Targeted Exome Sequencing Research Articles

Genomic alterations in the F8 gene correlating with severe hemophilia A in Egyptian patients.

BackgroundHemophilia A (HA) is an inherited X‐linked recessive coagulation disorder caused by factor VIII (F8) deficiency. F8 rearrangements involving intron 22 (int22) and intron 1 (int1) account for almost half of severe HA phenotype also a hotspot exon 14 provides numerous mutational patterns. This study aims to identify F8 gene mutations among Egyptian HA patients.MethodsDNA samples from 60 HA patients were screened for int22 and int1 rearrangements using simplified inverse shifting PCR (IS‐PCR) followed by exon 14 sequencing. Also, four uncharacterized patients were studied by targeted exome sequencing.ResultsIn 33.3% of the studied patients, we identified three int22 rearrangements, three exon 14 mutations (two frameshift; one novel (NM_000132.3:c.2734_2735delAA, p.(N912Ffs*6)), a second reported mutation (NM_000132.3:c.3091_3094delAGAA, p.(K1031Lfs*9)), and one nonsense mutation (NM_000132.3:c.2440C>T, p.(R814*)). All identified mutations were detected in patients with severe HA phenotype. Targeted exome sequencing could not detect any known pathogenic variants.ConclusionIntron 22 rearrangement and exon 14 mutations correlate with most severe hemophilia A Egyptian patients.

Early truncation of the N‐terminal variable region of EYA4 gene causes dominant hearing loss without cardiac phenotype

BackgroundAutosomal dominant hearing loss (ADHL) accounts for about 20% of all hereditary non‐syndromic HL. Truncating mutations of the EYA4 gene can cause either non‐syndromic ADHL or syndromic ADHL with cardiac abnormalities. It has been proposed that truncations of the C‐terminal Eya domain lead to non‐syndromic HL, whereas early truncations of the N‐terminal variable region cause syndromic HL with cardiac phenotype.MethodsThe proband and all the other hearing impaired members of the family underwent a thorough clinical and audiological evaluation. The cardiac phenotype was examined by ECG and echocardiography. Their DNA was subjected to target exome sequencing of 129 known deafness genes. The sequencing data were analyzed and the candidate variants were interpreted following the ACMG guidelines for clinical sequence interpretation. The effect of candidate variant on EYA4 gene expression was assessed by quantitative PCR and western blot of gene production in blood.ResultsWe report a Chinese family cosegregating post‐lingual onset, progressive ADHL with a novel nonsense mutation NM_004100.4:c.543C>G (p.Tyr181Ter) of EYA4. Two affected members show no cardiac abnormalities at least until now revealed by electrocardiography and echocardiography. The overall expression level of the EYA4 gene in the proband was lower than that in his unaffected relative.ConclusionThis report expands the mutational spectrum of the EYA4 gene and highlights the fact that more data are needed to elucidate the complex genotype–phenotype correlation of EYA4 mutations.

Targeted exome sequencing identified a novel USH2A mutation in a Chinese usher syndrome family: a case report

BackgroundUsher syndrome is a disease with a heterogeneous phenotype and genotype. Our purpose was to identify the gene mutation in a Chinese family with Usher syndrome type 2 and describe the clinical features.Case presentationA 23-year-old man complained of a 10-year duration of nyctalopia and a 3-year decline in visual acuity of both eyes accompanied by congenital dysaudia. To clarify the diagnosis, the clinical symptoms were observed and analysed in combination with comprehensive ophthalmologic examinations as well as genetic analysis (targeted exome sequencing, TES). A typical clinical presentation of Usher syndrome of the fundus was found, including a waxy yellow-like disc, bone-spicule formations and retinal vessel stenosis. Optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) showed loss of the ellipsoid zone and a reduction in paracaval vessel density in both eyes. Genetic analysis identified a novel homozygous c.8483_8486del (p.Ser2828*) mutation in USH2A. The mutation resulted in premature termination of translation and caused the deletion of 19 fibronectin type 3 domains (FN3), transmembrane (TM) region and PDZ-binding motif domain, which play an important role in protein binding. After combining the clinical manifestations and genetic results, the patient was diagnosed with Usher syndrome type 2.ConclusionWe found a novel c.8483_8486del mutation in the USH2A gene through TES techniques. The results broaden the spectrum of mutations in Usher syndrome type 2 and suggest that a combination of clinical information and molecular diagnosis via TES could help Usher syndrome patients obtain a better diagnosis.

