Rare Missense Research Articles

Mutations in the WLS are associated with dental anomalies, torus palatinus, and torus mandibularis.

Canonical and non-canonical WNT signaling are important for odontogenesis. WNT ligand secretion mediator (WLS; MIM611514) is required to transport lipid-modified WNT proteins from the Golgi to the cell membrane, where canonical and non-canonical WNT proteins are released into the extracellular milieu. Biallelic pathogenic variants in WLS are implicated in autosomal recessive Zaki syndrome (ZKS; MIM 619648), the only genetic condition known to be caused by pathogenic variants in WLS. To investigate molecular etiology of dental anomalies in 250 patients with or without oral exostoses. Clinical and radiographic examination, and whole exome sequencing, were performed in the case of 250 patients with dental anomalies with or without oral exostoses. Four extremely rare heterozygous missense variants (p.Ile20Thr, p.Met46Leu, p.Ser453Ile and p.Leu516Phe) in WLS were identified in 11 patients with dental anomalies. In five of these patients, a torus palatinus or a torus mandibularis was observed. We report for the first time the heterozygous WLS variants in patients with dental anomalies. Root maldevelopments in patients with WLS variants supports the role of canonical and non-canonical WNT signaling in root development. We also show that variants in WLS were implicated in torus palatinus and torus mandibularis. In addition, this is the first time that heterozygous carriers of WLS variants were found to manifest phenotypes. WLS variants were likely to have adverse effects on the concentration of WNT ligands delivered to the cell membrane, resulting in aberrant canonical and non-canonical WNT signaling, and subsequent phenotypes. Patient's positioning during the acquisition of panoramic radiography might have affected the appearance of the tooth structures. If we had all family members of each patient to study co-segregation between genotype and phenotype, it would have strengthened the association of WLS variants and the phenotypes.

Common and rare variants of EGF increase the genetic risk of Alzheimer's disease as revealed by targeted sequencing of growth factors in Han Chinese

Alzheimer's disease (AD) is the most common neurodegenerative disease with high heritability. Growth factors (GFs) might contribute to the development of AD due to their broad effects on neuronal system. We herein aimed to investigate the role of rare and common variants of GFs in genetic susceptibility of AD. We screened 23 GFs in 6324 individuals using targeted sequencing. A rare-variant-based burden test and common-variant-based single-site association analyses were performed to identify AD-associated GF genes and variants. The burden test showed an enrichment of rare missense variants (p = 6.08 × 10−4) in GF gene-set in AD patients. Among the GFs, EGF showed the strongest signal of enrichment, especially for loss-of-function variants (p = 0.0019). A common variant rs4698800 of EGF showed significant associations with AD risk (p = 3.24 × 10−5, OR = 1.26). The risk allele of rs4698800 was associated with an increased EGF expression, whereas EGF was indeed upregulated in AD brain. These findings suggested EGF as a novel risk gene for AD.

Predicting clinical phenotypes of metachromatic leukodystrophy based on the arylsulfatase A activity and the ARSA genotype? - Chances and challenges.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficiency of arylsulfatase A (ARSA). Subsequent accumulation of sulfatides leads to demyelination and neurodegeneration in the central and peripheral nervous system. To date MLD is classified based on the age at onset, however, especially for late onset forms this classification provides only limited projection regarding the clinical disease course. Moreover, evolving newborn screening approaches raise the need to predict the disease onset and course in pre-symptomatic individuals. Here, we correlate the ARSA activity and the ARSA-genotype with clinical parameters in a large cohort of 96 affected individuals. Clinical data of 96 affected individuals with genetically and/or biochemically confirmed MLD were collected from a national database. Leukocyte samples from 69 affected individuals were re-analyzed for the ARSA activity using p-nitrocatecholsulfate as substrate with a refined ARSA assay towards the lower limit of detection. For 84 individuals genetic sequencing was conducted by Sanger or next generation sequencing (NGS). The adapted ARSA assay revealed the discriminatory power to differentiate MLD subtypes as the residual enzyme activity was low in late infantile and early juvenile forms, and clearly higher in late juvenile and adult MLD (p < 0.001). A residual enzyme activity below 1% compared to controls predicted an early onset (late-infantile or early-juvenile) and rapid disease progression. A firm genotype-phenotype correlation was proven as reliable for bi-allelic protein-truncating variants in the ARSA gene resulting in minimal residual ARSA activity, an early onset of the disease and initial decline of motor functions. Although the impact of missense variants was equivocal, few variants with a recognizable clinical spectrum were identified. ARSA activity in leukocytes as well as the ARSA genotype can predict the age of disease onset and the dynamic of disease progression for most of the early onset forms. This knowledge is relevant for patient counseling and to guide treatment decisions, especially when identifying pre-symptomatic individuals, e.g., in newborn screening. However, due to the high cumulative frequency of rare disease-causing missense variants in the ARSA gene that lead to highly variable residual enzyme activity, reiterated biochemical and genetic studies are needed to improve disease course prediction.

