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Statistical learning (SL) refers to the ability to extract statistical regularities from the environment. Previous research has suggested that regularity extraction is modality-specific, occurring within but not between sensory modalities (Frost et al., 2015). The present study investigates the circumstances under which SL can occur between modalities. In the first experiment, participants were presented with a stream of meaningless visual fractals and synthetic sounds while performing an oddball detection task. Stimuli were grouped into unimodal (AA, VV) or crossmodal (VA, AV) pairs based on higher transitional probability between the elements. Using implicit and explicit measures of SL, we found that participants only learned the unimodal pairs. In a second experiment, we presented the pairs in separate unimodal (VVVV, AAAA) and crossmodal (AVAV, VAVA) blocks, allowing participants to anticipate which modality would be presented next. We found that SL for the crossmodal pairs outperformed that of unimodal pairs. This result suggests that modality predictability facilitates a correct crossmodal attention deployment that is crucial for learning crossmodal transitional probabilities. Finally, a third experiment demonstrated that participants can explicitly learn the statistical regularities between crossmodal pairs even when the upcoming modality is not predictable, as long as the pairs contain semantic information. This finding suggests that SL between crossmodal pairs can occur when sensory-level limitations are bypassed, and when learning can unfold at a supramodal level of representation. This study demonstrates that SL is not a modality-specific mechanism and compels revision of the current neurobiological model of SL in which learning of statistical regularities between low-level stimuli features relies on hard-wired learning computations that take place in their respective sensory cortices.

Hearing spoken words can enhance visual object recognition, detection and discrimination. Yet, the mechanisms that underpin this facilitation are incompletely understood. On one account, words do not bias early visual processing, but rather affect later semantic or decision-making stages. However, recent proposals suggest that words can alter early visual processes by activating category-specific priors in sensory regions. A prediction of this account is that top-down priors evoke changes in occipital areas in anticipation of visual stimuli. Here, we tested the hypothesis that neural oscillations serve as a mechanism to activate language-generated visual priors. Participants performed a cue-picture matching task where cues were either spoken words, in their native or second language, or natural sounds, while their EEG and reaction times were recorded. Behaviorally, we replicated the previously reported label-advantage effect, with images cued by words being recognized faster than those cued by natural sounds. A time-frequency analysis of cue-target intervals revealed that this behavioral label-advantage was associated with enhanced power in posterior alpha (9-11 Hz) and beta oscillations (17-19 Hz), both of which were larger when the image was preceded by a word compared to a natural sound. Importantly, object recognition performance improved with high alpha power but slowed down with enhancement of beta synchronization. These results suggest that alpha and beta rhythms play distinct functional roles to support language-mediated visual object recognition: alpha might function to amplify sensory priors in posterior regions, while beta may (re)activate the network states elicited by the auditory cue.

We investigated how native language experience shapes prediction mechanisms. Two groups of bilinguals (either Spanish or Basque natives) performed a word matching task (WMT) and a picture matching task (PMT). They indicated whether the stimuli they perceived matched with the noun they heard. Spanish noun endings were either diagnostic of the gender (transparent), or ambiguous (opaque). ERPs were time-locked to the gender-marked determiner preceding the predicted noun. In the WMT both groups showed a negative (~340 ms) effect. Basque natives displayed an earlier effect (~150 ms) for determiners preceding transparent nouns. In the PMT both groups showed an early effect (~160 ms) for determiners preceding opaque nouns. Transparent nouns' determiners elicited prediction at ~330 ms in Spanish natives, but at ~460 ms in Basque natives. It is concluded that bilinguals rely on the features of the L1 for predicting in the L2: native experience moulds prediction. Linguistic prediction is hierarchical, with different operation levels communicating at an interface stage.

