Accepted Article
1
Received date: 10/27/2014 Revised date: Accepted date: 11/19/2014 Original Article
Atypical hemolytic uremic syndrome: a Korean pediatric series1
Jiwon M. Lee1, MD; Young Seo Park3, MD, PhD; Joo Hoon Lee3, MD, PhD; Se Jin Park4 , MD, PhD; Jae Il Shin5, MD, PhD; Yong-Hoon Park6, MD, PhD; Kee Hwan Yoo7, MD, PhD; Min Hyun Cho8, MD, PhD; Su-Young Kim9, MD, PhD; Seong Heon Kim9, MD; Mee Kyung
Namgoong10, MD, PhD; Seung Joo Lee11, MD, PhD; Jun Ho Lee12, MD, PhD; Hee Yeon Cho13,
MD, PhD; Kyoung Hee Han14, MD, PhD; Hee Gyung Kang1,2, MD, PhD; Il Soo Ha1, MD, PhD; Jun-Seok Bae16,17, Nayoung K.D. Kim17, PhD; Woong-Yang Park17,18, MD, PhD; Hae Il Cheong1,2,15, MD, PhD
1
Department of Pediatrics, Seoul National University Children’s Hospital, Seoul, Korea
2
Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul,
Korea 3
Department of Pediatrics, Asan Medical Center, University of Ulsan, Seoul, Korea
4
Department of Pediatrics, Ajou University School of Medicine, Suwon, Korea
5
Department of Pediatrics, Severance Children’s Hospital, Yonsei University, Seoul, Korea
6
Department of Pediatrics, Yeungnam University College of Medicine, Daegu, Korea
This article has been accepted for publication and undergone full peer review but has not been through the copyed iting, typesetting, pagination and proofreading process, which may lead to differences between this version and th e Version of Record. Please cite this article as doi: 10.1111/ped.12549
This article is protected by copyright. All rights reserved.
Accepted Article
2
7
Department of Pediatrics, Korea University Guro Hospital, Seoul, Korea
8
Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea
9
Department of Pediatrics, Pusan National University Children’s Hospital, Yangsan, Korea
10
11
Department of Pediatrics, Wonju College of Medicine, Yonsei University, Wonju, Korea
Department of Pediatrics, Ehwa University Mokdong Hospital, Seoul, Korea
12
Department of Pediatrics, Bundang CHA Hospital, Seongnam, Korea
13
Department of Pediatrics, Samsung Medical Center, Seoul, Korea
14
Department of Pediatrics, Jeju University Hospital, Jeju, Korea
15
Kidney Research Institute, Medical Research Center, Seoul National University College of
Medicine, Seoul, Korea 16
Department of Health Sciences and Technology, Samsung Advanced Institute for Health
Sciences and Technology, Sungkyunkwan University, Seoul, Korea
17
Samsung Genome Institute, Samsung Medical Center, Seoul, Korea
18
Sungkyunkwan University School of Medicine, Seoul, Korea
Correspondence: Hae Il Cheong, M.D., Ph.D. Department of Pediatrics Seoul National University Children’s Hospital 101 Daehak-ro, Jongno-gu Seoul, 110-744, Korea Tel 82-2-2072-2810 Fax 82-2-743-3455
This article is protected by copyright. All rights reserved.
Accepted Article
3
E-mail [email protected]
Short title: A Korean pediatric cohort of aHUS
Number of text pages: 10
Number of words: 2950 Reference pages: 5
tables: 5
figures: 3
legends to figures: 3
Abstract
Background: Atypical hemolytic uremic syndrome (aHUS) is a rare disease with a genetic predisposition. Few studies have evaluated the disease in Asian population. We studied a Korean pediatric cohort to delineate the clinical characteristics and genotypes. Methods: A multicenter cohort of 51 Korean children with aHUS was screened for mutations by targeted exome sequencing covering 46 complement related genes. Anti-complement-factor-H autoantibodies (anti-CFH) titers were measured. Multiplex ligation dependent probe amplification assay was performed to detect deletions in the complement factor-H related protein genes (CFHRs). We grouped the patients according to the etiology and compared the clinical
This article is protected by copyright. All rights reserved.
