Showing papers in "Human Molecular Genetics in 1993"
TL;DR: A total of 20,000 parent-offspring transfers of alleles were examined through the genotyping within 40 CEPH reference families of 28 short tandem repeat polymorphisms (STRPs) located on chromosome 19, and the average mutation rate was nearly four times higher than the average rate for dinucleotide STRPs.
Abstract: A total of 20,000 parent-offspring transfers of alleles were examined through the genotyping within 40 CEPH reference families of 28 short tandem repeat polymorphisms (STRPs) located on chromosome 19. Forty-seven initial mutation events were detected in the STRPs using DNA from transformed lymphoblastoid cell lines, but less than half (39%) could be verified using DNA from untransformed cells. None of the cases where three alleles were observed in a single individual could be verified using DNA from untransformed cells. The average mutation rate for the chromosome 19 STRPs after correction for events which would not be detectable as Mendelian errors was 1.2 x 10(-3) per locus per gamete per generation. This rate may have been inflated by somatic as opposed to germline events. Observed mutation rates for individual STRPs ranged from 0 to 8 x 10(-3). The average mutation rate for tetranucleotide STRPs was nearly four times higher than the average rate for dinucleotide STRPs. For determination of the mode of mutation, events involving STRPs on other chromosomes were also examined. Of the events which were verified using DNA from untransformed lymphocytes or which were likely among those for which DNA from untransformed cells was not available: none were located at the sites of meiotic recombination, 91% involved the gain or loss of a single repeat unit, and 15 occurred in the male germline compared to 4 in the female germline (p = 0.01).
1,580 citations
TL;DR: Evidence is presented that sequence changes within the coding region of the RET proto-oncogene, a putative transmembrane tyrosine kinase, may be responsible for the development of neoplasia in these inherited disorders.
Abstract: Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are dominantly inherited conditions which predispose to the development of endocrine neoplasia. Evidence is presented that sequence changes within the coding region of the RET proto-oncogene, a putative transmembrane tyrosine kinase, may be responsible for the development of neoplasia in these inherited disorders. Single strand conformational variants (SSCVs) in exons 7 and 8 of the RET proto-oncogene were identified in eight MEN 2A and four FMTC families. The variants were observed only in the DNA of individuals who were either affected or who had inherited the MEN2A or FMTC allele as determined by haplotyping experiments. The seven variants identified were sequenced directly. All involved point mutations within codons specifying cysteine residues, resulting in nonconservative amino acid changes. Six of the seven mutations are located in exon 7. A single mutation was found in exon 8. Variants were not detected in four MEN 2B families studied for all exon assays available, nor were they detectable in 16 cases of well documented sporadic medullary thyroid carcinoma or pheochromocytoma that were tested for exon 7 variants. Coinheritance of the mutations with disease and the physical and genetic proximity of the RET proto-oncogene provide evidence that RET is responsible for at least two of the three inherited forms of MEN 2. Neither the normal function, nor the ligand of RET are yet known. However, its apparent involvement in the development of these inherited forms of neoplasia as well as in papillary thyroid carcinoma suggest an important developmental or cell regulatory role for the protein.
1,213 citations
TL;DR: The human dopamine D4 receptor contains a novel polymorphism within the putative third cytoplasmic loop of the protein, characterized by a varying number of direct imperfect 48-bp repeats in the gene.
Abstract: The human dopamine D4 receptor contains a novel polymorphism within the putative third cytoplasmic loop of the protein. The polymorphism is characterized by a varying number of direct imperfect 48-bp repeats in the gene. Pharmacological characterization has suggested that this receptor is the site through which the atypical neuroleptic clozapine exerts its antipsychotic action and that some polymorphic variants display different pharmacological properties. Further analysis of the repeat region using innovative technologies indicates that the alleles vary not only in the number of repeats (2-8 or 10 repeat units) but also in the sequence of the repeats and the order in which they appear. In 178 unrelated chromosomes we have identified 19 different repeats in 25 different haplotypes coding for 18 different predicted amino acid sequences, making this one of the most variable functional proteins currently described.
