Overlapping syndromes in laminopathies: a meta-analysis of the reported literature.

Mutations on the LMNA gene are responsible for an heterogeneous group of diseases. Overlapping syndromes related to LMNA gene alterations have been extensively reported. Study scope is to perform a systematic analysis of the overlapping syndromes so far described and to try to correlate the clinical features to the associated genetic alterations. We evaluated all the dominant overlapping syndromes reported by means of a PubMed search and by the analysis of the main databases containing the pathogenic LMNA gene variations and the associated diseases. Metabolic alterations in association to skeletal and/or cardiac alterations proved to be the most frequent overlap syndrome. Overlapping syndromes are mostly associated to inframe mutations in exons 1, 2, 8 and 9. These data further improve the understanding of the pathogenesis of laminopathies.

Introduction

The LMNA gene, placed on chromosome 1q21-22, spans 12 exons and codes via alternative splicing for the A type lamins (1). A type lamins, which belong to the type V intermediate filaments and include lamins A, C, (the major isoforms), C2 and A 10 (the minor isoforms) (2), are characterized by an N-terminal head domain, a central α-helical rod domain, and a COOH-terminal ''tail domain'' (3). The rod domain is constituted by 4 regions with a typical α-helical organization (1A, 1B, 2A, 2B), that are interconnected by 3 intervening regions with the role of linkers (L1, L12, and L2). The portion of A type lamins with an α-helical organization presents the repeated sequence a-b-c-d-e-f-g with a and d being predominantly apolar and e and g polar residues; the heptad repeat sequence facilitates the interaction between lamins monomers and the formation of dimers via non covalent interactions among apolar residues located on the rod domain of different lamins (4). A type lamins dimers are also predicted to interact in a "head to tail" fashion, via non covalent interactions between regions of lamins with a different charge (4); the regions of lamin molecules predicted to allow the head to tail interaction, include two positively charged segments (the first from 1 to 28 residue, the second from residue 386 to residue 402) and two or three negatively charged segments (essentially, the N terminal and C terminal parts of the ROD domain) (4). The LMNA gene exon 1 yields the head domain and the first tract of the rod domain; exons 2-6 encode for what remains of the rod domain; exons 7-9 code for the portion of COOH-tail domain shared by both A and C lamins, including the region of nuclear localization signal (NLS) and the portions of lamins binding directly to DNA; the exon 10 contains the splicing site alternatively activated/ silenced for the production of A and C lamins; also, exon 10 codes for the remaining portion of the COOH terminal head domain of lamins C whilst part of exon 10 and the whole exons 11 and 12 yield for the lamins A terminus portion (5).

These proteins take part in the constitution of the nuclear lamina, a complex network of proteins located underneath the inner nuclear membrane (1). Lamins interact with several partners including nuclear envelope constituents, nucleoplasmic actin, chromatin, DNA, regulators of genes expression and molecules implicated in signal transduction (6). Such a plethora of interactions explains why A type lamins play a central role in the physiologic processes of cell life, including formation and homeostasis of the nucleus (7), apoptosis (8), repair (9), replication and transcription of DNA (10), regulation of chromosomal positioning (10). They are also involved in other important processes including metabolic, biochemical and signal transduction pathways (11, 12). Mutations on the Lamin A/C gene cause several defined clinical conditions, commonly termed as laminopathies, consisting in a heterogeneous group of diseases which include: the autosomal dominant and recessive forms of Emery Dreifuss muscular dystrophy (EDMD2 and EDMD3); the limb girdle muscular dystrophy 1B (LGMD1B); the congenital muscular dystrophy-L (CMDL); the dilated cardiomyopathy with conduction defects (DCM1A); the heart hand syndrome of Slovenian type (HHS); a recessive form of sensory-motor peripheral neuropathy (CMT2B); the familial partial lipodystrophy of the Dunnigan type (FPLD2); the Hutchinson- Gilford progeria syndrome (HGPS); the atypical form of Werner syndrome (WS); the restrictive dermopathy (RD) and the mandibuloacral dysplasia (MADA) (13). Several clinical complex entities, obtained by the concomitant presence in the same subject of different diseases related to LMNA gene mutations, have also been reported (14-60). Diseases characterized by the compromise of skeletal muscles and/or the heart are associated to mutations spread throughout the gene (14), while diseases primarily affecting the peripheral nerves, the metabolism, the bones or causing alterations of the ageing mechanisms tend to be associated to particular mutations and to cluster to peculiar regions of the gene (62-65). A full correlation between genetic alterations and clinical manifestations has not been established; however, genetic studies demonstrated the presence of a non random association between clinical manifestations and Lamin A/C gene alterations (66), and the presence of a clustering among neuromuscular phenotypes (46); in particular, phenotypes characterized by skeletal and cardiac compromise tend to be associated to LMNA gene alterations placed upstream of the NLS, while clinical entities affecting the metabolism, the bones or causing premature ageing syndromes tend to be caused by alterations located downstream of the NLS (66). It has also been reported that frameshift and nonsense mutations are frequently associated to late onset cardiac and skeletal phenotypes; the possible pathogenic mechanism invoked is haploinsufficiency due to non-sense mediated mRNA decay or a rapid degradation of the aberrant transcript (46). On the other hand, early onset phenotypes affecting the skeletal muscles are mostly associated to alterations of the LMNA gene maintaining the reading frame; in this case, the pathogenic mechanism hypothesized is the poison peptide effect caused by the altered properties of mutated lamins (46). In the present paper, the authors showed the results of a meta-analysis study aimed at evaluating the pathogenic bases and the clinical manifestations of the overlapping syndromes related to Lamin A/C gene and identifying a possible relationship between the complex phenotypes producing the overlapping syndromes and the mutations of LMNA gene.

