Genetic Studies on HLH in Korea

Jong Jin Seo, MD, PhD

Asan Medical CenterSeoul, Korea

2019. 3. 15ICKSH & KSH Annual Meeting

I have no personal or financial interests to declare:

I have no financial support from an industry source at the current presentation.

대한혈액학회 Korean Society of Hematology

COI disclosureName of author ： Jong Jin Seo

Brief introduction on genetic aspects of familialHLH (FHL)

Previous Korean studies on the genetics of HLH Search for unidentified gene mutations in Korean

patients with HLH (preliminary result) Genotype-phenotype study of Korean FHL

patients (preliminary result)

Contents of presentation

Primary HLH is caused by a gene mutation, either at the FHL loci or in a gene responsible for one of several immunodeficiency syndromes

The incidence of 5 FHL types varies in different ethnic groups, some with founder effect

Patients with HLH gene mutation tend to present at a younger age than those without mutation

Previous studies showed that deleterious non-sense mutations are associated with earlier onset and a more severe clinical course suggesting possible genotype/ phenotype correlation

Genetic overview of FHL (1)

FHL is usually inherited in an autosomal recessive manner (ie, inheritance of a mutation at both alleles of a gene is required to manifest the disease)

Occasionally, patients may have compound heterozygotes (ie, a different mutation in each allele of the same gene), or they may show digenic inheritance(ie, separate mutations in 2 different genes)

The presence of homozygous, compound heterozygous, or digenic mutations is sufficient for diagnosis of FHL

Genetic overview of FHL (2)

Diagnostic criteria (HLH-2004)

Expanding spectrum of genes associated with primary HLH

J Pediatr 2013 163(5):1253-9

Locus Gene % Worldwide* % in Japanese‡ % in Korean

FHL1 N/A 4 consanguineous Pakistani families Not found

FHL2 PRF1 20%-30% worldwide(>50% in AA) 59 ?

FHL3 UNC13D ~20%-30% worldwide 34

FHL4 STX11 ~20% of Turkish/Kurdish familieslow in other ethnic groups Not found

FHL5 STXBP216% in Central Europeans, Turks, and Saudis~20% in North American

7

The incidence of FHL subtypes varies among different ethnic groups

* GeneReviews ‡ Plos One 2010;5(11):e14173

Genetic cornerstones of HLH

2005FHL4/STX11

2009FHL5/

STXBP2

1999FHL2/PRF1

1999of FHL1

2003FHL3/

UNC13D

2004 HLHdiagnostic

criteria

2010 Hematologica40 HLH pt.

8 FHL3 & 1 FHL2

2013 Ann Hematol72 HLH pt.

17 FHL3 & 3 FHL2Identification of

deep intronic mutation of UNC13D with

founder effect

2016 Clin GenetThe 1st FHL5

2016 Ann Lab MedThe 1st FHL4

Incorporation ofGenetic tests

1994 HLHdiagnostic

criteria

(International) (Korean)

2018 (Unpublished) Search for unidentified

genetic mutation Geno-phenotype

study

Genetic studies for HLH in Korea

1st

(2010, Haematologica)

• 40 pt with HLH

• FHL2 to FHL4 (PRF1,

UNC13D, STX11)

2nd

(2013, Ann Hematol)

• 32 new pt with HLH

• Pt without FHL

mutation / and

single mutation in

UNC13D (FHL3)

from the 1st set

• UNC13D deep

intronic mutation

3rd

(2016, Clin Genet)

• Pt without FHL

mutation from the

1st & 2nd set

• STXBP2 (FHL5)

(from the Korean Registry for Histiocytosis)

Genetic studies for HLH in Korea

1st

(2010, Haematologica)

• 40 pt with HLH

• FHL2 to FHL4 (PRF1,

UNC13D, STX11)

2nd

(2013, Ann Hematol)

• 32 new pt with HLH

• Pt without FHL

mutation / and

single mutation in

UNC13D (FHL3)

from the 1st set

• UNC13D deep

intronic mutation

3rd

(2016, Clin Genet)

• Pt without FHL

mutation from the

1st & 2nd set

• STXBP2 (FHL5)

(from the Korean Registry for Histiocytosis)

1st Set(n=40)

PRF1(n=1)

UNC13D(n=8)

No mutation(n=31)

Biallelic mutations (n=4)

Monoallelic mutation only(n=4)

