Biomedical subjects
Publications and source records attributed to J Loeffen.
A fumarase-deficient patient expressed a novel phenotype of congenital cerebral ventricular dilatation and periventricular cysts. The patient was a compound heterozygote for two mutations that are the only ones among the 12 published mutations that have been found in multiple, unrelated, fumarase-deficient patients.
Human complex I is built up and regulated by genes encoded by the mitochondrial DNA (mtDNA) as well as the nuclear DNA (nDNA). In recent years, attention mainly focused on the relation between complex I deficiency and mtDNA mutations. However, a high percentage of consanguinity and an autosomal-recessive mode of inheritance observed within our patient group as well as the absence of common mtDNA mutations make a nuclear genetic cause likely. The NDUFS2 protein is part of complex I of many pro- and eukaryotes. The nuclear gene coding for this protein is therefore an important candidate for mutational detection studies in enzymatic complex I deficient patients. Screening of patient NDUFS2 cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with direct DNA sequencing revealed three missense mutations resulting in the substitution of conserved amino acids in three families.
Deficiency of NADH:ubiquinone oxidoreductase, the first enzyme complex of the mitochondrial respiratory chain, is one of the most frequent causes of human mitochondrial encephalomyopathies. A relatively small percentage of human complex I deficiency is associated with mitochondrial DNA mutations. cDNA characterization and mutational analysis of the structural complex I genes in 19 complex I-deficient patients, in whom common mtDNA mutations have been excluded, has so far revealed five patients with alterations in evolutionary conserved nuclear-encoded proteins. In order to complete our knowledge about the expected 36 structural nuclear complex I genes, we characterized the NDUFB7 and the 17.2-kDa cDNA sequences of the hydrophobic (HP) fraction of the complex. Subsequently, we screened all subunits of this fraction for the presence of mutations in those 14 patients of our initial patient cohort in whom the underlying genetic cause had not been elucidated. Strikingly, no pathogenic mutations were found in the HP subunits that would explain the complex I deficiency in our patients. Other strategies are needed to unravel proteins involved in the pathogenesis of the complicated cellular network of transcription until correct assemblage of complex I.
We present the cDNA sequence of the human mitochondrial acyl carrier protein NDUFAB1, a nuclear-encoded subunit of complex I of the mitochondrial respiratory chain. We obtained the NDUFAB1 cDNA using the cDNA sequence of the bovine mitochondrial acyl carrier protein. The human cDNA contains two putative translation initiation codons. The human NDUFAB1 protein contains a phosphopantetheine attachment site (DLGLDSLDQVEIIMAM), unique for acyl carrier proteins, and an EF-hand calcium binding domain (DIDAEKLMCPQEI). Transcripts of this gene are found in a wide range of human tissues. The highests expression levels were observed, in descending order, in adult heart, skeletal muscle and fetal heart. We subjected NDUFAB1 fibroblast cDNA of 20 patients with an isolated enzymatic complex I deficiency to mutational detection. No mutations in the NDUFAB1 open reading frame were observed. Future studies will answer whether mutations in the NDUFAB1 promoter or transcription elements are responsible for the observed complex I deficiency.
We have cloned the cDNA of the NDUFS5 subunit (15 kDa) of the human mitochondrial respiratory chain complex NADH: ubiquinone oxidoreductase (complex I). The open reading frame consists of 321 base-pairs, coding for 106 amino acids, with a calculated molecular mass of 12.5 kDa. There is an 81.0% identity with the bovine equivalent on cDNA level and 74.5% identity on amino acid basis. PCR analysis of rodent-human somatic cell hybrids revealed that the human NDUFS5 gene maps to chromosome 1. The NDUFS5 mRNA is expressed ubiquitously in human tissues, with a relative higher expression in human heart, skeletal muscle, liver, kidney and fetal heart. A mutation detection study of twenty isolated enzymatic complex I-deficient patients revealed no mutations, nor polymorphisms.
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NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain can be fragmented in a flavoprotein (FP), iron-sulfur protein (IP), and hydrophobic protein (HP) subfraction. The IP subfraction is hypothesized to be significant, since it contains important prosthetic groups highly conserved among species. We cloned the cDNA of three remaining human NADH:ubiquinone oxidoreductase subunits of this IP fraction: the NDUFS2 (49 kDa), NDUFS3 (30 kDa), and NDUFS6 (13 kDa) subunits. All presented cDNAs include the complete open reading frame (ORF), which consist of 1392, 795, and 375 base pairs, coding for 463, 264, and 124 amino acids, respectively. The latter show 96, 90, and 83% homology with the corresponding bovine translation products. The 3' untranslated regions (UTR) are complete in all three cDNAs. Polymerase chain reaction performed with DNA isolated from somatic human-rodent cell hybrids containing defined human chromosomes as template gave a human-specific signal which mapped the NDUFS2 and NDUFS3 subunits to chromosomes 1 and 11, respectively. In the case of the NDUFS6 subunit a pseudogene may be present since signals were seen in the lanes containing chromosomes 5 and 6. The NDUFS2 contains a highly conserved protein kinase C phosphorylation site and the NDUFS3 subunit contains a highly conserved casein kinase II phosphorylation site which make them strong candidates for future mutation detection studies in enzymatic complex I-deficient patients.
