Homozygous Truncating Mutation Research Articles

ZNF142 mutation causes sex-dependent neurologic disorder

BackgroundSex-specific predilection in neurological diseases caused by mutations in autosomal genes is a phenomenon whose molecular basis is poorly understood. We studied females of consanguineous Bedouin kindred presenting with severe...

Hyperinsulinism/hyperammonemia syndrome caused by biallelic SLC25A36 mutation.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome has been known to be caused by dominant gain-of-function mutations in GLUD1, encoding the mitochondrial enzyme glutamate dehydrogenase. Pathogenic GLUD1 mutations enhance enzymatic activity by reducing its sensitivity to allosteric inhibition by GTP. Two recent independent studies showed that a similar HI/HA phenotype can be caused by biallelic mutations in SLC25A36, encoding pyrimidine nucleotide carrier 2 (PNC2), a mitochondrial nucleotide carrier that transports pyrimidine and guanine nucleotides across the inner mitochondrial membrane: one study reported a single case caused by a homozygous truncating mutation in SLC25A36 resulting in lack of expression of SLC25A36 in patients' fibroblasts. A second study described two siblings with a splice site mutation in SLC25A36, causing reduction of mitochondrial GTP content, putatively leading to hyperactivation of glutamate dehydrogenase. In an independent study, through combined linkage analysis and exome sequencing, we demonstrate in four individuals of two Bedouin Israeli related families the same disease-causing SLC25A36 (NM_018155.3) c.284 + 3A > T homozygous splice-site mutation found in the two siblings. We demonstrate that the mutation, while causing skipping of exon 3, does not abrogate expression of mRNA and protein of the mutant SLC25A36 in patients' blood and fibroblasts. Affected individuals had hyperinsulinism, hyperammonemia, borderline low birth weight, tonic-clonic seizures commencing around 6months of age, yet normal intellect and no significant other morbidities. Chronic constipation, hypothyroidism, and developmental delay previously described in a single patient were not found. We thus verify that biallelic SLC25A36 mutations indeed cause HI/HA syndrome and clearly delineate the disease phenotype.

A homozygous truncating mutation of FGL2 is associated with immune dysregulation

The type II transmembrane protein fibrinogen-like protein 2 (FGL2) plays critical roles in hemostasis and immune regulation. The C-terminal immunoregulatory domain of FGL2 can be secreted and is a mediator of regulatory T (Treg) cell suppression. Fgl2-/- mice develop autoantibodies and glomerulonephritis and have impaired Treg cell function. Our aim was to identify the genetic underpinning and immune function in a patient with childhood onset of leukocytoclastic vasculitis, systemic inflammation, and autoantibodies. Whole-exome sequencing was performed on patient genomic DNA. FGL2 protein expression was examined in HEK293 transfected cells by immunoblotting and in PBMCs by flow cytometry. T follicular helper cells and Treg cells were examined by flow cytometry. Treg cell suppression of T-cell proliferation was assessed invitro. The patient had a homozygous mutation in FGL2 (c.614_617del:p.V205fs), which led to the expression of a truncated FGL2 protein that preserves the N-terminal domain but lacks the C-terminal immunoregulatory domain. The patient had an increased percentage of circulating T follicular helper and Treg cells. The patient's Treg cells had impaired invitro suppressive ability that was rescued by the addition of full-length FGL2. Unlike full-length FGL2, the truncated FGL2V205fs mutant failed to suppress T-cell proliferation. We identified a homozygous mutation in FGL2 in a patient with immune dysregulation and impaired Treg cell function. Soluble FGL2 rescued the Treg cell defect, suggesting that it may provide a useful therapy for the patient.

Dual CDKN2A/MTAP loss compared to CDKN2A loss alone and response to immune-checkpoint inhibitors (ICI) in advanced solid tumors.

