A Novel LIPE Nonsense Mutation Found Using Exome Sequencing in Siblings With Late-Onset Familial Partial Lipodystrophy

Canadian Journal of Cardiology 30 (2014) 1649e1654

Clinical Research

A Novel LIPE Nonsense Mutation Found Using Exome Sequencing in Siblings With Late-Onset Familial Partial Lipodystrophy Sali M.K. Farhan, BSc,a,b John F. Robinson,b Adam D. McIntyre, BSc,b Maria G. Marrosu, MD,c Anna F. Ticca, MD,c Sara Loddo, BSc,d Nicola Carboni, MD, PhD,e Francesco Brancati, MD, PhD,f and Robert A. Hegele, MD, FRCPCa,b a b

Departments of Medicine and Biochemistry, Western University, London, Ontario, Canada

Robarts Research, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada c

Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Cagliari, Italy d

IRCCS Casa Sollievo della Sofferenza, Istituto Mendel, San Giovanni Rotondo, Italy e

f

Division of Neurology, Hospital San Francesco of Nuoro, Nuoro, Italy

Department of Medical, Oral and Biotechnological Sciences, Gabriele D’Annunzio University of Chieti-Pescara, Italy

ABSTRACT

  RESUM E

Background: Familial lipodystrophies are rare inherited disorders associated with redistribution of body fat and development of dyslipidemia, insulin resistance, and diabetes. We previously reported 2 siblings with unusual late-onset familial partial lipodystrophy in whom heretofore known causative genes had been excluded. We hypothesized they had a mutation in a novel lipodystrophy gene. Methods: Our approach centred on whole exome sequencing of the patients’ DNA, together with genetic linkage analysis and a bioinformatic prioritization analysis. All candidate variants were assessed in silico and available family members were genotyped to assess segregation of mutations. Results: Our prioritization algorithm led us to a novel homozygous nonsense variant, namely p.Ala507fsTer563 in the hormone sensitive lipase gene encoding, an enzyme that is differentially expressed in adipocytes and steroidogenic tissues. Pathogenicity of the mutation

Introduction : Les lipodystrophies familiales sont des troubles re ditaires rares associe s à une redistribution du gras corporel et un he veloppement de la dyslipide mie, une re sistance à l’insuline, et au de  ce demment rapporte  le cas rare de 2 memdiabète. Nous avons pre bres d’une même famille atteints d’une lipodystrophie familiale inhabituelle et partielle à apparition tardive pour laquelle les gènes sent avaient e  te  exclus. Nous responsables connus jusqu’à pre mettons l’hypothèse qu’ils avaient une mutation dans un nouveau e  à la lipodystrophie. gène lie thodes : Notre approche est centre e sur le se quençage de l’exome Me entier de l’ADN des patients, ainsi que sur une analyse de liaison  ne tique et une analyse bioinformatique de priorisation. Tous les ge  te e value s in silico et les membres variants des gènes candidats ont e  te  ge notype s pour e valuer la se gre gation de la famille disponibles ont e des mutations.

Studying rare genetic disorders associated with cardiovascular risk can provide an informative glimpse into pathological processes that might be more universally important.1 The study of rare human diseases has been made exponentially more efficient with the introduction of next-generation sequencing

methods; these have revolutionized human genetic research and are beginning to show clinical utility.2,3 Among metabolic risk factors for atherosclerotic cardiovascular disease, dyslipidemia, and diabetes or insulin resistance can be inherited as strong genetic traits in certain rare cases. A particularly strong form of inherited type 2 diabetes with insulin resistance is seen in families in which lipodystrophies are inherited across generations.4,5 Familial lipodystrophies are associated with redistribution of subcutaneous and visceral fat tissue depots, leading to dyslipidemia, insulin resistance, diabetes, and often premature cardiovascular disease events.6 The phenotype is typically detectable in adolescence or young adulthood, and can result from mutations in 1 of at least 10 different genes.5

