Defects of B-cell terminal differentiation in patients with type-1 Kabuki syndrome

Immune deficiencies, infection, and systemic immune disorders

Defects of B-cell terminal differentiation in patients with type-1 Kabuki syndrome Andrew W. Lindsley, MD, PhD,a,b Howard M. Saal, MD,b,c Thomas A. Burrow, MD,b,c Robert J. Hopkin, MD,b,c Oleg Shchelochkov, MD,d Pooja Khandelwal, MD,b,e Changchun Xie, PhD,f Jack Bleesing, MD, PhD,b,e Lisa Filipovich, MD,b,e Kimberly Risma, MD, PhD,a,b Amal H. Assa’ad, MD,a,b Phillip A. Roehrs, MD,g and Jonathan A. Bernstein, MDh Cincinnati, Ohio, Iowa City, Iowa, and Chapel Hill, NC Background: Kabuki syndrome (KS) is a complex multisystem developmental disorder associated with mutation of genes encoding histone-modifying proteins. In addition to craniofacial, intellectual, and cardiac defects, KS is also characterized by humoral immune deficiency and autoimmune disease, yet no detailed molecular characterization of the KS-associated immune phenotype has been reported. Objective: We sought to characterize the humoral immune defects found in patients with KS with lysine methyltransferase 2D (KMT2D) mutations. Methods: We comprehensively characterized B-cell function in a cohort (n 5 13) of patients with KS (age, 4 months to 27 years). Results: Three quarters (77%) of the cohort had a detectable heterozygous KMT2D mutation (50% nonsense, 20% splice site, and 30% missense mutations), and 70% of the reported mutations are novel. Among the patients with KMT2D mutations (KMT2DMut/1), hypogammaglobulinemia was detected in all but 1 patient, with IgA deficiency affecting 90% of patients and a deficiency in at least 1 other isoform seen in 40% of patients. Numbers of total memory (CD271) and classFrom the Divisions of aAllergy and Immunology, cHuman Genetics, and eBone Marrow Transplantation, Cincinnati Children’s Hospital Medical Center; bthe Department of Pediatrics and fthe Division of Biostatistics and Bioinformatics, Department of Environmental Health, University of Cincinnati; dthe Division of Genetics, Stead Department of Pediatrics, University of Iowa Hospitals and Clinics, Iowa City; gthe Division of Pediatric Hematology/Oncology, University of North Carolina, Chapel Hill; and h the Division of Immunology, Allergy and Rheumatology, Department of Internal Medicine, University of Cincinnati College of Medicine. A.W.L. received investigator-initiated funding from CSL Behring for this study. A.W.L. was also supported by National Institutes of Health K12 (HD028827) Child Health Research Career Development Award. Disclosure of potential conflict of interest: A. W. Lindsley has received research support from the National Institutes of Health (NIH)/the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD; K12 Career Development [HD028827]), CSL Behring (Investigator Initiated Research Grant), and the Cincinnati Children’s Research Foundation (Procter Scholarship Career Development Award). H. M. Saal has received research support and lecture fees from Alexion Pharmaceuticals and has provided expert testimony for Burg Simpson, Attorneys at Law. A. H. Assa’ad is employed by Cincinnati Children’s Hospital. J. A. Bernstein has received lecture fees from Baxter and CSL Bering. The rest of the authors declare that they have no relevant conflicts of interest. Received for publication January 7, 2015; revised May 28, 2015; accepted for publication June 2, 2015. Available online July 17, 2015. Corresponding author: Andrew W. Lindsley, MD, PhD, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229. E-mail: andrew.lindsley@ cchmc.org. The CrossMark symbol notifies online readers when updates have been made to the article such as errata or minor corrections 0091-6749/$36.00 Ó 2015 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaci.2015.06.002

switched memory B cells (IgM2) were significantly reduced in patients with KMT2DMut/1 mutations compared with numbers in control subjects (P < .001). Patients with KMT2DMut/1 mutations also had significantly reduced rates of somatic hypermutation in IgG (P 5 .003) but not IgA or IgM heavy chain sequences. Impaired terminal differentiation was noted in primary B cells from patients with KMT2DMut/1 mutations. Autoimmune pathology was observed in patients with missense mutations affecting the SET domain and its adjacent domains. Conclusions: In patients with KS, autosomal dominant KMT2D mutations are associated with dysregulation of terminal B-cell differentiation, leading to humoral immune deficiency and, in some cases, autoimmunity. All patients with KS should undergo serial clinical immune evaluations. (J Allergy Clin Immunol 2016;137:179-87.) Key words: Kabuki syndrome, KMT2D, KDM6A, hypogammaglobulinemia, memory B cells, CD21lo B cells, class-switch recombination, somatic hypermutation, PTIP, AICDA, AID, polymerase eta

Kabuki syndrome (KS) or Niikawa-Kuroki syndrome is a congenital syndrome characterized by a stereotypical set of facial features, skeletal anomalies, dermatoglyphic abnormalities, intellectual disability, and postnatal growth deficiency.1,2 Classic KS-associated facies include arched eyebrows with central notching, elongated palpebral fissures with eversion of the lateral lower eyelid, and large, prominent, cupped ears. Although consensus clinical diagnostic criteria have not been established for this rare disease, other frequently noted structural defects include congenital heart disease, cleft palate, gastrointestinal malformations, and ophthalmologic defects.3-5 Functional defects are also observed, including immune dysfunction (recurrent infections and autoimmune disorders), seizures, hearing loss, and endocrinopathies.6-9 Autosomal dominant mutations in 2 epigenetic regulatory genes, lysine methyltransferase 2D (KMT2D/MLL2/ALR/MLL4; KS-1, OMIM 147920) and lysine demethylase 6A (KDM6A/ UTX; KS-2, OMIM 300867), were recently found in large cohorts of patients with KS, suggesting that defects in histone-mediated regulation drive the diverse pathology of this syndrome.10,11 KMT2D modifies gene expression through methylation of histone 3, lysine 4 (H3K4), and mutations of this gene are the most common cause of KS (approximately 60% to 70% of cases).12,13 KMT2D encodes a large multidomain protein that forms the core of one of the 6 mammalian complex of proteins associated with SET1 (COMPASS) complexes.14 KDM6A is a cofactor physically associated with the KMT2D COMPASS complex and exhibits complementary demethylase activity at lysine 27 on histone 3 (H3K27).15 Together, the components of the KMT2D 179

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Abbreviations used AICDA: Activation-induced cytidine deaminase cKS: Clinical Kabuki syndrome (no identifiable KMT2D/ KDM6A mutations) COMPASS: Complex of proteins associated with SET1 CSR: Class-switch recombination CVID: Common variable immune deficiency H3K4: Histone 3, lysine 4 IGH: Immunoglobulin heavy chain ITP: Immune thrombocytopenia KDM6A: Lysine demethylase 6A KMT2D: Lysine methyltransferase 2D KMT2DMut/1: Autosomal dominant mutation in KMT2D KS: Kabuki syndrome MLPA: Multiplex ligation-dependent probe amplification PTIP: Paired box (PAX) transactivation domain–interacting protein SHM: Somatic hypermutation

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little about the underlying pathogenesis of the observed immune dysfunction. Despite a growing clinical interest in KS, the cellular characteristics and molecular mechanisms of KS-associated immune dysfunction remain poorly understood. However, a variety of recent studies have now shown that histone modifications regulate key events in terminal B-cell differentiation. Specifically, CSR and the related phenomenon of somatic hypermutation (SHM) are 2 critical B-cell processes subject to epigenetic regulation,27-29 but despite their importance to humoral immunity, these processes have never previously been evaluated in patients with KS. To address these knowledge gaps, we characterize herein the specific cellular and molecular B-cell defects found in a cohort of 13 patients with KS (8 pediatric and 5 adult patients). Our findings provide mechanistic insight into the humoral immune deficiency and autoimmunity found in this understudied patient population.

