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Dravet syndrome is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, comorbidities related to developmental, cognitive, and motor delays, and a high mortality burden due to sudden unexpected death in epilepsy (SUDEP). Most Dravet syndrome cases are attributed to SCN1A haploinsufficiency, with genetic modifiers and environmental factors influencing disease severity. Mouse models with heterozygous deletion of Scn1a recapitulate key features of Dravet syndrome, including seizures and premature mortality; however, severity varies depending on genetic background. Here, we refined two Dravet survival modifier (Dsm) loci, Dsm2 on chromosome 7 and Dsm3 on chromosome 8, using interval-specific congenic (ISC) mapping. Dsm2 was complex and encompassed at least two separate loci, while Dsm3 was refined to a single locus. Candidate modifier genes within these refined loci were prioritized based on brain expression, strain-dependent differences, and biological relevance to seizures or epilepsy. High priority candidate genes for Dsm2 include Nav2, Ptpn5, Ldha, Dbx1, Prmt3 and Slc6a5, while Dsm3 has a single high priority candidate, Psd3. This study underscores the complex genetic architecture underlying Dravet syndrome and provides insights into potential modifier genes that could influence disease severity and serve as novel therapeutic targets., (© 2024. The Author(s).)

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., (© 2024 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

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.

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., Competing Interests: Declaration of interests In the past year, through the offices of UCL Consultants Ltd., a wholly owned subsidiary of University College London, S.J.T. has undertaken consultancy services for Alnylam Pharmaceuticals, Annexon, Ascidian Therapeutics, Arrowhead Pharmaceuticals, Atalanta Therapeutics, Design Therapeutics, F. Hoffman-La Roche, HCD Economics, IQVIA, Iris Medicine, Latus Bio, LifeEdit, Novartis Pharma, Pfizer, Prilenia Neurotherapeutics, PTC Therapeutics, Rgenta Therapeutics, Takeda Pharmaceuticals, UniQure Biopharma, and Vertex Pharmaceuticals. In the past 12 months, University College London Hospitals NHS Foundation Trust, S.J.T.’s host clinical institution, received funding to run clinical trials for F. Hoffman-La Roche, Novartis Pharma, PTC Therapeutics, and UniQure Biopharma., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024 Peripheral Nerve Society.)

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.

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., (© 2024. The Author(s).)

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 NPC1 I1061T 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.

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.

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.

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.

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).

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., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

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., (© 2023. The Author(s).)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Donato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To nominate novel disease genes for obesity and type 2 diabetes (T2D), we recently generated two mouse backcross populations of the T2D-susceptible New Zealand Obese (NZO/HI) mouse strain and two genetically different, lean and T2D-resistant strains, 129P2/OlaHsd and C3HeB/FeJ. Comparative linkage analysis of our two female backcross populations identified seven novel body fat-associated quantitative trait loci (QTL). Only the locus Nbw14 (NZO body weight on chromosome 14) showed linkage to obesity-related traits in both backcross populations, indicating that the causal gene variant is likely specific for the NZO strain as NZO allele carriers in both crosses displayed elevated body weight and fat mass. To identify candidate genes for Nbw14, we used a combined approach of gene expression and haplotype analysis to filter for NZO-specific gene variants in gonadal white adipose tissue, defined as the main QTL-target tissue. Only two genes, Arl11 and Sgcg, fulfilled our candidate criteria. In addition, expression QTL analysis revealed cis-signals for both genes within the Nbw14 locus. Moreover, retroviral overexpression of Sgcg in 3T3-L1 adipocytes resulted in increased insulin-stimulated glucose uptake. In humans, mRNA levels of SGCG correlated with body mass index and body fat mass exclusively in diabetic subjects, suggesting that SGCG may present a novel marker for metabolically unhealthy obesity. In conclusion, our comparative-cross analysis could substantially improve the mapping resolution of the obesity locus Nbw14. Future studies will throw light on the mechanism by which Sgcg may protect from the development of obesity., (© The Author(s) 2022. Published by Oxford University Press.)

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.

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.

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.