Accepted Article
4
features using the Mann-Whitney U test and chi-square test. Results: Fifteen patients (Group A, 29.7%) had anti-CFH and mutations were detected in 11(Group B, 21.6%) patients, including one with combined mutations. Remaining 25(Group C, 49.0%) were neither–positive. Anti-CFH-association was more frequent than the world-wide prevalence. Group A showed older onset age than Group B, although did not significantly differ in the clinical manifestation. Group B showed worst renal outcome. Gene frequencies of homozygous CFHR1 deletion were 73.3%, 2.7% and 1% in Group A, Group B+C and the control, respectively. Conclusions: In our cohort, we observed a relatively high incidence of anti-CFH-association. Clinical outcomes largely conformed to the previous reports. Although the size is limited, this cohort provides a reassessment of clinicogenetic features of aHUS in Korean children.
Key Words: Anti-complement factor H autoantibody, Atypical hemolytic uremic syndrome, Complement factor H, Asian, Mutation
This article is protected by copyright. All rights reserved.
Accepted Article
5
Introduction Hemolytic uremic syndrome (HUS), characterized by the triad of microangiopathic hemolytic
anemia, thrombocytopenia and acute renal failure, is a microvascular occlusion disorder that belongs to the category of thrombotic microangiopathy (TMA)1-5. HUS may present primarily or due to secondary causes, such as bone marrow transplantation, medication (e.g., anticancer chemotherapy, calcineurin inhibitors and antiplatelet agents), pneumococcal or viral infections and autoimmune diseases6. In primary HUS, the atypical form (aHUS) is distinguished from the typical form by the absence of a prior verotoxin-producing Escherichia coli infection. Primary aHUS can further be divided into subgroups according to the etiology; complement alternative pathway dysregulation1,2,4,7, anti-complement factor H autoantibodies (anti-CFH)8, mutations in the coagulation pathway genes9-11, and also in combination of above12. To date, genetic or acquired abnormalities in the complement system have been documented in nearly 60% of patients with aHUS1,13; including mutations in the complement factor H (CFH)14,15, complement factor I (CFI)16, complement factor B (CFB)17, membrane cofactor protein (MCP/CD46)16,18, and
complement 3 (C3) genes19, as well as in their combinations6,12. More recently identified mutations on the coagulation pathway genes include THBD9, DGKE10, and PLG11. THBD encodes thrombomodulin, which functions as a cofactor for thrombin to reduce blood coagulation and also regulates CFI-induced C3b inactivation9,20. DGKE encodes diacylglycerol
kinase ε and is implicated in regulation of thrombus formation by modulating protein kinase C activity in endothelial cells and platelets9. PLG, a gene in the coagulation pathway encoding plasminogen which is converted into plasmin by a variety of enzymes on binding to clots, has emerged as a novel causative gene in the pathogenesis of aHUS11. Anti-CFH8 is an acquired cause of aHUS frequently presenting in association with homozygous deletion of CFHR1 which
This article is protected by copyright. All rights reserved.
Accepted Article
6
encodes complement factor H-related protein 121-25. Since genetic backgrounds largely underlie in the pathogenesis of aHUS, investigation on the
clinical and genetic characteristics in patients with different ethnic background may be of a clinical value. In fact, genetic variability among racial groups may account for medically important differences in disease outcomes26. In addition, racial disparities have been demonstrated in TMAs in the Oklahoma Thrombotic thrombocytopenic purpura (TTP)-HUS Registry, most recently27. We designed this study to 1) investigate the genetic etiologies and clinical outcomes of aHUS in
a nationwide Korean pediatric cohort, 2) compare the clinical features between the etiologic subgroups, and 3) if any, explore racial disparities in the disease.
Materials and Methods Study design, patients and sample collection From 1996 to 2013, 51 unrelated Korean children with aHUS were prospectively collected from
different medical centers throughout South Korea, including three previously reported patients with anti-CFH-HUS28 and three with CFH mutations29-31. For this study, we reviewed the clinical and laboratory data of the patients and enrolled those who presented with the triad: microangiopathic anemia, thrombocytopenia and acute kidney injury (i.e., serum creatinine levels greater than age-related norms) and were under the age of 18 at the time of onset. We excluded cases of typical HUS followed by verotoxin-producing E.coli infection, HUS associated with pneumococcal infection and other secondary cases of HUS. Peripheral blood samples from the patients were obtained during the acute stage of the disease
prior to any plasma therapy. Plasma and genomic DNA were purified and stored at -80°C until
This article is protected by copyright. All rights reserved.
