1/9. female haemophilia A in a family with seeming extreme bidirectional lyonization tendency: abnormal premature X-chromosome inactivation?We studied a female child with mild classical haemophilia A, presenting with a F VIII deficiency similar to that detected in her maternal grandfather. Investigations on several occasions showed that the obligate carrier mother of the proposita had normal VIII:C activity, whereas her likewise obligate carrier sister had a typical carrier VIII:C/vWf:Ag pattern. The child was a phenotypically normal female with normal karyotype. Her father had no clinical or biochemical signs of haemophilia A. RFLP-analysis using DX13 and St14 probes each elicited one allele (5.8 and 3.4 kb, respectively) segregating along with the affected F VIII gene from the hemizygous grandfather to both his daughters and further to the haemophilic female child. The paternity of the child was analyzed using various red cell and hla antigens and RFLP by p29C, a probe detecting polymorphic hypervariable TaqI and PstI fragments in the pseudoautosomal areas of the X- and Y-chromosomes. All results obtained were concordant with the declared paternity. RFLP-analysis, using single (Pst I) and double digestion (Pst I/Hha I) of DNA and a PGK probe, revealed a remarkable difference in hybridization fragments, strongly suggesting hypermethylation, and in consequence, preferential X-chromosome inactivation in the proposita. This points to extreme lyonization as the most plausible explanation for haemophilia A in this female child. A familial tendency to abnormal premature X-chromosome inactivation is speculated.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
2/9. Moderately severe hemophilia a resulting from GluGly substitution in exon 7 of the factor viii gene. To define the molecular basis of a TaqI site alteration in the factor viii gene of a patient with moderately severe hemophilia a, we used a combination of genomic amplification followed by direct sequencing and oligonucleotide hybridization, to demonstrate an A-to-G substitution in exon 7 (codon 291) of this gene. This mutation generates a Gly in place of Glu at amino acid 272 of the mature factor viii protein. The mutation arose de novo in a germ cell of the patient's mother.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
3/9. Haemophilia A in a female: study of a family using intragenic and extragenic restriction site polymorphisms.Restriction fragment length polymorphisms (RFLPs) were studied in a large Algerian family which includes 6 haemophiliacs and a previously described case of female haemophilia A. The female propositus is 66 years old with a normal karyotype. Her parents are first cousins. Her 3 sons are haemophiliacs and her 3 daughters with affected children are obligate carriers. The proband has an excessive bleeding tendency and markedly reduced levels of F.VIII (VIII C 0.03 U/ml, VIII Ag 0.01 U/ml) with elevated vWF Ag (2.30 U/ml), similar to the levels observed in affected males from the family. Four RFLPs can be identified by Southern blotting after digesting genomic DNA with the restriction enzymes Bcl I, Bgl I, Kpn I/Xba I and Taq I and hybridization with a 647 bp Stu I/Sca I F.VIII genomic probe, a 1.8 Kb EcoRI F.VIII cDNA probe, a 1.0 Kb EcoRI/Sst I fragment of intron 22 and the extragenic probe ST 14, respectively. With these four RFLPs, the propositus was found to be homozygous for the alleles segregating in this family with the abnormal X-chromosome. The carrier status was proven in a granddaughter and excluded in another. In conclusion, this RFLP linkage analysis is another argument to suggest that the propositus, a rare case of female haemophilia, is homozygous for the abnormal gene.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
4/9. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia a patient.In this paper we report the molecular characterization of a large deletion that removes the entire factor viii gene in a severe hemophilia a patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. in situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the y chromosome (Yp). In our patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the x chromosome. sequence analysis of the 3' breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
5/9. Twin pregnancy after preimplantation diagnosis for sex selection.A twin female pregnancy was obtained in a haemophilia carrier after two preimplantation diagnosis cycles. The embryonic sex of biopsied blastomeres was determined with the use of dual fluorescence in-situ hybridization with directly labelled dna probes specific for the X and y chromosome. A twin female pregnancy was confirmed by means of ultrasonography and amniocentesis at the 14th week of amenorrhoea. The patient delivered two healthy females by Caesarian section at the 37th week of pregnancy.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
6/9. sex determination of human embryos using the polymerase chain reaction and confirmation by fluorescence in situ hybridization.OBJECTIVE: To use fluorescence in situ hybridization to corroborate the polymerase chain reaction (PCR) preimplantation diagnosis of human embryos in three couples carrying a chromosome X-linked disease. SETTING: Clinical and research IVF laboratories. patients: Individuals undergoing preimplantation diagnosis. RESULTS: Four ETs were performed in couples undergoing preimplantation diagnosis by multiplex PCR or fluorescence in situ hybridization, resulting in the birth of two normal female twins. The result of another is pending. A total of 22 embryos were analyzed by PCR. Embryos that were diagnosed as being at risk of carrying the genetic abnormality (n = 8), embryos that failed diagnosis (n = 4), and genetically normal embryos that arrested development (n = 4) were further analyzed by fluorescence in situ hybridization. The sex of all 16 embryos was determined and confirmed the previous 12 preimplantation diagnoses by multiplex PCR. In addition, fluorescence in situ hybridization analysis allowed the detection of two aneuploid embryos, one XO and one XXY, previously diagnosed by PCR as a normal female and male. Two mosaics were also detected. CONCLUSION: polymerase chain reaction and fluorescence in situ hybridization are possible for preimplantation sex determination in cases of genetic sex-linked disease. fluorescence in situ hybridization, however, supplies additional information about sex chromosome aneuploidy and is not susceptible to contamination or misdiagnosis of monosomy X.- - - - - - - - - - ranking = 10keywords = hybridization (Clic here for more details about this article) |
7/9. Unsuspected varicella-zoster virus encephalitis in a child with acquired immunodeficiency syndrome.We report a case of progressive encephalitis caused by varicella-zoster virus (VZV) in an adolescent with hemophilia and acquired immunodeficiency syndrome but without cutaneous signs of VZV infection. magnetic resonance imaging of the brain demonstrated an abnormally increased periventricular signal in T2-weighted images. infection with VZV was proved by in situ hybridization and immunofluorescence staining of brain tissue, which showed histologic evidence of herpesvirus infection. encephalitis caused by infection with VZV is a potentially treatable complication of acquired immunodeficiency syndrome and requires a high index of suspicion for diagnosis.- - - - - - - - - - ranking = 1keywords = hybridization (Clic here for more details about this article) |
8/9. A novel DNA inversion causing severe hemophilia a.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.- - - - - - - - - - ranking = 2keywords = hybridization (Clic here for more details about this article) |
9/9. Variant of intron 22 inversions in the factor viii gene in severe hemophilia a.Recurrent DNA inversions, which disrupt the factor viii (FVIII) gene, 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. This report describes a patient with severe hemophilia a and a high level inhibitor with atypical hybridization patterns. A Bcl I Southern blot assay was altered to 17.5, 16, and 14 kb. His mother and two out of four aunts tested had normal and abnormal restriction patterns which led to a total of five different fragments, suggesting that they were carriers. The Xba I plus Kpn I restriction fragment-length polymorphism in intron 22 by Southern blotting using the same probe (probe a) yielded the 6.2 kb polymorphic band, with a clearly separated 6.6 kb band from the non-factor viii region; an alternative int22h hybridization probe (probe x) detected no additional fragment. These results suggest that probe a as well as probe x could recognize an intron-22-sized fragment. This report shows a variation in the number of int22h copies although we could not find the inversion junction.- - - - - - - - - - ranking = 2keywords = hybridization (Clic here for more details about this article) |