Clin Genet 2016: 89: 44–54 Printed in Singapore. All rights reserved

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12613

Original Article

X-chromosome inactivation in female patients with Fabry disease Echevarria L., Benistan K., Toussaint A., Dubourg O., Hagege A.A., Eladari D., Jabbour F., Beldjord C., De Mazancourt P., Germain D.P.. X-chromosome inactivation in female patients with Fabry disease. Clin Genet 2016: 89: 44–54. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 Fabry disease (FD) is an X-linked genetic disorder caused by the deficient activity of lysosomal α-galactosidase (α-Gal). While males are usually severely affected, clinical presentation in female patients may be more variable ranging from asymptomatic to, occasionally, as severely affected as male patients. The aim of this study was to evaluate the existence of skewed X-chromosome inactivation (XCI) in females with FD, its concordance between tissues, and its contribution to the phenotype. Fifty-six females with FD were enrolled. Clinical and biological work-up included two global scores [Mainz Severity Score Index (MSSI) and DS3], cardiac magnetic resonance imaging, measured glomerular filtration rate, and measurement of α-Gal activity. XCI was analyzed in four tissues using DNA methylation studies. Skewed XCI was found in 29% of the study population. A correlation was found in XCI patterns between blood and the other analyzed tissues although some punctual variability was detected. Significant differences in residual α-Gal levels, severity scores, progression of cardiomyopathy and deterioration of kidney function, depending on the direction and degree of skewing of XCI were evidenced. XCI significantly impacts the phenotype and natural history of FD in females. Conflict of interest

Nothing to declare.

L. Echevarriaa,b , K. Benistanb , A. Toussaintc , O. Dubourgd , A.A. Hagegee , D. Eladarif , F. Jabbourb , C. Beldjordc , P. De Mazancourtg and D.P. Germaina,b,g a Division of Medical Genetics, University of Versailles, Montigny, France, b Assistance Publique – Hôpitaux de Paris (AP-HP), Referral Center for Fabry Disease and Inherited Disorders of Connective Tissue, Garches, France, c Laboratory of Biochemistry and Molecular Biology, University Paris V Descartes, Paris, France, d Department of Cardiology, University of Versailles, Boulogne, France, e Department of Cardiology, HEGP (APHP), f Department of Physiology, HEGP (APHP), University Paris V Descartes, Paris, France, and g UFR des sciences de la santé, University of Versailles, Montigny, France

Key words: enzyme replacement therapy – Fabry disease – heterozygotes – phenotype – X-chromosome inactivation Corresponding author: Prof. Dominique P. Germain, MD PhD, Division of Medical Genetics, University of Versailles, 78180 Montigny, France. Tel.: +0033147104435; fax: +0033147104436; e-mail: [email protected] Received 25 November 2014, revised and accepted for publication 12 May 2015

Fabry disease (FD, OMIM #301500) is a rare, progressive X-linked genetic disease due to mutations in GLA (OMIM 300644), a 14-kb house-keeping gene that maps to Xq22.1 (GRCh38.p2 X:101,397,803–101,407,925 reverse strand) and encodes for α-galactosidase A (α-Gal, EC 3.2.1.22; Uniprot P06280), a ubiquitous lysosomal acid hydrolase. Males with severe mutations in GLA have virtually no residual α-Gal activity and develop classic FD with onset of symptoms (dysesthesia,

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gastrointestinal disturbances, angiokeratomas, autonomic dysfunction) in childhood and, with advancing age, increasing risk of developing life-threatening complications involving vital organs, including progressive renal failure, stroke and hypertrophic cardiomyopathy with myocardial fibrosis and arrhythmias (1–3). Due to the X-linked inheritance, heterozygous females have historically been described as asymptomatic carriers, but evolving knowledge has shown that females present a heterogeneous clinical spectrum that ranges