METHODS Patient cohort COMPASS complex remove inhibitory epigenetic marks and add activating marks, specifically H3K4 monomethylation, dimethylation, or trimethylation (H3K4me1, 2, or 3). KMT2D has been implicated in the regulation of hundreds of genes through regulation of enhancer and promoter elements, providing a rationale for how defects in this single gene and its binding partners can lead to a complex and heterogeneous congenital phenotype.12,16,17 KMT2D binds multiple cofactors, including the paired box (PAX) transactivation domain–interacting protein (PTIP), which plays a key role in B-cell terminal differentiation. PTIP has dual activities, binding regions of DNA double-strand breaks, such as those that occur during class-switch recombination (CSR), and interacting with PAX transcription factors. PTIP directly binds the B-cell master transcription factor PAX5, targeting KMT2Dmediated H3K4me3 methylation to switch regions within the immunoglobulin heavy chain (IGH) locus during CSR.18-20 B cell–specific knockout of Ptip in primary murine B cells dramatically impairs CSR.20 Studies of PTIP have demonstrated the important role of the KMT2D COMPASS complex in regulating the IGH locus during B-cell differentiation, thus linking the molecular functions of the 2 KS-associated genes with a central mechanism of humoral immunity. Immune dysfunction is a common feature of KS. Described as having a common variable immune deficiency (CVID)–like immune profile in early studies,7 this initial observation was later correlated with clinical findings in 3 larger cohorts of patients with heterozygous KMT2D mutations (KMT2DMut/1). Approximately half of the patients in these studies had clinical features of immune deficiency, including recurrent otitis media (47%)21 and frequent/repeat infections (41.9%22/64%23). A more recent study found recurrent otitis media to be a nearly universal clinical finding (85%) in patients with KMT2DMut/1 KS.24 In addition, autoimmune diseases, especially immune thrombocytopenia (ITP), hemolytic anemia, and vitiligo, have also been reported in patients with KS; however, all of these early cases were in patients with clinical diagnoses but without a defined genetic cause. However, 2 new case reports of ITP in genetically defined patients with KMT2DMut/1 mutations have strengthened the link between KS and autoimmunity.25,26 Together, these foundational studies provide a general outline of the KS immune phenotype but reveal

Patients with a clinical diagnosis of KS by a board-certified clinical geneticist were recruited to participate. All participants, their parents/guardians, or both provided written consent/assent for enrollment in this study (including explicit authorization to publish images of patients). The study protocol (no. 2012-4636) was approved by the Institutional Review Board at Cincinnati Children’s Hospital Medical Center. Phenotypic data were collected from medical record review and interviews with patients, guardians, and physicians.

KMT2D/KDM6A sequencing analysis and multiplex ligation-dependent probe amplification All 13 patients underwent KMT2D sequencing (reference no. NM_003482.3). KMT2D domains were mapped according to the UniProt Database (O14686). Per the stepwise approach to genetic diagnosis of KS in clinical practice, the 3 patients (patients A-C) in whom no KMT2D mutation was identified underwent multiplex ligation-dependent probe amplification (MLPA) analysis of 23 KMT2D exons, sequencing of the KDM6A coding region, and MLPA of 26 KDM6A exons. See the Methods section in this article’s Online Repository at www.jacionline.org for MLPA exon numbers.

Splice site analysis To confirm the presence of misspliced mutant variant transcripts, putative splice site variants were analyzed with NNSPLICE software.30 cDNA from patients 3 and 7, who have mutations in KMT2D splice sites, was generated from PBMCs. Primers flanking the predicted splice site mutation amplified regions of interest. Bands were purified, cloned, and sequenced.

Clinical laboratory evaluations Immunoglobulin levels, postvaccination serology, lymphocyte subpopulation data, and mitogen studies were derived from physician review of medical records. All clinical evaluations were performed at Clinical Laboratory Improvement Amendments–certified clinical laboratories and reported with age-matched normal ranges. B-cell flow cytometry was performed in the Diagnostic Immune Laboratory at Cincinnati Children’s Hospital Medical Center.

SHM analysis V(D)J libraries were generated from PBMC-derived cDNA, cloned, and sequenced. Briefly, we combined the BIOMED-2 FR1 pool of variable gene primers31 with individual antisense, constant region–specific (m, g, and a) primers to amplify pools of isotype-specific immunoglobulin-derived clones.32,33 After gel purification, these libraries were cloned (TOPO TA

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TABLE I. KS phenotype Subject no.: Age at enrollment

1

2

3

4

5

6

7

8

9

10

A

B

C

0 y, 4 mo

0 y, 5 mo

0 y, 11 mo

3 y, 8 mo

6 y, 10 mo

15 y, 5 mo

15 y, 6 mo

23 y, 0 mo

24 y, 10 mo

27 y, 2 mo

2 y, 11 mo

12 y, 0 mo

13 y, 6 mo

Cardinal features Typical facial 1 1 1 1 1 1 1 1 1 1 1 1 1 features Skeletal anomalies 2 1 1 1 1 2 1 1 2 1 1 1 1 Mild-to-moderate NA NA NA NA 1 1 1 1 1 1 1 1 1 intellectual disability Postnatal growth 1 2 1 1 2 1 1 1 1 1 2 1 1 deficiency Additional features Congenital heart 1 1 1 1 1 1 1 2 2 1 2 2 1 defects Genitourinary 2 1 2 1 2 1 1 1 1 1 2 1 1 anomalies Cleft lip and/or 1 2 1 1 1 2 1 2 1 2 2 2 2 palate Gastrointestinal 2 2 2 2 2 2 1 2 1 2 2 2 2 anomalies Ophthalmologic 1 1 2 2 2 1 1 1 2 2 2 1 1 anomalies Autoimmune 2 2 2 2 1 1 2 1 2 2 2 2 2 complications Recurrent 2/2/2 2/2/2 2/2/2 1/1/1 1/2/2 1/1/1 1/1/2 1/1/1 1/1/2 2/2/2 1/2/2 1/2/2 1/1/2 infections OM/PNA/UTI

Present in Metathis cohort analysis9

100%

87%

77% 100%*

ND ND

77%

70%

69%

46%

69%

44%

46%

37%

15%

ND

54%

ND

23%

ND

77%

61%

NA, Not available; ND, not done; OM, otitis media; PNA, pneumonia; UTI, urinary tract infection. *Percentage of subjects older than 5 years of age.

cloning; Life Technologies, Grand Island, NY) and screened for inserts, and 20 to 30 clones per library were sequenced. Immunoglobulin sequences were aligned with the human IGH locus by using the IgBLAST software and the IMTG database.34 Nonproductive clones were excluded from analysis. Mutation analysis was performed with SHMTool software.35

In vitro terminal B-cell differentiation B-cell differentiation was induced by using a modified version of the protocol described by Ettinger et al.36 See the Methods section in this article’s Online Repository for culture and staining details. Cells were analyzed after 3 or 7 days of culture by using flow cytometry (LSR-II flow cytometer; BD Biosciences, Franklin Lakes, NJ).

