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Germline mutations at the adenomatous polyposis (APC) gene are responsible for familial adenomatous polyposis (FAP), an inherited condition that predisposes to the development of hundreds to thousands benign adenomas in the colorectum. If not surgically removed, colorectal adenomas inevitably progress into malignant adenocarcinomas. To date, more than 450 germline mutations have been described at the APC gene, allowing the establishment of genotype-phenotype correlations between the site and type of the molecular defects and their morbid consequences. However, the function of the APC protein and its role in intestinal tumorigenesis are still elusive. Somatic mutations in the APC gene have also been found in the majority of early sporadic adenomas with similar frequencies as those reported in the more advanced colorectal tumors, clearly indicating that it represents an important determinant in the pathogenesis of this common cancer. Therefore, studies on the normal function of APC and the mechanisms by which its tumorigenic mutations lead to the development of cancer would have practical (i.e., clinical) as well as theoretical relevance. Here, we review the current literature on the relationship between APC mutations and their phenotypic consequences, with particular regard of the implications for the understanding of the function of this gene in homeostasis and tumorigenesis.

Germ-line mutations in the human adenomatous polyposis coli (APC) gene result in familial adenomatous polyposis, an autosomal dominant disorder characterized by the early onset of multiple adenomatous polyps in the large bowel with a high likelihood of developing colorectal carcinomas. To understand the role of APC in intestinal tumor formation, we have introduced a chain-termination mutation in the 15th exon of the mouse Apc gene and employed it to modify the endogenous gene by homologous recombination in embryonic stem cells. Mice which are heterozygous for the Apc gene modification progressively develop intestinal tumors in a manner that is similar to that observed in patients with familial adenomatous polyposis and in mice which carry a mutation called multiple intestinal neoplasia (Min). Our results indicate that the Apc gene modification is a critical event in the initiation of intestinal tumor formation and results in an autosomal dominant predisposition toward development of spontaneous colonic and intestinal tumors in mice.

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2-3 Mb in the region q12-q13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.

Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogenesis. For the identification of protein truncating mutations of the APC gene, we developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectiveness of this method.

The molecular analysis of genetic diseases relies on several technical approaches which allow genetic and physical mapping, characterization of the gene structure, expression studies, and identification of disease-causing mutations. Denaturing gradient gel electrophoresis (DGGE) allows the rapid screening for single base changes in enzymatically amplified DNA. The technique is based on the migration of double-stranded DNA molecules through polyacrylamide gels containing linearly increasing concentrations of a denaturing agent. In this review DGGE and the several modifications of the original protocol are presented. Moreover, its applications in human molecular genetics are summarized together with a preliminary comparison with other mutation detection technologies such as chemical cleavage, RNase protection, and single-strand conformation polymorphism.

An unselected series of 19 colorectal adenocarcinomas obtained from patients treated by surgery at Memorial Sloan-Kettering Cancer Center (MSKCC), New York, USA was investigated for mutations in exons 5, 6, 7 and 8 of the p53 gene. Ten of the tumors revealed at least one somatic mutation either in the exon 5 or 8. Two of them were found to carry two somatic mutations each. The DGGE pattern of one of these two tumors indicated that it contained two different clonal cell populations; a similar assessment was not possible in the other tumor. Sequence analysis of all the observed variants showed that eighty percent of the mutations were due to transitions and that half of them were at mutational hot spot codons, 175 and 273.

We searched for P53 mutations in gastric carcinoma by analyzing tumor DNAs from 29 patients. We detected 13 different somatic mutations in 15 patients (52%) and a biallelic polymorphism in exon 6 (5 heterozygous subjects). The somatic mutations were mainly localized in the sequences corresponding to the highly conserved domains of the protein. Twelve samples showed a single base change: 11 missense and 1 nonsense mutations. Three samples showed deletions leading to a frame shift, to the in-frame loss of 2 amino acids, and to the deletion of a splicing site. All point mutations, except one, were transitions, and 91% of them were G:C-->A:T changes. We previously analyzed this panel of tumors for allelic loss at the 17p13 chromosomal region, where the P53 gene had previously been located: the results showed an increasing incidence of allelic loss in late-stage tumors. On the contrary, in the present study no trend between P53 mutations and tumor stages was found. This observation indicates that mutation events precede allelic loss in gastric cancer. Half (54%) of the mutations occurred in samples without allelic loss, suggesting that specific mutated alleles, acting in a dominant negative fashion, can alter in vivo the P53 protein function.