Issue Online:21 September 2018 We investigated how native language experience shapes anticipatory language processing. Two groups of bilinguals (either Spanish or Basque natives) performed a word matching task (WordMT) and a picture matching task (PictureMT). They indicated whether the stimuli they visually perceived matched with the noun they heard. Spanish noun endings were either diagnostic of the gender (transparent) or ambiguous (opaque). ERPs were time-locked to an intervening gender-marked determiner preceding the predicted noun. The determiner always gender agreed with the following noun but could also introduce a mismatching noun, so that it was not fully task diagnostic. Evoked brain activity time-locked to the determiner was considered as reflecting updating/reanalysis of the task-relevant preactivated representation. We focused on the timing of this effect by estimating the comparison between a gender-congruent and a gender-incongruent determiner. In the WordMT, both groups showed a late N400 effect. Crucially, only Basque natives displayed an earlier P200 effect for determiners preceding transparent nouns. In the PictureMT, both groups showed an early P200 effect for determiners preceding opaque nouns. The determiners of transparent nouns triggered a negative effect at similar to 430 ms in Spanish natives, but at similar to 550 ms in Basque natives. This pattern of results supports a "retracing hypothesis" according to which the neurocognitive system navigates through the intermediate (sublexical and lexical) linguistic representations available from previous processing to evaluate the need of an update in the linguistic expectation concerning a target lexical item. Spanish Ministry of Economy and Competitiveness (MINECO), Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) (grant PSI2015‐65694‐P to N. M.), Spanish Ministry of Economy and Competitiveness “Severo Ochoa” Programme for Centres/Units of Excellence in R&D (grant SEV‐2015‐490)

Available online 3 April 2017 Language comprehension is largely supported by predictive mechanisms that account for the ease and speed with which communication unfolds. Both native and proficient non-native speakers can efficiently handle contextual cues to generate reliable linguistic expectations. However, the link between the variability of the linguistic background of the speaker and the hierarchical format of the representations predicted is still not clear. We here investigate whether native language exposure to typologically highly diverse languages (Spanish and Basque) affects the way early balanced bilingual speakers carry out language predictions. During Spanish sentence comprehension, participants developed predictions of words the form of which (noun ending) could be either diagnostic of grammatical gender values (transparent) or totally ambiguous (opaque). We measured electrophysiological prediction effects time-locked both to the target word and to its determiner, with the former being expected or unexpected. Event-related (N200– N400) and oscillatory activity in the low beta-band (15–17 Hz) frequency channel showed that both Spanish and Basque natives optimally carry out lexical predictions independently of word transparency. Crucially, in contrast to Spanish natives, Basque natives displayed visual word form predictions for transparent words, in consistency with the relevance that noun endings (post-nominal suffixes) play in their native language. We conclude that early language exposure largely shapes prediction mechanisms, so that bilinguals reading in their second language rely on the distributional regularities that are highly relevant in their first language. More importantly, we show that individual linguistic experience hierarchically modulates the format of the predicted representation. This work was partially supported by the Spanish Ministry of Economy and Competitiveness (grants PSI2012-32350 and PSI2015-65694-P to N.M. and grant PSI2014-54500-P to C.M. and S.C.), by the Basque Government (grant PI_2015_1_25 to C.M. and S.C.) and by the Ikerbasque Research Fellowships to N.M and C.M. Further support derived from the AThEME project funded by the European Commission 7th Framework Programme, the ERC- 2011-ADG-295362 from the European Research Council and the award ‘‘Centro de Excelencia Severo Ochoa SEV-2015-0490”.

One-third of the UK haemophilia A population was screened to establish a national database of mutations and pedigrees and advance knowledge of the disease. The following mutations were found: 131 intron 22- and 13 intron1-breaking inversions; 11 gross deletions and an insertion; 65 frameshifts; three in-frame deletions and one insertion; 46 nonsense; 30 intronic mutations affecting splice sites and four generating new sites; 469 non-synonymous mutations due to 203 different base substitutions of which four affected, and nine were predicted to affect, splicing; three promoter mutations; two synonymous exon 14 mutations possibly affecting splicing; two VWF mutations. Of the above mutations, 176 are not listed in the Haemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS). Four gross deletions arose by non-homologous end-joining; we detected unexpected splicing in some mutations; substitution of amino acids conserved for less than 90 million years are rare; the risk of developing inhibitors for patients with nonsense mutations is greater when the stop codon is in the 3' half of the mRNA; changes likely to generate splice sites causing frameshifts are over-represented among non-synonymous mutations associated with inhibitors; our data and those in HAMSTeRS enabled the size of the spectrum of specific mutations causing the disease to be estimated and to determine how much of it is known.

