Rare Polymorphisms Research Articles

DNA methylation changes in the genome of patients with hypogonadotropic hypogonadism

Although some Mendelian neurodevelopmental disorders have been shown to entail specific DNA methylation changes designated as epi-signatures, it remains unknown whether epi-signatures are consistent features of other genetic disorders. Here, we analyzed DNA methylation profiles of patients with hypogonadotropic hypogonadism (HH), a rare neuroendocrine disorder typically caused by monogenic or oligogenic mutations. First, we performed microarray-based genome-wide methylation analyses of nine patients with HH due to ANOS1, SOX2, or SOX10 variants and 12 control individuals. The results showed that 1118 probes were differentially methylated in one or more patients. The differentially methylated probes were highly variable among patients. No significant methylation changes were observed in genes functionally associated with ANOS1, SOX2, or SOX10. Then, we performed pyrosequencing of six selected CpG sites in the nine patients and 35 additional HH patients. The results of the patients were compared with those of 48 fertile men. There were no common methylation changes among these patients, with the exception of hypermethylation of two CpG sites in the ZNF245 promoter of three patients. Hypermethylation of the promoter has previously been reported as a very rare epigenetic polymorphism in the general population. These results indicate that genomes of HH patients have considerable DNA methylation changes; however, these changes are more likely to be physiological epigenetic variations than disease-specific epi-signatures. Our data suggest a possible association between hypermethylation of the ZNF254 promoter and HH, which needs to be examined in future studies.

Sulfotransferase 4A1 Coding Sequence and Protein Structure Are Highly Conserved in Vertebrates.

Cytosolic sulfotransferases (SULTs) are Phase 2 drug-metabolizing enzymes that catalyze the conjugation of sulfonate to endogenous and xenobiotic compounds, increasing their hydrophilicity and excretion from cells. To date, 13 human SULTs have been identified and classified into five families. SULT4A1 mRNA encodes two variants: (1) the wild type, encoding a 284 amino acid, ~33 kDa protein, and (2) an alternative spliced variant resulting from a 126 bp insert between exon 6 and 7, which introduces a premature stop codon that enhances nonsense-mediated decay. SULT4A1 is classified as an SULT based on sequence and structural similarities, including PAPS-domains, active-site His, and the dimerization domain; however, the catalytic pocket lid 'Loop 3' size is not conserved. SULT4A1 is uniquely expressed in the brain and localized in the cytosol and mitochondria. SULT4A1 is highly conserved, with rare intronic polymorphisms that have no outward manifestations. However, the SULT4A1 haplotype is correlated with Phelan-McDermid syndrome and schizophrenia. SULT4A1 knockdown revealed potential SULT4A1 functions in photoreceptor signaling and knockout mice display hampered neuronal development and behavior. Mouse and yeast models revealed that SULT4A1 protects the mitochondria from endogenously and exogenously induced oxidative stress and stimulates cell division, promoting dendritic spines' formation and synaptic transmission. To date, no physiological enzymatic activity has been associated with SULT4A1.

F2c.*C20209T mutation in patients with a history of thrombosis: A case report, retrospective 2 site-results and review of the literature.

G20210A (c.*97G>A) prothrombin gene variant, found in white population has been associated with an increased risk of venous thromboembolism (VTE). Other rare polymorphisms in F2 gene (C20209T) have been reported, more rare and touching black people, but its potential association with VTE remain uncertain. About a 69 years-old Caucasian woman presenting an unprovoked deep venous thrombosis of the leg, we analyzed retrospectively 25.000 thrombophilia tests on a 11-year period of time (2007-2018), at Nice and Marseille University Hospitals, and performed extensive review of the literature. Genetic determination included a similar PCR protocol and sequencing. Twenty-one heterozygous cases out of 25.585 determinations (0.08%) was found. The C20209T mutation detected in our Caucasian patient is rare, with a frequency that differed from what was reported in the previous literature, mainly in non-Caucasian patients (Africans, Africans-Americans, and Caribbeans). One hundred and thirteen patients with this mutation have been described in the literature, of which only one homozygous. This study is the most important on C20209T mutation performed at present, allowing to precise its frequency and its potential role in venous thromboembolism.

