Acta Diabetol DOI 10.1007/s00592-014-0687-7
ORIGINAL ARTICLE
Exome sequencing identifies novel ApoB loss-of-function mutations causing hypobetalipoproteinemia in type 1 diabetes Feng Gao • Hao Luo • Zhiyao Fu • Chun-Ting Zhang Ren Zhang
•
Received: 3 October 2014 / Accepted: 14 November 2014 Ó Springer-Verlag Italia 2014
Abstract Aim Diabetic patients commonly suffer from disturbances in production and clearance of plasma lipoproteins, known as diabetic dyslipidemia, resulting in an increased risk of coronary heart disease. The study aimed to examine the cause of hypobetalipoproteinemia in two patients with type 1 diabetes. Methods The Diabetes Control and Complications Trial (DCCT) is a study demonstrating that intensive blood glucose control delays the onset and progression of type 1 diabetes complications. Hypobetalipoproteinemia was present in two DCCT subjects, IDs 1427 and 1078, whose LDL-C levels were 36 and 28 mg/dL, respectively, and triglyceride levels were 20 and 28 mg/dL, respectively. We performed exome sequencing on genomic DNA from the two patients with hypobetalipoproteinemia. Results The subjects 1427 and 1078 had heterozygous loss-of-function mutations in the gene apolipoprotein B (ApoB), and these mutations resulted in premature stop codons at amino acid 1333 (ApoB-29) and 3680 (ApoB81), respectively. Indeed, the plasma ApoB level of subject 1427 (19 mg/dL) was the lowest and that of subject 1078 (26 mg/dL) was the second to the lowest among all the 1,441 DCCT participants. Sequencing genomic DNA of family members showed that probands 1427 and 1078
inherited the mutations from the father and the mother, respectively. Conclusions The identification of ApoB loss-of-function mutations in type 1 diabetic patients presents innovative cases to study the interaction between hypobetalipoproteinemia and insulin deficiency. Keywords ApoB DCCT Hypobetalipoproteinemia Type 1 diabetes Abbreviations ApoB Apolipoprotein B BMI Body mass index DCCT Diabetes control and complications trial EDIC Epidemiology of diabetes interventions and complications FHBL Familial hypobetalipoproteinemia HDL-C High-density lipoprotein cholesterol LDL Low-density lipoprotein LDL-C Low-density lipoprotein cholesterol Lp(a) Lipoprotein (a) NIDDK National Institute of Diabetes and Digestive and Kidney diseases SNP Single nucleotide polymorphism TAG Triacylglycerol
Managed by Massimo Porta.
Introduction F. Gao H. Luo C.-T. Zhang (&) Department of Physics, Tianjin University, Tianjin, China e-mail: [email protected] Z. Fu R. Zhang (&) Center for Molecular Medicine and Genetics, and the Cardiovascular Research Institute, School of Medicine, Wayne State University, Detroit, MI, USA e-mail: [email protected]
Lipoproteins are apolipoprotein–lipid complexes, which transport triacylglycerols (TAGs) and cholesterol through the lymphatic and circulatory systems. Chylomicrons and low-density lipoprotein (LDL) are lipoprotein particles that consist mainly of TAGs and cholesterols, respectively [1– 4]. Apolipoprotein B (ApoB), as the main apolipoprotein
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on chylomicrons and LDL, plays a pivotal role in the metabolism of lipids, especially TAGs and cholesterols. ApoB circulates in the plasma in two forms, ApoB-48 and ApoB-100, and the former and the latter are synthesized by the intestine and the liver, respectively [5–7]. In humans, ApoB loss-of-function mutations cause familial hypobetalipoproteinemia (FHBL), a condition characterized by lowdensity lipoprotein cholesterol (LDL-C) levels in the lowest fifth percentile (90 mg/dL) [8–12]. Both type 1 and type 2 diabetes are associated with an increased risk of coronary heart disease; for instance, more than 65 % of people with diabetes die from heart disease or stroke, representing the first cause of death in patients with diabetes [13–15]. Patients with diabetes commonly suffer from disturbances in production and clearance of plasma lipoproteins [13], known as diabetic dyslipidemia, which is characterized by increased TAGs, reduced high-density lipoprotein cholesterol (HDL-C), and postprandial lipemia [13, 16–18]. Correcting lipid abnormalities in diabetic patients has the potential to reduce cardiovascular complications in diabetes. The diabetes control and complications trial (DCCT) is a prominent study showing that intensive blood glucose control delays the onset and progression of type 1 diabetes complications [19–21]. The DCCT had 1,441 participants, and all of them had type 1 diabetes. The participants were divided into two groups—standard versus intensive control of blood glucose. Although both groups received insulin treatment, the intensive control group’s hemoglobin A1c levels were kept as close as possible to the normal value [19, 20]. Although diabetes is often associated with diabetic dyslipidemia, to the best of our knowledge cases of hypobetalipoproteinemia in type 1 diabetes have not been reported. From the DCCT participants, we identified two subjects who had consistent, extremely low levels of TAG and LDL-C. We then performed exome sequencing and identified loss-of-function mutations resulting in premature stop codons in ApoB in both subjects. This finding represents novel cases of hypobetalipoproteinemia in type 1 diabetes.
