Genetic Modifiers of Stroke in Patients with Sickle Cell Disease-A Scoping Review.

Sickle cell disease (SCD) clinically manifests itself with a myriad of complications. Stroke, both ischemic and hemorrhagic, as well as silent white matter changes, occurs at a relatively high prevalence. Understanding why and in whom stroke is most likely to occur is critical to the effective prevention and treatment of individuals with SCD. Genetic studies, including genome- and exome-wide association studies (GWAS and EWAS), have found several key modifiers associated with increased stroke/stroke risk in SCD via mechanisms including Hemoglobin F (HbF) modulation, inflammation, cellular adhesion, endothelial disruption, and hemolysis. We present a review on the modifiers that have most clearly demonstrated an association to date. More studies are needed to validate other potential polymorphisms and identify new ones. Incorporating gene-focused screenings in clinical care could provide avenues for more targeted, more effective, and less toxic prevention of stroke in this population. The data from this review will be used to inform the initial GWAS performed by the International Hemoglobinopathy Research Network (INHERENT) consortium.

Frequency of RPGRIP1 and MAP9 genetic modifiers of canine progressive retinal atrophy, in 132 breeds of dog.

Variants in RPGRIP1 and MAP9, termed RPGRIP1ins44 and MAP9del respectively, are both associated with a form of canine progressive retinal atrophy referred to as RPGRIP1-CRD and have both been demonstrated to modify the development and progression of this disease. In the current study both variants were genotyped in at least 50 dogs of 132 diverse breeds and the data reveal that both segregate in multiple breeds. Individually, each variant is common within largely non-overlapping subsets of breed, and there is a negative correlation between their frequencies within breeds that segregate both variants. The frequency of both variants exceeds 0.05 in a single breed only, the Miniature Longhaired Dachshund. These data indicate that both variants are likely to be ancient and predate the development and genetic isolation of modern dog breeds. That both variants are present individually at high frequency in multiple breeds is consistent with the hypothesis that homozygosity of either variant alone is not associated with a clinically relevant phenotype, whereas the negative correlation between the two variants is consistent with the application of selective pressure, from dog breeders, against homozygosity at both loci, probably due to the more severe phenotype associated with homozygosity at both loci.

Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease.

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.

Systematic Review of Genetic Modifiers Associated with the Development and/or Progression of Nephropathy in Patients with Sickle Cell Disease.

Sickle cell nephropathy (SCN) is a common complication of sickle cell disease (SCD) that significantly contributes to morbidity and mortality. In addition to clinical and life-style factors, genetic variants influence this risk. We performed a systematic review, searching five databases. Studies evaluating the effect of genetic modifiers on SCN were eligible. Twenty-eight studies (fair-to-good quality) were included: one genome-wide association study, twenty-six case-control studies, and one article combining both approaches. APOL1 was significantly associated with albuminuria and hyperfiltration in children and with worse glomerular filtration in adults. On the other hand, alpha-thalassemia protected patients against albuminuria and hyperfiltration, while BCL11A variants were protective against albuminuria alone. The HMOX1 long GT-tandem repeat polymorphism led to a lower glomerular filtration rate. No modifiers for the risk of hyposthenuria were identified. A genome-wide association approach identified three new loci for proteinuria (CRYL1, VWF, and ADAMTS7) and nine loci were linked with eGFR (PKD1L2, TOR2A, CUBN, AGGF1, CYP4B1, CD163, LRP1B, linc02288, and FPGT-TNNI3K/TNNI3K). In conclusion, this systematic review supports the role of genetic modifiers in influencing the risk and progression of SCN. Incorporating and expanding this knowledge is crucial to improving the management and clinical outcomes of patients at risk.

A study concept of expeditious clinical enrollment for genetic modifier studies in Charcot-Marie-Tooth neuropathy 1A.