An unselected series of colorectal adenocarcinomas together with their corresponding normal mucosa, derived from 24 Dutch patients, was investigated for the loss of a marker mapping close to the region of p53 on chromosome 17p and for mutations in exons 5, 6, 7 and 8 of the p53 gene. The observed loss of heterozygosity of the marker on chromosome 17p was 36% of the informative cases, while 58% of the tumors contained a mutation in p53. This might be an indication that the mutation in p53 precedes the loss involving p53 on the homologous chromosome. Four tumors showed the presence of two different missense mutations in the p53 gene; in one of them the mutations involved the first two nucleotides of one and the same codon, while in a second case they were found within the same exon. In the remaining two cases the assessment of their situation, cis or trans, was not feasible. Six of the 18 mutations observed during this study were base transversions, including 3 G- > T substitutions. The hotspot codons 248, 273 and 282, were found to be involved in a third of the mutations.

The human alpha-globin gene cluster (30 kb) is embedded in a GC-rich isochore very close to the telomere of Chromosome (Chr) 16p. The alpha-Locus Controlling Region (alpha-LCR) is located upstream of the adult alpha-globin genes and has been shown to be essential for their expression. In this study we have been looking for expressed genes in the region upstream of the alpha-globin cluster to understand the role of the LCR-like element in the expression and replication timing of flanking gene clusters. We show that the upstream alpha-globin region is conserved over a 75-kb range and includes at least two oppositely transcribed non-globin genes, here referred to as Mid1 and Dist1. Complementary DNA sequences of 250 bp and 2.5 kb from Mid1 (coordinate -68) and Dist1 (coordinate -90 to -99), respectively, were isolated from human and mouse. The deduced partial amino acid sequences of these cDNAs are 81% and 95% identical for the Mid1 and Dist1 gene respectively. We have cloned a mouse cosmid "contig" which includes Dist1, Mid1, and the entire murine alpha-globin cluster. The murine homolog of the alpha-LCR was mapped upstream of the mouse globin genes at approximately the same position as in the human locus. Our results indicate that, in mouse and human, the alpha-globin loci and their flanking sequences are homologous over a range of at least 130 kb. The structural homology of this region in both mammals suggests also a functional one and indicates the mouse as a potential model for studying the role of the alpha-LCR controlling element in the regulation of expression and replication timing of the flanking gene clusters.

In pancreatic neoplasias mutations in the first exon (codon 12) of K-ras gene occur at high frequency and seem to have a diagnostic significance. We set up the DGGE conditions to search for these mutations in pancreatic tumor sample DNAs. All samples were directly classified by simply comparing their DGGE patterns with those of control cell lines carrying known K-ras base substitutions. We found a mutation frequency of 73% in pancreatic adenocarcinoma, whereas no mutations were observed in benign lesions. The non-isotopic method we used turned out to be rapid and sensitive. DGGE could therefore be utilized for the detection of K-ras mutations in pancreatic lesions, to evaluate their actual or potential malignancy. In general, DGGE could be useful for K-ras gene screening on pathological tissue samples.

Familial adenomatous polyposis (FAP) is a dominantly inherited condition predisposing to colorectal cancer. The recent isolation of the responsible gene (adenomatous polyposis coli or APC) has facilitated the search for germ line mutations in affected individuals. Previous authors have used the RNase protection assay and the single-strand conformation polymorphisms procedure to screen for mutations. In this study we used denaturing gradient gel electrophoresis (DGGE). DGGE analysis of 10 APC exons (4, 5, 7, 8, 9, 10, 12, 13, 14, and part of 15) in 33 unrelated Dutch FAP patients has led to the identification of eight novel germ line mutations resulting in stop codons or frameshifts. The results reported here indicate that (1) familial adenomatous polyposis is caused by an extremely heterogeneous spectrum of point mutations; (2) all the mutations found in this study are chain terminating; and (3) DGGE represents a rapid and sensitive technique for the detection of mutations in the unusually large APC gene. An extension of the DGGE analysis to the entire coding region in a sufficient number of clinically well-characterized, unrelated patients will facilitate the establishment of genotype-phenotype correlations. On the other hand, the occurrence of an extremely heterogeneous spectrum of mutations spread throughout the entire length of the large APC gene among the FAP patients indicates that this approach may not be useful as a rapid presymptomatic diagnostic procedure in a routine laboratory. Nevertheless, the above DGGE approach has incidentally led to the identification of a common polymorphism in exon 13. Such intragenic polymorphisms offer a practical approach to a more rapid procedure for presymptomatic diagnosis of FAP by linkage analysis in informative families.