X-chromosome inactivation in Fabry disease from asymptomatic to clinical severity equal to that of males (4–6). Similarly, measurement of α-Gal activity in plasma or leukocytes, the reference method for the laboratory confirmation of the diagnosis in male patients, is often inconclusive in female patients who can have enzymatic activities ranging from low to normal values. The bases underlying the variability of the phenotype in females are still poorly understood, but a role of X-chromosome inactivation (XCI) has been hypothesized. XCI is the mechanism by which gene dosage equivalence is achieved between female eutherian mammals with two X chromosomes and male mammals with a single X chromosome (7). XCI occurs at random within females (8) resulting in an average inactivation ratio of 50:50 with normal distribution. Assuming a Gaussian distribution of maternal vs paternal X-inactivation, a percentage of females will have skewed XCI which is arbitrarily defined as more than 75% (80% for some authors) of cells showing, without any obvious explanation, a preferential inactivation of the same X chromosome (allele ratios >75:25) (9). Highly skewed patterns (allele ratios >90:10) occur but are uncommon (10). Skewed XCI has been described in other X-linked disorders in which disease severity appears to correlate with the direction and degree of skewing of inactivation (11, 12). In the case of FD, there have been conflicting results regarding the X-inactivation profiles of heterozygous females. Two early studies found X-inactivation to be a major factor in determining the clinical severity of heterozygous females (13, 14), whereas two subsequent studies reported that X-inactivation was random in heterozygotes for FD, ruling out its importance in explaining the phenotypic variability (15, 16). With the aim of further understanding the role of XCI in the phenotype of heterozygous females for FD, we have investigated the existence of tissular variation of XCI patterns in females with FD and have evaluated its contribution to the variability of disease manifestations and clinical expression. Patients and methods Subjects

Fifty-six consecutive female patients with a confirmed diagnosis of FD followed at the Division of Medical Genetics of the University of Versailles were enrolled. All patients gave written informed consent prior to participation. Physical examination

For each patient, disease severity was assessed by a thorough physical examination conducted by two senior physicians (DPG, KB) experienced in the care of FD patients. Two global severity scores, the Mainz Severity Score Index (MSSI) (17) and the DS3 (18), were calculated and used to assess the global burden of the disease. The presence of angiokeratomas was assessed

by physical examination (DPG) which allowed to classify their distribution into four categories: absent, rare, numerous or widespread. Renal function

Kidney function was assessed through measured glomerular filtration rate (mGFR) using the isotopic 51 Chrome Ethylenediaminetetraacetic acid (51 Cr EDTA) method. Cardiac structure

Left ventricular mass index (LVMi) was measured using cardiac magnetic resonance imaging. Measurement of α-Gal activity

Alpha-Gal activity was measured in peripheral blood leukocytes using the method previously described (19). Samples collection

Biological samples were obtained from all patients who gave written informed consent. All 56 patients gave written consent for urine sample and mouth swab collection, whereas 53 and 52 patients gave consent for blood sample and skin biopsy, respectively. Peripheral blood was drawn in tubes containing EDTA as anticoagulant. Mouth epithelial cells were collected through rubbing the patients’ cheeks inside the mouth with a cotton swab. Two skin biopsies were obtained and directly embedded in 200 μl RNAlater® solution (Qiagen, Hilden, Germany). One urine sample was obtained and centrifuged at 650 g for 5 min at 4∘ C. Cellular pellets were washed twice in phosphate buffered saline (PBS) 1X. All samples were stored at −80∘ C until use. XCI analysis

For each tissue, genomic DNA was extracted using QiaAmp DNA Mini Kit (Qiagen) following manufacturer’s instructions. X-inactivation patterns were determined by studying the methylation status of the polymorphic (CAG)n repeat region located within exon 1 of the human androgen receptor (HUMARA) gene (20) with minor modifications. The degree of XCI was determined from the area under the smaller (C1 ) and larger (C2 ) peaks as follows: peak area of [XC1 digested /XC1 nondigested /(XC1 digested /XC1 nondigested + XC2 digested /XC2 nondigested )]. Homozygous patients for HUMARA were studied for the polymorphic repeat region within the proprotein convertase subtilisin/kesin type 1 inhibitor gene (PCSK1N) (21). In females with skewed X-inactivation and available DNA sample from affected male relatives, informed consent was obtained from the latter and the predominant expression of the mutant or the wild-type GLA alleles was inferred from the study of the segregation of polymorphic markers on both sides of GLA (HUMARA,

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Echevarria et al. PCSK1N, and SLITRK4) (21) within families. To define XCI patterns, the study population was divided into three groups: (i) the R (Random) group included females who exhibited random X-inactivation in at least three or all four tissues, (ii) the W (Wild-type) group consisted of females whose skewed inactivation could be showed in at least two tissues with predominant expression of the wild-type allele and (iii) the M (Mutant) group represented females with skewed X-inactivation in at least two tissues and predominant expression of the mutant GLA allele. XIST minimal promoter mutation analysis

To detect potential mutations altering XCI, XIST minimal promoter was amplified and sequenced in patients with skewed X-inactivation patterns in all four tissues, using primers previously described (22). Statistical analysis