Materials and methods

We searched, by indicating in PubMed as keywords LMNA and Lamin A/C, for all papers reporting the overlapping syndromes related to LMNA gene mutations. We also looked at the UMD-LMNA mutations databases (14) [http://www.umd.be/LMNA/ (Universal Mutation Database The UMD-LMNA mutations database)] and Leiden muscular Dystrophy database (15) [http://www.dmd.nl/ (Leiden Muscular Dystrophy pages©)] in order to identify all the dominant LMNA gene mutations associated to overlapping syndromes and the papers cited in the references. We prepared a database containing the mutations identified and the complex phenotypes associated to the mutations, specifying the tissues and organs compromised; we also indicated any alterations of metabolisms or signs of premature ageing. Then, we considered the type of mutation, its position on the gene and on the protein, the effect on the aminoacidic sequence and the possible pathogenic role (haploinsufficiency, poison peptide effect) exerted by the mutations. We also calculated the frequency of the mutations per exon, associated to the overlapping syndromes. Finally, COILS software was applied to predict the coiled-coil forming and the heptad position for each aminoacidic substitution evaluated. Coils software gives a score from 0 to 1 (0: no possibility of coiled coil; 1: highest probability of coiled coil), according to the probability for the aminoacid to belong to the coiled-coil region (67).

Results

Table 1 shows the complex phenotypes related to dominant LMNA gene mutations and the characteristics of the genetic alterations. Of the identified syndromes, 69 cases are associated to 46 dominant mutations, 41 of them proved to be unique missense mutations located in 41 different positions; 31 of the 41 missense mutations involve a polar aminoacid residue, which is mutated in an apolar aminoacid in about 50% of cases; the remaining 10 missense mutations involve an apolar residue and determine in half of the cases a substitution with an aminoacid with the same polarity. Among the missense mutations, we decided to include c. 1698+13 C > T, p. Arg566 +5Cys observed in exon10; we considered the mutation position as a terminal part of the gene region coding for C lamin. A higher frequency of mutations causing overlapping syndromes per exon was observed in exons 1-2, 8 and 9 (Table 2). About half of the missense mutations are located in coiled coils regions (predicted by COILS with a probability higher than 0.5), involving in about 20% of cases the positions a and d of the heptad repeat. Six missense mutations are predicted to occur within the head-to- tail interaction region as defined by Strelkov (P4R, T101, R28W, E33D, E358K, R386T). Figure 1 also summarizes the clinical phenotypes of the overlapping syndromes associated to the reported LMNA A/C gene missense mutations, related to lamin structure and its main partners.

Table 1.

Characteristics of complex phenotypes caused by dominant LMNA gene mutations and of the related genetic alterations.

