Logo of ajhgLink to Publisher's site
Am J Hum Genet. Aug 2006; 79(2): 383–389.
Published online Jun 5, 2006. doi:  10.1086/505885
PMCID: PMC1559499

Sequencing of the Reannotated LMNB2 Gene Reveals Novel Mutations in Patients with Acquired Partial Lipodystrophy


The etiology of acquired partial lipodystrophy (APL, also called “Barraquer-Simons syndrome”) is unknown. Genomic DNA mutations affecting the nuclear lamina protein lamin A cause inherited partial lipodystrophy but are not found in patients with APL. Because it also encodes a nuclear lamina protein (lamin B2) and its genomic structure was recently reannotated, we sequenced LMNB2 as a candidate gene in nine white patients with APL. In four patients, we found three new rare mutations in LMNB2: intron 1 −6G→T, exon 5 c.643G→A (p.R215Q; in two patients), and exon 8 c.1218G→A (p.A407T). The combined frequency of these mutations was 0.222 in the patients with APL, compared with 0.0018 in a multiethnic control sample of 1,100 subjects (P=2.1×10-7) and 0.0045 in a sample of 330 white controls (P=1.2×10-5). These novel heterozygous mutations are the first reported for LMNB2, are the first reported among patients with APL, and indicate how sequencing of a reannotated candidate gene can reveal new disease-associated mutations.

Mutations in several genes have been found in patients with inherited lipodystrophies, including mutations in LMNA (MIM 150330), PPARG, and ZMPSTE24 in partial lipodystrophy and mutations in BSCL2 and AGPAT2 in congenital total lipodystrophy.14 However, the potential genetic contribution to acquired partial lipodystrophy (APL, also called “Barraquer-Simons syndrome” [MIM 608709]) and acquired generalized lipodystrophy (AGL) has not been established.5,6 A family history is usually absent in APL and AGL, whereas a wide range of secondary factors and conditions are often associated.5,6 For instance, systemic lupus erythematosis, dermatomyositis, hypocomplementemia, and membranoproliferative glomerulonephritis are sometimes seen in patients with APL,6 whereas panniculitis and various autoimmune diseases are sometimes seen in patients with AGL.5 An unresolved issue is whether acquired lipodystrophy syndromes have an underlying component of genetic susceptibility.6 Because such patients are sporadically affected and are typically not members of multigenerational families with multiple affected members, alternative strategies to classic positional cloning are required to find susceptibility genes.

Candidate-gene sequencing is one strategy to implicate a susceptibility gene through identification of rare DNA sequence mutations that are present in affected individuals and absent in healthy individuals. Putative biological function is a common criterion used to select a candidate gene for genomic DNA sequence analysis. For instance, because structural abnormalities in a nuclear envelope protein gene—namely, LMNA, encoding lamin A/C—cause familial partial lipodystrophy type 2 (FPLD2 [MIM 151660]), it is possible that mutations might be found in genes that encode related nuclear envelope proteins in other lipodystrophy syndromes. In 2001, we used the genomic information available at that time to develop amplification primers for candidate genes encoding nuclear envelope proteins, including LBR, LMNB1, and LMNB2 (MIM 150341), which encode lamin B receptor, lamin B1, and lamin B2, respectively.7 We then sequenced these candidate genes in patients with AGL or APL who had no mutations in LMNA.7 Although we identified several common polymorphisms, we found no disease-causing mutations and concluded that sequence variants affecting the nuclear lamina proteome were not likely to be associated with APL or AGL.7

Recent observations suggest that early versions of mammalian genome maps underestimated the total numbers of exons. For instance, new computational algorithms have revealed thousands of previously unrecognized exons in mammalian genomes.8 This understanding has necessitated significant reannotation of cDNA and genomic DNA databases by “stitching” newly identified exons into previously annotated genes. On the basis of this biological reality, we revisited the reannotated genomic structures of nuclear proteome genes7; in doing so, we found that LMNB2 had 6 exons identified in 2001 but 12 exons in 2005 (fig. 1A).

