Key Points

  • A definite genetic diagnosis was made in 10.4% of 1892 patients with suspected HLH by a panel approach including 15 HLH-associated genes.

  • HLH next-generation sequencing panels were ∼400 test orders per year; single-gene tests related to HLH have drastically decreased.

Abstract

This article explores the distribution and mutation spectrum of potential disease-causing genetic variants in hemophagocytic lymphohistiocytosis (HLH)–associated genes observed in a large tertiary clinical referral laboratory. Samples from 1892 patients submitted for HLH genetic analysis were studied between September 2013 and June 2018 using a targeted next-generation sequencing panel approach. Patients ranged in age from 1 day to 78 years. Analysis included 15 genes associated with HLH. A potentially causal genetic finding was observed in 227 (12.0%) samples in this cohort. A total of 197 patients (10.4%) had a definite genetic diagnosis. Patients with pathogenic variants in familial HLH genes tended to be diagnosed significantly younger compared with other genes. Pathogenic or likely pathogenic variants in the PRF1 gene were the most frequent. However, mutations in genes associated with degranulation defects (STXBP2, UNC13D, RAB27A, LYST, and STX11) were more common than previously appreciated and collectively represented >50% of cases. X-linked conditions (XIAP, SH2D1A, and MAGT1) accounted for 17.8% of the 197 cases. Pathogenic variants in the SLC7A7 gene were the least encountered. These results describe the largest cohort of genetic variation associated with suspected HLH in North America. Merely 10.4% of patients were identified with a clearly genetic cause by this diagnostic approach; other possible etiologies of HLH should be investigated. These results suggest that careful thought should be given regarding whether patients have a clinical phenotype most consistent with HLH vs other clinical and disease phenotypes. The gene panel identified known pathogenic and novel variants in 10 HLH-associated genes.

Introduction

Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome of hyperinflammation characterized by pathologic activation and proliferation of T cells and macrophages. Although HLH frequently affects infants, it is also observed in children and adults of all ages. HLH can occur as a typical or principal manifestation of several genetically heterogeneous disorders. A group of diseases known as familial HLH types 2-5 are caused by pathogenic variants in PRF1, UNC13D, STX11, and STXBP2, respectively, which are all critical for normal cytotoxic lymphocyte granule-mediated cytotoxicity.1-5  In addition, loss-of-function mutations in the LYST, RAB27A, and AP3B1 genes cause problems in the formation of the cytotoxic granules or transport of the granules through the cytoplasm6-8  and can also lead to HLH as well as other problems such as pigmentary dilution in the disorders known as Chediak-Higashi syndrome, Griscelli syndrome type 2, and Hermansky-Pudlak syndrome type 2, respectively. Other genetic disorders with more complex mechanisms of diseases that are associated with a high risk of HLH include X-linked lymphoproliferative syndrome type 1 and 2 (XLP1 and XLP2) caused by mutations in the SH2D1A9  and XIAP10  genes, respectively, X-linked immunodeficiency with magnesium defect, Epstein-Barr virus infection and neoplasia caused by defects in the MAGT1 gene,11  CD27 deficiency from loss of function in CD27,12  and interleukin-2 inducible T-cell kinase (ITK) deficiency from ITK dysfunction.13  Some metabolic disorders can also be complicated by the development of HLH, notably including lysinuric protein intolerance caused by mutations in the SLC7A7 gene.14 

Defects in these genes are sometimes indistinguishable from each other clinically. Historically, genetic investigations started with Sanger sequencing of the most commonly defective gene, PRF1. Sequential examination of other HLH-related genes was then pursued if PRF1 testing was negative. This process not only prolonged the diagnosis in many cases but was also expensive. Next-generation sequencing (NGS) technology has allowed the creation of targeted gene panels in which several genes can be interrogated at once in a time and cost-efficient manner. At Cincinnati Children’s Hospital Medical Center (CCHMC), an NGS-based HLH sequencing panel including 15 HLH-associated genes was launched in September 2013. In this study, we aimed to examine the impact of NGS HLH panel on genetic testing ordering patterns and examine the distribution and details of genetic variants observed in 1892 patient samples submitted for NGS HLH panel sequencing.

Materials and methods

Patient samples and clinical information

The present study was approved by the institutional review board at Cincinnati Children’s Hospital, Cincinnati, OH. A total of 1892 patient samples and submitted clinical information were analyzed and reviewed after HLH sequencing panel testing was submitted to the Molecular Diagnostic Laboratory at CCHMC between September 2013 and June 2018. Of these, 33 orders clearly stated that the reason for testing was for mutation carrier status evaluation and these were excluded from further analysis for molecular diagnosis. Of the remaining 1859 samples, 1632 had only variants classified as benign, likely benign, or variants of uncertain significance. At least 1 pathogenic or likely pathogenic variant was detected in 227 samples. Among them, 197 samples were identified with either homozygous or compound heterozygous pathologic variants in an autosomal recessive condition, or a hemizygous pathologic variant in an X-linked disorder. In addition, 30 patient samples were identified carrying only 1 heterozygous pathogenic or likely pathogenic variant in a recessive condition (supplemental Figure 1). Patients were referred by physicians from 300 institutions across North America and the included patients were either referred with a clinical diagnosis of HLH or suspected related conditions. Patient age at time of referral ranged from 1 day to 78 years. Forty-seven percent (895/1892) were female and 53% (997/1892) were male. The reported cohort of 197 patients ranged from 1 day to 57.8 years, including 44% (86/197) female and 56% (111/197) male. Fifty-eight percent (114/197) were younger than 2 years old, and 30% (59/197) were between 2 and 18 years old (Table 1). Clinical information was collected using a standardized clinical checklist completed by the ordering physician that captured information such as age of onset, and general clinical history such as fever, liver and spleen abnormalities, infections, skin abnormalities, laboratory findings, neurological abnormalities, family history, and results of previous testing. Our laboratory did not systematically confirm clinical characteristics or prior laboratory investigations of patients reported by referring clinicians.

Table 1.

Pathogenic or likely pathogenic variants identified in 197 HLH patients with a definite genetic diagnosis