562 citations
TL;DR: In this paper, the authors have cloned and characterized the rearranged EcoRI fragment of four unrelated Facioscapulohumeral Muscular Dystrophy (FSHD) patients, which strongly support the hypothesis that the FSHD associated rearrangements are due to deletions of integral copies of the 3.2 kb repeated unit.
Abstract: Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by progressive weakness of the facial, shoulder and upper arm muscles. The disease is associated with DNA rearrangements which are detectable using probe p13E-11 (D4F104S1) in DNA digested with EcoRI or other restriction enzymes. We have cloned and characterized the rearranged EcoRI fragment of four unrelated FSHD patients. Restriction fragment mapping and DNA sequence analysis showed that the proximal and distal parts of the EcoRI fragment, which flank a region of tandemly repeated 3.2 kb units, are identical in normal and rearranged EcoRI fragments. These results strongly support the hypothesis that the FSHD associated rearrangements are due to deletions of integral copies of the 3.2 kb repeated unit. Since these repeated units are likely to form part of the FSHD transcription unit, the variation in repeat unit number might affect the function of the gene product. Hence, our data confine the FSHD gene region and thus provide a starting point for cloning the FSHD gene.
501 citations
TL;DR: It is demonstrated that direct intramuscular injection of a plasmid vector encoding the Hepatitis B surface antigen will give rise to secretion of the viral surface protein into the circulation which leads to an appropriate antibody response.
Abstract: The possibility of inducing an immune response to a protein expressed directly from an introduced gene represents an alternative to classic vaccination. We evaluated the ability of plasmid-based eukaryotic expression vectors to produce the Hepatitis B surface antigen (HBsAg) after injection of pure DNA into mouse tibialis anterior muscles. DNA was injected into either normal mature muscle, or regenerating muscle following cardiotoxin-induced degeneration. The sera obtained from these animals contained significant levels of HBsAg as early as 10 days after gene transfer, at which time low levels of antibodies to HBsAg (anti-HBsAg) were already present. Between 15-60 d after DNA transfer, serum levels of anti-HBsAg steadily increased whereas those for HbsAg fell, most likely due to the neutralizing effect of the antibodies. Analysis of proportions of HBs-seropositive mice showed that within 2 wk of injection of 100 micrograms pCMV-HBs in regenerating muscle, 91% of the mice were seropositive [defined as having more than 1 milli-International Unit/ml (mIU/ml) of anti-HBsAg]. Even at that early time, 68% had titers of anti-HBsAg greater than 10 mlU/ml, a level that is recognized as being sufficient in humans to confer protection against natural Hepatitis B virus infection. The proportion of seropositive animals rose to 95% by 4 wk, and 100% by 8 wk, at which time all mice had greater than 100 mIU anti-HBsAg in their sera. We have thus demonstrated that direct intramuscular injection of a plasmid vector encoding the HBsAg will give rise to secretion of the viral surface protein into the circulation which leads to an appropriate antibody response.(ABSTRACT TRUNCATED AT 250 WORDS)
451 citations
TL;DR: The quality of DNA isolated from buccal cells collected in this manner has been sufficient to reproducibly support multiplex amplification of 5 exons within the CFTR gene, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.
Abstract: Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.
399 citations
TL;DR: This study provides the first comprehensive analysis of the fibrillin gene and relevant information for the full characterization of Marfan syndrome mutations and derived a physical map of the genetic locus.