Modulation of PC1/3 activity by a rare double-site homozygous mutation.

Preprotein convertase 1/3 deficiency is a rare autosomal recessive disorder in which patients present with malabsorptive diarrhea and a series of symptoms of endocrine disorders such as polydipsia, reactive hypoglycemia, growth hormone deficiency, hypothyroidism, adrenal insufficiency, and early onset obesity. In its essence, pituitary hormone deficiency is caused by insufficient cleavage of pituitary prohormones. Here, we describe a female child with a rare double-site homozygous mutation in PCSK1 (Proprotein convertase subtilisin/kexin-type 1) gene, and thereby intend to investigate the relationship between these novel mutation sites and changes in protein synthesis and function. We tested this patient's blood and urine fecal indicators of infection, blood electrolytes, and relevant endocrine hormone levels in the laboratory. Next Generation Sequencing was applied to screen the patient's DNA. Western Blot was performed to evaluate the mutant protein's expression. The enzymatic activity was measured as the rate of cleavage of a synthetic fluorogenic substrate in a specific solution. We found that this patient presented shortly after birth with uncorrectable diarrhea and symptoms of metabolic acidosis with hypothyroidism. Next Generation Sequencing revealed that a rare double-site homozygous missense mutation, c.763G > A (p.G255R) and c.758C > T (p.S253L), were detected in exon 7 of PCSK1 (Proprotein convertase subtilisin/kexin-type 1) gene on chromosome 5 of the patient. Western blotting revealed that there was no significant decrease in protein synthesis levels in the mutant phenotype compared to the wild type. Compared with WT type, the proteins expressed by the mutations showed a significant decrease in the enzyme activity towards the fluorescent substrates. However, neither the single site mutation p.S253L or p.G255R, nor the double-site mutation of both, all showed no significant differences from each other. These two missense mutations have not been reported before, and it is even rarer to find homozygous variation of two sites in one patient. This study identifies two novel mutations for the first time and further investigates the changes in protein synthesis and enzyme activity, providing a new pathway to continue to explore the pathogenesis of diseases associated with the function of PC1/3.

Functional Analysis of Missense Mutations in GLI2 and GLI3 Involved in Congenital Heart Disease

Objective: In this study, we aimed to explore the biological functions of 10 rare case-specific missense mutations in GLI2 and 4 in GLI3, which were previously screened in a cohort of 412 patients with congenital heart disease and 213 normal controls from Shandong Province, China. Methods: A dual-luciferase reporter assay was used to assess the effects of these mutations in GLI2 and GLI3 on the activity of the sonic Hedgehog signaling pathway in HEK293T cells. Differences in protein levels between mutant and wild-type GLI2 and GLI3 were detected in HEK293T cells using Western blotting. Results: The dual-luciferase reporter assay showed that compared to the wild-type GLI2 protein, p.A1113V significantly increased activation of the sonic Hedgehog signaling pathway, whereas p.H78P and p.I1451S did not have a significant effect. The other mutations largely reduced the activation effect. Compared with the wild-type GLI3 protein, only p.A286V, among the four mutations, significantly reduced the activation effect on the SHH signaling pathway. Western blot data showed reduced expression of GLI2 p.G716V, GLI2 p.K736N, GLI2 p.I1451S, and GLI3 p.A286V, whereas the remaining mutations had no significant effects. Conclusion: The mutations GLI2 c.2147G>T (p.G716V), GLI2 c.2208G>C (p.K736N), and GLI3 c.857C>T (p.A286V) involved in congenital heart disease affect the regulation of the sonic Hedgehog signaling pathway; thus, these rare missense mutations in GLI2 and GLI3 might increase the risk of congenital heart disease.