2622 Background: We previously showed that CDKN2A genomic alterations (GAs) are associated with resistance to ICI (Adib E, Clinical Cancer Research, 2021). The majority of such GAs are homozygous deletions, which commonly (̃50-80%) include MTAP, located 100kb telomeric of CDKN2A. MTAP loss leads to 5′-deoxy-5′-methylthioadenosine (MTA) accumulation and immunosuppressive effects in tumors. We examined combined CDKN2A/MTAP deletion vs. CDKN2A deletion/mutation alone as predictors of poor ICI response. Methods: We curated clinical data for cancer patients (pts) treated with ICI at Dana-Farber Cancer Institute through 6/2021, who had targeted panel sequencing. Inclusion criteria were: ICI in metastatic setting, ≥2 cycles, no concurrent systemic therapy, cancer type with > 50 pts treated. CDKN2A/ MTAP GAs were defined as a deep deletion affecting both genes; CDKN2A only GAs included both homozygous deletions and truncating mutations. Hazard ratios (HR) for overall survival (OS) and time-to-treatment failure (TTF) were derived using multivariable Cox regression, adjusted for prior lines of therapy, treatment type (single vs. combination ICI), tumor mutational burden and ECOG PS. We also used a machine learning approach to quantify the density of tumor-infiltrating lymphocytes (TILs) in digital whole-slide H&E images of 144 melanoma pts with available genomic data. Results: 921 pts with 6 cancer types were studied: non-small cell lung cancer (NSCLC, n = 366), melanoma (mel, n = 228), urothelial carcinoma (UC, n = 120), esophagogastric carcinoma (EGC,n = 90), head and neck squamous cell carcinoma (HNSCC, n = 58), and renal cell carcinoma (RCC, n = 59). UC pts with MTAP/ CDKN2A GAs had shorter OS and TTF than pts without GA in either gene (OS HR = 1.9[1.1-3.4], p = 0.005; TTF HR = 1.8[1.0-3.1], p = 0.0016) after adjusting for covariates. Similar results were seen for melanoma (OS HR = 2.5[1.4-2.6],p = 0.00065; TTF HR = 1.9[1.1-3.2],p = 0.018). There was no significant difference between pts with CDKN2A GA only and those without GA in either gene for OS or TTF in either UC or melanoma. CDKN2A/MTAP status was not associated with significantly shorter survival for NSCLC and EGC; while the analysis was confounded by HPV events for HNSCC, and underpowered for RCC. ML-based analysis of digital slides for melanoma, showed that tumors with CDKN2A GAs only (n = 42) had similar median density of TILs compared to tumors without GAs in either gene (n = 84; 920 vs. 943 TILs/mm2; p = 0.42). In contrast, tumors with co-occurring CDKN2A/ MTAP GAs had lower TIL density (529 TIL/mm2, n = 17 vs. 925 TIL/mm2, n = 126 (pooled); p = 0.018, Wilcoxon rank sum). Conclusions: In this study, we showed that co-occurrence of MTAP/CDKN2A GAs, but not CDKN2A GA only, was associated with worse outcomes in pts with UC and melanoma treated with ICI. Lower TIL density was also seen in melanoma tissue samples with combined MTAP/CDKN2A GA.

Clinical and molecular genetic analysis of early-onset myopathy with fatal cardiomyopathy: Novel biallelic M-line TTN mutation and review of the literature

Early-onset myopathy with fatal cardiomyopathy (EOMFC), also known as Salih myopathy (SALMY) is a rare, heterogeneous, and severe form of titinopathies with autosomal recessive inherited neuromuscular disorders that affects both skeletal and cardiac muscles. It was previously identified only in the Arab population with unknown incidence. Titin (TTN) mutations that have been reported in congenital myopathies are associated with a variety of phenotypic spectrum of titinopathies, which are scattered along with the 364 exons of the gene. In this study, we report a consanguineous Moroccan female child aged 29 months who presented with congenital myopathy associated to dilated cardiomyopathy. Clinical Exome Sequencing identified a novel homozygous truncating mutation c.106541delA p.(Asp35514Valfs*32) in exon 361 of the TTN gene. Sanger sequencing confirmed the mutation in a homozygous state in the proband and in a heterozygous state in both of her parents. Clinical and molecular data of the proband were correlated with 15 patients reported in the literature for congenital myopathy associated to a heart defect or development of dilated cardiomyopathy with at least one mutant allele in the M-band titin protein as inclusion criteria. In conclusion, the application of NGS in rare genetic heterogeneous forms as EOMFC provides more evidently an increasing proportion of congenital myopathies than currently recognized and expands the mutation spectrum of the TTN gene for better guiding the genetic diagnosis with adequate genetic counseling to the Moroccan families.