Patient no.SexAge at testing, y (unless indicated otherwise)EthnicityGeneVariantZygosityPopulation frequency (gnomAD*), %Symptoms/immunology testing/family history
Female 53 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Male 63 d Unknown PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Male 0.4 Middle Eastern PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Female 27 d Unknown PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression 
Female 32 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression 
Male 4 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression; sibling died of HLH 
Female 0.4 African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Male 6 d African American + European-white PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression 
Male 59 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
10 Male 13 d African PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
11 Female 7 d Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent NK cell function 
    PRF1 c.266C>T(p.Pro89Leu) Heterozygous ND 
12 Female 0.3 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.350_356delinsATGC (p.Val117_Arg119delinsAspAla) Heterozygous ND 
13 Male 0.4 Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 
14 Male 3.3 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression; brother with HLH 
    PRF1 c.527G>A(p.Cys176Tyr) Heterozygous ND 
15 Female 0.5 Latino-Hispanic PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression 
    PRF1 c.659G>A(p.Gly220Asp) Heterozygous 0.0008 
16 Female 0.2 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent NK cell function 
    PRF1 c.853_855del(p.Lys285del) Heterozygous 0.0056 
17 Male 66 d Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.895C>T(p.Arg299Cys) Heterozygous 0.0012 
18 Male 20.6 Latino-Hispanic PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.902C>T(p.Ser301Leu) Heterozygous ND 
19 Male 54 d African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Symptoms of HLH 
    PRF1 c.916G>T(p.Gly306Cys) Heterozygous ND 
20 Female 45 d African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression 
    PRF1 c.916G>T(p.Gly306Cys) Heterozygous ND 
21 Male 32 d Latino-Hispanic PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Symptoms of HLH 
    PRF1 c.985dup(p.Val329fs) Heterozygous ND 
22 Male 0.3 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression 
    PRF1 c.1385C>A(p.Ser462*Heterozygous ND 
23 Male 36.1 Unknown PRF1 c.116C>A(p.Pro39His) Heterozygous 0.00081 NA 
    PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 
24 Female 1.2 Asian-American PRF1 c.133G>A(p.Gly45Arg) Homozygous 0.0012 Absent NK cell function 
25 Female 37 d Non-Hispanic white PRF1 c.150del(p.Thr51fs) Heterozygous 0.0004 Absent perforin expression 
    PRF1 c.227G>A(p.Cys76Tyr) Heterozygous 0.00071 
26 Male 69 d Latino-Hispanic PRF1 c.218G>C(p.Cys73Ser) Homozygous 0.0004 Symptoms of HLH 
27 Female 20 European-American PRF1 c.227G>A(p.Cys76Tyr) Heterozygous 0.00071 Absent perforin expression 
    PRF1 c.626A>C(p.Gln209Pro) Heterozygous 0.0012 
28 Female 21.8 Unknown PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 Absent NK cell function, decreased perforin expression 
    PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 
29 Male 17 European-American PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 Absent perforin expression 
    PRF1 c.635A>C(p.Tyr212Ser) Heterozygous ND 
30 Female 41.2 European-American PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 Absent NK cell function, decreased perforin expression 
    PRF1 c.666C>A(p.His222Gln) Heterozygous 0.0039 
31 Female 8.3 European-American PRF1 c.443C>G(p.Ala148Gly) Heterozygous 0.0004 NA 
    PRF1 c.666C>A(p.His222Gln) Heterozygous 0.0039 
32 Male 2.6 Unknown PRF1 c.445G>A(p.Gly149Ser) Homozygous 0.014 Absent NK cell function 
33 Male 0.8 Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Homozygous 0.014 Symptoms of HLH 
34 Female Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Homozygous 0.014 NA 
35 Female 0.3 European-American PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 Family history of HLH 
    PRF1 c.614A>G(p.Asn205Ser) Heterozygous 0.0043 
36 Male 42 d Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 NA 
    PRF1 c.938A>T(p.Asp313Val) Heterozygous 0.0012 
37 Male 4.6 Unknown PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 Absent perforin expression 
    PRF1 c.1081A>T(p.Arg361Trp) Heterozygous 0.0011 
38 Female 32 d Middle Eastern PRF1 c.501C>G(p.Tyr167*Homozygous ND Symptoms of HLH 
39 Female 0.3 Unknown PRF1 c.512C>A(p.Thr171Asn) Homozygous 0.0028 Absent perforin expression 
40 Male 9.5 European-American PRF1 c.786_801del(p.Gln263fs) Heterozygous ND Absent NK cell function 
    PRF1 c.886T>C(p.Tyr296His) Heterozygous 0.0012 
41 Male 59 d Unknown PRF1 c.853_855del(p.Lys285del) Heterozygous 0.0057 NA 
    PRF1 c.921del(p.His308fs) Heterozygous 0.002 
42 Female 0.7 Middle Eastern PRF1 c.880del(p.Gln294fs) Homozygous ND Symptoms of HLH 
43 Female Middle Eastern PRF1 c.895C>T(p.Arg299Cys) Homozygous 0.0012 Symptoms of HLH 
44 Female 0.2 Latino-Hispanic PRF1 c.904G>T(p.Glu302*Homozygous ND Absent perforin expression 
45 Female 1.8 Unknown PRF1 c.949G>A(p.Gly317Arg) Homozygous 0.0008 Symptoms of HLH 
46 Female 10.3 European-American PRF1 c.973T>C(p.Tyr325His) Heterozygous ND Absent perforin expression 
    PRF1 c.1326_1328del(p.Phe443del) Heterozygous ND 
47 Male 1.1 Middle Eastern PRF1 c.1070G>C(p.Arg357Pro) Homozygous ND Symptoms of HLH 
48 Male 12.5 Middle Eastern PRF1 c.1081A>T(p.Arg361Trp) Homozygous 0.0011 Abnormal brain lesions and seizures 
49 Female 2.6 Unknown PRF1 c.1229_1230delinsCC (p.Arg410Pro) Homozygous ND NA 
50 Female 0.2 African American PRF1 c.1304C>T(p.Thr435Met) Heterozygous 0.0028 Absent perforin expression 
    PRF1 c.1314T>A(p.Tyr438*Heterozygous 0.0032 
51 Female 2.6 Latino-Hispanic PRF1 c.1337A>C(p.Gln446Pro) Homozygous 0.0016 NA 
52 Female 2.6 Unknown PRF1 c.1337A>C(p.Gln446Pro) Homozygous 0.0016 Symptoms of HLH 
53 Female 0.4 Middle Eastern STXBP2 c.37+2T>C Heterozygous ND Absent NK cell function 
    STXBP2 c.1430C>T(p.Pro477Leu) Heterozygous 0.00074 
54 Male 0.6 Unknown STXBP2 c.37+5G>A Heterozygous ND NA 
    STXBP2 c.1057T>C (p.Cys353Arg) Heterozygous 0.0004 
55 Female 63 d Asian-American STXBP2 c.193C>T(p.Arg65Trp) Homozygous 0.00071 Absent NK cell function 
56 Female 5.5 Unknown STXBP2 c.194G>A(p.Arg65Gln) Heterozygous 0.0028 Absent NK cell function 
    STXBP2 c.560C>T (p.Pro187Leu) Heterozygous 0.00064 
57 Male 4.1 European-American STXBP2 c.194G>A(p.Arg65Gln) Heterozygous 0.0028 Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
58 Female 4.2 European-American STXBP2 c.326-30_326-23del Heterozygous 0.0068 Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
59 Male 0.6 Latino-Hispanic STXBP2 c.389T>C(p.Leu130Ser) Homozygous 0.0032 Symptoms of HLH 
60 Male 45 d African American STXBP2 c.389T>C(p.Leu130Ser) Heterozygous 0.0032 Symptoms of HLH; family history of HLH 
    STXBP2 exon 14-19 deletion Heterozygous ND 
61 Male 0.7 Middle Eastern STXBP2 c.481del(p.Arg161fs) Homozygous ND Symptoms of HLH 
62 Male 0.4 Unknown STXBP2 c.481del(p.Arg161fs) Homozygous ND Symptoms of HLH 
63 Female 11 European-American STXBP2 c.539_540delinsAA(p.Cys180*Heterozygous ND Symptoms of HLH 
    STXBP2 c.1247-1G>C Heterozygous 0.02 
64 Male 0.3 Latino-Hispanic STXBP2 c.703C>G(p.Arg235Gly) Homozygous 0.00071 Absent NK cell function 
65 Female 52.7 European-American STXBP2 c.752C>T(p.Ala251Val) Heterozygous ND Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
66 Male 0.9 Unknown STXBP2 c.902+5G>A Heterozygous 0.0036 NA 
    STXBP2 c.1247-1G>C Heterozygous 0.02 
67 Male 3.1 Unknown STXBP2 c.1247-1G>C Homozygous 0.02 Symptoms of HLH 
68 Female 22.7 Latino-Spanish STXBP2 c.1247-1G>C Homozygous 0.02 Decreased NK cell function 
69 Male 26.4 European-American STXBP2 c.1247-1G>C Homozygous 0.02 Symptoms of HLH 
70 Male 25.6 European-American STXBP2 c.1247-1G>C Homozygous 0.02 Symptoms of HLH 
71 Female 29.7 European-American STXBP2 c.1247-1G>C Homozygous 0.02 NA 
72 Male European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Absent NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
73 Female 15.8 European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Absent NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
74 Female 19 Unknown STXBP2 c.1247-1G>C Heterozygous 0.02 Decreased NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
75 Female 26.9 European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
76 Female 57.8 European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Absent NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
77 Female 0.2 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
78 Female 0.3 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 NA 
79 Female 0.3 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
80 Female 0.6 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
81 Male 0.2 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 NA 
82 Male 0.7 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
83 Female 0.8 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
84 Male 0.8 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
85 Male 0.6 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Family history of HLH 
86 Male 1.6 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
87 Male 0.3 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
88 Female 63 d Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Family history of HLH 
89 Male 0.5 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
90 Male 0.2 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
91 Male 11.1 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Absent NK cell function 
92 Female 10.1 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Heterozygous 0.00074 Symptoms of HLH 
    STXBP2 c.1696+5G>T Heterozygous ND 
93 Male 0.5 Middle Eastern STXBP2 c.1452+1G>A Homozygous ND Symptoms of HLH 
94 Male Middle Eastern STXBP2 c.1452+1G>A Homozygous ND Abnormal NK cell function 
95 Female 49 d European-American UNC13D c.118-308C>T Heterozygous 0.019 Dysmorphic facies, decreased NK cell function 
    UNC13D c.2258_2267delinsTACCTTGTTCGA (p.Gly753fs) Heterozygous ND 
96 Male 0.7 European-American UNC13D c.118-308C>T Heterozygous 0.019 Decreased NK cell function 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
97 Male 0.2 European-American + Latino-Spanish UNC13D c.118-308C>T Heterozygous 0.019 Decreased NK cell function 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
98 Female 1.3 Non-Hispanic white UNC13D c.118-308C>T Heterozygous 0.019 Symptoms of HLH, seizures, normal NK cell function 
