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Introduction: Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition., Methods and Results: Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells., Conclusions: We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.

It is generally well accepted that the pubertal surge in estrogen is responsible for the rapid bone accretion that occurs during puberty and that this effect is mediated by an estrogen-induced increase in growth hormone (GH)/insulin-like growth factor (IGF) action. To test the cause and effect relationship between estrogen and GH/IGF, we evaluated the consequence of ovariectomy (OVX) in prepubertal mice (C57BL/6J mice at 3 wk of age) on skeletal changes and the GH/IGF axis during puberty. Contrary to our expectations, OVX increased body weight (12-18%), bone mineral content (11%), bone length (4%), bone size (3%), and serum, liver, and bone IGF-I (30-50%) and decreased total body fat (18%) at 3 wk postsurgery. To determine whether estrogen is the key ovarian factor responsible for these changes, we performed a second experiment in which OVX mice were treated with placebo or estrogen implants. In addition to observing similar results compared with our first experiment, estrogen treatment partially rescued the increased body weight and bone size and completely rescued body fat and IGF-I levels. The increased bone accretion in OVX mice was due to increased bone formation rate (as determined by bone histomorphometry) and increased serum procollagen peptide. In conclusion, contrary to the known estrogen effect as an initiator of GH/IGF surge and thereby pubertal growth spurt, our findings demonstrate that loss of estrogen and/or other hormones during the prepubertal growth period effect leads to an increase in IGF-I production and bone accretion in mice.

We have investigated the effect of tumor necrosis factor-alpha (TNF-alpha) on the production of extracellular matrix-degrading proteases in skeletal muscles. Using microarray, quantitative PCR, Western blotting, and zymography, we found that TNF-alpha drastically increases the production of matrix metalloproteinase (MMP)-9 from C2C12 myotubes. In vivo administration of TNF-alpha in mice increased the transcript level of MMP-9 in skeletal muscle tissues. Although TNF-alpha activated all the three MAPKs (i.e. ERK1/2, JNK, and p38), inhibition of ERK1/2 or p38 but not JNK blunted the TNF-alpha-induced production of MMP-9 from myotubes. Inhibition of Akt also inhibited the TNF-alpha-induced production of MMP-9. TNF-alpha increased the activation of transcription factors NF-kappaB and AP-1 but not SP-1 in myotubes. Overexpression of a dominant negative inhibitor of NF-kappaB or AP-1 blocked the TNF-alpha-induced expression of MMP-9 in myotubes. Similarly, point mutations in AP-1- or NF-kappaB-binding sites in MMP-9 promoter inhibited the TNF-alpha-induced expression of a reporter gene. TNF-alpha increased the activity of transforming growth factor-beta-activating kinase-1 (TAK1). Furthermore, overexpression of a dominant negative mutant of TAK1 blocked the TNF-alpha-induced expression of MMP-9 and activation of NF-kappaB and AP-1. Our results also suggest that TNF-alpha induces MMP-9 expression in muscle cells through the recruitment of TRAF-2, Fas-associated protein with death domain, and TNF receptor-associated protein with death domain but not NIK or TRAF-6 proteins. We conclude that TAK1-mediated pathways are involved in TNF-alpha-induced MMP-9 production in skeletal muscle cells.

