Identification of mutations in two families with sporadic hemophilia A

Summary Direct sequencing of segments of the factor VIII gene in 30 hemophiliacs with sporadic disease (32+ kb of sequence in total) revealed two missense transitions: glutamate 1704 to lysine (E¹⁷⁰⁴K) in a patient with severe hemophilia A and proline 2300 to serine (P²³⁰⁰S) in a patient with mild hemophilia. Both transitions are likely to be causative mutations because the amino acids affected were evolutionarily conserved. Haplotype and sequence analysis of the mother and grandparents of patient HA12 (E¹⁷⁰⁴K) indicate that the mutation arose in the grandfather who was 27 years old when his daughter was conceived. The origin of mutation in patient HA39 (P²³⁰⁰S) could not be determined. As mutations that cause mild disease can be found in seemingly unrelated families, 96 unrelated hemophiliacs were screened rapidly for the P²³⁰⁰S mutation with polymerase chain reaction (PCR) amplification of specific alleles (PASA). None of these patients had the mutation. PASA was also used to conveniently assess a polymorphic site in intron 7. The polymorphism is estimated to be informative in 13% of Korean females and in 23% of Western European females.     

Maternal duplication associated with gene deletion in sporadic hemophilia.

Sporadic occurrences of X-linked disorders can give insights into mutagenesis in man. In a case of sporadic hemophilia, associated with a partial deletion of the factor VIII gene, an unexpected inheritance pattern of gene rearrangements was observed. The factor VIII gene was found to be partially duplicated in the hemophiliac's mother. A pedigree analysis indicates that the mother has contributed both aberrant genes as well as the normal gene to her offspring. One simple model for the evolution of the deletion in this family is that the duplication is the precursor to the deletion.     

Molecular genetic study of the factor VIII gene in families from Bashkir with hemophilia A

The distribution of hemophilia A was studied in Bashkortostan. The factor VIII gene of the blood coagulation system was analyzed in 34 patients with hemophilia A and 48 of their close relatives. Inversion of intron 22 of the factor VIII gene was revealed in nine cases, which comprised 30% of the total sample analyzed. The type II and type III of this mutation occurred at a relatively high frequency, which may be explained by the founder effect and genetic drift. The allelic frequencies of the polymorphic locus HindIII at intron 19 were similar; a substantial allelic heterogeneity of both microsatellite (CA)-repeats at intron 13 and the DXS52 locus were found on normal and mutant X chromosomes. The molecular genetic analysis of (CA)-repeats and the loci HindIII and DXS52 in families with hemophilia A makes it possible to reveal up to 89% of the informative families.     

First experiences in application of RFLP analysis for carrier detection in preparation of prenatal diagnosis of hemophilia A in the GDR

The application of the intragenic probe F8 e16-19 of factor VIII gene is reported for carrier detection in preparation of prenatal diagnosis of hemophilia A in a family at risk. The proband's mother is heterozygous for the Bcl I polymorphism and prenatal diagnosis can be offered to this family.     

Linkage analysis of families with fragile-X mental retardation, using a novel RFLP marker (DXS 304).

A new polymorphic DNA marker U6.2, defining the locus DXS304, was recently isolated and mapped to the Xq27 region of the X chromosome. In the previous communication we describe a linkage study encompassing 16 fragile-X families and using U6.2 and five previously described polymorphic markers at Xq26-q28. One recombination event was observed between DXS304 and the fragile-X locus in 36 informative meioses. Combined with information from other reports, our results suggest the following order of the examined loci on Xq: cen-F9-DXS105-DXS98-FRAXA-DXS304-(DXS52-F8 -DXS15). The locus DXS304 is closely linked to FRAXA, giving a peak lod score of 5.86 at a corresponding recombination fraction of .00. On the basis of the present results, it is apparent that U6.2 is a useful probe for carrier and prenatal diagnosis in fragile-X families.     

Evaluation of DHPLC in the analysis of hemophilia A

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilitate genetic counseling and prediction of the risk of anti-FVIII antibody development, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons), mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP). These techniques are time consuming, require specific expertise and are limited to detection rates of 70-85%. In contrast, the recently introduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previously identified mutations in the FVIII gene. Applying empirically determined exon-specific melting profiles, a total of 150 mutations (96.2%) were readily detected. Five mutations (3.2%) could be identified after temperatures were optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyzed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleotide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemophilia A.     

