The open conformation of WASP regulates its nuclear localization and gene transcription in myeloid cells.

Mutations in the gene encoding the Wiskott-Aldrich syndrome protein (WASP) are responsible for Wiskott-Aldrich syndrome and WASP is a major actin regulator in the cytoplasm. Although rare gain-of-function mutations in the WASP gene are known to result in X-linked neutropenia (XLN), the molecular pathogenesis of XLN is not fully understood. In this study, we showed that all reported constitutively activating mutants (L270P, S272P and I294T) of WASP were hyperphosphorylated by Src family tyrosine kinases and demonstrated higher actin polymerization activities compared with wild-type (WT) WASP. Further analysis showed a tendency of activating WASP mutants to localize in the nucleus compared with WT or the Y291F mutant of WASP. In addition, we found that WASP could form a complex with nuclear RNA-binding protein, 54 kDa (p54nrb) and RNA polymerase II (RNAP II). ChIP assays revealed that WASP associated with DNA, although the affinity was relatively weaker than RNAP II. To determine whether gene transcription was affected by WASP mutation in myeloid cells, we performed microarray analysis and found different expression profiles between WT and L270P WASP-transfected K562 cells. Among the genes affected, granulocyte colony-stimulating factor receptor, Runx1, and protein tyrosine phosphatase receptor c were included. ChIP on chip analysis of genomic DNA showed WT and L270P WASP had a highly similar DNA-binding pattern but differed in binding affinity at the same locus. Therefore, our results suggest that the open conformation of WASP regulates its nuclear localization and plays requisite roles in regulating gene transcription that would contribute to the outcome in the nucleus of myeloid cells.

A case of infant botulism infection due to consumption of untreated well-water.

A 7-week-old boy with flaccid paralysis was diagnosed with infant botulism caused by Clostridium botulinum toxin type A. In this case of infant botulism, untreated well-water was identified as a potential source of this infection.

Selective expansion of donor-derived regulatory T cells after allogeneic bone marrow transplantation in a patient with IPEX syndrome.

IPEX syndrome is a rare and fatal disorder caused by absence of regulatory T cells (Tregs) due to congenital mutations in the Forkhead box protein 3 gene. Here, we report a patient with IPEX syndrome treated with RIC followed by allogeneic BMT from an HLA-matched sibling donor. We could achieve engraftment and regimen-related toxicity was well tolerated. Although the patient was in mixed chimera and the ratio of donor cells in whole peripheral blood remained relatively low, selective and sustained expansion of Tregs determined as CD4+CD25+Foxp3+ cells was observed. Improvement in clinical symptoms was correlated with expansion of donor-derived Tregs and disappearance of anti-villin autoantibody, which was involved in the pathogenesis of gastrointestinal symptoms in IPEX syndrome. This clinical observation suggests that donor-derived Tregs have selective growth advantage in patients with IPEX syndrome even in mixed chimera after allogeneic BMT and contribute to the control of clinical symptoms caused by the defect of Tregs.

T-cell receptor ligation causes Wiskott-Aldrich syndrome protein degradation and F-actin assembly downregulation.

BACKGROUND: Wiskott-Aldrich syndrome protein (WASP) links T-cell receptor (TCR) signaling to the actin cytoskeleton. WASP is normally protected from degradation by the Ca(++)-dependent protease calpain and by the proteasome because of its interaction with the WASP-interacting protein. OBJECTIVE: We investigated whether WASP is degraded after TCR ligation and whether its degradation downregulates F-actin assembly caused by TCR ligation. METHODS: Primary T cells, Jurkat T cells, and transfected 293T cells were used in immunoprecipitation experiments. Intracellular F-actin content was measured in splenic T cells from wild-type, WASP-deficient, and c-Casitas B-lineage lymphoma (Cbl)-b-deficient mice by using flow cytometry. Calpeptin and MG-132 were used to inhibit calpain and the proteasome, respectively. RESULTS: A fraction of WASP in T cells was degraded by calpain and by the ubiquitin-proteasome pathway after TCR ligation. The Cbl-b and c-Cbl E3 ubiquitin ligases associated with WASP after TCR signaling and caused its ubiquitination. Inhibition of calpain and lack of Cbl-b resulted in a significantly more sustained increase in F-actin content after TCR ligation in wild-type T cells but not in WASP-deficient T cells. CONCLUSION: TCR ligation causes WASP to be degraded by calpain and to be ubiquitinated by Cbl family E3 ligases, which targets it for destruction by the proteasome. WASP degradation might provide a mechanism for regulating WASP-dependent TCR-driven assembly of F-actin.