Loss-of-function mutations in the co-chaperone protein BAG5 cause dilated cardiomyopathy requiring heart transplantation.

Dilated cardiomyopathy (DCM) is a major cause of heart failure, characterized by ventricular dilatation and systolic dysfunction. Familial DCM is reportedly caused by mutations in more than 50 genes, requiring precise disease stratification based on genetic information. However, the underlying genetic causes of 60 to 80% of familial DCM cases remain unknown. Here, we identified that homozygous truncating mutations in the gene encoding Bcl-2–associated athanogene (BAG) co-chaperone 5 (BAG5) caused inherited DCM in five patients among four unrelated families with complete penetrance. BAG5 acts as a nucleotide exchange factor for heat shock cognate 71 kDa protein (HSC70), promoting adenosine diphosphate release and activating HSC70-mediated protein folding. Bag5 mutant knock-in mice exhibited ventricular dilatation, arrhythmogenicity, and poor prognosis under catecholamine stimulation, recapitulating the human DCM phenotype, and administration of an adeno-associated virus 9 vector carrying the wild-type BAG5 gene could fully ameliorate these DCM phenotypes. Immunocytochemical analysis revealed that BAG5 localized to junctional membrane complexes (JMCs), critical microdomains for calcium handling. Bag5-mutant mouse cardiomyocytes exhibited decreased abundance of functional JMC proteins under catecholamine stimulation, disrupted JMC structure, and calcium handling abnormalities. We also identified heterozygous truncating mutations in three patients with tachycardia-induced cardiomyopathy, a reversible DCM subtype associated with abnormal calcium homeostasis. Our study suggests that loss-of-function mutations in BAG5 can cause DCM, that BAG5 may be a target for genetic testing in cases of DCM, and that gene therapy may potentially be a treatment for this disease.

Severe neonatal MEGDHEL syndrome with a homozygous truncating mutation in SERAC1

In the diagnostic work-up of a newborn infant with a metabolic crisis, lethal multiorgan failure on day six of life, and increased excretion of 3-methylglutaconic acid, we found using whole genome sequencing a homozygous SERAC1 mutation indicating MEGDHEL syndrome (3-methylglutaconic aciduria with deafness-dystonia, hepatopathy, encephalopathy, and Leigh-like syndrome). The SERAC1 protein is located at the contact site between mitochondria and the endoplasmic reticulum (ER) and is crucial for cholesterol trafficking. Our aim was to investigate the effect of the homozygous truncating mutation on mitochondrial structure and function. In the patient fibroblasts, no SERAC1 protein was detected, the mitochondrial network was severely fragmented, and the cristae morphology was altered. Filipin staining showed uneven localization of unesterified cholesterol. The calcium buffer function between cytoplasm and mitochondria was deficient. In liver mitochondria, complexes I, III, and IV were clearly decreased. In transfected COS-1 cells the mutant protein with the a 45-amino acid C-terminal truncation was distributed throughout the cell, whereas wild-type SERAC1 partially colocalized with the mitochondrial marker MT-CO1. The structural and functional mitochondrial abnormalities, caused by the loss of SERAC1, suggest that the crucial disease mechanism is disrupted interplay between the ER and mitochondria leading to decreased influx of calcium to mitochondria and secondary respiratory chain deficiency.

Generation of a homozygous LRPAP1 knockout human embryonic stem cell line (FDCHDPe009-B) by CRISPR/Cas9 system

The homozygous autosomal recessive truncating mutations of LDL receptor related protein associated protein 1 (LRPAP1) is a possible reason for Nonsyndromic Extreme Myopia, patients with which show typical chorioretinal degeneration. We generated an LRPAP1 knockout FDCHDPe009-B embryonic stem cell line to study mechanisms of retinal degeneration underlying LRPAP1 deficiency with the help of the CRISPR/Cas9 system. Two distinct biallelic deletions in the cell line have been confirmed, which causing a frameshift and premature stop codons thus influence the translation of LRPAP1. FDCHDPe009-B has maintained normal stem cell morphology, pluripotent gene expression, parental karyotype, and ability to differentiate into three germ layers.