Genealogical analysis of cystic fibrosis families and chromosome 7q RFLP haplotypes in the Hutterite Brethren.

In the 100-year period 1880-1980 the Hutterite population increased from about 442 to 23,000 individuals in North America. There are three endogamous subdivisions in this Caucasian genetic isolate. A total of 11 cystic fibrosis (CF) families from Canada and the United States were investigated, including at least two families from each of the three subdivisions, the Dariusleut, Lehrerleut, and Schmiedeleut. A study of RFLPs for the loci D7S8, D7S23, MET, and D7S18 (also called D7S16) in the region of the CF gene in 10 families shows considerable genetic variability. There were three different extended CF gene-region haplotypes on CF chromosomes (CF haplotypes), and there were 13 different extended CF gene-region haplotypes on normal chromosomes (normal haplotypes). The three CF haplotypes have different D7S23 and MET haplotypes. Parents who have the same CF haplotype are, on the average, more closely related than parents who have different haplotypes, but only within the same subdivision. A marriage node graph of 11 families illustrates the complexity of Hutterite genealogies. The frequency distribution of CF haplotypes in the Hutterite sample differs notably from those of larger agglomerates of family data from collaborative studies, with respect to D7S8, MET haplotypes, and D7S23 haplotypes. We propose that there were at least three CF carriers among the founders of the Hutterite population and that copies of a particular CF haplotype in current individuals are identical by descent. The alternative that one or more genetically distinguishable CF haplotypes resulted from recombination since the founding of the population is considered to be less likely.     

Germ line origins of de novo mutations in hemophilia B families

The germ line of origin for 13 of 14 de novo hemophilia B mutations has been determined. When added to previous reports, the origin, assuming no mosaicism, occurred in 43 female and 33 male gametes. Mutation rate estimates are twofold higher in males than in females. The pooled data also indicate that male and female germ lines have different mutation rates depending upon the type of mutation.     

Segregation analysis of hemophilia A and B.

We analyzed a sample of 1,485 families with hemophilia A and B and with unknown diagnosis. The frequency of sporadic cases was estimated to be .166 and .078 for the two types of hemophilia, respectively. The sex ratio of mutation rates did not differ significantly from unity. The average age of maternal grandfathers of probands at birth of mothers with a single child, affected by hemophilia B, and of maternal grandfathers of probands at birth of mothers with more than one child affected by hemophilia B, was higher than the age in appropriate control groups.     

Molecular analysis of hemophilia A mutations in the Finnish population

We have examined the Finnish hemophilia A population for factor VIII gene mutations. This study included 83 unrelated patients and revealed 10 mutations associated with hemophilia. Using cloned cDNA, genomic, and oligonucleotide probes, we have identified three classes of mutations: five mutations causing the loss of TaqI restriction sites, a point mutation resulting in a new TaqI site, and four partial gene deletions. Although exons 5 and 6 were involved in three of the four partial gene deletions, the extent of the DNA lost differs in each case. The fourth deletion was located entirely within intron 1 and segregated with the disease in a large hemophilia pedigree. There was no history of hemophilia in eight of the 10 families. The origin of the mutation was determined in six of these pedigrees, two of which showed evidence for maternal mosaicism.     

Localization of the gene for amiloride binding protein on chromosome 7 and RFLP analysis in cystic fibrosis families

Summary The apical sodium channel is essential for sodium reabsorption by the kidney. Its activity is blocked by the diuretic amiloride. Using a human cDNA coding for the amiloride binding protein (ABP), the corresponding structural gene was mapped to human chromosome 7q34–q36 by in situ hybridization. This region flanks the region implicated in cystic fibrosis (7q32). Because an alteration of the amiloride sensitive sodium channel function has been suggested in cystic fibrosis, a possible link between the ABP gene and this disease was analyzed by restriction fragments length polymorphism (RFLP) analyses. From this study, it appears that the gene coding for ABP is not directly modified by mutations causing cystic fibrosis.     

