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Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K-Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three-gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at-risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified,nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255-4delGTA; c.1682-1683 delTA in CCM1; c.48A > G; c.82-83dupAG in CCM2; and c.395 + 1G > A in CCM3 genes [corrected].The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at-risk relatives.

Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. LS predisposes to high risk of early-onset colorectal, endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five MLH1 or MSH2 variants. Of these, MSH2-Q402X, MSH2-G322D, and MLH1-K618A had been previously reported, while the MSH2-E205Q and MSH2-V367I variants were novel. MSH2-Q402X is a known stop mutation and reported here for the first time here in association with PC. MLH1-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant. MSH2-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants MSH2-E205Q and MSH2-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for 3, and for the first time determined for 6 novel variants. Overall, based on our results we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely non pathogenic". This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting. This article is protected by copyright. All rights reserved.

Allelic variants of several genes are increasingly recognized as susceptibility factors in age-related macular degeneration (AMD). Because of its metabolic characteristics the macula is sensitive to oxidative damage, and supplementation with antioxidants has been shown to be effective in slowing the progression of disease in AMD patients. The oxisterol-binding-protein (OSBP2) gene is expressed mainly in the retinal pigmented epithelium underlying the macular region. Its product specifically binds and transports oxisterols, the cytotoxic effects of which may be involved in macular damage. The aim of this study was to search for allelic variants of OSBP2 gene, as well as to evaluate several risk factors in 24 patients with AMD; 17 with nonexudative (NE) and 7 with neovascular (NV) form. Total cholesterol was elevated in 66% of the patients, high-density lipoprotein (HDL) cholesterol was reduced in 12%; vitamin A or vitamin E deficiency was not observed. OSBP2 gene analysis was performed in AMD patients and in 110 control subjects by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Six allelic variants were detected: 2 nonpolymorphic unique exonic variants in 2 AMD subjects and 4 polymorphic variants (2 exonic and 2 intronic). These data indicate a possible role of OSBP2 gene in the pathogenesis of oxidative damage to the macula induced by oxysterols in AMD patients.

Mutation-predicting models can be useful when deciding on the genetic testing of individuals at risk and in determining the cost effectiveness of screening strategies at the population level. The aim of this study was to evaluate the performance of a newly developed genetic model that incorporates tumor microsatellite instability (MSI) information, called the AIFEG model, and in predicting the presence of mutations in MSH2 and MLH1 in probands with suspected hereditary non-polyposis colorectal cancer. The AIFEG model is based on published estimates of mutation frequencies and cancer penetrances in carriers and non-carriers and employs the program MLINK of the FASTLINK package to calculate the proband's carrier probability. Model performance is evaluated in a series of 219 families screened for mutations in both MSH2 and MLH1, in which 68 disease-causing mutations were identified. Predictions are first obtained using family history only and then converted into posterior probabilities using information on MSI. This improves predictions substantially. Using a probability threshold of 10% for mutation analysis, the AIFEG model applied to our series has 100% sensitivity and 71% specificity.

Hereditary Colorectal Tumor Registry, Istituto NazionaleTumori, Milan, ItalyCommunicated by Jing ChengLarge genomic rearrangements are recognized as playing a pathogenic role in an increasing number of humangenetic diseases. It is important to develop efficient methods for the routine detection and confirmation of thesegermline defects. Multiplex ligation-dependent probe amplification (MLPA) is considered an early step formolecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPAanalysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verifiedby two independent methods. In this study, we developed nonfluorescent multiplex-PCR coupled to high-performance liquid chromatography (NFMP-HPLC) andanalyzed whetherthe use of this method combined withMLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements.A total of nine nonfluorescent multiplex-PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons.Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number ofcycles (r25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The resultsobtained by NFMP-HPLC were highly reproducible. The combined use of MLPA and NFMP-HPLC identifiedand independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients withhereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and inthree cases multiple contiguous exons. These results were in agreement with breakpoint and complementaryDNA (cDNA) analyses. Considering that MLPA and NFMP-HPLC are unlikely to be affected by the sameartifacts, their combined use could also provide a robust and cost-effective strategy for routine screening andconfirmation of putative rearrangements in other genes, especially when a single exon is involved or a precisecharacterization of breakpoints is not achieved. Hum Mutat 27(10), 1047–1056, 2006.