Abstract: Marfan syndrome results from mutations in an extracellular matrix glycoprotein, fibrillin. Previous studies have characterized approximately 6.9-kb of the estimated 10-kb fibrillin transcript. We have now completed the primary structure of fibrillin, elucidated the exon/intron organization of the gene and derived a physical map of the genetic locus. Pre-fibrillin consists of 2,871 amino acids which, excluding the signal peptide, are arranged into five structurally distinct regions. The largest of these regions comprises about 75% of the entire protein and consists of numerous repeated cysteine-rich sequences homologous to the peptide motifs of the epidermal growth factor (EGF) and transforming growth factor-beta binding protein (TGF-bp). Forty-three of the forty-six EGF-like repeats contain a calcium binding consensus sequence (EGF-CB) conceivably mediating protein-protein interactions. Fibrillin exhibits a few additional cysteine-rich modules that are apparently unique to this macromolecule and may represent evolutionary variants of the EGF-CB and TGF-bp motifs. Almost all of the cysteine-rich repeats are encoded by single exons; consequently, the fibrillin gene is relatively large (approximately 110-kb) and highly fragmented (65 exons). This study provides the first comprehensive analysis of the fibrillin gene and relevant information for the full characterization of Marfan syndrome mutations.
367 citations
TL;DR: Molecular analysis of a newly-generated apo(a) allele of different length suggests that the high degree of length polymorphism is in part due to recombination between sister chromatids.
Abstract: Lipoprotein(a) [Lp(a)], an atherogenic lipoprotein, consists of an LDL particle complexed with a large glycoprotein, apolipoprotein(a) [apo(a)]. Apo(a) is one of the most polymorphic expressed sequences in the human genome. The glycoprotein varies in size over a approximately 500 kDa range due to length polymorphisms in the apo(a) gene. The region of the apo(a) gene responsible for this size polymorphism was cloned and characterized. Apo(a) alleles of different length were found to contain varying numbers of a subset of a tandemly-repeated, 5.5 kilobase, kringle IV-encoding sequences. A total of 34 apo(a) alleles and glycoproteins could be distinguished using pulsed-field gel electrophoresis and genomic blotting and immunoblotting. Molecular analysis of a newly-generated apo(a) allele of different length suggests that the high degree of length polymorphism is in part due to recombination between sister chromatids.
315 citations
TL;DR: PTT is a rapid and sensitive method based on a combination of RT-PCR, transcription and translation and selectively detects translation-terminating mutations that should be widely applicable diagnostically in any disease where early terminations contribute substantially to the disease cause.
Abstract: Most commonly used techniques to detect (point) mutations, e.g., SSCP, DGGE, chemical mismatch cleavage and heteroduplex analysis, are versatile techniques which have proven their usefulness (for references see other chapters in this book). Still, they all have their limitations. They use genomic DNA as starting material and reveal all sequence changes, including silent mutations and sequence variations in coamplified noncoding sequences. They can only be used to analyze small regions, 100–600 bp depending on the technique used. They do not pinpoint the site (except chemical mismatch cleavage) or the type of the mutation. Sequence changes identified in or close to, for example, splice sites or a promoter, require further analysis to verify their effect at a cellular level. Finally, several frequently mutated genes have been identified whose structure precludes an efficient analysis with the techniques mentioned; they are very large and split into many exonic fragments (sometimes over 70).
293 citations
TL;DR: The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCID X1 gene is IL2 RG, a candidate for the X- linked form of severe combined immunodeficiency at the SC IDX1 locus.
Abstract: The gene encoding the gamma chain of the lymphocyte interleukin-2 receptor has been cloned and shown to be required to associate with the beta chain in order for IL-2 internalization and cell activation to occur (1). We considered this gene, IL2RG, a candidate for the X-linked form of severe combined immunodeficiency at the SCIDX1 locus, in which affected males have impaired lymphocyte development. Using fluorescence in situ hybridization and PCR amplification of somatic cell hybrid DNAs, we mapped IL2RG to human Xq13.1, a location within the SCIDX1 critical region established by linkage analysis. The 4.2 kb IL2RG gene was sequenced, and its genomic organization was elucidated. Seven of 19 transformed B-lymphocyte cell lines with independent SCIDX1 mutations had absent or minimal IL2RG mRNA. Unique point mutations were documented to be specifically associated with the disease and the carrier state in four unrelated affected males and their family members: one in a boy with no detectable IL2RG mRNA, in which the mutation ablated a splice donor site; one causing premature chain termination; and two causing distinct amino acid changes. The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCIDX1 gene is IL2RG. Noguchi et al. (2) have independently published IL2RG mapping to Xq13 and discovery of mutations in three affected males. The specific pathogenesis of IL2RG mutations and approaches to gene therapy can now be addressed in the X-linked form of SCID.