Phenotype-driven mutagenesis approach in the mouse holds much promise as a method for revealing gene function. Earlier, we have described an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to create genome-wide dominant mutations in the mouse model. Using this approach, we describe identification of two high bone density mutants in C57BL/6J (B6) background. The mutants, named as 12184 and 12137, have been bred more than five generations with wild-type B6 mice, each producing >200 backcross progeny. The average total body areal bone mineral density (aBMD) was 13-17% higher in backcrossed progeny from both mutant lines between 6 and 10 weeks of age, as compared to wild-type (WT) B6 mice (n=60-107). At 3 weeks of age the aBMD of mutant progeny was not significantly affected as compared to WT B6 mice. Data from 10- and 16-week old progeny show that increased aBMD was mainly related to a 14-20% higher bone mineral content, whereas bone size was marginally increased. In addition, the average volumetric BMD (vBMD) was 5-15% higher at the midshaft tibia or femur, as compared to WT mice. Histomorphometric analysis revealed that bone resorption was 23-34% reduced in both mutant mice. Consistent with histomorphometry data, the mRNA expression of genes that regulate osteoclast differentiation and survival were altered in the 12137 mutant mice. To determine the chromosomal location of the ENU mutation, we intercrossed both mutant lines with C3H/HeJ (C3H) mice to generate B6C3H F2 mice (n=164 for line 12137 and n=137 F2 for line 12184). Interval mapping using 60 microsatellite markers and aBMD phenotype revealed only one significant or suggestive linkage on chromosome 4. Since body weight was significantly higher in mutant lines, we also used body weight as additive and interactive covariate for interval mapping; both analyses showed higher LOD scores for both 12137 and 12184 mutants without affecting the chromosomal location. The large phenotype in the mutant mice compared to generally observed QTL effects (<5%) would increase the probability of identifying the mutant gene.

Growth hormone (GH) is important in the development and maintenance of bone; however, the IGF-dependent and -independent molecular pathways involved remain to be established. We used microarray analysis to evaluate GH signaling pathways in 4-wk-old GH-deficient mice following a single injection of GH (4 mg/kg body wt) or PBS (n = 6/group) at 6 or 24 h after treatment. Six thousand one hundred sixty genes were differentially expressed at P

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.

To identify the genes and signal pathways responsible for mechanical loading-induced bone formation, we evaluated differential gene expression on a global basis in the tibias of C57BL/6J (B6) mice after four days of four-point bending. We applied mechanical loads to the right tibias of the B6 mice at 9 N, 2 Hz for 36 cycles per day, with the left tibias used as unloaded controls. RNA from the tibias was harvested 24 h after last stimulation and subjected to microarray. Of the 20,280 transcripts hybridized to the array, 346 were differentially expressed in the loaded bones compared to the controls. The validity of the microarray data was established with the increased expression of bone-related genes such as pleiotrophin, osteoglycin, and legumain upon four-point bending and confirmation of increased expression of selected genes by real-time PCR. The list of differentially expressed genes includes genes involved in cell growth, differentiation, adhesion, proteolysis, as well as signaling molecules of receptors for growth factors, integrin, Ephrin B2, endothelin, and adhesion G protein coupled receptor. Pathway analyses suggested that 28 out of the 346 genes exhibited a direct biological association. Among the biological network, fibronectin and pleitrophin function as important signaling molecules in regulating periosteal bone formation and resorption in response to four-point bending. Furthermore, some expressed sequence tags (ESTs) with no prior known function have been identified as potential mediators of mechanotransduction signaling pathways. Further studies on these previously unknown genes will improve our understanding of the molecular pathways and mechanisms involved in bone's response to mechanical stress., (2005 Wiley-Liss, Inc.)

Unlabelled: Using a mouse mutant that fractures spontaneously and dies at a very young age, we identified that a deletion of the GULO gene, which is involved in the synthesis of vitamin C, is the cause of impaired osteoblast differentiation, reduced bone formation, and development of spontaneous fractures., Introduction: A major public health problem worldwide, osteoporosis is a disease characterized by inadequate bone mass necessary for mechanical support, resulting in bone fracture. To identify the genetic basis for osteoporotic fractures, we used a mouse model that develops spontaneous fractures (sfx) at a very early age., Materials and Methods: Skeletal phenotype of the sfx phenotype was evaluated by DXA using PIXImus instrumentation and by dynamic histomorphometry. The sfx gene was identified using various molecular genetic approaches, including fine mapping and sequencing of candidate genes, whole genome microarray, and PCR amplification of candidate genes using cDNA and genomic DNA as templates. Gene expression of selected candidate genes was performed using real-time PCR analysis. Osteoblast differentiation was measured by bone marrow stromal cell nodule assay., Results: Femur and tibial BMD were reduced by 27% and 36%, respectively, in sfx mice at 5 weeks of age. Histomorphometric analyses of bones from sfx mice revealed that bone formation rate is reduced by >90% and is caused by impairment of differentiated functions of osteoblasts. The sfx gene was fine mapped to a 2 MB region containing approximately 30 genes in chromosome 14. By using various molecular genetic approaches, we identified that deletion of the gulonolactone oxidase (GULO) gene, which is involved in the synthesis of ascorbic acid, is responsible for the sfx phenotype. We established that ascorbic acid deficiency caused by deletion of the GULO gene (38,146-bp region) contributes to fractures and premature death because the sfx phenotype can be corrected in vivo by treating sfx mice with ascorbic acid and because osteoblasts derived from sfx mice are only able to form mineralized nodules when treated with ascorbic acid. Treatment of bone marrow stromal cells derived from sfx/sfx mice in vitro with ascorbic acid increased expression levels of type I collagen, alkaline phosphatase, and osteocalcin several-fold., Conclusion: The sfx is a mutation of the GULO gene, which leads to ascorbic acid deficiency, impaired osteoblast cell function, and fractures in affected mice. Based on these and other findings, we propose that ascorbic acid is essential for the maintenance of differentiated functions of osteoblasts and other cell types.

