Biallelic Loss Of Function Variants Research Articles

Biallelic variants in the calpain regulatory subunit CAPNS1 cause pulmonary arterial hypertension

PurposeThe aim of this study was to identify the monogenic cause of pulmonary arterial hypertension (PAH), a multifactorial and often fatal disease, in 2 unrelated consanguine families. MethodsWe performed exome sequencing and validated variant pathogenicity by whole-blood RNA and protein expression analysis in both families. Further RNA sequencing of preserved lung tissue was performed to investigate the consequences on selected genes that are involved in angiogenesis, proliferation, and apoptosis. ResultsWe identified 2 rare biallelic variants in CAPNS1, encoding the regulatory subunit of calpain. The variants cosegregated with PAH in the families. Both variants lead to loss of function (LoF), which is demonstrated by aberrant splicing resulting in the complete absence of the CAPNS1 protein in affected patients. No other LoF CAPNS1 variant was identified in the genome data of more than 1000 patients with unresolved PAH. ConclusionThe calpain holoenzyme was previously linked to pulmonary vascular development and progression of PAH in patients. We demonstrated that biallelic LoF variants in CAPNS1 can cause idiopathic PAH by the complete absence of CAPNS1 protein. Screening of this gene in patients who are affected by PAH, especially with suspected autosomal recessive inheritance, should be considered.

P180: Biallelic loss-of function variants in LDB3 deleting the long form of the protein lead to neonatal left ventricular noncompaction cardiomyopathy

P180: Biallelic loss-of function variants in LDB3 deleting the long form of the protein lead to neonatal left ventricular noncompaction cardiomyopathy

Clinical and genetic analyses in syndromic intellectual disability with primary microcephaly reveal biallelic and de novo variants in patients with parental consanguinity.

Syndromic intellectual disability (ID) with accompanying primary microcephaly is a group of rare neurodevelopmental disorders exhibiting extreme genetic and clinical heterogeneity. This layered heterogeneity can partially be resolved by unbiased genetic approaches targeting the genome with next generation sequencing (NGS) technologies, including exome sequencing (ES). This study was performed to dissect the clinical and genetic features in five distinct IDM cases. Singleton or trio ES approach followed by in-depth variant analysis using alternative inheritance models was performed. We have identified biallelic loss of function variants in genes WDR62 and AP4M1 in three families, together with de novo missense variants in genes SOX11 and TRIO in two families. ES based haplotype analysis in two cases upon identification of an identical WDR62 variant in the homozygous state in two cases was suggestive of a small shared haplotype of 0.1Mb. Additionally, we have shown a paternal origin for the de novo variant in TRIO via a polymorphic tag SNP, which enlightens the mutational mechanism for this variant. In populations with high parental consanguinity, an autosomal recessive inheritance pattern for data analysis is usually the most obvious choice. Therefore, heterozygous variants may be overlooked in standard NGS analyses in consanguineous families. Our findings underlie the importance of using multiple inheritance models in NGS data analysis.

Identification of three novel homozygous variants in COL9A3 causing autosomal recessive Stickler syndrome

BackgroundStickler syndrome (STL) is a rare, clinically and molecularly heterogeneous connective tissue disorder. Pathogenic variants occurring in a variety of genes cause STL, mainly inherited in an autosomal dominant fashion. Autosomal recessive STL is ultra-rare with only four families with biallelic COL9A3 variants reported to date.ResultsHere, we report three unrelated families clinically diagnosed with STL carrying different novel biallelic loss of function variants in COL9A3. Further, we have collected COL9A3 genotype–phenotype associations from the literature.ConclusionOur report substantially expands the molecular genetics and clinical basis of autosomal recessive STL and provides an overview about allelic COL9A3 disorders.

EP448: Multiple pregnancies with fetal akinesia deformation sequence caused by variants in MUSK

Fetal akinesia deformation sequence (FADS), also known as Pena-Shokier Syndrome, is a clinical manifestation of arthrogryposis, abnormal facial features, and pulmonary hypoplasia, and demonstrates a high degree of genetic heterogeneity. Variants within the genes that are involved in the neuromuscular system are most frequently involved. One gene in particular, MUSK, is involved in the formation and maintenance of the neuromuscular junction. Biallelic loss of function variants in this gene have been documented by 5 affected fetuses in a Swedish family and an affected fetus in a Dutch family.