Received for publication August 26, 2014. Accepted September 11, 2014. Corresponding author: Dr Robert A. Hegele, Blackburn Cardiovascular Genetics Laboratory, 4288A-1151 Richmond St N, Robarts Research Institute, University of Western Ontario, London, Ontario N6A 5K8, Canada. Tel.: þ1-519-931-5271; fax: þ1-519-931-5218. E-mail: [email protected] See page 1653 for disclosure information.

http://dx.doi.org/10.1016/j.cjca.2014.09.007 0828-282X/Ó 2014 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

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was supported in bioinformatic analyses and variant cosegregation within the family. Conclusions: We have identified a novel nonsense mutation in hormone sensitive lipase gene, which likely explains the lipodystrophy phenotype observed in these patients.

sultats : Notre algorithme de hie rarchisation nous a conduit à un Re nouveau variant non-sens homozygote, à savoir p.Ala507fsTer563 dans le gène de la lipase hormonosensible (LIPE), une enzyme rentiellement exprime e dans les adipocytes et les tissus diffe roïdogènes. La pathoge nicite  de la mutation a e  te e taye e par les ste gre gation du variant au sein analyses bioinformatiques et par la co-se de la famille.  une nouvelle mutation non-sens Conclusions : Nous avons identifie notype de la lidans LIPE, ce qui explique probablement le phe  chez ces patients. podystrophie observe

We recently described an atypical presentation of familial lipodystrophy of unknown genetic origin in 2 offspring of a consanguineous mating.7 A brother and sister were each noted relatively late in life to have fat redistribution, and metabolic features such as dyslipidemia and diabetes were not apparent until the fifth decade of life. The affected individuals had been screened for known lipodystrophy genes, with no pathogenic mutations identified. Here, we describe experiments combining whole-exome sequencing (WES), genome-wide linkage analysis, and bioinformatic analysis that have identified the likely cause of the lipodystrophy phenotype in these siblings, namely a homozygous nonsense variant p.Ala507fsTer563 in hormone sensitive lipase (LIPE), encoding the enzyme hormone sensitive lipase.8 To our knowledge, this is only the second documented human LIPE mutation, further implicating LIPE in lipid metabolism and the development of type 2 diabetes.9

Ontario (Illumina Inc., San Diego, CA). The SureSelect Target Enrichment kit v1 (Agilent Technologies) was used to enrich 40 Mbase of exome regions. Genome Analysis Toolkit was used to align sequence data to the human reference genome (Hg19) to produce a consensus sequence (Genome Analysis Toolkit, Boston, MA).14 Nonsynonymous variants within the autozygous region and with a low minor allele frequency (< 1%) according to in-house control exomes, NCBI dbSNP, 1000 Genomes, or NHLBI ESP Exome Variant Server, were subsequently analyzed. Candidate variants were analyzed in silico using PolyPhen-2 and SIFT to predict whether the amino acid change might disrupt protein function. ClustalW was used to determine the conservation of the wild type amino acid residue across a set of diverged species by aligning species-specific homologues. Variant validation: genotyping of family members

Methods Patients The family pedigree is shown in Figure 1A. Clinical features are shown in Table 1. Blood and tissue samples were collected from the family members with informed consent (Western University Institutional Review Board # 07920E). Routine blood tests were performed to determine the lipid profile of the 2 affected individuals, their mother, and their offspring. Autozygosity mapping The 2 affected individuals (V-3 and V-4) and their mother (IV-4) were genotyped for > 900,000 single nucleotide polymorphisms (SNPs). SNP genotypes were called using the Birdseed v2 algorithm housed in the Affymetrix Genotyping Console Software (version 4.1.1, Affymetrix, Santa Clara, CA).10,11 GeneSpring GT version 2.0 (Agilent Technologies, Santa Clara, CA) software was used to identify regions of homozygosity that follow identity by descent. Location scores were calculated using the summation of LOD scores of accumulated homozygous regions in the genome.12,13 Homozygous regions unique to the 2 affected individuals were prioritized. WES and variant calling Genomic DNA (gDNA) (4-6 mg) from the 2 affected individuals (V-3 and V-4) was subjected to WES on the Illumina HiSeq 2000 with 2  100 paired-end chemistry in accordance with protocols used at The Centre for Applied Genomics at the Hospital for Sick Children, Toronto,