    UNC13D c.2867C>T(p.Pro956Leu) Heterozygous ND 
99 Female Non-Hispanic white UNC13D c.118-308C>T Heterozygous 0.019 Absent NK cell function 
    UNC13D c.3193C>T(p.Arg1065*Heterozygous 0.0011 
100 Female 3.2 European-American UNC13D c.118-308C>T Heterozygous 0.019 Absent NK cell function 
    UNC13D 253Kb inversion Heterozygous ND 
101 Male 2.6 Unknown UNC13D 253Kb inversion Heterozygous ND Decreased NK cell function 
    UNC13D c.154-1G>C Heterozygous ND 
102 Male 0.2 Unknown UNC13D 253Kb inversion Heterozygous ND Symptoms of HLH 
    UNC13D c.551G>A(p.Trp184*Heterozygous 0.0011 
103 Female 0.2 European-American UNC13D 253Kb inversion Heterozygous ND NA 
    UNC13D c.1389+1G>A Heterozygous 0.0071 
104 Female 0.2 European-American UNC13D 253Kb inversion Heterozygous ND Absent NK cell function 
    UNC13D c.2447+1G>A Heterozygous 0.00051 
105 Female 0.6 Non-Hispanic white UNC13D 253Kb inversion Heterozygous ND Decreased NK cell function 
    UNC13D c.2695C>T(p.Arg899*Heterozygous 0.0018 
106 Male 11.4 Hispanic white UNC13D c.182A>G(p.Tyr61Cys) Heterozygous ND Symptoms of HLH 
    UNC13D c.778T>C(p.Trp260Arg) Heterozygous ND 
107 Male 0.4 European-American UNC13D c.262-1G>A Heterozygous ND Symptoms of HLH, abnormal NK cell function, family history of HLH 
    UNC13D c.766C>T(p.Arg256*Heterozygous 0.0025 
108 Male 0.4 Unknown UNC13D c.321+1_321+2del Heterozygous ND Decreased NK cell function 
    UNC13D c.753+1G>T Heterozygous 0.0044 
109 Male 0.3 Unknown UNC13D c.322-2A>T Heterozygous 0.0024 Symptoms of HLH, decreased NK cell function 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
110 Male 0.3 Unknown UNC13D c.419T>C(p.Ile140Thr) Heterozygous 0.0004 NA 
    UNC13D c.460C>T(p.Arg154Trp) Heterozygous 0.011 
111 Female 5.4 Middle Eastern UNC13D c.424dup(p.Gln142fs) Homozygous ND Symptoms of HLH 
112 Male 7.7 Latino-Hispanic UNC13D c.518C>T(p.Thr173Met) Heterozygous 0.0028 NA 
    UNC13D c.1803_1819dup(p.Arg607fs) Heterozygous ND 
113 Female 39 d European-American UNC13D c.551G>A(p.Trp184*Heterozygous 0.0011 Abnormal NK cell function 
    UNC13D c.766C>T(p.Arg256*Heterozygous 0.0025 
114 Male 4.9 European-American UNC13D c.570-2A>T Heterozygous ND Absent NK cell function 
    UNC13D c.3049G>A(p.Glu1017Lys) Heterozygous 0.00044 
115 Female 0.2 Middle Eastern UNC13D c.753+1G>T Homozygous 0.0044 Symptoms of HLH 
116 Female 0.6 European-American UNC13D c.766C>T(p.Arg256*Heterozygous 0.0025 Symptoms of HLH, abnormal NK cell function 
    UNC13D c.2447+1G>A Heterozygous 0.00051 
117 Female 1.2 Latino-Spanish UNC13D c.859del(p.Arg287fs) Homozygous ND Decreased NK cell function 
118 Female Non-Hispanic white UNC13D c.1055+1G>T Heterozygous ND Decreased NK cell function, family history of HLH 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
119 Male 0.7 European-American UNC13D c.1229_1230dup(p.Arg411fs) Heterozygous 0.00 Symptoms of HLH, hypertelorism 
    UNC13D c.2298+1G>T Heterozygous ND 
120 Female 0.2 European-American UNC13D c.1259_1260del(p.Ser420fs) Heterozygous ND symptoms of HLH, decreased NK cell function 
    UNC13D c.1848+1G>C Heterozygous ND 
121 Male 18.3 European-American UNC13D c.1387C>T(p.Gln463*Heterozygous ND symptoms of HLH, decreased NK cell function 
    UNC13D c.1820G>C(p.Arg607Pro) Heterozygous 0.011 
122 Female 58 d European-American + African American UNC13D c.1389+1G>A Heterozygous 0.0071 Symptoms of HLH 
    UNC13D c.1848+1G>C Heterozygous ND 
123 Female 0.2 Middle Eastern UNC13D c.1423C>T(p.Gln475*Homozygous ND NA 
124 Male 10 d Pacific Islander UNC13D c.2296C>T(p.Gln766*Homozygous ND Decreased NK cell function 
125 Female 13.2 Unknown UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 NA 
    UNC13D c.2588G>A(p.Gly863Asp) Heterozygous 0.029 
126 Female Unknown UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 NA 
    UNC13D c.3065T>C(p.Leu1022Pro) Heterozygous ND 
127 Female 0.2 Middle Eastern UNC13D c.2553+1G>T Homozygous ND Symptoms of HLH 
128 Female 0.3 Middle Eastern UNC13D c.2553+1G>T Homozygous ND Symptoms of HLH 
129 Female Asian UNC13D c.2588G>A(p.Gly863Asp) Homozygous 0.029 Decreased NK cell function 
130 Female 0.4 African American UNC13D c.2695C>T(p.Arg899*Homozygous 0.0018 Symptoms of HLH, absent NK cell function, dysmorphic facies 
131 Male 0.7 Non-Hispanic white UNC13D c.2819del(p.Leu940fs) Homozygous ND symptoms of HLH 
132 Female 0.6 Middle Eastern UNC13D c.3048dup(p.Glu1017fs) Homozygous ND NA 
133 Female 0.7 Unknown UNC13D c.3053C>A(p.Ala1018Asp) Homozygous 0.00088 2 affected siblings 
134 Male 13 European-American RAB27A c.121A>G(p.Thr41Ala) Heterozygous ND NA 
    RAB27A c.352C>T(p.Gln118*Heterozygous ND 
135 Male 9.6 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 NA 
136 Male 9.9 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 NA 
137 Female Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Failure to thrive, bone marrow failure 
138 Female 9.4 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Symptoms of HLH 
139 Male 10.6 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Symptoms of HLH 
140 Male 19.5 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Symptoms of HLH 
141 Female 0.3 Latino-Hispanic RAB27A c.335del(p.Asn112fs) Homozygous 0.0044 Symptoms of HLH 
142 Female 0.4 Middle Eastern RAB27A c.400A>C(p.Lys134Gln) Homozygous ND NA 
143 Female 53.9 Pacific Islander RAB27A c.476A>G(p.Tyr159Cys) Homozygous ND Symptoms of HLH, absent NK cell function 
144 Male 1.2 Middle Eastern RAB27A c.598C>T(p.Arg200*Homozygous 0.0004 NA 
145 Female 0.4 European-American RAB27A c.638_642del(p.Glu213fs) Homozygous 0.0008 Symptoms of HLH 
146 Female 0.7 African American LYST c.925C>T(p.Arg309*Heterozygous ND Oculocutaneous albinism, neutropenia 
    LYST c.2015dup(p.Tyr672*Heterozygous ND 
147 Male 3.4 Unknown LYST c.3194del(p.Leu1065*Homozygous 0.0004 NA 
148 Female 0.3 Middle Eastern LYST c.4159dup(p.Thr1387fs) Homozygous ND Premature gray hair, anemia 
149 Male 0.9 European-American LYST c.5715del(p.Asn1905fs) Heterozygous ND Oculocutaneous albinism, neutropenia, absent NK cell function 
    LYST c.8802-2A>G Heterozygous ND 
150 Female 6.9 Unknown LYST c.5784+1G>T Homozygous ND Oculocutaneous albinism, dysmorphic facies, neutropenia 
151 Male 3.2 Non-Hispanic white LYST c.6159_6160del(p.Met2053fs) Homozygous ND NA 
152 Male 1.8 Middle Eastern LYST c.7291del(p.Leu2431fs) Homozygous ND Hypopigmentation, anemia 
153 Male 1.2 African American LYST c.8770C>T(p.Gln2924*Heterozygous ND Silver hair, hypopigmented skin lesions, pancytopenia 
    LYST c.9844_9845del(p.Ser3282fs) Heterozygous ND 
154 Female 7.1 Middle Eastern LYST c.10776C>G(p.Tyr3592*Homozygous ND Abnormal pigmentation, neutropenia 
155 Female 1.2 Unknown STX11 c.73G>T(p.Glu25*Heterozygous 0.0004 Decreased NK cell function 
    STX11 c.748C>T(p.Gln250*Heterozygous 0.00081 
156 Female 5.6 Middle Eastern STX11 c.173T>C(p.Leu58Pro) Homozygous 0.0008 Symptoms of HLH, grayish hair 
157 Female 11.6 Middle Eastern STX11 c.173T>C(p.Leu58Pro) Homozygous 0.0008 NA 
158 Male 2.9 European-American + Latino-Hispanic STX11 c.462_463delinsA(p.Asp155fs) Heterozygous ND Decreased NK cell function 
    STX11 c.784C>T(p.Gln262*Heterozygous ND 
159 Male 1.4 Asian-Indian STX11 c.687dup(p.Gln230fs) Homozygous ND NA 
160 Female 12 d Middle Eastern SLC7A7 c.1429+1G>C Homozygous ND Family history of HLH 
161 Male 1.2 African American SLC7A7 c.701del(p.Ser234fs) Heterozygous 0.0016 NA 
    SLC7A7 c.895-1G>A Heterozygous ND  
162 Female 13.7 European-American SLC7A7 c.360_361delinsAA (p.Trp121Arg) Homozygous ND NA 
163 Male 5.4 African American XIAP c.145C>T(p.Arg49*Hemizygous ND Markedly decreased XIAP expression 
164 Male 18.7 European-American XIAP c.345C>G(p.Tyr115*Hemizygous ND Symptoms of HLH 
165 Male 30.3 European-American XIAP c.608G>T(p.Cys203Phe) Hemizygous ND NA 
166 Male European-American XIAP c.664C>T(p.Arg222*Hemizygous ND Symptoms of HLH 
167 Male 8.9 European-American XIAP c.738del(p.Asp247fs) Hemizygous ND NA 
168 Male 16 European-American XIAP c.738del(p.Asp247fs) Hemizygous ND Absent XIAP expression 
169 Male 3.6 African American XIAP c.889A>T(p.Lys297*Hemizygous ND NA 
170 Male 0.4 Unknown XIAP c.894_898del(p.Lys299fs) Hemizygous ND NA 
171 Male 17.2 European-American XIAP c.894_898del(p.Lys299fs) Hemizygous ND Symptoms of HLH 
172 Male 19.6 African American XIAP c.926_929del(p.Asp309fs) Hemizygous ND Symptoms of HLH 
173 Male 17.2 European-American XIAP c.969G>A(p.Trp323*Hemizygous ND Decreased XIAP expression 
174 Male 22.6 Unknown XIAP c.1021_1022del(p.Asn341fs) Hemizygous ND NA 
175 Male 4.5 Unknown XIAP c.1056+1G>A Hemizygous ND NA 
176 Male 2.7 Latino-Hispanic XIAP c.1141C>T(p.Arg381*Hemizygous ND NA 
177 Male 11.8 Unknown XIAP c.1141C>T(p.Arg381*Hemizygous ND Absent XIAP expression 
178 Male 1 d Pacific-Islander XIAP c.1239_1242dup(p.Val415fs) Hemizygous ND Markedly decreased XIAP expression 
179 Male 1.9 Unknown XIAP c.1239_1242dup(p.Val415fs) Hemizygous ND NA 
180 Male 1.1 Unknown XIAP c.1301-1G>A Hemizygous ND Symptoms of HLH 
181 Male 1.5 Unknown XIAP c.1445C>G(p.Pro482Arg) Hemizygous ND NA 
182 Male 35 European-American XIAP c.1456dup(p.Thr486fs) Hemizygous ND Symptoms of HLH 
183 Male 4.5 European-American SH2D1A c.20A>G(p.Tyr7Cys) Hemizygous ND Absent SAP in CD8+ T cells 
184 Male 1.8 Unknown SH2D1A c.117C>T(p.Gly39Gly) Hemizygous ND NA 
185 Male 3.5 Unknown SH2D1A c.130T>C(p.Cys44Arg) Hemizygous ND Absent SAP in CD8+ T cells 
186 Male 27 European-American SH2D1A c.163C>T(p.Arg55*Hemizygous ND History of pneumonia 
187 Male 8 d Unknown SH2D1A c.172C>T(p.Gln58*Hemizygous ND NA 
188 Male 6.8 African American SH2D1A c.172C>T(p.Gln58*Hemizygous ND Absent SAP in CD8+ T cells 
189 Male 4.7 Latino-Hispanic SH2D1A c.199_201+19del(p.Glu67del) Hemizygous ND Absent SAP in CD8+ T cells 
190 Male 1.3 African American SH2D1A c.201G>A(p.Glu67Glu) Hemizygous ND Absent SAP in CD8+ T cells 
191 Male 7 d European-American SH2D1A c.245del(p.Asn82fs) Hemizygous ND Absent SAP in CD8+ T cells 
192 Male 34 d European-American + Pacific-Islander SH2D1A c.295C>T(p.Gln99*Hemizygous ND Absent SAP in CD8+ T cells 
193 Male 5.4 Middle-Eastern MAGT1 c.154_161delinsC(p.Ile52fs) Hemizygous ND Symptoms of HLH, bone pain in low extremities 
194 Male 10.4 European-American MAGT1 c.223C>T(p.Gln75*Hemizygous ND NA 
195 Male 18.4 African American MAGT1 c.407G>A(p.Trp136*Hemizygous ND NA 
196 Male 27.6 European-American MAGT1 c.443_444del(p.Phe148fs) Hemizygous ND Symptoms of HLH 
197 Male 17.3 European-American MAGT1 c.774del(p.Phe258fs) Hemizygous ND NA 
Patient no.SexAge at testing, y (unless indicated otherwise)EthnicityGeneVariantZygosityPopulation frequency (gnomAD*), %Symptoms/immunology testing/family history