EP115: Two novel NPR2 variants in a patient with acromesomelic dysplasia - Maroteaux type

Acromesomelic dysplasia, Maroteaux type (AMDM) is a rare autosomal recessive skeletal dysplasia characterized by severe disproportionate short stature (adult height < 120 cm), dolichocephaly, acro- and mesomelic shortening of the limbs, short, broad fingers and flat feet. Radiographic findings include bowed radius with a short ulna, dolichocephaly, platyspondyly and narrowing of the lumbar interpedicular distances. Intelligence is unaffected. The condition is typically diagnosed at birth or in infancy, but features become more apparent over time because of the loss of linear growth. AMDM is caused by biallelic loss of function variants in the NPR2 gene which encodes the natriuretic peptide receptor-B (NPR-B). The NPR2 gene encodes a transmembrane receptor that is critical in endochondral bone formation. Forty four percent of variants in NPR2 occur in the ligand binding domain, 8% in the transmembrane domain, 26% in the protein kinase domain and 21% in the nucleotide cyclase domain. A seven-month-old was seen with clinical findings suggestive of a skeletal dysplasia. She was born at 36 weeks following a pregnancy complicated by maternal polysubstance use and poor prenatal care. Clinical findings were shortening of all limb segments, trident hands, weak grasp, and overlapping second and third toes bilaterally. Radiographic findings were shortening of all the long bones with metaphyseal flaring, short and broad metatarsals, metacarpals and phalanges also with mild metaphyseal widening, platyspondyly with mild anterior vertebral body beaking in the upper lumbar spine. Cervical CT examination revealed platyspondyly of the suboccipital vertebrae and decreased AP diameter of the spinal canal at C1 secondary to anterior bowing of the posterior arch. A multi-gene skeletal dysplasia panel identified twelve variants, including two variants in NPR2. NPR2 c.1699G>T was classified as pathogenic, causing a premature termination at amino acid 567 in the kinase homology domain of the transmembrane protein. While classified as a VUS, the c.1123G>A missense variant was predicted to cause a substitution at amino acid 375 and falls at the last nucleotide of exon 4, which is part of the consensus splice site for this exon. The patient’s mother tested positive for the pathogenic variant and negative for the VUS in NPR2. Acromesomelic dysplasia, Maroteaux type is a rare skeletal dysplasia with limited published phenotypic and molecular information available. The described patient presented a diagnostic conundrum because only one of the variants had been characterized as pathogenic. The variant of uncertain significance is located in the ligand binding domain of the NPR2 gene, in which the majority of reported variants in this gene are found. In silico algorithms predict this variant to be deleterious to the protein structure and function but these predictions have not been confirmed by functional studies. Furthermore, missense variants within the consensus splice sites commonly cause abnormal splicing. This variant is not found in population databases. While paternal samples are not available to confirm that the variants are in trans, the patient was given the diagnosis of AMDM based on her clinical presentation. Molecular analysis and clinical description of other patients with AMDM will continue to characterize disease causing variants and natural history of this rare disease.

PCDH12 variants are associated with basal ganglia anomalies and exudative vitreoretinopathy

PCDH12 is a member of the non-clustered protocadherins that mediate cell-cell adhesion, playing crucial roles in many biological processes. Among these, PCDH12 promotes cell-cell interactions at inter-endothelial junctions, exerting essential functions in vascular homeostasis and angiogenesis. However, its exact role in eye vascular and brain development is not completely understood.To date, biallelic loss of function variants in PCDH12 have been associated with a neurodevelopmental disorder characterized by the typical neuroradiological findings of diencephalic-mesencephalic junction dysplasia and intracranial calcifications, whereas heterozygous variants have been recently linked to isolated brain calcifications in absence of cognitive impairment or other brain malformations. Recently, the phenotypic spectrum associated with PCDH12 deficiency has been expanded including cerebellar and eye abnormalities.Here, we report two female siblings harboring a novel frameshift homozygous variant (c.2169delT, p.(Val724TyrfsTer8)) in PCDH12. In addition to the typical diencephalic-mesencephalic junction dysplasia, brain MRI showed dysmorphic basal ganglia and thalamus that were reminiscent of a tubulin-like phenotype, mild cerebellar vermis hypoplasia and extensive prominence of perivascular spaces in both siblings. The oldest sister developed profound and progressive monocular visual loss and the eye exam revealed exudative vitreoretinopathy. Similar but milder eye changes were also noted in her younger sister.In summary, our report expands the clinical (brain and ocular) spectrum of PCDH12-related disorders and adds a further line of evidence underscoring the important role of PCDH12 in retinal vascular and brain development.

Hypomorphic variant in TRNT1 induces a milder autoinflammatory disease with congenital cataracts and impaired sexual development.

Dear Editor, tRNA nucleotidyltransferase-1 (TRNT1) is a nucleotidyltransferase of ubiquitous expression, implicated in the maturation of cytosolic and mitochondrial tRNA. Its activity is the addition of a conserved CCA trinucleotide sequence of the 3′ terminus that acts as the binding site of the amino acid in the tRNA molecule [1]. In 2014, Chakraborty et al. [2] reported that bi-allelic loss of function variants in TRNT1 were the cause of sideroblastic anaemia, B cell immunodeficiency, periodic fevers and developmental delay (SIFD) syndrome. This severe clinical phenotype had been described only a year before in 12 patients who shared these common features, with unknown genetic aetiology [3]. Seven patients had died before 14 years of age and the most severely affected did not live beyond the third year of life. Among the surviving individuals, only one had reached adulthood at the time of publication, presenting...

Biallelic loss of function variants in STAG3 result in primary ovarian insufficiency

Research questionDoes a genetic condition underlie the diagnosis of primary ovarian insufficiency (POI) in a 21-year-old woman with primary amenorrhoea? DesignA karyotype and genetic testing for Fragile X syndrome was undertaken. A next-generation sequencing panel of 24 genes associated with syndromal and non-syndromal POI was conducted. ResultsA nonsense variant c.1336G>T, p.(Glu446Ter) and whole gene deletion in STAG3 were identified. ConclusionsBiallelic loss of function variants in STAG3 are associated with primary ovarian failure type 8 and are a rare cause of POI.

Genes affecting ionizing radiation survival identified through combined exome sequencing and functional screening.

The study of genetic syndromes characterized by sensitivity to DNA damaging agents has provided important insights into the mechanisms that maintain genome stability and identified novel targets for cancer therapies. Here, we used exome sequencing to study 51 unrelated individuals with previously reported hypersensitivity to ionizing radiation as well as a range of neurologic, immunologic, and developmental features, but who did not clearly fit any previously defined genetic syndrome. Based on the combination of variant identification, computational evidence of deleteriousness, and functional screening, we identified three groups of subjects. Two subjects carried the bi-allelic loss of function variants in causative genes for known DNA damage response syndromes. Eight subjects carried the single loss of function variants in causative genes for DNA damage response syndromes, six of whom also carried predicted deleterious variants in other genes with DNA damage-related functions. Three subjects carried deleterious mutations in genes without obvious roles in DNA damage responses. However, treatment of U2OS cells with small interfering RNA targeting these genes resulted in significantly increased radiation sensitivity. Our results suggest that gene-gene interaction may contribute to ionizing radiation sensitivity as well as highlighting possible roles for several genes not obviously involved in the response to DNA damage.

Biallelic deep intronic variant c.5457+81T>A in TRIP11 causes loss of function and results in achondrogenesis 1A.

Biallelic loss of function variants in TRIP11 encoding for the Golgi microtubule-associated protein 210 (GMAP-210) causes the lethal chondrodysplasia achondrogenesis type 1A (ACG1A). Loss of TRIP11 activity has been shown to impair Golgi structure, vesicular transport, and results in loss of IFT20 anchorage to the Golgi that is vital for ciliary trafficking and ciliogenesis. Here, we report four fetuses, two each from two families, who were ascertained antenatally with ACG1A. Affected fetuses in both families are homozygous for the deep intronic TRIP11 variant, c.5457+81T>A, which was found in a shared region of homozygosity. This variant was found to cause aberrant transcript splicing and the retention of 77 base pairsof intron 18. The TRIP11 messenger RNA and protein levels were drastically reduced in fibroblast cells derived from one of the affected fetuses. Using immunofluorescence we also detected highly compacted Golgi apparatus in affected fibroblasts. Further, we observed a significant reduction in the frequency of ciliated cells and in the length of primary cilia in subject-derivedcell lines, not reported so far in patient cells with TRIP11 null or hypomorphic variants. Our findings illustrate how pathogenic variants in intronic regions of TRIP11 can impact transcript splicing, expression, and activity, resulting in ACG1A.

Bi-allelic loss of function variants in COX20 gene cause autosomal recessive sensory neuronopathy.

Sensory neuronopathies are a rare and distinct subgroup of peripheral neuropathies, characterized by degeneration of the dorsal root ganglia neurons. About 50% of sensory neuronopathies are idiopathic and genetic causes remain to be clarified. Through a combination of homozygosity mapping and whole exome sequencing, we linked an autosomal recessive sensory neuronopathy to pathogenic variants in the COX20 gene. We identified eight unrelated families from the eastern Chinese population carrying a founder variant c.41A>G (p.Lys14Arg) within COX20 in either a homozygous or compound heterozygous state. All patients displayed sensory ataxia with a decrease in non-length-dependent sensory potentials. COX20 encodes a key transmembrane protein implicated in the assembly of mitochondrial complex IV. We showed that COX20 variants lead to reduction of COX20 protein in patient's fibroblasts and transfected cell lines, consistent with a loss-of-function mechanism. Knockdown of COX20 expression in ND7/23 sensory neuron cells resulted in complex IV deficiency and perturbed assembly of complex IV, which subsequently compromised cell spare respiratory capacity and reduced cell proliferation under metabolic stress. Consistent with mitochondrial dysfunction in knockdown cells, reduced complex IV assembly, enzyme activity and oxygen consumption rate were also found in patients' fibroblasts. We speculated that the mechanism of COX20 was similar to other causative genes (e.g. SURF1, COX6A1, COA3 and SCO2) for peripheral neuropathies, all of which are functionally important in the structure and assembly of complex IV. Our study identifies a novel causative gene for the autosomal recessive sensory neuronopathy, whose vital function in complex IV and high expression in the proprioceptive sensory neuron further underlines loss of COX20 contributing to mitochondrial bioenergetic dysfunction as a mechanism in peripheral sensory neuron disease.

Description of four patients with TRIP11 variants expand the clinical spectrum of odontochondroplasia (ODCD) and demonstrate the existence of common variants

More than two decades since the first clinical and radiological description of odontochondroplasia (ODCD) was reported, biallelic loss of function variants in the Thyroid hormone receptor interactor 11 gene (TRIP11) were identified, the same gene implicated in the lethal disorder achondrogenesis (ACG1A). Here we report the clinical and radiological follow-up of four ODCD patients, including two siblings and an adult who interestingly has the mildest form observed to date. Four TRIP11 variants were detected, two previously unreported. Subsequently, we review the clinical and radiological findings of the 14 reported ODCD patients. The majority of ODCD patients are compound heterozygotes for TRIP11 variants, 12/14 have a null allele and a splice variant whilst one is homozygous for an in-frame splicing variant, with the splice variants resulting in residual GMAP activity and hypothesized to explain why they have ODCD and not ACG1A. However, adult patient 4 has two potentially null alleles and it remains unknown why she has very mild clinical features. The c.586C>T; p.(Gln196*) variant, previously shown by mRNA studies to result in p.Val105_Gln196del, is the most frequent variant, present in seven individuals from four families, three from different regions of the world, suggesting that it may be a variant hotspot. Another variant, c.2993_2994del; p.(Lys998Serfs*5), has been observed in two individuals with a possible common ancestor. In summary, although there are clinical and radiological characteristics common to all individuals, we demonstrate that the clinical spectrum of TRIP11-associated dysplasias is even more diverse than previously described and that common genetic variants may exist.

Natural human knockouts and Mendelian disorders: deep phenotyping in Italian isolates

Whole genome sequencing (WGS) allows the identification of human knockouts (HKOs), individuals in whom loss of function (LoF) variants disrupt both alleles of a given gene. HKOs are a valuable model for understanding the consequences of genes function loss. Naturally occurring biallelic LoF variants tend to be significantly enriched in “genetic isolates,” making these populations specifically suited for HKO studies. In this work, a meticulous WGS data analysis combined with an in-depth phenotypic assessment of 947 individuals from three Italian genetic isolates led to the identification of ten biallelic LoF variants in ten OMIM genes associated with known autosomal recessive diseases. Notably, only a minority of the identified HKOs (C7, F12, and GPR68 genes) displayed the expected phenotype. For most of the genes, instead, (ACADSB, FANCL, GRK1, LGI4, MPO, PGAM2, and RP1L1), the carriers showed none or few of the signs and symptoms typically associated with the related diseases. Of particular interest is a case presenting with a FANCL biallelic LoF variant and a positive diepoxybutane test but lacking a full Fanconi anemia phenotypic spectrum. Identifying KO subjects displaying expected phenotypes suggests that the lack of correct genetic diagnoses may lead to inappropriate and delayed treatment. In contrast, the presence of HKOs with phenotypes deviating from the expected patterns underlines how LoF variants may be responsible for broader phenotypic spectra. Overall, these results highlight the importance of in-depth phenotypical characterization to understand the role of LoF variants and the advantage of studying these variants in genetic isolates.

Bi-allelic loss of function variants in SLC30A5 as cause of perinatal lethal cardiomyopathy

Perinatal mortality is a heavy burden for both affected parents and physicians. However, the underlying genetic causes have not been sufficiently investigated and most cases remain without diagnosis. This impedes appropriate counseling or therapy. We describe four affected children of two unrelated families with cardiomyopathy, hydrops fetalis, or cystic hygroma that all deceased perinatally. In the four patients, we found the following homozygous loss of function (LoF) variants in SLC30A5 NM_022902.4:c.832_836del p.(Ile278Phefs*33) and NM_022902.4:c.1981_1982del p.(His661Tyrfs*10). Knockout of SLC30A5 has previously been shown a cardiac phenotype in mouse models and no homozygous LoF variants in SLC30A5 are currently described in gnomAD. Taken together, we present SLC30A5 as a new gene for a severe and perinatally lethal form of cardiomyopathy.

Bi-allelic truncating mutations in VWA1 cause neuromyopathy

The von Willebrand Factor A domain containing 1 protein, encoded by VWA1, is an extracellular matrix protein expressed in muscle and peripheral nerve. It interacts with collagen VI and perlecan, two proteins that are affected in hereditary neuromuscular disorders. Lack of VWA1 is known to compromise peripheral nerves in a Vwa1 knock-out mouse model. Exome sequencing led us to identify bi-allelic loss of function variants in VWA1 as the molecular cause underlying a so far genetically undefined neuromuscular disorder. We detected six different truncating variants in 15 affected individuals from six families of German, Arabic, and Roma descent. Disease manifested in childhood or adulthood with proximal and distal muscle weakness predominantly of the lower limbs. Myopathological and neurophysiological findings were indicative of combined neurogenic and myopathic pathology. Early childhood foot deformity was frequent, but no sensory signs were observed. Our findings establish VWA1 as a new disease gene confidently implicated in this autosomal recessive neuromyopathic condition presenting with child-/adult-onset muscle weakness as a key clinical feature.

Evidence that autosomal recessive spastic cerebral palsy-1 (CPSQ1) is caused by a missense variant in HPDL.

A subset of individuals diagnosed with cerebral palsy will have an underlying genetic diagnosis. Previously, a missense variant in GAD1 was described as a candidate mutation in a single family diagnosed with autosomal recessive spastic cerebral palsy-1 (CPSQ1; OMIM 603513). Following the ascertainment of a further branch of the CPSQ1 kindred, we found that the previously reported GAD1 variant did not segregate with the neurological disease phenotype in the recently ascertained branch of the kindred. Following genetic linkage studies to map autozygous regions and whole-exome sequencing, a missense variant (c.527 T > C; p. Leu176Pro, rs773333490) in the HPDL gene was detected and found to segregate with disease status in both branches of the kindred. HPDL encodes a 371-amino acid protein (4-Hydroxyphenylpyruvate Dioxygenase Like) that localizes to mitochondria but whose function is uncertain. Recently, biallelic loss of function variants and missense substitution-causing variants in HPDL were reported to cause a childhood onset progressive spastic movement disorder with a variable presentation. These findings suggest that HPDL-related neurological disease may mimic spastic cerebral palsy and that GAD1 should not be included in diagnostic gene panels for inherited cerebral palsy.

Genetic Analysis of Korean Adult Patients with Nontuberculous Mycobacteria Suspected of Primary Ciliary Dyskinesia Using Whole Exome Sequencing.

PurposeNontuberculous mycobacteria (NTM) is ubiquitous in the environment, but NTM lung disease (NTM-LD) is uncommon. Since exposure to NTM is inevitable, patients who develop NTM-LD are likely to have specific susceptibility factors, such as primary ciliary dyskinesia (PCD). PCD is a genetically heterogeneous disorder of motile cilia and is characterized by chronic respiratory tract infection, organ laterality defect, and infertility. In this study, we performed whole exome sequencing (WES) and investigated the genetic characteristics of adult NTM patients with suspected PCD.Materials and MethodsWES was performed in 13 NTM-LD patients who were suspected of having PCD by clinical symptoms and/or ultrastructural ciliary defect observed by transmission electron microscopy. A total of 45 PCD-causing genes, 23 PCD-candidate genes, and 990 ciliome genes were analyzed.ResultsFour patients were found to have biallelic loss-of-function (LoF) variants in the following PCD-causing genes: CCDC114, DNAH5, HYDIN, and NME5. In four other patients, only one LoF variant was identified, while the remaining five patients did not have any LoF variants.ConclusionAt least 30.8% of NTM-LD patients who were suspected of having PCD had biallelic LoF variants, and an additional 30.8% of patients had one LoF variant. Therefore, PCD should be considered in patients with NTM-LD with symptoms or signs suspicious of PCD.

Epilepsy with eyelid myoclonias and Sotos syndrome features in a patient with compound heterozygous missense variants in APC2 gene

Epilepsy with eyelid myoclonias and Sotos syndrome features in a patient with compound heterozygous missense variants in APC2 gene

Boricua Founder Variant in FRRS1L Causes Epileptic Encephalopathy With Hyperkinetic Movements.

To describe a founder mutation effect and the clinical phenotype of homozygous FRRS1L c.737_739delGAG (p.Gly246del) variant in 15 children of Puerto Rican (Boricua) ancestry presenting with early infantile epileptic encephalopathy (EIEE-37) with prominent movement disorder. EIEE-37 is caused by biallelic loss of function variants in the FRRS1L gene, which is critical for AMPA-receptor function, resulting in intractable epilepsy and dyskinesia. A retrospective, multicenter chart review of patients sharing the same homozygous FRRS1L (p.Gly246del) pathogenic variant identified by clinical genetic testing. Clinical information was collected regarding neurodevelopmental outcomes, neuroimaging, electrographic features and clinical response to antiseizure medications. Fifteen patients from 12 different families of Puerto Rican ancestry were homozygous for the FRRS1L (p.Gly246del) pathogenic variant, with ages ranging from 1 to 25 years. The onset of seizures was from 6 to 24 months. All had hypotonia, severe global developmental delay, and most had hyperkinetic involuntary movements. Developmental regression during the first year of life was common (86%). Electroencephalogram showed hypsarrhythmia in 66% (10/15), with many older children evolving into Lennox-Gastaut syndrome. Six patients demonstrated progressive volume loss and/or cerebellar atrophy on brain magnetic resonance imaging (MRI). We describe the largest cohort to date of patients with epileptic encephalopathy. We estimate that 0.76% of unaffected individuals of Puerto Rican ancestry carry this pathogenic variant due to a founder effect. Children homozygous for the FRRS1L (p.Gly246del) Boricua variant exhibit a very homogenous phenotype of early developmental regression and epilepsy, starting with infantile spasms and evolving into Lennox-Gastaut syndrome with hyperkinetic movement disorder.