Intronic Deletion Research Articles

Analysis of exonic deletions in a large population study provides novel insights into NRXN1 pathology

The NRXN1 locus is a hotspot for non-recurrent copy number variants and exon-disrupting NRXN1 deletions have been associated with increased risk of neurodevelopmental disorders in case-control studies. However, corresponding population-based estimates of prevalence and disease-associated risk are currently lacking. Also, most studies have not differentiated between deletions affecting exons of different NRXN1 splice variants nor considered intronic deletions. We used the iPSYCH2015 case-cohort sample to obtain unbiased estimates of the prevalence of NRXN1 deletions and their associated risk of autism, schizophrenia, depression, and ADHD. Most exon-disrupting deletions affected exons specific to the alpha isoform, and almost half of the non-exonic deletions represented a previously reported segregating founder deletion. Carriage of exon-disrupting NRXN1 deletions was associated with a threefold and twofold increased risk of autism and ADHD, respectively, whereas no significantly increased risk of depression or schizophrenia was observed. Our results highlight the importance of using population-based samples in genetic association studies.

Mitochondrial trifunctional protein deficiency caused by a deep intronic deletion leading to aberrant splicing

AbstractTrifunctional protein deficiency (TFP) is a disorder of fatty acid beta‐oxidation associated with metabolic, cardiac, and liver dysfunction in severe forms. We present two siblings diagnosed by newborn screening and confirmed by biochemical testing at birth. Their clinical course was complicated by recurrent rhabdomyolysis, retinopathy, and hypoparathyroidism. Both siblings were also diagnosed with focal segmental glomerulosclerosis (FSGS) and bone marrow failure and ultimately died of hypoxemic respiratory failure. Initial sequencing of the TFP‐associated genes HADHA and HADHB showed only a paternally inherited variant in HADHB, NM_000183.3:c.1059del (p.Gly354AspfsTer10). Subsequent evaluation by the Undiagnosed Diseases Network with genome and transcriptome sequencing revealed a rare maternally inherited 17 base pair deletion in HADHB, NM_000183.3:c.1390‐515_1390‐499del, located in the final intron and resulting in a pseudoexon that harbors a premature termination codon. Both sisters were compound heterozygous for this and the paternal premature termination codon. No other variants were detected that were potentially causative for the FSGS and bone marrow failure on genome sequencing. A review of the literature at that time revealed several case reports of the uncommon clinical findings of FSGS, bone marrow failure, and pulmonary involvement in patients with TFP, confirming this clinical diagnosis as the complete explanation for these siblings.

Biotin-Thiamine-Responsive Basal Ganglia Disease Case report

Introduction Biotin-thiamine responsive basal ganglia disease (BTBGD) is a rare autosomal recessive neurometabolic disorder characterized by diverse and variable phenotypic features, which can make diagnosis challenging. However, prompt treatment with thiamine and biotin can effectively manage the condition. Diagnosis relies on the identification of biallelic pathogenic variants in the SLC19A3 gene. This case report describes two novel variants of uncertain significance in the SLC19A3 gene, which may be correlated with the phenotypic manifestations of BTBGD. Case presentation Our case is a 7-month-old female infant presented with a 3-week history of irritability, altered behavior, and refusal of newly introduced solid foods. Symptoms started with an upper respiratory tract infection, followed by lethargy, floppiness, and abnormal movements. The patient was admitted to the pediatric ward with a broad differential diagnosis. Extensive laboratory evaluations revealed lactic acidosis. MRI brain showed symmetric restricted diffusion affecting the bilateral basal ganglia, thalami, and cortical regions. Whole genome sequencing identified biallelic variants of the SLC19A3: a c.1364T>G p.Met455Arg missense variant in the maternal allele and a 2.3 kb deletion of intron 3 of the paternal allele. Both variants were identified as variants of uncertain significance. However, given the clinical picture, MRI brain findings, resolution of symptoms with empiric biotin and thiamine supplementation, and biallelic SLC19A3 variants of unknown significance, the patient most likely suffers from BTBGD. Patient continues to show sustained developmental progress on biotin and thiamine supplementation. Conclusion This case highlights the fact that genetic testing remains a vital but improvable tool for the diagnosis of BTBGD. As of yet, genetic testing and diagnosis of BTBGD continues to be limited by the knowledge of which SLC19A3 variants are established to be pathogenic variants. Thus, further research is required to study other SCL19A3 variants of unknown significance to further improve genetic testing and diagnosis of BTBGD in the future.

