J Clin Immunol (2013) 33:1150–1155 DOI 10.1007/s10875-013-9927-9
ASTUTE CLINICIAN REPORT
Combined De-Novo Mutation and Non-Random X-Chromosome Inactivation Causing Wiskott-Aldrich Syndrome in a Female with Thrombocytopenia Boonchai Boonyawat & Santhosh Dhanraj & Fahad al Abbas & Bozana Zlateska & Eyal Grunenbaum & Chaim M. Roifman & Leslie Steele & Stephen Meyn & Victor Blanchette & Stephen W. Scherer & Sabina Swierczek & Josef Prchal & Qili Zhu & Troy R. Torgerson & Hans D. Ochs & Yigal Dror Received: 21 February 2013 / Accepted: 22 July 2013 / Published online: 14 August 2013 # Springer Science+Business Media New York 2013
Abstract Objective Disorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome. Patients and Methods Clinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry.
Boonchai Boonyawat and Santhosh Dhanraj contributed equally to the work. B. Boonyawat : S. Dhanraj : B. Zlateska : S. Meyn : S. W. Scherer : Y. Dror Genetics and Genome Biology Program, Research Institute, University of Toronto, Toronto, Ontario, Canada B. Boonyawat : F. al Abbas : V. Blanchette : Y. Dror The Division of Haematology/Oncology, University of Toronto, Toronto, Ontario, Canada E. Grunenbaum : C. M. Roifman Division of Immunology, University of Toronto, Toronto, Ontario, Canada L. Steele Molecular Genetic Laboratory, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada S. Swierczek : J. Prchal Hematology Division, University of Utah, Salt Lake City, UT, USA Q. Zhu : T. R. Torgerson : H. D. Ochs Seattle Children’s Research Institute, University of Washington, Seattle, WA, USA Y. Dror (*) Division of Hematology Oncology, SickKids Hospital, 555 University Avenue, Toronto, Ontario M5G1X8, Canada e-mail:
[email protected]
X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome. Results A female with thrombocytopenia, eczema and mild Tlymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G>A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome. Conclusions Our study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history. Keywords Wiskott-Aldrich syndrome . X-linked thrombocytopenia . congenital thrombocytopenia . X-chromosome inactivation . WAS . female
Introduction Wiskott-Aldrich syndrome (WAS) is characterized by recurrent infections, eczema, thrombocytopenia, small platelets, and increased risk of autoimmune disorders and cancers [1–4]. WAS is caused by mutations in the WAS gene at Xp11.2 [5, 6], The encoded protein, WASp, is expressed in the cytoplasm of hematopoietic cells and is involved in synapse formation,
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signal transduction and cytoskeleton reorganization [7, 8]. Mutations in the WAS gene can also cause X-linked thrombocytopenia (XLT); a mild variant of WAS [9, 10]. Typically, affected hemizygous males present at an early age, whereas heterozygous females are asymptomatic. This is explained by nonrandom and preferential selection of the non-mutated Xchromosome to be unmethylated and active [11–13]. Rare cases of females with WAS have been reported. Herein, we thoroughly studied a female patient who presented with congenital thrombocytopenia. After extensive negative diagnostic testing and the development of eczema, a diagnosis of WAS/XLT was entertained. Molecular and clonality analysis revealed a spontaneous heterozygous mutation in the WAS gene on the paternally derived X chromosome and extremely skewed X-chromosome inactivation (XCI) with preferential selection of the maternally derived wild-type X-chromosome to be inactivated. Our study challenges the existing clinical practice where genes on the X-chromosome are excluded from testing in female patients, and exemplifies how genetic testing can be applied to prove a causal role of X-linked mutations in female.