SUMMARY The reactions to monochromatic radiation of the skin of ten patients with xeroderma pigmentosum were investigated, and eight were abnormal. The abnormalities consisted of papular and vesicular reactions and delay in the development of the minimal erythema dose reaction with wavelengths principally in the 290-320 nm range. Three patients were too young for full action spectra to be obtained but of those patients in whom all wavelengths were tested only one showed a reaction to radiation above 320 nm and this was at 340 nm only. In only one patient was the minimal erythema dose at 300 nm at 24 h lower than normal. In six patients the repair synthesis of deoxyribonucleic acid after ultraviolet radiation was estimated. Reduced levels were seen in five but repair was normal in one patient. The patient with normal repair also had normal reactions to monochromatic radiation. The abnormal reaction of the skin to artificial radiation and the abnormal deoxyribonucleic acid repair synthesis may enable the diagnosis of xeroderma pigmentosum to be made at a very early age. In one of the patients the diagnosis was made at the age of 6 months in a child with photosensitivity but with no other clinical signs of the disease. It is suggested that, by making the diagnosis as early as possible and by protecting the skin from natural sunlight, cutaneous malignancies may be prevented.

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.

Summary. We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid-phase fluorescent chemical cleavage of mismatch and examining additional non-coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1·4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.

SummaryA national strategy for optimising genetic services in haemophilia A has been initiated in the UK. Solid phase fluorescent chemical cleavage of mismatch is used to screen the entire coding region of factor VIII in six segments: four amplified from the trace of mRNA in blood lymphocytes and two from genomic DNA for the 3.4 kb exon 14 and flanking intron sequences. These segments are analysed in two threefold multiplexes so that the genes of 18 patients can be screened in a single ABI 377 gel. The promoter and polyadenylation signal region are amplified and sequenced directly. We have analysed 142 unrelated patients and identified 141 factor VIII mutations and one Normandy type von Willebrand homozygote. The former mutations include 89 missense, 10 nonsense, 5 frameshift, one 24 bp deletion and one splice signal defect. These comprise 71 different changes, of which 39 have not been previously observed.

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.

The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of

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.

Summary. The families of sporadic haemophilia A patients registered at the Royal London Hospital and with living grandparents were selected for study. Twelve of the 13 known families agreed to collaborate. Of these 11 had a patient with severe and one a patient with mild haemophilia. Five of the severely affected patients had inversions of type 1, that is involving int22h-1 and int22h-3, and two had inversions of type 2, that is involving int22h-1 and int22h-2. The remaining four patients with severe disease had single base substitutions causing two different non-sense (Gln592Stop; Trp2313Stop) and two different mis-sense mutations (His267Pro; Arg2209Gln). A single base substitution causing a mis-sense mutation (His1961Asp) was also found in the patient with mild haemophilia. The Arg2209Gln mutation has previously been found in other patients while the other single base substitutions have not hitherto been observed. Three mutations (all single base substitutions) appear to have arisen in the germline of the patient’s mother, while seven mutations originated in the gonad of one of the patient’s grandparents. In two of the latter families (both with inversion type 2) the origin of muta-tions could be clearly assigned to the grandpaternal gonad. Data on parental age at the onset of mutation were ob-tained in the families investigated. In addition, further evidence was obtained that the int-22h-related inversions arise by intrachromatid, intrachromosome homologous re-combination as the int22h repeat sequences of a patient were found to be identical to those of the maternal grand-father whose germline represents the origin of mutation.

