Cloning, characterization, and genomic structure of the mouse Ikbkap gene.

Our laboratory recently reported that mutations in the human I-kappaB kinase-associated protein (IKBKAP) gene are responsible for familial dysautonomia (FD). Interestingly, amino acid substitutions in the IKAP correlate with increased risk for childhood bronchial asthma. Here, we report the cloning and genomic characterization of the mouse Ikbkap gene, the homolog of human IKBKAP. Like its human counterpart, Ikbkap encodes a protein of 1332 amino acids with a molecular weight of approximately 150 kDa. The Ikbkap gene product, Ikap, contains 37 exons that span approximately 51 kb. The protein shows 80% amino acid identity with human IKAP. It shows very high conservation across species and is homologous to the yeast Elp1/Iki3p protein, which is a member of the Elongator complex. The Ikbkap gene maps to chromosome 4 in a region that is syntenic to human chromosome 9q31.3. Because no animal model of FD currently exists, cloning of the mouse Ikbkap gene is an important first step toward creating a mouse model for FD. In addition, cloning of Ikbkap is crucial to the characterization of the putative mammalian Elongator complex.

A physical and transcript map of the MCOLN1 gene region on human chromosome 19p13.3-p13.2.

Mutations in MCOLN1 have been found to cause mucolipidosis type IV (MLIV; MIM 252650), a rare autosomal recessive lysosomal storage disorder found primarily in the Ashkenazi Jewish population. As a part of the successful cloning of MCOLN1, we constructed a 1.4-Mb physical map containing 14 BACs and 4 cosmids that encompasses the region surrounding MCOLN1 on human chromosome 19p13.3-p13.2-a region to which linkage or association has been reported for multiple diseases. Here we detail the precise physical mapping of 28 expressed sequence tags that represent unique UniGene clusters, of which 15 are known genes. We present a detailed transcript map of the MCOLN1 gene region that includes the genes KIAA0521, neuropathy target esterase (NTE), a novel zinc finger gene, and two novel transcripts in addition to MCOLN1. We also report the identification of eight new polymorphic markers between D19S406 and D19S912, which allowed us to pinpoint the location of MCOLN1 by haplotype analysis and which will facilitate future fine-mapping in this region. Additionally, we briefly describe the correlation between the observed haplotypes and the mutations found in MCOLN1. The complete 14-marker haplotypes of non-Jewish disease chromosomes, which are crucial for the genetic diagnosis of MLIV in the non-Jewish population, are presented here for the first time.

Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.

Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

Cloning, genomic organization and expression of a putative human transmembrane protein related to the Caenorhabditis elegans M01F1.4 gene.

A novel human transcript CG-2 (C9ORF5), was isolated from the familial dysautonomia candidate region on 9q31 using a combination of cDNA selection and exon trapping. CG-2 was detected as a relatively abundant 8kb transcript in all adult and fetal tissues with the exception of adult thymus. Genomic analysis of CG-2 identified 18 exons that span more than 110kb. The gene encodes a 911-amino-acid protein with a predicted molecular weight of 101kDa and a hypothetical pI of 9.03. Sequence analysis of CG-2 indicates that it is likely to encode a transmembrane protein. Here, we assess CG-2 as a candidate for familial dysautonomia.

Cloning, mapping, and expression of two novel actin genes, actin-like-7A (ACTL7A) and actin-like-7B (ACTL7B), from the familial dysautonomia candidate region on 9q31.

Two novel human actin-like genes, ACTL7A and ACTL7B, were identified by cDNA selection and direct genomic sequencing from the familial dysautonomia candidate region on 9q31. ACTL7A encodes a 435-amino-acid protein (predicted molecular mass 48.6 kDa) and ACTL7B encodes a 415-amino-acid protein (predicted molecular mass 45. 2 kDa) that show greater than 65% amino acid identity to each other. Genomic analysis revealed ACTL7A and ACTL7B to be intronless genes contained on a common 8-kb HindIII fragment in a "head-to-head" orientation. The murine homologues were cloned and mapped by linkage analysis to mouse chromosome 4 in a region of gene order conserved with human chromosome 9q31. No recombinants were observed between the two genes, indicating a close physical proximity in mouse. ACTL7A is expressed in a wide variety of adult tissues, while the ACTL7B message was detected only in the testis and, to a lesser extent, in the prostate. No coding sequence mutations, genomic rearrangements, or differences in expression were detected for either gene in familial dysautonomia patients.

Precise genetic mapping and haplotype analysis of the familial dysautonomia gene on human chromosome 9q31.

Familial dysautonomia (FD) is an autosomal recessive disorder characterized by developmental arrest in the sensory and autonomic nervous systems and by Ashkenazi Jewish ancestry. We previously had mapped the defective gene (DYS) to an 11-cM segment of chromosome 9q31-33, flanked by D9S53 and D9S105. By using 11 new polymorphic loci, we now have narrowed the location of DYS to <0.5 cM between the markers 43B1GAGT and 157A3. Two markers in this interval, 164D1 and D9S1677, show no recombination with the disease. Haplotype analysis confirmed this candidate region and revealed a major haplotype shared by 435 of 441 FD chromosomes, indicating a striking founder effect. Three other haplotypes, found on the remaining 6 FD chromosomes, might represent independent mutations. The frequency of the major FD haplotype in the Ashkenazim (5 in 324 control chromosomes) was consistent with the estimated DYS carrier frequency of 1 in 32, and none of the four haplotypes associated with FD was observed on 492 non-FD chromosomes from obligatory carriers. It is now possible to provide accurate genetic testing both for families with FD and for carriers, on the basis of close flanking markers and the capacity to identify >98% of FD chromosomes by their haplotype.

Molecular cloning and developmental expression of a small ribonuclear protein in the mouse testis.

U1 RNP C polypeptide is a ubiquitous and highly conserved protein that is found associated to the U1 small nuclear ribonuclear particle (U1 snRNP). The U1 snRNP is involved in pre-mRNA splicing by defining introns and exons and by binding to consensus sequences within the pre-mRNA. In the present study we immunoscreened a mouse testicular phagemid cDNA library with an anti-Sm serum from patients with systemic lupus erythematosus. Sequence analysis of a positive clone containing a 0.75 kb cDNA insert revealed that it encodes the entire amino acid sequence of the U1 RNP C polypeptide. Northern blots of total RNA isolated from testes and various adult mouse tissues demonstrated that the 0.75 kb transcript is highly expressed in the testes and that it begins developmentally at day 18 postpartum, corresponding to the appearance of preleptotene spermatocytes. In situ hybridization confirmed the meiotic and post-meiotic expression of this transcript. LM immunoperoxidase staining with the anti-Sm serum localized spliceosome snRNPs predominantly in the nuclei of somatic and germinal testicular cells but not in elongated spermatids. EM immunogold labeling confirmed the LM observations but additionally showed that snRNP content peaked in the nuclei of pachytene spermatocytes and that 2 cytoplasmic components found exclusively in meiotic and post-meiotic germ cells were intensively reactive. Immunoblots of testicular homogenates probed with the anti-Sm serum revealed several reactive proteins, of which one, a 21 kDa polypeptide, could be the U1 RNP C based on its predicted molecular weight. In summary we report an isoform of U1 RNP C which is testis specific and which may play a role in mRNA splicing exclusively in meiotic and post-meiotic germ cells during spermatogenesis.