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Summary. Background: Intrachromosomal, homologous recombination of the duplicon int22h-1 with int22h-2 or int22h-3 causes inversions accounting for 45% of severe hemophilia A, hence the belief that int22h-2 and int22h-3 are in opposite orientation to int22h-1. However, inversions involving int22h-2 are five times rarer than those involving its virtually identical copy: int22h-3. Recent sequencing has indicated that int22h-2 and int22h-3 form the internal part of the arms of an imperfect palindrome so that int22h-2, in the centromeric arm, has the same orientation as int22h-1 and, upon recombination with int22h-1, should produce deletions and duplications but not inversions. Aim: This work aims to provide rapid tests for all the mutations that can result from recombinations between the int22h sequences and to investigate whether int22h-2-related inversions causing hemophilia A arise in chromosomes, where the arms of the palindrome have recombined so that int22h2a ndint22h-3 swap places and orientation. Patients/ methods: Twenty patients with int22h-related inversions were examined together with a control and inversion carriers using reverse transcription-polymerase chain reaction (RTPCR), long-range PCR and sequencing. Results and conclusions: Analysis of mRNA in patients and a control provided evidence confirming the palindromic arrangement of int22h2a ndint22h-3 and the proposed inversion polymorphism that allows int22h-2 to be in the telomeric arm of the palindrome and in opposite orientation to int22h-1. New long-range PCR reactions were used to develop a single tube test that detects and discriminates inversions involving int22h-2 or int22h-3 and a two-tube test that can distinguish inversions, deletions, and duplications due to recombination between int22h sequences.

Summary We estimated the rates per base per generation of specific types of mutations, using our direct estimate of the overall mutation rate for hemophilia B and information on the mutations present in the United Kingdom's population as well as those reported year by year in the hemophilia B world database. These rates are as follows: transitions at CpG sites 9.7×10 −8 , other transitions 7.3×10 −9 , transversions at CpG sites 5.4×10 −9 , other transversions 6.9×10 −9 , and small deletions/insertions causing frameshifts 3.2×10 −10 . By taking into account the ratio of male to female mutation rates, the above figures were converted into rates appropriate for autosomal DNA—namely, 1.3×10 −7 , 9.9×10 −9 , 7.3×10 −9 , 9.4×10 −9 , 6.5×10 −10 , where the latter is the rate for all small deletion/insertion events. Mutation rates were also independently estimated from the sequence divergence observed in randomly chosen sequences from the human and chimpanzee X and Y chromosomes. These estimates were highly compatible with those obtained from hemophilia B and showed higher mutation rates in the male, but they showed no evidence for a significant excess of transitions at CpG sites in the spectrum of Y-sequence divergence relative to that of X-chromosome divergence. Our data suggest an overall mutation rate of 2.14×10 −8 per base per generation, or 128 mutations per human zygote. Since the effective target for hemophilia B mutations is only 1.05% of the factor IX gene, the rate of detrimental mutations, per human zygote, suggested by the hemophilia data is ∼1.3.

We report the precise mapping and characterization of the genomic structure of the human homolog of the rat gene for the nucleolar protein NAP57, which has been reported to be responsible for X-linked dyskeratosis congenita (DKC). This single-copy gene, now called DKC, is transcribed from a CpG island 60 kb centromeric to the factor VIII gene in distal Xq28 and lies tail to tail with the palmitoylated erythrocyte membrane protein gene, MPP1. DKC comprises 15 exons spanning at least 16 kb and is transcribed into a widely expressed 2.6-kb message. Several functional motifs of DKC are assigned to coding sequences specified by individual exons. Analysis of normal female DNA revealed the presence of two polymorphisms in the DKC exons, while mutation analysis of a DKC patient identified a novel single amino acid missense mutation in exon 4. The latter together with exon 3 contain five of the six missense mutations reported so far in the DKC gene.

A single-copy, widely expressed gene of at least 30 kb and six exons was discovered via a hybrid mRNA resulting from an inversion causing hemophilia A. A segment of the 1.7-kb message of this gene has been shown by others to encode a protein (named VBP1) interacting with the product of the von Hippel-Lindau gene and thus is expected to participate in pathways involving this tumor suppressor gene. The mouse VBP1 message we isolated encodes a polypeptide of 160 residues absolutely identical to that of human. Even the 3' untranslated tails of the mRNAs show 68% conservation, and both use the unusual ATTAAA polyadenylation signal. The mouse gene has a single transcription start while the human homologue has two major starts and a minor start. This could result in amino-end extensions of the human protein. A polymorphism with 42% heterozygosity in the United Kingdom population was detected in the 3' tail of the message. VBP1 is unlike other known proteins but a consensus for tyrosine phosphorylation possibly suggests regulation by kinases.