Novel human neurodevelopmental and neurodegenerative disease associated with IRF2BPL gene variants-mechanisms and therapeutic avenues.

Recently a broad range of phenotypic abnormalities related to the neurodevelopmental and neurodegenerative disorder NEDAMSS (Neurodevelopmental Disorder with Regression, Abnormal Movements, Loss of Speech, and Seizures) have been associated with rare single-nucleotide polymorphisms (SNPs) or insertion and deletion variants (Indel) in the intron-less gene IRF2BPL. Up to now, 34 patients have been identified through whole exome sequencing carrying different heterozygous pathogenic variants spanning the intron-less gene from the first polyglutamine tract at the N-terminus to the C3HC4 RING domain of the C-terminus of the protein. As a result, the phenotypic spectrum of the patients is highly heterogeneous and ranges from abnormal neurocognitive development to severe neurodegenerative courses with developmental and seizure-related encephalopathies. While the treatment of IRF2BPL-related disorders has focused on alleviating the patient's symptoms by symptomatic multidisciplinary management, there has been no prospect of entirely relieving the symptoms of the individual patients. Yet, the recent advancement of CRISPR-Cas9-derived gene editing tools, leading to the generation of base editors (BEs) and prime editors (PEs), provide an encouraging new therapeutic avenue for treating NEDAMSS and other neurodevelopmental and neurodegenerative diseases, which contain SNPs or smaller Indels in post-mitotic cell populations of the central nervous system, due to its ability to generate site-specific DNA sequence modifications without creating double-stranded breaks, and recruiting the non-homologous DNA end joining repair mechanism.

D239Y germline variant of NTHL1 glycosylase increases susceptibility to colorectal cancer in mice

Introduction: Persistent genotoxic stress leads to spontaneous DNA damage, which occurs on the order of 104−105 events per cell per day. The base excision repair is the first responder in the repair mechanisms after DNA damage, with DNA glycosylase being the first step in the repair process by binding to the DNA, cleaving the N-glycosidic bond and removing the oxidized pyrimidine DNA lesions. NTHL1 is a pivotal bifunctional DNA glycosylase enzyme involved in base excision repair mechanism. NTHL1 tumor syndrome is characterized by an increased lifetime risk for colorectal cancer (CRC), breast cancer, and colorectal polyposis. A rare rs3087468 polymorphism (D239Y) exhibits no glycosylase activity and may act as a dominant-negative mutation. Hypothesis: NTHL1-D239Y variant conforms susceptibility to inflammation-associated colorectal cancer. Methods: 6-8 weeks/old male and female C57BL/6 wild-type (WT, NTHL1WT/WT), heterozygous (NTHL1WT/D239Y), or homozygous (NTHL1D239Y/D239Y) knock-in mice were injected with 7.5mg/kg azoxymethane (AOM) followed by three cycles of alternating 2.5% dextran sulfate sodium (DSS, 5 days) and normal drinking water (14 days). We monitored body weight, collected fecal samples for microbial 16S rRNA amplicon sequencing, and analyzed colon length and number/size of polyps at euthanasia. The histology score was assessed by an unbiased pathologist to evaluate inflammation and tumor development. Blood samples were used to evaluate micronuclei formation. Results: NTHL1WT/D239Y and NTHL1D239Y/D239Y mice showed more pronounced weight loss starting from the 2nd DSS cycle. The number and size of polyps in the distal colon of NTHL1WT/D239Y and NTHL1D239Y/D239Y male mice were increased compared to WT littermates. NTHL1D239Y/D239Y females, but not males, showed increased polyp number compared to NTHL1WT/D239Y mice. No statistical difference was observed in the polyps’ size. Tumors extending into the proximal colon were observed uniquely in NTHL1D239Y/D239Y females. On the other hand, only NTHL1WT/D239Y males developed high adenomas in the distal colon compared to WT males. Interestingly, only NTHL1D239Y/D239Y males showed statistical difference in micronucleated normochromatic erythrocytes compared to WT mice. NTHL1 genotype did not significantly affect fecal microbial α- or β-diversity or colitis score and colon length in either gender. Conclusion: Our data suggest that NTHL1-D239Y variant confers a complex, zygosity- and sex-dependent susceptibility risk for inflammation-associated colorectal cancer that may be intrinsic and independent of changes in the gut microbiota. In females, it promotes more proximal tumor location, while in males, it promotes the size and tumor progression. 5P30CA023074 (JBS), Phoenix Women's Board of the Steele Children's Research Center (FKG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alzheimer Disease Associated Loci: APOE Single Nucleotide Polymorphisms in Marmara Region.

