A haemophilia A and B molecular genetic diagnostic programme in Hungary: a highly informative and cost-effective strategy

Our aim was to set up a protocol in order to provide carrier and prenatal diagnosis to Hungarian haemophilia A (HA) and B (HB) patients and their relatives. For HA, a combination of direct mutation detection and some indirect marker analyses were used: the detection of the inversion mutation and analysis of three polymorphisms, BclI, IVS13 (CA)n and P39(CA)n. In severe cases, direct mutation detection was performed first. In inversion-negative severe cases and in moderate and mild cases, indirect methods were used. For carrier and prenatal diagnosis in HB, four polymorphisms, DdeI, TaqI, XmnI, and HhaI were examined. Our DNA bank contains samples from 50 HA families (34 severe, 15 moderate and one mild) and seven HB families from different parts of the country. In 100% of the HA cases either the gene inversion and/or at least one of the polymorphisms was found to be informative for carrier or prenatal diagnosis. In the HB cases, an informative marker was found in 95% of the cases (19 of 20). We conclude that these strategies are sufficient to make genetic diagnosis available to almost all HA and HB families in the region. This approach is highly informative and cost-effective, so it can be very useful in countries where direct sequencing of genes for factor VIII and IX is not available for routine diagnosis.     

Carrier detection in the hemophilias

The subject of carrier detection in the hemophilias has received new impetus in the past several years. Treatment complications arising from clotting factor concentrates have become more evident and earlier prenatal diagnosis and new genetic markers for the clotting factor genes have focused interest on this area. Until now, carrier diagnosis has relied upon standard pedigree analysis and clotting factor assays. The results obtained using these methods are probabilistic, and the coagulation tests are unavoidably influenced by the effects of random X chromosome inactivation and the inherent variability of the methods involved. With the cloning and characterization of both factor IX and factor VIII genes, has come the capability of using gene analysis to diagnose the carrier state. This usually involves the detection of restriction fragment length polymorphisms (RFLPs) and their use as linked markers for the defective clotting factor gene. In hemophilia A, the combined use of three intragenic RFLPs and two closely linked, highly polymorphic extragenic markers will make carrier information available to approximately 90% of kindred. In hemophilia B, phenotypic analysis has been complicated by the more heterogeneous expression of the gene defect. To date, five intragenic and one closely linked RFLP have been reported, as well as two protein polymorphisms detectable by monoclonal antibody immunoassays. With the combined use of these genetic markers it is likely that accurate carrier assignment will be available to more than 80% of hemophilia B families.     

Genetic counseling of hemophilia carriers

Basic analysis of a potential carrier includes calculation of the probability, or odds, for carriership based on pedigree and clotting factor analysis. Genotype assessment constitutes a more accurate method of carrier detection. Where circumstances permit, the genetic diagnosis of hemophilia should be based on the direct identification of the pathogenic mutation in the factor (F) VIII gene. Neutral mutations in the FVIII gene and the risk of mosaicism (a mixture of normal and mutation carrying cells) in sporadic families may cause misclassification. If it is not possible to use the mutation for diagnostic purposes, it may be possible to use linked polymorphic markers (restriction fragment length polymorphisms [RFLP]) to trace the inheritance of the hemophilia gene within a pedigree. Linkage analysis is limited because of uninformative patterns of polymorphic markers, ethnic variation, linkage disequilibrium, and the need for participation of family members, and it is not useful in sporadic families, which constitute more than half of the hemophilia families. Potential carriers of hemophilia should be offered qualified assistance in genetic information, testing, and counseling to help them to cope with the psychological and ethical problems related to carriership of a genetic disorder.     

Mutation detection in 65 families with a possible diagnosis of ornithine carbamoyltransferase deficiency including 14 novel mutations

The high new mutation rate and the wide spectrum of mutations found in patients with ornithine carbamoyltransferase (OCT) deficiency means that direct mutation analysis is essential for providing accurate carrier detection and prenatal diagnosis in affected families. We present our strategy for mutation detection in the OCT gene and summarize the results from 31 families with a confirmed diagnosis and 34 families with a suspected diagnosis of OCT deficiency, and describe 14 previously unreported mutations.     

