A case of Usher syndrome type IIA caused by a rare USH2A homozygous frameshift variant with maternal uniparental disomy (UPD) in a Chinese family

Usher syndrome encompasses a group of genetically and clinically heterogeneous autosomal recessive disorders with hearing deficiencies and retinitis pigmentosa. The mechanisms underlying the Usher syndrome are highly variable. In the present study, a Chinese family with Usher syndrome was recruited. Whole exome sequencing (WES), Sanger sequencing, homozygosity mapping, short tandem repeat (STR) analysis and segregation analysis were performed. Functional domains of the pathogenic variant for USH2A were analysed. We identified a homozygous frameshift variant c.99_100insT (p.Arg34Serfs*41) in the USH2A gene in the proband that showed discordant segregation in the father. Further homozygosity mapping and STR analysis identified an unusual homozygous variant of proband that originated from maternal uniparental disomy (UPD). The p.Arg34Serfs*41 variant produced a predicted truncated protein that removes all functional domains of USH2A. The variant was not included in the 1000 Human Genomes Project database, ExAC database, HGMD or gnomAD database, but was included in the ClinVar databases as pathogenic. Although USH2A is an autosomal recessive disease, the effects of UPD should be informed in genetic counselling since the recurrence risk of an affected child is greatly reduced when the disease is due to the UPD mechanism. To test potential patients, WES, combined with STR analysis and homozygosity mapping, provides an accurate and useful strategy for genetic diagnosis. In summary, our discoveries can help further the understanding of the molecular pathogenesis of Usher syndrome type IIA to advance the prevention, diagnosis and therapy for this disorder.     

Structural Basis of Regulation of von Willebrand Factor Binding to Glycoprotein Ib

Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state.     

miR‐125a‐5p increases cellular DNA damage of aging males and perturbs stage‐specific embryo development via Rbm38‐p53 signaling

An increasing number of men are fathering children at an older age than in the past. While advanced maternal age has long been recognized as a risk factor for adverse reproductive outcomes, the influence of paternal age on reproduction is incompletely comprehended. Herein, we found that miR‐125a‐5p was upregulated in the sperm of aging males and was related to inferior sperm DNA integrity as an adverse predictor. Moreover, we demonstrated that miR‐125a‐5p suppressed mitochondrial function and increased cellular DNA damage in GC2 cells. We also found that miR‐125a‐5p perturbed embryo development at specific morula/blastocyst stages. Mechanistically, we confirmed that miR‐125a‐5p disturbed the mitochondrial function by targeting Rbm38 and activating the p53 damage response pathway, and induced a developmental delay in a p21‐dependent manner. Our study revealed an important role of miR‐125a‐5p in sperm function and early embryo development of aging males, and provided a fresh view to comprehend the aging process in sperm.     

Two closely spaced missense COL3A1 variants in cis cause vascular Ehlers‐Danlos syndrome in one large Chinese family

Vascular Ehlers‐Danlos syndrome (vEDS) is a rare and severe hereditary connective tissue disease arising from a mutation in the type III collagen alpha I chain (COL3A1) gene, with a poor prognosis due to exceptional vascular ruptures and premature death. Herein, starting from a 36‐year‐old Chinese male patient with a complaint of upper abdominal pain, we collected clinical data of and performed a genetic analysis of a total of 20 family members. We identified two closely spaced COL3A1 missense variants in cis, p.Leu734Phe (c.2199_2200TC>AT) and p.Gly741Ser (c.2221G>A), as the cause of vEDS in this family. p.Gly741Ser, a glycine substitution mutation, has been previously reported, whereas p.Leu734Phe, a non‐glycine substitution mutation, is novel. We analysed their independent and combined effects on the COL3A1 level in transfected skin fibroblast cells by means of Western blotting. We found that both variants independently led to a reduced COL3A1 level and, when combined, led to an even more reduced COL3A1 level compared to the wild type. Thus, each missense variant can be independently classified as a pathogenic variant, albeit with a synergetic effect when occurring together. Moreover, our genetic findings provide an explanation for four previous sudden deaths and identified two high‐risk carriers in the family.     

