The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes

In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly. In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome. We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal. Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene. The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb. The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites. The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker. As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders. Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders. Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected. PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups.     

Genomic Structure ofPEX13,a Candidate Peroxisome Biogenesis Disorder Gene

The peroxisome biogenesis disorders (PBDs) are a set of lethal genetic diseases characterized by peroxisomal metabolic deficiencies, multisystem abnormalities, mental retardation, and premature death. These disorders are genetically heterogeneous and are caused by mutations in genes, termedPEXgenes, required for import of proteins into the peroxisomal matrix. We have previously reported the identification of humanPEX13,the gene encoding the docking factor for the PTS1 receptor, or PEX5 protein. As such, mutations inPEX13would be expected to abrogate peroxisomal protein import and result in PBD phenotypes. We report here the structure of the humanPEX13gene.PEX13spans approximately 11 kb on chromosome 2 and contains four exons, one more than previously thought. The corrected PEX13 cDNA is predicted to encode a protein product with a molecular mass of 44,312 Da. We examined the ability ofPEX13expression to rescue the peroxisomal protein import defects of fibroblast cells representing all known PBD complementation groups. No complementation was observed, suggesting that this gene is not mutated in any set of existing patients. However, given that complementation group assignments have been determined for only a subset of PBD patients, it is possible thatPEX13-deficient patients may exist at a low frequency within our existing PBD patient population or within ethnic groups underrepresented in our patient pool.     

Cloning and characterization of the human GFRA2 locus and investigation of the gene in Hirschsprung disease

The glial-cell-line-derived neurotrophic factor (GDNF) family receptors alpha (GFRalpha) are cell surface bound glycoproteins that mediate interactions of the GDNF ligand family with the RET receptor. These interactions are crucial to the development of the kidney and some peripheral nerve lineages. In humans, mutations of RET or RET ligands are associated with the congenital abnormality Hirschsprung disease (HSCR) in which nerves and ganglia of the hind gut are absent. As the GFRalpha family are required for normal activation of the RET receptor, they are also candidates for a role in HSCR. The GFRA2 gene, which is required for the development of the myenteric nerve plexus, is an excellent candidate gene for HSCR. In this study, we cloned the human GFRA2 locus, characterized the gene structure, and compared it with other GFRA family members. We further investigated the GFRA2 gene for mutations in a panel of HSCR patients. GFRA2 has nine coding exons that are similar in size and organization to those of other GFRA family genes. We identified six sequence variants of GFRA2, four of which did not affect the amino acid sequence of the GFRalpha-2 protein. Two further changes that resulted in amino acid substitutions were found in exon 9 and were predicted to lie in the amino acid sequence encoding the glycosylphosphatidylinositol-linkage signal of GFRalpha-2. There was no difference in frequency of any of the sequence variants between control and HSCR populations. Our data indicate that members of the GFRA gene family are closely related in intron/exon structure and in sequence. We have not detected any correlation between sequence variants of GFRA2 and the HSCR phenotype.     

Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family

Three genes from the human cystatin gene family of cysteine-proteinase inhibitors have been isolated from a bacteriophage lambda library containing HindIII digests of human genomic DNA. Two of the genes code for salivary cystatin SN and SA, the third is a pseudogene. The cloned genes were identified with a probe made from a salivary cystatin cDNA. The complete nucleotide sequence of the gene that codes for the precursor form of the neutral salivary protein, cystatin SN, was determined. The gene, which we name CST1, contains three exons and two intervening sequences. The expected CAT and ATA boxes are present in the 5'-flanking region of the gene. Partial nucleotide sequence determination of a second gene revealed that it codes for the precursor form of the acidic salivary protein, cystatin SA. This gene, which we name CST2, has the same gene organization as CST1. The complete nucleotide sequence of a third gene was determined. It does not contain a typical ATA box, and in addition, a premature stop codon and a frameshift deletion mutation occur within the gene. These inactivation mutations show that this gene, which we name CSTP1, is a cystatin pseudogene. These data combined with our genomic Southern-blot analyses show that the cystatin genes form a multigene family with at least seven members.     

