Inherited Copper Transport Disorders: Biochemical Mechanisms, Diagnosis, and Treatment

Copper is an essential trace element required by all living organisms. Excess amounts of copper, however, results in cellular damage. Disruptions to normal copper homeostasis are hallmarks of three genetic disorders: Menkes disease, occipital horn syndrome, and Wilson’s disease

Exon duplications in the ATP7A gene: Frequency and Transcriptional Behaviour

<p>Abstract</p> <p>Background</p> <p>Menkes disease (MD) is an X-linked, fatal neurodegenerative disorder of copper metabolism, caused by mutations in the <it>ATP7A </it>gene. Thirty-three Menkes patients in whom no mutation had been detected with standard diagnostic tools were screened for exon duplications in the <it>ATP7A </it>gene.</p> <p>Methods</p> <p>The <it>ATP7A </it>gene was screened for exon duplications using multiplex ligation-dependent probe amplification (MLPA). The expression level of <it>ATP7A </it>was investigated by real-time PCR and detailed analysis of the <it>ATP7A </it>mRNA was performed by RT-PCR followed by sequencing. In order to investigate whether the identified duplicated fragments originated from a single or from two different X-chromosomes, polymorphic markers located in the duplicated fragments were analyzed.</p> <p>Results</p> <p>Partial <it>ATP7A </it>gene duplication was identified in 20 unrelated patients including one patient with Occipital Horn Syndrome (OHS). Duplications in the <it>ATP7A </it>gene are estimated from our material to be the disease causing mutation in 4% of the Menkes disease patients. The duplicated regions consist of between 2 and 15 exons. In at least one of the cases, the duplication was due to an intra-chromosomal event. Characterization of the <it>ATP7A </it>mRNA transcripts in 11 patients revealed that the duplications were organized in tandem, in a head to tail direction. The reading frame was disrupted in all 11 cases. Small amounts of wild-type transcript were found in all patients as a result of exon-skipping events occurring in the duplicated regions. In the OHS patient with a duplication of exon 3 and 4, the duplicated out-of-frame transcript coexists with an almost equally represented wild-type transcript, presumably leading to the milder phenotype.</p> <p>Conclusions</p> <p>In general, patients with duplication of only 2 exons exhibit a milder phenotype as compared to patients with duplication of more than 2 exons. This study provides insight into exon duplications in the <it>ATP7A </it>gene.</p

Compound Heterozygous Mutations in SLC30A2/ZnT2 Results in Low Milk Zinc Concentrations: A Novel Mechanism for Zinc Deficiency in a Breast-Fed Infant.

Zinc concentrations in breast milk are considerably higher than those of the maternal serum, to meet the infant's requirements for normal growth and development. Thus, effective mechanisms ensuring secretion of large amounts of zinc into the milk operate in mammary epithelial cells during lactation. ZnT2 was recently found to play an essential role in the secretion of zinc into milk. Heterozygous mutations of human ZnT2 (hZnT2), including H54R and G87R, in mothers result in low (>75% reduction) secretion of zinc into the breast milk, and infants fed on the milk develop transient neonatal zinc deficiency. We identified two novel missense mutations in the SLC30A2/ZnT2 gene in a Japanese mother with low milk zinc concentrations (>90% reduction) whose infant developed severe zinc deficiency; a T to C transition (c.454T>C) at exon 4, which substitutes a tryptophan residue with an arginine residue (W152R), and a C to T transition (c.887C>T) at exon 7, which substitutes a serine residue with a leucine residue (S296L). Biochemical characterization using zinc-sensitive DT40 cells indicated that the W152R mutation abolished the abilities to transport zinc and to form a dimer complex, indicating a loss-of-function mutation. The S296L mutation retained both abilities but was extremely destabilized. The two mutations were found on different alleles, indicating that the genotype of the mother with low milk zinc was compound heterozygous. These results show novel compound heterozygous mutations in the SLC30A2/ZnT2 gene causing zinc deficiency in a breast-fed infant

Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRα) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRα protein level in the cell is low and the LXRα protein itself is very hard to detect. We have previously reported that the mRNA for LXRα is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRα protein in the human macrophage, we have established a monoclonal antibody against LXRα, K-8607. The binding of mAb K-8607 to the human LXRα protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRα antibody should prove to be a useful tool in the analysis of the human LXRα protein

Survey of Uterine Cervix Cancer Screening by Examination Car in Niigata Prefecture from 1995 to 2009