Female 53 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Male 63 d Unknown PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Male 0.4 Middle Eastern PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Female 27 d Unknown PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression 
Female 32 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression 
Male 4 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression; sibling died of HLH 
Female 0.4 African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
Male 6 d African American + European-white PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Absent perforin expression 
Male 59 d African American PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
10 Male 13 d African PRF1 c.50del(p.Leu17fs) Homozygous 0.033 Symptoms of HLH 
11 Female 7 d Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent NK cell function 
    PRF1 c.266C>T(p.Pro89Leu) Heterozygous ND 
12 Female 0.3 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.350_356delinsATGC (p.Val117_Arg119delinsAspAla) Heterozygous ND 
13 Male 0.4 Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 
14 Male 3.3 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression; brother with HLH 
    PRF1 c.527G>A(p.Cys176Tyr) Heterozygous ND 
15 Female 0.5 Latino-Hispanic PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression 
    PRF1 c.659G>A(p.Gly220Asp) Heterozygous 0.0008 
16 Female 0.2 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent NK cell function 
    PRF1 c.853_855del(p.Lys285del) Heterozygous 0.0056 
17 Male 66 d Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.895C>T(p.Arg299Cys) Heterozygous 0.0012 
18 Male 20.6 Latino-Hispanic PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 NA 
    PRF1 c.902C>T(p.Ser301Leu) Heterozygous ND 
19 Male 54 d African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Symptoms of HLH 
    PRF1 c.916G>T(p.Gly306Cys) Heterozygous ND 
20 Female 45 d African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression 
    PRF1 c.916G>T(p.Gly306Cys) Heterozygous ND 
21 Male 32 d Latino-Hispanic PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Symptoms of HLH 
    PRF1 c.985dup(p.Val329fs) Heterozygous ND 
22 Male 0.3 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent perforin expression 
    PRF1 c.1385C>A(p.Ser462*Heterozygous ND 
23 Male 36.1 Unknown PRF1 c.116C>A(p.Pro39His) Heterozygous 0.00081 NA 
    PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 
24 Female 1.2 Asian-American PRF1 c.133G>A(p.Gly45Arg) Homozygous 0.0012 Absent NK cell function 
25 Female 37 d Non-Hispanic white PRF1 c.150del(p.Thr51fs) Heterozygous 0.0004 Absent perforin expression 
    PRF1 c.227G>A(p.Cys76Tyr) Heterozygous 0.00071 
26 Male 69 d Latino-Hispanic PRF1 c.218G>C(p.Cys73Ser) Homozygous 0.0004 Symptoms of HLH 
27 Female 20 European-American PRF1 c.227G>A(p.Cys76Tyr) Heterozygous 0.00071 Absent perforin expression 
    PRF1 c.626A>C(p.Gln209Pro) Heterozygous 0.0012 
28 Female 21.8 Unknown PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 Absent NK cell function, decreased perforin expression 
    PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 
29 Male 17 European-American PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 Absent perforin expression 
    PRF1 c.635A>C(p.Tyr212Ser) Heterozygous ND 
30 Female 41.2 European-American PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 Absent NK cell function, decreased perforin expression 
    PRF1 c.666C>A(p.His222Gln) Heterozygous 0.0039 
31 Female 8.3 European-American PRF1 c.443C>G(p.Ala148Gly) Heterozygous 0.0004 NA 
    PRF1 c.666C>A(p.His222Gln) Heterozygous 0.0039 
32 Male 2.6 Unknown PRF1 c.445G>A(p.Gly149Ser) Homozygous 0.014 Absent NK cell function 
33 Male 0.8 Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Homozygous 0.014 Symptoms of HLH 
34 Female Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Homozygous 0.014 NA 
35 Female 0.3 European-American PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 Family history of HLH 
    PRF1 c.614A>G(p.Asn205Ser) Heterozygous 0.0043 
36 Male 42 d Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 NA 
    PRF1 c.938A>T(p.Asp313Val) Heterozygous 0.0012 
37 Male 4.6 Unknown PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 Absent perforin expression 
    PRF1 c.1081A>T(p.Arg361Trp) Heterozygous 0.0011 
38 Female 32 d Middle Eastern PRF1 c.501C>G(p.Tyr167*Homozygous ND Symptoms of HLH 
39 Female 0.3 Unknown PRF1 c.512C>A(p.Thr171Asn) Homozygous 0.0028 Absent perforin expression 
40 Male 9.5 European-American PRF1 c.786_801del(p.Gln263fs) Heterozygous ND Absent NK cell function 
    PRF1 c.886T>C(p.Tyr296His) Heterozygous 0.0012 
41 Male 59 d Unknown PRF1 c.853_855del(p.Lys285del) Heterozygous 0.0057 NA 
    PRF1 c.921del(p.His308fs) Heterozygous 0.002 
42 Female 0.7 Middle Eastern PRF1 c.880del(p.Gln294fs) Homozygous ND Symptoms of HLH 
43 Female Middle Eastern PRF1 c.895C>T(p.Arg299Cys) Homozygous 0.0012 Symptoms of HLH 
44 Female 0.2 Latino-Hispanic PRF1 c.904G>T(p.Glu302*Homozygous ND Absent perforin expression 
45 Female 1.8 Unknown PRF1 c.949G>A(p.Gly317Arg) Homozygous 0.0008 Symptoms of HLH 
46 Female 10.3 European-American PRF1 c.973T>C(p.Tyr325His) Heterozygous ND Absent perforin expression 
    PRF1 c.1326_1328del(p.Phe443del) Heterozygous ND 
47 Male 1.1 Middle Eastern PRF1 c.1070G>C(p.Arg357Pro) Homozygous ND Symptoms of HLH 
48 Male 12.5 Middle Eastern PRF1 c.1081A>T(p.Arg361Trp) Homozygous 0.0011 Abnormal brain lesions and seizures 
49 Female 2.6 Unknown PRF1 c.1229_1230delinsCC (p.Arg410Pro) Homozygous ND NA 
50 Female 0.2 African American PRF1 c.1304C>T(p.Thr435Met) Heterozygous 0.0028 Absent perforin expression 
    PRF1 c.1314T>A(p.Tyr438*Heterozygous 0.0032 
51 Female 2.6 Latino-Hispanic PRF1 c.1337A>C(p.Gln446Pro) Homozygous 0.0016 NA 
52 Female 2.6 Unknown PRF1 c.1337A>C(p.Gln446Pro) Homozygous 0.0016 Symptoms of HLH 
53 Female 0.4 Middle Eastern STXBP2 c.37+2T>C Heterozygous ND Absent NK cell function 
    STXBP2 c.1430C>T(p.Pro477Leu) Heterozygous 0.00074 
54 Male 0.6 Unknown STXBP2 c.37+5G>A Heterozygous ND NA 
    STXBP2 c.1057T>C (p.Cys353Arg) Heterozygous 0.0004 
55 Female 63 d Asian-American STXBP2 c.193C>T(p.Arg65Trp) Homozygous 0.00071 Absent NK cell function 
56 Female 5.5 Unknown STXBP2 c.194G>A(p.Arg65Gln) Heterozygous 0.0028 Absent NK cell function 
    STXBP2 c.560C>T (p.Pro187Leu) Heterozygous 0.00064 
57 Male 4.1 European-American STXBP2 c.194G>A(p.Arg65Gln) Heterozygous 0.0028 Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
58 Female 4.2 European-American STXBP2 c.326-30_326-23del Heterozygous 0.0068 Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
59 Male 0.6 Latino-Hispanic STXBP2 c.389T>C(p.Leu130Ser) Homozygous 0.0032 Symptoms of HLH 
60 Male 45 d African American STXBP2 c.389T>C(p.Leu130Ser) Heterozygous 0.0032 Symptoms of HLH; family history of HLH 
    STXBP2 exon 14-19 deletion Heterozygous ND 
61 Male 0.7 Middle Eastern STXBP2 c.481del(p.Arg161fs) Homozygous ND Symptoms of HLH 
62 Male 0.4 Unknown STXBP2 c.481del(p.Arg161fs) Homozygous ND Symptoms of HLH 
63 Female 11 European-American STXBP2 c.539_540delinsAA(p.Cys180*Heterozygous ND Symptoms of HLH 
    STXBP2 c.1247-1G>C Heterozygous 0.02 
64 Male 0.3 Latino-Hispanic STXBP2 c.703C>G(p.Arg235Gly) Homozygous 0.00071 Absent NK cell function 
65 Female 52.7 European-American STXBP2 c.752C>T(p.Ala251Val) Heterozygous ND Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
66 Male 0.9 Unknown STXBP2 c.902+5G>A Heterozygous 0.0036 NA 
    STXBP2 c.1247-1G>C Heterozygous 0.02 
67 Male 3.1 Unknown STXBP2 c.1247-1G>C Homozygous 0.02 Symptoms of HLH 
68 Female 22.7 Latino-Spanish STXBP2 c.1247-1G>C Homozygous 0.02 Decreased NK cell function 
69 Male 26.4 European-American STXBP2 c.1247-1G>C Homozygous 0.02 Symptoms of HLH 
70 Male 25.6 European-American STXBP2 c.1247-1G>C Homozygous 0.02 Symptoms of HLH 
71 Female 29.7 European-American STXBP2 c.1247-1G>C Homozygous 0.02 NA 
72 Male European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Absent NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
73 Female 15.8 European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Absent NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
74 Female 19 Unknown STXBP2 c.1247-1G>C Heterozygous 0.02 Decreased NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
75 Female 26.9 European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Symptoms of HLH 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
76 Female 57.8 European-American STXBP2 c.1247-1G>C Heterozygous 0.02 Absent NK cell function 
    STXBP2 c.1621G>A(p.Gly541Ser) Heterozygous 0.023 
77 Female 0.2 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
78 Female 0.3 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 NA 
79 Female 0.3 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
80 Female 0.6 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
81 Male 0.2 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 NA 
82 Male 0.7 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
83 Female 0.8 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
84 Male 0.8 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
85 Male 0.6 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Family history of HLH 
86 Male 1.6 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
87 Male 0.3 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
88 Female 63 d Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Family history of HLH 
89 Male 0.5 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
90 Male 0.2 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Symptoms of HLH 
91 Male 11.1 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Homozygous 0.00074 Absent NK cell function 
92 Female 10.1 Unknown STXBP2 c.1430C>T(p.Pro477Leu) Heterozygous 0.00074 Symptoms of HLH 
    STXBP2 c.1696+5G>T Heterozygous ND 
93 Male 0.5 Middle Eastern STXBP2 c.1452+1G>A Homozygous ND Symptoms of HLH 
94 Male Middle Eastern STXBP2 c.1452+1G>A Homozygous ND Abnormal NK cell function 
95 Female 49 d European-American UNC13D c.118-308C>T Heterozygous 0.019 Dysmorphic facies, decreased NK cell function 
    UNC13D c.2258_2267delinsTACCTTGTTCGA (p.Gly753fs) Heterozygous ND 
96 Male 0.7 European-American UNC13D c.118-308C>T Heterozygous 0.019 Decreased NK cell function 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
97 Male 0.2 European-American + Latino-Spanish UNC13D c.118-308C>T Heterozygous 0.019 Decreased NK cell function 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
