Estimation of plasma and saliva levels of coenzyme Q10 and influence of oral supplementation.

Coenzyme Q10 (CoQ(10)) levels in human saliva were measured by HPLC with a highly sensitive electrochemical detector (ECD) and a special concentration column. This HPLC system showed satisfactory analytical results within the standard range of 0.78-50 ng/ml. We also found a significant correlation between CoQ(10) levels in plasma and in saliva from parotid glands, while this correlation was lacking between plasma CoQ10 and CoQ10 in whole saliva. Unlike in plasma, there are some fluctuations of saliva CoQ(10) levels throughout the day. A good correlation was obtained by collecting parotid gland saliva at times between meals. The mean saliva CoQ(10) level for 55 healthy volunteers was 17.0 ng/ml (S.D. 6.8 ng/ml); approximately one fiftieth of that in plasma. Regarding the influence of oral supplementation, CoQ(10) was analyzed in plasma and parotid gland saliva from 20 healthy volunteers supplemented daily with 100 mg of CoQ(10) for the first week and 200 mg for the second. The plasma CoQ(10) levels of all volunteers increased to different extents in accordance with the CoQ(10) daily intake and the corresponding change in saliva showed almost the same trend.

Detection of HER-2/neu (c-erb B-2) DNA amplification in primary breast carcinoma. Interobserver reproducibility and correlation with immunohistochemical HER-2 overexpression.

BACKGROUND: Fluorescent in situ hybridization (FISH) has been shown to be one of the most reliable methods with which to estimate the status of the HER-2/neu (or c-erb B-2) oncogene at the DNA level. METHODS: To study interobserver reproducibility and to determine more clinically correlated criteria for HER-2/neu alterations, two observers independently estimated HER-2/neu DNA status. The correlation between the consensus HER-2/neu DNA status by FISH and HER-2/neu protein status detected by immunohistochemistry (IHC) using a polyclonal antibody was studied in 216 surgically resected breast carcinomas and 34 noncancerous tissues. RESULTS: According to the HER-2/CEP17 ratio and mean HER-2 copies per nucleus, agreement level of HER-2/neu amplification was shown to be nearly perfect between two observers (kappa statistic (kappa) = 0.94 and kappa = 0.84). Finally, 40 tumors (19%) were judged to have HER-2/neu DNA amplification, with 6 having low-level amplification (> or = 2 but < 3 folds) and 34 having high-level amplification (> or = 3 folds). One hundred seventy-six other tumors, including 3 tumors that only 1 of the observers determined to be low-level amplifiers, and 34 noncancerous tissues had no detected amplification. The DNA amplification status was concordant between invasive and intraductal components in 14 carcinomas. HER-2/neu protein overexpression of moderate (2+) or high (3+) intensity based on IHC was detected in 51 carcinomas (24%), and was 2+ in 20 carcinomas and 3+ in 31 carcinomas. The HER-2/CEP17 ratio of > or = 2 was concordant with IHC findings of 2+/3+ in 91% of carcinomas (195 of 215 carcinomas), with a sensitivity of 70% (35 of 50 carcinomas) and a specificity of 97% (160 of 165 carcinomas). High-level amplification was detected in 29 of 31 IHC 3+ cases (94%), but in only 5 of 20 IHC 2+ cases (25%) and 0 in 165 IHC 0/1+ cases. All 34 cases with high-level amplification showed an IHC score of 3+ (29 cases) or an IHC score of 2+ (5 cases), but only 1 case was found to have an IHC score of 3+ and the remainder were IHC 0/1+ in 6 low-amplification cases. The concordance rate of the high-level amplification with an IHC score of 3+ was 97% (208 of 215 cases), with a sensitivity of 94% (29 of 31 cases) and a specificity of 97% (179 of 184 cases). CONCLUSIONS: The results of the current study indicated that high-level HER-2/neu amplification and an IHC score of 3+ nearly optimally identified breast carcinomas with clinically and biologically significant HER-2/neu activation. Conversely, it was confirmed that careful interpretation of test results is required in the case of low-level amplification and/or an IHC score of 2+.

Dopa-responsive dystonia simulating spastic paraplegia due to tyrosine hydroxylase (TH) gene mutations.

