Nucleophilic A-type lamins A and C, usually referred to as lamin A/C, are integral components of the nuclear lamina, a multiprotein filamentous structure associated with the inner nuclear membrane. Because of this crucial structural role, lamins A/C are expressed in virtually all mammalian cells, being expressed in equal quantities in the nuclear lamina. Lamins A and C are encoded by the LMNA gene (12 exons on chromosome 1q22; transcript NM_170,707) and are generated by alternative splicing of exon 10. Prelamin A is extensively processed and cleaved at C-terminal residues by ZMPSTE24, leading to the mature lamin A protein (Dittmer and Misteli, 2011; Genschel and Schmidt, 2000; Casasola et al., 2016). Thus, lamin A (canonical sequence of 664 amino acids; NP_001393912.1; UniProtKB: P02545-1; https://www.uniprot.org/uniprot/P02545) and lamin C (572 amino acids; NP_001393913.1; UniProtKB: P02545-2) share their first 566 N-terminal amino acids, with lamin C having differences in sequence for residues 567–572 and lacking the residues 573–664 in comparison to lamin A.

Pathogenic gene variants in the LMNA gene cause a group of heterogeneous human diseases collectively termed laminopathies (Crasto et al., 2020). These disorders include cardiac diseases, Emery-Dreifuss muscular dystrophies, limb-girdle muscular dystrophy (renamed as Emery-Dreifuss muscular dystrophy 2; EDMD2, OMIM: #181350) (Straub et al., 2018), Charcot-Marie-Tooth type 2, progeroid syndromes, and familial partial lipodystrophies (Genschel and Schmidt, 2000; Nishiuchi et al., 2017; Florwick et al., 2017). In total, more than 600 LMNA disease-causing gene variants have been reported (https://www.hgmd.cf.ac.uk/), resulting in at least 15 different human pathological phenotypes (Crasto et al., 2020; Fernandez-Pombo et al., 2023a). Although the mechanisms by which LMNA variants result in such multiple phenotypes remain unknown, it is increasingly recognized that lamins A/C have biological functions exceeding mere structural roles in the nuclear lamina, including involvement in gene transcription regulation, chromatin organization, and DNA replication (Crasto et al., 2020).

Lipodystrophies are a group of rare diseases characterized by severe reduction and redistribution of adipose tissue. Depending on the extent of adipose paucity, lipodystrophies can be generalized or partial, and depending on their etiology, they have been classically classified as congenital, familial, or acquired (Brown et al., 2016; Cortés et al., 2014; Cortés and Santos, 2019). Congenital generalized lipodystrophy (CGL) is the most extreme form of human genetic leanness and is caused by homozygous pathogenic variants in the AGPAT2, BSCL2, CAV1, or PTRF (CAVIN1) genes (Cortés et al., 2014; Cortés et al., 2009; Santos and Cortés, 2021). Patients with CGL develop severe insulin resistance, diabetes, hepatic steatosis, hypertriglyceridemia, and hyperphagia (Akinci et al., 2018). On the other hand, familial partial lipodystrophy (FPLD) is characterized by loss of fat restricted to some anatomic regions, typically the extremities, with preservation or even enlargement of the remaining adipose depots, typically in the abdomen. Patients with FPLD also develop insulin resistance, diabetes mellitus, fatty liver, and dyslipidemia, but usually to a lesser severity than those with CGL (Vasandani et al., 2022) without exhibiting hyperphagia (Monteiro et al., 2017). FPLD has been linked to pathogenic variants in the LMNA, CIDEC, LIPE, PCYT1A, PPARG, AKT2, POLD1, and KCNJ6 genes (Brown et al., 2016). The commonest mode of inheritance of FPLD is autosomal dominant, with very rare cases of homozygous disease-causing variants reported in specific populations (Fernandez-Pombo et al., 2023a). The more frequent FPLD subtype is caused by heterozygous LMNA pathogenic variants, termed FPLD type 2 (FPLD2) or Dunnigan variety (Vasandani et al., 2022). This lipodystrophy is characterized by the absence of adipose tissue in the limbs, muscle hypertrophy and preserved or increased adiposity in the face, neck, and trunk. FPLD has been reported to be ∼3–4 times more frequent in women than in men (Akinci et al., 2018; Fernandez-Pombo et al., 2023b).