Analysis of mRNA in two haemophilic monozygotic twins offers novel information on the organisation of expressed sequences distal to the coagulation factor VIII gene. These patients show an inversion that, in contrast to the common inversions responsible for 1/5 of all haemophilia A, affects the first rather than intron 22 of the gene. This displaces the most telomeric of the factor VIII exons (exon 1) by approximately 100 kb towards the telomere, and close to the region of the C6.1A gene. This novel inversion creates two hybrid transcription units: one formed by the promoter and first exon of the factor VIII gene followed by a widely expressed sequence; the other by the promoter and coding region of the C6.1A gene plus most of the factor VIII gene (part of intron 1 and exons 2-26). Investigation of this transcription unit reveals that the C6.1A gene has an unsuspected intron in the region coding for the previously described 3'-untranslated tail of the message. Furthermore, exons located beyond the known C6.1A sequence and present in normal transcripts precede exons 2-26 of the factor VIII gene in the hybrid mRNA of the haemophilic twins. The factor VIII sequences in this hybrid mRNA are not expected to be expressed because they lack the first exon, encoding the prepeptide, and follow a translation stop in the C6.1A gene. Leukaemia-related translocations in the C6.1A region suggest that this region may be somewhat unstable.

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.

Almost half of all cases of severe hemophilia A are caused by recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene. These inversions generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. They are routinely detected by a Bcl I Southern blot assay in which the sizes of two of the three normal hybridization bands are characteristically altered. However, atypical hybridization patterns have been observed, and this report describes the first detailed analysis of a hemophilia A patient with such a pattern. The abnormal result was found to be caused by a novel FVIII inversion involving an extra copy of int22h from a site only 70 to 200 kb telomeric of the FVIII gene. Polymerase chain reaction (PCR) allowed one of the inversion junctions to be analyzed, showing that the int22h sequence at this inversion junction was truncated. This patient and his novel inversion provide further evidence that int22h is associated with instability in Xq28.

The sixth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of

SummaryIn order to obtain complete success in the carrier and prenatal diagnoses required for genetic counselling in haemophilia B a new strategy is being implemented in the UK. This entails the construction of a national confidential database of mutations, pedigrees and haema- tological data. This will allow the inefficient indirect tests based on the analysis of DNA polymorphisms to be abandoned and direct detection of the gene defect to be used instead. After two and a half years of nationwide collaboration, 702 samples have been collected from 313 families, representing more than half of the UK haemophilia B families, and 217 mutations have been characterised. The 141 diagnostic tests so far performed have clearly indicated that the new strategy not only allows virtually 100% diagnostic success, but also rapid results. This work on haemophilia B may represent a model for other diseases with high mutational heterogeneity.

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.

The two X-linked hemorrhagic diseases hemophilia A and hemophilia B have made an important contribution to the understanding of human genetics. The first historical record of the inheritance of a disease and of “genetics”-motivated advice relates to these diseases, as do the first description of the X-linked pattern of inheritance, the appreciation of equilibria between selection and mutation, and the first attempt to estimate mutation rates. In modern times, the hemophilias have clearly illustrated the successes and problems of replacement therapy and have provided some insight into the causes of immunological complication associated with the therapeutic administration of the missing gene products. It is therefore not surprising that nowadays they attract the attention of scientists and clinicians involved in the development of gene therapy. The hemophilias, which affect 1/5000 males in the population, are members of a large group of diseases characterized by very high mutational heterogeneity. This group is of particular importance because, on the one hand, the wealth of natural mutants that each member possesses allows accumulation of detailed information on the structural features that are important to the functions of the gene and gene product involved in the disease; on the other, this creates the least favorable situation for the development of fully efficient strategies for the provision of the carrier and prenatal diagnoses that are necessary for genetic counseling. Surprisingly, mutation analysis in hemophilia A has revealed that homologous intranemic recombination between duplicated sequences can represent an important cause of recurring gene inactivation. Keywords: CpG and CpG island; Founder Mutation; Frameshift; Male-driven Evolution; Missense; Mutational Load; Nonsense; Uninemic; Zygote

