Montenegro Junior et al. Diabetol Metab Syndr (2020) 12:28 Diabetology & https://doi.org/10.1186/s13098-020-00538-y Metabolic Syndrome RESEARCH Open Access Leu124Serfs*26, a novel AGPAT2 mutation in congenital generalized lipodystrophy with early cardiovascular complications Renan Magalhães Montenegro Junior1* , Grayce Ellen da Cruz Paiva Lima1, Virgínia Oliveira Fernandes1, Ana Paula Dias Rangel Montenegro1, Clarisse Mourão Melo Ponte1, Lívia Vasconcelos Martins1, Daniel Pascoalino Pinheiro2, Maria Elisabete Amaral de Moraes2, Manoel Odorico de Moraes Filho2 and Catarina Brasil d’Alva1 Abstract Background: Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by the near-total loss of subcutaneous adipose tissue soon after birth, resulting in ectopic fat deposition and severe meta- bolic disturbances. Most cases are caused by AGPAT2 or BSCL2 gene mutations. We aimed to report two unrelated CGL patients with a novel frameshift mutation in AGPAT2 (p.Leu124Serfs*26). Methods: Clinical features and laboratory were obtained by medical interview and medical records review. DNA was extracted, amplifed and sequenced. Mutation Taster was used to estimate the potential biological impact of the AGPAT2 mutations on the protein function. Results: Patient 1: a 30-year-old woman with lipodystrophy phenotype at birth and diagnosis of diabetes at age 13 presented with severe hypertriglyceridemia and pancreatitis at age 17, hypertension and albuminuria at age 18, prolif- erative diabetic retinopathy with visual loss at age 25, and an acute myocardial infarction due to multivessel coronary disease during a hospitalization for forefoot amputation at age 29. At this time, she required hemodialysis due to end- stage renal disease. Patient 2: a 12-year-old girl with lipodystrophy phenotype and hypertriglyceridemia detected in the frst year of life and abnormalities in the global longitudinal strain, evaluated by speckle-tracking echocardiogra- phy last year. Molecular analysis identifed a c.369_372delGCTC (p.Leu124Serfs*26) AGPAT2 mutation in both unrelated patients, a compound heterozygous mutation in Patient 1, and homozygous mutation in Patient 2. Conclusion: We describe two unrelated patients with type 1 CGL due to Leu124Serfs*26, a novel AGPAT2 frameshift mutation, presenting as early cardiovascular disease. These fndings suggest an association between Leu124Serfs*26 and a more aggressive phenotype. Keywords: Congenital generalized lipodystrophy, AGPAT2, Cardiovascular disease Background Congenital generalized lipodystrophy (CGL; OMIN 608594) is a rare autosomal recessive disorder charac- *Correspondence: [email protected] terized by the total or near-total loss of subcutaneous 1 Brazilian Group for the Study of Inherited and Acquired Lipodystrophies (BRAZLIPO), Faculdade de Medicina, Universidade Federal do Ceará, Rua adipose tissue soon after birth, resulting in ectopic fat Professor Costa Mendes, 1608, Rodolfo Teóflo, Fortaleza, Ceará 60416200, deposition and metabolic complications. Tese patients Brazil present severe insulin resistance (IR), diabetes mellitus, Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Montenegro Junior et al. Diabetol Metab Syndr (2020) 12:28 Page 2 of 9 hypertriglyceridemia, hepatic steatosis, and cardiovascu- Blood glucose, cholesterol, and triglyceride levels lar disease [1–3]. were obtained after overnight fast and determined CGL is classifed into four subtypes—1, 2, 3, and 4— according to standard methods using automated equip- according to the afected gene, AGPAT2, BSCL2, CAV-1, ment. Glycohemoglobin A1c values were determined and CAVIN1, respectively. Most cases (95%) are subtypes by ion exchange high-performance liquid chromatog- 1 (CGL1) or 2 (CGL2). AGPAT2 is located in chromo- raphy (HPLC), and serum insulin levels were measured some 9q34 and encompasses six exons [4]. Tis gene by immunoassays with reagents provided by Roche codes a homonymous protein (1-acylglycerol-3-phos- Diagnostics (Basel, Switzerland). Tyroid stimulat- phate acyltransferase-β) that triggers the synthesis of ing hormone (TSH) and thyroid hormones (fT4 and triglycerides inside the adipocyte, converting lysophos- T3) were measured using chemiluminescence (ADVIA phatidic acid to phosphatidic acid [5, 6]. Tis mutation Centaur® XP, Siemens). Plasma leptin levels were meas- infuences the peroxisome proliferator-activated receptor ured by an immunoassay using a commercial kit (DIA- gamma (PPARγ), the main regulator of adipocyte difer- source ImmunoAssays, Louvain-la-Neuve, Belgium). entiation [7–9]. HOMA–IR (homeostasis model assessment) was also Diferent AGPAT2 mutations have been described calculated (fasting glycaemia (mmol/L) × fasting insu- worldwide. Tis gene is expressed in various tissues, lin (μU/mL))÷22.5) [11]. including the gastrointestinal tract, heart, kidney, and Standard in-house protocols based on a salting-out liver, among others [10]. Descriptions of the clinical method were used to extract genomic DNA from periph- characteristics of patients carrying new mutations are eral blood leucocytes [12]. Te entire coding regions important since they may improve our understanding of and intron–exon junctions of AGPAT2 and BSCL2 were multiorgan diseases and adipose tissue physiology, and amplifed by polymerase chain reactions (PCR) using spe- this understanding may enable us to speculate about gen- cifc primers and were automatically sequenced; primer otype–phenotype associations. sequences are listed in Table 1. Amplifcation reactions In this study, we reported two unrelated CGL1 were executed in a fnal volume of 25 µL, comprising patients with a novel frameshift mutation in AGPAT2, 200 ng genomic DNA, 0.2 mM dNTPs, 1.5 mM PCR x NM_006412:p.Leu124Serfs*26. Enhancer Solution (Invitrogen), 0.6 pmol of each primer, ® 1× PCR bufer, and 1U GoTaq DNA polymerase (Pro- Methods mega, Madison, WI), and were executed for 35 cycles: Clinical features and laboratory and molecular data from denaturation at 95 °C for 30 s, annealing at 55–62 °C for two Brazilian patients from the state of Ceará, Northeast 30 s, extension at 72 °C for 1 min, and a fnal extension Brazil (Fig. 1), were obtained by medical interview and for 10 min at 72 °C. Te PCR products were checked on medical records review. 1% agarose gel electrophoresis, purifed, and automati- cally sequenced in an ABI PRISM™ 3100 Genetic Ana- lyzer automatic DNA sequencer (Applied Biosystems, Foster City, CA). All sequence variations were assessed on both strands and repeated in a second PCR. Table 1 Oligonucleotides used for AGPAT2 amplifcation and sequencing Primer AGPAT2-1F 5′-cgcaataaggggcctgag-3′ AGPAT2-1R 5′-ggaccccctcctgtgc-3′ AGPAT2-2F 5′-gggactctgtccgcttca-3′ AGPAT2-2R 5′-cagccctgtgtcctcgtc-3′ AGPAT2-3F 5′-ggtgctcagcagctgtcttc-3′ AGPAT2-3R 5′-tttctgccaaaaccaagtcac-3′ AGPAT2-4F 5′-aaaacaagacccccacatcat-3′ AGPAT2-4R 5′-gaggagtcccttgtgtgtcaag-3′ AGPAT2-5F 5′-cctcagctgtgcgtctcc-3′ AGPAT2-5R 5′-gagtcactcattcgccacat-3′ AGPAT2-6F 5′-ctagggagtccaggggaaga-3′ Fig. 1 CGL pa–tients’ hometowns (in red) in the state of Ceará, Brazil AGPAT2-6R 5′-agtgacagaaggggcttcct-3′ Montenegro Junior et al. Diabetol Metab Syndr (2020) 12:28 Page 3 of 9 Mutation Taster was used to estimate the potential Results biological impact of the AGPAT2 mutations on the Study subjects protein function. This is a web-based application that Patient 1 performs a battery of in silico tests on protein and Patient 1 was a 30-year-old woman (Fig. 2), non-smoker, DNA level to estimate the impact of a variant on the born full-term (unknown birth weight) as the third child gene product. It predicts the functional consequences of nonconsanguineous parents in the city of Cascavel, of amino acid substitutions as well as intronic and state of Ceará, Brazil (Fig. 1). Lipodystrophic phenotype synonymous alterations, short insertion and deletion was detected at birth and the diagnosis of CGL was given mutations, and variants spanning intro-exon borders. at the age of 6 months when she was hospitalized due to a The program filter harmless mutations from the dis- respiratory infection. Hypertriglyceridemia (223 mg/dL) ease-causing ones by comparison with integrated data- was also detected at that time. bases and perform tests to determine the nature of the She had no clinical follow-up until the age 13, when given variant, that comprises the amino acid substitu- she was diagnosed with diabetes mellitus and,