Identification of RUNX1T1 as a potential epigenetic modifier in small-cell lung cancer.

Small-cell lung cancer (SCLC) can be subgrouped into common 'pure' and rare 'combined' SCLC (c-SCLC). c-SCLC features a mixed tumor histology of both SCLC and non-small-cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c-SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c-SCLC, but in only 2 of 88 'pure' SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%-15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c-SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP-seq data in Kasumi-1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

Molecular portrait of squamous cell carcinoma of the bovine horn evaluated by high-throughput targeted exome sequencing: a preliminary report

BackgroundSquamous Cell Carcinoma of horn, also known as horn cancer, is a prevailing type of cancer in cattles especially Bos indicus. It is one of the most prevalent disease in Indian bullocks often resulting in death and huge economic losses to farmers. Here, we have reported the use of targeted exome sequencing to identify variants present in horn cancer affected horn mucosa tissue and blood of the same animal to identify some of the prevalent markers of horn cancer.ResultsWe have observed higher number of variants present in tissue as compared to blood as well as among cancer samples compared to samples from normal animals. Eighty six and 1437 cancer-specific variants were identified among the predicted variants in blood and tissue samples, respectively. Total 25 missense variants were observed distributed over 18 genes. KRT8 gene coding for Keratin8, one of the key constituents of horn, displayed 5 missense variants. Additionally, three other genes involved in apoptosis pathway and two genes involved in antigen presentation and processing also contained variants.ConclusionsSeveral genes involved in various apoptotic pathways were found to contain non-synonymous mutations. Keratin8 coding for Keratin, a chief constituent of horn was observed to have the highest number of mutations. In all, we present a preliminary report of mutations observed in horn cancer.

Variants in RCBTB1 are Associated with Autosomal Recessive Retinitis Pigmentosa but Not Autosomal Dominant FEVR

ABSTRACT Purpose Variants in RCBTB1 have been reported in autosomal recessive inherited retinal dystrophies and autosomal dominant familial exudative vitreoretinopathy (FEVR). This study aims to verify the correlation between RCBTB1 variations and phenotypes. Methods Variants in RCBTB1 were selected from 6303 unrelated families with different forms of eye conditions based on whole exome sequencing and targeted exome sequencing. Potential pathogenic truncation variants were filtered by multistep bioinformatics analysis and identified by Sanger sequencing. Segregation and enrichment analysis were used to evaluate the association of truncation variants in RCBTB1 with phenotypes. Results Biallelic damaging variants in RCBTB1 were found in one family while heterozygous truncation variants were detected in 28 unrelated families based on our in-house data from 6303 unrelated families. Totally, 11 variants were present in the 29 families, including seven frameshift variants, three splicing variants, and one nonsense variant. The biallelic variants, c.170delG and c.905_906insTT, were identified in an isolated case with retinitis pigmentosa (RP). Heterozygous truncation variants were distributed in different forms of eye conditions (including normal) without significant enrichment in FEVR, suggesting unrelatedness to specific eye diseases. The frequency and location of truncation variants in the 6303 samples are comparable with the East Asian data from gnomAD. Segregation analysis of available family members further demonstrated the nonpathogenic nature of heterozygous truncation variants. Conclusions Contrary to the previous report, heterozygous truncation variants in RCBTB1 are not associated with FEVR based on our data. The extreme rareness of biallelic truncation variants present in a singleton case with RP further suggests that variants in RCBTB1 are responsible for autosomal recessive RP. This result reminds us that variants of a gene with certain diseases should be thoroughly verified before the use of such information in clinical practice.

