Neurofibromatosis type 1 (NF1) is a neurocutaneous genetic disorder with a broad spectrum of associated signs and symptoms, including skeletal anomalies. The association of NF1 with anterior chest wall deformities has been recently reported, especially the pectus excavatum (PE). Over the years, several authors have suggested loss of heterozygosity (LOH) as the possible pathogenic mechanism underlying the development of the typical NF1 skeletal features. Here, we report a NF1 patient with severe chest deformity and harboring the germline heterozygous pathogenic NF1 variant NM_001042492.3: c.4271delC p.(Ala1424Glufs<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04990005pt" id="M1" height="10.1524pt" version="1.1" viewBox="-0.0498162 -10.1025 6.17869 10.1524" width="6.17869pt"><g transform="matrix(.0091,0,0,-0.0091,0,-5.741)"><path id="g50-43" d="M486 158C486 177 478 202 466 220C413 228 386 236 336 262C386 288 413 297 466 304C478 323 486 347 485 366C470 376 444 381 422 380C389 338 368 319 321 288C323 345 329 372 349 422C339 442 322 461 305 470C289 461 271 442 262 422C281 372 287 345 290 288C243 319 222 338 189 380C167 381 142 376 125 366C125 347 133 322 145 304C198 296 225 288 275 262C225 236 198 227 145 220C133 201 125 177 126 158C141 148 167 143 189 144C222 186 243 205 290 236C288 179 282 152 262 102C272 82 289 63 306 54C322 63 340 82 350 102C330 152 324 179 321 236C368 205 390 186 422 144C444 143 470 148 486 158Z"/></g></svg>4). Through next-generation sequencing (NGS), we investigated the affected cartilage from the PE deformity and identified the additional frameshift variant NM_001042492.3: c.2953delC p.(Gln985Lysfs<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04990005pt" id="M2" height="10.1524pt" version="1.1" viewBox="-0.0498162 -10.1025 6.17869 10.1524" width="6.17869pt"><g transform="matrix(.0091,0,0,-0.0091,0,-5.741)"><use xlink:href="#g50-43"/></g></svg>7), occurring as a somatic NF1 second hit mutation. Exome sequencing confirmed the absence of additional variants of potential pathogenic relevance. Western blot analysis showed the absence of wild-type NF1 protein in the cartilage of the patient, consistent with a somatic double inactivation (SDI) of NF1. Taken together, our findings support the role of SDI in NF1-related PE, widening the spectrum of the pathophysiological mechanisms involved in NF1-related skeletal features.

Cristina Chelleri

Marcello Scala

Patrizia De Marco

Vittorio Guerriero

Marzia Ognibene

Francesca Madia

Sara Guerrisi

Marco Di Duca

Michele Torre

Serena Tamburro

Paolo Scudieri

Gianluca Piccolo

Girolamo Mattioli

Francesca Buffelli

Paolo Uva

Diego Vozzi

Ezio Fulcheri

Pasquale Striano

Maria Cristina Diana

Federico Zara

Somatic Double Inactivation of NF1 Associated with NF1-Related Pectus Excavatum Deformity