Statistical analyses were carried out using GraphPad Prism version 5.03 for Windows. Categorical variables are expressed as mean ± standard deviation (SD). Pearson’s correlation coefficient (R) or Fisher exact test was used to make correlation analyses and compare XCI distribution patterns among the different tissues or group populations when appropriate. In addition, regression analysis was performed and 95% prediction intervals were generated for each correlation analysis. One-way analysis of variance (anova) followed by Bonferroni post hoc testing was used to compare groups of means ± SD. Statistical significance was assumed at p < 0.05. Results Patients’ demographics and clinical phenotype

Fifty-six heterozygous females [45.75 year ± 15.15 (mean ± SD), range 20–68 years] from 35 unrelated families participated in the study. Of them, 51 (91%) were informative for the CAG polymorphic region in the HUMARA gene. The five noninformative patients were subsequently analyzed using the PCSK1N locus (21) which left only one noninformative case. Skewed XCI was found in 16/53 (30.2%), 10/52 (19.2%), 14/49 (28.6%), and 12/52 (23.1%) of blood, buccal smears, urine and skin samples, respectively (Fig. 1a). Pair-wise correlation coefficients between blood and the three other analyzed tissues were calculated for all analyzed subjects (n = 55). Statistically significant correlations were found for all three pair-wise comparisons (r > 0.5, p < 0.0001). The correlation coefficient was more divergent between blood and skin (r = 0.57, p < 0.0001) than between blood and buccal or urinary epithelia (r = 0.71, p < 0.0001) (Fig. 1b–d). Concordance was generally observed in XCI patterns between blood and the other tissues within individuals. However, some tissular differences were detected for five patients (9%). Three patients (Table 1, P#6, P#46 and

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P#51) were found to have skewed XCI in blood while they were randomly inactivated in all other three tissues. In contrast, two patients (Table 1, P#10 and P#21) were found to be randomly inactivated in blood while they were skewed in at least two of the three other tissues. As a consequence, these five patients would have been wrongly classified as random (or skewed) if only blood XCI had been evaluated. It is worthy to note that no skewing in opposite directions was evidenced in any patient. The X-inactivation patterns of the different tissues were also analyzed according to age (40 years, n = 35). No differences in XCI distribution were observed for buccal smears or urinary epithelia between the two age groups (data not shown). In blood, there was a trend in favor of more frequent skewed X-inactivation patterns in women aged >40 (37.1%) than in younger ones (17.6%), although the difference was not statistically significant (p = 0.21), probably due to the limited number of samples. The mean total MSSI was 18.74 (n = 55, range 1–61), with 1 female (Table 1, P#27) aged 65, being severely affected (MSSI ≥ 40), 18 moderately affected (20 < MSSI < 40) females (57.2 years ± 9.85), and 36 mildly affected (MSSI ≤ 20) females (39y ± 13.46). The mean DS3 score was 6.42 (n = 55, range 0–26). Considering DS3 score, 5 females (63.8 years ± 4.3) were classified as severely affected (DS3 > 12); 12 females (54.25y ± 11.7) were moderately affected (8 ≤ DS3 ≤ 12), and 38 females (39.7y ± 13.6) were mildly affected (DS3 < 8). A strong positive correlation was found between MSSI and DS3 scores (Pearson’s r = 0.92, p < 0.0001). XCI studies and family segregation analysis yielded the following results: group R included 39 females (71%), group W consisted of 6 individuals (11%) and group M comprised 10 subjects (18%). Detailed demographics, biological, genetic and clinical characteristics are shown in Table 1. XIST minimal promoter mutation analysis

In the patients with skewed X-inactivation in all four tissues, sequence analysis of the XIST minimal promoter did not reveal any mutation in the amplified fragment (data not shown). α-Gal activity and X-inactivation patterns

Leukocyte α-Gal activity was available for 54 patients. Alpha-Gal activity was compared in the W, R, and M groups. Patients with randomly inactivated X chromosomes (R) had approximately 50% α-Gal enzyme activity compared to normal. Patients exhibiting skewed XCI ratios in leukocytes and mainly expressing the wild-type allele (W) showed residual α-Gal A activity levels between 80% and 100% of those of controls, while patients with skewed XCI ratios and predominant expression of the mutant GLA allele (M) had very low or absent residual enzyme activity (Fig. 2). These findings were confirmed when leukocyte α-Gal activity levels