Figure  1.
A, Genomic structure of LMNB2 as it was known in 2001 and 2005 (with respective reference sequence numbers indicated). Boxes indicate exons, with the exon number shown above. B, Electropherogram tracings of genomic DNA sequences of the LMNB2 gene from ...

Using new primers, we interrogated the coding regions of the reannotated LMNB2 gene in nine unrelated patients with APL (table 1) whose conditions were diagnosed according to established criteria.6 Specifically, subjects had gradual onset of bilateral subcutaneous fat loss from the head, neck, upper extremities, and thorax but not the lower extremities.6 In addition, subjects had supportive diagnostic criteria, including absence of a clear family history of lipodystrophy and onset of lipodystrophy before adulthood. Other types of lipodystrophies, including inherited varieties such as congenital generalized lipodystrophy (CGL [MIM 269700]), FPLD2, or familial partial lipodystrophy type 3 (FPLD3 [MIM 604367]) and acquired varieties such as AGL and HIV-associated lipodystrophy, were excluded. Other associated conditions in the patients with APL are shown in table 1. In addition, one patient had dermatomyositis and alopecia areata, one patient had bilateral carpal tunnel syndrome, one patient was hypothyroid, and the single patient with C3 deficiency and biopsy-proven membranoproliferative glomerulonephritis had received three renal transplants, with subsequent development of secondary and tertiary hyperparathyroidism. All patients were white. All study subjects provided informed consent for DNA analysis, and the Institutional Review Board of the University of Western Ontario approved the study (protocol 07920E).

Table 1.
Demographic Clinical Attributes of Nine Patients with APL[Note]

No coding sequence or promoter mutation in LMNA, PPARG, ZMPSTE24, BSCL2, or AGPAT2 had been found in genomic DNA sequence of any patient with APL by use of established protocols.911 LMNB2 was originally sequenced using primer pairs7 to amplify the five C-terminal exons of the presumed total of six exons from the 2001 reference sequence (fig. 1A; GenBank accession numbers M94362 and M94363). However, in 2005, the reannotated LMNB2 gene in chromosome 19 was shown to have 12 exons (fig. 1A; GenBank accession numbers NT_011255 and NM_032737). We made new primers to amplify all 12 exons plus >50 nt at each intron-exon boundary of LMNB2 (table 2). Standard amplification conditions were used for all reactions, including annealing at 60°C for each of 30 cycles. Sequences were read on a Model 3730 Automated DNA Sequencer (Applied Biosystems). Mutations were confirmed by a second independent sequencing reaction performed on a different day.

Table 2.
Amplification Primers for Reannotated LMNB2 Gene

The frequencies of LMNB2 mutations were determined in a multiethnic control sample of 1,100 healthy individuals comprising 330 individuals of white descent, 35 individuals of African descent, 375 individuals of East Indian descent, and 360 individuals of Chinese descent. Genotyping methods are shown in table 3. All genotyping was performed with blinding to affected status, and sequence-proven positive controls were run with each reaction. A random 5% of genotypes were repeated, and no disparities were found for any genotype call. All statistical analyses were performed using SAS version 8.1 (SAS Institute). Between-group comparisons of proportion of discrete variables and calculations of relative risk for carriers of the variants were made using χ2 contingency analysis. The nominal P value for significance was .05.

Table 3.
LMNB2 Genotyping Procedures

Among the nine subjects with APL described in table 1, we found four heterozygous genomic sequence variants of LMNB2 in five subjects (fig. 1B). These variants were exon 1 c.82G→A, intron 1 −6G→T, exon 5 c.643G→A (p.R215Q; in two patients), and exon 8 c.1218G→A (p.A407T). None of these LMNB2 variants had been previously reported in any database.

