Next-generation sequencing and analysis of consecutive patients referred for connective tissue disorders.

Heritable connective tissue disorders (HCTDs) consist of a wide array of genetic disorders such as Ehlers-Danlos syndrome, Marfan syndrome, and osteogenesis imperfecta. The diagnosis relies on clinical presentation and family history to guide genetic testing with next-generation sequencing (NGS) for identification of gene variants in HCTDs. NGS was performed on a cohort of 100 consecutive, unrelated patients referred for a connective tissue disorder at Fulgent Genetics, an accredited commercial laboratory. One hundred seventeen gene variants were found in 76 patients with 10 recognized pathogenic or likely pathogenic variants seen in nine patients. The remaining variants were grouped as unknown clinical significance with 36 meeting three out of four pathogenicity criteria, or potentially pathogenic, as defined in our study in 33 patients. They were judged as potentially pathogenic for clinical care and management with disease surveillance based on the specific gene and phenotypic presentation. Gene variants in collagen-related proteins were the most frequent with ZNF469 and ADAMTSL2 variants most often identified. Joint hypermobility was the most frequent clinical finding. Variants were found in 76% of patients who had distinct clinical features of a HCTD. The data were stratified to provide insight into frequency and types of variants, their classification, and clinical manifestations.

De novo nonsense mutations in KAT6A, a lysine acetyl-transferase gene, cause a syndrome including microcephaly and global developmental delay.

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.

Geleophysic dysplasia: 48 year clinical update with emphasis on cardiac care.

Geleophysic dysplasia is a rare skeletal dysplasia often complicated by progressive cardiac disease. Information about long-term outcomes is limited. A clinical update of the oldest surviving patient described with geleophysic dysplasia type 1 is provided. Special note is made in relation to the cardiac disease and interventions. Genetic testing of ADAMTSL2 revealed a previously reported missense mutation as well as a novel nonsense mutation, which can be added to the list of causative mutations in geleophysic dysplasia.

De novo loss-of-function variants in STAG2 are associated with developmental delay, microcephaly, and congenital anomalies.

The cohesin complex is an evolutionarily conserved multi-subunit protein complex which regulates sister chromatid cohesion during mitosis and meiosis. Additionally, the cohesin complex regulates DNA replication, DNA repair, and transcription. The core of the complex consists of four subunits: SMC1A, SMC3, RAD21, and STAG1/2. Loss-of-function mutations in many of these proteins have been implicated in human developmental disorders collectively termed "cohesinopathies." Through clinical exome sequencing (CES) of an 8-year-old girl with a clinical history of global developmental delay, microcephaly, microtia with hearing loss, language delay, ADHD, and dysmorphic features, we describe a heterozygous de novo variant (c.205C>T; p.(Arg69*)) in the integral cohesin structural protein, STAG2. This variant is associated with decreased STAG2 protein expression. The analyses of metaphase spreads did not exhibit premature sister chromatid separation; however, delayed sister chromatid cohesion was observed. To further support the pathogenicity of STAG2 variants, we identified two additional female cases from the DECIPHER research database with mutations in STAG2 and phenotypes similar to our patient. Interestingly, the clinical features of these three cases are remarkably similar to those observed in other well-established cohesinopathies. Herein, we suggest that STAG2 is a dosage-sensitive gene and that heterozygous loss-of-function variants lead to a cohesinopathy.

Novel association of familial testicular germ cell tumor and autosomal dominant polycystic kidney disease with PKD1 mutation.

Adolescent brothers were diagnosed with testicular germ cell tumors within the same month. Both were found to have multiple renal cysts on pretreatment imaging done for staging. The proband, his brother, and their mother, were all found to have a novel splice variant in intron 8 of the PKD1 gene by clinical exome sequencing. This is the second family reported with both familial testicular germ cell tumor (FTGCT) and autosomal dominant polycystic kidney disease (ADPKD), and the first described association of FTGCT with a splice variant in PKD1. We suggest that this novel variant in PKD1 may convey increased risk for FTGCT in addition to causing ADPKD.

An infant with MLH3 variants, FOXG1-duplication and multiple, benign cranial and spinal tumors: A clinical exome sequencing study.