X-chromosome inactivation in Fabry disease (a)

(b)

(c)

(d)

Fig. 1. Tissular X-chromosome inactivation (XCI) distribution in 55 female patients with Fabry disease (FD) and correlation studies within the four analyzed tissues. (a) Tissue-specific distribution of XCI ratios in four tissues (blood, buccal smears, urinary epithelia and skin) in heterozygous females for FD (n = 55). (b–d) Pair-wise comparisons of XCI-ratio correlations were analyzed between blood and mouth smears (b), urinary epithelia (c), and skin (d). XCI values were expressed for the first (smaller) allele. Black lines represent 95% prediction intervals. The coefficient of correlation (R) was obtained for each pair-wise comparison (p < 0.0001).

were compared in W, R and M patients according to their XCI patterns analyzed separately in blood, mouth or urinary cells. No correlation was found when compared to XCI patterns obtained in skin (Fig. S1, Supporting Information). In order to evaluate if GLA mutations influenced our results, the correlation between leukocyte α-Gal activity and the type of mutation was also evaluated. With this aim, GLA mutations were classified into three categories: “mild missense” (n = 3) [or late-onset as commonly accepted in the literature: p.Asn215Ser (25) and p.Arg301Gln (43)], missense, and others (nonsense, splice-site, frameshift, small insertions and small deletions) (Table 1). No statistically significant correlation was found between enzyme activity and the type of GLA mutation in this study population (Fig. S2). These results confirm that α-Gal enzyme activity variability in heterozygous patients is mainly associated with XCI. Clinical severity scores and tissular X-inactivation pattern in heterozygous females for FD

MSSI and DS3 were calculated for all females and compared between the three groups (Fig. 3a,b). Individuals from W group (n = 6) had milder disease severity with few clinical symptoms (MSSI 7.5 ± 4.7, DS3 3.0 ± 2.7).

Among them, only one patient (P#49), aged 66 years and with comorbidities (medical history of uncontrolled hypertension, obesity, and diabetes), presented with cardiac symptoms. In contrast, patients belonging to the M group (n = 10) showed significantly higher severity scores (MSSI 30.30 ± 13.65, DS3 11.14 ± 6). Six of them (60%) showed at least one major organ involvement. Lastly, patients with random XCI (R group, n = 39) showed intermediate MSSI and DS3 scores (MSSI 17.51 ± 10.78, DS3 5.73 ± 4.47). Thirteen of them (33%) presented at least one major organ involvement and were aged over 40 years. Statistical analysis showed significant differences in both MSSI and DS3 values between the R and M groups and between W and M groups. Although a difference was observed in the severity score values between W and R groups, it was not statistically significant, probably due to the limited number of individuals in group W (n = 6). MSSI and DS3 scores were plotted against age for the three groups (R, W and M). In all three groups, MSSI and DS3 scores increased with age in agreement with the relentless progression of lysosomal storage of undegraded substrates. However, both MSSI and DS3 scores were consistently higher and slopes were steeper for patients in M group than in the randomly inactivated patients. In contrast, patients with skewed

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48

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39

Patient No.

56 66 45 26 41 50 68 20 34 22 28 33 43 47 51 41 67 51 54 49 65 53 54 33 29 56 65 63 56 49 63 30 37 34 66 37 25 62 52

Age (years)

50 20 69 31 55 19 57 34 63 40 15 65 88 7 7 31 42 54 40 53 17 129 89 77 22 47 3 0, 84 20 38 58 45 0 31 33 27 46 52 79

α-Galactosidase activity (%) 34 16 22 2 9 13 37 9 1 3 15 4 12 19 29 15 28 7 18 17 37 7 3 15 17 17 61 22 25 27 40 1 29 18 38 8 10 25 6

MSSI 15.63 6.99 7.65 3 2.6 4.96 15.82 1.2 0 2 9.16 1.3 4.66 4.82 11.83 1.83 8.82 4.7 6.66 8.4 11.4 1.49 3.32 1.7 4.4 3.83 26 8.8 5.8 7.32 15.2 0 7.33 4.1 8.43 2.8 2.66 6.66 1.66