EGFRvIII is a constitutively activated truncated variant of the epidermal growth factor receptor (EGFR) which has been shown to increase tumorgenicity. There are conflicting reports on the extent of EGFRvIII expression in tissues which may in part stem from the use of different assay methodologies. We investigated the expression of both EGFRvIII and wild-type EGFR (EGFRwt) in cell lines and primary breast cancers. First, we used a RT-PCR assay that can simultaneously measure EGFRwt and EGFRvIII mRNA to screen 55 tumor cell lines. We show that except for EGFRvIII transfected cells, only EGFRwt was detected. We then validated a real-time PCR assay and used this to screen 170 formalin fixed paraffin-embedded primary breast cancers for evidence of EGFRwt and EGFRvIII expression. No samples were positive for EGFRvIII expression except for control transfectants and glioblastomas. In contrast, EGFRwt was expressed at varying levels in the majority of samples tested. We conclude that the expression of EGFRvIII is extremely rare in breast cancer and therefore it does not contribute to the malignant phenotype.

beta-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human beta-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as < or = 350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5' untranslated region (C-->G at position -44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C-->G SNP at position -44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.

Steroid hormones are important in the etiology and progression of prostate cancer, and expression of genes involved in hormone production may alter susceptibility. One such gene is CYP17, which encodes the cytochrome P450c17a enzyme responsible for the biosynthesis of testosterone. A T to C transition (A2 allele) in the 5' promoter region of the gene is hypothesized to increase the rate of gene transcription, increase androgen production, and thereby increase risk of prostate cancer. To test this hypothesis, germ-line DNA samples from a large population-based study of incident prostate cancer cases (n = 590) and controls (n = 538) of similar age without the disease were genotyped. The frequency of the A2 allele was similar in cases and controls. Compared with men with the A1/A1 genotype, the adjusted odds ratio was 0.81 for the A1/A2 and 0.87 for the A2/A2 genotype. Risk estimates did not vary substantially by age or race. However, stratification by family history of prostate cancer revealed that among white men with an affected first-degree relative, homozygotes for the A2 allele had a significant elevation in risk (odds ratio = 19.2; 95% confidence interval, 2.2-157.4) compared with men who were homozygous for the A1 allele (interaction P = 0.0005). These results suggest that the CYP17 A2/A2 genotype predicts susceptibility to prostate cancer in white men with a family history of the disease. It is also possible that CYP17 interacts with other genes that influence risk of familial prostate cancer.