Accepted Article
7
testing. In addition, blood samples were collected from 100 healthy adults (at routine healthcare
examination). Written informed consent was obtained from all of the subjects or their parents, as applicable.
This study was conducted according to the Declaration of Helsinki (2000) and was approved by the Institutional Review Board of Seoul National University Hospital (H-0812-002-264).
Serum complement measurements Serum concentrations of complement 3 (C3) and complement 4 (C4) were measured by
nephelometry at each hospital. The plasma levels of CFH, CFI and CFB were measured using commercial ELISA kits (USCN Life Science Inc., Wuhan, China).
Targeted exome sequencing and mutational Analyses Targeted exome sequencing covering 46 complement related genes (Supplemental Table 1) was
performed at Samsung Genome Institute. Genomic DNA was captured by the customized SureSelect enrichment system (Agilent, Santa Clara, CA) against 46 aHUS-associated genes and sequenced using MiSeq (Illumina, San Diego, CA). Reads were aligned to the human genome reference sequence (hg19) using BWA-v0.7.5 with the ‘MEM’ algorithm (default settings). SAMTOOLS v0.1.18, GATK v2.4-7 and Picard v1.93 were used for processing SAM/BAM files, local realignment, base recalibration, and duplicate marking. Variants were called by Unified Genotyper in GATK and were also recalibrated by GATK. The Perl script offered by ANNOVAR was used to annotate the variants. To identify causal variants, we firstly selected exonic and splicing variants including non-synonymous variants and small indels. The variants with allele frequency over 1% were discarded based on NHLBI-ESP 6500, 1000 Genome Project, and our
This article is protected by copyright. All rights reserved.
Accepted Article
8
in-house database consisting of exomes of 80 Korean individuals. For prioritization of the candidate variants, we implemented a Pathogenecity Score (PS, Supplemental Table 2 and 3) described in reference11. Briefly, we used each prediction score from SIFT, PolyPhen2, Phylop,
LRT, MutationTaster and GERP++, and characterized the variants using PS, which was the sum of the prediction values from the tools. In case the prediction value of the variants has not been assigned by the tools, we counted the value as ‘0’. The detected mutations were confirmed by traditional Sanger sequencing. In addition, mutations in the THBD and DGKE genes which were not included in the targeted exome sequencing were screened by traditional Sanger sequencing in
all of the patients. The ADAMTS13 gene causing congenital thrombotic thrombocytopenic purpura was also included in this study but not coagulation pathway genes including PLG.
Plasma anti-CFH assay All the patients were tested for plasma anti-CFH IgG using a commercially available enzyme-
linked immunosorbent assay (ELISA) kit (CFH IgG ELISA kit, Abnova, Heidelberg, Germany). The anti-CFH titer was expressed in arbitrary units per mL (AU/mL). Titers greater than five standard deviations above the mean value (>565 AU/mL) of the 100 control subjects were considered positive.
Multiplex ligation-dependent probe amplification (MLPA) assay Large deletions of the CFH, CFHR1, CFHR2 and CFHR3 genes were detected using a
commercial Multiplex ligation dependent probe amplification (MLPA) kit (SALSA MLPA probemix P236-A3 ARMD mix-1, MRC-Holland, Amsterdam, the Netherlands). The probemix included in the kit contained probes covering the region around the CFH gene on chromosome
This article is protected by copyright. All rights reserved.
Accepted Article
9
1q23. Included were 13 probes for the CFH gene, 8 for CFHR3, 5 for CFHR1 and 4 for CFHR2, as well as 5 probes for the flanking genes KCNT2 and CFHR5. For controls, we analyzed 100 healthy Korean adults with no previous history of renal disease, who underwent routine healthcare examinations at Seoul National University Hospital. Written informed consents were obtained from all of the subjects.
Statistical analyses Clinical measurements of the groups were compared by the Mann–Whitney U test for
continuous variables and the chi-square test or Fisher exact test for categorical variables. P values under T
p.Cys926Phe
30
aH-48
CFH
c.3415C>T
p.Gln1139*
31
c.3231T>G
p.Cys1077Trp
aH-49
CFH
c.1064A>C
p.Tyr355Ser
aH-42
CFH
c.2944C>T
p.Pro982Ser
aH-35
CFH
c.3572C>T
p.Ser1191Leu
aH-24
CFH
c.A3593T
p.Glu1198Val
aH-32
CFH
c.3644G>A
p.Arg1215Gln
CFI
c.G1649A
p.Cys550Tyr
aH-25
CFI
c.485G>C
p.Gly162Ala
aH-31
CD46
c.381T>A
p.Cys127*
CD46
c.685C>T
p.Arg229*
aH-36
CD46
c.565T>G
p.Tyr189Asp
aH-41
DGKE
c.790_791insA
p.Thr204Asnfs*4
DGKE
c.501C>G
p.Cys167Trp
This article is protected by copyright. All rights reserved.