We performed genotyping using gDNA extracted from available family members (IV-4, V-3, V-4, VI-1, VI-2, VI-3, and VI-4) for the candidate variants identified by WES using standard Sanger sequencing. Briefly, gDNA from family members was polymerase chain reaction-amplified using denaturation (95 C, 5 minutes; 30 cycles of 30 seconds), annealing (melting temperatures [Tm] available for each gene [Supplemental Table S1], 30 cycles for 30 seconds), and extension (72 C, 30 cycles for 30 seconds; 7 minutes). We then cleaned, purified, and sequenced the amplicons at the London Regional Genomics Centre using Sanger sequencing. Amplification primers are shown in Supplemental Table S1. Electropherograms produced were analyzed using Applied Biosystems SeqScape Software version 2.6 with the reference sequence of each gene obtained from the NCBI GenBank database (Life Technologies Inc, Carlsbad, CA). Results These 2 affected individuals were Italian siblings who were the products of a consanguineous mating (Fig. 1A).7 Initially, the proband (V-4) presented in her late 40s with abnormal fat distribution, and metabolic alterations including an abnormal lipid profile (Table 1) and type 2 diabetes. She also had muscle weakness and an increased serum creatine kinase level, suggesting mild muscular dystrophy; pathological changes consistent with a nonspecific muscular dystrophy were confirmed at muscle biopsy.7 Her brother (V-5) was subsequently evaluated and was also diagnosed in his late 40s with abnormal fat distribution, type 2 diabetes, and dyslipidemia. He has increased creatine kinase levels and his lower limb muscles show signs of

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Figure 1. Integrated approach to identify the genetic origin of late-onset partial lipodystrophy. (A) Affected individuals are shown in shaded squares (male) and circles (female). A consanguineous marriage is shown by a double line between 2 individuals. Horizontal dashes above symbols indicate individuals who underwent DNA analysis. (B) Genome-wide autozygosity mapping of the family generated large homozygous segments on autosomal chromosomes (x-axis) with their respective location scores (y-axis). (C) The variants were filtered using a homozygous, nonsynonymous, and rare variant analysis criteria, which generated 5 candidate variants, which were all on chromosome 19. (D) Protein sequence and structure of wild type and mutated LIPE. (E) Electropherograms the DNA sequence of a homozygous, affected individual and a heterozygous, unaffected individual. ab-H, alpha-beta hydrolase fold; CBLC, cbl proto-oncogene C, E3 ubiquitin protein ligase; fs, frameshift; fs/þ, heterozygous for the frameshift mutation; HLS_N, hormone-sensitive lipase N-terminus; LIPE, hormone-sensitive lipase; MEGF8, multiple EGF-like-domains 8; PRR19, proline rich 19; ZNF283, zinc finger protein 283.

skeletal muscle abnormalities, however, he does not have muscular dystrophy. Both patients are still alive; they reached sexual maturity, and each has children (Fig. 1A). Targeted candidate gene sequencing in 2013 revealed no mutations in known lipodystrophy genes.15 Extension of the family and further clinical evaluation indicated that these siblings were the only affected individuals. Since the original ascertainment and characterization, the probands’ children and mother were carefully evaluated and found to have no evidence suggesting a lipodystrophy. Because of the clinical presentation, we suspected an autosomal recessive mode of inheritance. We performed genome-wide autozygosity mapping to isolate candidate loci (Fig. 1B). The large homozygous segments unique to affected individuals were on chromosomes 8, 19, and 21 with location scores of 161, 56, and 76, respectively (Table 2). We subsequently applied a nonsynonymous, rare variant analysis to the WES data and identified 5 rare candidate variants: p.Arg192Leu in multiple epidermal growth factor-

