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OBJECTIVE: To investigate seven congenital myopathy patients from six families: one French Gypsy, one Spanish Gypsy, four British Pakistanis, and one British Indian. Three patients required mechanical ventilation from birth, five died before 22 months, one is ventilator-dependent, but one, at 30 months, is sitting with minimal support. All parents were unaffected. METHODS: The alpha-skeletal muscle actin gene (ACTA1) was sequenced. Available muscle biopsies were investigated by standard histological and electron microscopic techniques. The expression of various proteins was determined by immunohistochemistry, western blotting, or both. RESULTS: Three homozygous ACTA1 null mutations were identified: p.Arg41X in the French patient, p.Tyr364fsX in the Spanish patient, and p.Asp181fsX10 in all five British patients. An absence of alpha-skeletal muscle actin protein but presence of alpha-cardiac actin was shown in all muscle biopsies examined, with more alpha-cardiac actin in the biopsy from the child with the greatest muscle function. Muscle biopsies from all patients exhibited nemaline bodies whereas three also contained zebra bodies. INTERPRETATION: The seven patients have recessive nemaline myopathy caused by absence of alpha-skeletal muscle actin. The level of retention of alpha-cardiac actin, the skeletal muscle fetal actin isoform, may determine alpha-skeletal muscle actin disease severity. This has implications for possible future therapy. Ann Neurol 2006. [ABSTRACT FROM AUTHOR]

The location of the HYP gene, which determines X-linked hypophosphataemic rickets, has been refined considerably by linkage analysis, and three new microsatellite primers isolated, Cap32 (DXS7473), Cap29 (DXS7474) and 7v2 (DXS7475). The locations of four other markers have also been determined (DXS1226, AFMa176zb1, AFMa152wc5, and AFM346azc1). Markers Cap29 and Cap32 are the closest distal markers to the gene with zetamax=11.93, thetamax= 0.018 and zetamax=12.03, thetamax = 0.015 respectively. Both Cap29 and Cap32 are proximal to DXS365 and AFMa176zb1, as deduced by screening non-chimaeric yeast artificial chromosomes (YACs) from a contig spanning the HYP gene. A single crossover places AFMa176zbl distal to the disease gene. There are no recombinations between 7v2 and HYP (zetamax=12.9, thetamax=0.0), or between 7v2 and adjacent markers Cap32, Cap29, AFMa176zb1, DXS1683 and DXS365. However screening of YAC clones encompassing the HYP gene and also P1 clones localises 7v2 distal to Cap29 and Cap32, and proximal to DXS443. Marker DXS1226 is placed outside the region containing the gene, and is located proximal to DXS274 as confirmed by a crossover for this marker and DXS41 against HYP and its presence on YAC 83B05. Genetic mapping of CEPH pedigrees, and screening of YACs places AFMa152wc5 and AFMa346zcl between DXS1683 and DXS1052. The following gene marker map presents the best order for the HYP region: Xptel-DXS43-DXS999-DXS443-(DXS365/DXS74 75/AFMa176zb1)-(DXS7474/DXS7473)-HYP- DXS1683-(AFMa152wc5/AFMa346zc1)-DXS1052-DXS 274 -(DXS41/DXS1226)-Xcen. The distance between the cluster of distal flanking markers Cap29 (DXS7474), Cap32 (DXS7473), and DXS1683 is approximately 300 kb, as deduced from physical map data from a YAC contig spanning the gene. Thus the gene for HYP is contained within a single YAC (900AO472). Of further interest, is the location of a putative vitamin D response element (VDRE) on this YAC.

A Duchenne muscular dystrophy patient who displayed near-normal dystrophin staining at the sarcolemma with N-terminal, but not with C-terminal, anti-dystrophin monoclonal antibodies was found to have a frameshift deletion of exons 42 and 43. This deletion introduces an early termination codon, and a 225-kD protein was detected by western blotting with N-terminal antibodies only. The results suggest that an N-terminal truncated dystrophin fragment encoded by exon 1-41 is able to associate with the muscle cell membrane. The current idea that the C-terminal domains of dystrophin are important or essential for its integration with the sarcolemma may have to be reexamined in the light of these observations.

