A CA-repeat polymorphism close to the adenomatous polyposis coli (APC) gene offers improved diagnostic testing for familial APC.

Presymptomatic genetic testing for the presence of a mutant allele causing familial adenomatous polyposis coli (APC) has been difficult to perform effectively in the past because DNA markers surrounding the APC gene on chromosome 5q have not been very informative. We report results of genetic linkage studies on both research families and clinical families by using D5S346, a highly polymorphic dinucleotide (CA)-repeat locus 30-70 kb from the APC gene. Linkage analysis with this marker in a large APC pedigree showed an increase of at least 9.0 LOD units, in likelihood of linkage of the disease-causing allele to the APC locus, when compared with the highest LOD score attained with any other closely linked marker. When the first 14 APC families that requested genotypic analysis by the DNA Diagnostic Laboratory at the University of Utah were tested with D5S346, 20 of the 31 at-risk individuals were identified as either carriers or noncarriers of an APC-predisposing allele. We see this marker as an important tool for research studies and for the presymptomatic diagnosis of APC.     

Linkage studies in Italian families with familial adenomatous polyposis

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.     

Diagnosis of neurofibromatosis I by using tightly linked, flanking DNA markers.

We tested 132 individuals from 21 families segregating an allele for neurofibromatosis type 1 (NF-1), by using nine RFLPs tightly linked to the NF-1 locus. Family members had requested DNA testing either to determine whether "at risk" children were carrying the NF-1 allele or to determine whether their respective families would be informative for prenatal testing. Predictions about whether a child carries the NF-1 mutation were possible for all 32 at-risk offspring (greater than 98% accuracy based on the recombination estimates currently available for these DNA markers). At least one informative probe was available for all 23 matings in these 21 families; flanking markers were informative for 10 matings. Pairwise analysis showed that several of the polymorphisms were in tight linkage disequilibrium; few recombination events were observed with these markers in the families under study. We conclude that the DNA probes used in this study perform well for diagnostic testing of NF-1 in familial cases. A subset of five probe-enzyme systems (pHHH202/RsaI, p11-3C4.2/MspI, pTH17.19/Bg/II, p11-2C11.7/BamHI, and p11-2F9.8/TaqI) provide reliable linkage information for both clinical testing and prenatal diagnosis.     

Eight novel inactivating germ line mutations at the APC gene identified by denaturing gradient gel electrophoresis.

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.     

A new deletion polymorphism at D5S71 raises the linkage information on adenomatous polyposis coli: implications for presymptomatic diagnosis

Summary Two independent study-groups, one in Britain and the other in the United States, were the first to report linkage between APC and a TaqI restriction fragment length polymorphism (RFLP) at D5S71 (probe C11p11) on chromosome 5q. They found no recombinants in about 50 informative meioses. The same TaqI RFLP was found to be uninformative for linkage in 15 Dutch polyposis families. The recently reported four base-pair deletion polymorphism (DEL1) at D5S71 has raised the polymorphism information content of this marker from 0.17 to 0.40 in the Dutch population. Seven of 20 polyposis families screened for the DEL1 as well as the TaqI polymorphism gave a combined peak lod score of 5.68 with no recombinants in 37 informative meioses. These data, together with those so far reported in the literature, raise the peak lod score to 17.09 at a recombination fraction of 0.05, the 95% upper confidence limit being 0.09. In combination with the use of another informative marker, D5S81 (probe YN5.48) closely mapping on the other side of APC, the presymptomatic diagnosis of the disease can be made with more than 99.9% certainty. It has to be stressed, however, that the the possible existence of more than one polyposis locus cannot, as yet, be excluded.     

Genetic linkage map of six polymorphic DNA markers around the gene for familial adenomatous polyposis on chromosome 5.

A genetic linkage map of six polymorphic DNA markers close to the gene (APC) for familial adenomatous polyposis (FAP) on chromosome 5q is reported. One hundred fifty-five typed members of nine FAP kindred provided more than 90 meioses for linkage analysis. A number of crucial recombination events have been identified which are informative at three or more loci, allowing confident ordering of parts of the map. There was no evidence of genetic heterogeneity, with all families showing linkage of at least one chromosome 5 marker to the gene. Recombination data and two-point linkage analysis support a locus order of centromere-pi 227-C11P11-ECB27-L5.62-APC-EF5.44-YN5.48-telomer e, although EF5.44 could lie in the interval L5.62-APC or ECB27-L5.62. No recombinants were identified between APC and either EF5.44 or YN5.48, but published deletion mapping in colorectal carcinomas and linkage analysis in FAP suggest that YN5.48 is 1-3 cM from APC. The present study suggests that YN5.48 and L5.62 delineate a small region of chromosome 5 within which the EF5.44 locus lies very close to the APC gene. These data not only allow use of flanking markers for presymptomatic diagnosis of FAP but also provide a high-density map of the region for isolation of the APC gene itself and for further assessment of the role of chromosome 5 deletions in the biology of sporadic colorectal cancer.     

