DNA replication is carried out by the replisome and is essential for maintaining genome integrity and cell proliferation. Pathogenic variants in genes encoding various replisome components cause microcephalic primordial dwarfism (MPD), characterized by growth retardation, microcephaly, and developmental abnormalities. Here, we report bi-allelic hypomorphic variants in WDHD1 as a cause of MPD with a broad spectrum of additional abnormalities, including acute liver failure, in 17 subjects from 14 families. WDHD1 encodes a replisome scaffolding protein (also known as AND-1 and Ctf4), which is essential for replisome assembly, replication fork stability, and sister chromatid cohesion. We found aberrant splicing of WDHD1 pre-mRNAs for all intronic variants tested and markedly reduced WDHD1 protein levels in subject-derived fibroblasts. Fibroblasts with bi-allelic WDHD1 variants showed globally reduced replication fork speed and impaired replication control, accompanied by spontaneous DNA damage and a G1-to-S transition defect. Using various cell biology approaches, we show that subject fibroblasts displayed reduced proliferation, abnormal nuclear morphology, including micronuclei, multilobed, and enlarged nuclei, as well as an increased number of metaphases with premature sister chromatid separation. Together, our findings establish WDHD1 as a protein required for normal organismal growth and development in humans and underscore its multiple functions in maintaining genome integrity.

PurposeAnophthalmia and microphthalmia (A/M) are among the most severe developmental eye defects. The aim of this study is to describe the genetic landscape of fetal syndromic phenotypes that include A/M.MethodsWe recruited 31 fetuses who underwent prenatal ultrasound examination, postnatal assessment, quantitative fluorescent PCR (QF-PCR) and prenatal exome sequencing (pES).ResultsAll cases displayed A/M associated with at least one extra-ocular malformation and of these, cerebellar hypoplasia was the most common ultrasound finding, detected in 14 (45.2%) cases. Chromosomal aneuploidies were identified in seven cases. Among the other fetuses (n=24), pES identified single nucleotide variants (SNVs) in 21 and copy number variations (CNVs) in three. Recurrent genetic diagnoses within our cohort included muscular dystrophy-dystroglycanopathy type A-3 (MDDGA3; n=3), MDDGA1 (n=2), cerebro-oculo-facio-skeletal syndrome 3 (n=2), syndromic microphthalmia type 9 (n=2), and Fraser syndrome (n=2). We report on seven novel variants.ConclusionsThis study broadens the molecular spectrum of syndromic A/M with 19 distinct variants identified across 16 different genes. Furthermore, some variants were detected in genes that have been rarely, or not previously, linked to human A/M, thereby highlighting atypical clinical findings and suggesting a possible expansion of the phenotypic spectrum associated with these genes.

Small nuclear RNAs (snRNAs) are essential components of the spliceosome. De novo variants in snRNA genes RNU4-2 (ReNU syndrome), RNU5B-1 and RNU2-2 have been linked to dominant neurodevelopmental disorders (NDDs), revealing a large unexpected contribution of noncoding RNA genes to genetic diseases. Here, through international collaborations, we analyze systematically 200 potentially functional snRNA genes in a French cohort of 34,329 people with rare disorders. We report RNU2-2 variants in 141 individuals, including 35 with recurrent dominant pathogenic variants and 91 affected members from 73 families with biallelic variants. Recessive RNU2-2 NDD is at least twice as frequent as the dominant form and often involves a de novo variant in trans with an inherited allele, consistent with the high mutability of snRNA genes. Dominant and recessive RNU2-2 NDDs share overlapping clinical features, with frequent epilepsy. Blood transcriptomics and DNA methylation analyses revealed subtle, variant-specific effects on splicing and episignatures. Our results support a gradient-of-impact model bridging dominant and recessive inheritance, and establish RNU2-2 variants as a principal contributor to NDDs, nearly as prevalent as ReNU syndrome.

