to the number of loci and patients that can be tested. METHODS: We implemented multiplex amplifiable probe hybridisation (MAPH) to test areas known to be rearranged in MR patients (for example, subtelomeric/pericentromeric regions and those affected in microdeletion syndromes) and to look for new regions that might be related to MR. RESULTS: In this study, over 30 000 screens for duplications and deletions were carried out; 162 different loci tested in each of 188 developmentally delayed patients. The analysis resulted in the detection of 19 rearrangements, of which approximately 65% would not have been detected by conventional cytogenetic analysis. A significant fraction (46%) of the rearrangements found were interstitial, despite the fact that only a limited number of these loci have so far been tested. DISCUSSION: Our results strengthen the arguments for whole genome screening within this population, as it can be assumed that many more interstitial rearrangements would be detected. The strengths of MAPH for this analysis are the simplicity, the high throughput potential, and the high resolution of analysis. This combination should help in the future identification of the specific genes that are responsible for MR.

Genes homologous to the autosomal dominant polycystic kidney disease genes (PKD1 and PKD2).

Autosomal Dominant Polycystic Kidney Disease (ADPKD), a common inherited disease leading to progressive renal failure, can be caused by a mutation in either the PKD1 or PKD2 gene. Both genes encode for putative transmembrane proteins, polycystin-1 and polycystin-2, which show significant homology to each other and are believed to interact at their carboxy termini. To identify genes that code for related proteins we searched for homologous sequences in several databases and identified one partial cDNA and two genomic sequences with significant homology to both polycystin-1 and - 2. Further analysis revealed one novel gene, PKD2L2, located on chromosome band 5q31, and two recently described genes, PKD2L and PKDREJ, located on chromosome bands 10q31 and 22q13.3, respectively. PKD2L2 and PKD2L, which encode proteins of 613 and 805 amino acids, are approximately 65% similar to polycystin-2. The third gene, PKDREJ, encodes a putative 2253 amino acid protein and shows about 35% similarity to both polycystin-1 and polycystin-2. For all the genes expression was found in testis. Additional expression of PKD2L was observed in retina, brain, liver and spleen by RT-PCR. Analyses of five ADPKD families without clear linkage to either the PKD1 or PKD2 locus showed no linkage to any of the novel loci, excluding these genes as the cause of ADPKD in these families. Although these genes may not be involved in renal cystic diseases, their striking homology to PKD2 and PKD1 implies similar roles and may contribute to elucidating the function of both polycystin-1 and polycystin-2.

Submicroscopic deletion of chromosome region 16p13.3 in a Japanese patient with Rubinstein-Taybi syndrome.

In a series of 25 Japanese patients with Rubinstein-Taybi syndrome, we screened, by high-resolution GTG banding and fluorescence in situ hybridization of a cosmid probe (RT1, D16S237), for microdeletions associated with this syndrome. In one patient, a microdeletion was demonstrated by in situ hybridization, but none were detected by high-resolution banding.

Rubinstein-Taybi syndrome caused by a De Novo reciprocal translocation t(2;16)(q36.3;p13.3).

Rubinstein-Taybi syndrome (RTS) is a multiple congenital anomalies and mental retardation syndrome characterized by facial abnormalities, broad thumbs, and broad big toes. We have shown previously that disruption of the human CREB-binding protein (CBP) gene, either by gross chromosomal rearrangements or by point mutations, leads to RTS. Translocations and inversions involving chromosome band 16p13.3 form the minority of CBP mutations, whereas microdeletions occur more frequently (approximately 10%). Breakpoints of six translocations and inversions in RTS patients described thus far were found clustered in a 13-kb intronic region at the 5' end of the CBP gene and could theoretically only result in proteins containing the extreme N-terminal region of CBP. In contrast, in one patient with a translocation t(2;16)(q36.3;p13.3) we show by using fiber FISH and Southern blot analysis that the chromosome 16 breakpoint lies about 100 kb downstream of this breakpoint cluster. In this patient, Western blot analysis of extracts prepared from lymphoblasts showed both a normal and an abnormal shorter protein lacking the C-terminal domain, indicating expression of both the normal and the mutant allele. The results suggest that the loss of C-terminal domains of CBP is sufficient to cause RTS. Furthermore, these data indicate the potential utility of Western blot analysis as an inexpensive and fast approach for screening RTS mutations.

Chromosomal localization of the 5-HT1F receptor gene: no evidence for involvement in response to sumatriptan in migraine patients.