Localization of the genetic defect in familial adenomatous polyposis within a small region of chromosome 5

Familial adenomatous polyposis (FAP), a Mendelian disorder that includes familial polyposis coli (FPC) and Gardner syndrome (GS), has an autosomal dominant mode of inheritance. It is characterized by hundreds to thousands of adenomatous polyps that can progress to carcinoma of the colon, suggesting that the gene that harbors the FAP germ-line mutation may play an important role in the somatic genetic pathway to colon cancer. The defect responsible for FAP was recently mapped to the long arm of chromosome 5 by linkage between the FPC phenotype and a locus defined by DNA probe pC11p11 (D5S71), located at 5q21-22. Because an important next step in the paradigm for identification of a disease gene is to obtain a more precise localization, we isolated and mapped by linkage six additional polymorphic DNA markers in the FAP region. Subsequent linkage analysis in six pedigrees, three having the FPC phenotype and three segregating GS, placed the FAP locus very close to a new marker, YN5.48 (D5S81), that is approximately 17 centimorgans distal to C11p11 on the genetic map. The analysis revealed no evidence of genetic heterogeneity between the two phenotypes, a question that had not been clearly resolved by the earlier studies. The new set of markers in the near vicinity of the FAP locus represents a further step toward isolation of the genetic defect and provides the opportunity for preclinical diagnosis of risk status for colon cancer among individuals in families that are segregating adenomatous polyposis.     

Linkage analysis in familial adenomatous polyposis: order of C11P11 (D5S71) and pi 227 (D5S37) loci at the apc gene

Linkage analysis with DNA probes C11P11 and pi 227 is reported in six Scottish families with familial adenomatous polyposis. Two families were informative for C11P11 and all six were at least partly informative for pi 227. Two C11P11-apc and two pi 227-apc recombinants were identified and one of these was recombinant for both C11P11-apc and pi 227-apc. A further possible combined C11P11-apc and pi 227-apc recombination event was also identified. Peak lod score for linkage of C11P11 to apc was 5.80 at a recombination fraction (theta) of 0.069 (95% probability limits 0.012-0.191) and for linkage of pi 227 to apc was 3.19 at theta = 0.110 (95% probability limits 0.023-0.286). Peak lod score for linkage of C11P11 to pi 227 was 1.79 at theta = 0.00. The data support a gene order of pi 227-C11P11-apc.     

Detection by DGGE of a new polymorphism closely linked to the adenomatous polyposis coli region

Summary The EF5.44 locus is in close proximity to the chromosome 5 region to which the genetic defect responsible for familial adenomatous polyposis has been mapped. We have devised two oligonucleotides that promote the specific polymerase chain reaction (PCR) amplificiation of a 365-bp sequence in this region. Analysis by denaturing gradient gel electrophoresis of the resulting fragment has unravelled individual differences that could be identified as a single base pair change in aMnlI restriction site. This PCR assayable polymorphism increases the informativeness at this locus, and should be useful in the presymptomatic diagnosis of familial adenomatous polyposis.     

The familial adenomatous polyposis region exhibits many different haplotypes

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. Received: 5 November 1997 / Accepted: 3 February 1998     

Diagnosis of carriers of haemophilia A by RFLP analysis: Austrian results.

RFLP analysis with extragenic and intragenic probes was performed in 25 Austrian families with haemophilia A for the purpose of carrier diagnosis. Diagnosis could be established in 22 families, in 7 families with intragenic and extragenic probes, in 15 families with extragenic probes only. The remaining 3 families were not informative. During routine diagnosis we found three interesting cases which are described in the following paper. Firstly, we identified one deletion with Bcl I/p 114.12. Secondly, we observed an additional polymorphic band (5.2 kb) with Xba I/probe A. Thirdly, we saw one case where Bcl I/p 114.12 and Bgl I/1.8 were not in linkage disequilibrium.     

Presymptomatic direct detection of adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis

The recent identification of the familial adenomatous polyposis (FAP) gene (designated as APC) enables conclusive genetic testing of at-risk family members for the specific mutation in families in which the germline gene mutation has been characterized. Presymptomatic molecular diagnosis of FAP was performed by direct direction of mutations in lymphocyte DNA in four families. Each of the families has a different mutation of the APC gene. Twenty-seven offspring of affected individuals (a priori risk of 50%) were tested. Ten of the 27 had already developed clinical features of FAP. Of the remaining seventeen, two had had a negative colon exam at an early age, and nine had never had colon exams (mean age, 12.1±3.1 SD years). Six children from this group (54%) were found to carry their affected parent's mutation. No change in the conventional FAP colon screening regimen is recommended for these children. In contrast, when direct tests indicate that an individual does not have the FAP mutation, we recommended that screening be decreased. Reduction of uncertainty for at-risk FAP family members is an important benefit of genetic testing.     