DS3 47.3 61.9 98.4 80.1 NA 81.2 33 115.4 78.5 103.2 47.3 115.4 107.4 NA 5a 101.5 69.3 94.5 66.9 82.9 59.9 NA 104 97.4 61.5 77.9 5a 75.5 88.7 NA 5a 105 34.1 118.9 58.9 105 107.4 62.9 102.7

mGFR (ml/min/1.73 m2 ) 68.48 NA 67.61 62.41 85.22 49.71 120 NA 60.23 48.04 55.03 58.27 104.43 99 86.09 54.64 138.1 58.47 53.4 103.19 152.21 49.58 57 57.92 56.4 59 197 75.4 113.48 73.05 203.34 91 116.6 55.8 89.88 55 45 NA 68

LVMi (g/m2 ) R R R R R R R R R W M R R M M R R R R R M W W R R R M NA R R R R M R R R R R W

XCI status c.118C>T c.1081_1100del c.797A>T c.679C>T c.644A>G c.1025G>A c.1033-1034del c.884T>G c.770C>T c.713G>A c. 334C>T c.467C>A c.644>G c.1145_1149del c.679C>T c.1117G>A c.548G>A c.770C>T c.1065C>A c.154T>C c.770C>T c.770C>T c.797A>T c.916C>T c.154T>C c.194+1G>A c.901C>T c.782dupG c.884T>G c.1117G>A c.778G>A c.770C>T c.154T>C c.1232G>A c.547+398insGGTA c.593T>C c.593T>C c.548G>A c.983G>A

GLA c.DNA nucleotide change p.(Pro40Ser) p.(Gly361ArgfsX7) p.(Asp266Val) p.(Arg227ter) p.(Asn215Ser)b p.(Arg342Gln) p.(Ser345ArgfsX29) p.(Phe295Cys) p.(Ala257Val) p.(Ser238Asn) p.(Arg112Cys) p.(Ala156Asp) p.(Asn215Ser)b p.(Cys382TyrfsX15) p.(Ala227ter) p.(Gly373Ser) p.(Gly183Asp) p.(Ala257Val) p.(Asn355Lys) p.(Cys52Arg) p.(Ala257Val) p.(Ala257Val) p.(Asp266Val) p.(Gln306ter) p.(Cys52Arg) Unknown p.(Arg301ter) p.Trp262LeufsX3 p.(Phe295Cys) p.(Gly373Ser) p.(Gly260Arg) p.(Ala257Val) p.(Cys52Arg) p.(Gly411Asp) Unknown p.(Ile198Thr) p.(Ile198Thr) p.(Gly183Asp) p.(Gly328Glu)

Predicted effect on the protein

Table 1. Clinical phenotypes, α-Gal activities, GLA genotypes and X chromosome inactivation patterns in 56 heterozygous females affected with Fabry disease

(23) (24) (25) (26) (26) (27) (28) (29) This study (30) (31) This study (26) This study (26) (32) (33) This study (34) (35) This study This study (25) (36) (35) This study (37) (34) (29) (32) This study This study (35) (24) This study This study This study (33) (1)

Reference (GLA mutation)

Echevarria et al.

35 24 20 21 67 41 53 68 52 66 27 48 64 27 51 24 53

Age (years)

4 117 49 8 33 64 8 16 99 102 41 155 16 30 47 25 39

α-Galactosidase activity (%) 15 11 3 18 38 26 18 38 20 15 16 11 34 8 25 10 31

MSSI 5.99 1.33 0 5.66 17.3 9.5 8.1 15 3.9 8.7 2.9 2.3 10.4 3 11.16 2.9 9.2

DS3 98.3 102.9 96.8 110 53.9 96.9 74.1 55.2 94.2 79 146.3 116.7 65 97.1 130.1 84.4 5a

mGFR (ml/min/1.73 m2 ) 53 53.03 67.9 51.95 224.22 56.21 NA 141.07 86.9 73 NA 55.14 249.9 NA 74.6 46.1 NA

LVMi (g/m2 ) M W R R R M R M R W R R M R R R R

XCI status c.154T>C c.427G>A c.1072G>A c.606-607del c.1045T>C c.902CA c. 334C>T c.797A>T c.154T>C c.983G>A c.1117G>A c.606T>G c.154T>C c.1086-1098del c.59_73del c.59_73del

GLA c.DNA nucleotide change

p.(Cys52Arg) p.(Ala143Thr) p.(Glu358Lys) p.(Cys202ter) p.(Trp349Arg) p.(Arg301Gln)b p.(Arg342Gln) p.(Arg112Cys) p.(Asp266Val) p.(Cys52Arg) p.(Gly328Glu) p.(Gly373Ser) p.(Cys202Trp) p.(Cys52Arg) p. (Arg363SerfsX24) p.(Ala20_Trp24del) p.(Ala20_Trp24del)