STX11(n=0)

Haematologica 2010

► UNC13D is the predominant causative gene in the Korean population

Genetic subtypes of FHL in Korea (1st set)

Meeths M et al. Blood 2011;118:5783-5793

Genetic studies for HLH in Korea

1st

(2010, Haematologica)

• 40 pt with HLH

• FHL2 to FHL4 (PRF1,

UNC13D, STX11)

2nd

(2013, Ann Hematol)

• 32 new pt with HLH

• Pt without FHL

mutation / and

single mutation in

UNC13D (FHL3)

from the 1st set

• UNC13D deep

intronic mutation

3rd

(2016, Clin Genet)

• Pt without FHL

mutation from the

1st & 2nd set

• STXBP2 (FHL5)

(from the Korean Registry for Histiocytosis)

2nd Set(n=32)

1st Set(n=40)

PRF1(n=1)

UNC13D(n=8)

No mutation(n=31)

2 mutations (n=4)

1 mutation only(n=4)

Investigation for

c.118-308C>T

UNC13D STX11PRF1STX11(n=0)

1st set(n=40)

UNC13D mutation

(n=8)

No mutation

(n=31)

PRF1 mutation (n=1)

2 mutations

(n=4)

1 mutation only

(n=4)

c.118-308C>T in 3/4 (75%)

c.118-308C>T in 2 in homozygous status

23%(9/40)

28%(11/40)

Mutation detection rate

Predominance of FHL3 in Korea due to 2 recurrent mutations of UNC13D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

c.247C>TR83*

(3%; 1/34)

c.754-1G>C(26%; 9/34)

c.1993-2A>G(3%; 1/34)

c.1693delGE565Sfs*7(6%; 2/34)

c.2954+5G>A(6%; 2/34)

c.2367+1G>A(3%; 1/34)

c.118-308C>T(41%; 14/34)

*c.1055+1G>A(9%; 3/34)

c.292C>TQ98*

(3%; 1/34)

Haplotype analyses revealed significantly shared haplotypes in both mutations, indicating the

presence of “founder effect”

This explains the unusual predominance of FHL3 in Korea

Genetic studies for HLH in Korea

1st

(2010, Haematologica)

• 40 pt with HLH

• FHL2 to FHL4 (PRF1,

UNC13D, STX11)

2nd

(2013, Ann Hematol)

• 32 new pt with HLH

• Pt without FHL

mutation / and

single mutation in

UNC13D (FHL3)

from the 1st set

• UNC13D deep

intronic mutation

3rd

(2016, Clin Genet)

• Pt without FHL

mutation from the

1st & 2nd set

• STXBP2 (FHL5)

(from the Korean Registry for Histiocytosis)

Mutation (+)(n=20)

72 patients (1st & 2nd set)

UNC13D(n=17)

PRF1(n=3)

STX11(n=0)

Mutation (-)(n=52)

STXBP2 mutation analysis(n=50)

No DNA samples for 2 patients

1 patient with 2 causative mutations in STXBP2 Identified mutations

- c.184A>G (p.Asn62Asp), heterozygous- c.577A>C, heterozygous

Frequency of FHL5 in Korean patients with HLH- 5% (1/21) of patients with FHL

FHL5 is rare in Korea

Summary of this Study

Search for unidentified genetic mutation in Korean pediatric patients

with HLH using gene panel test

- Preliminary results -

Analysis using gene panel test in Korean patients with no definitive diagnosis of FHL

Genes included in 14-Gene Panel Test

Variants identified by HLH-associated gene panel test in 72 patients

P122

P154

P157

P158

P161

P162

P163

P167

P168

P169

P18

P19

P21

P22

P24

P25

P26

P27

P28

P30

P31

P32

P33

P34

P38

P39

P40

P46

P47

P48

P50

P52

P55

P58

P59

P62

P64

P67

P68

P74

P78

P83

P87

P91

P92

P93

P94

P96

P97

P99

P100

P102

P103

P106

P107

P110

P112

P118

P120

P121

P3

P7

P9

P10

P13

P13

P14

P15

P16

P17

P170

P8

AP3B1

BLOC1S6

CD27

ITK

LYST

MAGT1

PRF1

RAB27A

SH2D1A

SLC7A7

STX11

STXBP2

UNC13D

XIAP

HLH-associated mutation Variant of unknown significance

Distribution of the genetic defects in Korean patients with HLH (n=101)