Novel and recurrent LDLR gene mutations in Pakistani hypercholesterolemia patients

The majority of patients with the autosomal dominant disorder familial hypercholesterolemia (FH) carry novel mutations in the low density lipoprotein receptor (LDLR) that is involved in cholesterol regulation. In different populations the spectrum of mutations identified is quite different and to date there have been only a few reports of the spectrum of mutations in FH patients from Pakistan. In order to identify the causative LDLR variants the gene was sequenced in a Pakistani FH family, while high resolution melting analysis followed by sequencing was performed in a panel of 27 unrelated sporadic hypercholesterolemia patients. In the family a novel missense variant (c.1916T > G, p.(V639G)) in exon 13 of LDLR was identified in the proband. The segregation of the identified nucleotide change in the family and carrier status screening in a group of 100 healthy subjects was done using restriction fragment length polymorphism analysis. All affected members of the FH family carried the variant and none of the non-affected members nor any of the healthy subjects. In one of the sporadic cases, two sequence changes were detected in exon 9, one of these was a recurrent missense variant (c.1211C > T; p.T404I), while the other was a novel substitution mutation (c.1214 A > C; N405T). In order to define the allelic status of this double heterozygous individual, PCR amplified fragments were cloned and sequenced, which identified that both changes occurred on the same allele. In silico tools (PolyPhen and SIFT) were used to predict the effect of the variants on the protein structure, which predicted both of these variants to have deleterious effect. These findings support the view that there will be a novel spectrum of mutations causing FH in patients with hypercholesterolaemia from Pakistan.     

Structural characterization and chromosomal localization of the MAGE-E1 gene

Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.     

Cloning and characterization of avfA and omtB genes involved in aflatoxin biosynthesis in three Aspergillus species

A collection of lethal and semi-lethal P-element insertions in the 70CD region of chromosome 3 of Drosophila melanogaster was used to investigate genes and gene arrangements by a combination of genetic, cytological, functional and molecular methods. The 12 lethal insertions studied fall into seven complementation groups of six genes. Lethal phases, expression patterns and other phenotypic aspects of these genes were determined. The genes and additional available sequences were placed on cloned genomic DNA fragments and arranged in an EcoRI map of 150kb that covers approximately the bands 70C7-8 to 70D1. Determination of deficiency breakpoints links the genetic, physical and molecular data. The sequences adjacent to seven independent P-element insertions were established after plasmid rescue or polymerase chain reaction. Similarity searches allowed the assignment of the P-element insertions to known mutations, expressed sequence tags, sequence tagged sites, or homologous genes of other species. Among these were identified a putative transacylase, a putative cell cycle gene, and the gene responsible for the dominant Polycomb-suppressor phenotype of devenir. The genomic sequence of the l(3)70Ca/b gene reveals a novel heat shock protein (hsc70Cb). l(3)70Da was identified as a member of the CDC48/PEX1 ATPase family and its coding sequence was determined.     

A gene for rabbit synovial cell collagenase: member of a family of metalloproteinases that degrade the connective tissue matrix

We have determined the nucleotide sequence of a collagenase mRNA from rabbit synovial cells from which the primary structure of the encoded protein was deduced. This proteinase is 51% homologous to the enzyme that activates it from the zymogen form, rabbit synovial cell activator/stromelysin. Rabbit collagenase and activator/stromelysin thus share comembership in a gene family that includes human skin collagenase; the human and rabbit metalloproteinase, activator/stromelysin; and an oncogene-induced proteinase from rat named transin. The mRNA sequence of collagenase enabled us to completely map the structure of its gene, which is 9.1 kilobases and is composed of 10 exons and 9 introns. This is the first report of the structure of a collagenase gene. We show that it has striking similarity to additional members of this metalloproteinase gene family, transin genes I and II of rat. We have further sequenced genomic DNA flanking the collagenase gene and have identified nucleic acid elements of possible importance in gene regulation.     

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus. Received: 20 August 1996     

Sequence, genomic structure and tissue expression of Human BRI3, a member of the BRI gene family

The BRI3 gene is a member of the BRI gene family, made up of at least three different genes (BRI1-3). Previous studies established the cDNA sequence and structure of the human and mouse BRI1 and BRI2 genes and we recently reported that mutations in the BRI2 isoform, located on chromosome 13, are associated with dementia in humans. In the present work, we determine the complete cDNA sequence and genomic organization of the human BRI3 gene. BRI3 codes for a polypeptide of 267 amino acids, with a Mr of 30 KDa and a pI of 8.47. The amino acid sequence is 43.7% identical to the sequence of the human BRI2, and 38.3% identical to that of human BRI1, with the highest percentage of amino acid identity being concentrated on the C-terminal half of the molecules. In Northern blots, BRI3 cDNA hybridizes only one message of approximately 2.1 kilobases, which is predominantly present in the human brain. The BRI3 gene is localized on chromosome 2 and consists of six exons spanning more than 20 kb. Homology search of EST data banks retrieved a Caenorhabditis briggsae homolog of BRI, indicating that the BRI gene belongs to a strongly conserved gene family. These studies, aimed at characterizing the members of the BRI gene family, may provide valuable clues to the understanding of their normal function and how mutations in BRI2 can cause neurodegeneration and dementia similar to Alzheimer's disease.     

Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G

Zellweger cerebro-hepato-renal syndrome is a severe congenital disorder associated with defective peroxisomal biogenesis. At least 23 PEX genes have been reported to be essential for peroxisome biogenesis in various species, indicating the complexity of peroxisomal assembly. Cells from patients with peroxisomal biogenesis disorders have previously been shown to segregate into >/=12 complementation groups. Two patients assigned to complementation group G who had not been linked previously to a specific gene defect were confirmed as displaying a cellular phenotype characterized by a lack of even residual peroxisomal membrane structures. Here we demonstrate that this complementation group is associated with mutations in the PEX3 gene, encoding an integral peroxisomal membrane protein. Homozygous PEX3 mutations, each leading to C-terminal truncation of PEX3, were identified in the two patients, who both suffered from a severe Zellweger syndrome phenotype. One of the mutations involved a single-nucleotide insertion in exon 7, whereas the other was a single-nucleotide substitution eight nucleotides from the normal splice site in the 3' acceptor site of intron 10. Expression of wild-type PEX3 in the mutant cell lines restored peroxisomal biogenesis, whereas transfection of mutated PEX3 cDNA did not. This confirmed that the causative gene had been identified. The observation of peroxisomal formation in the absence of morphologically recognizable peroxisomal membranes challenges the theory that peroxisomes arise exclusively by growth and division from preexisting peroxisomes and establishes PEX3 as a key factor in early human peroxisome synthesis.     

Human XPMC2H: cDNA Cloning, Mapping to 9q34, Genomic Structure, and Evaluation as TSC1

XPMC2 is a Xenopus gene identified on the basis of its ability to correct a mitotic defect in fission yeast. Here we report the identification of cDNA clones for human XPMC2H, its mapping to the tuberous sclerosis gene TSC1 region on 9q34, determination of genomic structure, and identification of several coding region polymorphisms. The predicted protein has strong sequence similarity to the Xenopus gene. Through SSCP and heteroduplex analysis of genomic DNA, we found two intragenic polymorphisms but no evidence for significant mutations in patients with tuberous sclerosis in this gene.     

Identification of PEX7 as the second gene involved in Refsum disease

Patients affected with Refsum disease (RD) have elevated levels of phytanic acid due to a deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase (PhyH). In most patients with RD, disease-causing mutations in the PHYH gene have been identified, but, in a subset, no mutations could be found, indicating that the condition is genetically heterogeneous. Linkage analysis of a few patients diagnosed with RD, but without mutations in PHYH, suggested a second locus on chromosome 6q22-24. This region includes the PEX7 gene, which codes for the peroxin 7 receptor protein required for peroxisomal import of proteins containing a peroxisomal targeting signal type 2. Mutations in PEX7 normally cause rhizomelic chondrodysplasia punctata type 1, a severe peroxisomal disorder. Biochemical analyses of the patients with RD revealed defects not only in phytanic acid alpha-oxidation but also in plasmalogen synthesis and peroxisomal thiolase. Furthermore, we identified mutations in the PEX7 gene. Our data show that mutations in the PEX7 gene may result in a broad clinical spectrum ranging from severe rhizomelic chondrodysplasia punctata to relatively mild RD and that clinical diagnosis of conditions involving retinitis pigmentosa, ataxia, and polyneuropathy may require a full screen of peroxisomal functions.     

Detection of combined genomic variants in a Jordanian family with familial non-autoimmune hyperthyroidism

Five patients, four brothers and their paternal aunt, presented with a history of overt hyperthyroidism and goiter. Hyperthyroidism in this family was remarkable for its poor response to carbimazole (30–50 mg/d). The thyroid ultrasound showed a diffusely enlarged gland in all the affected members, and thyroid stimulating antibodies (TSAB) were negative. Screening for germline mutations in thyroid stimulating hormone (TSH) receptor (TSHR) gene was performed by direct sequencing of genomic DNA extracted from peripheral blood leukocytes of all family members. The sequence analysis of all TSHR gene exons and intron borders revealed two genomic variants. The first was a single nucleotide polymorphism (SNP) within exon seven (Asn187Asn), whereas the other was located in intron seven (IVS7+68T>G). All affected members, two asymptomatic brothers with sub-clinical hyperthyroidism, and their father were heterozygous for those two genomic variants. Anti-thyroid drug treatment for several months successfully relieved symptoms in one subject, whereas the remaining patients required total thyroidectomy to control their disease. This is the first Jordanian family with familial non-autoimmune hyperthyroidism, with mutations affecting the TSHR gene.     