Alzheimer's disease (AD) is a major global health challenge, especially among individuals aged 65 or older. According to population health studies, Turkey has the highest AD prevalence in the Middle East and Europe. To accurately determine the frequencies of common and rare APOE single nucleotide polymorphisms (SNPs) in the Turkish population residing in the Marmara Region, we conducted a retrospective study analyzing APOE variants in 588 individuals referred to the Bursa Uludag University Genetic Diseases Evaluation Center. Molecular genotyping, clinical exome sequencing, bioinformatics analysis, and statistical evaluation were employed to identify APOE polymorphisms and assess their distribution. The study revealed the frequencies of APOE alleles as follows: ε4 at 9.94%, ε2 at 9.18%, and ε3 at 80.68%. The gender-based analysis in our study uncovered a tendency for females to exhibit a higher prevalence of mutant genotypes across various SNPs. The most prevalent haplotype observed was ε3/ε3, while rare APOE SNPs were also identified. These findings align with global observations, underscoring the significance of genetic diversity and gender-specific characteristics in comprehending health disparities and formulating preventive strategies.

Current Insight on the Role of Glucokinase and Glucokinase Regulatory Protein in Diabetes.

The glucokinase regulator (GCKR) gene encodes an inhibitor of the glucokinase enzyme (GCK), found only in hepatocytes and responsible for glucose metabolism. A common GCKR coding variation has been linked to various metabolic traits in genome-wide association studies. Rare GCKR polymorphisms influence GKRP activity, expression, and localization. Despite not being the cause, these variations are linked to hypertriglyceridemia. Because of their crystal structures, we now better understand the molecular interactions between GKRP and the GCK. Finally, small molecules that specifically bind to GKRP and decrease blood sugar levels in diabetic models have been identified. GCKR allelic spectrum changes affect lipid and glucose homeostasis. GKRP dysfunction has been linked to a variety of molecular causes, according to functional analysis. Numerous studies have shown that GKRP dysfunction is not the only cause of hypertriglyceridemia, implying that type 2 diabetes could be treated by activating liver-specific GCK via small molecule GKRP inhibition. The review emphasizes current discoveries concerning the characteristic roles of glucokinase and GKRP in hepatic glucose metabolism and diabetes. This information has influenced the growth of directed molecular therapies for diabetes, which has improved our understanding of lipid and glucose physiology.

Loss of NDST1 N-sulfotransferase activity is associated with autosomal recessive intellectual disability.

Intellectual Disability (ID) is the major cause of handicap, affecting nearly 3% of the general population, and is highly genetically heterogenous with more than a thousand genes involved. Exome sequencing performed in two independent families identified the same missense variant, p.(Gly611Ser), in the NDST1 (N-deacetylase/N-sulfotransferase member 1) gene. This variant had been previously found in ID patients of two other families but has never been functionally characterized. The NDST1 gene encodes a bifunctional enzyme that catalyzes both N-deacetylation and N-sulfation of N-acetyl-glucosamine residues during heparan sulfate (HS) biosynthesis. This step is essential because it influences the downstream enzymatic modifications and thereby determines the overall structure and sulfation degree of the HS polysaccharide chain. To discriminate between a rare polymorphism and a pathogenic variant, we compared the enzymatic properties of wild-type and mutant NDST1 proteins. We found that the p.(Gly611Ser) variant results in a complete loss of N-sulfotransferase activity while the N-deacetylase activity is retained. NDST1 shows the highest and the most homogeneous expression in the human cerebral structures compared to the other members of the NDST gene family. These results indicate that a loss of NDST1 N-sulfation activity is associated with impaired cognitive functions.