Gene-specific application of computational prediction tools aids the classification of rare missense variants in the diagnosis of hereditary endocrine tumour syndromes

Gene-specific application of computational prediction tools aids the classification of rare missense variants in the diagnosis of hereditary endocrine tumour syndromes

Adult-onset KMT2B-related dystonia.

KMT2B-related dystonia (DYT-KMT2B, also known as DYT28) is an autosomal dominant neurological disorder characterized by varying combinations of generalized dystonia, psychomotor developmental delay, mild-to-moderate intellectual disability and short stature. Disease onset occurs typically before 10 years of age. We report the clinical and genetic findings of a series of subjects affected by adult-onset dystonia, hearing loss or intellectual disability carrying rare heterozygous KMT2B variants. Twelve cases from five unrelated families carrying four rare KMT2B missense variants predicted to impact protein function are described. Seven affected subjects presented with adult-onset focal or segmental dystonia, three developed isolated progressive hearing loss, and one displayed intellectual disability and short stature. Genome-wide DNA methylation profiling allowed to discriminate these adult-onset dystonia cases from controls and early-onset DYT-KMT2B patients. These findings document the relevance of KMT2B variants as a potential genetic determinant of adult-onset dystonia and prompt to further characterize KMT2B carriers investigating non-dystonic features.

Genetic analysis of and clinical characteristics associated with ANXA11 variants in a Chinese cohort with amyotrophic lateral sclerosis

BackgroundVariants in the annexin A11 gene (ANXA11) have been reported to be associated with amyotrophic lateral sclerosis (ALS). These variants may be involved in the pathogenesis of ALS by causing defects in intracellular protein trafficking. However, the genetic spectrum and clinical characteristics of ALS patients with ANXA11 variants are largely unknown. MethodsGenetic analysis was performed on 1587 Chinese patients with ALS. Eight software packages were used to predict the deleteriousness of missense variants. In addition, we searched PubMed, Embase, and Web of Science for relevant literature and meta-analysed variant frequencies. ResultsIn our ALS cohort, we identified 20 non-synonymous variants in 29 ALS patients, including one stop-gain, one frameshift, and 18 rare missense variants with seven predicted pathogenic variants. In a literature review of 11 reported studies that included 69 patients, 37 ANXA11 variants were reported, with a frequency of 1.7%, which was similar to that in our cohort (1.8%). Both our cohort and previous reports showed that ANXA11 carriers were more commonly males than females (12/17 vs. 19/31). Patients carrying ANXA11 variants affecting the C-terminal of the protein had earlier disease onset and shorter survival times than those carrying variants affecting the N-terminal. We found a relatively longer median survival time than that previously reported (53.6 months vs. 46.0 months). Additionally, Caucasian ANXA11 carriers were more likely to have cognitive impairment, typically frontotemporal dementia (FTD) than their Asian counterparts (20.0% vs. 14.3%). While more than half of the patients in our cohort had cognitive impairment, none had FTD. ConclusionIn our and previously published cases, ALS-associated ANXA11 variants predominantly affected the N- and C-terminal conserved domains. ANXA11 variant carriers are typically male and cognitively impaired. Our study extends the genotypic and phenotypic spectra of ALS patients with ANXA11 variants. Further expansion of the sample size is needed to analyse the clinical and non-motor symptom characteristics of patients and to deepen the understanding of the pathogenesis of ANXA11-associated ALS.

Las mutaciones missense en el dominio ROD2 de la filamina C muestran un fenotipo con miocardiopatía restrictiva/hipertrófica y miocardio en dientes de sierra