Methods The patient was enrolled on the Canadian Inherited Marrow Failure Registry after obtaining consent from the parents. The study was approved by our Institutional Research Ethics Board. Mutation Analysis All exons of WAS and flanking intronic regions were amplified from peripheral blood deoxyribonucleic acid (DNA) as described [10, 14]. For complementary DNA (cDNA) sequencing, peripheral blood T cells were expanded using phytohemagglutinin A and interleukin-2. Total ribonucleic acid (RNA) was extracted, reverse transcribed and amplified as described [10, 14]. Mutations were annotated according to the Human Genome Variation Society nomenclature (http://www. hgvs.org/mutnomen)
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Clonality Assay Genomic DNA from peripheral blood T-cells underwent genotyping of single nucleotide exonic polymorphisms for five X-chromosome genes [dbSNP1135363 (C/T) in BTK; dbSNP 9018 (G/A) in FHL1; dbSNP 1141608 (C/T) in IDS; dbSNP 2230037 (C/T) in G6PD; dbSNP 1126762 (G/T) in MPP using TaqMan allele-discrimination assays as described [16]. Total RNA from peripheral blood T cells was used to assess allelic usage ratio of informative X-chromosome polymorphic genes. DNA-free RNA underwent reverse-transcription and quantitative allele-specific PCR [16]. Allele-specific primers, containing both a mismatched nucleotide and a locked nucleic acid, were used to enhance discrimination between the polymorphic nucleotides. Allelic frequency of the expressed exonic polymorphisms was calculated as described [16] using previously reported mathematical formulas [16, 17].
Genome-Wide Analysis of Genetic Alterations Metaphase cytogenetics of blood lymphocytes was performed by a standard G-banding procedure as described [18]. For molecular karyotyping, DNA was processed, hybridized to Affymentrix Genome-Wide Human SNP 6.0 Array (Santa Clara, CA, USA) and scanned as described [19]. Genotyping calls were determined using the Birdseed v.2 algorithm. We used 132 healthy controls from The Center of Applied Genomics, (Hospital for Sick Children, Toronto, Canada) as baseline to determine hits. Copy number alterations longer than 1 k base along at least five consecutive positive probes were identified, and deemed true if they were detected by at least 2 of the 3 algorithms: Genotype Console, Partek and Birdseed.
Immune Evaluation Enumeration of lymphocyte populations, T cell responses to mitogens, immunoglobulin levels, antibodies to tetanus, T cell receptor excision circles and T-cell receptor diversity were determined as described [20].
X-Chromosome Inactivation Study The triplet repeat-containing AR gene on Xq11-q12 was amplified from peripheral blood cell DNA before and after digestion with methylation-sensitive restriction enzymes, HhaI, HpaII and BamHI, as described [15]. The methylated (inactive) DNA strand is not digested and is amplified during PCR. PCR products with fluorescent primers were electrophoresed and analyzed. The X-inactivation ratio was calculated by peak height difference for each parental allele.
Flow Cytometry Ficoll-purified peripheral blood mononuclear cells were fixed, permeabilized, incubated with rabbit anti-human WASp antibody 503 or normal rabbit IgG ( BD Bioscinces, San Diego, CA), stained with fluorescein isothiocyanate labeled goat antirabbit IgG antibody (Southern Biotech, Birmingham, Alabama) and analyzed by flow cytometry as described [21].