Predicted effect on the protein

(35) (37) (38) (39) (40) (41) (27) (31) (25) (35) (1) (32) (42) (35) This study (34) (34)

Reference (GLA mutation)

α-Gal A, alpha-galactosidase A; FD, Fabry disease; LVMi, left ventricular mass index; MSSI, Mainz Severity Score Index; M, skewed X-chromosome inactivation with predominant expression of the mutant GLA allele; mGFR, measured glomerular filtration rate; NA, not available; R, random X-chromosome inactivation; W, skewed X-inactivation with predominant expression of the wild-type GLA allele; XCI, X chromosome inactivation. a Kidney transplant. b Missense mutations known to cause a late-onset phenotype of FD.

P40 P41 P42 P43 P44 P45 P46 P47 P48 P49 P50 P51 P52 P53 P54 P55 P56

Patient No.

Table 1. Continued

X-chromosome inactivation in Fabry disease

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Echevarria et al. However, no conclusion could be drawn for the late-onset group due to small sample size (n = 2) (Fig. S6). LVMi and tissular X-inactivation pattern

LVMi assessed through cardiac magnetic resonance imaging (MRI) increased in an age-dependent manner in all three groups. LVMi was higher and progressed more rapidly in the group of females with skewed inactivation and predominant expression of the mutant GLA allele. In contrast, LVMi was normal in young patients and its progression minimal in the group of patients with skewed inactivation and predominant expression of the wild-type allele (p = 0.02) (Fig. 4b). LVMi increased with age in all evaluated patients regardless of their type of GLA mutation (Fig. S7). Discussion Fig. 2. α-Galactosidase (α-Gal) A activities of female patients affected with Fabry disease (FD) stratified on their X-chromosome inactivation (XCI) status. Enzymatic α-Gal A activities were normalized and expressed as percentage in patients with random (gray) or skewed XCI favoring the expression of the mutant (black) or wild-type (white) GLA alleles. Data represent mean values ± SD. Group comparisons were analyzed by one-way analysis of variance (anova) and post hoc Bonferroni’s multiple t-test.

inactivation and preferential expression of the wild-type allele (W) showed milder disease and little, if any, disease progression (Fig. 3c,d). A semi-quantitative study of angiokeratomas was performed in all patients showing a link between their distribution and the direction and the degree of XCI. This held true when XCI was analyzed in all four tissues or only focused on skin samples (Fig. S3). To analyze whether phenotypic variability in heterozygotes was more strongly correlated with XCI or the type of GLA mutation, MSSI and DS3 were stratified on the category of mutation for all patients (n = 56). Results showed no significant differences in clinical severity between patients harboring late-onset (n = 3), missense (n = 39) or other mutations (n = 14) (Figs. S4 and S5). Kidney function and tissular X-inactivation pattern

mGFR decreased in an age-dependent manner in all three groups. However, in females with skewed inactivation and predominant expression of the mutant GLA allele (M), mGFR values were consistently lower and the slope of deterioration of kidney function was steeper than in the other two groups. In contrast, mGFR was normal and renal progression minimal in the group of patients with skewed inactivation and predominant expression of the wild-type allele (Fig. 4a). These results were confirmed when XCI analysis was focused only on urinary cells (data not shown). In female patients with classic FD, stratification on the category of GLA mutation did not reveal any major difference in glomerular filtration rate slopes between patients with missense or other types of mutations.

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Heterozygous females for X-linked disorders have historically been considered as asymptomatic or mildly symptomatic due to random XCI leading to a mosaic of cell populations, the expression of the wild-type allele in approximately 50% of cells being usually sufficient to spare heterozygotes from the clinical manifestations of the disease (44). XCI has therefore been historically proposed as the main mechanism underlying penetrance and expressivity of X-linked diseases in heterozygous patients (45). Likewise, highly skewed XCI favoring the expression of the mutant allele has been proposed as a mechanism to explain the occasional development of clinical symptoms in heterozygous patients for certain X-linked diseases (12, 46). In our study, a large majority of heterozygous females (71%) showed an unskewed XCI pattern. These results are in accordance with those obtained from electron microscopy examination of cardiac and renal biopsies from symptomatic heterozygotes with FD showing two cell populations, one with and one without glycosphingolipid accumulation (47). The 16 remaining heterozygous females (29%) were found to exhibit skewed XCI. Among them, 6 females predominantly expressed the wild-type and 10 the mutant GLA allele, indicating that there are no selection mechanisms favoring the wild-type GLA allele. Altogether, our data fit the Gaussian distribution of XCI in the general population (8) and studies on other X-linked genetic diseases (48, 49). Given our findings that 71% of patients showed random inactivation, XCI studies cannot be a reliable molecular diagnostic test of heterozygosity for at-risk females. This contrasts with other X-linked genetic diseases, such as severe combined immune-deficiency (44) or focal dermal hypoplasia (50), in which nonrandom XCI with predominant expression of the wild-type allele might be needed to rescue normal gene function in heterozygous subjects. Mechanisms that may lead to skewing include chromosomal abnormalities, mutations conveying a proliferative advantage or disadvantage to a cell, monozygotic twins, and mutations within the X-inactivation center (XIC), such as XIST promoter mutations (51). In this latter