Introduction and objectivesMissense mutations in the filamin C (FLNC) gene have been reported as cause of inherited cardiomyopathy. Knowledge of the pathogenicity and genotype-phenotype correlation remains scarce. Our aim was to describe a distinctive cardiac phenotype related to rare missense FLNC variants in the ROD2 domain. MethodsWe recruited 21 unrelated families genetically evaluated because of hypertrophic cardiomyopathy (HCM)/restrictive cardiomyopathy (RCM) phenotype carrying rare missense variants in the ROD2 domain of FLNC (FLNC-mRod2). Carriers underwent advanced cardiac imaging and genetic cascade screening. Myocardial tissue from 3 explanted hearts of a missense FLNC carrier was histologically analyzed and compared with an FLNC-truncating variant heart sample and a healthy control. Plasmids independently containing 3 FLNC missense variants were transfected and analyzed using confocal microscopy. ResultsEleven families (52%) with 20 assessed individuals (37 [23.7-52.7]) years showed 15 cases with a cardiac phenotype consisting of an overlap of HCM-RCM and left ventricular hypertrabeculation (saw-tooth appearance). During a median follow-up of 6.49 years, they presented with advanced heart failure: 16 (80%) diastolic dysfunction, 3 heart transplants, 3 heart failure deaths) and absence of cardiac conduction disturbances or skeletal myopathy. A total of 6 families had moderate genotype-phenotype segregation, and the remaining were de novo variants. Differential extracellular matrix remodeling and FLNC distribution among cardiomyocytes were confirmed on histology. HT1080 and H9c2 cells did not reveal cytoplasmic aggregation of mutant FLNC. ConclusionsFLNC-mRod2 variants show a high prevalence of an overlapped phenotype comprising RCM, HCM and deep hypertrabeculation with saw-tooth appearance and distinctive cardiac histopathological remodeling.Full English text available from:www.revespcardiol.org/en

Identity-by-descent analysis of a large Tourette’s syndrome pedigree from Costa Rica implicates genes involved in neuronal development and signal transduction

Tourette Syndrome (TS) is a heritable, early-onset neuropsychiatric disorder that typically begins in early childhood. Identifying rare genetic variants that make a significant contribution to risk in affected families may provide important insights into the molecular aetiology of this complex and heterogeneous syndrome. Here we present a whole-genome sequencing (WGS) analysis from the 11-generation pedigree (>500 individuals) of a densely affected Costa Rican family which shares ancestry from six founder pairs. By conducting an identity-by-descent (IBD) analysis using WGS data from 19 individuals from the extended pedigree we have identified putative risk haplotypes that were not seen in controls, and can be linked with four of the six founder pairs. Rare coding and non-coding variants present on the haplotypes and only seen in haplotype carriers show an enrichment in pathways such as regulation of locomotion and signal transduction, suggesting common mechanisms by which the haplotype-specific variants may be contributing to TS-risk in this pedigree. In particular we have identified a rare deleterious missense variation in RAPGEF1 on a chromosome 9 haplotype and two ultra-rare deleterious intronic variants in ERBB4 and IKZF2 on the same chromosome 2 haplotype. All three genes play a role in neurodevelopment. This study, using WGS data in a pedigree-based approach, shows the importance of investigating both coding and non-coding variants to identify genes that may contribute to disease risk. Together, the genes and variants identified on the IBD haplotypes represent biologically relevant targets for investigation in other pedigree and population-based TS data.

Gene Sequencing Identifies Perturbation in Nitric Oxide Signaling as a Nonlipid Molecular Subtype of Coronary Artery Disease.

A key goal of precision medicine is to disaggregate common, complex diseases into discrete molecular subtypes. Rare coding variants in the low-density lipoprotein receptor gene (LDLR) are identified in 1% to 2% of coronary artery disease (CAD) patients, defining a molecular subtype with risk driven by hypercholesterolemia. To search for additional subtypes, we compared the frequency of rare, predicted loss-of-function and damaging missense variants aggregated within a given gene in 41 081 CAD cases versus 217 115 controls. Rare variants in LDLR were most strongly associated with CAD, present in 1% of cases and associated with 4.4-fold increased CAD risk. A second subtype was characterized by variants in endothelial nitric oxide synthase gene (NOS3), a key enzyme regulating vascular tone, endothelial function, and platelet aggregation. A rare predicted loss-of-function or damaging missense variants in NOS3 was present in 0.6% of cases and associated with 2.42-fold increased risk of CAD (95% CI, 1.80-3.26; P=5.50×10-9). These variants were associated with higher systolic blood pressure (+3.25 mm Hg; [95% CI, 1.86-4.65]; P=5.00×10-6) and increased risk of hypertension (adjusted odds ratio 1.31; [95% CI, 1.14-1.51]; P=2.00×10-4) but not circulating cholesterol concentrations, suggesting that, beyond lipid pathways, nitric oxide synthesis is a key nonlipid driver of CAD risk. Beyond LDLR, we identified an additional nonlipid molecular subtype of CAD characterized by rare variants in the NOS3 gene.