Hypomorphic and hypermorphic mouse models of Fsip2 indicate its dosage-dependent roles in sperm tail and acrosome formation.

Loss-of-function mutations in multiple morphological abnormalities of the sperm flagella (MMAF)-associated genes lead to decreased sperm motility and impaired male fertility. As an MMAF gene, the function of fibrous sheath-interacting protein 2 (FSIP2) remains largely unknown. In this work, we identified a homozygous truncating mutation of FSIP2 in an infertile patient. Accordingly, we constructed a knock-in (KI) mouse model with this mutation. In parallel, we established an Fsip2 overexpression (OE) mouse model. Remarkably, KI mice presented with the typical MMAF phenotype, whereas OE mice showed no gross anomaly except for sperm tails with increased length. Single-cell RNA sequencing of the testes uncovered altered expression of genes related to sperm flagellum, acrosomal vesicle and spermatid development. We confirmed the expression of Fsip2 at the acrosome and the physical interaction of this gene with Acrv1, an acrosomal marker. Proteomic analysis of the testes revealed changes in proteins sited at the fibrous sheath, mitochondrial sheath and acrosomal vesicle. We also pinpointed the crucial motifs of Fsip2 that are evolutionarily conserved in species with internal fertilization. Thus, this work reveals the dosage-dependent roles of Fsip2 in sperm tail and acrosome formation.

ATP1A2- and ATP1A3-associated early profound epileptic encephalopathy and polymicrogyria.

Constitutional heterozygous mutations of ATP1A2 and ATP1A3, encoding for two distinct isoforms of the Na+/K+-ATPase (NKA) alpha-subunit, have been associated with familial hemiplegic migraine (ATP1A2), alternating hemiplegia of childhood (ATP1A2/A3), rapid-onset dystonia-parkinsonism, cerebellar ataxia-areflexia-progressive optic atrophy, and relapsing encephalopathy with cerebellar ataxia (all ATP1A3). A few reports have described single individuals with heterozygous mutations of ATP1A2/A3 associated with severe childhood epilepsies. Early lethal hydrops fetalis, arthrogryposis, microcephaly, and polymicrogyria have been associated with homozygous truncating mutations in ATP1A2. We investigated the genetic causes of developmental and epileptic encephalopathies variably associated with malformations of cortical development in a large cohort and identified 22 patients with de novo or inherited heterozygous ATP1A2/A3 mutations. We characterized clinical, neuroimaging and neuropathological findings, performed in silico and in vitro assays of the mutations' effects on the NKA-pump function, and studied genotype-phenotype correlations. Twenty-two patients harboured 19 distinct heterozygous mutations of ATP1A2 (six patients, five mutations) and ATP1A3 (16 patients, 14 mutations, including a mosaic individual). Polymicrogyria occurred in 10 (45%) patients, showing a mainly bilateral perisylvian pattern. Most patients manifested early, often neonatal, onset seizures with a multifocal or migrating pattern. A distinctive, 'profound' phenotype, featuring polymicrogyria or progressive brain atrophy and epilepsy, resulted in early lethality in seven patients (32%). In silico evaluation predicted all mutations to be detrimental. We tested 14 mutations in transfected COS-1 cells and demonstrated impaired NKA-pump activity, consistent with severe loss of function. Genotype-phenotype analysis suggested a link between the most severe phenotypes and lack of COS-1 cell survival, and also revealed a wide continuum of severity distributed across mutations that variably impair NKA-pump activity. We performed neuropathological analysis of the whole brain in two individuals with polymicrogyria respectively related to a heterozygous ATP1A3 mutation and a homozygous ATP1A2 mutation and found close similarities with findings suggesting a mainly neural pathogenesis, compounded by vascular and leptomeningeal abnormalities. Combining our report with other studies, we estimate that ∼5% of mutations in ATP1A2 and 12% in ATP1A3 can be associated with the severe and novel phenotypes that we describe here. Notably, a few of these mutations were associated with more than one phenotype. These findings assign novel, 'profound' and early lethal phenotypes of developmental and epileptic encephalopathies and polymicrogyria to the phenotypic spectrum associated with heterozygous ATP1A2/A3 mutations and indicate that severely impaired NKA pump function can disrupt brain morphogenesis.