Herein, we describe a female patient with partial lipodystrophy, with adipose depletion in the extremities but preservation in the face/trunk, hypertriglyceridemia, and severe insulin resistance. Exome sequencing revealed two heterozygous missense de novo pathogenic variants in cis within the LMNA gene: p.Y481H and p.K486N. These rare single nucleotide variants have been previously associated with dominant limb-girdle muscular dystrophy (Emery-Dreifuss muscular dystrophy 2) (Brown et al., 2016; Kitaguchi et al., 2001) and partial lipodystrophy (Shackleton et al., 2000), and reported to affect lamin A/C function by in vitro studies (Anderson et al., 2021; Simon et al., 2013). Using molecular modeling, molecular dynamics simulation, and predictive bioinformatic analysis, we propose mechanisms by which these variants lead to lamin A/C Ig-like domain instability and affect lamin A/C function. This is the first report of a sporadic partial lipodystrophy syndrome of genetic origin caused by concurrent de novo pathogenic variants within the same gene.

This research is based on a case-parent trio ascertained via the proband, who is a 24-year-old female patient referred to our center because of extreme leanness, hyperinsulinemia, oligomenorrhea, and hypertriglyceridemia (Table 1). The patient was born to nonconsanguineous parents and works as an administrative assistant. She was concerned about their general health status and future implications but felt that her current clinical status did not interfere with her daily life. Physical exploration revealed a notorious deficiency of subcutaneous adipose tissue in both the upper and lower extremities, with preservation of truncal and facial adiposity. She presented intense acanthosis nigricans at the base of the neck and axillar folds, hirsutism, and diffuse acneiform skin lesions. Despite the patient’s regional lipoatrophy, body weight remained within the normal range. Both parents showed normal adiposity and did not present metabolic abnormalities. The patient had no family history of low body weight, diabetes, dyslipidemia, or cardiovascular or muscle disease. Because of oligomenorrhea, she was prescribed oral contraceptives (ethynyl estradiol 0.035 mg, cyproterone 2 mg). Clinical assessment revealed no muscle pain or weakness.

Body composition with dual-energy X-ray absorptiometry (DXA; GE-Healthcare Lunar DPX-NT scanner) revealed a fat mass of 15.4%. Android-to-gynoid (A/G) ratio (android fat mass/gynoid fat mass), trunk/limb, and trunk/leg ratios exceeded the upper limits of 90% reference intervals using the same DXA equipment (Imboden et al., 2017), indicating a fat distribution pattern characterized by central adiposity and excessive accumulation of fat in the trunk relative to the arms and legs. The bone mineral density was 1.08 g/cm² (z-score = 0). Abdominal ultrasonography revealed splenomegaly and diffuse liver enlargement associated with mild steatosis. Liver function tests were within the normal range, including transaminases, total bilirubin, and alkaline phosphatase levels.

Massively parallel sequencing (MPS) was used to interrogate gene variants in the proband´s exome from DNA isolated from blood leukocytes. We used SureSelect XT v6 (Agilent Technologies) for exome capture. Libraries were sequenced on the Illumina HiSeq equipment in Theragen-Etex (http://www.theragenetex.net/) to a raw average sequencing depth of >100X and an exome coverage of 10X >98%. Each of the exomes of the trio resulted in >76 million reads with >96% of the Q20 quality score (read length = 101 × 2). Alignments were made using the hg19 human genome assembly (GRCh37). Sequence primary data analysis, including read alignment and variant calling, was also performed by Theragen-Etex. Mutations were confirmed via Sanger sequencing.

