RFLP analysis in families with sporadic hemophilia A

Summary To investigate the sporadic occurrence of hemophilia A and to estimate the sex ratio of mutation rates directly, 17 families with isolated cases of the disorder were studied by RFLP analysis and by clotting assays. Three RFLPs, one intragenic and two with close linkage to hemophilia A, were used. In eight families the RFLP study excluded the carrier status of the maternal grandmothers. Since hemostatic studies showed that the eight mothers of these propositi were hemophilia carriers, the origin of the newly mutated genes was inferred from the RFLP patterns: six hemophilic genes derived from the normal maternal grandfathers and two, from maternal grandmothers. The data indicate a higher mutation rate in males than in females, as previously suggested by segregation analysis and coagulation studies. However the sex ratio indicated by the RFLP analysis is lower than previously reported and could explain previous conflicting estimates.     

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type-dependent sex ratio of mutation frequencies.

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients--all exclusively from sporadic families--were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9 percent) of 76 patients, mothers, comprising 3 of 16 de novo mutations in the patients mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patients mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold-higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types.     

Molecular analysis of hemophilia A mutations in the Finnish population

We have examined the Finnish hemophilia A population for factor VIII gene mutations. This study included 83 unrelated patients and revealed 10 mutations associated with hemophilia. Using cloned cDNA, genomic, and oligonucleotide probes, we have identified three classes of mutations: five mutations causing the loss of TaqI restriction sites, a point mutation resulting in a new TaqI site, and four partial gene deletions. Although exons 5 and 6 were involved in three of the four partial gene deletions, the extent of the DNA lost differs in each case. The fourth deletion was located entirely within intron 1 and segregated with the disease in a large hemophilia pedigree. There was no history of hemophilia in eight of the 10 families. The origin of the mutation was determined in six of these pedigrees, two of which showed evidence for maternal mosaicism.     

Recurrent nonsense mutations at arginine residues cause severe hemophilia B in unrelated hemophiliacs

Summary Direct sequencing of the regions of the factor IX gene of likely functional significance was performed in four patients with severe hemophilia B. In two of the individuals, a transition at the dinucleotide CpG caused a nonsense mutation at arginine 333. In the other two individuals, a transition at CpG caused a nonsense mutation at arginine 29. Since these patients are all unrelated, as shown by differing alleles of the TaqI polymorphism in intron four or extensive nonoverlapping pedigrees, the mutations arose independently. In addition, the origin of one arginine 333 mutation in one family has been traced to the germline of the maternal grandfather. The frequent occurrence of mutations at arginine codons that contain the sequence CGN can be explained by the dramatic elevation of transitions at CpG. As a result, approximately one in four individuals with hemophilia B is expected to have a mutation at arginine and nonsense mutations at one of six arginine residues should be common causes of severe hemophilia.     

Direct and indirect estimation of the sex ratio of mutation frequencies in hemophilia A.

Carrier detection tests were carried out in 119 families with hemophilia A by using the data obtained with current DNA techniques (e.g., RFLP analysis and direct identification of mutations), conventional carrier detection tests (e.g., factor VIII:C and von Willebrand factor antigen), and pedigree information. On the basis of this data, we estimated the sex ratio of mutation frequencies with three completely different methods and compared the results. Since the classical indirect method derived from Haldane is substantially influenced by reproductive fitness (f), the sex ratio of mutation frequencies was estimated for both f = .3 and f = .5, resulting in a male:female mutation ratio of 5.37 (95% confidence interval 2.16-13.02) and 3.26 (95% confidence interval 0.97-8.73), respectively. According to the equilibrium-independent indirect method formulated by Rosendaal et al., the male:female ratio was estimated to be 3.4 (95% confidence interval 1.18-8.81). Since current DNA techniques provide information on the grandparental origin of the patient''s X chromosome, we were twice able to estimate directly the male:female mutation ratio as 15:1, by using the quotients of mutation origin (maternal grandfather/maternal grandmother and maternal grandfather/patient''s mother, respectively). Application of the Fisher test shows that the male mutation rate is higher than the female rate (P = 8.55 x 10(-7). Since all three completely different approaches unequivocally showed a higher male than female mutation frequency, this should be considered to be an established fact in calculating the risk in hemophilia A families.     