Spastic paraplegia is not widely recognized to occur in dopa-responsive dystonia (DRD). The authors found a compound heterozygote for novel mutations of the human tyrosine hydroxylase (TH) gene (TH). The patient was initially diagnosed as having spastic paraplegia, but responded completely to levodopa therapy. Exercise-induced stiffness in the patient's father, who had a TH deletion, also responded to levodopa. The data expand the clinical spectrum of TH deficiency and suggest that TH mutations may account for some patients with DRD simulating spastic paraplegia.

Clinical and genetic studies of CLCN5 mutations in Japanese families with Dent's disease.

BACKGROUND: Dent's disease is an X-linked renal tubular disorder that is characterized by low molecular weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. The disease is caused by inactivation of a renal chloride channel gene, CLCN5, that encodes a 746-amino acid protein with 12 to 13 transmembrane domains. The Japanese variant of Dent's disease has been observed to be less severe, and we have investigated two unrelated Japanese families for CLCN5 mutations. METHODS: Six patients from two unrelated families were studied. Leukocyte DNA from probands was used with CLCN5-specific primers for polymerase chain reaction (PCR) amplification of the coding region and exon-intron boundaries, and the DNA sequences of the products were determined to identify abnormalities in the gene. RNA extracted from the kidney, leukocytes, or urine sediments was used to characterize further the effects of the identified mutations. RESULTS: beta2-microglobulinuria was detected in five patients, hypercalciuria in two patients, nephrolithiasis in three patients (2 of whom were females), and one 51-year-old man had renal failure. Two novel CLCN5 mutations consisting of an a to g transition at the invariant ag acceptor splice site of intron 5 and an intragenic deletion that encompassed the region between intron 3 and intron 6 were identified. The acceptor splice site mutation led to the utilization of two alternative cryptic splice sites in exon 6 that resulted in a frameshift or skipping of the exon 6. The deletional mutation, which resulted in a loss of exons 4, 5, and 6, is predicted to lead to a loss of domains 1 through 4. Both mutations predict truncated chloride channels that are likely to result in a functional loss. CONCLUSIONS: The observations of renal failure in one male and nephrolithiasis in two females represent important new findings in this Japanese variant of Dent's disease that is associated with CLCN5 mutations. In addition, our study is the first to demonstrate the use of urinary sediment cells and renal tissue for the detection of CLCN5 transcript abnormalities. These results help to expand the spectrum of CLCN5 mutations associated with Dent's disease.

Comparison of gyrA and parC mutations and resistance levels among fluoroquinolone-resistant isolates and laboratory-derived mutants of oral streptococci.

Laboratory-derived fluoroquinolone-resistant mutants were obtained by serial passage of Streptococcus sanguis and Streptococcus anginosus isolates on agar containing increasing concentrations of old and new fluoroquinolones, ofloxacin and DU-6859a, respectively. Sequencing of an S. sanguis isolate exposed to DU-6859a showed that resistance was associated with two mutations in the quinolone resistance determining region (QRDR) of the gyrA gene (Ser83-->Phe; Glu87-->Lys), and with a mutation in the parC gene (Ser79-->Ile). However, different mutations in the gyrA gene (Ser83-->Tyr) and parC gene (Ser79-->Phe) were found in a S. sanguis isolate exposed to ofloxacin. A fluoroquinolone-resistant isolate, QR-95101, from a dental infection, had a single mutation in the gyrA gene (Ser83-->Phe) and in the parC gene (Ser79-->Phe). Two fluoroquinolone-resistant mutants, QS-701OFm and QS-701DUm, were obtained from S. anginosus QS-701, by exposure to ofloxacin and DU-6859a, respectively. These mutants showed a common substitution at codon 83 (Ser-->Phe) in the gyrA gene but had different substitutions at codon 87 (QS-701OFm, Glu-->Gln; QS-701DUm, Glu-->Lys). They also had different substitutions at codons 79 and 135 in the parC gene (QS-701OFm, Ser79-->Leu but no change at Glu135; QS-701DUm, Ser79-->Ile and Glu135-->Gln). The resistance levels of the DU-6859a-selected resistant S. sanguis mutant QS-951DUm to DU-6859a, ofloxacin, ciprofloxacin and norfloxacin were higher than those of the ofloxacin-selected resistant mutant QS-951OFm. However, ampicillin susceptibilities of these mutants were not different from the parental strains. In S. anginosus, the DU-6859a-selected fluoroquinolone-resistant mutant QS-701DUm was resistant to all the fluoroquinolones tested, while the ofloxacin-selected mutant QS-701OFm was resistant to three fluoroquinolones, but not DU-6859a. The results indicate that different fluoroquinolones select distinct mutations in the QRDR of the gyrA and parC genes in oral streptococci. The gyrA or parC mutation in oral streptococci may determine the levels of fluoroquinolone resistance.