56 Biomarker-Based High Throughput Sperm Phenotyping: the Good, the Bad and the Ugly

Abstract Research in our laboratory addresses the unmet need for developing high throughput phenotyping for collecting comprehensive phenotypes of production, reproduction and fitness traits, a major emphasis area of the USDA Blueprint for Animal Genome Research 2022-2027. Our short- and long-term goal within this emphasis area is to identify specific genomic determinants of aberrant sperm quality and integrity that impart unique morphometric and optical properties on functionally defective spermatozoa that can be captured in next generation flow cytometry at a high speed/throughput, and without the need for costly, time-consuming sample processing. We identified a large set of deleterious gene variants associated with specific sperm defects in the genomes of 85 genetically valuable bulls with suboptimal artificial insemination (AI) fertility. Ongoing efforts focus on developing a second generation, label-free sperm phenotyping pipeline, eliminating variance and costs associated with the conventional, probe-based approaches, based on the specific genomic determinants of aberrant sperm quality resulting in unique morphometric and optical properties of spermatozoa, captured by the next-generation, brightfield image-based flow cytometry (IBFC) and translatable to high-precision phenotyping of heritable production traits. First step is to phenotype and validate rare deleterious polymorphisms associated with aberrant sperm head and tail morphologies in aforementioned cohort of 85 genome-sequenced bulls grouped based on prevailing sperm defect phenotype. Focus is on sperm defects predicted to be heritable and non-compensable by increasing sperm number per AI dose, such as knobbed acrosomes (sperm head caps), nuclear vacuoles and aplastic defects of sperm mitochondrial sheath (the major source of energy to fuel sperm movement). Other potentially heritable defects of interest may not be manifested by aberrant sperm morphologies, and only detectable at subcellular/molecular level by applying biomarkers validated by past and ongoing genome-to-phenotype studies. In the second phase, a label free sperm phenotyping pipeline for bull fertility testing will be developed, translatable to somatic cell phenotyping for other heritable traits by using the dedicated FeatureFinder IFBC software integrated with our machine learning pipeline, followed by validation assessing sperm structural integrity, “gentle” live cell sorting and metabolomics. This approach literary puts artificial intelligence in artificial insemination (AI in AI approach). Altogether, such genome-to-phenome efforts align the livestock genomic selection with state-of-the-art animal andrology, veterinary diagnostics and precision breeding, supporting the improvement of livestock industry efficiency through global approaches integrating production, reproduction, nutrition, health, and welfare.

Female meiotic drive shapes the distribution of rare inversion polymorphisms in Drosophila melanogaster.

In all species, new chromosomal inversions are constantly being formed by spontaneous rearrangement and then stochastically eliminated from natural populations. In Drosophila, when new chromosomal inversions overlap with a pre-existing inversion in the population, their rate of elimination becomes a function of the relative size, position, and linkage phase of the gene rearrangements. These altered dynamics result from complex meiotic behavior wherein overlapping inversions generate asymmetric dyads that cause both meiotic drive/drag and segmental aneuploidy. In this context, patterns in rare inversion polymorphisms of a natural population can be modeled from the fundamental genetic processes of forming asymmetric dyads via crossing-over in meiosis I and preferential segregation from asymmetric dyads in meiosis II. Here, a mathematical model of crossover-dependent female meiotic drive is developed and parameterized with published experimental data from Drosophila melanogaster laboratory constructs. This mechanism is demonstrated to favor smaller, distal inversions and accelerate the elimination of larger, proximal inversions. Simulated sampling experiments indicate that the paracentric inversions directly observed in natural population surveys of Drosophila melanogaster are a biased subset that both maximizes meiotic drive and minimizes the frequency of lethal zygotes caused by this cytogenetic mechanism. Incorporating this form of selection into a population genetic model accurately predicts the shift in relative size, position, and linkage phase for rare inversions found in this species. The model and analysis presented here suggest that this weak form of female meiotic drive is an important process influencing the genomic distribution of rare inversion polymorphisms.

Rare genetic variants underlie outlying levels of DNA methylation and gene-expression.