32
Accepted Article
27
Table 2. Clinical presentation and laboratory findings of the patients at acute stage
Mean onset age (yr)
P values Group A (n=15)
Group B (n=11)
Group C (n=25)
Total (n=51) Group A vs. B
Group A vs. C
Group B vs. C
8.0 [2.7~13.4]
3.1 [0~4.5]
5.7 [0~14.5]
5.8 [0~14.5]
0.002
0.106
0.080
4 : 11
7:4
15:10
26 : 25
0.109
0.041
1.000
diarrhea
2 (13%)
2 (18%)
8 (32%)
12 (31%)
1.000
0.026
0.142
respiratory infection
2 (13%)
3 (27%)
6 (24%)
11 (22%)
0.620
0.686
1.000
Hypertension
5 (33%)
8 (72%)
11 (44%)
24 (47%)
0.111
0.505
0.112
oligo-anuria
9 (60%)
5 (45%)
13 (52%)
27 (53%)
0.692
0.622
0.717
CNS symptoms
1 (7%)
4 (36%)
7 (24%)
11 (22%)
0.279
0.633
0.650
hepatitis*
1 (7%)
1 (9%)
3 (12%)
5 (10%)
1.000
1.000
1.000
pancreatitis**
1 (7%)
2 (18%)
2 (8%)
5 (10%)
0.556
1.000
0.570
Mean Hb nadir (g/dL)
7.0 [5.4~9.1]
7.8 [4.8~11.8]
7.9 [4.2~12.0]
7.6[4.2~12.0]
0.540
0.399
0.930
Mean PLT nadir (K/uL)
32 [12~82]
42 [9~93]
105 [21~142]
69 [9~142]
0.646
1
Received date: 10/27/2014 Revised date: Accepted date: 11/19/2014 Original Article
Atypical hemolytic uremic syndrome: a Korean pediatric series1
Jiwon M. Lee1, MD; Young Seo Park3, MD, PhD; Joo Hoon Lee3, MD, PhD; Se Jin Park4 , MD, PhD; Jae Il Shin5, MD, PhD; Yong-Hoon Park6, MD, PhD; Kee Hwan Yoo7, MD, PhD; Min Hyun Cho8, MD, PhD; Su-Young Kim9, MD, PhD; Seong Heon Kim9, MD; Mee Kyung
Namgoong10, MD, PhD; Seung Joo Lee11, MD, PhD; Jun Ho Lee12, MD, PhD; Hee Yeon Cho13,
MD, PhD; Kyoung Hee Han14, MD, PhD; Hee Gyung Kang1,2, MD, PhD; Il Soo Ha1, MD, PhD; Jun-Seok Bae16,17, Nayoung K.D. Kim17, PhD; Woong-Yang Park17,18, MD, PhD; Hae Il Cheong1,2,15, MD, PhD
1
Department of Pediatrics, Seoul National University Children’s Hospital, Seoul, Korea
2
Research Coordination Center for Rare Diseases, Seoul National University Hospital, Seoul,
Korea 3
Department of Pediatrics, Asan Medical Center, University of Ulsan, Seoul, Korea
4
Department of Pediatrics, Ajou University School of Medicine, Suwon, Korea
5
Department of Pediatrics, Severance Children’s Hospital, Yonsei University, Seoul, Korea
6
Department of Pediatrics, Yeungnam University College of Medicine, Daegu, Korea
This article has been accepted for publication and undergone full peer review but has not been through the copyed iting, typesetting, pagination and proofreading process, which may lead to differences between this version and th e Version of Record. Please cite this article as doi: 10.1111/ped.12549
This article is protected by copyright. All rights reserved.