Laboratory methods for the genetic diagnosis of bleeding disorders

Accurate carrier detection and pre-natal diagnosis in haemophilia A and B and in von Willebrand's disease (VWD) can be achieved by genetic analysis. The spectrum of mutations responsible for these three disorders is described. Methods for linkage analysis using intra genic diallelic and multiallelic factor VIII and IX gene polymorphisms are mentioned, and situations where their use in carrier detection is inappropriate or fails are discussed. Linkage analysis for examination of von Willebrand factor gene inheritance in families with VWD is also described. Screening for the factor VIII gene inversion in patients with severe haemo philia A and the use of the factor VIII binding assay as a discriminant test in patients with possible mild haemophilia A or VWD are described. Point mutation screening methods AMD, CSGE, DGGE, SSCP and UHG analysis are also detailed. The variety of possible analyses available to genetically diagnose haemophilia A, B and VWD is explored.     

Carrier detection and prenatal diagnosis of hemophilia in developing countries

Hemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of coagulation factor VIII (FVIII) or FIX, respectively. Hemophilia is prevalent worldwide, without ethnic or geographic limitations, and remains a life-threatening and often disabling condition. Advances in molecular medicine in this century have markedly improved hemophilia treatment. However, management is still largely inadequate in developing countries. Therefore, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children with hemophilia in developing countries where patients with this coagulation disorder rarely live beyond childhood. Carrier detection and prenatal diagnosis are possible by direct or indirect genetic analysis of the F8 or F9 genes. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection by prescreening techniques or direct mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article suggests the possibilities of genetic diagnosis and a feasible strategy for carrier detection and prenatal diagnosis in families with hemophilia A and B in developing countries.     

Analysis of factor VIII gene inversion mutations in 166 unrelated haemophilia A families: frequency and utility in genetic counselling

Summary. Haemophilia A is an X-linked recessive bleeding disorder of variable severity that is caused by a deficiency of coagulation factor VIII (FVIII). The disease results from mutations in the FVIII gene which are heterogenous both in type and position within the gene. Recently, however, inversion mutations were found to be common to patients with severe disease (Lakich et al. , 1993). These mutations result from intrachromosomal recombinations between DNA sequences in the A gene (located in intron 22 of the FVIII gene) and one of two A genes upstream to the FVIII gene. To determine the frequency of these inversions we performed Southern blot analysis on banked DNA from 166 consecutive, unrelated haemophilia A families previously referred for carrier or prenatal testing. In 57/166 (34%) families an inversion or other unique mutation was detected. The distal and proximal A genes lying upstream to the FVIII gene were involved in 79% and 18% of the mutations, respectively, but in 3% of the families the sequences involved in the mutation have not been identified. In 20/38 (53%) families with severe disease a mutation was detected. Interestingly, the relative risk of developing inhibitors in patients with FVIII gene inversions or other 3° mutations detected by this assay, as compared to patients with no detectable mutation by this assay, was 3.8. In families for which a mutation is detected, direct DNA testing is an accurate and inexpensive alternative to linkage analysis for prenatal or haemophilia A carrier testing.     

Screening of the Bruton Tyrosine Kinase (BTK) Gene Mutations in 13 Iranian Patients with Presumed X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (Btk) gene. In order to identify the mutations in Btk gene in Iranian patients with antibody deficiency, 13 male patients with an XLA phenotype from 11 unrelated families were enrolled as the subjects of investigation for Btk mutation analysis using PCR-SSCP followed by sequencing. Five different mutations were identified in 5 patients from 5 unrelated families. Three mutations had been reported previously including TTTG deletion in intron 15 (4 bps upstream of exon 16 boundary), nonsense point mutation (1896G>A) that resulted in a premature stop codon (W588X) in kinase domain, and nucleotide alteration in invariant splice donor site of exon12 (IVS12+1G>A). While 2 novel missense mutations (2084A>G, 1783T>C) were identified leading to amino acid changes (I651T, Y551H). The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier detection and prenatal diagnosis.     

Multiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of haemophilia A

Summary. Use of molecular genetic studies for carrier detection and prenatal diagnosis of haemophilia A will be informative in approximately 75% of kindreds with a prior history of the disorder if previously reported bi-allelic intragenic polymorphisms are used. In this study we report the use of two multi-allelic, microsatellite repeat polymorphisms within the factor VIII gene in genetic testing for this disease. These two, dinucleotide repeat, polymorphisms have been analysed using a multiplex polymerase chain reaction (PCR) protocol, and results can be available within 3 d of receipt of samples. At the intron 13 polymorphic locus we have confirmed the original observation of eight alleles, whilst at the intron 22 locus, in contrast to a preliminary report of this polymorphism, we have seen five alleles. We have analysed 32 families (174 subjects) with the two microsatellite repeat markers and have found that using these two polymorphisms alone, 81% of families are informative for linkage analysis. The intron 13 repeat was informative in 22/31 families tested (71%) and the intron 22 polymorphism was informative in 12/17 families (71%). We have also found that in 11/32 families tested (34%) the microsatellite repeats were the only informative intragenic markers. In view of these observations, we believe that the most effective strategy for initiating haemophilia A genetic testing is to combine the multiplex PCR analysis of the intron 13 and 22 dinucleotide repeats with the PCR analysis of the intron 18 BclI marker. In our population this protocol provided informative results in approximately 88% of families and results can be available within 3 d of receiving the samples for testing.     

Determination of haemophilia A carrier status by mutation analysis

A reliable method for determination of carrier status and genetic counselling is required for effective control of haemophilia. Linkage analysis is currently the most widely used method for this purpose; however, in cases where there is no prior family history and/or unavailability of informative polymorphic markers it is less applicable. Detection of a mutation characterized in each family may be an alternative method for determination of the carrier status. In this study, linkage analysis using four polymorphic DNA markers, and direct mutation analysis were compared to determine the carrier status in six unrelated Thai haemophilia A families, two with a family history and four without. In the two families with a family history of haemophilia A, the carrier and noncarrier statuses could readily be determined in eight females by either linkage or direct mutation analysis. In the four families without a family history, the polymorphic DNA markers for linkage analysis were informative in two families and uninformative in the other two. The carrier status could be excluded in all four female siblings of the patients in the former. However, the specific FVIII gene mutation was not observed in the mother of one patient, who should have carried the mutation. In the remaining two families with uninformative polymorphic DNA markers, the carrier and noncarrier statuses of four female members could only be determined by direct mutation analysis. Therefore, direct mutation analysis could circumvent the limitations of linkage analysis in the determination of haemophilia A carrier status in families without a previous history or informative polymorphic markers.     

Factor VIII gene inversions and polymorphisms in Brazilian patients with haemophilia A: carrier detection and prenatal diagnosis

In families afflicted with haemophilia A, genetic counselling is often requested. Inversion mutations and polymorphic sites of the FVIII gene have been examined in a Brazilian population, with the aim of developing a strategy that would be accurate and informative for carrier analysis and prenatal diagnosis in Brazil. Patients with haemophilia A and families were studied. Inversion mutations in the FVIII gene were detected in 39.4% of severely affected patients, 85% of them being of distal type. No inversions were observed in patients with mild or moderate forms of the disease. Two bi-allelic polymorphisms were studied. Intron 18 SSCP and intron 19 RFLP analyses indicated the presence of a restriction site in 39.5% and 42.9% of haemophilics, respectively. Two multiallelic microsatellite polymorphisms in introns 13 and 22 were also studied; eight different alleles were detected in each system with a heterozygosity rate of 83.08% and 78.77%, respectively. When all four intragenic loci were examined in linkage analysis, the cumulative reliability was 100%. In conclusion, inversion mutation analysis should be the first-line test for Brazilian patients with severe haemophilia A. In cases of severe haemophilia A where no inversion could be detected or in mild or moderate haemophilia A, the use of all four polymorphisms is very informative for linkage analysis and should be used for carrier detection and genetic counselling in the Brazilian population.     