The Nakano cataract mouse (NCT) manifests a wavy coat for their first hair as a genetic trait. In this study, we explored the molecular genetic basis of the wavy coat. We revealed by crossing experiments that the wavy coat is controlled by a major gene on chromosome 7 of NCT, homozygosity of which is a prerequisite for developing the wavy coat, and by a gene on chromosome 9 with a minor effect to reinforce the manifestation of the trait. In humans, a polymorphism of the protease, serine 53 (PRSS53) gene on the homologous chromosome is known to be associated with curly scalp hair. We then investigated the Prss53 gene and discovered that NCT has an insertion of an intracisternal A particle element in the first intron of the gene. Nevertheless, the expression of the Prss53 is not altered in the NCT skin both in transcript and protein levels. Subsequently, we created C57BL/6J-Prss53 em1 knockout mice and found that these mice manifest vague wavy coats. A portion of backcross and intercross mice between the C57BL/6J-Prss53 em1 and NCT manifested intense or vague wavy coats. These findings demonstrate the polygenic nature of the wavy coat of NCT and Prss53 knockout mice and highlight the similarity of the trait to the curly hair of humans associated with the PRSS53 alteration., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

Background: Variation in respiratory response to cystic fibrosis (CF) small molecule therapies is due in part to the contribution of CF lung disease modifier genes. Cultured human bronchial epithelia (HBE) is the gold standard respiratory model for assessing CF therapeutic efficacy but it is hard to access. Cultured human nasal epithelia (HNE) is proposed as a more accessible surrogate model but it is unknown whether the expression profile of the modifier genes are comparable between HNE and HBE which we assess here., Methods: RNA-sequencing was conducted on paired cultured and fresh HNE and HBE (n = 71 samples) collected from 21 individuals with CF. Genome-wide gene expression was first compared between cultured and fresh cells and then between cultured HNE and HBE based on an equivalence testing procedure we implemented. The co-expression relationships of CFTR and CF lung disease modifier genes were compared between cultured HNE and HBE to determine equivalent interactions., Results: The culturing process had little impact on the expression level of CF lung disease modifier genes. Over 90% of expressed genes showed significant equivalent expression level across cultured HNE and HBE (expression fold-change<2, FDR<0.1), including CFTR and CF lung disease modifier genes. The difference in co-expression relationships among these genes was not significant (p-value=0.99), suggesting their functional interactions are likely to be consistent in the two models., Conclusions: Cultured HNE recapitulates the expression profile of CF lung disease modifier genes in cultured HBE, suggesting the biological processes involving these genes are likely to be consistent across the two models., Competing Interests: Declaration of Competing Interest There is no conflict of interest to be declared., (Copyright © 2022. Published by Elsevier B.V.)

Epistatic interactions complicate the identification of variants involved in phenotypic effect. In-depth knowledge in modifiers and in pathogenic variants would benefit the mechanistic studies on the genetic basis of complex traits. We systematically compared the modifier variants which have evidence of modifier effect with the pathogenic variants from multiple angles. Our study found that genomic loci of modifier variations differ from pathogenic loci in many aspects, such as population genetics statistics, epigenetic features, evolutionary characteristics and functional properties of the variations. Genes containing modifier variation(s) exhibit higher probability of being haploinsufficient and higher probability of recessive disease causation, and they are relatively more important in network communication. Furthermore, we reinforced that co-expression analysis is an effective methodology to predict functional associations between modifier genes and their potential target genes. In many aspects, we detected statistically significant differences between modifier variants/genes and pathogenic variants/genes, and investigated relationships between modifiers and their potential targets. Our results offer some actionable insights that may provide appropriate guidelines to clinical genetics and researchers to elucidate the molecular mechanism underlying the human phenotypic variation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The mutations in modifier genes may contribute to some inherited diseases including Wilson disease (WD). This study was designed to identify potential modifier genes that contribute to WD. A total of 10 WD patients with single or no heterozygous ATP7B mutations were recruited for whole-exome sequencing (WES). Five hundred and thirteen candidate genes, of which the genetic variants present in at least two patients, were identified. In order to clarify which proteins might be involved in copper transfer or metabolism processes, the isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify the differentially expressed proteins between normal and CuSO4-treated cell lines. Thirteen genes/proteins were identified by both WES and iTRAQ, indicating that disease-causing variants of these genes may actually contribute to the aberrant copper ion accumulation. Additionally, the c.86C > T (p.S29L) mutation in the SLC31A2 gene (coding CTR2) has a relative higher frequency in our cohort of WD patients (6/191) than reported (0.0024 in gnomAD database) in our healthy donors (0/109), and CTR2S29L leads to increased intracellular Cu concentration and Cu-induced apoptosis in cultured cell lines. In conclusion, the WES and iTRAQ approaches successfully identified several disease-causing variants in potential modifier genes that may be involved in the WD phenotype., (© The Author(s) 2022. Published by Oxford University Press.)