Targeted gene panel test further identified 10 Korean

patients with primary HLH who were previously not

diagnosed with conventional FHL2-5 genetic tests

- 2 male patients with XLP type 1 and type 2

- 8 patients with 2 HLH-associated variants

(6 of them with digenic defects)

17% of the registered patients were found to have

monoallelic mutation

Summary of this study

Functional analysis of identified variants Clinical correlation according to the types of

mutation Identification of unknown genetic

background in patients not enrolled to Korean HLH Registry using gene panel test

More extended gene panel test (>14 genes) for genetically undiagnosed patients

Further workups

Identification of genetic mutation with gene panel test : The registry study of

Korean pediatric patients with hemophagocytic lymphohistiocytosis

1Hyery Kim, 2Ja Young Seo, 1Kyung Nam Koh, 1Ho Joon Im, 3Ji Won Lee, 3Kun Hee Yoo, 3Ki Woong Sung, 3Hong Hoe Koo, 4Jae Min Lee, 5Hyoung Jin

Kang, 5Hee Young Shin,1Jong Jin Seo

1Department of Pediatrics, University of Ulsan College of Medicine, Asan Medical Center, Republic of Korea2Department of Laboratory Medicine, Gachon University Gil medical center, Republic of Korea3Department of Pediatrics, Sungkyunkwan University School of Medicine, Republic of Korea4Department of Pediatrics, Yeungnam University College of Medicine, Republic of Korea5Department of Pediatrics, Seoul National University College of Medicine, Republic of Korea

(Abstract OP09-6, 3/15 )

- Preliminary results -

Nationwide retrospective multicenter study

by reviewing medical records

July, 2005~March, 2017

Inclusion criteria

- Patients diagnosed with HLH according to

the HLH-2004 diagnostic criteria, and

- Patients with identified gene mutation

Total 48 patients from 10 institutions

Median age at Dx : 3.2 months (7 days-13 yrs) 77.0% <1 yr (n=37), 89.6% <3 yr (n=43)

0

2

4

6

8

10

12

1 mon 2 mon 3 mon 4 mon 6 mon 7 mon 8 mon 9 mon 1 yr 2 yr 6 yr 7 yr 9 yr 13 yr

3

11

10

5

3

2 2

1

3 3

1

2

1 1

OS 68.0%

PRF1 (N=7)

UNC13D (N=37)

STX11 (N=1)

STXBP2 (N=2)

SH2D1A (N=2) P=0.870

71.4%

50.0%

Overall Survival (months)

63.1%

48.8%

PRF1 (N=7)UNC13D (N=36)STX11 (N=1)STXBP2 (N=2)SH2D1A (N=2)

P=0.228

58.3% 50.5%

Sex Age at Dx(mon) Mutation Mutation

DescriptionFamily

Hx HSCT Reactivation Survival OS(mon)