Characterized by developmental delay with severe speech delay, dental anomalies, cleft palate, skeletal abnormalities, and behavioral difficulties, SATB2-associated syndrome (SAS) is caused by pathogenic variants in SATB2. The SAS phenotype range of severity has been documented previously in large series. Using data from the SAS registry, we present the SAS severity score, a comprehensive scoring rubric that encompasses 15 different individual neurodevelopmental and systemic features. Higher (more severe) systemic and total (sum of neurodevelopmental and systemic scores) scores were seen for null variants located after amino acid 350 (the start of the CUT1 domain), the recurrent missense Arg389Cys variant (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" width="17.789pt" style="vertical-align:-0.2063904pt" id="M1" height="8.55521pt" version="1.1" viewBox="-0.0498162 -8.34882 17.789 8.55521"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-111" d="M495 86L479 114C446 82 419 66 409 66C401 66 401 72 406 97C420 166 436 231 453 297C489 435 454 448 428 448C406 448 384 439 354 422C305 394 222 327 161 247H159L183 345C200 415 194 448 173 448C143 448 82 410 23 351L38 325C64 349 95 371 105 371C111 371 116 365 109 336L25 -4L31 -12C50 -4 77 3 107 9C119 69 132 122 145 168C197 254 321 381 370 381C387 381 393 374 378 305L329 95C309 17 320 -12 345 -12C372 -12 430 19 495 86Z"/></g><g transform="matrix(.013,0,0,-0.013,10.158,0)"><path id="g117-34" d="M535 323V373H52V323H535ZM535 138V188H52V138H535Z"/></g></svg><svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" width="12.679pt" style="vertical-align:-0.2063904pt" height="8.55521pt" version="1.1" viewBox="21.3711838 -8.34882 12.679 8.55521"><g transform="matrix(.013,0,0,-0.013,21.421,0)"><path id="g113-50" d="M384 0V27C293 34 287 42 287 114V635C232 613 172 594 109 583V559L157 557C201 555 205 550 205 499V114C205 42 199 34 109 27V0H384Z"/></g><g transform="matrix(.013,0,0,-0.013,27.661,0)"><path id="g113-49" d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"/></g></svg>), intragenic deletions, and larger chromosomal deletions. The Arg389Cys variant had the highest cognitive, verbal, and sialorrhea severity scores, while large chromosomal deletions had the highest expressive, ambulation, palate, feeding and growth, neurodevelopmental, and total scores. Missense variants not located in the CUT1 or CUT2 domain scored lower in several subcategories. We conclude that the SAS severity score allows quantitative phenotype morbidity description that can be used in routine clinical counseling. Further refinement and validation of the SAS severity score are expected over time. All data from this project can be interactively explored in a new portal.

Yuri A. Zarate

Katherine Bosanko

Amrit Kannan

Ashlen Thomason

Beth Nutt

Nihit Kumar

Kirt Simmons

Aaron Hiegert

Larry Hartzell

Adam Johnson

Tabitha Prater

Eduardo Pérez-Palma

Tobias Brünger

Arthur Stefanski

Dennis Lal

Aisling R. Caffrey

Quantitative Phenotype Morbidity Description of SATB2-Associated Syndrome

SRRM2 encodes a splicing factor recently implicated in developmental disorders due to a statistical enrichment of de novo mutations. Using data from the 100,000 Genomes Project, four unrelated individuals with intellectual disability (ID) were identified, each harbouring de novo whole gene deletions of SRRM2. Deletions ranged between 248 and 482 kb in size and all distal breakpoints clustered within a complex 144 kb palindrome situated 75 kb upstream of SRRM2. Strikingly, three of the deletions were complex, with inverted internal segments of 45-94 kb. In one proband-mother duo, de novo status was inferred by haplotype analysis. Together with two additional patients who harboured smaller predicted protein-truncating variants (p.Arg632<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04990005pt" id="M1" height="10.1524pt" version="1.1" viewBox="-0.0498162 -10.1025 6.17869 10.1524" width="6.17869pt"><g transform="matrix(.0091,0,0,-0.0091,0,-5.741)"><path id="g50-43" d="M486 158C486 177 478 202 466 220C413 228 386 236 336 262C386 288 413 297 466 304C478 323 486 347 485 366C470 376 444 381 422 380C389 338 368 319 321 288C323 345 329 372 349 422C339 442 322 461 305 470C289 461 271 442 262 422C281 372 287 345 290 288C243 319 222 338 189 380C167 381 142 376 125 366C125 347 133 322 145 304C198 296 225 288 275 262C225 236 198 227 145 220C133 201 125 177 126 158C141 148 167 143 189 144C222 186 243 205 290 236C288 179 282 152 262 102C272 82 289 63 306 54C322 63 340 82 350 102C330 152 324 179 321 236C368 205 390 186 422 144C444 143 470 148 486 158Z"/></g></svg> and p.Ala2223Leufs<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" style="vertical-align:-0.04990005pt" id="M2" height="10.1524pt" version="1.1" viewBox="-0.0498162 -10.1025 6.17869 10.1524" width="6.17869pt"><g transform="matrix(.0091,0,0,-0.0091,0,-5.741)"><use xlink:href="#g50-43"/></g></svg>13), we estimate the prevalence of this condition in cohorts of patients with unexplained ID to be ~1/1300. Phenotypic blending, present for two cases with additional pathogenic variants in CASR/PKD1 and SLC17A5, hampered the phenotypic delineation of this recently described condition. Our data highlights the benefits of genome sequencing for resolving structural complexity and inferring de novo status. The genomic architecture of 16p13.3 may give rise to relatively high rates of complex rearrangements, adding to the list of loci associated with recurrent genomic disorders.