274 citations
TL;DR: Five couples at risk of producing offspring with X-linked recessive disease underwent in vitro fertilisation with a view to preimplantation determination of embryo sex and selective transfer of females, finding evidence of mitotic non-disjunction in one and of complete X monosomy in a second.
Abstract: Five couples at risk of producing offspring with X-linked recessive disease underwent in vitro fertilisation with a view to preimplantation determination of embryo sex and selective transfer of females. On day three postinsemination, one or two blastomeres were removed by embryo biopsy, and used for dual fluorescent in situ hybridisation with X and Y chromosome-specific DNA probes. In two cases, two female embryos were transferred and one pregnancy, (sex confirmed), is ongoing at 19 weeks. All eight embryos from one couple were of such poor quality that diagnosis was possible in one only. In the remaining two cases no embryos were transferred due to the detection of an abnormal number of X chromosome signals. Investigation of the biopsied embryos that were not transferred revealed evidence of mitotic non-disjunction in one and of complete X monosomy in a second. A surviving fetus with this latter constitution would have developed Turner syndrome and would also have been at high risk of X-linked disease. The use of fluorescent in situ hybridisation rather than the polymerase chain reaction allowed the detection of abnormal copy numbers of X chromosomes thus preventing the transfer of potentially abnormal zygotes.
TL;DR: The methylation deficiency affects classical satellite families built from distinct unit sequences but located in the same chromosomal region, which may have important implications for the mechanism of chromosomal rearrangements.
Abstract: ICF syndrome has been described as the association of variable immunodeficiency, facial anomalies and centromeric heterochromatin instability. Since the chromosome rearrangements seen in cells of ICF patients are reminiscent of the chromosomal changes induced by the undermethylating agent 5-azacytidine in the late S-phase, we have analyzed the methylation pattern of satellite sequences in four patients. These sequences are almost completely methylated in normal leukocyte DNA. When ICF DNA was tested with methyl-sensitive enzymes, several classical satellite families, but not alphoid sequences, showed a very low level of methylcytosine in leukocyte DNA, with an abnormal pattern compared to the normal germinal and extraembryonic methylation profile. The methylation deficiency affects classical satellite families built from distinct unit sequences but located in the same chromosomal region. This observation may have important implications for the mechanism of chromosomal rearrangements.
TL;DR: A rapid, inexpensive method to detect trisomy using polymorphic small tandem repeat (STR) markers and the polymerase chain reaction (PCR) to amplify amniocyte DNA and expects as few as three STR markers per chromosome should be over 99% informative.
Abstract: Pregnant women over 35 years of age are routinely offered screening tests and karyotyping to detect Down syndrome and certain other aneuploidies because the risk of these disorders increases exponentially with maternal age It is, however, only cost-effective to karyotype high-risk pregnancies and a substantial number of remaining aneuploidy cases go undetected We describe a rapid, inexpensive method to detect trisomy using polymorphic small tandem repeat (STR) markers and the polymerase chain reaction (PCR) to amplify amniocyte DNA STR patterns obtained on patients with trisomy 21, trisomy 18 and triplo-X syndromes are distinct from controls Polymorphic trisomy genotypes either show three fragment peaks of equal intensity or two fragments at a 2:1 dosage ratio In addition, Turner syndrome (45, X0) DNA can be distinguished from normal male DNA because it fails to amplify a Y-chromosome specific PCR marker yet contains only a single dose of X-specific STR markers Quantitative analysis of peak heights and areas from STR markers show that the two peak patterns separate into completely non-overlapping groups The high level of heterozygosity of most STR markers result in a predominance of heterozygous controls and trisomy patterns with multiple alleles, the easiest patterns to differentiate Homozygosity, and hence an uninformative STR pattern, is more common in controls than in trisomy samples We anticipate as few as three STR markers per chromosome should be over 99% informative
TL;DR: It is demonstrated 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.