A case series of CAEBV of children and young adults treated with reduced-intensity conditioning and allogeneic bone marrow transplantation: a single-center study.

BACKGROUND: Epstein-Barr virus (EBV)-infected T or NK cells cause chronic active EBV infection (CAEBV). Allogeneic hematopoietic stem cell transplantation (HSCT) is curative treatment for CAEBV patients. However, chemotherapy prior to HSCT and optimal conditioning regimen for allogeneic HSCT are still controversial. PATIENTS AND METHODS: We retrospectively analyzed five patients with CAEBV treated with reduced-intensity conditioning (RIC) consisted of fludarabine, cyclophosphamide, and low-dose total-body irradiation followed by allogeneic bone marrow transplantation in a single institute. Only one of five patients received chemotherapy prior to transplantation. We analyzed EBV-infected cells in a patient whose EBV load increased after HSCT by T-cell repertoire assay, separation of T-cell subpopulations, in situ hybridization and microsatellite analysis. RESULTS: All five patients achieved engraftment, complete chimera, and eradication of EBV load. All patients have been alive without any serious regimen-related toxicity for more than 16 months following HSCT. However, one patient transplanted from HLA-matched sibling donor developed clonal proliferation of CD4+ Vß3+ T cells caused by monoclonal EBV infection on day 99 after transplantation. Further analysis revealed that the CD4+ Vß3+ T cells selectively harbored EBV genome, and these infected cells were derived from donor T cells. CONCLUSIONS: Allogeneic HSCT with RIC is a safe and effective treatment for better overall survival and less regimen-related toxicity in patients with CAEBV. Our first pediatric case reported in the literature suggests that we should consider the possibility of persistent EBV infection in donor T cells as well as the relapse in recipient cells if EBV load increases after allogeneic HSCT.

Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome.

Hyper-immunoglobulin E syndrome (HIES) is a compound primary immunodeficiency characterized by a highly elevated serum IgE, recurrent staphylococcal skin abscesses and cyst-forming pneumonia, with disproportionately milder inflammatory responses, referred to as cold abscesses, and skeletal abnormalities. Although some cases of familial HIES with autosomal dominant or recessive inheritance have been reported, most cases of HIES are sporadic, and their pathogenesis has remained mysterious for a long time. Here we show that dominant-negative mutations in the human signal transducer and activator of transcription 3 (STAT3) gene result in the classical multisystem HIES. We found that eight out of fifteen unrelated non-familial HIES patients had heterozygous STAT3 mutations, but their parents and siblings did not have the mutant STAT3 alleles, suggesting that these were de novo mutations. Five different mutations were found, all of which were located in the STAT3 DNA-binding domain. The patients' peripheral blood cells showed defective responses to cytokines, including interleukin (IL)-6 and IL-10, and the DNA-binding ability of STAT3 in these cells was greatly diminished. All five mutants were non-functional by themselves and showed dominant-negative effects when co-expressed with wild-type STAT3. These results highlight the multiple roles played by STAT3 in humans, and underline the critical involvement of multiple cytokine pathways in the pathogenesis of HIES.

Utility of thallium-201 scintigraphy in Tolosa-Hunt syndrome.

Tolosa-Hunt syndrome (THS) is a rare disorder, especially in the pediatric population, characterized by unilateral painful ophthalmoplegia with a relapsing-remitting course. Because the diagnosis of THS is based on the exclusion of other causes of painful ophthalmoplegia, attention should be paid to possible alternative diagnoses. Thallium-201 chloride ((201)Tl) scintigraphy has been used to evaluate tissue histology in clinical oncology with a marker, the retention index (RI). A higher value indicates histological malignancy. Although its utility in pediatric THS has not been discussed, we suggest that (201)Tl scintigraphy may be informative as a marker in the diagnosis. We present an 11-year-old boy with THS who was evaluated with (201)Tl scintigraphy before treatment with corticosteroids, when he had headache, photophobia, and diplopia. The RI of (201)Tl indicated that the lesion would be benign. Although his clinical symptoms did not fulfill the THS criteria completely, his eye symptoms disappeared 2 weeks after corticosteroid treatment, which was not within the 72 h as in the diagnostic criteria of THS. He has been symptom-free for more than 2 years with only an initial 4-week corticosteroid therapy. This report not only shows the potential of (201)Tl scintigraphy to contribute to the correct diagnosis of pediatric THS but also suggests the possibility that the diagnosis of THS could be supported uniquely even in a pediatric THS-suspicious patient who did not fulfill the current THS criteria completely. In conclusion, we suggest that (201)Tl scintigraphy may be useful for making the diagnosis of THS, especially in pediatric patients.