Genome-wide copy number variation association study in anorexia nervosa.

This study represents the first large-scale investigation of rare (<1% population frequency) copy number variants (CNVs) in anorexia nervosa (AN). Large, rare CNVs are reported to be causally associated with anthropometric traits, neurodevelopmental disorders, and schizophrenia, yet their role in the genetic basis of AN is unclear. Using genome-wide association study (GWAS) array data from the Anorexia Nervosa Genetics Initiative (ANGI), which included 7414 AN case and 5044 controls, we investigated the association of 67 well-established syndromic CNVs and 178 pleiotropic disease-risk dosage-sensitive CNVs with AN. To identify novel CNV regions (CNVRs) that increase the risk of AN, we conducted genome-wide association studies with a focus on rare CNV-breakpoints (CNV-GWAS). We found no net enrichment of rare CNVs, either deletions or duplications, in AN, and none of the well-established syndromic or pleiotropic CNVs had a significant association with AN status. However, the CNV-GWAS found 21 nominally associated CNVRs that contribute to AN risk, covering protein-coding genes implicated in synaptic function, metabolic/mitochondrial factors, and lipid characteristics, like the CD36 (7q21.11) gene, which transports long-chain fatty acids into cells. CNVRs intersecting genes previously related to neurodevelopmental traits include deletions of NRXN1 intron 5 (2p16.3), IMMP2L (7q31.1), and PTPRD (9p23). Overall, given that our study is well powered to detect the CNV burden level reported for schizophrenia, we can conclude that rare CNVs have a limited role in the etiology of AN, as reported for bipolar disorder. Our nominal associations for the 21 discovered CNVRs are consistent with AN being a metabo-psychiatric trait, as demonstrated by the common genetic architecture of AN, and we provide association results to allow for replication in future research.

RNA-First Approach Identifies Deep Intronic PHEX Variants in X-Linked Hypophosphatemic Rickets.

Up to 20% of patients with X-linked hypophosphatemic rickets (XLH) have no causative variant identified on routine molecular diagnostic testing. To identify intronic variants causing PHEX mis-splicing in patients with XLH. The metabolic bone clinic of a paediatric orthopedic hospital. Four patients (age 6 to 12 years; 3 girls) with clinically diagnosed XLH and no PHEX variant on routine testing. RNA and DNA sequence analysis of PHEX. Urine-derived cells were cultured, and mRNA was extracted and transcribed to cDNA. PHEX cDNA was amplified by PCR, followed by sequencing of PCR products. Sequencing of PHEX intronic DNA regions was performed to identify variants causing mis-splicing observed on RNA analysis. PHEX mis-splicing was identified in 3 of the 4 participants, and an intronic variant was identified in all 3 cases. In a 12-year-old boy, transcript analysis showed skipping of PHEX exon 13, while sequencing of PHEX intronic regions revealed a de novo 18 bp deletion in intron 13. In a 7-year-old girl, a pseudoexon in PHEX intron 17 was found, associated with a de novo deep intronic variant (c.1768+173A>G) that activated a cryptic splice donor site. Finally, an 84 bp pseudoexon in PHEX intron 21 caused by a recurrent de novo deep intronic variant (c.2147+1197A>G) was identified in an 11-year-old girl. Analysis of RNA from urine-derived cells combined with sequencing of PHEX introns can identify deep intronic variants in individuals with XLH without a detectable PHEX variant in routine exon-centric molecular diagnosis.

A rare mutation, deletion of HBB intron 2 to HBG1 exon 3 gene regions (12.6 kb) along with c.27dup, leading to transfusion-dependent thalassemia

A rare mutation, deletion of HBB intron 2 to HBG1 exon 3 gene regions (12.6 kb) along with c.27dup, leading to transfusion-dependent thalassemia

Seed color patterns in domesticated common bean are regulated by MYB-bHLH-WD40 transcription factors and temperature.

