Construction and analysis of a sequence-ready map in 4q25: Rieger syndrome can be caused by haploinsufficiency of RIEG, but also by chromosome breaks approximately 90 kb upstream of this gene.

The autosomal dominant disorder Rieger syndrome (RIEG) shows genetic heterogeneity and has a phenotype characterized by malformations of the anterior segment of the eye, failure of the periumbilical skin to involute, and dental hypoplasia. The main locus for RIEG was mapped to the 4q25-q27 chromosomal segment using a series of cytogenetic abnormalities as well as by genetic linkage to DNA markers. Recently, a bicoid-related homeobox transcription factor gene called RIEG has been cloned, characterized, and proven to cause the 4q25 linked RIEG. Its mode of action in the pathogenesis of RIEG was not conclusively proven, since most etiological mutations detected in the RIEG sequence caused amino acid substitutions or splice changes in the homeodomain. Through FISH analysis of a 460-kb sequence-ready map (PAC contig) around RIEG that we report in this paper, we demonstrate that the 4q25 linked RIEG disorder can arise from the haploid, whole-gene deletion of RIEG, but also from a translocation break 90 kb upstream from the gene. The data provide conclusive evidence that physical or functional haploinsufficiency of RIEG is the pathogenic mechanism for Rieger syndrome. The map also defines restriction fragments bearing sequences with a potential key regulatory role in the control of homeobox gene expression.

Expand Long PCR for fragile X mutation detection.

Fragile X mutation detection by DNA analysis enables accurate diagnosis of the fragile X syndrome. The mutation involves the expansion of CGG repeats in the FMR1 gene and has been primarily detected by the Southern blotting method. In this study we present a novel, efficient and reliable PCR protocol that is more convenient for routine diagnosis of the fragile X syndrome. This method is based on the use of the Expand Long PCR System, which enables the amplification of normal, premutated and full-mutated alleles, and therefore provides complete CGG repeat analysis of the FMR1 gene. Normal alleles were easily detected by ethidium bromide staining of the agarose gels, suggesting that this assay could be used as a screening test for a large number of referrals. The amplified premutations and full mutations were identified by hybridization with a digoxigenin-labeled 5'-(CGG)5-3' probe, followed by chemiluminescent detection. The accuracy of our Expand Long PCR protocol was confirmed by Southern blot analysis, illustrating that the Expand Long PCR results concur with those of Southern blotting. In this paper we propose a new strategy for molecular diagnosis of the fragile X syndrome in which our Expand Long PCR assay is used as the first screening test for fragile X mutation detection.

Rieger syndrome locus: a new reciprocal translocation t(4;12)(q25;q15) and a deletion del(4)(q25q27) both break between markers D4S2945 and D4S193.

Rieger syndrome (RS) is an autosomal dominant disorder of morphogenesis characterised by malformation of the anterior segment of the eye, dental hypoplasia, and failure of the periumbilical skin to involute. RS has been mapped to the 4q25-q27 chromosomal segment by a series of cytogenetic studies as well as by genetic linkage to DNA markers. It was first localised to chromosome 4q based on an association with a constitutional deletion of 4q23-q27. In this paper we localise the proximal breakpoint of this deletion from the original patient, and we describe a new family with a de novo balanced reciprocal translocation t(4;12)(q25;q15) segregating with full RS in two generations. Using FISH and the P1 artificial chromosomes (PACs) as probes, we have physically localised both the deletion and the translocation breakpoints between genetic markers which are known to be strongly linked to RS. We have mapped both the proximal deletion breakpoint and the translocation breakpoint within a region between two groups of PACs bearing the markers D4S2945 (on the centromeric side) and D4S193 and D4S2940 (on the telomeric side). We believe that these recombinant bacterial clones derived directly from genomic DNA (not subcloned from YACs) will be valuable complementary tools in the efforts to clone the RS gene and to construct a full transcriptional and sequence ready map of this region.

[Eleven years of registration of congenital anomalies in Croatia associated with the EUROCAT international project]. / Jedanaestogodisnje registriranje kongenitalnih anomalija u Hrvatskoj u sklopu medunarodnog projekta EUROCAT.

Results of the eleven-year registration of congenital anomalies in Croatia are presented. Zagreb Registry as a part of international EUROCAT (European Registration of Congenital Anomalies) project covers four regional centers (Varazdin Rijeka, Pula and Koprivnica). The ascertainment, calculation of prevalence rates and statistical methods are based on EUROCAT method of investigation. Mean prevalence rate of 18.86/1000 births (1228 children with congenital anomalies per 65,100 births) was registered in the 1983-1993 period. The most frequently registered anomalies were congenital heart diseases, a heterogenous group of limb defects, oral clefts, central nervous system anomalies and chromosomal aberrations. The most frequent structural anomalies expressed as rates per 1000 births are ventricular septal defect (1.8), cleft lip +/- palate (1.1), atrial septal defect (0.8) polydactyly (0.8) and limb reduction defects (0.5). Statistical analysis shows a fluctuation of Down's syndrome prevalence rates during the monitored period. Statistically significant differences (p < 0.01) in prevalence rates among four regions of Croatia were established. The prevalence rates of marker anomalies (Down's syndrome, polydactyly, oral clefts) are tenfold higher compared to routine statistical data of Republic of Croatia. Acquired experience, collaboration and results obtained in this project represent a good basis for more rational planning of medical care and further investigation of this significant medical and public health problem.

Megalocornea-mental retardation syndrome: report of a new case.