Unusual ribbon-like periventricular heterotopia with congenital cataracts in a Japanese girl.

Periventricular heterotopia (PH), clumps of neurons mislocated beside the ventricle, is caused by failure to initiate migration during embryogenesis. We report on a 32-month-old Japanese girl with a unique subtype of PH, namely ribbon-like PH. The patient presented with severe psychomotor developmental delay, intractable epilepsy, and congenital cataracts and developed West syndrome phenotype. Brain magnetic resonance imaging revealed a unique undulating form of PH, categorized as ribbon-like PH, and other brain malformations including simplified gyri and dysgenesis of the corpus callosum. There was no evidence of prenatal TORCH infection or associated syndrome. Array-based comparative genomic hybridization revealed no chromosomal rearrangements. Genetic analyses of the FLNA, DCX, ARX, LIS1, and TUBA1A genes showed no mutations. Although little is known about ribbon-like PH, the clinical manifestations in our patient clearly differed from those in other reported patients.

A homozygous mutation of voltage-gated sodium channel ß(I) gene SCN1B in a patient with Dravet syndrome.

Dravet syndrome is a severe form of epileptic encephalopathy characterized by early onset epileptic seizures followed by ataxia and cognitive decline. Approximately 80% of patients with Dravet syndrome have been associated with heterozygous mutations in SCN1A gene encoding voltage-gated sodium channel (VGSC) α(I) subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene encoding VGSC ß(I) subunit was recently described in a patient with Dravet syndrome. To further examine the involvement of homozygous SCN1B mutations in the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in 286 patients with epileptic disorders, including 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an etiologic candidate underlying Dravet syndrome.

Copper-trafficking efficacy of copper-pyruvaldehyde bis(N4- methylthiosemicarbazone) on the macular mouse, an animal model of Menkes disease.

BACKGROUND: Menkes disease (MD) is a disorder of copper transport caused by ATP7A mutations. Although parenteral copper supplements are partly effective in treating MD, the copper level in the brain remains insufficient, whereas copper accumulates in the kidney. We investigated the copper-trafficking efficacy of copper-pyruvaldehyde bis(N4-methylthiosemicarbazone) (Cu-PTSM), a lipophilic copper complex, in macular mice, an animal model of MD. METHODS: Macular mice were treated with cupric chloride (CuCl2) or Cu-PTSM on postnatal days 4, 10, and 17. At 4 wk of age, the copper levels in major organs and cytochrome oxidase (CO) activity in brain tissue were measured. Hematology, blood biochemistry, and urinary ß2-microglobulin (ß2-M) secretion were also assessed. RESULTS: The copper levels in the brains of the Cu-PTSM-treated group remained low, but CO activity in the cerebral and cerebellar cortices in the Cu-PTSM-treated group were higher than those in the CuCl2-treated group. There were no significant differences in hematological or biochemical findings or in urinary ß2-M secretion among the groups. CONCLUSION: Although the copper-trafficking efficacy of Cu-PTSM was limited, the improved CO activity in the brain suggests that Cu-PTSM delivered copper more effectively to neuronal CO than did CuCl2. Reduced renal copper accumulation may be beneficial in prolonged copper supplementation.

Successful cord blood transplantation with reduced-intensity conditioning for childhood cerebral X-linked adrenoleukodystrophy at advanced and early stages.