BACKGROUND: Caused by duplications of the gene encoding peripheral myelin protein 22 (PMP22), Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common hereditary neuropathy. Despite this shared genetic origin, there is considerable variability in clinical severity. It is hypothesized that genetic modifiers contribute to this heterogeneity, the identification of which may reveal novel therapeutic targets. In this study, we present a comprehensive analysis of clinical examination results from 1564 CMT1A patients sourced from a prospective natural history study conducted by the RDCRN-INC (Inherited Neuropathy Consortium). Our primary objective is to delineate extreme phenotype profiles (mild and severe) within this patient cohort, thereby enhancing our ability to detect genetic modifiers with large effects. METHODS: We have conducted large-scale statistical analyses of the RDCRN-INC database to characterize CMT1A severity across multiple metrics. RESULTS: We defined patients below the 10th (mild) and above the 90th (severe) percentiles of age-normalized disease severity based on the CMT Examination Score V2 and foot dorsiflexion strength (MRC scale). Based on extreme phenotype categories, we defined a statistically justified recruitment strategy, which we propose to use in future modifier studies. INTERPRETATION: Leveraging whole genome sequencing with base pair resolution, a future genetic modifier evaluation will include single nucleotide association, gene burden tests, and structural variant analysis. The present work not only provides insight into the severity and course of CMT1A, but also elucidates the statistical foundation and practical considerations for a cost-efficient and straightforward patient enrollment strategy that we intend to conduct on additional patients recruited globally.

Genetic Modifiers of Sickle Cell Anemia Phenotype in a Cohort of Angolan Children.

The aim of this study was to identify genetic markers in the HBB Cluster; HBS1L-MYB intergenic region; and BCL11A, KLF1, FOX3, and ZBTB7A genes associated with the heterogeneous phenotypes of Sickle Cell Anemia (SCA) using next-generation sequencing, as well as to assess their influence and prevalence in an Angolan population. Hematological, biochemical, and clinical data were considered to determine patients' severity phenotypes. Samples from 192 patients were sequenced, and 5,019,378 variants of high quality were registered. A catalog of candidate modifier genes that clustered in pathophysiological pathways important for SCA was generated, and candidate genes associated with increasing vaso-occlusive crises (VOC) and with lower fetal hemoglobin (HbF) were identified. These data support the polygenic view of the genetic architecture of SCA phenotypic variability. Two single nucleotide polymorphisms in the intronic region of 2q16.1, harboring the BCL11A gene, are genome-wide and significantly associated with decreasing HbF. A set of variants was identified to nominally be associated with increasing VOC and are potential genetic modifiers harboring phenotypic variation among patients. To the best of our knowledge, this is the first investigation of clinical variation in SCA in Angola using a well-customized and targeted sequencing approach.

Genetic modifiers of rare variants in monogenic developmental disorder loci.

Rare damaging variants in a large number of genes are known to cause monogenic developmental disorders (DDs) and have also been shown to cause milder subclinical phenotypes in population cohorts. Here, we show that carrying multiple (2-5) rare damaging variants across 599 dominant DD genes has an additive adverse effect on numerous cognitive and socioeconomic traits in UK Biobank, which can be partially counterbalanced by a higher educational attainment polygenic score (EA-PGS). Phenotypic deviators from expected EA-PGS could be partly explained by the enrichment or depletion of rare DD variants. Among carriers of rare DD variants, those with a DD-related clinical diagnosis had a substantially lower EA-PGS and more severe phenotype than those without a clinical diagnosis. Our results suggest that the overall burden of both rare and common variants can modify the expressivity of a phenotype, which may then influence whether an individual reaches the threshold for clinical disease.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

Primary Ciliary Dyskinesia and Retinitis Pigmentosa: Novel RPGR Variant and Possible Modifier Gene.

We report a novel RPGR missense variant co-segregated with a familial X-linked retinitis pigmentosa (XLRP) case. The brothers were hemizygous for this variant, but only the proband presented with primary ciliary dyskinesia (PCD). Thus, we aimed to elucidate the role of the RPGR variant and other modifier genes in the phenotypic variability observed in the family and its impact on motile cilia. The pathogenicity of the variant on the RPGR protein was evaluated by in vitro studies transiently transfecting the mutated RPGR gene, and immunofluorescence analysis on nasal brushing samples. Whole-exome sequencing was conducted to identify potential modifier variants. In vitro studies showed that the mutated RPGR protein could not localise to the cilium and impaired cilium formation. Accordingly, RPGR was abnormally distributed in the siblings' nasal brushing samples. In addition, a missense variant in CEP290 was identified. The concurrent RPGR variant influenced ciliary mislocalisation of the protein. We provide a comprehensive characterisation of motile cilia in this XLRP family, with only the proband presenting PCD symptoms. The variant's pathogenicity was confirmed, although it alone does not explain the respiratory symptoms. Finally, the CEP290 gene may be a potential modifier for respiratory symptoms in patients with RPGR mutations.

Inherited Retinal Degeneration Caused by Dehydrodolichyl Diphosphate Synthase Mutation-Effect of an ALG6 Modifier Variant.

