Editor—Phenotypic expression of X linked disorders in females may be the result of an X chromosome anomaly or homozygosity for the mutated gene, but is probably most frequently the result of skewed X chromosome inactivation. Skewed X inactivation may be the result of a chance event, but may also be because of genetic factors.1 We report here the results of X inactivation analysis in a family with haemophilia B, which showed extremely skewed X inactivation both in a female haemophilia patient and in her non-carrier daughter, indicating a possible genetic influence on X chromosome inactivation in this family. Familial skewed X inactivation may interfere with carrier detection, since skewed X inactivation with the mutant gene on the inactive X chromosome may lead to a normal phenotype in a carrier.

The patient was a 40 year old female who belonged to a family with moderate haemophilia B2 (fig 1). Plasma factor IX (FIX) activity was 0.02-0.03 units/ml, which was identical to the FIX activity in the affected male relatives. She had suffered from bleeding episodes after tooth extraction as a child and heavy bleeding from the episiotomy after her first delivery. A C→T transition, causing mutation S360L in exon 8 of the factor IX gene, was found in the male relative with haemophilia using single strand conformation analysis (fig 1, II.4). Both the female haemophilia patient and her mother (III.1 and II.3) were heterozygous for the mutation, whereas her two daughters (IV.1 and IV.2) did not have the mutation.

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Figure 1

Pedigree of haemophilia family modified from Ørstavik et al.2 The mutation 360L was identified in II.4, II.3, and III.1, but not in IV.1 or IV.2.

The X chromosome inactivation pattern (XIP) was determined by PCR analysis of a polymorphic trinucleotide repeat in the first exon of the androgen receptor (AR) gene which, after digestion with the methylation sensitive enzyme HpaII, gives a PCR product from the inactive X chromosome only.3 XIP was measured as the ratio between the PCR products from the two X chromosomes and scored as random (ratios 50:50-80:20) or skewed (ratios >80:20-95:5). When one band only was visible, the pattern was scored as extremely skewed (ratios >95:5). DNA from a girl with a deletion within chromosome Xp22 who had previously been shown to have a skewed XIP was used as a control. Samples containing mixtures of DNA from two males with well separated bands were run as quantitative controls. The most extreme mixture that gave two visible bands was 95:5. The results of X inactivation analysis are shown in fig 2. Each allele at the AR locus gives two equally strong bands on a denaturing polyacrylamide gel owing to the two complementary DNA strands migrating slightly differently during electrophoresis. After digestion of the patient’s DNA withHpaII one double band only, the band inherited from the father, was visible. The patient therefore had an extremely skewed XIP with the maternally inherited X chromosome as the active X chromosome in the majority of the cells. Identical results were obtained with DNA from cultured fibroblasts. Thus, extremely skewed X inactivation is most probably the cause of haemophilia in this female. The patient’s oldest daughter also had an extremely skewed pattern, since after HpaII digestion the paternally inherited double band was the only visible band. The second daughter, the patient’s mother, and a paternal aunt all had a random pattern. No other females of the family were available for X inactivation analysis.

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Figure 2

X chromosome inactivation analysis at the androgen receptor locus. −=undigested DNA, +=HpaII digested DNA. Lane 1: patient’s mother (II.3, random XIP). Lane 2: patient’s father (II.2). Lane 3: patient (III.1, extremely skewed XIP). Lane 4: patient’s daughter (IV.2, random XIP). Lane 5: patient’s daughter (IV.1, extremely skewed XIP). Lane 6: patient, fibroblast DNA (extremely skewed XIP). Lane 7: control sample, girl with a deletion within chromosome Xp22 and a known skewed XIP. Lanes 8-12: mixture of DNA from two males in ratios 5:95, 20:80, 50:50, 80:20, 95:5.

The finding of two members of a family with an extremely skewed XIP could be a chance occurrence. However, an extremely skewed pattern is rare since it was not found in any of 148 blood donors.4 A skewed XIP may be caused by selection against an unidentified deleterious gene on the X chromosome, which has been claimed to be an important cause of familial skewed X inactivation.5 A greater opportunity for selection exists in blood cells with more cell divisions than in most other tissues. Since extreme skewing was also found in fibroblast DNA from our patient, a selection mechanism in this family does not seem likely, but cannot be excluded.

A skewed pattern may also be the result of a genetic influence on X inactivation, which is in agreement with a previous report of more than one affected female in a family with haemophilia B.6 TheXIST (X inactivation specific transcript) gene, located at Xq13, is expressed from the inactive X only and is a candidate gene for mutations giving rise to skewed X inactivation.7 Marker analysis along the X chromosome of DNA from the patient, her parents, and two daughters showed that the daughters had inherited identical alleles from their mother, which were the mother’s paternally inherited alleles, including the markers DXS559 located between AR andXIST, and DXS56 located distally toXIST (fig 1). An exception was the marker DXS987, located on the distal part of Xp, where the two sisters had inherited different alleles from their mother. No information was available distally to this marker, since the more distal marker DXS6807 was uninformative. Thus, the extremely skewed X inactivation in our family does not cosegregate with XIST. Furthermore, the AR allele on the active X chromosome in the haemophilia patient was her maternally inherited allele, whereas the AR allele on the active X chromosome in her daughter was the allele inherited from her healthy maternal grandfather.

The occurrence of extremely skewed XIP in a mother and her daughter in this family may be because of a genetic influence on skewing of X inactivation, but a chance event cannot be excluded.