Evaluation of a new semi-automated Hydragel 11 von Willebrand factor multimers assay kit for routine use

BackgroundAccurate diagnosis and classification of von Willebrand disease (VWD) are essential for optimal management. The von Willebrand factor multimers analysis (VWF:MM) is an integral part of the diagnostic process in the phenotypic classification, especially in discrepant cases. The aim of this study was to evaluate the performance of a new Hydragel 11VWF multimer assay (H11VW).MethodsAnalytical performance characteristics such as repeatability (intra-assay variability, in gel between track variation), reproducibility (inter-assay variability, between gel variation), sensitivity, EQA performance and differences between two commercially available VWF:MM kits (H5VW and H11VW) were analysed in healthy volunteers'' plasmas using in-house prepared reference plasma.ResultsRepeatability and reproducibility results of H11VW demonstrated acceptable and equivalent performance with previously verified H5VW. Participation in EQA was successful. No statistically significant difference was detected between H5VW and H11VW kits for different fractions of multimers: LMWM p=0.807; IMWM p=0.183; HMWM p=0.774.ConclusionsH11VW demonstrated acceptable analytical performance characteristics. H11VW kit conveniently offers a more significant number of samples on a single gel. H5VW and H11VW kits can be used in daily practice interchangeably.     

Structural analysis of new compound heterozygous variants in PEPD gene identified in a patient with Prolidase Deficiency diagnosed by exome sequencing

Prolidase Deficiency (PD) is an autosomal recessive rare disorder caused by loss or reduction of prolidase enzymatic activity due to variants in the PEPD gene. PD clinical features vary among affected individuals: skin ulcerations, recurrent infections, and developmental delay are common. In this study, we describe a 16-year-old boy with a mild PD phenotype comprising chronic eczema, recurrent infections and elevated IgE. Whole exome sequencing analysis revealed three PEPD variants: c.575T>C p.(Leu192Pro) inherited from the mother, and c.692_694del p.(Tyr231del) and c.1409G>A p.(Arg470His), both inherited from the father. The variant p.(Tyr231del) has been previously characterized by high-resolution X-ray structure analysis as altering protein dynamics/flexibility. In order to study the effects of the other two prolidase variants, we performed site directed mutagenesis purification and crystallization studies. A high-resolution X-ray structure could only be obtained for the p.(Arg470His) variant, which showed no significant structural differences in comparison to WT prolidase. On the other hand, the p.(Leu192Pro) variant led to significant protein destabilization. Hence, we conclude that the maternal p.(Leu192Pro) variant was likely causally associated with the proband´s disease, together with the known pathogenic paternal variant p.(Tyr231del). Our results demonstrated the utility of exome sequencing to perform diagnosis in PD cases with mild phenotype.     

Novel exon mutation in SYCE1 gene is associated with non‐obstructive azoospermia

Non‐obstructive azoospermia (NOA) is a common cause of male infertility, and genetic problems, such as chromosomal abnormalities and gene mutations, are important causes of NOA. Our centre received a case of NOA, in which no mature sperm was found during microdissection testicular sperm extraction. A postoperative pathological examination revealed that testicular spermatogenesis was blocked. Target region capture combined with high‐throughput sequencing was used to screen for male infertility‐related gene mutations. Sanger sequencing further confirmed that the SYCE1 gene, a central component of the synaptonemal complex (SC) during meiosis, had a homozygous deletion mutation in the tenth exon (c.689_690del; p.F230fs). Through molecular biological studies, we discovered altered expression and nuclear localization of the endogenous mutant SYCE1. To verify the effects in vitro, wild‐ and mutated‐type SYCE1 vectors were constructed and transfected into a human cell line. The results showed that the expression and molecular weight were decreased for SYCE1 containing c.689_690del. In addition, mutated SYCE1 was abnormally located in the cytoplasm rather than in the nucleus. In summary, our research suggests that the novel homozygous mutation (c.689_690del; p.F230fs) altered the SYCE1 expression pattern and may have disturbed SC assembly, leading to male infertility and to a barrier to gamete formation. We reported for the first time that a frameshift mutation occurred in the exon region of SYCE1 in an NOA patient. This study is beneficial for accurate NOA diagnosis and the development of corresponding gene therapy strategies.     