Prenatal diagnosis of Duchenne muscular dystrophy: A three-year experience in a rapidly evolving field

Application of molecular genetic techniques has greatly increased diagnostic possibilities of hereditary disorders. In 1983 the first linkage of Duchenne muscular dystrophy with flanking DNA probes was described, which made carrier detection possible in a limited number of cases. The first published prenatal diagnosis for Duchenne muscular dystrophy dates from 1985. DNA-analysis for Duchenne muscular dystrophy and Becker muscular dystrophy has become increasingly informative, firstly by the development of more flanking markers, followed by intragenic probes detecting deletions and, more recently, by the use of cDNA probes detecting a deletion or duplication mutation in over 60% of the Duchenne and Becker muscular dystrophy patients. Although these developments allow a highly reliable (>99%) carrier detection and prenatal diagnosis in over 90% of cases, the continuing introduction of new probes and/or technologies has necessitated constant reappraisal of many families to derive maximum information. During the past 3 years we applied prenatal diagnosis for Duchenne and Becker muscular dystrophies with DNA-analysis on 53 male fetuses in 47 families. Twenty-two healthy male babies were born, after being diagnosed to have a low Duchenne muscular dystrophy risk. Two pregnancies also diagnosed as low risk have not yet come to term. In the other cases a high risk for Duchenne muscular dystrophy was found and the parents chose abortion. Our studies also revealed a number of important diagnostic pitfalls, such as non-paternity, karyotypic anomalies and gonadal mosaicism.     

The contribution of DNA analysis to carrier detection and prenatal diagnosis of hemophilia A and B

Summary Developments in DNA technology have provided a novel means of carrier detection and prenatal diagnosis of hemophilia A and B. The collection of a large set of data has enabled us to evaluate the present feasibility and reliability of a diagnosis at the gene level and its contribution to methods already available.Since 1984, 533 potential and obligate carriers belonging to 170 families with hemophilia have been referred to us. By the combined use of pedigree analysis, coagulation assays, and DNA (RFLP) analysis, certainty about the carrier status has been markedly increased for the potential carriers. Although RFLP analysis revealed the possible origin of the mutation in many families with an isolated patient, uncertainty remained for quite a number of their female relatives because of the possible occurrence of germline mosaicism. Forty-one women requested prenatal diagnosis during one or more pregnancies. The short time interval between pregnancies, even after abortion of an affected fetus, proved that first-trimester prenatal diagnosis has become an acceptable option for women at risk. Recently, efficient methods for direct identification of mutations have been developed, and they may allow a definite diagnosis for all families with hemophilia in the near future.This work was financially supported byHet Praeventievonds, Grant no. 28–1244 and, from January 1988, by the combined Dutch Health Insurances.     

Factor VIII gene inversions and an XbaI polymorphism: nonradioactive detection and clinical usage

To simplify molecular diagnosis among families with severe haemophilia A, digoxigenin probe-labelling and chemiluminescent detection was introduced. Sensitivity was the same as with 32P; background was lower. Of 33 previously untested families with severe haemophilia A, 15 have a factor VIII gene inversion (14 distal, one proximal). In three of the 13 families with isolated occurrence, de novo origin of the mutation was assigned to the maternal grandfather (two by XbaI linkage). Within the Seattle series, 64 (45% of 143) families with severe haemophilia A have an inversion; 15 are proximal. Fifteen patients from 12 families have had inhibitors; nine of these were > 10 Bethesda units per millilitre. All 36 mothers examined from the 43 families with isolated occurrence and nearly half (30 of 63) the daughters of an inversion carrier were carriers. Of 24 families where XbaI polymorphic analysis was performed for carrier assignment, 14 were informative. In families with severe haemophilia A, a nonradioactive, digoxygenin-labelling system facilitates both factor VIII gene inversion and XbaI polymorphism analyses by Southern blots of digested, genomic DNA samples.     