Elimination of inhibitors in children with hemophilia A

In five hemophilic children an attempt was made to eradicate inhibitors by continuous treatment with low doses of cryoprecipitate. All patients were high responders with maximum titers of 25 and 600 U. In four patients the anamnestic response was prevented or diminished by simultaneous treatment with cyclophosphamide. In these patients the inhibitor did not return during the continuous replacement therapy, even not after cessation of cyclophosphamide administration and in three of them not after intensive substitution. In the other child the titer decreased continuously in the beginning, but only to values unsuitable for replacement effects. Attempts to exterminate the inhibitor should be occasionally made by continuous low dose substitution therapy. The importance of the combination with cyclophosphamide cannot be decided until now because of the small number of observations.     

Segregation analysis of 1885 DMD families: significant departure from the expected proportion of sporadic cases

Summary The proportion of sporadic cases of Duchenne muscular dystrophy has been estimated by classical segregation analysis in a pooled sample of 1885 sibships from 7 different countries. A significant departure from the theoretical expectations based on mutation-selection equilibrium is observed (segregation frequency = 0.439 ± 0.017; frequency of sporadic cases = 0.229 ± 0.026, at the maximum likelihood). The occurrence of germinal mosaicism in some of the mothers of Duchenne cases may account for this peculiar finding, although a possible role of inequality of mutation rates in the two sexes cannot be ruled out.     

Molecular diagnosis of hemophilia A and B. Report of five families from Costa Rica

Hemophilia A and B are X-chromosome linked bleeding disorders caused by deficiency of the respective coagulation factor VIII and IX. Affected individuals develop a variable phenotype of hemorrhage caused by a broad range of mutations within the Factor VIII or Factor IX gene. Here, were report the results of the molecular diagnosis in a five Costa Rican families affected with Hemophilia. Methods of indirect and direct molecular diagnosis are applied in three Hemophilia A and two Hemophilia B families from Costa Rica as well as preconditions, practicability and facilities of this diagnosis. In two families with Hemophilia A and both families with Hemophilia B the causative mutation could be detected by Southern blotting, polymerase chain reaction or sequence analysis. One Hemophilia A family could only analyzed by linkage analysis using genomic markers.     

RFLP analysis of genomic regions associated with cooked-kernel elongation in rice

As a result of earlier breeding efforts, portions of the genome of Basmati 370 have been introgressed into a rice breeding line, B8462T3-710. Cooked-kernel elongation was increased in this breeding line to a level equal to that of Basmati 370. The objective of this study was to identify and locate quantitative trait loci (QTLs) associated with cooked-kernel elongation in an F3 population derived from a cross between B8462T3710 and the reduced-elongation recurrent parent variety, Dellmont. DNA from the parental lines and Basmati 370 as a control, were screened for RFLPs using 170 clones chosen to cover the rice genome at intervals of 8 cM on average. Eighteen markers identified RFLPs common to Basmati 370 and B8462T3-710, but different from Dellmont, suggesting possible associations with kernel elongation. The B8462T3-710/Dellmont F3 population was analyzed for segregation of those RFLPs and for kernel elongation. Analysis of variance of the kernel elongation ratio revealed that two markers, 14.6 cM apart on chromosome 8, are significantly associated with this trait (RZ323 P 0.005, RZ562 P 0.05). Interval mapping suggests a single QTL with a close proximity to RZ323. This QTL was tested in F6 lines derived from the same cross and the presence of the B8462T3-710 segment detected by RZ323 caused a highly significant increase of the kernel elongation ratio (P 0.04). In addition, the QTL for kernel elongation and a gene for aroma, which are major components of the grain quality characteristics of Basmati-type rices, showed linkage. The availability of linked markers to the QTL may facilitate early selection for kernel elongation in rice breeding programs.     

Chronic thrombocytopenia in an immunodeficient patient with hemophilia A

Coincident hemophilia and idiopathic thrombocytopenia has been rarely observed. We report here on a young man with severe hemophilia A who was treated with concentrates of lyophilized antihemophilic factor for several years before he developed thrombocytopenia. An isolated thrombocytopenia coincident with reduced platelet survival, ample megakaryocytes in the bone marrow, elevated platelet-associated IgG, as well as remission after treatment with prednisone and splenectomy, suggest the idiopathic form of thrombocytopenia. In addition, defects in cellular immunity became obvious. A causal relationship between the application of blood-derived products and the development of the platelet disorder as well as the impairment of the T-cell system remain to be demonstrated.