We used publicly available bioinformatic tools (Alphamissense, SIFT, Polyphen-2, CADD, REVEL, MetaLR, and MutationAssessor) to predict the effect of missense variants p.Y481H and p.K486N on protein function (available at https://alphamissense.hegelab.org/ and https://www.ensembl.org/). Homology analysis was carried out with WebLogo (https://weblogo.berkeley.edu/), PhyloP (https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=cons100way), CONSURF (https://consurf.tau.ac.il/), and EVE (https://evemodel.org/proteins/LMNA_HUMAN) programs (Lindquist et al., 2022). The supplementary material shows extended methods for the molecular modeling and dynamics simulation of Lamin A/C mutants.

Herein, we report two concurrent de novo missense pathogenic variants within the LMNA gene (p.Y481H and p.K486N) in a patient with partial lipodystrophy. These represent a novel and intriguing genetic alteration that underscores the genetic complexity and diversity contributing to laminopathies. To the best of our knowledge, this is the first case of partial lipodystrophy syndromes of genetic origin caused by multiple simultaneous de novo pathogenic variants within the same gene. The pathogenicity of p.Y481H and p.K486N variants in the LMNA gene found in our study is supported by previous functional analysis (Vasandani et al., 2022) and our molecular modeling and DNA evolutionary analysis. It has been proposed that LMNA pathogenic variants cause partial lipodystrophy phenotypes via a) disruption of the structural integrity of the nuclear lamina, with special intensity in tissues exposed to frequent mechanical forces (such as muscle of limbs), and b) distortion of gene expression and chromatin organization, leading to multisystem damage, including FPLD phenotype (Xiao et al., 2023).

From a clinical perspective, our patient showed marked limb lipoatrophy with preserved adipose tissue in the face, neck, and trunk. Metabolic abnormalities included severe insulin resistance, hypertriglyceridemia, low HDL cholesterol, and fatty liver. DXA analysis revealed A/G, trunk/limb, and trunk/leg ratios indicating fat accumulation in the trunk relative to the limbs and a tendency toward central adiposity, all of which features of Dunnigan-type lipodystrophy. In contrast to patients with CGL, our patient had no glycemic disorders or hyperphagia, likely because of her remaining circulating leptin levels. Leptin deficiency seems to be the single most important determinant of the severity of metabolic abnormalities in patients with lipodystrophy and the main predictor of the clinical response to metreleptin therapy (Oral et al., 2019). LMNA p.R482X variants are the commonest cause of FPLD2 (Akinci et al., 2019), and their typical presentation includes limb lipoatrophy combined with preserved or excessive adiposity in the face, neck, interscapular space, intra-abdominal, and vulva. Patients with less frequent LMNA variants (p.R644C, p.R582H, p.R582C, p.T528M, p.N466D, p.D47N, p.R471G) are referred to as having an “atypical” FPLD and show a milder phenotype (Fernandez-Pombo et al., 2023a; Araújo-Vilar et al., 2021). Although we did not find reports on the clinical presentation of patients with FPLD2 due to the LMNA variants Y481H and K486N, it resembles classical FPLD2 due to p.R482 variants. This phenotypical similarity is in line with the structural consequences that we found in the molecular modeling between the double mutant LMNA Y481H/K486N and classical R482Q and R482W variants.

Although FPLD is typically attributed to single causative mutations, combined alterations such as those found in patients with compound heterozygous mutations in the LMNA gene, underscore the complex genotype/phenotype relationships in laminopathies. For example, patients with FPLD that simultaneously carries LMNA p.S583L and p.T528M mutations showed a much more severe lipoatrophy than those harboring LMNA p.S583L only, while carriers of p.T528M variant do show not lipodystrophy at all (Savage et al., 2004).