Childhood cerebral ALD is a rapidly progressive and neurodegenerative disorder for which HSCT is the curative therapy if carried out at early stages. We successfully treated two patients of childhood cerebral ALD by CBT with RIC. The proband was a seven-yr-old boy whose brain MRI severity score (Loes score) was 14.5. Unrelated CBT was performed in five wk. To minimize conditioning regimen-related neurotoxicity, the combination of fludarabine (125 mg/m(2)), melphalan (140 mg/m(2)), and 4 Gy of brain-sparing TBI was used. The second patient was a six-yr-old brother of the proband. Four wk after the detection of a single small lesion (Loes score 1), he received unrelated CBT with the same RIC as the proband. In both patients, the engraftment was fast and stable, and severe complications were not observed. Furthermore, gadolinium-enhanced inflammation on brain MRI rapidly disappeared after CBT. Now, 20 and 13 months have passed after CBT, respectively, and both patients are neurologically stable. The RIC we used was sufficient for stable engraftment of cord blood and also tolerable even to the patient with advanced ALD. RIC-CBT should be considered for the patients with cerebral ALD at advanced stages, as well as those at early stages.

Two novel mutations in the lactase gene in a Japanese infant with congenital lactase deficiency.

Intestinal lactase is required for the hydrolysis of lactose that is the most essential carbohydrate in milk and the primary diet source of newborn. Congenital lactase deficiency [CLD (MIM 223000)] is a severe gastrointestinal disorder and is characterized by watery diarrhea due to an extremely low or the lack of lactase activity in the intestinal wall from birth. CLD is a rare disease and occurs more frequently in Finland. Recent studies have shown that mutations in the coding region of the lactase (LCT) gene underlie CLD in patients from Finland and other European countries. Here, we report two novel mutations in the LCT gene in a Japanese female infant with clinical features consistent with those of CLD. She suffered from severe watery diarrhea from the age of 2 days on breast milk/lactose containing cow's milk formula. With the lactose-free hydrolyzed cow's milk formula, diarrhea was stopped, and she has now developed well on a lactose-free diet. She shows a lactose-intolerance pattern on the lactose challenge test. Sequence analysis revealed the two mutations in her LCT gene: c.4419C>G (p.Y1473X) in exon 10 transmitted from her mother and c.5387delA (p.D1796fs) in exon 16 transmitted from her father. Both mutations cause premature truncation of lactase polypeptide and are supposed to be responsible for CLD. To our knowledge, this is the first report on mutations in the LCT gene in Japan. We suggest that an increased awareness is required regarding CLD.

Post-transplantation lymphoproliferative disorder in living-donor liver transplantation: a single-center experience.

BACKGROUND: Post-transplantation lymphoproliferative disorder (PTLD) is a group of life-threatening complications of organ transplantation, which occurs most frequently in pediatric patients. This retrospective study evaluates a single-institution experience of five cases of PTLD after living-donor liver transplantation (LDLT). PATIENTS AND METHOD: We reviewed the records of 78 pediatric patients (<18 years old) and 54 adult patients, who underwent LDLT between July 1991 and December 2009. RESULT: PTLD was diagnosed in five pediatric patients, yielding an overall incidence of 3.8%. There were no significant differences between the pediatric patients with and those without PTLD in terms of their age, sex, reason for transplantation, calcineurin inhibitor, Epstein-Barr virus (EBV) serostatus, ABO compatibility, lymphocyte cross-matching, or episodes of biopsy proven rejection. Two patients with abdominal lymphadenopathy and one with gastrointestinal PTLD responded to a reduction in immunosuppression. Treatment with rituximab was necessary for another gastrointestinal PTLD patient. Diffuse large-B-cell lymphoma was diagnosed in one patient with mediastinal and lung masses. This patient was treated with chemotherapy and rituximab, followed by surgical resection. All patients survived and no evidence of recurrence has been found since. CONCLUSION: Although PTLD is potentially life-threatening, it can be managed by appropriate and prompt treatment, with a good outcome.

Prospective study of a therapeutic regimen with all-trans retinoic acid and anthracyclines in combination of cytarabine in children with acute promyelocytic leukaemia: the Japanese childhood acute myeloid leukaemia cooperative study.