MetaRNN: differentiating rare pathogenic and rare benign missense SNVs and InDels using deep learning

Multiple computational approaches have been developed to improve our understanding of genetic variants. However, their ability to identify rare pathogenic variants from rare benign ones is still lacking. Using context annotations and deep learning methods, we present pathogenicity prediction models, MetaRNN and MetaRNN-indel, to help identify and prioritize rare nonsynonymous single nucleotide variants (nsSNVs) and non-frameshift insertion/deletions (nfINDELs). We use independent test sets to demonstrate that these new models outperform state-of-the-art competitors and achieve a more interpretable score distribution. Importantly, prediction scores from both models are comparable, enabling easy adoption of integrated genotype-phenotype association analysis methods. All pre-computed nsSNV scores are available at http://www.liulab.science/MetaRNN. The stand-alone program is also available at https://github.com/Chang-Li2019/MetaRNN.

Narrowing Beta-myosin hot spots in its association with hypertrophic cardiomyopathy

Abstract Introduction MYH7 encodes to β-cardiac myosin heavy chain, a large and pleomorphic protein, conformed by different and well-functionally characterized domains. This gene has an enormous contribution to hypertrophic cardiomyopathy (HCM) but also to other cardiomyopathies such as dilated, non-compaction and restrictive cardiomyopathy (and overlapping phenotypes). There are specific and adapted recommendations for classifying variants in this gene regarding its association with HCM; one of the aspects was the location of variants in the head domain, encompassing 181–937 residues as moderate ACMG criteria (Kelli et al., 2018). Objectives The purpose of this analysis was to determine if more specific regions of MYH7are enriched in HCM cases. This analysis could help to better characterise those regions where identifying a novel missense variant could reinforce their pathogenicity. Methods We included in the analysis 26,929 consecutives unrelated probands in which MYH7 was sequenced by NGS, referred from different countries and centres. We performed an enrichment analysis comparing the prevalence of rare missense variants (pre-established MAF cut-off 0.004%) in cases with the diagnosis of HCM versus their frequency in non-cardiomyopathy cases (internal controls, OR_int, comprising aortic diseases, channelopathies and dyslipidaemias) and in gnomAD population (external controls, OR_ext). Comparison between groups was performed using the Student's t-test; p value <0.05 was considered to indicate statistical significance. The rationale of this grouping was to avoid skewed analysis, given the high degree of overlapping cardiomyopathies observed in this gene. Results HCM was the diagnosis in 10,064 cases. 8,975 probands with a non-cardiomyopathy phenotype were used as internal controls; 7,890 cases with other MYH7-related phenotypes (restrictive, dilated, non-compaction and skeletal myopathies) were not included in the analysis. Rare missense variants in MYH7 were enriched in HCM cases (OR_int = 11.15; OR_ext =12.44) and in the pre-established hot spot head domain (OR_int = 16.43; OR_ext = 24.53). However, the regions with the highest odds ratios in both external and internal comparison were the central converter domain (residues 711–755, OR_int = 44.39; OR_ext = 50.27) and actin-binding region (residues 647–664, OR_int =18.77; OR_ext = 69.32). Conversely, the tail region (both S2 subfragment and light meromyosin region, LMM), had the lowest OR in this analysis. Conclusions Previously reported hotspot (head) is significantly enriched in HCM cases, but some specific regions within this domain, such as central converter and acting-binding region, could have a relatively higher contribution. These results reinforce the current approach, but on the other hand, show an asymmetric contribution within the head domain. This fact may have an impact on the reassessment of the variant classification in this gene towards a more precise approach. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Health in Code

VP.79 Challenges in genetic diagnosis of LAMA2-MD - when the pieces do not fit

Pathogenic variants in the <i>LAMA2</i> gene can cause muscular dystrophy (MD) associated with variable degrees of severity. We describe an infant with a congenital phenotype: severe hypotonia and weakness at birth, elevated creatine kinase levels, brain white-matter alterations and reduced merosin expression in muscle biopsy. Next-generation sequencing (NGS) targeting a panel of genes involved in hereditary myopathies revealed a rare homozygous missense variant in exon 39 of the <i>LAMA2</i> gene. Detailed genotype-phenotype evaluation associated with an incongruent bioinformatics analysis led us to proceed to full <i>LAMA2</i> cDNA analysis. A partial retention of intron 14 was identified, thought to be the primary genetic defect accountable for the patient´s phenotype. Further investigation enabled us to conclude that this splicing event was instigated by the presence of a homozygous intronic substitution located 117bp downstream of exon 14, which created a cryptic splice site. To our knowledge, this splicing variant represents the first pathogenic variant located further from the <i>LAMA2</i> exon-intron boundaries. Besides the importance of cDNA studies, this work highlights the need for critical judgment of any identified variants of unknown significance (VUS) in the definition of the correct and causal genotype.

