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Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.

Objective: Dysfunction in glutamate signalling is thought to play a role in the pathophysiology of bipolar disorder (BD). There is evidence of associations between single nucleotide polymorphisms (SNPs) in GRM3, GRIN2B, and DAOA genes and the diagnosis of BD. In this pilot study, we investigated the frequency of SNP variants in these 3 genes within South African population groups, and assessed interactions between genes and phenotypes of BD disease severity., Method: Multiplex SNaPshotTM PCR was used to genotype 191 case and 188 control samples. Cases comprised of 191 individuals in a South African cohort of mixed ancestry and Caucasians, with BD Type 1. Phenotypes of BD disease severity were: age of onset, number of illness episodes, number of hospitalisations for depression or mania and history of psychotic symptoms., Results: There were no significant difference in SNP allele frequencies between cases and controls. In the case-only analysis, the GRM3 rs6465084 heterozygote was associated with a 4-fold increased risk of lifetime history of psychotic symptoms, and the specific variants within the gene pair, DAOA and GRIN2B, had a significant interaction with the number of hospitalisations for mania, with lowest admission rates associated with both pairs of ancestral alleles., Conclusion: In BD, variations in glutamatergic genes may influence phenotypes related to the severity of illness. Speculatively, newly derived genes associated with various evolutionary advantages, may also increase the risk for more severe BD. These preliminary findings deserve validation in a larger cohort.

Mutations in SCO2, a cytochrome c oxidase (COX) assembly gene, have been reported in nine infants with early onset fatal cardioencephalomyopathy and a severe COX deficiency in striated muscle. Studies on a yeast homolog have suggested that human Sco2 acts as a copper chaperone, transporting copper to the Cu(A) site on the Cox II subunit, but the mechanism of action remains unclear. To investigate the molecular basis of pathogenesis of Sco2 defects in humans we performed genetic and biochemical studies on tissues, myoblasts and fibroblasts from affected patients, as well as on a recombinant human C-terminal Sco2 segment (22 kDa), bearing the putative CxxxC metal-binding motif. Recombinant Sco2 was shown to bind copper with a 1:1 stoichiometry and to form homomeric complexes in vitro, independent of the metal-binding motif. Immunohistochemistry using antibodies directed against different COX subunits showed a marked tissue-specific decrease in the Cox II/III subunits that form part of the catalytic core, consistent with the differential tissue involvement, but a more uniform distribution of Cox Vab, a nuclear-encoded subunit. Sco2 was severely reduced in patient fibroblasts and myoblasts by immunoblot analysis. Patient fibroblasts showed increased (64)Cu uptake but normal retention values and, consistent with this, the copper concentration was four times higher in Sco2-deficient myoblasts than in controls. COX activity in patient myoblasts was completely rescued by transduction with a retroviral vector expressing the human SCO2 coding sequence, and more interestingly by addition of copper-histidine (300 microM) to the culture medium. Whether the latter is accomplished by the very low residual levels of Sco2 in the patient cells, direct addition of copper to the Cu(A) site, or by another copper-binding protein remains unknown. Whatever the mechanism, this result suggests a possible therapy for the early treatment of this fatal infantile disease.

Mutations of the ATP7A gene (OMIM 300011) lead to the Menkes disease (MD, OMIM 309400) involving impaired brain development, neurological degeneration, connective tissue abnormalities, and high lethality in early infancy. Occipital horn syndrome (OHS, OMIM 304150), a milder phenotype, is also caused by ATP7A gene mutations. In MD patients, an early copper-histidine treatment may prevent the neurological impairment and prolong survival leading to an OHS phenotype. To demonstrate the genotype/phenotype correlation, two male patients are reported with different ATP7A gene mutations and several phenotypes. In the first patient with the MD phenotype, a mutation within the exon 20 (Gln1288Ter) was found producing a stop codon just prior to the highly conserved ATP binding domain. The OHS phenotype of the second patient was caused by a splice site mutation involving the position +6 of intron 6 within a copper binding domain. Small amounts of correctly spliced ATP7A transcript were sufficient to develop the milder OHS phenotype in this patient (OMIM 30001.0006). In conclusion, mutations of the copper transporting P-type ATPase ATP7A gene cause distinct human diseases showing some genotype/phenotype correlation and implications for treatment.

More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.

Our knowledge about Menkes disease (MD) has expanded greatly since its description in 1962 as a new X-linked recessive neurodegenerative disorder of early infancy. Ten years later a defect in copper metabolism was established as the underlying biochemical deficiency. In the beginning of 1990s efforts were concentrated on the molecular genetic aspects. The disease locus was mapped to Xq13.3 and the gene has been isolated by means of positional cloning. This was the beginning of a series of new findings which have greatly enhanced our understanding of copper metabolism not only in human, but also in other species. This review will focus on the molecular genetic aspects of Menkes disease and its allelic form occipital horn syndrome. The mutations will be compared briefly with those described in the animal model mottled mouse, and in Wilson disease, the autosomal recessive disorder of copper metabolism.

