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Heterogeneity of Nuclear Lamin A Mutations in Dunnigan-Type Familial Partial Lipodystrophy¹

Robarts Research Institute and Department of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5K8

Address all correspondence and requests for reprints to: Robert A. Hegele, M.D., Blackburn Cardiovascular Genetics Laboratory, Robarts Research Institute, 406–100 Perth Drive, London, Ontario, Canada N6A 5K8. E-mail: robert.hegele{at}rri.on.ca

	Abstract

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We previously identified a novel mutation, namely LMNA R482Q, that was found to underlie Dunnigan-type partial lipodystrophy (FPLD) and diabetes in an extended Canadian kindred. We have since sequenced LMNA in five additional Canadian FPLD probands and herein report three new rare missense mutations in LMNA: V440M, R482W, and R584H. One severely affected subject was a compound heterozygote for both V440M and R482Q. The findings indicated that 1) a spectrum of LMNA mutations underlies FPLD; 2) aberrant lamin A, and not lamin C, is likely to underlie FPLD, as R584H occurs within LMNA sequence that is specific for lamin A; 3) the V440M mutation may not cause lipodystrophy on its own; 4) compound heterozygosity for V440M and R482Q is associated with a relatively more severe FPLD phenotype, but not with complete lipodystrophy; and 5) variation in the severity of the phenotype might be related to environmental factors.

	Introduction

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PATIENTS WITH autosomal dominant Dunnigan-type partial lipodystrophy (FPLD; OMIM 151660) are born with normal fat distribution, but after puberty they lose sc fat from the extremities, trunk, and gluteal region (1, 2, 3). Also, excess fat may become deposited within the face, neck, back, and labia majora (1, 2, 3). Furthermore, patients with FPLD have normal stores of im, intraabdominal, intrathoracic, and bone marrow fat (1, 2, 3). Additional phenotypic findings are variable and include prominent musculature (especially noticeable in affected women), acanthosis nigricans, hirsutism, menstrual abnormalities, and polycystic ovarian disease (1, 2, 3). The biochemical hallmark of FPLD subjects is insulin resistance with hyperinsulinemia. FPLD subjects often develop diabetes, which can require large doses of insulin for glycemic control. FPLD subjects can also present with dyslipidemia and early coronary heart disease (1, 2, 3).

We first identified the LMNA R482Q mutation in several Canadian FPLD index cases (4), who have since been to shown to have a common ancestral pair. The genetic arguments that favored a causal relationship between LMNA R482Q and FPLD included its complete cosegregation with the FPLD phenotype, its presence in 22 FPLD subjects, its absence from 23 unaffected family members, its absence from 2000 alleles from normal subjects, and the conservation of the R482 residue throughout evolution (4). Interestingly, mutations affecting other residues of LMNA have been shown to cause the distinct clinical entities of autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) (5) and familial dilated cardiomyopathy (DCM) (6). Such findings suggest that mutations in different residues of lamin A/C have selective effects on the involved types of cells, tissues, and organs, resulting in profoundly variable clinical phenotypes. We have subsequently sequenced LMNA in five new Canadian FPLD probands, and herein report three novel rare LMNA missense mutations, namely V440M, R482W, and R584H.

	Subjects and Methods

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Patients

After informed consent had been obtained, we performed clinical evaluations and drew blood samples from five FPLD probands, namely New Brunswick subject NBFPLD-4–2, Alberta subject AFPLD-1–1, and Ontario subjects OFPLD-3–1, OFPLD-3–3, and OFPLD-4–1, all indicated by arrows in Fig. 1. Two of these independently ascertained probands, namely OFPLD-3–1 and OFPLD-3–3, were subsequently shown to be members of the same Ontario family. To determine the chromosomal phase of the two mutations found in subject NBFPLD-4–2, we later obtained a sample from her affected first cousin, NBFPLD-4–1, and her apparently unaffected mother, NBFPLD-4–3. We also obtained a sample from AFPLD-1–2, who was the clinically unaffected 3-yr-old daughter of AFPLD-1–1. All families were of northern European origin, whose ancestors had lived in Canada for several generations. Each adult family member was assessed for characteristic physical attributes of FPLD and provided a fasting serum sample for biochemical determinations, including insulin, C peptide, and creatine kinase.

