Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A-->G, which predicts amino acid substitution K194E, and 982T-->C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R-->H and 304R-->Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain, in contrast to the p63 mutations responsible for EEC syndrome, which reside in amino acid residues that directly interact with the DNA.

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Hypochondroplasia is a genetic disorder of disproportionate short stature. Linkage analysis provisionally placed hypochondroplasia in the chromosome 4p 16.3 region, a location to which the FGFR3 gene has been mapped. The genotyping of a three-generation family showed no recombinants between the hypochondroplasia phenotype and three highly polymorphic markers flanking the FGFR3 gene. Mutation analysis was performed by RT-PCR and direct sequencing. Primers covering most of the coding sequence of the FGFR3 gene were used for RT-PCR of FGFR3 mRNA and PCR amplification of genomic DNA. A C-->A transversion was detected in nucleotide 1659 predicting an N540K substitution in exon 11 which encodes part of the TK1 domain. The same mutation was found in an individual suspected to be an achondroplasia/hypochondroplasia compound phenotype and affected individuals from three other unrelated families. A second mutation, a C-->G transversion, also in nucleotide 1659 was detected in all affected individuals of another family. The latter also predicts an N540K substitution. These findings establish that a common mutation in the FGFR3 gene underlies hypochondroplasia.

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope s robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope s based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Regulation of transcription of the human insulin gene appears to involve a series of DNA sequences in the 5' region. Hypersensitivity to DNA structural probes has previously been demonstrated in regulatory regions of cloned genomic DNA fragments, and been correlated with gene activity. To investigate the structure of the DNA in the human insulin gene, bromoacetaldehyde and S1 nuclease were reacted with a supercoiled plasmid containing a 5kb genomic insulin fragment. Both probes revealed the human insulin gene linked polymorphic region (ILPR), a region (-363) upstream of the transcriptional start site which contains multiple repeats of a 14-15mer oligonucleotide with the consensus sequence ACAGGGGT(G/C)(T/C)GGGG, as the major hypersensitive site. Fine mapping and electron microscopic analysis both show a very different behaviour of the two DNA strands in the region of the ILPR and suggest the G-rich strand may be adopting a highly structured conformation with the complementary strand remaining largely single stranded.

SUMMARY Various developmental abnormalities can give rise to the clinical syndrome of autism, and some are due to chromosomal anomalies. One syndrome has been identified in which behavioural disorder is associated with the clinical features of epilepsy and ataxia, and with the chromosomal anomaly of an extra marker chromosome containing a duplication of 15q11‐13. The authors report a boy with autism, epilepsy, ataxia and an interstitial duplication of 15q, in whom molecular analysis reveals duplication of the GABRA5 and GABRB3 genes on the maternally derived chromosome. RÉSUMÉ Duplication de la région 15q11‐13 chez un patient présentant un tableau associant autisme, épilepsie et ataxie Des anomalies développcmentales variées peuvent conduire à un syndrome clinique ďautisme, et quelques unes peuvent être liees à des anomalies chromosomiques. Un syndrome a été identifié avec association ďun trouble comportemental, des caractéristiques cliniques ďune épilepsie et ďune ataxic, ainsi que ďune anomalie chromosomique ďun chromosome marqué contenant une duplication de 15q11 ‐ 13. Les auteurs décrivent le cas ďun garcon présentant un autisme, une épilepsie, une ataxie et une duplication interstitielle de 15q chez qui ľanalyse moléculaire révéla la duplication des gènes GABRA5 et GABRB3 dans un chromosome ďorigine maternelle. ZUSAMMENFASSUNG Duplikation von 15q11‐13 bei einetn Patienten mil dem Syndrom Autismus, Epilepsie und Ataxie Verschiedene Entwicklungsstörungen können das klinische Syndrom des Autismus auslösen, einige davon beruhen auf chromosomalen Veränderungen. Es ist ein Syndrom beschrieben worden, bei dem Verhaltensstörungen mit klinischen Befunden der Epilepsie und Ataxie und der Chromosomen‐anomalie eines extra Marker Chromosoms, das eine Duplikation von 15q 11 ‐ 13 enthält, verbunden sind. Die Autoren berichten uber einen Jungen mit Autismus, Epilepsie, Ataxie und einer imerstitiellen Duplikation von 15q, bei dem die Molekularanalyse eine Duplikation von GABRA5 und GABRB3 Genen in dem von der Mutter stammenden Chromosom ergab. Duplication de la region 15q11‐13 en un paciente con síndrome de autismo, epilepsia y ataxia Algunas anomalias del desarrollo pueden dar lugar al síndrome clinico del autismo y algunas se deben a alteraciones cromosómicas. Se ha identificado un sindrome en el que la alteración del comportamiento está asociada a epilepsia y ataxia y una anomalia cromosómica con un contenido cromosòmico marcador extra, con una duplicación de 15q11–13. Los autores aportan el caso de un niflo con autismo, epilepsia y ataxia y una duplicación intersticial de 15q en el cual el análisis molecular reveló una duplicación de los genes del GABRA5 y del GABRB3 en el cromosoma de origen materno.

The capacity of six sequences with different numbers and orientations of APT pairs flanked by alternating C'G pairs to adopt left-handed structures was evaluated in recombinant plasmids. A series of synthetic oligodeoxynucleotides were cloned into the BamHI site of pRW790, a small plasmid (=%2 kilobases) prepared especially for conformational studies of this type. Supercoil relaxation studies by twodimensional gel electrophoresis on topoisomers of each plasmid revealed the energetics and structures of the left-handed helices. Also, the presence of supercoil-induced altered DNA conformations within the inserts of topoisomer populations of the plasmids was detected by reaction with S1 nuclease followed by restriction mapping of the cleavage sites. We conclude that consecutive TEA base pairs, whether alternating (TATA) or contiguous (TTTT), can adopt a left-handed conformation (presumably Z) when flanked by reasonably short runs of alternating (C-G). (n = 3-5).Thus, these results substantially broaden the range of DNA sequences that can adopt left-handed Z conformations.