Materials and methods The DCCT was a multi-center clinical study involving 1,441 participants with type 1 diabetes in the United States and Canada. The participants were randomly divided into two groups, standard group versus experimental group, where the latter received more insulin to have hemoglobin A1C levels more tightly controlled [19, 20]. Clinical characteristics and genomic DNA were obtained from the National Institute of Diabetes and Digestive and Kidney
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Diseases (NIDDK) data repository. Exome sequencing was performed by Axeq Technologies (Rockville, MD) with SureSelect Target Enrichment Solutions (Agilent Technologies, Santa Clara, CA). For sequencing the exomes of patient 1427, the mappable read number was 39,416,432 (99.8 % out of total reads), with an average throughput depth of 78.0. For sequencing the exomes of the patient 1078, the mappable read number was 36,468,420 (99.8 % out of total reads), with an average throughput depth of 72.1. Sequencing reads were mapped against University of California Santa Cruz hg19 with BWA [22], and single nucleotide polymorphisms (SNPs) and indels were detected by SAMtools [23]. Mutated regions were amplified by polymerase chain reaction (PCR) with primers below: apob-1-f, 50 -GG CAAATCCTCCAGAGATCTAAA-30 ; apob-1-r, 50 -AGT CAGCCTTCATGTGGTAAC-30 ; apob-2-f, 50 -CCGGATT CATTCTGGGTCTT-30 ; apob-2-r, 50 -CACGAAGATGC TGTCTCCTAC-30 . Amplified PCR fragments were purified from gel with gel purification kit (Qiagen, Hilden, Germany) and sequenced by the Sanger sequencing method (Genewiz Inc., South Plainfield, NJ). The study protocol was approved by the Institutional Review Boards of Wayne State University School of Medicine and the Detroit Medical Center, and all studies were carried out in accordance with the approved guidelines.
Results Lipid profiles of two subjects with hypobetalipoproteinemia The DCCT was conducted from 1983 to 1993, and upon completion, more than 90 % of participants continued to be recruited into the follow-up study, the epidemiology of diabetes interventions and complications (EDIC) study [24–27]. Therefore, the baseline data for the DCCT and EDIC studies was about 10 years apart. Because there are variations when measuring lipid profiles even in the same subjects, we identified subjects who had extremely low lipid levels at baselines of both the DCCT and EDIC studies. The subject 1427 was a 28-year-old male with a BMI of 22.62 at the time of recruitment into the DCCT study. The TAG levels were 20 and 14 mg/dL at the DCCT and EDIC baselines, respectively. At both the DCCT and EDIC baselines, subject 1427’s TAG level ranked the lowest among all participants. The LDL-C level of the subject 1427 was 36 and 30 mg/dL at DCCT and EDIC baselines, respectively, and ranked 6th lowest and 2nd lowest in DCCT and EDIC, respectively (Fig. 1). Total cholesterol
Acta Diabetol Fig. 1 Distribution of triacylglycerol and LDL-C levels among DCCT/EDIC participants and positions of subjects 1427 and 1078. Distribution of triacylglycerol levels at a DCCT and b EDIC baselines. Distribution of LDLC levels at c DCCT and d EDIC baselines. Measurement of blood parameters were performed at the DCCT baseline and 10 years later at the EDIC baseline. The patients 1427 and 1078 persistently had extremely low triglyceride and LDL-C levels. Arrows indicate the position of the two subjects
Table 1 Clinical characteristics of the two patients with ApoB loss-of-function mutations and percentile ranks among DCCT/EDIC participants IDa 1427
IDa 1078
DCCT Value Age
28
Sex
Male
BMI (kg/m2) TAG (mg/dL)
22.62 20
EDIC PR
Value
DCCT PR
Value
EDIC PR
Value
PR
16 Male 42.16 0.07
22.54 14
15.47 0.07
24.70 28
69.76 0.49
27.56 17
64.52 0.14
CHOL (mg/dL)
103
0.49
87
0.07
115
2.08
118
1.12
HDL-C (mg/dL)
63
82.94
54
49.06
81
97.99
70
80.34
30
45
LDL-C (mg/dL)
36
0.42
HbA1c (%)
8.8
52.43
6.9
0.14
28
0.14
12.74
8.4
33.91
8.1
0.49 47.45
CHOL, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; PR, percentile rank; TAG, triacylglycerol a
ID is identification of masked patient ID, MASK_PAT in the DCCT/EDIC data file. DCCT and EDIC have 1,442 and 1,429 participants, respectively
levels were also low at 103 and 87 mg/dL at DCCT and EDIC, respectively, while HDL-C levels were normal (Table 1). The subject 1078 was a 16-year-old male with a BMI of 24.70 at the DCCT baseline. The TAG levels were 28 and 17 mg/dl at the DCCT and EDIC baseline, respectively. At the DCCT and EDIC baselines, the TAG levels ranked 7th and 2nd lowest. The LDL-C levels of subject 1078 were 28 and 45 mg/dL at the DCCT and EDIC baselines,
respectively, and ranked 2nd lowest and 7th lowest in DCCT and EDIC, respectively (Fig. 1). The total cholesterol levels were 115 and 118 mg/dL, which tended to be low, ranked 2 and 1 %, respectively, in the DCCT and EDIC, while HDL-C levels were normal (Table 1). Therefore, the two subjects consistently measured extremely low in TAG and LDL-C levels, with normal HDL-C levels. Hypobetalipoproteinemia is defined as a condition with a LDL-C level in the lowest fifth percentile,
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corresponding to 90 mg/dL [12]. Therefore, the two subjects clearly were affected with hypobetalipoproteinemia.