Abstract: 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.
TL;DR: Direct sequencing of PCR products derived from individuals homozygous at D11S527 shows that these products vary in length due solely to variations in the length of the dinucleotide repeat tract, ruling out PCR recombination and support slipped strand mispairing as the major mechanism for generation of shadow bands.
Abstract: Dinucleotide repeat polymorphisms ('microsatellites') are usually typed by resolving the products of PCR amplification on denaturing acrylamide gels. With this methodology, an allele consists not of a single fragment, but rather of a ladder of fragments, typically separated by intervals of 2nt. Mechanisms that have been invoked to explain the generation of these 'shadow bands' include slipped strand mispairing occurring during the PCR and artefactual 'recombination' caused by out-of-register annealing of truncated PCR products. The D11S527 locus contains the microsatellite sequence (GT)n(CTGT)m. By performing direct sequencing of PCR products derived from individuals homozygous at D11S527, we show that these products vary in length due solely to variations in the length of the dinucleotide repeat tract. These results rule out PCR recombination and support slipped strand mispairing as the major mechanism for generation of shadow bands.
TL;DR: The phenotype of the patient and the expression of SOX3 gene in neuronal tissues raises the possibility that this gene is a candidate gene for Borjeson-Forssman-Lehmann, an X-linked mental retardation syndrome.
Abstract: The mammalian genome contains a family of genes that are related to SRY, the mammalian sex determining gene. The homology is restricted to the region of SRY that encodes a DNA binding motif of the HMG-box class. These genes have been named SOX genes (SRY-related HMG-box genes). We have cloned and characterised SOX3, a member of the human SOX gene family. SOX3 maps to the X chromosome in the region Xq26-27. A mentally retarded male patient with haemophilia B is deleted for both the Factor IX gene and SOX3. This suggests that SOX3 is not essential for testis formation. The phenotype of the patient and the expression of SOX3 gene in neuronal tissues raises the possibility that this gene is a candidate gene for Borjeson-Forssman-Lehmann, an X-linked mental retardation syndrome.
TL;DR: The sex of the affected parent had no effect in the authors' sample beyond the effect of the length of the repeat, and the age of onset cannot be predicted from the size of the triplet repeat, particularly if the number of repeats is in the smaller end of the expanded range.
Abstract: Huntington's disease (HD) is an autosomal dominant disorder with a variable age of onset that is influenced by the sex of the affected parent. The recent recognition that HD is caused by an expanded triplet repeat suggests the possibility that the onset age may be predicted by the length of the repeat. This hypothesis was tested by assaying the length of the repeat in 114 individuals who were clinically diagnosed with HD and had a known onset age. Every individual had an expanded allele. The range was from 36 to 82 repeats (mean = 48.4 +/- 9.51) and larger than the normal range (6 to 31). The size of the expanded allele was correlated with the age of onset (r = -0.65 p < .0001). Despite the highly significant correlation, the repeat size explains less than half of the variance in onset age. Furthermore, the age of onset cannot be predicted from the size of the triplet repeat, particularly if the number of repeats is in the smaller end of the expanded range. If the repeat is < or = 50 triplets, the amount of variation in the age of onset explained by the length of the triplet repeat is less than 10%. As an illustration, the onset age of individuals with 39 repeats ranges from 30 to 65 years old. The sex of the affected parent had no effect in our sample beyond the effect of the length of the repeat. Affected offspring of affected fathers had longer repeats and a larger variance in allele size than offspring of affected mothers, perhaps reflecting greater instability in paternal transmission.
TL;DR: Cloned and sequenced the human cDNA encoding microsomal triglyceride transfer protein, which exists as a complex with protein disulphide isomerase in the endoplasmic reticulum, and elucidate a key process in the packaging of apolipoprotein B with lipid.