We report the cloning of the genomic and cDNA of the human 51 kDa subunit (NDUFV1) of mitochondrial complex I. The 6 kbp NDUFV1 gene is composed of 10 exons. All intron-exon boundaries comply to the consensus sequence for splice donor and acceptor sites. Within the 5' flanking region we identified a putative binding site for NRF-2, a GATA- and GC-box element. Canonical TATA- or CCAAT-boxes were absent, the transcriptional start site, however, lies within a CpG island, which is consistent with the "housekeeping" function of the gene. Within the coding sequence we detected consensus motifs for NADH, FMN, and iron-sulfur binding sites. The amino acid sequence homology between human and cow is 96.9%. Surprisingly we found a 48 bp long complete antisense homology between the 3'UTR of the NDUFV1-mRNA and the 5'UTR of the mRNA for the gamma-interferon inducible protein precursor (IP-30). This finding is intriguing since both genes lie on different chromosomes. The exact function of IP-30 is not yet known, but it may play a role in gamma-interferon mediated immune reactions. The NDUFV1-mRNA might act as an antisense suppresser, thus restraining translation of IP-30 in tissues with high energy demand. This finding could be a molecular link between complex I deficiency and inflammatory myopathy which have been repeatedly described to occur together.
Bovine NADH:ubiquinone oxidoreductase (complex 1) of the mitochondrial respiratory chain consists of about 36 nuclear-encoded subunits. We review the current knowledge of the 15 human complex I subunits cloned so far, and report the 598-bp cDNA sequence, the chromosomal localization and the tissue expression of an additional subunit, the B17 subunit. The cDNA open reading frame of B17 comprises 387 bp and encodes a protein of 128 amino acids (calculated Mr 15.5 kDa). There is 82.7% and 78.1% homology, respectively, at the cDNA and amino acid level with the bovine counterpart. The gene of the B17 subunit has been mapped to chromosome 2. Multiple-tissue dot-blots showed ubiquitous expression of the mRNA with relatively higher expression in tissues known for their high energy demand. Of these, kidney showed the highest expression. Mutational analysis of the subunit revealed no mutations or polymorphisms in 20 patients with isolated enzymatic complex I deficiency in cultured skin fibroblasts.
We report the cloning of the cDNA sequence of the nuclear-encoded NDUFA8 subunit of NADH: ubiquinone oxidoreductase, the first mitochondrial respiratory chain complex. The NDUFA8 open reading frame (ORF) includes 519 bp and encodes 172 amino acids (Mr=20.1 kDa). The human cDNA sequence shows 86.2% identity with the bovine sequence, whereas the human NDUFA8 amino acid sequence is 87.8% similar to its bovine PGIV protein counterpart. Both human and bovine NDUFA8 contain a conserved cysteine motif. Polymerase chain reaction analysis of rodent/human somatic cell hybrids maps the human NDUFA8 gene to chromosome 9. A multiple tissue blot has revealed the highest NDUFA8 mRNA expression in human heart, skeletal muscle, and fetal heart. Mutation analysis of the NDUFA8 fibroblast cDNA in 20 patients with an isolated enzymatic complex I deficiency in cultured skin fibroblasts has revealed two polymorphisms, one within the ORF and the other in the 3' untranslated region of the NDUFA8 cDNA sequence. The allelic frequency of both polymorphisms was similar in controls and complex-I-deficient patients.
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We report the cDNA cloning, chromosomal localization, and a mutation in the human nuclear gene encoding the 18-kD (AQDQ) subunit of the mitochondrial respiratory chain complex I. The cDNA has an open reading frame of 175 amino acids and codes for a protein with a molecular mass of 23.2 kD. Its gene was mapped to chromosome 5. A homozygous 5-bp duplication, destroying a consensus phosphorylation site, in the 18-kD cDNA was found in a complex I-deficient patient. The patient showed normal muscle morphology and a remarkably nonspecific fatal progressive phenotype without increased lactate concentrations in body fluids. The child's parents were heterozygous for the mutation. In 19 other complex I-deficient patients, no mutations were found in the 18-kD gene.
Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain. The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2. The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids. Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome. The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H). Both mutations were absent in 70 control alleles and cosegregated within the family. A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient. In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA. This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.