The nonsense-mediated mRNA decay (NMD) pathway is a translation-dependent mechanism that maintains cellular RNA homeostasis by eliminating mRNAs containing a premature termination codon (PTC). NMD also targets 15

BACKGROUND:Heterozygous pathogenic variants in the central region (exon 23-34) of FBN2 cause a hereditary connective tissue disorder named congenital contractural arachnodactyly, which presents with obligatory skeletal features but rarely with vascular manifestations. Scarce data exist on the association between FBN2 variants and aortic disease. This study aimed to investigate whether the location of FBN2 variants correlates with distinct clinical features, including aortic disease. METHODS:In this case-controlled cohort study, we ascertained clinical features, sequenced 62 (candidate) disease genes, and classified variants according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines in 392 patients with suspected connective tissue or thoracic aortic diseases. We summarized our results and published data and compared clinical manifestations between patients with variants outside and within the central region of FBN2. RESULTS:Heterozygous FBN2 variants outside the central region were identified in 10 patients from 5 families. Two variants were of uncertain significance, 1 was likely pathogenic, and 2 were pathogenic. A total of 60% of these patients had thoracic aortic disease, but only 20% were diagnosed with congenital contractural arachnodactyly according to an established clinical scoring system. Combined data from the literature and this study revealed that patients with FBN2 variants outside the central region presented with aortic dilatation (55.0% versus 9.9%; P<0.001) more often and had less pronounced musculoskeletal manifestations (congenital contractural arachnodactyly score, 5.6±5.1 versus 9.8±3.6; P=0.011) compared with those with central region variants. CONCLUSIONS:Our results suggest that heterozygous FBN2 variants outside the central region predispose individuals to thoracic aortic disease and are less associated with the typical clinical presentation of congenital contractural arachnodactyly than pathogenic variants in the FBN2 central region.

Congenital mirror movements (CMMs) are involuntary movements of one side of the body that mirror intentional movements of the opposite side. DCC, NTN1, RAD51, ARHGEF7, and DNAL4 have been associated with CMMs. Two-thirds of CMM-affected individuals remain without a genetic diagnosis, indicating that variants in additional genes need to be discovered. We report on a 27-year-old female with CMMs of the hands. Trio exome sequencing in the proband and healthy parents did not reveal a likely pathogenic variant in one of the CMM-associated genes but rather a de novo heterozygous frameshift variant c.523dup (p.Ser175Lysfs∗8) in the candidate RBM15. The variant results in only partial nonsense-mediated mRNA decay of RBM15 transcripts in the proband's lymphoblastoid cells. RBM15 encodes an RNA-binding protein involved in alternative splicing as well as other processes. Dcc alternative splicing generates Dcclong and Dccshort isoforms, which are important for commissural axon midline crossing. We tested whether Rbm15 regulates Dcc alternative splicing by using an in vitro minigene assay. Ectopic expression of Rbm15, similar to the splicing factors Nova1 and Nova2, promotes the production of Dcclong transcripts. The possible link between Rbm15 and Dcc supports a role for Rbm15 in CMMs.

A 16-yr-old male with a genetically undiagnosed neurodevelopmental disorder (NDD) was admitted to our outpatient clinic for skeletal assessment. DXA and HR-pQCT showed a severely reduced BMD and a pronounced reduction of trabecular and cortical bone mass. Lateral vertebral assessment identified multiple previously unrecognized vertebral fractures of the thoracic and lumbar spine. Laboratory tests indicated an activated bone turnover, which was confirmed by an increased number of osteoclasts and osteoblasts in an undecalcified tibia biopsy of the patient. Treatment of the severe osteoporosis was initiated with neridronate. Trio exome sequencing in the patient and healthy parents did not uncover a genetic cause of the disease. Importantly, however, targeted sequencing of the RNU4-2 gene, which encodes the U4 small nuclear RNA (a major component of the splicing machinery), identified a heterozygous causative variant in the patient. This led to the molecular diagnosis of ReNU syndrome. RNU4-2 pathogenic variants underlie a NDD with multisystemic involvement, including skeletal abnormalities. Therefore, this case not only underlines the relevance of osteologic assessment and therapy in individuals with NDDs, but also highlights the necessity of future research efforts to elucidate the bone pathologies in ReNU syndrome.