Table 3 Loss-of-function mutations of ApoB in two patients with type 1 diabetes Patient ID
Exome sequencing of genomic DNA from the two hypobetalipoproteinemia subjects We hypothesized that the extremely low TAG and LDL-C levels in subjects 1427 and 1078 were due to single genemediated Mendelian disorders. Exome sequencing is a powerful approach to identify variations in protein-coding genes, and therefore, we performed exome sequencing using genomic DNA of the two subjects. One lg of genomic DNA was used for exome sequencing. The numbers of sequencing reads were 39,507,012 and 36,559,302 for subjects 1427 and 1078. For both subjects, 99.8 % out of the total reads could be mapped onto the human genome. The mean read depth of protein-coding regions were 49.7 and 46.5 for 1427 and 1078, respectively. There were 61,199 and 61,287 SNPs in 1427 and 1078, among which 31 % are in coding regions. Among coding SNPs, there were 8,753 and 8,759 nonsynonymous SNPs for subjects 1427 and 1078, respectively (Table 2). Both subjects had mutations that led to premature stop codons in the ApoB gene. Novel loss-of-function mutations in ApoB The gene ApoB has 29 exons, and subjects 1427 and 1078 had mutations in exon 25 and 26, respectively. The subject 1427 had a C to T mutation in chromosome 2, position 21013379. This mutation is at position 4125 of the coding sequence, leading to the codon CGA changed to TGA, a stop codon at the amino acid of 1333. Subject 1078 had a T Table 2 Descriptive statistics of the exome sequencing output Patient ID
1427
1078
Total reads
39,507,012
36,559,302
Total yield (bp)
3,990,208,212
3,692,489,502
Read length (bp)
101
101
Average throughput depth of target regions Reads mapped to human genome
78
72.1
39,416,432
36,468,420
Reads uniquely mapped to human genome
37,271,762
34,546,263
On-target reads (mapped to target regions)
29,871,877
27,867,334
Mean read depth of target regions
49.7
46.5
No. of SNPs
61,199
61,287
No. of coding SNPs
19,098
19,274
No. of synonymous SNPs No. of nonsynonymous SNPs
9,915 8,753
10,057 8,759
BP, base pair; Patient ID, MAST_PAT in the DCCT/EDIC dataset; SNP, single nucleotide polymorphism
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1427
1078
Alleles
C/T
T/G
Wild type
C
T
Mutant
T
G
Coordinates
Chr 2: 21013379
Chr 2: 21228700
Position in transcript Position in CDS
4125 3997
11168 11040
Alleles
Position
Exon
25
26
Position in exon
155
6824
Position in protein
1333
3680
Consequence
Stop gain
Stop gain
Codons
CGA ? TGA
TAT ? TAG
Amino acids
Arg ? Ter
Tyr ? Ter
Consequence
The human genome assembly version GRCh37 was used CDS, coding sequence; Chr, chromosome; Patient ID, MAST_PAT in the DCCT/EDIC dataset; Ter, stop codon
to G mutation in chromosome 2, position 21228700. This mutation is at position 11040 of the coding sequence, leading to the codon TAT changed to TAG, a stop codon at the amino acid of 3680 (Table 3). To confirm the exome sequencing result, we performed Sanger sequencing on DNA fragment amplified by PCR. The primer pair apob-1 amplifies a 274-bp fragment containing the mutation in subject 1427, and apob-2 amplifies a 200-bp fragment containing the mutation in subject 1078. Indeed, subjects 1047 and 1078 both had a wild-type allele and a mutant allele; therefore, both had heterozygous mutations (Fig. 2). The premature stop codon in subjects 1427 and 1078 resulted in truncation of the ApoB protein in which only 29 and 81 % was retained. Consequently, according to the centile nomenclature [12], the ApoB variants in subjects 1427 and 1078 was named ApoB-29 and ApoB-81, respectively. Extremely low plasma ApoB levels in the two subjects with ApoB mutations Because subjects 1427 and 1078 had heterozygous mutations resulting in premature codons in ApoB, we hypothesized that the plasma levels of ApoB were low in these two patients. The lipid profiles including ApoB and Lipoprotein (a) [Lp(a)] were measured during the DCCT [19, 27, 28]. The ApoB levels of subjects 1427 and 1078 were 19 and 26 mg/ dL, respectively. The ApoB levels of all the DCCT subjects were normally distributed, and subjects 1427 and 1078 were
Acta Diabetol Fig. 2 Results of Sanger sequencing of the affected regions. Chromatogram for sequencing wild-type alleles (a, b), and for sequencing corresponding mutations at positions c 3997 and d 11040 in ApoB
Fig. 3 The pedigree chart for probands 1427 and 1078. a The father of proband 1427 carried the mutation, while the mother and a male sibling were not affected. b Both the mother and a female sibling of proband 1078 carried the mutation. The genomic DNA of the father was unavailable
the lowest and second to the lowest in ApoB levels among all participants. The distribution of Lp(a) was skewed, and in contrast to ApoB, the Lp(a) levels were ranked 7 and 46 % among all the DCCT participants. Therefore, consistent with loss-of-function mutations in ApoB, subjects 1427 and 1078 had extremely low levels of plasma ApoB. Pedigree of the affected ApoB family members We examined the corresponding genomic DNA from family members of probands 1427 and 1078. For 1427, we obtained genomic DNA from his father, mother, and a male sibling. For 1078, we obtained genomic DNA from his mother and a female sibling. Then, we performed PCR to amply the genomic fragment using the primer pairs of
apob-1 and apob-2. Purified fragments were then sequenced by Sanger sequencing. Proband 1427’s mother carried two wild-type alleles, and his father was heterozygous for the mutation. His male sibling had two wild-type alleles. Therefore, proband 1427 inherited one mutant allele from his father. Both proband 1078’s mother and female sibling were heterozygous for the mutant alleles; thus, he inherited the mutant allele from his mother (Fig. 3).
Discussion Sequencing technology has seen dramatic progress in the past decades, in terms of cost, dependability, and speed
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[29–31]. It is likely that in the near future, sequencing the entire human genome could cost less than $1,000 and take only hours. But currently whole genome sequencing is still impractical for clinical use due to the expense, technical obstacles, and subsequent sequence analysis. The goal of exome sequencing is to only sequence the protein-coding genes in a genome [32–34]. By focusing only on exons, sequencing effort is greatly reduced. Exons constitute about 1 % (about 30 Mb) of the human genome, but disease-causing mutations in exons are much more likely than in other parts of the human genome, such as introns and repeat elements [32–34]. Exome sequencing was used in this study to reliably identify loss-of-function mutations in two hypobetalipoproteinemia subjects. The low cost, reliability, and high speed in sequencing exons make this technique to have a deep impact on clinical use, medical research, and the biotechnology industry. There have been some reports on hypobetalipoproteinemia in type 2 diabetes [35–39]; nevertheless, to the best of our knowledge, cases of hypobetalipoproteinemia caused by ApoB loss-of-function mutations in type 1 diabetes have not been reported. Most patients with FHBL are caused by mutations in ApoB. These patients often present with nonalcoholic fatty liver disease due to reduced capacity to export lipids from the liver into the circulation [8, 40]. The unavailability of liver phenotypes and parameters of the two patients is one limitation of the current study. Judging by their extremely low levels of TAG and LDL-C, we hypothesize that the two patients likely have fatty liver, which, in more severe cases, may progress to cirrhosis. Another limitation is although we identified the mutations in family members of the two probands, the lipid phenotypes of the affected family members were not available. It is likely that affected family members present with similar patterns of lipid profiles as the two probands. In the two patients with ApoB mutations, the presence of type 1 diabetes did not mask hypobetalipoproteinemia, and therefore, it would be interesting to examine whether diabetic patients with hypobetalipoproteinemia are protected from diabetic dyslipidemia and its associated cardiovascular compilations. In summary, we have identified novel heterozygous ApoB loss-of-function mutations that cause hypobetalipoproteinemia in two DCCT participants (DCCT ID: 1427 and 1078), who exhibited extremely low levels of both TAG and LDL-C. These mutations cause premature stop codons at ApoB amino acid 1333 (ApoB-29) and 3680 (ApoB-81), and indeed, among all DCCT participants, the lowest circulating ApoB levels were found in these two patients. The identification of ApoB loss-of-function mutations in two type 1 diabetic patients presents innovative cases to study the interaction between hypobetalipoproteinemia and insulin deficiency.
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Acknowledgments The current study was supported in part by a fund from Wayne State University to R.Z., NNSF of China (90408028) to C.T.Z. and (31171238) to F.G. and the China National 863 High-Tech Program (2015AA020101) to F.G. Conflict of interest Feng Gao, Hao Luo, Zhiyao Fu, Chun-Ting Zhang and Ren Zhang declare that they have no conflict of interest. Human and Animal Rights disclosure All procedures followed were in accordance with the ethical standards of the Institutional Review Boards of Wayne State University School of Medicine and of the Detroit Medical Center, and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent disclosure Informed consent was obtained from all participants being included in the study.
