P. De Mazancourt

Y-STR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa

Identification of spermatozoa is the biological evidence most often sought in specimens from rape victims. Absence of spermatozoa usually terminates biological investigations, and the victims testimony can be contested. We assessed the utility and reliability of PCR amplification using Y-chromosomal STR polymorphisms in specimens from female victims of sexual assault with negative cytology. One hundred and four swabs without spermatozoa detected by cytology were collected from 79 alleged sexually assaulted female victims and amplification of Y-STR and of amelogenin was performed.Overall, Y-chromosome was detected and evidenced sexual penetration in 28.8% of swabs. In the population of victims examined more than 48 h after the sexual assault, Y-STR were still evidenced in 30% of the cases. These results show that swabs should be taken from victims for Y-chromosome DNA typing even after long delays between sexual assault and medical examination.

Thrombin generation in patients with factor XI deficiency and clinical bleeding risk.

Summary.  Factor XI (FXI) deficiency is a rare bleeding disorder. Most patients with FXI deficiency are mild bleeders but certain patients with similar FXI activity exhibit different bleeding phenotype. Routine laboratory assays do not help physicians to estimate the individual bleeding risk in these patients. Thrombin generation test (TGT) is a more comprehensive, global function test of the clotting system. We investigated whether or not the bleeding tendency of patients with FXI deficiency is correlated with features of the TGT. Twenty‐four patients with FXI deficiency were divided in two groups: (i) severe bleeders (n = 9) and (ii) mild or non‐bleeders (n = 15). All severe bleeders had a personal history of surgery‐related severe bleeding. Thrombin generation (TG) was measured in platelet‐rich plasma (PRP) using a low concentration of tissue factor 0.5 pm. In patients exhibiting severe bleeding tendency, independently of their FXI level, a dramatically impaired TG was observed. For example, despite a low plasma FXI = 1 IU dl−1, a clinically non‐bleeding individual exhibited normal TG results whereas another patient with severe bleeding history and FXI = 40 IU dl–1 had a very low TG capacity. Low velocity and delayed TG were the main parameters suggesting a higher bleeding risk. DNA analysis of patients reported eight novel mutations of the FXI gene but neither mutation location nor secretion or not of the variant correlated with the bleeding tendency. The results of this study suggest that TG measurement in PRP may be a useful tool to predict bleeding risk in FXI deficiency and should be studied further in larger prospective clinical studies.

Fibrinogen angers with a new deletion (γ GVYYQ 346‐350) causes hypofibrinogenemia with hepatic storage

Summary.  Introduction: This study reports a family with chronically abnormal blood liver function tests (LFT) and congenital hypofibrinogenemia. The proposita had cirrhosis initially related to alcohol abuse and chronic viral hepatitis C (HCV), but abnormal LFT persisted even when alcohol intake was stopped and despite HCV treatment was efficient based on serum RNA negative testing.Results: Needle biopsy specimens of the proposita and her brother showed eosinophilic intra‐cytoplasmic inclusions that reacted strongly with fibrinogen antisera on direct immunofluorescence. Electron microscopic examination showed that the rough endoplasmic reticulum was filled with inclusions that consisted of densely packed, curved tubular structures arranged in a fingerprint‐like pattern. Coagulation studies revealed low functional and antigenic fibrinogen concentrations suggestive of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous deletion of the a7690 to g7704 nucleotides of the γ chain gene in the 3′end of exon 8 (g 7690_7704del14; Genbank access M10014); this deletion encompassed the splicing site at position 7703 and predicts in a new putative consensus splicing sequence (AATGgtatgtt). RNA was extracted from a liver specimen from the proposita’s brother. The cDNA obtained by reverse transcription polymerase chain reaction confirmed the usage of a newly generated donor site at position 7688 of the genomic sequence resulting in an in‐frame heterozygous 5 amino acid deletion (GVYYQ 346‐350; p.G372_Q376del) and that this mutation is responsible for a new splicing site at position 7688 of the genomic sequence.Conclusion: we suggest that the molecular defect in fibrinogen Angers results in an impaired assembly and causes defective secretion and hepatic storage of fibrinogen.

