Independent constitutional germline mutations occurring in the RB1 gene in cousins with bilateral retinoblastoma

The inheritance of a genetic susceptibility to the development of retinoblastoma generally follows an autosomal mode of inheritance with high penetrance. Rare families, however, show evidence of incomplete penetrance where individuals can transmit the mutant gene without being affected themselves. In these families formal proof of this dogma requires the identification of the predisposing mutation. In this study we have identified the mutations in cousins with bilateral (hereditary) disease. Using SSCP and DNA sequencing, different constitutional mutations were detected in the affected cousins in this pedigree. One cousin carries a C-->T mutation in exon 8 generating a stop codon directly which was also present in his affected mother whereas the other cousin carries an 8 base pair deletion in exon 20. Neither half of the family carried the same mutation as the other. The mother of the patient with the 8 bp deletion carried neither of the mutations. Thus, we have demonstrated that the retinoblastomas in this family have developed as a result of independent, sporadic genetic events which occurred coincidentally in the same extended family rather than being due to a common mutation which manifests as incompletely penetrant. These observations have important implications for genetic counselling in this type of family.     

Somatic mutations of the ret protooncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are associated with tumor recurrence

Germline point mutations in exons 10, 11, and 16 of the ret protooncogene have been identified as causative in multiple endocrine neoplasia type 2 and in familial medullary thyroid carcinoma (MTC). Somatic point mutations of the same gene, exclusively associated with codon 918 of exon 16, have also been reported in few cases of sporadic medullary thyroid carcinoma. We analyzed the blood and tumor DNA of 19 patients with sporadic MTC and 6 patients with primary parathyroid adenoma for point mutations at exons 10, 11, and 16 of the ret protooncogene by restriction analysis of the PCR-amplified product and by sequence analysis of exons 10 and 11. A Cys634-->Tyr mutation was found in both the tumoral and blood DNA of one patient, indicating that he was affected by an hereditary form of MTC, erroneously considered sporadic. In the other 18 patients with MTC, somatic point mutations of ret were found in 8 cases (44.4%). In 5 cases the mutation affected exon 16 (Met918-->Thr), and in 3 cases it affected exon 11 (Cys634-->Arg in 1 and Cys634-->Trp in 2); these 3 mutations were confirmed by sequence analysis. The remaining 10 patients had no mutation in exon 10 by either restriction analysis or sequence analysis. Clinical data showed that 75% of the patients whose tumor carried ret mutation had tumor recurrence and/or increased serum calcitonin concentrations during the postsurgical follow-up period as opposed to 10% of the patients without mutations (P < 0.02, by chi2 analysis). No ret mutation was found in the tumoral DNA from parathyroid adenomas. Our findings indicate that the somatic ret point mutation frequently found in sporadic MTC may affect not only exon 16 but also exon 11 and is associated with less favorable clinical outcome.     

Mutational analysis of the Jagged 1 gene in Alagille syndrome families

Alagille syndrome (AGS) is an autosomal dominant disease characterized by five major abnormalities in the liver, heart, face, vertebrae and eye. The responsible gene has been recently identified as the human Jagged 1 (JAG1) gene, which encodes a ligand for the Notch receptor. We analyzed the JAG1 gene in eight AGS families, including affected and unaffected individuals, at the genomic DNA level, mainly by single-strand conformational polymorphism (SSCP) and DNA sequencing analysis. Four categories of mutations were identified: (i) four frameshift mutations in exons 9, 22, 24 and 26 were exhibited respectively in affected individuals of four AGS families, which resulted in moving the translational frame of JAG1; (ii) one nonsense mutation, a 1 bp substitution in exon 5 of the EGF-like repeat domain, was detected in two unrelated AGS families, which altered codon 235 from arginine to stop; (iii) one acceptor splice site mutation of exon 5 was revealed in a sporadic patient; and (iv) a 1.3 Mb deletion, which included the entire JAG1 gene, was found in another patient. Our results further demonstrate that AGS is a dominant disease and suggest that the JAG1 gene exerts a fundamental role in regulating genes involved in development.     