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ORIGINAL ARTICLE
Exome sequencing identifies novel ApoB loss-of-function mutations causing hypobetalipoproteinemia in type 1 diabetes Feng Gao • Hao Luo • Zhiyao Fu • Chun-Ting Zhang Ren Zhang
•
Received: 3 October 2014 / Accepted: 14 November 2014 Ó Springer-Verlag Italia 2014
Abstract Aim Diabetic patients commonly suffer from disturbances in production and clearance of plasma lipoproteins, known as diabetic dyslipidemia, resulting in an increased risk of coronary heart disease. The study aimed to examine the cause of hypobetalipoproteinemia in two patients with type 1 diabetes. Methods The Diabetes Control and Complications Trial (DCCT) is a study demonstrating that intensive blood glucose control delays the onset and progression of type 1 diabetes complications. Hypobetalipoproteinemia was present in two DCCT subjects, IDs 1427 and 1078, whose LDL-C levels were 36 and 28 mg/dL, respectively, and triglyceride levels were 20 and 28 mg/dL, respectively. We performed exome sequencing on genomic DNA from the two patients with hypobetalipoproteinemia. Results The subjects 1427 and 1078 had heterozygous loss-of-function mutations in the gene apolipoprotein B (ApoB), and these mutations resulted in premature stop codons at amino acid 1333 (ApoB-29) and 3680 (ApoB81), respectively. Indeed, the plasma ApoB level of subject 1427 (19 mg/dL) was the lowest and that of subject 1078 (26 mg/dL) was the second to the lowest among all the 1,441 DCCT participants. Sequencing genomic DNA of family members showed that probands 1427 and 1078
inherited the mutations from the father and the mother, respectively. Conclusions The identification of ApoB loss-of-function mutations in type 1 diabetic patients presents innovative cases to study the interaction between hypobetalipoproteinemia and insulin deficiency. Keywords ApoB DCCT Hypobetalipoproteinemia Type 1 diabetes Abbreviations ApoB Apolipoprotein B BMI Body mass index DCCT Diabetes control and complications trial EDIC Epidemiology of diabetes interventions and complications FHBL Familial hypobetalipoproteinemia HDL-C High-density lipoprotein cholesterol LDL Low-density lipoprotein LDL-C Low-density lipoprotein cholesterol Lp(a) Lipoprotein (a) NIDDK National Institute of Diabetes and Digestive and Kidney diseases SNP Single nucleotide polymorphism TAG Triacylglycerol
Managed by Massimo Porta.
Introduction F. Gao H. Luo C.-T. Zhang (&) Department of Physics, Tianjin University, Tianjin, China e-mail: [email protected] Z. Fu R. Zhang (&) Center for Molecular Medicine and Genetics, and the Cardiovascular Research Institute, School of Medicine, Wayne State University, Detroit, MI, USA e-mail: [email protected]
Lipoproteins are apolipoprotein–lipid complexes, which transport triacylglycerols (TAGs) and cholesterol through the lymphatic and circulatory systems. Chylomicrons and low-density lipoprotein (LDL) are lipoprotein particles that consist mainly of TAGs and cholesterols, respectively [1– 4]. Apolipoprotein B (ApoB), as the main apolipoprotein
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on chylomicrons and LDL, plays a pivotal role in the metabolism of lipids, especially TAGs and cholesterols. ApoB circulates in the plasma in two forms, ApoB-48 and ApoB-100, and the former and the latter are synthesized by the intestine and the liver, respectively [5–7]. In humans, ApoB loss-of-function mutations cause familial hypobetalipoproteinemia (FHBL), a condition characterized by lowdensity lipoprotein cholesterol (LDL-C) levels in the lowest fifth percentile (90 mg/dL) [8–12]. Both type 1 and type 2 diabetes are associated with an increased risk of coronary heart disease; for instance, more than 65 % of people with diabetes die from heart disease or stroke, representing the first cause of death in patients with diabetes [13–15]. Patients with diabetes commonly suffer from disturbances in production and clearance of plasma lipoproteins [13], known as diabetic dyslipidemia, which is characterized by increased TAGs, reduced high-density lipoprotein cholesterol (HDL-C), and postprandial lipemia [13, 16–18]. Correcting lipid abnormalities in diabetic patients has the potential to reduce cardiovascular complications in diabetes. The diabetes control and complications trial (DCCT) is a prominent study showing that intensive blood glucose control delays the onset and progression of type 1 diabetes complications [19–21]. The DCCT had 1,441 participants, and all of them had type 1 diabetes. The participants were divided into two groups—standard versus intensive control of blood glucose. Although both groups received insulin treatment, the intensive control group’s hemoglobin A1c levels were kept as close as possible to the normal value [19, 20]. Although diabetes is often associated with diabetic dyslipidemia, to the best of our knowledge cases of hypobetalipoproteinemia in type 1 diabetes have not been reported. From the DCCT participants, we identified two subjects who had consistent, extremely low levels of TAG and LDL-C. We then performed exome sequencing and identified loss-of-function mutations resulting in premature stop codons in ApoB in both subjects. This finding represents novel cases of hypobetalipoproteinemia in type 1 diabetes.