Identification of genomic deletions of the APC gene in familial adenomatous polyposis by two independent quantitative techniques

Large deletions in the APC (adenomatous polyposis coli) gene, causing familial adenomatous polyposis (FAP), cannot easily be detected by conventional mutation-detection techniques. Therefore, we have developed two independent quantitative methods for the detection of large deletions, encompassing one or more exons, of APC. Multiplex ligation-dependent probe amplification (MLPA) is performed in one reaction for the initial quantification of all APC exon copy numbers. Subsequently, quantitative real-time PCR (QRT-PCR) is used to verify the results obtained in the MLPA reaction. The identification of a deletion of the whole APC gene in a patient with classical FAP is described. The mutation was detected with the two quantitative methods and further verified on chromosomal level by the use of FISH (fluorescence in situ hybridization) on metaphase spreads. Furthermore, a large deletion covering exons 11-13 of the APC gene was detected in two apparently unrelated families. This deletion was further verified and characterized with long-range PCR. The MLPA test ensures a sensitive high-throughput screening for large deletions of the APC gene and can easily be implemented in the diagnostic testing for FAP.     

Diagnosis of susceptibility to malignant hyperthermia with flanking DNA markers.

OBJECTIVE--To define the region on human chromosome 19 carrying the gene for malignant hyperthermia susceptibility and to evaluate the use of flanking DNA markers in diagnosing susceptibility. DESIGN--Prospective molecular genetic linkage studies in a large malignant hyperthermia pedigree. SETTING--Irish malignant hyperthermia testing centre. SUBJECTS--A large Irish malignant hyperthermia pedigree. MAIN OUTCOME MEASURES--Routine diagnosis of susceptibility to malignant hyperthermia with in vitro contracture test on muscle biopsy specimens and genetic linkage between susceptibility and polymorphic DNA markers in a malignant hyperthermia family. RESULTS--Genetic typing of polymorphic DNA markers in a large Irish malignant hyperthermia pedigree generated a lod score of greater than 3 for the marker D19S9 and showed that the gene for susceptibility is flanked by the markers D19S9 and D19S16. These tightly linked flanking markers allowed non-invasive presymptomatic diagnosis of susceptibility in five untested subjects in the large pedigree with an accuracy of greater than 99.7%. CONCLUSIONS--DNA markers flanking the gene for susceptibility to malignant hyperthermia can be used with high accuracy to diagnose susceptibility in subjects in large known malignant hyperthermia pedigrees and may replace the previous in vitro contracture test for diagnosing this inherited disorder in large families with malignant hyperthermia.     

家族性腺瘤息肉病的遗传病因学研究进展

家族性腺瘤息肉病(FAP)是第二常见的遗传性结直肠癌综合征,多在青春期发病,发病率约1/10000,主要临床表现为大肠中多发的腺瘤性息肉,是一种结直肠癌的癌前病变,如果不予治疗,几乎100%的患者会发展成为结直肠癌。一直以来,FAP被认为是一种常染色体显性遗传疾病,发病由APC基因胚系突变引起。根据临床特点的不同,FAP患者可以分为经典型FAP(CFAP)和轻表型FAP(AFAP)。然而近年来,在一些无APC基因胚系突变的FAP患者中发现了Mut YH基因的双等位基因突变。这种由于Mut YH基因双等位基因突变而无APC生殖突变所引起的临床综合征定义为Mut YH基因相关性息肉病[2](MAP)。MAP为常染色体隐性遗传,是一种特殊类型的FAP。另外,很多研究表明,APC基因的突变位点与结肠腺瘤病的严重程度、癌变的风险程度和某些肠外表现相关。MAP的发现和对FAP基因型-表型相关性的研究,完善了对FAP遗传病因学的认识,对于FAP患者及高危亲属的合理防治和预后具有重要的意义。     

Isolation of chromosome-21-specific DNA probes and their use in the analysis of nondisjunction in Down syndrome