like-domains 8; p.Gly74Ser in cbl proto-oncogene C, E3 ubiquitin protein ligase (CBLC); p.Arg50Trp in proline rich 19 (PRR19); p.Thr363Ile in zinc finger protein 283 (ZNF283); and p.Ala507fsTer563 in LIPE (Table 3). Interestingly, all 5 candidate variants were within the homozygous segment on chromosome 19 (Fig. 1C); there were no candidate variants within the other linked homozygous blocks and microarray analysis ruled out copy number variation in these regions. Furthermore, these 5 candidate variants were not reported in in-house control exomes, NCBI dbSNP, 1000 Genomes, HGMD, or NHLBI ESP Exome Variant Server, which demonstrates their rarity. In silico predictions of pathogenicity using 3 different algorithms were inconsistent (Table 3). All 5 variants cosegregated with disease status in the family members who underwent genotyping (Fig. 1A and E, Table 3). Both patients were homozygous for all 5 variants, and unaffected individuals were heterozygous for all 5 variants. Interestingly, according to The Human Protein Atlas, a

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Table 1. Clinical presentations of individuals with LIPE mutations Study

This Study, Patient 1 (V-4), Pretreatment

This Study, Patient 1 (V-4) After Treatment

This Study, Patient 2 (V-5) After Treatment

Albert et al.,3 Median Values of 4 Patients

51 p.Ala507fsTer563 þ þ þ þ 43 þ 5.70 5.67 ND 0.85 10.21 438 ND ND ND 3



50 p.Ala507fsTer563 þ þ  þ  þ 5.78 1.95 3.78 1.06 5.66 479 0.56 0.81 <0.37 2

48 p.V767GfsTer102 þ þ  þ 46 þ ND 1.94 ND 1.19 9.10 ND ND ND ND 6

Current age, years LIPE mutation Abnormal subcutaneous fat distribution Reduced lower limb subcutaneous fat distribution Muscular dystrophy Type 2 diabetes Age at diagnosis of type 2 diabetes Hepatic steatosis Total cholesterol (normal range, 3.9-6.2), mmol/L Triglycerides (normal range, 0.3-2.0), mmol/L LDL cholesterol (normal range, 1.5-3.9), mmol/L HDL cholesterol (normal range, 1.1-1.9), mmol/L Glucose (normal range, 4-5.9), mmol/L Creatine kinase (normal range, 30-170), m/L Apo-A (normal range, 0.8-1.2), g/L Apo-B (normal range, 1.0-1.4), g/L CRP (normal range, <1.0), mg/dL Offspring, n

þ þ þ þ  þ 6.27 2.50 4.07 1.06 4.61 ND 1.16 1.35 <0.03

Median values from Albert et al. are reported from homozygous individuals only. þ, present; , absent; Apo, apolipoprotein; CRP, C-reactive protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LIPE, hormone-sensitive lipase; ND, not determined or reported.

public database that holds information on protein and transcript expression profiles based on immunohistochemistry from 44 different normal human tissues, only LIPE transcript and protein products were expressed in adipose tissue, which is the most compromised tissue in these patients (Table 2). Furthermore, a different homozygous LIPE nonsense mutation had recently been reported in a family with atypical lipodystrophy.9 In addition, the other putative candidate genes were prioritized as less likely to be causative for various reasons. For instance, multiple epidermal growth factor-likedomains 8 had been implicated in a completely unrelated pediatric developmental disorder, namely Carpenter syndrome type 2 (MIM 614976).16 Also, CBLC deficiency has been associated with disturbed vitamin B12 metabolism, but normal fat metabolism.17 Finally, there are no direct functional studies providing evidence for either a PRR19- or a ZNF283-mediated human deficiency state. LIPE p.Ala507fsTer563 is novel as demonstrated by its complete absence from genome databases. The frameshift begins in the hormone-sensitive lipase domain (302-616 amino acids) and affects 2 downstream domains: a/b hydrolase 3 (647-809) and (935-1028) achieving a stop codon at position 563, approximately a 50% net loss of the original polypeptide; whether this truncation affects lipase activity is still being investigated (Fig. 1D). The adipose tissue-specific LIPE is 775 amino acids, however, both LIPE isoforms have been studied to determine their distinct tissue-specific

