CD45-Deficient Severe Combined Immunodeficiency Caused By

CD45-deﬁcient severe combined immunodeﬁciency caused by uniparental disomy

Joseph L. Robertsa, Rebecca H. Buckleya,b,1, Biao Luoc,d, Jianming Peid, Alla Lapidusc, Suraj Peric, Qiong Weie, Jinwook Shina, Roberta E. Parrotta, Roland L. Dunbrack, Jr.e, Joseph R. Testad,f, Xiao-Ping Zhonga,b, and David L. Wiestf

aDepartment of Pediatrics and bDepartment of Immunology, Duke University Medical Center, Durham, NC 27710; and dCancer Biology Program, eDevelopmental Therapeutics Program, fBlood Cell Development and Cancer Keystone, Immune Cell Development and Host Defense Program, and cCancer Genome Institute, Fox Chase Cancer Center, Philadelphia, PA 19111

Contributed by Rebecca H. Buckley, March 16, 2012 (sent for review November 16, 2011) Analysis of the molecular etiologies of SCID has led to important mesoderm-derived lymphocytes and ectoderm-derived buccal epi- insights into the control of immune cell development. Most cases of thelial cells, suggesting that the duplication occurred before germ- SCID result from either X-linked or autosomal recessive inheritance layer speciﬁcation. of mutations in a known causative gene. However, in some cases, the molecular etiology remains unclear. To identify the cause of Results SCID in a patient known to lack the protein-tyrosine phosphatase SCID Patient Lacks CD45 Expression. Flow cytometric analysis of the CD45, we used SNP arrays and whole-exome sequencing. The patient’s peripheral blood lymphocytes at presentation at age 10 patient’s mother was heterozygous for an inactivating mutation mo revealed normal numbers of B and NK cells but dramatically in CD45 but the paternal alleles exhibited no detectable mutations. reduced numbers of T cells (Table 1). Moreover, the few T cells The patient exhibited a single CD45 mutation identical to the ma- present were found to be nonfunctional as they were unresponsive ternal allele. Patient SNP array analysis revealed no change in copy to mitogenic stimulation (Fig. 1B). Serum IgM, IgA, and IgE were number but loss of heterozygosity for the entire length of chromo- undetectable or low (Table 1). Cell-surface CD45 protein expres- some 1 (Chr1), indicating that disease was caused by uniparental sion was lacking on all leukocytes following staining with Abs disomy (UPD) with isodisomy of the entire maternal Chr1 bearing recognizing CD45 RA, CD45RO, or all CD45 isoforms (Fig. 1A

CD45 IMMUNOLOGY the mutant allele. Nonlymphoid blood cells and other meso- and Table 1), consistent with the previous finding of absent bone derm- and ectoderm-derived tissues retained UPD of the entire marrow leukocyte CD45 expression at the referring hospital. CD45 maternal Chr1 in this patient, who had undergone successful bone is essential for T-cell development and T-cell receptor (TCR) marrow transplantation. Exome sequencing revealed mutations in signal transduction (9, 10), and has previously been identified in two seven additional genes bearing nonsynonymous SNPs predicted to fi fatal cases of SCID (3, 4). The patient in this report underwent a have deleterious effects. These ndings are unique in representing successful T-cell–depleted haploidentical maternal bone marrow a reported case of SCID caused by UPD and suggest UPD should be stem-cell transplant without preconditioning or posttransplantation considered in SCID and other recessive disorders, especially when graft-versus-host disease (GVHD) prophylaxis at age 10 mo and the patient appears homozygous for an abnormal gene found in currently has normal numbers of B and NK cells, as well as normal only one parent. Evaluation for alterations in other genes affected numbers of CD45-expressing, functional T cells at 5 y posttrans- by UPD should also be considered in such cases. plantation (Fig. 1B and Table 1). The loss of CD45 expression in the patient was not the result of T lymphocyte | T cell receptor | signaling a defect in transcription, because the level of CD45 mRNA in the patient was only slightly decreased relative to that of controls (Fig. CID is a syndrome characterized by absent T- and B-lympho- 1C). Sanger sequencing revealed that the patient’s mother was Scyte function that is uniformly fatal in infancy without immune heterozygous for a nonsense mutation at position 1618 (1618A > reconstitution (1, 2). Mutations in several different genes impor- T) of the coding sequence in exon 14 of the CD45 gene that cre- tant for normal T-cell development, function, or survival have been ated a stop codon at amino acid 540 (K540X); however, no mu- shown to cause SCID, with a majority of reported cases caused tations were observed in the coding region of either paternal CD45 IL2RG IL7RA ADA JAK3 RAG1 RAG2 by mutations in , , , , , ,or allele (Fig. 2). Surprisingly, the patient was homozygous for the DCLRE1C (1, 2). Rare defects in six other genes have also been 1618A > T mutation observed in the maternal allele (Fig. 2). This described, including two fatal cases caused by mutations in the finding suggested that either one copy of paternal Chr1 bore a gene encoding the CD45 protein tyrosine phosphatase (1, 3, 4). microdeletion eliminating the CD45 locus, or the patient inherited Uniparental disomy (UPD) refers to the inheritance of two two copies of the mutant maternal CD45 allele. copies of a chromosome, or segment of a chromosome, from one parent. UPD was first observed in 1988 in a patient with cystic SCID Is Caused by Duplication of the Mutant Maternal CD45 Allele fibrosis who had inherited two maternal copies of chromosome 7 Because of UPD of Chr1. To distinguish these possibilities, SNP bearing a mutant CFTR allele (5, 6). Since that report, UPD has arrays were performed on genomic DNA from EBV lines derived been found to underlie a number of diseases, including Prader– from B lymphocytes of the parents and patient. These analyses Willi, Angelman, and Beckwith–Wiedermann syndromes (7). UPD causes a genetic disorder either through inheritance of two mutant copies of a gene, thereby enabling a recessive mutation to manifest, Author contributions: J.L.R., R.H.B., J.R.T., and D.L.W. designed research; J.S. and R.E.P. or through inheritance of two silenced copies of an intact allele (8). performed research; B.L., J.P., A.L., S.P., Q.W., R.L.D., and X.-P.Z. contributed new re- UPD has not previously been reported as a mechanism of in- agents/analytic tools; J.L.R., R.H.B., B.L., J.P., A.L., S.P., Q.W., R.L.D., J.R.T., X.-P.Z., and D.L.W. analyzed data; and J.L.R. and D.L.W. wrote the paper. heritance in SCID. In the present report, we are unique in de- fl scribing an example of SCID caused by UPD, in which the first The authors declare no con ict of interest. surviving CD45-deficient SCID patient inherited two complete Data deposition: The data reported in this paper have been deposited in the Gene Ex- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE35674) copies of a single maternal Chr1 bearing a nonsense mutation in and the Database of Genotypes and Phenotypes (dbGaP), www.ncbi.nlm.nih.gov/gap re- the exodomain of CD45. This complete isodisomy of Chr1 with pository (accession no. phs000479.v1.p1). resultant loss of heterozygosity (LOH) was present in both 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1202249109 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Table 1. Patient Immune phenotype and function Patient

