Distribution of copper in the Atlantic and Pacific Oceans using green turtles (Chelonia mydas) as a bioindicator.

Marine pollution by trace elements is a global concern due to potential toxicity to species and ecosystems. Copper is a fundamental trace element for many organisms; however, it becomes toxic at certain concentrations. The green turtle (Chelonia mydas) is a good sentinel species, due to its circumglobal distribution, long life cycle, coastal habits when juvenile, and is subject to environmental pollution. Quantifying and comparing copper levels makes it possible to understand the availability of this trace element in nature. During this research, comparisons were made between the levels of copper found in the liver, kidneys, and muscles of 35 turtles, from the United States (Hawaii and Texas), Brazil, and Japan. Copper was found in all specimens. In the liver, animals from Hawaii (91.08 µg g-1), Texas (46.11 µg g-1), and Japan (65.18 µg g-1) had statistically equal means, while those from Brazil (16. 79 µg g-1) had the lowest means. For the kidney, copper means were statistically equal for all Hawaii (3.71 µg g-1), Texas (4.83 µg g-1), Japan (2.47 µg g-1), and Brazil (1.89 µg g-1). In muscle, the means between Texas (0.75 µg g-1) and Japan (0.75 µg g-1) were the same, and the mean for Brazil (0.13 µg g-1) was the lowest. Among the organs, the highest levels of copper were found in the liver (28.33 µg g-1) followed by the kidney (2.25 µg g-1) and with the lowest levels in the muscle (0.33 µg g-1). This is the first study of copper levels among marine vertebrates in distant parts of the globe using similar comparative filters between different locations. Similar levels in turtles from such distant locations may indicate that there is a pantropical pattern of copper distribution in the biota, and that these animals are subject to the process of bioavailability of this metal in the environment and metabolic regulation.

Pantropical distribution of zinc in green turtles (Chelonia mydas): marine vertebrates as sentiel species.

Pollution is one of the biggest threats to marine life and trace elements are among the most toxic pollutants in this environment. Zn is an essential trace element for biota but becomes toxic at high concentrations. Sea turtles are good bioindicators of trace element pollution, due to their longevity and cosmopolitan distribution that allow bioaccumulation for years in their tissues. Determining and comparing Zn concentrations in sea turtles from faraway places is relevant for conservation due to the lack of knowledge of geographically broader distribution patterns of Zn in vertebrates. In this study, comparative analyses of bioaccumulation in the liver, kidney, and muscles of 35 C. mydas from Brazil, Hawaii, the USA (Texas), Japan, and Australia of statistically equal sizes were performed. Zn was found in all specimens, with the highest levels in the liver and kidneys. Specimens from Australia (30.58 µg g-1), Hawaii (31.91 µg g-1), Japan (29.99 µg g-1), and the USA (33.79 µg g-1) showed statistically equal means in the liver. Kidney levels were the same in Japan (35.09 µg g-1) and the USA (37.29 µg g-1) and the same in Australia (23.06 µg g-1) and Hawaii (23.31 µg/g). Specimens from Brazil had the lowest means in both organs (12.17 µg g-1 in the liver and 9.39 µg g-1 in the kidney). The pattern of equal Zn values for most specimens in the liver is an important finding, demonstrating that there are pantropical patterns in the distribution of this metal even in regions so far from each other. A possible explanation is due to the essential nature of this metal linked to metabolic regulation, in addition to the bioavailability for biological absorption in marine environments, such as RS in Brazil, with a lower standard bioavailability also found in other organisms. Therefore, factors such as metabolic regulation and bioavailability indicate that there is a pantropical distribution of Zn in marine biota and green turtles can be a useful model as a sentinel species.

New host records of three Kudoa spp. (K. yasunagai, K. thalassomi, and K. igami) with notable variation in the number of shell valves and polar capsules in spores.

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

Cadmium in tissues of green turtles (Chelonia mydas): A global perspective for marine biota.