Recurrent mutations in the factor IX gene: founder effect or repeat de novo events

The investigation of 114 unrelated patients, representing about half the sample of the German haemophilia B population, enabled us to delineate the causative mutation in 103 (90.4%) haemophilic factor IX genes. Of these 103 cases 84 (81.6%) turned out to be unique molecular events, the remainder being repeats. Haplotype analysis revealed that the great majority, if not all, of these recurrent observations occurred independently. This conclusion is supported by our finding that three de novo mutations could be demonstrated at two sites of frequent mutation. A further 20 de novo events could be established in an unselected sample of 37 families with sporadic haemophilia B and 37 families with a history of the disease. Altogether, the germ line of origin could be determined in 21 of these 23 cases, thereby indicating a ratio of male to female mutation rates close to 2. On the basis of the data available, it is becoming clear that rearrangements in the factor IX gene (35.4% of de novo cases) are responsible for haemophilia B at a higher frequency than has been observed today (12.3%). More than two-thirds of the de novo cases cause the severe form of the disease, thereby reflecting the deficit of these haemophilic genes in the actual gene pool because of excess mortality in the past. In addition 40% (12/30) of the de novo single-base mutations were transitions at CpG dinucleotides. Compared with the expected at-random frequency, this observation indicates an 83-fold enhancement of mutation at CpG.     

Segregation analysis of hemophilia A and B.

We analyzed a sample of 1,485 families with hemophilia A and B and with unknown diagnosis. The frequency of sporadic cases was estimated to be .166 and .078 for the two types of hemophilia, respectively. The sex ratio of mutation rates did not differ significantly from unity. The average age of maternal grandfathers of probands at birth of mothers with a single child, affected by hemophilia B, and of maternal grandfathers of probands at birth of mothers with more than one child affected by hemophilia B, was higher than the age in appropriate control groups.     

Parental origin and germline mosaicism of deletions and duplications of the dystrophin gene: a European study

Summary Knowledge about the parental origin of new mutations and the occurrence of germline mosaicism is important for estimating recurrence risks in Duchenne (DMD) and Becker muscular dystrophy (BMD). However, there are problems in resolving these issues partly because not all mutations can as yet be directly detected, and additionally because genetic ratios are very sensitive to ascertainment bias. In the present study, therefore, analysis was restricted to currently detectable mutations (deletions and duplications) in particular types of families which tend to be rare. In order to obtain sufficient data we pooled results from 25 European centers. In mothers of affected patients who were the first in their family with a dystrophin gene deletion or duplication, the ratio between the paternal and the maternal origin of this new mutation was 32:49 (binomial test P = 0.075) for DMD. In five BMD families the ratio between paternal and maternal origin of new mutations was 32. Recurrence risk because of maternal germline mosaicism was studied in sisters or subsequent sibs of isolated cases with an apparently new detectable mutation. In 12 out of 59 (0.20; 95% CI 0.10–0.31) transmissions of the risk haplotype the DMD mutation was transmitted as well. No recurrences were found in nine BMD families.     

The pattern of factor IX germ-line mutation in Asians is similar to that of Caucasians.

To begin documenting the pattern of germ-line mutations in different human races, we have delineated the mutation in nine Korean families with hemophilia B by direct genomic sequencing of the regions of likely functional significance in the factor IX gene. An evaluation of these mutations in combination with previously described point mutations in the factor IX gene of Asians indicates that transitions predominate followed by transversions and microdeletions/insertions. Transitions at the dinucleotide CpG are a dramatic hot spot of mutation. This pattern of mutation is very similar to that observed in Caucasians with hemophilia B, despite the many differences between Asians (mostly Koreans) and Caucasians in diet, environment and cultural life-styles. The similarity may reflect the predominance of endogenous processes or ubiquitous mutagens rather than specific mutagens in the environment. The following additional conclusions emerge: (1) The missense mutations in Asians occur at evolutionarily conserved amino acids. When combined with the previous data this makes it likely that more than two-thirds of the missense mutations which could possibly occur at nonconserved amino acids do not cause hemophilia B. (2) Surprisingly, a change in the sixth base of the intron 2 donor splice-junction sequence is associated with severe disease in HB 74/77. (3) Direct carrier testing of nine Korean families demonstrates that the stability of DNA at ambient temperature in blood with the anticoagulant ACD solution B makes it feasible for a diagnostic laboratory to perform such testing at a distance of 7,000 miles. Carrier testing revealed that the mutation in HB78 arose in his mother's germ-line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Germ-line origins of mutation in families with hemophilia B: the sex ratio varies with the type of mutation.