Materials and methods The DCCT was a multi-center clinical study involving 1,441 participants with type 1 diabetes in the United States and Canada. The participants were randomly divided into two groups, standard group versus experimental group, where the latter received more insulin to have hemoglobin A1C levels more tightly controlled [19, 20]. Clinical characteristics and genomic DNA were obtained from the National Institute of Diabetes and Digestive and Kidney
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Diseases (NIDDK) data repository. Exome sequencing was performed by Axeq Technologies (Rockville, MD) with SureSelect Target Enrichment Solutions (Agilent Technologies, Santa Clara, CA). For sequencing the exomes of patient 1427, the mappable read number was 39,416,432 (99.8 % out of total reads), with an average throughput depth of 78.0. For sequencing the exomes of the patient 1078, the mappable read number was 36,468,420 (99.8 % out of total reads), with an average throughput depth of 72.1. Sequencing reads were mapped against University of California Santa Cruz hg19 with BWA [22], and single nucleotide polymorphisms (SNPs) and indels were detected by SAMtools [23]. Mutated regions were amplified by polymerase chain reaction (PCR) with primers below: apob-1-f, 50 -GG CAAATCCTCCAGAGATCTAAA-30 ; apob-1-r, 50 -AGT CAGCCTTCATGTGGTAAC-30 ; apob-2-f, 50 -CCGGATT CATTCTGGGTCTT-30 ; apob-2-r, 50 -CACGAAGATGC TGTCTCCTAC-30 . Amplified PCR fragments were purified from gel with gel purification kit (Qiagen, Hilden, Germany) and sequenced by the Sanger sequencing method (Genewiz Inc., South Plainfield, NJ). The study protocol was approved by the Institutional Review Boards of Wayne State University School of Medicine and the Detroit Medical Center, and all studies were carried out in accordance with the approved guidelines.
Results Lipid profiles of two subjects with hypobetalipoproteinemia The DCCT was conducted from 1983 to 1993, and upon completion, more than 90 % of participants continued to be recruited into the follow-up study, the epidemiology of diabetes interventions and complications (EDIC) study [24–27]. Therefore, the baseline data for the DCCT and EDIC studies was about 10 years apart. Because there are variations when measuring lipid profiles even in the same subjects, we identified subjects who had extremely low lipid levels at baselines of both the DCCT and EDIC studies. The subject 1427 was a 28-year-old male with a BMI of 22.62 at the time of recruitment into the DCCT study. The TAG levels were 20 and 14 mg/dL at the DCCT and EDIC baselines, respectively. At both the DCCT and EDIC baselines, subject 1427’s TAG level ranked the lowest among all participants. The LDL-C level of the subject 1427 was 36 and 30 mg/dL at DCCT and EDIC baselines, respectively, and ranked 6th lowest and 2nd lowest in DCCT and EDIC, respectively (Fig. 1). Total cholesterol
Acta Diabetol Fig. 1 Distribution of triacylglycerol and LDL-C levels among DCCT/EDIC participants and positions of subjects 1427 and 1078. Distribution of triacylglycerol levels at a DCCT and b EDIC baselines. Distribution of LDLC levels at c DCCT and d EDIC baselines. Measurement of blood parameters were performed at the DCCT baseline and 10 years later at the EDIC baseline. The patients 1427 and 1078 persistently had extremely low triglyceride and LDL-C levels. Arrows indicate the position of the two subjects
Table 1 Clinical characteristics of the two patients with ApoB loss-of-function mutations and percentile ranks among DCCT/EDIC participants IDa 1427
IDa 1078
DCCT Value Age
28
Sex
Male
BMI (kg/m2) TAG (mg/dL)
22.62 20
EDIC PR
Value
DCCT PR
Value
EDIC PR
Value
PR
16 Male 42.16 0.07
22.54 14
15.47 0.07
24.70 28
69.76 0.49
27.56 17
64.52 0.14
CHOL (mg/dL)
103
0.49
87
0.07
115
2.08
118
1.12
HDL-C (mg/dL)
63
82.94
54
49.06
81
97.99
70
80.34
30
45
LDL-C (mg/dL)
36
0.42
HbA1c (%)
8.8
52.43
6.9
0.14
28
0.14
12.74
8.4
33.91
8.1
0.49 47.45
CHOL, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; PR, percentile rank; TAG, triacylglycerol a
ID is identification of masked patient ID, MASK_PAT in the DCCT/EDIC data file. DCCT and EDIC have 1,442 and 1,429 participants, respectively
levels were also low at 103 and 87 mg/dL at DCCT and EDIC, respectively, while HDL-C levels were normal (Table 1). The subject 1078 was a 16-year-old male with a BMI of 24.70 at the DCCT baseline. The TAG levels were 28 and 17 mg/dl at the DCCT and EDIC baseline, respectively. At the DCCT and EDIC baselines, the TAG levels ranked 7th and 2nd lowest. The LDL-C levels of subject 1078 were 28 and 45 mg/dL at the DCCT and EDIC baselines,
respectively, and ranked 2nd lowest and 7th lowest in DCCT and EDIC, respectively (Fig. 1). The total cholesterol levels were 115 and 118 mg/dL, which tended to be low, ranked 2 and 1 %, respectively, in the DCCT and EDIC, while HDL-C levels were normal (Table 1). Therefore, the two subjects consistently measured extremely low in TAG and LDL-C levels, with normal HDL-C levels. Hypobetalipoproteinemia is defined as a condition with a LDL-C level in the lowest fifth percentile,
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corresponding to 90 mg/dL [12]. Therefore, the two subjects clearly were affected with hypobetalipoproteinemia.