Summary Thirteen single-copy, chromosome-21-specific DNA probes were isolated from a recombinant library made from flow-sorted chromosome 21 DNA and regionally mapped using a panel of somatic cell hybrids. Five probes mapped in the 21q21-q22.1 region, six to the 21q22.1-qter region, and one to each of the regions 21q22.1-q22.2 and 21q22.3. Two of these probes, one of which maps in the critical region for Down syndrome, have recently been shown to be expressed at high levels in Down syndrome brain tissue (Stefani et al. 1988). Following preliminary screening for restriction fragment lenght polymorphisms (RFLPs), five polymorphisms were discovered with four of the chromosome 21 DNA probes. A frequent MspI polymorphism detected by one of the probes was used in conjunction with four previously described polymorphic chromosome 21 probes to analyse the origin of nondisjunction in 33 families with a child or fetus with trisomy 21. The parental origin of the additional chromosome 21 was determined in 12 cases: in 9 (75%) of these it was derived from the mother and in the other 3 cases (25%) it was of paternal origin. Cytogenetic analysis of Q-banding heteromorphisms was informative in three of five families tested, and in each case the RFLP results were confirmed. The meiotic stage of nondisjunction was defined with confidence in five families, the results being obtained with pericentromeric RFLP or cytogenetic markers. Recombination between two nondisjoined chromosomes was demonstrated in one family and is consistent with the view that a lack of recombination between chromosome 21 homologues or failure of their conjunction is not the invariable cause of trisomy 21.     

Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile-X locus (FRAXA)

We report the validation and use of a cell hybrid panel which allowed us a rapid physical localization of new DNA probes in the vicinity of the fragile-X locus (FRAXA). Seven regions are defined by this panel, two of which lie between DXS369 and DXS296, until now the closest genetic markers that flank FRAXA. Of those two interesting regions, one is just distal to DXS369 and defined by probe 2-71 (DXS476), which is not polymorphic. The next one contains probes St677 (DXS463) and 2-34 (DXS477), which are within 130 kb and both detect TaqI RFLPs. The combined informativeness of these two probes is 30%. We cloned from an irradiation-reduced hybrid line another new polymorphic probe, Do33 (DXS465; 42% heterozygosity). This probe maps to the DXS296 region, proximal to a chromosomal breakpoint that corresponds to the Hunter syndrome locus (IDS). The physical order is thus Cen-DXS369-DXS476-(DXS463,DXS477)-(DXS296, DXS465)-IDS-DXS304-tel. We performed a linkage analysis for five of these markers in both the Centre d'Etude du Polymorphisme Humain families and in a large set of fragile-X families. This establishes that DXS296 is distal to FRAXA. The relative position of DXS463 and DXS477 with respect to FRAXA remains uncertain, but our results place them genetically halfway between DXS369 and DXS304. Thus the DXS463-DXS477 cluster defines presently either the closest proximal or the closest distal polymorphic marker with respect to FRAXA. The three new polymorphic probes described here have a combined heterozygosity of 60% and represent a major improvement for genetic analysis of fragile-X families, in particular for diagnostic applications.     

Presymptomatic detection or exclusion of prion protein gene defects in families with inherited prion diseases.

The identification of defects in the prion protein (PrP) gene in families with inherited Creutzfeldt-Jakob disease or Gerstmann-Straussler syndrome allows presymptomatic diagnosis or exclusion of these disorders in subjects at risk. After counseling, PrP gene analysis was performed in three such individuals: two from families with a 144-bp insert and one with a point mutation at codon 102 in the PrP gene. The presence of a PrP gene defect was confirmed in one and excluded in two. Despite the potential problems of using PrP gene analysis in genetic prediction - specifically, uncertainty about penetrance and, generally, problems of presymptomatic testing in any inherited late-onset neurodegenerative disorder - we conclude that it has a role to play in improved genetic counseling for families with inherited prion diseases.     

山东省假性肥大型肌营养不良的RFLP分析 Ⅰ．可疑携带者的基因型确定

本文选用10个DMD基因内部和桥探针,对山东省9个地区的21个DMD／BMD家系的173名成员进行RFLP分析,其中可疑携带者55人,多数家系应用1－3个探针,有基因重组家庭选用4－5个探针,便可完成多态分析,RFLP分析可以确定85．45％的可疑携带者（≥95％可信限）,即13人确定为携带者,30人排除携带者,另有4人风险大幅提高,通过这些探针的群体多态检测和不同家庭结构和类型的应用分析,我们提出了在中国人群中DMD／SMD基因诊断的基本程序。     

Screening for an inherited susceptibility to colorectal cancer

The principal Mendelian disorders predisposing to colorectal cancer are familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). FAP is caused by mutations in the adenomatous polyposis coli (APC) gene. HNPCC is caused by a mutation in one of at least five mismatch repair genes. It is important to identify individuals with these conditions because colon cancer will occur in at least 80% and onset is earlier than in the general population. Potential benefits of identification include improved compliance with recommended surveillance, early detection of polyps, reduction in cancer mortality, and reassurance for relatives found to be negative with attendant savings in the time and expense of surveillance. For classic FAP, the large number of polyps readily identifies affected persons. For HNPCC, identification of individuals meriting DNA sequencing requires either recognition of a suspect family history or finding high microsatellite instability in a tumor. Individuals accepting the offer of genetic counseling and DNA testing often have more cancers in their family, are motivated to inform relatives, have a larger social network, and have more confidence in their coping ability. Individuals who decline are often concerned about their own or their family's emotional reaction or fear discrimination.