A.F. Markham

Mutation in the gene encoding ferritin light polypeptide causes dominant adult-onset basal ganglia disease

We describe here a previously unknown, dominantly inherited, late-onset basal ganglia disease, variably presenting with extrapyramidal features similar to those of Huntingtons disease (HD) or parkinsonism. We mapped the disorder, by linkage analysis, to 19q13.3, which contains the gene for ferritin light polypeptide (FTL). We found an adenine insertion at position 460–461 that is predicted to alter carboxy-terminal residues of the gene product. Brain histochemistry disclosed abnormal aggregates of ferritin and iron. Low serum ferritin levels also characterized patients. Ferritin, the main iron storage protein, is composed of 24 subunits of two types (heavy, H and light, L) which form a soluble, hollow sphere. Brain iron deposition increases normally with age, especially in the basal ganglia, and is a suspected causative factor in several neurodegenerative diseases in which it correlates with visible pathology, possibly by its involvement in toxic free-radical reactions. We found the same mutation in five apparently unrelated subjects with similar extrapyramidal symptoms. An abnormality in ferritin strongly indicates a primary function for iron in the pathogenesis of this new disease, for which we propose the name neuroferritinopathy.

Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis.

Papillon-Lefèvre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients. Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. Some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset. The PLS locus has been mapped to chromosome 11q14–q21 (refs 7, 8, 9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

BackgroundReal-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called TaqMan approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.ResultsWe have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.ConclusionOur assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Cyclooxygenase 2 is up-regulated and localized to macrophages in the intestine of Min mice.

SummaryExpression of cyclooxygenase 2 (COX-2) is believed to play an important role in adenoma formation in murine polyposis models, and inhibition of COX-2 activity may, at least, partly explain the chemopreventative activity of non-steroidal anti-inflammatory drugs against colorectal cancer in humans. However, the mechanism by which COX-2 acts in intestinal tumorigenesis remains unresolved because of conflicting data on the cellular localization of COX-2 in intestinal mucosa. Using immunohistochemistry with specific COX-2 antiserum, we have shown that COX-2 protein is localized to interstitial cells at the base of and within adenomas of the small and large intestine of multiple intestinal neoplasia (Min) mice. No COX-2 staining was observed in dysplastic epithelial cells within adenomas or in histologically normal epithelium. Moreover, COX-2 staining was observed in lamina propria cells of histologically normal intestine of Min mice. No staining was demonstrated in wild-type littermates. The rat monoclonal antibody F4/80 was used to show that COX-2-positive cells represented a subset of the macrophage population present in the intestine of Min mice. Localization of COX-2 to macrophages implies a paracrine effect of COX-2 function on epithelial cells in adenomas and also on histologically normal epithelium. Up-regulation of COX-2 expression in lamina propria macrophages may precede loss of the second functional Apc allele in epithelial cells before adenoma formation in the Min mouse model of intestinal tumorigenesis.

Mutations in a protein target of the Pim-1 kinase associated with the RP9 form of autosomal dominant retinitis pigmentosa

The RP9 form of autosomal dominant retinitis pigmentosa (adRP) maps to a locus on human chromosome 7p14. We now report two different disease associated mutations in a previously unidentified human gene, the mouse orthologue of which has been characterised by its interaction with the Pim-1 oncogene. In the original linked family we identified the missense mutation H137L. A second missense mutation, D170G, was found in a single RP patient. The putative RP9 gene appears to be expressed in a wide range of tissues, but its function is unknown and a pathogenic mechanism remains to be determined.

Deletion of the OPA1 gene in a dominant optic atrophy family: evidence that haploinsufficiency is the cause of disease

Dominant optic atrophy (DOA) is the most common form of autosomally inherited optic neuropathy.1 The disease typically presents in childhood with slow bilateral loss of visual acuity, visual field defects, abnormal colour discrimination, and pallor of the optic discs. The majority of DOA families published to date have shown linkage to a major locus on chromosome 3q28 ( OPA1 ). The OPA1 gene was recently identified and found to encode a ubiquitously expressed, dynamin related GTPase.2,3 In order to determine the mutation spectrum of OPA1 in DOA, we and others have screened the coding exons and their flanking splice sites in large patient cohorts.4–7 Over 60 different mutations have been reported, most of which are specific to individual families. It has been speculated that haploinsufficiency is the cause of disease,3–6 but to date there has been no evidence to prove that this mechanism, rather than aberrant function of mutated proteins, is responsible for the disease.nnUsing a combined approach of single stranded conformational polymorphism and heteroduplex analysis, we detected mutations in 57% (20/35) of our affected DOA patients.5 Additional DNA samples from an Australian family, in which no OPA1 mutation was identified in the above mutation screen, have recently been obtained enabling further investigation of the cause of disease in this family. All available family members underwent clinical evaluation to determine their disease status. This included best corrected visual acuity (BCVA), assessment of colour vision with Isihara plates and/or City University testing, automated perimetry (Humphrey Field Analyzer, San Leandro, CA), and dilated fundus examination with stereo disc photography. The ocular phenotype observed was typical of DOA with visual acuity reduction ranging from mild to moderate, mild to moderate colour vision disturbance, and visual field analysis ranging from normal to mild paracentral …

Death from early colorectal cancer is predicted by the presence of transcripts of the REG gene family.