Table 3 Loss-of-function mutations of ApoB in two patients with type 1 diabetes Patient ID
Exome sequencing of genomic DNA from the two hypobetalipoproteinemia subjects We hypothesized that the extremely low TAG and LDL-C levels in subjects 1427 and 1078 were due to single genemediated Mendelian disorders. Exome sequencing is a powerful approach to identify variations in protein-coding genes, and therefore, we performed exome sequencing using genomic DNA of the two subjects. One lg of genomic DNA was used for exome sequencing. The numbers of sequencing reads were 39,507,012 and 36,559,302 for subjects 1427 and 1078. For both subjects, 99.8 % out of the total reads could be mapped onto the human genome. The mean read depth of protein-coding regions were 49.7 and 46.5 for 1427 and 1078, respectively. There were 61,199 and 61,287 SNPs in 1427 and 1078, among which 31 % are in coding regions. Among coding SNPs, there were 8,753 and 8,759 nonsynonymous SNPs for subjects 1427 and 1078, respectively (Table 2). Both subjects had mutations that led to premature stop codons in the ApoB gene. Novel loss-of-function mutations in ApoB The gene ApoB has 29 exons, and subjects 1427 and 1078 had mutations in exon 25 and 26, respectively. The subject 1427 had a C to T mutation in chromosome 2, position 21013379. This mutation is at position 4125 of the coding sequence, leading to the codon CGA changed to TGA, a stop codon at the amino acid of 1333. Subject 1078 had a T Table 2 Descriptive statistics of the exome sequencing output Patient ID
1427
1078
Total reads
39,507,012
36,559,302
Total yield (bp)
3,990,208,212
3,692,489,502
Read length (bp)
101
101
Average throughput depth of target regions Reads mapped to human genome
78
72.1
39,416,432
36,468,420
Reads uniquely mapped to human genome
37,271,762
34,546,263
On-target reads (mapped to target regions)
29,871,877
27,867,334
Mean read depth of target regions
49.7
46.5
No. of SNPs
61,199
61,287
No. of coding SNPs
19,098
19,274
No. of synonymous SNPs No. of nonsynonymous SNPs
9,915 8,753
10,057 8,759
BP, base pair; Patient ID, MAST_PAT in the DCCT/EDIC dataset; SNP, single nucleotide polymorphism
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1427
1078
Alleles
C/T
T/G
Wild type
C
T
Mutant
T
G
Coordinates
Chr 2: 21013379
Chr 2: 21228700
Position in transcript Position in CDS
4125 3997
11168 11040
Alleles
Position
Exon
25
26
Position in exon
155
6824
Position in protein
1333
3680
Consequence
Stop gain
Stop gain
Codons
CGA ? TGA
TAT ? TAG
Amino acids
Arg ? Ter
Tyr ? Ter
Consequence
The human genome assembly version GRCh37 was used CDS, coding sequence; Chr, chromosome; Patient ID, MAST_PAT in the DCCT/EDIC dataset; Ter, stop codon
to G mutation in chromosome 2, position 21228700. This mutation is at position 11040 of the coding sequence, leading to the codon TAT changed to TAG, a stop codon at the amino acid of 3680 (Table 3). To confirm the exome sequencing result, we performed Sanger sequencing on DNA fragment amplified by PCR. The primer pair apob-1 amplifies a 274-bp fragment containing the mutation in subject 1427, and apob-2 amplifies a 200-bp fragment containing the mutation in subject 1078. Indeed, subjects 1047 and 1078 both had a wild-type allele and a mutant allele; therefore, both had heterozygous mutations (Fig. 2). The premature stop codon in subjects 1427 and 1078 resulted in truncation of the ApoB protein in which only 29 and 81 % was retained. Consequently, according to the centile nomenclature [12], the ApoB variants in subjects 1427 and 1078 was named ApoB-29 and ApoB-81, respectively. Extremely low plasma ApoB levels in the two subjects with ApoB mutations Because subjects 1427 and 1078 had heterozygous mutations resulting in premature codons in ApoB, we hypothesized that the plasma levels of ApoB were low in these two patients. The lipid profiles including ApoB and Lipoprotein (a) [Lp(a)] were measured during the DCCT [19, 27, 28]. The ApoB levels of subjects 1427 and 1078 were 19 and 26 mg/ dL, respectively. The ApoB levels of all the DCCT subjects were normally distributed, and subjects 1427 and 1078 were
Acta Diabetol Fig. 2 Results of Sanger sequencing of the affected regions. Chromatogram for sequencing wild-type alleles (a, b), and for sequencing corresponding mutations at positions c 3997 and d 11040 in ApoB
Fig. 3 The pedigree chart for probands 1427 and 1078. a The father of proband 1427 carried the mutation, while the mother and a male sibling were not affected. b Both the mother and a female sibling of proband 1078 carried the mutation. The genomic DNA of the father was unavailable