Functional tests to guide management in an adult with loss of function of type-1 angiotensin II receptor

BackgroundGenetic loss of function of AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), or AGTR1 (type-1 angiotensin II receptor) leads to renal tubular dysgenesis (RTD). This syndrome is almost invariably lethal. Most surviving patients reach stage 5 chronic kidney disease at a young age.MethodsHere, we report a 28-year-old male with a homozygous truncating mutation in AGTR1 (p.Arg216*), who survived the perinatal period with a mildly impaired kidney function. In contrast to classic RTD, kidney biopsy showed proximal tubules that were mostly normal. During the subsequent three decades, we observed evidence of both tubular dysfunction (hyperkalemia, metabolic acidosis, salt-wasting and a urinary concentrating defect) and glomerular dysfunction (reduced glomerular filtration rate, currently ~30 mL/min/1.73 m2, accompanied by proteinuria). To investigate the recurrent and severe hyperkalemia, we performed a patient-tailored functional test and showed that high doses of fludrocortisone induced renal potassium excretion by 155%. Furthermore, fludrocortisone lowered renal sodium excretion by 39%, which would have a mitigating effect on salt-wasting. In addition, urinary pH decreased in response to fludrocortisone. Opposite effects on urinary potassium and pH occurred with administration of amiloride, further supporting the notion that a collecting duct is present and able to react to fludrocortisone.ConclusionsThis report provides living proof that even truncating loss-of-function mutations in AGTR1 are compatible with life and relatively good GFR and provides evidence for the prescription of fludrocortisone to treat hyperkalemia and salt-wasting in such patients.

Primary cutaneous SMARCB1‐deficient carcinoma

SMARCB1-deficient malignancies can arise in various sites. We describe a novel primary SMARCB1-deficient carcinoma of skin (SDCS) and characterize SMARCB1 mutations in non-melanoma skin cancers (NMSC). Cases underwent immunophenotyping and targeted exome sequencing (MSK-IMPACT) assay interrogating somatic mutations in 468 cancer-related genes. The MSK-IMPACT database from 2014 to 2020 encompassing 55, 000 cases was searched for NMSC with SMARCB1 mutations. SDCS arose on the scalp of an 18-year-old woman showing homozygous SMARCB1 deletion with a LATS2 G963E variant. Another case arose on the temple of a 76-year-old man harboring a SMARCB1 W206* mutation associated with loss of heterozygosity (LOH), 59 concurrent mutations, and a UV mutation signature (UV-MS). Both tumors exhibited INI1 loss, positive CK5/6, p40, p63, and claudin-4 with negative CD34. Of 378 NMSC cases, including 370 carcinomas, 7 SMARCB1-mutated tumors were identified: 3 squamous cell, 3 Merkel cell, and one basal cell carcinoma. Six showed UV-MS. Five INI1-interrogated cases retained protein expression suggesting they were SMARCB1-proficient. SDCS can be clinically aggressive, harbor SMARCB1 homozygous deletions or truncating SMARCB1 mutations associated with LOH, and can occur with or without UV-MS. Overall, SMARCB1 mutations in NMSC are rare with most being of undetermined significance and associated with retained INI1 and UV-MS.

U-Fiber Leukoencephalopathy Due to a Novel Mutation in the TACO1 Gene.

Translational activator of cytochrome c oxidase I (TACO1) is a mitochondrial translation factor involved in mitochondria-encoded cytochrome c oxidase subunit I (MT-CO1) synthesis.1,2 Loss-of-function mutations in the TACO1 gene cause respiratory chain complex IV deficiency. Clinically heterogeneous human diseases are due to cytochrome c oxidase (COX) deficiency, ranging from Leigh syndrome to myopathy, deafness, or ataxia. Recently, 2 different TACO1 mutations have been identified in 3 families with late-onset Leigh syndrome and a leukoencephalopathy involving predominantly basal ganglia and cystic changes.3,4 Here, we report a subject carrying a novel homozygous truncating mutation in the TACO1 gene and presenting an adult-onset slowly progressive spastic paraparesis with cognitive impairment and a subcortical U-fiber leukoencephalopathy. The authors thank the patient and her family for participating in the study and Valentina Baderna for the technical support in functional studies.