A mouse strain intercross between Apoe -/- AKR/J and DBA/2J mice identified three replicated atherosclerosis quantitative trait loci (QTLs). Our objective was to fine map mouse atherosclerosis modifier genes within a genomic region known to affect lesion development in apoE-deficient ( Apoe -/- ) mice. We dissected the Ath28 QTL on the distal end of chromosome 2 by breeding a panel of congenic strains and measuring aortic root lesion area in 16-week-old male and female mice fed regular laboratory diets. The parental congenic strain contained ~9.65 Mb of AKR/J DNA from chromosome 2 on the DBA/2J genetic background, which had lesions 55% and 47% smaller than female and male DBA/2J mice, respectively ( p < 0.001). Seven additional congenic lines identified three separate regions associated with the lesion area, named Ath28.1 , Ath28.2 , and Ath28.3 , where the AKR/J alleles were atherosclerosis-protective for two regions and atherosclerosis-promoting for the other region. These results were replicated in both sexes, and in combined analysis after adjusting for sex. The congenic lines did not greatly impact total and HDL cholesterol levels or body weight. Bioinformatic analyses identified all coding and non-coding genes in the Ath28.1 sub-region, as well as strain sequence differences that may be impactful. Even within a <10 Mb region of the mouse genome, evidence supports the presence of at least three atherosclerosis modifier genes that differ between the AKR/J and DBA/2J mouse strains, supporting the polygenic nature of atherosclerosis susceptibility.

CFTR encodes for a chloride and bicarbonate channel expressed at the apical membrane of polarized epithelial cells. Transepithelial sodium transport mediated by the amiloride-sensitive sodium channel ENaC is thought to contribute to the manifestation of CF disease. Thus, ENaC is a therapeutic target in CF and a valid cystic fibrosis modifier gene. We have characterized SCNN1B as a genetic modifier in the three independent patient cohorts of F508del- CFTR homozygotes. We could identify a regulatory element at SCNN1B to the genomic segment rs168748-rs2303153-rs4968000 by fine-mapping (Pbest = 0.0177), consistently observing the risk allele rs2303153-C and the contrasting benign allele rs2303153-G in all three patient cohorts. Furthermore, our results show that expression levels of SCNN1B are associated with rs2303153 genotype in intestinal epithelia ( p = 0.003). Our data confirm that the well-established biological role of SCNN1B can be recognized by an association study on informative endophenotypes in the rare disease cystic fibrosis and calls attention to reproducible results in association studies obtained from small, albeit carefully characterized patient populations.

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31 , however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.

Calpain-mediated proteolysis has been proposed to modulate the pathogenesis of spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), a disorder due to a CAG repeat expansion (CAGexp) at ATXN3. We aimed to investigate if single-nucleotide polymorphisms (SNPs) at calpain gene CAPN2 and at calpastatin gene CAST modulate the age at onset (AO) and disease progression in SCA3/MJD. A total of 287 SCA3/MJD symptomatic subjects (151 families) were included. AO was analyzed and controlled by the CAG repeat length of expanded allele and family. Candidate polymorphisms were chosen based on the literature and on a priori criteria. The CAG repeat length and SNPs were genotyped according to standard methods. AO of carriers of AA and AG + GG rs1559085 genotypes in CAST and with the median value of 75 repeats at the expanded allele were 34.23 (33.07-35.38) and 36.42 years (34.50-38.34), respectively (p = 0.049, mixed model treating the expanded CAG repeat size as fixed effect and family as random effect). Carriers of haplotype C rs27852 /G rs1559085 had mean AO of 37.23 (12.76) and 33.42 years (12.20) (p = 0.047, Student's t test). Our data suggest an association between allele G rs1559085 and haplotype C rs27852 /G rs1559085 at CAST and variations in the AO of SCA3/MJD subjects, independent from the effects of the CAGexp and family. The present results support the potential role of calpain cleavage pathway over modulation of SCA3/MJD phenotype., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Breast cancer is the most common tumor among women with inherited variants in the TP53 tumor suppressor, but onset varies widely suggesting interactions with genetic or environmental factors. Rodent models haploinsufficent for Trp53 also develop a wide variety of malignancies associated with Li-Fraumeni syndrome, but BALB/c mice are uniquely susceptible to mammary tumors and is genetically linked to the Suprmam1 locus on chromosome 7. To define mechanisms that interact with deficiencies in p53 to alter susceptibility to mammary tumors, we fine mapped the Suprmam1 locus in females from an N2 backcross of BALB/cMed and C57BL/6J mice. A major modifier was localized within a 10 cM interval on chromosome 7. The effect of the locus on DNA damage responses was examined in the parental strains and mice that are congenic for C57BL/6J alleles on the BALB/cMed background (SM1-Trp53 +/- ). The mammary epithelium of C57BL/6J-Trp53 +/- females exhibited little radiation-induced apoptosis compared to BALB/cMed-Trp53 +/- and SM1-Trp53 +/- females indicating that the Suprmam1 B6/B6 alleles could not rescue repair of radiation-induced DNA double-strand breaks mostly relying on non-homologous end joining. In contrast, the Suprmam1 B6/B6 alleles in SM1-Trp53 +/- mice were sufficient to confer the C57BL/6J-Trp53 +/- phenotypes in homology-directed repair and replication fork progression. The Suprmam1 B6/B6 alleles in SM1-Trp53 +/- mice appear to act in trans to regulate a panel of DNA repair and replication genes which lie outside the locus., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Patients with a homozygous β 0 -thalassemia mutation usually have a transfusion-dependent β-thalassemia major phenotype. However, some β-thalassemia patients present with a relatively mild and even normal phenotype and always have a high level of Hb F induced by genetic modifiers., Methods: In this study, we identified a homozygous β 0 -thalassemia mutation (HBB: c.126&#95;129delCTTT) in a 36-year-old pregnant woman. She had not presented any clinical symptoms of β-thalassemia since birth. To investigate her unexpected mild phenotype, known genetic modifiers that ameliorate the severity of β-thalassemia were analysed. Besides, we described the haematological changes during pregnancy., Results: Two genetic modifiers (a heterozygous KLF1: c.519&#95;525dup mutation; and two homozygous HBS1L-MYB locus SNP variants: rs7776054 and rs9399137) were identified. However, she showed a gradually decreased level of Hb during pregnancy, and serious transfusion complication of hyperhaemolysis was induced and complicated the pregnancy., Conclusion: This report is in accordance with previous findings that genetic modifiers can ameliorate the clinical severity of β-thalassemia, even without obvious clinical symptoms in a prolonged steady state. However, the steady state can be disrupted during pregnancy. In addition, raising awareness of hyperhaemolysis among clinicians treating patients with thalassemia is necessary., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Abstract: One of the biggest challenges in clinical genetics is establishing associations between specific germline mutations and the resulting spectrum of phenotypes. The careful characterization of clinical presentations continues to be a tool for establishing these genotype phenotype correlations. The authors intend, by presenting a case study, proposing that the concomitant occurrence of a combinations of mild structural anomalies in the same individual may be due to changes in genes that can be linked by related pathways. A new born with cleft lip and palate was referred at the Cleft Lip and Palate Center. The anamnese was performed and collected data of familiar history, parental consanguinity, and information about pregnancy period. The careful characterization of clinical presentations and the genetic pathways was studied. It is possible that there is no single mutation that can be clearly identified as the etiology of the combination of the defects displayed in the present case., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 by Mutaz B. Habal, MD.)