Accepted Article
2
7
Department of Pediatrics, Korea University Guro Hospital, Seoul, Korea
8
Department of Pediatrics, Kyungpook National University Hospital, Daegu, Korea
9
Department of Pediatrics, Pusan National University Children’s Hospital, Yangsan, Korea
10
11
Department of Pediatrics, Wonju College of Medicine, Yonsei University, Wonju, Korea
Department of Pediatrics, Ehwa University Mokdong Hospital, Seoul, Korea
12
Department of Pediatrics, Bundang CHA Hospital, Seongnam, Korea
13
Department of Pediatrics, Samsung Medical Center, Seoul, Korea
14
Department of Pediatrics, Jeju University Hospital, Jeju, Korea
15
Kidney Research Institute, Medical Research Center, Seoul National University College of
Medicine, Seoul, Korea 16
Department of Health Sciences and Technology, Samsung Advanced Institute for Health
Sciences and Technology, Sungkyunkwan University, Seoul, Korea
17
Samsung Genome Institute, Samsung Medical Center, Seoul, Korea
18
Sungkyunkwan University School of Medicine, Seoul, Korea
Correspondence: Hae Il Cheong, M.D., Ph.D. Department of Pediatrics Seoul National University Children’s Hospital 101 Daehak-ro, Jongno-gu Seoul, 110-744, Korea Tel 82-2-2072-2810 Fax 82-2-743-3455
This article is protected by copyright. All rights reserved.
Accepted Article
3
E-mail [email protected]
Short title: A Korean pediatric cohort of aHUS
Number of text pages: 10
Number of words: 2950 Reference pages: 5
tables: 5
figures: 3
legends to figures: 3
Abstract
Background: Atypical hemolytic uremic syndrome (aHUS) is a rare disease with a genetic predisposition. Few studies have evaluated the disease in Asian population. We studied a Korean pediatric cohort to delineate the clinical characteristics and genotypes. Methods: A multicenter cohort of 51 Korean children with aHUS was screened for mutations by targeted exome sequencing covering 46 complement related genes. Anti-complement-factor-H autoantibodies (anti-CFH) titers were measured. Multiplex ligation dependent probe amplification assay was performed to detect deletions in the complement factor-H related protein genes (CFHRs). We grouped the patients according to the etiology and compared the clinical
This article is protected by copyright. All rights reserved.
Accepted Article
4
features using the Mann-Whitney U test and chi-square test. Results: Fifteen patients (Group A, 29.7%) had anti-CFH and mutations were detected in 11(Group B, 21.6%) patients, including one with combined mutations. Remaining 25(Group C, 49.0%) were neither–positive. Anti-CFH-association was more frequent than the world-wide prevalence. Group A showed older onset age than Group B, although did not significantly differ in the clinical manifestation. Group B showed worst renal outcome. Gene frequencies of homozygous CFHR1 deletion were 73.3%, 2.7% and 1% in Group A, Group B+C and the control, respectively. Conclusions: In our cohort, we observed a relatively high incidence of anti-CFH-association. Clinical outcomes largely conformed to the previous reports. Although the size is limited, this cohort provides a reassessment of clinicogenetic features of aHUS in Korean children.
Key Words: Anti-complement factor H autoantibody, Atypical hemolytic uremic syndrome, Complement factor H, Asian, Mutation
This article is protected by copyright. All rights reserved.
Accepted Article
5
Introduction Hemolytic uremic syndrome (HUS), characterized by the triad of microangiopathic hemolytic
anemia, thrombocytopenia and acute renal failure, is a microvascular occlusion disorder that belongs to the category of thrombotic microangiopathy (TMA)1-5. HUS may present primarily or due to secondary causes, such as bone marrow transplantation, medication (e.g., anticancer chemotherapy, calcineurin inhibitors and antiplatelet agents), pneumococcal or viral infections and autoimmune diseases6. In primary HUS, the atypical form (aHUS) is distinguished from the typical form by the absence of a prior verotoxin-producing Escherichia coli infection. Primary aHUS can further be divided into subgroups according to the etiology; complement alternative pathway dysregulation1,2,4,7, anti-complement factor H autoantibodies (anti-CFH)8, mutations in the coagulation pathway genes9-11, and also in combination of above12. To date, genetic or acquired abnormalities in the complement system have been documented in nearly 60% of patients with aHUS1,13; including mutations in the complement factor H (CFH)14,15, complement factor I (CFI)16, complement factor B (CFB)17, membrane cofactor protein (MCP/CD46)16,18, and
complement 3 (C3) genes19, as well as in their combinations6,12. More recently identified mutations on the coagulation pathway genes include THBD9, DGKE10, and PLG11. THBD encodes thrombomodulin, which functions as a cofactor for thrombin to reduce blood coagulation and also regulates CFI-induced C3b inactivation9,20. DGKE encodes diacylglycerol
kinase ε and is implicated in regulation of thrombus formation by modulating protein kinase C activity in endothelial cells and platelets9. PLG, a gene in the coagulation pathway encoding plasminogen which is converted into plasmin by a variety of enzymes on binding to clots, has emerged as a novel causative gene in the pathogenesis of aHUS11. Anti-CFH8 is an acquired cause of aHUS frequently presenting in association with homozygous deletion of CFHR1 which
This article is protected by copyright. All rights reserved.