Abstract: Abetalipoproteinemia is an inherited disorder of lipoprotein metabolism. Affected individuals produce virtually no circulating apolipoprotein B-containing lipoproteins (chylomicrons, very low density lipoprotein, low density lipoprotein and lipoprotein (a)). Malabsorption of the antioxidant vitamin E occurs, leading to spinocerebellar and retinal degeneration. Biochemical and genetic studies show that abetalipoproteinemia is not a defect of lipid biosynthesis or of the apolipoprotein B gene. Instead a microsomal triglyceride transfer protein, which exists as a complex with protein disulphide isomerase in the endoplasmic reticulum, has been implicated. We have cloned and sequenced the human cDNA encoding microsomal triglyceride transfer protein. The predicted amino acid sequence shows extensive homology to vitellogenin, the precursor of the lipovitellin complex, which has been shown by X-ray crystallography to contain a large lipid storage cavity. Microsomal triglyceride transfer protein is expressed in ovary, testis and kidney, in addition to liver and small intestine. A homozygous mutation that disrupts splicing has been identified in affected siblings with classical abetalipoproteinemia. These results elucidate a key process in the packaging of apolipoprotein B with lipid, and should increase our understanding of the processes regulating the production of atherogenic lipoproteins.
TL;DR: The data suggest that muscle and non-muscle isoforms of dystroglycan differ by carbohydrate moieties but not protein sequence, and hypothesize that variable glycosylation of the conserved protein core might modulate laminin binding.
Abstract: Dystroglycan is a novel laminin binding component of the dystrophin-glycoprotein complex which provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. Here we report the cDNA and genomic structure of human dystroglycan. The human dystroglycan is encoded by a single gene (DAG1) mapped to chromosome 3 band p21. The coding sequence is organized into two exons, separated by a large intron. The predicted amino acid sequence of human and rabbit dystroglycan are 93% identical with predicted glycosylation sites being conserved. Human dystroglycan is expressed in a variety of fetal and adult tissues. Our data suggest that muscle and non-muscle isoforms of dystroglycan differ by carbohydrate moieties but not protein sequence. Therefore, we hypothesize that variable glycosylation of the conserved protein core might modulate laminin binding. The relationship of dystroglycan to human diseases is discussed.
TL;DR: Immunocytochemical studies showed a cytoplasmic localization of huntingtin in various cell types including neurons in most of the neuronal cells, and no difference in molecular mass or intracellular localization was found between normal and mutant cells.
Abstract: The recent identification of the Huntington's disease (HD) gene, enabled us to synthesize oligopeptides corresponding with the carboxy-terminal end of the predicted HD-gene (IT15) product. Immunobiochemcial studies with polyclonal antibodies directed against this synthetic peptide (position 3114-3141) on lymphoblastoid cells from normal individuals and patients with Huntington disease, revealed the presence of a protein (huntingtin) with a molecular mass of approximately 330 kDa. Immunocytochemical studies showed a cytoplasmic localization of huntingtin in various cell types including neurons. In most of the neuronal cells the protein was also present in the nucleus. No difference in molecular mass or intracellular localization was found between normal and mutant cells.
TL;DR: It is demonstrated that the sex of transmitting parent is the major influence on trinucleotide expansion and clinical features in juvenile Huntington disease.
Abstract: Juvenile Huntington disease (HD), characterised by onset of symptoms before the age of 20 with rigidity and intellectual decline, is associated with a predominance of affected fathers. In order to investigate the molecular basis for the observed parental effect, we have analysed the CAG trinucleotide repeat within the HD gene in 42 juvenile onset cases from 34 families. A highly significant correlation was found between the repeat length and age of onset (r = -0.86, p < 10(-7) and it was determined that the sex of transmitting parent was the major influence on CAG expansion leading to earlier onset. Neither the size of the parental upper allele, the age of parent at conception of juvenile onset child, nor the grandparental sex conferred a significant effect upon expansion. Affected sib pair analysis of CAG repeat length, however, revealed a high correlation (r = 0.91, p < 10(-7). Furthermore, analysis of nuclear and extended families showed a familial predisposition to juvenile onset disease. This study demonstrates that the sex of transmitting parent is the major influence on trinucleotide expansion and clinical features in juvenile Huntington disease.