At presentation Most recent Controls

Serum Ig level* IgG† 516 604 192–515 IgA 0 12 12–31 IgM 9 9 39–92 ‡ Lymphocyte subpopulation CD45+ 4 (0.4) 581 (51.9) 1,500–7,000 + CD3 31 (2.9) 596 (53.2) 1,111–5,183 + Percent of CD3 that are CD45RO (%) ND (55.8) (24.9–42.5) Percent of CD3 that are CD45RA+ (%) ND (22.8) (27.6–46.2) CD4+ 24 (2.3) 273 (24.4) 675–3151 + Percent of CD4 that are CD45RO (%) ND (58.1) (24.9–42.5) + Percent of CD4 that are CD45RA (%) ND (21.2) (27.6–46.2) CD8+ 3 (0.3) 174 (15.5) 431–2012 Percent of CD8 that are CD45RO+ (%) ND (41.1) (24.9–42.5) + Percent of CD8 that are CD45RA (%) ND (32.5) (27.6–46.2) + TCRαβ 21 (2) 468 (41.8) 1,855–3,199 CD20+ 854 (80.9) 516 (46.1) 144–671 + CD16 71 (6.7) 31 (2.8) 152–709 Proliferative stimulus§ Medium 128 228 693 ± 825 Candida ND 3,539 5,937–59,291 Tetanus ND 8,486 13,004–68,696

ND, not determined. *Values are expressed as mg/dL (IgG, IgA, IgM) or U/mL (IgE). Normal values are the 95% conﬁdence intervals for 9- to 12-mo-old control subjects. † The patient was receiving intravenous immune globulin when the IgG levels were measured. ‡ Values are expressed as cells/mm3 or (percentage of lymphocytes). Control values are the 95% conﬁdence intervals for 1,550 normals. §Values are cpm [3H]thymidine incorporation. Controls values are the mean ± SD of responses in 167 normals.