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Result and Discussion Clinical Characteristics A French-Canadian girl was prematurely (35 week of gestation) born by normal vaginal delivery. Due to neonatal jaundice, complete blood count was done, which revealed platelets count of 34×109/L, hemoglobin of 179 g/L, white blood cells 8.7×109/L, absolute neutrophil count 5×109/L. Mean platelet volume without previous platelet transfusions was 7.5 fL (normal range: 4.0–14.0 fL). Initial blood smears showed teardrop-shape red blood cells and schistocytes, which gradually disappeared. The platelet count spontaneously increased to 72×109/L, but later on slowly but persistently decreased. She started requiring platelet transfusion at 9 months of age due to platelets of less than 10×109/L or bleeding. Her hemoglobin progressively dropped to below 70 g/L at 3.5 months of age, requiring transfusions; but then spontaneously normalized. Direct and indirect antiglobulin tests were negative. Testing for anti-platelet antibodies has not been done. The patient did not suffer from unusual or recurrent infections. Bone marrow evaluations at the age of 3.5 and 9.5 months revealed dyserythropoiesis, dysplastic megakaryopoiesis, reduced megakarypoiesis and erythropoiesis. Cytogenetics was normal. The patient underwent hematopoietic stem cell transplantation at the age of 15 months. Conditioning comprised of cyclophosphamide 200 mg/kg, busulfan 16 mg/kg and rabbit antithymocyte globulin 7.5 mg/kg. At last follow-up, two years post transplant, she was well and had normal blood counts. She has maintained stable mixed chimerism of 50– 70 %. In addition to hematological manifestations, the patient had feeding difficulty, reduced height and weight (below the 3rd percentile) and mild motor and mental developmental delay. At the age of 9 months she developed mild to moderate eczema. Family history revealed two healthy, non-consanguineous parents and four healthy siblings. The paternal age and maternal age were 37 and 39 years, respectively. The mother had one stillbirth due to hypoplastic left heart, one miscarriage at 22 week and another miscarriage at 12 week gestational age. There was no history of blood disorders or immune deficiency in the family. Investigations to rule out various acquired and inherited conditions were done. The results of SBDS, DKC1, TINF2, TERC, NHP2, NOP10 and TERT sequence analysis were negative. The Patient Manifested Mild Immunological Abnormalities Immunological evaluation at 10 months of age revealed normal serum immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin E, production of anti-tetanus
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antibodies, total lymphocyte counts and CD4+ T cells, but reduced number of CD8+ T cells (280/μL) and increased CD4/CD8 ratio. The stimulation index response of T cells to phytohemaglutinins was 269, compared to 579 in a control, while the response to anti-CD3 was similar to control. T cell repertoire demonstrated the presence of all V-beta families without clonal expansion, with reduced percentage of Vb 14 at 0.8 (normal 3.5+/−1.4) and Vb 17 at 2.4 (normal 5.1+/ −1.3) (Fig. 1a). TREC were 1,565 copies/0.5 μg of DNA (normal >400). WAS Gene Analysis Revealed a Mutation that is Typically Associated with a Severe Phenotype The patient suffered from congenital and progressive thrombocytopenia, mild-moderate eczema and mild growth and developmental delay. Although these abnormalities are not specific to WAS/XLT, they have been reported in males with these disorders [22–24]. Therefore, direct sequencing of WAS in genomic DNA was done, which revealed a heterozygosity mutation c.397G>A that is predicted to change the glutamic acid to lysine, p.E133K (Fig. 1b). Both parents had normal WAS sequence. The c.397G>A mutation was previously reported in male patients with severe WAS [14] and in a female with WAS [25]. The Patient Expresses Only the Mutant WAS Allele To study whether the heterozygous mutation was pathogenic, we sequenced cDNA from the patient’s T-cells. This revealed expression of only the mutant c.397G>A allele, demonstrating that only the abnormal X-chromosome of the proband is being transcribed (Fig. 1c). To investigate the consequences of exclusive expression of the mutant mRNA, we analyzed the WASp protein level. We found no WASp expression in lymphocytes (Fig. 1d) and in granulocytes (data not shown). Patients with no expression or expression of only truncated WASp (“WASp-negative”) usually present with classic WAS phenotype [2, 14, 21]. The Patient AR Locus Showed Evidence of Skewed Inactivation of the X-Chromosome Heterozygous females for a mutant gene on the X chromosome are asymptomatic due to either random XCI or skewed inactivation in favor of the normal X chromosome caused by selective advantage [11, 26, 27]. In very rare cases female may manifest clinical WAS due to predominance transcription of the mutant allele [25, 28]. To assess whether the patient has non-random XCI, we studied the amplified products of the AR gene before and after methylation-sensitive restriction enzyme digestion. The father had a 17-repeat allele for his AR gene, whereas the mother had 18-repeat alleles in both X chromosomes (Fig. 1e).