In childhood acute promyelocytic leukaemia (APL), the efficacy of therapy combining cytarabine with all-trans retinoic acid (ATRA) and anthracyclines remains unclear in terms of long-term prognosis. Between August 1997 and March 2004, 58 children with APL (median age: 11 years) were enrolled into an acute myeloid leukaemia (AML) study (AML99-M3) and followed up for a median time of 86 months. The regimen included ATRA and anthracyclines combined with cytarabine in both induction and consolidation. In induction, two patients died of haemorrhage and four patients developed retinoic acid syndrome. Of 58 patients, 56 (96·6%) achieved complete remission, two of whom relapsed in the bone marrow after 15 and 19 months respectively. Sepsis was a major complication, with an incidence of 5·6-10·9% in the consolidation blocks, from which all but one of patients recovered. Consequently, 7-year overall and event-free survival rates were 93·1% and 91·4% respectively, and cumulative incidence of relapse plateaued at 3·6% after 2 years. Follow-up survey of 54 patients revealed no patients with late cardiotoxicity or secondary malignancy, except one with asymptomatic prolongation of QTc interval. This study suggests that the combination of cytarabine with ATRA and anthracycline-based therapy may have useful implications in the perspective of long-term prognosis and late adverse effects for childhood APL.

Nationwide survey of patients with primary immunodeficiency diseases in Japan.

To determine the prevalence and clinical characteristics of patients with in Japan, we conducted a nationwide survey of primary immunodeficiency disease (PID) patients for the first time in 30 years. Questionnaires were sent to 1,224 pediatric departments and 1,670 internal medicine departments of Japanese hospitals. A total of 1,240 patients were registered. The estimated number of patients with PID was 2,900 with a prevalence of 2.3 per 100,000 people and homogenous regional distribution in Japan. The male-to-female ratio was 2.3:1 with a median age of 12.8 years. Adolescents or adults constituted 42.8% of the patients. A number of 25 (2.7%) and 78 (8.5%) patients developed malignant disorders and immune-related diseases, respectively, as complications of primary immunodeficiency disease. Close monitoring and appropriate management for these complications in addition to prevention of infectious diseases is important for improving the quality of life of PID patients.

A genome-wide association study identifies RNF213 as the first Moyamoya disease gene.

Moyamoya disease (MMD) shows progressive cerebral angiopathy characterized by bilateral internal carotid artery stenosis and abnormal collateral vessels. Although ∼ 15% of MMD cases are familial, the MMD gene(s) remain unknown. A genome-wide association study of 785,720 single-nucleotide polymorphisms (SNPs) was performed, comparing 72 Japanese MMD patients with 45 Japanese controls and resulting in a strong association of chromosome 17q25-ter with MMD risk. This result was further confirmed by a locus-specific association study using 335 SNPs in the 17q25-ter region. A single haplotype consisting of seven SNPs at the RNF213 locus was tightly associated with MMD (P = 5.3 × 10(-10)). RNF213 encodes a really interesting new gene finger protein with an AAA ATPase domain and is abundantly expressed in spleen and leukocytes. An RNA in situ hybridization analysis of mouse tissues indicated that mature lymphocytes express higher levels of Rnf213 mRNA than their immature counterparts. Mutational analysis of RNF213 revealed a founder mutation, p.R4859K, in 95% of MMD families, 73% of non-familial MMD cases and 1.4% of controls; this mutation greatly increases the risk of MMD (P = 1.2 × 10(-43), odds ratio = 190.8, 95% confidence interval = 71.7-507.9). Three additional missense mutations were identified in the p.R4859K-negative patients. These results indicate that RNF213 is the first identified susceptibility gene for MMD.

[Clinical features of Japanese pediatric patients with anti-aquaporin 4 antibody].

Recently, a disease-specific antibody was found in serum from patients with neuromyelitis optica (NMO), and its target antigen was identified as aquaporin 4 (AQP4) water channel protein. There is no clinical picture of pediatric cases with anti-AQP4 antibody, except one report from North America. Here, we report the clinical features of 18 Japanese anti-AQP4-antibody-positive patients with childhood-onset of NMO. Of the 2000 patients who had been examined for anti-AQP4 antibody at Tohoku University Hospital up until 2008, 60 were under 15 years of age at onset, and 18 of them were positive for anti-AQP4 antibody. We analyzed the clinical information on those patients. There were 14 girls and 4 boys (M:F ratio = 1:3.5). The age of onset ranged from 3 to 15 years old (median 13). The clinical diagnoses of the 18 patients before the anti-AQP4 antibody tests were: NMO in 8 (44%), MS in 7 (39%), and opticospinal MS (OSMS) in 3 (17%). Nine patients developed only optic neuritis at onset. At the last follow-up, brain magnetic resonance imaging (MRI) was abnormal in 14 patients, nine had monocular or binocular blindness, and the expanded disability status scale score was 6.0 or higher (cannot walk without support) in eight patients. NMO has a poor prognosis, as seen in adult cases, suggesting the importance of an early diagnosis using the anti-AQP4 antibody test to institute effective immunosuppressive treatment.

