Jorge Di Paola, Assistant Professor of Pediatrics, University of Iowa, 200 Hawkins Dr, Iowa City, IA 52242, USA. Tel.: +1 319 384 4597; fax: +1 319 356 7659; e-mail: firstname.lastname@example.org
Summary. Hemophilia B is an X-linked bleeding disorder caused by the deficiency of coagulation factor (F)IX, with an estimated prevalence of 1 in 30 000 male births. It is almost exclusively seen in males with rare exceptions. We report a girl who was diagnosed with severe (<1%) FIX deficiency at 4 months of age. Cytogenetic studies in the patient showed a balanced translocation between one of the X-chromosomes and chromosome 14, with breakpoints at bands Xq27.1 and 14q32.3. Both parents were found to have normal chromosomes. Late replication studies by incorporation of 5-bromodeoxyuridine showed non-random inactivation of the normal X-chromosome, a phenomenon frequently seen in balanced X/autosome translocations. To map the breakpoint, fluorescent in-situ hybridization was performed. A PAC DNA probe, RP6-88D7 (which contains the FIX gene) hybridized only on the normal chromosome X as well as onto the derivative 14. Using a PAC DNA probe, RP11-963P9 that is located proximal to the FIX gene, we obtained signals on the normal and derivative X and also on the derivative 14. We conclude that the breakpoint is located within the DNA sequence of this clone mapping proximal to the FIX gene. Since the FIX gene seems to be intact in the derivative 14, the breakpoint may affect an upstream regulatory sequence that subjects the gene to position effect variegation (PEV).
Coagulation factor (F)IX deficiency (hemophilia B) is an X-linked recessive disorder with a prevalence of 1 : 30 000 male live births. Severity of bleeding usually correlates with levels of coagulation FIX activity and recurrent bleeding episodes in joints and muscles may lead to chronic arthropathy and incapacitating disease.
Phenotypic expression in females is extremely rare and depends on different genetic mechanisms. Homozygosity for the recessive allele, although in theory possible, has not been reported. Female heterozygotes for mutations in the FIX gene, also known as ‘carriers’, may present with bleeding symptoms due to lower than expected levels as a consequence of skewed X-inactivation of the normal X-chromosome [1–3]. Girls with Turner syndrome (TS) and mosaics TS with only one copy of a mutated FIX gene have also been described [4,5]. It has also been related to several other chromosomal abnormalities such as testicular feminization, or X/autosome translocations. Previous reports have described two females with clinical hemophilia due to X/autosome translocations [6,7]. The first report by Schroeder describes a girl with a translocation t(X;15) that presented with clinical bleeding and a FIX level of < 1%. In their chromosome analysis they found that the FIX gene was located in the derivative X and demonstrated skewed inactivation of the normal X-chromosome. In a similar report Krepischi-Santos et al. described a girl with severe hemophilia B as a result of a translocation t(X;1) and deletion of the FIX gene. Here we describe a girl diagnosed at 4 months of age with severe FIX deficiency as a result of a t(X:14) (q27.1;q32.3).
Materials and methods
The patient, now a 6-year-old girl, was diagnosed at 4 months of age due to excessive bruising. FIX level in plasma was found to be < 1%. FIX levels in both parents were within the normal range. No history of hemophilia or consanguinity in the family was found. The patient does not have any dysmorphic physical features.
During the first years of life she had multiple episodes of bleeding in different joints.
She is currently on prophylactic recombinant FIX infusions three times a week in order to prevent serious bleeding. Informed consent for this study was obtained from the parents following institutional guidelines.
The FIX level was measured in the patient and parents with a one-stage clotting assay using a FIX-deficient substrate plasma (Precision Biologicals, Darmouth, Nova Scotia, Canada) .
Cytogenetic studies and fluorescence in-situ hybridization
Peripheral blood samples were processed for high-resolution chromosome study using standard protocols. Twenty G-banded metaphases were analyzed in the patient and the parents. To map the breakpoint, fluorescent in-situ hybridization (FISH) was performed. Several DNA clones were used according to their physical proximity to the FIX gene and the breakpoint: a PAC DNA clone RP6-88D7 that contains the FIX gene, and a PAC DNA probe RP11-963P9, that maps distal to the FIX gene.
We also used BAC DNA probes RP11-35F15 and RP13-206I2 that are located proximal to the FIX gene. All clones were obtained from the human genomic library at Children's Hospital of Oakland Research Institute, BacPac Resource Center (Oakland, CA, USA). All clones were labeled for FISH analysis using the Nick translation kit according to the manufacturer's instructions (Vysis, Inc., Downers Grove, IL, USA). IgH (14q32.3) probe and an X centromeric probe were used as controls to identify chromosomes 14 and X, respectively. All clones are located in contig NT011786 (GenBank: http://www.ncbi.nlm.nih.gov/mapview/map).