Most families with haemophilia A or B carry gene defects of independent origin. Haemophilia B is mostly due to small changes in the factor IX gene affecting either its transcription, mRNA maturation, mRNA translation or the fine structure of factor IX. Only 2-3% of patients show gross deletions or rearrangements. The great variety of missense mutations reported to cause haemophilia B indicates that this multidomain protein is highly constrained. Less is known about the factor VIII gene as fully efficient mutation detection procedures only became available in 1991. This, however, led to the discovery that almost half the severe cases of haemophilia A or a fifth of all cases are due to frequently occurring inversions caused by homologous intra-chromosome (-chromatid) recombination between repeated sequences 9.5 kb long. Of the three repeats one is in intron 22 of the factor VIII gene and two are 400-500 kb more telomeric. They are 99.8% similar to each other. The spectrum of the other haemophilia A mutations is similar to that of haemophilia B. Since 1983 mounting evidence has shown that in both haemophilias the nature of the mutation is important in predisposing to the inhibitor complication.

To the Editor:Ruiz-Pesini et al. (2000xHuman mtDNA haplogroups associated with high or reduced spermatozoa motility. Ruiz-Pesini, E, Lapena, A-C, Diez-Sanchez, C, Perez-Martos, A, Montoya, J, Alvarez, E, Diaz, M, Urries, A, Montoro, L, Lopez-Perez, MJ, and Enriquez, J. Am J Hum Genet. 2000; 67: 682–696Abstract | Full Text | Full Text PDF | PubMed | Scopus (300)See all References2000) cleverly show that extant human mtDNA variation affects sperm function. They find that mitochondrial haplogroup T is overrepresented in asthenozoospermic populations and shows reduced sperm oxidative phosphorylation pathway (OXPHOS) activity, relative to the H haplogroup that is overrepresented in nonasthenozoospermic populations. These authors—as well as Moore and Reijo-Pera (2000xMale sperm motility dictated by mother's mtDNA. Moore, FL and Reijo-Pera, RA. Am J Hum Genet. 2000; 67: 543–548Abstract | Full Text | Full Text PDF | PubMed | Scopus (40)See all References2000), in the accompanying invited editorial—stress that, because of the exclusive matrilinear inheritance of mitochondria, mutations of mtDNA purely affecting male fertility are not selected against and therefore can become fixed. The absence of a direct check against mitochondrial mutations that affect male fertility is unfortunate and begs the question of why such a pattern became established.In keeping with an earlier suggestion (Giannelli 1986xDNA maintenance and its relation to human pathology. Giannelli, F. J Cell Science Suppl. 1986; 4: 383–416Crossref | PubMedSee all References1986), I propose that the exclusion of sperm mitochondria from the zygote is part of a scheme enabling mitochondria to provide an indirect measure of sperm quality and, hence, to favor fertilization by optimal spermatozoa while avoiding the risk of passing on mtDNA exposed to high physiological stress and, hence, potential damage. This would clearly have adaptive value and could help justify the establishment of matrilineal mitochondrial inheritance.There is evidence that mitochondria have a role in germ-cell selection. Krakauer and Mira (1999xMitochondria and germ-cell death. Krakauer, DC and Mira, A. Nature. 1999; 400: 125–126Crossref | PubMed | Scopus (95)See all References1999), in a phylogenetic study, note that species producing fewer offspring have fewer egg mitochondria and experience greater ovarian atresia, and these authors conclude that lower numbers of mitochondria offer greater opportunities for variation in mitochondrial function and, thus, for elimination of eggs with poor mitochondria. This results in purifying selective pressure on mitochondrial genomes. Some proof of a mitochondrial role in ovarian atresia exists, as microinjection of 5 × 103 mitochondria from nonapoptotic follicular granulosa rescues the oocytes from FVB-strain mice that undergo an inherently high rate of apoptosis in vitro (Perez et al. 2000xMitochondria and the death of oocytes. Perez, GI, Trbovich, AM, Gosden, RG, and Tilly, JL. Nature. 