Seed colors and color patterns are critical for the survival of wild plants and the consumer appeal of crops. In common bean, a major global staple, these patterns are also essential in determining market classes, yet the genetic and environmental control of many pigmentation patterns remains unresolved. In this study, we genetically mapped variation for several important seed pattern loci, including T, Bip, phbw, and Z, which co-segregated with candidate genes PvTTG1, PvMYC1, PvTT8, and PvTT2, respectively. Proteins encoded by these genes are predicted to work together in MYB-bHLH-WD40 (MBW) complexes, propagating flavonoid biosynthesis across the seed coat as observed in Arabidopsis. Whole-genome sequencing of 37 accessions identified mutations, including seven unique parallel mutations in T (PvTTG1) and non-synonymous SNPs in highly conserved residues in bipana (PvMYC1) and z (PvTT2). A 612 bp intron deletion in phbw (PvTT8) eliminated motifs conserved since the Papilionoideae origin and corresponded to a 20-fold reduction in transcript abundance. In multi-location field trials of seven varieties with partial seed coat pigmentation patterning, the pigmented seed coat area correlated positively with ambient temperature, with up to 11-fold increases in the pigmented area from the coolest to the warmest environments. In controlled growth chamber conditions, an increase of 4°C was sufficient to cause pigmentation on an average additional 21% of the seed coat area. Our results shed light on key steps of flavonoid biosynthesis in common bean. They will inform breeding efforts for seed coat color/patterning to improve consumer appeal in this nutritious staple crop.

An Improved Helper Plasmid Containing Deletions Within the E4 and E2a Genes Results in Increased Adeno-Associated Virus Productivity.

The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in ∼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.

Are COVID-19 Polymorphisms in ACE and ACE2 Prognosis Predictors?

Regardless of the containment of the SARS-CoV-2 pandemic, it remains paramount to comprehensively understand its underlying mechanisms to mitigate potential future health and economic impacts, comparable to those experienced throughout the course of the pandemic. The angiotensin-converting enzyme 2 (ACE2) provides anchorage for SARS-CoV-2 binding, thus implicating that ACE and ACE2 might contribute to the variability in infection severity. This study aimed to elucidate predisposing factors influencing the disease course among people infected by SARS-CoV-2, focusing on angiotensin-converting enzyme (ACE) and ACE2 polymorphisms. Notably, despite similar demographics and comorbidities, COVID-19 patients exhibit substantial differences in prognosis. Genetic polymorphisms in ACE and ACE2 have been implicated in disease progression, prompting our investigation into their role in COVID-19 evolution. Using next-generation sequencing (NGS), we analyzed ACE and ACE2 genes in a sample group comprising six subjects infected by SARS-CoV-2. Our findings revealed a correlation between specific polymorphisms and COVID-19 outcomes. Specifically, ACE and ACE2 intronic deletions were observed in all deceased patients, suggesting a potential association with mortality. These results highlight the significance of genetic factors in shaping the clinical course of COVID-19, emphasizing the importance of further research into the impact of genetic variations on COVID-19 severity.

Novel splicing variant and gonadal mosaicism in DYRK1A gene identified by whole-genome sequencing in multiplex autism spectrum disorder families.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with considerable genetic heterogeneity. The disorder is clinically diagnosed based on DSM-5 criteria, featuring deficits in social communication and interaction, along with restricted and repetitive behaviours. Here, we performed whole-genome sequencing (WGS) on four individuals with ASD from two multiplex families (MPX), where more than one individual is affected, to identify potential single nucleotide variants (SNVs) and structural variants (SVs) in coding and non-coding regions. A rigorous bioinformatics pipeline was employed for variant detection, followed by segregation analysis. Our investigation revealed an unreported splicing variant in the DYRK1A gene (c.-77 + 2T > C; IVS1 + 2T > C; NM_001396.5), in heterozygote form in two affected children in one of the families (family B), which was absent in the healthy parents and siblings. This finding suggests the presence of gonadal mosaicism in one of the parents, representing the first documented instance of such inheritance for a variant in the DYRK1A gene associated with ASD. Furthermore, we identified a 50bp deletion in intron 9 of the DLG2 gene in two affected patients from the same family, confirmed by PCR and Sanger sequencing. In Family A, we identified potential candidate variants associated with ASD shared by the two patients. These findings enhance our understanding of the genetic landscape of ASD, particularly in MPX families, and highlight the utility of WGS in uncovering novel genetic contributions to neurodevelopmental disorders.