[Case of a pharyngeal-cervical-brachial variant of Guillain-Barré syndrome without pharyngeal palsy].

We report a case of a 3-year-old boy with acute muscle weakness that initially affected neck and all four limbs but later vanished from the lower limbs. Pharyngeal palsy was not observed during the course. All deep tendon reflexes were absent. Peripheral nerve conduction studies showed a demyelination pattern in each limb. The patient received intravenous high-dose corticosteroid hormone, followed by two immunoglobulin therapies. His muscle strength gradually improved after treatment and was almost completely restored four months later. We ultimately diagnosed the condition as the pharyngeal-cervical-brachial variant of Guillain-Barré syndrome, in consideration of the patient's muscle weakness of the neck and four limbs, the greater degree of weakness of the upper limbs versus the lower limbs. His clinical presentation was atypical for the pharyngeal-cervical-brachial variant of Guillain-Barré syndrome, as he presented no pharyngeal muscle weakness or anti GT1a antibodies, typical manifestations of the condition.

Deletions of SCN1A 5' genomic region with promoter activity in Dravet syndrome.

Mutations involving the voltage-gated sodium channel alpha(I) gene SCN1A are major genetic causes of childhood epileptic disorders, as typified by Dravet syndrome. Here we investigated the upstream regions of the SCN1A 5' noncoding exons and found two major regions with promoter activity. These two major promoters were simultaneously active in various brain regions and in most neurons. Using multiplex ligation-dependent probe amplification (MLPA) assays with probes for the 5' noncoding exons, their upstream regions, and all coding exons of SCN1A, we investigated 130 epileptic patients who did not show any SCN1A mutations by sequence analysis of all coding exons and exon-intron boundaries. Among 71 Dravet syndrome patients, we found two patients with heterozygous microdeletions removing the 5' noncoding exons and regions with promoter activity but not affecting the coding exons. We also identified four patients with deletions/duplication in the coding region. One patient with symptomatic focal epilepsy also showed a deletion in the coding region. This study provides the first case of microdeletion limited to the SCN1A 5' promoter region with the coding sequence preserved, and indicates the critical involvement of this upstream region in the molecular pathology of Dravet syndrome.

Molecular and clinical analysis of RAF1 in Noonan syndrome and related disorders: dephosphorylation of serine 259 as the essential mechanism for mutant activation.

Noonan syndrome (NS) and related disorders are autosomal dominant disorders characterized by heart defects, facial dysmorphism, ectodermal abnormalities, and mental retardation. The dysregulation of the RAS/MAPK pathway appears to be a common molecular pathogenesis of these disorders: mutations in PTPN11, KRAS, and SOS1 have been identified in patients with NS, those in KRAS, BRAF, MAP2K1, and MAP2K2 in patients with CFC syndrome, and those in HRAS mutations in Costello syndrome patients. Recently, mutations in RAF1 have been also identified in patients with NS and two patients with LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome. In the current study, we identified eight RAF1 mutations in 18 of 119 patients with NS and related conditions without mutations in known genes. We summarized clinical manifestations in patients with RAF1 mutations as well as those in NS patients withPTPN11, SOS1, or KRAS mutations previously reported. Hypertrophic cardiomyopathy and short stature were found to be more frequently observed in patients with RAF1 mutations. Mutations in RAF1 were clustered in the conserved region 2 (CR2) domain, which carries an inhibitory phosphorylation site (serine at position 259; S259). Functional studies revealed that the RAF1 mutants located in the CR2 domain resulted in the decreased phosphorylation of S259, and that mutant RAF1 then dissociated from 14-3-3, leading to a partial ERK activation. Our results suggest that the dephosphorylation of S259 is the primary pathogenic mechanism in the activation of RAF1 mutants located in the CR2 domain as well as of downstream ERK.