Modern advances in disease genetics have uncovered numerous modifier genes that play a role in the severity of disease expression. One such class of genetic conditions is known as inherited retinal degenerations (IRDs), a collection of retinal degenerative disorders caused by mutations in over 300 genes. A single missense mutation (K42E) in the gene encoding the enzyme dehydrodolichyl diphosphate synthase (DHDDS), which is required for protein N-glycosylation in all cells and tissues, causes DHDDS-IRD (retinitis pigmentosa type 59 (RP59; OMIM #613861)). Apart from a retinal phenotype, however, DHDDS-IRD is surprisingly non-syndromic (i.e., without any systemic manifestations). To explore disease pathology, we selected five glycosylation-related genes for analysis that are suggested to have disease modifier variants. These genes encode glycosyltransferases (ALG6, ALG8), an ER resident protein (DDOST), a high-mannose oligosaccharyl transferase (MPDU1), and a protein N-glycosylation regulatory protein (TNKS). DNA samples from 11 confirmed DHDDS (K42E)-IRD patients were sequenced at the site of each candidate genetic modifier. Quantitative measures of retinal structure and function were performed across five decades of life by evaluating foveal photoreceptor thickness, visual acuity, foveal sensitivity, macular and extramacular rod sensitivity, and kinetic visual field extent. The ALG6 variant, (F304S), was correlated with greater macular cone disease severity and less peripheral rod disease severity. Thus, modifier gene polymorphisms may account for a significant portion of phenotypic variation observed in human genetic disease. However, the consequences of the polymorphisms may be counterintuitively complex in terms of rod and cone populations affected in different regions of the retina.

A Role for Genetic Modifiers in Tubulointerstitial Kidney Diseases.

With the increased availability of genomic sequencing technologies, the molecular bases for kidney diseases such as nephronophthisis and mitochondrially inherited and autosomal-dominant tubulointerstitial kidney diseases (ADTKD) has become increasingly apparent. These tubulointerstitial kidney diseases (TKD) are monogenic diseases of the tubulointerstitium and result in interstitial fibrosis and tubular atrophy (IF/TA). However, monogenic inheritance alone does not adequately explain the highly variable onset of kidney failure and extra-renal manifestations. Phenotypes vary considerably between individuals harbouring the same pathogenic variant in the same putative monogenic gene, even within families sharing common environmental factors. While the extreme end of the disease spectrum may have dramatic syndromic manifestations typically diagnosed in childhood, many patients present a more subtle phenotype with little to differentiate them from many other common forms of non-proteinuric chronic kidney disease (CKD). This review summarises the expanding repertoire of genes underpinning TKD and their known phenotypic manifestations. Furthermore, we collate the growing evidence for a role of modifier genes and discuss the extent to which these data bridge the historical gap between apparently rare monogenic TKD and polygenic non-proteinuric CKD (excluding polycystic kidney disease).

Susceptibility and disease modifier genes in amyotrophic lateral sclerosis: from genetic associations to therapeutic implications.

PURPOSE OF REVIEW: Amyotrophic lateral sclerosis (ALS) is a severe disease characterized by the degeneration of motor neurons. Large-scale genetic studies have now identified over 60 genes that are associated with ALS, which in large part have also been functionally characterized. The purpose of this review is to outline how these advances are being translated into novel therapeutic strategies. RECENT FINDINGS: The emergence of techniques that allow the specific therapeutic targeting of a (mutant) gene, in particular antisense oligonucleotide therapy (ASOs), have led to the first successful gene therapy for SOD1-ALS and multiple other gene-targeted trials are underway. This includes genetic variants that modify the disease phenotype as well as causal mutations. SUMMARY: Technological and methodological advances are enabling researchers to unravel the genetics of ALS. Both causal mutations and genetic modifiers are viable therapeutic targets. By performing natural history studies, the phenotype-genotype correlations can be characterized. In conjunction with biomarkers for target engagement and international collaboration, this makes performing gene-targeted trials ALS feasible. The first effective treatment has now been developed for SOD1-ALS and, with multiple studies underway, it seems realistic that more therapies will follow.

Exome sequencing of choreoacanthocytosis reveals novel mutations in VPS13A and co-mutation in modifier gene(s).