Based on the results obtained from 1995 to 2009, we explored the current activity statuses of the uterine cervix cancer screening by examination car conducted in Niigata Prefecture. A total of 318,580 women between age 20 and 85 (60,215 initial examinees including examinees who received cancer screening at interval of more than 3 or 5 years, and 258,365 re-examinees who received the screening more than twice within the past 3 or 5 years) were screened during this 15-year period. The mean consultation rate (proportion of examinees to the target population) was 4.10%, showing the highest rate 5.50% in 1996 and the lowest rate 1.98% in 2007. By the cancer screening, 255 cancer patients were detected in the 15 years (mean detection rate: 0.08%, range: 0.04% -1.30%). The mean cancer detection rate in the initial examinees (0.28%, 167 cases) was 8.1 times higher than that in the re-examinees (0.03%, 88 cases). Furthermore, the frequency of examinees diagnosed with dysplasia in the initial examinees was 5.7 times higher than that in the re-examinees (0.51% vs. 0.09%). Examinees requiring detailed examination (3.27%), or examinees diagnosed with dysplasia (0.18%), were detected at the highest rate in their twenties. However, the overall cancer detection rate in their twenties (0.17%) was the secondhighest during the study period, after that in their thirties (0.24%). The cancer detection rate leveled off at about 0.08% during the 15 years, and higher detection rates were found in the initial examinees, especially in their twenties and thirties. We believe the increase in cancer screening examinees of these generations is related to the increase in detection rates of dysplasia or cancer, and may be implicated in the future decrease in the cervical cancer death rate

Juvenile Granulosa Cell Tumor with an Unusual Clinical Course: A Late-onset and Late Recurrent Case

Juvenile granulosa cell tumors (JGCTs) are rare ovarian tumors with overall good prognoses. They differ from adult granulosa cell tumors (AGCTs), which are well known for late recurrence. Most JGCTs (～97%) occur in individuals G missense point mutation of FOXL2 gene (characteristic of AGCT but absent in JGCT) allowed differentiation from AGCT. This is the first comprehensive report of JGCT with late recurrence. Although rare, late recurrence of JGCT can occur; long-term surveillance is suggested

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4α (HNF4α; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4α causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4α in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4α protein, due, in part, to the limited availability of human HNF4α-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4α fusion proteins as the immunizing agent. The mouse anti-human HNF4α monoclonal antibody (K9218) generated against human HNF4α1/α2/α3 amino acids 3–49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4α proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4α protein, but HEK293 showed no expression of HNF4α protein. Nuclear-specific localization of the HNF4α protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4α protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4α protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4α in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4α mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4α and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4α isoforms in humans and in several other mammalian species

Bioorganic synthesis of a recombinant HIV-1 fusion inhibitor, SC35EK, with an N-terminal pyroglutamate capping group.

The bioorganic synthesis of an end-capped anti-HIV peptide from a recombinant protein was investigated. Cyanogen bromide-mediated cleavage of two Met-Gln sites across the target anti-HIV sequence generated an HIV-1 fusion inhibitor (SC35EK) analog bearing an N-terminal pyroglutamate (pGlu) residue and a C-terminal homoserine lactone (Hsl) residue. The end-capped peptide, pGlu-SC35EK-Hsl, had similar bioactivity and biophysical properties to the parent peptide, and an improved resistance to peptidase-mediated degradation was observed compared with the non-end-capped peptide obtained using standard recombinant technology

Maternal Undernutrition and Breast Milk Macronutrient Content Are Not Associated with Weight in Breastfed Infants at 1 and 3 Months after Delivery

This study examined whether maternal nutritional intake and breast milk macronutrient content influence the weight of breastfed infants. We investigated 129 healthy mothers with singleton babies born from July 2016 to December 2017 in a university hospital in Tokyo, Japan. Information was obtained by a self-administered food frequency questionnaire at 1 (valid response n = 92; mean age, 34 years) and 3 (n = 57) months after delivery. Breast milk was sampled at 1 and 3 months and the macronutrient contents were analyzed. The average pre-pregnancy body mass index and weight gain during pregnancy were 20.7 +/- 2.6 kg/m(2) and 9.6 +/- 3.7 kg, respectively. At 1 month, average maternal calorie intake was 1993 +/- 417 kcal/day, which was lower than the intake recommended by Japanese Dietary Reference Intakes for breastfeeding mothers. There were no significant differences with regard to maternal calorie and protein intake, and breast milk macronutrient content between breastfed infants with weight above and below the 25th percentile of its distribution at both 1 and 3 months. This study suggests that suboptimal calorie intake by breastfeeding mothers and breast milk macronutrient content were not associated with weight of their infants at 1 and 3 months after delivery

Radiation exposure and circulatory disease risk: Hiroshima and Nagasaki atomic bomb survivor data, 1950-2003

Objective To investigate the degree to which ionising radiation confers risk of mortality from heart disease and stroke