demonstrated that there was no change in copy number across the To identify such alleles, exome sequencing was performed on CD45 locus on Chr1 (Fig. 3A), indicating that the patient had DNA samples from mother, father, and patient. Using the ma- either inherited two entire copies of maternal Chr1 or two copies ternal alleles for UPD analysis, we identified 36 homozygous of the region encompassing the maternal CD45 mutation. The SNPs, all of which were located on Chr1 (Table 2). Conversely, no allele profiles from the whole-genome array analysis revealed paternal alleles of UPD from any chromosome were identified. LOH over the entire length of Chr1 (Fig. 3A), demonstrating Consistent with the Sanger sequencing analysis, the maternal PTPRC CD45 isodisomy. Isodomy was found to be restricted to Chr1, as there ( ) nonsense allele was homozygous in the patient. fi was no evidence for LOH on any of the other chromosomes in the The identi ed maternal UPD SNP alleles are distributed patient (Fig. 3B). Whole-genome array analysis revealed that throughout Chr1, in agreement with the results of the whole-ge- isodisomy was also evident in other primary blood cell types nome array indicating UPD of the entire maternal Chr1. Among the 36 SNP alleles on Chr1, we found 7 other mutated genes in (lymphocytes and neutrophils), which are derived from embry- PTPRC onic mesoderm, as well as in buccal epithelium, which is derived addition to bearing nonsynonymous SNPs that were predicted to have deleterious effects on gene function (Table 2). from embryonic ectoderm (Fig. 3C). Taken together, these data Although several of these were found in genes of unknown func- suggest that the events leading to the isodisomy of maternal Chr1 tion, others have been shown to function in cell-cell contact occurred very early in development, probably before the separa- (LGALSG), binding bacterial cell wall components (PGLYRP3), tion of the germ layers, and that isodisomy is likely manifested in replication of DNA (ORC1L), and in binding seratonin (HTR1D) most if not all tissues. (12–15). Collectively, these SNPs appear to have no deleterious effects on early development, because aside from SCID caused by Additional Genes on Chr1 Exhibit Changes in Coding Sequence. Humans the CD45 mutation, the 5-y-old male patient appears to be oth- are estimated to have in excess of 200 recessive disease-causing erwise phenotypically normal. Nevertheless, it remains possible mutations distributed throughout their genomes (>100 per haploid these mutations may manifest later. genome) (11). Because the patient inherited two complete copies of maternal Chr1, which represents ∼5% of the entire genome Discussion = = (both Chr1 homologs 9%, maternal copy 4.5% of entire ge- We are unique in describing a case of SCID caused by UPD. This nome), the patient would be predicted to have on the order of 18 child is also the first reported surviving CD45-deficient SCID pa- recessive disease-causing alleles in both copies of Chr1 (9 per each tient. The first reported SCID patient lacking CD45 expression parental copy). Importantly, because isodisomy in this patient died from a CMV infection 55 d after a matched unrelated bone appeared to arise before separation of the embryonic germ layers, marrow transplant and was subsequently shown to be homozygous these mutations would be expected to persist even after bone for an in-frame 6-bp deletion in exon 11 of the CD45 gene that marrow transplantation in both nonlymphoid hematopoietic tissue caused deletion of residues E339 and Y340 in the extracellular and in nonhematopoietic tissues. domain of the protein (3, 16). Both of her consanguineous parents

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1202249109 Roberts et al. Downloaded by guest on September 29, 2021 Fig. 1. Patient CD45 expression and Im- mune data. (A) The CD45 expression on whole-blood samples from the patient and healthy volunteer as measured by four-color ﬂow cytometry on electroni- − cally gated CD14 cells. The anti-CD45 Ab recognizes all CD45 isoforms (clone HI30, solid lines). A FITC-labeled isotype control Ab is indicated by dashed lines. (B) The patient’s T-cell function was measured by [3H]thymidine incorporation on PBMC exposed to the indicated stimulus and plotted over time. (C) The expression of mRNA encoding CD45 as measured by SYBR green real-time PCR on cDNA prepared from sorted patient and normal volunteer PBMC. Levels of CD45 mRNA were normalized to those of β-actin for

each sample and are presented as arbi- IMMUNOLOGY trary unit (a.u.) of fold change calculated –ΔΔ using the 2 CT method. Values are mean ± SEM of triplicate determinations. Con A, concanavalin A; pokeweed mito- gen, PWM.

were heterozygous for this deletion. The other reported patient transplant that led to chimerism in several lineages (eosinophils, with CD45-deficient SCID inherited a large deletion in the 3′ end neutrophils, and B cells) in addition to T cells. of the CD45 gene from his mother. The patient’s second CD45 UPD has been reported to cause disease either through in- gene mutation was a G > A substitution at the donor splice site of heritance of two silent, imprinted alleles from the same parent – IVS 13, leading to aberrant splicing that was not present in either (e.g., Prader Willi Syndrome) or through inheritance of a duplicated recessive, mutant allele (8). The case of SCID described parent (4). The child died from a B-cell lymphoma at age 2 y. The here resulted from inheritance of two entire copies of a maternal patient in the present report underwent successful bone marrow Chr1 bearing a nonsense mutation in the exodomain of CD45, transplantation without pretransplant chemotherapy and with a which eliminated its expression and abrogated T-cell function. rigorously T-cell–depleted maternal bone marrow stem-cell Because both SCID (<1/40,000 births) and UPD (1/3,500 births) are rare, it is unlikely that our identification of UPD underlying SCID is an isolated occurrence (17). UPD can occur through a variety of mechanisms including mistakes in meiosis that produce aneuploid gametes or postfertilization errors in a normal zygote (mitotic disjunction) and these distinct causes produce UPD with different inheritance patterns (17). The case of UPD described here manifested as complete duplication of an entire maternal allele of Chr1 and was present in tissues arising from both embryonic mesoderm (blood) and ectoderm (buccal epithelium). Accordingly, this was unlikely to be the result of a postfertilization error, which typi- cally produces chimeric representation of UPD involving chro- mosomal fragments. The presence of Chr1 duplication in at least two of the three embryonic germ layers also suggests that this event occurred very early in development, perhaps involving the gametes. It is estimated that nearly 20% of eggs and 3–4% of sperm are aneuploid, raising the possibility that fertilization Fig. 2. Sequence analysis of the CD45 alleles of the patient and parents. The events involving aneuploid gametes occur relatively frequently schematic depicts the domains of CD45, including the alternatively spliced (18). Accordingly, the isodisomy we observed could have been exons that give rise to the RA and RO isoforms, the fibronectin III-like (FNIII) caused by fertilization of a disomic gamete (egg) by a nullisomic domain, transmembrane domain (TM), and phosphatase domains (PTP1 and -2). The position of the nonsense mutation is marked by an asterisk. The male gamete, a process known as gamete complementation, or change in nucleotide sequence (Top) and amino acid sequence (Bottom)in by monosomy rescue, when a monosomic gamete is fertilized by the patient (Mut hCD45) are listed (Middle). The Sanger sequence trace on a nullisomic gamete, following which the chromosome is dupli- genomic DNA from parents and patient are depicted (Bottom). cated (17). In cases involving the division of cells with a normal