Decreased p53 is associated with a decline in asymmetric stem cell self‐renewal in aged human epidermis

With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self‐renewal in multiple murine tissues. The fate of SC self‐renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self‐renewal has been observed in inflammatory‐mediated hyperproliferation, while increased symmetric SC self‐renewal has been observed in cancers. We analyzed SC self‐renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age‐dependent decrease in epidermal basal layer divisions. The balance of SC self‐renewal shifted toward symmetric SC self‐renewal, with a decline in asymmetric SC self‐renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self‐renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin‐3 (a p53 activator) returned p53 activity as well as asymmetric SC self‐renewal divisions to adult levels. Nutlin‐3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin‐3 (p53)‐induced asymmetric SC self‐renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53‐induced asymmetric SC divisions.     

Identification of a case of Y:18 translocation using a Y-specific repetitive DNA probe

Summary We have used a recombinant DNA clone derived from the Y-specific 3,4-kb repeats for in situ chromosome hybridization and Southern blotting analysis to identify a case of de novo Y;18 translocation. The proband has a chromosome complement of 46,XY and a variant chromosome 18 with a Q-bright and C-positive short arm. The father has a normal male karyotype of 46,XY. The mother has a female karyotype of 46,XX and an unusually large Q-bright satellite on one chromosome 22. In situ hybridization with the 3,4-kb probe to the metaphase preparations of family members indicated that the additional Q-bright material in the proband's variant chromosome 18 derived from the Y chromosome of his father, and not from the variant chromosome 22 of his mother. On Southern hybridization, the proband had approximately twice the amount of 3,4-kb repeats per cell as his father. These observations suggest a de novo genetic rearrangement in the proband which probably occurred during the father's spermatogenesis.     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Analysis and pharmacological modulation of senescence in human epithelial stem cells

Human epithelial stem cells (ESCs) are characterized by long‐term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly‐ectodermal dysplasia‐clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ‐secretase inhibitor; N‐[N‐(3, 5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine T‐butyl ester). All cells were able to differentiate in vitro but exhibited variable self‐renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up‐ or down‐regulated during their lifetime, thus allowing to identify druggable gene networks and off‐the‐shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.     

A recessive ryanodine receptor 1 mutation in a CCD patient increases channel activity

Ryanodine receptors plays a crucial role in skeletal muscle excitation–contraction coupling by releasing calcium ions required for muscle contraction from the sarcoplasmic reticulum. At least three phenotypes associated with more than 100 RYR1 mutations have been identified; in order to elucidate possible pathophysiological mechanisms of RYR1 mutations linked to neuromuscular disorders, it is essential to define the mutation class by studying the functional properties of channels harbouring clinically relevant amino acid substitutions. In the present report we investigated the functional effects of the c.7304G > T RYR1 substitution (p.Arg2435Leu) found in a patient affected by central core disease. Both parents were heterozygous for the substitution while the proband was homozygous. We characterized Ca²⁺ homeostasis in myoD transduced myotubes from controls, the heterozygous parents and the homozygous proband expressing the endogenous mutation. We also expressed the recombinant mutant channels in heterologous cells and characterized their [³H]ryanodine binding and single channel properties. Our results show that the p.Arg2435Leu substitution affects neither the resting [Ca²⁺], nor the sensitivity of the ryanodine receptor to pharmacological activators, but rather reduces the release of Ca²⁺ from intracellular stores induced by pharmacological activators as well as by KCl via the voltage sensing dihydropyridine receptor.     