Novel neuroclinical findings of autosomal recessive primary microcephaly 15 in a consanguineous Iranian family

Major facilitator superfamily domain-containing 2A (MFSD2A) is required for brain uptake of Docosahexaenoic acid and Lysophosphatidylcholine, both are essential for the normal neural development and function. Mutations in MFSD2A dysregulate the activity of this transporter in brain endothelial cells and can lead to microcephaly. In this study, we describe an 11-year-old male who is affected by autosomal recessive primary microcephaly 15. This patient also shows severe intellectual disability, recurrent respiratory and renal infections, low birth weight, and developmental delay. After doing clinical and neuroimaging evaluations, due to heterogeneity of neurogenetic disorders, no narrow clinical diagnosis was possible, therefore, we utilized targeted-exome sequencing to identify any causative genetic factors. This revealed a homozygous in-frame deletion (NM_001136493.1: c.241_243del; p.(Val81del)) in the MFSD2A gene as the most likely disease-susceptibility variant which was confirmed by Sanger sequencing. Neuroimaging revealed lateral ventricular asymmetry, corpus callosum hypoplasia, type B of cisterna magna, and widening of Sylvian fissures. All of these novel phenotypes are associated with autosomal recessive primary microcephaly-15 (MCPH15). According to the genotype-phenotype data, p.(Val81del) can be considered a likely pathogenic variant leading to non-lethal microcephaly. However, further cumulative data and molecular approaches are required to accurately identify genotype-phenotype correlations in MFSD2A.

TMOD-34. PATIENT-DERIVED XENOGRAFT AND CELL LINE MODELS FACILITATE NOVEL TREATMENT DISCOVERY IN CENTRAL NERVOUS SYSTEM LYMPHOMAS

Abstract Lymphomas of the central nervous system (CNSL) are rare tumors with few treatment options; however, recent genomic studies have uncovered several druggable targets. To facilitate novel treatment discovery, we sought to establish patient-derived xenograft (PDX) models and cell lines from patients with newly diagnosed or recurrent CNSL. PDX models were attempted from fresh tumor samples from 18 consented patients for whom rich clinical annotation was available. Orthotopic intracranial injections were performed via stereotactic injection. Cell line generation was attempted from both primary tumors (PDCL) and PDX samples (PDXCL). Characterization included histopathology, targeted exome sequencing, low-pass whole-genome sequencing for copy number evaluation, and expression profiling of matched patient tumors and PDXs. Drug response testing was performed using CellTiterGlo. From 18 samples attempted, 8 PDX models were successfully generated: 2 primary CNS diffuse large B-cell lymphoma (DLBCL), 5 secondary CNS DLBCL, and 1 secondary cutaneous T-cell lymphoma (CTCL). Mean time to moribund was 66 days. All 8 PDXs grew successfully as orthotopic models, and 4 also grew in subrenal capsule. Additionally, 6 PDCL and 2 PDXCL were established (> P3). Models faithfully represented primaries, showing nearly identical histopathology, immunoprofiles, and genomic signatures. To support expanded and rapid preclinical use of these models in drug testing we documented that PDX could be explanted to create short-term cell preparations or long-term cell lines with a 100% and 40% success rate, respectively. Proof of principle testing showed that explanted DLBCL PDX cells and cell lines showed sensitivity to copanlisib and venetoclax, which were not efficacious in the CTCL PDX. These findings showcase a diverse collection of CNSL models demonstrating high fidelity to primary tumors at the genomic and phenotypic levels, emphasizing their utility for preclinical studies and as patient avatars to rapidly determine sensitivities to existing and novel regimens prior to initiation of therapy.

Loss of histone H3 trimethylation on lysine 27 and nuclear expression of transducin-like enhancer 1 in primary intracranial sarcoma, DICER1-mutant.

Primary intracranial sarcoma, DICER1-mutant is a recently described central nervous system tumour with specific genomic and DNA-methylation profiles. Although some of its histological features (focal spindle-cell morphology, intracytoplasmic eosinophilic granules, and focal heterologous differentiation) are common across most reported cases, the presence of significant histological variability and the lack of differentiation pose diagnostic challenges. We aim to further define the immunoprofile of this tumor. We reviewed the clinical history and performed immunohistochemistry for glial fibrillary acidic protein, oligodendrocyte transcription factor 2, SOX2, SOX10, S100, histone H3 trimethylated on lysine 27 (H3K27me3), desmin, myogenin, CD99, epithelial membrane antigen (EMA) and transducin-like enhancer of split 1 (TLE1) on six primary intracranial sarcomas, DICER1-mutant, with appropriate controls. Targeted exome sequencing was performed on all cases. The sarcomas showed diffuse (n=4), mosaic (n=1) or minimal (≤5%, n=1) loss of H3K27 trimethylation and nuclear TLE1 expression (n=6). Four had immunohistochemical evidence of myogenic differentiation. SOX2, SOX10, S100 and EMA were negative; CD99 expression ranged from focal cytoplasmic (n=4) to crisp diffuse membranous (n=2). One tumour had focal cartilaginous differentiation. Similar immunohistochemical findings were observed in a pleuropulmonary blastoma (albeit with focal TLE1 expression), a DICER1-related pineoblastoma, and an embryonal tumour with a multilayered rosette-like DICER1-related cerebellar tumour. Targeted exome sequencing confirmed the presence of pathogenic biallelic DICER1 mutations in all tumours included in this study. We conclude that H3K27me3 and TLE1 immunostains, when utilised in combination, can be helpful diagnostic markers for primary intracranial sarcoma, DICER1-mutant.