Roberts et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Fig. 3. Copy number and allele peak analysis of vari- ous cell populations from the parents and patient. (A) Analysis of copy number and allele peaks in EBV-im- mortalized B cells is depicted. The y axis depicts DNA copy number (not log2 ratio) (Upper) and the allele peak (Lower). Allele peak panels normally show three “bands,” representing all homozygous (Top and Bot- tom bands) and heterozygous (Middle band) allele calls. Note that the copy number panels reveal two copies of each gene on Chr1, but show only two bands, with loss of the Middle (heterozygous) band (arrow) across the entire chromosome, indicative of UPD. (B) Chromosome coordinates for each autosome and the X chromosome are shown on the x axis. (C) Copy number and allele peak analysis are depicted for Chr1 in neutrophils, lymphocytes, and buccal epithelium from the patient. All analyses were performed using Affymetrix Cytogenetics Whole-Genome arrays as described in Materials and Methods.

karyotype, the occurrence of UPD is generally considered to be genes on Chr1 bearing homozygous mutations predicted to be a de novo genetic alteration with negligible risk of recurrence deleterious to function (Table 1). Moreover there are 15 genes on (8). Nevertheless, the mother of our patient had a miscarriage in Chr1 that are predicted to be affected by imprinting (20, 21); the year before his birth of a 7-wk-old embryo with trisomy of all therefore, some of these genes may be silenced in the majority of chromosomes (Fig. 4). This finding suggests a predisposition to the patient’s tissues. Thus, it would seem prudent for those pro- aneuploidy in one of the parents. Sequencing of the maternal viding care to such patients be aware that additional complica- exome revealed no obvious mutations in genes regulating mei- tions might arise, and monitor accordingly. osis; notably, however, the father was found to be heterozygous Our findings are unique in representing a reported case of SCID for a K326X nonsense mutation in BRCA2. BRCA2 mutations caused by UPD. Although UPD has been implicated in the de- have been associated with increased UPD in breast cancer (19). velopment of a number of inherited syndromes, it is not widely Accordingly, it will be of interest to carefully investigate both considered as an inheritance mechanism and has only been de- parental exome sequences for potential mutations in genes reg- scribed in isolated cases of five other primary immune-deficiency ulating meiosis, as well as to determine if the father’s sperm cells syndromes, including C4 deficiency (22), cartilage-hair hypoplasia exhibit increased levels of aneuploidy compared with that of without associated SCID (23), Chediak–Higashi syndrome (24), controls. Taken together, these findings raise the possibility that familial hemophagocytic lymphohistiocytosis (25), and IFN-γ re- in some cases, a predisposition to UPD may exist. ceptor 1 deficiency (26). Disease in our patient was because of In this patient, SCID was successfully treated by bone marrow isodisomy of Chr 1, and of the 23 other reported cases of this type transplantation. Nevertheless, our analysis revealed that the non- of UPD (27), only one was associated with immune deficiency (24). lymphoid blood cells as well as other mesoderm- and ectoderm- Our results indicate that UPD is readily detectable by SNP derived tissues retained UPD of the entire maternal Chr1. There- array and, although our findings represent a unique instance of fore, this patient may be at risk for additional consequences SCID caused by UPD, we suggest that UPD should be regarded resulting from the complete LOH of this chromosome. Indeed, as a novel third mode of inheritance in cases of SCID, as well as exome sequencing revealed the presence of seven additional any other inherited recessive disorder in which the patient

Table 2. Analysis of homozygous mutations resulting from UPD of chromosome 1 Mutation Gene Chromosome: position Function type/position Uniprot Change in AA Seq. SVM prediction*