Missense gene variants in the lamin A/C C-terminal end as well as pathogenic variants in the Ig domain (amino acid positions 430–545) that prevent appropriate protein folding and stability have been associated with muscular dystrophies and lipodystrophies (Dhe-Paganon et al., 2002; Krimm et al., 2002; Östlund et al., 2001; Scharner et al., 2014). LMNA p.Y481H was first found in a Japanese patient with juvenile-onset proximal muscle weakness who belonged to a large family with multiple relatives affected by cardiac arrhythmias, suggesting an autosomal dominant pattern of inheritance (Kitaguchi et al., 2001). This disease was initially termed limb-girdle muscular dystrophy type 1B (LGMD1B) and subsequently renamed as Emery-Dreifuss muscular dystrophy 2 (EDMD2) (Straub et al., 2018). Interestingly, p.R482Q was reported in Emery-Dreifuss muscular dystrophy type 3 (OMIM: #616516) (Wiltshire et al., 2013), and in five Canadian probands affected with FPLD2 (OMIM: # 151660) (Cao and Hegele, 2000). Inherited disease-causing pathogenic variants in heterozygosity in the LMNA gene were detected for FPLD2 by positional cloning and identified in a tight cluster in different families as p.R482W, p.R482Q, p.R482L and p.K486N18. However, all the pathogenic variants reported until now were found individually across different families. In contrast, the case reported herein shows two simultaneous de novo mutations in the LMNA gene in cis (p.Y481H and p.K486N). The concurrence of these double de novo pathogenic variants represents a new genetic finding in FPLD2.

Both p.Y481H and p.K486N were previously evaluated through in vitro functional assays. The functional effect of p.Y481H was also assessed in HEK293 cells, which showed misfolding of lamin A with no protein aggregation (Anderson et al., 2021). In vitro analysis of p.K486N showed reduced interaction between the lamin A tail and its major posttranslational modifier ubiquitin-like modifier-1 (encoded by SUMO1 gene) in Cos-7 cells (Anderson et al., 2021; Simon et al., 2013). Our dynamic simulation analyses suggest that p.Y481H and p.K486N, individually or combined, decrease the structural stability of the lamin A/C Ig-like domain, supporting the hypothesis that such variants compromise lamin A/C function and possibly the structural integrity of the nuclear lamina. Our findings suggest that the double mutation Y481H/K486N induces significant structural alterations in the protein, exceeding the effects of isolated single mutations. Increased RoG indicates a less compact and more expanded protein conformation, while elevated SASA at both protein and residue levels suggests enhanced solvent exposure and potential destabilization of the protein structure. The similarity of these structural changes to those observed in the pathogenic controls R482W and R482Q, also associated with FPLD, suggests that the double mutant Y481H/K486N reported herein has pathogenic implications.

The 500–520 loop plays a crucial role in maintaining the structural integrity and functional capacity of the Ig-like fold within the tail domain of lamin A/C. This region supports key interactions necessary for lamin polymerization and its association with nuclear components, including chromatin (Magracheva et al., 2009). Specifically, Pro508 is part of a groove essential for disulfide bond involving Cys522, which is critical for lamin A/C dimerization, which is necessary for the structural integrity of lamin A/C and its interaction with nuclear components (Ahn et al., 2022). In addition to its structural role, the 500–520 loop contributes indirectly to DNA binding and chromatin interactions by stabilizing the protein’s architecture. This domain has been shown to participate in DNA binding, and residues such as Lys515, Thr519, and Gln517 are key for interacting with DNA (Stierlé et al., 2003; Khromova et al., 2019). Therefore, disruption or mutations in this loop can compromise these interactions, leading to structural defects in the nuclear lamina and the onset of laminopathies, such as FPLD. Our results suggest that the Y481H and K486N mutations synergistically exacerbate structural destabilization, which could contribute to disease phenotypes (Krimm et al., 2002; Magracheva et al., 2009; Bidault et al., 2013). While bioinformatic functional prediction algorithms also support this conclusion, further functional studies are warranted to explore the biological implications of these structural changes.