Missense variants in ANKRD11 cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein.

Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants. We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants through in silico analyses and cell-based experiments. We identified 20 unique, mostly de novo, ANKRD11 missense variants in 29 individuals, presenting with syndromic neurodevelopmental disorders similar to KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in repression domain 2 at the ANKRD11 C-terminus. Of the 10 functionally studied missense variants, 6 reduced ANKRD11 stability. One variant caused decreased proteasome degradation and loss of ANKRD11 transcriptional activity. Our study indicates that pathogenic heterozygous ANKRD11 missense variants cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges because the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping.

De novo variants in FRMD5 are associated with developmental delay, intellectual disability, ataxia, and abnormalities of eye movement

Proteins containing the FERM (four-point-one, ezrin, radixin, and moesin) domain link the plasma membrane with cytoskeletal structures at specific cellular locations and have been implicated in the localization of cell-membrane-associated proteins and/or phosphoinositides. FERM domain-containing protein 5 (FRMD5) localizes at cell adherens junctions and stabilizes cell-cell contacts. To date, variants in FRMD5 have not been associated with a Mendelian disease in OMIM. Here, we describe eight probands with rare heterozygous missense variants in FRMD5 who present with developmental delay, intellectual disability, ataxia, seizures, and abnormalities of eye movement. The variants are de novo in all for whom parental testing was available (six out of eight probands), and human genetic datasets suggest that FRMD5 is intolerant to loss of function (LoF). We found that the fly ortholog of FRMD5, CG5022 (dFrmd), is expressed in the larval and adult central nervous systems where it is present in neurons but not in glia. dFrmd LoF mutant flies are viable but are extremely sensitive to heat shock, which induces severe seizures. The mutants also exhibit defective responses to light. The human FRMD5 reference (Ref) cDNA rescues the fly dFrmd LoF phenotypes. In contrast, all the FRMD5 variants tested in this study (c.340T>C, c.1051A>G, c.1053C>G, c.1054T>C, c.1045A>C, and c.1637A>G) behave as partial LoF variants. In addition, our results indicate that two variants that were tested have dominant-negative effects. In summary, the evidence supports that the observed variants in FRMD5 cause neurological symptoms in humans.

P.157 Dominant HSPB6 mutation in a myopathy patient

HSPB6 (also known as Hsp20) is a small heat-shock protein (sHSP) showing highest expression is skeletal, cardiac and smooth muscles. In contrast to most sHSPs, HSPB6 does not readily assemble into large oligomers on its own. However, it heterodimerizes with HSPB1 and hetero-oligomerizes with HSPB1 and αB-crystallin (CRYAB, HSPB5), modulating their oligomer size and chaperone activity. HSPB6 may also be involved in BAG3-mediated autophagy. Mutations in several sHSPs lead to neuromuscular disease. While rare missense variants in <i>HSPB6</i> have been associated with cardiomyopathy, the gene has not been previously linked to skeletal muscle disease. We describe the likely pathogenic heterozygous <i>HSPB6</i> variant c.464delC (p.Pro155Argfs*25) in a 62-year-old man with onset of the disease at 43 years of age characterized by a proximal lower limb weakness and a respiratory insufficiency. At 60 years of age, he walks with 2 canes. Of note, he underwent a cataract surgery at 28 years of age. The single-nucleotide deletion causes a frameshift predicted to replace of the six C-terminal amino acid residues of the HSPB6 protein with 24 erroneous residues. Western blotting of patient biopsy showed the normal and extended HSPB6 proteins at a 50:50 ratio, indicating that the mutant protein is expressed and stable. Histological findings included infrequent central HSPB6 accumulation in the myofibers, and myotilin-positive cytoplasmic bodies. In cell culture experiments, mutant HSPB6 showed reduced solubility compared to the wild-type protein and affected the solubility of coexpressed wild-type HSPB6, HSPB1 and CRYAB. Immunofluorescence microscopy revealed altered CRYAB localization patterns in cells expressing mutant HSPB6. Our results suggest that the <i>HSPB6</i> c.464delC (p.Pro155Argfs*25) mutation may cause muscle disease through a gain-of-function mechanism interfering with the sHSP machinery.