The retinoblastoma protein-interacting zinc finger gene RIZ (PRDM2) is a member, by sequence homology, of a nuclear protein-methyltransferase (MTase) superfamily involved in chromatin-mediated gene expression. The gene produces two protein products, RIZ1 that contains a conserved MTase domain and RIZ2 that lacks the domain. RIZ1 gene expression is frequently silenced in human cancers, and the gene is also a common target of frameshift mutation in microsatellite-unstable cancers. We now report studies of mice with a targeted mutation in the RIZ1 locus. The mutation inactivates RIZ1 but not RIZ2. These RIZ1 mutant mice were viable and fertile but showed a high incidence of diffuse large B-cell lymphomas (DLBL) and a broad spectrum of unusual tumors. RIZ1 deficiency also accelerated tumorigenesis in p53 heterozygous mutant mice. Finally, several missense mutations of RIZ1 were found in human tumor tissues and cell lines; one of these was particularly common in human DLBL tumors. These missense mutations, as well as the previously described frameshift mutation, all mapped to the MTase functional domains. All abolished the capacity of RIZ1 to enhance estrogen receptor activation of transcription. These data suggest a direct link between tumor formation and the MTase domain of RIZ1 and describe for the first time a tumor susceptibility gene among methyltransferases.

A set of 90 nonpolypotic colon cancer families in which germ-line mutations of MSH2 and MLH1 had been excluded were screened for mutations in two additional DNA mismatch repair genes, MSH6 and MSH3. Kindreds fulfilling and not fulfilling the Amsterdam I criteria, showing early and late onset colorectal (and other) cancers, and having microsatellite stable and unstable tumors were included. Two partly parallel approaches were used: genetic linkage analysis (19 large families) and the protein truncation test (85, mostly smaller, families). Whereas MSH3 was not involved in any family, a large Amsterdam-positive, late-onset family showed a novel germ-line mutation in MSH6 (deletion of CT at nucleotide 3052 in exon 4). The mutation was identified through genetic linkage (multipoint lod score 2.4) and subsequent sequencing of MSH6. Furthermore, the entire MSH6 gene was sequenced exon by exon in families with frameshift mutations in the (C)8 tract in tumors, previously suggested as a predictor of MSH6 germ-line mutations; no mutations were found. We conclude that germ-line involvement of MSH6 and MSH3 is rare and that other genes are likely to account for a majority of MSH2-, MLH1-mutation negative families with nonpolypotic colon cancer.

We have analyzed the survival motor neuron gene (SMN1) dosage in 100 parents of children with homozygous SMN1 deletions. Of these parents, 96 (96%) demonstrated the expected one-copy SMN1 carrier genotype. However, four parents (4%) were observed to have a normal two-copy SMN1 dosage. The presence of two intact SMN1 genes in the parent of an affected child indicates either the occurrence of a de novo mutation event or a situation in which one chromosome has two copies of SMN1, whereas the other is null. We have separated individual chromosomes from two of these parents with two-copy SMN1 dosage by somatic cell hybridization and have employed a modified quantitative dosage assay to provide direct evidence that one parent is a two-copy/ zero-copy SMN1 carrier, whereas the other parent had an affected child as the result of a de novo mutation. These findings are important for assessing the recurrence risk of parents of children with spinal muscular atrophy and for providing accurate family counseling.

We hypothesize that loss of imprinting (LOI) of the insulin-like growth factor II (IGF2) gene is associated with a predisposition to sporadic colorectal cancer. We confirmed a previously known strong correlation between LOI and microsatellite instability and showed that LOI was not a consequence of microsatellite instability or mismatch repair deficiency. LOI of IGF2 correlated strongly with biallelic hypermethylation of a core of five CpG sites in the insulator region of IGF2/H19, which is a known CTCF-binding element. As this methylation-dependent LOI was present in both tumors and normal colonic mucosa, it is possible that hypermethylation creates a field defect predisposing to cancer.

PMS2 is one of a complex of genes encoding DNA repair proteins that includes MSH2, MLH1, MSH6 and MSH3. Mutation of any of these DNA mismatch repair genes leads to impairment of DNA repair and can lead to tumorigenesis. Germline mutation of PMS2 has been reported as a rare cause of hereditary nonpolyposis colorectal cancer (HNPCC) and Turcot's syndrome. The PMS2 gene is located on chromosome 7p22 and consists of 15 exons. Within exon 11 of PMS2 is a coding repeat of eight adenosines. This study reports on the finding of a nonexpressed pseudogene that is highly homologous to the PMS2 gene in this region. The pseudogene is polymorphic for two alterations in the repeat region: a 3 bp delAAA at a site corresponding to nucleotide 1231 in PMS2; and an AA-->GG change at nucleotide 1238. Due to the high homology in both intronic and exonic sequences, polymorphisms in this pseudogene could be mistaken for mutations in the PMS2 gene and erroneously thought to be a cause of HNPCC and/or Turcot's syndrome., (Copyright 2000 Wiley-Liss, Inc.)

