Comprehensive genetic screening for vascular Ehlers–Danlos syndrome through an amplification-based next-generation sequencing system

Vascular Ehlers–Danlos syndrome (vEDS) is a hereditary connective tissue disorder (HCTD) characterized by arterial dissection/aneurysm/rupture, sigmoid colon rupture, or uterine rupture. Diagnosis is confirmed by detecting heterozygous variants in COL3A1. This is the largest Asian case series and the first to apply an amplification-based next-generation sequencing through custom panels of causative genes for HCTDs, including a specific method of evaluating copy number variations. Among 429 patients with suspected HCTDs analyzed, 101 were suspected to have vEDS, and 33 of them (32.4%) were found to have COL3A1 variants. Two patients with a clinical diagnosis of Loeys–Dietz syndrome and/or familial thoracic aortic aneurysm and dissection were also found to have COL3A1 variants. Twenty cases (57.1%) had missense variants leading to glycine (Gly) substitutions in the triple helical domain, one (2.9%) had a missense variant leading to non-Gly substitution in this domain, eight (22.9%) had splice site alterations, three (8.6%) had nonsense variants, two (5.7%) had in-frame deletions, and one (2.9%) had a multi-exon deletion, including two deceased patients analyzed with formalin-fixed and paraffin-embedded samples. This is a clinically useful system to detect a wide spectrum of variants from various types of samples.     

Congener-specific data on polychlorinated biphenyls in tissues of common porpoise from Puck Bay,Baltic Sea

Individual congeners of polychlorinated biphenyls (PCBs), including the highly toxic non-ortho coplanar 3,3,4,4-tetrachlorobiphenyl (IUPAC No. 77), 3,3,4,4,5-pentachlorobiphenyl (IUPAC No. 126), and 3,3,4,4,5,5-hexachlorobiphenyl (IUPAC No. 169), and their mono- and di-ortho analogs, have been identified and quantified in the blubber, liver, and muscles of three female common porpoise Phocoena phocoena collected from the Puck Bay (inner Gulf of Gdask, Poland) in 1989–1990, to elucidate actual concentrations and toxic potential. The total 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalent for 13 coplanar PCBs in blubber was 1,500±470 pg/g wet wt. 2,3,4,4,5-Pentachlorobiphenyl (IUPAC No. 118) was the most contributing individual and occupied between 57 and 67% in total toxic equivalent of coplanar PCBs in blubber, while 2,2,4,4,5,5-hexachlorobiphenyl (IUPAC No. 153), 2,3,3,4,4-pentachlorobiphenyl (IUPAC No. 105) and 2,2,3,4,4,5-hexachlorobiphenyl (IUPAC No. 138) comprised between 9.5–14, 7.6–11.5, and 7.2–11.0%, respectively (totally 82–95%), and 2,3,3,4,4,5-hexachlorobiphenyl (IUPAC No. 156) was absent. A potentially most toxic non-ortho PCB members such as 3,3,4,4,5-pentachlorobiphenyl, 3,3,4,4,5,5-hexachlorobiphenyl and 3,3,4,4-tetrachlorobiphenyl were only minor contributors, altogether occupying between 1.0 and 14.5% in total TEQ of coplanar PCBs. Concentrations of total PCBs in lipids of the blubber ranged from 26 to 47 g/g and were comparable or lower than reported earlier for common porpoises from the Baltic Sea, North Sea, and North Atlantic by other authors.     

Hyposensitivity of peripheral α-adrenoceptors in respiratordependent amyotrophic lateral sclerosis assessed by intravenous norepinephrine infusion

Intravenous norepinephrine infusion test was performed in eight patients with amyotrophic lateral sclerosis (ALS) supported by respirators and nine control subjects, to examine -adrenoceptor function of peripheral resistant blood vessels. Baseline plasma norepinephrine concentrations in ALS patients were significantly higher than those in control subjects, indicating basal sympathetic hyperactivity (normal 218.2 ± 59.7 pg/ml; ALS 450.0 ± 288.4 pg/ml). The stimulus-response curves in the patients were similar to those in control subjects, and there were no significant differences between mean gains of the stimulus—response curves in both groups (normal 18.7 ± 5.5; ALS 15.2 ± 11.2). However, three ALS patients, two of whom had circulatory fluctuation and sympathetic hyperactivity, revealed lower gain levels than the mean minus 2 SD in control subjects (4.7, 1.1 and 3.7). This indicates hyposensitivity or down-regulation of the -adrenoceptor function of peripheral blood vessels in these ALS patients. For early detection of sympathetic hyperactivity and prediction of circulatory collapse, it would be useful to measure the plasma norepinephrine concentration and the gain of the norepinephrine infusion curve in respirator-dependent ALS patients.     