Choreoacanthocytosis, one of the forms of neuroacanthocytosis, is caused by mutations in vacuolar protein sorting-associated protein A (VPS13A), and is often misdiagnosed with other form of neuroacanthocytosis with discrete genetic defects. The phenotypic variations among the patients with VPS13A mutations significantly obfuscates the understanding of the disease and treatment strategies. In this study, two unrelated cases were identified, exhibiting the core phenotype of neuroacanthocytosis but with considerable clinical heterogeneity. Case 1 presented with an additional Parkinsonism phenotype, whereas seizures were evident in case 2. To decipher the genetic basis, whole exome sequencing followed by validation with Sanger sequencing was performed. A known homozygous pathogenic nonsense mutation (c.799C > T; p.R267X) in exon 11 of the VPS13A gene was identified in case 1 that resulted in a truncated protein. A novel missense mutation (c.9263T > G; p.M3088R) in exon 69 of VPS13A identified in case 2 was predicted as pathogenic. In silico analysis of the p.M3088R mutation at the C-terminus of VPS13A suggests a loss of interaction with TOMM40 and may disrupt mitochondrial localization. We also observed an increase in mitochondrial DNA copy numbers in case 2. Mutation analysis revealed benign heterozygous variants in interacting partners of VPS13A such as VAPA in case 1. Our study confirmed the cases as ChAc and identified the novel homozygous variant of VPS13A (c.9263T > G; p.M3088R) within the mutation spectrum of VPS13A-associated ChAc. Furthermore, mutations in VPS13A and co-mutations in its potential interacting partner(s) might contribute to the diverse clinical manifestations of ChAc, which requires further study.

Application of an F0-based genetic assay in adult zebrafish to identify modifier genes of an inherited cardiomyopathy.

Modifier genes contribute significantly to our understanding of pathophysiology in human diseases; however, effective approaches to identify modifier genes are still lacking. Here, we aim to develop a rapid F0-based genetic assay in adult zebrafish using the bag3 gene knockout (bag3e2/e2) cardiomyopathy model as a paradigm. First, by utilizing a classic genetic breeding approach, we identified dnajb6b as a deleterious modifier gene for bag3 cardiomyopathy. Next, we established an F0-based genetic assay in adult zebrafish through injection of predicted microhomology-mediated end joining (MMEJ)-inducing single guide RNA/Cas9 protein complex. We showed that effective gene knockdown is maintained in F0 adult fish, enabling recapitulation of both salutary modifying effects of the mtor haploinsufficiency and deleterious modifying effects of the dnajb6b gene on bag3 cardiomyopathy. We finally deployed the F0-based genetic assay to screen differentially expressed genes in the bag3 cardiomyopathy model. As a result, myh9b was identified as a novel modifier gene for bag3 cardiomyopathy. Together, these data prove the feasibility of an F0 adult zebrafish-based genetic assay that can be effectively used to discover modifier genes for inherited cardiomyopathy.

The impact of modifier genes on cone-rod dystrophy heterogeneity: An explorative familial pilot study and a hypothesis on neurotransmission impairment.

Cone-rod dystrophies (CORDs) are a heterogeneous group of inherited retinopathies (IRDs) with more than 30 already known disease-causing genes. Uncertain phenotypes and extended range of intra- and interfamilial heterogenicity make still difficult to determine a precise genotype-phenotype correlation. Here, we used a next-generation sequencing approach to study a Sicilian family with a suspected form of CORD. Affected family members underwent ophthalmological examinations and a proband, blind from 50 years, underwent whole genome and exome sequencing. Variant analysis was enriched by pathway analysis and relevant variants were, then, investigated in other family members and in 100 healthy controls from Messina. CORD diagnosis with an intricate pattern of symptoms was confirmed by ophthalmological examinations. A total of about 50,000 variants were identified in both proband's genome and exome. All affected family members presented specific genotypes mainly determined by mutated GUCY2D gene, and different phenotypical traits, mainly related to focus and color perception. Thus, we looked for possible modifier genes. According to relationship with GUCY2D, predicted functional effects, eye localization, and ocular disease affinity, only 9 variants, carried by 6 genes (CACNG8, PAX2, RXRG, CCDC175, PDE4DIP and LTF), survived the filtering. These genes encode key proteins involved in cone development and survival, and retina neurotransmission. Among analyzed variants, CACNG8c.*6819A>T and the new CCDC175 c.76C>T showed extremely low frequency in the control group, suggesting a key role on disease phenotypes. Such discovery could enforce the role of modifier genes into CORD onset/progression, contributing to improve diagnostic test towards a better personalized medicine.

Assessment of Rare Genetic Variants to Identify Candidate Modifier Genes Underlying Neurological Manifestations in Neurofibromatosis 1 Patients.