TL;DR: Nine point mutations were identified at separate locations in four exons and one splice site, providing strong evidence for heterogeneity in mutations responsible for the X-linked Kallmann syndrome.
Abstract: Kallmann syndrome represents the association of hypogonadotropic hypogonadism with anosmia. Three modes of transmission, X chromosome-linked, autosomal recessive and autosomal dominant, have been described. The KAL gene, responsible for the X-linked form of the disease, has been isolated and its intron-exon organization recently determined. We have searched for mutations of the KAL gene in 21 unrelated males affected by familial Kallmann syndrome. In these families, segregation of the disease was suggestive of the X-linked mode of transmission. In 2 families, large Xp22.3 deletions, both including the entire KAL gene, have been detected by Southern blot analysis. Here we report the sequence analysis of the entire coding region of the KAL gene in the 19 remaining patients. The approach consisted of sequencing each of the 14 coding exons and splice site junctions. Each exon was amplified by PCR on the genomic DNA, using oligonucleotides from the flanking intronic sequences as specific primers. Nine point mutations were identified at separate locations in four exons and one splice site, providing strong evidence for heterogeneity in mutations responsible for the X-linked Kallmann syndrome. In addition, the high frequency of unilateral renal aplasia in X-linked Kallmann patients (6 out of 11 males with identified alterations of the KAL gene) should be emphasized.
TL;DR: It is shown that in human adult testis SRY transcription involves multiple start sites, and two human cell lines, NTERA-2 cl.D1 (NT2/D1) and Hep G2, have been identified which express SRY at similar levels to adultTestis.
Abstract: SRY encodes the Y-linked testis-determining factor in humans. A predominant 900 bp transcript originates from a single exon and encompasses the putative SRY coding sequence. We show that in human adult testis SRY transcription involves multiple start sites. In addition to a previously defined major initiation site, transcripts originating at least 410 bp upstream of this site were detected. Using a cDNA specific RT-PCR assay, embryonic and adult human tissues were screened for SRY expression. In humans, SRY transcription is not restricted to the presumptive and the mature gonadal tissues in the embryo and the adult respectively but can be detected in a range of other locations. Two human cell lines, NTERA-2 cl.D1 (NT2/D1) and Hep G2, have been identified which express SRY at similar levels to adult testis. The NT2/D1 SRY transcripts appear to have the same structure as those in adult testis. HMBA-induced differentiation of NT2/D1 cells results in a diminution of SRY mRNA, while transcription of SRY in retinoic acid differentiated NT2/D1 is unaffected.
TL;DR: A technique which allows the detection and chromosomal localization of DNA sequence copy number changes in solid tumor genomes from frozen sections and paraffin embedded, formalin fixed specimens is presented.
Abstract: We present a technique which allows the detection and chromosomal localization of DNA sequence copy number changes in solid tumor genomes from frozen sections and paraffin embedded, formalin fixed specimens. Based on comparative genomic hybridization and on universal DNA amplification procedures this technique is possible even if only a few tumor cells are available. We demonstrate the feasibility of this method to visualize complete and partial chromosome gains and losses and gene amplifications in archived solid tumor samples.
TL;DR: In all cases the length of the expansion seen in DNA isolated from muscle was larger than that seen in lymphocytes from the same patient, and there was no progression over a period of 10-15 years in muscle samples from two patients even though there had been significant progression of the severity of symptoms during that time.