HTR1D 1:23392180 Seratonin receptor Missense P28221 R374W Deleterious (96%) C1ord94 1:34440323 Unknown Missense Q6P1W5 N251I Deleterious (85%) ORC1L 1:52627549 Origin recognition complex Missense Q13415 A372V Deleterious (55%) PGLYRP3 1:151546220 Peptidoglycan recognition Missense Q96LB9 R68Q Deleterious (60%) PTPRC 1:196954019 CD45 phosphatase Nonsense P08575 K540* Deleterious* DDX59 1:198886387 Dead-box polypeptide 59; unknown Missense Q5T1V6 L368P Deleterious (55%) SIPA1L2 1:230628143 Signal-induced Proliferation Missense Q9P2F8 S1482W Deleterious (80%) associated like 2; unknown LGALS8 1:234773522 Galectin family; adhesion Missense O00214-2 P239S Deleterious (80%)

*Prediction of the likelihood a mutation will be deleterious to protein function using SVM. The number in parenthesis quantiﬁes the likelihood that a mutation will disrupt protein function. A detailed description is available in Materials and Methods.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1202249109 Roberts et al. Downloaded by guest on September 29, 2021 a miscarriage in the year before the patient’s birth of a 7-wk embryo with triploidy of all chromosomes (Fig. 4).