Statistical analysis Patient-derived B-cell data were compared with data from age-matched control subjects by using a linear regression model in which the P value was calculated based on the F-test (GLM procedure; version 9.4, SAS Institute, Cary, NC). All other statistical analyses were performed with an unpaired Student t test. A P value of less than .05 was considered statistically significant.

RESULTS Cohort characteristics Each patient’s KS phenotype was evaluated for the presence of 4 of the 5 cardinal manifestations of KS (dermatoglyphic analysis not done, Table I).37 Images of each patient’s face and right ear are provided (see Fig E1 in this article’s Online Repository at www. jacionline.org). Additional KS-associated structural and

functional anomalies were also evaluated. Frequencies of selected traits in our cohort were calculated and compared with the metaanalysis of B€ ogershausen and Wollnik (4 studies, total n 5 287 patients).9 All but 2 categories of KS-associated traits showed similar frequency rates between our cohort and the metaanalysis; congenital heart disease and genitourinary anomalies were more common in our cohort than in the meta-analysis cohorts. Adults with KS also exhibit a variety of CVID-associated comorbidities, especially splenomegaly and pulmonary disease (see Table E1 in this article’s Online Repository at www. jacionline.org).

Molecular genetic evaluation All of the patients underwent KMT2D gene sequencing, and 10 (77%) of 13 were found to have mutations predicted to be pathogenic (see Fig E2 in this article’s Online Repository at www.jacionline.org), a rate consistent with prior reports.9,10 Seven of these KMT2D mutations were novel. Half of the 10 identified patients with KMT2DMut/1 mutations harbored nonsense/frameshift mutations predicted to generate a truncated protein or induce nonsense-mediated decay of the mutated transcript.38 Two patients (patients 3 and 7) had splice site mutations identified (see Fig E2). Bioinformatic analysis of patient 3’s mutation predicted a deleterious effect (loss of the intron 26 acceptor site). This prediction was confirmed by means of PCR amplification of a mutant transcript fragment from patient-derived PBMC cDNA (see Fig E3, A, in this article’s Online Repository at www. jacionline.org). Cloning and sequencing of this aberrant band

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revealed retained intronic sequence encoding a premature stop codon. Patient 7 was also found to have a splice site mutation (adjacent to the donor site of intron 29), and bioinformatic analysis predicted reduced confidence in the functionality of the site. Similar PCR analysis of PBMC-derived cDNA from patient 7 did not detect a mutant band (see Fig E3, B). Three patients (patients 5, 6, and 8) had missense mutations in the 39 end of the KMT2D locus (exons 48 and 51) within highly conserved functional domains (see Fig E2). The previously reported missense mutation identified in patient 8 (c.16294C>T) was recently shown to disrupt the Kabuki interaction surface, which forms a secondary active site in the COMPASS complex.39 The 3 patients (patients A-C) who were given clinical diagnoses of KS but in whom no KMT2D mutation was detected (clinical Kabuki syndrome [cKS]) also underwent KMT2D exon copy number variant testing (MLPA) and KDM6A sequencing and copy number testing (MLPA). In all 3 cases these additional studies did not detect a pathogenic mutation, insertion, or deletion in these 2 previously described KS-associated genes.

Autoimmune disease clinical histories Autoimmune disease is commonly associated with humoral immune deficiency and observed in approximately 20% of patients with CVID.40 Three of our patients have a history of marked autoimmune disease (patients 5, 6, and 8; 23% of the total cohort; detailed clinical histories are provided in Fig E4 in this article’s Online Repository at www.jacionline. org). All 3 patients experienced ITP, and in 2 cases, ITP developed within several months of contracting EBV (patients 5 and 8). Patient 6 also had vitiligo, which required phototherapy. Patient 8 has had recurrent ITP, autoimmune neutropenia, autoimmune hepatitis, and insulin-dependent diabetes mellitus. Interestingly, these patients all harbor similar missense mutations in the 39 end of the KMT2D gene near the SET domain region (see Fig E2). Immune evaluations Lymphocyte evaluations. Peripheral lymphocyte subpopulations were evaluated for 12 of the 13 patients, and the majority of patients had normal or minor deviations from agematched control values (see Table E2 in this article’s Online Repository at www.jacionline.org).41 Similarly, mitogen studies were performed in 11 of 13 subjects, with more than 80% having essentially normal results. Two patients (patients 6 and 8) had diminished but not absent mitogen responses (see Table E2). With regard to lymphocyte subpopulations, 4 (33%) of 12 patients were found to have a reduced percentage of CD191 B cells (vs age-matched control subjects). Four patients (patients 2, 5, 6, and 8) had lymphopenia and abnormal lymphocyte subpopulations at the time of enrollment. Patient 2 has a large retroperitoneal lymphatic malformation, resulting in significant chylous ascites and loss of peripheral lymphocytes, especially T cells. Also, patient 2’s absolute B-cell count was low (556 cells/mL vs the age-adjusted normal range of 700-2500 cells/mL), despite an increased B-cell percentage because of preferential loss of T cells. Patient 5 underwent thymectomy at 3 days of life, with intermittent lymphopenia noted since that time. Patient 6 was noted to have lymphocyte