Cadmium (Cd) is a metal of toxicological interest because of its potential high toxicity to organisms and ability to biomagnify. Evaluating concentrations of Cd in organisms on a large spatial scale can provide insights to its global distribution. This study examined Cd concentrations in kidney and liver tissues of 137 specimens of green turtles (Chelonia mydas) collected in Australia, Brazil, Hawaii, Japan, and the continental United States (Gulf of Mexico). We used comparative analyses of kidney and liver of 35 individuals, of which seven turtles from each locality belong to the same size class for comparison purposes between their ocean of origin. Cd was detected in all samples, with the highest bioconcentration in kidneys. Specimens originating from the Pacific Ocean had significantly higher mean Cd levels in liver (13.24 µg/g) and kidney (34.17 µg/g) than the specimens collected in the Atlantic Ocean with lower mean values in liver (1.00 µg/g) and kidney (4.04 µg/g). Furthermore, Cd concentrations in turtle tissues were generally greater than concentrations found in other marine organisms, for example dolphins. This result was unexpected because dolphins occupy a higher trophic level than green turtles which are only herbivorous. A possible explanation is a change in feeding habits of green turtles, in which juveniles feed in near shore habitats, potentially resulting in greater Cd accumulation in juveniles compared to adults. This global distribution trend has also been observed in other marine organisms (e.g., insects, birds, and mammals) and indicates that global factors may be more important than regional factors in determining Cd concentrations of marine organisms. CAPSULE: Global factors are more relevant than local factors in the distribution of cadmium in biota, using green turtle as a sentinel species.

Morphological and molecular genetic characterization of Kudoa konishiae n. sp. (Myxosporea: Multivalvulida) in the muscle of Japanese Spanish mackerel (Scomberomorus niphonius).

The Japanese Spanish mackerel (Scomberomorus niphonius; Perciformes: Scombridae) is widely distributed in the continental shelves of the northwestern Pacific Ocean around Japan, Sea of Japan, and East China Sea. In the present study, small, spindle-shaped myxosporean plasmodia (0.15-0.53 mm by 0.04-0.13 mm) were frequently encountered in the myofiber of trunk muscles of two Japanese Spanish mackerels; one fished in the Sea of Japan off western Japan and the other in the northwestern Pacific Ocean off southern Japan in the autumn of 2016. Isolated myxospores of Kudoa konishiae n. sp. (Myxosporea: Multivalvulida) from these two fish were stellate with six equal shell valves and polar capsules, 8.1-9.7 µm in width, 7.1-8.8 µm in thickness, and 7.1-8.8 µm in length. The polar capsules were teardrop-shaped, 2.7-4.7 µm by 1.2-2.5 µm. The lateral view of spores revealed a drawstring-pouch shape. The nucleotide sequences of the 18S and 28S ribosomal RNA gene (rDNA) were distinct from any recorded species. Phylogenetic trees demonstrated a close relationship of the present new species with Kudoa spp. with stellate spores with five or more shell valves/polar capsules, recorded in scombrid fishes. To clarify the phylogenetic relationships between three closely related species, i.e., Kudoa konishiae n. sp., Kudoa hexapunctata, and Kudoa neothunni, three mitochondrial DNA genes (cytochrome c oxidase subunit 1 gene (cox-1) and the small and large subunits of the ribosomal RNA gene (rns-rnl)) of two isolates of the new species, six isolates of K. hexapunctata, and 13 isolates of K. neothunni were sequenced. The interspecific and intraspecific variations of the newly obtained cox-1 and rns-rnl nucleotide sequences of K. hexapunctata, K. neothunni, and K. konishiae n. sp. were clarified for the first time.

Rapid detection of gene mutations responsible for non-syndromic aortic aneurysm and dissection using two different methods: resequencing microarray technology and next-generation sequencing.

Aortic aneurysm and/or dissection (AAD) is a life-threatening condition, and several syndromes are known to be related to AAD. In this study, two new technologies, resequencing array technology (ResAT) and next-generation sequencing (NGS), were used to analyze eight genes associated with syndromic AAD in 70 patients with non-syndromic AAD. Eighteen sequence variants were detected using both ResAT and NGS. In addition one of these sequence variants was detected by ResAT only and two additional variants by NGS only. Three of the 18 variants are likely to be pathogenic (in 4.3% of AAD patients and in 8.6% of a subset of patients with thoracic AAD), highlighting the importance of genetic analysis in non-syndromic AAD. ResAT and NGS similarly detected most, but not all, of the variants. Resequencing array technology was a rapid and efficient method for detecting most nucleotide substitutions, but was unable to detect short insertions/deletions, and it is impractical to update custom arrays frequently. Next-generation sequencing was able to detect almost all types of mutation, but requires improved informatics methods.

Exome sequencing reveals a homozygous SYT14 mutation in adult-onset, autosomal-recessive spinocerebellar ataxia with psychomotor retardation.