98 Female 1.3 Non-Hispanic white UNC13D c.118-308C>T Heterozygous 0.019 Symptoms of HLH, seizures, normal NK cell function 
    UNC13D c.2867C>T(p.Pro956Leu) Heterozygous ND 
99 Female Non-Hispanic white UNC13D c.118-308C>T Heterozygous 0.019 Absent NK cell function 
    UNC13D c.3193C>T(p.Arg1065*Heterozygous 0.0011 
100 Female 3.2 European-American UNC13D c.118-308C>T Heterozygous 0.019 Absent NK cell function 
    UNC13D 253Kb inversion Heterozygous ND 
101 Male 2.6 Unknown UNC13D 253Kb inversion Heterozygous ND Decreased NK cell function 
    UNC13D c.154-1G>C Heterozygous ND 
102 Male 0.2 Unknown UNC13D 253Kb inversion Heterozygous ND Symptoms of HLH 
    UNC13D c.551G>A(p.Trp184*Heterozygous 0.0011 
103 Female 0.2 European-American UNC13D 253Kb inversion Heterozygous ND NA 
    UNC13D c.1389+1G>A Heterozygous 0.0071 
104 Female 0.2 European-American UNC13D 253Kb inversion Heterozygous ND Absent NK cell function 
    UNC13D c.2447+1G>A Heterozygous 0.00051 
105 Female 0.6 Non-Hispanic white UNC13D 253Kb inversion Heterozygous ND Decreased NK cell function 
    UNC13D c.2695C>T(p.Arg899*Heterozygous 0.0018 
106 Male 11.4 Hispanic white UNC13D c.182A>G(p.Tyr61Cys) Heterozygous ND Symptoms of HLH 
    UNC13D c.778T>C(p.Trp260Arg) Heterozygous ND 
107 Male 0.4 European-American UNC13D c.262-1G>A Heterozygous ND Symptoms of HLH, abnormal NK cell function, family history of HLH 
    UNC13D c.766C>T(p.Arg256*Heterozygous 0.0025 
108 Male 0.4 Unknown UNC13D c.321+1_321+2del Heterozygous ND Decreased NK cell function 
    UNC13D c.753+1G>T Heterozygous 0.0044 
109 Male 0.3 Unknown UNC13D c.322-2A>T Heterozygous 0.0024 Symptoms of HLH, decreased NK cell function 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
110 Male 0.3 Unknown UNC13D c.419T>C(p.Ile140Thr) Heterozygous 0.0004 NA 
    UNC13D c.460C>T(p.Arg154Trp) Heterozygous 0.011 
111 Female 5.4 Middle Eastern UNC13D c.424dup(p.Gln142fs) Homozygous ND Symptoms of HLH 
112 Male 7.7 Latino-Hispanic UNC13D c.518C>T(p.Thr173Met) Heterozygous 0.0028 NA 
    UNC13D c.1803_1819dup(p.Arg607fs) Heterozygous ND 
113 Female 39 d European-American UNC13D c.551G>A(p.Trp184*Heterozygous 0.0011 Abnormal NK cell function 
    UNC13D c.766C>T(p.Arg256*Heterozygous 0.0025 
114 Male 4.9 European-American UNC13D c.570-2A>T Heterozygous ND Absent NK cell function 
    UNC13D c.3049G>A(p.Glu1017Lys) Heterozygous 0.00044 
115 Female 0.2 Middle Eastern UNC13D c.753+1G>T Homozygous 0.0044 Symptoms of HLH 
116 Female 0.6 European-American UNC13D c.766C>T(p.Arg256*Heterozygous 0.0025 Symptoms of HLH, abnormal NK cell function 
    UNC13D c.2447+1G>A Heterozygous 0.00051 
117 Female 1.2 Latino-Spanish UNC13D c.859del(p.Arg287fs) Homozygous ND Decreased NK cell function 
118 Female Non-Hispanic white UNC13D c.1055+1G>T Heterozygous ND Decreased NK cell function, family history of HLH 
    UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 
119 Male 0.7 European-American UNC13D c.1229_1230dup(p.Arg411fs) Heterozygous 0.00 Symptoms of HLH, hypertelorism 
    UNC13D c.2298+1G>T Heterozygous ND 
120 Female 0.2 European-American UNC13D c.1259_1260del(p.Ser420fs) Heterozygous ND symptoms of HLH, decreased NK cell function 
    UNC13D c.1848+1G>C Heterozygous ND 
121 Male 18.3 European-American UNC13D c.1387C>T(p.Gln463*Heterozygous ND symptoms of HLH, decreased NK cell function 
    UNC13D c.1820G>C(p.Arg607Pro) Heterozygous 0.011 
122 Female 58 d European-American + African American UNC13D c.1389+1G>A Heterozygous 0.0071 Symptoms of HLH 
    UNC13D c.1848+1G>C Heterozygous ND 
123 Female 0.2 Middle Eastern UNC13D c.1423C>T(p.Gln475*Homozygous ND NA 
124 Male 10 d Pacific Islander UNC13D c.2296C>T(p.Gln766*Homozygous ND Decreased NK cell function 
125 Female 13.2 Unknown UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 NA 
    UNC13D c.2588G>A(p.Gly863Asp) Heterozygous 0.029 
126 Female Unknown UNC13D c.2346_2349del(p.Arg782fs) Heterozygous 0.01 NA 
    UNC13D c.3065T>C(p.Leu1022Pro) Heterozygous ND 
127 Female 0.2 Middle Eastern UNC13D c.2553+1G>T Homozygous ND Symptoms of HLH 
128 Female 0.3 Middle Eastern UNC13D c.2553+1G>T Homozygous ND Symptoms of HLH 
129 Female Asian UNC13D c.2588G>A(p.Gly863Asp) Homozygous 0.029 Decreased NK cell function 
130 Female 0.4 African American UNC13D c.2695C>T(p.Arg899*Homozygous 0.0018 Symptoms of HLH, absent NK cell function, dysmorphic facies 
131 Male 0.7 Non-Hispanic white UNC13D c.2819del(p.Leu940fs) Homozygous ND symptoms of HLH 
132 Female 0.6 Middle Eastern UNC13D c.3048dup(p.Glu1017fs) Homozygous ND NA 
133 Female 0.7 Unknown UNC13D c.3053C>A(p.Ala1018Asp) Homozygous 0.00088 2 affected siblings 
134 Male 13 European-American RAB27A c.121A>G(p.Thr41Ala) Heterozygous ND NA 
    RAB27A c.352C>T(p.Gln118*Heterozygous ND 
135 Male 9.6 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 NA 
136 Male 9.9 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 NA 
137 Female Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Failure to thrive, bone marrow failure 
138 Female 9.4 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Symptoms of HLH 
139 Male 10.6 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Symptoms of HLH 
140 Male 19.5 Middle Eastern RAB27A c.244C>T(p.Arg82Cys) Homozygous 0.0016 Symptoms of HLH 
141 Female 0.3 Latino-Hispanic RAB27A c.335del(p.Asn112fs) Homozygous 0.0044 Symptoms of HLH 
142 Female 0.4 Middle Eastern RAB27A c.400A>C(p.Lys134Gln) Homozygous ND NA 
143 Female 53.9 Pacific Islander RAB27A c.476A>G(p.Tyr159Cys) Homozygous ND Symptoms of HLH, absent NK cell function 
144 Male 1.2 Middle Eastern RAB27A c.598C>T(p.Arg200*Homozygous 0.0004 NA 
145 Female 0.4 European-American RAB27A c.638_642del(p.Glu213fs) Homozygous 0.0008 Symptoms of HLH 
146 Female 0.7 African American LYST c.925C>T(p.Arg309*Heterozygous ND Oculocutaneous albinism, neutropenia 
    LYST c.2015dup(p.Tyr672*Heterozygous ND 
147 Male 3.4 Unknown LYST c.3194del(p.Leu1065*Homozygous 0.0004 NA 
148 Female 0.3 Middle Eastern LYST c.4159dup(p.Thr1387fs) Homozygous ND Premature gray hair, anemia 
149 Male 0.9 European-American LYST c.5715del(p.Asn1905fs) Heterozygous ND Oculocutaneous albinism, neutropenia, absent NK cell function 
    LYST c.8802-2A>G Heterozygous ND 
150 Female 6.9 Unknown LYST c.5784+1G>T Homozygous ND Oculocutaneous albinism, dysmorphic facies, neutropenia 
151 Male 3.2 Non-Hispanic white LYST c.6159_6160del(p.Met2053fs) Homozygous ND NA 
152 Male 1.8 Middle Eastern LYST c.7291del(p.Leu2431fs) Homozygous ND Hypopigmentation, anemia 
153 Male 1.2 African American LYST c.8770C>T(p.Gln2924*Heterozygous ND Silver hair, hypopigmented skin lesions, pancytopenia 
    LYST c.9844_9845del(p.Ser3282fs) Heterozygous ND 
154 Female 7.1 Middle Eastern LYST c.10776C>G(p.Tyr3592*Homozygous ND Abnormal pigmentation, neutropenia 
155 Female 1.2 Unknown STX11 c.73G>T(p.Glu25*Heterozygous 0.0004 Decreased NK cell function 
    STX11 c.748C>T(p.Gln250*Heterozygous 0.00081 
156 Female 5.6 Middle Eastern STX11 c.173T>C(p.Leu58Pro) Homozygous 0.0008 Symptoms of HLH, grayish hair 
157 Female 11.6 Middle Eastern STX11 c.173T>C(p.Leu58Pro) Homozygous 0.0008 NA 
158 Male 2.9 European-American + Latino-Hispanic STX11 c.462_463delinsA(p.Asp155fs) Heterozygous ND Decreased NK cell function 
    STX11 c.784C>T(p.Gln262*Heterozygous ND 
159 Male 1.4 Asian-Indian STX11 c.687dup(p.Gln230fs) Homozygous ND NA 
160 Female 12 d Middle Eastern SLC7A7 c.1429+1G>C Homozygous ND Family history of HLH 
161 Male 1.2 African American SLC7A7 c.701del(p.Ser234fs) Heterozygous 0.0016 NA 
    SLC7A7 c.895-1G>A Heterozygous ND  
162 Female 13.7 European-American SLC7A7 c.360_361delinsAA (p.Trp121Arg) Homozygous ND NA 
163 Male 5.4 African American XIAP c.145C>T(p.Arg49*Hemizygous ND Markedly decreased XIAP expression 
164 Male 18.7 European-American XIAP c.345C>G(p.Tyr115*Hemizygous ND Symptoms of HLH 
165 Male 30.3 European-American XIAP c.608G>T(p.Cys203Phe) Hemizygous ND NA 
166 Male European-American XIAP c.664C>T(p.Arg222*Hemizygous ND Symptoms of HLH 
167 Male 8.9 European-American XIAP c.738del(p.Asp247fs) Hemizygous ND NA 
168 Male 16 European-American XIAP c.738del(p.Asp247fs) Hemizygous ND Absent XIAP expression 
169 Male 3.6 African American XIAP c.889A>T(p.Lys297*Hemizygous ND NA 
170 Male 0.4 Unknown XIAP c.894_898del(p.Lys299fs) Hemizygous ND NA 
171 Male 17.2 European-American XIAP c.894_898del(p.Lys299fs) Hemizygous ND Symptoms of HLH 
172 Male 19.6 African American XIAP c.926_929del(p.Asp309fs) Hemizygous ND Symptoms of HLH 
173 Male 17.2 European-American XIAP c.969G>A(p.Trp323*Hemizygous ND Decreased XIAP expression 
174 Male 22.6 Unknown XIAP c.1021_1022del(p.Asn341fs) Hemizygous ND NA 
175 Male 4.5 Unknown XIAP c.1056+1G>A Hemizygous ND NA 
176 Male 2.7 Latino-Hispanic XIAP c.1141C>T(p.Arg381*Hemizygous ND NA 
177 Male 11.8 Unknown XIAP c.1141C>T(p.Arg381*Hemizygous ND Absent XIAP expression 
178 Male 1 d Pacific-Islander XIAP c.1239_1242dup(p.Val415fs) Hemizygous ND Markedly decreased XIAP expression 
179 Male 1.9 Unknown XIAP c.1239_1242dup(p.Val415fs) Hemizygous ND NA 
180 Male 1.1 Unknown XIAP c.1301-1G>A Hemizygous ND Symptoms of HLH 
181 Male 1.5 Unknown XIAP c.1445C>G(p.Pro482Arg) Hemizygous ND NA 
182 Male 35 European-American XIAP c.1456dup(p.Thr486fs) Hemizygous ND Symptoms of HLH 
183 Male 4.5 European-American SH2D1A c.20A>G(p.Tyr7Cys) Hemizygous ND Absent SAP in CD8+ T cells 
184 Male 1.8 Unknown SH2D1A c.117C>T(p.Gly39Gly) Hemizygous ND NA 
185 Male 3.5 Unknown SH2D1A c.130T>C(p.Cys44Arg) Hemizygous ND Absent SAP in CD8+ T cells 
186 Male 27 European-American SH2D1A c.163C>T(p.Arg55*Hemizygous ND History of pneumonia 
187 Male 8 d Unknown SH2D1A c.172C>T(p.Gln58*Hemizygous ND NA 
188 Male 6.8 African American SH2D1A c.172C>T(p.Gln58*Hemizygous ND Absent SAP in CD8+ T cells 
189 Male 4.7 Latino-Hispanic SH2D1A c.199_201+19del(p.Glu67del) Hemizygous ND Absent SAP in CD8+ T cells 
190 Male 1.3 African American SH2D1A c.201G>A(p.Glu67Glu) Hemizygous ND Absent SAP in CD8+ T cells 
191 Male 7 d European-American SH2D1A c.245del(p.Asn82fs) Hemizygous ND Absent SAP in CD8+ T cells 
192 Male 34 d European-American + Pacific-Islander SH2D1A c.295C>T(p.Gln99*Hemizygous ND Absent SAP in CD8+ T cells 
193 Male 5.4 Middle-Eastern MAGT1 c.154_161delinsC(p.Ile52fs) Hemizygous ND Symptoms of HLH, bone pain in low extremities 
194 Male 10.4 European-American MAGT1 c.223C>T(p.Gln75*Hemizygous ND NA 
195 Male 18.4 African American MAGT1 c.407G>A(p.Trp136*Hemizygous ND NA 
196 Male 27.6 European-American MAGT1 c.443_444del(p.Phe148fs) Hemizygous ND Symptoms of HLH 
197 Male 17.3 European-American MAGT1 c.774del(p.Phe258fs) Hemizygous ND NA 