F 71.9 c.[1693delG(p.Asp565Serfs*7)];[2954+5G>A] Frameshift/Splicing No Yes No Dead in CR 5.3M 5.1 c[1693delG(p.Asp565Serfs*7)];[2954+5G>A] Frameshift/Splicing Yes Yes Before HSCT Alive 135.6F 85.5 c.[1693delG(p.Asp565Serfs*7)];[2954+5G>A] Frameshift/Splicing Yes Yes No Alive 94.9F 108.8 c.[1693delG];[2954+5G>A] Frameshift/Splicing Yes Yes No Alive 51.6F 1.2 c.[118-308C>T];[2447+1G>A] Regulatory/? No No Before HSCT DOD 3.9F 1.9 c.[118-308C>T];[766C>T] Regulatory/Nonsense No Yes No Alive 52M 1.9 c.[118-308C>T];[2695C>T(p.Arg899*)] Regulatory/Nonsense No Yes After HSCT Dead in CR 8.6M 0.3 c.[292C>T(p.Gln98*)];[118-308C>T] Regulatory/Nonsense No Yes Before HSCT Alive 116.8F 26.1 c.[118-308C>T];[2695C>T(p.Arg899*)] Regulatory/Nonsense Yes Yes Before HSCT Alive 33.1F 6.9 c.[118-308C>T];[247C>T(p.Arg83*)] Regulatory/Nonsense No Yes Before HSCT Alive 77.7F 1.7 c.[118-308C>T];[118-308C>T] Regulatory/Regulatory No Yes No Alive 73.8M 2.1 c.[118-308C>T];[118-308C>T] Regulatory/Regulatory No Yes No Alive 47.5F 1.3 c.[118-308C>T];[754-1G>C] Regulatory/Splicing No Yes Before HSCT Alive 102.8F 1.6 c.[118-308C>T];[754-1G>C] Regulatory/Splicing No Yes No Alive 50.4M 3.2 c.[118-308C>T];[754-1G>C] Regulatory/Splicing No Yes No Alive 47.9F 3.8 c.[118-308C>T];[754-1G>C] Regulatory/Splicing No Yes No Alive 99.8F 2.1 c.[118-308C>T];[754-1G>C] Regulatory/Splicing No Yes After HSCT DOD 39.7F 11.6 c.[118-308C>T];[754-1G>C] Regulatory/Splicing Yes Yes No Dead in CR 9.8M 7.4 c.[118-308C>T];[1055+1G>A] Regulatory/Splicing No Yes No Dead in CR 8.2M 12.7 c.[118-3C>A];[602A>G(p.His201Arg)] Splicing/Missense No Yes No Alive 41.5M 2.5 c.[754-1G>C];[1055+1G>A] Splicing/Splicing No Yes Before HSCT Alive 113.4F 31.3 c.[754-1G>C];[754-1G>C] Splicing/Splicing No No Before HSCT DOD 9.5M 2.9 c.[754-1G>C];[2367+1G>A] Splicing/Splicing No No Before HSCT DOD 11.5F 3.9 c.[754-1G>C];[1993-2A>G] Splicing/Splicing No No No DOD 1.6M 5.2 c.[754-1G>C];[754-1G>C] Splicing/Splicing No Yes No DOD 3.7

Overall Survival (months) Reactivation free survival (months)

P=0.101

Biallelic splicing mutations(N=5)

58.7% Others (N=21)

Biallelic splicing mutations (N=5)

71.1% Others (N=21)

P=0.03920.0%

51.2% Total (N=26)

61.3% Total (N=26)

The prevalence of UNC13D was 75.5% of Korean FHL patients, and it was followed by PRF1 mutation (14.3%)

There was no statistical difference in the phenotype at diagnosis between genotype groups in Korean patients with FHL

Approximately 35% of patients showed reactivation, and there was no difference in reactivation rates by genotype

There was no difference in the post-transplant OS rates by genotype

Patients with biallelic splicing mutations showed significantly lower OS and lower reactivation free survival rates compared to other patients

This Korean study needs further analysis in larger cohort

Locus Gene % Worldwide* % in Japanese‡ % in Korean

FHL1 N/A4 consanguineous Pakistani families Not found Not found

FHL2 PRF1 20%-30% worldwide(>50% in AA) 59 ~15

FHL3 UNC13D ~20%-30% worldwide 34 75~80

FHL4 STX11 ~20% of Turkish/Kurdish familieslow in other ethnic groups Not found ~2 (1 case)

FHL5 STXBP216% in Central Europeans, Turks, and Saudis~20% in North American

7 ~5 (2 cases)

Frequencies of FHL Gene Mutations

* GeneReviews ‡ Plos One 2010;5(11):e14173

Genetic heterogeneity and expanding spectrum of HLH necessitates advanced molecular diagnostic technique

Multi-gene panel test may become more readily available in expanded spectrum of genes

Genotype-phenotype correlation of FHL patients needs to be clarified with further studies

Comprehensive molecular testing can provide detailed genetic information, and thereby may help to reveal unanswered molecular pathophysiology which may improve the treatment outcome of HLH

Prospects

Acknowledgement

AMC PHO Ho Joon ImKyung Nam KohHyery KimAMC LabChan-Jeoung ParkSeongsoo JangYoung-Uk Cho

SNUH PHOHyoung Jin Kang

Kyung Hee Univ. MC PHOHoi Soo Yoon

Young Nam Univ. MC PHOJae Min Lee

SMC PHO/LabKeon Hee Yoo Hee-Jin Kim

Gil MC LabJa-Young Seo

CMC PHONack Gyun Chung

Severance Hospital PHOChuhl Joo Lyu

Chonnam NUH PHOHoon KookHee Jo Baek

Thanks to all the members of Korea Histiocytosis WP

Thank you for your attention!