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of

Inversions breaking the 1041 bp int1h-1 or the 9.5-kb int22h-1 sequence of the F8 gene cause hemophilia A in 1/30,000 males. These inversions are due to homologous recombination between the above sequences and their inverted copies on the same DNA molecule, respectively, int1h-2 and int22h-2 or int22h-3. We find that (1) int1h and int22h duplicated more than 25 million years ago; (2) the identity of the copies (>99%) of these sequences in humans and other primates is due to gene conversion; (3) gene conversion is most frequent in the internal regions of int22h; (4) breakpoints of int22h-related inversions also tend to involve the internal regions of int22h; (5) sequence variations in a sample of human X chromosomes defined eight haplotypes of int22h-1 and 27 of int22h-2 plus int22h-3; (6) the latter two sequences, which lie, respectively, 500 and 600 kb telomeric to int22h-1 are five-fold more identical when in cis than when in trans, thus suggesting that gene conversion may be predominantly intrachromosomal; (7) int1h, int22h, and flanking sequences evolved at a rate of about 0.1% substitutions per million years during the divergence between humans and other primates, except for int1h during the human-chimpanzee divergence, when its rate of evolution was significantly lower. This is reminiscent of the slower evolution of palindrome arms in the male specific regions of the Y chromosome and we propose, as an explanation, that intrachromosomal gene conversion and cosegregation of the duplicated regions favors retention of the ancestral sequence and thus reduces the evolution rate.

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Hemophilia A is an X-linked disorder that leads to a defect in blood coagulation. This is caused by mutations in the factor VIII gene, which results in its activity being reduced or abolished in the blood-clotting cascade. The factor VIII gene is 186 kb long with 26 exons, varying from 69 bp (exon 5) to 3106 bp (exon 14) (1). The factor VIII mRNA is 9028 bases in length with a 7053 nucleotides long coding region (2).

The messenger RNA (mRNA) from 5 of 69 patients with severe hemophilia A did not support amplification of complementary DNA containing the first few exons of the factor VIII (F8) gene but supported amplification of mRNA containing exon 1 of F8 plus exons of the VBP1 gene. This chimeric mRNA signals an inversion breaking intron 1 of the F8 gene. Using an inversion patient, one deleted for F8 exons 1 to 6, and cosmids mapped 70 to 100 kb telomeric of the F8 gene, this study shows that this break strictly affects a sequence (int1h-1) repeated (int1h-2) about 140 kb more telomerically, between the C6.1A and VBP1 genes. The 1041-base pair repeats differ at a single nucleotide (although int1h-2 also showed one polymorphism) and are in opposite orientation. The results demonstrate that they cause inversions by intrachromosome or intrachromatid homologous recombination. The genomic structure of the inversion region shows that transcription traverses intergenic spaces to produce the 2 chimeric mRNAs containing the F8 sequences and characteristic of the inversion. This observation prompts the suggestion that nature may use such extended transcription to test whether the addition of novel domains from neighboring genes creates desirable new genes. A rapid polymerase chain reaction test was developed for the inversion in both patients and carriers. This has identified 10 inversions, affecting F8 genes with 5 different haplotypes for the BclI, introns 13 and 22 VNTR polymorphism, among 209 unrelated families with severe hemophilia A. This indicates a prevalence of 4.8% and frequent recurrence of the inversion. This should result in absence of F8, and one inversion patient is known to have inhibitors. (Blood. 2002;99:168-174)