Familial pulmonary Mycobacterium avium complex disease.

We report two Japanese families affected by pulmonary Mycobacterium avium complex (MAC) disease, involving an older brother and younger sister in one family and two brothers in the second family. We investigated whether defects in the natural resistance-associated macrophage protein gene (NRAMP1) underlay susceptibility to MAC in these cases. All of the patients had computed tomographic findings of peripheral nodules and bronchiectasis. Pulse-field gel electrophoresis patterns of mycobacterial genomic DNA restriction fragments revealed that none of the MAC strains isolated from the patients was epidemiologically related to any of the others. Direct sequencing of the complementary DNA of the patients' NRAMP1 revealed a nonconservative missense mutation at codon 419 in one patient, which was heterozygous and was not seen in his affected sibling. No variations similar to those found in mice that show susceptibility to MAC were found. The results suggest an underlying genetic defect in host defense rather than exposure to an unusually virulent strain of MAC as the pathogenetic factor in MAC disease; however, alterations in the coding region of NRAMP1 do not appear to explain the susceptibility to MAC.

Dopa-responsive dystonia due to a large deletion in the GTP cyclohydrolase I gene.

Although it is assumed that most patients with autosomal dominant dopa-responsive dystonia (DRD) have a GTP cyclohydrolase I dysfunction, conventional genomic DNA sequencing of the gene (GCH1) coding for this enzyme fails to reveal any mutations in about 40% of DRD patients, which makes molecular genetic diagnosis difficult. We found a large heterozygous GCH1 deletion, which cannot be detected by the usual genomic DNA sequence analysis, in a three-generation DRD family and conclude that a large genomic deletion in GCH1 may account for some "mutation-negative" patients with dominantly inherited DRD.

Mutations of the WASP gene in 10 Japanese patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency, and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study, we identified mutations of the WASP gene in 10 Japanese patients from 9 unrelated families with WAS/XLT. All XLT patients (n = 3) and one WAS patient had a missense mutation at the PH domain of WASP. Two WAS patients had nonsense mutations. One WAS patient had exon 8 skipping caused by one nucleotide deletion at the acceptor site of intron 7. Three WAS patients had genomic deletions; one of the three had a large genomic deletion involving exons 3 to 7. Codons 45 and 86 seem to be the hot spots of the WASP mutation, because missense mutations in these codons have been reported previously in several WAS/XLT patients in addition to the patients in this report, and patients with the same mutation show a similar clinical phenotype. All other mutations are novel, indicating that the mutations of WASP are heterogeneous. EB virus-transformed cell lines from XLT patients expressed nearly normal amounts of WASP, whereas those from typical WAS patients expressed almost undetectable amounts of WASP. We conclude that the analysis of gene mutation and protein expression of WASP are useful together in assessing the severity of WAS.

Striatal biopterin and tyrosine hydroxylase protein reduction in dopa-responsive dystonia.

OBJECTIVE: To determine the mechanism leading to striatal dopamine (DA) loss in dopa-responsive dystonia (DRD). BACKGROUND: Although mutations in the gene GCH1, coding for the tetrahydrobiopterin (BH4) biosynthetic enzyme guanosine triphosphate-cyclohydrolase I, have been identified in some patients with DRD, the actual status of brain BH4 (the cofactor for tyrosine hydroxylase [TH]) is unknown. METHODS: The authors sequenced GCH1 and measured levels of total biopterin (BP) and total neopterin (NP), TH, and dopa decarboxylase (DDC) proteins, and the DA and vesicular monoamine transporters (DAT, VMAT2) in autopsied brain of two patients with typical DRD. RESULTS: Patient 1 had two GCH1 mutations but Patient 2 had no mutation in the coding region of this gene. Striatal BP levels were markedly reduced (<20% of control subjects) in both patients and were also low in two conditions characterized by degeneration of nigrostriatal DA neurons (PD and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated primate), whereas brain NP concentrations were selectively decreased (<45%) in the DRD patients. In the putamen, both DRD patients had severely reduced (<3%) TH protein levels but had normal concentrations of DDC protein, DAT, and VMAT2. CONCLUSIONS: The data suggest that 1) brain BH4 is decreased substantially in dopa-responsive dystonia, 2) dopa-responsive dystonia can be distinguished from degenerative nigrostriatal dopamine deficiency disorders by the presence of reduced brain neopterin, and 3) the striatal dopamine reduction in dopa-responsive dystonia is caused by decreased TH activity due to low cofactor concentration and to actual loss of TH protein. This reduction of TH protein, which might be explained by reduced enzyme stability/expression consequent to congenital BH4 deficiency, can be expected to limit the efficacy of acute BH4 administration on dopamine biosynthesis in dopa-responsive dystonia.