X-chromosome inactivation in Fabry disease (a)

(b)

(c)

(d)

Fig. 3. Clinical severity in female patients heterozygotes for Fabry disease (FD) in relation to their X-chromosome inactivation (XCI) status. Mainz Severity Score Index (MSSI) (a) and DS3 (b) severity scores were evaluated in randomly inactivated patients (R, gray) and patients with skewed XCI favoring the expression of either the wild-type (W, white) or the mutant GLA allele (M, black). Data represent mean values ± standard deviation (SD). Group comparison were analyzed by one-way analysis of variance (anova) and post hoc Bonferroni’s multiple t-test. MSSI (c) and DS3 severity scores (d) were plotted against age for patients belonging to R (n = 39, gray circles, gray line), W (n = 6, white squares, dotted line) and M (n = 10, black circles, black line) groups.

(a)

(b)

Fig. 4. Renal function and cardiac geometry in female patients affected with Fabry disease (FD) according to their XCI status. (a) mGFR [51 Chrome ethylenediaminetetraacetic acid (51 C EDTA) measured glomerular filtration rate] and (b) left ventricular mass index (LVMi) were plotted against age in patients from R (random) group (gray circles, gray line, n = 39), W group (white squares, dotted line, n = 6) and M group (black circles, black line, n = 10).

case, females are not mosaic due to mutations affecting the initial process that chooses the active X chromosome, and skewed XCI patterns run within families (52, 53). In our study, no mutation in the XIST promoter minimal region was found in those individuals showing unexplained skewed XCI patterns in all four tissues.

There were no monozygotic twin patients in our study population. Because tissues affected by FD may be inaccessible (e.g., brain) or difficult to access (e.g., heart, kidney), it is important to determine to what extent easily accessible tissues (e.g., blood or buccal smears) accurately reflect

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Echevarria et al. the XCI pattern in other tissues (9). Concordance of XCI among different tissues has been studied in the general population with varying results. In one study, XCI in blood was comparable to patterns obtained from other tissues (54). In contrast, previous works have shown X-inactivation patterns to vary widely within the different tissues of a subject (10, 55, 56), making it difficult to extrapolate XCI status only from blood. To date, only one previous study has evaluated the intraindividual differences in XCI patterns in heterozygous females for FD. The results did not show significant variations among tissues (14). Our data, which are the first to incorporate results obtained from skin, showed a significant correlation between XCI results in blood and the other three investigated tissues, although some discrepancies were found for a limited number of patients. On the basis of our findings, we recommend to study XCI in at least one other tissue in addition to leukocytes, preferentially buccal smears because of noninvasiveness. In our study, skewed X-inactivation patterns in leukocytes were more common in elderly than in young women. The difference did not reach statistical significance, probably due to the limited size of the study population, but the trend is in line with data from the literature and needs to be borne in mind when retrospectively interpreting data on the natural history of FD in an elderly woman with apparently skewed XCI in leukocytes, specifically in cases where random X-inactivation is found in other analyzed tissues (57). The measurement of α-Gal activity as a laboratory confirmation of a clinical diagnosis of FD in females has historically been reported as inconclusive. Among the four previous studies on XCI in FD, three did not investigate α-Gal activities (13–15) whereas the fourth one did not find any correlation between residual α-Gal activities and XCI patterns (16). In a case report on a young female with skewed XCI and predominant expression of the mutant allele who had experienced early-onset stroke, low residual α-Gal activity was reported (58). Our data show that residual α-Gal activity strongly correlates with the direction and the degree of XCI. This held true both when XCI was studied in leukocytes only and when results from all four tissues were pooled together. Therefore, our results suggest that a low level of leukocyte residual α-Gal activity in heterozygous females is a surrogate marker of higher disease severity and poor outcome in relation to predominant expression of the mutant GLA allele. However, one patient did not fit that model warranting evaluation of a larger cohort of females to document the relevance of residual α-Gal activity further as an independent predictor of the clinical severity of FD in female patients. In females with random XCI, the overall burden of FD assessed by MSSI or DS3 strongly correlated with age. This supports the hypothesis that, in female patients, cells expressing the mutant GLA allele proliferate unhindered, leading to the progressive detrimental effects of lysosomal storage and subsequent pathogenic cascades over time (59, 60). Our data show that most female patients with random XCI, although they exhibit on average 50% of residual α-Gal activity in leukocytes,