A 4-month-old male infant presented with severe developmental delay, cerebellar, brainstem, and cutaneous hemangiomas, bilateral tumors (vestibular, hypoglossal, cervical, and lumbar spinal), and few café-au-lait macules. Cerebellar and lumbar tumor biopsies revealed venous telangiectasia and intraneural perineuroma, respectively. Sequencing NF1, NF2, and RASA1 (blood), and NF2 and SMARCB1 (lumbar biopsy) was negative for pathogenic mutations. Clinical exome sequencing (CES), requested for tumor syndrome diagnosis, revealed two heterozygous missense variants, c.359T>C;p.Phe120Ser and c.3344G>A;p.Arg1115Gln, in MLH3 (NM_001040108.1), a DNA mismatch repair (MMR) gene, Polyphen-predicted as probably damaging, and benign, respectively. Sanger sequencing confirmed both variants in the proband, and their absence in the mother; biological father unavailable. Both biopsied tissues were negative for microsatellite instability, and expressed MLH1, MSH2, PMS2, MSH6, and MLH3 immunohistochemically. Chromosomal microarray showed a 133 kb segment copy number duplication of 14q12 region encompassing FOXG1, possibly explaining the developmental delay, but not the tumors. The presence of MLH3 variants with multiple benign neural and vascular tumors was intriguing for their possible role in the pathogenesis of these neoplasms, which were suspicious for, but not diagnostic of, constitutional MMR deficiency. However, functional assays of non-neoplastic patient-derived cells showed intact base-base MMR function. Also, no previous FOXG1-aberrant patient was reported with tumors. We now report a 3-year-old FOXG1-duplicated patient with a yet undescribed tumor syndrome with clinical features of neurofibromatosis types I and II, where several validation studies could not ascertain the significance of CES findings; further studies may elucidate precise mechanisms and diagnosis for clinical management, including tumor surveillance.

Analysis of the ABCA4 genomic locus in Stargardt disease.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses. The entire 140 kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation revealing, on average, 200 intronic variants per sample. Filtering of these data resulted in 141 candidates for new mutations. Two variants were detected in four samples, two in three samples, and 20 variants in two samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 12 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. No copy number variation (large deletions and insertions) was detected in any patient suggesting that it is a very rare event in the ABCA4 locus. Many variants were excluded since they were not conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants. The sequence variability in the ABCA4 locus is extensive and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Defining disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and extensive analyses by a combination of various approaches.

TOX3 is expressed in mammary ER(+) epithelial cells and regulates ER target genes in luminal breast cancer.

BACKGROUND: A breast cancer susceptibility locus has been mapped to the gene encoding TOX3. Little is known regarding the expression pattern or biological role of TOX3 in breast cancer or in the mammary gland. Here we analyzed TOX3 expression in murine and human mammary glands and in molecular subtypes of breast cancer, and assessed its ability to alter the biology of breast cancer cells. METHODS: We used a cell sorting strategy, followed by quantitative real-time PCR, to study TOX3 gene expression in the mouse mammary gland. To study the expression of this nuclear protein in human mammary glands and breast tumors, we generated a rabbit monoclonal antibody specific for human TOX3. In vitro studies were performed on MCF7, BT474 and MDA-MB-231 cell lines to study the effects of TOX3 modulation on gene expression in the context of breast cancer cells. RESULTS: We found TOX3 expression in estrogen receptor-positive mammary epithelial cells, including progenitor cells. A subset of breast tumors also highly expresses TOX3, with poor outcome associated with high expression of TOX3 in luminal B breast cancers. We also demonstrate the ability of TOX3 to alter gene expression in MCF7 luminal breast cancer cells, including cancer relevant genes TFF1 and CXCR4. Knockdown of TOX3 in a luminal B breast cancer cell line that highly expresses TOX3 is associated with slower growth. Surprisingly, TOX3 is also shown to regulate TFF1 in an estrogen-independent and tamoxifen-insensitive manner. CONCLUSIONS: These results demonstrate that high expression of this protein likely plays a crucial role in breast cancer progression. This is in sharp contrast to previous studies that indicated breast cancer susceptibility is associated with lower expression of TOX3. Together, these results suggest two different roles for TOX3, one in the initiation of breast cancer, potentially related to expression of TOX3 in mammary epithelial cell progenitors, and another role for this nuclear protein in the progression of cancer. In addition, these results can begin to shed light on the reported association of TOX3 expression and breast cancer metastasis to the bone, and point to TOX3 as a novel regulator of estrogen receptor-mediated gene expression.

Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory.

PURPOSE: Sanger sequencing is currently considered the gold standard methodology for clinical molecular diagnostic testing. However, next-generation sequencing has already emerged as a much more efficient means to identify genetic variants within gene panels, the exome, or the genome. We sought to assess the accuracy of next-generation sequencing variant identification in our clinical genomics laboratory with the goal of establishing a quality score threshold for confirmatory Sanger-based testing. METHODS: Confirmation data for reported results from 144 sequential clinical exome-sequencing cases (94 unique variants) and an additional set of 16 variants from comparable research samples were analyzed. RESULTS: Of the 110 total single-nucleotide variants analyzed, 103 variants had a quality score ≥Q500, 103 (100%) of which were confirmed by Sanger sequencing. Of the remaining seven variants with quality scores

Whole exome sequencing detects homozygosity for ABCA4 p.Arg602Trp missense mutation in a pediatric patient with rapidly progressive retinal dystrophy.

BACKGROUND: A pediatric patient presented with rapidly progressive vision loss, nyctalopia and retinal dystrophy. This is the first report of homozygosity for the p.Arg602Trp mutation in the ABCA4 gene. The child became legally blind within a period of 2 years. CASE PRESENTATION: An eight year-old Hispanic female presented with bilateral decreased vision following a febrile gastrointestinal illness with nausea and vomiting. Extensive workup involved pediatric infectious disease and rheumatology consultations.Initial visual acuity was 20/60 at distance and 20/30 at near in both eyes. Rapidly progressive vision loss occurred during a 2-year period resulting in visual acuities of 20/200 at distance in both eyes. Fundus exam disclosed attenuated vessels and multiple subretinal blister-like elevations. Optical coherence tomography showed far more lesions than were clinically evident with different levels of elevation. Autofluorescence imagery showed dramatic and widespread geographic areas of atrophy. The deposits that appeared drusen-like on clinical exam were hyperfluorescent, consistent with lipofuscin deposits containing A2e (N-retinylidene-N-retinylethanolamine) indicative of RPE cell dysfunction. Electroretinography was consistent with cone dystrophy, with relative preservation of rod function. Blood analysis and rheumatology evaluation found no evidence of a diffuse post-infectious/inflammatory process. The unique and rapid progression of her subretinal blister-like lesions was documented by fluorescein angiography, optical coherence tomography, autofluorescence imagery, and fundus photography. Family pedigree history disclosed consanguinity, her parents being first cousins. DNA analysis by whole exomic sequencing revealed homozygosity of p.Arg602Trp in the ABCA4 gene. CONCLUSION: The pediatric patient presented with a striking clinical appearance and dramatic rate of progression that was clinically more characteristic of an infectious or inflammatory process. This case expands the diverse range of phenotypes attributed to ABCA4 mutations and further supports the role of whole exome sequencing as a powerful new tool available to aid clinicians in establishing diagnosis for challenging cases.

De Novo variants in the KMT2A (MLL) gene causing atypical Wiedemann-Steiner syndrome in two unrelated individuals identified by clinical exome sequencing.

BACKGROUND: Wiedemann-Steiner Syndrome (WSS) is characterized by short stature, a variety of dysmorphic facial and skeletal features, characteristic hypertrichosis cubiti (excessive hair on the elbows), mild-to-moderate developmental delay and intellectual disability. [MIM#: 605130]. Here we report two unrelated children for whom clinical exome sequencing of parent-proband trios was performed at UCLA, resulting in a molecular diagnosis of WSS and atypical clinical presentation. CASE PRESENTATION: For patient 1, clinical features at 9 years of age included developmental delay, craniofacial abnormalities, and multiple minor anomalies. Patient 2 presented at 1 year of age with developmental delay, microphthalmia, partial 3-4 left hand syndactyly, and craniofacial abnormalities. A de novo missense c.4342T>C variant and a de novo splice site c.4086+G>A variant were identified in the KMT2A gene in patients 1 and 2, respectively. CONCLUSIONS: Based on the clinical and molecular findings, both patients appear to have novel presentations of WSS. As the hallmark hypertrichosis cubiti was not initially appreciated in either case, this syndrome was not suspected during the clinical evaluation. This report expands the phenotypic spectrum of the clinical phenotypes and KMT2A variants associated with WSS.