Introduction: An intronic germline mutation in the MSH2 gene, A-->T at nt942+3, interferes with the exon 5 donor splicing mechanism leading to a mRNA lacking exon 5. This mutation causes typical hereditary non-polyposis colorectal cancer (HNPCC) and has been observed in numerous probands and families world wide. Recurrent mutations either arise repeatedly de novo or emanate from ancestral founding mutational events. The A-->T mutation had previously been shown to be enriched in the population of Newfoundland where most families shared a founder mutation. In contrast, in England, haplotypes failed to suggest a founder effect. If the absence of a founder effect could be proven world wide, the frequent de novo occurrence of the mutation would constitute an unexplored predisposition., Methods: We studied 10 families from England, Italy, Hong Kong, and Japan with a battery of intragenic and flanking polymorphic single nucleotide and microsatellite markers., Results: Haplotype sharing was not apparent, even within the European and Asian kindreds. Our marker panel was sufficient to detect a major mutation arising within the past several thousand generations., Discussion: As a more ancient founder is implausible, we conclude that the A-->T mutation at nt942+3 of MSH2 occurs de novo with a relatively high frequency. We hypothesise that it arises as a consequence of misalignment at replication or recombination caused by a repeat of 26 adenines, of which the mutated A is the first. It is by far the most common recurrent de novo germline mutation yet to be detected in a human mismatch repair gene, accounting for 11% of all known pathogenic MSH2 mutations.

The distal portion of chromosome 1p is one of the most commonly affected regions in human cancer. In this study of hereditary and sporadic colorectal cancer, a region of frequent deletion was identified at 32.2 centimorgans from 1ptel. Deletion breakpoints clustered in the vicinity of or inside the gene RIZ, which encodes a retinoblastoma protein-interacting zinc finger protein. Sequence analysis revealed frequent frameshift mutations of the RIZ gene. The mutations consisted of 1- or 2-bp deletions of a coding (A)(8) or (A)(9) tract and were confined to microsatellite-unstable colorectal tumors, being present in 9 of 24 (37.5%) primary tumors and in 6 of 11 (54.5%) cell lines; in 2 cell lines the mutation was homozygous/hemizygous. The mutations apparently were selected clonally in tumorigenesis, because similar poly(A) tracts in other genes were not affected. Two alternative products of the gene exist, RIZ1, which contains a PR (PRDI-BF1-RIZ1) domain implicated in tumor suppressor function, and RIZ2, which is lacking this motif. Furthermore, the C-terminal region, which contains the poly(A) tracts, includes a PR-binding motif, possibly mediating interactions with other proteins or with RIZ itself (oligomerization). Four of eleven microsatellite-unstable colorectal cancer cell lines, three of which had frameshifts, showed reduced or absent mRNA expression of RIZ1. In a cell line that is homozygous/hemizygous for the typical frameshift mutation, immunoblotting showed truncated RIZ protein, whereas adenovirus-mediated RIZ1 expression caused G(2)/M arrest and apoptosis. We propose that RIZ is a target of the observed 1p alterations, with impairment of the PR domain-mediated function through either frameshift mutation or genomic deletion.

The role of genetic factors in the etiology of colorectal cancers (CRCs) has recently been elucidated with the discovery of the mismatch repair. These genes are responsible for less than 5% of all cases of CRCs in Caucasian series. In this pilot study, tumors from 5 randomly ascertained CRC patients were subjected to microsatellite analysis, and two were microsatellite unstable. Both of these two patients had germline mutations in MSH2. If this finding can be confirmed in a larger series of patients, it suggests that MMR genes play an important role in the etiology of CRCs in Africa.