The sodium/proton exchanger SLC9C1 (sNHE) is essential for human sperm motility and fertility

Asthenozoospermia, defined by the absence or reduction of sperm motility, constitutes the most frequent cause of human male infertility. This pathological condition is caused by morphological and/or functional defects of the sperm flagellum, which preclude proper sperm progression. While in the last decade many causal genes were identified for asthenozoospermia associated with severe sperm flagellar defects, the causes of purely functional asthenozoospermia are still poorly defined. We describe here the case of an infertile man, displaying asthenozoospermia without major morphological flagellar anomalies and carrying a homozygous splicing mutation in SLC9C1 (sNHE), which we identified by whole-exome sequencing. SLC9C1 encodes a sperm-specific sodium/proton exchanger, which in mouse regulates pH homeostasis and interacts with the soluble adenylyl cyclase (sAC), a key regulator of the signalling pathways involved in sperm motility and capacitation. We demonstrate by means of RT-PCR, immunodetection and immunofluorescence assays on patient's semen samples that the homozygous splicing mutation (c.2748 + 2 T > C) leads to in-frame exon skipping resulting in a deletion in the cyclic nucleotide-binding domain of the protein. Our work shows that in human, similar to mouse, SLC9C1 is required for sperm motility. Overall, we establish a homozygous truncating mutation in SLC9C1 as a novel cause of human asthenozoospermia and infertility.

Recessive Mutations in SYNPO2 as a Candidate of Monogenic Nephrotic Syndrome

IntroductionMost of the approximately 60 genes that if mutated cause steroid-resistant nephrotic syndrome (SRNS) are highly expressed in the glomerular podocyte, rendering SRNS a “podocytopathy.”MethodsWe performed whole-exome sequencing (WES) in 1200 nephrotic syndrome (NS) patients.ResultsWe discovered homozygous truncating and homozygous missense mutation in SYNPO2 (synaptopodin-2) (p.Lys1124∗ and p.Ala1134Thr) in 2 patients with childhood-onset NS. We found SYNPO2 expression in both podocytes and mesangial cells; however, notably, immunofluorescence staining of adult human and rat kidney cryosections indicated that SYNPO2 is localized mainly in mesangial cells. Subcellular localization studies reveal that in these cells SYNPO2 partially co-localizes with α-actinin and filamin A−containing F-actin filaments. Upon transfection in mesangial cells or podocytes, EGFP-SYNPO2 co-localized with α-actinin-4, which gene is mutated in autosomal dominant SRNS in humans. SYNPO2 overexpression increases mesangial cell migration rate (MMR), whereas shRNA knockdown reduces MMR. Decreased MMR was rescued by transfection of wild-type mouse Synpo2 cDNA but only partially by cDNA representing mutations from the NS patients. The increased mesangial cell migration rate (MMR) by SYNPO2 overexpression was inhibited by ARP complex inhibitor CK666. SYNPO2 shRNA knockdown in podocytes decreased active Rac1, which was rescued by transfection of wild-type SYNPO2 cDNA but not by cDNA representing any of the 2 mutant variants.ConclusionWe show that SYNPO2 variants may lead to Rac1-ARP3 dysregulation, and may play a role in the pathogenesis of nephrotic syndrome.