Overlapping syndrome	LMNA Exon	Gene mutation	Protein mutation	Mutation type	Position in protein	Aminoacid substitution	COILS probability	Heptad position	Skeletal muscle phenotype	Cardiac phenotype	Nervous system (peripheral or central)	Metabolism	Ageing mechanisms	Bone/skeletal	Skin	Other	Mutation position
4	1	c. 3-12 del		Deletion	2		0		Yes		Neuropathy				-		Head
1	1	c.11C>G	P4R	Missense	4	P=apolare R=polare	0					Metabolic disturbances	Progerioid features	Bones abnormalities	Yes		Head
2	1	c.29C>T	T10I	Missense	10	T=polare I=apolare	0				-	High triglycerides + glycemia, lipoatrophy	Progerioid features		Thinned skin	Short stature	Head
2	1	c.29C>T	T10I	Missense	10	T=polare I=apolare	0					High triglycerides + glycemia, lipoatrophy	Progerioid features		Thinned skin	Short stature	Head
3	1	c.82C>T	R28W	Missense	28	R=polare W=apolare	0,997	a	Yes	Yes		FLPD2					Head
3	1	c.82C>T	R28W	Missense	28	R=polare W=apolare	0,997	a		Yes		FLPD2			-		Head
3	1	c.82C>T	R28W	Missense	28	R=polare W=apolare	0,997	a		Yes		FPLD			-		Head
4	1	c.99G>T	E33D	Missense	33	E=polare D=polare	1	f	Yes	Yes	Neuropathy				-		Head
4	1	c.99G>T	E33D	Missense	33	E=polare D=polare	1	f	Yes	Yes	Axonal neuropathy					Leukonichia	Head
4	1	c.99G>T	E33D	Missense	33	E=polare D=polare	1	f	Yes	Yes	Neuropathy				-	Leukonichia	Head
5	1	c.169G>C	A57P	Missense	57	A=polare P=apolare	1	b		Yes		Partial lipodystrophy	Atypical WS			Slopping shoulders hypogonadism (ovarian failure)	c-Fos binding domain 1
5	1	c.176T>G	L59R	Missense	59	L=apolare R=polare	1	d		Yes		Partial lipodystrophy	Atypical WS			Slopping shoulders hypogonadism (ovarian failure)	c-Fos binding domain 1
6	1	c.176T>G, de novo	L59R	Missense	59	L=apolare R=polare	1	d		Yes		-	Werner S		-		c-Fos binding domain 1
7	1	c.176T>G	L59R	Missense	59	L=apolare R=polare	1	d					Progerioid features	MADA			c-Fos binding domain 1
3	1	c.178C>G	R60G	Missense	60	R=polare G=apolare	1	e		Yes		Fat accumulation on face and neck and lipoatrophy on limbs			-		c-Fos binding domain 1
13	1	c.178C>G	R60G	Missense	60	R=polare G=apolare	1	e		Yes	Axonal peripheral neuropathy	Fat accumulation on face and neck and lipoatrophy on limbs			-		c-Fos binding domain 1
3	1	c.178C>G	R60G	Missense	60	R=polare G=apolare	1	e		Yes		FPLD2			-		c-Fos binding domain 1
3	1	c.184C>G	R62G	Missense	62	R=polare G=apolare	0,998	g		Yes		FPLD2			-		c-Fos binding domain 1
3	1	c.184C>G	R62G	Missense	62	R=polare G=apolare	0,998	g		Yes		FPLD2					c-Fos binding domain 1
3	1	c.274C>T	L92F	Missense	92	L=apolare F=apolare	1	a	Yes		-	FPLD			-		coil 1b
1	1	c.331G>A	E111K	Missense	111	E=polare K=polare	1	f				Metabolic disturbances	Progerioid features	Bones abnormalities	Yes		coil 1b
3	2	c. 398 G>T	R133L	Missense	133	R=polare L=apolare	1	g		Yes		Lipodystrophy+ hepatic steatosis+ high triglycerides		-	Skin changes		coil 1b
3	2	c.398G>T	R133L	Missense	133	R=polare L=apolare	1	g		Yes		Lipoatrophy, diabetes, liver steatosis			-	Leukomelanodermic papules	coil 1b
1	2	c.406G>C	D136H	Missense	136	D=polare K=polare	1	c				Metabolic disturbances	Progerioid features	Bones abnormalities			coil 1b
1	2	c.412G>A	E138K	Missense	138	E=polare K=polare	1	e				Metabolic disturbances	Progerioid features	Bones abnormalities			coil 1b
7	2	c. 412 G>A	E138K	Missense	138	E=polare K=polare	1	e					Progeria syndrome	MADA			coil 1b
6	2	428 C>T de novo	S143F	Missense	143	S=polare F=apolare	0,999	c	Yes	Yes		-	Progeria		-	Contractures	coil 1b
8	2	428 C>T de novo	S143F	Missense	143	S=polare F=apolare	0,999	c	Yes	Yes		-	Progeria	Ospteolysis, ospeopenia, midface hypoplasia		Leukomelanodermic papules	coil 1b
2	2	c.433 G>A	E145K	Missense	145	E=polare K=polare	0,999	e				Alterations of subcutaneous fat distribution	Atypical HGPS		-	Persisting coarse hair	coil 1b
4	2	c. 471 G>A	T157T	Synonymous	157		1	c	Yes		Neuropathy				-		coil 1b
1	2	c.475G>A	E159K	Missense	159	E=polare K=polare	1	e				Metabolic disturbances	Progerioid features	Bones abnormalities			coil 1b