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subpopulation levels within normal age-matched ranges at initial evaluation (5.5 years of age) but subsequently had lymphopenia with onset of autoimmune disease (see Fig E4). Similarly, patient 8 had a normal absolute lymphocyte count at 13 years of age before contracting EBV, but since that time, the patient has had chronic borderline lymphopenia (see Fig E4). Immunoglobulin levels. We found that 90% of our patients with KMT2DMut/1 mutations had IgA deficiency, whereas our 3 patients with cKS (patients A-C) had normal or above-normal serum IgA levels (Fig 1, A). Among patients greater than 5 years of age, IgA levels were significantly decreased in patients with KMT2DMut/1 mutations compared with patients with cKS (patients A-C, P 5 .003; Fig 1, A, right panel). Low serum IgM levels were noted in 40% of patients with KMT2DMut/1 mutations (patients 4, 6, 8, and 9) but in none of the patients with cKS (Fig 1, B). Low IgG levels were also noted in 40% of patients with KMT2D mutations (patients 6, 8, and 9) and in 1 patient with cKS (Fig 1, C). Three patients with KMT2DMut/1 mutations (patients 6, 8, and 9) had panhypogammaglobulinema. Specific antibody titers against tetanus were also evaluated for all patients greater than 2 years of age, except patients 4 and 5. All patients had protective tetanus titers, except patient 6. One month after revaccination, patient 6 mounted a 10-fold increase in her anti-tetanus titers (still less than the protective level), but this level returned to her previously low baseline by 1 year later. An alternative measure of vaccine responsiveness was available for patient 5, who mounted protective responses to hepatitis A and B vaccinations at 6 years of age. Of note, 5 adults with KMT2DMut/1 mutations (patients 6-10) underwent prevaccination and postvaccination pneumococcal titer testing, and 3 (60%) of 5 did not mount a normal response (defined as a 2-fold increase in 70% of tested titers, see Table E3 in this article’s Online Repository at www. jacionline.org).42 B-cell subpopulations. Given our finding of hypogammaglobulinemia in the majority of our patients with KMT2DMut/1 mutations, we next evaluated peripheral B-cell subpopulations in 12 of 13 patients in our cohort (patient 5, no data; patient 6, not all data available). Consistent with our lymphocyte subpopulation analysis, 4 (33%) of 12 of our patients had a low percentage of total B cells (CD191/CD201; Fig 2, A); all 4 had KMT2DMut/1 mutations (4/9 [44%]). We next evaluated total memory B cells (CD271) and found that 89% (8/9) of patients with KMT2DMut/1 mutations were deficient in this cell type, a feature shared with 67% (2/3) of patients with cKS (Fig 2, B). When compared with control subjects, we found that patients with KMT2DMut/1 mutations had a significant reduction in memory B-cell numbers after adjusting for age (P 5 .0005). Classswitched memory B cells (IgM2, IgD2, CD271) were also low in 5 patients with KMT2DMut/1 mutations (63%) and in 1 patient lacking a KMT2D mutation (Fig 2, C). Compared with ageadjusted control subjects, we found that patients with KMT2DMut/1 mutations had a significant reduction in classswitched memory B-cell numbers (P 5 .0077). Finally, we evaluated CD191CD21lo B cells, a cell population previously associated with CVID in adults,43 and found that 4 patients with KMT2DMut/1 mutations had an expanded population of these cells; no expansion of these cells was seen in patients with cKS (Fig 2, D). When compared with control subjects,

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FIG 1. Serum immunoglobulin levels. Patients are arranged by ascending age. Gray shading represents the age-matched normal range (2.5th-97.5th percentile). A, IgA levels in patients with KMT2DMut/1 mutations (110, solid circles) and patients with cKS (A-C, open squares). B, IgA values (bar 5 mean) are significantly reduced in patients with KMT2DMut/1 mutations older than 5 years versus patients with cKS. *P < .05. C and D, IgM and IgG levels. IgG values for patients 2, 6, C, and 9 (^) are the last levels acquired before intravenous immunoglobulin/subcutaneous immunoglobulin treatment.

patients with KMT2DMut/1 mutations had a significant expansion in CD191CD21lo B cells after adjusting for age (P < .0001). Collectively, these findings indicate that many patients with KS exhibit abnormal terminal B-cell differentiation. SHM rates. A critical step in terminal B-cell differentiation is postactivation SHM of the IGH locus. Therefore we quantified the rate of SHM in V(D)J transcripts cloned from KMT2DMut/1 B cells to investigate whether this process was disrupted by KMT2D genetic lesions. We focused our analysis on our 4 adult patients with KMT2DMut/1 mutations (patients 7-10) because of the availability of age-matched control subjects. Using a set of diverse variable gene primers (BioMED2 FR1) paired with a constant region primer (IgM, pan-IgG, or IgA specific), we isolated and sequenced patient-specific V(D)J clones and quantified their divergence from germline sequence. SHM rates for IgM and IgA V(D)J clones from patients with KMT2DMut/1 mutations did not significantly differ from rates derived from control subject clones (Fig 3, A and B) and were consistent with previously published rates of SMH in adults.44-47 Interestingly, the SHM rate for IgG V(D)J clones was significantly lower in 3 of 4 patients with KMT2DMut/1 mutations when compared with control subject clones (P < .003; Fig 3, C). These findings show that a subset of adult patients with KS have diminished IgG affinity

maturation in circulating memory B cells. IgG affinity maturation appears to be more susceptible to KMT2D insufficiency than IgA SHM, possibly the result of the divergent cytokine signaling mechanisms and cell trafficking that drive mucosal (IgA) versus systemic (IgG) humoral immunity.48 To explore the mechanism driving this reduction in the SHM rate, we next performed mutation analysis on IgG clones derived from patients with KMT2DMut/1 mutations (see Table E4 in this article’s Online Repository at www.jacionline.org). SHM-associated mutations are targeted to the V(D)J sequence and occur in nonrandom patterns, with certain nucleotide motifs being preferentially targeted (‘‘hotspot’’) or ignored (‘‘coldspot’’). We observed no differences in the fraction of transition or transversion mutations detected in V(D)J sequences derived from patients with KMT2DMut/1 mutations versus those from healthy control subjects. Likewise, the percentage of total V(D)J mutations occurring at activationinduced cytidine deaminase (AICDA, also known as AID) ‘‘hotspot’’ and ‘‘coldspot’’ motifs also did not differ between patients with KMT2DMut/1 mutations and control subjects. In contrast, we observed a trend toward reduced use of the preferred polymerase h (eta) motif in patients with KMT2DMut/1 mutations (KS average: 9.01% vs control average: 15.96%, P 5 .058). This approximately 40%

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FIG 2. B-cell subset analysis. Patients are arranged by ascending age; gray shading represents the agematched normal range (5th-95th percentile). A, Total B cells (CD191CD201) in patients with KMT2DMut/1 mutations (solid circles) and patients with cKS (open squares). B, Total memory B cells (CD191, CD271). C, Class-switched memory B cells (CD191CD271IgM2IgD2). D, CD191CD21lo B cells. #Data were not available for patient 6 (n 5 11). *Received anti-CD20 (rituximab) antibody therapy 3 years before analysis and had a normal total B-cell count before therapy.

reduction was seen in all patients with KMT2DMut/1 mutations examined, including patient 10, who had a normal overall SHM rate. In vitro terminal B-cell differentiation. To clarify whether the humoral defects seen in many of our patients with KS were the result of intrinsic B-cell defects (cell autonomous or nonautonomous), we induced in vitro CSR in B cells isolated from an adult patient with a KMT2DMut/1 mutation with hypogammaglobulinemia but no prior exposure to rituximab or other immunosuppressive medication (patient 9). Total B cells were isolated from patient 9 and 2 agematched control subjects and incubated in medium alone or medium plus germinal center differentiation factors/stimuli (IL-21, anti-CD40 antibody, and anti-IgM antibody). Cells were analyzed after 3 and 7 days of incubation by means of flow cytometry. Control cells exposed to the differentiation factors efficiently downregulated IgD and induced CD38 (Fig 4), a marker of memory and plasma cell differentiation.36 B cells derived from patient 9 appeared to initiate normal differentiation at day 3 of culture by downregulating surface IgD but did not upregulate CD38 on IgD2 cells by day 7 of culture (2.8% vs 36.3% IgD2CD381 in control cells). CD27 was also examined and followed the same pattern as CD38 (data not shown). These findings suggest a specific role for

KMT2D signaling during germinal center–mediated terminal differentiation.