2000; 403: 500–501Crossref | PubMed | Scopus (98)See all References2000). With regard to spermatozoa, Ruiz-Pesini et al. (2000xHuman mtDNA haplogroups associated with high or reduced spermatozoa motility. Ruiz-Pesini, E, Lapena, A-C, Diez-Sanchez, C, Perez-Martos, A, Montoya, J, Alvarez, E, Diaz, M, Urries, A, Montoro, L, Lopez-Perez, MJ, and Enriquez, J. Am J Hum Genet. 2000; 67: 682–696Abstract | Full Text | Full Text PDF | PubMed | Scopus (300)See all References2000) provide direct evidence that mitochondrial function is critical to their motility and hence presumably to their success in fertilization.Nevertheless, egg and sperm differ dramatically in their mitochondrial complement, since the human oocyte has 106 mtDNA molecules, whereas mature spermatozoa have only 100 (Cummings 1998xMitochondrial DNA in mammalian reproduction. Cummings, J. Rev Reprod. 1998; 3: 172–182Crossref | PubMed | Scopus (106)See all References1998). It follows that spermatozoa should be exquisitely sensitive to mitochondrial malfunction.I suggest that this allows mitochondria to become both a sensitive meter of genetic damage to sperm and the means of selecting, at fertilization, spermatozoa derived from the germ-cell lines that have best preserved the quality of their genome during postzygotic life. However, reliance on a small complement of mitochondria to produce very high levels of kinetic energy in conditions of unusually high oxygen tension in the female genital tract exposes sperm mtDNA to extraordinary risks from oxygen radicals, and, therefore, the disposal of mtDNA at zygote formation also seems advantageous. To provide a sensitive, indirect measure of the quality of the sperm genome, the mitochondrial complement of a spermatozoon must meet two conditions: it should not afford a high degree of functional redundancy, and it must offer a target for deleterious mutations larger than the nuclear genome.The results of Ruiz-Pesini et al. (2000xHuman mtDNA haplogroups associated with high or reduced spermatozoa motility. Ruiz-Pesini, E, Lapena, A-C, Diez-Sanchez, C, Perez-Martos, A, Montoya, J, Alvarez, E, Diaz, M, Urries, A, Montoro, L, Lopez-Perez, MJ, and Enriquez, J. Am J Hum Genet. 2000; 67: 682–696Abstract | Full Text | Full Text PDF | PubMed | Scopus (300)See all References2000) suggest that the former is true, because a mitochondrial haplogroup associated with modest OXPHOS deficit is associated with asthenozoospermia. More-direct experimental evidence on this point would require the inactivation of a proportion of the mitochondria and subsequent examination of sperm function. Such evidence is not available, but, since each human spermatozoon competes with 108 colleagues for a single oocyte, it seems probable that a sperm needs all of the energy its 100 mitochondria can produce.It is reasonable to conclude that the mitochondrial complement of a spermatozoon offers a sufficiently large target for deleterious mutations, for the following reasons. The germinal mutation rate for mtDNA is 50-fold greater than that for genomic DNA. Human mitochondria have no introns, intergenic sequences, or complex, large centromeres and telomeres, and they have nonredundant gene sequences that even show some overlap. In contrast, nuclear coding sequences are highly dispersed (International Human Genome Sequencing Consortium 2001xInitial sequencing and analysis of the human genome. International Human Genome Sequencing Consortium. Nature. 2001; 409: 860–921Crossref | PubMed | Scopus (12719)See all References2001) and show some degree of functional redundancy. Therefore, the essential information content of mtDNA can be considered to be at least 100-fold greater than that of a nuclear DNA segment of similar length. It follows that the effective target for deleterious mutations presented by a mtDNA molecule should be equivalent to 16,569 bp × 50 × 100 = 83 Mb of genomic DNA. This is ∼1/36 of the haploid genome. Therefore, the 100 mtDNA molecules of a spermatozoon offer a target for deleterious mutation equivalent to 2.8 haploid genomes. Thus, if the functional redundancy of the mitochondrial complement of a spermatozoon, in terms of its competition with 108 other spermatozoa, is

An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 x 120-mm filters representing a total of approximately 20,000 individual clones in approximately 3 h. High-density arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies.