Accepted Article
6
encodes complement factor H-related protein 121-25. Since genetic backgrounds largely underlie in the pathogenesis of aHUS, investigation on the
clinical and genetic characteristics in patients with different ethnic background may be of a clinical value. In fact, genetic variability among racial groups may account for medically important differences in disease outcomes26. In addition, racial disparities have been demonstrated in TMAs in the Oklahoma Thrombotic thrombocytopenic purpura (TTP)-HUS Registry, most recently27. We designed this study to 1) investigate the genetic etiologies and clinical outcomes of aHUS in
a nationwide Korean pediatric cohort, 2) compare the clinical features between the etiologic subgroups, and 3) if any, explore racial disparities in the disease.
Materials and Methods Study design, patients and sample collection From 1996 to 2013, 51 unrelated Korean children with aHUS were prospectively collected from
different medical centers throughout South Korea, including three previously reported patients with anti-CFH-HUS28 and three with CFH mutations29-31. For this study, we reviewed the clinical and laboratory data of the patients and enrolled those who presented with the triad: microangiopathic anemia, thrombocytopenia and acute kidney injury (i.e., serum creatinine levels greater than age-related norms) and were under the age of 18 at the time of onset. We excluded cases of typical HUS followed by verotoxin-producing E.coli infection, HUS associated with pneumococcal infection and other secondary cases of HUS. Peripheral blood samples from the patients were obtained during the acute stage of the disease
prior to any plasma therapy. Plasma and genomic DNA were purified and stored at -80°C until
This article is protected by copyright. All rights reserved.
Accepted Article
7
testing. In addition, blood samples were collected from 100 healthy adults (at routine healthcare
examination). Written informed consent was obtained from all of the subjects or their parents, as applicable.
This study was conducted according to the Declaration of Helsinki (2000) and was approved by the Institutional Review Board of Seoul National University Hospital (H-0812-002-264).
Serum complement measurements Serum concentrations of complement 3 (C3) and complement 4 (C4) were measured by
nephelometry at each hospital. The plasma levels of CFH, CFI and CFB were measured using commercial ELISA kits (USCN Life Science Inc., Wuhan, China).
Targeted exome sequencing and mutational Analyses Targeted exome sequencing covering 46 complement related genes (Supplemental Table 1) was
performed at Samsung Genome Institute. Genomic DNA was captured by the customized SureSelect enrichment system (Agilent, Santa Clara, CA) against 46 aHUS-associated genes and sequenced using MiSeq (Illumina, San Diego, CA). Reads were aligned to the human genome reference sequence (hg19) using BWA-v0.7.5 with the ‘MEM’ algorithm (default settings). SAMTOOLS v0.1.18, GATK v2.4-7 and Picard v1.93 were used for processing SAM/BAM files, local realignment, base recalibration, and duplicate marking. Variants were called by Unified Genotyper in GATK and were also recalibrated by GATK. The Perl script offered by ANNOVAR was used to annotate the variants. To identify causal variants, we firstly selected exonic and splicing variants including non-synonymous variants and small indels. The variants with allele frequency over 1% were discarded based on NHLBI-ESP 6500, 1000 Genome Project, and our
This article is protected by copyright. All rights reserved.
Accepted Article
8
in-house database consisting of exomes of 80 Korean individuals. For prioritization of the candidate variants, we implemented a Pathogenecity Score (PS, Supplemental Table 2 and 3) described in reference11. Briefly, we used each prediction score from SIFT, PolyPhen2, Phylop,
LRT, MutationTaster and GERP++, and characterized the variants using PS, which was the sum of the prediction values from the tools. In case the prediction value of the variants has not been assigned by the tools, we counted the value as ‘0’. The detected mutations were confirmed by traditional Sanger sequencing. In addition, mutations in the THBD and DGKE genes which were not included in the targeted exome sequencing were screened by traditional Sanger sequencing in
all of the patients. The ADAMTS13 gene causing congenital thrombotic thrombocytopenic purpura was also included in this study but not coagulation pathway genes including PLG.