Editor—Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disorder characterised by the development of hundreds to thousands of adenomatous polyps in the colon and rectum. If left untreated, there is a very high risk of colorectal cancer. Adenomatous polyps may also develop proximally in the stomach and the distal part of the duodenum. FAP is also associated with a variety of extracolonic benign and malignant manifestations, including congenital hypertrophy of the retinal pigment epithelium (CHRPE), dental abnormalities, desmoid tumours, osteomas, epidermoid cysts, hepatoblastoma, and thyroid neoplasia.1 Germline mutations of the APC gene localised on chromosome 5q21.22 are responsible for FAP.2 3 APC is a tumour suppressor gene encoding a 2843 amino acid protein, which contains multiple functional domains and which mediates growth regulatory signals by its association with a variety of cytoplasmic proteins. More than 300 different APC mutations have so far been identified distributed throughout the whole gene, with a higher concentration in the 5′ part of exon 15 (codons 713-1597).4 5 The majority of mutations are predicted to introduce premature termination signals resulting from single nucleotide alterations, small insertions or deletions, or splice site mutations that lead to truncation of the normal protein product.4 Missense mutations have rarely been reported and their functional implications are often unclear.6 7 Larger deletions and insertions have been described, as well as genomic rearrangements resulting from recombinations mediated by Alu elements which cause inappropriate exon splicing.8-10 Isoforms of APC transcripts lacking exon 9, exon 10A, and exon 14 encoded sequences have been reported.2 11-13 Isoforms lacking exon 9 or exon 14 owing to splice site mutations have also been associated with a FAP phenotype.14-17 In this study, we describe a G→T transversion at nucleotide position 1869 in exon 14 which gives …

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

More than 100 different mutations in the adenomatous polyposis coli (APC) gene have been identified; virtually all lead to the production of a truncated protein. Clinical details of patients with missense mutations undoubtedly cosegregating with the disease have not been reported and may be relevant in understanding the APC protein function.In one family with familial adenomatous polyposis (FAP) the APC gene was analyzed by SSCP and sequencing of the aberrant SSCP band.A missense mutation in exon 15 at nucleotide 4921 segregating with the disease was observed. This predicts a tryptophan instead of an arginine at amino acid 1641 of the APC protein. No such mutation was present in 100 control subjects.In this family the colonic manifestations are as expected for classical FAP. However, the occurrence of congenital hypertrophy of the retinal pigment epithelium is unusual, owing to the inconsistency of this manifestation between family members and because congenital hypertrophy of the retinal pigment epithelium is generally absent when mutations are after codon 1387.

Summary Familial Adenomatous Polyposis (FAP) is a premalignant disease of the gastrointestinal tract inherited as an autosomal dominant trait assigned to chromosome 5q21. The 15 exons of the APC gene responsible for the defect were amplified from the DNA of one FAP patient. SSCP analysis of the amplified DNA revealed a variant conformer of exon 10. The sequencing of the cloned PCR product showed a 1 base insertion at position 1370, creating a stop codon four nucleotides downstream. SSCP analysis of 20 family members and nucleotide sequencing of exon 10 in three affected members confirmed the Mendelian inheritance of the mutant allele.

Sixty-eight patients affected by Philadelphia chromosome (Ph) positive chronic myelogenous leukemia (CML) underwent allogeneic bone marrow transplantation (BMT) and were successfully studied from a cytogenetic point of view, before and after the BMT. Nineteen had evidence of cytogenetic and clinical relapse. Cytogenetic analyses of 14 patients who, after the relapse, showed progression to the accelerated or blastic phase of the disease, are presented. Five of these cases had only the Ph chromosome without karyotype evolution; in one case Ph duplication without other anomalies was detected, while in the remaining eight cases cytogenetic analysis showed apparently random clonal structural abnormalities (translocations, inversions, deletions, and marker formations). Therefore, the classical “non-random” abnormalities (+8, i(17q), +Ph, +19, +21) were not as common as in conventionally treated Ph+ CML. From our data, karyotype evolution during advanced phases in Ph+ CML patients after BMT differs from the evolution seen in conventionally treated patients, by the presence of numerous structural unusual abnormalities, possibly related to radiochemotherapy conditioning to BMT. Therefore, BMT treatment is not always able to eradicate the Ph+ clone but can reduce the incidence of the formation and/or expansion of Ph+ clones with additional non-random abnormalities.

Familial cerebral cavernous malformation (CCM) exhibits autosomal dominant inheritance and is characterized by vascular disorders of the brain, which can lead to seizures, focal neurological deficits, hemorrhagic stroke, and migraine. Three CCM loci have been mapped, but the gene for only one locus--KRIT1 coding for Krev-1/rap1 interaction trapped 1 (KRIT1) protein, which is responsible for more than 40% of familial cases--has been identified. To date, a total of 72 mutations have been described, with one founder effect in the Mexican/Hispanic community. We report the case of an Italian family with CCM that has a novel KRIT1 gene mutation leading to a truncated KRIT1 protein. The protein truncation test (PTT) has been used as a rapid method of identifying germline mutations in the KRIT1 gene.

