Rare Homozygous Missense Variants Research Articles

A homozygous missense variant in YTHDC2 induces azoospermia in two siblings.

Male infertility is a complex multifactorial reproductive disorder with highly heterogeneous phenotypic presentations. Azoospermia is a medically non-manageable cause of male infertility affecting ∼1% of men. Precise etiology of azoospermia is not known in approximately three-fourth of the cases. To explore the genetic basis of azoospermia, we performed whole exome sequencing in two non-obstructive azoospermia affected siblings from a consanguineous Pakistani family. Bioinformatic filtering and segregation analysis of whole exome sequencing data resulted in the identification of a rare homozygous missense variant (c.962G>C, p. Arg321Thr) in YTHDC2, segregating with disease in the family. Structural analysis of the missense variant identified in our study and two previously reported functionally characterized missense changes (p. Glu332Gln and p. His327Arg) in mice showed that all these three variants may affect Mg2+ binding ability and helicase activity of YTHDC2. Collectively, our genetic analyses and experimental observations revealed that missense variant of YTHDC2 can induce azoospermia in humans. These findings indicate the important role of YTHDC2 deficiency for azoospermia and will provide important guidance for genetic counseling of male infertility.

Homozygous missense variants in YKT6 result in loss of function and are associated with developmental delay, with or without severe infantile liver disease and risk for hepatocellular carcinoma

PurposeYKT6 plays important roles in multiple intracellular vesicle trafficking events but has not been associated with Mendelian diseases. MethodsWe report 3 unrelated individuals with rare homozygous missense variants in YKT6 who exhibited neurological disease with or without a progressive infantile liver disease. We modeled the variants in Drosophila. We generated wild-type and variant genomic rescue constructs of the fly ortholog dYkt6 and compared their ability in rescuing the loss-of-function phenotypes in mutant flies. We also generated a dYkt6KozakGAL4 allele to assess the expression pattern of dYkt6. ResultsTwo individuals are homozygous for YKT6 [NM_006555.3:c.554A>G p.(Tyr185Cys)] and exhibited normal prenatal course followed by failure to thrive, developmental delay, and progressive liver disease. Haplotype analysis identified a shared homozygous region flanking the variant, suggesting a common ancestry. The third individual is homozygous for YKT6 [NM_006555.3:c.191A>G p.(Tyr64Cys)] and exhibited neurodevelopmental disorders and optic atrophy. Fly dYkt6 is essential and is expressed in the fat body (analogous to liver) and central nervous system. Wild-type genomic rescue constructs can rescue the lethality and autophagic flux defects, whereas the variants are less efficient in rescuing the phenotypes. ConclusionThe YKT6 variants are partial loss-of-function alleles, and the p.(Tyr185Cys) is more severe than p.(Tyr64Cys).

Case report: Identification of a rare homozygous missense variant in the PKLR gene reported for the first time in transfusion-dependent Saudi Patient

Red cell pyruvate kinase deficiency is one of the most common erythrocytic glycolytic pathway defects connected with congenital non-spherocytic anemia. The condition inherited as an autosomal recessive Mendelian trait is caused by mutations in the PKLR gene located on chromosome 1q21. Pyruvate kinase enzyme is crucial in the energy-producing glycolysis pathway that provides red blood cells with the primary source of energy (ATP). We report here a case of a Saudi female patient that was initially diagnosed at a few months old with beta-thalassemia major and was treated with regular blood transfusions and iron overload management. At the time of our sample collection, the patient was recently transferred to King Abdul Aziz University Hospital. Genetic testing was performed to identify the disease-contributing variant of beta-thalassemia using TaqMan genotyping of six common beta-thalassemia variants (negative results). NGS targeted HBB gene sequencing which did not reveal any related variants. MLPA was performed to rule out alpha thalassemia diagnosis. The use of whole genome sequencing revealed a rare missense variant in the PKLR gene c.1015G>A (D339N) in a homozygous state that correlates to her severe phenotype. Documenting this incident will aid medical staff in providing appropriate care to similar cases and highlights the importance of following up with the diagnosis investigation process to minimize misdiagnosis incidences.

Identification of a missense variant of MND1 in meiotic arrest and non-obstructive azoospermia.

Meiotic arrest is a common pathologic phenotype of non-obstructive azoospermia (NOA), yet its genetic causes require further investigation. Meiotic nuclear divisions 1 (MND1) has been proved to be indispensable for meiotic recombination in many species. To date, only one variant of MND1 has been reported associated with primary ovarian insufficiency (POI), yet there has been no report of variants in MND1 associated with NOA. Herein, we identified a rare homozygous missense variant (NM_032117:c.G507C:p.W169C) of MND1 in two NOA-affected patients from one Chinese family. Histological analysis and immunohistochemistry demonstrated meiotic arrest at zygotene-like stage in prophase I and lack of spermatozoa in the proband's seminiferous tubules. In silico modeling demonstrated that this variant might cause possible conformational change in the leucine zippers 3 with capping helices (LZ3wCH) domain of MND1-HOP2 complex. Altogether, our study demonstrated that the MND1 variant (c.G507C) is likely responsible for human meiotic arrest and NOA. And our study provides new insights into the genetic etiology of NOA and mechanisms of homologous recombination repair in male meiosis.