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T cell expression (%)
a
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d
12
8
Patient 4
b e
VB 23
VB 22
VB 21.3
VB 20
VB 18
VB 17
VB 16
VB 14
VB 13.2
VB 13.6
VB 13.1
VB 11
VB 12
VB 9
VB 7.2
VB 8
VB 7.1
VB 5.3
VB 5.1
VB 5.2
VB 4
VB 3
VB 2
VB 1
0
Control Paternal AR gene
Maternal AR gene
Genomic DNA sequencing: Heterozygous mutation c.397G>A (E133K)
c PCR before digest
cDNA sequencing in T cells: Exclusive expression of the mutant c.397G>A allele (E133K)
PCR after digest
Fig. 1 a Analysis of T-cell repertoire. Flow cytometry analysis revealed the presence of all TCRVβ families without clonal expansion, and also demonstrated the reduction (arrows) in the percentages of the patient’s (solid bars) TCRVβ 14 and TCRVβ 17 in comparison to age matched normal controls (open bars) b Direct sequence analysis of exon 4 of the WAS gene in the female patient. Direct sequence analysis revealed double peak signals for both the wild-type guanine (G) and the mutant adenine (A) at the position 397 in exon 4 [(397G>N(A/G)], indicating a heterozygous missense mutation (E133K) from glutamic acid (GAG) to lysine (AAG) of the WAS gene in the patient’s genomic DNA c Direct sequence analysis of WAS cDNA of the patient revealed only the G to A transition at the position 397 (397G>A), suggesting the absence of the normal WAS cDNA d WASp protein expression. Nucleated blood cells were separated, stained with an anti-WASp antibody and analyzed by flow cytometry. Dot plot figures of the patient
(upper panel) and a control (lower panel) e X-chromosome inactivation analysis of the patient and her parents. A. Results obtained at the androgen receptor (AR) locus, the blue peaks correspond to the PCR products both before and after methylation sensitive enzyme digestion. The top of each histogram demonstrate the length of the PCR fragments which indicate the size and number of triplet repeat sequences. The histogram revealed the father had only 17-repeat fragment for his only one active X allele, the mother is homozygous for 18-repeat alleles which one of them was transmitted to the daughter. The histogram of the patient revealed both 17-repeat and 18-repeat allele in which 18-repeat allele was much greater than 17repeat allele after restriction enzyme digestion, indicating a skewed Xchromosome inactivation toward the maternally derived-X allele. After Xinactivation ratio was calculated, the result indicated that the maternal derived allele was 94 % inactive (~100 % inactive)
The patient’s histogram showed 2 amplification peaks: a 17-repeat fragment (paternal), and an 18-repeat fragment (maternal). Nevertheless, the 18-repeat allele was predominantly (94 %) detected after restriction enzyme digestion, indicating the non-random and an extremely skewed inactivation pattern in the patient’s DNA. The patient’s mother was homozygous for 18-repeat alleles of the AR locus; hence, this method could not be used to determine her X-inactivation pattern.
