EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion-Almeida type.

Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.

Thomas HB ，Wood KA ，Buczek WA ，Gordon CT ，Pingault V ，Attié-Bitach T ，Hentges KE ，Varghese VC ，Amiel J ，Newman WG ，O'Keefe RT ... -  《-》

Mandibulofacial Dysostosis with Microcephaly: Mutation and Database Update.

Mandibulofacial dysostosis with microcephaly (MFDM) is a multiple malformation syndrome comprising microcephaly, craniofacial anomalies, hearing loss, dysmorphic features, and, in some cases, esophageal atresia. Haploinsufficiency of a spliceosomal GTPase, U5-116 kDa/EFTUD2, is responsible. Here, we review the molecular basis of MFDM in the 69 individuals described to date, and report mutations in 38 new individuals, bringing the total number of reported individuals to 107 individuals from 94 kindreds. Pathogenic EFTUD2 variants comprise 76 distinct mutations and seven microdeletions. Among point mutations, missense substitutions are infrequent (14 out of 76; 18%) relative to stop-gain (29 out of 76; 38%), and splicing (33 out of 76; 43%) mutations. Where known, mutation origin was de novo in 48 out of 64 individuals (75%), dominantly inherited in 12 out of 64 (19%), and due to proven germline mosaicism in four out of 64 (6%). Highly penetrant clinical features include, microcephaly, first and second arch craniofacial malformations, and hearing loss; esophageal atresia is present in an estimated ∼27%. Microcephaly is virtually universal in childhood, with some adults exhibiting late "catch-up" growth and normocephaly at maturity. Occasionally reported anomalies, include vestibular and ossicular malformations, reduced mouth opening, atrophy of cerebral white matter, structural brain malformations, and epibulbar dermoid. All reported EFTUD2 mutations can be found in the EFTUD2 mutation database (http://databases.lovd.nl/shared/genes/EFTUD2).

Huang L ，Vanstone MR ，Hartley T ，Osmond M ，Barrowman N ，Allanson J ，Baker L ，Dabir TA ，Dipple KM ，Dobyns WB ，Estrella J ，Faghfoury H ，Favaro FP ，Goel H ，Gregersen PA ，Gripp KW ，Grix A ，Guion-Almeida ML ，Harr MH ，Hudson C ，Hunter AG ，Johnson J ，Joss SK ，Kimball A ，Kini U ，Kline AD ，Lauzon J ，Lildballe DL ，López-González V ，Martinezmoles J ，Meldrum C ，Mirzaa GM ，Morel CF ，Morton JE ，Pyle LC ，Quintero-Rivera F ，Richer J ，Scheuerle AE ，Schönewolf-Greulich B ，Shears DJ ，Silver J ，Smith AC ，Temple IK ，UCLA Clinical Genomics Center ，van de Kamp JM ，van Dijk FS ，Vandersteen AM ，White SM ，Zackai EH ，Zou R ，Care4Rare Canada Consortium ，Bulman DE ，Boycott KM ，Lines MA ... -  《-》

Disease modeling of core pre-mRNA splicing factor haploinsufficiency.

The craniofacial disorder mandibulofacial dysostosis Guion-Almeida type is caused by haploinsufficiency of the U5 snRNP gene EFTUD2/SNU114. However, it is unclear how reduced expression of this core pre-mRNA splicing factor leads to craniofacial defects. Here we use a CRISPR-Cas9 nickase strategy to generate a human EFTUD2-knockdown cell line and show that reduced expression of EFTUD2 leads to diminished proliferative ability of these cells, increased sensitivity to endoplasmic reticulum (ER) stress and the mis-expression of several genes involved in the ER stress response. RNA-Seq analysis of the EFTUD2-knockdown cell line revealed transcriptome-wide changes in gene expression, with an enrichment for genes associated with processes involved in craniofacial development. Additionally, our RNA-Seq data identified widespread mis-splicing in EFTUD2-knockdown cells. Analysis of the functional and physical characteristics of mis-spliced pre-mRNAs highlighted conserved properties, including length and splice site strengths, of retained introns and skipped exons in our disease model. We also identified enriched processes associated with the affected genes, including cell death, cell and organ morphology and embryonic development. Together, these data support a model in which EFTUD2 haploinsufficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on gene expression, including altering the expression of ER stress response genes and genes involved in the development of the craniofacial region. The increased burden of unfolded proteins in the ER resulting from mis-splicing would exceed the capacity of the defective ER stress response, inducing apoptosis in cranial neural crest cells that would result in craniofacial abnormalities during development.

Wood KA ，Rowlands CF ，Qureshi WMS ，Thomas HB ，Buczek WA ，Briggs TA ，Hubbard SJ ，Hentges KE ，Newman WG ，O'Keefe RT ... -  《-》

Delineation of EFTUD2 haploinsufficiency-related phenotypes through a series of 36 patients.

Mandibulofacial dysostosis, Guion-Almeida type (MFDGA) is a recently delineated multiple congenital anomalies/mental retardation syndrome characterized by the association of mandibulofacial dysostosis (MFD) with external ear malformations, hearing loss, cleft palate, choanal atresia, microcephaly, intellectual disability, oesophageal atresia (OA), congenital heart defects (CHDs), and radial ray defects. MFDGA emerges as a clinically recognizable entity, long underdiagnosed due to highly variable presentations. The main differential diagnoses are CHARGE and Feingold syndromes, oculoauriculovertebral spectrum, and other MFDs. EFTUD2, located on 17q21.31, encodes a component of the major spliceosome and is disease causing in MFDGA, due to heterozygous loss-of-function (LoF) mutations. Here, we describe a series of 36 cases of MFDGA, including 24 previously unreported cases, and we review the literature in order to delineate the clinical spectrum ascribed to EFTUD2 LoF. MFD, external ear anomalies, and intellectual deficiency occur at a higher frequency than microcephaly. We characterize the evolution of the facial gestalt at different ages and describe novel renal and cerebral malformations. The most frequent extracranial malformation in this series is OA, followed by CHDs and skeletal abnormalities. MFDGA is probably more frequent than other syndromic MFDs such as Nager or Miller syndromes. Although the wide spectrum of malformations complicates diagnosis, characteristic facial features provide a useful handle.

Lehalle D ，Gordon CT ，Oufadem M ，Goudefroye G ，Boutaud L ，Alessandri JL ，Baena N ，Baujat G ，Baumann C ，Boute-Benejean O ，Caumes R ，Decaestecker C ，Gaillard D ，Goldenberg A ，Gonzales M ，Holder-Espinasse M ，Jacquemont ML ，Lacombe D ，Manouvrier-Hanu S ，Marlin S ，Mathieu-Dramard M ，Morin G ，Pasquier L ，Petit F ，Rio M ，Smigiel R ，Thauvin-Robinet C ，Vasiljevic A ，Verloes A ，Malan V ，Munnich A ，de Pontual L ，Vekemans M ，Lyonnet S ，Attié-Bitach T ，Amiel J ... -  《-》

Mandibulofacial dysostosis Guion-Almeida type caused by novel EFTUD2 splice site variants in two Asian children.

Yu KPT ，Luk HM ，Gordon CT ，Fung G ，Oufadem M ，Garcia-Barcelo MM ，Amiel J ，Chung BHY ，Lo IFM ，Tiong YT ... -  《-》