X/autosome translocation in three generations ascertained through an infant with trisomy 16p due to failure of spreading of X-inactivation.

We report on a reciprocal translocation t(X;16)(q28;p12) detected in a newborn girl with clinical manifestations of partial trisomy 16p. A balanced translocation was found in the mother and in the maternal grandmother. Replication studies on lymphocytes and fibroblasts showed nonrandom X-inactivation in both the patient and her mother. In the mother, the derivative X (der(X)) was active, whereas the normal X was late replicating. In contrast, in the patient the der(X) was late replicating, and there was no spreading of X-inactivation onto the autosomal segment, thus giving an explanation for the full clinical picture of partial trisomy 16p.

XX-agonadism in a fetus with multiple dysraphic lesions: a new syndrome.

We report on a 19-week-old fetus with a 46,XX karyotype, normal female external genitalia, complete gonadal agenesis, large encephalocele, spina bifida, and omphalocele. We postulate a new syndrome. Hitherto no consistent malformation patterns have been observed in agonadism patients. True agonadism, including even the unusual finding of an XX gonosomal status, is obviously not as rare as suggested.

Genomic structure and mutation screening of the E2F4 gene in human tumors.

E2F4, a member of the E2F family of transcription factors, is abundant in non-proliferating and differentiated cells where it plays an important role in the suppression of proliferation-associated genes. The E2F4 gene spans 6 kb and has 10 exons. It contains a serine (CAG) repeat tract in exon 7, which is unstable in gastrointestinal tumors. To further investigate a possible role of this gene in tumorigenesis we performed mutational analysis and expression studies in different tumors. Primary human tumor tissue of the stomach, colon, breast and lung (28), metastatic tumors of the colon (3) and small cell lung tumor cell lines (18) were screened for somatic mutations in the coding region of E2F4. No mutation was found. Microsatellite instability of the CAG repeat, however, was documented in primary stomach and colon tumors. Northern blot analysis revealed upregulated E2F4 transcript levels in tumor cell lines. Our data suggest that a direct involvement of E2F4 in tumorigenesis is unlikely, although increased E2F4 expression may be associated with human cancer.

How does inactivation change timing of replication in the human X chromosome?

The kinetics of replication of the inactive (late replicating) X chromosome (LRX) were studied in karyotypically normal lymphocytes and human amniotic fluid cells. Both cell types were successively pulse labeled with 1-h or 1/2-h thymidine pulses in an otherwise BrdU-substituted S phase after partial synchronization of the cultures at G1/S. For the first time with this technique, the entire sequence of replication was analyzed for the LRX from the beginning to the end of the S phase, with special reference to mid S (R-band to G-band transition replication). The inactive X is the last chromosome of the metaphase to start replication, with a delay of 1 or 2 h, after which time a thymidine pulse results in R-type patterns. In mid S, the inactive X is the first chromosome to switch to G-type replication (without overlapping of both types and without any detectable replication pause). Until the end of S, a thymidine pulse results in G-type patterns. To rule out artifacts that might arise by the synchronization of cultures in these experiments, controls were carried out with BrdU pulses and the BrdU antibody technique without synchronization. In the course of replication, no fundamental difference was seen between the two different cell types examined. In contrast to studies using continuous labeling, this study did not reveal an interindividual difference of replication kinetics in the LRXs of the seven individuals studied; thus it is concluded that the inactive X chromosome shows only one characteristic course of replication.

Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability.

