Five new subjects with ring chromosome 22.

Ring chromosome 22, a rare cytogenetic finding, was first described by Weleber et al. in 1968. Since then approximately 50 patients have been reported in the medical literature. We describe five previously unreported subjects with ring chromosome 22 syndrome, summarize the clinical findings of reported patients from the literature and discuss the involvement of the ring chromosome and clinical outcome. Our subjects demonstrated the prominent features of this syndrome including mental retardation, hypotonia, motor delay, lack of speech, full eyebrows, and large ears. In addition, two of our subjects had central nervous system malformations and regression. The lack of consistent physical abnormalities in our subjects further supports no consistent phenotype manifestations in this cytogenetic syndrome. The variable clinical manifestations seen in ring chromosome 22 subjects may be associated with loss of chromosome 22 sequences near the telomere or attributed to the genetic background of each subject. Similarly, recessive alleles unmasked by the deletion could also contribute to the phenotype.

Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors.

The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure-function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12-13 in human. Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development.

Molecular and cytogenetic analysis of familial Xp deletions.

Five families in which an Xp deletion is segregating and two families in which an X chromosome rearrangement including a deletion of the short arm is segregating were ascertained for study. Normal fertility was seen in all families. Members from 5 of the 7 families manifested short stature (height <5th centile), while normal height was present in two families. Studies of both the FMR-1 and the androgen receptor loci using PCR based X-inactivation analysis demonstrated that in all families analyzed, there is preferential inactivation of one X chromosome. Molecular cytogenetic analysis showed that members of 3 of the 7 families share a common breakpoint in an approximate 2-3 Mb region at Xp22.12, suggesting a possible hotspot for chromatin breakage. Previous genotype-phenotype correlations and deletion mapping have indicated that a gene for stature resides within the pseudoautosomal region in Xp22.33. Our findings indicate that the loss of this region is not always associated with short stature, suggesting that other factors may be involved.

The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4.

TBC1D1 is the founding member of a family of related proteins with homology to tre-2/UPS6, BUB2, and cdc16 and containing the tbc box motif of 180-220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1-->4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1-->4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.

Sex chromosome markers: characterization using fluorescence in situ hybridization and review of the literature.

Fluorescence in situ hybridization (FISH) using biotin labeled X- and Y-chromosome DNA probes was utilized in the analysis of 23 sex chromosome-derived markers. Specimens were obtained through prenatal diagnosis, because of a presumptive diagnosis of Ullrich-Turner syndrome, mental retardation, and minor anomalies or ambiguous genitalia; three were spontaneous abortuses. Twelve markers were derived from the X chromosome and eleven from the Y chromosome; this demonstrates successfully the value and necessity of FISH utilizing DNA probes in the identification of sex chromosome markers. Both fresh and older slides, some of which had been previously G-banded, were used in these determinations. We have also reviewed the literature on sex chromosome markers identified using FISH.

Reciprocal translocation 4;11 with both adjacent-1 segregants viable within a family.

We describe a family carrying a balanced 4;11 translocation in which both adjacent-1 segregants are viable. The proband had an unbalanced karyotype: 46,XY,der(11)t(4;11)(q34.3;q23.1)mat. At 8.5 years of age he showed trigonocephaly, hypertelorism, epicanthal folds, down-slanting palpebral fissures, low-set ears, anteverted nares, down-turned carp-shaped mouth, and bilateral fifth finger clinodactyly. His maternal aunt was also dysmorphic with high-arched palate, short philtrum and mild developmental delay. Her karyotype was 46,XX,der(4)t(4;11)(q34.3;q23.1)pat. Other relatives who likely carried a chromosomally unbalanced segregant were identified from photographs and medical records. We compare the clinical findings in our family with descriptions of other similar karyotypic abnormalities from previous case reports.

Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain.