The structure and organization of the human carnitine/acylcarnitine translocase (CACT1) gene2

One hundred and eighty-one families with multiple endocrine neoplasia type 2A (MEN-2A) or familial medullary thyroid carcinoma (FMTC) have been investigated for mutations in the ret protooncogene in Germany. In 8 families with FMTC or MEN-2A, no mutation could be detected in the cysteine-rich domain encoded in exons 10 and 11 of the ret protooncogene. DNA sequencing of additional exons (no. 13-15) revealed rare noncysteine mutations in 3 families (codons 631, 768, and 844). In contrast to these rare events, heterozygous missense mutations in exon 13, codons 790 and 791, were found in 5 families (4 with MTC only; 1 family with MTC and pheochromocytoma) and 11 patients with apparently sporadic tumors. Two different mutations in codon 790 (TTG-->TTT, TTG-->TTC; Leu790Phe) and one mutation in codon 791 (TAT-->TTT; Tyr791Phe) created a phenylalanine residue. We conclude that codons 790 and 791 of the ret protooncogene represent a new hot spot for FMTC/MEN-2A causing mutations. With the discovery of these considerably common mutations in codons 790 and 791 and the identification of some rare mutations, 100% of the German FMTC/MEN-2A families could be characterized by a mutation in the ret protooncogene.     

Somatic mosaicism: a common cause of classic disease in tumor-prone syndromes? Lessons from type 2 neurofibromatosis

Blood samples from 125 families with classic type 2 neurofibromatosis with bilateral vestibular schwannomas were analyzed for mutations in the NF2 gene. Causative mutations were identified in 52 families. In five families, the first affected individual in the family (the index case) was a mosaic for a disease-causing mutation. Only one of nine children from the three mosaic cases with children are affected. Four of these nine children inherited the allele associated with the disease-causing mutation yet did not inherit the mutation. NF2 mutations were identified in only 27/79 (34%) of sporadic cases, compared with 25/46 (54%) of familial cases (P<.05). In 48 families in which a mutation has not been identified, the index cases have had 125 children, of whom only 29 are affected with NF2 and of whom only a further 21 cases would be predicted to be affected by use of life curves. The 50/125 (40%) of cases is significantly less than the 50% expected eventually to develop NF2 (P<.05). Somatic mosaicism is likely to be a common cause of classic NF2 and may well account for a low detection rate for mutations in sporadic cases. Degrees of gonosomal mosaicism mean that recurrence risks may well be <50% in the index case when a mutation is not identified in lymphocyte DNA.     

CNS tissue Cu/Zn superoxide dismutase (SOD1) mutations in motor neurone disease (MND)

DNA extracted from CNS tissue of 79 cases of motor neurone disease (MND) was screened by single strand conformation analysis (SSCA) and heteroduplex analysis (HA) for mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The aims were to determine whether somatic mutations of SOD1 may underlie some cases of MND and to characterize the genetic abnormalities by sequencing, for subsequent correlation with the molecular pathological phenotype. In 3 cases a point mutation was found in exon 4: E100G in one familial case, and I113T in two cases (one familial, one sporadic). Two cases had previously undescribed mutations in the 3' untranslated region (3'UTR) of SOD1 and one case had a single base substitution in the intronic sequence upstream from exon 2. None of these patients had a positive family history. Non-CNS tissue was available for 3 out of the 6 cases in whom changes were found. In all 3 the same changes were consistently found in both CNS and non-CNS tissue, excluding the presence of somatic mutations in SOD1. We investigated many MND blood samples and normal controls for the presence of the 3'UTR deletions. We found the 4 bp deletion in 1/90 sporadic MND patients and 1/209 non-MND controls. If the 3'UTR deletions are pathogenic, they would have to operate via a loss of the function mechanism, and further work is necessary to define their significance.     

Transnasal endoscopic repair of congenital choanal atresia: long-term results

Somatic mutations in the RET proto-oncogene are involved in the pathogenesis of an important subset (40-60%) of sporadic medullary thyroid carcinomas (MTCs) and less frequently (0-31%) in benign, sporadic phaeochromocytomas. Since limited data exist regarding the significance of somatic RET mutations in malignant phaeochromocytomas, we analysed a multicentre series of proven malignant (i.e., metastasised) phaeochromocytomas. Analogous with MTCs, where RET mutations lead to an aggressive behaviour, we hypothesised that somatic mutations would occur more frequently in malignant than in benign phaeochromocytomas. Paraffin-embedded tissue was available from 29 malignant and 27 benign phaeochromocytomas. Exons 10, 11 and 16 were analysed by non-radioactive single-strand conformation polymorphism, heteroduplex gel electrophoresis, restriction enzyme digestion and aberrant band patterns by non-isotopic sequencing. In only 1 of 29 malignant phaeochromocytomas was a mis-sense mutation found (at codon 634 of exon 11), whereas in 15% (4/27) of the benign tumours a point mutation was detected (in 3 tumours in exon 16 at codon 918 and in 1 tumour in exon 10 at codon 618). Absence of these mutations in non-tumourous DNA proved their somatic origin. Contrary to what has been reported for MTCs, oncogenic RET mutations are not associated with an aggressive tumour behaviour in sporadic phaeochromocytomas.     

Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations

The aim of this study was to evaluate the prevalence of simple sequence variation in the BRCA2 gene. To this end, 71 breast and breast-ovarian cancer (HBC/HBOC) families along with 95 control individuals from a wide range of ethnicities were analyzed by means of denaturing high-performance liquid chromatography (DHPLC) and direct sequence analysis. In the coding (10 257 bp) and non-coding (2799 bp) sequences of BRCA2, 82 sequence variants were identified. Three different, apparently disease-associated BRCA2 mutations were found in six HBC/HBOC families (8%): two splice site mutations in introns 5 and 21, and one frameshift mutation in exon 11. In the coding region, 53 simple sequence variants were found: 35 missense mutations, one 2 bp deletion (CT) resulting in a stop at codon 3364, one nonsense mutation with a stop at codon 3326, one deletion of a complete codon (AAA) resulting in the loss of leucine, and 15 silent mutations. In the non-coding region, 26 polymorphisms were detected. Of the 79 sequence variants that were not obviously disease-associated, eight were detected only in HBC/HBOC families. The remaining 71 variants were identified in both HBC/HBOC families and control individuals. Sixty three sequence variants (80%) were specific for a continent. Forty two percent (33 out of 79) of the sequence variants were detected exclusively in Africa, though only 13% of the 332 chromosomes screened were of African origin. Our data indicate that, in BRCA2, simple sequence variation is frequent [in the coding region 1 in 194 bp (straight theta = 2.2 x 10(-4)), and in the non-coding region 1 in 108 bp (straight theta = 4.4 x 10(-4)), respectively].     

A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype

We have used single strand conformation polymorphism analysis to study the 27 exons of the RB1 gene in individuals from a family showing 'mild' expression of the retinoblastoma phenotype. In this family affected individuals developed unilateral tumors and, as a result of linkage analysis, unaffected mutation carriers were also identified within the pedigree. A single band shift using SSCP was identified in exon 21 which resulted in a missense mutation converting a cys-->arg at nucleotide position 28 in the exon. The mutation destroyed an NdeI restriction enzyme site. Analysis of all family members demonstrated that the missense mutation co-segregated with patients with tumors or who, as a result of linkage analysis had been predicted to carry the predisposing mutation. These observations point to another region of the RB1 gene where mutations only modify the function of the gene and raise important questions for genetic counseling in families with these distinctive phenotypes.     

Haplotype analysis of two recurrent CDKN2A mutations in 10 melanoma families: evidence for common founders and independent mutations

Germ-line mutations in CDKN2A have been shown to predispose to cutaneous malignant melanoma. We have identified 2 new melanoma kindreds which carry a duplication of a 24bp repeat present in the 5' region of CDKN2A previously identified in melanoma families from Australia and the United States. This mutation has now been reported in 5 melanoma families from 3 continents: Europe, North America, and Australasia. The M53I mutation in exon 2 of CDKN2A has also been documented in 5 melanoma families from Australia and North America. The aim of this study was to determine whether the occurrence of the mutations in these families from geographically diverse populations represented mutation hotspots within CDKN2A or were due to common ancestors. Haplotypes of 11 microsatellite markers flanking CDKN2A were constructed in 5 families carrying the M53I mutation and 5 families carrying the 24bp duplication. There were some differences in the segregating haplotypes due primarily to recombinations and mutations within the short tandem-repeat markers; however, the data provide evidence to indicate that there were at least 3 independent 24bp duplication events and possibly only 1 original M53I mutation. This is the first study to date which indicates common founders in melanoma families from different continents.     