DISCUSSION On the basis of our results and accumulating evidence from multiple model systems,27,49,50 we believe that the hypogammaglobulinemia, reduced memory B-cell numbers, and impaired SHM observed in our patients with KMT2DMut/1 mutations are likely the result of disrupted epigenetic regulation of the IGH locus. During B-cell terminal differentiation, the IGH locus undergoes a variety of dynamic stepwise epigenetic modifications. In activated naive mature B cells, H3K4me3 modifications accumulate throughout the V(D)J region and IGH switch regions. These targeted H3K4me3 marks are associated with an open chromatin state and initiate a cascade of secondary H3 histone modifications that drive AICDA/AID recruitment to the switch regions and ultimately induce recombination of the IGH locus.51 The V(D)J sequence also is subject to similar epigenetic regulation during SHM, with initial H3K4me3 accumulation at the V(D)J coding sequence leading to secondary histone modifications, recruitment of AICDA/ AID and the error-prone DNA polymerase-h (pol-h), and subsequent mutation-driven affinity maturation of the antibody.49

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FIG 3. SHM rate. Isoform-specific V(D)J clones analyzed in adult patients with KMT2DMut/1 mutations (circles) and age-matched adult control subjects (triangles). Results are presented as percentage divergent from germline IGH sequence. IgM (A), IgG (B), and IgA (C) mutation rates in patients with KMT2DMut/1 mutations and control subjects are shown. IgG mutation rates (Fig 3, B) were significantly lower in 3 of 4 patients with KMT2DMut/1 mutations than in control subjects. *P < .05. All data are presented as means 6 SDs.

Impaired H3K4 trimethylation of the IGH locus reduces both CSR and SHM efficiency,27,50 linking these independent but simultaneous processes to epigenetic regulatory mechanisms. These findings are highly relevant to KS-associated immune dysfunction because KMT2D catalyzes H3K4 methylation at the IGH locus by binding its COMPASS cofactor PTIP,20 therefore supporting a direct link between KMT2D function and CSR/ SHM (see Fig E5 in this article’s Online Repository at www. jacionline.org). The CVID-like phenotype observed in our patients with KMT2DMut/1 mutations implicates altered epigenetic signaling as a cause for human B-cell dysfunction. Multiple knockout murine models have shown that a variety of chromatin modifier genes (Ezh2, Suv39h1, and Spt6) have important roles in terminal B-cell differentiation, CSR, and SHM.27-29 Future investigations into the cause of CVID might benefit from closer inspection of B-cell epigenetic regulation. Other important aspects of B-cell differentiation can also be affected in some patients with KMT2DMut/1 mutations, such as impaired deletion of autoreactive B-cell clones. One intriguing observation is that all 3 of our patients with KMT2DMut/1 mutations with autoimmune disease were found to have missense point mutations in the terminal carboxyl end of KMT2D gene. Of the 2 other patients with KMT2DMut/1 mutations reported in the literature to have had ITP, 1 patient exhibited a highly similar missense mutation in the same gene region (exon 52, c.16384G>C, p.D5462H).26 A recent enzymatic study of the KMT2D pre-

SET domain region modeled 5 previously reported KS missense mutations and found that all clustered onto a solvent-exposed surface termed the Kabuki interaction surface.39 These mutations interfered with KMT2D’s formation of a second active site, with critical protein cofactors rendering the protein only capable of performing histone monomethylation. Additional studies, perhaps using animal models that recapitulate human genetic lesions, are needed to clarify the in vivo effects of specific KMT2D point mutations on the development of autoreactive B cells. KS is a complex and heterogeneous disease, and patients are at increased risk for recurrent sinopulmonary infections and serious autoimmune sequelae, as seen in our adult patients with KMT2DMut/1 mutations (see Tables E2 and E3). Unfortunately, no disease-specific clinical treatments are currently approved for use in patients with KS, although this might change now that the underlying gene defects have been identified. Several lysine demethylase (KDM) inhibitors have recently been described and might prove useful in directly targeting H3K4 methylation defects.52 B cells are interesting initial targets for therapeutic epigenetic treatment given their lifelong ongoing differentiation from stem cells, providing a broad developmental window for aberrant epigenetic signaling to be normalized by pharmacologic intervention. In summary, we show that greater than 80% of patients with KMT2DMut/1 mutations have hypogammaglobulinemia and diminished memory B-cell populations. In addition, we present

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FIG 4. In vitro B-cell differentiation. Isolated total B cells cultured in medium alone or medium plus differentiation factors. Flow cytometry was performed on days 3 and 7 of culture. Studies were performed in duplicate, with representative flow plots shown.

evidence associating KMT2D mutations with expansion of the CVID-associated CD21lo B-cell population, impaired SHM in IgG transcripts, and disrupted terminal B-cell differentiation. We also note a clustering of missense mutations in the terminal region of the KMT2D gene and that such mutations might increase the risk for autoimmune disease in patients with KMT2DMut/1 mutations. In summary, this report reveals that epigenetic dysregulation of the B-cell compartment can lead to clinically relevant primary immune deficiency and autoimmunity in human subjects.

Clinical implications: KMT2DMut/1 KS causes IgA deficiency in nearly all patients, although additional humoral defects (memory B-cell deficiency and IgG hypogammaglobulinemia) have variable expressivity. Missense mutations in terminal domains might increase autoimmunity risk. REFERENCES 1. Niikawa N, Matsuura N, Fukushima Y, Ohsawa T, Kajii T. Kabuki make-up syndrome: a syndrome of mental retardation, unusual facies, large and protruding ears, and postnatal growth deficiency. J Pediatr 1981;99:565-9.