Autosomal-recessive cerebellar ataxias (ARCAs) are clinically and genetically heterogeneous disorders associated with diverse neurological and nonneurological features that occur before the age of 20. Currently, mutations in more than 20 genes have been identified, but approximately half of the ARCA patients remain genetically unresolved. In this report, we describe a Japanese family in which two siblings have slow progression of a type of ARCA with psychomotor retardation. Using whole-exome sequencing combined with homozygosity mapping, we identified a homozygous missense mutation in SYT14, encoding synaptotagmin XIV (SYT14). Expression analysis of the mRNA of SYT14 by a TaqMan assay confirmed that SYT14 mRNA was highly expressed in human fetal and adult brain tissue as well as in the mouse brain (especially in the cerebellum). In an in vitro overexpression system, the mutant SYT14 showed intracellular localization different from that of the wild-type. An immunohistochemical analysis clearly showed that SYT14 is specifically localized to Purkinje cells of the cerebellum in humans and mice. Synaptotagmins are associated with exocytosis of secretory vesicles (including synaptic vesicles), indicating that the alteration of the membrane-trafficking machinery by the SYT14 mutation may represent a distinct pathomechanism associated with human neurodegenerative disorders.

[Acute lymphoblastic leukemia in a pediatric patient with Marfan's syndrome].

We report a rare case of acute lymphoblastic leukemia (ALL) in a 7-year-old boy with Marfan's syndrome. He was diagnosed as having Marfan's syndrome by clinical findings at the age of 2 years, and the diagnosis was confirmed by the detection of gene mutation in FBN1. He was referred to our hospital because of the swelling of cervical lymph nodes at the age of 7 years. Findings on bone marrow examination demonstrated T lymphoblastic ALL. He obtained complete remission after induction therapy, and had no serious side effects including cardiotoxicity during chemotherapy. He has remained in continuous complete remission for 34 months following diagnosis. To our knowledge, only three cases of leukemia in patients with Marfan's syndrome were reported previously. We speculate that increased activity of TGF-ß, which is known as a tumor suppressor factor, in patients with Marfan's syndrome may diminish the risk of developing leukemia, although such a thesis was not proven in this case.

De novo 19q13.42 duplications involving NLRP gene cluster in a patient with systemic-onset juvenile idiopathic arthritis.

Systemic-onset juvenile idiopathic arthritis (s-JIA) is a rare inflammatory disease classified as a subtype of chronic childhood arthritis, manifested by spiking fever, erythematous skin rash, pericarditis and hepatosplenomegaly. The genetic background underlying s-JIA remains poorly understood. To detect disease-related copy number variations (CNVs), we performed single-nucleotide polymorphism array analysis in 50 patients with s-JIA. We detected many CNVs, but most of them were inherited from either of normal-phenotype parents. However, in one patient, we could identify two de novo microduplications at 19q13.42 with the size of 77 and 622 kb, separated by a 109-kb segment of normal copy number. The duplications encompass NLRP family (NLRP2, NLRP9 and NLRP11) as well as IL11 and HSPBP1, all of which have an important role in inflammatory pathways. These genes may significantly contribute to the pathogenesis of s-JIA.

Functional characterization of the zebrafish WHSC1-related gene, a homolog of human NSD2.

Methylation of specific lysine residues of histone H3 and H4 has been reported to be important in the structuring of chromatin and for the transcription of certain genes. Proteins with SET domains have been shown to methylate specific lysine residues of histone H3 and H4. We isolated a SET domain-containing gene from the zebrafish (Danio rerio). The gene has the highest sequence similarity to human NSD2, also known as Wolf-Hirschhorn syndrome candidate 1 or WHSC1, and therefore, was named DrWhsc1. DrWhsc1 mRNA is expressed in various tissues with the highest level in testis. Morpholino oligonucleotides for the DrWhsc1 gene affected early embryogenesis in zebrafish, such as endbrain enlargement, abnormal cartilage, marked reduction of bone, and incomplete motor neuron formation. Such developmental abnormalities are also observed in Wolf-Hirschhorn syndrome patients and Whsc1-deficient mice. In addition, suppression of the DrWhsc1 gene or defect in the SET domain of DrWhsc1 resulted in impairment of di-methylation of histone H3K36 at early embryogenesis. These results indicate that DrWhsc1 is a functional homolog of WHSC1 and that the SET domain of DrWhsc1 is essential for di-methylation of histone H3K36 in zebrafish.

Analysis of an insertion mutation in a cohort of 94 patients with spinocerebellar ataxia type 31 from Nagano, Japan.