Alterations in neural activation in the ventral frontoparietal network during complex magnocellular stimuli in developmental dyslexia associated with READ1 deletion

BackgroundAn intronic deletion within intron 2 of the DCDC2 gene encompassing the entire READ1 (hereafter, READ1d) has been associated in both children with developmental dyslexia (DD) and typical readers (TRs), with interindividual variation in reading performance and motion perception as well as with structural and functional brain alterations. Visual motion perception -- specifically processed by the magnocellular (M) stream -- has been reported to be a solid and reliable endophenotype of DD. Hence, we predicted that READ1d should affect neural activations in brain regions sensitive to M stream demands as reading proficiency changes.MethodsWe investigated neural activations during two M-eliciting fMRI visual tasks (full-field sinusoidal gratings controlled for spatial and temporal frequencies and luminance contrast, and sensitivity to motion coherence at 6%, 15% and 40% dot coherence levels) in four subject groups: children with DD with/without READ1d, and TRs with/without READ1d.ResultsAt the Bonferroni-corrected level of significance, reading skills showed a significant effect in the right polar frontal cortex during the full-field sinusoidal gratings-M task. Regardless of the presence/absence of the READ1d, subjects with poor reading proficiency showed hyperactivation in this region of interest (ROI) compared to subjects with better reading scores. Moreover, a significant interaction was found between READ1d and reading performance in the left frontal opercular area 4 during the 15% coherent motion sensitivity task. Among subjects with poor reading performance, neural activation in this ROI during this specific task was higher for subjects without READ1d than for READ1d carriers. The difference vanished as reading skills increased.ConclusionsOur findings showed a READ1d-moderated genetic vulnerability to alterations in neural activation in the ventral attentive and salient networks during the processing of relevant stimuli in subjects with poor reading proficiency.

A 9.5-kb deletion in the 1st intron of OsMADS51 enhances temperature sensitivity in rice

Temperature is an important environmental factor affecting heading date of rice. Despite its importance, genes responsible for temperature-sensitive heading in rice have remained elusive. Our previous study identified a quantitative trait locus qHd1 which advances heading date under high temperatures. A 9.5-kb insertion was found in the first intron of OsMADS51 in indica variety Zhenshan 97 (ZS97). However, the function of this natural variant in controlling temperature sensitivity has not been verified. In this study, we used CRISPR/Cas9 to knock out the 9.5-kb insertion in ZS97. Experiments conducted under cotrolled conditions in phytotrons confirmed that deletion increased temperature sensitivity and advanced heading by downregulating the expression level of OsMADS51. One-hybrid assays in yeast, ChIP-quantitative polymerase chain reaction, electrophoretic mobility shift, and luciferase-based transient transactivation assays collectively confirmed that OsMADS51 affects heading date by regulation of heading date gene Ehd1. We further determined that the long non-coding RNA HEATINR is generated from the first intron of OsMADS51, offering an explanation for how the 9.5-kb insertion affects temperature sensitivity. We also found that OsMADS51 was strongly selected in early/late-season rice varieties in South China, possibly accounting for their strong temperature sensitivity. These insights not only advance our understanding of the molecular mechanisms underlying the temperature-responsive regulation of heading date in rice but also provide a valuable genetic target for molecular breeding.

An axonemal intron splicing program sustains Plasmodium male development

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint

Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of β-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD intron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBB exon 1 β-thalassemia variant that strengthens a cryptic donor (NM_000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt.