Biallelic variants in NUP107 cause isolated or syndromic steroid-resistant nephrotic syndrome (SRNS), characterised by proteinuria, hypoalbuminaemia and focal segmental glomerulosclerosis that progresses to end-stage renal disease. Patients with syndromic SRNS have microcephaly, developmental delay or intellectual disability and short stature. Simplified gyration is observed in some individuals. We report on a 2-year-old girl with novel biallelic NUP107 variants, c.2606G>T; p.(Gly869Val) and c.1576+1G>A, proteinuria and a severe neurodevelopmental disorder with microcephaly, developmental delay, early-onset seizures, sensorineural hearing loss and brain structural anomalies, including simplified gyral pattern and hypoplasia of the corpus callosum, pons, brainstem and cerebellum. NUP107 is part of the NUP107-160 complex, which, together with other proteins termed nucleoporins, forms the nuclear pore complex (NPC). The NPC regulates nucleocytoplasmic transport and other cellular processes. In patient-derived fibroblasts, we identified aberrantly spliced NUP107 mRNAs with a frameshift and premature stop codon leading to non-sense-mediated mRNA decay, reduced levels of NUP107 transcripts, reduced NUP107 and NUP133 proteins, and a reduced NPC number. In addition, an abnormal nucleolar morphology was found in patient-derived cells. Our functional data support the conclusion that the NUP107 variants underlie the patient's phenotype, thereby broadening the clinical spectrum associated with NUP107 variants to include abnormal brain development.

Background Marfan syndrome (MFS) guidelines recommend optimal pharmacological therapy (OPT) and replacement of the ascending aorta (RAA) at 5.0cm diameters to prevent acute type A aortic dissection (ATAAD) and death. The effect of early MFS diagnosis and initiation of therapy on outcomes is not known. Objective To evaluate the effect of age at MFS diagnosis and therapy initiation on delayed RAA and death. Methods This retrospective observational cohort study with long-term follow-up included consecutive patients with MFS, pathogenic FBN1 variant, and regular visits to a European Reference Network Center. We considered MFS diagnosis at age ≥21 years late, and OPT initiation at age <21 years early. Outcomes were delayed RAA with aneurysm diameter >5.0cm or ATAAD, and death from all causes. We used landmark design starting at age 21 years to determine associations with outcomes. Results The study group consisted of 288 patients (45.1% male), including 169 patients with late MFS diagnosis (58.7%) and 63 with early OPT (21.9%). During mean follow-up of 25±14.7 years, 78 patients had delayed RAA, with 42 operations for ATAAD and 36 for aneurysms ≥5.0cm. There were 33 deaths, including 11 deaths late after ATAAD. All deaths were cardiovascular. Late diagnosis, but not early OPT, showed univariate association with delayed RAA (P<0.001) and death (P=0.025). Multivariate Cox regression analysis confirmed late diagnosis as predictor of delayed RAA (hazard ratio (HR)=8.01; 95% confidence interval (95%CI) 2.52-25.45; P<0.001) and death (HR=4.68; 95%CI 1.17-18.80; P=0.029). Conclusions Late diagnosis of Marfan syndrome is associated with delayed surgery and death.

We report two rare homozygous variants, including a recurrent missense and intronic variant, in the EIF3K gene in four unrelated individuals with global developmental delay, microcephaly, proportionate short stature, dysmorphic craniofacial features, digit flexion deformities, and the cardiac anomaly, patent ductus arteriosus. Three individuals, who were all of Puerto Rican descent, were homozygous for the NM_013234.3:c.128A>G; p.(Asp43Gly) variant in EIF3K and homozygous for a missense variant in SYNE4 (NM_001039876.2:c.355C>T; p.(Arg119Trp)). SYNE4 is associated with autosomal recessive bilateral sensorineural hearing loss, which was also reported in these probands. Analysis of our dataset confirmed these EIF3K and SYNE4 variants were in linkage disequilibrium in affected individuals, suggesting a possible common ancestor and founder event. A fourth individual from Egypt harbored the homozygous intronic variant c.355-13A>G in EIF3K, which segregated with the phenotype in the family and led to aberrant splicing of EIF3K pre-mRNAs, as shown by insertion of 12 intronic base pairs, skipping of 2 exons, and significantly reduced EIF3K protein levels in skin fibroblasts. Through genetic and functional approaches, we suggest that biallelic EIF3K variants are associated with an autosomal recessive syndromic neurodevelopmental disorder with growth retardation, microcephaly, congenital heart defect, and other anomalies.