We have isolated a cDNA encoding a novel human intracytoplasmic tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). In addition, we have cloned and characterized the murine homolog of the human RAFTK cDNA. Comparison of the deduced amino acid sequences of human RAFTK and murine Raftk cDNAs revealed 95% homology, indicating that RAFTK is highly conserved between these species. The RAFTK cDNA clone, encoding a polypeptide of 1009 amino acids, has closest homology (48% identity, 65% similarity) to the focal adhesion kinase (pp125FAK). Comparison of the deduced amino acid sequences also indicates that RAFTK, like pp125FAK, lacks a transmembrane region, myristylation sites, and SH2 and SH3 domains. In addition, like pp125FAK, RAFTK contains a kinase domain flanked by large N-terminal (426 residues) and C-terminal (331 residues) domains, and the C-terminal region contains a predicted proline-rich stretch of residues. In fetal tissues, RAFTK expression was abundant in brain, and low levels were observed in lung and liver. In adult tissues, it was less restricted, indicating that RAFTK expression is developmentally up-regulated. Expression of RAFTK was also observed in human CD34+ marrow cells, primary bone marrow megakaryocytes, platelets, and various areas of brain. The human RAFTK gene was assigned to human chromosome 8 using genomic DNAs from human/rodent somatic cell hybrid lines. The mouse Raftk gene was mapped to chromosome 14, closely linked to gonadotropin-releasing hormone. Using specific antibodies for RAFTK, a approximately 123-kDa protein from the human megakaryocytic CMK cell line was immunoprecipitated. Treatment of the megakaryocytic CMK cells with thrombin caused a rapid induction of tyrosine phosphorylation of RAFTK protein. The structural features of RAFTK suggest that it is a member of the focal adhesion kinase gene family and may participate in signal transduction in human megakaryocytes and brain as well as in other cell types.

Assignment of FGF8 to human chromosome 10q25-q26: mutations in FGF8 may be responsible for some types of acrocephalosyndactyly linked to this region.

Emerging evidence suggests that Fgf8, a recently identified member of the fibroblast growth factor family, plays an important role in outgrowth and patterning of the face, limbs, and central nervous system of the vertebrate embryo. We report the mapping of FGF8 to human chromosome 10q25-q26, using Southern blot analyses of genomic DNAs from rodent/human somatic cell hybrid lines. Apert, Crouzon, Jackson-Weiss, and Pfeiffer syndromes are craniosynostoses genetically linked in part to 10q25-q26 and are associated with point mutations in the extracellular domain of FGFR2. Given the assignment to the same chromosomeal band(s) as FGFR2 and the probable ligand-receptor relationship of the gene products of FGF8 and FGFR2, we hypothesize that some cases of these craniosynostoses linked to 10q25-q26 that do not have mutations in FGFR2 may involve mutations in FGF8.

Evidence for a distinct region causing a cat-like cry in patients with 5p deletions.

The cri-du-chat syndrome is a contiguous gene syndrome that results from a deletion of the short arm of chromosome 5 (5p). Patients present with a cat-like cry at birth, which is usually considered diagnostic of this syndrome. Additional features of the syndrome include failure to thrive, microcephaly, hypertelorism, epicanthal folds, hypotonia, and severe mental retardation. We report on four families in which patients with 5p deletions have only the characteristic cat-like cry, with normal to mildly delayed development. The precise locations of the deletions in each family were determined by FISH using lambda phage and cosmid clones. All of the deletion breakpoints map distal to a chromosomal region that is implicated with the facial features and severe mental and developmental delay in the cri-du-chat syndrome. DNA clones mapping in the chromosomal region associated with the cat-like cry feature will be useful diagnostic tools. They will allow for the distinction between 5p deletions that will result in the severe delay observed in most cri-du-chat syndrome patients and those deletions that result in the isolated cat-like cry feature, which is associated with a better prognosis.

Assignment of the transcription factor GATA4 gene to human chromosome 8 and mouse chromosome 14: Gata4 is a candidate gene for Ds (disorganization).

