The mutation c.346-1G > a in SOHLH1 impairs sperm production in the homozygous but not in the heterozygous condition

Nonobstructive azoospermia (NOA) is an important cause of male infertility, and the genetic pathogenesis is still incompletely understood. The previous study reported that heterozygous mutation of c.346-1G > A in SOHLH1 was identified in two NOA patients and suggested it is the pathogenic factor for NOA. However, in our research, this heterozygous mutation was confirmed in three Chinese infertile patients who were suffered from teratozoospermia, but they had normal sperm number. Intriguingly, a homozygous mutation of c.346-1G > A in SOHLH1 was detected in a severe oligozoospermia (SOZ) patient, characterized with severely decreased sperm count. Notably, we unprecedently revealed this homozygous mutation of c.346-1G > A in SOHLH1 lead to the sharp decrease in various germ cells and spermatogenesis dysfunction, which is similar with the phenotype of Sohlh1 knockout male mice. Moreover, western blotting confirmed that the homozygous mutation declined SOHLH1 protein expression. Additionally, we correlated the good prognosis of intracytoplasmic sperm injection (ICSI) in the patients carrying the mutation of c.346-1G > A in SOHLH1. Thus, we suggested that the heterozygous mutation of c.346-1G > A in SOHLH1 is responsible for teratozoospermia, and this homozygous mutation in SOHLH1 impairs spermatogenesis and further leads to the reduced sperm count, eventually causing male infertility, which unveils a new recessive-inheritance pattern of SOHLH1-associated male infertility initially.

The Recruitment-Secretory Block (“R-SB”) Phenomenon and Endoplasmic Reticulum Storage Diseases

In this article, we review the biological and clinical implication of the Recruitment-Secretory Block (“R-SB”) phenomenon. The phenomenon refers to the reaction of the liver with regard to protein secretion in conditions of clinical stimulation. Our basic knowledge of the process is due to the experimental work in animal models. Under basal conditions, the protein synthesis is mainly carried out by periportal (zone 1) hepatocytes that are considered the “professional” synthesizing protein cells. Under stimulation, midlobular and centrolobular (zone 2 and 3) hepatocytes, are progressively recruited according to lobular gradients and contribute to the increase of synthesis and secretion. The block of secretion, operated by exogenous agents, causes intracellular retention of all secretory proteins. The Pi MZ phenotype of Alpha-1-antitrypsin deficiency (AATD) has turned out to be the key for in vivo studies of the reaction of the liver, as synthesis and block of secretion are concomitant. Indeed, the M fraction of AAT is stimulated for synthesis and regularly exported while the Z fraction is mostly retained within the cell. For that reason, the phenomenon has been designated “Recruitment-Secretory Block” (“R-SB”). The “R-SB” phenomenon explains why: (a) the MZ individuals can correct the serum deficiency; (b) the resulting immonohistochemical and electron microscopic (EM) patterns are very peculiar and specific for the diagnosis of the Z mutation in tissue sections in the absence of genotyping; (c) the term carrier is no longer applicable for the heterozygous condition as all Pi MZ individuals undergo storage and the storage predisposes to liver damage. The storage represents the true elementary lesion and consequently reflects the phenotype-genotype correlation; (d) the site and function of the extrahepatic AAT and the relationship between intra and extracellular AAT; (e) last but not least, the concept of Endoplasmic Reticulum Storage Disease (ERSD) and of a new disease, hereditary hypofibrinogenemia with hepatic storage (HHHS). In the light of the emerging phenomenon, described in vitro, namely that M and Z AAT can form heteropolymers within hepatocytes as well as in circulation, we have reviewed the whole clinical and experimental material collected during forty years, in order to evaluate to what extent the polymerization phenomenon occurs in vivo. The paper summarizes similarities and differences between AAT and Fibrinogen as well as between the related diseases, AATD and HHHS. Indeed, fibrinogen gamma chain mutations undergo an aggregation process within the RER of hepatocytes similar to AATD. In addition, this work has clarified the intriguing phenomenon underlying a new syndrome, hereditary hypofibrinogenemia and hypo-APO-B-lipoproteinemia with hepatic storage of fibrinogen and APO-B lipoproteins. It is hoped that these studies could contribute to future research and select strategies aimed to simultaneously correct the hepatocytic storage, thus preventing the liver damage and the plasma deficiency of the two proteins.

A Novel Heterozygous Missense Mutation in COL7A1 Gene in Dystrophic Epidermolysis Bullosa

ABSTRACT Hereditary Epidermolysis Bullosa is categorized by blister formation on the skin which includes Dystrophic Epidermolysis Bullosa as one of its types. It is characterized by blister formation on hands, feet, knees, and elbows. The severe condition leads to vision loss and blemishes. The objectives included the identification of COL7A1 gene mutation for DEB susceptibility through sequence analysis of hot spot regions of 73, 74 and 75 exons. The experimental design included the enrolment of DEB-affected two families. The genetic analysis techniques included inorganic DNA extraction, Polymerase Chain Reaction and Sanger sequencing method. The study inferred the identification of novel missense mutation in exon 75 of COL7A1 gene at (c. 6223 G>T) where the aspartic acid is converted into tyrosine in the heterozygous condition in affected families. The recognized novel missense mutation is silent (heterozygous) which becomes severe when homozygous. However, whole-exome sequencing strategy may identify the causative mutations.