X-linked dystonia-parkinsonism is a neurodegenerative disorder caused by a founder retrotransposon insertion, in which a polymorphic hexanucleotide repeat accounts for ~50% of age at onset variability. Employing a genome-wide association study to identify additional factors modifying age at onset, we establish that three independent loci are significantly associated with age at onset (p < 5 × 10 -8 ). The lead single nucleotide polymorphisms collectively account for 25.6% of the remaining variance not explained by the hexanucleotide repeat and 13.0% of the overall variance in age at onset in X-linked dystonia-parkinsonism with the protective alleles delaying disease onset by seven years. These regions harbor or lie adjacent to MSH3 and PMS2, the genes that were recently implicated in modifying age at onset in Huntington's disease, likely through a common pathway influencing repeat instability. Our work indicates the existence of three modifiers of age at onset in X-linked dystonia-parkinsonism that likely affect the DNA mismatch repair pathway.

Congenital microcephaly is the clinical presentation of significantly reduced head circumference at birth. It manifests as both non-syndromic-microcephaly primary hereditary (MCPH)-and syndromic forms and shows considerable inter- and intrafamilial variability. It has been hypothesized that additional genetic variants may be responsible for this variability, but data are sparse. We have conducted deep phenotyping and genotyping of five Pakistani multiplex families with either MCPH ( n = 3) or Seckel syndrome ( n = 2). In addition to homozygous causal variants in ASPM or CENPJ , we discovered additional heterozygous modifier variants in WDR62, CEP63, RAD50 and PCNT -genes already known to be associated with neurological disorders. MCPH patients carrying an additional heterozygous modifier variant showed more severe phenotypic features. Likewise, the phenotype of Seckel syndrome caused by a novel CENPJ variant was aggravated to microcephalic osteodysplastic primordial dwarfism type II (MOPDII) in conjunction with an additional PCNT variant. We show that the CENPJ missense variant impairs splicing and decreases protein expression. We also observed centrosome amplification errors in patient cells, which were twofold higher in MOPDII as compared to Seckel cells. Taken together, these observations advocate for consideration of additional variants in related genes for their role in modifying the expressivity of the phenotype and need to be considered in genetic counseling and risk assessment.