Abstract: The discovery that there is an expansion of a CTG repeat underlying myotonic dystrophy has led to new approaches in diagnosis and genetic counselling for this disorder. The size of the expansion correlates to a reasonable degree with the clinical symptoms within a given family. We report comparisons of the length of the expansion seen in lymphocytes and muscle samples from eight patients. In all cases the length of the expansion seen in DNA isolated from muscle was larger than that seen in lymphocytes from the same patient. There was no progression of the expansion over a period of 10-15 years in muscle samples from two of these patients even though there had been significant progression of the severity of symptoms during that time.
TL;DR: The elucidation of the exon composition of the FMR1 gene and its flanking region will enhance detection of coding sequence mutations possible in fragile X phenocopy individuals.
Abstract: The fragile X syndrome is due to a CGG triplet expansion in the first exon of FMR1, resulting in hypermethylation and extinction of gene expression. To further our understanding of the gene's involvement in the syndrome, we report the physical structure of this locus. A high resolution restriction map of the FRAX(A) locus has been prepared encompassing approximately 50 kb. Using exon-exon PCR and restriction analysis, the FMR1 gene has been determined to consist of 17 exons spanning 38 kb of Xq27.3. Each intron-exon boundary has been sequenced. In general, the splice donors and acceptors located in the 5' portion of the gene demonstrate greater adherence to consensus than those in the 3' end, providing a possible explanation for the finding of alternative splicing in FMR1. The elucidation of the exon composition of the FMR1 gene and its flanking region will enhance detection of coding sequence mutations possible in fragile X phenocopy individuals.
TL;DR: The chromosomal localization of one form of CMT4 will have important implications in clarifying the nosology of this complex group of disorders.
Abstract: Autosomal recessive Charcot-Marie-Tooth (CMT) disease (CMT4) is a complex group of severe childhood motor and sensory neuropathies, characterized by an early age of onset with rapidly progressive distal limb weakness and atrophy. One subgroup designated CMT4 type A (CMT4A) was selected from a series of Tunisian CMT4 families according to the following electrophysiological and pathological criteria: slow motor nerve conduction velocity (MCV), severe hypomyelination upon nerve biopsy with basal lamina onion bulbs and no myelin outfolding. In an attempt to localize the CMT4A locus, we studied four inbred families with 13 affected patients. Significant evidence for linkage was found for several markers from chromosome 8q13-21.1 (D8S279, D8S164, D8S286, D8S84, D8S275 and D8S167). An overall two point peak lod score of z(theta) = 9.19 at theta = 0.00 (95% confidence limit 0.00-0.08) was obtained for D8S164. No evidence of genetic heterogeneity was found. The chromosomal localization of one form of CMT4 will have important implications in clarifying the nosology of this complex group of disorders.
TL;DR: The length variation of the repeat in 513 non-HD chromosomes from normal individuals and HD patients showing 23 alleles with 11 to 33 repeats is demonstrated, indicating the repeat number in the DNA of blood lymphocytes is probably representative of all tissues in a patient.
Abstract: The gene causing Huntington's disease, an autosomal dominantly inherited, neurodegenerative disorder, has been identified recently. The corresponding mutation is involving an expansion in the number of (CAG)n repeats in the coding region of the Huntington's disease gene on chromosome 4. In this report, we demonstrate the length variation of the repeat in 513 non-HD chromosomes from normal individuals and HD patients showing 23 alleles with 11 to 33 repeats. Analyzing the inheritance of the (CAG)n stretch we found meiotic instability for HD alleles ([CAG]40 to [CAG]75) with a mutation frequency of approximately 0.7, while in 431 meioses of normal alleles only two expansions were identified. The risk of expansion during spermatogenesis is enhanced compared to oogenesis explaining juvenile onset by transmission from affected fathers. Further, the number of (CAG)n copies in an affected individual in relation to the sex of the transmitting parent was evaluated and no significant differences were found. No mosaicism or differences in the repeat lengths were observed in the DNA from different tissues including brain and lymphocytes of two HD patients indicating mitotic stability of the mutation. Therefore, the determination of the repeat number in the DNA of blood lymphocytes is probably representative of all tissues in a patient.