Immunologic Phenotype Analysis. Serum Ig levels were determined by nephelometry. Standard four-color flow cytometry of whole blood was performed with labeled Abs to CD3ε, CD4, CD8, CD14, CD16, CD20, CD25, CD45 (clone HI30 recognizing all isoforms), CD45 RA, CD45 RO, CD56, CD62L, TCRαβ, and TCRγδ purchased from BD Biosciences. Lymphocyte proliferation was assessed by measuring [3H]thymidine incorporation into mononuclear cells following culture with optimal concentrations of the indicated stimuli as previously described (28). This analysis is not part of a clinical trial. All studies were performed with the approval of the Duke University Health System’s Institutional Review Board for Clinical Investigations, and written informed consent by the patient’s parents. Patient immune phenotype and function data were analyzed by J.L.R., R.H.B., and R.E.P. CD45 expression data analysis was performed by J.L.R., J.S., and X.-P.Z. Fig. 4. Triploid karyotype in 7-wk-old embryo. Chromosome spreads were produced from tissue obtained from an embryo spontaneously aborted at Cell Sorting. Patient and healthy volunteer cells used for real-time PCR and 7 wk of gestation. The miscarriage occurred in the year before the birth of indicated cytochip analyses were isolated from frozen peripheral-blood the SCID patient. mononuclear cells (PBMC) using a FACS Vantage SE (Becton Dickinson) by sorting cells incubated with unlabeled mouse antibody to HLA-DR11 (Life- span Biosciences) followed by incubation with normal goat serum and appears homozygous for an abnormal gene found in only one staining with RPE-goat anti-mouse Ig (Lifespan Biosciences). Sorting was parent. If UPD is present, one should then consider performing required because available patient frozen PBMC had been obtained 3 mo postbone marrow transplantation but before T-cell development and con- high-throughput sequence analyses to determine if additional tained 9.3% CD45+ maternal (HLA-DR11−) lymphocytes. Sorted patient (HLA- + genes affected by UPD exhibit deleterious homozygous muta- DR11 ) cells stained brighter for HLA-DR11 plus secondary antibody than − tions or are predicted to be affected by imprinting and monitor with secondary antibody alone. Similarly sorted HLA-DR11 normal volun- patients accordingly. Finally, although UPD is generally regarded teer cells were collected to control for potential effects of cell sorting in as a de novo genetic alteration, the finding that the mother of the subsequent experiments. patient had a previous miscarriage that exhibited triploidy (Fig. 4) IMMUNOLOGY suggests that in some cases of UPD, a predisposition to chromo- Real-Time PCR Analysis of CD45 mRNA Expression. RNA isolated from sorted some missegregation may exist. patient and healthy volunteer PBMC (TRIzol; Invitrogen) was used for cDNA synthesis (Superscript III; Invitrogen). CD45 and β-actin transcripts were then fi Materials and Methods quanti ed by real-time PCR with a Mastercycler realplex and SYBR green master mix (Eppendorf). Three sets of CD45 primers (sequences available on fl Subject. The patient presented at age 6 mo with severe GE re ux and failure to request) were used for amplification of regions of exons 1–2 and exons 13– thrive and developed Pneumocystis jiroveci pneumonia at age 10 mo. He was 14 upstream of the patient exon 14 mutation, and exons 21–23 downstream noted to have pancytopenia, hypogammaglobulinemia, normal B cells, and of the patient mutation. NK cells but almost no T cells on flow cytometry, and he had an absent lymphocyte proliferative response to phytohemagglutinin (PHA). A bone marrow CD45 Sequence Analysis. RNA isolated from PBMC (RNeasy Mini Kit; Qiagen) biopsy showed normal cellularity but no detectable CD45 on his leukocytes. He was used for synthesis of cDNA templates (Superscript II; Invitrogen) that were was referred to Duke University Medical Center, where the diagnosis of CD45 PCR-amplified with primer pairs (sequences available upon request) that deficient SCID was confirmed (Fig. 1 A and B, and Table 1), and he received an generated overlapping products spanning the full length CD45 variant 1 unconditioned, rigorously T-cell–depleted haploidentical maternal bone transcript coding sequence (NM002838.3). Genomic DNA templates isolated marrow stem-cell transplant without posttransplantation GVHD prophylaxis. from whole blood (DNeasy Tissue Kit; Qiagen) were also PCR-amplified with An adenovirus infection was diagnosed on admission with positive stool and primer pairs (sequences available upon request) spanning alternatively spliced respiratory secretion cultures, and he was treated with Cidofivir for the first 4 CD45 exons 4 through 6 and surrounding intron splice sites. PCR products were mo. Shortly after the medication was stopped he developed fever, leukocy- purified (Qiaex II Gel Extraction Kit; Qiagen) and used as templates in se- tosis (43,800/mm3), eosinophilia (24,528/mm3), elevated serum IgG (1,740 mg/ quencing reactions (Big Dye Terminator Cycle Sequencing System; PerkinElmer dL) and IgE (1,700 IU/mL) levels, and diffuse lymphadenopathy with negative Life Sciences). Sequencing reactions representing both strands were analyzed routine cultures. An axillary lymph-node biopsy performed on day 139 post- using an ABI 377 Prism DNA (PerkinElmer) instrument and software. The transplantation showed necrotizing granulomatous lymphadenitis with detected 1618A > T mutation was further evaluated by sequence analysis of + CD45 (bone marrow donor) myeloid (neutrophils and eosinophils) and lym- genomic DNA obtained from the patient and his parents using exon 14 spe- fi phoid (primarily B cells, but also T cells) cells with no evidence of malignancy or ci c primers. Nucleotide numbers refer to the published cDNA sequence of full = GVHD and negative stains for microorganisms. length CD45 variant 1 transcript (NM0002838.3) with start codon 1. CD45 Sanger sequencing data were analyzed by J.L.R. Karyotype analysis of the biopsied node showed engraftment with 10% female donor cells and the patient’s peripheral blood T-cell proliferative response to PHA of 71,316 cpm at the time also demonstrating the presence of Cytogenetics Whole-Genome Array. Total genomic DNA (100 ng) from each test sample was whole genome-amplified. Individual amplified DNA samples T-cell function. Serum immunofixation studies showed a monoclonal IgG-κ were each purified, fragmented, biotin labeled, and hybridized to an Affy- component with a lambda light chain. A repeat stool adenovirus culture was metrix Cytogenetics Whole-Genome 2.7 M array, according to the manu- positive at 5 mo posttransplantation and Cidofivir therapy was reinitiated facturer’s protocol. The hybridized arrays were washed using an Affymetrix and continued until 8 mo posttransplantation and cultures have remained GeneChip Fluidics 450 apparatus and then scanned with a GeneChip Scanner negative to date. The patient subsequently developed diarrhea at 7 mo 3000 7G. Probe hybridization intensities were analyzed using Affymetrix posttransplantation and Clostridium difficile toxin was found in colonoscopy GeneChip Command Console, and DNA copy number and allele analysis samples. He responded to metronidazole treatment. were performed using Affymetrix Chromosome Analysis Suite software, ’ The patient s T-cell proliferative responses normalized at 4 mo post- which compares all hybridization intensities against a built-in reference data transplantation (Fig. 1B). His fevers, elevated white cell counts, increased set (Cytogenetics_Array.na31.v1.REF_MODEL). All tiny focal copy number serum Ig levels, and adenopathy resolved by 12 mo posttransplantation and peaks were checked against the Database of Genomic Variants of the Hos- he is currently doing well clinically 5 y posttransplantation with normal T-cell pital for Sick Children, Toronto, to rule out the possibility that a focal peak is function (Fig. 1B and Table 1) while receiving subcutaneous IgG replacement a known copy number variant (polymorphism). The Cytogenetics Whole- therapy. The patient was the child of unrelated Caucasian parents. There Genome array was performed and analyzed by J.P. and J.R.T. All data were was no known family history of immunodeficiency, and the patient has a submitted to the National Center for Biotechnology Information (NCBI) healthy older sister and younger brother. However, his mother had Gene Expression Omnibus (GEO) repository, accession no. GSE35674.