View larger version (75K): [in this window] [in a new window]   Figure 1. FPLD family structures. Structure of study families and genotyping of LMNA mutations in codons 440, 482, and 584. Arrowheads flanking gels indicate the positions of mutant bands. Solid symbols indicate subjects who were definitely affected on clinical grounds. Arrowheads within the pedigrees indicate probands. For the codon 440 mutation in exon 7, the wild-type allele, V440, produced two BstUI fragments with sizes of 530 and 123 bp, whereas the mutant allele, M440 was not digested by BstUI. The invariant 123-bp fragment was not seen on this gel. For the codon 482 mutations in exon 8, the wild-type allele, R482, produced two variant MspI fragments with sizes of 480 and 69 bp, in addition to invariant fragments with sizes of 381, 81, and 59 bp. In addition, either mutant allele, W482 or Q482, produced a single MspI fragment with a size of 549 bp in addition to the invariant fragments. The invariant 81- and 59-bp fragments are not seen on this gel. DNA sequencing confirmed that the subjects NBFPLD-4–1 and -2 each had the R482Q mutation and that the subjects AFPLD-1–1 and -2 and OFPLD-3–1, -2, -3, -4, -5, and -6 each had the R482W mutation. For the codon 584 mutation in exon 11, the wild-type allele, R584, produced two BstUI fragments with sizes of 294 and 106 bp, whereas the mutant allele, H584, was not digested by BstUI.

DNA analysis

DNA was extracted from all family members. DNA was sequenced in subjects with a certain diagnosis of FPLD and in an unrelated, unaffected normal control subject. Primers for DNA amplification and sequencing were derived using published sequence information for all 12 exons, all intron-exon boundaries, and the 5'- and 3'-untranslated regions of LMNA (7). For each identified LMNA mutation, a rapid genotyping assay was developed.

The LMNA V440M mutation disrupted a BstUI recognition site; the diagnostic PCR method involved amplification of a 653-bp fragment that contained exon 7, using primers 5'-TCC TTC CCC ATA CTT AGG GC-3' and 5'-GTC TTG CCA CTC TCT CCC TG-3', followed by digestion of the amplification products and electrophoresis in 2% agarose. The wild-type allele, V440, produced two BstUI fragments with sizes of 530 and 123 bp, whereas the mutant allele, M440, was not digested by BstUI.

Both the LMNA R428W and R482Q mutations disrupted a MspI recognition site; the diagnostic PCR method involved amplification of a 1069-bp fragment that contained exon 8, using primers 5'-GCA AGA TAC ACC CAA GAG CC-3' and 5'-ACA CCT GGG TTC CCT GTT C-3', followed by digestion of the amplification products with MspI and electrophoresis in 2% agarose. For both mutations, the wild-type allele, R482, produced two variant MspI fragments with sizes of 480 and 69 bp, in addition to invariant fragments with sizes of 381, 81, and 59 bp. However, either mutant allele, W482 or Q482, produced a single MspI fragment with a size of 549 bp in addition to the invariant fragments. DNA sequencing was used to confirm the specific nucleotide change in each family member.

The LMNA R584H mutation disrupted a BstUI recognition site; the diagnostic PCR method involved amplification of a 400-bp fragment that contained exon 11, using primers 5'-AGT GGT CAG TCC CAG ACT CG-3' and 5'-CAA GGC CAC CTC GTC CTA C-3', followed by digestion of the amplification products and electrophoresis in 2% agarose. The wild-type allele, R584, produced two BstUI fragments with sizes of 294 and 106 bp, whereas the mutant allele, H584, was not digested by BstUI.

	Results

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Genetic studies

We evaluated five probands and selected relatives from four Canadian kindreds with FPLD (Fig. 1). DNA sequencing revealed that all probands with FPLD had a rare sequence variant in their genomes, each of which was predicted to alter one amino acid in the lamin A protein. Subject NBFPLD-4–2 was a compound heterozygote for both a rare GA change at codon 440 in exon 7, which predicted the replacement of valine (GTG) by methionine (ATG) (Fig. 2a) and a GA change at codon 482 in exon 8, which predicted the replacement of arginine (CGG) by glutamine (CAG; Fig. 2c). Sequencing showed that subject NBFPLD-4–1 was a simple heterozygote for the previously reported GA change at codon 482 in exon 8 (4), which predicted the replacement of arginine (CGG) by glutamine (CAG; Fig. 2a), and had normal sequence at codon 440. Sequencing showed that subject NBFPLD-4–3 was a simple heterozygote for the GA change at codon 440 in exon 7, which predicted the replacement of valine (GTG) by methionine (ATG; Fig. 2a), and had normal sequence at codon 482. This confirmed that the V440M and R482Q mutations in subjects NBFPLD-4–2 were heteroallelic on opposite chromosomes.

View larger version (28K): [in this window] [in a new window]   Figure 2. DNA sequencing of LMNA mutations in FPLD. Direct DNA sequencing for LMNA V440M, R482W, R482Q, and R584H mutations is shown. The particular study subject was NBFPLD-4–2 in a and c, OFPLD-3–1 in b, and OFPLD-4–1 in d.