NA, no data; ND, no data; NK, natural killer.

*

gnomAD v2.1.1 total population frequency.

HLH hemophagocytic lymphohistiocytosis, symptoms of HLH reported included any or all of the following “fever, hepatosplenomegaly, anemia/cytopenias, neutropenia/leukopenia, elevated ferritin/triglycerides, and/or decreased fibrinogen.”

According to the ACMG guideline, c.272C>T(p.Ala91Val) in PRF1 was classified as a variant of unknown significance.

HLH NGS panel

Fifteen genes that have been associated with HLH or HLH-like conditions were included in our HLH NGS panel: PRF1, UNC13D, STX11, STXBP2, ITK, CD27, SH2D1A, XIAP, MAGT1, LYST, RAB27A, AP3B1, BLOC1S6, SLC7A7, and GATA2. Their associated OMIM diseases, transcripts, and inheritance pattern are listed in supplemental Table 1. A typical turnaround time for this clinical testing is 4 weeks. Expedited turnaround time is available upon request.

NGS, data analysis, and Sanger confirmation

NGS was performed on the genomic DNA isolated from the patient samples using microdroplet polymerase chain reaction technology (RainDance Technologies Inc.) and sequenced on an Illumina HiSeq2500 instrument (Illumina Inc.). All exons, flanking intronic (±20 base pairs) and 5′ and 3′ untranslated regions of the 15 genes in the HLH panel (supplemental Table 1) were captured. Data for each sample were assessed for quality and confirmed they had at least 20× read depth at every target base. Sanger sequencing was performed to rescue all low coverage (<20× read depth) regions. Variants within those regions were identified and evaluated using a validated, custom bioinformatic pipeline. The American College of Medical Genetics and Genomics (ACMG) guidelines for sequence variant classification were used to categorize variants. All reported variants were confirmed by Sanger sequencing. In addition, allele-specific analysis for the 253-kb inversion as well as targeted analysis of the c.118-308 and c.118-307 regions in the UNC13D gene were performed for each sample because these variants have been reported to disrupt UNC13D transcription in lymphocytes and abolish Munc13-4 expression.15 

We reviewed the results of the 1892 patient samples, excluded potential carriers based on clinical information provided, and reported the number of samples that were abnormal with pathogenic or likely pathogenic variants associated with HLH. Samples were classified according to the genes affected, types of mutations, and predicted impact on protein sequencing or structure. Pathogenic or likely pathogenic variants were identified in 10 genes: PRF1, STXBP2, UNC13D, LYST, RAB27A, STX11, SLC7A7, XIAP, SH2D1A, and MAGT1.

Results

At CCHMC, a 15-gene NGS panel for the molecular diagnosis of HLH disorders was offered from September 2013. Since then, the number of orders for traditionally sequential single-gene tests related to HLH disorders drastically decreased. As shown in Figure 1, from 2013 to 2018, the orders for single-gene Sanger sequencing such as PRF1, UNC13D, STXBP2, RAB27A, XIAP, and SH2D1A decreased from 308, 302, 277, 249, 132, and 104 in 2013 to 21, 3, 4, 1, 9, and 10 in 2018, respectively. On the other hand, the orders of HLH NGS panel jumped and maintained ∼400 test orders per year from 2014 to 2018.

Figure 1.

Volume of HLH-related single gene and HLH panel testing in Cincinnati Children’s Hospital Medical Center from 2013 to 2018.

Figure 1.

Volume of HLH-related single gene and HLH panel testing in Cincinnati Children’s Hospital Medical Center from 2013 to 2018.

A total of 1892 HLH panel testing results were analyzed, and clearly pathogenic and likely pathogenic variants were identified in 227 patients. Of these, 197 samples had a definite molecular genetic diagnosis: 87 samples with homozygotes and 75 with compound heterozygotes observed in a recessive condition, respectively, and 35 samples with hemizygotes observed in an X-linked disorder. This resulted in a positive molecular diagnostic rate of 10.4% (supplemental Figure 1). Table 1 lists the genetic variants identified in these 197 patients. Pathogenic or likely causal variants in the PRF1 gene were the most frequent and were identified in 26.4% (52/197) of patients (Figure 2A). Mutations in the genes associated with degranulation defects were more common than previously appreciated: 21.3% (42/197) of the patients had pathogenic or likely causal variants in STXBP2, 19.8% (39/197) in UNC13D, 6.1% (12/197) in RAB27A, 4.6% (9/197) in LYST, and 2.5% (5/197) in STX11. Pathogenic variants in the lysinuric protein intolerance gene SLC7A7 were identified in 1.5% (3/197) of the patients, the least frequent group of patients in our cohort. X-linked conditions accounted for 17.8% (35/197) of the patients: 20 (10.2%) patients had pathogenic or suspected diagnostic variants in XIAP, 10 (5.1%) in SH2D1A, and 5 (2.5%) in MAGT1 (Figure 2A). In addition, 30 of 227 patients with clinically suspected HLH were identified to carry only 1 pathogenic or likely pathogenic variant in a recessive condition by this panel approach. Among them, 50% (15/30) had a PRF1 pathogenic variant, 3 had a STXBP2 pathogenic variant, 4 had a UNC13D pathogenic variant, 5 had a RAB27 pathogenic variant, 2 had a LYST pathogenic variant, and 1 patient had a STX11 pathogenic variant. Of these 30 patients, 3 (cases S18, S23, and S30) also carried another common PRF1 variant c.272C>T (p.Ala91Val) in the heterozygous state (Table 2).

Figure 2.

Characteristics of genetic findings and age ranges for 197 HLH patients. (A) Distribution of genetic findings in 197 HLH patients with a definite genetic diagnosis. (B) Whisker-box plot of the age ranges at referral for 197 HLH patients.

Figure 2.

Characteristics of genetic findings and age ranges for 197 HLH patients. (A) Distribution of genetic findings in 197 HLH patients with a definite genetic diagnosis. (B) Whisker-box plot of the age ranges at referral for 197 HLH patients.

Table 2.

