Genomic structure of karyopherin alpha2 ( KPNA2) within a low-copy repeat on chromosome 17q23-q24 and mutation analysis in patients with Russell-Silver syndrome.

Human karyopherin alpha2 (KPNA2), a member of the karyopherin alpha family, plays a key role in the nuclear import of proteins with a classical nuclear localization signal (NLS). KPNA2, as part of a karyopherin alpha-beta heterodimer, directly binds to the NLS of proteins and functions as an adaptor that binds NLS-containing proteins via karyopherin beta to the nuclear pore complex. The NLS protein-receptor complex is translocated through the pore by an energy-dependent mechanism. Recently, we have identified and mapped the gene for KPNA2 in close proximity to a translocation breakpoint within 17q23-q24 associated with Russell-Silver syndrome (RSS). Therefore, we considered KPNA2 as a positional candidate gene for this heterogeneous disorder. RSS is mainly characterized by pre- and postnatal growth retardation, lateral asymmetry, and other dysmorphic features. Here, we present the genomic organization of the human KPNA2 gene with 11 exons spanning approximately 10 kb on chromosome 17q23-q24. Screening for mutations within all exons and adjacent intronic sequences from 31 unrelated RSS patients revealed three single nucleotide polymorphisms (SNPs) in exons 1, 5, and 7, and five SNPs in introns 1, 4 (2 SNPs), 8, and 9, respectively. No disease-related mutation was identified by comparing the sequence data of the RSS patients with their clinically normal parents and controls.

Kabuki syndrome-like features associated with a small ring chromosome X and XIST gene expression.

Although clinical features in Kabuki syndrome (KS; Niikawa-Kuroki syndrome) have been well defined, the underlying genetic mechanism still remains unclear. We report a 9-year-old girl with typical KS-like facial appearance, skeletal and dermatoglyphic abnormalities, severe mental retardation, and growth deficiency. In 60 of 100 GTG-banded metaphases from peripheral blood lymphocytes, a ring chromosome smaller than a G group chromosome was found, which, according to reverse painting, consisted of Xq11.1q13. The proband's karyotype was described as mos45,X/46,X,+r(X). Several loci were analyzed with fluorescence in situ hybridization (FISH) and microsatellite markers revealing that one r(X) breakpoint mapped proximal to DXS422 (Xp11.21) and the second mapped distal to XIST gene, between loci DXS128E and DXS441 (Xq13.2). Uniparental disomy for X and r(X) was excluded and the paternal origin of r(X) was identified. XIST expression was demonstrated by nested reverse transcription polymerase chain reaction (RT-PCR) using primers spanning exons 5, 6i, and 6 in RNA prepared from lymphocytes. The observation of XIST expression is in contrast to two other cases in which the XIST gene was either not present on r(X) or not expressed. To our knowledge, this is the first case of Kabuki-like syndrome manifestations with r(X) and XIST expression.

Parental mosaicism of JAG1 mutations in families with Alagille syndrome.

The Alagille syndrome (AGS), a congenital disorder affecting liver, heart, skeleton and eye in association with a typical face, is an autosomal dominant disease with nearly complete penetrance and variable expression. AGS is caused by mutations in the developmentally important JAG1 gene. In our mutation screening, where 61 mutations in JAG1 were detected, we identified five cases where mosaicism is present. Our results point to a significant frequency of mosaicism for JAG1 mutations in AGS of more than 8.2%. Because mosaicism may be associated with a very mild phenotype, the appropriate diagnosis of AGS and consequently the determination of the recurrence risk can be complicated.

Specific binding of a 47-kilodalton protein to the 3' untranslated region of rat transition protein 2 messenger ribonucleic acid.