The Schizosaccharomyces pombe DEAD-box protein Mss116 is required for mitoribosome assembly and mitochondrial translation

DEAD-box helicases are important players in mitochondrial gene expression, which is necessary for mitochondrial respiration. In this study, we characterized Schizosaccharomyces pombe Mss116 (spMss116), a member of the family of DEAD-box RNA helicases. Deletion of spmss116 in a mitochondrial intron-containing background significantly reduced the levels of mitochondrial DNA (mtDNA)-encoded cox1 and cob1 mRNAs and impaired mitochondrial translation, leading to a severe respiratory defect and a loss of cell viability during stationary phase. Deletion of mitochondrial introns restored the levels of cox1 and cob1 mRNAs to wide-type (WT) levels but could not restore mitochondrial translation and respiration in Δspmss116 cells. Furthermore, deletion of spmss116 in both mitochondrial intron-containing and intronless backgrounds impaired mitoribosome assembly and destabilization of mitoribosomal proteins. Our findings suggest that defective mitochondrial translation caused by deletion of spmss116 is most likely due to impaired mitoribosome assembly.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

Loss-of-function of activity-dependent neuroprotective protein (ADNP) by a splice-acceptor site mutation causes Helsmoortel-Van der Aa syndrome.

Mutations in ADNP result in Helsmoortel-Van der Aa syndrome. Here, we describe the first de novo intronic deletion, affecting the splice-acceptor site of the first coding ADNP exon in a five-year-old girl with developmental delay and autism. Whereas exome sequencing failed to detect the non-coding deletion, genome-wide CpG methylation analysis revealed an episignature suggestive of a Helsmoortel-Van der Aa syndrome diagnosis. This diagnosis was further supported by PhenoScore, a novel facial recognition software package. Subsequent whole-genome sequencing resolved the three-base pair ADNP deletion c.[-5-1_-4del] with transcriptome sequencing showing this deletion leads to skipping of exon 4. An N-terminal truncated protein could not be detected in transfection experiments with a mutant expression vector in HEK293T cells, strongly suggesting this is a first confirmed diagnosis exclusively due to haploinsufficiency of the ADNP gene. Pathway analysis of the methylome indicated differentially methylated genes involved in brain development, the cytoskeleton, locomotion, behavior, and muscle development. Along the same line, transcriptome analysis identified most of the differentially expressed genes as upregulated, in line with the hypomethylated CpG episignature and confirmed the involvement of the cytoskeleton and muscle development pathways that are also affected in patient cell lines and animal models. In conclusion, this novel mutation for the first time demonstrates that Helsmoortel-Van der Aa syndrome can be caused by a loss-of-function mutation. Moreover, our study elegantly illustrates the use of EpiSignatures, WGS and Phenoscore as novel complementary diagnostic tools in case a of negative WES result.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

A retrospective study on the clinical and molecular outcomes of calpainopathy in a Turkish patient cohort.

Calpainopathy, also known as limb-girdle muscular dystrophy recessive type 1, is a progressive muscle disorder that impacts the muscles around the hips and shoulders. The disease is caused by defects in the CAPN3 gene and can be inherited in both recessive and dominant forms. In this retrospective study, we aimed to evaluate the clinical and molecular results of our patients with calpainopathy and to examine the CAPN3 variants in Turkish and global populations. Molecular analyses were performed using the next-generation sequencing (NGS) method. CAPN3 variants were identified through the examination of various databases. In this retrospective study, the cohort consisted of seven patients exhibiting the CAPN3 (NM_000070.3) mutation and a phenotype compatible with calpainopathy at a single center in Türkiye. All patients displayed high CK levels and muscle weakness. We report a novel missense c.2437G>A variant that causes the autosomal dominant form of calpainopathy. Interestingly, the muscle biopsy report for the patient with the novel mutation indicated sarcoglycan deficiency. Molecular findings for the remaining individuals in the cohort included a compound heterozygous variant (frameshift and missense), one homozygous nonsense, one homozygous intronic deletion, and three homozygous missense variants. The most common variant in the Turkish population was c.550del. In both populations, pathogenic variants were most frequently located in exon 21, according to exon length. Variants were stochastically distributed based on consequences in CAPN3 domains. Therefore, the NGS method proves highly effective in diagnosing rare diseases characterized by clinical heterogeneity. Assessing variants based on ethnicity holds significance in the development of precise therapies.

Genomic full-length sequence of the novel HLA-DQB1*04:01:01:04 allele.

The sequence of HLA-DQB1*04:01:01:04 differs from HLA-DQB1*04:01:01:03 by four nucleotide deletion in intron 2.