Previous work involving the characterizing of a factor IX promoter mutation (-26 G-->C) of a 21-year-old patient with severe haemophilia B suggested that an androgen response element (ARE) was present in the wild-type factor IX promoter but was disrupted in this patient. However, other theories not involving this ARE have been suggested for the mechanism of recovery in the more typical (Leyden) promoter patients, so that the ARE hypothesis requires further evidence if it is to be accepted. We now present a case history and functional data on another 48-year-old severe haemophilia B patient (UK232) with a different (G-->A) mutation at the same position, -26. This mutation impairs transactivation of the minimal factor IX promoter region (-220-->+43) by HNF4 in transient transfection experiments in HepG2 and HeLa cells. It disrupts binding of both androgen receptor (AR) and HNF4 to oligonucleotides spanning this region (-40-->-9) in competition gel mobility shift assays. It impairs AR/testosterone transactivation of these oligonucleotides (-40-->-9) when tetramerized upstream of a CAT reporter gene in cotransfection assays in HeLa cells. And, finally, no clinical recovery has occurred since puberty. These results strengthen the evidence for the importance of nucleotide -26, both for the normal transcription of the gene in response to HNF4 and for the proposed Leyden recovery mechanism in response to AR and testosterone acting directly through the factor IX ARE.

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

The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments (approximately 1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5' or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microtiter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5' labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost two-fold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day.

A region of intron 22 of the factor VIII gene, which contains factor VIII-associated gene A (F8A), is repeated twice more nearer the Xq telomere. It has been proposed that intrachromosomal homologous recombination occurs between the intron 22 repeat and either of the two extragenic copies, resulting in the recurrent inversions that cause almost half of all cases of severe haemophilia A. We have precisely defined the repeated region as 9.5 kb of DNA which we have termed int22h (intron 22 homologous region). The junctions of the inversions examined were shown to represent precise exchanges between the int22h repeats, thus providing conclusive evidence for homologous recombination. The three copies of int22h were compared along 8 kb of their length, using chemical mismatch analysis, and found to be 99.9% similar. The presence of such long, almost identical inverted repeats near the Xq telomere could account for the high frequency at which the inversions occur.

Publisher Summary An accurate account of hemophilia and the X-linked nature of the disease that affects males and is transmitted by females were published by Otto in 1803. This description, of course, precedes Mendel's discovery of the laws of inheritance by half a century and that of X-linkage by more than a century. In 1952, the discovery of complementation of the coagulation defect in mixtures of plasma from unrelated patients clearly indicated the existence of two different hemophilias. In the United Kingdom, the two hemophilias were named “classical hemophilia” and “Christmas disease,” while in the United States the terms “hemophilia A” and “hemophilia B” were used. The coagulation factors deficient in hemophilia A and B are now called factors VIII and IX, respectively. The latter is a serine protease, while the former is a cofactor of factor IX; both circulate in the blood in an inactive form. When activated, factors IX and VIII cooperate to cleave and activate factor X, thus together contributing to the proteolytic cascade that controls the conversion of fibrinogen to fibrin. Because hemophilia A and B affect the same blood coagulation step, patients with either type of hemophilia have essentially the same features. These include spontaneous bleeding, especially into joints and muscles, prolonged bleeding from minor cuts, and severe bleeding from lacerations. The expression of cloned genes has led to the introduction of recombinant factor VIII in the treatment of hemophilia A, and the genes for both factors VIII and IX are being used to develop successful approach toward gene therapy.

Chemical mismatch detection when combined with PCR or RT-PCR amplification represents one of the most efficient mutation screening methods. This procedure has been used for the analysis of both large populations of mutants and large and complex genes because it detects and locates virtually all sequence variations in segments of DNA of up to 1.7 kb cleaving at, and/or adjacent to, mismatches in heteroduplexes formed by target and probe DNA. By using fluorescent tags and an automatic gel scanning system, the efficiency of the method has been increased fourfold with gains also in the safety and quality control of probes and in the flexibility of the procedure. Using 12 hemophilia B patients, 8 with known mutations and 4 chosen at random, we show how the chemical mismatch cleavage of up to four DNA segments can be multiplexed and examined in a single gel lane and how the increase in efficiency thus obtained can be used either totally to maximize the DNA screened per lane or partly to locate the mutation unequivocally so that only one sequencing reaction is needed to characterize it fully.

This article describes the direct sequencing of PCR-amplified DNA, a technique that bypasses the problem of replication errors sometimes associated with other PCR procedures. The direct sequencing procedure produces an “average sequence” of all the copies of the target. Any miscopied molecule usually represents only a small proportion of the total. The technique described here is based on the “traditional” ddNTP sequencing method of Sanger et al.