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.

Monosomy for the X chromosome in humans creates a genetic Achilles' heel for nature to deal with. We report that the human X chromosome appears to have one-third the density of the coding sequence of the autosomes and, because of partial shielding from the high mutation rate of the male sex, that it should also have a lower mutation rate than the autosomes (i.e., .73). Hence, the X chromosome should contribute one quarter (.33×.73=.24) of the deleterious mutations expected from its DNA content. In this way, selection has possibly moderated risks from mutation in X-linked genes that are thought to have been fixed in their syntenic state since the onset of the mammalian lineage. The unexpected difference in the density of coding sequences indicates that our recent, hemophilia B–based estimate of the rate of deleterious mutations per zygote should be increased from 1.3 to 4 (1.3×3).

Six fibroblast strains sensitive to long wavelength ultraviolet radiation (UVA) and one control strain were used to see if cooperation between the different cell strains could modify the abnormally high yield of single-strand DNA breaks (SSB) in the sensitive strains caused by UVA irradiation in complete Dulbecco's MEM. The sensitive strains were established from individuals showing proneness to different types of light-induced skin damage (actinic reticuloid, familial actinic keratoses with internal malignancies, and unusual frequency of basal cell carcinomata). When sensitive and normal cells were cocultivated, the UVA-induced SSB decreased in the sensitive cells and increased in the normal ones by amounts proportional to the ratio of the two types of cells in the mixtures. Furthermore the regression of SSB, in the sensitive cells, on the proportion of normal cells in the mixture extrapolated to normal levels of SSB when the proportion of normal cells increased to one. Cocultivation of different sensitive cells did not reduce the UVA-induced SSB to levels below those of the less sensitive cell strains. From these results we conclude that substances, present in limiting amounts, even in normal cells, can be transferred from cell to cell, presumably by metabolic cooperation, and modify the yield of SSB caused by UVA radiation. The abnormal yields of SSB in the sensitive cells appear to be entirely attributable to deficits in the substances responsible for the intercellular cooperation. We suggest that such substances are small molecular weight scavengers of active oxygen species.

In an attempt to replace the existing, DNA-based, 50% effective, carrier and prenatal diagnoses of haemophilia A with the 100% successful direct detection of defective genes, a new procedure was developed to screen and identify mutations in all the essential regions of the factor VIII gene (putative promoter, coding sequence, and the cleavage and polyadenylation region). Genomic DNA and cDNA obtained by reverse transcription of the "leaky" mRNA found in peripheral lymphocytes were amplified by means of the polymerase chain reaction to yield a set of eight segments comprising the essential gene sequences. The segments were then screened individually for mutations by the amplification mismatch detection method, which detects and locates any type of sequence discrepancy between the test DNA and the control probe by cleavage of the probe at the site of mismatches. Two haemophilia A patients were studied. The first showed two single-base changes: one (substitution of tryptophan 2229 by cysteine in the C2 domain) is the probable cause of the disease, since it affects a conserved residue of factor VIIIa, whereas the other (the conservative substitution of aspartic acid at position 1241 by glutamic acid) occurs in a domain (B) irrelevant to factor VIII activity. The second patient showed a complete failure of pre-mRNA splicing due to a single-base substitution that changes the obligatory AG acceptor splice site of intron 5 to GG. The method characterises the gene defect in 10 days or less and should lead to the rapid accumulation of information on the molecular biology of haemophilia A.

Two varieties of an inversion breaking the factor VIII gene (F8) and causing half of all severe hemophilia A have previously been described. These were thought to involve the intron 22 sequence: int22h-1 [1] interacting with either the proximal (int22h-2), or the distal (int22h-3) of two extragenic and telomeric copies. However, new sequence data show that only int22h-3 should be involved in these inversions [2]. We explain this discrepancy and note that the new sequence data predict a confounding complication for mutation detection in some families with hemophilia A. In 1993, it was shown that int22h-3 is in inverse orientation to int22h-1 and the same was thought to be true for int22h-2 because BclI blots indicated that both int22h-2 and int22h-3 recombined with int22h-1 to cause inversions [3]. By contrast, the new sequence of the human X chromosome [2] shows that int22h-1 is in the same orientation as int22h-2 so that their interaction should lead to deletion and duplication rather than inversion. These data show that int22h-2 and int22h-3 form the arms of an imperfect palindrome separated by a 65 kb