Molecular cloning and sequence of the gene for p9Ka. A cultured myoepithelial cell protein with strong homology to S-100, a calcium-binding protein

The gene for p9Ka, a protein of molecular weight 9000 that is expressed in cultured rat mammary myoepithelial cells, has been isolated from a normal rat genomic library in bacteriophage lambda, by its ability to hybridize to a cloned complementary DNA corresponding to p9Ka mRNA. The cloned rat genomic DNA fragment hybridized to translatable p9Ka mRNA. A nucleotide sequence of 2340 base-pairs of genomic DNA surrounding the p9Ka cDNA sequence has been obtained; the gene contains one intervening sequence of 675 nucleotides. The 3' end of the p9Ka mRNA has been identified on the gene sequence to be 13 nucleotides downstream from a poly(A) addition signal AATAAA. The gene contains an open reading frame of 101 amino acid residues, which is the only open reading frame in the entire gene long enough to encode a protein of molecular weight at least 9000. This hypothetical protein sequence shows greater than 40% homology to rat or bovine S-100 protein and over 30% homology to bovine intestinal calcium-binding protein. The results suggest that p9Ka may be related to a family of low molecular weight calcium-binding proteins.     

Genetic and molecular characterization of the maize rp3 rust resistance locus

In maize, the Rp3 gene confers resistance to common rust caused by Puccinia sorghi. Flanking marker analysis of rust-susceptible rp3 variants suggested that most of them arose via unequal crossing over, indicating that rp3 is a complex locus like rp1. The PIC13 probe identifies a nucleotide binding site-leucine-rich repeat (NBS-LRR) gene family that maps to the complex. Rp3 variants show losses of PIC13 family members relative to the resistant parents when probed with PIC13, indicating that the Rp3 gene is a member of this family. Gel blots and sequence analysis suggest that at least 9 family members are at the locus in most Rp3-carrying lines and that at least 5 of these are transcribed in the Rp3-A haplotype. The coding regions of 14 family members, isolated from three different Rp3-carrying haplotypes, had DNA sequence identities from 93 to 99%. Partial sequencing of clones of a BAC contig spanning the rp3 locus in the maize inbred line B73 identified five different PIC13 paralogues in a region of approximately 140 kb.     

Human NDUFB9 gene: genomic organization and a possible candidate gene associated with deafness disorder mapped to chromosome 8q13

Human NADH dehydrogenase (ubiquinone) 1beta-subcomplex, 9 (NDUFB9) is a nuclear encoded mitochondrial protein with the respiratory electron transport chain. It has been physically mapped to a 1-Mb deletion at chromosome 8q13 which also contains the gene for branchio-oto-renal (BOR) syndrome. BOR syndrome is characterized by branchial and renal abnormalities with hearing impairment. Since several hereditary deafness disorders have been associated with mitochondrial mutations, NDUFB9 was considered a candidate gene for BOR syndrome. Recently, EYA1 gene has been identified in the region which underlies the BOR syndrome but majority of BOR families did not show mutations in the EYA1 gene. Here we have determined the genomic structure of the NDUFB9 gene, including the nucleotide sequence, organization and the boundaries of the four coding exons. PCR primers were designed from the adjacent intron sequences that allow amplification of the four exons that encode the complete open reading frame. To identify whether mutations in NDUFB9 are involved in causing the BOR syndrome, we screened 9 BOR families which did not show mutations in the EYA1 gene by heteroduplex analysis; however, no mutations were found.     

Characterization of duplicated Dunaliella viridis SPT1 genes provides insights into early gene divergence after duplication

The sodium-dependent phosphate transporter gene from unicellular green algae Dunaliella viridis, DvSPT1, shares similarity with members of Pi transporter family. Sequencing analysis of D. viridis BAC clone containing the DvSPT1 gene revealed two inverted duplicated copies of this gene (DvSPT1 and DvSPT1-2 respectively). The duplication covered most of both genes except for their 3' downstream region. The duplicated genomic sequences exhibited 97.9% identity with a synonymous divergence of Ks=0.0126 in the coding region. This data indicated very recent gene duplication in D. viridis genome, providing an excellent opportunity to investigate sequence and expression divergence of duplicated genes at an early stage. Scatted point mutations and length polymorphism of simple sequence repeats (SSRs) were predominant among the sequence divergence soon after gene duplication. Due to sequence divergence in the 5' regulatory regions and a swap of the entire 3' downstream regions (3'-UTR), DvSPT1 and DvSPT1-2 showed expression divergence in response to extra-cellular NaCl concentration changes. According to their expression patterns, the two diverged gene copies would provide better adaptation to a broader range of extra-cellular NaCl concentration. Furthermore, Southern blot analysis indicated that there might be a large phosphate transporter gene family in D. viridis.