the lowest and second to the lowest in ApoB levels among all participants. The distribution of Lp(a) was skewed, and in contrast to ApoB, the Lp(a) levels were ranked 7 and 46 % among all the DCCT participants. Therefore, consistent with loss-of-function mutations in ApoB, subjects 1427 and 1078 had extremely low levels of plasma ApoB. Pedigree of the affected ApoB family members We examined the corresponding genomic DNA from family members of probands 1427 and 1078. For 1427, we obtained genomic DNA from his father, mother, and a male sibling. For 1078, we obtained genomic DNA from his mother and a female sibling. Then, we performed PCR to amply the genomic fragment using the primer pairs of
apob-1 and apob-2. Purified fragments were then sequenced by Sanger sequencing. Proband 1427’s mother carried two wild-type alleles, and his father was heterozygous for the mutation. His male sibling had two wild-type alleles. Therefore, proband 1427 inherited one mutant allele from his father. Both proband 1078’s mother and female sibling were heterozygous for the mutant alleles; thus, he inherited the mutant allele from his mother (Fig. 3).
Discussion Sequencing technology has seen dramatic progress in the past decades, in terms of cost, dependability, and speed
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[29–31]. It is likely that in the near future, sequencing the entire human genome could cost less than $1,000 and take only hours. But currently whole genome sequencing is still impractical for clinical use due to the expense, technical obstacles, and subsequent sequence analysis. The goal of exome sequencing is to only sequence the protein-coding genes in a genome [32–34]. By focusing only on exons, sequencing effort is greatly reduced. Exons constitute about 1 % (about 30 Mb) of the human genome, but disease-causing mutations in exons are much more likely than in other parts of the human genome, such as introns and repeat elements [32–34]. Exome sequencing was used in this study to reliably identify loss-of-function mutations in two hypobetalipoproteinemia subjects. The low cost, reliability, and high speed in sequencing exons make this technique to have a deep impact on clinical use, medical research, and the biotechnology industry. There have been some reports on hypobetalipoproteinemia in type 2 diabetes [35–39]; nevertheless, to the best of our knowledge, cases of hypobetalipoproteinemia caused by ApoB loss-of-function mutations in type 1 diabetes have not been reported. Most patients with FHBL are caused by mutations in ApoB. These patients often present with nonalcoholic fatty liver disease due to reduced capacity to export lipids from the liver into the circulation [8, 40]. The unavailability of liver phenotypes and parameters of the two patients is one limitation of the current study. Judging by their extremely low levels of TAG and LDL-C, we hypothesize that the two patients likely have fatty liver, which, in more severe cases, may progress to cirrhosis. Another limitation is although we identified the mutations in family members of the two probands, the lipid phenotypes of the affected family members were not available. It is likely that affected family members present with similar patterns of lipid profiles as the two probands. In the two patients with ApoB mutations, the presence of type 1 diabetes did not mask hypobetalipoproteinemia, and therefore, it would be interesting to examine whether diabetic patients with hypobetalipoproteinemia are protected from diabetic dyslipidemia and its associated cardiovascular compilations. In summary, we have identified novel heterozygous ApoB loss-of-function mutations that cause hypobetalipoproteinemia in two DCCT participants (DCCT ID: 1427 and 1078), who exhibited extremely low levels of both TAG and LDL-C. These mutations cause premature stop codons at ApoB amino acid 1333 (ApoB-29) and 3680 (ApoB-81), and indeed, among all DCCT participants, the lowest circulating ApoB levels were found in these two patients. The identification of ApoB loss-of-function mutations in two type 1 diabetic patients presents innovative cases to study the interaction between hypobetalipoproteinemia and insulin deficiency.
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Acknowledgments The current study was supported in part by a fund from Wayne State University to R.Z., NNSF of China (90408028) to C.T.Z. and (31171238) to F.G. and the China National 863 High-Tech Program (2015AA020101) to F.G. Conflict of interest Feng Gao, Hao Luo, Zhiyao Fu, Chun-Ting Zhang and Ren Zhang declare that they have no conflict of interest. Human and Animal Rights disclosure All procedures followed were in accordance with the ethical standards of the Institutional Review Boards of Wayne State University School of Medicine and of the Detroit Medical Center, and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent disclosure Informed consent was obtained from all participants being included in the study.
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