A novel homozygous ALMS1 protein truncation mutation (c.2938dupA) revealed variable clinical expression among Saudi Alström syndrome patients

IntroductionAlström syndrome, ALMS (OMIM 203800) is a rare multi-systemic disease. The characteristic clinical features include blindness due to progressive cone-rod dystrophy, sensorineural hearing loss, type 2 diabetes mellitus, dilated cardiomyopathy, and childhood obesity. The aim of this study was to identify the genetic cause of Alström syndrome in patients who presented with variable clinical characteristics.Material and methodsClinical phenotyping and whole exome sequencing were performed in Saudi Alström syndrome patients. The Sanger sequencing was done to ascertain the segregation of Alström syndrome causative mutation in the family members. The rare prevalence of this mutation was further established by sequencing an additional 100 healthy Saudi controls.ResultsWhole exome sequencing analysis revealed that Alström syndrome patients have inherited a novel homozygous protein truncating mutation (c.2938dupA) in the ALMS1 gene segregated in an autosomal recessive fashion. This variant was absent in healthy controls. Genotype-phenotype analysis showed its interesting association with intra-familial clinical variability with regards to vision abnormalities, age at onset of dilated cardiomyopathy (DCM), obesity and hearing loss symptoms in the Alström syndrome patients.ConclusionsOur findings indicate that the atypical presentation of Alström syndrome, even within siblings, could sometimes lead to clinical misdiagnosis. Hence, the present study emphasizes the utility of exome sequencing to support the clinical diagnosis of Alström syndrome patients.

The First Report of Biallelic Missense Mutations in the SFRP4 Gene Causing Pyle Disease in Two Siblings.

BackgroundPyle disease is a rare autosomal recessive bone dysplasia characterized by the broadening of metaphyses with generalized cortical thinning. Homozygous truncating mutations in secreted frizzled-related protein 4 (SFRP4) were, to date, the only known variants causative for this type of skeletal disorder. SFRP4 controls cortical and trabecular bone remodeling by differential regulation of the canonical and non-canonical WNT signaling in both bone compartments. Loss-of-function mutations in the SFRP4 gene lead to the protein deficiency causing skeletal phenotype typical for Pyle disease.ResultsHerein, we report on the first SFRP4 missense mutations that occurred in compound heterozygosity in two siblings affected by Pyle disease, and which we have identified using a whole-genome sequencing approach followed by a comprehensive in silico pathogenicity assessment. The variants we have found were extremely rare and evaluated to be disease-causing by several online available tools and software.ConclusionWith this paper, we have shown that Pyle disease may be related not only to SFRP4 truncating mutations but also to other loss-of-function alterations that possibly impair the protein capacity to bind WNT ligands. As we have expanded here, the range of deleterious variants underlying Pyle disease, we contribute to the knowledge on the pathogenesis of this rare skeletal disorder.

Novel CAPN1 mutations extend the phenotypic heterogeneity in combined spastic paraplegia and ataxia

ObjectiveRecessive mutations in the CAPN1 gene have recently been identified in spastic paraplegia 76 (SPG76), a complex hereditary spastic paraplegia (HSP) that is combined with cerebellar ataxia, resulting in an ataxia‐spasticity disease spectrum. This study aims to assess the influence of CAPN1 variants on the occurrence of SPG76 and identify factors potentially contributing to phenotypic heterogeneity.MethodsWe screened a cohort of 240 unrelated HSP families for variants in CAPN1 using high‐throughput sequencing analysis. We described in detail the clinical and genetic features of the SPG76 patients in our cohort and summarized all reported cases.ResultsSix unreported CAPN1‐associated families containing eight patients with or without cerebellar ataxia were found in our cohort of HSP cases. These patients carried three previously reported homozygous truncating mutations (p.V64Gfs*103, c.759+1G>A, and p.R285*), and three additional novel compound heterozygous missense mutations (p.R481Q, p.P498L, and p.R618W). Lower limbs spasticity, hyperreflexia, and Babinski signs developed in about 94% of patients, with ataxia developing in 63% of cases. In total, 33 pathogenic mutations were distributed along the three reported functional domains of calpain‐1 protein, encoded by CAPN1, with no hotspot region. A comparison of gender distribution between the two groups indicated that female SPG76 patients were significantly more likely to present with complicated HSP than male patients (P = 0.015).InterpretationOur study supports the clinically heterogeneous inter‐ and intra‐family variability of SPG76 patients, and demonstrates that gender and calpain‐1 linker structure may contribute to clinical heterogeneity in SPG76 cases.