Previous epidemiological and biochemical studies have generated conflicting estimates of the sex ratio of mutation. Direct genomic sequencing in combination with haplotype analysis extends previous analyses by allowing the precise mutation to be determined in a given family. From analysis of the factor IX gene of 260 consecutive families with hemophilia B, we report the germ-line origin of mutation in 25 families. When combined with 14 origins of mutation reported by others and with 4 origins previously reported by us, a total of 25 occur in the female germ line, and 18 occur in the male germ line. The excess of germ-line origins in females does not imply an overall excess mutation rate per base pair in the female germ line. Bayesian analysis of the data indicates that the sex ratio varies with the type of mutation. The aggregate of single-base substitutions shows a male predominance of germ-line mutations (P < .002). The maximum-likelihood estimate of the male predominance is 3.5-fold. Of the single-base substitutions, transitions at the dinucleotide CpG show the largest male predominance (11-fold). In contrast to single-base substitutions, deletions display a sex ratio of unity. Analysis of the parental age at transmission of a new mutation suggests that germ-line mutations are associated with a small increase in parental age in females but little, if any, increase in males. Although direct genomic sequencing offers a general method for defining the origin of mutation in specific families, accurate estimates of the sex ratios of different mutational classes require large sample sizes and careful correction for multiple biases of ascertainment. The biases in the present data result in an underestimate of the enhancement of mutation in males.     

CG dinucleotide transitions in the factor IX gene account for about half of the point mutations in hemophilia B patients: a Seattle series

Summary Hemophilia B is due to multiple molecular defects in the factor IX gene. Over 80% of mutations are single base substitutions. By amplification and direct sequencing, 51 single base substitutions were found in the transcribed sequence of the factor IX genes of patients from 50 distinct families with hemophilia B. These include 30 mutations in 29 families not previously reported by us; of these, 12 are novel, i.e., not previously published in other series. Of the 51 substitutions in our overall series 23 (45%) occurred as C-to-T or G-to-A transitions at 11 sites within CG dinucleotides. It is estimated that CG transitions occur from one to two orders of magnitude more frequently than mutations in nucleotides that are not within a CG pair. More than one family had identical defects for 6 of the CG mutations. At 4 of these sites, most patients had different haplotypes compatible with distinct mutations. Non-CG-type mutations occurred thoughout the coding regions with only one mutation in more than one family. The latter included 7 families with a 397 Ile-to-Thr defect that all share a rare haplotype, suggesting a common ancestor.     

Mutations in btk in patients with presumed X-linked agammaglobulinemia.

In 1993, two groups showed that X-linked agammaglobulinemia (XLA) was due to mutations in a tyrosine kinase now called Btk. Most laboratories have been able to detect mutations in Btk in 80%-90% of males with presumed XLA. The remaining patients may have mutations in Btk that are difficult to identify, or they may have defects that are phenotypically similar to XLA but genotypically different. We analyzed 101 families in which affected males were diagnosed as having XLA. Mutations in Btk were identified in 38 of 40 families with more than one affected family member and in 56 of 61 families with sporadic disease. Excluding the patients in whom the marked decrease in B cell numbers characteristic of XLA could not be confirmed by immunofluorescence studies, mutations in Btk were identified in 43 of 46 patients with presumed sporadic XLA. Two of the three remaining patients had defects in other genes required for normal B cell development, and the third patient was unlikely to have XLA, on the basis of results of extensive Btk analysis. Our techniques were unable to identify a mutation in Btk in one male with both a family history and laboratory findings suggestive of XLA. DNA samples from 41 of 49 of the mothers of males with sporadic disease and proven mutations in Btk were positive for the mutation found in their son. In the other 8 families, the mutation appeared to arise in the maternal germ line. In 20 families, haplotype analysis showed that the new mutation originated in the maternal grandfather or great-grandfather. These studies indicate that 90%-95% of males with presumed XLA have mutations in Btk. The other patients are likely to have defects in other genes.     