Studies of effects of anticancer agents in combination with/without hyperthermia on metastasized human bladder cancer cells in chick embryos using the polymerase chain reaction technique

Cultivated T24 cells derived from a human bladder cancer were inoculated into the chorioallantoic membrane vein of chick embryos. Hyperthermic treatment was performed following injection of anticancer agents 3 days after the inoculation of the T24 cells. DNA samples were obtained from the livers of the chick embryos, and the polymerase chain reaction technique was used to amplify a DNA fragment specific to the human -globin gene. The Southern hybridization method was used to evaluate the inhibitory effects of anticancer agents in combination with/without hyperthermia on T24 cells metastasized to the liver. The hyperthermia exerted an inhibitory effect on the growth of the T24 cells in the livers of the chick embryos, and this was dependent on the thermal dose. The antitumor effects of hyperthermia performed at 42.5° C for 20 min and at 43.0° C for 10 min were evidenced by 69.2% an 82.0% inhibition of the growth of the metastasized T24 cells, respectively, as compared with the growth of untreated T24 cell. Hyperthermia performed at 42.5° C for 10 min alone produced 26.7% tumor growth inhibition, and these conditions for hyperthermia were subsequently used as a criterion for evaluating the effects of its combination with various anticancer agents. Adriamycin (20 g/egg) alone, mitomycin C (10 g/egg) alone, carboplatin (10 g/egg) alone, and cisplatin (10 g/egg) alone produced 13.5%, 58.9%, 27.3%, and 29.1% tumor growth inhibition, respectively. Adriamycin and mitomycin C applied in combination with hyperthermia showed additive inhibitory effects on the growth of the metastasized T24 cells in this chick embryo model.     

Studies on karyotype evolution in higher primates in relation to human chromosome 14 and 9 by comparative mapping of immunoglobulin C epsilon genes with fluorescence in situ hybridization

Karyotypic homologies in relation to human chromosome 14 and 9 were studied through comparative mapping of the immunoglobulin C epsilon genes in higher primates by fluorescence in situ hybridization (FISH) technique. The C epsilon genes will be suitable probes for the analysis of evolutionary rearrangements due to that the multiple recombinational events such as gene duplications and deletions have occurred repeatedly in the immunoglobulin CH gene family (IGH@) during the course of primate evolution. IGH@ locating on the terminal region of human chromosome 14 (HSA14), at band HSA14q32.33, has generated multiple pseudogenes and among subclasses of IGH@ the C epsilon genes have shown most dynamic changes with generating both truncated type (C epsilon 2) and processed type (C epsilon 3) pseudogenes. In this study, chromosomal homologies and rearrangements on HSA14 (C epsilon 1) and HSA9 (C epsilon 3) in relation to the evolutionary genesis of their primate homologous chromosomes in speciation were investigated by comparative mapping with FISH and chromosome painting (ZOO-FISH) techniques. Comparative mapping of the C epsilon 1 gene at HSA14q32.33 was carried out in seven species of nonhuman primates: common chimpanzee (PTR), pygmy chimpanzee (PPA), gorilla (GGO), orangutan (PPY), white-handed gibbon (HLA), agile gibbon (HAG), and Japanese macaque (MFU). The C epsilon 1 gene was assigned to the telomeric region of HSA14 homologues in each species, namely, PTR15q32, PPA15q32, GGO18q16, PPY15q32, HLA17qter, HAG17qter, and MFU7q29, respectively. These results suggested that HSA14 has high degree of syntenic organization with its primate homologues confirmed by ZOO-FISH. Concerning HSA9, comparative mapping of the C epsilon 3 gene at HSA9p24.2-->p24.1 was performed. The mapped positions indicated the HSA9 homologous regions detected by ZOO-FISH in each species, namely, PTR11q34, PPA11q34, GGO13q22, PPY13q16, HLA8qter, HAG8qter, and MFU14q22, respectively, suggesting that several dynamic chromosomal rearrangements including at least twice pericentric inversions have occurred during the course of hominoid evolution. The comparison of syntenic groups and painting results has provided a hypothesis of the evolutionary genesis of HSA9 and its homologues with defined breakpoints on the present chromosomes. Likewise, studies on karyotype evolution will be promoted by combining comparative mapping with ZOO-FISH that can more clearly define the chromosomal rearrangements among species.     