The Duchenne muscular dystrophy locus is remarkable in that it shows a high mutation rate and the majority of mutations found are deletions. These deletions are generated as meiotic as well as mitotic events and occur preferentially in the central region of the gene. Nothing is known so far about the mechanisms involved. This paper reports the first sequencing of deletion junctions in the dystrophin gene. The data from a study of two patients with deletions in the central region of dystrophin show the breakpoints to lie in regions of introns in which stretches of dA-dT are seen. The relationship between these observations and possible mechanisms for the mutations is discussed.

As part of a study into the molecular basis of attenuation and reversion to neurovirulence in the Sabin poliovirus vaccines, we have determined the complete nucleotide sequence of a cloned DNA copy of the genome of P3/Leon/37, the neurovirulent progenitor of the type 3 Sabin vaccine strain, P3/Leon 12a1b. Comparison of the sequence with that which we previously obtained for the vaccine strain [Stanway, G., Cann, A. J., Hauptmann, R., Hughes, P., Clarke, L. D., Mountford, R. C., Minor, P. D., Schild, G. C. & Almond, J. W. (1983) Nucleic Acids Res. 11, 5629-5643] indicates that attenuation has been brought about by a maximum of 10 point mutations, at least 5 of which are likely to be of minor significance. Predicted amino acid sequences of all the known virus-encoded proteins show that amino acid substitutions have occurred at only three positions. Two of these are in structural proteins (i.e., Ser----Phe in VP3 and Lys----Arg in VP1), and the third, Thr----Ala, is in the nonstructural protein P2-3b. The distribution and nature of nucleotide and amino acid sequence differences suggest that a single base substitution may be responsible for the attenuated phenotype of the vaccine strain.

Three closely related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque-purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA . cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.

The genomes of two neurovirulent strains of poliovirus type 3, wild type P3/Leon/37 and a vaccine revertant P3/119/70, have been cloned in E. coli. The cDNA: RNA hybrid method used was efficient and may have wide applicability for cDNA cloning. Overlapping clones spanning the entire genome were obtained for each strain. These have been used to produce full-length DNA copies of the two genomes each within a single plasmid.

The complete nucleotide sequence of the genome of the Sabin vaccine strain of poliovirus type 3 (P3/Leon 12 a1 b) has been determined from cDNA cloned in E. coli. The genome comprises a 5' non-coding region of 742 nucleotides, a large open reading frame of 6618 nucleotides (89% of the sequence) and a 3' non-coding region of 72 nucleotides. There is 77.4% base-sequence homology and 89.6% predicted amino-acid homology between types 1 and 3. Conservation of all glutamine-glycine and tyrosine-glycine cleavage sites suggests a mechanism of polyprotein processing similar to that established for poliovirus type 1.

Overlapping cDNA clones representing the entire genome of poliovirus type 3 have been prepared in E. coli by two separate methods. Cloning of RNA . cDNA hybrids produced a more comprehensive set of clones with generally larger cDNA inserts than cloning of double - stranded cDNA. A restriction map of the entire genome and the nucleotide sequence of 2003 bases from the 3' terminus, comprising the region encoding the protease and polymerase proteins, are presented.

Hypophosphataemic rickets is commonly an X linked dominant hereditary disorder associated with a renal tubular defect in phosphate transport and bone deformities. The gene causing this disorder has been mapped to Xp22.31----p21.3 by using cloned human X chromosome sequences identifying restriction fragment length polymorphisms (RFLPs) in linkage studies of affected families. The hypophosphataemic rickets gene locus (HPDR) was previously mapped distal to the X linked polymorphic locus DXS41 (99.6) but its position in relation to the distal loci DXS43 (D2) and DXS85 (782) was not established. In order to obtain a precise mapping of the disease locus in relation to these genetic loci, additional affected families informative for these X linked markers have been investigated. The combined results from the two studies have established linkage with the loci DXS41 (99.6) and DXS43 (D2); peak lod score for DXS41 (99.6) = 7.35, theta = 0.09, and peak lod score for DXS43 (D2) = 4.77, theta = 0.16. Multilocus linkage analysis mapped the hypophosphataemic rickets gene distal to the DXS41 (99.6) locus and proximal to the DXS43 (D2) locus, thereby revealing two bridging genetic markers for the disease.