Alistair T. Pagnamenta

Jing Yu

Tracey A. Willis

Mona Hashim

Eleanor G. Seaby

Susan Walker

Jiaqi Xian

Emily W. Y. Cheng

Ana Lisa Taylor Tavares

Francesca Forzano

Helen Cox

Tabib Dabir

Angela F. Brady

Neeti Ghali

Santosh S. Atanur

Sarah Ennis

Diana Baralle

Jenny C. Taylor

A Palindrome-Like Structure on 16p13.3 Is Associated with the Formation of Complex Structural Variations and SRRM2 Haploinsufficiency

The vast majority of patients at risk of hereditary breast and/or ovarian cancer (HBOC) syndrome remain without a molecular diagnosis after routine genetic testing. One type of genomic alteration that is commonly missed by diagnostic pipelines is mobile element insertions (MEIs). Here, we reanalyzed multigene panel data from suspected HBOC patients using the MEI detection tool Mobster. A novel Alu element insertion in ATM intron 54 (ATM:c.8010+30_8010+31insAluYa5) was identified as a potential contributing factor in seven patients. Transcript analysis of patient-derived RNA from three heterozygous carriers revealed exon 54 skipping in 38% of total ATM transcripts. To manifest the direct association between the Alu element insertion and the aberrant splice pattern, HEK293T and MCF7 cells were transfected with wild-type or Alu element-carrying minigene constructs. On average, 77% of plasmid-derived transcripts lacked exon 54 in the presence of the Alu element insertion compared to only 4.7% of transcripts expressed by the wild-type minigene. These results strongly suggest ATM:c.8010+30_8010+31insAluYa5 as the main driver of ATM exon 54 skipping. Since this exon loss is predicted to cause a frameshift and a premature stop codon, mutant transcripts are unlikely to translate into functional proteins. Based on its estimated frequency of up to 0.05% in control populations, we propose to consider ATM:c.8010+30_8010+31insAluYa5 in suspected HBOC patients and to clarify its role in carcinogenesis through future epidemiological and functional analyses. Generally, the implementation of MEI detection tools in diagnostic sequencing pipelines could increase the diagnostic yield, as MEIs are likely underestimated contributors to genetic diseases.

Janin Klein

Aldrige B. Allister

Gunnar Schmidt

Annette Otto

Kai Heinecke

Jördis Bax-Knoche

Carmela Beger

Sarah Becker

Stephan Bartels

Tim Ripperger

Jens Bohne

Thilo Dörk

Brigitte Schlegelberger

Winfried Hofmann

Doris Steinemann

A Novel Alu Element Insertion in ATM Induces Exon Skipping in Suspected HBOC Patients

Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification are essential for reliable diagnosis of DICER1-related tumor predisposition and the identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP) to create DICER1-specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known pathogenic/likely pathogenic (P/LP) variants, 12 known benign/likely benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