SURF1 related Leigh syndrome: Clinical and molecular findings of 16 patients from Turkey

IntroductionPathogenic variants in SURF1, a nuclear-encoded gene encoding a mitochondrial chaperone involved in COX assembly, are one of the most common causes of Leigh syndrome (LS). Material-methodsSixteen patients diagnosed to have SURF1-related LS between 2012 and 2020 were included in the study. Their clinical, biochemical and molecular findings were recorded. 10/16 patients were diagnosed using whole-exome sequencing (WES), 4/16 by Sanger sequencing of SURF1, 1/16 via targeted exome sequencing and 1/16 patient with whole-genome sequencing (WGS). The pathogenicity of SURF1 variants was evaluated by phylogenetic studies and modelling on the 3D structure of the SURF1 protein. ResultsWe identified 16 patients from 14 unrelated families who were either homozygous or compound heterozygous for SURF1 pathogenic variants. Nine different SURF1 variants were detected The c.769G > A was the most common variant with an allelic frequency of 42.8% (12/28), c.870dupT [(p.Lys291*); (8/28 28.5%)], c.169delG [(p.Glu57Lysfs*15), (2/24; 7.1%)], c.532 T > A [(p.Tyr178Asn); (2/28, 7.1%)], c.653_654delCT [(p.Pro218Argfs*29); (4/28, 14.2%)] c.595_597delGGA [(p.Gly199del); (1/28, 3.5%)], c.751 + 1G > A (2/28, 4.1%), c.356C > T [(p.Pro119Leu); (2/28, 3.5%)] were the other detected variants. Two pathogenic variants, C.595_597delGGA and c.356C > T, were detected for the first time. The c.769 G > A variant detected in 6 patients from 5 families was evaluated in terms of phenotype-genotype correlation. There was no definite genotype – phenotype correlation. ConclusionsTo date, more than 120 patients of LS with SURF1 pathogenic variants have been reported. We shared the clinical, molecular data and natural course of 16 new SURF1 defect patients from our country. This study is the first comprehensive research from Turkey that provides information about disease-causing variants in the SURF1 gene. The identification of common variants and phenotype of the SURF1 gene is important for understanding SURF1 related LS. SynopsisSURF1 gene defects are one of the most important causes of LS; patients have a homogeneous clinical and biochemical phenotype.

Delineation of the genetic and clinical spectrum, including candidate genes, of monogenic diabetes: a multicenter study in South Korea.

Monogenic diabetes includes a group of heterogeneous diabetes types. We aimed to identify the frequency, clinical and molecular features of monogenic diabetes in a Korean pediatric cohort. A retrospective cohort and multicenter study of Korean children suspected to have monogenic diabetes, managed by four pediatric endocrine centers in the southeast region of South Korea, from February 2016 to February 2020. We recruited 27 pediatric Korean patients suspected to have monogenic diabetes who had at least two of the following three criteria (age at diagnosis, family history, and clinical presentation). Targeted exome sequencing was conducted in these patients. The functional consequences of the variants were predicted by bioinformatics and protein structure analysis. Molecular genetic analysis identified 16 patients (59.3%) with monogenic diabetes. We identified a total ofeight unique variants, including five novel variants (HNF4A c.1088C>T, CEL c.1627C>T and c.1421C>T, PAX4 c.538+8G>C, INS c.71C>T). We also identified two potential candidate gene variants for monogenic diabetes, namely c.650T>C in the SLC2A2 gene and c.629G>A in the PTF1A gene. Other variants were identified in the WFS1and NPHP3 genes in two rare genetic disorders. Variant-positive individuals had a lower presence of autoantibody positivity at the time of diagnosis and higher glycosylated hemoglobin levels at last follow-up when compared to variant-negative patients (p<0.001 and p=0.029, respectively). These results further expand the spectrum of known variants as well as potential candidate gene variants associated with monogenic diabetes in Korea.