Genotyping confirmed that subject NBFPLD-4–1 was a simple Q482/R482 heterozygote and that subject NBFPLD-4–3 was a simple M440/V440 heterozygote, whereas subject NBFPLD-4–2 was a compound heterozygote for both M440/V440 and Q482/R482 (Fig. 1). This indicated that the two mutations in subject NBFPLD-4–2 indeed segregated independently. Subjects AFPLD-1–1 and -2 and OFPLD-3–1, -2, -3, -4, and -5 were each simple heterozygotes for W482/R482. Subject OFPLD-4–1 was a simple heterozygote for H584/R584 (Fig. 1). In sharp contrast, all normal control alleles were homozygous for normal restriction digestion patterns at codons 440, 482, and 584. This provided strong statistical evidence (by Fisher’s exact test, P < 10^-10) that these mutations were not simply common polymorphisms. The as yet clinically unaffected 3-yr-old child, AFPLD-1–2, was clearly a carrier of the LMNA W482 allele by genotyping (Fig. 1).

Phenotypic characteristics in subjects with LMNA mutations

Clinical data from the five probands and six adult relatives are shown in Table 1. There was variability in the clinical expression among family members with the same mutation. For example, in OFPLD-3, most subjects with definite FPLD and LMNA R482W had diabetes and dyslipidemia, except for OFPLD-3–2, who had been a lifelong athlete and still walked 5 km daily, and OFPLD-3–6, a previously undiagnosed male. Also, subject NBFPLD-4–2, who was a compound heterozygote for LMNAQ and V440M, had a much more severe phenotype than simple heterozygotes for R482Q, including profound insulin resistance, severe diabetes, aggressive cardiovascular disease, and the imminent need for lower limb amputation.

View this table: [in this window] [in a new window]   Table 1. Clinical and biochemical attributes of FPLD probands and selected family members from Fig. 1

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We present evidence that mutations in LMNA cause FPLD, associated with diabetes and hyperlipidemia. Mutations were found in all four Canadian families studied, and each mutation was absent from more than 2000 normal chromosomes. The data indicate that a variety of mutations affecting specific residues in the tail domain of LMNA underlie the clinical phenotype of FPLD. The data confirm an important role for nuclear membrane proteins in adipocyte differentiation and abnormal metabolism, in addition to the pivotal function for these proteins in skeletal and cardiac myocytes that has been suggested by the mutations in EDMD-AD and DCM (5, 6).

The FPLD mutations in LMNA are instructive for several reasons. For example, 1 of them, LMNA R584H, occurs specifically within the sequence encoding lamin A (Fig. 3). Alternative splicing of the 12 exons of LMNA results in messenger ribonucleic acid species that encodes at least 4 closely related proteins, including lamins A, A10, C, and C2 (8). Lamin A and C are coexpressed in the nuclear envelope of many tissues, including adipocytes. The first 566 amino acids of lamins A and C (encoded by exons 1–10) are identical, whereas the carboxyl-termini of these proteins differ in length and amino acid sequence. The LMNA R584H mutation in exon 11 selectively altered the carboxyl-terminus of lamin A. In contrast, the DCM R571S mutation in LMNA exon 10 selectively altered the carboxyl-terminus of lamin C (6). This suggests that altered lamin A specifically underlies FPLD, whereas altered lamin C specifically underlies DCM (Fig. 3). It is possible that mutations in the carboxyl-terminus could perturb lamin stability or prevent head to tail polymerization of lamin filaments. Selective cellular involvement might be due to restricted tissue expression of the LMNA R584H and R571S mutations and/or to distinctive functions of lamins A and C.

View larger version (9K): [in this window] [in a new window]   Figure 3. LMNA genomic map and disease mutations. LMNA encodes different lamin isoforms, including lamins A and C, through alternative splicing of the 12 exons. Exons 1–10 contain sequences that are represented in all isoforms (solid shading). Exon 10 also contains sequences that encode lamin C specifically (brick shading). Exons 11 and 12 contain sequences that encode lamin A specifically (horizontal bars). The 3'-untranslated region becomes part of lamin A messenger ribonucleic acid. Also shown the positions of the mutations, indicated by the single letter amino acid codes, that are found in FPLD (above the linear map) and EDMD-AD and DCM (below the linear map).