Forensic and police identification of “X” bodies. A 6-years French experience

The identification of X bodies is an everyday preoccupation in forensic pathology. This retrospective analysis studied all methods of identification and characteristics of unidentified bodies arrived in the Department of Forensic Medicine and Pathology (University Hospital R. Poincaré, Garches, France) during a 6-years period (2003-2009). The aim was to determine the identification methods used during all the forensic investigations, but also to study causes and manner of death in this sample of the population. A total of 9.1% of all autopsies were on X cadavers (217 cases out of 2384). On this total, only 134 of them have been included in our series after exclusion of archaeological and animal samples, but also of unidentified individuals or incomplete data available. Almost 28% of them have been identified with molecular biology (DNA), 23% with odontological examination, 7.5% with fingerprinting and 6.7% with autopsy data. Manner of death was mainly suicide (40.3%) especially by asphyxia following drowning, then accidental death (17.9%) especially consecutive to multiple trauma after traffic accident, acute carbon monoxide intoxication or carbonization in a fire. A total of 11.9% natural deaths were found (50% of them being of cardio-vascular origin) and 11.2% of homicides (with the use of firearm in a third of them). For 18.7% of X cadavers, the mode of death was undetermined. 46.4% of all unidentified bodies in our series were only identified by the police investigations, using physical recognition (direct or with photographs) or personal effects or identity documents in close relationship with the body. Our study highlights the fact that quite half of all unidentified bodies are inhumed with an identity not scientifically proved. Bodies which remained unidentified after all investigations represent 10.2% of X cadavers (if we consider a group of 176 cases composed of our study sample of 134 cases plus 24 subjects identified just before the autopsy and the 18 cases which remained unidentified) and 0.8% of all autopsies performed in the department.

Recurrent thromboembolism in a patient with β-thalassemia major associated with double heterozygosity for factor V R506Q and prothrombin G20210A mutations

Double heterozygosity for factor V R506Q and prothrombin G20210A mutations was identified in a 24-year-old man with beta-thalassemia major. The patient experienced a first thrombotic event at the age of 19 years and three recurrent thromboses in a short time interval, the third occurring while the patient was receiving long-term anticoagulant treatment. This case suggests that patients with major thalassemia and congenital thrombophilic mutations need intensive and long-lasting anticoagulant treatment. Thus, even if thrombotic events could be explained by a hypercoagulable state observed in patients with major thalassemia, after a first thrombotic event has occurred these patients should be screened for acquired and congenital thrombophilia.

LETTERS TO THE EDITOR: Four cases of hypofibrinogenemia associated with four novel mutations

parturition in a patient with severe Glanzmann’s thrombasthenia. Scand J Haematol 1981; 27: 159–64. 4 Boval B, Bellucci S, Boyer-Neumann C, d’Oiron R, Ciraru-Vigneron N, Adibert F,Wautier JL. Glanzmann thrombasthenia and pregnancy: clinical observations and management of four affected women. Thromb Haemost 2001; 86: P1154 [abstract]. 5 Kiefel V, Santoso S, Weisheit M, Mueller-Eckardt C. Monoclonal antibody-specific immobilization of platelet antigens (MAIPA): a new tool for the identification of platelet-reactive antibodies. Blood 1987; 70: 1722–6. 6 Bierling P, Fromont P, Elbez A, Duedari N, Kieffer N. Early immunization against platelet glycoprotein IIIa in a newborn with Glanzmann type 1 patient. Vox Sang 1988; 55: 109–13. 7 Laurian Y, Girma JP, Allain JP, Verroust F, Larrieu MJ. Absence of anamnestic response after transfusion of washed red blood cells in haemophilia A patients with antibody to factor VIII. Scand J Haematol 1982; 28: 233–7. 8 Meryman HT. Red cell freezing by the American National Red Cross. Am J Med Technol 1975; 41: 265–82.

Fibrinogen Paris IX: a case of symptomatic hypofibrinogenemia with Bβ Y236C and Bβ IVS7‐1G→C mutations