2	2	c. 407A>G	D163G	Missense	163	D=polare G=apolare	1	b				Lipodystrophy, insulin resistence	Progeroid facies		Achantosis nigricans		coil 1b
4	5	c.864-867del; fs*190	H289fsX	Frameshift	190	H=polare A=apolare	1	e	Yes	Yes	Myopathic and neurogenic features, at muscle biopsy				-		E 1B 19K
3	3	c. 575 A>T	D192V	Missense	192	D=polare V=apolare	1	c		Yes		FPLD2			-		coil 1b
3	3	c. 575 A>T	D192V	Missense	192	D=polare V=apolare	1	c		Yes		FPLD			-		coil 1b
9	5	c.832 G>A	A278T	Missense	278	A=apolare T=polare	0,63	a	Yes	Yes					Achantosis nigricans		E 1B 19K
3	6	c. 1001-1003 del GCC p.Ser334- Ser334 del	S334del	Deletion	334		1	d	Yes			FPLD			-		Local interaction site
10	6	c. 1003 C>T	R335W	Missense	335	R=polare W=apolare	1	e	Yes	Yes		High triglycerides		Acro-osteolysis			Local interaction site
3	6	c.1045 C>T	R349W	Missense	349	R=polare W=apolare	1	e	Yes			Lipodystrophy			-		Local interaction site
3	6	c. 1045 C>T	R349W	Missense	349	R=polare W=apolare	1	e		Yes		FPLD			-		Local interaction site
10	6	c. 1072 G>A	E358K	Missense	358	E=polare K=polare	1	c	Yes	Yes		FPLD2 like phenotype		Midfacial hypoplasia; short stature		Broad nasal bridge, limited eye closure, uterine fibroids; Respiratory failure	Local interaction site
3	6	c.1157G >C	R386T	Missense	386	R=polare T=polare	0,638	g		Yes		FPLD			-		Emerin binding domain
3	7	c.1262 T>C	L421P	Missense	421	L=apolare P=apolare	0		Yes			IRS			-		NLS
3	7	c.1262 T>C	L421P	Missense	421	L=apolare P=apolare	0			Yes		Met syndrome			-		NLS
3	7	c.1315 C>T	R439C	Missense	439	R=polare C=polare	0		Yes			FPLD			-		PCNA interaction site
3	7	c.1315 C>T	R439C	Missense	439	R=polare C=polare	0		Yes			Met syndrome and fat distribution abnormalities			-		PCNA interaction site
14	7	c. 1318 G>A	V440M	Missense	440	V=apolare M=apolare	0		Yes					MADA			PCNA interaction site
3	7	c.1357 C>T	R453W	Missense	453	R=polare W=apolare	0		Yes			FPLD			-		c-Fos binding domain 2
14	8	c. 1411 C>T	R471C	Missense	471	R=polare C=polare	0		Yes					MADA			Actin binding domain (1)
3	8	c.1411C>G	R471G	Missense	471	R=polare G=apolare	0		Yes			FPLD2			-		Actin binding domain (1)
11	8	c.1444 C>T	R482W	Missense	482	R=polare W=apolare	0			-	Akinetohypertonic syndrome	FPLD2			-	Multinodular goiter, primary hyperaldosteronism	Actin binding domain (1)
3	8	c.1444 C>T	R482W	Missense	482	R=polare W=apolare	0		Yes			Lipodystrophy			-		Actin binding domain (1)
3	8	c.1444 C>T	R482W	Missense	482	R=polare W=apolare	0		Yes			FPLD			-		Actin binding domain (1)
2	8	c.1454C>G	P485R	Missense	485	P=apolare R=polare	0					FPLD	WS Actin binding domain		-		Actin binding domain (1)
4	9	c. 1496delC fsX49	A499V	Missense	499	A=apolare V=apolare	0		Yes	Yes	Neuropathy						PKC Alpha Binding site
3	9	c. 1516 C>G	H506D	Missense	506	H=polare D=polare	0		Yes		-	FPLD			-		PKC Alpha Binding site
4	9	c. 1535 T>C	L512P	Missense	512	L=apolare P=apolare	0		Yes		HNPP + Axonal Loss						PKC Alpha Binding site
12	9	c.1551G>A	Q517Q	Synonymous	517		0				Neuropathy	FPLD2					PKC Alpha Binding site
12	9	c. 1551G>A	Q517Q	Synonymous	517		0				Neuropathy	PLD					PKC Alpha Binding site
3	9	c.1580G>C	R527P	Missense	527	R=polare P=apolare	0		Yes	Yes		FPLD2					PKC Alpha Binding site
3	9	c.1580G>C	R527P	Missense	527	R=polare P=apolare	0		Yes	Yes		Lipoatrophy of trunk and proximal limbs					PKC Alpha Binding site
12	10	c. 1683 G>C	L561L	Synonymous	561		0				Neuropathy	FPLD2					PKC Alpha Binding site
13	11	c. 1711 A >T	S571C	Missense	571	S=polare C=apolare	0		Yes		Neuropathy	PLD					Lamin A tail
1	11	c.1762T>C	C588R	Missense	588	C=polare R=polare	0					Metabolic disturbances	Progerioid features	Bones abnormalities			Lamin A tail
3	11	c. 1772 G>T	C591F	Missense	591	C=polare F=apolare	0			Yes		FPLD2, liver steatosis			-		Lamin A tail
3	11	c.1772G>T	C591F	Missense	591	C=polare F=apolare	0		Yes	Yes		FPLD2				Polymenorrhea	Lamin A tail
3	11	c. 1804 G>A	G602S	Missense	602	G=apolare S=polare	0		Yes			IRS					Lamin A tail
1	11	c.1930C>T	p.R644C	Missense	644	R=polare C=polare	0					Metabolic disturbances	Progerioid features	Bones abnormalities			Lamin A tail