Research into haemophilia has resulted in important advances in both the theory and practice of human genetics. In 1935, Haldane showed that haemophilia, an X-linked recessive condition, was maintained in the population by an equilibrium between the loss of haemophilia genes, due to the reduced chance that affected males have of reproducing, and the gain of new haemophilia genes, due to mutation. This scenario results in allelic heterogeneity and explains why disease severity varies, patients are frequently sporadic, and mutations are usually different and of independent origin in every family. The small size of modern western families allows haemophilia mutations to go undetected for some generations, and artificially increases the proportion of sporadic patients. In 1952, locus heterogeneity was discovered. Thus, deficiency of coagulation factor VIII was called haemophilia A and that of factor IX named haemophilia B. This knowledge eventually led to treatments based on the administration of specific gene products missing in patients. These substances were provided by networks of haemophilia centres, and the mean life expectancy of haemophilia patients in the UK seemed to approach that of the general population. Unfortunately, however, blood-derived therapeutic agents were later shown to be the vehicle of dangerous viral infections, such as HIV-1 and hepatitis, a situation that is now compounded by concerns about transmission of prions for bovine spongiform encephalopathy. Investigators should learn from these adversities, and new therapies should be preceded and accompanied by thorough risk assessment. A proportion of patients with either haemophilia A or B become refractory to treatment because of an immune reaction against the therapeutic coagulant factor. The occurrence of this reaction, called inhibitor formation, is strongly affected by the patient’s gene defect. Thus, large gene deletions or nonsense mutations cause strong predisposition, presumably because they prevent the synthesis in the patient’s cells of any immunologically recognisable factor IX protein. Therefore, the normal gene product, absent in the patient, seems foreign to their immune system. Direct or indirect replacement therapy can result in adverse immune reactions in patients who have had no chance of developing immune tolerance to the therapeutic agent. Research is needed to learn how to prevent or overcome such reactions. The relatives of patients with haemophilia are often worried about having the disease, for although haemophilia is, by and large, manageable, treatments often cause adverse reactions. There is, therefore, demand for genetic screening, which usually involves general genetic counselling, carrier diagnosis, and, more rarely, prenatal diagnoses. Carrier tests are usually required by patients’ sisters or more distant female relatives, but in 50% of families the mother of an apparently sporadic patient will want to know if she is a carrier, and hence at high risk of having more sons with haemophilia. Concern about haemophilia depends on individual circumstances, including severity of disease in the family—some patients bleed excessively only after major trauma, whereas others bleed frequently and spontaneously. In patients with haemophilia B there is good correlation between phenotype and gene defect, so that information about the gene defect has prognostic value. So far, the phenotypic consequences of 750 different mutations have been recorded. Mutations that seriously affect the structure of factor IX result in a large reduction in the amount of circulating factor IX, whereas mutations that alter its catalytic centre or its ability to interact with factor VIII and factor X (the substrate of the complex formed by factors VIII and IX) tend to cause pronounced loss of factor IX coagulant activity. The large variety and recent origin of most haemophilia mutations complicates the genetic screening process. The best way to diagnose carriers is to test for the gene defect specific to their family. Thus, in order to provide a good screening service for those with haemophilia B, a new strategy has been introduced. This is based on the construction of a national, confidential database of gene mutations and pedigrees. The mutations are identified in one patient or carrier in every family and recorded. Thus, a family member can be screened quickly, accurately, and at a reduced cost, since only a small part of the haemophilia gene needs to be checked, dependent on which family the individual belongs to. This strategy is now in full use in the UK and in Sweden, and it represents a model for the screening of other inherited diseases characterised by a varied spectrum of genetic defects, including haemophilia A. In the UK the construction of a confidential database of haemophilia A mutations and pedigrees is well on the way. Descendants of Queen Victoria (arrow), showing inheritance of haemophilia Patients are shown as red squares, and known carriers as purple circles.

Using an mRNA-based method to examine haemophilia A mutations we provide an explanation for the puzzling report that half of the mutations causing severe disease are not detected by analysis of the putative promoter, exons, and most exon/intron boundaries of the factor VIII gene. An unusual cluster of mutations involving regions of intron 22 not examined earlier leads to defective joining of exons 22 and 23 in the mRNA and caused haemophilia A in 10/24 severely affected UK patients.