Instability in the repeat size of microsatellite sequences has been described in both hereditary nonpolyposis and sporadic colorectal cancers. Tumors expressing microsatellite instability are identified through the comparison of the repeat sizes at multiple microsatellite loci between tumor and matched normal tissue DNA. The use of a five-marker panel including two mononucleotide repeat microsatellites, BAT-25 and BAT-26, has recently been suggested for the clinical determination of tumor microsatellite instability. The BAT-25 and BAT-26 loci included in this panel have both demonstrated sensitivity to microsatellite instability and normal quasimonomorphic allelic patterns, which has simplified the distinction between normal and unstable alleles. However, in this study, we identified allelic variations in the size of the poly(A) tract at BAT-26 in 12.6% of 103 healthy African-Americans screened. In addition, 18.4% exhibited allelic size variations in the poly(T) tract at BAT-25. Finally, 2.9% showed variant alleles at both BAT-25 and BAT-26 loci. Screening a small population of Nigerians confirmed the polymorphic nature of both loci and the ethnic origin of alleles not identified in other populations studied thus far. Our results dispute the quasimonomorphic nature of both BAT-25 and BAT-26 in all populations and support the need for thorough population studies to define the different allelic profiles and frequencies at microsatellite loci.

Megaloblastic anaemia 1 (MGA1, OMIM 261100) is a rare, autosomal recessive disorder characterized by juvenile megaloblastic anaemia, as well as neurological symptoms that may be the only manifestations. At the cellular level, MGA1 is characterized by selective intestinal vitamin B12 (B12, cobalamin) malabsorption. MGA1 occurs worldwide, but its prevalence is higher in several Middle Eastern countries and Norway, and highest in Finland (0.8/100,000). We previously mapped the MGA1 locus by linkage analysis in Finnish and Norwegian families to a 6-cM region on chromosome 10p12.1 (ref. 8). A functional candidate gene encoding the intrinsic factor (IF)-B12 receptor, cubilin, was recently cloned; the human homologue, CUBN, was mapped to the same region. We have now refined the MGA1 region by linkage disequilibrium (LD) mapping, fine-mapped CUBN and identified two independent disease-specific CUBN mutations in 17 Finnish MGA1 families. Our genetic and molecular data indicate that mutations in CUBN cause MGA1.

Background & Aims: Colorectal cancer is one of the most frequent cancers in humans. Recently, a germline missense mutation, I1307K, was identified in the adenomatous polyposis coli (APC) gene that was suggested to increase cancer predisposition in Ashkenazi Jews. However, a second study indicated that the I1307K mutation did not contribute greatly to the risk of colon cancer in Ashkenazi breast-ovarian cancer families, and a role of mismatch repair deficiency was suggested. This study investigated the frequency of the I1307K mutation in several non-Ashkenazi Jewish populations. We also compared the distribution and frequency of APC mutations from colon tumors that were positive and negative for the I1307K mutation. Finally, the association between the presence of mutations in the I1307K region and mismatch repair deficiency was studied., Methods: We tested for I1307K in 345 patients who were not Ashkenazi Jews using a heteroduplex screen. We also performed an extensive mutational analysis in this region of the APC gene on DNA extracted from 240 Italian, Finnish, and Hawaiian-Japanese colon tumors and determined replication error status., Results: The I1307K mutation was not found among 345 non-Ashkenazis. Somatic mutations occurred at a lower frequency and were more randomly distributed when the I1307K allele was not present. The most common characteristic somatic mutation occurring around codon 1307 in I1307K-positive patients did not occur in tumors negative for the I1307K mutation. An association between mutations in the region around APC codon 1307 and mismatch repair deficiency was not found., Conclusions: Our findings support the hypothesis that the I1307K mutation is unique to the Ashkenazi Jews, contributes to tumor predisposition in colorectal cancer, and is unrelated to mismatch repair deficiency.