Screening methods in genetic diagnosis of hereditary protein C deficiency

Genomic analysis and detailed blood coagulation examinations of 22 family members of 18 families with repeatedly low protein C activity have been performed. Blood coagulation examinations: INR, fibrinogen, plasminogen, alpha-2-antiplasmin, lupus anticoagulant, APC resistance test, protein C activity and antigen, protein S activity and antithrombin activity. Genetic examinations: the presence of FII G20210A alle and FV:Q506 Leiden mutation were examined and for the mutation screening in the protein C gene combination of polymerase chain reaction (PCR) with denaturing gradient gelelectrophoresis (DGGE) or with single-strand conformation polymorphism (SSCP) analysis has been performed. The amplified DNA fragments with aberrant migration during DGGE and SSCP analysis were sequenced. Nine family members of seven families were identified carrying mutations in the protein C gene: one nonsense mutation in exon VII (Arg 157-Stop), two types of missense mutations in four patients in exon IXA (230 Arg-Lys, 254 Thr-Ile, the latter is a new mutation, Protein C Pécs), one missense mutation in two patients in exon IXB (325 Val-Ala), one missense mutation in exon IXC (359 Asp-Asn) and a rare frameshift deletion in exon IXC (364 Met-Trp, 378 Stop). Nine families were evaluated carrying no mutation in their protein C gene, but other genetic or blood coagulation disturbances have been identified, eight of them had borderline decrease in their protein C activity (60-70%). The presence of FV:Q506 mutation could be diagnosed in eight families (in 3 cases homozygous, in 5 cases heterozygous form), among them combination of the defects could be proved in three of the eight families: FV:Q506 Leiden mutation with antiphospholipoid antibodies in 2 families and the presence of Leiden mutation with prothrombin gene mutation in 1 family. Protein S deficiency in combination with prothrombin gene mutation has been identified in 1 family. There were 2 families where no genetic or blood coagulation alterations could be detected in the background of the repeatedly low protein C activity. Large deletions or insertions which are not detectable by our screening methods could not be excluded in these families and therefore sequencing of the total protein C gene had been performed with negative results. According to the literature and our experience the screening methods that were administered in this study are suitable for the detection of mutations in the protein C gene.     

Evolution of precipitates in lead-implanted aluminum: A backscattering and channeling study

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Mutation of the transforming growth factor-beta type II receptor gene is a rare event in human sporadic gastric carcinomas

Mutations of the transforming growth factor-beta type II receptor (TGF-beta RII) gene have been detected in several human cancers. However, mutation analysis of coding sequences of TGF-beta RII in gastric carcinomas has not yet been fully elucidated. We performed PCR-SSCP analysis and direct DNA sequencing of the entire coding region of TGF- RII in 38 human sporadic gastric cancers and 8 gastric cancer cell lines. Mutations of the TGF-beta RII were detected in two tumors and three cell lines. Two tumors had one base deletion in the polyadenine tract in exon 3, the cystein-rich extracellular domain. Three cell lines had a silent mutation in the kinase domain located in exon 4. Polymorphisms were detected in introns 2 and 3. An a/g polymorphism was observed at the seventh base in intron 2 and an a/t polymorphism was observed at the fourth to last base in intron 3. There were no mutations in exons 1, 2, 5, 6 and 7. These results indicate that the polyadenine tract in the TGF-beta RII is a mutational hot spot in human gastric cancer. However, these results also suggest that mutations of the gene are rare events in human sporadic gastric cancer.     

A frequent activated smoothened mutation in sporadic basal cell carcinomas

Basal-cell carcinomas (BCCs) are the most common cancer in Caucasians. It has been reported that the patched gene is inactivated in 30-40% sporadic BCCs and 20% sporadic medulloblastomas via loss of heterozygosity and nonsense mutations. Recently, two activating smoothened mutations have been found in the sporadic basal cell carcinomas. One, at base pair 1604 (G-to-T transversion) of exon 9, changes codon 535 from tryptophan to leucine, and the other, at base pair 1685 (G-to-A transition) of exon 10, changes codon 562 from arginine to glutamine (Xie et al., 1998). In our study, 1604G-->T was found in 20 out of 97 (20.6%) sporadic BCCs. The high prevalence indicates that 1604G is the mutation hot spot in our tumor samples. This mutation was detected in all three histological subtypes of BCCs, suggesting that smoothened mutation is an early event during the development of the tumor. Our finding of a high smoothened mutation rate, together with high frequent patched gene mutations reported recently, indicates that activation of the hedgehog signal transduction pathway is the most common and early event in the development of sporadic BCCs. Additionally, to determine whether smoothened, like patched, is also involved in the carcinogenesis of medulloblastomas, we screened medulloblastoma samples for these two mutations by restriction analysis. We have found the 1604G-->T mutation in 1 out of 21 medulloblastomas. This result confirmed smoothened gene involvement in the carcinogenesis of medulloblastoma.     