To determine how specific these variants were to our sample of patients with APL, we genotyped our nonlipodystrophic control samples (table 4). Genotyping conditions are described in table 3. The c.82G→A variant had frequencies of 0.0091, 0.0, 0.008, and 0.067 in the white, African, East Indian, and Chinese samples, respectively, and an overall allele frequency of 0.027 in the multiethnic control sample. The intron 1 −6C→T and p.A407T variants were not seen in the multiethnic control sample. The p.R215Q variant was seen in 3 of the 330 white controls (allele frequency 0.0045) and in 1 of the 375 East Indian controls (allele frequency 0.0013) but was absent from the other control samples (overall allele frequency 0.0018). Allele frequencies were then compared between the APL and control samples. The observation of one LMNB2 c.82G→A heterozygote among nine subjects with APL indicated an allele frequency (0.056) that was not significantly different from the allele frequency in the multiethnic control sample and the white subsample (0.027 and 0.0091, respectively; both P>.05). This suggested that, although the c.82G→A variant was relatively uncommon in normal subjects, it was not significantly associated with APL in this sample; the relative risk (RR) of APL in carriers of the variant was 2.1 (95% CI 0.35–12.9; P>.05).

Table 4.
Allele Frequencies in Controls of New Mutations and Variants Found in the Reannotated LMNB2 Gene among Patients with APL

However, the combined frequency of the LMNB2 mutations intron 1 −6C→T, p.R215Q, and p.A407T in the nine subjects with APL was 0.222 (4 of 18 alleles) compared with 0.0018 (4 of 2,200 alleles) in all controls (P=2.1×10-7) and 0.0045 (3 of 660 alleles) in white controls (P=1.2×10-5). Thus, the RR of APL for carriers of these variants compared with all control subjects was 110.1 (95% CI 35.9–271.0); compared with white control subjects, it was 37.9 (95% CI 12.7–83.3). The clinical features of the four subjects with APL with these three LMNB2 mutations are shown in table 5.

Table 5.
Clinical Features of Subjects with APL and Rare LMNB2 Mutations[Note]

When the three mutations were considered individually, the presence of p.A407T in one of nine subjects with APL, together with its complete absence from 1,100 multiethnic and 330 white control subjects, was significant; the RRs of APL for carriers were 138.5 (95% CI 26.4–138.5) and 42.3 (95% CI 8.1–42.3) compared with all controls and the white controls, respectively. The intron 1 −6C→T mutation had the same RRs of APL for carriers compared with multiethnic and white control samples. The recurrence of p.R215Q in two of nine unrelated patients with APL compared with 3 of 330 reference white subjects was highly significant; the RR of APL for white carriers of the variant was 19.1 (95% CI 4.9–47.7). These two APL-affected patients who were heterozygous for p.R215Q had some similarities (e.g., both were women with long-term diabetes and dyslipidemia) but also some phenotypic differences (e.g., one had retinopathy and xanthomata, whereas the other had dermatomyositis, hepatosplenomegaly, osteoporosis, hirsutism, and alopecia). Interestingly, p.R215Q was also present, albeit at a very low frequency, in the control East Indian sample but not in the other ethnic groups; RR of APL for carriers compared with the overall multiethnic control group was 52.5 (95% CI 13.2–147.6). However, we recognize the limitations of RR calculations for rare alleles and a rare disease phenotype, especially for a small cohort of patients. Control subjects with these mutations had been randomly ascertained from healthy populations and did not have diabetes, hypertension, or dyslipidemia.

The findings indicate how resequencing of a candidate gene that has been reannotated at the genomic level can reveal new disease-associated mutations. The new LMNB2 mutations include two coding-sequence variants—p.R215Q in two patients with APL and p.A407T in one patient with APL—and one variant near a newly defined intron-exon boundary (intron 1 −6C→T). The high prevalence of the mutations in the nine patients with APL and the very low frequency of the mutations in the control samples indicates an extremely strong, nonrandom association between the mutations and the APL phenotype.