The T-type voltage-gated calcium channel CaV3.3 is expressed in GABAergic neurons of the thalamic reticular nucleus (TRN), where its pacemaking activity controls sleep spindle rhythmogenesis during the non-rapid eye movement (NREM) phase of natural sleep. Previously, we established CACNA1I, the gene coding for CaV3.3, as a disease gene for neurodevelopmental disease with or without epilepsy. Here we report three newly identified activation-gate-modifying heterozygous missense variants of CACNA1I, found in four unrelated patients with neurodevelopmental disease with or without seizures. One of these variants, p.(Met1425Val), is an amino-acid substitution at the same position as previously published variant p.(Met1425Ile). Notably, the other two variants studied here are also a pair of two different substitutions of the same amino acid: p.(Ala398Val) and p.(Ala398Glu). By using site-directed mutagenesis, voltage-clamp electrophysiology, computational modelling of neuronal excitability, and structure modelling, we found that the two substitutions of M1425 both result in a gain of channel function including left-shifted voltage-dependence of activation and inactivation, slowed inactivation and deactivation kinetics, and increased neuronal excitability. Remarkably, the two substitutions of A398 show opposite effects on channel function. While substitution A398E leads to a gain of channel function, A398V results in decreased current density, accelerated gating kinetics, and a decreased neuronal excitability. The lack of seizures in the two independent p.(Ala398Val) patients correlates with the absence of increased neuronal excitability in this variant. This is the first report of a gate-modifying CaV3.3 channel variant with partial loss-of-function effects associated with developmental delay and intellectual disability without seizures. Our study corroborates the role of CaV3.3 dysfunction in the etiology of neurodevelopmental disorders. Moreover, our data suggest that substantial gain-of-function of CaV3.3 leads to the development of seizures, whereas both gain- and loss-of-function variants of CACNA1I can cause neurodevelopmental disease.

Ribosomes are ribonucleoproteins that are responsible for protein synthesis. They consist of ribosomal proteins and ribosomal RNAs (rRNAs). Pre-rRNAs are co-transcriptionally processed and chemically modified. The 2'-O-methylation of rRNAs is guided by box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which are composed of a box C/D snoRNA and the core proteins NOP56, NOP58, SNU13, and the methyltransferase fibrillarin. Catalytically active box C/D snoRNPs function in nucleoli. We performed trio whole-exome sequencing in a proband with a severe neurodevelopmental disorder including global developmental delay, microcephaly, seizures, and ophthalmological and brain abnormalities and his healthy parents and identified the homozygous synonymous variant c.516G>A; p.Leu172= in NOP58. In fibroblasts of the proband, we demonstrated skipping of exon 7 in most NOP58 mRNAs, while ∼20% canonically spliced NOP58 transcripts were detected in the proband compared with control cells. NOP58 protein levels were reduced to ∼12% in proband cells that concomitantly reduced fibrillarin levels. Analysis of nucleoli in proband-derived fibroblasts revealed changes in the number of nucleolar condensates and in nucleolar morphology. We found reduced levels of three box C/D snoRNAs required for 2'-O-methylation and of one box C/D snoRNA important for 2'-O-methylation and pre-rRNA processing. Analysis of pre-rRNA maturation by RT-qPCR revealed increased 45S and 21S pre-rRNA levels, whereas the amplification signal for the 47S, 32S, and 26S pre-rRNAs was substantially decreased in proband compared with control cells. Together, our data unveil that the homozygous NOP58 variant c.516G>A represents a hypomorphic allele and underlies the neurodevelopmental phenotype in the proband, likely by impairing pre-rRNA maturation.