Menkes disease (MD) is caused by a defect in copper homeostasis and has a recognised mouse model, mottled (Atp7aMo). Copper uptake and retention assays performed on fibroblast cultures have been used successfully for pre- and postnatal diagnosis of Menkes disease. We report here the results of these assays applied to primary fibroblast cultures established from nine independent mottled alleles associated with phenotypes of varying severity maintained on identical genetic backgrounds. No significant differences were found between the different alleles, or between the mottled cultures and fibroblasts established from MD patients. Thus, in the mouse, the data obtained for copper retention/uptake at the cellular level do not correlate with the severity of the phenotype.

Genomic DNA of 41 unrelated patients affected with the classical severe form of Menkes disease was investigated for point mutations in the ATP7A gene (previously designated as the "MNK" gene). Using SSCP analysis and direct sequencing of the exons amplified by PCR, we identified 41 different mutations, including 19 insertions/deletions, 10 nonsense mutations, 4 missense mutations, and 8 splice-site alterations. Approximately 90% of the mutations were predicted to result in the truncation of the protein (ATP7A). In 20 patients the mutations were within exons 7-10, and half of these mutations affected exon 8. Furthermore, five alterations were observed within the 6-bp sequence at the splice-donor site of intron 8, which would be predicted to affect the efficiency of splicing of exon 8. Although a specific function has not been attributed to the protein region encoded by this exon, this region may be important in serving as a "stalk" joining the metal-binding domains and the ATPase core. The present findings not only help us in understanding the underlying genetic defect but are invaluable data especially for carrier detection and prenatal diagnosis of this lethal disorder.

The gene defective in Menkes disease, an X-linked recessive disturbance of copper metabolism, has been isolated and predicted to encode a copper-binding P-type ATPase. We determined the complete exon-intron structure of the Menkes disease gene, which spans about 150 kb of genomic DNA. The gene contains 23 exons, and the ATG start codon is in the second exon. All of the exon-intron boundaries were sequenced and conformed to the GT/AT rule, except for the 5' splice site of intron 9. A preliminary comparison demonstrated a striking similarity between the exon structures of the Menkes and Wilson disease genes, giving insight into their evolution.

To evaluate the short-term effects of growth hormone (GH), insulin and different levels of glycemia on insulin-like growth factors (IGF) I and II and IGF binding proteins (IGFBP) 1, 2 and 3, we studied six GH-deficient adolescents during a night and the following day in the postabsorptive (basal) state followed by sequential euglycemic (5 mmol/l) and hypoglycemic (3 mmol/l) glucose clamps concomitant with an intravenous infusion (starting at 24.00 h) of GH (35 micrograms/h) or saline. Current GH therapy was withdrawn 24 h prior to each study. Nocturnal levels of IGF-I, IGF-II, IGFBP-2 and IGFBP-3 remained stable during both studies. Nocturnal serum IGFBP-1 increased and correlated inversely with insulin in both studies. Regression analysis revealed a significant inverse correlation between mean nocturnal IGFBP-2 and IGFBP-3 levels. During the daytime, serum IGF-I declined slowly during saline infusion, whereas serum IGF-II remained stable in both studies. Serum IGFBP-1 displayed a gradual significant decline during the basal state and the euglycemic and hypoglycemic clamps seemed to be unaffected by GH levels. By contrast, serum IGFBP-2 remained stable during the same period in both the GH and the saline study. Serum IGFBP-3 declined insignificantly during the daytime in the saline study.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific radioimmunoassay for human IGFBP-2 was developed using a polyclonal antiserum directed against a partial sequence (hIGFBP-2(176-190)). The tracer was prepared by radioiodination of a [Tyr]o-hIGFBP-2(176-190) derivative. The assay was used to study IGFBP-2 levels in numerous clinical and experimental situations. There was little circadian fluctuations of serum level which showed a marked age-dependence with high levels at birth and senescence and low levels during puberty. Decreased IGFBP-2 levels were present in untreated insulin-dependent diabetes mellitus (IDDM), in acromegaly and during dexamethasone suppression test. GH deficiency, fasting, IGF-I administration to patients with GH receptor deficiency, hepatic failure and insulinomas caused a moderate increase of serum IGFBP-2. Markedly elevated levels were found in chronic renal failure, non-islet cell tumour induced hypoglycemia and leukaemias. The fact that all possible relationships between insulin secretion and IGFBP-2 levels could be identified suggests that insulin is not a major regulator. In general, there was an inverse relationship with serum IGFBP-3 (except IDDM) and IGFBP-2 levels were high in situations where free IGF-II should be expected to be high. The tentative conclusion would therefore be that free IGF-II is a major regulator of circulating IGFBP-2.

Menkes disease is a lethal-X linked recessive disorder associated with copper metabolism disturbance. We have recently mapped two chromosome breakpoints related to this disease in a 1 megabase yeast artificial chromosome contig at Xq13.3. We now report the construction of a phage contig and the isolation of candidate partial cDNAs for the Menkes disease gene. The candidate gene expresses an 8 kb message in all investigated tissues, and deletions were detected in 16% of 100 unrelated Menkes patients. The deduced partial protein sequence shared the GMTCXXC motif with bacterial metal resistance operons, suggesting a potential heavy metal binding protein. These findings should lead to more accurate prenatal diagnosis of this severe disease and a better understanding of the cellular homeostasis of essential heavy metals.