Testing the effect of rare variants on phenotypic variation is difficult due to the need for extremely large cohorts to identify associated variants given expected effect sizes. An alternative approach is to investigate the effect of rare genetic variants on DNA methylation (DNAm) as effect sizes are expected to be larger for molecular traits compared with complex traits. Here, we investigate DNAm in healthy ageing populations-the Lothian Birth Cohorts of 1921 and 1936-and identify both transient and stable outlying DNAm levels across the genome. We find an enrichment of rare genetic single nucleotide polymorphisms (SNPs) within 1kb of DNAm sites in individuals with stable outlying DNAm, implying genetic control of this extreme variation. Using a family-based cohort, the Brisbane Systems Genetics Study, we observed increased sharing of DNAm outliers among more closely related individuals, consistent with these outliers being driven by rare genetic variation. We demonstrated that outlying DNAm levels have a functional consequence on gene expression levels, with extreme levels of DNAm being associated with gene expression levels toward the tails of the population distribution. This study demonstrates the role of rare SNPs in the phenotypic variation of DNAm and the effect of extreme levels of DNAm on gene expression.

Factor VII Padua in Iran: clinical and laboratory findings of three unrelated patients.

The congenital factor VII (FVII) deficiency with an estimated incidence of one per 300 000 is the most common rare congenital bleeding disorder. The heterogeneous clinical pictures, including asymptomatic to life-threatening manifestations, are seen in patients with FVII deficiency. A variety of gene variants throughout the FVII ( F7 ) gene have been reported so far. In this setting, very rare FVII Padua polymorphism provokes an interesting condition in which results of prothrombin time and FVII activity are different based on the thromboplastin sources used in these tests. The current study aimed to report the phenotype and genotyping of patients with Padua variant. During the workup of the laboratory for FVII deficiency for diagnosis of FVII Padua, all patients with FVII deficiency who had prolonged prothrombin time, normal activated partial thromboplastin time, and variable FVII activity results using different sources of thromboplastin were included. Demographic data and clinical findings were recorded. For the molecular study, the F7 gene sequencing was performed using the Sanger sequencing technique. Five patients with FVII Padua and a history of mild-to-moderate bleeding, including easy bruising, epistaxis, gingivorrhagia, and bleeding after surgical challenges (including dental extraction and tonsillectomy), were detected during the study. DNA sequencing revealed a heterozygote CGG to CAG (Arg364Gln) variant in exon 9 at nucleotide position 1091, consistent with the genetic variant of FVII Padua. Timely diagnosis of FVII Padua is vital to avoid unnecessary exposure of patients to replacement therapy.

Pharmacogenetics and pharmacogenomics in glaucoma therapeutics: the way to personalized therapy.

Pharmacogenetics and pharmacogenomics in glaucoma therapeutics: the way to personalized therapy.

Self-Hybridization in Leishmania major.

Genetic exchange between different Leishmania strains in the sand fly vector has been experimentally demonstrated and is supported by population genetic studies. In nature, opportunities for Leishmania interstrain mating are restricted to flies biting multiply infected hosts or through multiple bites of different hosts. In contrast, self-mating could occur in any infected sand fly. By crossing two recombinant lines derived from the same Leishmania major strain, each expressing a different drug-resistance marker, self-hybridization in L. major was confirmed in a natural sand fly vector, Phlebotomus duboscqi, and in frequencies comparable to interstrain crosses. We provide the first high resolution, whole-genome sequencing analysis of large numbers of selfing progeny, their parents, and parental subclones. Genetic exchange consistent with classical meiosis is supported by the biallelic inheritance of the rare homozygous single nucleotide polymorphisms (SNPs) that arose by mutation during the generation of the parental clones. In contrast, heterozygous SNPs largely failed to be transmitted in Mendelian ratios for reasons not understood. SNPs that were heterozygous in both parents, however, recombined to produce homozygous alleles in some hybrids. For trisomic chromosomes present in both parents, transmittal to the progeny was only altered by self-hybridization, involving a gain or loss of somy in frequencies predicted by a meiotic process. Whole-genome polyploidization was also observed in the selfing progeny. Thus, self-hybridization in Leishmania, with its potential to occur in any infected sand fly, may be an important source of karyotype variation, loss of heterozygosity, and functional diversity. IMPORTANCE Leishmania are parasitic protozoa that cause a wide spectrum of diseases collectively known as the leishmaniases. Sexual reproduction in Leishmania has been proposed as an important source of genetic diversity and has been formally demonstrated to occur inside the sand fly vector midgut. Nevertheless, in the wild, opportunities for genetic exchange between different Leishmania species or strains are restricted by the capacity of different Leishmania strains to colonize the same sand fly. In this work, we report the first high resolution, whole-genome sequence analysis of intraclonal genetic exchange as a type of self-mating in Leishmania. Our data reveal that self-hybridization can occur with comparable frequency as interstrain mating under experimental lab conditions, leading to important genomic alterations that can potentially take place within every naturally infected sand fly.