Molecular epidemiology of IDH2 hotspot mutations in cancer and immunohistochemical detection of R172K, R172G, and R172M variants

IDH1/2 hotspot mutations occur in glioma, cholangiocarcinoma, chondrosarcoma, sinonasal carcinoma, and T-cell lymphoma and have diagnostic, prognostic, and/or therapeutic value. Availability of immunohistochemistry (IHC) protocols for specific IDH2 mutation detection is limited. A targeted exome sequencing assay MSK-IMPACT cohort comprising >38,000 cancer cases was explored for the presence of IDH1/2 mutations in solid malignancies and select T-cell lymphomas. Seventy-four formalin-fixed paraffin-embedded IDH1/2-mutated (n=62) and wild-type (n=12) samples were used for testing and optimization of anti-IDH2 monoclonal antibodies (mAbs) 14H7, 3C11, and MMab1 targeting R172K, R172G, and R172M mutant proteins, respectively. IDH1/2 mutations were common in glioma (26.8% and 1.6%), intrahepatic cholangiocarcinoma (23.1% and 5.7%), chondrosarcoma (19.4% and 10.7%), sinonasal undifferentiated/large-cell neuroendocrine carcinoma (0% and 84.2%), angioimmunoblastic T-cell lymphoma (0% and 22%), and peripheral T-cell lymphoma (0 and 5.1%). In other cancers, IDH2 mutations were rare. IDH2 R172 variants included R172K (39%), R172S (29%), R172W (12%), R172G (10%), R172M (5%), and R172T (4%). 14H7, 3C11, and MMab1 detected all IDH2 R172K, R172G, and R172M, respectively, and produced a crisp, granular cytoplasmic staining pattern. 3C11 was also positive in 5 of 6 IDH1 R132G mutants showing a homogeneous, smooth cytoplasmic staining. All 3 mAbs were negative in other IDH1/2 mutant or wild-type cases. IHC using mAbs 14H7, 3C11, and MMab1 can facilitate molecular diagnosis as a reliable, fast, and inexpensive alternative for specific IDH2 variant detection. Given the distinct distribution of IDH2 R172 mutations in cancers, these mAbs could also serve as useful pathologic diagnostic markers.

Glaucoma phenotype in a large Chinese family with myocilin Val251Ala mutation

Family study is an effective way to identify disease-causing mutations (DCMs) and characterize the clinical phenotype of genetic diseases. In this study we recruited a Chinese primary open-angle glaucoma (POAG) family spanning six generations and consisting 112 individuals, in which 63 were participated in. Targeted exome sequencing on the proband identified a heterozygous mutation (c.752T>C, p.Val251Ala) in MYOC gene. Sanger sequencing performed on all participants found that fourteen family members carried this mutation. Ten (71.4%) of them were diagnosed with POAG, two (14.3%) with ocular hypertension (OHT) and two (14.3%) without manifestations of glaucoma. According to the results of ophthalmic examinations of the family members and their medical history, we found that the Val251Ala mutation was associated with clinical phenotype including intermediate penetrance, high intraocular pressure (IOP), severe visual defects and requirement of surgery.

FAT1 biallelic truncating mutation causes a non-syndromic proteinuria in a child

The identification of monogenic causes in patients with proteinuria has revealed that the encoded proteins functionally participate in distinct cellular tasks and signaling pathways in the slit diaphragms of the glomerular basement membrane. FAT1 is a member of a small family of vertebrate-cadherin-like genes, which is a crucial component in slit diaphragms and has a vital role in tubular regeneration. Only 5 cases with glomerulonephritis having FAT1 gene biallelic variants have been reported. However, only one had the biallelic truncating variant, and others had missense variants. Therefore, we need further evidence of this gene being responsible for steroid-resistant nephrotic syndrome (SRNS) or glomerulonephritis. Here we describe a 5-year-old boy in who proteinuria was detected at the age of 3years without any extrarenal symptom. The pathological findings were examined, and targeted exome sequencing was performed. We also conducted reviews for all previously-reported cases of glomerulonephritis possessing FAT1 biallelic gene variants. We found two novel truncating variants in FAT1 (NM_005245.3), c.12867dup in exon 10, and, c.5480_5483del in exon 25. Our case showed mild proteinuria compared to previously-reported cases who showed SRNS and extrarenal symptoms that might have been because the latter variant in our patient was located on out of cadherin domains; however, our follow up period is short and we further need careful follow up. Our findings corroborate the evidence that individuals with FAT1-truncating variants can show isolated mild proteinuria. Further studies are needed to investigate the genotype-phenotype correlation in this disease. Therefore, our case will provide vital information regarding this rare condition.