Polymorphisms of the human homologue of the Drosophila white gene are associated with mood and panic disorders

The Drosophila white gene is a member of the ATP-binding cassette (ABC) transporter superfamily and is involved in the cellular uptake of tryptophan. Its human homologue gene (hW) has been mapped to chromosome 21q22.3. Tryptophan is the precursor for the neurotransmitter serotonin, which has been implicated in the regulation of mood and anxiety. The locus 21q22.3 has also been reported to be associated with mood disorders. The 3'-untranslated region (3'-UTR) in the hW gene has been shown to contain a polymorphic poly(T) region. We have identified a new polymorphism G2457A in the 3'-UTR in the present study. We examined the relationship between these polymorphisms and mood and panic disorders, and a significant association between the poly(T) polymorphisms and mood disorders was detected (P=0.039 (allele frequency)). Associations were found between the polymorphisms and mood (poly(T) polymorphism: P=0.047 (allele frequency), G2457A: P=0.040 (allele frequency), P=0.044 (genotype frequency)) and panic disorders (G2457A: P=0.026 (allele frequency), P=0.011 (genotype frequency)) in males, but not in females. These findings suggest that the hW gene may be an important gene in the control of mood and anxiety as well as one of the genetic factors related to mood disorders and panic disorder in males. The statistical significance of the association remains relatively low and larger materials facilitating further dissection of the clinical phenotype will be needed to confirm and independently validate this finding and to evaluate its significance.     

Problems of intravenous lidocaine treatment in status epilepticus or clustering seizures in childhood

Lidocaine was administered intravenously as a substitute for diazepam, to 12 patients with status epilepticus or clustering seizures aged 26 days to 11 years. The medication was very effective in 3 cases with acute convulsions, which disappeared immediately after infusion of lidocaine without relapse. The medication was effective only temporarily in 4 patients; they experienced relapsing seizures during drip infusion of lidocaine intravenously for maintenance. All the relapsing seizures were secondarily generalized ones with diffuse ictal discharges. In 2 cases of localization-related epilepsy, complex partial seizures evolved to secondarily generalized seizures immediately after administration of lidocaine. It must be noticed that in a relatively large number of cases lidocaine is ineffective or even harmful.     

Total Esophagectomy versus Proximal Esophagectomy for Esophageal Cancer at the Cervicothoracic Junction

To investigate the adequate extent of esophagectomy and lymphadenectomy for an esophageal cancer localized at the cervicothoracic junction, the mortality and morbidity rates, survival rates, and patterns of recurrence were retrospectively analyzed in two groups—14 patients who underwent total esophagectomy with or without laryngectomy and 15 patients who underwent proximal esophagectomy with or without laryngectomy—at Kurume University Hospital from 1981 to 1996. Proximal esophagectomy with or without laryngectomy resulted in a lower hospital mortality rate and better overall survival for patients who underwent curative esophagectomy compared with total esophagectomy with or without laryngectomy. Multivariate analysis indicated that the extent of esophagectomy (total esophagectomy versus proximal esophagectomy) was not a prognostic factor. The incidence of recurrence was not different between the two groups. Lymph node metastasis or recurrence from such esophageal cancers was localized to the neck and upper mediastinum. For an esophageal cancer localized at the cervicothoracic junction, therefore, proximal esophagectomy with or without laryngectomy and with cervical and upper mediastinal lymphadenectomy could be better indicated for preselected patients.     

Multidisciplinary management of metastatic colorectal cancer

When colorectal cancer metastasizes to distant organs, usually multiple sites are involved and treatment consists primarily of systemic chemotherapy and supportive care. Chemotherapeutic agents effective against metastatic colorectal cancer include 5-fluorouracil, often used in combination with leucovorin or methotrexate, and irinotecan (CPT-11). Median survival with optimal chemotherapy regimens ranges from 10 to 15 months. Less frequently, colorectal cancer metastasizes only to the liver or lung. In a minority of these cases, surgical resection can be performed and results in a median survival of 28-46 months for hepatic resections and 24-25 months for pulmonary resections. Five-year survival rates range from 24 to 38% and 21 to 44% for hepatic and pulmonary resections, respectively. For isolated liver metastases that are not surgically resectable, other regional therapies that can be considered are hepatic cryosurgery, radiofrequency ablation, and hepatic arterial infusion chemotherapy. Median survival following cryosurgery is between 26 and 30 months, while median survival following radiofrequency ablation has not been established in large series. Hepatic arterial infusion chemotherapy, especially with newer combination drug regimens, may increase survival in patients with isolated liver metastases compared to systemic chemotherapy, but this must be confirmed in randomized, prospective trials. Colorectal cancer metastases to the brain can be treated with radiation therapy or surgical resection, but median survival with treatment is less than one year.