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.

Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications

PPFIBP1 encodes for the liprin-β1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.

A Novel Homozygous Missense Variant in the LRRC32 Gene Is Associated With a New Syndrome of Cleft Palate, Progressive Vitreoretinopathy, Growth Retardation, and Developmental Delay.

Cleft lip and/or cleft palate are a common group of birth defects that further classify into syndromic and non-syndromic forms. The syndromic forms are usually accompanied by additional physical or cognitive abnormalities. Isolated cleft palate syndromes are less common; however, they are associated with a variety of congenital malformations and generally have an underlying genetic etiology. A single report in 2019 described a novel syndrome in three individuals, characterized by cleft palate, developmental delay and proliferative retinopathy due to a homozygous non-sense mutation in the LRRC32 gene encoding glycoprotein A repetitions predominant (GARP), a cell surface polypeptide crucial for the processing and maturation of transforming growth factor β (TGF-β). We describe a patient who presented with cleft palate, prenatal and postnatal severe growth retardation, global developmental delay, dysmorphic facial features and progressive vitreoretinopathy. Whole exome sequencing (WES) revealed a very rare homozygous missense variant in the LRRC32 gene, which resulted in substitution of a highly conserved isoleucine to threonine. Protein modeling suggested this variant may negatively affect GARP function on latent TGF-β activation. In summary, our report further expands the clinical features of cleft palate, proliferative retinopathy and developmental delay syndrome and emphasizes the association of LRRC32 pathogenic variants with this new syndrome.

ADAMTS1, MPDZ, MVD, and SEZ6: candidate genes for autosomal recessive nonsyndromic hearing impairment

Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.

Prediction and interpretation of rare missense variant in OTOG associated with hearing loss

OTOG encodes for otogelin, a component of the tectorial membrane. This gene is associated with nonprogressive mild-to-moderate hearing loss. However, no studies have yet identified the association between OTOG variation and severe-to-profound hearing loss. Therefore, to address this issue, a family-based whole-exome sequencing strategy (WES) was carried out. Two unrelated Iranian families with non-syndromic hearing loss were identified, and WES was conducted on one selected candidate from each family. As a result, a rare homozygous missense variant, OTOG (c.C2383T:p.R795C), was detected in both of the subjected probands, and segregation analysis confirmed the c.C2383T variant in seven cases of severe-to-profound hearing loss. Additionally, the results from the protein modeling demonstrated that the altered position of a few disulfide bonds in the TIL domain may have a deleterious impact on protein stability and normal functionality. In conclusion, it seems that the homozygosity of the OTOG c.C2383T mutation sheds light on hearing loss pathobiology. Nevertheless, further studies are required to unravel the precise function of OTOG mutation, which is potentially associated with severe-to-profound hearing loss.

Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA), the lack of spermatozoa in semen due to impaired spermatogenesis affects nearly 1% of men. In about half of cases, an underlying cause for NOA cannot be identified. This study aimed to identify novel variants associated with idiopathic NOA. We identified a nonconsanguineous family in which multiple sons displayed the NOA phenotype. We performed whole-exome sequencing in three affected brothers with NOA, their two unaffected brothers and their father, and identified compound heterozygous frameshift variants (one novel and one extremely rare) in Telomere Repeat Binding Bouquet Formation Protein 2 (TERB2) that segregated perfectly with NOA. TERB2 interacts with TERB1 and Membrane Anchored Junction Protein (MAJIN) to form the tripartite meiotic telomere complex (MTC), which has been shown in mouse models to be necessary for the completion of meiosis and both male and female fertility. Given our novel findings of TERB2 variants in NOA men, along with the integral role of the three MTC proteins in spermatogenesis, we subsequently explored exome sequence data from 1495 NOA men to investigate the role of MTC gene variants in spermatogenic impairment. Remarkably, we identified two NOA patients with likely damaging rare homozygous stop and missense variants in TERB1 and one NOA patient with a rare homozygous missense variant in MAJIN. Available testis histology data from three of the NOA patients indicate germ cell maturation arrest, consistent with mouse phenotypes. These findings suggest that variants in MTC genes may be an important cause of NOA in both consanguineous and outbred populations.

The homozygous variant c.245G > A/p.G82D in PNPLA2 is associated with arrhythmogenic cardiomyopathy phenotypic manifestations.