Study on syndromic deafness caused by novel pattern of compound heterozygous variants in the CDH23 gene

Objective: To explore the pathogenic variants of a family with syndromic deafness by high-throughput sequencing. Methods: The family was from Puyang City, Henan Province, and had four members, including two with syndromic deafness. The proband and his sister had congenital deafness, and their parents had normal phenotypes. The clinical phenotype of the family was characterized using clinical examinations and pedigree analysis. The clinical examinations included imaging examination, audiometry (pure tone audiometry, acoustic immittance, brainstem auditory evoked potential, and otoacoustic emission), vestibular function test, and ophthalmic examination (visual acuity test, visual field test, fundus examination, visual evoked potential, and electroretinogram). Target exome sequencing of 129 known deafness genes and bioinformatics analysis were used to screen suspected pathogenic variants. Sanger sequencing and minigene assay were used to verify and functionally investigate the mutation detected, respectively. According to the standards and guidelines for interpreting genetic variants proposed by the American College of Medical Genetics and Genomics, the variants c.6049G>A and c.8699A>G were classified as pathogenic/likely pathogenic, and the variant c.9856C>G was classified as variants of uncertain significance. Results: The probands and his sister had severe sensorineural hearing loss with decreased binocular vision, night blindness, decreased peripheral visual field sensitivity and partial visual field defect, and normal vestibular function. Both of them had three CDH23 mutations, including CDH23 (NM_022124.5) c.6049G>A (p.Gly2017Ser),c.9856C>G (p.His3286Asp), and c.8699A>G (p. Asp2900Gly), The first two were inherited from the father, and the last one was from the mother. The missense variants c.9856C>G and c.8699A>G were not included in the gnomad database. The missense mutation c.6049G>A was located in the last position of exon 46 and was predicted to affect splicing by bioinformatics software. The minigene experiment showed that the mutation cause exon skipping of exon 46, resulting in an abnormal protein. Conclusions: Compound heterozygous variations of the CDH23 are the leading cause of USH1D in the family. This study confirms that the compound heterozygosity of splicing and missense variants of the CDH23 gene could lead to USH1D.

Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness

PurposeSeveral hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology. MethodsExome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions. ResultsWe identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1, were also found. The remaining well-characterized unsolved patients (48%) need further investigation. ConclusionUsing our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes.

A family of Melnick-Needles syndrome: a case report

BackgroundMelnick-Needles syndrome (MNS) is an extremely rare osteochondrodysplasia caused by a mutation of FLNA, the gene encoding filamin A. MNS is inherited in an X-linked dominant manner. In this study, we describe three members of the same family with MNS, who exhibited different phenotypic severity despite having an identical FLNA gene mutation.Case presentationThe patient was 16 months old, with a history of delayed physical development, multiple upper respiratory infections and otitis media episodes. She was referred to our orthopedic clinic because of bowed legs and an abnormal plain chest radiograph. Both upper and lower extremities were bowed. Plain X-rays showed thoracolumbar kyphoscoliosis, with anterior and posterior vertebral scalloping, and thin, wavy ribs. Hypoplasia of the pubis and ischium, with bilateral coxa valga, were also noted. Target exome sequencing revealed a heterozygous mutation of FLNA, c.3578 T > C, p.Lys1193Pro, which confirmed the diagnosis of MNS. Her older sister and mother had minimal deformities of the axial and extremity skeleton, but genetic analyses revealed the same FLNA mutation as the patient. The mutation identified in this family has not been previously reported.ConclusionThis report illustrates the potential inherited nature of MNS and the phenotypic variability of clinicoradiologic characteristics. In patients with traits suggestive of MNS, a careful medical and family history should be obtained, and genetic testing should be performed for the patient, as well as all family members.