An intrinsic component of colorectal carcinogenesis may be the capacity to activate regenerative responses simultaneously with inhibition of apoptosis. Since apoptosis is known to be inhibited in colorectal cancer, this study sought evidence for the activation of the REG family of genes which are considered to be activated during regeneration of intestinal mucosa. Transcripts for the REG gene were found in 53% of colorectal cancers and for the PAP gene in 60% of colorectal cancers, by RT-PCR. Using in situ hybridization, the REG transcripts were found to be present in the tumour cells themselves rather than inflammatory or stromal cells. There were no significant correlations between the expression of these two genes and tumour stage, age or sex of the patient population or tumour site. However, in patients with non-metastatic disease who underwent ostensibly curative surgery, the expression of REG alone and co-expression of REG with PAP had a highly significantly adverse effect on survival. These data provide support for the concept that, in some tumours, carcinogenesis involves a regenerative process which co-exists with apoptotic inhibition and may provide a valuable selective indicator of the need for adjuvant therapy in those patients with early-stage colorectal cancer whose disease is destined to recur after curative surgery.

Ketohexokinase: Expression and Localization of the Principal Fructose-metabolizing Enzyme

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.

Investigation of chromosome 9q22.3-q31 DNA marker loss in odontogenic keratocysts

Multiple basal cell carcinomas and odontogenic keratocysts of the jaws are a feature of the inherited naevoid basal cell carcinoma syndrome (NBCCS), although both occur more commonly as single, sporadic cases. The NBCCS gene has been mapped to chromosome 9q22.3-q31 and loss of heterozygosity for DNA markers from this region has been observed in familial and sporadic basal cell carcinomas. Based on these observations, we undertook a pilot study to determine if a similar pattern of chromosome loss occurs in odontogenic keratocysts. DNA extracted from microdissected odontogenic keratocyst epithelium was examined for loss of heterozygosity for six polymorphic DNA markers mapping to human chromosome 9q22.3-q31. Allelotype loss was detected in epithelium from three, single, sporadic odontogenic keratocysts. These results implicate homozygous inactivation of the NBCCS gene in the initiation and progression of the odontogenic keratocyst.

Expression of the Sonic Hedgehog receptor ‘PATCHED’ in basal cell carcinomas and odontogenic keratocysts

Basal cell carcinoma (BCC) is a common invasive skin lesion in Caucasians. Odontogenic keratocysts (OKs) are developmental, non‐inflammatory oral cysts. They can be sporadic and/or multiple and are locally destructive. Basal cell naevus syndrome (BCNS) comprises both multiple BCCs and multiple OKs, in addition to several other systemic manifestations. The genetic defect underlying this autosomal dominant syndrome is a germ line mutation in the Sonic Hedgehog receptor PATCHED (PTCH) gene. For this study, a rabbit anti‐peptide PTCH antiserum was produced. Immunohistochemistry procedures were performed using PTCH antibody and commercially produced GLI‐1 antibody (downstream member in the hedgehog pathway) to stain 11 BCNS‐OKs, eight sporadic OKs, two BCNS‐BCCs, and six sporadic BCCs. Most of these lesions had been previously screened for PTCH mutation. Most BCCs (n=7) demonstrated moderate staining, with the heaviest staining in the outer palisading cell layer, except a BCNS‐BCC which had mutation proximal to the sequence used for production of immunogenic peptide; this demonstrated only weak staining. Although moderate to heavy staining with PTCH antibody was demonstrated in the epithelium of both types of OK (n=19), a quite different pattern of staining of the basal cell layer was observed in the two patient groups. In BCNS, OK staining was heaviest in basal epithelial layers. In contrast, staining in non‐BCNS odontogenic keratocysts was exclusively located in the superficial epithelial layers. Up‐regulation of PTCH and GLI‐1 protein was demonstrated in both BCCs and OKs. The pattern of PTCH expression matched the PTCH transcript pattern previously reported in BCCs and appeared sufficiently characteristic in OKs to allow differentiation between syndromic and non‐syndromic cysts. Copyright