Arrhythmogenic cardiomyopathy (ACM) is a familial cardiomyopathy featured by fibrofatty replacement of cardiomyocytes. Responsible genetic factors are not discernible in approximately one-third of ACM probands. To investigate this further, we performed whole genome sequencing in 14 mutation-negative ACM probands who underwent cardiac transplantation, and we identified one ACM proband with a rare homozygous missense variant in PNPLA2 (c.245G > A, p.G82D), a rate-limiting enzyme that hydrolyzes triglycerides into fatty acids and diacylglycerol. Bioinformatic analysis suggested that this missense variant may lead to loss of function and therefore impair lipid catabolism. Genetic screening in this proband's family also inferred that the homozygous variant cosegregated with disease. To validate the pathogenicity of this variant and confirm its association with ACM, we established a knockin mouse model carrying the orthologous human homozygous PNPLA2 variant. Interestingly, mice with the homozygous variant presented with arrhythmias and significant cardiac dysfunction at 12 weeks, whereas heterozygous mice were not affected. Moreover, those homozygous mice suffered sudden death and/or heart failure by the age of 14 weeks. Pathological examination showed that extensive lipogenesis in cardiomyocytes and cardiac fibrosis were prominent in the myocardium. Herein, our data demonstrated that the homozygous missense variant PNPLA2 (c.245G > A, p.G82D) associated with a recessive form of ACM.

Dysfunction of the Cerebral Glucose Transporter SLC45A1 in Individuals with Intellectual Disability and Epilepsy

Glucose transport across the blood brain barrier and into neural cells is critical for normal cerebral physiologic function. Dysfunction of the cerebral glucose transporter GLUT1 (encoded by SLC2A1) is known to result in epilepsy, intellectual disability (ID), and movement disorder. Using whole-exome sequencing, we identified rare homozygous missense variants (c.526C>T [p.Arg176Trp] and c.629C>T [p.Ala210Val]) in SLC45A1, encoding another cerebral glucose transporter, in two consanguineous multiplex families with moderate to severe ID, epilepsy, and variable neuropsychiatric features. The variants segregate with the phenotype in these families, affect well-conserved amino acids, and are predicted to be damaging by insilico programs. Intracellular glucose transport activity of the p.Arg176Trp and p.Ala210Val SLC45A1 variants, measured in transfected COS-7 cells, was approximately 50% (p = 0.013) and 33% (p = 0.008) lower, respectively, than that of intact SLC45A1. These results indicate that residues at positions 176 and 210 are critical for the glucose transport activity of SLC45A1. All together, our data strongly suggest that recessive mutations in SLC45A1 cause IDand epilepsy. SLC45A1 thus represents the second cerebral glucose transporter, in addition to GLUT1, to be involved in neurodevelopmental disability. Identification of additional individuals with mutations in SLC45A1 will allow better definition of the associated phenotypic spectrum and the exploration of potential targeted treatment options.

RETRACTED: Generalized verrucosis and abnormal T cell activation due to homozygous TAOK2 mutation

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. The authors have notified the Editor of a serious error in their initial assumptions and, therefore, the overall conclusions presented in this article. The causative mutation is essential for the analysis and, therefore, it is difficult to correct part of the article. Had the Editor been aware of the issues flagged by the authors, the article would not have been accepted for publication. The authors have requested that the article is retracted because their data and conclusions are incorrect, and the Editor has agreed to retract the article.

Whole genome analyses reveal no pathogenetic single nucleotide or structural differences between monozygotic twins discordant for amyotrophic lateral sclerosis

The contribution of genetic and environmental factors to the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) remains unclear. To investigate the genetic component of the disease, we performed whole genome sequencing on ALS discordant monozygotic twins. Illumina whole genome sequencing on white blood cell DNA of five ALS-discordant monozygotic twin pairs (10 samples in total) yielded ∼30x coverage per individual. All single nucleotide variants, indels, and structural variants (copy number variants, inversions and translocations) were called and evaluated for functional consequence, evolutionary conservation, population frequency and overlap with known ALS associated variants and genes. Results showed that no validated discordant coding or regulatory single nucleotide variants or indels were found, and nor were any genome-wide discordant structural variants detected. Concordant variants of particular interest were: 1) two rare, highly-conserved heterozygous non-synonymous variants in SYT9 and EWSR1, genes previously associated with ALS (out of 2044 rare heterozygous variants detected); 2) three rare homozygous missense variants; and 3) three novel copy number deletions that overlapped genes. In conclusion, no convincing coding or regulatory nucleotide or genome-wide structural differences were found between ALS discordant monozygotic twins. The results suggest that more work is needed to elucidate possible environmental, epigenetic, oligogenic and somatic genetic factors that could underlie susceptibility to sporadic ALS.