Preimplantation genetic diagnosis of hemophilia A

Mutation spectrum of the F8 gene, genotyping strategies, and possible PGD approaches

Since the publication of the sequence of the F8 in 1984, more than 2000 gene mutations causing HA have been described and these are catalogued in the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk/ac/index.php) and Factor VIII Variant Database (http://www.factorviii-db.org/). In 2008, we had first published the mutation spectrum in the Taiwanese population [6]. Of 31 unrelated HA patients (19 severe and 10 moderate/mild males, and 2 severe females), 12 (38.7 %) and 1 (3.2 %) severe males were genotyped with INV22 and INV1 respectively. The F8 defects in the remaining 18 inversion-negative patients cover a wide spectrum, in which 17 different mutations were identified (10 missense and 3 nonsense mutations, and 2 small and 2 large deletions). Eleven of these mutations are novel and unique, confirming a high diversity of molecular defects in HA [6]. A systematic review for data from 30 studies on 5383 patients had been reported and showed 45 % of HA had INV22, 2 % INV1, 3 % large deletions, 16 % small deletions or insertions, and 28 % point mutations (15 % missense mutations, 10 % nonsense mutations, and 3 % splicing site mutations). In 4.6 % of patients, the mutation was unknown [12]. Overall, with the exceptions of recurrent INV22 and INV1, no mutation hot spots have been identified.

There are a number of different approaches for the genotyping of HA (Table 1). For reasons of rapid and smart screening, however, targeted mutation analysis for the recurrent INV22 and INV1 has become the first test assessed in patients (particularly in severely affected hemophiliacs). INV22 can be detected by Southern blotting or, more time- and labor-saving choice, by long-distance polymerase chain reaction (long-distance PCR) or inverse PCR (I-PCR) [13, 14]. INV1 is typically detected by multiplex PCR [15]. Other mutations responsible for HA are mostly point mutation and small deletion/insertion in the F8 gene and their spectrum is quite complex. In these cases, mutation can be detected by PCR with a number of screening methods (e.g., single strand conformational polymorphism, conformation sensitive gel electrophoresis, amplification and mismatch detection, denaturing gradient gel electrophoresis) followed by direct DNA sequencing [16–21]. For female patients with only one mutation detected and also in those females suspected to be carriers but no mutation could be found, gene dosage assays such as multiplex ligation-dependent probe amplification (MLPA) should be applied to screen for the underlying exon deletions since deletions in single allele usually escape detection by the PCR-based analysis, due to the masking of the non-deleted allele. In Fig. 1, we exemplified the MLPA finding of a female HA patient who was karyotyped as 45,X [22]/46,X,idic(X)(q21) [8] mosaicism. Her aberrant X-chromosome (idic(X)(q21)) do not contain the Xq22q28 (and thus F8 gene) and familiar follow-up studies demonstrate this anomaly is of de novo. PCR amplification for exon1-22 of the F8 is failure in patient but is successful in her parents. Through the MLPA analyses, it is evidenced that the patient carries an exon 1–22 deletion in the allele on her “morphologically-normal” X-chromosome, which is inherited from her mother (Fig. 1).

Genotype-phenotype relationship, genetic testing and preimplantation genetic diagnosis (PGD) in hemophilia A

MLPA multiplex ligation-dependent probe amplification, I-PCR inverse polymerase chain reaction, ARMS amplification refractory mutation system, NA not available

^aSee the review in Gouw et al., [12]

^bHA patients are clinically divided into three different severities based on the residual FVIII coagulant activity (FVIII:C): severe (FVIII:C??1 % of normal level), moderate (FVIII:C is 1–5 % of normal level) and mild (FVIII:C is 5–30 % of normal level)

Given the marked morbidity associated with severe HA, PGD has become a feasible option for couples at risk of having a child with HA since it reduces the risk of termination of affected pregnancies. Gender selection by fluorescence in situ hybridization (FISH) and transfer of only female embryos is a simple strategy for X-linked recessive disorders, such as HA, and has been adopted in many clinics [11]. However, in practice, gender selection is illegal in some countries (e.g., Taiwan) and methods allowing the correct and more definitive diagnosis of the HA status of every embryo are more desirable because the number of embryos available for transfer is increased. PGD involving whole genome amplification (WGA) step was broadly applied to mutation detection strategies, but the high rate of amplification bias renders WGA an imperfect option [22]. Recently, co-amplification of polymorphic microsatellite markers, linked with the targeted mutation, had been the gold-standard genotyping strategy for PGD [4, 23–26] (Table 1). A linkage approach using polymorphic markers located near the mutation allows monitoring the occurrence of allele dropout, a known problem associated with PCR amplification bias in PGD. Below, we describe our experience with two HA families seeking for PGD.