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develop clinical symptoms in an age-dependent manner with an increase of disease burden over the years. Remarkably, heterozygous females for Hunter disease (mucopolysaccharidosis type II), another X-linked lysosomal storage disease due to iduronate sulfatase deficiency, are clinically unaffected, except in the very rare instances where the mutant IDS allele is solely expressed (61). In heterozygous females with FD, cells with an active wild-type GLA allele produce a fully functional enzyme, which in theory is partly secreted and can be taken up through the mannose-6-phosphate receptors (M6PR) at the plasma membrane of α-Gal-deficient cells. However, our data suggest a poor efficiency of this metabolic cooperation due to either insufficient secretion of α-Gal from cells expressing the wild-type GLA allele, glycolysation defect(s) of the secreted enzyme, or deficient M6PR-mediated uptake of secreted α-Gal by contiguous or more distant cells. This is in agreement with previously published results showing that, in FD, normal fibroblasts are unable to cross-correct fibroblasts filled with Gb3 due to an insufficient secretion of α-Gal (62). This suggests that most female patients with FD and random XCI will develop increasing symptoms with age and should be annually or bi-annually monitored for disease progression. In females with skewed XCI, disease progression was correlated with the direction of skewing. Predominant expression of the mutant GLA allele was strongly associated with higher MSSI and DS3 scores, more rapid progression of left ventricular hypertrophy and deterioration of renal function, although this was not the case for one of the 10 patients belonging to this category. In contrast, all female patients with predominant expression of the wild-type GLA allele consistently had a milder phenotype and little, if any, disease progression with age. One limitation of this study is that enzyme replacement therapy (ERT) was not considered in the analyses. Long-term ERT might have halted the progression of the disease. However, no patient belonging to the W group received ERT at any time, excluding a role of ERT in artificially lowering severity scores. Moreover, the hypothesis that ERT may have contributed to lower severity scores in some patients in the R and M groups would actually further support our conclusions. Nevertheless, future studies should ideally focus on ERT-naive females. Another limitation is the relatively small size of the study population. Like many genetic disorders, FD has a significant phenotypic variability, which may in part be explained by several parameters such as age or the nature of the GLA mutation. Our results show that disease expression appears to be more strongly correlated with XCI patterns than with the type of GLA mutation, although the latter might also have an influence on residual α-Gal activity and clinical phenotype (e.g., the p.(Asn215Ser) missense mutation which has been associated with a late-onset, primarily cardiac, phenotype). Finally, modifier genes (63), a concomitant genetic disease, as well as yet undiscovered epigenetic or environmental factors may also contribute, in addition to XCI status, to the clinical variability in females with FD.

X-chromosome inactivation in Fabry disease Conclusion

In females with random X chromosome inactivation, Fabry disease relentlessly worsens with age. Young female patients are generally mildly affected while older females exhibit gradually increasing MSSI and DS3 scores, progression of left ventricular hypertrophy, and deterioration of renal function over time. This supports the hypothesis of inefficient cross-correction between healthy and affected cells in heterozygotes. Female patients with skewed XCI profiles have a different disease course, which depends on the predominantly expressed allele. Expression of the wild-type GLA allele is associated with a mild phenotype and little progression over the years. In contrast, most female patients with predominant expression of the mutant GLA allele have early-onset disease, rapid progression with age and a poorer prognosis, requiring closer monitoring and early therapeutic intervention. Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Acknowledgements This work was supported by the Plan National Maladies Rares (French Ministry of Health), Genzyme and Shire. D.P.G. has received research grants, honoraria and speaker fees from Genzyme, Sanofi and Shire. A.A.H. has received speaker honoraria from Genzyme.

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