We have determined a major antigenic site for virus neutralization on the capsid protein VP1 of poliovirus type 3. Antigenic mutant viruses selected for resistance to individual monoclonal antibodies had point mutations concentrated in a region 277-294 bases downstream from the start of the region of viral RNA coding for VP1. These findings provide the basis for an improved understanding of the molecular basis of virus neutralization.

We describe a family in which two boys with Norrie disease have a deletion of the DXS7 locus. The deletion does not extend as far distally as the OTC or DXS84 loci. A full clinical description of the patients is given to help establish the range of manifestations of Norrie disease. There is no evidence of any other X linked disease in our patients.

Eleven families with X-linked dominant hypophosphataemic rickets (HPDR) have been typed for a series of X chromosome markers. Linkage with probe 99.6 (DXS41) was demonstrated with a peak lod score of 4.82 at 10% recombination. Multilocus linkage analysis showed that HPDR maps distal to 99.6; this probe has previously been located at Xp22.31-p21.3 by in situ hybridisation. In the mouse hypophosphataemia (Hyp) maps to the distal part of the X chromosome; our location in man is consistent with a scheme which relates the mouse and human X chromosomes by two rearrangements. No marker has yet been found which shows no recombination with HPDR.

The complete nucleotide sequence of the single-stranded RNA genome of human rhinovirus 14, one of the causative agents of the common cold, has been determined from cDNA cloned in E. coli. The genome is typical of the picornaviridae family, comprising a 5' non-coding region of 624 nucleotides, a long open reading frame of 6537 nucleotides (90.8% of the genome) and a 3' non-coding region of 47 nucleotides. Comparison of the nucleotide sequence and the predicted amino acid sequence with those of the polioviruses reveals a surprising degree of homology which may allow recognition of regions of antigenic importance and prediction of the virus polyprotein cleavage sites. The results presented here imply a closer genetic relationship between the rhinovirus and enterovirus genera than previously suspected.

Deletions in the gene sequence for Duchenne (DMD) and Becker (BMD) muscular dystrophy were detected in affected males with four cDNA probes, Cf56a, Cf23a, Ca1A, and Cf27. Most of the deletions were seen with only one of the probes. Cf23a detected all BMD deletions seen with Cf56a and some that were not. The same markers also detected restriction fragment length polymorphisms for those cases where deletions were not evident. The probes were also used successfully for prenatal diagnosis in two families each with two DMD affected males. In DMD families successive application of probes Cf56a, Ca1A, and Cf27 will give a 70% chance of detecting the mutation. BMD families should first be screened with the Cf23a probe.

A panel of patients with Duchenne and Becker muscular dystrophy (DMD and BMD) has been screened with the cDNA probes Cf56a and Cf23a, which detect exons in the central part of the DMD gene. One or more exons were deleted in 60% of patients. The deletions were mapped and prove to be heterogeneous in size and extent, particularly in DMD. Deletions specific to DMD and to BMD are described. Half of all BMD patients have a deletion of one particular small group of exons; smaller deletions within this same group produce the more severe DMD.

A total of 102 families with Duchenne muscular dystrophy has been studied with linked DNA polymorphisms as an aid to estimating carrier risks for female relatives. Early work using probes RC8, L1.28, and pXUT23 gave very little clinically useful information because of the high recombination rates between these probes and Duchenne muscular dystrophy and the low proportion of women who were heterozygous. Clinically useful results were obtained using probes 99-6, 754, and particularly pERT87. Examples are given of deductions which can be made using these probes. The importance of a genetic register is stressed as a tool for long term contact with the families and other professionals.