Allele frequencies of three factor VIII gene polymorphisms in Iranian populations and their application in hemophilia A carrier detection

Hemophilia A is an X-linked recessive bleeding disorder caused by a quantitative or qualitative deficiency of blood coagulation factor VIII (FVIII). ARMS (amplification refractory mutation system) primers were designed to determine allele frequencies of three FVIII gene linked markers, IVS7 nt 27 G/A SNP, BclI/intron 18, and HindIII/intron 19 among 85 normal Iranian women from unrelated families. Then same method was applied to perform carrier detection for hemophilia A families. The allele frequencies of IVS7 nt 27 "G"/"A" allele, BclI "T"/"A" allele, and HindIII "C"/"T" allele among normal women were 0.88/0.12, 0.52/0.48, and 0.48/0.52, respectively. The three polymorphisms were found to be in strong linkage disequilibrium, which decreased the overall heterozygosity to 51%. Twenty-one women from 15 unrelated hemophilia A families were referred to us for hemophilia A carrier detection. Taking advantage of these three biallelic polymorphisms in conjunction with multiallelic St14 VNTR (locus DXS52), IVS13 (CA)n STR, and IVS22 (CA)n STR, carrier status was determined in 16 women (16/21 or 76% of the at-risk women) from 11 families (11/15 or 73% of the families). The used ARMS methods are rapid and can easily be applied in conjunction with other FVIII gene linked polymorphisms for indirect mutation detection of hemophilia A where they are informative.     

Family studies of the Lesch-Nyhan syndrome: the use of a restriction fragment length polymorphism (RFLP) closely linked to the disease gene for carrier state and prenatal diagnosis

We have investigated the usefulness of a full-length cDNA probe for the hypoxanthine phosphoribosyltransferase, HPRT gene in the diagnosis of the carrier state for the Lesch-Nyhan syndrome in 13 families. The products of DNA digestion by 21 different restriction enzymes were examined by Southern analysis. The only useful polymorphism detected was a three allele polymorphism withBam HI; others were found withMsp I andHind III but they were not linked to the disease. None of the boys with the Lesch-Nyhan syndrome had a unique polymorphism and therefore a detectable gene deletion or other major structural alteration. A DNA polymorphism capable of giving additional diagnostic information with respect to carrier status was present in 8 of the 13 families, but not for all of the family members at risk. The cDNA probe and restriction enzyme analysis gave anunequivocal diagnosis to 20 women out of 28 who might have been carriers and for whom it would not have been available on the basis of pedigree analysis and biochemical tests alone. If a linked polymorphism is to be useful for carrier detection it must be present in only one of the mother's X-chromosomes (i.e. the mother must carry a different allele on each X-chromosome). Although this polymorphism is detected by an HPRT probe it is not at the site of the specific gene lesion. In spite of considerable genetic heterogeneity, most cases of the Lesch-Nyhan syndrome appear to be due to either single base substitutions or small deletions which are not detectable by Southern analysis. None of the propositi in these families was due to a new mutation arising in the maternal germ line. One family was identified in which the evidence was compatible with a mutation having arisen in the X-chromosome which the mother of the propositus inherited from her father, and in another family the mutation arose in the maternal grandmother. A case illustrating the application of this restriction fragment length polymorphism to the first trimester prenatal diagnosis of the Lesch-Nyhan syndrome is also reported. This study illustrates the limitations as well as the value of using a linked DNA polymorphism for carrier state diagnosis in X-linked diseases.     