Biallelic mutations in WDR12 are associated with male infertility with tapered-head sperm.

Teratozoospermia is a rare disease associated with male infertility. Several recurrent genetic mutations have been reported to be associated with abnormal sperm morphology, but the genetic basis of tapered-head sperm is not well understood. In this study, whole-exome sequencing (WES) identified a homozygous WD repeat domain 12 (WDR12; p.Ser162Ala/c.484T>G) variant in an infertile patient with tapered-head spermatozoa from a consanguineous Chinese family. Bioinformatic analysis predicted this mutation to be a pathogenic variant. To verify the effect of this variant, we analyzed WDR12 protein expression in spermatozoa of the patient and a control individual, as well as in the 293T cell line, by Western blot analysis, and found that WDR12 expression was significantly downregulated. To understand the role of normal WDR12, we evaluated its mRNA and protein expression in mice at different ages. We observed that WDR12 expression was increased in pachytene spermatocytes, with intense staining visible in round spermatid nuclei. Based on these results, the data suggest that the rare biallelic pathogenic missense variant (p.Ser162Ala/c.484T>G) in the WDR12 gene is associated with tapered-head spermatozoa. In addition, after intracytoplasmic sperm injection (ICSI), a successful pregnancy was achieved. This finding indicates that infertility associated with this WDR12 homozygous mutation can be overcome by ICSI. The present results may provide novel insights into understanding the molecular mechanisms of male infertility.

The p190 RhoGAPs, ARHGAP35, and ARHGAP5 are implicated in GnRH neuronal development: Evidence from patients with idiopathic hypogonadotropic hypogonadism, zebrafish, and in vitro GAP activity assay

PurposeThe study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). MethodsA cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase–activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. ResultsRare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. ConclusionThis study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.

The impact of Mendelian sleep and circadian genetic variants in a population setting.

Rare variants in ten genes have been reported to cause Mendelian sleep conditions characterised by extreme sleep duration or timing. These include familial natural short sleep (ADRB1, DEC2/BHLHE41, GRM1 and NPSR1), advanced sleep phase (PER2, PER3, CRY2, CSNK1D and TIMELESS) and delayed sleep phase (CRY1). The association of variants in these genes with extreme sleep conditions were usually based on clinically ascertained families, and their effects when identified in the population are unknown. We aimed to determine the effects of these variants on sleep traits in large population-based cohorts. We performed genetic association analysis of variants previously reported to be causal for Mendelian sleep and circadian conditions. Analyses were performed using 191,929 individuals with data on sleep and whole-exome or genome-sequence data from 4 population-based studies: UK Biobank, FINRISK, Health-2000-2001, and the Multi-Ethnic Study of Atherosclerosis (MESA). We identified sleep disorders from self-report, hospital and primary care data. We estimated sleep duration and timing measures from self-report and accelerometery data. We identified carriers for 10 out of 12 previously reported pathogenic variants for 8 of the 10 genes. They ranged in frequency from 1 individual with the variant in CSNK1D to 1,574 individuals with a reported variant in the PER3 gene in the UK Biobank. No carriers for variants reported in NPSR1 or PER2 were identified. We found no association between variants analyzed and extreme sleep or circadian phenotypes. Using sleep timing as a proxy measure for sleep phase, only PER3 and CRY1 variants demonstrated association with earlier and later sleep timing, respectively; however, the magnitude of effect was smaller than previously reported (sleep midpoint ~7 mins earlier and ~5 mins later, respectively). We also performed burden tests of protein truncating (PTVs) or rare missense variants for the 10 genes. Only PTVs in PER2 and PER3 were associated with a relevant trait (for example, 64 individuals with a PTV in PER2 had an odds ratio of 4.4 for being "definitely a morning person", P = 4x10-8; and had a 57-minute earlier midpoint sleep, P = 5x10-7). Our results indicate that previously reported variants for Mendelian sleep and circadian conditions are often not highly penetrant when ascertained incidentally from the general population.