Complement Factor I (CFI) Deficiency Is Linked to Alterations in Humoral Immunity and Accelerated Development of JAK2V617F-Dependent Phenotype

Background: Inflammation is an important phenomenon in hematologic malignancies, though its drivers are not well defined. Within the hematopoietic system, the complement cascade plays a complex role in regulating hematopoietic stem and progenitor cell (hSPC) homing and exit to systemic circulation (Lenkiewicz et al, Frontiers in Immunology 2019). The role the complement system plays in both normal and malignant hSPC egress from the bone marrow has been recognized, though its role in pathogenesis of hematologic malignancies is not well established. Complement cascade contributions to inflammation-mediated pathologies expanded the traditional view of the cascade from an acute process at the site of injury or pathogen invasion, to a mediator of chronic inflammation (Markiewskiet al, American Journal of Pathology 2007). We previously uncovered a rare single nucleotide polymorphism in an inhibitor of the complement cascade Complement Factor I - CFI (CFIG119R). This loss-of-function mutation was detected using whole genome sequencing in 20% of patients with primary myelofibrosis (PMF). In a separate PMF patient cohort, we observed an increase in global complement activity through elevated CH50 levels and decrease in C3 levels (Sadler et al, Blood 2021). There remains a gap in the understanding of how the complement cascade in general and CFI specifically contribute to development and progression of hematologic malignancies. Therefore, we established a murine model of CFI deficiency to determine mechanistic details of complement cascade contribution to both systemic and local inflammation and development of JAK2V617F-dependent pathologies. Methods: To determine the effect of CFI deficiency on homeostasis of hematopoietic system a CFI KO mouse model was generated by floxing exon 2 of the Cfi gene and removing it using CRISPR/Cas9-driven cre recombination. Inactivation of Cfi was confirmed by genotyping. Frequencies of granulocytes, B cells, plasmacytes, T cells, macrophages/monocytes, platelets, erythroid cells were assessed by FACS within the fraction of bone marrow mononuclear cells in 4-week-old CFIWT, HET (n=8) and KO (n=8) mice using established profiling protocols (Chorzalska et al, Blood 2018). Complete blood counts (CBCs) were performed on peripheral blood of 4-week-old Cfi WT (n=20), HET (n=26) and KO (n=10) mice. To determine the effect of CFI deficiency on JAK2V617F-driven phenotypes we performed colony forming assay using CFI WT (n=4, 2F, 2M)) or HET (n=4, 2F, 2M) bone marrows that were transduced with JAK2 V617F encoding retrovirus. Results: CFI KO and HET animals were viable and fertile. CBC analyses of CFI KO, HET vs. WT 4-week-old mice indicated no significant changes with an exception of a significant increase in red blood cell width distribution (RDW) in KO vs. WT and KO vs. HET peripheral blood samples suggestive of a mild anemia. Trends towards granulocytosis and monocytosis as well as leukopenia, erythropenia and decrease in hemoglobin levels were noted. Changes within the red blood cell compartment were confirmed by FSCS. In addition, flow cytometry data showed a significant decrease in CD19+ B-cells in CFI HET vs. WT animals, suggestive of dysregulation of B-cell maturation. Finally, colony forming unit assays showed 59% increase in number of colonies formed by CFI HET/JAK2 V617F+ vs. CFI WT/JAK2 V617F+ bone marrow cells indicative that loss of one copy of Cfi is sufficient to exacerbate JAK2V617F-dependent cellular phenotype. Conclusions: Early characterization of our newly generated CFI KO mouse model shows a phenotype indicative of CFI-loss induced changes within the homeostasis of hematopoietic system affecting red blood cell and B-cell compartments. Our data also indicate that loss of one copy of Cfi may accelerate cell growth and proliferation characteristics of JAK2 V617F-dependent myeloproliferative neoplasms. Detailed analysis of the CFI KO model may uncover novel therapeutically targetable mechanisms that link the pro-inflammatory and myeloproliferative phenotypes of some stem cell disorders, including JAK2 V617F-dependent MPNs.