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2. Kuroki Y, Suzuki Y, Chyo H, Hata A, Matsui I. A new malformation syndrome of long palpebral fissures, large ears, depressed nasal tip, and skeletal anomalies associated with postnatal dwarfism and mental retardation. J Pediatr 1981;99: 570-3. 3. Matsumoto N, Niikawa N. Kabuki make-up syndrome: a review. Am J Med Genet C Semin Med Genet 2003;117C:57-65. 4. Armstrong L, Abd El Moneim A, Aleck K, Aughton DJ, Baumann C, Braddock SR, et al. Further delineation of Kabuki syndrome in 48 well-defined new individuals. Am J Med Genet A 2005;132A:265-72. 5. Schrander-Stumpel CT, Spruyt L, Curfs LM, Defloor T, Schrander JJ. Kabuki syndrome: Clinical data in 20 patients, literature review, and further guidelines for preventive management. Am J Med Genet A 2005;132A:234-43. 6. Kawame H, Hannibal MC, Hudgins L, Pagon RA. Phenotypic spectrum and management issues in Kabuki syndrome. J Pediatr 1999;134:480-5. 7. Hoffman JD, Ciprero KL, Sullivan KE, Kaplan PB, McDonald-McGinn DM, Zackai EH, et al. Immune abnormalities are a frequent manifestation of Kabuki syndrome. Am J Med Genet A 2005;135:278-81. 8. Ming JE, Russell KL, McDonald-McGinn DM, Zackai EH. Autoimmune disorders in Kabuki syndrome. Am J Med Genet A 2005;132A:260-2. 9. Bogershausen N, Wollnik B. Unmasking Kabuki syndrome. Clin Genet 2013;83: 201-11. 10. Ng SB, Bigham AW, Buckingham KJ, Hannibal MC, McMillin MJ, Gildersleeve HI, et al. Exome sequencing identifies MLL2 mutations as a cause of Kabuki syndrome. Nat Genet 2010;42:790-3. 11. Lederer D, Grisart B, Digilio MC, Benoit V, Crespin M, Ghariani SC, et al. Deletion of KDM6A, a histone demethylase interacting with MLL2, in three patients with Kabuki syndrome. Am J Hum Genet 2012;90:119-24. 12. Issaeva I, Zonis Y, Rozovskaia T, Orlovsky K, Croce CM, Nakamura T, et al. Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol Cell Biol 2007;27:1889-903. 13. Ansari KI, Mandal SS. Mixed lineage leukemia: roles in gene expression, hormone signaling and mRNA processing. FEBS J 2010;277:1790-804. 14. Smith E, Lin C, Shilatifard A. The super elongation complex (SEC) and MLL in development and disease. Genes Dev 2011;25:661-72. 15. Lee MG, Villa R, Trojer P, Norman J, Yan KP, Reinberg D, et al. Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science 2007;318:447-50. 16. Guo C, Chang CC, Wortham M, Chen LH, Kernagis DN, Qin X, et al. Global identification of MLL2-targeted loci reveals MLL2’s role in diverse signaling pathways. Proc Natil Acad Sci U S A 2012;109:17603-8. 17. Hu D, Gao X, Morgan MA, Herz HM, Smith ER, Shilatifard A. The MLL3/ MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers. Mol Cell Biol 2013;33:4745-54. 18. Cho YW, Hong T, Hong S, Guo H, Yu H, Kim D, et al. PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex. J Biol Chem 2007;282:20395-406. 19. Schwab KR, Patel SR, Dressler GR. Role of PTIP in class switch recombination and long-range chromatin interactions at the immunoglobulin heavy chain locus. Mol Cell Biol 2011;31:1503-11. 20. Daniel JA, Santos MA, Wang Z, Zang C, Schwab KR, Jankovic M, et al. PTIP promotes chromatin changes critical for immunoglobulin class switch recombination. Science 2010;329:917-23. 21. Li Y, Bogershausen N, Alanay Y, Simsek Kiper PO, Plume N, Keupp K, et al. A mutation screen in patients with Kabuki syndrome. Hum Genet 2011;130:715-24. 22. Micale L, Augello B, Fusco C, Selicorni A, Loviglio MN, Silengo MC, et al. Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients. Orphanet J Rare Dis 2011;6:38. 23. Banka S, Veeramachaneni R, Reardon W, Howard E, Bunstone S, Ragge N, et al. How genetically heterogeneous is Kabuki syndrome?: MLL2 testing in 116 patients, review and analyses of mutation and phenotypic spectrum. Eur J Hum Genet 2012;20:381-8. 24. Lin JL, Lee WI, Huang JL, Chen PK, Chan KC, Lo LJ, et al. Immunologic assessment and KMT2D mutation detection in Kabuki syndrome. Clin Genet 2014 [Epub ahead of print]. 25. Brackmann F, Krumbholz M, Langer T, Rascher W, Holter W, Metzler M. Novel MLL2 mutation in Kabuki syndrome with hypogammaglobulinemia and severe chronic thrombopenia. J Pediatr Hematol Oncol 2013;35:e314-6. 26. Giordano P, Lassandro G, Sangerardi M, Faienza MF, Valente F, Martire B. Autoimmune haematological disorders in two Italian children with Kabuki syndrome. Ital J Pediatr 2014;40:10. 27. Begum NA, Stanlie A, Nakata M, Akiyama H, Honjo T. The histone chaperone Spt6 is required for activation-induced cytidine deaminase target determination through H3K4me3 regulation. J Biol Chem 2012;287: 32415-29.

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28. Bradley SP, Kaminski DA, Peters AH, Jenuwein T, Stavnezer J. The histone methyltransferase Suv39h1 increases class switch recombination specifically to IgA. J Immunol 2006;177:1179-88. 29. Beguelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, et al. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer cell 2013;23:677-92. 30. Reese MG, Eeckman FH, Kulp D, Haussler D. Improved splice site detection in Genie. J Comput Biol 1997;4:311-23. 31. van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4CT98-3936. Leukemia 2003;17:2257-317. 32. Berkowska MA, Driessen GJ, Bikos V, Grosserichter-Wagener C, Stamatopoulos K, Cerutti A, et al. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways. Blood 2011;118:2150-8. 33. Ruminy P, Etancelin P, Couronne L, Parmentier F, Rainville V, Mareschal S, et al. The isotype of the BCR as a surrogate for the GCB and ABC molecular subtypes in diffuse large B-cell lymphoma. Leukemia 2011;25:681-8. 34. Ye J, Ma N, Madden TL, Ostell JM. IgBLAST: an immunoglobulin variable domain sequence analysis tool. Nucleic Acids Res 2013;41(Web Server issue):W34-40. 35. Maccarthy T, Roa S, Scharff MD, Bergman A. SHMTool: a webserver for comparative analysis of somatic hypermutation datasets. DNA Repair 2009;8: 137-41. 36. Ettinger R, Sims GP, Fairhurst AM, Robbins R, da Silva YS, Spolski R, et al. IL21 induces differentiation of human naive and memory B cells into antibodysecreting plasma cells. J Immunol 2005;175:7867-79. 37. Niikawa N, Kuroki Y, Kajii T, Matsuura N, Ishikiriyama S, Tonoki H, et al. Kabuki make-up (Niikawa-Kuroki) syndrome: a study of 62 patients. Am J Med Genet 1988;31:565-89. 38. Micale L, Augello B, Maffeo C, Selicorni A, Zucchetti F, Fusco C, et al. Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients. Hum Mutat 2014;35:841-50. 39. Shinsky SA, Hu M, Vought VE, Ng SB, Bamshad MJ, Shendure J, et al. A nonactive-site SET domain surface crucial for the interaction of MLL1 and the RbBP5/Ash2L heterodimer within MLL family core complexes. J Mol Biol 2014;426:2283-99. 40. Cunningham-Rundles C. Autoimmune manifestations in common variable immunodeficiency. J Clin Immunol 2008;28(suppl 1):S42-5. 41. Comans-Bitter WM, de Groot R, van den Beemd R, Neijens HJ, Hop WC, Groeneveld K, et al. Immunophenotyping of blood lymphocytes in childhood. Reference values for lymphocyte subpopulations. J Pediatr 1997; 130:388-93. 42. Orange JS, Ballow M, Stiehm ER, Ballas ZK, Chinen J, De La Morena M, et al. Use and interpretation of diagnostic vaccination in primary immunodeficiency: a working group report of the Basic and Clinical Immunology Interest Section of the American Academy of Allergy, Asthma & Immunology. J Allergy Clin Immunol 2012;130(suppl):S1-24. 43. Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood 2008; 111:77-85. 44. Weill JC, Weller S, Reynaud CA. Human marginal zone B cells. Annu Rev Immunol 2009;27:267-85. 45. Fecteau JF, Cote G, Neron S. A new memory CD27-IgG1 B cell population in peripheral blood expressing VH genes with low frequency of somatic mutation. J Immunol 2006;177:3728-36. 46. Chong Y, Ikematsu H, Yamaji K, Nishimura M, Kashiwagi S, Hayashi J. Agerelated accumulation of Ig V(H) gene somatic mutations in peripheral B cells from aged humans. Clin Exp Immunol 2003;133:59-66. 47. Klein U, Kuppers R, Rajewsky K. Evidence for a large compartment of IgMexpressing memory B cells in humans. Blood 1997;89:1288-98. 48. Bemark M, Boysen P, Lycke NY. Induction of gut IgA production through T celldependent and T cell-independent pathways. Ann N Y Acad Sci 2012;1247: 97-116. 49. Borchert GM, Holton NW, Edwards KA, Vogel LA, Larson ED. Histone H2A and H2B are monoubiquitinated at AID-targeted loci. PLoS One 2010;5:e11641. 50. Stanlie A, Aida M, Muramatsu M, Honjo T, Begum NA. Histone3 lysine4 trimethylation regulated by the facilitates chromatin transcription complex is critical for DNA cleavage in class switch recombination. Proc Natl Acad Sci U S A 2010;107:22190-5. 51. Li G, Zan H, Xu Z, Casali P. Epigenetics of the antibody response. Trends Immunol 2013;34:460-70. 52. Helin K, Dhanak D. Chromatin proteins and modifications as drug targets. Nature 2013;502:480-8.