Roberts et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 Exome Sequencing and Bioinformatic Analysis. DNA libraries were prepared from the Uniprot Web site. We used PSI-BLAST (33) to search for homologs from 2 μg genomic DNA using modified Illumina Genomic PE Sample Prep of the Uniprot sequence in the Uniref100 database and to calculate a posi- Kit protocol (Illumina) where all DNA purification steps were performed tion-specific scoring matrix (PSSM) for each sequence. The homologs with with AMPure SPRI bead purification (Bechman Coulter Genomics). Coding greater than 35% sequence identity to the queries were aligned with the sequences were captured using the Agilent SureSelect Target Enrichment program Muscle (34) and the program AL2CO (35) was used to calculate System with the Human All Exon Kit targeting 50 Mb of sequence. The a conservation score for each sequence position. We used the program captured DNA libraries were PCR-amplified using the supplied paired-end Disopred (36) to predict whether each mutation position was present in PCR primers and sequenced in one lane of an Illumina Genome Analyzer IIx. ordered regions or intrinsically disordered regions of the proteins. We also Sample preparation and sequencing was performed by Expresssion Analysis. used the Pfam Web site to determine whether the mutations were located Sequence reads were mapped to the reference genome (hg18; http://www. in defined domains according to Pfam (37). Finally, mutations were mapped ncbi.nlm.nih.gov/) using the SamTools package (29) and BWA aligner (30). to protein structures using profile-profile alignment search methods de- Duplicated reads were removed with Picard (http://picard.sourceforge.net/). veloped by us (38). Recalibration of base quality and indel realignment were performed using For the mutations with available structures, we used a support vector the GATK package (31). Single-nucleotide variants and indel variants were machine (SVM) trained on 4,600 disease-associated mutations in 1,491 human fi fi identi ed using the Uni ed Genotyper caller of GATK package from mul- proteins and 4,600 mutations between human proteins and primate tiple samples, including samples from other projects. Mutations were orthologs. The features used included surface accessible area in the bi- annotated with SeattleSeq Annotation (http://gvs.gs.washington.edu/ ological assemblies and monomers of known structures, the PSSM scores of SeattleSeqAnnotation/). A SQL database was created from the annotated the wild-type and mutant amino acids, and the conservation scores. For dataset. For maternal UPD analysis, alleles that fulfill the following those mutations not present in any structure and predicted to be ordered, requirements were identified: (i) heterozygous in mother’s sample; (ii) ho- we used a separate SVM without the structural features. For those muta- mozygous for wild-type allele in father’s sample; (iii) homozygous for wild- tions without structures and predicted to be disordered, we used a third type allele in other unrelated samples in the dataset; (iv) homozygous for SVM trained on 463 mutations in disordered regions of 261 different human alternative allele in child’s sample. For paternal UPD analysis, alleles that proteins. Table 2 shows the predicted phenotypes of the mutations. If the fulfill the following requirements are identified: (i) heterozygous in father’s deleterious probability is greater than 50%, then the prediction is that the sample; (ii) homozygous for wild-type allele in mother’s sample; (iii) ho- mutation is deleterious; otherwise, the mutation is predicted to be neutral. mozygous for wild-type allele in other unrelated samples in the dataset; (iv) The analysis predicting the likelihood that mutations are deleterious to homozygous for alternative allele in child’s sample. Manual examination protein function was performed by Q.W. and R.L.D. was conducted with TViewer (http://tviewer.sourceforge.net/) of SamTools to identify high confidence mutations from the raw sequence data. Analysis of the exome sequence data were performed by A.L., B.L., and S.P. All data ACKNOWLEDGMENTS. We thank Drs. Arvil Burks, Dietmar Kappes, and Maureen Murphy for critical review of the manuscript, and Dr. Haydar Frangoul were submitted to the NCBI Database of Genotypes and Phenotypes (dbGaP) of Vanderbilt University for referral of the patient. This work was supported in repository, accession no. phs000479.v1.p1. part by the Institute of Personalized Medicine at Fox Chase Cancer Center To predict the phenotype of missense mutations, we obtained sequence- through Grants AI047605 and AI042951; National Institutes of Health Challenge based and structure-based information on each mutation. The mutations Grant RC1 HL099617 and Core Grant P01CA06927; Center Grant P30-DK-50306; were mapped to Uniprot sequences (32), and these sequences were obtained and an appropriation from the Commonwealth of Pennsylvania.