Missense mutations and the magnitude of functional deficit: the example of factor IX

Summary Some missense changes are compatible with normal protein function while others compromise essential aspects of protein maturation, specific activity, or stability. For those missense changes that alter function in the intact organism, how likely is it for the mutated protein to retain appreciable residual activity? By genetic analysis of patients with hemophilia B of known severity, this question can be addressed for missense mutations that reduce factor IX activity by fourfold or more below the average. We estimate that missense changes cause only 59% of moderate and severe disease, but these mutations are almost always (95%) of independent origin. In contrast, missense mutations are found in virtually all (97%) families with mild disease, but only a minority of these (41%) are of independent origin. From the aggregate data, we estimate that most (71%) of the independent deleterious missense mutations cause at least a 20-fold decrease in factor IX activity.     

Sex differences in mutational rate and mutational mechanism in the NF1 gene in neurofibromatosis type 1 patients

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a prevalence of around 1 in 3500, affecting all ethnic groups. The clinical manifestations of the disease are variable, even among members of the same family, and affect a variety of tissues and cell types, including skin, iris, central and peripheral nervous systems and skeletal system. It has been reported that the majority of sporadic mutations in NF1 arise in paternally inherited alleles. We present here a collaborative study of the parental origin and type of mutation in individuals with de novo NF1, who account for up to a half of all cases of clinically diagnosed NF1. We have studied intragenic and extragenic markers in 470 NF1 families. In 32 of these families it was possible to assess the parental origin of a de novo NF1 mutation either by linkage analysis (in families with three generations) or by the detection of an intragenic deletion in a sporadic NF1 case. Eleven of these 32 families have three generations (the second and third generation being affected), with the mutation (not a large deletion) being of paternal origin in 82% of them (P < 0.05). In the other 21 families an intragenic deletion was detected, in 76% being in the maternal chromosome and in 24% in the paternal one (P < 0.05). Our results suggest that in NF1 the majority of deletions occur in oogenesis, while other types of mutations should account for the paternally derived NF1 mutations. Received: 26 June 1996 / Revised: 1 August 1996     

Mutation rates in humans. I. Overall and sex-specific rates obtained from a population study of hemophilia B

A population-based study of hemophilia B mutations was conducted in the United Kingdom in order to construct a national confidential database of mutations and pedigrees to be used for the provision of carrier and prenatal diagnoses based on mutation detection. This allowed the direct estimate of overall (micro), male (v), and female (u) mutation rates for hemophilia B. The values obtained per gamete per generation and the 95% confidence intervals are micro;=7.73 (6. 29-9.12&parr0;x10-6; v=18.8 (14.5-22.9&parr0;x10-6; and u=2.18 (1. 44-3.16&parr0;x10-6. The ratio of male-to-female mutation rates is 8. 64, with a 95% confidence interval of 5.46-14.5. The higher male rate was not caused by a much higher rate of transition at CpG sites in the male. Attempts to detect evidence of gonadal mosaicism for hemophilia B mutation in suitable families did not detect any instances of ovarian mosaicism in any of 47 available opportunities. This suggests that the risk of a noncarrier mother manifesting as a gonadal mosaic by transmitting the mutation to a second child should be <0.062.     

Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.     