Jessica N. Hatton

Megan N. Frone

Hannah C. Cox

Stephanie B. Crowley

Susan Hiraki

Noriko N. Yokoyama

Noura S. Abul-Husn

James F. Amatruda

Michael J. Anderson

Xavier Bofill-De Ros

Ann G. Carr

Elizabeth C. Chao

Kenneth S. Chen

Shuo Gu

Cecilia Higgs

Jerry Machado

Deborah Ritter

Kris Ann P. Schultz

Emily R. Soper

Mona K. Wu

Jessica L. Mester

Jung Kim

William D. Foulkes

Leora Witkowski

Douglas R. Stewart

Specifications of the ACMG/AMP Variant Classification Guidelines for Germline DICER1 Variant Curation

The sarcoglycanopathies are autosomal recessive limb-girdle muscular dystrophies (LGMDs) caused by the mutations in genes encoding the α, β, γ, and δ proteins which stabilizes the sarcolemma of muscle cells. The clinical phenotype is characterized by progressive proximal muscle weakness with childhood onset. Muscle biopsy findings are diagnostic in confirming dystrophic changes and deficiency of one or more sarcoglycan proteins. In this study, we summarized 1,046 LGMD patients for which a precise diagnosis was identified using targeted sequencing. The most frequent phenotypes identified in the patients are LGMDR1 (19.7%), LGMDR4 (19.0%), LGMDR2 (17.5%), and MMD1 (14.5%). Among the reported genes, each of CAPN3, SGCB, and DYSF variants was reported in more than 10% of our study cohort. The most common variant SGCB p.Thr182Pro was identified in 146 (12.5%) of the LGMD patients, and in 97.9% of these patients, the variant was found to be homozygous. To understand the genetic structure of the patients carrying SGCB p.Thr182Pro, we genotyped 68 LGMD patients using a whole genome microarray. Analysis of the array data identified a large ~1 Mb region of homozygosity (ROH) (chr4:51817441-528499552) suggestive of a shared genomic region overlapping the recurrent missense variant and shared across all 68 patients. Haplotype analysis identified 133 marker haplotypes that were present in ~85.3% of the probands as a double allele and absent in all random controls. We also identified 5 markers (rs1910739, rs6852236, rs13122418, rs13353646, and rs6554360) which were present in a significantly higher proportion in the patients compared to random control set (<svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" width="17.789pt" style="vertical-align:-0.2063904pt" id="M1" height="8.55521pt" version="1.1" viewBox="-0.0498162 -8.34882 17.789 8.55521"><g transform="matrix(.013,0,0,-0.013,0,0)"><path id="g113-111" d="M495 86L479 114C446 82 419 66 409 66C401 66 401 72 406 97C420 166 436 231 453 297C489 435 454 448 428 448C406 448 384 439 354 422C305 394 222 327 161 247H159L183 345C200 415 194 448 173 448C143 448 82 410 23 351L38 325C64 349 95 371 105 371C111 371 116 365 109 336L25 -4L31 -12C50 -4 77 3 107 9C119 69 132 122 145 168C197 254 321 381 370 381C387 381 393 374 378 305L329 95C309 17 320 -12 345 -12C372 -12 430 19 495 86Z"/></g><g transform="matrix(.013,0,0,-0.013,10.158,0)"><path id="g117-34" d="M535 323V373H52V323H535ZM535 138V188H52V138H535Z"/></g></svg><svg xmlns:xlink="http://www.w3.org/1999/xlink" xmlns="http://www.w3.org/2000/svg" width="18.943pt" style="vertical-align:-0.2063904pt" height="8.55521pt" version="1.1" viewBox="21.3711838 -8.34882 18.943 8.55521"><g transform="matrix(.013,0,0,-0.013,21.421,0)"><path id="g113-50" d="M384 0V27C293 34 287 42 287 114V635C232 613 172 594 109 583V559L157 557C201 555 205 550 205 499V114C205 42 199 34 109 27V0H384Z"/></g><g transform="matrix(.013,0,0,-0.013,27.661,0)"><path id="g113-51" d="M412 140C382 77 369 73 315 73H129L270 222C362 320 402 379 402 466C402 571 322 635 234 635C177 635 130 609 99 576L42 495L64 475C90 514 133 568 201 568C274 568 318 519 318 435C318 349 255 267 193 193C144 135 87 78 32 23V0H405C417 45 427 89 440 131L412 140Z"/></g><g transform="matrix(.013,0,0,-0.013,33.901,0)"><path id="g113-57" d="M249 635C141 635 70 555 70 471C70 401 114 353 179 316C143 294 106 267 90 252C68 231 45 202 45 157C45 50 130 -12 237 -12C322 -12 435 52 435 169C435 256 372 304 303 343C349 374 375 398 383 407C401 429 411 458 411 487C411 569 344 635 249 635ZM238 603C285 603 337 567 337 482C337 422 310 385 276 358C205 393 145 426 145 500C145 552 179 603 238 603ZM248 20C183 20 125 70 125 163C125 218 158 268 206 300C284 261 355 217 355 143C355 66 308 20 248 20Z"/></g></svg>) and the population database. Of note, admixture analysis was suggestive of greater proportion of West Eurasian/European ancestry as compared to random controls. Haplotype analysis and frequency in the population database indicate a probable event of founder effect. Further systematic study is needed to identify the communities and regions where the SGCB p.Thr182Pro variant is observed in higher proportions. After identifying these communities and//or region, a screening program is needed to identify carriers and provide them counselling.