Turcot syndrome (TS) is a rare genetic disease in which brain tumors occur in association with colonic polyposis. Since Turcot's original description in 1959, there have been disagreements about the mode of inheritance as well as the clinical expression of this condition. Some investigators maintain that TS is a phenotypic variant of the autosomal dominant familial adenomatous polyposis (FAP), while others observe that there are clinical differences between TS and FAP, and that the pattern of inheritance of TS is autosomal recessive. The distribution of persons with colonic lesions in a family with a patient of colonic polyposis and a brain tumor, described in this report, favored the recessive hypothesis. In this family, the involvement of the FAP gene on chromosome 5q21-q22 could be excluded by a linkage study using a panel of FAP-linked DNA markers. This finding, which indicates the occurrence of another polyposis gene elsewhere in the genome, will have consequences for the presymptomatic diagnosis of FAP by linked DNA markers. We conclude that TS is a distinct clinical-genetical entity with the triad of atypical polyposis coli, CNS tumors, and a recessive mode of inheritance.

We have outlined a fast, non-radioactive strategy to identify point mutations in the 5' flanking region of the gamma-globin genes using denaturing gradient gel electrophoresis (DGGE) of amplified DNA. In a sample of previously characterized carriers of non deletion-type hereditary persistence of fetal haemoglobin (HPFH) the different point mutations in both gamma gene promoters could be easily identified by DGGE of a 327 bp fragment. A 4 bp deletion at position -225 to -222 of the A gamma globin gene was unexpectedly found in several samples and shown to represent a frequent polymorphism. Analysis of a 681 bp fragment specific for the 5' region of the A gamma gene, showed that this can be used to determine the haplotype of the chromosome under study. This technique may be useful in the study of sequence variations associated with high Hb F expression in physiological and pathological conditions.

A mild, non-transfusion dependent, beta thalassaemia phenotype is described in a Dutch patient homozygous for a mutation in the cleavage-polyadenylation sequence of the beta globin gene. The molecular basis of the mutation, AATAAA greater than AATGAA, was determined using denaturing gradient gel electrophoresis (DGGE) and direct sequencing of genomic DNA amplified by the polymerase chain reaction (PCR). Different fragments of the beta globin gene were amplified and analysed on DGGE for the presence of mutations. The fragment with an abnormal melting behaviour was reamplified and the base substitution in the polyadenylation sequence was identified by direct sequencing.

We report two different disorders of the beta-globin gene cluster segregating in a Belgian family: a novel deletion that results in (G) gamma + ((A) gamma delta beta)(0)-thalassemia (thal) and a heterocellular hereditary persistence of foetal hemoglobin of the Swiss type linked to a delta(0)-thal gene (delta (0)-HPFH). Heterozygosity for the heterocellular HPFH brings about a moderate (3.4% to 8.24%) increase of hemoglobin (Hb) F having a G gamma/A gamma ratio of 4:1, whereas carriers of the G gamma + ((A) gamma delta beta)(0)-thal deletion show in their peripheral blood a considerably higher (15%) percentage of Hb F. Both defects interact in the compound heterozygotes for G gamma + ((A) gamma delta beta)(0)-thal and delta(0)-HPFH producing a further increase (up to 24%) of fetal Hb consisting entirely of G gamma chains. Molecular characterization of the (G) gamma + ((A) gamma delta beta)(0)-thal by means of Southern analysis showed that the deletion spans about 50 kb, removing the 3' end of the A gamma-gene, the psi beta-, delta-, and beta-genes. A number of possible mechanisms leading to the overproduction of Hb F in HPFH and (G) gamma + ((A) gamma delta beta)(0)-thal will be discussed.