The synthesis of transition protein (TNP) 2, one of the predominant nuclear proteins of mammalian spermatids, was shown to be posttranscriptionally regulated, by storing the untranslated mRNA for about 3-5 days in the cytoplasm of differentiating spermatids. It has been proposed that binding of a cytoplasmic protein to a conserved motif of 8 nucleotides (nt) in the 3' untranslated region (3'UTR) of TNP2 mRNA is involved in this translational control mechanism. In this report, we show that deletion or variation of the conserved 8-nt motif (GCCAT-CAC) in rat TNP2-3'UTR abolishes the capacity of the in vitro-transcribed RNA to reconstitute specific RNA-protein complexes in RNA bandshift assays. Using Northwestern analysis, we identified specific binding to the TNP2-3'UTR of four proteins of 45, 47, 49, and 60 kDa, all of which are stage-specifically regulated in male germ cell differentiation. Deletion of the 8-nt motif in the 3'UTR specifically prevented binding of the 47-kDa protein, the interaction of which is thought to be mediated by the RNA secondary structure. Analysis of the RNA secondary structure revealed that the 8-nt motif is an essential element of a specific stem-loop structure that is predicted for rat wild-type TNP2-3'UTR. Therefore we assume that the 47-kDa protein plays an important role in specific RNA-protein complex formation of rat TNP2-3'UTR that may be a central event in the translational control of rat TNP2 mRNA.

Stage and developmental specific gene expression during mammalian spermatogenesis.

Spermatogenesis is a complex developmental process which involves amplification of germinal stem cells, their differentiation into spermatocytes, meiotic division and finally transformation into mature spermatozoa. Therefore, spermatogenesis provides an interesting system for examining the regulation of gene expression during development and differentiation. The genes expressed during spermatogenesis can be divided into two main groups: diploid and haploid expressed genes. In this review, we report about the regulation of expression of a diploid expressed gene, namely the proacrosin gene, and that of a haploid expressed gene, the transition protein 2 gene.

Sequence analysis of the conserved protamine gene cluster shows that it contains a fourth expressed gene.

Structural data are presented on the protamine gene cluster (PGC) of human, mouse, rat, and bull. By restriction mapping we demonstrate that the organization of the protamine cluster is conserved throughout all four species, i.e., the genes are situated in a head to tail arrangement in the order: protamine 1-protamine 2-transition protein 2. Further, we established the nucleotide sequence of the entire human PGC (25 kb in total) and the 3' portion of the rat protamine cluster (PRM2 and TNP2 genes and intergenic region). In addition, a 1 kb fragment of the bovine and murine protamine cluster, situated between PRM2 and TNP2, was sequenced. This fragment is conserved regarding sequence, position, and orientation in all species examined, and was classified as likely coding region by gene recognition program GRAIL. Using the rat fragment as a probe in RNA blots, we detected a testis-specific signal of about 0.5 kb. Finally, we demonstrate a high density of Alu elements, both full and fragmented copies, in the human PGC and discuss their localization with respect to evolutionary and functional aspects.

Disturbances of nuclear condensation in human spermatozoa: search for mutations in the genes for protamine 1, protamine 2 and transition protein 1.

During spermiogenesis, the successive replacement of the somatic histones by basic proteins, the transition proteins and protamines, allows normal sperm nuclear condensation. It was suggested that disturbances in nuclear condensation may result in male infertility. Here we report the first molecular analysis of the structure of three genes which code for germ cell-specific nuclear proteins, namely protamine 1 (PRM1), protamine 2 (PRM2) and transition protein 1 (TNP1) in infertile men with disturbed sperm chromatin condensation. In 36 infertile men whose spermatozoa showed a positive reaction with aniline blue, which is an indication for the presence of histones in the nuclei, the complete nucleotide sequences of the coding regions and 5' and 3' untranslated regions of the three genes were evaluated. In addition, 10 infertile patients with oligoasthenoteratozoospermia were studied in the same way, as well as nine infertile patients whose spermatozoa showed a reduction of the protamine 2 content. We did not detect any mutation in the three genes in any of the patients. We assume that the disturbances in the sperm chromatin condensation of our patients, and those described in the literature, are not primarily due to mutations in the genes for PRM1, PRM2 and TNP1.

A conserved 8 bp motif (GCYATCAY) in the 3'UTR of transition protein 2 as a putative target for a transcript stabilizing protein factor.

The 3'UTR of the transition protein 2 gene of several mammalian species was sequenced and the transcript level of this gene was determined by Northern blots. In all species tested so far, a Northern blot detectable transcript level was associated with the presence of a conserved 8 bp motif (GCYATCAY) 50 bp downstream of the stop codon. RNA-bandshift experiments indicate that this region is capable of binding a cytosolic protein factor from rat testis. These findings support our hypothesis that the low transcript level of the transition protein 2 gene in human is due to insufficient storage of the mRNA as ribonucleic/protein particle.