Late replication studies
Late-replication studies to determine the X-inactivation pattern were carried out using standard protocol. Briefly, phytohemagglutinin-stimulated lymphocytes were cultured for 72 h at 37 °C in RPMI media supplemented with 20% fetal bovine serum. Six hours prior to harvest of cells, 5-bromodeoxyuridine (BrdU) was added to the cultures. Following arrest of cell division with colcemid (final concentration 0.1 µg mL−1), chromosomes were stained by the fluorescence plus Giemsa banding methods. Thirty cells were analyzed. The staining difference between active and inactive X-chromosomes permits the identification of the late replicating X. FISH analysis of the same cells with probes to the derivative and normal X allowed precise chromosome identification.
Sequencing of the FIX gene of the patient's genomic DNA was performed at the Beckman Research Institute, City of Hope National Medical Center. Genomic DNA was isolated from peripheral mononuclear cells. FIX DNA (4.2 kb) was sequenced in both directions. The sequence included the putative promoter region, all the coding regions, the splice junctions, and the 3′ terminal untraslated segment of the mRNA that contains the poly A addition sequence.
FIX activity level in the patient was measured as < 1%. FIX level in both parents was within the normal range.
Cytogenetic studies of the patient's cells showed an apparently balanced translocation, in all cells, between chromosome X and chromosome 14 with breakpoints at bands Xq27 and 14q32.3 (Fig. 1). Both parents had normal karyotypes.
The PAC DNA probe, RP6-88D7 (which contains the FIX gene) hybridized on the normal chromosome X as well as in the derivative 14. We obtained identical results with two BAC DNA probes RP11-35F15 and RP11-206I21 derived from a region that is distal to the FIX gene, indicating the presence of a more centromeric breakpoint.
Using a PAC DNA probe, RP11-963P9 that is located proximal to the FIX gene, we obtained three signals, on the normal and derivative X and also on the derivative 14 indicating that the breakpoint is within the sequence of this clone (Fig. 2). Since the FIX gene is transcribed toward the telomere, the proximal breakpoint is upstream of the FIX gene.
Late replication studies
Replication studies with 5-BrdU were performed in 30 cell preparations. All of them showed early replication of the derivative X and late replication of the normal chromosome X, demonstrating that the latter is the inactive one of the pair. The chromosomes were identified by FISH analysis (Fig. 3). To our knowledge, this methodology has been employed for the first time in X/autosomal translocations.
No mutations were found in the DNA sequence of the patient's FIX gene.
Although hemophilia B is a disorder seen almost exclusively in males, there are several reports of girls with clinical hemophilia in the medical literature [9–12]. Here, and in two of those cases, this occurred as a result of an X/autosome translocation and subsequent inactivation of the normal X-chromosome. It is well known that X inactivation usually follows a non-random pattern in cases of X/autosome translocations with the normal X inactivated.
In a report of a 104 cases from the UK, Waters et al. found in the majority of the cases the normal X-chromosome was preferentially inactivated, supporting the concept that either functional autosomal monosomy or X disomy may give rise to an abnormal phenotype and therefore is naturally avoided .
In our patient with a de novo balanced translocation t(X:14) (q27.1;q32.3), we have demonstrated by FISH and DNA sequence analysis that the entire FIX gene is translocated into the derivative chromosome 14. The actual breakpoint is located within the sequence of the clone RP11-963p9 that is approximately 1 Mb closer to the centromere than the FIX gene. Where the breakpoint in this balanced translocation is proximal to FIX, the breakpoints for the previous hemophilia-causing translocations were distal, suggesting that the entire locus may be unstable. We also demonstrated by 5-BrdU incorporation that the normal chromosome X is preferentially inactivated. In the two previous reports with similar translocations leading to hemophilia B in females, the inactivation studies showed similar results.
In the report by Schroeder , where no mutations were found in the FIX gene, the reason for gene malfunction was attributed to either positional effect secondary to placement of the gene into a new highly heterochromatic region, represented by the translocated tip of chromosome 15, or by a possible disruption of a regulatory region. In the second report by Krepischi-Santos , the gene was transected leading to the FIX deficiency. In our case, since the normal X-chromosome is inactivated, we first ruled out a mutation in the FIX gene on the derivative 14.
Genomic DNA sequence of her two copies of the FIX gene yielded normal results. It has been postulated that the translocation of a gene to a heterochromatic region may affect gene expression. However, in this case the region of chromosome 14 where the FIX gene was translocated is not highly heterochromatic. Therefore the breakpoint may be affecting an unidentified upstream regulatory sequence of the gene such as a silencer, enhancer or locus control region (LCR). LCRs are sequences of DNA that lie upstream from the gene and are essential for full gene expression by recruiting the transcriptional apparatus onto the gene promoter. Abnormalities in LCRs have been described in certain cases of β-thalasemias where deletions of the upstream regions of the β-globin gene locus caused absence of gene expression . Moreover, Miao et al. have shown a 65-fold increase of FIX levels in vivo, in a murine model of hemophilia B when a hepatic LCR was included in the transfected plasmids . This finding underscores the importance of this regulatory element in gene expression and may give a plausible molecular explanation for our case. Further studies in this patient and particularly in this chromosomal region may lead to a better understanding of the FIX gene regulation.
The authors thank Margaret Malik for her excellent technical support and Jeff Murray for help and suggestions. This work was supported by grants from the National Institute of Health K23 HL04460-01A1 and K12-HD27748.