Abstract 1183: Race, copy number variation, and local hog concentration in breast cancer mortality

Abstract Purpose of study: Female breast cancer is the second leading cause of cancer death in U.S. women, and the incidence of new breast cancer cases continues to increase. Despite slightly lower breast cancer incidence rates among African American (AA) compared to European American (EA) women in the U.S., AA women experience significantly higher breast cancer mortality. To better understand the extent to which environmental and genetic factors might help explain higher mortality rates among AA women, we examined the association between a measure of exposure to chemical pollutants and genomic profiling with breast cancer mortality among women with breast cancer in North Carolina. Procedures: Using a molecular epidemiologic approach, we examined the association of county hog concentration (a marker of environmental exposure to chemical pollutants) and genomic variations identified via targeted exome sequencing with age-adjusted breast cancer mortality rates among 146 women in North Carolina, stratified by race (AA vs. EA). The odds of breast cancer-specific mortality were estimated as a function of county hog concentration quintiles. Results: Overall, breast cancer mortality among AA women was higher than among EA women (OR=1.4, p&amp;lt;0.0001). Although there was no effect of hog concentration on breast cancer mortality of EA women, there was an increase in deaths from breast cancer in African American women in the highest quintile of hog concentration (OR=1.18, p=0.03), compared to the lower quintiles. In the lowest quintile of hog concentration, breast cancer mortality remained higher among AA versus EA women (OR=1.38, p=0.0002). This suggests that along with environmental factors, biological factors also contribute to disparities. Next, we performed targeted exome sequencing of breast cancer samples collected from a cohort in eastern North Carolina. AA breast cancer samples showed a high frequency of CNVs (copy number variations) of COL11A2, COL12A1, CYP21A2, LYRM2, COL9A1, DNAH11, and DST. EA breast cancer samples showed a high frequency of CNVs of PRG4, PLB1, CHRM5, OCA2, HMCN1, and MUC5B. Among these, HMCN1, MUC5B (among EA), and COL12A1, DNAH11, DST (among AA) were genes with significantly high CNVs identified from The Cancer Genome Atlas. Conclusions: Our findings suggest that living in a county with a high concentration of hogs may increase breast cancer mortality risk among AA women, and the high frequency of CNVs varies by race; both CNV and county hog concentration may contribute to racial disparities in breast cancer mortality among AA and EA women in North Carolina. Citation Format: Jung S. Byun, Sean Lee, Sijung Yun, Eliseo J. Perez-Stable, Anna M. Napoles, Paul Strickland, Kevin L. Gardner. Race, copy number variation, and local hog concentration in breast cancer mortality [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 1183.

Abstract 5789: A novel role of APC-partnering mutations in the sidedness of colorectal cancer survival

Abstract Left-sided colorectal cancer (CRC) tumors have been reported to be more favorably associated with better survival than right-sided tumors. A molecular basis of the laterality of prognosis, however, is still poorly understood. We have recently developed a multi-gene mutation classification system from targeted exome sequencing of 1,321 cancer-related genes on 468 CRC tumor specimens (Schell et al, Nat Commun, 2016). We found that among 17 significantly mutated genes, four (BRAF, APC, KRAS, TP53) demonstrated pairwise, statistically significant, correlations, with APC-partnering mutations playing a central role in predicting overall survival. APC might assume 0, 1, or 2 identified truncating mutations, each with a striking differential impact on survival. Tumors lacking any APC mutation carried a worse prognosis than single mutation tumors, but the triply-mutated tumors (APC, KRAS, TP53) with two APC mutations conferred substantially worse survival. Thus, we hypothesized that APC-partnering mutations might also have a significant role in the sidedness effect on CRC outcome. We carried out various Left (L) vs Right (R) survival analyses on 464 tumors that had the primary tumor location data. When individual driver mutations (BRAF, APC, KRAS, TP53) were analyzed separately, although a statistically significant association with overall survival was seen in left-sided tumors for APC mutations and BRAF (V600E) for all patients and for MSS patients, no significant survival difference was observed when each of four driver mutations was (left vs right) compared. However, when APC-partnering mutations (i.e. APC WT; APC only; APC/KRAS; APC/TP53; APC/KRAS/TP53) were analyzed, left-sided APC/KRAS tumors were found to be associated with dramatically better survival than their right-sided counterparts (HR=0.37 (0.17-0.82), logrank p=0.0078 for all patients; HR=0.059 (0.021-0.162), logrank p=0.0001 for MSS patients). Analysis of APC hit mutation (0-hit, 1-hit, 2-hit) groups reveals that left-sided APC 2-hit tumors had significantly better survival than right-sided counterparts in all patients and MSS patients. However, surprisingly, we also identified that several mutation subgroups conferred better survival in right-sided tumors than left-sided tumors. For example, APC-only mutation tumors were associated with better survival than their left-sided counterparts (logrank p=0.030) in MSS patients (n=403); Right-sided all wild-type (BRAF, APC, KRAS, TP53) tumors conferred much better survival (HR=0.12 (0.038-0.368), logrank p=0.0135) in APC WT tumors (n=35). In conclusion, our findings identified a novel role of APC-partnering mutation subgroups associated with the sidedness effect, suggesting that routine clinical APC and TP53 mutation assessment, in addition to BRAF and KRAS, may refine and improve predicting outcomes in left vs right-sided CRC patients. Citation Format: Mingli Yang, Michael Schell, Andrey Loboda, Michael Nebozhyn, W. Jack Pledger, Timothy Yeatman. A novel role of APC-partnering mutations in the sidedness of colorectal cancer survival [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 5789.