Clinical exome sequencing for genetic identification of rare Mendelian disorders.

IMPORTANCE: Clinical exome sequencing (CES) is rapidly becoming a common molecular diagnostic test for individuals with rare genetic disorders. OBJECTIVE: To report on initial clinical indications for CES referrals and molecular diagnostic rates for different indications and for different test types. DESIGN, SETTING, AND PARTICIPANTS: Clinical exome sequencing was performed on 814 consecutive patients with undiagnosed, suspected genetic conditions at the University of California, Los Angeles, Clinical Genomics Center between January 2012 and August 2014. Clinical exome sequencing was conducted as trio-CES (both parents and their affected child sequenced simultaneously) to effectively detect de novo and compound heterozygous variants or as proband-CES (only the affected individual sequenced) when parental samples were not available. MAIN OUTCOMES AND MEASURES: Clinical indications for CES requests, molecular diagnostic rates of CES overall and for phenotypic subgroups, and differences in molecular diagnostic rates between trio-CES and proband-CES. RESULTS: Of the 814 cases, the overall molecular diagnosis rate was 26% (213 of 814; 95% CI, 23%-29%). The molecular diagnosis rate for trio-CES was 31% (127 of 410 cases; 95% CI, 27%-36%) and 22% (74 of 338 cases; 95% CI, 18%-27%) for proband-CES. In cases of developmental delay in children (<5 years, n = 138), the molecular diagnosis rate was 41% (45 of 109; 95% CI, 32%-51%) for trio-CES cases and 9% (2 of 23, 95% CI, 1%-28%) for proband-CES cases. The significantly higher diagnostic yield (P value = .002; odds ratio, 7.4 [95% CI, 1.6-33.1]) of trio-CES was due to the identification of de novo and compound heterozygous variants. CONCLUSIONS AND RELEVANCE: In this sample of patients with undiagnosed, suspected genetic conditions, trio-CES was associated with higher molecular diagnostic yield than proband-CES or traditional molecular diagnostic methods. Additional studies designed to validate these findings and to explore the effect of this approach on clinical and economic outcomes are warranted.

Development of a comprehensive cardiovascular disease genetic risk assessment test.

Background: Despite monogenic and polygenic contributions to cardiovascular disease (CVD), genetic testing is not widely adopted, and current tests are limited by the breadth of surveyed conditions and interpretation burden. Methods: We developed a comprehensive clinical genome CVD test with semi-automated interpretation. Monogenic conditions and risk alleles were selected based on the strength of disease association and evidence for increased disease risk, respectively. Non-CVD secondary findings genes, pharmacogenomic (PGx) variants and CVD polygenic risk scores (PRS) were assessed for inclusion. Test performance was modeled using 2,594 genomes from the 1000 Genomes Project, and further investigated in 20 previously tested individuals. Results: The CVD genome test is composed of a panel of 215 CVD gene-disease pairs, 35 non-CVD secondary findings genes, 4 risk alleles or genotypes, 10 PGx genes and a PRS for coronary artery disease. Modeling of test performance using samples from the 1000 Genomes Project revealed ~6% of individuals with a monogenic finding in a CVD-associated gene, 6% with a risk allele finding, ~1% with a non-CVD secondary finding, and 93% with CVD-associated PGx variants. Assessment of blinded clinical samples showed complete concordance with prior testing. An average of 4 variants were reviewed per case, with interpretation and reporting time ranging from 9-96 min. Conclusions: A genome sequencing based CVD genetic risk assessment can provide comprehensive genetic disease and genetic risk information to patients with CVD. The semi-automated and limited interpretation burden suggest that this testing approach could be scaled to support population-level initiatives.

Evaluation of autosomal dominant retinal dystrophy genes in an unaffected cohort suggests rare or private missense variants may often be benign.

BACKGROUND: Many genes have been reported as harboring autosomal dominant mutations causing retinal dystrophy. As newly available gene panel sequencing and whole exome sequencing will open these genes up to greater scrutiny, we assess the rate of rare coding variation in these genes among unaffected individuals to provide context for variants that will be discovered when clinical subjects are sequenced. METHODS: Publicly available data from the Exome Variant Project were analyzed, focusing on 36 genes known to harbor mutations causing autosomal dominant macular dystrophy. RESULTS: Rates of rare (minor allele frequency ≤0.1%) and private missense variants within autosomal dominant retinal dystrophy genes were found to occur at a high frequency in unaffected individuals, while nonsense variants were not. CONCLUSIONS: We conclude that rare missense variations in most of these genes identified in individuals with retinal dystrophy cannot be confidently classified as disease-causing in the absence of additional information such as linkage or functional validation.