One of the most widely cited hypotheses to explain the evolutionary maintenance of genetic recombination states that the reshuffling of genotypes at meiosis increases the efficiency of natural selection by reducing interference among selected loci. However, and despite several decades of theoretical work, a quantitative estimation of the possible selective advantage of a mutant allele increasing chromosomal map length (the average number of cross-overs at meiosis) remains difficult. This article derives a simple expression for the strength of selection acting on a modifier gene affecting the genetic map length of a whole chromosome or genome undergoing recurrent mutation. In particular, it shows that indirect selection for recombination caused by interference among mutations is proportional to [Formula: see text], where [Formula: see text] is the effective population size, U is the deleterious mutation rate per chromosome, and R is the chromosome map length. Indirect selection is relatively insensitive to the fitness effects of deleterious alleles, epistasis, or the genetic architecture of recombination rate variation and may compensate for substantial costs associated with recombination when linkage is tight. However, its effect generally stays weak in large, highly recombining populations., Competing Interests: The author declares no competing interest.

Background: Clinical phenotypes of hypertrophic cardiomyopathy (HCM) vary greatly even among patients with the same gene mutations. This variability is largely regulated by unidentified modifier loci. The purpose of the study is to identify modifier genes for cardiac fibrosis-a major phenotype of HCM-using the BXD family, a murine cohort., Methods: The relative severity of cardiac fibrosis was estimated by quantitation of cardiac collagen volume fraction (CCVF) across 66 members of the BXD family. Quantitative trait locus (QTL) mapping for cardiac fibrosis was done using GeneNetwork. Candidate modifier loci and genes associated with fibrosis were prioritized based on an explicit scoring system. Networks of correlation between fibrosis and cardiac transcriptomes were evaluated to generate causal models of disease susceptibility., Results: CCVF levels varied greatly within this family. Interval mapping identified a significant CCVF-related QTL on chromosome (Chr) 2 in males, and a significant QTL on Chr 4 Mb in females. The scoring system highlighted two strong candidate genes in the Chr 2 locus-Nek6 and Nr6a1. Both genes are highly expressed in the heart. Cardiac Nek6 mRNA levels are significantly correlated with CCVF. Nipsnap3b and Fktn are lead candidate genes for the Chr 4 locus, and both are also highly expressed in heart. Cardiac Nipsnap3b gene expression correlates well with CCVF., Conclusion: Our study demonstrated that candidate modifier genes of cardiac fibrosis phenotype in HCM are different in males and females. Nek6 and Nr6a1 are strong candidates in males, while Nipsnap3b and Fktn are top candidates in females., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The consequence of a mutation can be influenced by the context in which it operates. For example, loss of gene function may be tolerated in one genetic background, and lethal in another. The extent to which mutant phenotypes are malleable, the architecture of modifiers and the identities of causal genes remain largely unknown. Here, we measure the fitness effects of ~ 1,100 temperature-sensitive alleles of yeast essential genes in the context of variation from ten different natural genetic backgrounds and map the modifiers for 19 combinations. Altogether, fitness defects for 149 of the 580 tested genes (26%) could be suppressed by genetic variation in at least one yeast strain. Suppression was generally driven by gain-of-function of a single, strong modifier gene, and involved both genes encoding complex or pathway partners suppressing specific temperature-sensitive alleles, as well as general modifiers altering the effect of many alleles. The emerging frequency of suppression and range of possible mechanisms suggest that a substantial fraction of monogenic diseases could be managed by modulating other gene products., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Our understanding of cystic fibrosis (CF) has grown exponentially since the discovery of the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene in 1989. With evolving genetic and genomic tools, we have come to better understand the role of CFTR genotypes in the pathophysiology of the disease. This, in turn, has paved the way for the development of modulator therapies targeted at mutations in the CFTR , which are arguably one of the greatest advances in the treatment of CF. These modulator therapies, however, do not target all the mutations in CFTR that are seen in patients with CF and, furthermore, a variation in response is seen in patients with the same genotype who are taking modulator therapies. There is growing evidence to support the role of non-CFTR modifiers, both genetic and environmental, in determining the variation seen in CF morbidity and mortality and also in the response to existing therapies. This review focusses on key findings from studies using candidate gene and genome-wide approaches to identify CF modifier genes of lung disease in cystic fibrosis and considers the interaction between modifiers and the response to modulator therapies. As the use of modulator therapies expands and we gain data around outcomes, it will be of great interest to investigate this interaction further. Going forward, it will also be crucial to better understand the relative influence of genomic versus environmental factors. With this understanding, we can truly begin to deliver personalised care by better profiling the likely disease phenotype for each patient and their response to treatment.