Surprisingly half of all severe haemophilia A patients have no mutation in the promoter, coding sequences and normal RNA processing signals of the factor VIII gene. Instead they manifest a unique mRNA defect that prevents the amplification of the message across the boundary between exon 22 and 23. This locates the defect to internal regions of intron 22. Novel sequences 3' to exon 22 were isolated from the 9 available patients with the above abnormality by combining RACE and vectorette amplifications on trace amounts of mRNA. This showed that exons 1-22 of the factor VIII mRNA had become part of a hybrid message containing new multi exonic sequences expressed in normal cells. The novel sequences were not located in a YAC covering the whole factor VIII gene. Southern blots from patients probed by novel sequences and clones covering intron 22 showed no obvious abnormalities. This suggested inversions involving intron 22 repeated sequences. Screening of 3 YAC libraries with the novel sequences located them at least 200 kb telomeric (5') to factor VIII and pulsed field gel analysis detected abnormal bands in patients. This demonstrates that the mutations in the patients are inversions of long DNA regions possibly involving the repeated sequences and occurring at the surprising rate of approximately 4 x 10(-6) per gene per gamete per generation.

The vertical integration of cosmid and yeast artificial chromosome (YAC) resources is of particular importance in the development of high-resolution maps of selected regions of the human genome. A resource of approximately 95,000 cosmids constructed using DNA from primary fibroblasts of karyotype 49,XXXXX was validated by detailed characterization of a 200-kb cosmid contig spanning exons 8-20 of the dystrophin gene. This resource was used to construct contigs in 0.65 Mb of Xq26 by hybridization of gel-purified YAC DNA to high-density gridded arrays of the cosmid library; positive cosmids were overlapped by finger-printing. Contigs were oriented and ordered relative to existing YACs in the region using cross-hybridization. The overlaps between a representative set of cosmids define 54 intervals of 5-20 kb and were used to construct a high-resolution cosmid interval map of the region, locating markers, dinucleotide repeats, and candidate CpG islands. This approach can be applied rapidly to large regions of the genome and without recourse to subcloning of individual YACs.

Haemophilia A is a mutationally heterogeneous disease caused by defects in the large and complex factor VIII gene. Recent studies examining the putative promoter, all exons and most intron/exon boundaries have failed to detect mutations in half the patients with severe disease leading to hypotheses such as mutations in remote controlling regions or even in genes other than factor VIII. We have amplified the factor VIII gene (putative promotor, coding region and polyadenylation/cleavage signal region) in 8 fragments from reverse transcribed mRNA and genomic DNA. Any mutation is then located by chemical mismatch detection and characterised by direct sequencing. This rapid and efficient method has been fully successful and has revealed an unusual cluster of mutations causing severe disease. Of the 28 patients we have reported, 5 had mild or moderate disease and all had a missense mutation. Twenty-three patients were severely affected and 13 of these had different detrimental mutations that were fully characterised at the genomic DNA level. The remaining 10 patients all had mRNA with exon 22 not contiguous to exon 23. Since all exons were normal and so were the splice sites of intron 22, the mutation in these patients should be in the regions of intron 22 that were not screened. These results prove that all haemophilia A cases are due to mutations of the factor VIII gene where, unexpectedly, intron 22 seems to be the target of approximately 40% of the mutations causing severe haemophilia A.

The structure of the gene coding for iduronate sulphate sulphatase (IDS) has been determined. We have used exon to exon and vectorette PCR to identify 9 exons within the IDS gene and to characterise the surrounding intron sequences. The results of this study will be useful for the complete analysis of many mutations giving rise to Hunter syndrome. IDS is the first member of the group of lysosomal nonarylsulphatase genes for which the gene structure has been determined. It bears no relationship to the exon organisation of steroid sulphatase, despite the homology between these two proteins. This suggests that the division of the sulphatases into the two subgroups on the basis of substrate specificity is also reflected at the level of gene structure.