Expression Patterns of cDNA Clones from the X-Chromosome were Consistent with Non-Random Inactivation in the Child and Random Inactivation in the Mother Since we could not determine the mother’s XCI status using the AR locus, we performed family studies using cDNA clonal expression of several other genes on the X-chromosome. We
1154 Table I Genotyping of genes on the X-chromosome and allelic frequency of the expressed messenger ribonucleic acid
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Samples
Mother sample 1 Mother sample 2 Patient sample 1
Results based on quantitative transcription based assay. Values shown are the mean of duplicate determinations
Patient sample 2
Genotyped X-Chromosome polymorphisms determined to be Heterozygous
Allelic frequencies (percent) of expressed exonic polymorphisms in T-cells ribonucleic acid
MPP1 G/T
FHL1 C/T
IDS C/T
G6PD C/T
BTK C/T
MPP1 G/T
FHL1 C/T
57/43
40/60
57/43
40/60
+
+
−
−
−
−
−
−
−
+
+
−
−
−
−
−
−
+
−
−
−
−
−
−
−
+
−
−
−
screened genomic DNA from the patient and her mother for heterozygosity loci on genes on the X-chromosome that could inform about the pattern of inactivation. We found herozygosity sites in the MPP1 and FHL1 genes in the mother and in the MPP1 gene in the patient (Table I). These heterozygousity sites were selected for studying the transcribed RNA. The mother produced 2 types of transcripts, but the patient produces only one (Table I). This indicates that the mother’s X-chromosomes were randomly inactivated, while the child`s X-chromosomes were not randomly inactivated. These experiments showed that the patient XCI pattern was not inherited from the mother, but occurred de-novo in the X-chromosome that does not carry the mutation. This also led to the conclusion that the de novo mutation spontaneously occurred on the paternal X-chromosome during spermatogenesis and caused the disease in our patient. Skewing of the XCI was Not Due to Macroscopic Chromosomal Abnormalities or Submicroscopic Copy Number Alterations Karyotypic abnormalities (e.g., deletions) might lead to predominant inactivation of the structurally abnormal chromosome; thereby, predominant expression of a mutated allele on the other chromsome. To investigate the etiology for skewed inactivation, standard karyotyping and genome wide analysis of the genetic alterations were performed. The standard cytogenetic analysis revealed normal female karyotype for both patient and her mother and normal male karyotype for her father. Molecular karyotyping by genome wide-analysis of copy number variations did not reveal amplifications or deletions in the patient’s DNA. Thus, skewed XCI pattern in our patient was unlikely due to chromosomal abnormalities. Nevertheless, submicroscopic structural aberrations that cannot be detected by metaphase cytogenetics or SNP array, such as small X-autosome translocation, can result in exclusive survival of
− −
−
− − −
−
− − −
0/100 0/100
−
cells that manifest inactivation of the normal X chromosome, allowing correct expression of the autosome. This phenomenon can lead to the manifestation of X-linked recessive female carriers if the derivative X-chromosome carries a recessive disease allele or the translocation disrupts a disease causing gene. Other possible explanation for XCI skewing in female carriers of X-linked recessive disease include selection against deleterious alleles and genetic defects of the X-inactivation process such as XIST (X-inactivation-specific transcript) promoter mutation. The cumulative clinical and molecular findings demonstrate how a female carrier with heterozygous WAS mutation can express an X-linked WAS/XLT disease. The cause of the reduced height and weight in the patient is unclear. Also, the association of WAS and hypoplastic left heart in this family remained to be clarified, but this might be related to a defect of methylation that also affected an X-linked or autosomal gene involved in cardiogenesis. Our study also demonstrates the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of family history. It is noteworthy that a clinical phenotype that resembles WAS in girls can also be caused by biallelic mutations in the autosomal WIP gene [29]. Mutations in this gene are rare, but should be considered in the differential diagnosis of males and female with such a WAS phenotype and negative testing for mutations in the WAS gene. Financial Disclosure The authors acknowledge the support to this work from the C17 Council and the support they receive from Childhood Cancer Canada, and Coast to Coast Against Cancer Foundation, the Nicola’s Kids Triathlon and Canadian Institute for Health Research (funding reference 102528). Conflict of Interest The authors have no conflicts of interest relevant to this article to disclose.
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Contributor’s Statement BB designed and performed experiments, analyzed data, wrote the manuscript; SD designed and performed experiments, analyzed data, wrote the manuscript; FAA design laboratory investigation; BZ analyzed data, wrote the manuscript; EG performed experiments, analyzed data, wrote the manuscript; CMR designed experiments; LS designed and performed experiments; SM design laboratory investigation, data interpretation; VB design laboratory investigation, SWS provided control data and technical support, SS performed experiments, JP designed experiments, QZ designed experiments, TRT designed experiments, HDO, designed experiments, analyzed data, wrote the manuscript; YD conceived and designed research, analyzed data and wrote the paper.