Neurological phenotypes such as intellectual disability occur in almost half of patients with neurofibromatosis 1 (NF1). Current genotype-phenotype studies have failed to reveal the mechanism underlying this clinical variability. Despite the presence of pathogenic variants of NF1, modifier genes likely determine the occurrence and severity of neurological phenotypes. Exome sequencing data were used to identify genetic variants in 13 NF1 patients and 457 healthy controls, and this information was used to identify candidate modifier genes underlying neurological phenotypes based on an optimal sequence kernel association test. Thirty-six genes were identified as significant modifying factors in patients with neurological phenotypes and all are highly expressed in the nervous system. A review of the literature confirmed that 19 genes including CUL7, DPH1, and BCO1 are clearly associated with the alteration of neurological functioning and development. Our study revealed the enrichment of rare variants of 19 genes closely related to neurological development and functioning in NF1 patients with neurological phenotypes, indicating possible modifier genes and variants affecting neurodevelopment. Further studies on rare genetic variants of candidate modifier genes may help explain the clinical heterogeneity of NF1.

Modifier Factors of Cystic Fibrosis Phenotypes: A Focus on Modifier Genes.

Although cystic fibrosis (CF) is recognized as a monogenic disease, due to variants within the CFTR (Cystic Fibrosis Transmembrane Regulator) gene, an extreme clinical heterogeneity is described among people with CF (pwCF). Apart from the exocrine pancreatic status, most studies agree that there is little association between CFTR variants and disease phenotypes. Environmental factors have been shown to contribute to this heterogeneity, accounting for almost 50% of the variability of the lung function of pwCF. Nevertheless, pwCF with similar CFTR variants and sharing the same environment (such as in siblings) may have highly variable clinical manifestations not explained by CFTR variants, and only partly explained by environmental factors. It is recognized that genetic variants located outside the CFTR locus, named "modifier genes", influence the clinical expression of the disease. This short review discusses the latest studies that have described modifier factors associated with the various CF phenotypes as well as the response to the recent CFTR modulator therapies.

Candidate Modifier Genes for the Penetrance of Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease caused by mitochondria DNA (mtDNA) mutation. It is characterized by acute and subacute visual loss predominantly affecting young men. The mtDNA mutation is transmitted to all maternal lineages. However, only approximately 50% of men and 10% of women harboring a pathogenic mtDNA mutation develop optic neuropathy, reflecting both the incomplete penetrance and its unexplained male prevalence, where over 80% of patients are male. Nuclear modifier genes have been presumed to affect the penetrance of LHON. With conventional genetic methods, prior studies have failed to solve the underlying pathogenesis. Whole exome sequencing (WES) is a new molecular technique for sequencing the protein-coding region of all genes in a whole genome. We performed WES from five families with 17 members. These samples were divided into the proband group (probands with acute onset of LHON, n = 7) and control group (carriers including mother and relative carriers with mtDNSA 11778 mutation, without clinical manifestation of LHON, n = 10). Through whole exome analysis, we found that many mitochondria related (MT-related) nuclear genes have high percentage of variants in either the proband group or control group. The MT genes with a difference over 0.3 of mutation percentage between the proband and control groups include AK4, NSUN4, RDH13, COQ3, and FAHD1. In addition, the pathway analysis revealed that these genes were associated with cofactor metabolism pathways. Family-based analysis showed that several candidate MT genes including METAP1D (c.41G > T), ACACB (c.1029del), ME3 (c.972G > C), NIPSNAP3B (c.280G > C, c.476C > G), and NSUN4 (c.4A > G) were involved in the penetrance of LHON. A GWAS (genome wide association study) was performed, which found that ADGRG5 (Chr16:575620A:G), POLE4 (Chr2:7495872T:G), ERMAP (Chr1:4283044A:G), PIGR (Chr1:2069357C:T;2069358G:A), CDC42BPB (Chr14:102949A:G), PROK1 (Chr1:1104562A:G), BCAN (Chr 1:1566582C:T), and NES (Chr1:1566698A:G,1566705T:C, 1566707T:C) may be involved. The incomplete penetrance and male prevalence are still the major unexplained issues in LHON. Through whole exome analysis, we found several MT genes with a high percentage of variants were involved in a family-based analysis. Pathway analysis suggested a difference in the mutation burden of MT genes underlining the biosynthesis and metabolism pathways. In addition, the GWAS analysis also revealed several candidate nuclear modifier genes. The new technology of WES contributes to provide a highly efficient candidate gene screening function in molecular genetics.

LTBP4, SPP1, and CD40 Variants: Genetic Modifiers of Duchenne Muscular Dystrophy Analyzed in Serbian Patients.

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.