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METHODS MLPA analysis The following exons were analyzed in patients with cKS: KMT2D exons —1, 4, 7, 10, 11, 14, 15, 19, 22, 26, 30, 31, 33, 34, 35, 39, 41, 43, 45, 47, 49, 51, and 54; KDM6A exons—1 to 4, 7 to 27, and 29.

In vitro terminal B-cell differentiation Total peripheral B cells were isolated with RosetteSep Human B Cell Enrichment Cocktail (STEMCELL Technologies, Vancouver, British Columbia, Canada), according to the manufacturer’s instructions. Postisolation flow cytometry revealed greater than 95% CD191 enrichment. Cells were

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cultured in RPMI with 10% FBS, 100 mg/mL streptomycin, 100 U/mL penicillin, and L-glutamine (2 mmol/L). Differentiation medium was as above but supplemented with 100 ng/mL recombinant human IL-21 (Life Technologies), 1 mg/mL goat anti-human CD40 antibody (R&D Systems, Minneapolis, Minn), and 5 mg/mL donkey anti-human IgM F(ab)2 (Jackson ImmunoResearch, West Grove, Pa). Cells were stained with the following anti-human antibodies: anti-CD19–Brilliant violet 421 (Clone HIB19; BioLegend, San Diego, Calif), anti-IgD–fluorescein isothiocyanate (Life Technologies), anti-CD38–PE-Cy7 (Clone HB7; BD PharMingen, San Jose, Calif), and anti-CD27–PerCP-Cy5.5 (Clone M-T271, BD PharMingen). Viability staining was performed (LIVE/DEAD-UV, Life Technologies), and cells were analyzed immediately.

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FIG E1. Facial features of patients with KS. Representative images of patients at the time of enrollment arranged by ascending age and designated 1 to 10 (patients with KMT2DMut/1 mutations) and A to C (patients with no identified KMT2D or KDM6A mutations). Inset depicts the patient’s right ear.

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FIG E2. KMT2D locus structure, domains, and mutation sites. KMT2D mutations of the patients depicted in Fig 1 are arranged along the gene schematic. Red lettering indicates nonsense/frameshift mutations, green lettering indicates splice acceptor site mutations, and black lettering indicates missense mutations. Asterisks mark previously reported mutations (Pt 3,23 Pt 5,21 and Pt851). CC, Coiled coil domains; FYRC, FY-rich domain C terminal; FYRN, FY-rich domain N terminal; HMG, high-mobility group box domain; JSA, Joubert syndrome–associated domain; LXXLL, leucine interaction motif; PHD, plant homeobox domain; PRR, proline-rich region; Post-SET, post-SET domain; Pt, patient; SET, Su(var)3-9, enhancer-ofzeste, trithorax domain; WIN, WDR5 interaction motif.

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FIG E3. Splice site mutation analysis. A, Amplification of PBMC-derived KMT2D transcripts from patient 3 showed a mutant band (MUT) not seen in the healthy control subject (CTL). Sequencing of the mutant band revealed a retained intron 26 sequence creating a stop codon. B, Amplification of PBMC-derived KMT2D transcripts from patient 7 only revealed the expected wild-type (WT) band, suggesting that reduced splicing efficiency, as opposed to generation of an abnormal alternatively spliced transcript, drives the KS phenotype in this patient. Ex, Exon; In, intron; Pt, patient.

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FIG E4. Clinical summaries: patients with autoimmunity. Detailed clinical histories of 3 patients with KMT2D mutations and autoimmune disease are provided. Autoimmune diagnoses are shown in boldface. A, Patient 5 had prenatally diagnosed hypoplastic left heart syndrome (HLHS) and underwent staged repairs during the first 2.5 years of life. Starting at approximately 6 months of age, she had recurrent acute otitis media and has required multiple rounds of tympanostomy tube placement since that time. She contracted EBV at approximately 6 years of age and had ITP 6 months later. B, Patient 6 also required cardiac repair early in life, had recurrent acute otitis media and sinopulmonary infections, and was given a diagnosis of hypogammaglobulinemia at 5.5 years of age. She subsequently had recurrent ITP and vitiligo and was started on intravenous immunoglobulin at approximately 8.5 years of age. C, Patient 8 has a long history of recurrent sinopulmonary infections and autoimmune disease. He contracted EBV at 13 years of age and shortly thereafter had signs of liver disease. He was given a diagnosis of hypogammaglobulinemia at 14 years of age and started on intravenous immunoglobulin. He later had seronegative arthritis, recurrent ITP, and autoimmune hepatitis requiring liver transplantation at approximately 22 years of age. He had EBV reactivation with tacrolimus treatment and subsequently had insulin-dependent diabetes mellitus and nodular regenerative hyperplasia in his liver graft. ASD/VSD, Atrial septal defect/ventricular septal defect; GT, gastric tube; IDDM, insulin-dependent diabetes mellitus; IVIG, intravenous immunoglobulin; PE, pressure equalization; TPM/SMX, trimethoprim sulfamethoxazole.

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FIG E5. Proposed model of KMT2D insufficiency in B cells. A model of the post-V(D)J recombined human IGH locus is depicted as it undergoes CSR and affinity maturation through SHM. A, The resting naive mature B cell actively transcribes both the full-length IgM transcript (V[D]J-Cm) and a germline Cm noncoding transcript originating from the m switch region (red arrows). Each of these regions harbors activating H3K4me3 histone modifications, whereas repressive H3K27me3 histone modifications mark the switch regions of all other IGH constant genes (from Cg3 to Ca2). B, On B-cell activation and receiving specific costimulation signals, multiple transcription factors, including PAX5, are recruited to the promoter regions of select constant genes (Cg2 in this example), initiating Cg2 germline transcription (red arrow). The KMT2D complex, guided by PTIP interaction with PAX5, is recruited to the constant region, the UTX (KDM6A, KS-2) subunit removes the inhibitor H3K27me3 mark, and the SET domain of KMT2D adds the activating H3K4me mark, initiating a cascade of secondary signaling events that lead to AID recruitment and ultimately CSR. The V(D)J region also acquires additional activating histone modifications and ultimately recruits AID, which initiates targeted SHM. C, The postswitch IGH locus recombines, excising the intervening sequencing (blue highlighting), fusing the Cm and Cg2 switch regions into a new open reading frame with the V(D)J region, and generating an episomal switch circle. During repair of AID-associated DNA damage to the V(D)J sequence, the error-prone DNA polymerase-h preferentially introduces mutations at select motifs downstream of AID deamination sites. We propose that KMT2D insufficiency (red text) results in diminished H3K4 methylation of key IGH locus sites during B-cell activation and terminal differentiation, resulting in reduced CSR and SHM efficiency and ultimately impaired memory B-cell production. AID, Activation-induced cytidine deaminase; Pol-h, DNA polymerase-h; UNG, uracil-DNA glycosylase.