A Novel missense mutation of COL2A1 gene in a large family with stickler syndrome type I

Stickler syndrome type I (STL1, MIM 108300) is characterized by ocular, auditory, skeletal and orofacial manifestations. Nonsyndromic ocular STL1 (MIM 609508) characterized by predominantly ocular features is a subgroup of STL1, and it is inherited in an autosomal dominant manner. In this study, a novel variant c.T100>C (p.Cys34Arg) in COL2A1 related to a large nonsyndromic ocular STL1 family was identified through Exome sequencing (ES). Bioinformatics analysis indicated that the variant site was highly conserved and the pathogenic mechanism of this variant may involve in affected structure of chordin‐like cysteine‐rich (CR) repeats of ColIIA. Minigene assay indicated that this variant did not change alternative splicing of exon2 of COL2A1. Moreover, the nonsyndromic ocular STL1 family with 16 affected members showed phenotype variability and certain male gender trend. None of the family members had hearing loss. Our findings would expand the knowledge of the COL2A1 mutation spectrum, and phenotype variability associated with nonsyndromic ocular STL1. Search for genetic modifiers and related molecular pathways leading to the phenotype variation warrants further studies.     

Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population

ABSTRACT: BACKGROUND: Oculocutaneous albinism (OCA) is caused by a group of genetically heterogeneous inherited defects that result in the loss of pigmentation in the eyes, skin and hair. Mutations in the TYR, OCA2, TYRP1 and SLC45A2 genes have been shown to cause isolated OCA. No comprehensive analysis has been conducted to study the spectrum of OCA alleles prevailing in Pakistani albino populations. METHODS: We enrolled 40 large Pakistani families and screened them for OCA genes and a candidate gene, SLC24A5. Protein function effects were evaluated using in silico prediction algorithms and ex vivo studies in human melanocytes. The effects of splice-site mutations were determined using an exon-trapping assay. RESULTS: Screening of the TYR gene revealed four known (p.Arg299His, p.Pro406Leu, p.Gly419Arg, p.Arg278*) and three novel mutations (p.Pro21Leu, p.Cys35Arg, p.Tyr411His) in ten families. Ex vivo studies revealed the retention of an EGFP-tagged mutant (p.Pro21Leu, p.Cys35Arg or p.Tyr411His) tyrosinase in the endoplasmic reticulum (ER) at 37degreesC, but a significant fraction of p.Cys35Arg and p.Tyr411His left the ER in cells grown at a permissive temperature (31degreesC). Three novel (p.Asp486Tyr, p.Leu527Arg, c.1045-15T>G) and two known mutations (p.Pro743Leu, p.Ala787Thr) of OCA2 were found in fourteen families. Exon-trapping assays with a construct containing a novel c.1045-15T>G mutation revealed an error in splicing. No mutation in TYRP1, SLC45A2, and SLC24A5 was found in the remaining 16 families. Clinical evaluation of the families segregating either TYR or OCA2 mutations showed nystagmus, photophobia, and loss of pigmentation in the skin or hair follicles. Most of the affected individuals had grayish-blue colored eyes. CONCLUSIONS: Our results show that ten and fourteen families harbored mutations in the TYR and OCA2 genes, respectively. Our findings, along with the results of previous studies, indicate that the p.Cys35Arg, p.Arg278* and p.Gly419Arg alleles of TYR and the p.Asp486Tyr and c.1045-15T>G alleles of OCA2 are the most common causes of OCA in Pakistani families. To the best of our knowledge, this study represents the first documentation of OCA2 alleles in the Pakistani population. A significant proportion of our cohort did not have mutations in known OCA genes. Overall, our study contributes to the development of genetic testing protocols and genetic counseling for OCA in Pakistani families.     

Altered sperm tsRNAs in aged male contribute to anxiety‐like behavior in offspring

Parental age at first pregnancy is increasing worldwide. The offspring of aged father has been associated with higher risk of several neuropsychiatric disorders, such as schizophrenia and autism, but the underlying mechanism remains elusive. Here we report that advanced paternal age in mice alters the profile of transfer RNA‐derived small RNAs (tsRNAs). Injection of sperm tsRNAs from aged male mice into zygotes induced anxiety‐like behaviors in F1 males. RNA sequencing of the cerebral cortex and hippocampus of those F1 male mice altered the gene expression of dopaminergic synapse and neurotrophin. tsRNAs from aged male mice injection also altered the neuropsychiatry‐related gene expression in two‐cell and blastocyst stage embryos. More importantly, the sperm tsRNA profile changes significantly during aging in human. The up‐regulated sperm tsRNA target genes were involved in neurogenesis and nervous system development. These results suggest that aging‐related changes of sperm tsRNA may contribute to the intergenerational transmission of behavioral traits.     