Xeroderma pigmentosum (x.p.) was diagnosed prenatally by detecting, in amniotic cells cultured in vitro, the defect in the repair of ultraviolet-damaged D.N.A. which is pathognomonic of X.P. The procedure adopted is simple and reliable and can, therefore, be put to routine use for the prevention of X.P. The efficacy of preventive measures in X.P. is at least partly dependent upon the early postnatal detection of index cases as exemplified by the history of the family which is the subject of this report.

The unscheduled DNA synthesis (UDS) induced by ultraviolet radiations (UVR) in fibroblasts from 5 patients with Bloom Syndrome (BS) has been studied. Since often a high proportion of BS cells has large, probably polyploid nuclei, care was taken to select cells of normal size. Furthermore, UDS was usually measured over constant nuclear areas by a photometric method and each cell strain was tested on several different occasions. This showed that each BS cell strain had levels of UDS exceeding control values, on average, by 19-29%. The difference between the UDS of BS and control cells did not change with UVR doses from 5 to 100 J . m-2 but, unirradiated BS cells showed no evidence of "spontaneous" UDS. Also erythemal UVR (greater than 295 nm) elicited excessive UDS in the BS cell strain which was exposed to such radiations. When BS and control fibroblasts were incubated at low (32.5 degrees C) and high temperature (40.5 degrees C) before and after UVR doses of 5, 25 and 100 J . m-2, it was found that at the lowest dose the temperature of incubation did not modify the difference in UDS between normal and BS cells while, at the highest dose, BS cells incubated at 32.5 degrees C did not show more UDS than the controls. Finally, BS and control fibroblasts were fused with xeroderma pigmentosum (XP) cells of complementation groups C and D, which are complemented slowly by their partners, and it was than found that BS cells may transfer their tendency to high levels of UVR-induced UDS to their fusion partners. In keeping with these findings, UV-irradiated fibroblasts from BS heterozygotes seemed to show more UDS than normal cells. The anomalous behaviour of BS cells exposed to UVR is unexplained but trivial factors such as differences in cell geometry do not seem to account for our findings. Therefore, since an abnormally small endogenous pool of thymidine and "spontaneous" UDS in BS were not observed, it seems possible that the greater UDS performed during the first 1.5 h of repair by BS cells may be due to: greater numbers of damaged sites repaired; greater incorporation of thymidine per site repaired; or finally, both. In fact, the cell-fusion experiments suggest that BS cells may contain a diffusible factor which influences at least the initial rate of UDS. If such a factor were the product of the BS allele it could be argued that the BS mutations are not amorph and that the BS gene product may compete with that of the normal allele and modify the initial rate of UDS induced by UVR. It is hoped that our observations and their may possible interpretations will stimulate further work on BS.

Xeroderma pigmentosum of groups A, C and D shows complementation differing in kinetics, dependence on the dose of wild-type alleles and dependence on protein synthesis. Such differences suggest that XP-A, -C and -D carry mutations at different loci. The product of the first of these loci (factor A) is present in significant excess in normal fibroblasts, seems to turn over rapidly and may be a dimer or higher polymer. The products of the other two loci (factors C and D) do not seem to be present in significant excess in the cytoplasm of normal fibroblasts, but factor C may accumulate abnormally in XP-D. Factors C and D turn over slowly (D more than C) and they do not move freely from the cell nucleus. Factors A and C, at least, seem to act directly and not via gene regulation.

DNA from 33 healthy White subjects was analysed with a 2·5 kilobase subgenomic DNA probe derived from the gene for coagulation factor IX, containing the exon "d" region of that gene. Intragenic Taq I restriction-fragment length polymorphism was revealed, with allelic frequencies estimated at 0·65 and 0·35 (SE = 0·06), also detectable by a cDNA probe. The genomic DNA probe is technically superior to the cDNA probe and has been used in three families with haemophilia B (factor IX deficiency). The polymorphic marker segregates with the deleterious mutation, allowing the identification or exclusion of carriers. The allelic frequencies of the Taq I polymorphism are virtually ideal. Therefore, such a polymorphism should be helpful both in genetic counselling of approximately 40% of affected families and in prenatal diagnosis.