In the present study, we used five different polymorphic markers to construct the haplotype at the adenomatous polyposis coli (APC) locus in families with familial adenomatous polyposis (FAP) and in the normal Italian population. Non-ambiguous haplotypes were reconstructed from 246 normal chromosomes and 65 FAP chromosomes. In the control population, the four polymorphisms intragenic to APC gave rise to 16 haplotypes, the most common of which (II and XV) accounted for over 50% of all chromosomes. In FAP patients, 13 haplotypes were found but their distribution was not statistically different from normal subjects. Eighty complete chromosomal haplotypes (many fewer than the theoretical maximum of 208) for the five polymorphic sites assayed were observed in the control population, 35 being found in the FAP patients. We compared the distribution of these haplotypes within the two groups; no statistically significant differences between normal and FAP chromosomes were found. The elevated heterogeneity of FAP chromosomes was clearly confirmed by the observation that 19 patients who carried one or other of the two most common APC mutations (nt 3183 and nt 3927) showed 18 different haplotypes. On the basis of these results, we were not able to identify a founder FAP chromosome. Various mechanisms are presented to explain this observation.

Linkage analysis was performed on 188 subjects belonging to 18 Italian families segregating for familial adenomatous polyposis (FAP) using 7 polymorphic markers (5 restriction fragment length and 2 dinucleotide repeat polymorphisms) mapping in 5q21. A two-point linkage analysis performed with the LINKAGE program gave significant lod scores (> 3) between the Pi227, C11p11, YN5.64, YN5.48 probes and the disease, whereas the ECB27, CB83 and EF5.44 markers showed lower lod scores. Some 11 recombination events were identified from the analysis of 101 meioses. The best map that we could determine confirmed that reported in previous studies. The location of the new marker, CB83, lying between YN5.64 and YN5.48, remains imprecise. No genetic heterogeneity was detected, with all the families showing linkage for at least one of the probes. One 34-year-old individual having an affected haplotype was however classified as healthy after clinical examinations. The results confirm the applicability of the linkage approach for presymptomatic diagnosis of FAP.

Familial adenomatous polyposis (FAP) is a common hereditary syndrome characterized by early development of colorectal cancer consequent to extensive adenomatous polyps of the colon. In addition to the colonic manifestations the syndrome presents several extracolonic features including polyps of the upper gastrointestinal tract, congenital hypertrophy of the retinal pigment, jaw cysts, osteomata and desmoid tumors. In this study the entire APC coding region has been analysed for mutation in a panel of one Turcot and 33 unrelated Italian FAP patients using SSCP analysis, PTT and DNA sequencing. We detected APC mutations in 23 of them and identified nine which, to our knowledge were not previously reported. All of these novel mutations are in exon 15, including two nonsense mutations, 6 deletions or insertions leading to premature termination of the protein and one missense mutation (7697G>A). This last mutation occurs in the EB1-binding domain of the APC protein and segregates in four relatives of the patient with three of them presenting 2-3 adenomatous polyps.

Forty-eight consecutive patients affected by chronic myelogenous leukemia (CML) Philadelphia (Ph) chromosome positive during the chronic phase of the disease underwent allogeneic bone marrow transplantation (BMT). Thirty-five patients had a follow-up over 12 months and were included in a cytogenetic study in order to evaluate the Ph clone eradication. In 25 cases, the Ph chromosome disappeared in all cytogenetic studies, and their hematologic picture is at present apparently normal. Ten patients showed cytogenetic relapse. In one case, the cytogenetic relapse was transitory without any clinical sign of the disease; in three cases, after a transitory cytogenetic relapse, a persistent relapse with clinical picture of progression of the disease occurred; in six cases cytogenetic and a clinical relapse were coincident. Structural chromosomal abnormalities other than Ph were temporarily seen in three cases. The so-called “nonrandom” chromosomal changes typical of the blastic phase were never detected. The reappearing Ph-positive clone spontaneously disappeared in three patients, and their hematologic picture reverted to complete chimerism. The present study confirms that the eradication of the Ph clone is often defective with BMT, and cytogenetic analysis can detect the competition between donor and residual host marrow. Furthermore, the karyotype evolution is different from that found in CML patients treated with conventional chemotherapy.