Plasma anti-CFH assay All the patients were tested for plasma anti-CFH IgG using a commercially available enzyme-
linked immunosorbent assay (ELISA) kit (CFH IgG ELISA kit, Abnova, Heidelberg, Germany). The anti-CFH titer was expressed in arbitrary units per mL (AU/mL). Titers greater than five standard deviations above the mean value (>565 AU/mL) of the 100 control subjects were considered positive.
Multiplex ligation-dependent probe amplification (MLPA) assay Large deletions of the CFH, CFHR1, CFHR2 and CFHR3 genes were detected using a
commercial Multiplex ligation dependent probe amplification (MLPA) kit (SALSA MLPA probemix P236-A3 ARMD mix-1, MRC-Holland, Amsterdam, the Netherlands). The probemix included in the kit contained probes covering the region around the CFH gene on chromosome
This article is protected by copyright. All rights reserved.
Accepted Article
9
1q23. Included were 13 probes for the CFH gene, 8 for CFHR3, 5 for CFHR1 and 4 for CFHR2, as well as 5 probes for the flanking genes KCNT2 and CFHR5. For controls, we analyzed 100 healthy Korean adults with no previous history of renal disease, who underwent routine healthcare examinations at Seoul National University Hospital. Written informed consents were obtained from all of the subjects.
Statistical analyses Clinical measurements of the groups were compared by the Mann–Whitney U test for
continuous variables and the chi-square test or Fisher exact test for categorical variables. P values under T
p.Cys926Phe
30
aH-48
CFH
c.3415C>T
p.Gln1139*
31
c.3231T>G
p.Cys1077Trp
aH-49
CFH
c.1064A>C
p.Tyr355Ser
aH-42
CFH
c.2944C>T
p.Pro982Ser
aH-35
CFH
c.3572C>T
p.Ser1191Leu
aH-24
CFH
c.A3593T
p.Glu1198Val
aH-32
CFH
c.3644G>A
p.Arg1215Gln
CFI
c.G1649A
p.Cys550Tyr
aH-25
CFI
c.485G>C
p.Gly162Ala
aH-31
CD46
c.381T>A
p.Cys127*
CD46
c.685C>T
p.Arg229*
aH-36
CD46
c.565T>G
p.Tyr189Asp
aH-41
DGKE
c.790_791insA
p.Thr204Asnfs*4
DGKE
c.501C>G
p.Cys167Trp
This article is protected by copyright. All rights reserved.
32
Accepted Article
27
Table 2. Clinical presentation and laboratory findings of the patients at acute stage
Mean onset age (yr)
P values Group A (n=15)
Group B (n=11)
Group C (n=25)
Total (n=51) Group A vs. B
Group A vs. C
Group B vs. C
8.0 [2.7~13.4]
3.1 [0~4.5]
5.7 [0~14.5]
5.8 [0~14.5]
0.002
0.106
0.080
4 : 11
7:4
15:10
26 : 25
0.109
0.041
1.000
diarrhea
2 (13%)
2 (18%)
8 (32%)
12 (31%)
1.000
0.026
0.142
respiratory infection
2 (13%)
3 (27%)
6 (24%)
11 (22%)
0.620
0.686
1.000
Hypertension
5 (33%)
8 (72%)
11 (44%)
24 (47%)
0.111
0.505
0.112
oligo-anuria
9 (60%)
5 (45%)
13 (52%)
27 (53%)
0.692
0.622
0.717
CNS symptoms
1 (7%)
4 (36%)
7 (24%)
11 (22%)
0.279
0.633
0.650
hepatitis*
1 (7%)
1 (9%)
3 (12%)
5 (10%)
1.000
1.000
1.000
pancreatitis**
1 (7%)
2 (18%)
2 (8%)
5 (10%)
0.556
1.000
0.570
Mean Hb nadir (g/dL)
7.0 [5.4~9.1]
7.8 [4.8~11.8]
7.9 [4.2~12.0]
7.6[4.2~12.0]
0.540
0.399
0.930
Mean PLT nadir (K/uL)
32 [12~82]
42 [9~93]
105 [21~142]
69 [9~142]
0.646