The LMNA V440M variant appeared to have a more subtle relationship with the FPLD phenotype than the other three LMNA mutations in FPLD. For instance, subject NBFPLD-4–3, who had LMNA V440M alone, had not been diagnosed with lipodystrophy or diabetes and was apparently healthy. However, the LMNA V440M variant was also a very rare mutation, as it was absent from all normal chromosomes. A possible phenotypic influence of LMNA V440M was suggested by the observation in subject NBFPLD-4–2, who was a compound heterozygote for both LMNA R482Q and V440M. Compared to simple heterozygotes for R482Q, such as subject NBFPLD-4–1 and all of the affected subjects in our previous report (4), subject NBFPLD-4–2 was more severely affected, with profound insulin resistance, diabetes, aggressive vascular disease, and the imminent need for lower limb amputation. These findings suggest that LMNA V440M may not function through a dominant negative mechanism like the other mutations, but might instead modulate the severity of the phenotype in a subject with another LMNA mutation. However, the other three LMNA mutations in FPLD, the four reported LMNA mutations in EDMD-AD, and the five reported LMNA mutations in DCM each appeared to function through a dominant negative mechanism (5, 6). In particular, the point mutations in the rod and tail domains of lamin that lead to changes in charge or hydrophobicity would be expected to disrupt specific functions, but not necessarily to act as null alleles (5, 6). We are in the process of extending kindred NBFPLD-4 to find more subjects with LMNA V440M to to determine its associated phenotype.

The FPLD phenotype appears to be modulated by nongenetic factors. For example, in OFPLD-3, all subjects with definite FPLD and LMNA R482W had diabetes and dyslipidemia, except for OFPLD-3–2, who had been a lifelong athlete and still walked 5 km daily. This suggested that environmental factors modulate susceptibility to the metabolic complications in FPLD in carriers of mutant LMNA and could have therapeutic implications for mutation carriers who are identified early in the course of the disease. Other therapeutic strategies for carriers of LMNA mutations for FPLD are suggested from animal experiments, in which the administration of leptin attenuated the development of lipodystrophy in induced-mutant mice with susceptibility to this phenotype (9). Such interventions might have future relevance for subjects such as AFPLD-1–2, who is a mutation carrier at the age of 3 yr, but is probably years away from developing the obvious clinical manifestations of FPLD.

A complicating attribute in the relationship between phenotype and genotype in FPLD is that puberty is clearly related to the onset of adipocyte degeneration (1, 2, 3). This suggests that changes in the hormonal or metabolic milieu might trigger the expression of the specific histological and anatomical changes in carriers of the mutant LMNA. Such complexity might overwhelm current in vitro functional assays of lamin A interactions with other laminar proteins to fully explain the phenotypic changes observed in FPLD; other in vitro models of lamin function might be required.

In summary, we report that FPLD results from a variety of missense mutations in LMNA, which had in common their impact specifically on the tail domain of lamin A. We observed heterogeneity in the FPLD phenotype and have provided some evidence that both genetic and nongenetic factors can affect the severity of clinical expression of the metabolic complications of FPLD in subjects with the same mutation in LMNA. The observations confirm that mutations affecting a nuclear membrane protein can underlie adipocyte wasting and metabolic phenotypes. Taken together with the observations from EDMD-AD and DCM patients with LMNA mutations, it would appear that the position of the particular mutation in LMNA is a critical determinant of the affected cell, tissue, and organ types. This raises the possibility that LMNA mutations could underlie other diseases characterized by the wasting of specific cell types.

	Acknowledgments

 
We thank Drs. W. Dykeman, M. C. McSween, and P. Grundy for referring their patients to us.

	Footnotes

 
¹ This work was supported by grants from the Medical Research Council of Canada (MT13430) and the Canadian Genetic Diseases Network.

² Career Investigator with the Heart and Stroke Foundation of Ontario.

Received December 16, 1999.

Revised January 26, 2000.

Accepted February 15, 2000.

	References

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4. Cao H, Hegele RA. 2000 Nuclear lamin A/C R482Q mutation in Canadian kindreds with Dunnigan-type familial partial lipodystrophy. Hum Mol Genet. 9:109–211.[Abstract/Free Full Text]
5. Bonne G, DiBarletta MR, Varnous S, et al. 1999 Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat Genet. 21:285–288.[CrossRef][Medline]
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7. Lin F, Worman HJ. 1993 Structural organization of the human gene encoding nuclear lamin A and nuclear lamin C. J Biol Chem. 268:16321–16326.[Abstract/Free Full Text]
8. Stuurman N, Heins S, Aebi U. 1998 Nuclear lamins: their structure, assembly and interactions. J Struct Biol. 122:42–66.[CrossRef][Medline]
9. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL. 1999 Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature. 401:73–76.[CrossRef][Medline]

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