Objectives: Myeloproliferative neoplasms (MPN) comprising polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) follow a bi-phasic course of disease with fibrotic and/or blastic progression. At presentation in the chronic phase, currently there are only insufficient tools to predict the risk of progression in individual cases., Methods: In this study, chronic phase MPN (16 PMF, 11 PV, and 11 MPN unclassified) with blastic transformation during course of disease (n = 38, median follow-up 5.3 years) were analyzed by high-throughput sequencing. MPN cases with a comparable follow-up period and without evidence of blast increase served as control (n = 63, median follow-up 5.8 years)., Results: Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) found at presentation were not significantly associated with blastic transformation. By contrast, mutations of SRSF2, U2AF1, and IDH1/2 at first presentation were frequently observed in the progression cohort (13/38, 34.2%) and were completely missing in the control group without blast transformation during follow-up (P = .0007 for SRSF2; P = .0063 for U2AF1 and IDH1/2)., Conclusion: Unlike frequent ARCH/CHIP alterations (TET2, ASXL1, DNMT3A), mutations in SRSF2, IDH1/2, and U2AF1 when manifest already at first presentation provide an independent risk factor for rapid blast transformation of MPN., (© 2021 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Collagen type IV alpha 1 and alpha 2 (COL4A1 and COL4A2) are major components of almost all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that can affect any organ but typically involves the cerebral vasculature, eyes, kidneys and skeletal muscles. In recent years, patient advocacy and family support groups have united under the name of Gould syndrome. The manifestations of Gould syndrome are highly variable, and animal studies suggest that allelic heterogeneity and genetic context contribute to the clinical variability. We previously characterized a mouse model of Gould syndrome caused by a Col4a1 mutation in which the severities of ocular anterior segment dysgenesis (ASD), myopathy and intracerebral hemorrhage (ICH) were dependent on genetic background. Here, we performed a genetic modifier screen to provide insight into the mechanisms contributing to Gould syndrome pathogenesis and identified a single locus [modifier of Gould syndrome 1 (MoGS1)] on Chromosome 1 that suppressed ASD. A separate screen showed that the same locus ameliorated myopathy. Interestingly, MoGS1 had no effect on ICH, suggesting that this phenotype could be mechanistically distinct. We refined the MoGS1 locus to a 4.3 Mb interval containing 18 protein-coding genes, including Fn1, which encodes the extracellular matrix component fibronectin 1. Molecular analysis showed that the MoGS1 locus increased Fn1 expression, raising the possibility that suppression is achieved through a compensatory extracellular mechanism. Furthermore, we found evidence of increased integrin-linked kinase levels and focal adhesion kinase phosphorylation in Col4a1 mutant mice that is partially restored by the MoGS1 locus, implicating the involvement of integrin signaling. Taken together, our results suggest that tissue-specific mechanistic heterogeneity contributes to the variable expressivity of Gould syndrome and that perturbations in integrin signaling may play a role in ocular and muscular manifestations., Competing Interests: Competing interests M.J. is an employee of Genentech, a member of the Roche group. K.H. is an employee of Centrillion Technologies., (© 2021. Published by The Company of Biologists Ltd.)

Background: Inherited cardiomyopathy associates with a range of phenotypes, mediated by genetic and nongenetic factors. Noninherited cardiomyopathy also displays varying progression and outcomes. Expression of cardiomyopathy genes is under the regulatory control of promoters and enhancers, and human genetic variation in promoters and enhancers may contribute to this variability., Methods: We superimposed epigenomic profiling from hearts and cardiomyocytes, including promoter-capture chromatin conformation information, to identify enhancers for 2 cardiomyopathy genes, MYH7 and LMNA . Enhancer function was validated in human cardiomyocytes derived from induced pluripotent stem cells. We also conducted a genome-wide search to ascertain genomic variation in enhancers positioned to alter cardiac expression and correlated one of these variants to cardiomyopathy progression using biobank data., Results: Multiple enhancers were identified and validated for LMNA and MYH7 , including a key enhancer that regulates the switch from MYH6 expression to MYH7 expression. Deletion of this enhancer resulted in a dose-dependent increase in MYH6 and faster contractile rate in engineered heart tissues. We searched for genomic variation in enhancer sequences across the genome, with a focus on nucleotide changes that create or interrupt transcription factor binding sites. The sequence variant, rs875908, disrupts a T-Box Transcription Factor 5 binding motif and maps to an enhancer region 2 kilobases from the transcriptional start site of MYH7. Gene editing to remove the enhancer that harbors this variant markedly reduced MYH7 expression in human cardiomyocytes. Using biobank-derived data, rs875908 associated with longitudinal echocardiographic features of cardiomyopathy., Conclusions: Enhancers regulate cardiomyopathy gene expression, and genomic variation within these enhancer regions associates with cardiomyopathic progression over time. This integrated approach identified noncoding modifiers of cardiomyopathy and is applicable to other cardiac genes.