Spinocerebellar ataxia type 31 (SCA31) is a recently defined subtype of autosomal dominant cerebellar ataxia (ADCA) characterized by adult-onset, pure cerebellar ataxia. The C/T substitution in the 5'-untranslated region of the puratrophin-1 gene (PLEKHG4) or a disease-specific haplotype within the 900-kb SCA31 critical region just upstream of PLEKHG4 has been used for the diagnosis of SCA31. Very recently, a disease-specific insertion containing penta-nucleotide (TGGAA)(n) repeats has been found in this critical region in SCA31 patients. SCA31 was highly prevalent in Nagano, Japan, where SCA31 accounts for approximately 42% of ADCA families. We screened the insertion in 94 SCA31 patients from 71 families in Nagano. All patients had a 2.6- to 3.7-kb insertion. The size of the insertion was inversely correlated with the age at onset but not associated with the progression rate after onset. (TAGAA)(n) repeats at the 5'-end of the insertion were variable in number, ranging from 0 (without TAGAA sequence) to 4. The number of (TAGAA)(n) repeats was inversely correlated to the total size of the insertion. The number of (TAGAA)(n) repeats was comparatively uniform within patients from the three endemic foci in Nagano. Only one patient, heterozygous for the C/T substitution in PLEKHG4, had the insertions in both alleles; they were approximately 3.0 and 4.3 kb in size. Sequencing and Southern hybridization using biotin-labeled (TGGAA)(5) probe strongly indicated that the 3.0-kb insertion, but not the 4.3-kb insertion, contained (TGGAA)(n) stretch. We also found that 3 of 405 control individuals (0.7%) had the insertions from 1.0 to 3.5 kb in length. They were negative for the C/T substitution in PLEKHG4, and neither of the insertions contained (TGGAA)(n) stretch at their 5'-end by sequencing. The insertions in normal controls were clearly detected by Southern hybridization using (TAAAA)(5) probe, while they were not labeled with (TGGAA)(5) or (TAGAA)(5) probe. These data indicate that control alleles very rarely have a nonpathogenic large insertion in the SCA31 critical region and that not only the presence of the insertion but also its size is not sufficient evidence for a disease-causing allele. We approve of the view that (TGGAA)(n) repeats in the insertion are indeed related to the pathogenesis of SCA31, but it remains undetermined whether a large insertion lacking (TGGAA)(n) is nonpathogenic.

A locus for ophthalmo-acromelic syndrome mapped to 10p11.23.

Ophthalmo-acromelic syndrome (OAS, OMIM %206920) is a rare autosomal recessive disease, presenting with clinical anophthalmia and limb anomalies. We recruited three OAS families including a Japanese family with two affected patients and two consanguineous Lebanese families each having an affected. Homozygosity mapping was performed using the 50K SNP chip and additional informative markers. A locus for OAS was mapped to the 422-kb region at 10q11.23, based on the results from the two consanguineous families as well as the consistent data from the Japanese non-consanguineous family. The 422-kb region only contained one gene, MPP7. Although we could not detect any pathological mutations in OAS families analyzed, MPP7 could remain a candidate as aberrant changes might exist beyond our mutation detection methods. Further families are needed to confirm this candidate locus.

Severity and progression rate of cerebellar ataxia in 16q-linked autosomal dominant cerebellar ataxia (16q-ADCA) in the endemic Nagano Area of Japan.

16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA) is a recently defined subtype of ADCA identified by a disease-specific C/T substitution in the 5' untranslated region of the puratrophin-1 gene. In Nagano, the central mountainous district of the main island of Japan, 16q-ADCA and spinocerebellar ataxia type 6 (SCA6) are the most and second most prevalent subtypes of ADCA, respectively. Both subtypes are classified into Harding's ADCA III, but little attention has been given to the differences in the severity and progression rate of cerebellar ataxia between 16q-ADCA and SCA6. We investigated the clinical severity and progression rate of cerebellar ataxia of 16q-ADCA patients using international cooperative ataxia rating scale and scale for the assessment and rating of ataxia and compared them with those of SCA6 patients. The age at onset was much higher in 16q-ADCA patients (60.1 +/- 9.8 years, n = 66) than in SCA6 patients (41.1 +/- 8.7 years, n = 35). Clinical features of 16q-ADCA were basically consistent with pure cerebellar ataxia, as well as in SCA6, but gaze-evoked nystagmus was observed less frequently in 16q-ADCA patients than in SCA6 patients. When compared at almost the same disease duration after onset, the severity of cerebellar ataxia was a little higher, and the progression rate seemed more rapid in 16q-ADCA patients than in SCA6 patients, but the differences were not significant.

Paternal somatic mosaicism of a TGFBR2 mutation transmitting to an affected son with Loeys-Dietz syndrome.

We report on somatic mosaicism of a TGFBR2 missense mutation, c.1336G>A (D446N). The affected son with the heterozygous mutation was previously reported [Sakai et al. (2006); Am J Med Genet A 140A:1719-1725]. Further evaluation indicates his clinical condition is Loeys-Dietz syndrome. Parental blood samples were studied to confirm whether the propositus' mutation was a de novo change, and suggested a trace of the mutation in the father. DNAs extracted from blood leukocytes, buccal cells, hair root cells, and nails in the father indicated 52%, 25%, 0%, and 35% of cells harbored the mutation, respectively. This is the first detailed report of somatic mosaicism of a TGFBR2 mutation.