Although it has been recognized as a clinical entity for almost a century, the etiology of APL has been unclear.6 APL was first described as a form of progressive lipodystrophy.6,1214 Most reported APL cases have been sporadic, and the female:male affected ratio is ~4:1.6 This is similar to the female bias (or at least clinical recognition) of the expression of FPLD2.13 APL does not segregate as a classic simple Mendelian trait in families with an affected proband, nor does it typically cluster in families.6,1517 Discordance for APL in MZ twins has been noted.18,19 In addition, the presence of dermatomyositis, systemic lupus erythematosis, C3 hypocomplementemia, and membranoproliferative glomerulonephritis in some patients with APL6 suggests that such factors might trigger expression of the disease. These associations are consistent with the idea that APL represents a complex phenotype, possibly with a component of genetic susceptibility that requires the presence of environmental factors or acquired disorders to be expressed. The fact that not all subjects with APL had LMNB2 mutations indicates possible molecular heterogeneity; if other associated genes are identified, it might be appropriate to divide APL into molecular subtypes, analogous to FPLD2 and FPLD3.2 Furthermore, the fact that a few carriers of LMNB2 mutations, particularly p.R215Q, were found among apparently healthy controls is consistent with a complex etiology for APL, in which a susceptibility allele may not be sufficient for expression of the phenotype, instead requiring the presence of additional factors.

LMNB2 was selected as a candidate gene for genomic sequence analysis in patients with APL because, like LMNA, which is the causative gene for FPLD2, it encodes a nuclear envelope constituent. The implication of mutant LMNB2 in APL widens the range of disorders associated with mutations in intermediate filament proteins20 and, in particular, increases to four the number of nuclear envelope proteins that are associated with human diseases.21 Other nuclear envelope constituents in which mutations are associated with disease include lamin A/C (numerous laminopathies22), emerin (Emery-Dreifuss muscular dystrophy [MIM 310300]), and the lamin B receptor (Pelger-Huet anomaly [MIM 169400] and hydrops-ectopic calcification-moth-eaten skeletal dysplasia [MIM 215140]).

The p.R215Q mutation occurs within the rod domain of lamin B2 (fig. 1C) and alters an arginine residue that is conserved not only in human lamins A/C and B1 but also in mouse and zebrafish lamin B2 genes. Furthermore, certain missense mutations affecting this domain in LMNA result in a dilated cardiomyopathy phenotype—namely, p.E203K and p.L215Q—and in Emery-Dreifuss muscular dystrophy—namely, p.H222P.22 In contrast, the p.A407T mutation occurs within the much less evolutionarily conserved tail domain of LMNB2 (fig. 1C); the analogous residue to A407 is a threonine in both lamin A/C and lamin B1. Furthermore, there are no missense mutations affecting this residue in lamin A/C, although mutations in proximal residues p.R386K and p.R401C underlie Emery-Dreifuss muscular dystrophy.22 The presence of the intron 1 −6C→T variant near an intron-exon boundary suggests that it might affect RNA splicing. However, functional assessment of any of these mutations would be difficult, given that the function of lamin B2 is not precisely defined,2326 particularly in comparison with lamin B1.27 The range of potential roles of lamin B2 includes supporting the inner side of the nuclear envelope, stabilizing the nucleus and chromatin, and regulating gene expression.28 Lamin B2 replicates in the 1st min of S-phase.28 The LMNB2 origin of replication is very well characterized, accommodates the defined start site of DNA synthesis, binds to origin-recognition complexes in living cells and in vitro, and supports the initiation of DNA synthesis at ectopic chromosomal locations.28 It is thus possible that mutations that alter the function of lamin B2 might have an effect on the rate of DNA replication.

In summary, we have demonstrated human disease-associated mutations in newly defined coding sequences of a recently reannotated candidate gene, and, in doing so, we found the first human mutations in LMNB2 and the first mutations in APL. From these studies, APL appears to behave like a complex trait with a component of genetic susceptibility, which, in some patients, is mediated by LMNB2, with the further requirement for any one of several secondary associated illnesses or conditions. The findings are consistent with possible molecular genetic heterogeneity for APL, since five of nine patients did not have an LMNB2 mutation. The findings extend the range of phenotypes associated with mutations in nuclear envelope components and increase the number of nuclear envelope components that are associated with disease. The findings also implicate a second nuclear lamin gene in a human lipodystrophy syndrome and suggest that other acquired lipodystrophy phenotypes, such as HIV-related or drug-induced lipodystrophies, may be associated with mutations affecting the nuclear lamina or interacting proteins. Finally, the findings suggest that reannotation of known genes will disclose new coding sequences that should be examined by genomic sequence analysis to search for new disease-associated mutations.