VP.81 In vivo modulation of novel genetic modifiers for LAMA2-RD

Merosin-deficient congenital muscular dystrophy (LAMA2-RD) is caused by mutations in the LAMA2 gene, which encodes for the α2 subunit of laminin-211. Patients with complete laminin-211 deficiency show a clinically homogeneous and severe phenotype. To date, only one atypical patient with complete laminin-211 deficiency and a remarkably mild phenotype has been reported. Strikingly, the atypical patient and its severely affected sibling carry the same <i>LAMA2</i> loss-of-function mutation, and both lack laminin-211 expression in muscle. Since laminin-211 is not present in either sibling, we hypothesize that there is a rare genetic modifier in the atypical sibling that compensates for laminin-211 absence in muscle via a novel mechanism. We performed unbiased muscle RNA-sequencing and identified genes differentially expressed in the atypical patient compared to unrelated LAMA2-RD patients and healthy controls. Transcriptome data were complemented by the analysis of whole genome sequencing data of both siblings. We identified differentially expressed genes potentially regulated by very rare DNA polymorphisms uniquely present in the atypical sibling. These genes affect pathways commonly dysregulated in LAMA2-RD, such as energy metabolism, regeneration, inflammation and fibrosis, and represent candidate modifier gene(s)/pathway(s) that could provide a novel therapeutic approach and ameliorate LAMA2-RD severity. We are currently testing the most promising candidates in vivo in a LAMA2-knockout zebrafish model that recapitulates LAMA2-RD. In particular we are assessing the potential for these protective modifier(s) to improve muscle morphology and improve the phenotype in the diseased animals. Outcomes of this project will add new knowledge about the molecular aspects of this disorder, shed light on novel mechanisms involved in attenuating LAMA2-RD severity and potentially lead to the development of new therapeutic strategies.

Noncoding rare variants in PANX3 are associated with chronic back pain.

Back pain is the leading cause of years lived with disability worldwide, yet surprisingly, little is known regarding the biology underlying this condition. The impact of genetics is known for chronic back pain: its heritability is estimated to be at least 40%. Large genome-wide association studies have shown that common variation may account for up to 35% of chronic back pain heritability; rare variants may explain a portion of the heritability not explained by common variants. In this study, we performed the first gene-based association analysis of chronic back pain using UK Biobank imputed data including rare variants with moderate imputation quality. We discovered 2 genes, SOX5 and PANX3 , influencing chronic back pain. The SOX5 gene is a well-known back pain gene. The PANX3 gene has not previously been described as having a role in chronic back pain. We showed that the association of PANX3 with chronic back pain is driven by rare noncoding intronic polymorphisms. This result was replicated in an independent sample from UK Biobank and validated using a similar phenotype, dorsalgia, from FinnGen Biobank. We also found that the PANX3 gene is associated with intervertebral disk disorders. We can speculate that a possible mechanism of action of PANX3 on back pain is due to its effect on the intervertebral disks.

Functional significance of the rare rs35667974 IFIH1 gene polymorphism, associated with multiple autoimmune diseases, using a structural biological approach

