Takeshi Taketani

Development and Testing of New Screening Method for Keratan Sulfate in Mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA), a progressive lysosomal storage disease, causes skeletal dysplasia through excessive storage of keratan sulfate (KS). We developed an ELISA-sandwich assay that used a MAb specific to KS. Forty-five blood and 59 urine specimens from MPS IVA patients (ages 1–65 y) were analyzed to determine whether KS concentration is a suitable marker for early diagnosis and longitudinal assessment of disease severity. Blood specimens were obtained from patients categorized as phenotypically severe (n = 36) and milder (n = 9). Urine specimens were also analyzed from patients categorized as severe (n = 56) and milder (n = 12), respectively. Blood KS levels (101–1525 ng/mL) in MPS IVA patients were two to eight times higher than those in age-matched controls (15–323 ng/mL). It was found that blood KS level varied with age and clinical severity. Blood KS levels in both MPS IVA and controls peaked between 5 and 10 y of age (mean, 776 versus 234 ng/mL, respectively). Blood levels in severe MPS IVA were 1.5 times higher than in the milder form. In contrast to blood, urine KS levels in both MPS IVA and controls peaked between 1 and 5 y (15.3 versus 0.26 mg/g creatinine), and thereafter declined with age. Urine KS level also varied with age and clinical severity, and the severe MPS IVA phenotype was associated with 6.7 times greater urine KS excretion than the milder one. These findings indicate that the new assay for blood or urine KS may be suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

New Bone Formation by Allogeneic Mesenchymal Stem Cell Transplantation in a Patient with Perinatal Hypophosphatasia

Mesenchymal stem cells (MSCs) can show osteogenic differentiation capability when implanted in vivo, as well as cultured in vitro; therefore we attempted to use allogeneic MSCs for an 8-month-old patient with hypophosphatasia. MSCs were obtained by culture expansion of fresh marrow from the patients father. Some of the MSCs were further cultured under osteogenic conditions on a culture dish or porous hydroxyapatite ceramics, resulting in cultured osteoblasts and osteogenic constructs, respectively. The MSCs and osteoblasts were injected into the patient, and the constructs were implanted locally. After traditional bone marrow transplantation, the MSCs, osteoblasts, and osteogenic constructs were used for treatment and to improve the patients respiratory condition and skeletal abnormality. The condition worsened again, and an MSC booster shot was administered. At the same time, the construct was retrieved. The respiratory condition improved, and the retrieved construct showed de novo bone derived from both donor and patient cells. We demonstrated the importance of allogeneic MSC transplantation for hypophosphatasia and the constructs as an alternative to bone fragments that provided further osteogenic capability in the patient.

Effective ex vivo expansion of hematopoietic stem cells using osteoblast‐differentiated mesenchymal stem cells is CXCL12 dependent

Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine‐differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast‐differentiated B10 (Ost‐B10) as a feeder layer and examined ex vivo expansion of CD34+CD38− HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost‐B10 cells exhibited similar effects on total HSC expansion; however, Ost‐B10 demonstrated a higher potency in CD34+CD38− cell‐specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony‐forming cell assay and long‐term culture initiating cell assay revealed that Ost‐B10 displayed multipotent differentiation and enabled long‐term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost‐B10 cells compared with the B10 cells. CD34+CD38− cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost‐B10, whereas expansion of CD34+CD38− cells was decreased in Ost‐B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast‐differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway.

Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: a study of the Japanese childhood AML Cooperative Study Group.

Mixed‐lineage leukemia (MLL)‐partial tandem duplication (PTD) is associated with poor prognosis in adult acute myeloid leukemia (AML), but its relationship to pediatric AML is unknown.

Fusion of an AF4-related gene, LAF4, to MLL in childhood acute lymphoblastic leukemia with t (2 ; 11) (q11 ; q23)

We showed that the LAF4 gene on 2q11.2–12 was fused to the MLL gene on 11q23 in a pediatric patient with CD10 positive acute lymphoblastic leukemia (ALL) having t(2;11)(q11;q23). The LAF4 gene, which encodes a lymphoid nuclear protein of 1227 amino acids with transactivation potential, is thought to have a role in early lymphoid development. The LAF4 protein was homologous to AF4 and AF5q31 proteins that are fused to MLL in infant early pre-B ALL and the breakpoint of LAF4 was located within the region homologous to the transactivation domain of AF4 and AF5q31. Expression of the 8.5-kb LAF4 transcript was detected in the adult heart, brain, and placenta and in the fetal brain. LAF4 expression was found to be higher in ALL cell lines than in AML and Epstein–Barr virus-transformed B-lymphocyte cell lines. These findings suggest that LAF4, AF4 and AF5q31 might define a new family particularly involved in the pathogenesis of 11q23-associated ALL.