Table 2.

Distribution and frequency of the mutations causing the complex phenotypes distributed per exon.

Exon	Unique mutations	% unique mutations	Total mutations	% total mutations	Protein exon length	Total frequency normalized by exon length
1	11	23.91	21	30.43	119	25.58
2	8	17.39	11	15.94	52	30.66
3	1	2.17	2	2.90	42	6.90
4	0	0	0	0	57	0
5	2	4.35	2	2.90	42	6.90
6	4	8.70	5	7.25	73	9.93
7	4	8.70	6	8.70	73	11.91
8	4	8.70	7	10.14	36	28.18
9	5	10.87	7	10.14	40	25.36
10	2	4.35	2	2.90	30	9.66
11	5	10.87	6	8.70	80	10.87
12	0	0	0	0	9	0
TOT	46		69

Figure 1.

Causative missense mutations in the context of the lamin A/C protein organization and related overlapping syndromes.

Discussion

We report a meta-analysis describing the clinical features of all overlapping syndromes related to dominant LMNA gene mutations so far published and the possible relationship with the underlying genetic alterations. We identified at least 14 different overlapping syndromes due to dominant mutations on the Lamin A/C gene. As shown in tables 1 and 2, LMNA gene mutations may be associated to complex phenotypes obtained by the variable association of different phenotypes including metabolism disturbances, premature ageing syndromes, dermatologic changes, skeletal and cardiac compromise, nervous system alterations. The most frequent overlapping syndrome linked to LMNA gene alterations is the association between metabolic alterations and skeletal and/or cardiac involvement caused by inframe mutations spread throughout the gene. It is likely that the pathogenic mechanism underlying this condition is the poison peptide effect: as a matter of fact, all the mutations so far identified alter the biochemical properties of A type lamins, either perturbing their stability or modifying the possible interactions with the numerous binding partners (54).

The overlapping syndrome characterized by the association of skeletal and/or cardiac compromise with neuropathy and inconstant dermatologic abnormalities are caused by mutations spread throughout the gene; a possible pathogenic effect should be either a dominant negative or even a haploinsufficiency secondary to the production of un unstable mRNA or of a mutated protein, lacking the typical structure of intermediate filaments. For the third and fourth group of complex phenotypes, obtained by the variable association among muscle and/or heart disease, peripheral neuropathy, metabolism disturbances and concomitant presence of lipodystrophy, the few reports so far published do not consent any final correlation. However, the presence of either missense or silent mutations suggest that a dominant negative effect may play a major role in the pathogenesis of these two entities. For overlapping syndromes with variable association of MADA/bones alterations, metabolism abnormalities and premature ageing syndromes and other clinical entities such as dermatologic abnormalities, skeletal and/or cardiac diseases, the paucity of reports again do not consent any correlation with the mutation's position. Furthermore any direct correlation between clinical manifestations and LMNA gene mutations is hampered by the pleiotropic effect possibly exerted by Lamin A/C gene mutations (17-18, 36, 39, 53, 55, 69-70).

However, we can speculate that overlapping syndromes are mostly associated to inframe mutations able to alter the stability of A type lamins and the interactions with the numerous partners (54), causing a perturbation of the physiologic processes regulated by lamins on the different tissues. These data contribute to further improve the understanding of the pathogenic mechanisms of laminopathies.