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TABLE E1. Immune phenotype summary: CVID-associated sequelae in adult patients with KMT2DMut/1 mutations Subject ID:

6

7

8

9

10

Splenomegaly Hepatomegaly/hepatitis Pulmonary

1 1

2 2

1 1

1 2

2 2

2

1

2

1

1

2 2

2 2

1 2

2 1

2 2

1 2

2 2

1 2

2 2

2 2

2/2

2/2

2/2

2/2

2/2

Obstructive disease/ nonatopic asthma Granulomatous disease Lymphoid interstitial pneumonitis Autoimmune disease Recurrent gastrointestinal infections Lymphoproliferative/ lymphoma

Subject ID:

1

2*

3

4

5y

Absolute 4.32 (2.5-16.5 Y1.26 (2.5-16.5 8.38 (3-13.5 7.80 (4-10.5 Y1.3 (1.5-7 lymphocyte K/mL) k/mL) k/mL) K/mL) k/mL) count T-cell function NT NT PHA WNL WNL WNL ConA WNL WNL WNL PWM WNL WNL WNL CD3 WNL Y33% (50% WNL WNL Y45% (61% to 77%) to 86%) CD4 Y29% (33% Y19% (33% WNL WNL Y27% (34% to 58%) to 58%) to 58%) CD8 [30% (11% Y12% (13% WNL WNL WNL to 25%) to 28%) CD4/CD8 Y1 (1.7-3.9) WNL WNL WNL WNL CD19 WNL [51% (13% WNL Y7% (14% WNL to 35%) to 44%) CD16/CD56 [20% (2% [15% (2% WNL WNL [29% (1% to 14%) to 13%) to 27%)

6z

7

8

Y1.13 (1.5-6.5 3.16 (1.5-6.5 Y0.99 (1-4.8 k/mL) k/mL) k/mL) Y 74% 80% 78% [91% (52% to 78%) [77% (25% to 48%) WNL [7 (0.9-3.4) Y5% (8% to 24%) Y3% (6% to 27%)

WNL WNL WNL WNL

Y 53% 42% 57% WNL

WNL WNL WNL WNL

9

1.26 (1-4.8 k/mL)

10

A

B

C

2.38 (1-4.8 5.04 (3-9.5 1.76 (1.5-6.8 3.16 (1.5-6.5 k/mL) k/mL) k/mL) k/mL)

WNL WNL WNL WNL

WNL WNL WNL WNL

WNL WNL WNL WNL

WNL WNL WNL WNL

WNL

Y/WNL 43% WNL WNL [89.8% (59.3% to 88.6%) WNL

WNL

WNL

WNL

WNL

WNL

WNL

WNL

WNL

WNL

WNL WNL

[43% (14% to 33%) Y0.6 (0.9-2.9) WNL

WNL WNL

WNL WNL

WNL

WNL

WNL

WNL

WNL WNL Y6% (6% Y3% (6% to 19%) to 19%) [29% (6% Y7% (7% NT to 27%) to 31%)

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TABLE E2. Lymphocyte evaluations

Arrows and boldface text indicate whether the value is greater than or less than the age-matched normal range. Mitogens are reported as normal or as a percentage of the lower limit of normal. ConA, Concanavalin A; k/mL, thousands of cells per microliter; NT, not tested; PWM, pokeweed mitogen; WNL, within normal age-matched limits. *Patient 2 has lymphopenia secondary to a large retroperitoneal lymphatic malformation. The absolute total B-cell count was low (556 cells/mL; age-adjusted normal range, 700-2500 cells/mL).  Patient 5 underwent cardiac surgery with thymectomy at 3 days of life, resulting in chronic mild lymphopenia. àPatient 6 had normal absolute lymphocyte count and lymphocyte subpopulations at initial evaluation (5.5 years of age). She subsequently had lymphopenia and associated reduction in numbers B cells and natural killer cells shown above after onset of autoimmunity.

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TABLE E3. Immune phenotype summary: immune deficiency phenotype Subject ID:

6

7

8

9

10

Recurrent infections, OM/PNA/UTI 1/1/1 1/1/2 1/1/1 1/1/2 2/2/1 Low IgA 1 1 1 1 1 Low IgG 1 2 1 1 1 Low IgM 1 2 1 1 2 Low pneumococcal titers 1 2 2 1 1 Low total B-cell numbers 1 2 2 1 2 Low memory B-cell 1 2 1 1 1 numbers Low class-switched NT 2 1 1 1 B-cell numbers NT 1 1 1 2 Expanded CD21lo Low SHM rate NT 1 1 1 2 Autoimmunity 1 2 1 2 2 Tested features 100 30 80 90 50 present (%) NT, Not tested; OM, otitis media; PNA, pneumonia; UTI, urinary tract infection.

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TABLE E4. V(D)J mutation analysis Subject ID:

Mutation rate (per aligned bp) Transversions Transitions AID motifs GYW  WRC  SYCà GRSà Poly-h motifs WA  TW 

Control subjects

7

8

9

10

5.74%

3.76%*

3.41%*

2.12%*

7.11%

44.86% 55.13%

47.40% 52.59%

49.13% 50.86%

43.28% 56.71%

50.53% 49.46%

21.29% 14.06% 0.76% 1.90%

19.48% 15.58% 0% 3.89%

29.31% 9.48% 2.58% 2.60%

24.62% 16.41% 0.74% 1.49%

24.38% 15.19% 0.70% 1.41%

15.96% 7.98%

10.38% 14.28%

9.48% 6.03%

5.22% 9.70%

10.95% 7.42%

IgG transcripts were analyzed for mutation rate (per aligned base pair), frequency of mutation type (transversion or transition), and utilization rate of AICDA/AID or polymerase h ‘‘hotspot’’ and ‘‘coldspot’’ motifs. No significant differences were noted in these parameters between control subject–derived sequences and sequences derived from patients with KS; however, a trend toward reduced use of the polymerase h motif WA (with W indicating adenosine or thymidine) was noted in all assessed patients with KS (KS average: 9.01% vs control average: 15.96%, P 5 .058). Nucleotide abbreviations used in motif sequences: A, Adenosine; C, cytosine; G, guanine; S, guanine (G) or cytosine (C); T, thymidine; W, adenosine (A) or thymidine (G); Y, cytosine (C) or thymidine (G). *P < .05, 2-sided unpaired Student t test.  ‘‘Hotspots.’’ à‘‘Coldspots.’’