This work was supported by Canadian Institutes of Health Research operating grant MT13430, the Heart and Stroke Foundation of Ontario, Genome Canada, and the Canadian Diabetes Association. R.A.H. is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Edith Schulich Vinet Canada Research Chair (Tier I) in Human Genetics and the Jacob J. Wolfe Distinguished Medical Research Chair.

Web Resources

Accession numbers and URLs for data presented herein are as follows:

GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for LMNB2 2001 reference sequence [accession numbers M94362 and M94363] and 2005 reference sequence [accession numbers NT_011255 and NM_032737])
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for LMNA, APL, FPLD2, LMNB2, CGL, FPLD3, Emery-Dreifuss muscular dystrophy, Pelger-Huet anomaly, and hydrops-ectopic calcification-moth-eaten skeletal dysplasia)


1. Garg A (2004) Acquired and inherited lipodystrophies. N Engl J Med 350:1220–1234 [PubMed] [Cross Ref]10.1056/NEJMra025261
2. Hegele RA (2004) Phenomics, lipodystrophy, and the metabolic syndrome. Trends Cardiovasc Med 14:133–137 [PubMed] [Cross Ref]10.1016/j.tcm.2004.02.001
3. Capeau J, Magre J, Lascols O, Caron M, Bereziat V, Vigouroux C, Bastard JP (2005) Diseases of adipose tissue: genetic and acquired lipodystrophies. Biochem Soc Trans 33:1073–1077 [PubMed] [Cross Ref]10.1042/BST20051073
4. Hegele RA (2005) Lessons from human mutations in PPARγ. Int J Obes (Lond) Suppl 1 29:S31–S35 [PubMed]
5. Misra A, Garg A (2003) Clinical features and metabolic derangements in acquired generalized lipodystrophy: case reports and review of the literature. Medicine (Baltimore) 82:129–146 [PubMed]
6. Misra A, Peethambaram A, Garg A (2004) Clinical features and metabolic and autoimmune derangements in acquired partial lipodystrophy: report of 35 cases and review of the literature. Medicine (Baltimore) 83:18–34 [PubMed]
7. Hegele RA, Yuen J, Cao H (2001) Single-nucleotide polymorphisms of the nuclear lamina proteome. J Hum Genet 46:351–354 [PubMed] [Cross Ref]10.1007/s100380170072
8. Frey BJ, Mohammad N, Morris QD, Zhang W, Robinson MD, Mnaimneh S, Chang R, Pan Q, Sat E, Rossant J, Bruneau BG, Aubin JE, Blencowe BJ, Hughes TR (2005) Genome-wide analysis of mouse transcripts using exon microarrays and factor graphs. Nat Genet 37:991–996 [PubMed] [Cross Ref]10.1038/ng1630
9. Cao H, Hegele RA (2000) Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy. Hum Mol Genet 9:109–112 [PubMed] [Cross Ref]10.1093/hmg/9.1.109
10. Bhayana S, Siu VM, Joubert GI, Clarson CL, Cao H, Hegele RA (2002) Cardiomyopathy in congenital complete lipodystrophy. Clin Genet 61:283–287 [PubMed] [Cross Ref]10.1034/j.1399-0004.2002.610407.x
11. Hegele RA, Cao H, Frankowski C, Mathews ST, Leff T (2002) PPARG F388L, a transactivation-deficient mutant, in familial partial lipodystrophy. Diabetes 51:3586–3590 [PubMed]
12. Barraquer-Ferre L (1935) Lipodystrophie progressive: syndrome de Barraquer-Simons. Presse Med 86:1672–1674
13. Langhof H, Zabel R (1960) On lipodystrophia progressiva (follow-up study of the case published by A. Simons in 1911 for the first time in German literature) [in German]. Arch Klin Exp Dermatol 210:313–321 [PubMed]