The presence of several polymorphic markers along the alpha-globin gene complex allows the identification of haplotypes associated with alpha-thalassemia determinants. These are found at very high frequencies in geographic areas where malaria is or has been endemic which suggests a positive selective role by the parasitic disease in favour of alpha(+)-thalassemia mutants. A population survey among forest tribal communities from Andhra Pradesh, India, revealed the prevalence and molecular heterogeneity of alpha(+)-thalassemia determinants presumably due to a long backdated malaria endemicity among these populations. Analysis of the tribal alpha-thalassemia haplotypes has shown a great degree of genetic heterogeneity which can be explained as the result of multiple recombination events in the presence of natural selection by malaria.

Various types of thalassemia or hereditary persistence of fetal hemoglobin (HPFH) are caused by deletions at the human beta-globin gene cluster. Many of these molecular lesions show a clear clustering as far as size and location of their breakpoints are concerned. This might indicate common recombination mechanisms responsible for the generation of these deletions. The Belgian G gamma+(A gamma delta beta)zero-thalassemia results from a large deletion spanning the beta-globin gene cluster 3' of the A gamma gene. The extent of this deletion, analyzed by field-inversion gel electrophoresis, is approximately 50 kb and is very similar to that of the Indian HPFH (G gamma A gamma HPFH III) previously characterized by P. S. Henthorn et al. (1986). Proc. Natl. Acad. Sci. USA 83: 5194-5198. Isolation of the deletion junction of the Belgian G gamma+(A gamma delta beta)zero-thalassemia by means of inverse polymerase chain reaction confirmed a very close relationship between these two independent deletions. The 3' breakpoint of the Belgian deletion is located at the midpoint of a 160-bp palindrome, only four nucleotides 5' from the correspondent endpoint of the Indian HPFH.

The analysis of polymerase chain reaction (PCR)-amplified beta-globin DNA with allele-specific oligonucleotide (ASO) probes reveals a very heterogeneous spectrum of beta-thalassaemia in the Netherlands. However about 20% of the beta-thalassemia mutations cannot be identified with this approach. The combination of specific amplification of certain regions of the beta-globin gene with denaturing gradient gel electrophoresis (DGGE) allowed us to rapidly localize several of these mutations to specific regions of the gene, which were again amplified and directly sequenced. We believe that the combination of DGGE and the direct sequence determination of PCR amplified genomic DNA represents a valid alternative to the 'ASO probes' approach, especially in countries where a very heterogeneous spectrum of beta-thalassaemia mutations occurs.

We report the characterization of a new hemoglobin variant having a single amino acid substitution (Lys----Thr) at position 61 of the alpha chain. In addition to the structural analysis, we also describe the strategy used for the identification of the base substitution and the localization of the defect at the gene level using polymerase chain reaction and hybridization with allele-specific oligonucleotides.

We describe here the screening of a small group of apparently healthy individuals belonging to the tribal communities of Koya Dora and Konda Reddi. A remarkably high incidence of deletion and nondeletion alpha + thalassemia mutants has been found with allele frequencies and distributions characteristic to each tribe. We have confirmed the strict relationship between Hb S levels and the number of alpha globin genes in double heterozygotes for the S gene and alpha thalassemia. In this population sample we did not find either heterozygous carriers of alpha 0 thalassemia (deletion of both alpha genes in "cis") or individuals showing hemolytic anemia due to inactivation of three alpha-globin genes (Hb H disease). Selection by malaria is most probably responsible for the prevalence of the various alpha + thalassemia haplotypes among the two tribal populations of Andhra Pradesh.

We describe a novel mutation in the delta globin gene of a compound heterozygote for delta o thalassemia and a deletion type G gamma + (A gamma delta beta) zero thalassemia. The delta was amplified using the polymerase chain reaction (PCR), and the amplified material was used in a direct sequencing experiment. The nucleotide sequence of the mutant delta gene showed that the insertion of an extra nucleotide at the third position of codon 91 in the second exon, which gives rise to a premature stop codon at position 94, leads to the silencing of this gene. The presence of the mutation in the carriers of delta-thalassemia in this family was confirmed by dot blot hybridization. A possible model for the insertion of the extra nucleotide is discussed.