Background: Genetic disorders that predispose people to colorectal cancer include the polyposis syndromes and hereditary nonpolyposis colorectal cancer. In contrast to the polyposis syndromes, hereditary nonpolyposis colorectal cancer lacks distinctive clinical features. However, a germ-line mutation of DNA mismatch-repair genes is a characteristic molecular feature of the disease. Since clinical screening of carriers of such mutations can help prevent cancer, it is important to devise strategies applicable to molecular screening for this disease., Methods: We prospectively screened tumor specimens obtained from 509 consecutive patients with colorectal adenocarcinomas for DNA replication errors, which are characteristic of hereditary colorectal cancers. These replication errors were detected through microsatellite-marker analyses of tumor DNA. DNA from normal tissue from the patients with replication errors was screened for germ-line mutations of the mismatch-repair genes MLH1 and MSH2., Results: Among the 509 patients, 63 (12 percent) had replication errors. Specimens of normal tissue from 10 of these 63 patients had a germ-line mutation of MLH1 or MSH2. Of these 10 patients (2 percent of the 509 patients), 9 had a first-degree relative with endometrial or colorectal cancer, 7 were under 50 years of age, and 4 had had colorectal or endometrial cancer previously., Conclusions: In this series of patients with colorectal cancer in Finland, at least 2 percent had hereditary nonpolyposis colorectal cancer. We recommend testing for replication errors in all patients with colorectal cancer who meet one or more of the following criteria: a family history of colorectal or endometrial cancer, an age of less than 50 years, and a history of multiple colorectal or endometrial cancers. Patients found to have replication errors should undergo further analysis for germ-line mutations in DNA mismatch-repair genes.

Loss of heterozygosity (LOH) and replication error (RER) are important phenomena in tumor development, with diagnostic and prognostic relevance. Therefore, screening for LOH and RER is a desirable first step in the molecular analysis of tumors. We used semiautomated procedures based on multicolor fluorescently labeled microsatellite markers and an automated sequencer for PCR amplification, electrophoresis of PCR products, and allele detection with a set of 16 microsatellites in 56 colorectal tumors. We improved existing software for computer-assisted assessment of LOH and RER. A comparison of these results with those of a conventional, radioactive technique and visual interpretation shows a high degree of correlation between the two methods. The detection rates of LOH and RER are similar to those reported previously. The main advantages of the semiautomated fluorescence-based typing are in the objective, observer-unrelated, easy, and rapid computer-based scoring, and the resulting quantitative assessment of RER.

We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.

A crucial factor in the success of positional cloning efforts is the ability to screen rapidly many different candidate genes for mutations. By modifying standard software, we have improved the detection of heterozygous base positions in PCR products sequenced by cycle sequencing. A key element of the method is the incorporation of a modified heterozygote detection algorithm that permits the use of DNA sequence data derived from PCR and sequencing reactions that have not been fully optimized. This allows sequencing runs of average quality to be used. We demonstrate that the sensitivity and specificity of the method are well suited to mutation detection applications such as positional cloning.

The distal portion of the rat insulin I gene 5'-flanking DNA contains two sequence elements, the Far and FLAT elements, that can function in combination, but not separately, as a beta-cell-specific transcriptional enhancer. We have isolated several cDNAs encoding proteins that bind to the FLAT element. Two of these cDNAs, cdx-3 and lmx-1, represent homeo box containing mRNAs with restricted patterns of expression. The protein encoded by lmx-1 also contains two amino-terminal cysteine/histidine-rich "LIM" domains. Both cdx-3 and lmx-1 can activate transcription of a Far/FLAT-linked gene when expressed in a normally non-insulin-producing fibroblast cell line. Furthermore, in fibroblasts expressing transfected beta-cell lmx-1, the addition of the Far-binding, basic helix-loop-helix protein shPan-1 (the hamster equivalent of human E47) causes a dramatic synergistic activation. ShPan-1 causes no activation in fibroblasts expressing transfected cdx-3 or the related LIM-homeodomain protein isl-1. Deletion of one or both of the LIM domains from the 5' end of the lmx-1 cDNA removes this synergistic interaction with shPan-1 without any loss of basal transcriptional activation. We conclude that beta-cell lmx-1 functions by binding to the FLAT element and interacting through the LIM-containing amino terminus with shPan-1 bound at the Far element. These proteins form the minimal components for a functional minienhancer complex.