Autoimmune diseases, which affect approximately 5% of human population, are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Recent genome wide association studies (GWAS) have successfully identified novel autoimmune disease-associated loci, with many of them shared by multiple disease-associated pathways but much of the genetics and pathophysiological mechanisms remain still obscure. Considering that most of the potential causal variants are still unknown, many studies showed that the missense variant rs35667974 at interferon-induced with helicase C domain 1 (IFIH1) gene is protective for type 1 diabetes (T1D), psoriasis (PS) and psoriatic arthritis (PsA). Recently, this variant was found to be also associated with ankylosing spondylitis (AS), Crohn’s disease (CD) and ulcerative colitis (UC). The IFIH1 gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5) that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. In the present study we sought to investigate the association of the rare rs35667974 variant of IFIH1 gene, which resides in exon 14 and changes a conserved isoleucine at position #923 to valine, in the development of various autoimmune diseases and give a reason for the selectivity affecting different autoimmune diseases. Evolutionary studies and three-dimensional (3 D) homology modelling were employed on the MDA5 protein product, through its association with dsRNA, recognition factor controlling cytokine and chemokine signalling, to investigate the protective role of the MDA5 variant for certain autoimmune diseases.

Polymorphic variant Asp239Tyr of human DNA glycosylase NTHL1 is inactive for removal of a variety of oxidatively-induced DNA base lesions from genomic DNA

Base excision repair is the major pathway for the repair of oxidatively-induced DNA damage, with DNA glycosylases removing modified bases in the first step. Human NTHL1 is specific for excision of several pyrimidine- and purine-derived lesions from DNA, with loss of function NTHL1 showing a predisposition to carcinogenesis. A rare single nucleotide polymorphism of the Nthl1 gene leading to the substitution of Asp239 with Tyr within the active site, occurs within global populations. In this work, we overexpressed and purified the variant NTHL1-Asp239Tyr (NTHL1-D239Y) and determined the substrate specificity of this variant relative to wild-type NTHL1 using gas chromatography-tandem mass spectrometry with isotope-dilution, and oxidatively-damaged genomic DNA containing multiple pyrimidine- and purine-derived lesions. Wild-type NTHL1 excised seven DNA base lesions with different efficiencies, whereas NTHL1-D239Y exhibited no glycosylase activity for any of these lesions. We also measured the activities of human glycosylases OGG1 and NEIL1, and E. coli glycosylases Nth and Fpg under identical experimental conditions. Different substrate specificities among these DNA glycosylases were observed. When mixed with NTHL1-D239Y, the activity of NTHL1 was not reduced, indicating no substrate binding competition. These results and the inactivity of the variant D239Y toward the major oxidatively-induced DNA lesions points to the importance of the understanding of this variant’s role in carcinogenesis and the potential of individual susceptibility to cancer in individuals carrying this variant.

Metabolic and Genetic Evaluation in Children with Nephrolithiasis.

To evaluate metabolic and genetic abnormalities in children with nephrolithiasis attending a referral center in North India. The patients aged 1-18 y old with nephrolithiasis underwent biochemical evaluation and whole-exome sequencing. The authors evaluated for monogenic variants in 56 genes and compared allele frequency of 39 reported polymorphisms between patients and 1739 controls from the GenomeAsia 100K database. Fifty-four patients, aged 9.1 ± 3.7 y were included. Stones were bilateral in 42.6%, familial in 33.3%, and recurrent in 25.9%. The most common metabolic abnormalities were hypercalciuria (35.2%), hyperoxaluria (24.1%), or both (11.1%), while xanthinuria (n = 3), cystinuria (n = 1), and hyperuricosuria (n = 1) were rare. Exome sequencing identified an etiology in 6 (11.1%) patients with pathogenic/likely pathogenic causative variants. Three variants in MOCOS and one in ATP7B were pathogenic; likely pathogenic variants included MOCOS (n = 2), AGXT, and SLC7A9 (n = 1, each). Causality was not attributed to two SLC34A1 likely pathogenic variants, due to lack of matching phenotype and dominant family history. Compared to controls, allele frequency of the polymorphism TRPV5 rs4252402 was significantly higher in familial stone disease (allele frequency 0.47 versus 0.53; OR 3.2, p = 0.0001). The chief metabolic abnormalities were hypercalciuria and hyperoxaluria. A monogenic etiology was identified in 11% with pathogenic or likely pathogenic variants using a gene panel for nephrolithiasis. Heterozygous missense variants in the sodium-phosphate cotransporter SLC34A1 were common and required evaluation for attributing pathogenicity. Rare polymorphisms in TRPV5 might increase the risk of familial stones. These findings suggest that a combination of metabolic and genetic evaluation is useful for determining the etiology of nephrolithiasis.