Exome Sequencing in 53 Sporadic Cases of Schizophrenia Identifies 18 Putative Candidate Genes

Schizophrenia (SCZ) is a severe, debilitating mental illness which has a significant genetic component. The identification of genetic factors related to SCZ has been challenging and these factors remain largely unknown. To evaluate the contribution of de novo variants (DNVs) to SCZ, we sequenced the exomes of 53 individuals with sporadic SCZ and of their non-affected parents. We identified 49 DNVs, 18 of which were predicted to alter gene function, including 13 damaging missense mutations, 2 conserved splice site mutations, 2 nonsense mutations, and 1 frameshift deletion. The average number of exonic DNV per proband was 0.88, which corresponds to an exonic point mutation rate of 1.7×10⁻⁸ per nucleotide per generation. The non-synonymous-to-synonymous mutation ratio of 2.06 did not differ from neutral expectations. Overall, this study provides a list of 18 putative candidate genes for sporadic SCZ, and when combined with the results of similar reports, identifies a second proband carrying a non-synonymous DNV in the RGS12 gene.     

Adaptive co‐evolution of mitochondria and the Y‐chromosome: A resolution to conflict between evolutionary opponents

In most species with motile sperm, male fertility depends upon genes located on the Y‐chromosome and in the mitochondrial genome. Coordinated adaptive evolution for the function of male fertility between genes on the Y and the mitochondrion is hampered by their uniparental inheritance in opposing sexes: The Y‐chromosome is inherited uniparentally, father to son, and the mitochondrion is inherited maternally, mother to offspring. Preserving male fertility is problematic, because maternal inheritance permits mitochondrial mutations advantageous to females, but deleterious to male fertility, to accumulate in a population. Although uniparental inheritance with sex‐restricted adaptation also affects genes on the Y‐chromosome, females lack a Y‐chromosome and escape the potential maladaptive consequences of male‐limited selection. Evolutionary models have shown that mitochondrial mutations deleterious to male fertility can be countered by compensatory evolution of Y‐linked mutations that restore it. However, direct adaptive coevolution of Y‐ and mitochondrial gene combinations has not yet been mathematically characterized. We use population genetic models to show that adaptive coevolution of Y and mitochondrial genes are possible when Y‐mt gene combinations have positive effects on male fertility and populations are inbred.     

46,XX, der(15),t(Y;15)(q12;p11) karyotype in an azoospermic male

We report on a Yq/15p translocation in a 23-year-old infertile male referred for Klinefelter Syndrome testing, who had azoospermia and bilateral small testes. Hormonal studies revealed hypergonadotropic hypogonadism. Conventional cytogenetic procedures giemsa trypsin giemsa (GTG) and high resolution banding (HRB) and molecular cytogenetic techniques Fluorescence In Situ Hybridization (FISH) performed on high-resolution lymphocyte chromosomes revealed the karyotype 46,XX, t(Y;15)(q12;p11). SRY-gene was confirmed to be present by classical Polymerase Chain Reaction (PCR) methods. His father carried de novo derivative chromosome 15 [45,X, t(Y;15)(q12;p11)] and was fertile; the karyotype of the father using G-band technique confirmed a reciprocal balanced translocation between chromosome Y and 15. In the proband, the der (15) has been inherited from the father because the mother had a normal karyotype (46,XX). In the proband, the der (15) could have produced genetic imbalance leading to unbalanced robertson translocation between chromosome Y and 15, which might have resulted in azoospermia and infertility in the proband. The paternal translocation might have lead to formation of imbalanced ova, which might be resulted infertility in the proband. Sister''s karyotypes was normal (46,XX) while his brother was not analyzed.