Molecular analysis of the eighteen most frequent mutations in the BRCA1 gene in 63 Chilean breast cancer families

BRCA1 gene mutations account for nearly all families with multiple cases of both early onset breast and/or ovarian cancer and about 30% of hereditary breast cancer. Although to date more than 1,237 distinct mutations, polymorphisms, and variants have been described, several mutations have been found to be recurrent in this gene. We have analyzed 63 Chilean breast/ovarian cancer families for eighteen frequent BRCA1 mutations. The analysis of the five exons and two introns in which these mutations are located was made using mismatch PCR assay, ASO hybridization assay, restriction fragment analysis, allele specific PCR assay and direct sequentiation techniques. Two BRCA1 mutations (185delAG and C61G) and one variant of unknown significance (E1250K) were found in four of these families. Also, a new mutation (4185delCAAG) and one previously described polymorphism (E1038G) were found in two other families. The 185delAG was found in a 3.17% of the families and the others were present only in one of the families of this cohort. Therefore these mutations are not prominent in the Chilean population. The variant of unknown significance and the polymorphism detected could represent a founder effect of Spanish origin.     

Human germline mutation in the factor IX gene

The molecular epidemiology of factor IX germline mutations in patients with hemophilia B has been studied in detail because it is an advantageous model for analyzing recent germline mutations in humans. It is estimated that mutations have been defined in the majority of nucleotides that are the target for mutation. The likelihood that a factor IX missense mutation will cause disease correlates with the degree of evolutionary conservation of the amino acid. Mutation rates per base-pair have been estimated after careful consideration and correction for biases, predicting about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes. The male-to-female sex ratio of mutation varies with the type of mutation. There is evidence for a maternal age effect and an excess of non-CpG G:C to A:T transitions. The factor IX mutation pattern is similar among geographically, racially and ethnically diverse human populations. The data support primarily endogenous mechanisms of germline mutation in the factor IX gene. Mutations at splice junctions are compatible with simple rules for predicting disease causing mutations.     

The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence.

Synthetic tandem repeats (STRs) of oligonucleotides have previously been shown to detect polymorphic loci in the human genome. Here, we report results from the use of three such probes to screen a human cosmid library. Nine of the 45 positive clones that were analyzed appear to contain highly polymorphic minisatellite or VNTR loci. The degree of enrichment for minisatellite sequences varied with the choice of STR: one provided a 15- to 20-fold enrichment (4 polymorphic loci among 10 clones), whereas 2 others gave a 3- to 5-fold enrichment (5 polymorphic probes in a total of 35 clones) compared to random screening. The 9 VNTR markers have been localized by linkage analysis in the CEPH panel and/or by in situ hybridization. Eight probes identify new loci, one of which maps to an interstitial region. One of the VNTR loci (identified by probe CEB1) was found to be hypermutable, with 52 mutation events identified among 310 children characterized in 40 CEPH families. The parental origin of the mutation could be identified in all instances, and only one mutation was found to be of maternal origin. The mutation rate in males was estimated to be approximately 15%. Segregation analysis of flanking markers suggests that mutations are not associated with crossing over. As the only previously described hypermutable minisatellite loci in humans have equal rates of male and female mutations, these observations establish that a second type of hypermutable minisatellite exists in the human genome. In neither case does the generation of new alleles appear to be associated with unequal crossing over.     

粘多糖贮积症Ⅱ型患者IDS基因的2个新突变

为了研究粘多糖贮积症II型(MPS Ⅱ)患者发病的分子遗传学机制, 采用PCR扩增艾杜糖-2-硫酸酯酶(IDS)基因突变热点区(外显子2、3、5、7、8和9)、DNA测序分析和限制性内切酶图谱分析的方法, 对2个粘多糖贮积症Ⅱ型家系进行了遗传突变分析。结果表明, 2个家系患者的IDS 基因分别出现IVS 6-1g→a和c.1587~1588 ins T 2个新突变。前者属于单碱基替换, 位于内含子6的3′端剪接位点, 导致跨外显子剪接; 后者属于插入突变, 插入点位于外显子9的cDNA 1,587和1,588碱基之间, 是迄今为止报道的人类IDS基因插入突变中最接近肽链末端的突变, 导致移码突变和转录提前终止。经限制性酶切分析, 证实2个家系中的患者母亲是突变基因的携带者, 符合该病X染色体隐性遗传的规律。另外,在对随机抽取的50名正常人及另外6名不相关的粘多糖病人的测序分析中, 未检测到这2个突变, 说明不是多态性。对于筛查所得的2个新突变是否是患者的致病原因, 尚需进一步证实。