Molecular diagnosis of putative Stargardt Disease probands by exome sequencing.

BACKGROUND: The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. METHODS: We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. RESULTS: Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. CONCLUSIONS: Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.

Assessing Variant Causality and Severity Using Retinal Pigment Epithelial Cells Derived from Stargardt Disease Patients.

Purpose: Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods: Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results: Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions: Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance: This "Disease in a Dish" approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.

A Streamlined Approach to Prader-Willi and Angelman Syndrome Molecular Diagnostics.

Establishing or ruling out a molecular diagnosis of Prader-Willi or Angelman syndrome (PWS/AS) presents unique challenges due to the variety of different genetic alterations that can lead to these conditions. Point mutations, copy number changes, uniparental isodisomy (i-UPD) 15 of two subclasses (segmental or total isodisomy), uniparental heterodisomy (h-UPD), and defects in the chromosome 15 imprinting center can all cause PWS/AS. Here, we outline a combined approach using whole-exome sequencing (WES) and DNA methylation data with methylation-sensitive multiplex ligation-dependent probe amplification (MLPA) to establish both the disease diagnosis and the mechanism of disease with high sensitivity using current standard of care technology and improved efficiency compared to serial methods. The authors encourage the use of this approach in the clinical setting to confirm and establish the diagnosis and genetic defect which may account for the secondary genetic conditions that may be seen in those with isodisomy 15, impacting surveillance and counseling with more accurate recurrence risks. Other similarly affected individuals due to other gene disorders or cytogenetic anomalies such as Rett syndrome or microdeletions would also be identified with this streamlined approach.

Disruption of CTNND2, encoding delta-catenin, causes a penetrant attention deficit disorder and myopia.

Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable neurodevelopmental disorder with poorly understood pathophysiology and genetic mechanisms. A balanced chromosomal translocation interrupts CTNND2 in several members of a family with profound attentional deficit and myopia, and disruption of the gene was found in a separate unrelated individual with ADHD and myopia. CTNND2 encodes a brain-specific member of the adherens junction complex essential for postsynaptic and dendritic development, a site of potential pathophysiology in attentional disorders. Therefore, we propose that the severe and highly penetrant nature of the ADHD phenotype in affected individuals identifies CTNND2 as a potential gateway to ADHD pathophysiology similar to the DISC1 translocation in psychosis or AUTS2 in autism.

The Value of Parental Testing by Next-Generation Sequencing Includes the Detection of Germline Mosaicism.

When a potential disease-causing variant is detected in a proband, parental testing is used to determine the mode of inheritance. This study demonstrates that next-generation sequencing (NGS) is uniquely well suited for parental testing, in particular because of its ability to detect clinically relevant germline mosaicism. Parental variant testing by NGS was performed in a clinical laboratory for 1 year. The detection of mosaicism by NGS was compared with its detection by Sanger sequencing. Eight cases of previously unrevealed mosaicism were detected by NGS across eight different genes. Mosaic variants were differentiated from sequencing noise using custom bioinformatics analyses in combination with familial inheritance data and complementary Sanger sequencing. Sanger sequencing detected mosaic variants with allele fractions ≥8% by NGS, but could not detect mosaic variants below that level. Detection of germline mosaicism by NGS is invaluable to parents, providing a more accurate recurrence risk that can alter decisions on family planning and pregnancy management. Because NGS can also confirm parentage and increase scalability, it simultaneously streamlines and strengthens the variant curation process. These features make NGS the ideal method for parental testing, superior even to Sanger sequencing for most genomic loci.

Congenital myasthenic syndrome caused by a frameshift insertion mutation in GFPT1.

OBJECTIVE: Description of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families. METHODS: Muscle biopsies, EMG, and whole-exome sequencing were performed. RESULTS: All 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the GFPT1 gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pathology. CONCLUSIONS: These results expand on the spectrum of known loss-of-function GFPT1 mutations in CMS12 and in one family demonstrate a novel mode of inheritance due to UPD.