14. Jeune M, Freycon MT, Hermier M, Lamit HJ, Abboud N, Brunat N (1965) Nephropathies in progressive lipodystrophy or Barraquer-Simons disease [in French]. Ann Pediatr (Paris) 12:749–758 [PubMed]
15. Biasi D, Caramaschi P, Carletto A, Bambara LM (1999) A case of acquired partial lipodystrophy associated with localized scleroderma and undifferentiated connective tissue disease. Rheumatol Int 19:75–76 [PubMed] [Cross Ref]10.1007/s002960050106
16. Quecedo E, Febrer I, Serrano G, Martinez-Aparicio A, Aliaga A (1996) Partial lipodystrophy associated with juvenile dermatomyositis: report of two cases. Pediatr Dermatol 13:477–482 [PubMed]
17. Ferrarini A, Milani D, Bottigelli M, Cagnoli G, Selicorni A (2004) Two new cases of Barraquer-Simons syndrome. Am J Med Genet A 126:427–429 [PubMed] [Cross Ref]10.1002/ajmg.a.20623
18. Reichel W, Kobberling J, Fischbach H, Scheler F (1976) Membranoproliferative glomerulonephritis with partial lipodystrophy: discordant occurrence in identical twins. Klin Wochenschr 54:75–81 [PubMed] [Cross Ref]10.1007/BF01468772
19. Bier DM, O’Donnell JJ, Kaplan SL (1978) Cephalothoracic lipodystrophy with hypocomplementemic renal disease: discordance in identical twin sisters. J Clin Endocrinol Metab 46:800–807 [PubMed]
20. Omary MB, Coulombe PA, McLean WH (2004) Intermediate filament proteins and their associated diseases. N Engl J Med 351:2087–2100 [PubMed] [Cross Ref]10.1056/NEJMra040319
21. Worman HJ (2005) Components of the nuclear envelope and their role in human disease. Novartis Found Symp 264:35–42 [PubMed]
22. Hegele R (2005) LMNA mutation position predicts organ system involvement in laminopathies. Clin Genet 68:31–34 [PubMed]
23. Tribioli C, Biamonti G, Giacca M, Colonna M, Riva S, Falaschi A (1987) Characterization of human DNA sequences synthesized at the onset of S-phase. Nucleic Acids Res 15:10211–10232 [PMC free article] [PubMed]
24. Hoger TH, Zatloukal K, Waizenegger I, Krohne G (1990) Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin. Chromosoma 99:379–390 [PubMed] [Cross Ref]10.1007/BF01726689
25. Biamonti G, Giacca M, Perini G, Contreas G, Zentilin L, Weighardt F, Guerra M, Della Valle G, Saccone S, Riva S, Falaschi A (1992) The gene for a novel human lamin maps at a highly transcribed locus of chromosome 19 which replicates at the onset of S-phase. Mol Cell Biol 12:3499–3506 [PMC free article] [PubMed]
26. Giacca M, Zentilin L, Norio P, Diviacco S, Dimitrova D, Contreas G, Biamonti G, Perini G, Weighardt F, Riva S, Falaschi A (1994) Fine mapping of a replication origin of human DNA. Proc Natl Acad Sci USA 91:7119–7123 [PMC free article] [PubMed] [Cross Ref]10.1073/pnas.91.15.7119
27. Vergnes L, Peterfy M, Bergo MO, Young SG, Reue K (2004) Lamin B1 is required for mouse development and nuclear integrity. Proc Natl Acad Sci USA 101:10428–10433 [PMC free article] [PubMed] [Cross Ref]10.1073/pnas.0401424101
28. Kusic J, Kojic S, Divac A, Stefanovic D (2005) Noncanonical DNA elements in the lamin B2 origin of DNA replication. J Biol Chem 280:9848–9854 [PubMed] [Cross Ref]10.1074/jbc.M408310200

Articles from American Journal of Human Genetics are provided here courtesy of American Society of Human Genetics
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...