List of 30 patients in whom only 1 heterozygous pathogenic or likely pathogenic variant was identified

Patient no.SexAge at testing, y (unless indicated otherwise)EthnicityGeneVariantZygosityPopulation frequency (gnomAD*), %Symptoms/immunology testing/family history
S01 Female 28.3 Unknown PRF1 c.35_46del(p.Leu12_Leu15del) Heterozygous ND NA 
S02 Male 20 d Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Decreased NK cell function and perforin expression 
S03 Female 0.4 Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Symptoms of HLH 
S04 Male 7.8 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Decreased perforin expression 
S05 Female 11.2 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent NK cell function 
S06 Male 52 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 HLH, lymphoma 
S07 Male 13.3 Native American PRF1 c.112G>A(p.Val38Met) Heterozygous 0.0073 Symptoms of HLH 
S08 Male 16.6 Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 Symptoms of HLH 
S09 Female 9.9 Unknown PRF1 c.563C>T(p.Pro188Leu) Heterozygous 0.013 Symptoms of HLH 
S10 Male 17.8 Unknown PRF1 c.853_855del(p.Lys285del) Heterozygous 0.0057 Thrombocytopenia, absent NK cell function 
S11 Female 3.1 European-American PRF1 c.1066C>T(p.Arg356Trp) Heterozygous 0.0014 NA 
S12 Female Unknown PRF1 c.1117C>T(p.Arg373Cys) Heterozygous 0.0051 Symptoms of HLH; normal NK cell function 
S13 Female 16.7 Unknown PRF1 c.1117C>T(p.Arg373Cys) Heterozygous 0.0051 Absent NK cell function 
S14 Female 12 d Middle Eastern PRF1 c.1122G>A(p.Trp374*Heterozygous 0.0016 NA 
S15 Female 9.7 Malaysian-Chinese PRF1 c.1349C>T(p.Thr450Met) Heterozygous 0.0028 History of HLH 
S16 Female 0.5 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Heterozygous 0.00074 Symptoms of HLH 
S17 Female 0.5 Latino-Hispanic STXBP2 c.1717C>T(p.Pro573Ser) Heterozygous ND NA 
S18 Male 37 d European-American STXBP2 c.1717C>T(p.Pro573Ser) Heterozygous ND Absent NK cell function 
    PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 
S19 Male 14.9 Asian-American UNC13D c.118-307G>A Heterozygous ND Absent NK cell function 
S20 Male 20.3 Unknown UNC13D c.247C>T(p.Arg83*Heterozygous 0.0004 NA 
S21 Female 62.9 European-American UNC13D c.1759C>T(p.Arg587Cys) Heterozygous 0.019 Symptoms of HLH 
S22 Female 13.1 European-American UNC13D c.2037_2038insG(p.Arg680fs) Heterozygous ND Symptoms of HLH, one sibling deceased due to HLH 
S23 Male 0.8 Middle Eastern RAB27A c.148_149delinsC(p.Arg50fs) Heterozygous ND Gray hair, suspected for GS, consanguinity 
    PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 
S24 Male European-American RAB27A c.240-47_240delins20 Heterozygous ND Rash, neutropenia 
S25 Male 11.1 Indian RAB27A c.244C>T(p.Arg82Cys) Heterozygous 0.0016 Symptoms of HLH 
S26 Female 24.9 Latino-Hispanic RAB27A c.335del(p.Asn112fs) Heterozygous 0.0044 Symptoms of HLH 
S27 Male 2.1 Unknown RAB27A c.400_401del(p.Lys134fs) Heterozygous 0.0004 Abnormal brain MRI, decreased NK cell function 
S28 Male 29 European-American LYST c.465_466del(p.Asp157fs) Heterozygous ND Pancytopenia, increased ferritin level 
S29 Male 6.4 Middle Eastern LYST c.4159dup(p.Thr1387fs) Heterozygous ND Gray hair 
S30 Female 23.2 Unknown STX11 c.650T>A(p.Leu217Gln) Heterozygous 0.0004 NA 
    PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 
Patient no.SexAge at testing, y (unless indicated otherwise)EthnicityGeneVariantZygosityPopulation frequency (gnomAD*), %Symptoms/immunology testing/family history
S01 Female 28.3 Unknown PRF1 c.35_46del(p.Leu12_Leu15del) Heterozygous ND NA 
S02 Male 20 d Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Decreased NK cell function and perforin expression 
S03 Female 0.4 Unknown PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Symptoms of HLH 
S04 Male 7.8 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Decreased perforin expression 
S05 Female 11.2 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 Absent NK cell function 
S06 Male 52 African American PRF1 c.50del(p.Leu17fs) Heterozygous 0.033 HLH, lymphoma 
S07 Male 13.3 Native American PRF1 c.112G>A(p.Val38Met) Heterozygous 0.0073 Symptoms of HLH 
S08 Male 16.6 Latino-Hispanic PRF1 c.445G>A(p.Gly149Ser) Heterozygous 0.014 Symptoms of HLH 
S09 Female 9.9 Unknown PRF1 c.563C>T(p.Pro188Leu) Heterozygous 0.013 Symptoms of HLH 
S10 Male 17.8 Unknown PRF1 c.853_855del(p.Lys285del) Heterozygous 0.0057 Thrombocytopenia, absent NK cell function 
S11 Female 3.1 European-American PRF1 c.1066C>T(p.Arg356Trp) Heterozygous 0.0014 NA 
S12 Female Unknown PRF1 c.1117C>T(p.Arg373Cys) Heterozygous 0.0051 Symptoms of HLH; normal NK cell function 
S13 Female 16.7 Unknown PRF1 c.1117C>T(p.Arg373Cys) Heterozygous 0.0051 Absent NK cell function 
S14 Female 12 d Middle Eastern PRF1 c.1122G>A(p.Trp374*Heterozygous 0.0016 NA 
S15 Female 9.7 Malaysian-Chinese PRF1 c.1349C>T(p.Thr450Met) Heterozygous 0.0028 History of HLH 
S16 Female 0.5 Middle Eastern STXBP2 c.1430C>T(p.Pro477Leu) Heterozygous 0.00074 Symptoms of HLH 
S17 Female 0.5 Latino-Hispanic STXBP2 c.1717C>T(p.Pro573Ser) Heterozygous ND NA 
S18 Male 37 d European-American STXBP2 c.1717C>T(p.Pro573Ser) Heterozygous ND Absent NK cell function 
    PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 
S19 Male 14.9 Asian-American UNC13D c.118-307G>A Heterozygous ND Absent NK cell function 
S20 Male 20.3 Unknown UNC13D c.247C>T(p.Arg83*Heterozygous 0.0004 NA 
S21 Female 62.9 European-American UNC13D c.1759C>T(p.Arg587Cys) Heterozygous 0.019 Symptoms of HLH 
S22 Female 13.1 European-American UNC13D c.2037_2038insG(p.Arg680fs) Heterozygous ND Symptoms of HLH, one sibling deceased due to HLH 
S23 Male 0.8 Middle Eastern RAB27A c.148_149delinsC(p.Arg50fs) Heterozygous ND Gray hair, suspected for GS, consanguinity 
    PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 
S24 Male European-American RAB27A c.240-47_240delins20 Heterozygous ND Rash, neutropenia 
S25 Male 11.1 Indian RAB27A c.244C>T(p.Arg82Cys) Heterozygous 0.0016 Symptoms of HLH 
S26 Female 24.9 Latino-Hispanic RAB27A c.335del(p.Asn112fs) Heterozygous 0.0044 Symptoms of HLH 
S27 Male 2.1 Unknown RAB27A c.400_401del(p.Lys134fs) Heterozygous 0.0004 Abnormal brain MRI, decreased NK cell function 
S28 Male 29 European-American LYST c.465_466del(p.Asp157fs) Heterozygous ND Pancytopenia, increased ferritin level 
S29 Male 6.4 Middle Eastern LYST c.4159dup(p.Thr1387fs) Heterozygous ND Gray hair 
S30 Female 23.2 Unknown STX11 c.650T>A(p.Leu217Gln) Heterozygous 0.0004 NA 
    PRF1 c.272C>T(p.Ala91Val) Heterozygous 2.92 
*

gnomAD v2.1.1 total population frequency.

HLH hemophagocytic lymphohistiocytosis, symptoms of HLH reported included any or all of the following “fever, hepatosplenomegaly, anemia/cytopenias, neutropenia/leukopenia, elevated ferritin/triglycerides, and/or decreased fibrinogen.”

According to the ACMG guideline, c.272C>T(p.Ala91Val) in PRF1 was classified as a variant of unknown significance.

When the patients were divided based on age ranges, the diagnostic rates in patients aged 0 to 12 months, 1 to 5 years, 5 to 12 years, 12 to 18 years, and older than 18 years are 28.6% (95/332), 11.3% (43/380), 6.7% (25/371), 3.3% (10/304), and 4.8% (24/505), respectively. Moreover, patients with a molecular diagnosis in familial HLH type 2-5 genes (PRF1, UNC13D, STX11, and STXBP2; supplemental Table 1) tended to be referred and diagnosed at an earlier age compared with other genes (median age, 0.7 years [4 days-57.8 years] vs 4.5 years [1 day-53.9 years]; P = .009). Patients with X-linked conditions (XIAP, SH2D1A, and MAGT1) were referred and diagnosed at relatively older ages (median age, 5.4 years [1 day-35 years]) (Figure 2B).

Ten of 15 genes in the HLH sequencing panel were identified with pathogenic or likely pathogenic variants in our patient cohort, with the highest allele number and unique variants in the PRF1 gene (115 alleles; 45 unique variants). The majority of the identified PRF1 variants (66.7%; 30/45) were missense changes that were distributed along the exons without a particular hot spot. The most frequent pathogenic variant identified in our cohort was c.50del (p.Leu17fs), which appeared in 37 alleles in 27 patients including 10 homozygotes, 12 compound heterozygotes, and 5 heterozygotes. Of the 27 patients carrying this variant, 16 were African American, 3 were Latino-Spanish, 1 was Middle Eastern, and 7 were unknown. The other frequently detected variant was c.445G>A (p.Gly149Ser), which was identified in 19.2% (10/52) of the patients including 3 homozygotes, 6 compound heterozygotes, and 1 heterozygote. Among these 10 patients, 4 were Latino-Spanish, 1 was European-American, and 5 were unknown. (Tables 1 and 2; Figures 3 and 4A).

Figure 3.

Distribution of unique pathogenic or likely pathogenic variants identified in 10 HLH-associated genes in our patient cohort.

Figure 3.

Distribution of unique pathogenic or likely pathogenic variants identified in 10 HLH-associated genes in our patient cohort.

Figure 4.