Abstract A22: STAG2, a novel genetic regulator of bladder cancer

Abstract STAG2 is known as a cohesin complex component, and frequent loss-of-function mutations in bladder cancer (BC) suggest its importance in tumorigenesis. While STAG2 functions in chromatid segregation and colocalizes with CTCF to organize chromatin, we provide evidence that STAG2 may have roles independent of this canonical function. Importantly, unique STAG2 binding sites may correspond to tissue-specific enhancers. We hypothesize that STAG2 acts as a transcription factor that influences BC. Whole- and targeted-exome sequencing of muscle-invasive (MIBC) patient tissues (n=119) revealed a STAG2 mutation in over 10%. Tumor immunohistochemistry confirmed loss of STAG2 protein expression. We observed that patients (n=330) with no STAG2 expression had prolonged median overall (34 vs. 24.5 months, p=.049) and progression-free survival (23 vs. 13.5 months, p=.016) compared to STAG2-positive tumors. To evaluate the functional impact of STAG2 status, we stably knocked down STAG2 in T24 cells. We observed decreased invasion in shSTAG2 cells via transwell migration assays and a significant increase in cisplatin sensitivity. To determine STAG2 interactions with chromatin, we conducted ChIP-seq (WT STAG2) and RNA-seq (upon STAG2 knockdown) in T24 cells. We identified STAG2-dependent dysregulation of epithelial-to-mesenchymal transition genes including MMP2, MMP9, SLUG, and SNAIL, consistent with STAG2's role in invasion. Cistrome pipeline integration of ChIP-seq and RNA-seq data suggests a repressive role for STAG2 (p=.000503). Overlap of STAG2 targets from Cistrome with GeneHancer revealed 2,811 enhancers that STAG2 may regulate. Finally, STAG2 rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) identified 154 potential transcription cofactors that immunoprecipitated with STAG2 in the context of chromatin. As expected, we identified known components of the cohesin complex, such as RAD21, SMC1A, and SMC3. However, many of the proteins identified through RIME have not been previously found to interact with STAG2 as cohesins. Interestingly, some of these STAG2-interacting proteins have been implicated in the regulation of gene expression, such as EIF4A1 and NONO, further supporting our hypothesis that STAG2 is part of a transcriptional repression complex that is important for BC. Our ongoing analyses of STAG2 will reveal if STAG2 enhancer binding is independent of its role in chromatin organization. We suggest that STAG2 may bind to specific enhancers and repress transcription in BC. Loss of STAG2 may push disease towards a less invasive phenotype and may also confer sensitivity to cisplatin treatment. These results are consistent with the idea that loss of STAG2 may be beneficial in a subset of MIBC patients. Citation Format: Nithya Krishnan, Lanni Aquila, Sofia Lage Vickers, Swathi Ramakrishnan, Kristopher Atwood, Qiang Hu, Eduardo Gomez Cortes, Jianmin Wang, Anna Woloszynska. STAG2, a novel genetic regulator of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A22.