In a patient with chronic myelocytic leukemia chromosome analysis showed a translocation (22;22) (q13;q11). Chromosomes 9 were apparently not involved. Using somatic cell hybrids and a v-abl probe, we demonstrated the translocation of c-abl sequences from chromosome 9 to chromosome 22q-. This confirms the hypothesis that the translocation of c-abl oncogene is essential for the development of Ph1 positive CML.

Seven cases of acute nonlymphoblastic leukemia showing t(8;21)(q22;q22) at diagnosis are described. Involvement of a sex chromosome was found in all patients (in six cases as a loss, and in one as a Y duplication), thus, confirming the suggestion of a correlation between these two chromosomal abnormalities. The constitutional folic acid-sensitive fragile site fra(8)(q22) was not detected, in spite of careful analysis of over 300 metaphases of cells grown in folic acid-free medium. Morphologic diagnoses and clinical aspects are briefly discussed.

Chromosome analysis showed a t(9;9)(p13;q34) in a patient with chronic myeloid leukemia (CML) without a Philadelphia (Ph) chromosome in all examined cells. Southern blot analysis of leukocyte DNA revealed rearrangement of breakpoint cluster region (bcr) within the 5.8-kb bcr sequences as in Ph-positive CML patients. The findings confirm that the 9q34 and 22q11 bands are always involved in CML independent of the chromosomal evidence. It is suggested that Ph-negative bcr-positive CML may have variant translocations, as in the case of the t(9;9) reported here.

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.

Favism is a severe, acute haemolytic anaemia which occurs in about 20% of G6PD deficient subjects after ingestion of fava beans. Since not all G6PD deficient subjects are sensitive to fava beans, the possibility has been suggested that extra erythrocytic factors may play an important role in the susceptibility to haemolytic favism. To test the hypothesis that an autosomal enzyme is involved in the pathogenesis of favism, we carried out a beta-glucosidase assay in small intestine biopsies from normal subjects and G6PD deficient subjects with or without favism. Beta-glucosidase might be involved in the absorption and metabolism of fava beans and a quantitative polymorphism could explain the different susceptibility to fava beans of G6PD deficient subjects. Our observation showed no consistent quantitative polymorphism of beta-glucosidase in the subjects examined.

We report two cases of myeloproliferative syndromes in which the only karyotypic abnormality was an isochromosome of the long arm of chromosome 17. Because i(17q) is a nonrandom structural aberration found in nearly 12% of cases of Philadelphia (Ph)-positive chronic myelogenous leukemia (CML), we carried out a molecular analysis of the breakpoint cluster region (bcr) to verify the presence of genomic rearrangements characteristic of CML. The interest of the study was strengthened by the fact that i(17q) is frequently seen in CML and by recent reports showing that genomic changes of c-abl and bcr genes can be present even in the absence of a Ph chromosome. One of the two patients showed the presence of a rearranged fragment within the bcr, suggesting a Ph-positive CML diagnosis.

Editor—Hereditary non-polyposis colorectal cancer (HNPCC) is a heterogeneous autosomal dominant disease with incomplete penetrance. The frequency is estimated at 1:200/1:1000. HNPCC results from constitutional mutation in one of the five human mismatch repair genes (MMR) that have so far been implicated: hMSH2 , hMLH1 , hPMS1 , hPMS2 , and hMSH6 .1 2 hMSH2 and hMLH1 account for the majority of mutations found in HNPCC families (25-70%). The function of MMR genes is to maintain genetic stability and tumour DNA from HNPCC patients shows an accumulation of replication errors exhibiting an instability phenotype at microsatellite loci (MSI).3 4 Standard criteria have been established to define HNPCC clinically. These are known as the “Amsterdam criteria” and require that at least two generations be affected by colorectal cancer (CRC), that three or more relatives with histologically verified CRC be present, one of whom is a first degree relative of the other two, and at least one case of CRC be diagnosed before the age of 50.5 The syndrome is characterised by synchronous and metachronous CRC tumours and by association of certain types of extracolonic tumours, especially endometrial neoplasia.6 In 45-86% of cases,7 genetic testing characterises the mutation in the index patient for each HNPCC family identified and predicts a possible predisposition to colon and/or related syndrome cancer in at risk subjects of the same kindred. In clinical practice, however, besides HNPCC families, there are families with multiple CRC patients that do not fulfil the Amsterdam criteria, or with a single case of early onset CRC, or with multiple primary tumours. These features can suggest an HNPCC syndrome. In these instances, the percentage of germline mutations of MMR genes is low and the lifetime incidence of colorectal cancer is lower than in Amsterdam families.8 9 …