We report the mapping of the human and mouse genes for transcription factor GATA-4, a newly identified member of DNA-binding proteins involved in lineage determination. The human GATA4 gene was assigned to the short arm of human chromosome 8 using genomic DNAs from human-rodent somatic cell hybrid lines. Southern blot analyses indicated the presence of a human-specific 7.6-kb fragment that was observed only in DNA from the hybrid cells containing human chromosome 8 or the proximal region of its short arm. The mouse Gata4 gene was mapped to chromosome 14, closely linked to Clu (clusterin), using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x M. spretus backcross. This mapping assignment places the Gata4 gene in the vicinity of the mouse Ds (disorganization) locus, a dominant gain-of-function mutation affecting embryonic development. We speculate that Ds is caused by a mutation in the Gata4 gene, ectopic expression of GATA-4, or a mutation in another lineage determination gene closely linked to Gata4.

Deletions of the long arm of chromosome 10.

Patients with a partial deletion of the long arm of chromosome 10 are rare. We report eight new cases involving various segments of 10q: one terminal deletion (10q26), four (8;10) translocations resulting in terminal deletions (10q26) and duplications (8q24.3), a de novo interstitial deletion (10q23), an interstitial deletion due to a (10;13) translocation (10q11.2----10q22.1), and a ring (10p15----10q26).

Sex steroids in the umbilical circulation of fetal rhesus monkeys from the time of gonadal differentiation.

Male rhesus monkey fetuses have significantly more testosterone (T) in their circulation than females on days 35--50 of gestation (P less than 0.01; n = 6 males and 6 females). However, we found no sex differences for androstenedione (delta 4). T concentrations remained significantly higher in male fetuses than in females later in gestation, e.g. days 79--84, 100--133, and 140--160. Levels of delta 4 differed between the sexes only on days 79--84, and dihydrotestosterone concentrations were significantly higher in male fetuses than in females on days 100--133 and 140--163. The fact that delta 4 concentrations were not different between the sexes at the earliest period studied (days 35--50) indicates that systemic concentrations of this hormone in the fetus probably are not important for sexual differentiation, especially of the central nervous system. Quantification of three steroids (T, delta 4, and dihydrotestosterone) in umbilical arterial and venous plasma from five male and nine female fetuses (days 35--100) revealed significant arterial/venous differences only for T in males (arterial greater than venous). These data, which suggest that fetal testes secrete T during morphological differentiation, lend credence to the hypothesis that endogenous T partially regulates sexual differentiation.

Somatic cell hybrids derived from terminally differentiated rhesus cells and established mouse cell lines.

The mechanisms of normal cell differentiation in vivo may be related to some features of cellular aging in vitro in that both are considered to be under genetic control. Diploid rhesus choroidal melanocytes and purified peripheral lymphocytes were fused by means of inactivated Sendai virus with three long-term murine cell lines which lacked either hypoxanthine-guanine phosphoribosyl transferase or thymidine kinase. Cell hybrids were selected by their growth in medium containing hypoxanthine, aminopterin, thymidine, and glycine. G-banded chromosomes were analyzed and elements from both the rhesus and the established mouse cell lines were identified in all metaphases. Hybrids derived from choroid X mouse cells contained more than one chromosome set from the mouse, but those between lymphocytes and established cell lines had only one. However, in every combination continuously replicating hybrids were produced; most of them have undergone more than 40 subculture passages. Our results demonstrate not only that DNA synthesis can be re-initiated in postreplicative cells, but also that DNA continues to replicate in a manner consistent with the life span of the long-term cell line parent.

In vivo hybridisation of cultured melanoma cells and isogenic normal mouse cells.

Somatic cell hybrids were derived by fusing tumourigenic and melanogenic melanoma (PAZG) cells with normal diploid male mouse cells in vivo. Their chromosomal composition was equivalent to the sum of both parental genomes and included a Y chromosome lacking in the melanoma parent. Our study showed that in PAZG X C57BL hybrids (MP), tumourigenicity was suppressed but pigmentation was expressed.

Mouse satellite DMA in noncentromeric heterochromatin of cultured cells.

Specific chromosomes in several mouse lines have interstitial C-bands. In situ hybridization studies indicate that these interstitial bands contain typical mouse satellite DNA.