Table 2. Autozygous blocks identified in affected individuals Chromosome 8 19 21

dbSNP

Chromosome Location

Location Score of Chromosome

Start: rs16904315 End: rs6991482 Start: rs12978323 End: rs2116891 Start: rs2831491 End: rs16987645

Start: 131616893 End: 133149967 Start: 41756038 End: 46117413 Start: 29476742 End: 32427232

161 56 76

activities.18-21 Importantly, a recent report by Albert and colleagues described a novel homozygous null mutation in LIPE in 4 individuals affected with insulin resistance, abnormal subcutaneous fat distribution, increased level of plasma triglycerides, and type 2 diabetes, features present in the study patients, and summarized in Table 1.9 Discussion We report the second human mutation in LIPE, a homozygous nonsense variant, namely p.Ala507fsTer563, that appears to be the proximal cause of a late-onset form of familial partial lipodystrophy. Other genes known to cause familial partial lipodystrophy, such as lamin A/C (LMNA) or polymerase I and transcript release factor (PTRF) had been excluded, allowing us to hypothesize that the phenotype described in these siblings was due to a mutation in a novel gene. The variant was found using WES with an accelerated, automated variant calling approach; narrowing of the likely pathogenic region was accomplished using genome-wide linkage analysis. Our prioritization strategy demonstrated absence of the variant from public databases, and a range of in silico and biological evidence, including previous implication of LIPE in other patients with a rare presentation of familial partial lipodystrophy, made the nonsense mutation in LIPE the most likely candidate. Because of inconsistencies with phenotypes in patients with previously reported variants, we were able to exclude the other genes and mutations found in these samples (Table 3). In particular, CBLC and ZNF283 are not reported to be involved in lipid biosynthesis or metabolism, but rather are involved in cell signalling through ubiquitination and regulation of tyrosine kinases, and according to gene ontology (GO) annotation, DNA binding, and zinc ion binding, respectively.22 Furthermore, abnormal CBLC activity is associated with the development of irregular vitamin B12 (cobalamin) metabolism and even human glioblastoma.17,23 Also, the precise molecular functions and mechanisms of

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Table 3. Variants identified using whole-exome sequencing

Gene

Chromosome Location

Amino Acid Change

Type of Mutation

Effect

In Silico Prediction

Cosegregation in Family

Expressed in Adipose Tissue?

MEGF8 CBLC

19q12 19q13.2

p.Arg192Leu p.Gly74Ser

SNP SNP

Missense Missense

Benign Damaging/benign

Yes Yes

RNA detected Not detected

PRR19 ZNF283 LIPE

19q13.2 19q13.31 19q13.2

p.Arg50Trp p.Thr363Ile p.Ala507fsTer563

SNP SNP Indel

Missense Missense Frameshift

Damaging Damaging/benign d

Yes Yes Yes

Not detected RNA detected RNA and protein detected

Associated With a Human Disease? Carpenter syndrome type 2 Disturbed cobalamin metabolism No No Insulin resistance, impaired lipolysis, and type 2 diabetes

MEGF8, multiple epidermal growth factor-like-domains 8; CBLC, cbl proto-oncogene C, E3 ubiquitin protein ligase; PRR19, proline rich 19; ZNF283, zincfinger protein 283; LIPE, hormone-sensitive lipase; Indel, insertion/deletion.

