Alexander F. Markham

Identification of Microcephalin, a Protein Implicated in Determining the Size of the Human Brain

Primary microcephaly (MIM 251200) is an autosomal recessive neurodevelopmental condition in which there is a global reduction in cerebral cortex volume, to a size comparable with that of early hominids. We previously mapped the MCPH1 locus, for primary microcephaly, to chromosome 8p23, and here we report that a gene within this interval, encoding a BRCA1 C-terminal domain-containing protein, is mutated in MCPH1 families sharing an ancestral 8p23 haplotype. This gene, microcephalin, is expressed in the developing cerebral cortex of the fetal brain. Further study of this and related genes may provide important new insights into neocortical development and evolution.

Localization of cyclooxygenase-2 in human sporadic colorectal adenomas.

A putative target for the anti-colorectal cancer action of nonsteroidal anti-inflammatory drugs is the inducible isoform of cyclooxygenase (COX), COX-2. COX-2 is expressed within intestinal adenomas in murine polyposis models, but expression has been poorly characterized in human colorectal neoplasms. Therefore, we investigated the localization of the COX-2 protein in human sporadic colorectal adenomas. Immunohistochemistry for COX-2 and CD68 (a tissue macrophage marker) was performed on formalin-fixed, paraffin-embedded (n = 52) and frozen, acetone-fixed (n = 6) sections of human sporadic colorectal adenomas. Forty of 52 (77%) formalin-fixed adenomas expressed immunoreactive COX-2. COX-2 was localized to superficial interstitial macrophages in 39 cases (75%) and to deep interstitial macrophages in 9 cases (17%). COX-2 staining of dysplastic epithelial cells was observed in 15 cases (29%). A logistic regression analysis identified the adenoma site (P = 0.012) and histological type (P = 0.001) as independent predictors of superficial macrophage COX-2 expression. There was no relationship between the number of macrophages within an adenoma and macrophage COX-2 expression. These results indicate that COX-2 is expressed predominantly by interstitial macrophages within human sporadic colorectal adenomas. If COX-2 does indeed play a role in the early stages of colorectal carcinogenesis in man, these data suggest COX-2-mediated paracrine signaling between the macrophages and epithelial cells within adenomas.

EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse re-distribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.

Reduced expression of oestrogen receptor β in invasive breast cancer and its re-expression using DNA methyl transferase inhibitors in a cell line model

To gain insights into the possible role of oestrogen receptor (ER) β in breast carcinogenesis, immunohistochemical analysis of ER β was performed on 512 breast specimens encompassing normal (n = 138), pure ductal carcinoma in situ (n = 16), invasive cancers (n = 319), lymph node metastases (n = 31), and recurrences (n = 8). Real‐time polymerase chain reaction (PCR) was used to investigate the methylation status of the ER β gene in the ER β negative breast cancer cell lines SkBr3 and MDA‐MB‐435. A gradual reduction in, but not a complete loss of, ER β expression was observed during the transition from normal and pre‐invasive lesions to invasive cancers, where ER β was lost in 21% of cases. This was more pronounced in invasive ductal than in lobular carcinomas, a significantly higher proportion of which were ER β‐positive (74% compared with 91%, respectively, p = 0.0004). Examination of paired primary cancers with their axillary lymph node metastases showed that if ER β was present in the primary tumour, it persisted in the metastasis. Treatment of ER β‐negative cell lines with DNA methyl transferase inhibitors restored ER β expression, providing experimental evidence that silencing of ER β in breast carcinomas could be due to promoter hypermethylation. These results suggest that loss of ER β expression is one of the hallmarks of breast carcinogenesis and that it may be a reversible process involving methylation. Copyright

Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy

Digital clubbing, recognized by Hippocrates in the fifth century BC, is the outward hallmark of pulmonary hypertrophic osteoarthropathy, a clinical constellation that develops secondary to various acquired diseases, especially intrathoracic neoplasm. The pathogenesis of clubbing and hypertrophic osteoarthropathy has hitherto been poorly understood, but a clinically indistinguishable primary (idiopathic) form of hypertrophic osteoarthropathy (PHO) is recognized. This familial disorder can cause diagnostic confusion, as well as significant disability. By autozygosity methods, we mapped PHO to chromosome 4q33–q34 and identified mutations in HPGD, encoding 15-hydroxyprostaglandin dehydrogenase, the main enzyme of prostaglandin degradation. Homozygous individuals develop PHO secondary to chronically elevated prostaglandin E2 levels. Heterozygous relatives also show milder biochemical and clinical manifestations. These findings not only suggest therapies for PHO, but also imply that clubbing secondary to other pathologies may be prostaglandin mediated. Testing for HPGD mutations and biochemical testing for HPGD deficiency in patients with unexplained clubbing might help to obviate extensive searches for occult pathology.