Aims: In long QT syndrome (LQTS) patients, modifier genes modulate the arrhythmic risk associated with a disease-causing mutation. Their recognition can improve risk stratification and clinical management, but their discovery represents a challenge. We tested whether a cellular-driven approach could help to identify new modifier genes and especially their mechanism of action., Methods and Results: We generated human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) from two patients carrying the same KCNQ1-Y111C mutation, but presenting opposite clinical phenotypes. We showed that the phenotype of the iPSC-CMs derived from the symptomatic patient is due to impaired trafficking and increased degradation of the mutant KCNQ1 and wild-type human ether-a-go-go-related gene. In the iPSC-CMs of the asymptomatic (AS) patient, the activity of an E3 ubiquitin-protein ligase (Nedd4L) involved in channel protein degradation was reduced and resulted in a decreased arrhythmogenic substrate. Two single-nucleotide variants (SNVs) on the Myotubularin-related protein 4 (MTMR4) gene, an interactor of Nedd4L, were identified by whole-exome sequencing as potential contributors to decreased Nedd4L activity. Correction of these SNVs by CRISPR/Cas9 unmasked the LQTS phenotype in AS cells. Importantly, the same MTMR4 variants were present in 77% of AS Y111C mutation carriers of a separate cohort. Thus, genetically mediated interference with Nedd4L activation seems associated with protective effects., Conclusion: Our finding represents the first demonstration of the cellular mechanism of action of a protective modifier gene in LQTS. It provides new clues for advanced risk stratification and paves the way for the design of new therapies targeting this specific molecular pathway., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Novel genetic variants exist in patients with hereditary neuromuscular disorders (NMD), including muscular dystrophy. These patients also develop cardiac manifestations. However, the association between these gene variants and cardiac abnormalities is understudied. To determine genetic modifiers and features of cardiac disease in NMD patients, we have reviewed electronic medical records of 651 patients referred to the Muscular Dystrophy Association Care Center at the University of Cincinnati and characterized the clinical phenotype of 14 patients correlating with their next-generation sequencing data. The data were retrieved from the electronic medical records of the 14 patients included in the current study and comprised neurologic and cardiac phenotype and genetic reports which included comparative genomic hybridization array and NGS. Novel associations were uncovered in the following eight patients diagnosed with Limb-girdle Muscular Dystrophy, Bethlem Myopathy, Necrotizing Myopathy, Charcot-Marie-Tooth Disease, Peripheral Polyneuropathy, and Valosin-containing Protein-related Myopathy. Mutations in COL6A1, COL6A3, SGCA, SYNE1, FKTN, PLEKHG5, ANO5, and SMCHD1 genes were the most common, and the associated cardiac features included bundle branch blocks, ventricular chamber dilation, septal thickening, and increased outflow track gradients. Our observations suggest that features of cardiac disease and modifying gene mutations in patients with NMD require further investigation to better characterize genotype-phenotype relationships.

