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Genetic diagnosis of haemophilia and other inherited bleeding disorders F. PEYVANDI,* G. JAYANDHARAN,   M.CHANDY,   A.SRIVASTAVA,   S. M. NAKAYA, à M. J. JOHNSON, à A. R. THOMPSON, à A. GOODEVE, § I. GARAGIOLA,* S. LAVORETANO,* M. MENEGATTI,* R. PALLA,* M. SPREAFICO,* L. TAGL IABUE,* R. ASSELTA, – S. DUGA – and P. M. MANNUCCI* *Department of Medicine and Medical Specialities, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Luigi Villa Foundation, University of Milan, Milan, Italy;  Department of Haematology, Christian Medical College, Vellore, Tamil Nadu, India; àPuget Sound Blood Center and University of Washington, Seattle, WA, USA; §Division of Genomic Medicine, University of Sheffield, Sheffield, UK; and –Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy

Summary. Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand’s disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500.000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies

rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for 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 reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand’s disease and of rare bleeding disorders.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency (less factor/more bleeding). Haemophilia A and B are the most frequent inherited bleeding disorders. Together with von Willebrand disease (VWD), a defect of primary

haemostasis associated with a secondary defect in coagulation factor VIII (FVIII), these X-linked disorders include 95–97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits in both sexes, are rare, with prevalence of the presumably homozygous forms in the general population with prevalence of 1:500 000 for FVII deficiency and 1 in 2–3 million for prothrombin (FII) and FXIII deficiency. Carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children with coagulation disorders in developing countries where patients with these deficiencies

Correspondence: Flora Peyvandi, MD, PhD, Department of Medicine and Medical Specialities, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Luigi Villa Foundation, University of Milan, Milan, Italy. Tel.: +39 02 54125707; Fax: +39 02 54100125; e-mail: [email protected]

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Keywords: haemophilia A and B, genetic diagnosis, carrier detection and prenatal diagnosis, von Willebrand’s disease, inhibitor development, rare bleeding disorders


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rarely live beyond childhood and where management is still largely inadequate. 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. Dr G. Jayandharan describes the approaches to the genetic evaluation of haemophilia for better understanding its biology, detection of carriers and prenatal diagnosis. The authors suggest a feasible strategy and the recent advances that should facilitate genetic diagnosis of haemophilia for carrier detection and prenatal diagnosis in families with haemophilia A and B based on their expertise and experiences in molecular diagnosis. Dr A. R. Thompson reports the genotype characterization in 150 haemophilic patients with inhibitors from 135 families. This retrospective series suggests that genotype influences response to immune tolerance induction with 1 to 28 years).

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Immune tolerance induction (ITI) by frequent infusions of FVIII will often, after a few to several months, result in a clinical remission [17]. The relationship of genotype to response to ITI was suggested in an earlier report [18]; herein this retrospective series is updated. Genotypes were determined in 153 haemophilic patients with inhibitors from 138 families; 44 were low titre. For the 109 patients from 105 families with high-titre inhibitors (5–9585 BU mL)1), the clinical courses of their immune responses are summarized in Table 1. Of 30 on ITI with an inversion, 19 had a complete response, one had a partial response to a persistent low titre, five are pending and five failed. Of 12 others with an inversion and no ITI, two had spontaneous remissions and 10 had long-term persistence. Of 10 with high titres and large deletions, the sole responder of seven completing ITI had an inframe deletion of exon 24 and a peak titre of 12 BU mL)1. Overall, eight of nine with large gene deletions have long-term persistence. Of 10 with frameshift genotypes, two of the three responses involved runs of several As as did one of three with long-term persistence and no ITI; three failed ITI. The one failure of four with splice junction genotypes had a 300 BU mL)1 peak titre, the other three were C mutation was frequent among Jews originating from the island of Djerba [29]. Our preliminary results on 161 families suggest a founder effect for (i) the LMAN1 gene: the Met1Thr mutation occurring in Italian patients, responsible of combined FV + FVIII deficiency; (ii) the FVII gene: Gln100Arg frequent in Europe; (iii) the FXIII gene: Arg77His frequent in Iran and (iv) the FX gene: Gly222Asp occurring in Turkish patients coming from Iran, Turkey and Germany. Nonetheless, other studies are necessary to confirm these observations. Genotype–phenotype relationships The complete absence of a coagulation factor probably occurs only with large gene deletions [30]. ÔNullÕ mutations predicting the production of truncated proteins or of unstable mRNAs (partial deletions, out-of-frame insertions, splicing abnormalities, nonsense mutations) are usually associated with very low or undetectable plasma factors and severe clinical manifestations. The effect of missense mutations is less homogenous: while in some instances they lead to severe factor deficiency, in others they are associated with partial deficiencies and milder clinical manifestations [31]. Some missense mutations could even cause the production of mutant proteins with heightened procoagulant properties associated with thrombotic phenotypes, usually transmitted as autosomal dominant traits. A better knowledge on the mutational spectrum of RBDs and the establishment, whenever possible, of genotype–phenotype correlations can represent a useful tool to prevent them through prenatal diagnosis and to develop new treatment strategies. Expression studies Ex vivo expression studies of a number of novel mutations and characterization of the trafficking and secretion of the corresponding recombinant proteins have significantly helped to describe the mechanism of the deficiencies. In some instances – as, for example, missense mutations in the genes coding for fibrinogen [32], FVII [33], FX [34] and FXI [35] – mutant proteins are produced normally but not secreted because of impaired folding and/or conformational changes of recombinant protein, causing their retention by the quality control system of the secretory pathway, eventually leading to intracellular degradation or accumulation [36,37]. In others, the

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mutant recombinant protein is fully secreted with abnormal functional properties. Ex vivo expression studies have proved to be an invaluable tool to understand the nature of the genetic defect and to unravel the underlying molecular mechanism of the deficiencies, an essential prerequisite for future innovative therapeutical approaches.

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