A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR.

The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion.

The polymorphic 43Thr bcl-2 protein confers relative resistance to autoimmunity: an analytical evaluation.

We have found a novel polymorphic (Ala43Thr; ACC-->GCC) bcl-2 allele in a Japanese population. An in vitro expression study with a mouse IL-7-dependent pre-B cell line has revealed that inhibition of the programmed cell death function of 43Thr bcl-2 protein is suppressed compared with that of normal 43Ala bcl-2 protein. Since bcl-2 expression in B-lymphoid cells elicits autoimmune disease in mice, we have investigated the possibility of whether a bcl-2 polymorphism has a different susceptibility to autoimmune disease. To evaluate the clinical impact of this polymorphism, the frequency of bcl-2 polymorphism was investigated in 221 children with insulin-dependent diabetes mellitus (IDDM), 237 adults with autoimmune disease (105 with rheumatoid arthritis, 57 with systemic lupus erythematosus, 55 with Sjögren's syndrome, and 20 others), and 290 healthy Japanese children and adults. The frequency of the 43Thr bcl-2 allele, either homozygous or heterozygous, was 14.5% in normal controls, 6.8% (P<0.01) in children with IDDM, and 8.0% (P<0.025) in adults with autoimmune disease. These results suggest that the 43Thr allele of bcl-2 confers resistance to autoimmune disease. The different anti-apoptotic function resulting from the different expression of bcl-2 protein in lymphocytes seems to be associated with the development of autoimmune disease, indicating that the bcl-2 gene affects human autoimmune disease.

Dystonia with motor delay in compound heterozygotes for GTP-cyclohydrolase I gene mutations.

Mutations in the GTP-cyclohydrolase I (GCH) gene have been identified as a cause of two disorders: autosomal dominant hereditary progressive dystonia/dopa-responsive dystonia (HPD/DRD) and autosomal recessive GCH-deficient hyperphenylalaninemia (HPA). Detailed clinical descriptions and genetic analysis of patients with phenotypes intermediate between HPD/DRD (mild) and GCH-deficient HPA (severe) have not been reported. We conducted genomic DNA sequencing of the GCH gene in two patients (Cases 1 and 2) manifesting generalized dystonia responsive to levodopa and severe developmental motor delay. In the pedigree of Patient 1, there were HPD/DRD patients in three generations preceding the index case. Patients 1 and 2 were compound heterozygotes with maternally and paternally transmitted mutations in the coding region of the GCH gene. In both compound heterozygotes, tetrahydrobiopterin (BH4) levels in cerebrospinal fluid were lower than those in HPD/DRD. Administration of BH4, in addition to levodopa, further improved the symptomatology of Patient 1. Our data demonstrate a new phenotype of GCH deficiency associated with compound heterozygosity for GCH gene mutations and suggest the usefulness of combined BH4 and levodopa therapy for this disorder.

Oculocerebrorenal syndrome of Lowe: three mutations in the OCRL1 gene derived from three patients with different phenotypes.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked multisystem disorder with major abnormalities of eyes, nervous system, and kidneys. Clinical manifestations include congenital cataract, mental retardation, and renal tubular dysfunction. A gene (OCRL1) responsible for OCRL was identified by positional cloning and its product OCRL-1 protein was shown to be a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] 5-phosphatase localized to the Golgi apparatus. We describe three mutations in OCRL1, one in a patient with severe phenotype and two in patients with moderate phenotype (degree of mental retardation and musculoskeletal abnormalities). The patient with severe phenotype had a G-to-A transition at nucleotide (nt) 1,739, causing an Arg-to-Gln substitution at amino acid 577, and one patient with moderate phenotype had a C-to-G transversion at nt 1,812, leading to a His-to-Gln substitution at amino acid 601. Both Arg-577 and His-601 are encoded by exon 15 and are probably important for proper function of this protein, since these are conserved in various enzymes catalyzing dephosphorylation of inositol compounds. In the other patient with the moderate phenotype, there was a G-to-A transition at nt 2,797 located at the 3'-end of exon 22. This substitution led to a skip of the same exon as well as conversion of codon-930 from GCT (Ala) to ACT (Thr) in the normal-size transcript. When we measured the enzyme activity in skin fibroblasts from the three patients, the activity was less than 10%, compared to findings in normal controls. Western blot analysis showed absence or severe decrease in OCRL-1 protein in cell lysates derived from these patients.