Bezafibrate can be a new treatment option for mitochondrial fatty acid oxidation disorders: Evaluation by in vitro probe acylcarnitine assay

BACKGROUND The number of patients with mitochondrial fatty acid oxidation (FAO) disorders is recently becoming larger with the spread of newborn mass screening. Despite the advances in metabolic and molecular characterization of FAO disorders, the therapeutic studies are still limited. It was reported recently that bezafibrate (BEZ), an agonist of peroxisome proliferating activator receptor (PPAR), can restore FAO activity in cells from carnitine palmitoyltransferase-2 (CPT2) and very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies as well as clinical symptoms in the adult patients. METHODS In this study, the therapeutic effect of BEZ was determined by in vitro probe acylcarnitine (IVP) assay using cultured fibroblasts and tandem mass spectrometry on various FAO disorders. The clinical trial of BEZ treatment for a boy with the intermediate form of glutaric acidemia type 2 (GA2) was also performed. RESULTS The effect of BEZ was proven in cells from various FAO disorders including GA2, deficiencies of VLCAD, medium-chain acyl-CoA dehydrogenase, CPT2, carnitine acylcarnitine translocase and trifunctional protein, by the IVP assay. The aberrantly elevated long- or medium-chain acylcarnitines that are characteristic for each FAO disorder were clearly corrected by the presence of BEZ (0.4 mmol/L) in culture medium. Moreover, daily administration of BEZ in a 2-year-old boy with GA2 dramatically improved his motor and cognitive skills, accompanied by sustained reduction of C4, C8, C10 and C12 acylcarnitines in blood, and normalized the urinary organic acid profile. No major adverse effects have been observed. CONCLUSION These results indicate that BEZ could be a new treatment option for FAO disorders.

Clinical and genetic aspects of hypophosphatasia in Japanese patients

Objective We examined the clinical and genetic features of hypophosphatasia (HPP) in Japanese patients. HPP is a rare metabolic bone disorder of bone mineralisation caused by mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene, which encodes tissue-non-specific alkaline phosphatase isoenzyme. Methods We retrospectively investigate the incidence and clinical features of 52 patients with paediatric HPP who were born between 1999 and 2010. Mutations of the ALPL gene were analysed in 31 patients. Results The annual incidence of perinatal lethal HPP (PLH) was estimated to be 2–3/1 000 000 births. The most frequent clinical type was PLH followed by prenatal benign. In addition to bone symptoms, cerebral manifestations were frequently observed including convulsion, mental retardation, deafness and short stature with growth hormone deficiency. Respiratory failure was the most significant predictor of a poor prognosis for PLH. The first and second most frequent mutations in the ALPL gene were c.1559delT and c.T979C (p.F327L), respectively. The c.1559delT homozygous mutation was lethal with respiratory failure. Patients with the p.F327L compound heterozygous mutation had the different non-lethal type with short stature and a gradual improvement in ALP level and bone mineralisation. Conclusions The most frequent clinical type was the PLH type with prognosis related to respiratory failure, biochemical/radiological changes and ALPL mutations. Cerebral manifestations frequently occurred. Genotype–phenotype correlations were associated with specific outcomes in the PLH type, whereas different clinical features were associated with the same genotype in the non-lethal type.

NUP98–NSD3 fusion gene in radiation-associated myelodysplastic syndrome with t(8;11)(p11;p15) and expression pattern of NSD family genes

Chromosomal 11p15 abnormality of therapy-related myelodysplastic syndrome (t-MDS)-acute myeloid leukemia (AML) is rare. NUP98-NSD3 fusion transcripts have been detected previously in one patient with AML and one patient with t-MDS having t(8;11)(p11;p15). Here we present the case of a 60-year-old man with radiation-associated MDS (r-MDS) carrying chromosome abnormalities, including t(8;11)(p11;p15) and del(1)(p22p32). Fluorescence in situ hybridization analysis demonstrated that the NUP98 gene at 11p15 was split by the translocation. Southern blot analysis of bone marrow cells showed both rearrangements of NUP98 and NSD3 genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) followed by sequence analysis revealed the presence of both NUP98-NSD3 and NSD3-NUP98 fusion transcripts. Expression analysis by RT-PCR showed that NSD3 as well as NSD1 and NSD2 was ubiquitously expressed in leukemic cell lines and Epstein-Barr virus transformed B lymphocyte cell lines derived from the normal adult lymphocytes examined. Two isoforms of NSD3, NSD3S and NSD3L (but not NSD3L2), were expressed in leukemic cell lines and were fused to NUP98 in our patient, suggesting that qualitative change of these two isoforms of NSD3 by fusion with NUP98 might be related to leukemogenesis, although the function of each isoform of the NSD3 gene remains unclear.

High frequencies of simultaneous FLT3 -ITD, WT1 and KIT mutations in hematological malignancies with NUP98 -fusion genes

High frequencies of simultaneous FLT3 -ITD, WT1 and KIT mutations in hematological malignancies with NUP98 -fusion genes

Familial outbreak of Yersinia enterocolitica serotype O9 biotype 2.

In Japan, infection with Yersinia enterocolitica of the pathogenic serobiogroup serotype O9 biotype 2 (O9/2) has rarely occurred, and familial outbreaks of Y. enterocolitica are also infrequently reported. We found a familial outbreak of Y. enterocolitica O9/2. Y. enterocolitica O9/2 was detected from stools collected from three persons in the same family. Two patients (an 11-month-old girl and her 68-year-old grandmother) contemporaneously suffered from enterocolitis, and the third person, a carrier (a 5-year-old girl), manifested no symptoms. This bacteria was not detected from other family members or from their pet hamster. All the bacteria obtained from the three people were genetically identified by pulsed-field gel electrophoresis (PFGE). The infection route was likely to have been via food, because Y. enterocolitica was not found from the pet hamster. This is the first report of a familial outbreak of Y. enterocolitica O9/2 genetically identified by PFGE in Japan.