Ectodermal dysplasia is a genetically and clinically heterogeneous condition that is caused by developmental defects of hair, teeth, nails, and certain glands. Biallelic KREMEN1 variants cause severe tooth agenesis and mild ectodermal features. KREMEN1 encodes a transmembrane receptor that binds DKK1 and LRP5/6 that are implicated in the WNT/β-catenin pathway. We report on 3 patients from 2 families with oligodontia and sparse scalp hair and eyebrows with the, to our knowledge, previously unreported homozygous KREMEN1 variants c.497G>A; p.(Gly166Asp) and c.136C>T; p.(Gln46∗). To gain insight into the functional consequences of KREMEN1 pathogenic variants, we ectopically expressed C-terminally Flag-tagged KREMEN1 wild-type and the protein variants Cys111Ser, Gly166Asp, Phe209Ser, and Phe258_Pro259del in human embryonic kidney 293T cells. KREMEN1 wild type was extensively N- and O-glycosylated, whereas the 4 protein variants showed a significant reduction in glycosylation, in particular of O-glycans. Ternary complex formation of all 4 KREMEN1 protein variants with DKK1 and LRP6 was reduced compared with that of wild type, whereas formation of the KREMEN1-LRP6 complex was not affected by the nonsynonymous variants. Primary fibroblasts from the 3 patients with KREMEN1 pathogenic variants showed a higher WNT pathway activity under starved culture condition that was followed by attenuated signaling response to WNT3A, suggesting a more general WNT pathway deregulation.

Pregnancy loss is a major problem in clinical medicine with devastating consequences for families. Next generation sequencing has improved our ability to identify underlying molecular causes, though over half of all cases lack a clear etiology. Here, we began with clinical evaluation combined with exome sequencing across independent families to identify bi-allelic candidate genetic variants in the Programmed Cell Death 2 (PDCD2) gene in multiple fetuses with nonimmune hydrops fetalis (NIHF). PDCD2 is an evolutionarily conserved protein with no prior association with monogenic disorders. PDCD2 is known to act as a molecular chaperone for the ribosomal protein uS5, and this complex formation is important for incorporation of uS5 into the 40S subunit, a crucial step in ribosome biogenesis. Primary fibroblasts from an affected fetus and cell lines expressing PDCD2 patient variants demonstrated reduced levels of PDCD2, reduced PDCD2 binding to uS5, and altered ribosomal RNA processing. Xenopus tadpoles with Pdcd2 knockdown demonstrated developmental defects and edema, reminiscent of the NIHF seen in affected fetuses, and showed altered ribosomal RNA processing. Through genetic, biochemical, and in vivo approaches, we provide evidence that bi-allelic PDCD2 variants cause an autosomal recessive ribosomal biogenesis disorder resulting in pregnancy loss.

Biallelic variants in COL25A1 have been associated with isolated congenital cranial dysinnervation disorders (CCDDs) and arthrogryposis multiplex congenital (AMC) with or without CCDD. COL25A1 encodes collagen XXV that belongs to the subfamily of membrane-associated collagens with interrupted triple helices. COL25A1 contains four non-collagenous and three collagenous domains. Three alternatively spliced COL25A1 transcript variants are known. In mice, Col25a1 is required for intramuscular motor innervation and cranial motor neuron development. We report seven subjects with novel biallelic COL25A1 pathogenic variants, including three AMC-affected individuals, one of whom died in infancy, and four unrelated fetuses. We expand the associated phenotypic spectrum as fetuses showed lethal phenotypes including reduced or no movement, contractures, and hydrops in three and growth retardation and skeletal abnormalities in one. The molecular spectrum includes two microdeletions encompassing several 5′ or 3′ exons, two missense, one nonsense, one frameshift, and one variant affecting splicing. In fibroblasts of the subject who was compound heterozygous for the c.367G > C and c.1198G > T variants, we identified skipping of exon 3 in COL25A1 mRNAs due to the G-to-C change. These aberrantly spliced transcripts were subject to nonsense-mediated mRNA decay. Analysis of transcriptome sequencing data from primary human fibroblasts without COL25A1 pathogenic variants revealed novel COL25A1 exon-exon junctions and 13 not previously annotated alternatively spliced in-frame exons. We hypothesized that interindividual variation in the splicing of COL25A1 exons in different tissues may underlie the variable phenotypes in the affected individuals.