Megalocornea-mental retardation syndrome (MMR) is a rare autosomal recessive disorder presenting with megalocornea, mental and motor retardation, hypotonia, seizures, short stature, and characteristic dysmorphic traits (MIM 249310). We present a new case in order to delineate with more accuracy the typical phenotype.

[Mucolipidosis type III (case report)]. / Mukolipidoza tip III (prikaz bolesnika).

A six-year-old boy with mucolipidosis type III or pseudo Hurler polydystrophy is described. The disease is manifested by multiple progressive joint contractures, especially of fingers, presenting as claw hands. The diagnosis of mucolipidosis was established after exclusion of rheumatoid arthritis and mucopolysaccharidosis. In serum and medium of cultured skin fibroblasts, high catalytic activities of several lysosomal enzymes with strikingly decreased values in fibroblast homogenate were found. In most lysosomal diseases gene mutations cause reduced or absent activity of a specific enzyme. In mucolipidosis type III, a basic biochemical disorder is the absence of mannose-6-phosphate, a marker that enables lysosomal membrane receptors to recognize lysosomal enzymes. The transport of lysosomal enzymes across the lysosomal membrane is therefore defective. These and several other metabolic diseases are thus categorized as the disorders of the lysosomal enzyme transport. A genetic and biochemical heterogeneity, as well as clinical manifestations, are described in more detail.

Cytogenetic analysis in ataxia telangiectasia with malignant lymphoma.

We present the results of cytogenetic analysis in a brother and sister with ataxia telangiectasia (AT), one of whom had malignant T-cell lymphoma. In both children, cytogenetic analysis of phytohemagglutinin (PHA)-stimulated lymphocytes showed chromosomal instability and inv(7) in 10% of the cells examined. The malignant lymphoma was analyzed cytogenetically on slides obtained from short-term culture of the lymph node cells; 64 cells were analyzed. A heterogeneous cell population was noted. Fourteen cells (21.9%) had a normal male karyotype; t(7;14)(p14;q12) and inv(7)(p14q35) were observed in 6.3% and 3.1% of metaphases. Owing to low frequency, these cells are probably a characteristic of the basic disease and have no features of malignant cells. Forty cells (62.5%) had a pseudodiploid karyotype 46,XY,dup(1)(p22p36),del(5)(q33),del(12)(p11), without cytogenetically evident aberrations of chromosomes 7 and 14. The results of these investigations suggest that the cells with rearrangements of chromosomes 1, 5, and 12 are malignant cells and did not originate by transformation of cells with inv(7) and t(7;14).

Application of the monodimensional electrophoresis for the separation of glycosaminoglycans on Cellogel cellulose acetate strip.

A monodimensional electrophoretic method for the separation of glycosaminoglycans on Titan III Zip Zone cellulose acetate plate based on their different electrophoretic mobilities in barium acetate and different solubilities in ethanol was applied to the Chemetron electrophoretic equipment. Improved timing of individual steps of electrophoretic run, additional cooling and pressure must be introduced for optimal separation of glycosaminoglycans mixture (dermatan sulphate, heparan sulphate, hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate and keratan sulphate) resulting in five well separated sharp bands. By all these changes in the original procedure of Hopwood and Harrison, the separation of chondroitin-4-sulphate and chondroitin-6-sulphate was not achieved. The modified procedure on Cellogel strip is suitable for the screening of mucopolysaccharidoses.

[Incidence of congenital anomalies in 2 communities in Croatia before and after the Chernobyl nuclear accident]. / Incidencija kongenitalnih anomalija u dvije opcine SR Hrvatske prije i nakon Cernobilske nuklearne nescrece.

The Institute for Medical Protection of Mothers and Children, being regional centre of European registry of congenital malformations (EUROCAT) since 1982, registers congenital anomalies in municipals of Varazdin and Rijeka. Following the nuclear disaster of Chernobyl, there were numerous articles published mainly in daily newspapers, pointing to the increased number of malformations, particularly to Down's syndrome, due to additional irradiation imposed on population. Through this study we wanted to find out whether in Varazdin and Rijeka, following the Chernobyl's accident, there has been any increase of congenital anomalies and whether our regional and EUROCAT registry have been adequate to find out genetic effects of small doses of ionizing radiation. The total incidence of registered congenital anomalies in Varazdin and Rijeka in previous four-year period, amounted to 12.97%, while following Chernobyl, it amounted to 12.7%. Not even nine marker malformations, including Down's syndrome, show any statistically significant increased number of malformations, a year after this nuclear accident. In 18 EUROCAT registries, on almost half a million of newly born children and foetuses, conceived before and after May 1, 1986, the frequency of Down's syndrome and congenital malformations of central nervous system and eyes has been compared. There have been no important differences between two compared groups, and the rate of Down's syndrome was 1.26% before, and 0.91% after the accident. Anticipated stochastic genetic effects of measured and estimated additional doses of radiation imposed to our and Western European populations are too small to be found out neither by regional nor by EUROCAT registries.

Cortical heterotopia in Aicardi's syndrome--CT findings.

A case of 5-month-old female infant with Aicardi's syndrome is presented. The main clinical features were severe developmental retardation and intractable epileptic seizures. Ophthalmoscopic examination revealed pathognomonic chorioretinopathy. Ultrasonic examination of the brain detected agenesis of the corpus callosum, whereas CT showed a coexisting malformation of the brain, i.e. cortical heterotopia of the gray matter. Agenesis of the corpus callosum is an entity well-recognized by sonography. However ultrasonography is an insufficient modality for the visualization of cortical heterotopia which is common to all cases of Aicardi's syndrome. Therefore, in cases of suspected Aicardi's syndrome CT is recommended, as it enables the diagnosis of cortical heterotopia.