ZNF283 and PRR19 have not been described; these candidate genes were unlikely to be involved in the phenotype of these patients. Of the candidate genes identified by our WES and prioritization strategy, only LIPE had a transcript and a protein product that were both fully expressed in adipocytes, the tissue most affected in these patients. Further, a recent report described a novel homozygous null mutation in LIPE, namely p.V767GfsTer102, in patients with impaired lipolysis, insulin resistance, inflammation, and type 2 diabetes.9 Clinical features of those subjects compared with those of our 2 affected subjects are shown in Table 1. The frameshift mutation reported by Albert et al.9 occurred further downstream than the one observed in our patients, although it still had a catastrophic effect on lipolytic activity. The mutation seen in our patients would be predicted to result in an even more prematurely truncated enzyme, with likely comparable effects on lipolytic activity and might explain the abnormal lipid profiles. Interestingly, the muscular dystrophy phenotype was observed only in the sister however, her brother did have some skeletal muscle abnormalities including increased creatine kinase levels. This disparity between the patients described by Albert et al. and the ones in our study might be explained by allelic variation giving rise to different clinical phenotypes. Specifically, the location of the mutation could generate multiple phenotypes causing overlapping syndromes, which has been observed in various laminopathy phenotypes.24,25 Alternatively, the myopathy in our proband could be the result of a sporadic dominant mutation. However, we found no mutations in known muscular dystrophy genes (data not shown); this contingency could not be further investigated even with full WES data. Hormone-sensitive lipase was characterized initially by Canadian scientists and was found to hydrolyze stored triglycerides in adipose tissue to release free fatty acids.26,27 Interestingly, > 240 variants comprised of SNPs (minor allele frequency > 1%) and mutations (minor allele frequency < 1%) have been observed in LIPE, 57 of which have been classified as “probably damaging” according to in silico predictive software. However, these mutations were not found in patients with lipodystrophy and were not functionally tested. Some studies have suggested that a common polymorphism in LIPE, namely g.60C>G in the promoter region, when coupled with obesity and insulin resistance, is a risk factor in the development of fatty liver and can also modulate lipid levels.10,19

Because hormone-sensitive lipase regulates lipolysis free fatty acid flux from adipose tissue, it is not surprising that disruption of its activity might be related to a lipodystrophy phenotype.28 Interestingly, induced mutation of LIPE in rodents causes deficient spermatogenesis, azoospermia, and ultimately, infertility in male mice.20 Specifically, the long LIPE isoform is primarily expressed in the testes, where it converts cholesteryl esters to free cholesterol for steroid hormone synthesisda process disrupted in hormone-sensitive lipase-deficient male mice.20 The dual role of hormone-sensitive lipase in reproduction and lipid homeostasis is likely related to its specific tissue expression. Hormone-sensitive lipase deficiency in male steroidogenic tissue can confer susceptibility to infertility and deficiency in adipose tissue might lead to obesity or partial lipodystrophy. In our study, the affected male sibling was fertile with 2 biological children, indicating that there might be species differences in tissue expression of proteins of redundant function that can rescue this particular phenotype. In summary, we have identified a novel LIPE-mediated human disease in patients with familial partial lipodystrophy. The frameshift mutation truncates nearly 50% of LIPE and might be responsible for the abnormal lipid profile and ultimately the phenotype in these patients. The findings extend the range of mutations that underlie these interesting metabolic disorders. Acknowledgements The authors thank the family members for their participation in our study. Funding Sources This study was supported by the Canadian Institutes of Health Research (project grants MOP-13430, MOP-79523, CTP-79853 to R.A.H.), the Government of Canada through Genome Canada, and the Ontario Genomics Institute (OGI-049). S.M.K.F. is supported by the Ontario Graduate Scholarship. Disclosures The authors have no conflicts of interest to disclose. References 1. Farhan SM, Hegele RA. Genetics 101 for cardiologists: rare genetic variants and monogenic cardiovascular disease. Can J Cardiol 2013;29:18-22.

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2. Farhan SM, Hegele RA. Sequencing: the next generationewhat is the role of whole-exome sequencing in the diagnosis of familial cardiovascular diseases? Can J Cardiol 2014;30:152-4.

17. Fischer S, Huemer M, Baumgartner M, et al. Clinical presentation and outcome in a series of 88 patients with the CBLC defect. J Inherit Metab Dis 2014;37:831-40.

3. Farhan SM, Hegele RA. Exome sequencing: new insights into lipoprotein disorders. Curr Cardiol Rep 2014;16:507-17.

18. Hsiao PJ, Chen ZC, Hung WW, et al. Risk interaction of obesity, insulin resistance and hormone-sensitive lipase promoter polymorphisms (LIPE-60 C > G) in the development of fatty liver. BMC Med Genet 2013;14:54.