A Yeast-Based Screening Unravels Potential Therapeutic Molecules for Mitochondrial Diseases Associated with Dominant ANT1 Mutations

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

Bardet–Biedl Syndrome

The Bardet–Biedl syndrome is a rare autosomal recessive disease of the group of ciliopathies with polymorphic clinical symptoms including the retinal degeneration, obesity, polydactyly, mental retardation, hypogonadism, and renal dysfunction. The Pleiotropic effects are caused by the defects in genes encoding the proteins responsible for the functioning of cilia. The Article addresses the issues of the clinical features, diagnosis, differential diagnosis and treatment of this disease. The clinical case demonstrates the patient with Bardet–Biedl syndrome, manifested by the retinal degeneration, obesity, brachydactylia, syndactyly and clinodactyly, hypogenitalism, mental retardation and concomitant hypothyroidism. As per results of the molecular genetic testing, the child was found having the mutations in exon 2 of BBS10 gene c.271dupT and c.583G> A (p.G180E) in the compound heterozygous condition, inherited from the father and mother, respectively, that are the healthy carriers.

Characterization of HA-tagged α9 and α10 nAChRs in the mouse cochlea

Neurons of the medial olivary complex inhibit cochlear hair cells through the activation of α9α10-containing nicotinic acetylcholine receptors (nAChRs). Efforts to study the localization of these proteins have been hampered by the absence of reliable antibodies. To overcome this obstacle, CRISPR-Cas9 gene editing was used to generate mice in which a hemagglutinin tag (HA) was attached to the C-terminus of either α9 or α10 proteins. Immunodetection of the HA tag on either subunit in the organ of Corti of adult mice revealed immunopuncta clustered at the synaptic pole of outer hair cells. These puncta were juxtaposed to immunolabeled presynaptic efferent terminals. HA immunopuncta also occurred in inner hair cells of pre-hearing (P7) but not in adult mice. These immunolabeling patterns were similar for both homozygous and heterozygous mice. All HA-tagged genotypes had auditory brainstem responses not significantly different from those of wild type littermates. The activation of efferent neurons in heterozygous mice evoked biphasic postsynaptic currents not significantly different from those of wild type hair cells. However, efferent synaptic responses were significantly smaller and less frequent in the homozygous mice. We show that HA-tagged nAChRs introduced in the mouse by a CRISPR knock-in are regulated and expressed like the native protein, and in the heterozygous condition mediate normal synaptic function. The animals thus generated have clear advantages for localization studies.

A clinical case of cystic fibrosis patient with pathogenic N1303K genotype variant with assessment of the CFTR channel function by intestinal current measurement and forskolin-induced swelling in rectal organoids

Rationale: Cystic fibrosis is a common monogenic disease related to pathogenic nucleotide sequence variants in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) (ABCC7) gene. The CFTR gene consists of 27 exons and is located in the 31.1 region on the long arm of chromosome 7 (7q31.1). The use of the sequencing method has led to the accumulation of new information about the diversity of genetic variants in cystic fibrosis. This information is important considering approaches to the development of targeted therapy for the disease, based on an individual genotype. No targeted therapy has been developed for the N1303K class II genetic variant. The function of the chloride channel in this mutation has not been compared with that in class II mutations like F508del.Materials and methods: We have analyzed medical files of a patient with cystic fibrosis and F508del/N1303K CFTR genotypes, including the results of rectal biopsy samples. The assessments included measurement of the intestinal potential difference and forskolin-induced swelling assay (FIS) in rectal organoids, with the results being analyzed in relation to the clinical data.Results: The results of intestinal current measurements (ICM) confirm that the N1303K genetic variant is “severe” and leads to the loss of the working CFTR protein, which is consistent with the clinical manifestations. The mean short circuit currency density (ΔISC) in response to amiloride (sodium channel stimulation) was -39±1.22 µA/cm2 , to forskolin (chloride channel stimulation) 3.83±1.43 µA/cm2 , to carbachol 6±2.47 µA/cm2 , and to histamine 8.5±3.02 µA/cm2 . FIS results indicate that the VX-770 potentiator and the VX-809 corrector have a weak effect on the stimulation of organoids by forskolin in the genetic variant N1303K: organoid swelling was non-significant (about 20% from their baseline size).Conclusion: The use of the ICM method and FIS assay in human intestinal organoids makes it possible to quantify the work of the CFTR protein and determine the in vitro effectiveness of targeted therapy in patients with cystic fibrosis. CFTR modulators are ineffective in patients with N1303K mutation in the compound-heterozygous condition with F508del, despite both pathogenic variants belong to class II.

Arabica coffee accessions originated from Ethiopia with resistance to nematode Meloidogyne paranaensis

The aim of this study was to evaluate the resistance of Ethiopian Coffea arabica from IAPAR’s germplasm bank to Meloidogyne paranaensis. Two experiments were carried out in a greenhouse in IAPAR, Londrina, Paraná State, Brazil. A completely randomized design was used with 15 treatments, eight replications and one plant per plot. 14 accessions of C. arabica from Ethiopia were evaluated and the cultivar Mundo Novo IAC 376-4 was used as a susceptible control. Seedlings with three to four pairs of leaves were transplanted into plastic cups with a capacity of 700 mL and 1200 eggs and/or J2 of M. paranaensis (IP) were inoculated after one month. The evaluations were performed 130 days after inoculation, when the data of the number of eggs and J2 per gram of roots, the final population of nematodes (FP) were obtained and the reproduction factor (RF) was calculated using the formula: RF = IP / FP. To classify the resistance levels of the genotypes, the reduction of the reproduction factor (RRF) was used. The Ethiopian accessions were classified from highly resistant to highly susceptible. Most of the Ethiopian coffees were highly resistant to M. paranaensis. The genotype Geisha also showed resistance, but is still in heterozygous condition.

Allosteric control of hemoglobin S fiber formation by oxygen and its relation to the pathophysiology of sickle cell disease

The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

The Cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation

The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from caput epididymis of CB1-knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from caput and cauda epididymis of wild-type and CB1-knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of CB1-gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that CB1-gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of Cdyl and Brdt mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms.