Clear evidence of LAMA5 gene biallelic truncating variants causing infantile nephrotic syndrome

Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only 7 patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome and one SRNS case with biallelic LAMA5 missense variants. Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. Using targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results: Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in 3 patients from 2 families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.(Arg3078*)) and a splice site variant (c.1282+1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.(Arg2720*)) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of eight and carried compound heterozygous missense variants (c.1493C>T, p.(Ala498Val) and c.8399G>A, p.(Arg2800His)). Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathological characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in congenital/infantile nephrotic syndrome patients.

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Clinical Characteristics of POC1B-Associated Retinopathy and Assignment of Pathogenicity to Novel Deep Intronic and Non-Canonical Splice Site Variants

International Journal of Molecular Sciences ◽

10.3390/ijms22105396 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5396

Author(s):

Nicole Weisschuh ◽

Pascale Mazzola ◽

Miriam Bertrand ◽

Tobias B. Haack ◽

Bernd Wissinger ◽

...

Keyword(s):

Genome Sequencing ◽

Splice Site ◽

Age Of Onset ◽

Splice Variants ◽

Exon Skipping ◽

Cone Dystrophy ◽

Compound Heterozygous ◽

Pathogenic Variants ◽

Splice Site Variant

Mutations in POC1B are a rare cause of inherited retinal degeneration. In this study, we present a thorough phenotypic and genotypic characterization of three individuals harboring putatively pathogenic variants in the POC1B gene. All patients displayed a similar, slowly progressive retinopathy (cone dystrophy or cone-rod dystrophy) with normal funduscopy but disrupted outer retinal layers on optical coherence tomography and variable age of onset. Other symptoms were decreased visual acuity and photophobia. Whole genome sequencing revealed a novel homozygous frameshift variant in one patient. Another patient was shown to harbor a novel deep intronic variant in compound heterozygous state with a previously reported canonical splice site variant. The third patient showed a novel nonsense variant and a novel non-canonical splice site variant. We aimed to validate the effect of the deep intronic variant and the non-canonical splice site variant by means of in vitro splice assays. In addition, direct RNA analysis was performed in one patient. Splicing analysis revealed that the non-canonical splice site variant c.561-3T>C leads to exon skipping while the novel deep intronic variant c.1033-327T>A causes pseudoexon activation. Our data expand the genetic landscape of POC1B mutations and confirm the benefit of genome sequencing in combination with downstream functional validation using minigene assays for the analysis of putative splice variants. In addition, we provide clinical multimodal phenotyping of the affected individuals.

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Novel Splice-Site and Missense Variant of PNPLA6 in an Austrian Family Causing Spastic Paraplegia-39 with Cerebellar Oculomotor Disorder

10.21203/rs.3.rs-1142999/v1 ◽

2022 ◽

Author(s):

Sebastian Viertauer ◽

Ingo Kurth ◽

Katja Eggermann ◽

Christian Eggers

Keyword(s):

Splice Site ◽

Lower Limb ◽

Clinical Symptoms ◽

Hypogonadotropic Hypogonadism ◽

Missense Variant ◽

Compound Heterozygous ◽

Spastic Paraplegia ◽

Meaningful Improvement ◽

Pathogenic Variants ◽

Splice Site Variant

Abstract Background The term hereditary spastic paraplegia comprises an ever-expanding array of neurological disorders with distinct aetiologies. Spastic paraplegia 39 is one of the many variants with additional features of other organs and neurological systems. We describe a large kindred with two hitherto undescribed mutations of PNPLA6 and a novel clinical phenotype as, in addition to spastic paraplegia, affected subjects suffered from a prominent cerebellar oculomotor dysfunction. Methods Three of five genetically tested family members of a large kindred were affected by spastic gait and cerebellar oculomotor dysfunction. Clinical, imaging, laboratory and electrophysiological data were analyzed. Genetic analysis was done using next-generation sequencing. Segregation analyses were performed by Sanger sequencing. To assess the pathogenicity of genetic variants on the encoded protein, in silico assessments were carried out. Results Two hitherto unknown sequence variants in the PNPLA6 gene, a splice-site variant c.1635+3G>T and a missense variant c.3401A>T, p.(Asp1134Val), were detected. Compound-heterozygous siblings presented with lower limb spasticity and a marked cerebellar oculomotor disorder accompanied by moderate hypogonadotropic hypogonadism in the female. A paternal uncle being homozygous for the splice-site variant of PNPLA6 presented with increased lower limb reflexes, an unstable gait and cerebellar oculomotor dysfunction. Treatment with 4-aminopyridin, a potassium channel blocker, lead to meaningful improvement of clinical symptoms. Conclusion PNPLA6 gene variants are associated with a broad phenotypic spectrum to which we add cerebellar oculomotor dysfunction. In our kindred, the full clinical manifestation only occurred in compound-heterozygous subjects indicating that biallelic pathogenic variants lead to more serious and earlier onset of symptoms. Our findings emphasize the role of PNPLA6 in different neurodegenerative disorders.

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Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

Genetics in Medicine ◽

10.1038/s41436-021-01158-1 ◽

2021 ◽

Author(s):

Paolo Zanoni ◽

Katharina Steindl ◽

Deepanwita Sengupta ◽

Pascal Joset ◽

Angela Bahr ◽

...

Keyword(s):

Loss Of Function ◽

Mild Phenotype ◽

Psychological Issues ◽

Missense Variants ◽

Mild Developmental Delay ◽

Pathogenic Variants ◽

In Silico Modeling ◽

And Function ◽

Methylation Activity

Abstract Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.

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MO1027EFFICACY OF CALCINEURIN INHIBITORS IN CHILDREN WITH MONOGENIC STEROID-RESISTANT NEPHROTIC SYNDROME

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab108.0024 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Larisa Prikhodina ◽

Svetlana Papizh ◽

Inna Povolotskaya

Keyword(s):

Nephrotic Syndrome ◽

Calcineurin Inhibitors ◽

Compound Heterozygous ◽

Target Level ◽

Steroid Resistant ◽

Steroid Resistant Nephrotic Syndrome ◽

Pathogenic Variants ◽

Nail Patella Syndrome

Abstract Background and Aims Monogenic causes of steroid-resistant nephrotic syndrome (SRNS) have been reported for up to one-third of children depending on age of the disease onset. Immunosuppressive treatment of genetic SRNS with calcineurin inhibitors (CNIs) is still controversial. The aim of the study was to investigate the efficacy of CNIs with focus on inducing remission and long-term kidney function in children with monogenic SRNS. Method Retrospective analysis of efficacy CNIs in five children (2M/3F) with monogenic SRNS was performed. Kidney biopsy prior CNIs revealed FSGS (n=4) and MCD (n=1). The initial cyclosporine (CsA) dose was 5 mg/kg/24h to keep a target level of 80-150 ng/ml and tacrolimus (TAC) - 0.1 mg/kg/24h to achieve a target level of 5-10 ng/ml. CsA took all 5 patients with subsequent switching to TAC in 2 children due to cosmetic side effects. The median follow-up period was 165.0 (IQR: 59.0; 185.5) months. Next generation sequencing (NGS) was used for identification of pathogenic variants in all patients. Results The median age at onset of monogenic SRNS was 33.0 (IQR: 16.5; 63.0) months. 2/5 (40%) patients presented with acute SRNS, 1/5 (20%) child with infantile NS, 1/5 (20%) - with isolated nephrotic range proteinuria with hypoalbuminemia and 1/5 (20%) - with NS and extrarenal features of Nail-Patella syndrome. NGS identified previously described pathogenic variants in all 5 children, including NPHS2 homozygous c.28dup (p.Glu87Ter) (n=1), NPHS2 compound heterozygous c.868G>A (p.Val290Met) in combination with c.686G>A (p.Arg229Gln) (n=1), LMX1B heterozygous c.788T>G (p.Val263Gly) (n=1), LMX1B heterozygous c.737G>A (p.Arg246Gln) (n=1), and COL4A3 heterozygous c.2962G>A (p.Gly988Arg) variant (n=1). The median time from diagnosis to initiation of CNIs treatment was 72.0 (IQR: 33.0; 93.0) months. CNIs induced complete remission in 2/5 (40%) patients, presented with acute SRNS, including one girl with MCD due to NPHS2 compound heterozygous variants with mutation-dependent pathogenicity of one (p.R229Q) of them and one boy with FSGS due to COL4A3 heterozygous variant (n=1). Partial remission was induced by CNIs in 2/5 (40%) girls with FSGS due to LMX1B heterozygous variants with isolated SRNS (n=1) and Nail-Patella syndrome (n=1). The median duration of CNIs treatment to obtain complete or partial remission was 13.5 (IQR: 6.8; 15.8) months. Acute CNIs-associated nephrotoxicity had 2 patients with LMX1B variants. At the last follow up full and partial responders to CNIs treatment aged of 16.5 (IQR: 11.8; 17.5) years had CKD-1 (n=3) and CKD-2 (n=1). 1/5 (20%) boy with NPHS2-associated infantile NS was CNI resistant and developed CKD-5 at the age of 6.5 years with subsequent living related kidney transplantation. Conclusion We found that 4/5 (80%) children with monogenic SRNS demonstrated partial or full response to CNIs treatment with stable long-term kidney function. We assume that CNIs might improve podocyte function by stabilization of their cytoskeleton disrupted in patients with monogenic SRNS.

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Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males

Human Genetics ◽

10.1007/s00439-021-02412-x ◽

2021 ◽

Author(s):

Hans-Jürgen Kreienkamp ◽

Matias Wagner ◽

Heike Weigand ◽

Allyn McConkie-Rossell ◽

Marie McDonald ◽

...

Keyword(s):

De Novo ◽

Functional Characterization ◽

Monozygotic Twins ◽

Rna Recognition Motif ◽

Neurodevelopmental Delay ◽

Missense Variants ◽

Mild Developmental Delay ◽

Splicing Regulators ◽

Pathogenic Variants

AbstractBain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype–phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

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Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-019-1241-z ◽

2019 ◽

Vol 14 (1) ◽

Author(s):

Niu Li ◽

Yufei Xu ◽

Yi Zhang ◽

Guoqiang Li ◽

Tingting Yu ◽

...

Keyword(s):

Postnatal Growth ◽

Compound Heterozygous ◽

Loss Of Function ◽

Atrioventricular Canal ◽

Multiple Systems ◽

Feeding Difficulties ◽

Functional Studies ◽

Pathogenic Variants ◽

Compound Heterozygous Variants

Abstract Background Gain-of-function pathogenic variants of the Erb-B2 receptor tyrosine kinase 3 (ERBB3) gene contribute to the occurrence and development of a variety of human carcinomas through activation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) signaling. ERBB3 gene homozygous germline variants, whose loss of function may cause autosomal recessive congenital contractural syndrome, were recently identified. This study aims to identify the disease-causing gene in a Chinese pedigree with variable phenotypes involving multiple systems, including developmental delay, postnatal growth retardation, transient lower limb asymmetry, facial malformations, atrioventricular canal malformation, bilateral nystagmus and amblyopia, feeding difficulties, immunodeficiency, anemia, and liver damage, but without congenital contracture. Methods Trio-whole exome sequencing (WES) was performed to identify the disease-causing gene in a 24-month-old Chinese female patient. The pathogenicity of the identified variants was evaluated using in silico tools and in vitro functional studies. Results Trio-WES revealed compound heterozygous variants of c.1253 T > C (p.I418T) and c.3182dupA (p.N1061Kfs*16) in the ERBB3 gene. Functional studies showed that p.I418T resulted in normal expression of ERBB3, which was capable of interacting with ERBB2. However, the variant impaired ERBB3 phosphorylation, consequently blocking ERBB2 phosphorylation and AKT and ERK activation. The truncated protein resulting from the c.3182dupA variant also lacked the capacity to activate downstream signaling pathways. Conclusions We report the first patient with a novel multisystem syndrome disorder without congenital contracture resulting from biallelic loss-of-function variants of ERBB3.

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Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families

Genes ◽

10.3390/genes11111329 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1329

Author(s):

Julia Doll ◽

Barbara Vona ◽

Linda Schnapp ◽

Franz Rüschendorf ◽

Imran Khan ◽

...

Keyword(s):

Hearing Loss ◽

Exome Sequencing ◽

Splice Site ◽

Genetic Heterogeneity ◽

Bioinformatics Analysis ◽

Molecular Genetic ◽

Compound Heterozygous ◽

Pathogenic Variants ◽

Syndromic Hearing Loss ◽

Pakistani Families

The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes.

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Germline variants in cancer genes in high-risk non-BRCA patients from Puerto Rico

Scientific Reports ◽

10.1038/s41598-019-54170-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Julie Dutil ◽

Jamie K. Teer ◽

Volha Golubeva ◽

Sean Yoder ◽

Wei Lue Tong ◽

...

Keyword(s):

Breast Cancer ◽

Puerto Rico ◽

High Risk ◽

Cancer Patients ◽

Significant Proportion ◽

Pathogenic Variant ◽

Breast Cancer Patients ◽

Missense Variants ◽

Pathogenic Variants

AbstractInherited pathogenic variants in genes that confer moderate to high risk of breast cancer may explain up to 50% of familial breast cancer. This study aimed at identifying inherited pathogenic variants in breast cancer cases from Puerto Rico that were not linked to BRCA1 or BRCA2. Forty-eight breast cancer patients that met the clinical criteria for BRCA testing but had received a negative BRCA1/2 result were recruited. Fifty-three genes previously implicated in hereditary cancer predisposition were captured using the BROCA Agilent cancer risk panel followed by massively parallel sequencing. Missense variants of uncertain clinical significance in CHEK2 were evaluated using an in vitro kinase assays to determine their impact on function. Pathogenic variants were identified in CHEK2, MUTYH, and RAD51B in four breast cancer patients, which represented 8.3% of the cohort. We identified three rare missense variants of uncertain significance in CHEK2 and two variants (p.Pro484Leu and p.Glu239Lys) showed markedly decreased kinase activity in vitro comparable to a known pathogenic variant. Interestingly, the local ancestry at the RAD51B locus in the carrier of p.Arg47* was predicted to be of African origin. In this cohort, 12.5% of the BRCA-negative breast cancer patients were found to carry a known pathogenic variant or a variant affecting protein activity. This study reveals an unmet clinical need of genetic testing that could benefit a significant proportion of at-risk Latinas. It also highlights the complexity of Hispanic populations as pathogenic factors may originate from any of the ancestral populations that make up their genetic backgrounds.

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X-Linked Retinitis Pigmentosa Caused by Non-Canonical Splice Site Variants in RPGR

International Journal of Molecular Sciences ◽

10.3390/ijms22020850 ◽

2021 ◽

Vol 22 (2) ◽

pp. 850

Author(s):

Friederike Kortüm ◽

Sinja Kieninger ◽

Pascale Mazzola ◽

Susanne Kohl ◽

Bernd Wissinger ◽

...

Keyword(s):

Retinitis Pigmentosa ◽

Splice Site ◽

Exon Skipping ◽

Open Reading Frame ◽

Aberrant Splicing ◽

Reading Frame ◽

Pathogenic Variants ◽

Nucleotide Addition ◽

Intron 2

We aimed to validate the effect of non-canonical splice site variants in the RPGR gene in five patients from four families diagnosed with retinitis pigmentosa. Four variants located in intron 2 (c.154 + 3_154 + 6del), intron 3 (c.247 + 5G>A), intron 7 (c.779-5T>G), and intron 13 (c.1573-12A>G), respectively, were analyzed by means of in vitro splice assays. Splicing analysis revealed different aberrant splicing events, including exon skipping and intronic nucleotide addition, which are predicted to lead either to an in-frame deletion affecting relevant protein domains or to a frameshift of the open reading frame. Our data expand the landscape of pathogenic variants in RPGR, thereby increasing the genetic diagnostic rate in retinitis pigmentosa and allowing patients harboring the analyzed variants to be enrolled in clinical trials.

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Biallelic Variants in the COLGALT1 Gene Causes Severe Congenital Porencephaly

Neurology Genetics ◽

10.1212/nxg.0000000000000564 ◽

2021 ◽

Vol 7 (2) ◽

pp. e564

Author(s):

Mariel W.A. Teunissen ◽

Erik-Jan Kamsteeg ◽

Suzanne C.E.H. Sallevelt ◽

Maartje Pennings ◽

Noel J.C. Bauer ◽

...

Keyword(s):

Splice Site ◽

Sanger Sequencing ◽

Small Vessel Disease ◽

Lymphocyte Culture ◽

Reverse Transcription Pcr ◽

Pathogenic Variants ◽

Spastic Quadriplegia ◽

Splice Site Variant ◽

A Minor ◽

Cryptic Splice Sites

ObjectiveWe describe a third patient with brain small vessel disease 3 (BSVD3), being the first with a homozygous essential splice site variant in the COLGALT1 gene, with a more severe phenotype than the 2 children reported earlier.MethodsAnalysis of whole exome sequencing (WES) data of the child and parents was performed. We validated the missplicing of the homozygous variant using reverse transcription PCR and Sanger sequencing of the mRNA in a lymphocyte culture.ResultsThe patient presented antenatally with porencephaly on ultrasound and MRI. Postnatally, he showed a severe developmental delay, refractory epilepsy, spastic quadriplegia, and a progressive hydrocephalus. WES revealed a homozygous canonical splice site variant NM_024656.3:c.625-2A>C. PCR and Sanger sequencing of the mRNA demonstrated that 2 cryptic splice sites are activated, causing a frameshift in the major transcript and in-frame deletion in a minor transcript.ConclusionsWe report a third patient with biallelic pathogenic variants in COLGALT1, confirming the role of this gene in autosomal recessive BSVD3.

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