Genes, patients, families, doctors—Mutation analysis in clinical practice

Summary  Developments in mutation analysis have led to significant benefits for patients with inherited metabolic disorders and their families. This is particularly the case where new methodologies have prevented the need for invasive tissue biopsies or have allowed carrier detection or first trimester prenatal testing to be undertaken. Whereas in the past it may have only been possible to identify specific ‘common’ mutations, the availability of techniques, such as automated sequencing, and novel technologies including mutation scanning techniques, multiplex ligation dependent probe amplification, and array technologies, have vastly improved the diagnostic efficiency of molecular testing. Competing interests: None declared This paper is based on the European Metabolic Group (EMG) Lecture given at the 40th EMG Meeting in Heidelberg, Germany, on June 1 2008 - organized and sponsored by Milupa Metabolics.     

Detection of carriers of haemophilia A: use of bioassays and restriction fragment length polymorphisms (RFLP)

Abstract Background: Haemophilia A is a sex-linked bleeding disorder carried by unaffected females. Currently, the two main methods used for the determination of carrier status in women from families with haemophilia A are bioassays and DNA-based assays using restriction fragment length polymorphisms (RFLP).
Aim: The aim of this paper was to assess the current usefulness of these two methods.
Methods: Bioassays measured factor VIII coagulation activity by a two-stage coagulation assay and von Willebrand antigen by immunoelectrophoresis. RFLP were determined with two intragenic probes (pi 14 and p486) and two linked probes (Stl4 and DX13). Data were analysed using a Bayesian analysis to allow for all possible recombination events. We also incorporated an estimate for the risk of mosaicism into calculations in isolated haemophilia families. Both bioassays and RFLP were used to determine carrier status in 63 women, 31 from known haemophilia families and 32 from families of isolated cases. The techniques were assessed for their ability to classify the patients as normal (p<0.2) or carrier (p>0.7). Where applicable, intron 22 inversion was also tested.
Results: In the known families, six women could not be classified after bioassay, but all could be classified by RFLP. Of the 32 women from families of isolated cases, eight were unclassified by bioassay and 12 were not definitely classified using RFLP. However, RFLP was useful in determining that a recent mutation had occurred in six of the eight families in which DNA from the grandparents was available.
Conclusion: For diagnosis of carriers of haemophilia, RFLP is the preferred method in familial haemophilia, but is less useful in isolated haemophilia.     

Bruton's tyrosine kinase is present in normal platelets and its absence identifies patients with X-linked agammaglobulinaemia and carrier females

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk) and is characterized by an arrest of B-cell development. We analysed Btk protein expression in platelets using flow cytometry and found that normal platelets express large amounts of Btk. Assessment of affected males from 45 unrelated XLA families revealed that platelets of the majority of the patients (37 out of 45 families) had decreased or absent Btk expression, and that platelets from carrier females of these families had both normal and mutated Btk expression, indicating that megakaryocytes in XLA carriers undergo random X-chromosome inactivation. These observations demonstrate that Btk is not crucial for maturation of megakaryocytes and the production of platelets. No correlation between Btk expression in platelets and clinical phenotype was observed in this study. Flow cytometric evaluation using platelets is a simple and rapid method to test Btk expression. It may be used as a screening test for XLA and for carrier detection, followed, if necessary, by more expensive mutation analyses.     

Gonadal mosaicism in a family with adrenoleukodystrophy: Molecular diagnosis of carrier status among daughters of a gonadal mosaic when direct detection of the mutation is not possible

Summary Adrenoleukodystrophy is a severe, X-linked neurological disease that has been shown to be linked to DNA markers from Xq28. We tested several families with these markers and, in one family, found two apparent recombination events between DXS52 and the disease. Expansion of the study to include other tests and several others markers from Xq28 led us to conclude that recombination probably had not occurred and that, instead, the mutation in this family had a mitotic origin and that the grandmother was a gonadal mosaic. For genes that have been cloned, it is often possible to demonstrate the presence or absence of a specific mutation in such families and to determine carrier status on that basis. This is not possible when the gene has not been cloned. We therefore describe a method that can be employed by a molecular diagnostic laboratory to discriminate between people who inherit the same RFLP halpotype, with or without the mutation, from a parent with gonadal mosaicism in diseases where direct gene analysis is not yet possible.