De novo somatic or germline pathogenic variants are a wide category of genetic variations distinct from inherited variants occurring during gametogenesis and post-zygotically (Acuna-Hidalgo et al., 2016; Mohiuddin et al., 2022). Approximately 6.5% of de novo variants are not germline, while mosaics may occur post-zygotically derived from low-level mosaicism already present in parents (Acuna-Hidalgo et al., 2015). Our analysis suggests that both p.Y481H and p.K486N in the LMNA gene are germline de novo pathogenic variants with no mosaicism. The estimated single-nucleotide variant (SNV) mutation rate in humans leading to de novo variants is estimated to be ∼1.2 × 10⁻⁸ mutations per position per haploid genome, yielding approximately 70 new mutations in the diploid human genome, with only 1 or 2 mutations occurring within the exome (Besenbacher et al., 2016). On a megabase scale, germline mutation rates are determined by the parental age at conception, sequence context, methylation status, accessibility of the global DNA repair machinery, compaction of heterochromatin, and replication timing (Besenbacher et al., 2016; Gonzalez-Perez et al., 2019; Ségurel et al., 2014). Interestingly, late-replicating loci are typically clustered in heterochromatin and localize to the nuclear periphery, accumulating more germline and somatic mutations compared to early-replication loci, which are located near the nucleus center (Gonzalez-Perez et al., 2019; Du et al., 2019). Additional factors defining chromatin features (nucleosomes, transcription factors, and gene structure) are also important determinants of de novo germline mutations (Gonzalez-Perez et al., 2019). In this evolutionary context, hypermutability events within a gene may represent a mechanism with the potential to generate multiple pathogenic gene variants (Crow, 1997). Multiple closely spaced inherited and de novo pathogenic variants separated by <100 nucleotides have been reported (Chen et al., 2009) and referred as “mutational clusters” as opposed to multiple mutational events occurring in the same genome location in different individuals (referred to as “mutational hotspots”) (Acuna-Hidalgo et al., 2016).

Although single de novo pathogenic variants have been previously reported in two patients with a neonatal-onset type of lipodystrophy (Garg, 2011), the case described herein is appealing from a genetic standpoint because it occurs with two de novo pathogenic variants separated by only 17 nucleotides within exon 8 of the LMNA gene (5 amino acid residues in the lamin A/C protein). The generation of de novo variants may occur either as independent events or in clusters close to each other more frequently than expected by chance. Approximately 3% of human de novo single nucleotide variants are part of multinucleotide regions spanning a median distance of 525 bp (Besenbacher et al., 2016). A case of Ullrich congenital muscular dystrophy caused by two de novo pathogenic variants in cis (27 nucleotides apart) within the COL6A3 gene was recently reported (Shimomura et al., 2019). De novo pathogenic variants can be involved in multiple diseases and are, on average, more deleterious than inherited genetic variations (Veltman and Brunner, 2012). Interestingly, multiple de novo missense mutations have also been reported in oncogenes of cancer patients’ tumors (Saito et al., 2021).

In summary, herein we describe the clinical and genetic characteristics of a patient with partial lipodystrophy due to two simultaneous de novo missense pathogenic variants in cis located in exon 8 of the LMNA gene: p.Y481H and p.K486N. Both variants were previously but independently and separately reported in muscle dystrophies and partial lipodystrophies and were causally related to lamin A/C dysfunction through in vitro studies. Molecular dynamics simulations provide additional insights into how these specific pathogenic variants may lead to structural instability of the lamin A/C Ig-like domain. Importantly, the clinical presentation of our patient does not correspond to the classic form of acquired partial lipodystrophy (Barraquer-Simmonds syndrome), whose etiopathogenesis remains unknown; and thus we propose to refer to this case as sporadic partial lipodystrophy of genetic origin. This sporadic form of disease caused by multiple de novo pathogenic variants expands the genetic and phenotypic spectrum of partial lipodystrophy syndromes and laminopathies.