Regulation and function of the interaction between the APC tumour suppressor protein and EB1

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.

Immunohistochemical detection of the anti-apoptosis protein, survivin, predicts survival after curative resection of stage II colorectal carcinomas.

Background:This study examined the role of Survivin protein, a novel inhibitor of apoptosis, in determining prognosis after curative resection of stage II colorectal carcinomas.Methods: Expression of Survivin, P53, and BCL-2 was evaluated immunohistochemically in stage II colorectal carcinomas from 49 patients who were followed for up to 9 years after operation. The Cox proportional hazards regression model was used to examine the predictive value of several covariates.Results: The patients comprised 33 men and 16 women with a median age of 71 years. There were 32 colonic and 17 rectal cancers comprising 40 T3 and nine T4 primary tumors. Survivin was expressed in 30 (61.2%), P53 in 30 (61.2%), and BCL-2 in 21 (42.9%) tumors. Expression of Survivin was independent of P53 or BCL-2 expression and histopathological characteristics of the tumor. The 5-year survival rate of patients with Survivin-positive tumors was significantly lower than that of patients with Survivin-negative tumors (52.5% vs. 94.1%, respectively; P = .01). On multivariate analysis, expression of Survivin (Hazard Ratio [HR] = 9; P = .03), and rectal origin of cancer (HR = 3; P = .05) were the only factors which independently predicted an increased risk of death from recurrent cancer.Conclusion: Survivin expression within the tumor can identify patients with stage II colorectal carcinoma who are at increased risk of death from recurrent disease and might particularly benefit from adjuvant therapy.

The Ubiquitin-conjugating Enzymes UbcH7 and UbcH8 Interact with RING Finger/IBR Motif-containing Domains of HHARI and H7-AP1

Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitrobinding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.

Fcγ receptor type IIIA is associated with rheumatoid arthritis in two distinct ethnic groups

OBJECTIVE To investigate a possible association between a functional polymorphism in the intermediate-affinity receptor for IgG called Fc-gamma receptor type IIIA (FcgammaRIIIA [CD16]) and rheumatoid arthritis (RA). METHODS This was an allelic association study in which a single nucleotide polymorphism in FcgammaRIIIA was examined as a susceptibility and/or severity factor for RA. The FcgammaRIIIA-158V/F polymorphism was genotyped by direct sequencing in 2 well-characterized ethnic groups, UK Caucasians (141 RA patients and 124 controls) and North Indians and Pakistanis (108 RA patients and 113 controls). RESULTS The FcgammaRIIIA-158V/F polymorphism was associated with RA in both ethnic groups (P = 0.028 for UK Caucasians, P = 0.050 for North Indians and Pakistanis, and P = 0.003 for both groups combined). FcgammaRIIIA-158VF and -158W individuals had an increased risk of developing RA in both populations (UK Caucasians odds ratio [OR] 1.6, P = 0.050; North Indians and Pakistanis OR 1.9, P = 0.023; and combined groups OR 1.7, P = 0.003). In the UK Caucasian group, the highest risk was for nodular RA, a more severe disease subset, associated with homozygosity for the FcgammaRIIIA-158V allele (OR 4.4, P = 0.004). There was also evidence for an interaction between the RA-associated HLA-DRB1 allele and the presence of at least 1 FcgammaRIIIA-158V allele in predicting susceptibility to RA (OR 5.5, P = 0.000). CONCLUSION We have demonstrated that the FcgammaRIIIA-158V/F polymorphism is a susceptibility and/or severity marker for RA in 2 distinct ethnic groups. This finding may ultimately provide additional insights into the pathogenesis of RA and other autoantibody/immune complex-driven autoimmune diseases.

Mutations in TSPAN12 cause autosomal-dominant familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Although mutations in three genes (LRP5, FZD4, and NDP) are known to cause FEVR, these account for only a fraction of FEVR cases. The proteins encoded by these FEVR genes form part of a signaling complex that activates the Norrin-beta-catenin signaling pathway. Recently, through a large-scale reverse genetic screen in mice, Junge and colleagues identified an additional member of this signaling complex, Tspan12. Here, we report that mutations in TSPAN12 also cause autosomal-dominant FEVR. We describe seven mutations identified in a cohort of 70 FEVR patients in whom we had already excluded the known FEVR genes. This study provides further evidence for the importance of the Norrin-beta-catenin signaling pathway in the development of the retinal vasculature and also indicates that more FEVR genes remain to be identified.