Distributions and frequencies of pathogenic or likely pathogenic variants in the most frequently affected genes associated with HLH in our cohort. (A) Distributions and frequencies of pathogenic or likely pathogenic variants in PRF1. (B) Distributions and frequencies of pathogenic or likely pathogenic variants in STXBP2. (C) Distributions and frequencies of pathogenic or likely pathogenic variants in UNC13D. Note: the 253-kb inversion is not shown in the graph. (D) Distributions and frequencies of pathogenic or likely pathogenic variants in XIAP. (A-D) The total number of alleles affected with each variant is indicated in the circles. *Novel variants.

Figure 4.

Distributions and frequencies of pathogenic or likely pathogenic variants in the most frequently affected genes associated with HLH in our cohort. (A) Distributions and frequencies of pathogenic or likely pathogenic variants in PRF1. (B) Distributions and frequencies of pathogenic or likely pathogenic variants in STXBP2. (C) Distributions and frequencies of pathogenic or likely pathogenic variants in UNC13D. Note: the 253-kb inversion is not shown in the graph. (D) Distributions and frequencies of pathogenic or likely pathogenic variants in XIAP. (A-D) The total number of alleles affected with each variant is indicated in the circles. *Novel variants.

The second most frequently mutated gene in our cohort was the STXBP2 gene but with fewer unique variants (87 alleles, 20 unique variants). One-half (10/20) of unique STXBP2 variants were missense changes. As shown in Figure 4B, the missense variant c.1430C>T (p.Pro477Leu) and the splicing variant c.1247-1G>C were the 2 most frequent variants identified in STXBP2, accounting for 33 alleles in 18 patients and 17 alleles in 12 patients, respectively. Of the 18 patients carrying the c.1430C>T (p.Pro477Leu) variant, 13 were Middle Eastern and 5 were unknown. This result implies that this particular mutation in the STXBP2 gene identified in these patients might be identical by descent (ie, they might be inherited from a common ancestry). On the other hand, among the 12 patients who carry at least 1 splicing variant c.1247-1G>C, 8 were European-American, 1 was Latino-Spanish, and 3 were unknown (Tables 1 and 2; Figures 3 and 4B).

UNC13D was identified with a similar number of causal alleles to STXBP2 but with the doubled number of unique variants (82 alleles, 45 unique variants). Fourteen of 45 unique UNC13D variants were splicing mutations. The intronic variant c.118-308C>T and the 253-kb inversion were each identified in 6 patients in the compound heterozygous state along with a diverse second mutation, much higher than a recently reported Chinese cohort16  (Table 1; Figures 3 and 4C). As shown in Table 1, among the 39 patients with the UNC13D pathogenic or likely pathogenic variants, besides common HLH symptoms, cases 95 and 130 were also reported to have dysmorphic facies, case 98 seizures, and case 119 hypertelorism. Eleven mutated alleles and 7 unique alterations were identified in the STX11 gene in our cohort. Although most unique variants (5/7) are truncating variants, the missense variant c.173T>C (p.Leu58Pro) was the most frequently identified in STX11, appearing as 2 homozygotes in 2 patients with unknown relationship. Twenty-nine and 20 pathogenic or likely causal variants representing 11 and 13 unique genetic alterations were also identified in RAB27A and LYST, respectively. Most patients (7/9; 77.8%) with a molecular variation finding in the LYST gene were reported to have an albinism phenotype including silver hair, abnormal pigmentation, and oculocutaneous albinism (Tables 1 and 2; Figure 3; supplemental Figure 2).

Thirty unique pathogenic and likely pathogenic variants were identified in the 3 genes associated with an X-linked condition: 16, 9, and 5 unique variants in XIAP, SH2D1A, and MAGT1 were identified in 20, 10, and 5 male patients, respectively. Of these unique variants, 83% (25/30) were truncating variants which would presumably result in loss of function of the protein products (Table 1; Figures 2A, 3, and 4D; supplemental Figure 2).

Discussion

In this study, we analyzed 1892 samples tested for a panel of 15 HLH-associated genes, which were received between September 2013 and June 2018 at CCHMC. Both known and novel pathogenic or likely pathogenic variants have been identified in this population, and their frequency and distributions were analyzed. We found that 12% of the patients had at least 1 pathogenic or likely pathogenic variant in 1 of the 15 genes, and 10.4% of the patients had a definite molecular diagnosis in an HLH-associated gene. To our knowledge, this is the largest cohort of genetically diagnosed HLH, with the highest number of targeted HLH disease-causing genes tested simultaneously.

Patients with a definite genetic diagnosis were significantly younger than patients with only 1 heterozygote finding, with a median age of 14 months (197 cases) compared with 10 years (30 cases) (P = .007). When including all patients analyzed, we found that samples from younger patients were more likely to result in a genetic diagnosis (see "Results"). This observation in our cohort is consistent with previous reports in other similar studies.16-18 

Together, the genes responsible for degranulation defects (UNC13D, STXBP2, STX11, LYST, RAB27A) accounted for the majority of cases (107/197; 54.3%). As seen in southern Europe,17  FHL2 and FHL3 account for a significant proportion of our cases (91/197; 46.2%). However, whereas FHL5 accounted for a minor proportion of HLH in Europeans, it is much more prevalent in our cohort (42/197; 21.3%). PRF1 was the most frequent causal gene (52/197; 26.4%). Although this finding is not surprising, other groups have described PRF1 defects in 40% to 60% of their cases.19,20  Nevertheless, all cohorts reported to date used different methods and criteria to define their cases; some included the variants of unknown significance, and others reported only the known significant biallelic or hemizygous variants. These inconsistent criteria make it difficult to compare the results among different groups. The lower percentage of PRF1 gene mutations described in our cohort may possibly reflect the use of immunological screening (perforin detection by flow cytometry) before sending for genetic diagnosis.

Compared with other large cohorts previously reported,16,17,21  our results show an extremely wide range of different mutations, reflecting the vast multiethnic population living in North America. Among the 227 cases, mutations were found in 10 different genes (PRF1, UNC13D, STX11, STXBP2, RAB27A, LYST, XIAP, SH2D1A, MAGT1, and SLC7A7), with a total of 175 different mutations. Of these unique mutations, 105 were found in only 1 patient. Only 4 variants were observed in more than 7 patients (PRF1: c.50del, c.445G>A; STXBP2: c.1247-1G>C, c.1430C>T). The well-described frameshift variant commonly found in patients with African American background, c.50del in PRF1,19,22  was the most common variant found in our cohort. The second most common mutation in PRF1 was c.445G>A, which has been previously described by multiple groups in different ethnic backgrounds, including Caucasian, Hispanic, Portuguese, German, and Chinese,16,19,20,23,24  reflecting the multiethnic population in North America. Interestingly, the commonly reported mutation in the Turkish population,20,25,26  c.1122G>A, was found in only 1 patient in our cohort. Similarly, we have only 1 case with c.1349C>T, the most common PRF1 mutation among the Chinese population,16  and no cases of c.1090_1091del or c.207del, variants commonly found in the Japanese population.27,28  One of the most prevalent mutations in our cohort, c.1247-1G>C in STXBP2, was previously described by other groups and found in multiple ethnic background including Caucasian, Turkish, Northern European, and Pakistani.5,29,30 

Splicing mutations accounted for an important proportion of our UNC13D variants, with 14 of 45 unique mutations,31,32  which represents a different mutation spectrum of UNC13D variation from a Chinese cohort.16  We observed 3 cases of c.753+1G>T, a predominant mutation found in the German population20  as well as other parts of Europe (Italy33  and Croatia15 ). The deep intronic variant c.118-308C>T was observed in 6 unrelated patients, being one of the most frequent UNC13D mutations in our cohort. This is one of the most commonly reported mutations among the Korean population, but interestingly we had no cases of c.754-1G>C, the other very common mutation in Korea.32,34 

The NGS-based approach is highly accurate in capturing point mutations and small deletions and insertions (such as <10 base pair deletions, duplication or insertion), which are the vast majority of sequence changes causing HLH in this 15-gene panel. However, our NGS-based diagnostic pipeline could not reliably identify large structural variations such as large deletions, duplications, or insertions occurring in the genes in this panel because of the limitation of sensitivity and specificity. Better algorithms that could use enriched NGS data to reliably identify these types of variation are warranted. It may be helpful for making a definite genetic diagnosis in the 30 patients in whom only 1 suspicious heterozygous variant in the genes associated with autosomal recessive conditions was identified.

Overall, a genetic diagnosis could be made in 10.4% of the patients using this HLH panel approach. The relatively low diagnostic rate can be explained by multiple factors. Complete clinical information and fulfillment of HLH clinical criteria is crucial to understanding our genetic results. As a heterogeneous disease with different disease mechanisms, many HLH-associated genes have yet to be discovered. In addition, some newly discovered HLH-associated genes such as NLRC435,36  and CDC4237  were not included in our panel and therefore could cause a missing genetic diagnosis for some of those negative cases. Some additional primary immune deficiencies have been reported to present with HLH, for example severe combined immunodeficiency, DiGeorge syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, and STAT1 gain of function, among others.18,38-40  Several metabolic diseases also predispose to the development of HLH.41-44  With sequencing costs and analysis times going down, whole exome sequencing and even whole genome sequencing will provide more comprehensive solutions for detecting underlying genetic causes of HLH. Whole exome and whole genome sequencing would also identify genetic disorders that are associated with clinical phenotypes that mimic HLH. Indeed, a broader use of whole exome sequencing has already been recommended for patients with HLH.18  On the other hand, a significant proportion of the 1859 patients in this cohort likely had a secondary form of acquired HLH that developed in the context of malignancy, autoimmune disease, or infection, and whole exome and whole genome sequencing may not be a cost-effective approach for all patients with HLH at present. As the field of genetics continues to make clinical advances, clinicians should continue to weigh the ease, cost, completeness, and timeliness of genetic panel testing options for patients with HLH.

Data sharing requests can be e-mailed to the corresponding authors, Miao Sun (miao.sun@cchmc.org) or Rebecca A. Marsh (rebecca.marsh@cchmc.org).

Acknowledgments

The authors thank the physicians and genetic counselors that referred patients to our laboratory for HLH testing and the patients and their families that participated in this testing.

Authorship

Contribution: K.A.R., R.A.M., and M.S. designed the research; V.G.-L., L.D., R.S., J.C., K.Z., K.A.R., R.A.M., and M.S. analyzed data; V.G.-L. and M.S. interpreted results; V.G.-L., J.C., R.A.M., and M.S. wrote the manuscript; and all authors edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Miao Sun, Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, 3333 Burnet Ave, Cincinnati, OH 45229; e-mail: miao.sun@cchmc.org; and Rebecca A. Marsh, Division of Bone Marrow Transplant and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, 3333 Burnet Ave, Cincinnati, OH 45229; e-mail: rebecca.marsh@cchmc.org.

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