4. Hegele RA, Joy TR, Al-Attar SA, et al. Thematic review series: adipocyte biology. Lipodystrophies: windows on adipose biology and metabolism. J Lipid Res 2007;48:1433-44. 5. Garg A. Clinical review: lipodystrophies: genetic and acquired body fat disorders. J Clin Endocrinol Metab 2011;96:3313-25.

19. Vatannejad A, Khodadadi I, Amiri I, et al. Genetic variation of hormone sensitive lipase and male infertility. Syst Biol Reprod Med 2011;57: 288-91.

6. Hegele RA. Premature atherosclerosis associated with monogenic insulin resistance. Circulation 2001;103:2225-9.

20. Wang SP, Wu JW, Bourdages H, et al. The catalytic function of hormone-sensitive lipase is essential for fertility in male mice. Endocrinology 2014;155:3047-53.

7. Carboni N, Brancati F, Cocco E, et al. Partial lipodystrophy associated with muscular dystrophy of unknown genetic origin. Muscle Nerve 2013;49:928-30.

21. Watt MJ, Carey AL, Wolsk-Petersen E, et al. Hormone-sensitive lipase is reduced in the adipose tissue of patients with type 2 diabetes mellitus: influence of IL-6 infusion. Diabetologia 2005;48:105-12.

8. Kraemer FB, Shen WJ. Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis. J Lipid Res 2002;43: 1585-94.

22. Takeshita K, Tezuka T, Isozaki Y, et al. Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c. J Biochem 2012;152:487-95.

9. Albert JS, Yerges-Armstrong LM, Horenstein RB, et al. Null mutation in hormone-sensitive lipase gene and risk of type 2 diabetes. N Engl J Med 2014;370:2307-15.

23. Mizoguchi M, Nutt CL, Louis DN. Mutation analysis of CBL-C and SPRED3 on 19q in human glioblastoma. Neurogenetics 2004;5:81-2.

10. Rabbee N, Speed TP. A genotype calling algorithm for affymetrix snp arrays. Bioinformatics 2006;22:7-12. 11. Korn JM, Kuruvilla FG, McCarroll SA, et al. Integrated genotype calling and association analysis of SNPs, common copy number polymorphisms and rare CNVs. Nat Genet 2008;40:1253-60. 12. Lander ES, Botstein D. Homozygosity mapping: a way to map human recessive traits with the DNA of inbred children. Science 1987;236: 1567-70. 13. Broman KW, Weber JL. Long homozygous chromosomal segments in reference families from the centre d’etude du polymorphisme humain. Am J Hum Genet 1999;65:1493-500. 14. DePristo MA, Banks E, Poplin R, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011;43:491-8. 15. Johansen CT, Dube JB, Loyzer MN, et al. Lipidseq: a next-generation clinical resequencing panel for monogenic dyslipidemias. J Lipid Res 2014;55:765-72. 16. Begum S, Khatun N, Rayhan SM, et al. Carpenter syndrome: a case report. Mymensingh Med J 2012;21:547-9.

24. Hegele R. LMNA mutation position predicts organ system involvement in laminopathies. Clin Genet 2005;68:31-4. 25. Wiltshire KM, Hegele RA, Innes AM, et al. Homozygous lamin A/C familial lipodystrophy R482Q mutation in autosomal recessive emery Dreifuss muscular dystrophy. Neuromuscul Disord 2013;23:265-8. 26. Hollenberg CH, Raben MS, Astwood EB. The lipolytic response to corticotropin. Endocrinology 1961;68:589-98. 27. Vaughan M, Berger JE, Steinberg D. Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue. J Biol Chem 1964;239: 401-9. 28. Osuga J, Ishibashi S, Oka T, et al. Targeted disruption of hormonesensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad Sci U S A 2000;97:787-92.

Supplementary Material To access the supplementary material accompanying this article, visit the online version of the Canadian Journal of Cardiology at www.onlinecjc.ca and at http://dx.doi.org/10. 1016/j.cjca.2014.09.007.