Genetic basis of SMARCB1 protein loss in 22 sinonasal carcinomas

SMARCB1-deficient sinonasal carcinoma (SNC) is an aggressive malignancy characterized by INI1 loss mostly owing to homozygous SMARCB1 deletion. With the exception of a few reported cases, these tumors have not been thoroughly studied by massive parallel sequencing (MPS). A retrospective cohort of 22 SMARCB1-deficient SNCs were studied by light microscopy, immunohistochemistry, fluorescence in situ hybridization (n = 9), targeted exome MPS (n = 12), and Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) (n = 10), a bioinformatics pipeline for copy number/zygosity assessment. SMARCB1-deficient SNC was found in 13 (59%) men and 9 (41%) women. Most common growth patterns were the basaloid pattern (59%), occurring mostly in men (77%), and plasmacytoid/eosinophilic/rhabdoid pattern (23%), arising mostly in women (80%). The former group was significantly younger (median age = 46 years, range = 24–54, vs 79 years, range = 66–95, p < 0.0001). Clear cell, pseudoglandular, glandular, spindle cell, and sarcomatoid features were variably present. SMARCB1-deficient SNC expressed cytokeratin (100%), p63 (72%), neuroendocrine markers (52%), CDX-2 (44%), S-100 (25%), CEA (4/4 cases), Hepatocyte (2/2 cases), and aberrant nuclear β-catenin (1/1 case). SMARCB1 showed homozygous deletion (68%), hemizygous deletion (16%), or truncating mutations associated with copy neutral loss of heterozygosity (11%). Coexisting genetic alterations were 22q loss including loss of NF2 and CHEK2 (50%), chromosome 7 gain (25%), and TP53 V157F, CDKN2A W110∗, and CTNNB1 S45F mutations. At 2 years and 5 years, the disease-specific survival and disease-free survival were 70% and 35% and 13% and 0%, respectively. SMARCB1-deficient SNCs are phenotypically and genetically diverse, and these distinctions warrant further investigation for their biological and clinical significance.

Abstract 5869: Characterization and therapeutic implications of SMARCA4 mutations in cancer

Abstract Genomic studies performed in cancer patients have identified a high frequency of alterations in the core catalytic subunit of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex, SMARCA4. Cells with inactivating SMARCA4 mutations rely on its paralog, SMARCA2, making it an attractive therapeutic target. Here we report the results of genomic profiling of 131,668 cases from patients with solid tumors and identified 8,588 cases with one or more SMARCA4 alterations. We observed a high prevalence of homozygous SMARCA4 truncating and non-truncating mutations in certain tumor types, notably non-small cell lung cancer (NSCLC) and cancers of unknown primary. We found that homozygous SMARCA4 mutations in NSCLC were mutually exclusive with other mutations in mSWI/SNF and oncogenic drivers, including KRAS and EGFR. A retrospective study of NSCLC patients who were treated in the Flatiron Health network (&amp;gt;265 oncology practices in the U.S.) and underwent FoundationOne® or FoundationOne CDx® tumor sequencing as part of routine care revealed that real-world patients with homozygous truncating SMARCA4 mutations had significantly reduced overall survival. The large sample size of our cohort also elucidated novel hotspot missense mutations within the helicase domain of SMARCA4. Functional modeling of these mutations using biochemical and cell-based (ATAC-seq) assays demonstrated that all evaluated mutations have significantly reduced nucleosome remodeling activity upon in vitro reconstitution in SMARCA4-deficient cells. We also addressed whether SMARCA4 missense mutations could rescue the cell growth defects and loss in chromatin accessibility induced upon SMARCA2 loss. Interestingly, we observe that a few missense mutants were able to either partially or fully rescue growth, suggesting hypomorphic activity under the selective pressure of SMARCA2 loss. Overall, these studies highlight NSCLC patients with homozygous truncating SMARCA4 mutations as a novel population with an unmet need who may benefit from SMARCA2-targeted therapy and underscore that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations. Citation Format: Tharu M. Fernando, Robert Piskol, Russell Bainer, Ethan Sokol, Sally Trabucco, Qing Zhang, Huong Trinh, Sophia Maund, Marc Kschonsak, Subhra Chaudhuri, Tom Januario, Zora Modrusan, Robert L. Yauch. Characterization and therapeutic implications of SMARCA4 mutations in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5869.