Recombinant adeno-associated virus-mediated gene transfer into human leukemia cell lines.

Adeno-associated virus (AAV)-based vector is a promising gene transfer vehicle by virtue of the characteristics of wild-type AAV:tropism to a wide range of human tissues and locus-specific integration at chromosome 19q13.3. To elucidate the nature of the recombinant AAV (rAAV), transduction of neomycin phosphotransferase enzyme gene (NeoR gene) into seven human leukemia cell lines was performed. Transduction efficiencies were assessed by colony formation assay and limiting dilution assay. The results suggested that both assays are comparable. Transduction efficiencies of the NeoR gene into K-562, MEG-O1, Raji, MOLT-3, HL-60, U937 and NKM-1 at a multiplicity of infection (MOI) of 0.1 were 0.27, 0.25, 0.015, 0.009, < 0.0025 and < 0.0025%, respectively. After purification and concentration of rAAV, 27% efficiency was observed in K562 at an MOI of 7 and a linear relationship between MOI and efficiency was confirmed, suggesting that this system may be useful for gene transduction into leukemia cells. Integration of the NeoR gene into the host genome was detected by Southern blotting analysis, which showed various sizes of digested fragments. A fluorescent in situ hybridization (FISH) study was carried out on 11 clones, in all of which the NeoR gene was integrated out of chromosome 19q13.3. In five of the clones, whole chromosome painting probes revealed that the integration sites were chromosomes 1q, 2q, 2q, 11p, 12p and 13q.

Gender-related penetrance and de novo GTP-cyclohydrolase I gene mutations in dopa-responsive dystonia.

We evaluated the influence of gender on penetrance of GTP-cyclohydrolase I (GCH) gene mutations in hereditary progressive dystonia/dopa-responsive dystonia (HPD/DRD) and determined whether some apparently sporadic HPD/DRD patients owe their disorder to a de novo mutation of the GCH gene. Previous clinical investigations of HPD/DRD have shown a predominance of affected women, with approximately half of HPD/DRD patients being sporadic. We conducted genomic DNA sequencing of the GCH gene in five HPD/DRD families having at least two generations of affected members and in four apparently sporadic cases and all of their parents. In the nine HPD/DRD pedigrees, we found independent mutations of the GCH gene (five deletions, one insertion, one nonsense mutation, and two point mutations at splice acceptor sites). The female-to-male ratio of the HPD/DRD patients was 4.3 with the penetrance of GCH gene mutations in women being 2.3 times higher than that in men (87% versus 38%, p = 0.026). There was no significant difference in the penetrance between maternally and paternally transmitted offspring. All of the four sporadic cases had de novo mutations because none of their parents were carriers. The results demonstrate gender-related incomplete penetrance of GCH gene mutations in HPD/DRD and suggest that this may not be due to genomic imprinting. Our data also suggest a relatively high spontaneous mutation rate of the GCH gene in this autosomal dominant disorder.

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.

Familial lethal inheritance of a mutated paternal gene in females causing X-linked ornithine transcarbamylase (OTC) deficiency.

A Leu148Phe substitution of the ornithine transcarbamylase (OTC) gene was identified in a 2-year-old girl with OTC deficiency (14% of control). Her two elder sisters died in childhood of hyperammonemia, and the patient also died of OTC deficiency. Enzyme activity in Cos1 cells transfected by the mutant cDNA was undetectable, thereby indicating a definite pathogenic mutation. Familial gene analysis showed that the mother had wild-type OTC alleles on both X-chromosomes and the father was a mosaic for the mutant allele in his lymphocytes and spermatozoa. This clinical case shows that a somatic and germline mosaicism for a single-gene disorder led to an unusual pattern of X-linked inheritance in the family, and all three daughters in the family died of OTC deficiency. The possibility that inherited factors will lead to skewed X-inactivation needs to be considered.