The 5-HT1F receptor, which is present in both human vascular and neuronal tissue, may mediate the therapeutic effect and/or side-effects of sumatriptan. We investigated the chromosomal localization of the 5-HT1F receptor gene and the relation between eventually existing polymorphisms and the clinical response to sumatriptan in migraine patients. The 5-HT1F receptor gene was localized using a monochromosomal mapping panel, followed by a radiation-reduced hybrid mapping and fluorescent in situ hybridization. The results of these techniques show that the 5-HT1F receptor gene is localized at 3p12. We investigated the presence of polymorphisms by single strand conformation polymorphism analysis in 14 migraine patients who consistently responded well to sumatriptan, 12 patients who consistently experienced recurrence of the headache after initial relief, 12 patients with no response to sumatriptan, and in 13 patients who consistently experienced chest symptoms after use of sumatriptan. No polymorphisms were detected in any of the patients. We therefore conclude that genetic diversity of the 5-HT1F receptor gene is most probably not responsible for the variable clinical response to sumatriptan.

Rubinstein-Taybi syndrome with deletions of FISH probe RT1 at 16p13.3: two UK patients.

We report two patients with Rubinstein-Taybi syndrome out of a total of 16 tested who have a deletion of the region visualised by the cosmid probe RT1. These results further confirm this as a locus for Rubinstein-Taybi syndrome.

A case of de novo interstitial deletion of chromosome 5(q33q34).

The present paper describes a girl with a small de novo deletion of chromosome 5(q33q34). Fluorescence in situ hybridisation with locus specific probes was used to define the extent of this deletion. Clinical features in this patient are microcephaly, dysmorphic facial features such as epicanthus, small biparietal distance and retrognathia, four-finger lines on both hands and mild mental retardation.

CBFB/MYH11 fusion in a patient with AML-M4Eo and cytogenetically normal chromosomes 16.

We present a unique case of acute myeloid leukemia M4Eo (AML-M4Eo) with a CBFB/MYH11 fusion transcript and a trisomy 22, but in whom cytogenetic analyses did not disclose an inv(16). Fluorescence in situ hybridization (FISH) analysis with chromosome arm-specific painting probes as well as with the c40 and c36 cosmids also revealed no evidence for an inv(16), whereas the application of locus-specific probes confirmed the presence of a masked inv(16). The results of our comprehensive FISH investigations indicate that the events leading to this masked inv(16) were complex and concurred with deletions on both the long and short arms. The most likely explanation for the formation of the relevant CBFB/MYH11 fusion is an insertion of parts of the MYH11 into the CBFB gene, although it is also possible that it was formed by a double inversion.

Fish mapping of 250 cosmid and 26 YAC clones to chromosome 4 with special emphasis on the FSHD region at 4q35.

Facioscapulohumeral muscular dystrophy (FSHD) is located on chromosome 4q35, close to the telomere. FSHD patients carry deletions within a cluster of tandemly repeated DNA. Although expression of a functional FSHD gene will be altered in patients, the sequence itself may be unaffected by this deletion. Hence, the FSHD gene could lie outside of the deleted region. This study employs fluorescent in situ hybridization using chromosome 4-specific cosmid and YAC clones to rapidly saturate chromosome 4 with new markers. Some 250 cosmids and 26 YACs were regionally mapped, of which 5 YACs and 55 cosmids mapped to the distal portion of 4q. Only one of these clones (D4S1454) mapped telomerically to a translocation breakpoint specified by D4S187. Using two-color interphase mapping, the following marker order was obtained: Cen-D4S187-D4S1454-HSPCAL2-D4S163-D4S139-D4F35S1. Absence of additional markers mapping distal to D4F35S1 indicates that the linkage group containing the FSHD gene lies extremely close to the 4q telomere.

Fish mapping of 250 cosmid and 26 YAC clones to chromosome 4 with special emphasis on the FSHD region at 4q35.

Facioscapulohumeral muscular dystrophy (FSHD) is located on chromosome 4q35, close to the telomere. FSHD patients carry deletions within a cluster of tandemly repeated DNA. Although expression of a functional FSHD gene will be altered in patients, the sequence itself may be unaffected by this deletion. Hence, the FSHD gene could lie outside of the deleted region. This study employs fluorescent in situ hybridization using chromosome 4-specific cosmid and YAC clones to rapidly saturate chromosome 4 with new markers. Some 250 cosmids and 26 YACs were regionally mapped, of which 5 YACs and 55 cosmids mapped to the distal portion of 4q. Only one of these clones (D4S1454) mapped telomerically to a translocation breakpoint specified by D4S187. Using two-color interphase mapping, the following marker order was obtained: Cen-D4S187-D4S1454-HSPCAL2-D4S163-D4S139-+ ++D4F35S1. Absence of additional markers mapping distal to D4F35S1 indicates that the linkage group containing the FSHD gene lies extremely close to the 4q telomere.

Two-colour FISH detection of the inv(16) in interphase nuclei of patients with acute myeloid leukaemia.