Biallelic variants in PISD cause a phenotypic spectrum ranging from short stature with spondyloepimetaphyseal dysplasia (SEMD) to a multisystem disorder affecting eyes, ears, bones, and brain. PISD encodes the mitochondrial-localized enzyme phosphatidylserine decarboxylase. The PISD precursor is self-cleaved to generate a heteromeric mature enzyme that converts phosphatidylserine to the phospholipid phosphatidylethanolamine. We describe a 17-year-old male patient, born to unrelated healthy parents, with disproportionate short stature and SEMD, featuring platyspondyly, prominent epiphyses, and metaphyseal dysplasia. Trio genome sequencing revealed compound heterozygous PISD variants c.569C>T; p.(Ser190Leu) and c.799C>T; p.(His267Tyr) in the patient. Investigation of fibroblasts showed similar levels of the PISD precursor protein in both patient and control cells. However, patient cells had a significantly higher proportion of fragmented mitochondria compared to control cells cultured under basal condition and after treatment with 2-deoxyglucose that represses glycolysis and stimulates respiration. Structural data from the PISD orthologue in Escherichia coli suggest that the amino acid substitutions Ser190Leu and His267Tyr likely impair PISD's autoprocessing activity and/or phosphatidylethanolamine biosynthesis. Based on the data, we propose that the novel PISD p.(Ser190Leu) and p.(His267Tyr) variants likely act as hypomorphs and underlie the pure skeletal phenotype in the patient.

VLDLR cerebellar hypoplasia is characterized by intellectual disability, non-progressive cerebellar ataxia, and seizures. The characteristic MRI findings include hypoplasia of the inferior portion of the cerebellar vermis and hemispheres, simplified cortical gyration, and a small brain stem. Biallelic VLDLR pathogenic variants cause loss-of-function of the encoded very low-density lipoprotein receptor. VLDLR exons 4 and 16 are alternatively spliced, resulting in the expression of four transcript variants, including two exon 4-lacking mRNAs expressed in the human brain. Previously reported VLDLR pathogenic variants affect all four transcript variants. Here we report on two sisters with facial dysmorphism, microcephaly, intellectual disability, and normal brain imaging. Exome sequencing in one patient identified the homozygous VLDLR nonsense variant c.376C>T; p.(Gln126*) in exon 4; her similarly affected sister also carried the homozygous variant and parents were heterozygous carriers. VLDLR transcript analysis identified mRNAs with and without exon 4 in patient fibroblasts, while exon 4-containing VLDLR mRNAs were predominantly detected in control fibroblasts. We found significantly reduced VLDLR mRNA levels in patient compared to control cells, likely caused by nonsense-mediated mRNA decay of exon 4-containing VLDLR transcripts. Expression of neuronal VLDLR isoforms produced from exon 4-lacking transcripts may have protected both patients from developing the cerebellar hypoplasia phenotype.

Homozygous VPS50 variants have been previously described in two unrelated patients with a neurodevelopmental disorder with microcephaly, seizures and neonatal cholestasis. VPS50 encodes a subunit that is unique to the heterotetrameric endosome-associated recycling protein (EARP) complex. The other subunits of the EARP complex, such as VPS51, VPS52 and VPS53, are also shared by the Golgi-associated retrograde protein complex. We report on an 18-month-old female patient with biallelic VPS50 variants. She carried a paternally inherited heterozygous nonsense c.13A>T; p.(Lys5*) variant. By long-read genome sequencing, we characterised a structural variant with a 4.3 Mb inversion flanked by deletions at both breakpoints on the maternal allele. The ~428 kb deletion at the telomeric inversion breakpoint encompasses the entire VPS50 gene. We demonstrated a deficiency of VPS50 in patient-derived fibroblasts, confirming the loss-of-function nature of both VPS50 variants. VPS53 and VPS52 protein levels were significantly reduced and absent, respectively, in fibroblasts of the patient. These data show that VPS50 and/or EARP deficiency and the associated functional defects underlie the phenotype in patients with VPS50 pathogenic variants. The VPS50-related core phenotype comprises severe developmental delay, postnatal microcephaly, hypoplastic corpus callosum, neonatal low gamma-glutamyl transpeptidase cholestasis and failure to thrive. The disease is potentially fatal in early childhood.