We have analysed the mitotic behaviour of expanded CTG repeats in somatic tissues and cultured somatic cells from myotonic dystrophy (DM) fetuses using indirect and direct methods. Heterogeneity of expansions between fetal tissues was demonstrated in a 16 week old fetus whereas there was no evidence for such a somatic heterogeneity in a 13 week old fetus. Dilution plating of cultured cells from an adult patient and a fetus resulted in isolation of clones showing single expanded restriction fragments when the donor showed a heterogeneous smear of expansions or a single expanded fragment. During proliferation in vitro to 45 doublings, DM cells experienced highly synchronous further repeat expansion which first became evident at approximately 15 cell generations and reached a plateau of maximum expansion at approximately 200 days. When mathematically expressed as a function of culture time the dynamics of expansion of restriction fragments followed a sigmoid curve. This unstable behaviour of CTG repeat expansions in DM was compared to the mitotically stable patterns of full mutation in fragile X fetal tissues and led to the hypothesis that methylation of CpGs within the repeat sequence is a stabilizing factor of largely expanded CGG and GCC repeats allowing for efficient methyl-directed strand-specific DNA mismatch repair.

The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat.

Using methylation-sensitive restriction enzymes, we characterized the methylation pattern on the 5' side of the CTG repeat in the DMPK gene of normal individuals and of patients affected with myotonic dystrophy, showing expansions of the repetitive sequence. The gene segment analyzed corresponds to the genomic SacI-HindIII fragment carrying exons 11-15. There is constitutive methylation in intron 12 at restriction sites of SacII and HhaI, localized 1,159-1,232 bp upstream of the CTG repeat, whereas most, if not all, of the other sites of SacII, HhaI, and HpaII in this region are unmethylated, in normal individuals and most of the patients. In a number of young and severely affected patients, however, complete methylation of these restriction sites was found in the mutated allele. In most of these patients, the onset of the disease was congenital. Preliminary in vivo footprinting data gave evidence for protein-DNA contact in normal genes at an Sp1 consensus binding site upstream of the CTG repeat and for a significant reduction of this interaction in cells with a hypermethylated DMPK gene.

Developmental and tissue-specific expression of the Q5k gene.

Expression of the Q5k gene was examined by northern blot analysis and polymerase chain reaction (PCR) in the AKR mouse and various cell lines, each of the H-2k haplotype. Our results show that Q5k mRNA is present during the whole postimplantational development of the AKR embryo/fetus (gestation day 6 to 15). In the juvenile mouse (week 2 to 4) transcription of the Q5k gene persisted in all organs examined. In contrast, in the adult animal expression of the Q5k gene was limited to the thymus and uterus of the pregnant mouse. Upon malignant transformation, the amount of Q5k-specific mRNA increased dramatically in thymus and could also be observed in the spleen of thymoma bearing animals. Expression of the Q5k gene was also detectable in several transformed mouse cell lines. Mitogen stimulation or treatment with cytokines induced Q5k expression in primary spleen cell cultures. A possible explanation for the tissue-restricted expression in the adult AKR mouse is discussed.

Organization of the AKR Qa region: structure of a divergent class I sequence, Q5k.

We established the organization of the AKR Qa region and determined the sequence of the Q4 and Q5 genes. Restriction mapping and genomic Southern blot analysis revealed that the AKR strain codes for only three H-2K homologous genes in this region. The AKR Q5 gene is not homologous to the Q5 gene of the C57BL strain, but is presumably allelic to the Q5 gene isolated from Balb/c. The organization and structure of the AKR Qa family is virtually identical to the Qa genes of the C3H mouse. The AKR Q5 gene, in contrast to other H-2K homologous Qa region genes, codes for a typical transmembrane region, and upon transfection into BHK cells, a 1.6 kb Q5 transcript is detected.

Multipoint mapping of the central core disease locus.

A linkage analysis with 12 DNA markers from proximal 19q was performed in eight families with central core disease (CCO). Two-point analysis gave a peak lod score of Z = 4.95 at theta = 0.00 for the anonymous marker D19S190 and of Z = 2.53 at theta = 0.00 for the ryanodine receptor (RYR1) candidate gene. Multipoint linkage data place the CCO locus at 19q13.1, flanked proximally by D19S191/D19S28 and distally by D19S47. This map location includes the RYR1 gene. The results of the linkage study present no evidence for genetic heterogeneity of CCO.

Characterization of FMR1 promoter elements by in vivo-footprinting analysis.

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.