1. Buckley RH (2011) Transplantation of hematopoietic stem cells in human severe 20. Morison IM, Paton CJ, Cleverley SD (2001) The imprinted gene and parent-of-origin combined immunodeficiency: Longterm outcomes. Immunol Res 49:25–43. effect database. Nucleic Acids Res 29:275–276. 2. Fischer A, et al. (2005) Severe combined immunodeficiency. A model disease for 21. Horsthemke B, Buiting K (2008) Genomic imprinting and imprinting defects in hu- molecular immunology and therapy. Immunol Rev 203:98–109. mans. Adv Genet 61:225–246. 3. Kung C, et al. (2000) Mutations in the tyrosine phosphatase CD45 gene in a child with 22. Welch TR, Beischel LS, Choi E, Balakrishnan K, Bishof NA (1990) Uniparental isodisomy severe combined immunodeficiency disease. Nat Med 6:343–345. 6 associated with deficiency of the fourth component of complement. J Clin Invest 86: 4. Tchilian EZ, et al. (2001) A deletion in the gene encoding the CD45 antigen in a pa- 675–678. tient with SCID. J Immunol 166:1308–1313. 23. Sulisalo T, et al. (1997) Uniparental disomy in cartilage-hair hypoplasia. Eur J Hum 5. Engel E (1980) A new genetic concept: Uniparental disomy and its potential effect, Genet 5:35–42. – isodisomy. Am J Med Genet 6:137 143. 24. Dufourcq-Lagelouse R, et al. (1999) Chediak-Higashi syndrome associated with ma- 6. Spence JE, et al. (1988) Uniparental disomy as a mechanism for human genetic dis- ternal uniparental isodisomy of chromosome 1. Eur J Hum Genet 7:633–637. – ease. Am J Hum Genet 42:217 226. 25. Al-Jasmi F, Abdelhaleem M, Stockley T, Lee KS, Clarke JT (2008) Novel mutation of the 7. Ledbetter DH, et al. (1981) Deletions of chromosome 15 as a cause of the Prader-Willi perforin gene and maternal uniparental disomy 10 in a patient with familial hemo- – syndrome. N Engl J Med 304:325 329. phagocytic lymphohistiocytosis. J Pediatr Hematol Oncol 30:621–624. 8. Yamazawa K, Ogata T, Ferguson-Smith AC (2010) Uniparental disomy and human 26. Prando C, et al. (2010) Paternal uniparental isodisomy of chromosome 6 causing disease: An overview. Am J Med Genet C Semin Med Genet 154C:329–334. a complex syndrome including complete IFN-gamma receptor 1 deficiency. Am J Med 9. Kishihara K, et al. (1993) Normal B lymphocyte development but impaired T cell Genet A 152A:622–629. maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell 74: 27. Nimmo G, et al. (2010) Rhizomelic chrondrodysplasia punctata type 2 resulting from 143–156. paternal isodisomy of chromosome 1. Am J Med Genet A 152A:1812–1817. 10. Koretzky GA, Picus J, Thomas ML, Weiss A (1990) Tyrosine phosphatase CD45 is es- 28. Buckley RH, et al. (1986) Development of immunity in human severe primary T cell sential for coupling T-cell antigen receptor to the phosphatidyl inositol pathway. deficiency following haploidentical bone marrow stem cell transplantation. JIm- Nature 346:66–68. munol 136:2398–2407. 11. Anonymous; 1000 Genomes Project Consortium (2010) A map of human genome 29. Li H, et al.; 1000 Genome Project Data Processing Subgroup (2009) The Sequence variation from population-scale sequencing. Nature 467:1061–1073. Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079. 12. Ideo H, Matsuzaka T, Nonaka T, Seko A, Yamashita K (2011) Galectin-8-N-domain 30. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler recognition mechanism for sialylated and sulfated glycans. J Biol Chem 286: – 11346–11355. transform. Bioinformatics 25:1754 1760. 13. Lu X, et al. (2006) Peptidoglycan recognition proteins are a new class of human 31. McKenna A, et al. (2010) The Genome Analysis Toolkit: A MapReduce framework for – bactericidal proteins. J Biol Chem 281:5895–5907. analyzing next-generation DNA sequencing data. Genome Res 20:1297 1303. 14. Bicknell LS, et al. (2011) Mutations in ORC1, encoding the largest subunit of the origin 32. Bairoch A, et al. (2005) The Universal Protein Resource (UniProt). Nucleic Acids Res – recognition complex, cause microcephalic primordial dwarfism resembling Meier- 33(Database issue):D154 D159. Gorlin syndrome. Nat Genet 43:350–355. 33. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein – 15. Brown KM, et al. (2007) Further evidence of association of OPRD1 & HTR1D poly- database search programs. Nucleic Acids Res 25:3389 3402. morphisms with susceptibility to anorexia nervosa. Biol Psychiatry 61:367–373. 34. Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high 16. Cale CM, et al. (1997) Severe combined immunodeficiency with abnormalities in ex- throughput. Nucleic Acids Res 32:1792–1797. pression of the common leucocyte antigen, CD45. Arch Dis Child 76:163–164. 35. Pei J, Grishin NV (2001) AL2CO: Calculation of positional conservation in a protein 17. Robinson WP (2000) Mechanisms leading to uniparental disomy and their clinical sequence alignment. Bioinformatics 17:700–712. consequences. Bioessays 22:452–459. 36. Ward JJ, McGuffin LJ, Bryson K, Buxton BF, Jones DT (2004) The DISOPRED server for 18. Guttenbach M, Engel W, Schmid M (1997) Analysis of structural and numerical the prediction of protein disorder. Bioinformatics 20:2138–2139. chromosome abnormalities in sperm of normal men and carriers of constitutional 37. Finn RD, et al. (2010) The Pfam protein families database. Nucleic Acids Res 38(Da- chromosome aberrations. A review. Hum Genet 100:1–21. tabase issue):D211–D222. 19. Makishima H, Maciejewski JP (2011) Pathogenesis and consequences of uniparental 38. Wang G, Dunbrack RL, Jr. (2004) Scoring profile-to-profile sequence alignments. disomy in cancer. Clin Cancer Res 17:3913–3923. Protein Sci 13:1612–1626.

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