The inv(16)(p13q22) and t(16;16)(p13;q22) in acute myeloid leukaemia are associated with a relatively good prognosis but are difficult to detect using classic cytogenetics. We have designed a two-colour fluorescence in situ hybridization approach that uses two DNA probes that map close to and on either side of the inv(16) p-arm breakpoint region. This new strategy clearly detected the inv(16)(p13q22)/t(16;16)(p13;q22) on both metaphase chromosomes and in interphase nuclei, even when they are of poor quality. This procedure also detected the inv(16) in cases with an additional deletion of sequences proximal to the 16p-arm breakpoint which is present in 20% of all cases.

Cloning of Mycobacterium bovis BCG DNA and expression of antigens in Escherichia coli.

A gene bank of Mycobacterium bovis BCG DNA in Escherichia coli was constructed by cloning Sau3A-cleaved mycobacterium DNA fragments into the lambda vector EMBL3. The expression of mycobacterial antigens was analyzed by Western blotting with hyperimmune rabbit sera. Among 770 clones tested, several were found that produced various mycobacterial antigens in low amounts, with concentrations generally close to the detection limit. One particular clone was chosen for further investigation. This clone produced a 64-kilodalton (kDa) antigen. By placing the lambda promoter PL in front of the structural gene of this antigen, an overproducing E. coli strain was obtained. Rocket-line immunoelectrophoresis experiments showed that antigens cross-reacting with the 64-kDa protein are present in a wide variety of mycobacteria and also in so-called purified protein derivatives which are routinely used for skin tests. Preliminary experiments indicate the presence of antibodies against the 64-kDa antigen in sera from tuberculosis patients.

Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenne Muscular Dystrophy gene. All exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single exon trap product rather than small single exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of exon trap material.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon.

Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer cell lines that cover a range of drug-resistance levels, and we have analyzed the MRP amplicon. In the SW-1573-derived, weakly resistant cell line 30.3M, MRP mRNA is elevated 3-fold in the absence of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due to transcriptional activation. In the highly resistant GLC4/ADR and COR-L23/R cells, MRP gene amplification predominates, whereas in the moderately resistant MOR/R cells, gene amplification is combined with a mechanism resulting in an additional increase in the level of MRP mRNA. Fluorescence in situ hybridization shows that, in the GLC4/ADR cells, amplified MRP sequences are present both in double minute chromosomes (DM) and in homogeneously staining regions (HSR). By pulsed-field gel electrophoresis we show that the MRP-containing DM are 1 Mb in length. Chromosome-16-specific repetitive sequences adjacent to the MRP gene are also present in the DM and HSR, compatible with the involvement of these sequences in recombination events underlying MRP gene amplification. Our results show that low levels of drug resistance may arise by transcriptional activation of the MRP gene, whereas at high levels of drug resistance amplification of the MRP gene predominates, possibly facilitated by the presence of recombination-prone sequences.

Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP.

The Rubinstein-Taybi syndrome (RTS) is a well-defined syndrome with facial abnormalities, broad thumbs, broad big toes and mental retardation as the main clinical features. Many patients with RTS have been shown to have breakpoints in, and microdeletions of, chromosome 16p13.3 (refs 4-8). Here we report that all these breakpoints are restricted to a region that contains the gene for the human CREB binding protein (CBP), a nuclear protein participating as a co-activator in cyclic-AMP-regulated gene expression. We show that RTS results not only from gross chromosomal rearrangements of chromosome 16p, but also from point mutations in the CBP gene itself. Because the patients are heterozygous for the mutations, we propose that the loss of one functional copy of the CBP gene underlies the developmental abnormalities in RTS and possibly the propensity for malignancy.

Molecular, cytogenetic, and phenotypic studies of a constitutional reciprocal translocation t(5;10)(q22;q25) responsible for familial adenomatous polyposis in a Dutch pedigree.

Familial adenomatous polyposis (FAP) is an inherited predisposition to colorectal cancer caused by germline mutations in the adenomatous polyposis coli (APC) gene located on chromosome segment 5q21-q22. We detected a germline rearrangement of the APC gene in a Dutch FAP family by screening genomic DNA samples with APC cDNA probes. Subsequent molecular and cytogenetic studies revealed a constitutional reciprocal translocation t(5;10)(q22;q25) that resulted in the disruption of the APC gene. Southern blot and polymorphic marker analysis indicated that part of the APC gene had been deleted. Analysis of the APC protein product indicated that the translocation breakpoint did not lead to the formation of a detectable truncated APC protein but apparently resulted in a null allele. Evaluation of the clinical phenotypes in the patients suggested that they exhibited features of an unusual form of FAP characterized by a slightly delayed age of onset of colorectal cancer and a reduced number of colorectal polyps. The latter were mainly sessile and were located predominantly in the proximal colon. To our knowledge, this is the first description of FAP caused by a reciprocal translocation disrupting the APC gene.