V. Manjunath

S. G. Thenral

B. R. Lakshmi

Atchayaram Nalini

A. Bassi

K. Priya Karthikeyan

K. Piyusha

R. Menon

A. Malhotra

L. S. Praveena

R. M. Anjanappa

S. M. Sakthivel Murugan

Kiran Polavarapu

Mainak Bardhan

V. Preethish-Kumar

Seena Vengalil

Saraswati Nashi

S. Sanga

M. Acharya

R. Raju

V. R. Pai

V. L. Ramprasad

R. Gupta

Large Region of Homozygous (ROH) Identified in Indian Patients with Autosomal Recessive Limb-Girdle Muscular Dystrophy with p.Thr182Pro Variant in SGCB Gene

Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase-activating protein (GAP) for RAS that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Obtaining a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci, and uncertainty over the effects of some NF1 and SPRED1 variants on pre-mRNA splicing and/or protein expression and function. To improve NF1 and SPRED1 variant classification and establish pathogenicity for NF1 and SPRED1 variants identified in individuals with NF1 or LS, we analyzed patient RNA by RT-PCR and performed in vitro exon trap experiments and estimated NF and SPRED1 protein expression, RAS GAP activity, and interaction. We obtained evidence to support pathogenicity according to American College of Medical Genetics guidelines for 73/114 variants tested, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.

Hannie Douben

Marianne Hoogeveen-Westerveld

Mark Nellist

Jesse Louwen

Marian Kroos-de Haan

Mattijs Punt

Babeth van Ommeren

Leontine van Unen

Peter Elfferich

Esmee Kasteleijn

Yolande van Bever

Margreethe van Vliet

Rianne Oostenbrink

Jasper J. Saris

Anja Wagner

Yvette van Ierland

Tjakko van Ham

Rick van Minkelen

Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for Individuals with Neurofibromatosis Type 1 and Legius Syndrome

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Human Mutation&nbsp;provides a unique forum for the exchange of ideas, methods, and applications of interest to molecular, human, and medical geneticists in academic, industrial, and clinical research settings worldwide.

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Human Mutation provides a unique forum for the exchange of ideas, methods, and applications of interest to molecular, human, and medical geneticists in academic, industrial, and clinical research settings worldwide.

Human Mutation