Craniosynostosis in a patient with a de novo 15q15-q22 deletion.

Interstitial deletions involving the chromosomal band 15q15 are very rare. A total of five cases were previously reported. Here another case of a 15q15.2-q22.2 deletion is reported, presenting with severe craniosynostosis of coronary, metopic, and sagittal sutures. The chromosome 15 with the 17.7-Mb deletion was of the paternal origin. A critical region for craniosynostosis may be located at the 734-kb segment at 15q15.2. Interestingly, the entire FBN1 gene was deleted in this patient.

FBN2, FBN1, TGFBR1, and TGFBR2 analyses in congenital contractural arachnodactyly.

FBN2, FBN1, TGFBR1, and TGFBR2 were analyzed by direct sequencing in 15 probands with suspected congenital contractural arachnodactyly (CCA). A total of four novel FBN2 mutations were found in four probands (27%, 4/15), but remaining the 11 did not show any abnormality in either of the genes. This study indicated that FBN2 mutations were major abnormality in CCA, and TGFBR and FBN1 defects may not be responsible for the disorder. FBN2 mutations were only found at introns 30, 31, and 35 in this study. Thus analysis of a mutational hotspot from exons 22 to 36 (a middle part) of FBN2 should be prioritized in CCA as previously suggested.

Comprehensive genetic analysis of relevant four genes in 49 patients with Marfan syndrome or Marfan-related phenotypes.

In order to evaluate the contribution of FBN1, FBN2, TGFBR1, and TGFBR2 mutations to the Marfan syndrome (MFS) phenotype, the four genes were analyzed by direct sequencing in 49 patients with MFS or suspected MFS as a cohort study. A total of 27 FBN1 mutations (22 novel) in 27 patients (55%, 27/49), 1 novel TGFBR1 mutation in 1 (2%, 1/49), and 2 recurrent TGFBR2 mutations in 2 (4%, 2/49) were identified. No FBN2 mutation was found. Three patients with either TGFBR1 or TGFBR2 abnormality did not fulfill the Ghent criteria, but expressed some overlapping features of MFS and Loeys-Dietz syndrome (LDS). In the remaining 19 patients, either of the genes did not show any abnormalities. This study indicated that FBN1 mutations were predominant in MFS but TGFBRs defects may account for approximately 5-10% of patients with the syndrome.

A -16C>T substitution in the 5' UTR of the puratrophin-1 gene is prevalent in autosomal dominant cerebellar ataxia in Nagano.

The molecular bases of autosomal dominant cerebellar ataxia (ADCA) have been increasingly elucidated, but 17-50% of ADCA families still remain genetically undefined in Japan. In this study we investigated 67 genetically undefined ADCA families from the Nagano prefecture, and found that 63 patients from 51 families possessed the -16C>T change in the puratrophin-1 gene, which was recently found to be pathogenic for 16q22-linked ADCA. Most patients shared a common haplotype around the puratrophin-1 gene. All patients with the -16C>T change had pure cerebellar ataxia with middle-aged or later onset. Only one patient in a large, -16C>T positive family did not have this change, but still shared a narrowed haplotype with, and was clinically indistinguishable from, the other affected family members. In Nagano, 16q22-linked ADCA appears to be much more prevalent than either SCA6 or dentatorubral-pallidoluysian atrophy (DRPLA), and may explain the high frequency of spinocerebellar ataxia.

Complete hydatidiform mole and normal live birth following intracytoplasmic sperm injection.

A twin pregnancy with complete hydatidiform mole (HM) and preterm birth of a normal female infant after intracytoplasmic sperm injection (ICSI) conception was experienced. ICSI due to severe oligozoospermia was performed on three ova, and three embryos with confirmed two proneclei (2PN) were subsequently transferred to the uterus. At 7 weeks of gestation, molar pregnancy as well as a viable fetus was recognized. At 33 weeks, the pregnancy was terminated due to preterm labor. Dichorionic pregnancy consisting of a normal fetus and placenta in one chorionic membrane and complete HM in the other was recognized. Cytomolecular analysis indicated that the complete HM genome was derived from duplication of a single sperm, and a normal neonate was from biparental genomes. It should be noted that ICSI can avoid incomplete HM (mostly triploid) due to multi-sperm fertilization but might not be able to avoid complete HM (paternal diploid) although such a risk is very low. This is the second report of this condition and is accompanied by the first well-described molecular analysis.