Fibrinogen is a 340-kDa glycoprotein circulating in the plasma as an assembly of three duplicate chains (Aa, Bb and c) [1]. As a result of the activation of the coagulation cascade, fibrinogen molecules are cleaved into fibrin monomers, which interact with each other and are subsequently cross-linked to form a fibrin network [2]. The three chains are encoded by three different genes, clustered in a 50 kb region on chromosome 4 (region 4q31.3). Low levels of fibrinogen are known as hypofibrinogenemia, and non-detectable levels correspond to afibrinogenemia [3]. Fewer mutations registered in the GEHT database as of February 2005 (release 20), have been described in the former cases (21 cases, 19 unique mutations) than in the latter (70 cases, 40 unique mutations) [4], possibly because hypofibrinogenemia is often unrecognized. Constitutional hypofibrinogenemia may be a consequence of frameshift mutations, missense mutations, or large deletions [5]. Afibrinogenemia is a consequence of either homozygosity or compound heterozygosity [6]. We report here on a patient with hemorrhagic, thrombotic and pregnancy complications and hypofibrinogenemia associated with two previously undescribed mutations. The patient was a 30-year-old woman who was initially referred because of a prolonged prothrombin time. She had recurrent epistaxis during childhood, a life-long history of easy bruising and menorrhagia. However she had appendicectomy at age 22 and teeth extractions without bleeding. Testing revealed a low fibrinogen level in both functional (70 mg dL) and immunologic assay (85 mg dL). Functional fibrinogen was determined by clotting method of Clauss with STA-fibrinogen reagent on STA analyser from Diagnostic Stago, (Asnières, France), immunologic assay was performed by radial immunodiffusion method with NOR-Partigen Fibrinogen Dade Behring (Marburg, Germany). Low level of fibrinogen was associated with a short prolongation of thrombin time (patient 26 s, control 20 s) and reptilase time (patient 25 s, control 22 s). At age 36 she presented a placental abruption at 20 weeks of pregnancy with a fibrinogen level of 40 mg dL. She delivered a dead fetus by emergency Caesarean section, under infusion of frozen plasmas, without hemorrhagic complications. She developed a distal vein thrombosis 10 days after the Caesarean section, treated with low molecular weight heparin during 3 weeks. The father of the patient suffered frommyocardial infarction at age 49 and died of a recurrence at age 60. The mother, 77 years old, had no history of spontaneous and no bleeding after appendicectomy and cholecystectomy. She had three pregnancies: her first pregnancy was complicated by postpartum hemorrhage without transfusion of blood products and death of the baby at 2 days of life. The second pregnancy had an unremarkable course, and preeclampsia occurred at 8 months of the third pregnancy. Her level of fibrinogen was 287 mg dL (functional assay) and 325 mg dL (immunologic assay). She had no personal history of venous thrombosis. The purified DNA was used for amplification by a polymerase chain reaction (PCR) of all the exons of the Aa, Bb and c chain genes with primers allowing intron–exon boundaries sequencing. Dideoxysequencing was performed with the DYEnamic ET Terminator Cycle sequencing kit (Amersham Biosciences, Piscataway, NJ, USA). The sequence numbering used in this report refers to Genbank accession number M64983 (Bb gene). Unless stated in the text, the amino acid numbering refers to the circulating chain (cleavage of the 30 N-terminal residues before secretion). The mother of the patient bears a heterozygous a5909g transition on the exon 5 of the Bb-chain gene responsible for a heterozygous Y236C missense mutation (Y266C when referring to the translation numbering). All coding sequences and intron–exon boundaries have been sequenced, and no other mutation was found. The patient inherited the heterozygous a5909g transition responsible for the Y236C mutation on the circulating protein, and she also bears a heterozygous Bb IVS7-1G fi C transversion. This mutation possibly affects exon 8 splicing. Correspondence: Philippe de Mazancourt, Laboratoire de Biochimie et Biologie Moléculaire, Hôpital Poincaré, 104 Bd Poincaré, 92380 Garches, France. Fax: +33 1 47 10 79 23; e-mail: philippe.de-mazancourt@rpc. ap-hop-paris.fr

A new electrophoretic variant of fibrinogen associated with venous thromboembolism, fibrinogen Bordeaux Aα Arg439→Cys

M. HA NSS , * C. VERGNES , L . RUGER I , P . FFRENCH* and P . DE MAZANCOUR T§ *Laboratoire d Hématologie, Hospices Civils de Lyon, CBPE, Lyon; Laboratoire d Hématologie, Hôpital Haut-Lévêque, Bordeaux; Service des Maladies du Sang, Hôpital Edouard Herriot, Lyon; and §Laboratoire de Biologie, CHI de Poissy-St-Germain, Poissy, and UVSQ EA2493, Faculté de Médecine Paris Ile de France-Ouest, Versailles, France

Molecular characterization of a novel mutation in the factor XIII A subunit gene associated with a severe defect and an adulthood diagnosis

N. TRIGUI,* C. FRÈRE, C. D’ERCOLE,§ H. CHAMBOST,– N. CHAPUIS,* C. POUYMAYOU, P. MORANGE and P. DE MAZANCOURT** *Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital Raymond Poincaré, Garches; Laboratoire d’HématologieHémostase, CHU Timone, Marseille; UMR 626, Faculté de médecine de la Timone, Marseille; §Service de gynécologieobstétrique, CHU Nord, Marseille; –Service de Pédiatrie et d’Hématologie Pédiatrique, CHU Timone, Marseille; and **EA2493, Faculté de médecine Paris-Ile de France Ouest, UVSQ, Versailles, France

A Bβ 14 Arg → Cys fibrinogen variant in a patient with thrombotic complications (fibrinogen St‐Germain III)

incidence of VTE events. As previously discussed [1], events that occurred at home and were diagnosed out of hospital were included in our studies. The incidence rate we observed in western Brittany was indeed higher than the rate found in a US community-based study [3] that used only hospital records, but was consistent, as regards DVT incidence, with a communitybased study performed in Sweden [10]. Our data raise questions about prevention in patients with cancer and hospitalized patients [8,9]. Further investigations are warranted to specify whether a preventive therapy was effectively implemented or not. To some extent, it hopefully was and obviously failed, and the next question is why? In conclusion, a straightforwardly recognizable situation, i.e. hospitalization and/or history of cancer, was present in 41.5% of VTE cases. In those situations, optimization of preventive therapies should be a priority.