Trace amine-associated receptor 1 (Taar1) impacts methamphetamine (MA) intake. A mutant allele (Taar1 m1J ) derived from the DBA/2J mouse strain codes for a non-functional receptor, and Taar1 m1J/m1J mice consume more MA than mice possessing the reference Taar1 + allele. To study the impact of this mutation in a genetically diverse population, heterogeneous stock-collaborative cross (HS-CC) mice, the product of an eight-way cross of standard and wild-derived strains, were tested for MA intake. HS-CC had low MA intake, so an HS-CC by DBA/2J strain F2 intercross was created to transfer the mutant allele onto the diverse background, and used for selective breeding. To study residual variation in MA intake existing in Taar1 m1J/m1J mice, selective breeding for higher (MAH) vs lower (MAL) MA intake was initiated from Taar1 m1J/m1J F2 individuals; a control line of Taar1 +/+ individuals (MAC) was retained. The lines were also examined for MA-induced locomotor and thermal responses, and fluid and tastant consumption. Taar1 m1J/m1J F2 mice consumed significantly more MA than Taar1 +/+ F2 mice. Response to selection was significant by generation 2 and there were corresponding differences in fluid consumed. Fluid consumption was not different in non-MA drinking studies. Taar1 m1J/m1J genotype (MAL or MAH vs MAC mice) was associated with heighted MA locomotor and reduced hypothermic responses. MAL mice exhibited greater sensitization than MAH mice, but the selected lines did not consistently differ for thermal or tastant phenotypes. Residual variation among high-risk Taar1 m1J/m1J mice appears to involve mechanisms associated with neuroadaptation to MA, but not sensitivity to hypothermic effects of MA., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Wilms tumor is the most common pediatric renal cancer. However, genetic bases behind Wilms tumor remain largely unknown. H19 is a critical maternally imprinted gene. Previous studies indicated that single nucleotide polymorphisms (SNPs) in the H19 can modify the risk of several human malignancies. Epigenetic errors at the H19 locus lead to biallelic silencing in Wilms tumors. Genetic variations in the H19 may be related to Wilms tumor susceptibility., Methods: We conducted a four-center study to investigate whether H19 SNP was a predisposing factor to Wilms tumor. Three polymorphisms in the H19 (rs2839698 G > A, rs3024270 C > G, rs217727 G > A) were genotyped in 355 cases and 1070 cancer-free controls, using Taqman method. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the associations., Results: We found that all of these three polymorphisms were significantly associated with Wilms tumor risk alterations. The rs2839698 G > A polymorphism (AG vs. GG: adjusted OR = 0.74, 95% CI = 0.57-0.96, p = 0.024; AA vs. GG: adjusted OR = 1.52, 95% CI = 1.05-2.22, p = 0.027), the rs3024270 C > G polymorphism (CG vs. CC: adjusted OR = 0.61, 95% CI = 0.46-0.81, p = 0.0007; and the rs217727 polymorphism (AG vs. GG: adjusted OR = 0.76, 95% CI = 0.58-0.99, p = 0.035). The Carriers of 1, 2, and 1-2 risk genotypes were inclined to develop Wilms tumor compared with those without risk genotype (adjusted OR = 1.36, 95% CI = 1.02-1.80, p = 0.037; adjusted OR = 1.84, 95% CI = 1.27-2.67, p = 0.001; adjusted OR = 1.50, 95% CI = 1.17-1.92, p = 0.002, respectively). The stratified analysis further revealed that rs2839698 AA, rs217727 AA, and 1-2 risk genotypes could strongly increase Wilms tumor risk among children above 18 months of age, males, and with clinical stage I+II disease., Conclusion: Our findings indicate that genetic variations in the H19 may confer Wilms tumor risk., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Huntington's disease (HD) is caused by an expanded (>35) CAG trinucleotide repeat in huntingtin (HTT). Age-at-onset of motor symptoms is inversely correlated with the size of the inherited CAG repeat, which expands further in brain regions due to somatic repeat instability. Our recent genetic investigation focusing on autosomal SNPs revealed that age-at-onset is also influenced by genetic variation at many loci, the majority of which encode genes involved in DNA maintenance/repair processes and repeat instability., Objective: We performed a complementary association analysis to determine whether variants in the X chromosome modify HD., Methods: We imputed SNPs on chromosome X for ∼9,000 HD subjects of European ancestry and performed an X chromosome-wide association study (XWAS) to test for association with age-at-onset corrected for inherited CAG repeat length., Results: In a mixed effects model XWAS analysis of all subjects (males and females), assuming random X-inactivation in females, no genome-wide significant onset modification signal was found. However, suggestive significant association signals were detected at Xq12 (top SNP, rs59098970; p-value, 1.4E-6), near moesin (MSN), in a region devoid of DNA maintenance genes. Additional suggestive signals not involving DNA repair genes were observed in male- and female-only analyses at other locations., Conclusion: Although not genome-wide significant, potentially due to small effect size compared to the power of the current study, our data leave open the possibility of modification of HD by a non-DNA repair process. Our XWAS results are publicly available at the updated GEM EURO 9K website hosted at https://www.hdinhd.org/ for browsing, pathway analysis, and data download.

Methods employed in genome-wide association studies are not feasible ways to explore genotype-phenotype associations in rare disorders due to limited statistical power. An alternative approach is to examine relationships among specific single nucleotide polymorphisms (SNPs), selected a priori, and behavioural characteristics. Here, we adopt this strategy to examine relationships between three SNPs (5-HTTLPR, MAOA, COMT) and specific clinically-relevant behaviours that are phenotypic of fragile X syndrome (FXS) but vary in severity and frequency across individuals. Sixty-four males with FXS participated in the current study. Data from standardised informant measures of challenging behaviour (defined as physical aggression, property destruction, stereotyped behaviour, and self-injury), autism symptomatology, attention-deficit-hyperactivity-disorder characteristics, repetitive behaviour and mood/interest and pleasure were compared between each SNP genotype. No association was observed between behavioural characteristics and either 5-HTTLPR (serotonin) or MAOA (monoamine oxidase) genotypes. However, compared to the COMT (dopamine) AG and GG genotypes, the AA genotype was associated with greater interest and pleasure in the environment, and with reduced risk for property destruction, stereotyped behaviour and compulsive behaviour. The results suggest that common genetic variation in the COMT genotype affecting dopamine levels in the brain may contribute to the variability of challenging and repetitive behaviours and interest and pleasure in this population. This study identifies a role for additional genetic risk in understanding the neural and genetic mechanisms contributing to phenotypic variability in neurodevelopmental disorders, and highlights the merit of investigating SNPs that are selected a priori on a theoretical basis in rare populations.