Analysis of RET proto-oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) may occur either as a sporadic or familial (FMTC) disease. Multiple endocrine neoplasia (MEN) type 2, inherited as an autosomal dominant disease, is characterized by coexistence of MTC with other endocrine neoplasia. Activating mutations of the RET proto-oncogene, involving the somatic or the germinal cell lineage, are found in both inherited and acquired forms. In this study, RET mutations were screened in 47 individuals either affected by MTC or belonging to families with hereditary MTC. Exons 10, 11, 13, 14, 15 and 16 of the RET gene were amplified by polymerase chain reaction and examined by DNA sequence and/or restriction enzyme analysis to detect mutations in purified amplicons. Six MEN 2A families with a germline mutation at codon 634, one FMTC family carrying a mutation at codon 618 and two MEN 2B families with a mutation at codon 918 were identified. In affected members of a MEN 2A family no known RET mutations were observed. Besides, we identified a germline mutation in a patient with apparently sporadic MTC and in two out of three sons, indicating the presence of a sporadic misclassified familial disease. In all of the families examined we were able to distinguish the affected vs unaffected (not at risk) members. A somatic mutation of codon 918 was detected in three out of ten patients with apparently sporadic MTC.     

Statistics of malignant neoplasms in children in Russia

Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In a population of 172 healthy people (average age, 34; mutant frequency, 10.3 x 10(-6)), deletion/insertion mutations constituted 41% (89) of the 217 independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among +/- 1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions of 3-200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by -1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both endpoints were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), possibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 deletions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation.     

Mutation analysis of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human colon cancer using genomic DNA and intron primers

Mutations in the transforming growth factor beta type II receptor (TGFbeta RII) gene have been detected in several human cancers exhibiting microsatellite instability. To extend analyses of this gene, we previously investigated the exon-intron organization of the TGFbeta RII gene and defined seven exons and flanking intron sequences. In this study, we further determined the nucleotide sequences surrounding these seven exons and designed eight sets of intron-based primers to examine the entire coding region of the TGFbeta RII gene. Using these primers, we screened genomic DNA sequences from 30 sporadic colorectal cancers for mutations of the TGFbeta RII gene. TGFbeta RII mutations were detected in two of 30 tumors and both displayed microsatellite instability. One had a deletion in a polyadenine tract in exon 3 and the other had a point mutation in the kinase domain located in exon 7. There were no mutations in exons 1, 2, 4, 5 and 6. These results further implicate the polyadenine tract and kinase domain as mutational hotspots in the TGFbeta RII gene in cells with genomic instability and suggest that TGFbeta RII gene mutations occur rarely in cells lacking genomic instability.     

A complex nine base pair deletion in RET exon 11 common in sporadic medullary thyroid carcinoma

Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.     

Mutations in haemophilia A

In the present study DNA from 281 unrelated haemophilia A patients including 15 inhibitor patients has been analysed by Southern blotting technique. Using various restriction enzymes, cloned factor VIII cDNA probes and genomic fragments we have identified 14 mutations. Six of the mutations are novel partial factor VIII gene deletions. One deletion affects exon 1, two deletions concern exon 6, another deletion, of which breakpoints are sequenced, takes part of exon 16 and two deletions affect exon 26. Besides the deletions, eight point mutations have been found at the TaqI restriction sites of exons 18, 24 and 26. Five C-->T mutations resulted in nonsense mutations, one in exon 18, one in exon 26 and three in exon 24. Two G-->A mutations caused a missense mutation in exon 24 leading to an arginine/glutamine exchange. Although two patients showed this mutation, their clinical phenotypes were different, possibly due to an additional unidentified sequence polymorphism. A G-->T mutation in exon 26 substituted the arginine with leucine. All deletions and seven of the point mutations are associated with severe disease with a detectable inhibitor in the patient with the TaqI-point mutation in exon 18. One of the G-->A mutations is associated with mild haemophilia but the patient also has developed an inhibitor. Amongst these mutations the origin of the mutation could be determined in four kindred, one of which showed maternal mosaicism.