Citation: Kosac, A.; Pesovic, J.; Radenkovic, L.; Brkusanin, M.; Radovanovic, N.; Djurisic, M.; Radivojevic, D.; Mladenovic, J.; Ostojic, S.; Kovacevic, G.; et al. LTBP4, SPP1, and CD40 Variants: Genetic Modifiers of Duchenne Muscular Dystrophy Analyzed in Serbian Patients. Genes 2022, 13, 1385. https://doi.org/10.3390/ genes13081385 Academic Editor: Allison D. Ebert Received: 28 June 2022 Accepted: 30 July 2022 Published: 4 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). genes G C A T T A C G G C A T Article LTBP4, SPP1, and CD40 Variants: Genetic Modifiers of Duchenne Muscular Dystrophy Analyzed in Serbian Patients Ana Kosac 1, *, Jovan Pesovic 2 , Lana Radenkovic 2 , Milos Brkusanin 2 , Nemanja Radovanovic 2 , Marina Djurisic 3 , Danijela Radivojevic 3 , Jelena Mladenovic 1 , Slavica Ostojic 4 , Gordana Kovacevic 4 , Ruzica Kravljanac 4,5 , Dusanka Savic Pavicevic 2 and Vedrana Milic Rasic 5 1 Department of Neurology, Clinic of Neurology and Psychiatry for Children and Youth, 11000 Belgrade, Serbia 2 Centre for Human Molecular Genetics, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia 3 Laboratory of Medical Genetics, Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, 11000 Belgrade, Serbia 4 Department of Neurology, Mother and Child Health Care Institute of Serbia “Dr Vukan Cupic”, 11000 Belgrade, Serbia 5 Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia * Correspondence: kosacana@gmail.com; Tel.: +381-11-2658-355 Abstract: Background: Clinical course variability in Duchenne muscular dystrophy (DMD) is partially explained by the mutation location in the DMD gene and variants in modifier genes. We assessed the effect of the SPP1, CD40, and LTBP4 genes and DMD mutation location on loss of ambulation (LoA). Methods: SNPs in SPP1-rs28357094, LTBP4-rs2303729, rs1131620, rs1051303, rs10880, and CD40-rs1883832 were genotyped, and their effect was assessed by survival and hierarchical cluster analysis. Results: Patients on glucocorticoid corticosteroid (GC) therapy experienced LoA one year later (p = 0.04). The modifying effect of SPP1 and CD40 variants, as well as LTBP4 haplotypes, was not observed using a log-rank test and multivariant Cox regression analysis. Cluster analysis revealed two subgroups with statistical trends in differences in age at LoA. Almost all patients in the cluster with later LoA had the protective IAAM LTBP4 haplotype and statistically significantly fewer CD40 genotypes with harmful T allele and “distal” DMD mutations. Conclusions: The modifying effect of SPP1, CD40, and LTBP4 was not replicated in Serbian patients, although our cohort was comparable in terms of its DMD mutation type distribution, SNP allele frequencies, and GC-positive effect with other European cohorts. Cluster analysis may be able to identify patient subgroups carrying a combination of the genetic variants that modify LoA. Keywords: Duchenne muscular dystrophy; single-nucleotide polymorphisms; LTBP4; SPP1; CD40 1. Introduction Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and has a progressive devastating course. It is caused by a complete deficiency of protein dystrophin due to loss-of-function mutations in the DMD gene. The clinical course of the disease is usually clearly defined as follows: muscle weaknesses leading to loss of independent ambulation (LoA) by the teenage years, followed by the loss of the hand function and unavoidable complete immobility. Cardiomyopathy and respiratory fail- ure progress in the advanced phases of the disease, leading to the lethal outcome. The multidisciplinary care approach has prolonged life expectancy in DMD patients [1–5]. However, variability in the disease progression is documented, and we are now seeing patients with a prolonged period of mobility, delayed onset of cardiomyopathy, and need for respiratory support [6–9]. Duchenne muscular dystrophy phenotype variability is influenced by standards of care, therapy with glucocorticoid corticosteroids (GCs), and the genetic architecture of the patient, including the type and location of DMD mutations and genetic modifiers [10–13]. Genes 2022, 13, 1385. https://doi.org/10.3390/genes13081385 https://www.mdpi.com/journal/genes